Milk casein-derived tripeptides: A special focus on blood pressure and vascular function

Pauliina Ehlers (ne Jkl)

Institute of Biomedicine, Pharmacology University of Helsinki

ACADEMIC DISSERTATION

To be presented by kind permission of the Medical Faculty of the University of Helsinki for public examination in Lecture Hall 3, Biomedicum Helsinki, Haartmaninkatu 8, on September 24th, 2010, at 12 noon.

Helsinki 2010

SUPERVISORS Professor Heikki Vapaatalo, MD, PhD Institute of Biomedicine, Pharmacology University of Helsinki Helsinki, Finland Professor Riitta Korpela, PhD Institute of Biomedicine, Pharmacology University of Helsinki Helsinki, Finland

REVIEWERS Docent Risto Kerkel, MD, PhD Institute of Biomedicine, Department of Pharmacology and Toxicology University of Oulu Oulu, Finland Professor Hannu J. Korhonen, PhD Biotechnology and Food Research MTT Agrifood Research Finland Jokioinen, Finland

OPPONENT Professor Ilkka Prsti, MD, PhD Medical School Internal Medicine University of Tampere Tampere, Finland

ISBN 978-952-92-7829-9 (paperback) ISBN 978-952-10-6430-2 (PDF) http://ethesis.helsinki.fi Helsinki University Print 2010 Helsinki 2010

To Henkka

TABLE OF CONTENTS

List of original publications

Abbreviations .......................................................................................................................................... 7

Abstract ................................................................................................................................................... 9

1. Introduction ................................................................................................................................ 11 2. Review of the literature.............................................................................................................. 13 2.1. Circulatory system ................................................................................................................. 13 2.2. Arterial structure and function.............................................................................................. 14 2.2.1. Vascular smooth muscle .............................................................................................. 15 2.2.2. Endothelium ................................................................................................................ 18 2.3. Regulation of blood pressure ................................................................................................ 27 2.3.1. Nervous system ........................................................................................................... 27 2.3.2. Renin-angiotensin system (RAS) .................................................................................. 30 2.3.3. Kallikrein-kinin system (KKS)........................................................................................ 36 2.4. Hypertension and vascular disease ....................................................................................... 37 2.4.1. Experimental models of hypertension ........................................................................ 39 2.4.2. Arterial stiffness........................................................................................................... 41 2.4.3. Endothelial dysfunction ............................................................................................... 45 2.4.4. Pharmacological treatment ......................................................................................... 49 2.4.5. Non-pharmacological treatment ................................................................................. 52 2.5. Antihypertensive effect of milk and its components ............................................................ 53 2.5.1. Minerals ....................................................................................................................... 54 2.5.2. Protein ......................................................................................................................... 55 2.5.3. Milk as a source of bioactive peptides ........................................................................ 56 2.5.4. Antihypertensive peptides from milk proteins............................................................ 58 2.6. Casein-derived tripeptides Ile-Pro-Pro and Val-Pro-Pro ....................................................... 59 2.6.1. Experimental studies ................................................................................................... 59 2.6.2. Clinical studies ............................................................................................................. 64 2.6.3. Biochemical aspects..................................................................................................... 67

3. Aims of the study ........................................................................................................................ 69 4. Materials and methods .............................................................................................................. 71 4.1. Experimental animals and study designs .............................................................................. 71 4.2. Subjects and study design (study V) ...................................................................................... 72 4.3. Study products....................................................................................................................... 74 4.4. Compounds ........................................................................................................................... 77 4.5. Methods ................................................................................................................................ 77 4.5.1. Blood pressure measurement ..................................................................................... 77 4.5.2. Vascular function assessment ..................................................................................... 78 4.5.3. Collection of the samples ............................................................................................ 80 4.5.4. Biochemical determinations ........................................................................................ 81 4.6. Statistical analysis .................................................................................................................. 83 4.7. Ethics ..................................................................................................................................... 84 5. Results ........................................................................................................................................ 85 5.1. Intake of tripeptides Ile-Pro-Pro, Val-Pro-Pro, minerals and plant sterols ........................... 85 5.2. Blood pressure....................................................................................................................... 85 5.3. Vascular function and arterial stiffness ................................................................................. 88 5.3.1. In vitro study (study I) .................................................................................................. 88 5.3.2. Long-term experimental studies (studies II, III and IV)................................................ 89 5.3.3. Clinical study ................................................................................................................ 91 5.4. Renin-angiotensin system and L-arginine-NO pathway ......................................................... 92 6. Discussion ................................................................................................................................... 93 6.1. Methodological aspects ........................................................................................................ 93 6.2. Antihypertensive effects of Ile-Pro-Pro and Val-Pro-Pro ...................................................... 97 6.2.1. Experimental studies ................................................................................................... 97 6.2.2. Clinical study .............................................................................................................. 100 6.3. Protective effect of Ile-Pro-Pro and Val-Pro-Pro on vascular function ............................... 101 6.4. Mechanistic insights into the beneficial effects of Ile-Pro-Pro and Val-Pro-Pro ................. 105 6.5. Clinical relevance and open questions ................................................................................ 107 7. Summary and conclusions ........................................................................................................ 109 8. Acknowledgements .................................................................................................................. 111 9. References ................................................................................................................................ 113

Original publications ........................................................................................................................... 145

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original publications (Studies I-V) and some unpublished data.

Jkl P, Jauhiainen T, Korpela R, Vapaatalo H. Milk protein-derived bioactive tripeptides IlePro-Pro and Val-Pro-Pro protect endothelial function in vitro in hypertensive rats. J Functional Foods 2009; 1: 266-273.

II

Jkl P, Pere E, Lehtinen R, Turpeinen A, Korpela R, Vapaatalo H. Cardiovascular activity of milk casein-derived tripeptides and plant sterols in spontaneously hypertensive rats. J Physiol Pharmacol 2009; 60: 11-20.

III

Jkl P, Hakala A, Turpeinen AM, Korpela R, Vapaatalo H. Casein-derived bioactive tripeptides Ile-Pro-Pro and Val-Pro-Pro attenuate the development of hypertension and improve endothelial function in salt-loaded Goto-Kakizaki rats. J Functional Foods 2009; 1: 366-374.

IV

Jkl P, Turpeinen AM, Rajakari K, Korpela R, Vapaatalo H. Biological effects of caseinderived tripeptide powders are not affected by fermentation process. Int Dairy J 2010; 20: 366-370.

Turpeinen AM, Ehlers P, Hakala A, Jrvenp S, Filler I, Wiegert E, Jnchen E, Vapaatalo H, Korpela R, Wagner F. Ile-Pro-Pro and Val-Pro-Pro tripeptide-containing milk product has acute blood pressure lowering effects in mildly hypertensive subjects. Submitted.

The original publications are reprinted with the kind permission of the copyright holders.

ABBREVIATIONS

ACE ACE2 AIx Ang I Ang II Ang (1-7) AT1 AT2 BKCa cGMP COX DBP dTGR EC EDCF EDHF eNOS GK IKCa Ile-Pro-Pro NO PGI2 PP PWV PWA RAS ROS SBP sGC SHR SKCa Val-Pro-Pro VSMC WKY

angiotensin-converting enzyme angiotensin-converting enzyme 2 augmentation index angiotensin I angiotensin II angiotensin 1-7 angiotensin II type 1 receptor angiotensin II type 2 receptor large conductance calcium-activated potassium channel cyclic guanosine monophosphate cyclo-oxygenase diastolic blood pressure double transgenic rat endothelial cell endothelium-derived contracting factor endothelium-derived hyperpolarizing factor endothelial nitric oxide synthase Goto-Kakizaki rat intermediate conductance calcium-activated potassium channel isoleucine-proline-proline nitric oxide prostacyclin pulse pressure pulse-wave velocity pulse-wave analysis renin-angiotensin system reactive oxygen species systolic blood pressure soluble guanylyl cyclase spontaneously hypertensive rat small conductance calcium-activated potassium channel valine-proline-proline vascular smooth muscle cell Wistar-Kyoto rat

ABSTRACT
Increased consumption of low-fat milk products is inversely associated with the risk of hypertension. The beneficial effect of milk on blood pressure is attributed to high calcium and potassium content but also to specific peptide sequences, which are cleaved from milk protein during gastrointestinal digestion, fermentation of milk with proteolytic starter cultures or enzymatic hydrolysis. Milk products fermented with Lactobacillus helveticus contain casein-derived tripeptides isoleucineproline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro), which have been shown to possess antihypertensive effects in humans and in experimental animals. The aim of the present series of studies was to investigate the effects of tripeptides Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them on vascular function and blood pressure and to elucidate the mechanisms behind them by using different experimental models of hypertension. Another aim was to characterize the acute effects of tripeptides on blood pressure and arterial stiffness in mildly hypertensive humans. Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them attenuated the development of hypertension in two experimental models of hypertension, spontaneously hypertensive rat (SHR) and type 2 diabetic Goto-Kakizaki (GK) rat fed with high-salt diet. Significant differences in systolic blood pressure (SBP) were seen after 8 weeks treatment with tripeptidecontaining products compared to control product. Plant sterols did not enhance this effect. Two differently produced tripeptide powders produced a similar attenuating effect on SBP in SHR. In mildly hypertensive subjects, a single administration of tripeptide- and plant sterol-containing fermented milk product decreased both SBP and diastolic blood pressure (DBP) over a period of 8 hours. Protective effect of tripeptides Ile-Pro-Pro and Val-Pro-Pro and fermented milk products containing them on vascular function was demonstrated in in vitro studies and long-term experimental studies. The effect was shown to be endothelium-dependent and possibly involving endothelium-derived hyperpolarizing factor (EDHF). In the clinical study, single administration of tripeptide-containing fermented milk product did not affect measures of arterial stiffness. Long-term treatment with fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro inhibited angiotensin-converting enzyme (ACE) and decreased aldosterone levels thus showing beneficial effects on the reninangiotensin system (RAS) in SHR and GK. No changes in the components of RAS were observed by the single administration of the same product in mildly hypertensive subjects. Increased levels of cGMP, NOX and citrulline suggest increased nitric oxide (NO) production by the tripeptides.

Taken together, Ile-Pro-Pro and Val-Pro-Pro -containing products attenuate the development of hypertension after long-term treatment in experimental models of hypertension and possess an acute antihypertensive effect in mildly hypertensive subjects. In addition, these tripeptides show endothelium-mediated beneficial effects on vascular function. Attenuation of blood pressure increase by the tripeptides in experimental animals involves RAS, but its role in the antihypertensive effect in humans remains to be elucidated.

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1. INTRODUCTION
Increased blood pressure is one of the leading risk factors for cardiovascular diseases and cardiovascular events. According to the general definition of hypertension [systolic blood pressure (SBP) , W , up to 30% of the world adult population had hypertension in 2000 (Kearney et al. 2005). In Finland, 37% of men and 28% of women were estimated to have increased SBP in 2007 (Peltonen et al. 2008). Hypertension often coexists with other risk factors, including hypercholesterolemia, insulin resistance, metabolic syndrome and arterial stiffness. Altogether these conditions increase the cardiovascular morbidity and mortality. Thus, hypertension is an important public-health challenge, of which prevention, early identification and treatment should receive high attention. Blood vessels contribute to the blood pressure regulation by controlling the vascular resistance. Due to aging, increased blood pressure or other pathophysiological factors, arteries stiffen and gradually lose their ability to adjust to the blood pressure changes (for review, see Ghiadoni et al. 2009). Aortic pulse-wave velocity (PWV), a measure of arterial stiffness, is a strong predictor of cardiovascular events and all-cause mortality (Inoue et al. 2009, Vlachopoulos et al. 2010). In addition, endothelial dysfunction is often observed in the presence of cardiovascular diseases and risk factors, such as hypertension (for review, see Taddei et al. 2000). Endothelial dysfunction can be defined as reduced vasodilatating response to endothelial stimuli. In humans and experimental animals, it can be observed by impaired endothelium-dependent relaxation. Several abnormalities account for this and are often related to the strong endogenous vasodilator, nitric oxide (NO) (for review, see Versari et al. 2009). Besides pharmacological therapy, lifestyle and nutritional factors play a significant role in the prevention and treatment of hypertension and related disorders. Low saturated fat and sodium intake and increased consumption of potassium, calcium and soluble fiber positively affect blood pressure (Krousel-Wood et al. 2004). In addition, both epidemiological and intervention studies suggest that consumption of low-fat dairy products is inversely related to the risk of hypertension (McCarron et al. 1984, Appel et al. 1997, Toledo et al. 2009). Milk is rich in calcium and potassium, but contains also large amount of physiologically active peptides encrypted in the protein sequences. At present, milk proteins are considered the most important source of bioactive peptides (for review, see Korhonen 2009). These peptides may be released during gastrointestinal digestion or dairy food processing and once liberated, cause various physiological effects. Tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) have been found in milk products fermented with Lactobacillus helveticus. They have been shown to possess 11

antihypertensive effects both in humans (for meta-analyses, see Pripp 2008, Xu et al. 2008) and in experimental animals (Sipola et al. 2001, Sipola et al. 2002a, Jauhiainen et al. 2010a). In previous experimental studies, tripeptides Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them have decreased SBP of spontaneously hypertensive rats (SHR) after single oral administration (Nakamura et al. 1995a) and attenuated the development of hypertension in young SHR (Sipola et al. 2001, Jauhiainen et al. 2005a). In humans, they have been shown to decrease blood pressure after long-term treatment (Pripp 2008, Xu et al. 2008). Also beneficial effects on arterial stiffness have been demonstrated (Jauhiainen et al. 2010b). Inhibition of angiotensinconverting enzyme (ACE), a pivotal enzyme in the renin-angiotensin system (RAS), has been suggested to be the main mechanism of action of tripeptides (for review, see Jauhiainen and Korpela 2007). However, as not all studies support this, other modes of action may exist as well. The purpose of the present series of studies was to investigate the effects of tripeptides Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them on vascular function and blood pressure and to elucidate the mechanisms behind them by using different experimental models of hypertension. To better understand the vascular effects of tripeptides in humans, another aim was to characterize the acute effects of tripeptides on arterial stiffness by a clinical study.

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2. REVIEW OF THE LITERATURE

2.1. CIRCULATORY SYSTEM The circulation can be divided into the peripheral (or systemic) circulation and the pulmonary circulation (for review, see Guyton and Hall 2006a). As regards peripheral circulation, arteries, beginning from the thoracic aorta, transport blood under high pressure from the left ventricle of the heart to the tissues. Arterioles, small branches of the arterial system, release blood into the capillaries, which exchange nutrients, electrolytes, hormones and fluid between the blood and the interstitial fluid. Venules collect blood from the capillaries and merge gradually into larger veins. Vena cava transports blood back to the right atrium of the heart. From the entire blood volume, about 84% is in the peripheral circulation and the rest in the heart and lungs. The cardiac cycle, the cardiac events that occur between two heartbeats, consists of a period of relaxation (diastole) and a period of contraction (systole) (for review, see Guyton and Hall 2006a). During diastole, the heart is filled with blood, which during systole is pushed forward to the aorta. Because of this pulsatile mode of operation, two pressure levels can be detected; systolic and diastolic pressure (SBP and DBP, respectively). The difference between these two pressures is called the pulse pressure (PP). Cardiac output (CO) is defined as the quantity of blood pumped into the aorta each minute (for review, see Guyton and Hall 2006b). Respectively, venous return is the quantity of blood returning to the heart each minute from peripheral circulation. In humans, the average CO in resting state is approximately 5 l/min. As the heart pumps blood continuously into the aorta, the blood pressure in the aorta is high (mean pressure approximately 100 mmHg). However, as the blood flows through the peripheral circulation, pressure falls progressively to about 0 mmHg in vena cava. Vascular compliance describes the total quantity of blood that can be stored in a given part of the circulation for each mmHg pressure rise. Owing to the compliance of the arterial system, the pulsatile mode of blood flow is almost totally attenuated by the time the blood reaches the capillaries. Total arterial compliance (TAC) is an estimate of the compliance in the entire arterial system and its measurement can be used to identify patients at risk of a cardiovascular event (Haluska et al. 2010).

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Blood flo & W between the two ends of the vessel and vascular resistance (R), the impediment of the blood vessel to blood flow (for review, see Guyton and Hall 2006a). Ohms law connects these parameters as follows: & WZ Another form of the same law, but in the level of the entire circulatory system, is often used to describe the connection of blood pressure (BP), total peripheral resistance (TPR) and cardiac output (CO): BP = CO x TPR. Circulation can be divided into macro- and microcirculation based on the size and function of the arteries. Macrocirculation consists of large, proximal, elastic-type arteries and medium-sized, more distal muscular arteries (for review, see Feihl et al. 2009). They transport blood and oxygen from the aorta to organs and buffer pressure oscillations generated by left ventricular ejection. Microcirculation designates small arteries (with diameter less than 150-300 m), arterioles, capillaries and venules. These resistance vessels ensure that the mean intracapillary pressure remains within an optimal range. Superior mesenteric artery is widely used in experimental studies to model the function and characteristics of a resistance artery. It is an unpaired vessel, which originates from the abdominal aorta (Geboes et al. 2001). Branches of the superior mesenteric arteries supply blood to the abdominal organs, namely jejunum and ileum of the small intestine, some parts of the large intestine, liver and pancreas. They diverge and anastomose several times forming arcades.

2.2. ARTERIAL STRUCTURE AND FUNCTION Arteries and veins are usually composed of three layers: tunica intima, tunica media and tunica adventitia. The innermost layer, tunica intima, consists of a single layer of endothelial cells, basal lamina made up by thin collagen fibers and internal elastic membrane (for review, see Renkin and Curry 1982). The central layer, tunica media, is a thick layer consisting mainly of vascular smooth muscle cells. External elastic membrane separates tunica media from tunica adventitia, which is a layer of connective tissue containing collagen and elastic fibers. The tunica adventitia contains also small blood vessels (vasa vasorum), which supply nutrients to the outer wall of the blood vessel, and

14

small nerves, representing fibers of the autonomic nervous system, which innervate the smooth muscle of the blood vessel. Tunica adventitia has been previously considered to be only a structural support for the media, but recent evidence indicates that it also participates in the blood vessel function. The adventitia is the primary site of superoxide production (Wang et al. 1998, Berry et al. 2000) and abundant of angiotensin II type 2 (AT2)-receptors e.g. in renal arteries (Zhuo et al. 1996, Zhuo et al. 1998). It has been shown to play a key role in the angiotensin II (Ang II)-induced responses by counterbalancing basal superoxide production with the enhancement of NO release through AT2-dependent mechanisms (Somoza et al. 2005). Also dopamine D2-like receptors can be found from tunica adventitia; they participate in blood pressure regulation by affecting ion and water transport in kidney and mediating vasorelaxation (for review, see Zeng et al. 2007). Capillaries consist only of endothelial cells, which are supported by basal lamina (for review, see Pries and Kuebler 2006). They are the principal vessel type (in addition to venules) where the exchange of water, oxygen, carbon dioxide, nutrients and waste compounds between blood and interstitium of surrounding tissues takes place. Mechanisms of transfer of compounds include filtration, osmosis and diffusion (Moore and Ruska 1957). In certain types of capillaries, small indentations develop at the interior or exterior plasma membrane, pinch off to form vesicles and join with the opposite plasma membrane to release their content.

2.2.1. Vascular smooth muscle The principal function of vascular smooth muscle cells (VSMCs) in mature animals is contraction and relaxation and consequently regulation of blood vessel tone and diameter, blood pressure and blood flow distribution (for review, see Owens et al. 2004). VSMCs within adult blood vessels proliferate at a very low rate, exhibit very low synthetic activity and express an array of contractile proteins, ion channels, receptors and signal-transducing molecules to carry out their functions. However, VSMCs retain remarkable plasticity and can undergo changes in phenotype in response to changes in local environmental signals. During vascular development, VSMCs play a key role in morphogenesis of the blood vessel and exhibit high rates of proliferation, migration and production of extracellular matrix components. Similarly, in response to vascular injury, VSMCs increase their rate of proliferation and migration and play a critical role in vascular repair. Unfortunately, because of the high degree of plasticity, VSMCs respond also to abnormal environmental signals that can lead to adverse phenotypic switching and consequently to development or progression of vascular disease. 15

Phenotypic switching plays a major role in a number of diseases such as atherosclerosis, cancer and hypertension (for review, see Rzucidlo et al. 2007).

Vascular smooth muscle contraction Vascular smooth muscle contraction can be initiated by mechanical, electrical or chemical stimuli. A change in transmural pressure in the blood vessel may cause contraction that originates from the smooth muscle itself and is therefore termed myogenic response (for review, see Davis and Hill 1999). Membrane depolarization and a number of chemical stimuli such as noradrenaline, Ang II, endothelin-1 (ET-1), vasopressin and thromboxane A2 (TXA2) by binding to their receptors can cause contraction of the vascular smooth muscle (for review, see Wynne et al. 2009). The primary regulator of vascular smooth muscle contraction is the Ca2+ signal. The contractile stimuli trigger an elevation of the cytosolic Ca2+ concentrations ([Ca2+i) by activating either the release of Ca2+ from the intracellular stores (sarcoplasmic reticulum) or the Ca2+ influx from the extracellular space via voltage-dependent L-type Ca2+ channels (for review, see Karaki et al. 1997). Ca2+ and calmodulin interact and activate myosin light chain kinase (MLCK), which phosphorylates myosin regulatory light chain (MLC). Phosphorylated MLC interacts with actin to induce smooth muscle contraction.

Potassium channels VSMCs are electrically excitable, and potassium ion (K+) channel activity is one of the major determinants of VSMC membrane potential and thus vascular tone. Membrane hyperpolarization due to an efflux of K+ is a result of opening of K+ channels in vascular smooth muscle (for review, see Ko et al. 2008). This is followed by the closure of voltage-dependent Ca2+ channels, reduction in Ca2+ entry and vasodilatation. At present, four distinct types of K+ channels have been identified in vascular smooth muscle: voltage-dependent K+ (Kv) channels, Ca2+ activated (BKCa) channels, ATPsensitive K+ (KATP) channels and inward rectifier K+ (Kir) channels (Figure 1). In response to depolarization of the membrane potential, Kv channels open to allow an efflux of K+, which results in repolarization (for review, see Nelson and Quayle 1995). Thus, Kv channels function to limit membrane depolarization and to maintain resting vascular tone. 4-Aminopyridine blocks Kv channels and is used in many studies in order to separate Kv current from BKCa current, which is also activated by membrane depolarization (for review, see Ko et al. 2008). In addition, BKCa channels are activated by changes in the intracellular Ca2+ concentration. They are believed to contribute to the

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maintenance of the membrane potential in small myogenic vessels. Blockers of BKCa channels include tetraethylammonium (TEA) and iberiotoxin (Milesi et al. 1999).

Figure 1. Major signaling pathways mediating vasodilatation. EC, endothelial cell; VSMC, vascular smooth muscle cell; CaM, calmodulin; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; sGC, soluble guanylyl cyclase; GTP, guanosine triphosphate; cGMP, cyclic guanosine monophosphate; PKG, protein kinase G; Cx, connexin; PLA2, phospholipase A2; COX, cyclo-oxygenase; PGI2, prostacyclin; SKCa, small conductance Ca2+-activated K+ channel; IKCa, intermediate conductance Ca2+activated K+ channel; BKCa, large conductance Ca2+-activated K+ channel; Kv, voltage-dependent K+ channel; KATP, ATP-sensitive K+ channel; Kir, inward rectifying K+ channel; VDCC, voltage-dependent Ca2+ channel. Modified from Fltou 2009.

KATP channels were first identified in cardiac myocytes (Noma 1983) but, thereafter, also from various other cells including VSMCs. Several endogenous agonists (such as calcitonin gene-related peptide, adenosine) activate KATP channels leading to hyperpolarization and relaxation (for review, see Teramoto 2006). In contrast, various neurotransmitters [noradrenaline, 5-hydroxytryptamine (5,d and vasoconstrictors (Ang II, ET-1) inhibit KATP channels leading to membrane depolarization and

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vasoconstriction. Thus, KATP channels contribute to the maintenance of resting potential and local blood flow, which is critical for the regulation of blood pressure. Kir channels are abundant in the smooth muscle of small-diameter resistance vessels such as coronary and cerebral arteries (Knot et al. 1996, Quayle et al. 1996) but poorly expressed in mesenteric arteries (Smith et al. 2008). Kir channels are important in modulating basal arterial tone (for review, see Chrissobolis and Sobey 2003). They conduct K+ more readily into the cell than out of the cell over a wide range of membrane potentials. Small increases in the extracellular K+ concentration activate Kir channels and enhance K+ efflux through them causing vascular hyperpolarization and relaxation. Ba2+, a specific inhibitor of Kir channels, attenuates the ability of K+ channel activators to produce vasodilatation (Smith et al. 2008) VSMCs express a wide variety of receptors, of which the most important as regards blood pressure regulation and vascular tone are alpha1- and alpha2-adrenergic receptors, beta2-adrenergic receptors, angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors, bradykinin type 2 receptor (B2), endothelin receptor ETA and prostanoid receptors (DP, EP, FP, IP and TP) (for reviews, see Bouallegue et al. 2007, Norel 2007, Bylund et al. 2010, Lemari and Schiffrin 2010). Recent evidence indicates that also dopamine receptors participate in the regulation of systemic blood pressure (for review, see Zeng et al. 2007). From the dopamine receptor subtypes, D1-like receptors (D1 and D5) are located mainly in the tunica media (VSMCs).

2.2.2. Endothelium Differentiated epithelial cells, endothelial cells form a thin layer, endothelium, which lines the entire circulatory and lymphatic systems (Fishman 1982). This organ with a large surface (350 m2) was formerly thought to be only an inert semipermeable barrier determining the exchange of substances between blood and tissues (for review, see Pries and Kuebler 2006). However, the understanding of its structure and function has evolved, and endothelium is now considered to have an important role in maintaining the balance between vasodilatation and vasoconstriction (for review, see Fltou and Vanhoutte 2009). It controls also the proliferation and migration of VSMCs and adhesion and aggregation of platelets. Endothelial cells synthesize and release factors that regulate vascular tone and permeability, angiogenesis and inflammatory responses. Regional differences exist in the biologic function and significance of the endothelial layer: capillary walls consists almost nothing but endothelial cells whereas the endothelium of the aorta is only the

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innermost layer of a thick vessel wall (Fishman 1982). Transport of plasma proteins and solutes across the endothelium involves two different routes: one paracellular, through interendothelial junctions, and the other transcellular, via caveolae-mediated vesicular transport (for review, see Komarova and Malik 2010). Two types of interendothelial junctions present in the endothelium, tight junctions and adherens junctions, contribute to maintenance of the endothelial barrier. They restrict the transport of plasma proteins of the size of albumin from the vessel lumen to stroma. By contrast, transcellular pathway is responsible for the transport of albumin, albumin-bound ligands and hormones across the endothelial barrier.

Endothelium-derived vasoactive factors In response to various substances released by autonomic and sensory nerves or platelets, circulating hormones, autacoids, cytokines, drugs, as well as to chemical or physical stimuli (changes in pressure, shear stress, pH), endothelial cells synthesize and release various factors that modulate vascular tone and permeability (Fltou and Vanhoutte 2006). These vasoactive factors include both relaxing [nitric oxide (NO), prostacyclin (PGI2), epoxyeicosatrienoic acids (EETs), adenosine, C EW racting [TXA2, isoprostanes, superoxide anion (O2-), ET-1, Ang II factors. Endothelium acts as a major regulator of local vascular homeostasis by maintaining the balance between vasodilatation and vasoconstriction. Upsetting this balance leads to endothelial dysfunction.

Nitric oxide (NO) The most important vasorelaxing factor is NO, previously termed endothelium-derived relaxing factor (EDRF). Its role in vascular function was understood in 1980s, when Furchgott and Zawadzki (1980) first discovered that acetylcholine requires the presence of endothelial cells to produce vasodilatation. They concluded that endothelial cells must synthesize a substance, which diffuses to the vascular smooth muscle. EDRF was later found to have a very short biological half life (few seconds) (Rubanyi et al. 1985), to stimulate soluble guanylyl cyclase (sGC) in the VSMCs (Ignarro et al. 1986) and to be scavenged by superoxide anions (Rubanyi and Vanhoutte 1986). The EDRF was finally identified to be NO by several researchers at the same time (Ignarro et al. 1987, Palmer et al. 1987).

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Thereafter, the enzymes responsible for NO production were discovered. NO is formed from Larginine by nitric oxide synthases (NOS), of which three distinct isoforms are known to exist: constitutively expressed neuronal NOS (nNOS, NOS1) and endothelial (eNOS, NOS3), and inducible NOS (iNOS, NOS2) (for review, see Shimokawa and Tsutsui 2010). In the vasculature, eNOS is the main source of NO in physiological conditions. Interestingly, also platelets (Mehta et al. 1995) and erythrocytes (Chen and Mehta 1998) have been described to produce NO. All NOS isoforms are synthesized as monomers, but normal function of eNOS requires dimerization of the enzyme (for review, see Frstermann and Mnzel 2006). In intact eNOS, a C-terminal reductase domain is linked to the N-terminal oxygenase domain of the other monomer. The reductase domain binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), whereas the oxygenase domain carries a prosthetic heme group and binds also (6R-)5,6,7,8-tetrahydrobiopterin (BH4), O2 and the substrate L-arginine. The enzyme catalyzes flavin-mediated electron transfer from the C-terminally bound NADPH to the heme on the N terminus. At the heme, electrons are used to reduce and activate O2. To synthesize NO, eNOS hydroxylates L-arginine to N- hydroxy-L-arginine, which is further oxidized to L-citrulline and NO. If the electron transfer chain is disturbed, O2- is generated instead of NO. This is referred to as eNOS uncoupling, which is known to occur parallel to endothelial dysfunction in hypertensive, diabetic and aged blood vessels (Li et al. 2006, Wenzel et al. 2008, Yang et al. 2009).

As NO has a major role in controlling vascular tone in most blood vessels, its production by eNOS is tightly regulated by multiple mechanisms and signaling pathways. Ca2+-calmodulin -complex is essential for activating eNOS (Busse and Mlsch 1990). eNOS activity can also be regulated by phosphorylation of the enzyme by protein kinase B (Akt), protein kinase A (PKA), cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), or calmodulin-dependent kinase II on Ser1177 in response to various stimuli (for review, see Dudzinski and Michel 2007). In contrast, protein kinase C decreases eNOS activity by increasing the phosphorylation on Thr495. Other posttranslational modifications include nitrosylation and acylation. eNOS is localized in caveolae, small invaginations of the cell membrane, which are enriched with cholesterol and sphingolipids (Shaul et al. 1996). Numerous receptors and signaling molecules are centralized in caveolae thus facilitating eNOS to receive signals from upstream mediators and to communicate with downstream signaling pathways.

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Although only few vessels are innervated by cholinergic nerves, muscarinic receptors can be found from the endothelium. In general, five subtypes of muscarinic receptors (M1-M5) have been identified, of which M3 seems to have a major role in the endothelium mediating endotheliumdependent relaxations and contractions (Bruning et al. 1995, Attina et al. 2008). Therefore, muscarinic receptor agonist acetylcholine is frequently used as a pharmacological tool to evoke endothelium-dependent, NO-mediated relaxations in experimental studies. However, a number of more physiological stimuli (e.g. bradykinin, histamine, 5-HT, adenosine diphosphate) are able to cause endothelium-dependent NO release as well (for review, see Michel and Vanhoutte 2010). In conduit arteries, NO is the main component of endothelium-dependent relaxation (Lscher et al. 1986, Shimokawa et al. 1996).

After synthesis in the endothelium, NO diffuses to the underlying vascular smooth muscle (Figure 1). Interestingly, it has been shown that NO may also be transported across endothelial and vascular smooth muscle membranes by aquaporin-1 (Herrera et al. 2006). NO activates sGC in vascular smooth muscle (Ignarro et al. 1986). Stimulation of sGC leads to the conversion of guanosine triphosphate (GTP) to cGMP. Accumulation of cGMP activates PKG, which causes vascular smooth muscle relaxation by decreasing intracellular Ca2+ (Archer et al. 1994).

Prostanoids and endothelium-derived contracting factor (EDCF) Prostanoids are a subclass of eicosanoids, which all derive from polyunsaturated fatty acids containing 20 carbon atoms, predominantly arachidonic acid (AA) (for review, see Mbonye and Song 2009). They are lipid hormones that act locally in an autocrine or paracrine fashion through Gprotein coupled receptors to induce multiple physiological and pathophysiological responses. Cyclooxygenase (COX) plays a pivotal role in the metabolism of AA and is the rate-limiting step in the formation of prostanoids (Vane et al. 1998). In the first stage, COX converts arachidonic acid to endoperoxide prostaglandin G2 (PGG2). Next PGG2 is transformed by COX to PGH2, which is converted to prostaglandins such as prostaglandin E2, F, D2 and I2 (PGI2, prostacyclin) as well as thromboxane A2 (TXA2) by their selective synthases. COX exists in two isoforms, COX-1 and COX-2, of which COX-1 is constitutively expressed in many mammalian tissues (for review, see Mbonye and Song 2009). In contrast, COX-2 is selectively expressed in some tissues in an inducible and transient manner. For example, COX-2 expression is increased in atherosclerosis, in which it is predominantly localized in macrophages within atherosclerotic lesions (Schnbeck et al. 1999, Stemme et al. 2000),

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and in diabetes (Nacci et al. 2009). However, there is some evidence indicating that COX-2 is not just an inducible enzyme; it is known to be constitutively expressed in certain cells and organs, e.g. kidney (Harris et al. 1994) and brain (Breder et al. 1995). The expression of COX-1 is augmented in SHR aorta, particular in endothelial cells, as compared to blood vessels of normotensive WKY rats (Tang and Vanhoutte 2008). In addition to COX-1 and COX-2, a variant of COX-1, COX-3 has been described in canine and human cerebral cortex and heart (Chandrasekharan et al. 2002).

Both endothelial and vascular smooth muscle cells contain COX, however, endothelial cells contain 20 times more of the enzyme than smooth muscle cells (DeWitt et al. 1983). Endothelial cells express high levels of COX-1 but do not usually express COX-2 with the exception of kidney in which both vascular and nonvascular cells express COX-2 (Khan et al. 1998). As the most abundant prostanoid synthase in endothelial cells is PGI synthase, the majority of the endothelial COX-1derived endoperoxides are transformed to PGI2 (Tand and Vanhoutte 2008). PGI2 is a functional antagonist to TXA2, which in the vascular system is generated predominantly by platelets and stimulates platelet aggregation and vasoconstriction (for review, see Fltou et al. 2009). In contrast, PGI2, produced predominantly by the endothelium, inhibits platelet aggregation and vasoconstriction. Via activation of M3-receptors, acetylcholine increases endothelial intracellular Ca2+ concentration, which induces phospholipase A2 (PLA2)-dependent mobilization of arachidonic acid, COX activation and thus formation of prostanoids. In vascular smooth muscle cells, TXA2, PGI2 and other prostanoids interact with G-protein coupled receptors, which are classified in five subtypes DP, EP, FP, IP and TP according to their sensitivity to the five primary prostanoids, prostaglandins E2, F, D2, I2 and TXA2, respectively (for review, see Tsuboi et al. 2002). Each receptor type exerts its actions via different mechanisms; for example PGI2 binds to IP receptor in the vascular smooth muscle cells and activates adenylate cyclase (AC), which leads to an increase in intracellular cyclic adenosine monophosphate (cAMP) and vascular relaxation (Wise and Jones 1996). TP receptor communicates mainly with Gq- and G13-proteins, and the receptor activation leads to phospholipase C activation and guanine nucleotide exchange factor of the small G protein Rho (RhoGEF) activation, respectively (for review, see Nakahata 2008).

A phenomenon that is closely related to endothelium-derived prostanoids is endotheliumdependent contractions. Endothelium-derived contracting factor (EDCF) has been detected in experimental studies using hypertensive rats. Namely, in the quiescent arterial rings of spontaneously hypertensive rats (SHR), acetylcholine produces endothelium-dependent contractions that are amplified in the presence of a NOS inhibitor (Auch-Schwelk et al. 1992, Iwama et al. 1992). 22

The same is observed in aged blood vessels of normotensive Wistar-Kyoto rats (WKY) (Koga et al. 1989, Iwama et al. 1992). Inhibitors of COX inhibit endothelium-dependent contractions; thus endothelium-dependent contractions involve diffusible contractile COX derivatives, which originate from endothelium and oppose the relaxing effect of NO.

Besides acetylcholine, also other stimuli can cause the generation of EDCF, such as adenosine triphosphate (ATP) (Koga et al. 1989, Mombouli and Vanhoutte 1993), vascular endothelial growth factor (VEGF) (Liu et al. 2001) and calcium ionophore A23187 (Tang et al. 2007). As A23187 increases membrane permeability to Ca2+ and consequently Ca2+ influx, increase in intracellular Ca2+ seems to be involved in the production of endothelium-dependent contractions. Accordingly, acetylcholine causes a rapid increase in intracellular Ca2+ that is significantly greater in SHR than in WKY (Tang et al. 2007).

Endothelium-dependent contractions are blocked by antagonists of the TP receptors but not by inhibitors of TXA2 synthase, indicating that TXA2 is not the EDCF. The role of other prostanoids (PGE2, PGF) in eliciting endothelium-dependent contractions is still controversial (Fltou et al. 2009), but they may act as EDCF by a compensatory mechanism when PGI synthase is inhibited. PGI2 is released in larger amounts in the aorta of SHR than WKY and although it generally is described as an endothelium-dependent vasodilator, it paradoxically acts as a contracting prostanoid in SHR and aged WKY (Gluais et al. 2005). There are several observations that speak for PGI2 to be involved at least partly in endothelium-dependent contractions. For example, PGI2 and endothelium-dependent contractions both involve TP receptors, the contractions evoked by PGI2 mimic the endotheliumdependent contractions produced by acetylcholine both in terms of duration and amplitude, and the release of PGI2 correlates with the amplitude of the endothelium-dependent contraction over the full concentration range in both SHR and WKY (Gluais et al. 2005).

Endothelium-dependent contractions have been studied most extensively in SHR aorta, but the phenomenon has been demonstrated also in various other animal models of cardiovascular diseases and different vascular beds, at least in rat mesenteric arteries (Sipola et al. 2002b, Paulis et al. 2008, Matsumoto et al. 2009), rat femoral arteries (Paulis et al. 2008), rat renal arteries (Michel et al. 2008) and mouse mesenteric arteries (Morikawa et al. 2005). Increased production of EDCF is present also in human essential hypertension and contributes to endothelial dysfunction (Taddei et al. 1993, Taddei et al. 1997).

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Calcium-activated potassium channels and endothelium-derived hyperpolarizing factor (EDHF) In healthy arteries, large conductance calcium-activated potassium channels (BKCa) are preferentially expressed in VSMCs, whereas small and intermediate conductance potassium channels (SKCa and IKCa) are preferentially located in endothelial cells (Figure 1) (for review, see Fltou 2009). They are the key molecules regulating electrical events in the membrane. In endothelial cells, potassium channels contribute to the effects of endothelium-derived hyperpolarizing substances and endothelium-derived hyperpolarizing factor (EDHF)-mediated responses. In resistance-sized arteries, endothelium-dependent relaxation is predominantly mediated by EDHF (Garland and McPherson 1992, Onaka et al. 1998) and the importance of EDHF increases as the vessel size decreases (Shimokawa et al. 1996). SKCa and IKCa channels differ in the spatial distribution in endothelial cells. For example, in the rat mesenteric artery SK Ca are preferentially located in caveolin-rich domains, at sites of endothelial gap junctions and are associated with various connexins (gap junction proteins) whereas IKCa are localized at the sites of endothelial projections towards the underlying smooth muscle cells (Sandow et al. 2006, Absi et al. 2007). Tetraethylammonium is a non-specific antagonist of all calciumactivated potassium channels; specific blockers of SKCa and IKCa include bee toxin apamin and analogue of clotrimazole, TRAM-34, respectively (for review, see Fltou 2009). A scorpion toxin charybdotoxin blocks both IKCa and BKCa. Increase in endothelial intracellular Ca2+ concentration activates SKCa and IKCa (for review, see Fltou and Vanhoutte 2009). Opening of these channels leads to hyperpolarization of the endothelial cells and cause endothelium-dependent hyperpolarization of vascular smooth muscle. Already in the early 1980s it was observed that a non-NO- and non-PGI2-mediated component of endothelium-dependent relaxation exists. These endothelium-derived hyperpolarizing factor (EDHF)mediated dilatations have been ascribed to lipoxygenase products (Faraci et al. 2001, Miller et al. 2003), metabolites of arachidonic acid (for review, see Campbell and Falck 2007), C-type natriuretic peptide (Chauhan et al. 2003, Villar et al. 2007) and hydrogen peroxide (for review, see Shimokawa and Morikawa 2005). In addition to the concept of EDHF being a diffusible factor, there is also evidence for this type of relaxation to be a biophysical phenomenon. Two scenarios have been suggested. First, hyperpolarization could be mediated via myoendothelial gap junctions, which electrically couple endothelium and vascular smooth muscle (Sandow et al. 2002, Dora et al. 2003, Sokoya et al. 2006). However, myoendothelial coupling does not seem to correlate EDHF-responses in all vascular beds (Siegl et al. 2005). Secondly, K+ release through the channels may cause a small increase in K+ in the intracellular space between endothelium and vascular smooth muscle activating 24

Kir channels and/or Na+/K+-ATPase, which finally cause vascular smooth muscle membrane hyperpolarization (Edwards et al. 1998, Bssemaker et al. 2002). Hyperpolarization closes the voltage-gated Ca2+ channels leading to a decrease in intracellular Ca2+ concentration and finally relaxation. The two proposed biophysical pathways may also act in parallel or synergistically.

Endothelial dysfunction In vascular diseases and aging, endothelium undergoes functional and structural alterations, thus losing its protective role and becoming a proatherosclerotic structure (Vanhoutte 1989). The loss of normal endothelial function is referred as to endothelial dysfunction, which is characterized by impaired NO bioavailability (for review, see Versari et al. 2009). This can be a consequence of either a reduced production of NO by eNOS or an increased breakdown of NO by reactive oxygen species (ROS). When NO bioavailability is significantly reduced, endothelium activates several compensatory physiological pathways. The production and release of endothelium-derived vasodilators other than NO (prostanoids, EDHF) is increased, thereby partly maintaining the endothelium-dependent vasodilatation. However, dysfunctional endothelium produces also mediators that have detrimental effects on the arterial wall, namely ET-1, TXA2, prostaglandin H2 and ROS. Accordingly, endothelial dysfunction has been associated with a number of pathophysiological processes, including e.g. hypertension, atherosclerosis, diabetes, heart and renal failure, hypercholesterolemia, inflammation and smoking (Fltou and Vanhoutte 2006). Although the mechanisms underlying endothelial dysfunction may be multifactorial, oxidative stress is one of the main mechanisms contributing to the phenomenon. The term oxidative stress describes the conditions involving chronically elevated ROS levels (for review, see Paravicini and Touyz 2006). Vascular ROS production is increased in different experimental models of hypertension and is often accompanied with impaired endothelium-dependent vasodilatation (Zrov-Nedelcevov et al. 2006, Miyagawa et al. 2007, Tang et al. 2007). ROS include NO, superoxide anion O2-, hydroxyl radical OH, hydrogen peroxide H 2O2 and peroxynitrite ONOO- (for review, see Paravicini and Touyz 2006). ROS production has been shown to occur in all vascular cell types. The most relevant sources of ROS as regards vascular disease and hypertension seem to be xanthine oxidase, uncoupled eNOS and NAD(P)H oxidase. ROS can inhibit the three major endothelium-dependent pathways, that is, NO, prostacyclin and EDHF (Fltou and Vanhoutte 2006). O2- reduces the bioavailability of NO and inhibits its target, soluble guanylyl cyclase (sGC) (Rubanyi and Vanhoutte 1986). It reacts rapidly with NO to form the 25

highly reactive ONOO-. ONOO- uncouples eNOS (Zou et al. 2002) and has been shown to inactivate PGI synthase (Zou and Ulrich 1996). The activity of Ca2+-activated K+ channels is decreased by O2- (Kusama et al. 2005). In addition, ROS enhance the contractility of vascular smooth muscle by facilitating the mobilization of Ca2+ and increasing the sensitivity of the contractile proteins to Ca2+ ions (Fltou and Vanhoutte 2006). Endothelium-dependent relaxations are impaired in various experimental models of hypertension, such as spontaneously hypertensive rat (SHR) (Mkynen et al. 1995, Sipola et al. 2002b, Bagnost et al. 2008), stroke-prone spontaneously hypertensive rat (SHRSP) (Intengan and Schiffrin 2000, Shimamura et al. 2000), Ang II-induced hypertension (Dal-Ros et al. 2009, Kane et al. 2010), double transgenic rat harbouring human renin and angiotensinogen genes (dTGR) (Mervaala et al. 2001) and salt-loaded type 2 diabetic Goto-Kakizaki (GK) rat (Cheng et al. 2001, Oniki et al. 2006). The direct measurement of vascular contraction and relaxation has been widely used to test the endothelial dysfunction in experimental animals (for review, see Bernatova et al. 2009). The presence and the severity of endothelial dysfunction are highly dependent on the vascular bed studied (conduit or resistance arteries) and the age of the animal, and is complicated by several methodological aspects. In isolated arterial preparations, endothelium-dependent relaxation and thus endothelial function is usually evaluated by administering acetylcholine, bradykinin or substance P to a precontracted arterial preparation and calculating the degree of relaxation as a percentage of the precontraction. In normotensive rats, the degree of endothelium-dependent relaxation is usually near 100%, whereas in hypertensive animals endothelium-dependent relaxation can be severely impaired reaching only 20-40%. In the aorta of SHR, impaired endothelium-dependent relaxations are associated with the generation of EDCF (for review, see Fltou et al. 2009). There seems to be no or little alteration in the production of NO. The same applies to smaller, i.e. renal and mesenteric, arteries as well. However, in these resistance arteries where endothelium-dependent hyperpolarizations participate in endothelium-dependent relaxations, a marked attenuation in the EDHF-mediated component is observed in SHR (Onaka et al. 1998, Bssemaker et al. 2003, Michel et al. 2008). The decrease in EDHF-mediated response has been associated with a change in the expression profile of gap junctions in endothelial cells (for review, see Figueroa and Duling 2009). Expression of connexins 37 and 40 is lower in the arteries of SHR than that of WKY (Kansui et al. 2004). In addition, increased activity of Ca2+-dependent chloride channels may be responsible for the impairment of EDHFmediated responses in SHR (Goto et al. 2007).

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2.3. REGULATION OF BLOOD PRESSURE It has been widely accepted that kidneys play the major role in blood pressure regulation. The elegant study by Rettig et al. (1990) showed that if kidneys of spontaneously hypertensive rats (SHR) are transplanted to normotensive rats and vice versa, rats with new, hypertensive kidneys develop hypertension while genetically hypertensive rats with normotensive kidneys remain in normal blood pressure levels. Particularly the long-term regulation of blood pressure is closely related to kidney function and body fluid volume homeostasis while the short-term control of blood pressure has been attributed to sympathetic nervous system (for review, see Wyss and Carlson 2001). However, sympathetic nervous system overactivity seems to underlie some forms of hypertension and thus nervous system may contribute to the long-term control of blood pressure as well.

2.3.1. Nervous system Nervous system controls blood pressure almost totally through the autonomic nervous system. Sympathetic nervous system accounts for most of the blood pressure regulation; however, parasympathetic nervous system contributes to the regulation of heart function and this way affects blood pressure as well (for review, see Olshansky et al. 2008). In most tissues, all the blood vessels (except capillaries) are innervated by sympathetic nerves, which carry a large number of vasoconstrictor nerve fibers and only a few vasodilator fibers (for review, see Guyton and Hall 2006c). Vasomotor centre, which is located in the brain stem, transmits sympathetic impulses through the spinal cord and peripheral sympathetic nerves to arteries, arterioles and veins in the whole body and, likewise, parasympathetic impulses through the vagus nerves to the heart. Vasomotor tone, partial state of contraction in the blood vessels, is maintained by continuous, slow firing of sympathetic vasoconstrictor nerve fibers.

Alpha- and beta-adrenergic regulation Sympathetic impulses are transmitted to the adrenal medulla parallel to the transmission to the blood vessels. They stimulate medulla to secrete both adrenaline and noradrenaline, which after reaching the blood are able to act directly on blood vessels and cause mainly vasoconstriction. Noradrenaline and adrenaline are agonists of alpha-adrenergic receptors, which are located both pre- and postsynaptically (for review, see Langer and Hicks 1984). In blood vessels, the presynaptic receptor, which inhibits the release of noradrenaline, corresponds to the alpha2-subtype. Both the 27

alpha1- and alpha2-adrenergic receptor subtypes are present postsynaptically in the vascular smooth muscle, where they mediate vasoconstriction. There are differences between vascular beds on which subtype is postsynaptically predominant; in most peripheral vascular beds either the alpha1-subtype predominates (e.g. renal vascular bed) or both alpha1- and alpha2-adrenergic receptors are present (e.g. mesenteric vascular bed). However, distribution and function of the subtypes seems to be dependent on the diameter of the blood vessel, the species and the hypertensive state of the subject; for example, in SHR, vascular smooth muscle alpha2-adrenergic receptors mediate vasoconstrictor responses to exogenous noradrenaline and to sympathetic stimulation to a greater extent than in normotensive Wistar-Kyoto (WKY) rats (Medgett et al. 1984, Miyagawa et al. 2007). Thus, postsynaptic vascular alpha2-adrenergic receptors may play an important role in the pathophysiology of hypertension and also contribute to the increased vascular reactivity to noradrenaline observed in hypertensive states. Agonists of alpha-adrenergic receptors differ in their selectivity to different subtypes. Noradrenaline and adrenaline stimulate both alpha1- and alpha2-subtypes, while for example methoxamine and phenylephrine are more selective for alpha1-subtype (for review, see Langer and Hicks 1984). Noradrenaline infusion to humans increases blood pressure, the effect that is more pronounced in low-renin hypertensives than in normotensives or normal-renin hypertensives (Nishimura et al. 1988). Alpha1-subtypes (alpha1A, alpha1B and alpha1D) are mainly coupled to Gq/11-protein to stimulate phospholipase C activity (for review, see Guimares and Moura 2001). This enzyme promotes the hydrolysis of phosphatidylinositol bisphosphate producing inositol triphosphate and diacylglycerol, which act as second messengers mediating Ca2+ release from intracellular stores and activating protein kinase C, respectively. In contrast, alpha2-subtypes (alpha2A, alpha2B and alpha2C) are predominantly coupled to the inhibitory heterotrimeric GTP-binding protein inhibiting the activity of AC, inhibiting the opening of voltage-gated Ca2+ channels and activating K+ channels. Alpha-adrenergic receptor-mediated effects predominate in the vast majority of vascular tissues (for review, see Guimares and Moura 2001). However, beta-adrenergic receptor-mediated vasodilatation plays an important role in the regulation of vascular tone. Total of four subtypes of beta-adrenergic receptors have been identified so far (Bylund et al. 2010), of which beta1-subtype predominantly regulates cardiac contractility and heart rate, beta2-subtype predominantly mediates the vasodilatation evoked by sympathomimetic agonists and beta3-subtype predominantly controls lipolysis in adipose tissue. Accumulating evidence suggests also the existence of beta4-subtype, localized in cardiac tissues. Adrenaline and noradrenaline have a similar affinity to beta1-adrenergic receptors, whereas adrenaline activates beta2-adrenergic receptors by smaller concentrations than

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noradrenaline (Bylund et al. 2010). Beta3-subtype is more sensitive to noradrenaline than to adrenaline. It has a low affinity for conventional beta-adrenergic receptor antagonists. All betaadrenergic receptor subtypes signal by coupling to the stimulatory G-protein G leading to activation of AC and accumulation of the second messenger cAMP (for review, see Guimares and Moura 2001). cAMP activates protein kinase A (PKA), which phosphorylates L-type Ca2+ channels facilitating Ca2+ entry. In cardiac myocytes, PKA regulates Ca2+ transport by phosphorylating phospholamban, an inhibitor of Ca2+-ATPase (SERCA2a) (Stein et al. 1996). The groundbreaking study of Lands et al. (1967) divided beta-adrenergic receptors into two groups, of which beta2 was responsible for the vasodilatation. However, since then, several studies have shown that also other subtypes participate in the relaxation of blood vessels (Graves and Poston 1993, Huang et al. 1998, Briones et al. 2005). The involvement of one or more beta-subtypes in vasodilatation varies with vascular bed and species in question (for review, see Guimares and Moura 2001). Recent evidence indicates that it may not be only the VSMCs that are responsible for the beta-adrenergic receptor-mediated relaxation, but also endothelium plays a role (Georcescu et al. 2005, Figueroa et al. 2009).

Baroreceptor reflex Baroreceptor reflex mechanisms contribute importantly to the short-term regulation of heart rate, sympathetic tone and blood pressure. A few baroreceptors are located in the wall of almost all large arteries but are extremely abundant in the wall of carotid sinus and aortic arch (for review, see Guyton and Hall 2006c). They are stimulated when stretched and transmit signals through Herings and glossopharyngeal nerves or vagus nerves (carotid sinus and aortic arch, respectively) to the tractus solitarius of the medulla in the brain stem. Thereafter, secondary signals inhibit the vasoconstrictor center of the medulla and excite the vagal parasympathetic center. As a consequence, veins and arterioles dilate and heart rate and heart contraction strength decreases. Both a decrease in vascular resistance and a decrease in cardiac output cause arterial pressure to decrease. Normally arterial baroreflex opposes increases in blood pressure as described, but chronic increases in blood pressure impair arterial baroreflex by increasing threshold for activation and by reducing sensitivity (for review, see Narkiewicz and Grassi 2008).

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Interactions between nervous system and renin-angiotensin system (RAS) The renin-angiotensin system (RAS) and sympathetic nervous system interact at various levels to influence vascular function. For example, renal sympathetic nerves stimulate renin release via beta1adrenergic receptors (Holmer et al. 1997). Numerous studies have demonstrated that Ang II may enhance the activity of the sympathetic nervous system in many ways. Ang II, either exogenous or endogenous, has been shown to stimulate sympathetic neuronal activity in the central nervous system, to facilitate noradrenaline release from synaptic nerve terminals, to potentiate the postsynaptic effects of noradrenaline and to affect noradrenaline uptake and synthesis (for review, see Reid 1992). Also circulating Ang II may produce central effects, as area postrema, the most probable site for Ang II action, has fenestrated capillaries, high density of Ang II receptors and direct connections with medullary centers (Papas et al. 1990). Part of the vasoconstrictor effects of Ang II may thus result from the increased sympathetic tone. This may be the case especially in essential hypertension, where sympathetic hyperactivity is playing an important pathophysiological role. Ang II facilitates the release of noradrenaline in the periphery through presynaptically located AT1receptors (Balt et al. 2001). This effect is enhanced in blood vessels of hypertensive rats (Nagase et al. 1996, Byku et al. 2008). Angiotensin (1-7) [Ang (1-, which is suggested to be an endogenous inhibitor of Ang II, inhibits both Ang I- and Ang II-induced facilitation of noradrenaline release from the perfused rat kidneys of both normotensive and hypertensive rats (Stegbauer et al. 2004). Also in SHR mesenteric arterial bed, Ang (1-7) decreased the nerve-stimulated overflow of noradrenaline and neuropeptide Y (Byku et al. 2010). In rat hypothalamic preparations, Ang (1-7) decreased noradrenaline release, with a greater effect in hypertensive than in normotensive rats (Gironacci et al. 2004). The effects of Ang (1-7) to noradrenaline release are suggested to be mediated via the AT2and Mas-receptors and ultimately bradykinin/nitric oxide-dependent pathways, which stimulate cGMP release.

2.3.2. Renin-angiotensin system (RAS) Renin-angiotensin system (RAS) was originally described as a circulating hormone system and a main regulator of cardiovascular functions. The scientific history of the RAS began in 1898 by Robert Tigerstedt and Per Bergman as they discovered that injection of renal homogenate from one rabbit to another caused an acute elevation of blood pressure (Tigerstedt and Bergman 1898). They proposed that the kidney secretes a hormone with vasopressor properties. The hormone, later found to be an enzyme, was named renin based on its origin. The findings of Tigerstedt and Bergman 30

were not widely noticed at that time, and it took almost 40 years before more effectors of the reninangiotensin cascade were deciphered (Braun-Menendez et al. 1940). Major components of classical circulating RAS were found in the early 1970s, when it was understood that RAS has an important role in the regulation of fluid balance and blood pressure (Peart 1975). However, the understanding of the role of RAS in blood pressure regulation has gradually increased. In addition to circulating RAS, local tissue RAS has been found in most organs and tissues studied (for review, see Fyhrquist and Saijonmaa 2008). Thus, RAS is not only an endocrine, but also a paracrine and an intracrine system. It regulates more physiological functions than was previously thought and involves mediators that have been newly discovered or are still being unveiled.

Renin The first step in the RAS effector cascade is renin, an aspartyl-protease enzyme, which is produced and activated within the juxtaglomerular cells of afferent arteriole in the kidney (for review, see Beierwaltes 2010). Renin secretion is controlled through a complex interaction of at least four different regulatory pathways. First, renal baroreceptors respond to increases and decreases in blood pressure in preglomerular vessels inhibiting and stimulating renin release, respectively. Second, sympathetic activation of beta1-adrenergic receptors in juxtaglomerular cells enhances renin secretion (Holmer et al. 1997). Third, change in tubular NaCl delivery to the macula densa, an area of specialized epithelial cells adjacent to the juxtaglomerular cells, creates a signal to modify renin release (for review, see Davis and Freeman 1976). Fourth, certain hormones and autacoids affect renin release, e.g. ATP by inhibiting it (Yao et al. 2003). Renin acts only on the leucyl-leucine (in mouse and rat) or leucyl-valine (in humans) bond of its single substrate, angiotensinogen (for review, see Beierwaltes 2010) (Figure 2). Angiotensinogen is expressed in multiple tissues, including liver, adipose tissue, heart, vessel wall, brain and kidney (for review, see Dickson and Sigmund 2006). Although angiotensinogen is produced also locally, most of the angiotensinogen belonging to the circulating RAS is derived from the liver (Stec et al. 1999). The renin-angiotensinogen enzymatic reaction is the rate-limiting step of the RAS cascade (Gould and Green 1971). Renin releases decapeptide angiotensin I (Ang I; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-HisLeu) from N-terminal of angiotensinogen (for review, see Dickson and Sigmund 2006). Ang I is further cleaved to octapeptide angiotensin II (Ang II; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) by endothelial membrane-bound angiotensin-converting enzyme (ACE).

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Figure 2. Renin-angiotensin system. ACE, angiotensin-converting enzyme; ACE2, angiotensinconverting enzyme 2; ACEi, angiotensin-converting enzyme inhibitors; ARB, angiotensin receptor 1 blockers. AT1, AT2 and AT4, angiotensin II receptor 1, 2 and 4; BK1/BK2, bradykinin receptors 1 and 2; NEP, neutral endopeptidase; RPR, renin/prorenin receptor; Modified from Fyhrquist and Saijonmaa 2008 and Sharma 2009.

Renin/prorenin receptors were identified and cloned rather recently (Nguyen et al. 2002). Renin receptors are abundant in heart, brain and placenta, as lower levels are found in kidney and liver (for review, see Nguyen 2010). Binding of prorenin to the renin/prorenin receptors leads to activation of prorenin to active renin, which is able to generate Ang I from angiotensinogen thereafter. Receptor activation leads also to activation of mitogen-activated protein kinases p44/42 and TGF-beta, ultimately increasing contractility, hypertrophy and fibrosis.

Angiotensin-converting enzyme (ACE) Two distinct forms of ACE (dipeptidyl-carboxypeptidase I/kininase II) are expressed in humans, a somatic form that is particularly abundant on the endothelial surface of lung vessels but also 32

expressed in all other endothelial cell types as well as some smooth muscle cells, adipocytes and monocytes, and a germinal form that is found only in testis (for review, see Fleming 2006). Somatic ACE is a type I membrane protein that comprises two homologous extracellular catalytic domains, the N- and C-terminal domains, each of which contains an active site (Soubrier et al. 1988). Testicular ACE contains only the C-terminal domain (Ehlers et al. 1989). Both C- and N-terminal domains require zinc to be active, are activated by chloride and are sensitive to competitive ACEinhibitors (Wei et al. 1991). Both domains cleave Ang I to Ang II (Figure 2), although Ang I conversion is reported to take place preferentially within the C-terminal domain (van Esch et al. 2005). Besides cleaving Ang I to Ang II, ACE hydrolyzes bradykinin, a vasoactive peptide that promotes the generation of NO and other endothelium-derived vasodilators (for review, see Fleming 2006). Both the C- and N-terminal domains of ACE contribute to the degradation of bradykinin. N-domain of ACE hydrolyzes also angiotensin 1-7 [Ang (1-7), which inhibits the enzymatic activity of the C-terminal domain (Deddish et al. 1998). Also a soluble form of ACE exists; it is present in plasma, serum and other body fluids (for reviews, see Dzau et al. 2002, Fleming 2006). It accounts for less than 10% of the total ACE and is derived from the membrane-bound form through the action of the ACE secretase. Elevated plasma ACE levels have been suggested to represent a risk factor for coronary artery disease and myocardial infarction. Expression of endothelial ACE is upregulated in response to injury and in disease conditions (e.g. hypercholesterolemia, hypertension and diabetes). Increased accumulation of ACE in atherosclerotic blood vessel walls has been reported as well (Diet et al. 1996, Ohishi et al. 1997).

Angiotensin II (Ang II) Ang II exerts its actions via G-protein coupled angiotensin II type 1 and angiotensin II type 2 receptors (AT1 and AT2, respectively, Figure 2) (for review, see Lemari and Schiffrin 2010). In principle, these two receptors mediate opposite functions, of which AT1 activation has harmful consequences and AT2 activation results in protective actions. Although the sequence homology between AT1 and AT2 is only 30 % and the receptors differ in their affinity to pharmacological agents, Ang II does not distinguish between the two receptors (for review, see de Gasparo et al. 2010). In rodents, two types of AT1 receptor have been reported, AT1A and AT1B. The AT1 receptor is ubiquitously expressed in vascular smooth muscle, liver, kidney, heart, lung, adrenal cortex, pituitary and brain, whereas the expression of the AT2 receptor is developmentally regulated: it is highly expressed in various foetal tissues and at lower levels in adult adrenal medulla, brain and

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reproductive tissues. However, AT2 receptor expression is upregulated in various pathological conditions associated with tissue remodeling or inflammation, including hypertension, diabetes and renal injury (Ruiz-Ortega et al. 2003, Lee et al. 2008, Yayama and Okamoto 2008). In the vasculature, endothelium seems to be the most important site for the AT2 receptor expression (for review, see Paul et al. 2006). Binding of Ang II to AT1 receptor leads to activation of Gq/G11 and/or Gi/Go proteins and to the stimulation of several intracellular signaling pathways (for review, see de Gasparo et al. 2010). The signal transduction mechanisms of the AT1 receptor depend on at least five different effectors including phospholipase C, voltage-dependent Ca2+ channels, phospholipases D and A2 and AC. AT1 receptor activation increases intracellular Ca2+, which activates acute responses such as VSMC contraction. In contrast, the signal transduction mechanisms of AT2 receptor are still poorly understood, but three main mechanisms have been described: activation of protein phosphatases causing protein dephosphorylation, activation of bradykinin/NO/cGMP pathway and the stimulation of phospholipase A2 and release of arachidonic acid (for review, see Lemari and Schiffrin 2010). However, as regards physiological effects, AT2 receptor counterbalances the effects of the AT1 receptor. AT1 receptor-mediated effects of Ang II include vasoconstriction, cellular growth, vascular and cardiac hypertrophy, stimulation of aldosterone and generation of oxidative stress and inflammation, whereas AT2 receptor activation is involved in vasodilatation, inhibition of cellular growth, natriuresis and neuronal activity. Upregulation of AT2 receptors in the vasculature in pathological conditions seems to be a compensatory mechanism by which the blood vessels counteract Ang II-induced AT1 receptor-mediated vasoconstriction. AT1 receptor antagonists exert their effects on the one hand via blockade of activation of detrimental signaling pathways mediated by the AT1 receptors, on the other hand by stimulation of renin release (via negative feedback) and increased production of Ang II, which acts on unblocked AT2 receptors.

Other peptides in RAS Angiotensin 2-8 (Ang III) is generated from Ang II by aminopeptidase A (Figure 2) (Zini et al. 1996). Ang III binds to both AT1 and AT2 receptors, has similar actions as Ang II and is a main effector in the control of vasopressin release via AT1 receptor activation. Angiotensin 3-8 (Ang IV) is generated from Ang III by aminopeptidase M and binds to the insulin-regulated amino peptidase receptor (IRAP), also called as AT4 (for review, see Chai et al. 2004). IRAP-mediated actions of Ang IV include renal 34

vasodilatation, hypertrophy and activation of nuclear factor-kappa B (NF-). It seems that Ang IV is involved in the vascular inflammatory responses and therefore may have a pathophysiological role in cardiovascular diseases. In addition to the ACE-Ang II-AT1/AT2-receptor pathway, Ang I can be converted also to Ang (1-7) by angiotensin-converting enzyme 2 (ACE2) (for review, see Ferrario et al. 2005). ACE2 is a membraneassociated carboxypeptidase, which cleaves one residue from Ang I to generate angiotensin 1-9 ([Ang (1-9) and one residue from Ang II to generate Ang (1-7). ACE2 is reported to be highly expressed in vascular endothelium of heart, kidney, aortic wall and testis. The catalytic efficiency of ACE2 is far higher with Ang II as a substrate than with Ang I, suggesting that the production of Ang (1-7) is preferred to Ang (1-9). Ang (1-7) potentiates the hypotensive and vasodilatory effect of bradykinin either through the inhibition of ACE or by inhibiting the desensitization of bradykinin receptors (Tom et al. 2001, Greco et al. 2006). However, Ang (1-7) itself exerts also a direct vasodilatory effect through G-protein coupled Mas receptors (Santos et al. 2003), which are abundantly expressed in brain and testis and at lower levels in other tissues including heart and kidney (Metzger et al. 1995). In addition, infusion of Ang (1-7) to rats decreases blood pressure (Benter et al. 1995) but, interestingly, the effect is seen only in hypertensive rats. Activation of Mas receptors by Ang (1-7) stimulates NO production in endothelial cells via PI3K/Akt-dependent pathways, which induce changes in eNOS phosphorylation (Sampaio et al. 2007). Thus, Ang (1-7) has actions opposing those of Ang II and may act as a counterregulatory peptide for Ang II. Renin-angiotensin system (RAS) interacts with the sympathetic nervous system, and these two systems regulate the blood pressure in concert. Besides having direct vasoconstrictive or -dilative effects, the main mediators of RAS [Ang II, Ang (1- noradrenaline release and utilization (Balt et al. 2001, Stegbauer et al. 2004).

Local RAS Local RAS systems express all components necessary for the production of Ang II and other angiotensin peptides, namely angiotensinogen, renin, ACE, AT1 and AT2 receptors (for review, see Paul et al. 2006). However, almost all renin found in local RAS is derived from renal renin. Local RAS systems have been identified in most organs and tissues studied. In some organs such as adrenal glands and brain, local RAS system operates independently of the circulating RAS, whereas for example in heart and kidney, they operate in close interaction, in a complementary fashion. The 35

circulating RAS is seen as a regulator of systemic volume, electrolyte balance and blood pressure homeostasis, as local RAS systems account for local effects including proliferation, growth and protein synthesis (for review, see Fyhrquist and Saijonmaa 2008).

2.3.3. Kallikrein-kinin system (KKS) In addition to renin-angiotensin system (RAS), another system, kallikrein-kinin system (KKS), participates in blood pressure regulation. KKS involves several pharmacologically active polypeptides, kinins, which are released in the tissues and body fluids as a result of enzymatic action of kallikreins on kininogens (Figure 2) (for review, see Sharma 2009). The kinin family includes bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), kallidin (Lys- Arg-Pro-Pro-Gly-Phe-Ser-Pro-PheArg) and methionyl-lysyl-bradykinin (Met-Lys-Arg-Pro-Pro-Gly-Phe-Arg). Kinins exert their pharmacologic actions through the activation of two G-protein coupled receptors, B1 and B2. Under physiological conditions, vascular actions of bradykinin are mediated via B2 receptor, which is constitutively expressed by many cell types (for review, see Bhoola et al. 1992). B2 receptor localization in different parts of the vasculature is dependent on the artery size (Figueroa et al. 2001). In rat mesenteric arteries, B2 receptors are mainly expressed in the endothelial cell layer. In contrast to B2 receptors, B1 receptors are rarely expressed in physiological situations, but seem to be upregulated in pathological states associated with injury or inflammation (for review, see McLean et al. 2000). By stimulating B2 receptors, bradykinin induces endothelium-dependent relaxation via multiple mechanisms involving nitric oxide (NO), prostacyclin (PGI2) and endothelium-derived relaxing factor (EDHF) (Ohlmann et al. 1997, Tirapelli et al. 2007). The pharmacologic action of bradykinin in the regulation of systemic blood pressure involves vasodilatation in most areas of circulation, a reduction of total peripheral resistance and regulation of sodium excretion (for review, see Sharma 2009). In general, bradykinin-induced blood pressure lowering effect is mediated by B2 receptors. ACE (kininase II) inhibitors inhibit the degradation of bradykinin and thus potentiate its effects (Ausch-Schwelk et al. 1992). Therefore, part of the antihypertensive and vasoprotective effects of ACE inhibitors may come from the accumulation of bradykinin with subsequent release of NO and PGI2. B2 receptors may also participate in AT2 receptor-mediated vasodilatation. Indeed, abdominal aortic banding induces upregulation of AT2 receptors, which leads to attenuation of Ang II-induced contractile responses in mouse aorta (Hiyoshi et al. 2004). The contractile responses are restored in

36

the presence of icatibant, a specific B2 receptor antagonist, and L-NAME, inhibitor of NO synthase (Yayama and Okamoto 2008). This suggests that B2 receptors are activated in Ang II-induced, AT2 receptor-mediated vasodilatation.

2.4. HYPERTENSION AND VASCULAR DISEASE Hypertension is a known risk factor for cardiovascular diseases, including coronary heart disease, peripheral artery disease and stroke. Because of high prevalence and severe consequences, hypertension poses an important, world-wide health challenge. Any reduction in blood pressure, however small, is meaningful; systolic blood pressure (SBP) reduction of 10-12 mmHg and diastolic blood pressure (DBP) reduction of 5 mmHg may reduce the risk of stroke by 40%, coronary heart disease by 16% and all-cause mortality by 13% (Collins and MacMahon 1994). Classification of blood pressure according to the European Society of Hypertension and the European Society of Cardiology (Mancia et al. 2007) is presented in Table 1. The primary goal of treatment of hypertension is to achieve maximum reduction in the long-term total risk of cardiovascular disease. In grade 1 hypertensives at low and moderate risk, drug therapy should be started after a suitable period with lifestyle changes (Mancia et al. 2009). Earlier initiation of treatment is recommended, if grade 1 hypertension is associated with a high level of risk, or if hypertension is grade 2 or 3. There is no evidence from clinical trials on treatment benefits in patients with high normal blood pressure without diabetes or previous cardiovascular events. However, there is sufficient evidence to recommend that SBP and DBP should be lowered below 140/90 mmHg in all hypertensive patients. Guidelines also recommend that in diabetic patients and in patients at very high cardiovascular risk (previous cardiovascular events, renal dysfunction, proteinuria), the goal is set for 130/80 mmHg (Mancia et al. 2007). Despite this recommendation, there are no trials in which decreasing SBP below 130 mmHg in diabetic patients would have brought any proven benefits (Mancia et al. 2009).

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Table 1. Classification of blood pressure levels (mmHg). Modified from Mancia et al. 2007. Category Optimal Normal High normal Grade 1 hypertension Grade 2 hypertension Grade 3 hypertension Isolated systolic hypertension Systolic <120 120-129 130-139 140-159 160-179 and and/or and/or and/or and/or and/or and Diastolic <80 80-84 85-89 90-99 100-109 <90

Besides pharmacological therapy, nutritional factors play a significant role in the prevention and treatment of hypertension. Dietary efforts to decrease saturated fat and sodium and increase potassium, calcium and soluble fiber intake affect positively blood pressure (Krousel-Wood et al. 2004). Although drug therapy is needed for most hypertensive patients, changes in diet and lifestyle (physical activity, weight reduction, moderation of alcohol consumption, smoking cessation) may be enough for some people with mild hypertension to decrease the blood pressure to the desired level. To enhance preventive and early treatment, a new classification of hypertension was established in 2003 by the USA Joint National Committee Guidelines (JNC 7). According to this, individuals with a blood pressure between normal levels and established hypertension (SBP 120-139 mmHg and DBP 80-89 mmHg) are categorized as having prehypertension (Chobanian et al. 2003). Also in some references grade 1 hypertension is described as mild hypertension. In Finland, cardiovascular risk factors have been studied with population-based health surveys since 1972 at five years intervals. Blood pressure has declined from 1970s, but the decline seems to be leveled off during the past years; most probably due to increased prevalence of obesity and increased alcohol consumption (Peltonen et al. 2008). In 2007, blood pressure was 136/81 and 131/77 mmHg for men and women aged 25-74 years, respectively. In this age group, 37% of men and 28% of women had SBP over 140 mmHg. Despite the decline in blood pressure levels, Finland ranks high in international surveys (Wolf-Maier et al. 2003). Finnish Current Care guidelines (2009) involve recommendations for the management of hypertension, which correspond to that of the European Society of Hypertension and the European Society of Cardiology (Mancia et al. 2007).

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2.4.1. Experimental models of hypertension Experimental models of hypertension have become an essential part of the research in hypertension-related fields. They enable studies where novel agents are used, toxicological profile is characterized or where tissue sampling is necessary. For example, to study vascular function and mechanisms leading to vasodilatation and -constriction, blood vessel samples are needed. Human skin and adipose tissue biopsies and blood vessels from them may be utilized in some studies, but often vessel samples from the main arteries of the body (aorta, carotid artery) are needed. Although various mouse models of hypertension have been generated (e.g. model of renovascular hypertension) (Harris et al. 2007) as well as transgenic mouse models relevant to cardiovascular diseases, rat models are used most frequently in hypertension research. The spontaneously hypertensive rat (SHR) is probably the most widely used animal model of essential hypertension. The strain was developed in Kyoto, Japan in 1960s, when Wistar-Kyoto (WKY) male rats with marked elevation of blood pressure were mated to female with slightly elevated blood pressure (Okamoto and Aoki 1963). SHR start to develop hypertension in the age of 5 to 6 weeks and males exhibit average SBP over 200 mmHg by 3-4 months of age. SHR develop many features of hypertensive end-organ damage such as cardiac hypertrophy, cardiac failure and renal dysfunction (for review, see Pinto et al. 1998). Under specific environmental conditions, such as when fed a high-fructose diet, SHR also develop additional features related to metabolic syndrome, including insulin resistance and dyslipidemia (Oron-Herman et al. 2008). SHR show also marked endothelial dysfunction of conduit and resistance arteries (for review, see Bernatova et al. 2009). Age of the animal is an important factor affecting vascular function in SHR as well as in normotensive rats (such as the WKY rat, which is usually used as the control for SHR). Endothelium-dependent relaxation is reduced in aged SHR, whereas alterations in vascular function are not present in young SHR, although the blood pressure might be already elevated. Therefore, it appears that endothelial dysfunction might not be an initiating factor in the development of hypertension and the decrease in endothelium-dependent relaxations might be the consequence rather than the cause of high blood pressure. Stroke-prone spontaneously hypertensive rat (SHRSP) was obtained by selective breeding of spontaneously hypertensive rat for stroke phenotype (Okamoto et al. 1975). SHRSP develop progressive hypertension from normal levels at 4 weeks of age to a plateau of mean blood pressure >220 mmHg by approximately 20 weeks of age in male and 25-30 weeks in female (Yamori et al.

39

1976). The phenotype is polygenic and associated with overactivity of the renin-angiotensin system (RAS). Multiple cerebrovascular lesions occur spontaneously in > 80% of the rats. Goto-Kakizaki (GK) rat is a non-obese Wistar substrain that develops adult onset type 2 diabetes early in life. The model was developed in the 1970s in Sendai, Japan by selective breeding of Wistar rats with highest blood glucose levels measured by oral glucose tolerance test (Goto and Kakizaki 1981). GK rat is characterized by impaired glucose-induced insulin secretion, abnormal glucose regulation, insulin resistance and polyuria (for review, see stenson and Efendic 2007). GK rats are normotensive or exhibit a slightly higher blood pressure than age-matched Wistar controls (Oniki et al. 2006, Vahtola et al. 2008). However, GK rats have been shown to develop hypertension if fed with high-salt diet (Cheng et al. 2001, Grnholm et al. 2005, Pilvi et al. 2006). Other experimental models of hypertension include angiotensin II (Ang II)-induced hypertension (Dal-Ros et al. 2009, Kane et al. 2010), in which chronic administration of Ang II induces e.g. a significant increase in arterial blood pressure and endothelial dysfunction. Double transgenic rat harbouring human renin and angiotensinogen genes (dTGR) develop hypertension with severe organ damage and do not live more than 7 or 8 weeks (Park et al. 2009, Jauhiainen et al. 2010a). Dahl saltsensitive rat, when fed with high-salt diet, shows marked increase in blood pressure, which results in left ventricular hypertrophy, cardiac failure and eventually death (Inoko et al. 1994). Dahl saltsensitive rat fed with high-salt diet is a useful model to investigate rapidly developing congestive heart failure.

Measurement of blood pressure in experimental animals Both indirect and direct methods to assess blood pressure in rats and mice exist. Three methods are used most frequently: tail-cuff plethysmography, intra-arterial catheters and radiotelemetry (for review, see Plehm et al. 2006). The tail-cuff method is a widely used, indirect method, which is invaluable especially when large groups of animals need to be measured and/or blood pressure is followed over a long time period (for review, see van Vliet et al. 2000). It is noninvasive, simple and relatively inexpensive. However, the main disadvantage of the tail-cuff method is that it requires physical restraint of the animal and some degree of warming to ensure that the tail blood flow is sufficient for the measurements. These factors may lead to overestimation of the blood pressure. In the tail-cuff method, animals are placed in restrainer tubes and kept in warming chambers (temperature 32-37C, depending on the system). A tail-cuff sensor is placed around the tail of the rat and connected to the system, which involves a cuff pump, amplifier and chart recorder. With the 40

tail-cuff method, SBP can be read when pulse/flow disappears during cuff inflation or when it reappears during deflation. Although SBP can be detected in both events, pulse/flow appearance is the value often considered as representative of SBP (Fritz and Rinaldi 2008). Different types of sensors exist; photoelectric and piezoelectric pulse detectors are the most frequently used at present. In direct methods, arterial blood pressure is measured directly with the aid of a sensor device implanted invasively within the arterial system (for review, see van Vliet et al. 2000). The most widely used sensor device has been a saline-filled catheter, whose distal end is connected to a calibrated pressure transducer. This method provides rather inexpensive and precise measurement of blood pressure, but has a limited dynamic response, which makes detection of the systolic and diastolic pressures challenging in small animals with high heart rates. Higher dynamic response is obtained by the transducer-tipped catheter, which contains a miniature pressure transducer at the tip (for review, see van Vliet et al. 2000). The use of transducer-tipped catheters is limited to shortterm applications, because the calibration of the devices drifts with time. Radiotelemetric blood pressure measurement allows continuous recordings from conscious, freely moving animals. Blood pressure telemetry systems consist of a blood pressure sensor, such as a catheter and transducer, a transmitter device, and the electronics for receiving and processing the signal (for review, see van Vliet et al. 2000). In rats, the catheter is usually placed into the lumen of abdominal aorta. Although the method has several advantages, it is rather expensive.

2.4.2. Arterial stiffness Arterial stiffness describes the capacity of arteries to dilate and contract during cardiac cycle. Other closely related terms include arterial compliance and arterial distensibility. During systole, left ventricle increases the pressure in large blood vessels, which may store a significant part of the left ventricle ejection volume because of their elastic properties (for review, see Feihl et al. 2009). During diastole, the blood is pushed towards the periphery. Arterial stiffness can be expressed as either arterial compliance (C), which is the capability of arteries to adjust their diameter according to the changes in blood pressure, or arterial distensibility (D), which describes compliance relative to the initial diameter of the artery, according to the following equations:

41

 W

and

 W

umen cross- W in pressure. Loss of arterial compliance leads to stiffening of the arteries. The two major determinants of arterial stiffness are aging and increased blood pressure, but also low physical activity, smoking and type 2 diabetes contribute to the arterial stiffness (for review, see Laurent and Boutouyrie 2007). The physiological properties of arterial walls are highly dependent on elastin and collagen, and the elasticity of the large arteries is a result of the high elastin to collagen ratio (for review, see Gkaliagkousi and Douma 2009). However, the orderly arrangement of elastic fibers and laminae is lost gradually over time (for review, see Laurent et al. 2006). The degeneration of elastic fibers is associated with an increase in collagenous material and in ground substance, often accompanied by increase in calcium content. Arterial stiffness has probably also a genetic component, which is independent of the influence of blood pressure and other cardiovascular risk factors. In addition, chronic low-grade inflammation may play a role in arterial stiffness. Indeed, a relationship between the presence of various inflammatory markers [e.g. high-sensitive C-reactive protein (hs-CRP), tumor necrosis factor-alpha (TNF- -6 (IL- (Amar et al. 2005, Mahmud and Feely 2005).

Left ventricle contraction generates a pulse wave that travels from the aorta to the peripheral arteries (for review, see Ghiadoni et al. 2009). The speed of the pulse wave is mainly determined by the artery wall stiffness and lumen diameter. Pulse wave is reflected from bifurcations and any impediments of the arterial tree and retrograde waves are generated. This accounts for the secondary fluctuations of the pressure waveform and can contribute to the increased pulse pressure (PP) and SBP. With increased arterial stiffness, pulse wave travels more rapidly, as the arterial compliance is reduced and the ability of arteries to adjust to the pressure change is disabled. The elastic properties of conduit arteries vary along the arterial tree proximal arteries being more elastic and distal arteries stiffer. This is reflected to the pulse-wave velocity (PWV); in humans, the PWV increases from 45 m/s in the ascending aorta to 56 m/s in the abdominal aorta and to 89 m/s in the iliac and femoral arteries (Latham et al. 1985). In addition, the difference in elastic properties along the arterial tree has important physiological and pathophysiological consequences. A pressure wave encountering no reflection sites is attenuated, whereas a pressure wave propagating in an artery with numerous branches and impediments is progressively amplified from central to distal conduit arteries (for review, see Laurent et al. 2006). Consequently, the reflected 42

waves are added onto the forward wave and the amplitude of the pressure wave is higher. This is called the amplification phenomenon.

Assessment of arterial stiffness The measurement of pulse-wave velocity (PWV) is generally accepted to be the most simple, noninvasive, robust and reproducible method to determine arterial stiffness (for review, see Laurent et al. 2006). Carotid-femoral PWV, which is measured along the aortic and aorto-iliac pathway, is the most clinically relevant, as aorta and its first branches are responsible for most of the pathophysiological effects of arterial stiffness. Carotid-femoral PWV is thus considered to be the gold standard measurement of arterial stiffness (for review, see Laurent et al. 2006). PWV can be measured from various different waveforms (e.g. pressure), that are usually obtained transcutaneously at the common carotid artery and the femoral artery (for review, see Laurent et al. 2006). PWV is calculated as the ratio of the distance between the two recording sites (D) to the time delay (Dt or transit time) measured between the feet of the two waveforms: PWV = D (meters) / Dt (seconds). In healthy and elastic vessels, PWV is low and the reflected waves arrive back to the aortic root during diastole. However, if arteries are stiff, PWV rises and the reflected waves arrive back earlier, add to the forward wave and augment systolic blood pressure. This phenomenon can be quantified using the augmentation index (AIx), which is defined as the difference between the second and first systolic peaks (P2-P1) and is expressed as a percentage of the pulse pressure (Mackenzie et al. 2002) (Figure 3).

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Figure 3. Pressure waveform and calculation of the augmentation index (AIx). PP, pulse pressure; DBP, diastolic blood pressure; SBP, systolic blood pressure; AP, augmentation pressure. Modified from Laurent et al. 2006 and Ghiadoni et al. 2009.

Optimally, pulse-wave analysis (PWA) should be obtained at the central level (at the site of carotid artery or the ascending aorta) and either directly recorded or computed from the radial artery waveform using a transfer function (for review, see Laurent et al. 2006). AIx is a relative measurement and can be thus calculated without calibration, but for the estimation of absolute values of pulse pressure, augmentation pressure and systolic blood pressure, blood pressure needs to be measured from a reference artery (usually brachial artery). However, it should be noted that central pressure, AIx and PWV cannot be used interchangeably as indexes of arterial stiffness. PWV is a direct measure of arterial stiffness whereas central pressure and AIx are only indirect, surrogate measures. However, they provide additional information concerning wave reflections. Optimally, central PWA and aortic PWV measurements should be coupled to determine the aortic stiffness. The 2007 Guidelines for the management of arterial hypertension by the European Society of Hypertension and the European Society of Cardiology included arterial stiffness, measured as carotid-femoral PWV, as an intermediate end point in evaluating target organ damage (Mancia et al. 2007). This is justifiable, as a number of longitudinal studies show that aortic stiffness expressed as aortic PWV is a strong predictor of future cardiovascular events and all-cause mortality (Inoue et al. 2009, Vlachopoulos et al. 2010). The predictive ability of arterial stiffness is higher in subjects with a higher baseline cardiovascular risk. Although the relationship between aortic stiffness and 44

cardiovascular events is continuous, a threshold >12 m/s for PWV has been suggested as an estimate of significant alterations of aortic function in middle-aged hypertensives (Mancia et al. 2007). In recent studies, arterial stiffness has been shown to be associated with left ventricular hypertrophy (Gosse et al. 2010) and increased risk of cardiovascular events such as myocardial infarction, stroke and heart failure (Mitchell et al. 2010).

2.4.3. Endothelial dysfunction Endothelial dysfunction, defined as reduced vasodilatating response to endothelial stimuli, is often observed in the presence of cardiovascular diseases or risk factors such as hypertension (for review, see Taddei et al. 2000), type I and type II diabetes (for review, see Rask-Madsen and King 2007), hypercholesterolemia (Kawano et al. 2002, Vladimirova-Kitova et al. 2009), metabolic syndrome (Ghiadoni et al. 2008) and chronic smoking (Heitzer et al. 1996, Poredos et al. 1999). Conversely, the presence of endothelial dysfunction is also suggested to increase the susceptibility to develop hypertension (Rossi et al. 2004). Endothelial dysfunction plays also a role in the progression of atherosclerotic process; increased intima-media thickness of common carotid artery, a non-invasive marker of atherosclerosis, is directly related to the impairment of endothelial function in the peripheral circulation (Ghiadoni et al. 1998). In secondary hypertensive patients (primary aldosteronism or renovascular hypertension), the normalization of arterial blood pressure restores endothelium-dependent relaxation, indicating that endothelial dysfunction is a consequence of the high blood pressure (for review, see Taddei et al. 2000). However, in essential hypertension, endothelial dysfunction seems to precede the onset of high blood pressure as in young, normotensive offspring of essential hypertensive patients, the response to acetylcholine is already reduced (Taddei et al. 1996). As antihypertensive agents do not improve endothelial function to the same extent despite similar reduction in blood pressure (for review, see Tang and Vanhoutte 2010), altogether these data indicate that endothelial dysfunction in essential hypertension is not directly related to blood pressure values and also a genetic component may exist. Several potential abnormalities can account for the reductions in endothelium-dependent relaxation, including changes in the activity and/or expression of the eNOS, decreased sensitivity of VSMCs to NO or decreased bioavailability of NO due to degradation by reactive oxygen species (ROS) (for review, see Mnzel et al. 2008). Reduced NO bioavailability is partly compensated by the activation of alternative pathways, including the production and release of endothelium-derived 45

hyperpolarizing factor (EDHF) (for review, see Grgic et al. 2009). However, in some disease states also defects in EDHF system have been associated with endothelial dysfunction. In addition, hypertensive patients show an augmented vasoconstriction to endothelin-1 (ET-1), which exerts its actions via ETA and ETB receptors (for review, see Penna et al. 2006). ETA receptors are mainly localized in VSMCs and mediate vasoconstriction whereas ETB receptors are found both from VSMCs and endothelium and mediate NO release. Thus, in the presence of endothelial dysfunction, activation of ETB receptors is not able to increase NO-mediated vasodilatation and the contracting effect of ET-1 via ETA receptors is enhanced. A lack of the eNOS substrate L-arginine by enhanced activity of vascular arginase has been suggested to play a role in endothelial dysfunction as well (Zhang et al. 2004).

Assessment of endothelial function in humans The current gold standard for diagnosing endothelial dysfunction is the assessment of endothelial function in coronary arteries via administration of acetylcholine to the coronary blood vessel system, combined with a measurement of coronary blood vessel flow (for review, see Mnzel et al. 2008). However, this invasive procedure is not reasonable to use in conventional clinical settings. As nitric oxide (NO) has a very short half-life (Rubanyi et al. 1985), the direct quantification of the gaseous molecule is difficult. Therefore, NO bioavailability in humans is indirectly estimated from its vasodilatating effect after endothelial stimulation either with mechanical or pharmacological tools. Accordingly, the intracoronary infusion of acetylcholine would be the most valuable approach, but it is very invasive and has several other limitations (for review, see Mnzel et al. 2008). In this method, the change in vessel diameter by acetylcholine is measured by quantitative coronary angiography. A much less invasive approach is to measure the flow-mediated dilation (FMD) of the brachial artery by using high-resolution ultrasound (Celermajer et al. 1992). Brachial artery diameter is measured before and after an increase in shear stress induced by reactive hyperemia. A sphygmomanometer cuff is placed proximal to brachial artery and inflated up to 200 mmHg for 5 minutes and when the cuff is released, the reactive, flow-dependent dilatation of the brachial artery is recorded. FMD is measured as the percentage change in brachial artery diameter from baseline in response to the increased flow. The degree of dilatation reflects endothelial function and NO bioavailability. The measurement is usually followed by testing the dilatation in response to sublingual nitroglycerin to assess the total vasodilatative capacity of the vessel. Evidence for endothelial dysfunction is seen when FMD is below 8% (Gokce et al. 2002, Akcakoyun et al. 2008).

46

Forearm plethysmography is used to quantify changes in forearm blood flow in response to intrabrachial infusion of acetylcholine or other endothelium-dependent vasodilators (for review, see Mnzel et al. 2008). Infusion of nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) has been used to assess the contribution of basally released NO. Endothelial function may be assessed also using finger-pulse plethysmography, in which changes of the pulse wave amplitude are recorded using pneumatic finger probes (Bonetti et al. 2004). Besides the assessment of arterial stiffness, pulse-wave analysis can be used to evaluate endothelial function as well. In this case, beta2-adrenoceptor agonist salbutamol, which causes endothelial release of NO but only minor changes of blood pressure, is given by inhaler at standard doses and pulse-wave analysis by radial artery tonometry is performed (Hayward et al. 2002). Augmentation index (AIx) is used to quantify the changes in arterial waveform. The method seems to correlate with FMD but has larger within subject variation; thus, FMD is the preferred method to detect effects of interventions on endothelial function (Rambaran et al. 2008). A proper control of confounding factors applies to all the methods used for the assessment of endothelial function: time of investigation, food intake, hormonal factors, stress and sleep deprivation all influence endothelial function and affect the results (for review, see Mnzel et al. 2008). Endothelial function is usually assessed by using the aforementioned techniques. However, measurement of circulating biomarkers could provide an indirect option to evaluate endothelial function and risk for cardiovascular events. Several factors have been proposed to serve as such potential markers: nitrites and nitrosylated proteins partly reflect endothelial generation of NO (Rassaf et al. 2004) whereas E-selectin, P-selectin and adhesion molecules (VCAM-1, ICAM-1) represent the endothelial cell activation and inflammatory status of the vascular wall (for review, see Savoia and Schiffrin 2007). Elevated levels of apoptotic circulating endothelial cells and circulating endothelial progenitor cells have been reported in patients with cardiovascular diseases and may thus be potential markers of endothelial function (Goon et al. 2005). An interesting marker is asymmetric dimethyl L-arginine, ADMA, an endogenous competitive antagonist of NO synthase. Levels of ADMA are elevated in subjects with cardiovascular risk factors or diseases such as hypercholesterolemia (Bger et al. 1998) and hypertension (Wang et al. 2009). Increased levels are associated with a reduction in NO bioavailability (for review, see Blackwell et al. 2010). ADMA has been shown to be an independent predictor of cardiovascular events and mortality. However, due to difficulty, expensiveness and biological variability, these measures have only a very limited role in the assessment of individual patients but can provide important information regarding mechanisms and severity of endothelial dysfunction in clinical research settings. 47

Assessment of endothelial function in experimental animals Methods for both in vivo and in vitro assessment of endothelial function in experimental animals exist. Using anesthetized animals, changes in arterial diameter in response to different agonists can be monitored using intravital microscopy (Schuschke et al. 1991, Kelsall et al. 2001). Pharmacological agents are usually administered topically or intra-arterially. Some of the experimental systems enable also simultaneous recording of blood pressure of the animals (McGown et al. 2010). Isolated arteries from rats and mice are often used to determine the effects of an in vivo treatment on vascular function or to see if different compounds possess vasoactive properties in vitro. After isolation, arteries are cleaned of adherent connective tissue and cut into sections or rings. Endothelium may be left intact or rubbed off to obtain endothelium-free preparations. The basic experimental setup for the measurement of arterial wall force generation involves placing two stainless steel hooks or stainless steel or tungsten wires through the lumen of an arterial section (for review, see Matheson and Garrison 2005). One hook or wire is anchored in a tissue bath containing physiological salt solution as the other is attached to a sensitive force transducer. Great care is needed to avoid damage to the endothelium in dissecting the artery, placing the wires or hooks through the lumen of the artery and mounting the arterial preparation in the bath. To quantificate the arterial contractility and dilatation, arteries must be investigated under a known mechanical load (for review, see Mulvany and Aalkjaer 1990). Before the actual experiments, arterial rings are stretched with a certain preload, of which degree depends on the size of the artery. Standard organ bath chambers, in which hooks are used, allow the study of large-sized arteries such as aorta, but also smaller arteries (e.g. superior mesenteric arteries) may be suitable for this system. Wire and pressure myographs enable the investigation of smaller arteries, such as branches of mesenteric artery and coronary arteries. In wire myograph (also known as Mulvany myograph), arteries are mounted as isometric preparations between two stainless steel or tungsten wires that are fastened to a force transducer and a micrometer (for review, see Mulvany and Aalkjaer 1990). In pressure myograph, an artery is cannulated on size-matched cannulae and pressurized, thus being an isobaric preparation (Coats and Hillier 1999). The lumen diameter is then either determined from light microscopy or measured electronically or optically. It has been suggested that the spontaneous basal tone is more easily reproduced in vitro in pressurized arteries than in wire-mounted arteries. In addition, there are differences in the sensitivity of the arteries to alpha-adrenergic agonists and steepness of the concentration-response relation between the two methods (Buus et al. 1994). It seems that pressure myograph corresponds more closely to the in vivo situation, as also the pressure component is involved. 48

In all above-mentioned systems, different pharmacological agents are introduced to the arterial preparations and following responses determined by computerized systems. The increase or decrease in tension or the diameter of the artery is calculated and used in further analysis.

2.4.4. Pharmacological treatment The main benefits of antihypertensive treatment are due to blood pressure lowering effect per se, and largely independent of the drugs used (Mancia et al. 2007). At present, five major classes of antihypertensive agents are recommended for the initiation and maintenance of antihypertensive treatment, including beta-blockers, thiazide diuretics, calcium antagonists, angiotensin-converting enzyme (ACE)-inhibitors and angiotensin II receptor 1 (AT1) antagonists. However, some other drug classes (imidazoline receptor agonists, alpha2-adrenoceptor agonists) may be used as well. Although the antihypertensive treatment is usually initiated with one drug, combination treatment is needed in the majority of patients to control the blood pressure. Each of the classes has advantages and limitations, which should be taken into account when planning the therapy. The blood pressure lowering effect should last 24 hours. This can be checked by office or home blood pressure measurements or 24 hour ambulatory blood pressure monitoring.

Beta-blockers By blocking beta1-receptors, beta-blockers reduce the heart rate and myocardial contractility, thus lowering cardiac output and arterial blood pressure (Frishman and Silverman 1979). Beta-blockers also inhibit renin release in the kidney, inhibit central nervous sympathetic outflow, reduce venous return and plasma volume and improve vascular compliance (for review, see Che et al. 2009). They can be classified in three categories: 1) nonselective beta-blockers, which block both beta1- and beta2-receptors (propranolol, timolol), 2) selective beta-blockers, which specifically block beta1receptors alone (atenolol, bisoprolol, metoprolol) and 3) beta-blockers with additional peripheral vasodilatatory effects (carvedilol, nebivolol). Administration of beta-blockers has been shown to be beneficial especially in patients with angina pectoris, heart failure and a recent myocardial infarction (Mancia et al. 2007). As beta-blockers have adverse effects on lipid metabolism and increase the incidence of new onset diabetes, they should not be preferred in hypertensives with several risk factors of metabolic syndrome. However, vasodilator beta-blockers such as carvedilol and nebivolol, have less or no dysmetabolic action. Nebivolol is an interesting, novel selective beta-blocker, which

49

causes vasodilatation through activation of L-arginine/nitric oxide pathway (Cockcroft et al. 1995, Georgescu et al. 2005). It has been shown to improve endothelial dysfunction in essential hypertensive patients; an effect that is probably related to decreased levels of circulating asymmetric dimethyl L-arginine (ADMA, endogenous competitive antagonist of NO synthase) (Pasini et al. 2008).

Thiazide diuretics Thiazide diuretics were the first tolerated efficient antihypertensive drugs that showed a significant reduction in cardiovascular mortality and morbidity in placebo-controlled studies (for review, see Salvetti and Ghiadoni 2006). Thiazide (hydrochlorothiazide, trichlormethiazide) or thiazide-like (indapamide) diuretics act by inhibiting the renal Na+/Cl- cotransporter (for review, see Hughes 2004). This leads to a reduction in interstitial fluid and plasma volume, decreased venous return and cardiac output and thus a decrease in blood pressure. However, in long-term use, interstitial and plasma volume return to the normal level but the antihypertensive effect is preserved. If used at high doses, thiazide diuretics may have dyslipidaemic and diabetogenic effects (Weidmann et al. 1983, Manrique et al. 2010). Accordingly, they are not recommended to patients with metabolic syndrome. Thiazide diuretics are often used in combination with other antihypertensive agents.

Calcium antagonists Calcium antagonists used in the treatment of hypertension can be divided into dihydropyridines (nifedipine, amlodipine, felodipine), diltiazem and verapamil. They all share the common feature of inhibiting cellular entry of calcium through voltage-dependent L- and T-type calcium channels, but differ in inotrophic effects and vascular selectivity (for review, see Nathan et al. 2005). Namely, dihydropyridines cause vasodilatation in peripheral arteries as diltiazem and verapamil have more effects on cardiac function (negative chronotrophy). Calcium antagonists appear to be beneficial in slowing down the progression of carotid hypertrophy and atherosclerosis and if antihypertensive treatment is needed during pregnancy, they are the drugs of choice (for review, see Mancia et al. 2007).

50

ACE-inhibitors and AT1 antagonists ACE-inhibitors (captopril, ramipril, enalapril) inhibit the conversion of angiotensin I to angiotensin II (Ang II) and thus prevent the vasoconstrictive and hypertrophic effects of Ang II. They also inhibit the degradation of bradykinin, a potent vasodilator, and decrease serum aldosterone levels. AT1 antagonists (losartan, telmisartan, candesartan) act directly on the AT1 receptors by inhibiting the actions of Ang II. During the last decade, several large clinical trials have shown that ACE-inhibitors and AT1 antagonists reduce the risk of cardiovascular events. These two classes of antihypertensive agents seem to be comparable in many ways. For example, in the HOPE study ACE-inhibitor ramipril reduced the risk of myocardial infarction by 20%, stroke by 32% and cardiovascular mortality by 26% in patients at high risk of cardiovascular events (Yusuf et al. 2000). In the ONTARGET study, the combination of AT1 antagonist telmisartan and ramipril was investigated; cardiovascular death, nonfatal myocardial infarction, stroke or the development of heart failure did not differ between telmisartan + ramipril or telmisartan alone compared to ramipril (ONTARGET investigators 2008). In addition, two meta-analyses on the antihypertensive effect of either ACE-inhibitors or AT1 antagonists concluded that both classes of drugs decrease SBP and DBP by 8 and 5 mmHg, respectively (Heran et al. 2008a, Heran et al. 2008b). The evidence also suggests that there are no significant differences between different ACE-inhibitors and AT1 antagonists within the classes. However, there are more adverse effects (cough, angioneurotic edema) associated with ACEinhibitors than with AT1 antagonists. Both ACE-inhibitors and AT1 antagonists have been reported to be particularly effective in reducing left ventricular hypertrophy, microalbuminuria and proteinuria, and in preserving renal function and delaying renal disease (for review, see Mancia et al. 2007).

Renin inhibitors A new class of antihypertensive agents was introduced recently. Aliskiren is a direct renin inhibitor at the site of its activation and has been shown to be effective in lowering SBP and DBP when given as monotherapy (Mancia et al. 2009). It is effective also in combination with several other antihypertensive agents. Aliskiren combined to AT1 antagonists has been shown to be superior to AT1 antagonist alone in various endpoints and it appears to have a favorable tolerance profile (Oparil et al. 2007, Parving et al. 2008). The role of aliskiren in the treatment of hypertension remains to be elucidated in the near future.

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2.4.5. Non-pharmacological treatment Lifestyle changes are an essential part of the treatment of hypertension in all patients, including patients with high normal blood pressure and those requiring medication. The purpose is to lower blood pressure, to control other risk factors (such as metabolic syndrome) and to reduce the number of doses of antihypertensive drugs.

Nutrition Dietary salt intake contributes to the development and prevalence of hypertension (Law 1997, Johnson et al. 2001). It also increases the risk of stroke and other cardiovascular events (Berry and Beevers 1992, Tuomilehto et al. 2001). According to Finnish Current Care guidelines (2009), daily intake of salt should be lowered below 5 g. However, this is not currently achieved as daily intake of salt was 9-10 and 7-8 g in men and women, respectively, in 2007 (Paturi et al. 2008). According to a meta-analysis of 28 trials, a modest reduction in salt intake for a duration of 4 weeks or more reduces systolic and diastolic blood pressure (SBP and DBP) by 5.0 and 2.7 mmHg, respectively, in hypertensive patients (He and MacGregor 2004). Increased intakes of potassium, calcium and magnesium have been shown to lower SBP by 1 to 3 mmHg (Whelton et al. 1997, Dickinson et al. 2006, van Mierlo et al. 2006). Compliance to DASH diet (a diet rich in fruits, vegetables and low-fat dairy products) decreases blood pressure as well (Appel et al. 1997). There are also studies reporting that high-dose omega-3 polyunsaturated fatty acid (PUFA) supplements lower blood pressure in hypertensive individuals. Authors of a meta-analysis that collected data from 17 trials concluded that diet supplementation with more than 3 g/day of PUFAs can lead to clinically relevant blood pressure reductions in untreated hypertensives (Appel et al. 1993). Plant sterols and stanols effectively lower low-density lipoprotein (LDL) cholesterol, but do not seem to lower blood pressure as such (Tapola et al. 2004, Hallikainen et al. 2006). However, through their cholesterol-lowering action, they might have indirect, long-term effects on blood pressure and arterial stiffness, as has been observed with intensive cholesterol reduction by statins (Glorioso et al. 1999, Ferrier et al. 2002).

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Other factors There is a considerable number of evidence from observational studies documenting that body weight is directly associated with blood pressure (Daniels et al. 1996). Weight reduction lowers blood pressure in obese patients and has beneficial effects on associated risk factors such as insulin resistance, diabetes, hyperlipidaemia and obstructive sleep apnea. For example, weight reduction by 4% lowers blood pressure in overweight hypertensives by 6/3 mmHg and reduces the need of medication (Neter et al. 2003, Horvath et al. 2008). A large number of epidemiological studies have demonstrated the positive association between alcohol intake and blood pressure (for review, see Keil et al. 1998). High levels of alcohol consumption are also associated with high risk of stroke (Wannamethee and Shaper 1996), especially in binge drinking. Moderation of alcohol consumption has beneficial effects on blood pressure; reduction of heavy drinking by 3-4 drinks per day decreases blood pressure by 3/2 mmHg (Finnish Current Care guidelines 2009) Smoking is a strong cardiovascular risk factor and cessation of smoking is probably the single most effective lifestyle change for the prevention of cardiovascular diseases. Both normotensive and untreated hypertensive smokers have higher daily blood pressure values than non-smokers (Mann et al. 1991, Verdecchia et al. 1995). Also lack of physical activity has a powerful influence in cardiovascular disease prognosis. Aerobic exercise lowers blood pressure significantly; a metaanalysis involving 72 trials and 3936 subjects showed that endurance training induced significant reductions in both resting and daytime ambulatory blood pressure, 3/2 and 3/4 mmHg, respectively (Cornelissen and Fagard 2005). The reduction in resting blood pressure was even more in hypertensive subjects.

2.5. ANTIHYPERTENSIVE EFFECT OF MILK AND ITS COMPONENTS Epidemiological studies have suggested that consumption of dairy products is inversely related to the risk of hypertension. The first National Health and Nutrition Examination Survey (NHANES I) showed that lower consumption of milk products was associated with higher blood pressure (McCarron et al. 1984). Intake of dairy products, particularly low-fat products, has consistently been associated with lower blood pressure levels and reduced risk of hypertension also in other observational studies. For example, a nine years follow-up study of 6912 white, normotensive men and women showed that subjects consuming three or more servings of low-fat milk per day had 2.7 mmHg lower increase of SBP compared to those consuming less than one serving per week (Alonso 53

et al. 2009). In addition, a longitudinal 12-month analysis of PREDIMED trial participants (men and women aged 55-80 and 60-80 years, respectively, at high cardiovascular risk) and their diet showed the inverse association of low-fat dairy products and blood pressure (Toledo et al. 2009). Interestingly, a prospective study of 3157 young adults (age 18-30 years) followed for 10 years found that the incidence of elevated blood pressure was inversely associated with total dairy food intake, if subjects had body mass index (BMI) more than 25 kg/m2 but not in normal weight subjects (Pereira et al. 2002). The association of dairy consumption and blood pressure has been shown also in intervention studies. The Dietary Approaches to Stop Hypertension (DASH) trial with 459 normotensive or mildly hypertensive subjects showed that a diet rich in fruits, vegetables and low-fat dairy products lowers blood pressure significantly (Appel et al. 1997). This combination diet (DASH diet) decreased SBP and DBP by 5.5 and 3.0 mmHg, respectively, more than the control diet whereas a diet containing fruits and vegetables alone produced blood pressure reductions of approximately half of this. However, other dietary alterations (e.g. reduced saturated fat) were also incorporated into the DASH diet, so the greater reduction in SBP cannot be ascribed to the milk products only. Thereafter, data from other trials evaluating the effect of dietary interventions on several clinical outcomes, including blood pressure, has been published. It seems that translating the results from original DASH trials to broader populations is more difficult, as blood pressure reductions have not been of a similar degree or no differences have been detected (Appel et al. 2003, Howard et al. 2006, Folsom et al. 2007). However, the DASH diet has been incorporated into dietary guidelines world-wide and the dietary pattern is recommended to be used for the prevention and management of hypertension (Khan et al. 2008, Harnden et al. 2010, National Heart Lung and Blood Institute 2010). Nonetheless, other intervention studies have as well demonstrated a relationship between the intake of milk products and reduction in blood pressure (van Beresteijn et al. 1990, Buonopane et al. 1992, Hilary Green et al. 2000), although not all studies have been able to show this (Barr et al. 2000).

2.5.1. Minerals Milk is rich in calcium, potassium and magnesium (National Institute for Health and Welfare 2009). According to the FINDIET 2007 survey, about 60% of the calcium and 20 % of potassium intake was derived from milk and dairy products in Finnish adult population (Paturi et al. 2008).

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A meta-analysis of 33 randomized controlled clinical trials (total 2609 subjects) showed that increased intake of potassium reduces SBP and DBP by 3.1 mmHg and 2.0 mmHg, respectively (Whelton et al. 1997). Potassium has been shown to lower blood pressure even more in hypertensive subjects (Geleijnse et al. 2003). There is also a considerable amount of evidence for the effect of calcium on blood pressure. According to a meta-analysis of 40 studies (2492 subjects), calcium supplementation (mean 1200 mg/day) decreased SBP by 1.9 mmHg and DBP by 1.0 mmHg (van Mierlo et al. 2006). Thus, electrolyte content may partly explain the inverse association between consumption of milk products and blood pressure. Milk is low in sodium and consequently favorable as concerns the need for the decrease of salt intake in controlling elevated blood pressure. The role of sodium was also acknowledged in the DASH study, as the blood pressure lowering effects of the DASH diet became even more effective, if sodium was restricted even lower than usual recommendations (Sacks et al. 2001). Studies with experimental models of hypertension have also shown the beneficial effect of minerals on blood pressure. High-calcium diet attenuates almost uniformly the development of hypertension in young spontaneously hypertensive rats (SHR) (Prsti et al. 1992, Mkynen et al. 1995, Tolvanen et al. 1998). Also potassium supplementation has an antihypertensive effect in rats (Tobian et al. 1985, Tolvanen et al. 1998, Pere et al. 2000), although the effect has been less consistent than with calcium. In different studies, calcium supplementation has decreased systolic blood pressure or attenuated the development of hypertension by 10 to 50 mmHg depending on the strain and duration of the study (for review, see Hatton and McCarron 1994).

2.5.2. Protein Besides minerals, the antihypertensive effect of milk has been related to milk protein. Most observational studies suggest that increased intake of protein is associated with lower blood pressure and attenuated blood pressure increase over time (Obarzanek et al. 1996, He and Whelton 1999). Although the number of randomized, controlled clinical trials large enough is limited, mainly positive effects of protein intake on blood pressure have been reported (Burke et al. 2001, Appel et al. 2005, He et al. 2005). Bovine milk contains about 32 g/l of protein, of which caseins account for 80 % and whey proteins constitute 20 % (for review, see Haug et al. 2007). Caseins are defined as those phosphoproteins that precipitate from raw skim milk by acidification to pH 4.6 at 20C (Farrell et al. 2004). Caseins can s1- s2- - -casein. The whey fraction, the fluid that remains soluble in 55

milk after caseins are removed, - -lactalbumin, but contains also various other proteins, e.g. immunoglobulins, serum albumin and lactoferrin in smaller quantities.

2.5.3. Milk as a source of bioactive peptides Milk contains the nutrients that are needed for growth and development and is a rich source of lipids, proteins, minerals and vitamins. Besides being an essential requirement for neonates and a traditional food product, milk contains large amount of physiologically active peptides encrypted in the protein sequences. Bioactive peptides have been defined as specific protein fragments that have a positive effect on body functions or conditions and may ultimately influence health (Kitts and Weiler 2003). At present, milk proteins are considered the most important source of bioactive peptides (Korhonen 2009). Milk-derived peptides, both from whey and casein, possess a variety of physiological effects. Numerous known peptide sequences exhibit antihypertensive (for review, see Saito 2008), immunomodulatory (Gauthier et al. 2006), osteoprotective and mineral-binding (Mller et al. 2008), antioxidative (Pihlanto 2006), antimicrobial (Tomita et al. 1991, Zucht et al. 1995), antithrombotic (Jolls et al. 1986) or opioid activities (Antila et al. 1991, Nurminen et al. 2000). Accordingly, milk-derived peptides are potential candidates to be incorporated into food products and to be targeted at cardiovascular, skeletal or digestive system or to improve immune defence or mood. Peptides may be deliberated from their parent proteins by enzymatic hydrolysis during gastrointestinal digestion, fermentation of milk with proteolytic starter cultures or hydrolysis by enzymes obtained from microorganisms (for review, see Phelan et al. 2009). It is also possible to produce peptides by chemical synthesis, recombinant DNA technology or enzymatic synthesis, when the structure of the peptide is known (Gill et al. 1996).

Gastrointestinal digestion During gastrointestinal digestion, physiologically active peptides may be produced from several milk proteins (Chabance et al. 1998, Parrot et al. 2003, Hernndez-Ledesma et al. 2004). Hydrolysis may occur in various stages after ingestion of the dietary protein. In the stomach, pepsin initiates the process of protein digestion (for review, see Guyton and Hall 2006d). After entering the small intestine, pancreatic enzymes trypsin and chymotrypsin further hydrolyse proteins producing 56

peptides of various lengths. Brush border peptidases (aminopolypeptidase and several dipeptidases) in the membrane of enterocytes split the remaining larger polypeptides to tripeptides, dipeptides and some of them to amino acids. Amino acids and remaining peptides are transported to the interior of the enterocyte, where the final digestion occurs. They pass on through the other side of the enterocyte and finally into the circulation. However, some proteins may be resistant to proteinases and remain intact. Peptides may express a variety of functions either at the gastrointestinal tract, at the intestinal epithelium or after systemic absorption into the circulation (for review, see Shimizu 2004).

Fermentation of milk with proteolytic starter cultures The peptidase systems of lactic acid bacteria (LAB) utilize milk protein by converting peptides to amino acids, which are further used for protein synthesis, generation of metabolic energy and recycling of reduced cofactors (for review, see Christensen et al. 1999). LAB, such as Lactobacillus helveticus and Lactococcus lactis, are traditionally used in dairy food processing. Therefore, in the fermentation process of milk, bioactive peptides can be generated by dairy starter cultures and significant amounts of them may be found from the final product. Tripeptides isoleucine-prolineproline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) have been found from sour milk fermented with L. helveticus CP790 and Saccharomyces cerevisiae (Nakamura et al. 1995a). Also several cheeses of Swiss origin contain the same tripeptides (Meyer et al. 2009). The concentration of Ile-Pro-Pro and Val-Pro-Pro seems to increase in the course of ripening process, reaching 100 mg/kg after 4-7 months. Whey fraction of a yoghurt-like product fermented by L. helveticus CPN4 was found to contain a dipeptide Tyr-Pro, which produced a significant antihypertensive effect in SHR (Yamamoto et al. 1999).

Enzymatic hydrolysis Gastrointestinal enzymes, such as pepsin and trypsin may be utilized to generate bioactive peptides from whole proteins (Pihlanto-Leppl et al. 2000). Also enzyme combinations can be used. Proteolytic enzymes of LAB, such as the cell-wall associated serine protease, may be isolated, purificated and used to produce bioactive peptides from casein of different species (Minervini et al. 2003). In functional food production, use of commercially available microbial-derived proteinases

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and ultrafiltration membranes is cost-effective and increases product yields (Korhonen and Pihlanto 2003).

2.5.4. Antihypertensive peptides from milk proteins Although both casein and whey fractions of milk have been shown to decrease blood pressure as such (Pal and Ellis 2009), especially several peptides encrypted in the parent milk protein possess antihypertensive effects. Perhaps the most extensively studied are casein-derived tripeptides IlePro-Pro and Val-Pro-Pro. However, other antihypertensive peptides have been found from casein as well. The hydrolysis of isoelectric casein with pepsin generates peptides corresponding to s1-casein f(90-94) (Arg-Tyr-Leu-Gly-Arg), s1-casein f(143-149) (Ala-Tyr-Phe-Tyr-Pro-Glu-Leu) and s2-casein f(89-95) (Tyr-Gln-Lys-Phe-Pro-Gln-Tyr), which exerted antihypertensive activity after oral administration to SHR (del Mar Contreras et al. 2009). These peptides inhibited ACE by IC50 values of 0.7, 6.6 and 20.1 D / -casein f(133-138) peptide (Leu-His-Leu-Pro-LeuPro), identified from milk fermented with Enterococcus faecalis, showed a significant antihypertensive effect in SHR (Quirs et al. 2007). -Lactorphin (Tyr-Gly-Leu-Phe) and -lactorphin (Tyr-Leu-Leu-Phe) can be released from milk whey proteins -lactalbumin and -lactoglobulin, respectively, in enzymatic proteolysis by gastric and pancreatic enzymes (Antila et al. 1991). -Lactorphin has been shown to produce a transient, dosedependent blood pressure lowering effect, which is abolished by a specific opioid receptor antagonist, naloxone (Nurminen et al. 2000). Both tetrapeptides improved vascular function of isolated rat mesenteric arteries in vitro (Sipola et al. 2002b). Another tetrapeptide from lactoglobulin, -lactosin B (Ala-Leu-Pro-Met) showed strong antihypertensive effect in SHR as well (Murakami et al. 2004). Also proteinase K-digested whey of cheese origin was shown to decrease blood pressure in SHR after single-dose administration (Abubakar et al. 1998). From the digest, the peptide showing the strongest antihypertensive activity was found to be tripeptide Ile-Pro-Ala, originating from -lactoglobulin.

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2.6. CASEIN-DERIVED TRIPEPTIDES ILE-PRO-PRO AND VAL-PRO-PRO

2.6.1. Experimental studies Both acute and long-term effects of tripeptides Ile-Pro-Pro and Val-Pro-Pro and fermented milk products containing them have been studied using normotensive Wistar-Kyoto rats (WKY) and different models of hypertension, such as spontaneously hypertensive rat (SHR) and double transgenic rat (dTGR) harbouring human renin and angiotensinogen genes. Only a few studies have addressed the effects of Ile-Pro-Pro and Val-Pro-Pro on vascular function so far. The results of animal studies have been summarized in Table 2. In addition, several in vitro and in vivo studies have been performed to get more insight into mechanisms of action and bioavailability of casein-derived tripeptides.

Antihypertensive effects The antihypertensive effect of milk casein-derived peptides was first demonstrated by casein hydrolysate formed by purified proteinase from L. helveticus CP790 and milk fermented with the same bacteria (Yamamoto et al. 1994). Acute blood pressure lowering effect after oral administration was observed in SHR but not in normotensive Wistar-Kyoto (WKY) rats. It was concluded that the peptides deliberated from casein by extracellular proteinase were responsible for the antihypertensive activity. Thereafter, angiotensin-converting enzyme (ACE)-inhibitory substances were found to be produced during fermentation of milk with L. helveticus and Saccharomyces cerevisiae (Nakamura et al. 1995b). After isolation, the sequences were identified to be Ile-Pro-Proand Val-Pro-Pro, which inhibited ACE by IC50-values of 5 and 9 M, respectively. However, IC50-values of Ile-Pro-Pro and Val-Pro-Pro can be even lower depending on the substrate concentration used in the in vitro experiments (Lehtinen et al. 2010). Also a third tripeptide, leucine-proline-proline (LeuPro-Pro), has been shown to inhibit ACE (Lehtinen et al. 2010).

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Table 2. Published experimental studies on the effects of tripeptides Ile-Pro-Pro and Val-Pro-Pro on blood pressure and vascular function.
DOSE SYSTOLIC BLOOD PRESSURE VASCULAR FUNCTION

REFERENCE

DURATION

STUDY CHARACTERISTICS

Acute experiments -22 mmHg after 6 h -35 mmHg after 8 h -22 mmHg after 6 h -

Yamamoto et al. 1994

Casein hydrolysate L. helveticus CP790 fermented milk

Nakamura et al. 1995a

L. helveticus and S. cerevisae fermented milk

15 mg/kg peptides 15 mg/kg peptides 0.3 mg/kg Ile-Pro-Pro, 0.6 mg/kg Val-Pro-Pro

Long-term experiments not specified -19 mmHg vs. control diet -12 mmHg vs. control -17 mmHg vs. control -21 mmHg vs. control -10 mmHg vs. control -8 mmHg vs. control -13 mmHg vs. control -17 mmHg vs. control -3 mmHg vs. control -19 mmHg vs. control -

Nakamura et al. 1996

16 wk

Diet containing 2.5% lyophilized sour milk

Ile-Pro-Pro and Val-Pro-Pro in water

Sipola et al. 2001

12 wk

L. helveticus fermented milk

60 60

L. helveticus fermented milk

Sipola et al. 2002a

14 wk

L. helveticus and S. cerevisiae fermented milk

Ile-Pro-Pro and Val-Pro-Pro in water

Jauhiainen et al. 2005a

9 wk

Ile-Pro-Pro, Val-Pro-Pro and minerals in water

L. helveticus fermented milk

ACh SNP ACh SNP ACh SNP -

Ile-Pro-Pro and Val-Pro-Pro in water

Jauhiainen et al. 2010a

3 wk

L. helveticus fermented milk

2.5-3.5 mg/kg/d Ile-ProPro + Val-Pro-Pro 2.5-3.5 mg/kg/d Ile-ProPro + Val-Pro-Pro 0.4 mg/kg/d Ile-Pro-Pro, 0.6 mg/kg/d Val-Pro-Pro 0.2 mg/kg/d Ile-Pro-Pro, 0.3 mg/kg/d Val-Pro-Pro 2.0 mg/kg/d Ile-Pro-Pro +Val-Pro-Pro 1.7 mg/kg/d Ile-Pro-Pro +Val-Pro-Pro 1.5 mg/kg/d Ile-Pro-Pro +Val-Pro-Pro 10.9 mg/kg/d Ile-Pro-Pro +Val-Pro-Pro 5.4 mg/kg/d Ile-Pro-Pro +Val-Pro-Pro

Spontaneously hypertensive rats (SHR) were used in all studies except in Jauhiainen et al. 2010a, in which double transgenic rats (dTGR) were used. ACh, acetylcholine; SNP, sodium nitroprusside.

The amino acid sequences corresponding to Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro are found in the primary structure of bovine -casein (74-76 Ile-Pro-Pro, 84-86 Val-Pro-Pro, 161-163 Leu-Pro-Pro) and -casein (108-110 Ile-Pro-Pro) (Farrell et al. 2004). Both pure tripeptides and fermented milk products containing them have been shown to lower blood pressure in experimental animals (Table 2). Interestingly, the effect is observed only in hypertensive animals. Furthermore, Ile-Pro-Pro and Val-Pro-Pro were detected from solubilized fraction of abdominal aorta of SHR but not of WKY after the rats had received a single dose of fermented milk containing the tripeptides (Masuda et al. 1996). As ACE activity is significantly higher in aortas of SHR compared to WKY (Nakata et al. 1987, Nakamura et al. 1988), there may have not been sufficient amounts of ACE in WKY aorta to capture tripeptides so that they would retain in the aortic tissue to be detected in the analysis. This would perhaps explain why the antihypertensive effect of tripeptides is seen only in hypertensives. Long-term studies have been initiated with young animals, which still have blood pressure at normal level (Table 2). This has enabled to study the possible attenuating effect of tripeptides on the development of hypertension. The development of hypertension has attenuated significantly in rats receiving either pure Ile-Pro-Pro and Val-Pro-Pro in water or fermented milk products containing them (Table 2). However, pure tripeptides have not produced as strong antihypertensive effect as the milk products containing them. Minerals (calcium, potassium) present in the milk product have likely contributed to the more pronounced antihypertensive effect. In addition, the bioavailability of peptides may be better from milk in comparison to water and improved by other milk components. Long-term treatment with fermented milk products containing Ile-Pro-Pro and Val-Pro-Pro has shown to have effects on renin-angiotensin system. In SHR, plasma renin activity increased after treatment with tripeptides (Sipola et al. 2002a). It seems that the antihypertensive effect of tripeptides is dose-related, as shown in the study of Sipola et al. (2002a), in which two fermented milk products containing different amounts of tripeptides were studied. Furthermore, if the treatment either with pure tripeptides or fermented milk product is terminated, the blood pressure of treated rats gradually increases to the same level as with the control rats (Sipola et al. 2001). By using the DNA microarray technique, administration of Ile-Pro-Pro and Val-Pro-Pro to SHR for five days increased eNOS and connexin 40 gene expressions in the aorta (Yamaguchi et al. 2009). Ile-ProPro and Val-Pro-Pro also slightly increased COX-1 expression and decreased both the nuclear factor kappa B subunit (NF-B) gene and the peroxisome proliferator activator receptor gamma (PPAR) gene expressions. Therefore, the antihypertensive effect of tripeptides may involve also other pathways and systems besides renin-angiotensin system.

61

Effects on vascular function Effect of long-term intake of tripeptides Ile-Pro-Pro and Val-Pro-Pro on vascular function has been assessed only in one study after the blood pressure monitoring has been completed (Table 2). In the study of Jauhiainen et al. (2005a), mesenteric arteries and aortas of rats that had received minerals and tripeptides in their drinking fluid for 8 weeks showed improved endothelium-dependent relaxation. No differences were observed in endothelium-independent relaxation. Angiotensin I (Ang I)-induced contraction was attenuated in mesenteric artery rings of rats that had received fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro. In a functional bioassay using normotensive Wistar rat mesenteric arteries, Ile-Pro-Pro dosedependently (1-3.3 mM) inhibited the Ang I-induced contraction after 15 min preincubation (Sipola et al. 2001). Val-Pro-Pro did not show any inhibitory activity. Inhibition of Ang I-induced contraction was seen also in another study, in which Ile-Pro-Pro and Val-Pro-Pro were administered together (0.33 mM of each) on normotensive Sprague-Dawley rat mesenteric arteries (Jauhiainen et al. 2010a).

Enzyme-inhibitory activity Already the first studies by Nakamura et al. (1995b) showed that fermentation of milk by L. helveticus produces ACE-inhibitory substances, which were later identified to Ile-Pro-Pro and ValPro-Pro. Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro inhibit ACE competitively at micromolar level, but do not inhibit angiotensin-converting enzyme 2 (ACE2) in physiologically relevant concentrations (Lehtinen et al. 2010). Inhibitory effect of tripeptides on ACE but not on ACE2 has been shown using porcine ocular tissues as well (Luhtala et al. 2009). As regards other enzymes, Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro show inhibitory activity towards arginase, IC50 being 1.2 mM, 10.1 mM and 13.4 mM, respectively (Lehtinen et al. 2010). Although it is highly unlikely to reach such concentrations in vivo after intake of fermented milk products, even a minor inhibition may cause a significant increase in the substrate availability for nitric oxide (NO) synthesis. As concerns NO synthesis, the interplay between nitric oxide synthase (NOS) and arginases plays a crucial role (for review, see Morris 2007). While arginases break down L-arginine to produce ornitine and urea, NOS use L-arginine to produce NO and citrulline. The affinity of NOS for L-arginine is -fold greater than is the affinity of arginases, but the Vmax of the arginases is -fold greater than that of the NOS (Wu and Morris 1998). Thus, at physiological L-arginine concentrations, arginases are able to compete from the substrate and limit NO production. Several 62

amino acids, namely branched-chain amino acids (leucine, isoleucine and valine) and proline, have been shown to inhibit arginase as well (Carvajal and Cederbaum 1986, Dabir et al. 2006).

Bioavailability Bioavailability of milk-derived bioactive peptides has been questioned. Normally, peptides are rapidly metabolized to the constituent amino acids by brush border membrane peptidases after oral administration and the absorption and bioavailability remains very low. However, there are data demonstrating that at least di- and tripeptides may absorb intact, enter the circulation and produce systemic effects (Foltz et al. 2008, Miguel et al. 2008). Bioactive peptides may be absorbed via carrier-mediated transport or paracellular diffusion (for review, see Shimizu 2004). Apparently, short tripeptides are actively transported via a specific transporter (PepT1) and oligopeptides via the paracellular route. As regards casein-derived tripeptides, Foltz et al. (2008) investigated the transport of Ile-Pro-Pro and Val-Pro-Pro by using three different absorption models and demonstrated that these tripeptides are transported in small amounts intact across the barrier of the intestinal epithelium. The major transport mechanisms of Ile-Pro-Pro and Val-Pro-Pro were demonstrated to be paracellular transport and passive diffusion. Also a study conducted in humans showed that Ile-Pro-Pro and LeuPro-Pro were detectable from plasma after ingestion of tripeptide-enriched yoghurt (Foltz et al. 2007). The Cmax of Ile-Pro-Pro rose up to almost nanomolar level and the Tmax was reached after ca 40 min. Interestingly, measurable plasma Ile-Pro-Pro concentrations were also found after ingestion of a placebo yogurt beverage without added tripeptides. This has been suggested to be due to the generation of Ile-Pro-Pro in the intestinal tract from milk proteins by luminal or brush border peptidases. However, Ile-Pro-Pro and Val-Pro-Pro were not generated from -casein by gastrointestinal enzymes in an in vitro study, suggesting that the fermentation step is necessary to produce the tripeptides (Ohsawa et al. 2008). In conscious pigs, Ile-Pro-Pro, Val-Pro-Pro and Leu-ProPro reached the blood circulation intact after intragastric administration (4.0 mg/kg of each tripeptide) and maximal plasma concentrations were about 10 nmol/l (van der Pijl et al. 2008). Halflives of absorption and elimination were only few minutes. Many discovered ACE-inhibitory peptides have a proline or proline-proline (Pro-Pro) residue at the Cterminal end (Nakamura et al. 1995b, Yamamoto et al. 1999). It has been reported that tripeptides containing a C-terminal Pro-Pro bond are usually resistant to human proteolytic enzymes (for review, see Vanhoof et al. 1995). Consequently, there is a strong possibility that these peptides 63

reach the circulation and target sites intact and exert also systemic effects. As regards casein-derived Ile-Pro-Pro, Jauhiainen et al. (2007a) used radiolabelled tripeptide and showed that it absorbed partly intact from the gastrointestinal tract after a single oral dose to rats. Considerable amounts of radioactivity were found from several tissues, e.g. liver, kidney and aorta. The excretion of Ile-ProPro was slow; even after 48 hours the radiolabelled peptide had not been completely excreted. IlePro-Pro did not bind to albumin or other plasma proteins in vitro. Considering this and the longlasting retention of the radioactivity in the tissues, accumulation of Ile-Pro-Pro may occur in daily administration in sufficient concentrations to cause blood pressure lowering effects e.g. by ACEinhibition in the vascular wall.

2.6.2. Clinical studies

Antihypertensive effects Effects of fermented milk products containing tripeptides Ile-Pro-Pro and Val-Pro-Pro on blood pressure have been studied in a number of clinical studies. Recently, two meta-analyses on antihypertensive peptides derived from different food sources have been performed (Pripp 2008, Xu et al. 2008). Pripp (2008) included 15 clinical trials in the analysis, of which 13 trials concerned milkderived peptides. Casein-derived tripeptides Ile-Pro-Pro and Val-Pro-Pro were studied in 9 of them. Xu et al. (2008) had 12 trials in the analysis and tripeptides were involved in the intervention in all of them. Significant decreases of 4.8 mmHg in SBP and 2.2 mmHg in DBP were found in the meta-analysis of Xu et al. (2008). When a stratified meta-analysis of trials with tripeptides were performed in the study of Pripp (2008), the result was very similar; 4.6 and 2.2 mmHg in SBP and DBP, respectively. All the trials included in these analyses had lasted at least 4 weeks, were performed in Finland or Japan and involved subjects that had high normal blood pressure or grade 1 hypertension. The two analyses provide evidence that tripeptides Ile-Pro-Pro and Val-Pro-Pro have antihypertensive effects in prehypertensive and mildly hypertensive subjects after long-term treatment. In addition, several newer clinical studies report hypotensive effects by treatment with the products containing tripeptides; SBP decreases of 3 mmHg (de Leeuw et al. 2009), 6 mmHg (Yoshizawa et al. 2009, Turpeinen et al. 2009) and as large as 16 mmHg have been observed (Nakamura et al. 2009a). However, recent studies conducted now also in the Netherlands by Engberink et al. (2008), van der 64

Zander et al. (2008a) and van Mierlo et al. (2009) and one study conducted in Finland (Jauhiainen et al. 2010b) have not found any significant effect either on SBP or DBP by treatment with tripeptidecontaining products. The effect of tripeptides on blood pressure has been studied mainly in long-term clinical trials. Only one study has addressed the acute effects of tripeptides so far. In the study of van der Zander et al. (2008b), a significant decrease of 2 mmHg in SBP was observed over a period of 8 hours after ingestion of tripeptide-containing milk product. Other studies have lasted for 4 to 21 weeks. The hypotensive effect of lactotripeptide-containing products becomes more obvious as the intervention is lengthened (Xu et al. 2008). In general, no significant blood pressure lowering effects have been seen after 2 weeks of intervention, but already after 4 weeks the SBP has decreased significantly from the baseline values. The blood pressure lowering effect seems to be greater in patients with higher baseline blood pressure levels. Studies involving a follow-up period show that when the treatment is terminated, blood pressure returns gradually to the baseline within 2-4 weeks (Kajimoto et al. 2001, Seppo et al. 2002, Nakamura et al. 2004). In contrast to the published data from animal studies in which tripeptides have shown clear antihypertensive effects in different hypertension models, human data are more contradictory. There may be several reasons for this. For example, tripeptides have been given in several product forms in different studies. In most clinical studies, the test products have consisted of sour milk prepared by fermenting skim milk with L. helveticus and/or S. cerevisiae. As a placebo regular sour milk or artificially acidified milk has been used. In some studies, test products have consisted of powdered fermented milk incorporated into tablets (Aihara et al. 2005, Mizuno et al. 2005) or casein hydrolysate incorporated into capsules (Hirota et al. 2007). Also fruit juice (Sano et al. 2005) and spread (Turpeinen et al. 2009) has been used as a carrier. Corresponding placebo products have been prepared without the active ingredients. The components of a milk product may influence the peptide absorption, act directly on blood pressure (e.g. minerals) or contain other bioactive peptides in addition to Ile-Pro-Pro and Val-Pro-Pro. Therefore the results from different trials with different products are not directly comparable, especially when the effects on a biological variable are small. Three different kinds of test products containing Ile-Pro-Pro and Val-Pro-Pro have been used in clinical studies: directly fermented milk (Mizushima et al. 2004, Jauhiainen et al. 2005b), powdered fermented milk in tablets (Aihara et al. 2005) and powdered enzymatically hydrolyzed tripeptides (Sano et al. 2005, van der Zander et al. 2008a). In addition, different bacteria (L. helveticus, S. cerevisiae, A. oryzae) have been used. The dose has varied between the studies; the lowest dose that has been shown to be effective in humans is 2.6 mg of tripeptides/day (Hata et al. 1996) as the 65

highest tested dose has been 52.5 mg of tripeptides/day (Jauhiainen et al. 2005b). All these discrepancies may explain the controversial results obtained in the clinical studies. In the analysis of the active components from the final product, attention has often been paid only to Ile-Pro-Pro and Val-Pro-Pro. Therefore, there may be also other components in the test products that have effects on blood pressure. Although the antihypertensive effect of tripeptide-containing products is lesser than that of the most effective antihypertensive drugs (e.g. ACE-inhibitors, AT1 antagonists), they provide an option to people with high normal blood pressure before pharmacological therapy is required.

Effects on vascular function Besides blood pressure, endothelial dysfunction and arterial stiffness are important risk factors for cardiovascular diseases and are often associated with hypertension (for reviews, see Ghiadoni et al. 2009, Tang and Vanhoutte 2010). Effects of casein-derived tripeptides on these variables have been evaluated in a few clinical studies. Index of endothelial function, reactive hyperemia measured by plethysmography, was significantly increased after one week administration of casein hydrolysate containing Ile-Pro-Pro and Val-ProPro to grade 1 hypertensive subjects (Hirota et al. 2007). Interestingly, no changes were detected in systemic blood pressure during the same period, indicating that the improvement of endothelial function attributable to Ile-Pro-Pro and Val-Pro-Pro was independent of hemodynamic changes. In contrast, Jauhiainen et al. (2010b) observed no effects on endothelial function, assessed by pulse wave reflection response to sublingual nitroglycerin and salbutamol inhalation, after 12 weeks intervention with a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro in grade 1 hypertensive subjects. Effects of casein-derived tripeptides on arterial stiffness have been evaluated by arterial compliance measurement, pulse-wave analysis and from ambulatory blood pressure measurements. In the study of Yoshizawa et al. (2009), carotid artery compliance was significantly increased in sedentary, postmenopausal women after eight weeks intervention with tablets containing casein hydrolysate. The improvements in arterial compliance were associated with reductions in arterial blood pressure and plasma angiotensin II concentrations. This is rather surprising, as the subjects had almost optimal blood pressure at baseline (SBP 114 and 122 mmHg, placebo and treatment groups, respectively), and as in previous studies the antihypertensive effect of tripeptides has not been observed in normotensive subjects. 66

Ten weeks intervention with L. helveticus fermented milk product containing Ile-Pro-Pro and ValPro-Pro significantly improved the ambulatory arterial stiffness index (AASI), which was calculated from the ambulatory blood pressure recordings, in subjects with high normal blood pressure (Jauhiainen et al. 2007b). The same product decreased augmentation index (AIx) significantly in another study, in which grade 1 hypertensive subjects received the treatment for 12 weeks (Jauhiainen et al. 2010b). However, no effects either on AIx or pulse-wave velocity (PWV) were observed after 10 weeks intervention with tripeptide- and plant sterol-containing spread in subjects with high normal blood pressure (Turpeinen et al. 2009). Even so, SBP decreased significantly by the active treatment in this study.

2.6.3. Biochemical aspects Many of the antihypertensive peptides possess ACE-inhibitory activity. The majority of milk-derived ACE-inhibitory peptides have relatively low molecular mass and short chain. This is consistent with findings of Natesh et al. (2003), which demonstrated that large peptide molecules do not fit into the active site of ACE. It seems that peptide binding to ACE is strongly influenced by the C-terminal tripeptide sequence of the peptide. Many ACE-inhibitory peptides have hydrophobic amino acid residues at each of the three C-terminal positions and proline at the C-terminal end (for review, see Meisel 2005). However, in vitro observed ACE-inhibitory activity of a peptide is not always directly related to the hypotensive effect in vivo, which may be due to degradation in the gastrointestinal tract. Vice versa, active, ACE-inhibitory fragments may be generated in the body from longer precursor peptides, which do not inhibit ACE as such. Although ACE-inhibitory effect of a hypotensive peptide could be demonstrated in vitro, question on the true antihypertensive mechanism may still remain. For example, after a 7-day administration of a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro, no changes were detected in plasma AcSDKP (a marker of the specific ACE-activity of the N-terminal domain), ACE activity or active renin concentrations in humans (Wuerzner et al. 2009). A small, transient increase was detected in urine AcSDKP. The authors concluded that neither plasma nor endothelial ACE was inhibited and no specific effect was seen on N-terminal or C-terminal ACE-domains. However, it should be taken into account that the study was conducted in normotensive, healthy individuals, who normally do not respond either to antihypertensive peptides or ACE-inhibitory drugs. Interesting structural connections between casein-derived Ile-Pro-Pro and snake venom-derived bradykinin-potentiating peptides can be found. Namely, these proline-rich, ACE-inhibitory peptides 67

(e.g. Bj-BPP-7a and Bj-BPP-10c) contain Ile-Pro-Pro-sequence in their C-terminal (Ianzer et al. 2007). They show antihypertensive effects, which are reported to be unrelated to their ability to inhibit ACE or potentiate bradykinin. This raises a question if also other compounds possessing ACE-inhibitory properties lower blood pressure by mechanisms independent of ACE-inhibition. The antihypertensive effect of casein-derived tripeptides has been repeatedly shown, but some clinical studies have failed to support ACE-inhibition as a mechanism of action. Therefore, other possible mechanisms of action should be considered and investigated as well.

68

3. AIMS OF THE STUDY

Besides pharmacological treatment, nutrition plays a central role in the prevention and treatment of hypertension. Increased consumption of low-fat milk products has been associated with lower blood pressure levels and reduced risk of hypertension. Favorable mineral composition of milk (low in sodium, high in calcium and potassium) contributes to this. Specific antihypertensive casein-derived tripeptide sequences, isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) have been detected from fermented milk products. In addition, these peptides seem to have beneficial effects on vascular function. Although angiotensin-converting enzyme (ACE)-inhibition has been suggested to be the main antihypertensive mechanism of the tripeptides, not all studies have supported this. Thus, other mechanisms are likely to contribute to the effects as well. The aim of this series of studies was to evaluate the effects of Ile-Pro-Pro and Val-Pro-Pro and fermented milk products containing them on blood pressure and vascular function, and by means of this clarify the antihypertensive mechanisms of tripeptides. The specific aims were: 1. To find out the possible beneficial effects of casein-derived tripeptides Ile-Pro-Pro and ValPro-Pro on vascular function in normo- and hypertensive rats in vitro (study I) 2. To investigate how long-term treatment with fermented milk products inhibits the development of hypertension and affects vascular function in an animal model of essential hypertension (study II) 3. To elucidate whether long-term treatment with fermented milk products affects blood pressure and vascular function in an animal model of type 2 diabetes (study III) 4. To evaluate whether plant sterols and casein-derived tripeptides have any synergistic effects on cardiovascular function in animal models of hypertension (study II, III) 5. To compare the biological effects of Ile-Pro-Pro and Val-Pro-Pro produced by different fermentation processes (study IV) 6. To characterize the acute effects of a fermented milk product containing Ile-Pro-Pro and ValPro-Pro on blood pressure and arterial stiffness in mildly hypertensive humans (study V)

69

70

4. MATERIALS AND METHODS

4.1. EXPERIMENTAL ANIMALS AND STUDY DESIGNS In the in vitro study (study I), male normotensive Wistar-Kyoto (WKY) rats and male spontaneously hypertensive rats (SHR) were used. WKY rats were from Taconic (Germantown, NY, USA) and SHR from Charles River Laboratories (Sulzfeld, Germany). Long-term studies were performed with male SHR (studies II and IV) (Charles River) and male Goto-Kakizaki (GK) rats (study III) (Taconic, Ejby, Denmark). Details of the experimental animals are presented in Table 3. The rats were housed four to five to a cage in a standard experimental animal laboratory (illuminated from 7 a.m. to 7 p.m., room temperature 22-24 C, relative humidity 55 15%). In the study I, rats received Harlan Teklad Global 19% protein extruded rodent diet 2019 (Madison, WI, USA) and tap water ad libitum. In the studies II and IV, the rats had free access to standard rat food pellet [CRM (P) 801722, SDS Special D ^ t  h< containing 0.6% NaCl. The rats in the study III received a similar rat pellet but containing 8% NaCl [CRM (P) 829521, SDS Special Diet Services, Witham, Essex, h<. In the long-term studies (studies II, III and IV), consumption of food was recorded weekly. The drinking fluids given to the rats are presented in Table 3 and described in greater detail below. Table 3. Experimental animals and study characteristics.
Study I II Strain WKY SHR SHR N 50 60 9 9 9 9 10 10 10 10 10 5 8 9 8 Age (wk) 10-15 9-12 6 Duration (wk) 8 Baseline SBP (mmHg, mean SEM) 141.8 1.3 98.7 2.9 98.0 2.7 98.4 2.5 98.4 2.2 135.8 3.5 135.4 2.4 135.5 2.4 136.8 2.3 136.0 3.0 133.4 4.3 98.1 3.1 98.5 3.0 98.5 2.5 Drinking fluid Water Water Standard product Test product Test product +sterols Control product Standard product Test product Test product +sterols Control product Water Water Powder A Powder B Water Food Normal Normal Normal Normal Normal Normal High-salt High-salt High-salt High-salt High-salt Normal Normal Normal Normal

III

GK

7-8

IV

SHR

WKY, Wistar-Kyoto rat; SHR, spontaneously hypertensive rat; GK, Goto-Kakizaki rat; SBP, systolic blood pressure.

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In vitro study (study I) The in vitro effects of tripeptides Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro on vascular function was studied by incubating the arteries in the presence or absence of tripeptides for different time periods and performing vascular reactivity experiments thereafter. Captopril was used as a reference ACE-inhibitor. Isolated mesenteric arteries of both normo- and hypertensive rats were cleaned of adherent connective tissue and stored either in pure, oxygenated (O2/CO2, 95%/5%) Krebs solution (pH 7.4-7.6, composition in mM: NaCl 119.0, NaHCO3 25.0, glucose 11.1, CaCl2 1.6, KCl 4.7, KH2PO4 1.2, MgSO4 1.2), Krebs-based tripeptide solution or captopril solution at +4 C for 1, 12, 24 or 48 h. Tripeptides Ile-Pro-Pro, Val-Pro-Pro or Leu-Pro-Pro were diluted in a freshly prepared Krebs solution to prepare a 10 M, 100 M or 1 mM solution. Similarly, a 100 M captopril solution was also prepared. After incubation, the effect of tripeptides on vascular function was investigated using the standard organ bath chamber. Two 3 mm sections of each artery were used.

Long-term experimental studies (studies II, III, IV) At the beginning of each long-term study, baseline systolic blood pressures (SBP) were measured from each rat. Rats were randomized into the treatment groups by baseline SBP and body weight (Table 3). Study products were given as the drinking fluid in plastic rodent bottles. Bottles were changed and the consumption of the products was monitored daily. The intake of tripeptides was kept constant during the study by increasing the amount of the study products according to the weight gain of the rats (3.0-6.0 mg/kg/d depending on the study). Also water bottles were provided during the study to avoid dehydration of the rats.

4.2. SUBJECTS AND STUDY DESIGN (STUDY V) The study was conducted as a single centre, randomized, double-blind, placebo-controlled crossover study. Subjects were recruited through local newspaper advertisements. Twenty-five subjects (15 males, 10 females) were included based on the following inclusion criteria: mild hypertension, also classified as grade 1 hypertension (SBP 140-159 mmHg, DBP 90-99 mmHg) and age 30-65 years. Subjects with antihypertensive or lipid lowering medication, secondary hypertension, type 1 diabetes, milk allergy or body mass index >30 were excluded from the study. Smoking, alcohol abuse 72

(> 4 drinks/day), pregnancy and breast feeding were exclusion criteria as well. The participants were 52 years old and had BMI 27 kg/m2 in average. The mean SBP and DBP were 155 and 90 mmHg, respectively. After screening, subjects meeting the inclusion criteria were randomized to start either with a fermented milk product containing tripeptides Ile-Pro-Pro and plant sterols (active treatment) or a fermented milk product without the active components (placebo) during the first of two 24 h inhouse periods. After a washout period of two weeks, the subjects crossed over to the second 24 h in-house period to receive the other treatment (Figure 4).

Figure 4. Design of the clinical study (study V). ABPM, ambulatory blood pressure measurement; PWA, pulse-wave analysis. Subjects were admitted to the research unit on the evening of day 0, remained in-house for a period of 24 h and were released on the evening of day 1 of each period. In the morning of day 1, subjects consumed 250 g of study product or equivalent amount of placebo one hour before a light breakfast. They were required to fast from 10 pm in the evening of day 0 prior to dosing on the morning of day 1. During this period water was permitted ad libitum. Subject compliance (consumption of the study products) was immediately monitored. 73

The subjects were instructed to maintain their usual dietary habits throughout the study. Caffeine and xanthine consumption as well as moderate intake of alcohol (maximum of 1 and 1-2 glass of beer or wine, women and men, respectively) were allowed, except for the 24 h prior to screening and prior to and during the in-house part of the study, when products containing alcohol, caffeine or xanthine were not permitted. During the two 24 h in-house periods, a standard breakfast, lunch and dinner was served at approximately 1 h, 5 h and 9 h after ingestion of the study product, respectively. 24 h ambulatory blood pressure measurement (ABPM) was started on the evening of day 0 (12 h before dosing) and continued until release on the evening of day 1. Pulse-wave analysis was performed prior to and 2, 4 and 8 hours after the study product dosing. Blood samples were taken prior to and 2, 4 and 8 hours after the study product, as urine samples were taken prior to the study product and thereafter collected over 8 hours.

4.3. STUDY PRODUCTS

Long-term experimental studies (studies II, III and IV) All study products were produced by Valio Ltd., Helsinki, Finland. In studies II and III three different Lactobacillus helveticus fermented milk products (low-fat, low-lactose) were used: A) Standard product: L. helveticus fermented milk product containing tripeptides (Ile-Pro-Pro and Val-Pro-Pro, 0.02 mg/ml). B) Test product: milk product containing tripeptides Ile-Pro-Pro and Val-Pro-Pro (0.02 mg/ml, 30-50 % produced enzymatically and the rest by fermentation with L. helveticus). C) Test product +sterols: milk product containing tripeptides Ile-Pro-Pro and Val-Pro-Pro (0.02 mg/ml, 30-50 % produced enzymatically and the rest by fermentation with L. helveticus) and plant sterols (8 mg/ml in total). Plant sterol mixture (Cognis, Monheim, Germany) was obtained by esterification of free plant sterols with fatty acids obtained from vegetable oil and contained mainly -sitosterol (69%), campesterol (15%), -sitostanol (8%) and brassicasterol (3%). OR D) Control product: fermented milk product containing neither tripeptides nor plant sterols.

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All products (except the control product) contained also minor amounts of a third tripeptide, leucine-proline-proline (Leu-Pro-Pro) ( d sterols in study products in different studies are presented in Table 4. In the study IV, two differently produced tripeptide powders (powder A and powder B) diluted in the water were given to rats. Essentially, the manufacturing process of powders A and B proceeded similarly. Skimmed milk (Valio Ltd.) was fermented with L. helveticus Lc 1936 according to the patent application FI 20065064 (Rajakari and Tossavainen 2006). In the fermentation process of the powder B, also a proline specific endoprotease (DSM, Heerlen, Netherlands) was used to enhance the breakdown of Ile-Pro-Pro from -casein. Thus, in the final powder of powder B, 30-50 % of tripeptides were of enzymatic origin and the rest originated from fermentation. In the powder A, all the tripeptides were derived by fermentation. Peptide powders were standardized by adding maltodextrin. Standardized peptide powder, calcium phosphate, tripotassium citrate and sugar were mixed and diluted in purified water. Tripeptides in standard product correspond to powder A as tripeptides in test product and test product +sterols correspond to powder B.

Clinical study (study V) The active product was a L. helveticus fermented milk product that contained tripeptides Ile-Pro-Pro and Val-Pro-Pro 25 mg/250 g and 2 g/250 g plant sterol esters. The placebo product was a similar fermented milk product, but did not contain the tripeptides or plant sterols.The product was low-fat and low-lactose. Nutritional content (/100g) was as follows: energy 290 kJ, proteins 3.7 g, carbohydrates 10 g (lactose <1g) and fat 0.6 g.

All study products were produced in two weeks intervals. The contents of energy nutrients, minerals, tripeptides Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro and plant sterols of the products were analyzed by R&D in Valio Ltd. Tripeptide analysis was performed with Waters Aquity UPLC Micromass ZQ2000 Mass Spectrometry System (Waters, Milford, MA). Total plant sterol content of the products was analyzed by the method of Laakso (2005) with minor modifications.

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Table 4. Contents of minerals, tripeptides and plant sterols in the study products.
Calcium (mg/100 g) Magnesium (mg/100 g) Ile-Pro-Pro (mg/100 ml) Val-Pro-Pro (mg/100 ml) Leu-Pro-Pro (mg/100 ml) Sterols (g/100 g)

Sodium (mg/100 g)

Potassium (mg/100 g)

STUDY II 266 260 260 268 164 13 <0.01 <0.01 <0.01 160 13 0.81 0.77 0.03 162 14 0.84 0.81 0.03 0.7 162 13 0.72 1.03 0.06 -

Standard product

41

Test product

50

Test product +sterols

46

Control product

44

STUDY III 312 262 253 262 162 12 <0.01 160 13 1.1 1.2 <0.01 162 14 1.1 1.1 160 12 1.2 1.6 0.1 <0.01 0.1 <0.01 0.8 -

Standard product

39

76
242 236 154 1.2 156 1.5 0.7 0.9 1.05 0.89 210 185 125 145 na na <0.1

Test product

47

Test product +sterols

42

Control product

41

STUDY IV 0.05 0.37 -

Powder A

1.8

Powder B

1.6

STUDY V 8.6 (tripeptides in total) 0.8 0.0

Active product

na

Placebo

na

na: not assayed.

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4.4. COMPOUNDS Tripeptides isoleucine-proline-proline, valine-proline-proline and leucine-proline-proline were purchased from Bachem AG (Bubendorf, Switzerland) (study I). Acetylcholine chloride, angiotensin I human acetate hydrate, angiotensin II human acetate, apamin, captopril, diclofenac sodium salt, L-( )-noradrenaline (+)-bitartrate salt monohydrate, N-nitro-L-arginine methyl ester hydrochloride (LNAME), sodium nitroprusside, TRAM-34 (1-[(2--1H-pyrazole) and ()verapamil hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA) (studies, I, II, III and IV). Apamin, diclofenac and verapamil were dissolved in distilled water and TRAM-34 in dimethyl sulphoxide (DMSO), while all the other compounds were dissolved in Krebs solution.

4.5. METHODS

4.5.1. Blood pressure measurement In experimental studies (studies I, II, III and IV), systolic blood pressure (SBP) of the rats was measured using the tail-cuff method. Measurements were performed in randomized order in the beginning of the experiment and thereafter every week (studies II and IV) or every two weeks (study III). In the study I, blood pressure was measured only in the beginning of the experiment. Rats were placed on restrainer tubes and warmed in a heated chamber (temperature 32-34C) for 20 min to make the pulsations of the tail artery detectable. The measurements were performed with Apollo2AB Blood Pressure Analyzer, model 179-2AB (IITC Life Science, Woodland Hills, CA, USA), which uses a photoelectric sensor for detection of blood pressure pulses. After obtaining three consecutive and successful recordings without disturbance of the signal, the average of the values was regarded as the SBP. Diastolic blood pressure (DBP) values were not collected, as reliable DBP values are not obtained by the method used. If the rat was restless and no solid recordings were obtained, the measurement was stopped and repeated later. In the clinical study (study V), the effect of treatment on SBP and diastolic blood pressure (DBP) was investigated using 24 h ambulatory blood pressure measurement. The method is regarded as the most reliable non-invasive method for blood pressure evaluation in humans. The cuff of the system was placed on the non-dominant upper arm. Recordings were made every 15 min at daytime (from 6.00 a.m. to 21.59 p.m.) and every 30 min at night-time (from 22.00 p.m. to 5.59 a.m.) and were evaluated centrally (Spacelabs 90207 Ambulatory Blood Pressure Monitor, Spacelabs Healthcare, 77

Issaquah, Washington, DC, USA). Ambulatory blood pressure data at baseline and at time points 2, 4 and 8 hours after study product dosing were analyzed. Values 30 min of the time point were included in the analysis.

4.5.2. Vascular function assessment

Preparation and mounting of the arteries (studies I, II, III and IV) Superior mesenteric arteries (studies I, II, III and IV) and thoracic aortas (study II) of the rats were carefully cleaned of adherent connective tissue on a petri dish containing oxygenated Krebs buffer (composition as above) under a light microscope. For the mesenteric arteries, a 5 mm section from the proximal end of mesenteric artery-aorta junction was cut off and the following 3 mm section was used in the experiments. For the aortas, a 3 mm section after the aortic arch was used. The endothelium-intact rings were placed between stainless steel hooks (diameter 0.15 mm and 0.2 mm, mesenteric arteries and aortas, respectively) and mounted in a standard organ bath chamber (volume 10 ml) in Krebs solution (+37C), and oxygenated with O2/CO2 (95%/5%). The rings were initially equilibrated for 1 h with a resting tension of 1 g for the mesenteric arteries and 1.5 g for the aortas. The force of contraction was measured with an isometric force-displacement transducer using a computerized system (EMKA Technologies, Paris, France). The method enables the follow-up of the responses online.

Vascular reactivity measurements (studies I, II, III and IV) The vascular reactivity of arteries was studied by adding vasoactive agents directly into the chambers either cumulatively [acetylcholine (ACh), noradrenaline (NA), sodium nitroprusside (SNP) or as a single dose [(ACh, angiotensin I (Ang I), angiotensin II (Ang II), NA, <. When the compounds were added into the chambers cumulatively, the following dose was not added until a stable response by the previous dose was obtained. The concentrations henceforth are the final concentrations in the organ chamber. The rings were equilibrated for 20-30 min between different experiments and washed 2-3 times with Krebs solution during this period. After the initial equilibration period of the arterial rings, a preliminary test was performed to see the endothelial function of the arteries (studies I, II, III and IV). Arterial rings were precontracted with 1 78

M NA and after a plateau was reached, rings were relaxed by 1 M ACh. To determine endothelium-dependent and -independent relaxations of the arteries, cumulative concentrations of ACh (1 nM-10 M) and SNP (1 nM-10 M) were added in NA-precontracted arterial rings, respectively. The contractile responses were investigated by constructing a cumulative concentration-response curve to NA (1 nM-1 M) or replacing NaCl in Krebs solution with KCl by equimolar basis (30 or 60 mM of KCl). A tracing of a representative protocol is presented in Figure 5.

Figure 5. Tracing of a representative protocol in an endothelium-intact arterial ring. NA, noradrenaline; ACh, acetylcholine; KCl, potassium chloride; Ang I, angiotensin I; SNP, sodium nitroprusside.

The role of cyclo-oxygenases (COX) and nitric oxide synthases (NOS) in the relaxation responses was elucidated in the presence of their inhibitors diclofenac (3 M) and L-NAME (100 M), respectively. After 15 min preincubation with these inhibitors the rings were precontracted with NA and a cumulative-response curve to ACh was constructed. In the study I, ACE-inhibitory activity of the tripeptides was assessed by adding 0.1 M of either Ang I or Ang II to the consecutive sections of the same artery. A ratio of EC50 of Ang I-induced contraction to EC50 of Ang II-induced contraction in grams (ratio R) was calculated to represent the ACE activity in the arteries. Ang I-induced contractions (0.1 M) were investigated also in the study II. In the study III, the contribution of calcium-activated potassium channels (Ca2+-activated K+channels, KCa) in relaxation responses was investigated. Cumulative-response curves to ACh were first performed in the presence of diclofenac and L-NAME, and thereafter also in the presence of apamin (inhibitor of small conductance KCa, SKCa, 0.1 M) and apamin and TRAM-34 (inhibitor of

79

intermediate conductance KCa, IKCa, 1 M). The inhibitors were introduced to the rings 30 min prior to the relaxation studies. Also the contribution of L-type calcium channels in relaxation responses was elucidated in the study III. Some of the arterial rings were preincubated with verapamil (50 nM) for 15 min prior to cumulative concentration-response curves to ACh.

Pulse-wave analysis (study V) Arterial stiffness was assessed using an investigator-independent oscillometric method for measuring pulse-wave velocity (PWV), pulse pressure (PP) and augmentation index (AIx) (Arteriograph TensioClinic, TensioMed, Budapest, Hungary) (study V). The Arteriograph analyses oscillometric pressure curves recorded with a cuff at the upper arm. The method is based on plethysmography and records pulsatile pressure changes in the artery. Measurements were performed by the same investigator in individual subjects, in a supine position after at least 15 min in rest, in the same room throughout the study with a constant temperature of 22 C and unaffected by external environmental influences. Before the measurement, the suprasternal notch (jugulum) pubic bone (symphysis) distance was measured. This parameter is necessary for calculating the PWV.

4.5.3. Collection of the samples

Metabolic cages (studies II, III and IV) During the last week of treatment, rats were housed individually in metabolic cages for 24 h, where they received the same study product and food as during the experiment ad libitum. The consumption of feed and drinking fluid was measured. Urine and faeces were collected and urinary volume measured. Urine samples were centrifuged at 200 G for 10 min and stored at -20C until analysis. The amount of faeces was weighed and the samples stored at -20C.

80

Collection of the blood and tissue samples (studies II, III and IV) As the treatment was completed, rats were rendered unconscious with CO2/O2 (30%/70%, AGA, Riihimki, Finland) and decapitated. Blood samples, with and without ethylenediaminetetraacetic acid (EDTA, 12 mM) as an anticoagulant, were collected for biochemical measurements for plasma and serum samples, respectively. Blood samples were centrifuged at 1800 G for 20 min at +4C and stored either at -20C or -80C (for plasma 8-iso-prostaglandin F2) until analysis. The hearts, left kidneys and adrenal glands were excised, washed with ice-cold saline, blotted dry and weighed. The left ventricle was separated and weighed. Aortas (study II) and superior mesenteric arteries (studies II, III and IV) were excised and placed in ice-cold oxygenated Krebs buffer (composition as above) for vascular reactivity studies. One 5 mm section from the abdominal aorta was taken for the superoxide measurement (study II). In the study I, only superior mesenteric arteries were isolated and used for vascular function studies.

Collection of blood and urine samples (study V) Blood samples were taken in the morning after an overnight fast before consumption of the study product and at three time points (2, 4 and 8 h) after the study product dosing. The samples were centrifuged at 1000 G for 15 min at +4C and stored at -20C. Heparin was used as an anticoagulant for ACE and aldosterone analysis and ethylenediaminetetraacetic acid (EDTA) for angiotensin II analysis. An inhibitor cocktail [1,10-phenanthroline 0.44 mM, EDTA 25 mM, 4-

(hydroxymercuri)benzoic acid 1 mM, pepstatin A 0.12 mM was used with the samples intended for angiotensin II analysis. Urine samples were collected for 8 h after the study product consumption.

4.5.4. Biochemical determinations

Long-term experimental studies (studies II, III and IV) After the blood pressure measurement of each rat, non-fasting blood glucose was measured with a commercial glucometer (Ascensia CONTOUR, Bayer Diagnostics, Dublin, Ireland) from the top of the hindpaw by pricking the vein with a 27 G injection needle (study III). 81

Plasma and serum samples were analyzed for renin-angiotensin system markers as well as other hypertension-related parameters. Plasma renin activity (PRA) (Angiotensin I RIA kit, IBL International GmbH, Hamburg, Germany), plasma 8-iso-prostaglandin F2 (Direct 8-iso-Prostaglandin F2 Enzyme Immunoassay Kit, Assay Designs, Ann Arbor, MI, USA), plasma or serum aldosterone (Aldosterone EIA Kit, Cayman Chemicals, Ann Arbor, MI, USA), serum ACE (Quantikine ACE Immunoassay, R&D Systems, Minneapolis, MN, USA) (study III) and serum insulin (Ultra Sensitive Rat Insulin ELISA kit, Crystal Chem Inc., Downers Grove, IL, USA) (study III) were determined according to the manufacturers instructions. Total plasma nitrate and nitrite (NOX) was measured by Nitrate/Nitrite Fluorometric Assay Kit (Cayman Chemicals) (study II). Serum ACE-activity was measured by a fluorometric assay using the substrate Hip-His-Leu as described by Schwager et al. (2006) (study II). Serum antioxidant activity was determined by a ABTS radical cation decolorization assay, described by Re et al. (1999). Plasma cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triacylglycerols (TAG) were analysed by Modular P800 -analysator (Roche Diagnostics GmbH, Mannheim, Germany) (study III). Total cholesterol, HDL and TAG were measured enzymatically, while LDL was calculated by the Friedewald equation (Friedewald et al. 1972). Plant sterols were determined from serum samples by a GLC analysis as their trimethylsilylderivatives using a 50-meter-long Ultra 2 capillary column (Agilent Technologies, Wilmington, DE, USA) (Miettinen 1988) (study II). The concentrations of squalene, cholesterol, cholestanol, desmosterol, lathosterol, campesterol, sitosterol, sitostanol and avenasterol were measured in the increasing order of retention time during the GLC procedure. Amino acids were determined from plasma samples by HPLC (Shimadzu Scientific Instruments, Kyoto, Japan) using C18-HC -column (Waters, Elstree, Hertsfordshire, UK) and detected by fluorescence (Shimadzu RF-10Ax1 detector) (study IV). Urinary samples were analyzed for urinary albumin (ELISA for Determination of Albumin, CellTrend GmbH, Luckenwalde, Germany) and cyclic guanosine 3,5-monophosphate (cGMP) by cyclic GMP EIA kit (Cayman Chemicals) (study II). Urinary electrolytes were measured using ion selective electrodes (Roche/Hitachi Modula Analytic, Roche Diagnostics GmbH) for sodium and potassium and o-cresolphthalein complexone -method (Roche Diagnostics GmbH) for calcium. Superoxide (O2-) content was determined from the abdominal aortic samples by lucigenin-enhanced chemiluminescence as described by Ketonen et al. (2005) (study II).

82

Clinical study (study V) Blood samples from study V were analyzed for plasma ACE (Quantikine Human ACE, R&D systems, Wiesbaden-Nordenstadt, Germany), aldosterone (Aldosterone EIA Kit, Cayman Chemicals) and angiotensin II (Angiotensin II Enzyme Immunoassay Kit, SPIbio, Montigny le Bretonneux, France). Analysis of the urine samples was performed for aldosterone, cyclic guanosine 3,5-monophosphate (cGMP, ParameterTM cGMP, R&D Systems, Abingdon, UK), total nitrite/nitrate (NOx, ParameterTM Total NO/Nitrite/Nitrate, R&D Systems), 6-ketoprostaglandin F1 (6-keto Prostaglandin F1 EIA kit, Cayman Chemicals) and creatinine (ParameterTM Creatinine, R&D Systems).

4.6. STATISTICAL ANALYSIS

Experimental studies (studies I, II, III and IV): All data are presented as means standard error of the mean (SEM) or means 95 percent confidence intervals (CI). Statistical analysis was performed using GraphPad Prism software (version 4.02) or SPSS Statistics (version 17.0). Students t-test, one-way analysis of variance (ANOVA) followed by Tukeys or Dunnetts Multiple Comparison Test were used to compare groups. When the data consisted of repeated observations at successive drug concentrations or blood pressure values, ANOVA for repeated measurements was applied in order to investigate between-group differences. Cumulative concentration-response curves for acetylcholine were fitted to a logistic function by using a non-linear regression analysis (sigmoidal dose-response model, standard slope) to estimate EC50 values. The EC50 values are expressed as the negative logarithm of the molar concentration (pD2 values) at which 50% of the maximal relaxation effect has been reached. To calculate the area under the curve (AUC) for relaxation curves, area of positive peaks was included. The response for blood pressure was calculated as AUC divided by the total time of the study. Human study (study V): Data are presented as means with standard deviations (SD) and 95% CI. The CIs for the changes in plasma markers of the renin-angiotensin system were obtained by biascorrected bootstrapping (5000 replications) due to their skewed distributions. The individual time points or changes to individual time points were tested by paired samples t-test or permutation test. The information from different time points was combined by calculating the AUC divided by the total time of the study. The effect of the intervention on the AUC of the response was analyzed using

83

linear mixed models. The study design (including sequence, period and treatment effect) was taken into account and the baseline measurement was added into the model as a covariate.

4.7. ETHICS The protocols for experimental studies were approved by National Animal Experimentation Committee according to EC Directive 86/609/EEC and Finnish Experimental Animal Act 62/2006. The protocol for clinical study (study V) was approved by the Ethics Committee of the Charit Research Organization (Berlin, Germany) and was conducted in accordance with Good Clinical Practices (GCP) and the guiding principles of the Declaration of Helsinki. Written informed consent was obtained from each participant before they entered the study.

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5. RESULTS

5.1. INTAKE OF TRIPEPTIDES ILE-PRO-PRO AND VAL-PRO-PRO, MINERALS AND PLANT STEROLS The amount of study products given to rats in long-term experimental studies was elevated in the course of the experiments based on the weight gain of the rats. The intakes were 30-65, 60-75 and 30-65 ml in the studies II, III and IV, respectively. These amounts corresponded to intake of 2.8-4.4, 4.6-6.6 and 3.2-5.2 mg tripeptides Ile-Pro-Pro + Val-Pro-Pro + Leu-Pro-Pro /kg/day. The intake of plant sterol esters was 0.42 and 0.55 g/kg/day in the studies II and III, respectively, in those rats receiving test products +sterols. In the clinical study, subjects received 21.5 mg of tripeptides (IlePro-Pro + Val-Pro-Pro + Leu-Pro-Pro) and 2.0 g plant sterol esters from the active product (single administration). The weight gain of the rats did not differ between the treatment groups in any of the experiments (studies II, III and IV). High-salt fed GK rats in the study III received 545 29, 550 47, 430 56 and 671 44 mg of NaCl daily in standard product, test product, test product +sterols and control product groups, respectively (P<0.01 for test product +sterols group vs. other groups). Plant sterols were shown to be absorbed from the fermented milk product. Serum levels of D7- and D8-lathosterol, campesterol, sitosterol, sitostanol, avenasterol and squalene were increased after 8 weeks treatment with plant sterol containing milk product (P<0.01 test product +sterols vs. test product) as cholestanol levels were decreased (P<0.001) (study II). Serum total cholesterol was slightly but not statistically significantly lower in test product +sterols group vs. test product group (P=0.10).

5.2. BLOOD PRESSURE

Experimental studies In long-term experimental studies performed with SHR (studies II and IV), the blood pressure of the rats started to increase shortly after beginning of the experiment. However, fermented milk products containing Ile-Pro-Pro and Val-Pro-Pro attenuated the development of hypertension. At the end of the 8 weeks treatment period, systolic blood pressure (SBP) was 164.9 2.3, 166.9 2.7, 85

171.7 3.3 and 179.0 3.2 mmHg in standard product, test product, test product +sterols and control product groups, respectively (study II). The difference in SBP after the treatment was statistically significant in standard product and test product groups compared to control product group (Table 5). Also tripeptide powders diluted in water attenuated the blood pressure increase significantly (study IV, Table 5). At the end of the 8 weeks treatment, SBP was 170.0 2.0, 169.4 2.5 and 183.6 3.8 mmHg in powder A, powder B and water (control) groups, respectively. In high-salt fed type 2 diabetic Goto-Kakizaki (GK) rats (study III), the blood pressure increase was not as strong as with SHR in other studies. Even so fermented milk products inhibited the blood pressure increase. After 8 weeks treatment, SBP was 141.8 2.2, 140.6 1.6, 142.9 2.4 and 153 2.4 mmHg in standard product, test product, test product +sterols and control product groups, respectively. All groups treated with fermented milk products differed significantly from the control product group in SBP at the end of the treatment (Table 5).

Table 5. Attenuation of the development of hypertension by long-term treatment with tripeptidecontaining products in rats. Study II (SHR) Study product Standard product Test product Test product +sterols Control product Standard product Test product Test product +sterols Control product Powder A Powder B Water (control) Difference in SBP vs. control (mmHg, mean) -14.1 -12.1 -7.3 -11.2 -12.4 -10.1 -13.6 -14.2 Significance P < 0.01 P < 0.05 P ns. P < 0.01 P < 0.01 P < 0.05 P < 0.01 P < 0.01

III (GK)

IV (SHR)

n=8-10 in each group. SHR, spontaneously hypertensive rat; GK, Goto-Kakizaki rat; SBP, systolic blood pressure.

High-salt diet induced a strong hypertensive effect in GK rats given only water as drinking fluid (Ehlers et al., unpublished data). As their food consumption and therefore also salt intake were higher than in the rats receiving milk products, no direct comparisons to other groups could be made as regards blood pressure. SBP rose up to 176.4 2.1 mmHg in this group after the 8 weeks treatment.

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Clinical study The differences in SBP and diastolic blood pressure (DBP) between active product and placebo at baseline and 2, 4 and 8 hours after the study product consumption are presented in Figure 6. The mean SD AUC of SBP was 140 12 mmHg in the active product and 142 10 mmHg in the placebo group; the baseline adjusted treatment effect was -2.1 mmHg (95 % CI: -4.1 to -0.1, P=0.045) in favour of the active product. The corresponding values for DBP were 89 8 mmHg in the active product and 91 8 mmHg in the placebo group; the baseline adjusted treatment effect was -1.6 mmHg (95% CI: -3.1 to -0.1, P=0.03). Also the AUC of mean arterial pressure (MAP) favoured the active product; 107 9 mmHg in the active group and 108 8 mmHg in the placebo. The baselineadjusted treatment effect was -1.9 mmHg (95 % CI: -3.3 to -0.4, P=0.014).

Figure 6. Differences in SBP and DBP after single administration of the study products (n=25).

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5.3. VASCULAR FUNCTION AND ARTERIAL STIFFNESS

5.3.1. In vitro study (study I) In general, prolonged incubation of isolated mesenteric arteries with the tripeptides at +4C resulted in better preserved endothelial function in comparison to control arteries. Storage affected the arterial function; after 48 h incubation, endothelium-dependent relaxations assessed by acetylcholine were already greatly impaired (unpublished data) and no differences could be detected between the groups. However, tripeptides Ile-Pro-Pro and Val-Pro-Pro protected endothelial function of both SHR and WKY mesenteric arteries indicated as more pronounced endothelium-dependent relaxation. This could be observed most clearly with SHR mesenteric arteries incubated for 24 h (Figure 7A). Captopril did not cause a similar effect. No differences were observed in endothelium-independent relaxation between the groups (Figure 7B).

Figure 7. A) Endothelium-dependent relaxation of SHR mesenteric arteries after 24 h incubation with one of the tripeptides, captopril or without any of the compounds. Symbols denote means SEM, n=4-6 in each group. P<0.001 Ile-Pro-Pro vs. control; Ile-Pro-Pro vs. captopril, P<0.01 Val-Pro-Pro vs. control. B) Endotheliumindependent relaxation of the same arteries as in Figure 7A. P ns.

The protective effect of Ile-Pro-Pro and Val-Pro-Pro was observed only with 1 mM concentration. No differences in endothelium-dependent relaxations between the groups were seen with lower concentrations (100 M, 10 M) of the tripeptide (P ns. vs. control arteries). Incubation with all three tripeptides together did not cause any significant additional effect either (data not shown).

88

5.3.2. Long-term experimental studies (studies II, III and IV)

Endothelium-dependent relaxation Endothelium-dependent relaxations were impaired in both SHR and high-salt fed GK rats. In general, treatment with tripeptide-containing products produced only mild changes in endotheliumdependent relaxation. In SHR, aortic rings of rats that had received standard product showed poorer relaxation compared to control rats (P<0.01) (study II). Endothelium-dependent relaxation of mesenteric arteries of same rats did not greatly differ, however, the relaxation was decreased in the arteries of rats treated with test product +sterols (P<0.05). Mesenteric arteries of SHR treated with tripeptide powders diluted in water (powders A and B) did not differ significantly from control in endothelium-dependent relaxations (study IV). In GK rats, endothelium-dependent relaxations of mesenteric artery rings were strongly impaired in all groups (study III). However, arterial rings of rats treated either with standard product, test product or test product +sterols had improved relaxation compared to rats treated with control product (P<0.01 standard product and test product +sterols vs. control product group, P<0.05 test product vs. control product group).

Endothelium-independent relaxation Endothelium-independent relaxation assessed by sodium nitroprusside-induced relaxation did not differ between the groups in any of the studies (II, III and IV). The relaxation was near 100% in all arteries indicating that vascular smooth muscle function was unchanged despite the hypertensive state of the animals.

Contractility In SHR, mesenteric arterial rings of rats that received standard product and test product contracted significantly more by noradrenaline than those of test product +sterols and control product groups (P<0.05) (study II). No differences were observed in arterial rings of rats that were treated with

89

tripeptide powders compared to water (study IV). Contraction induced by KCl did not differ between the groups either (studies II and IV). In GK rats, no differences were observed in contractile responses induced either by KCl or noradrenaline (study III).

Role of COX, NOS, Ca2+-activated K+-channels and L-type Ca2+channels In SHR, inhibition of cyclooxygenase by diclofenac and nitric oxide synthase by L-NAME in aortic rings resulted in strongly attenuated acetylcholine-induced endothelium-dependent relaxation (study II). However, aortic rings of rats treated with test product +sterols relaxed significantly better than aortic rings of other groups (P<0.001). In GK rats, COX- and NOS-inhibition in mesenteric arterial rings almost totally abolished the endothelium-dependent relaxation (study III). Even so there was some residual relaxation left in the arterial rings of test product group rats (P<0.05 vs. other groups). As also Ca2+-activated K+-channels were inhibited, no relaxations were observed in any of the arterial rings. Blocking of L-type Ca2+ channels by verapamil almost normalized the endothelium-dependent relaxation, which was greater in test product and test product +sterols groups than in the other groups (P<0.05).

90

5.3.3. Clinical study Analysis of vascular function was conducted at baseline and 2, 4 and 8 h after the study product consumption. No significant differences between the active product and placebo treatment were observed in pulse-wave velocity (PWV) or aortic augmentation index (AIx aortic) (Figure 8). Also other variables (brachial AIx, pulse pressure or aortic SBP) remained similar between the groups.

Figure 8. Differences between active product and placebo in pulse-wave velocity (PWV) and aortic augmentation index (AIx aortic) after single administration of the study products (n=25).

91

5.4. RENIN-ANGIOTENSIN SYSTEM AND L-ARGININE-NO PATHWAY Plasma, serum and urinary samples from long-term experimental studies (studies, II, III and IV) and clinical study (study V) were analyzed for markers of renin-angiotensin system and other relevant, hypertension-associated variables. In experimental studies, changes in serum ACE-activity or amount of ACE, aldosterone, urinary albumin and cGMP were detected after the 8 weeks treatment with different tripeptide-containing products (Table 6). Table 6. Biochemical analysis of plasma, serum and urine samples after long-term treatment with tripeptide containing products (studies II, III and IV).
Variable 8-Iso-PGF ACE ACE-activity Aldosterone Albumin AOA cGMP NOx Renin activity Standard product SHR GK Test product SHR GK Test product +sterols SHR GK Powder A SHR Powder B SHR -

Arrows denote if there was a statistically significant difference (increase or decrease) between the treatment group and control group. One arrow denotes for P<0.05, two for P<0.01 and three for P<0.001. ACE, angiotensin-converting enzyme; AOA, antioxidative activity; cGMP, cyclic guanosine monophosphate; NOX, total plasma nitrate and nitrite.

An extensive plasma amino acid analysis demonstrated that the intake of tripeptide powders diluted in water (study IV) slightly increased the amino acid levels in general. However, no differences in tripeptide components isoleucine and valine were detected between the groups. The concentration of citrulline, a byproduct of NO synthesis, was higher in the rats that had received powder A (P<0.05) or powder B (P ns.) compared to water (control) group. In the clinical study, analyses from plasma samples were performed at baseline and 8 hours after the study product consumption. No significant differences between the active treatment and placebo groups were found in ACE, aldosterone or angiotensin II (P=0.27 for ACE, P=0.93 for aldosterone and P=0.66 for angiotensin II). Eight hour urinary samples were used for analysis of cGMP, NOx and 6keto-PGF excretion and were adjusted for urinary creatinine excretion. From these, cGMP excretion was significantly higher in the active treatment group vs. placebo (P=0.0093). Also creatinine-adjusted urinary NOx concentrations tended to be higher in the active treatment group (P=0.13). 92

6. DISCUSSION

Previous clinical and experimental studies have shown that tripeptides Ile-Pro-Pro and Val-Pro-Pro or milk products containing them have antihypertensive effects in humans and animal models of hypertension. In vitro studies have demonstrated that Ile-Pro-Pro and Val-Pro-Pro inhibit angiotensin-converting enzyme (ACE), to which the antihypertensive effect often is attributed. However, effects of Ile-Pro-Pro and Val-Pro-Pro on vascular function and mechanisms behind them are not well known. Moreover, there is not much knowledge of the acute effects of Ile-Pro-Pro and Val-Pro-Pro in humans. This series of studies investigated the effects of Ile-Pro-Pro and Val-Pro-Pro on vascular function and blood pressure in experimental models of hypertension and in humans. The potential synergistic effect of plant sterols and influence of different fermentation processes to produce the tripeptides were also studied.

6.1. METHODOLOGICAL ASPECTS

Experimental studies Hypertension is a strong risk factor for cardiovascular diseases and related to other risk factors such as hypercholesterolemia, insulin resistance and arterial stiffness. Several experimental models of hypertension exist; in the present studies, spontaneously hypertensive rat (SHR) and Goto-Kakizaki (GK) rat fed with high-salt diet were used. Also normotensive Wistar-Kyoto (WKY) rats were used. SHR is a widely used and well-described rat model of human essential hypertension. In SHR, systolic blood pressure increases over 200 mmHg by 3-4 months of age (Okamoto and Aoki 1963). Although SHR have shorter life expectancy than normotensive rats, they are suitable for long-term studies (> 4 weeks) if compared e.g. to double transgenic rats (dTGR) harbouring human renin and angiotensinogen genes, which die in the age of 7-8 weeks (Park et al. 2009). As regards antihypertensive treatment, not only therapy of hypertension but also prevention can be studied, if the intervention is started in young, still normotensive SHR. Although the use of the model has been criticized for heterogeneity between various colonies and inadequate use of control strains (Rapp et al. 1987), it was considered to be a suitable model for these studies due to its availability, simplicity (no need for surgical operations), and as no effects on mortality by the treatments were expected 93

(no need to have a model with a very short life expectancy). Previous studies investigating the effect of casein-derived tripeptides on blood pressure have been performed mainly with SHR (e.g. Nakamura et al. 1996, Sipola et al. 2001, Jauhiainen et al. 2005a). Hypertension often coexists with metabolic syndrome and type 2 diabetes. GK rat was used in the study to investigate the effects of tripeptides on blood pressure and vascular function in an animal model of type 2 diabetes. The rats were fed with high-salt diet (8% NaCl), which has been shown to induce the development of hypertension (Cheng et al. 2001, Pilvi et al. 2006). GK rats exhibit fasting hyperglycemia, of which evaluation was complicated by the nutritional treatment in this study, however. GK rats do not exhibit obesity, which is a drawback as regards the research on metabolic syndrome, but may ease the handling of the animals. GK has not been used before in investigating the effects of tripeptides Ile-Pro-Pro and Val-Pro-Pro on blood pressure. In long-term studies (studies II, III and IV), it was noted that when the rats (SHR or GK) received fermented milk products, their food consumption decreased as they got energy also from the drinking fluid. However, as control groups received also milk products, there were no major differences between the treatment and control groups in the food consumption, energy intake and weight gain. In addition, as the composition of the control products (including mineral composition) was similar to active products except for tripeptides or tripeptides and plant sterols, the effects observed can be directly attributed to these active components. In the analysis of study product components, fat content of test product +sterols was higher than in the other milk products. This is, however, mainly due to the plant sterols, which do not, at least in humans, absorb almost at all (Salen et al. 1970, Ostlund et al. 2002). Control milk products (both in experimental and the clinical study) contained no detectable amounts of either Ile-Pro-Pro, Val-Pro-Pro or Leu-Pro-Pro. It is also previously shown that regular milk and sour milk do not contain these tripeptides (Jauhiainen et al. 2005b, Foltz et al. 2007). The tail-cuff method was used to measure blood pressure of the rats in all studies. Although being non-invasive and simple, the method may overestimate blood pressure levels because of the stress and inconvenience the animal may experience. However, as regards the number of the animals studied and the length of the long-term experimental studies, this method was regarded as the most reasonable for the present study purposes. The vascular function of isolated arteries was assessed by standard organ bath chamber in in vitro studies (study I) and after long-term experiments (studies II, III and IV). The method is widely used to assess e.g. the effects of pharmacological agents on vasodilatation or -contraction, their direct

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effects on vascular function, the pathophysiology of vascular disorders and the treatment- or disease-induced changes in arterial reactivity. The standard organ bath system enables the study of isolated large- and medium-sized arteries. In the present studies, mainly superior mesenteric artery (resistance artery) but also aorta (conductance artery) was used. The use of mesenteric arteries is reasonable, as resistance arteries contribute to blood pressure regulation by regulating peripheral resistance (Mulvany 2002). However, even smaller arteries, namely the branches of superior mesenteric artery, should be used to ideally detect changes in endothelium-derived hyperpolarizing factor (EDHF)-mediated responses (Shimokawa et al. 1996). To perform this, small vessel myography needs to be used. After all, also aorta as a large artery was included in the studies (study II) to investigate the effect of tripeptides on mainly nitric oxide-mediated endothelium-dependent relaxations. The problem counteracted in long-term studies was that it was possible to take only one arterial ring from each animal, as the studies need to be completed in as short time period as possible. If the number of arterial rings taken from the animals was increased, it would be possible to perform more extensive vascular reactivity experiments by using various agonists, antagonists and inhibitors, since receptor desensitization and irreversible binding of inhibitors would not hinder the experiments that much. In previous studies, acute effects of Ile-Pro-Pro and Val-Pro-Pro on vascular function of isolated arteries have been rather weak (Sipola et al. 2001). It has not been possible to show e.g. any direct vasodilatating effect if tripeptides have been added directly to the arterial preparations in the organ bath (unpublished data). Consequently, the in vitro study (study I) was performed after long-term incubation of the isolated mesenteric arteries with tripeptides at +4C. Different time periods were used for evaluation of time dependency. In organ transplantation and cardiovascular surgery, cold storage is often used to maintain the organ viability. As regards isolated arteries, smaller (e.g. thirdorder mesenteric) seem to survive better than larger (e.g. thoracic) arteries and contractibility is maintained at normal level longer than dilatation when stored in cold (Trk et al. 1993, McIntyre et al. 1998, Rinaldi 2001). Also endothelium-independent relaxation has been shown to be less susceptible to deterioration in comparison to endothelium-dependent relaxation. However, reports on the preservation of the vascular function are little controversial, but may be explained by varying experimental conditions (different preservation or physiological salt solutions, various vascular beds etc.). In this study, Krebs solution was used in the incubation as also the actual vascular reactivity experiments were performed with it.

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Clinical study The clinical study was conducted as a randomized, double-blind, placebo-controlled cross-over study. The sample size was rather small, but as the cross-over design was employed, the influence of confounding factors was reduced and therefore fewer subjects were required. In addition, the study was carried out in hospital in highly standardized conditions. The two weeks wash-out period between the two 24 h in-house periods could have been longer, as in long-term clinical studies the antihypertensive effect of tripeptide-containing products has lasted for 2-4 weeks (Kajimoto et al. 2001, Seppo et al. 2002). However, as the study product was given as a single dose, it seems unlikely that the effect would carry over. The subject compliance was high and the study products were well tolerated. Total of four adverse effects were reported, none of which could be considered to be related to the study products. The effect of treatment on blood pressure was evaluated using 24 h ambulatory blood pressure measurement, which is reported to measure the extent of treatment-induced blood pressure reduction more accurately than office blood pressure measurement (Mancia et al. 2007). It also reduces the white coat and placebo effect. The subjects were accustomed to the device by starting the measurement in the preceding evening (12 h before the study product consumption). Pulse-wave analysis (PWA) was performed by an oscillometric method to assess arterial stiffness. By this method, pulse-wave velocity (PWV), the gold standard measurement of arterial stiffness, can be measured. PWV has been shown to be an independent predictor of cardiovascular events in several studies (Inoue et al. 2009, Sehestedt et al. 2009). Another measure, augmentation index (AIx), is also a useful marker of cardiovascular risk, but as an indirect parameter may not be as closely associated to the cardiovascular risk as PWV (Song et al. 2009). PWA was carried out before the study product consumption and 2, 4 and 8 hours afterwards. As the maximum plasma concentrations (Cmax) of Ile-Pro-Pro has been reported to appear during the first 30 to 60 min after consumption of a tripeptide-containing milk product (Foltz et al. 2007), in the present study there could have been one measurement more, e.g. after 1 h, to better characterize the acute effects of tripeptides on arterial stiffness.

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Taken together, the present series of studies have been conducted by using generally accepted, current methods suitable for the research questions. Clinical study was carried out in a very standardized manner and employed the most reliable methods to evaluate the acute effects of treatment on arterial stiffness and blood pressure. Animal models used are appropriate for investigating the attenuating effect of treatment on the development of hypertension. Vascular function studies were designed to extensively investigate the role of different components of vasodilatation and effects of tripeptides on them. However, more knowledge may have been gained if it were possible to increase the number of arterial rings studied in long-term experimental studies and also if smaller resistance arteries were used.

6.2. ANTIHYPERTENSIVE EFFECTS OF ILE-PRO-PRO AND VAL-PRO-PRO

6.2.1. Experimental studies All tripeptide-containing milk products (standard product, test product, test product +sterols) in SHR and GK and tripeptide powders diluted in water (powder A and powder B) in SHR attenuated the blood pressure development of the hypertensive rats. To our groups knowledge, the study III was the first experimental study which investigated the effects of tripeptide-containing fermented milk products on blood pressure in an animal model of type 2 diabetes. Previous long-term experimental studies investigating the effects of tripeptide-containing milk products on blood pressure in SHR and one study in dTGR have shown 8-19 mmHg lower SBP values compared to the control groups. In the present studies, treatment attenuated the blood pressure development by 7-14 mmHg depending on the study product. It is noteworthy that in the previous studies, the effect of tripeptidecontaining products has often been compared to a group of rats that has received water as drinking fluid. This approach may overestimate the antihypertensive effect of tripeptides, as other components (mainly minerals) likely contribute to the blood pressure as well. Indeed, this was shown in the study of Jauhiainen et al. (2005a), in which L. helveticus fermented milk produced the strongest attenuating effect on SBP (-17 mmHg), as Ile-Pro-Pro, Val-Pro-Pro and minerals in water and Ile-Pro-Pro and Val-Pro-Pro in water resulted in milder decrease (-13 and -8 mmHg, respectively), although the differences were not statistically significant. Thus, in the present study, the difference between rats treated with tripeptide-containing milk products and control rats in SBP 97

would have been perhaps even larger if the blood pressure values were directly compared to waterreceiving rats. All long-term experimental studies lasted for 8 weeks. The beneficial effect of tripeptide-containing products on blood pressure was seen only after 5-6 weeks of treatment. No acute effect on blood pressure was observed; most probably because the rats ingested the products during the night and blood pressure measurements were performed several hours afterwards. In acute experiments with SHR, the maximal antihypertensive effect of fermented milk or casein hydrolysate containing Ile-ProPro and Val-Pro-Pro has been observed after 6 to 8 hours of ingestion (Yamamoto et al. 1994, Nakamura et al. 1995a). In the present studies, this effect may have not been detected. In addition, no dose-response relationship could be observed in the present studies, as they were designed so that the intake of Ile-Pro-Pro and Val-Pro-Pro was similar in all groups. Although GK rats received more tripeptides per kilogram per day compared to SHR, difference to the control group in SBP was of similar degree in both SHR and GK rats. Different strain and background of hypertension may certainly explain this. However, a clear dose-response of the tripeptides was observed in the study of Sipola et al. (2002a), which showed that doubling the daily amount of Ile-Pro-Pro and Val-Pro-Pro led to a 2-fold stronger effect on blood pressure in SHR. The two differently produced tripeptide powders did not differ in their effect to attenuate blood pressure development (study IV). The question concerning the influence of different manufacturing processes of tripeptides has arosen from clinical studies, as in a majority of studies an antihypertensive effect has been observed but in a few studies no effect has been seen. Furthermore, at least Ile-Pro-Pro may exist in different conformations (Valjakka 2010, personal communication), which could theoretically affect e.g. binding to an enzyme. Tripeptides in the powder A were produced totally by fermentation; a method that has been used in the majority of studies published so far. In the powder B, tripeptides originated mainly from enzymatic hydrolysis, which has been employed to produce the milk products used in several studies with zero result (Engberink et al. 2008, van der Zander et al. 2008a, van Mierlo et al. 2009). Thus, the present results suggest that the manufacturing process does not play a role in the antihypertensive effect of the tripeptides Ile-Pro-Pro and Val-Pro-Pro.

Plant sterols In studies II and III, standard product and test product attenuated the development of hypertension by somewhat similar degree. Test product +sterols was not as effective as the other two fermented 98

milk products, although there was no statistically significant difference between these three groups. Plant sterols are not thought to affect blood pressure in general, but through their cholesterol lowering action, plant sterols may have indirect effects on blood pressure as well. No effects on blood pressure were observed in a study where SHR were fed with a diet fortified with phytosteryl esters for 9 weeks (Kim et al. 2007). In the same study, however, the rats treated with phytosterols had decreased baroreflex activity. Salt-loaded SHRSP (stroke-prone SHR) treated with soybean oil diet containing phytosterols had higher SBP after three weeks than those who had received a diet without phytosterols (Ogawa et al. 2003). Also the same treatment group had a marked decrease in lifespan. These reports and data from the present studies on the potentially harmful effects of plant sterols on cardiovascular function of the rat are perhaps not directly applicable to humans, as the body weight-adjusted doses are far higher than the usual recommended doses for humans (0.8 g/day in rats, 2.0 g/day in humans in the present studies). In addition, in the study II, the differences in serum sterol levels were multifold between test product and test product +sterols groups, e.g. campesterol and sitosterol levels were 4.7- and 5.8-fold, respectively, indicating that plant sterols absorbed in significant amounts. This does not occur in humans, at least to the same extent, as serum campesterol levels were found to increase by 0.7- and sitosterol levels by 0.4-fold after 10 weeks intervention with sterol ester-containing spread (Hallikainen et al. 2006). Furthermore, the study of Ikeda et al. (2001) showed that certain rat strains, namely WKY and SHRSP but not e.g. Wistar rat, deposit plant sterols in the body by enhancing the absorption and lowering the excretion of plant sterols. Accordingly, care should be taken when these observations are extrapolated to humans. Besides plant sterols, also plant stanols, saturated forms of sterols, are often used either alone or in combination with sterols in products intended for lowering low-density lipoprotein (LDL) cholesterol (Katan et al. 2003). As the effects of plant sterols and stanols on blood pressure were compared in SHRSP during a 5 weeks treatment, no differences were seen in SBP but DBP was increased by 2fold in plant sterol and 3-fold in plant stanol group compared to the control group (Chen et al. 2009). There are also other reports suggesting that plant sterols and stanols may not be equal as regards other biological effects than LDL-cholesterol reduction (Ratnayake et al. 2003). In the present studies, the plant sterol mixture contained almost only plant sterols (92%). However, plant sterols and stanols are generally considered to be safe in humans (for review, see Tikkanen 2005). The superiority of plant sterols over stanols in the cardiovascular effects remains to be clarified.

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6.2.2. Clinical study In humans, effects of tripeptide-containing milk products on blood pressure have been investigated mainly in long-term studies. The present study (study V), was designed to characterize the acute effects of tripeptide- and plant sterol-containing fermented milk product. A significant decrease in SBP, DBP and mean arterial pressure (MAP) was observed within eight hours after ingestion of the study product containing 21.5 mg tripeptides and 2.0 g plant sterols. A similar effect was reported in the study of van der Zander et al. (2008b), in which ingestion of fermented milk product (8.7 mg tripeptides) resulted in 2 mmHg decrease in SBP over a period of 8 hours. Interestingly, in their study, no significant effects on blood pressure were seen in subjects with baseline SBP <130 mmHg, whereas the acute effect was -3.5 mmHg in subjects with SBP >130 mmHg. In the screening of the present study, the mean SBP of the subjects was 155 mmHg, but at the moment of the in-house periods, the baseline SBP was far lower. The general finding is that blood pressure lowering effect of tripeptide-containing products is greater in subjects with higher baseline blood pressure levels (Xu et al. 2008). Thus, if the subjects in the present study had had higher baseline blood pressure, more pronounced decrease in blood pressure would perhaps have been seen. However, as antihypertensive medication was included in the exclusion criteria, it could have been difficult to find such otherwise healthy patients but with severe hypertension. Considering the present results, findings by van der Zander et al. (2008a) and data from experimental studies, intake of tripeptide-containing products several times a day would possibly provide more efficient blood pressure lowering effect. That is, if tripeptides were ingested e.g. in 6 or 8 hours intervals (in the morning, afternoon and evening), the blood pressure would stay longer on the desired level and would perhaps provide more constant antihypertensive therapy. As regards pharmacological therapy, antihypertensive drugs are usually administered 1-3 times a day to keep the plasma concentrations of the drugs at the steady state level. For example, maximal plasma concentrations of ACE-inhibitor captopril are reached within 1 to 2 hours after oral administration and the hypotensive effect has been observed to last for 2 to 6 hours (Finnish Medicine Agency 2010). Although tripeptide-containing milk products should not be considered as drugs, parallels may be drawn with pharmacological agents on how to take into account pharmacokinetics (and dynamics) and how to get the most optimal antihypertensive response with tripeptides as well. For these reasons, pharmacokinetics of the tripeptides needs further investigation.

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In aggregate, long-term treatment with tripeptide-containing fermented milk products attenuated the development of hypertension in spontaneously hypertensive rats (SHR) and type 2 diabetic Goto-Kakizaki rats. The fermented milk product containing also plant sterols was slightly less effective compared to the other tripeptide-containing products. Differently produced tripeptide powders produced a similar attenuating effect on systolic blood pressure of SHR. An acute blood pressure lowering effect was observed in mildly hypertensive subjects after single administration of tripeptide- and plant sterol-containing fermented milk product. Findings from experimental and clinical studies indicate that despite the acute effect, tripeptides needs to be used several weeks to achieve a constant blood pressure lowering effect. Optimization of the dosing pattern might be beneficial for a more effective antihypertensive response.

6.3. PROTECTIVE EFFECT OF ILE-PRO-PRO AND VAL-PRO-PRO ON VASCULAR FUNCTION

Experimental studies The present in vitro study (study I) showed that tripeptides Ile-Pro-Pro and Val-Pro-Pro protect endothelial function in a time-dependent manner. Acetylcholine-induced endothelium-dependent relaxation was better preserved after incubation of the isolated mesenteric arteries with tripeptides. The effect was seen both in normotensive Wistar-Kyoto (WKY) rat and SHR arteries, although it was more pronounced in SHR. Incubation with tripeptides did not affect endothelium-independent relaxation. Vascular response studies performed after long-term experimental studies provided evidence on the protective effect of tripeptide-containing products on vascular function as well. However, the results were not fully consistent. In the study III, endothelium-dependent relaxation in mesenteric arteries of GK rats was improved with rats that had received tripeptide- or tripeptide- and plant sterolcontaining fermented milk products. In contrast, tripeptide-containing products did not improve endothelium-dependent relaxations neither in aorta nor mesenteric artery in the studies II and IV performed with SHR. Also in the study of Jauhiainen et al. (2005a), 9 weeks treatment with fermented milk product containing tripeptides did not increase endothelium-dependent relaxation

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of mesenteric or aortic rings of SHR, but minerals and tripeptides diluted in water significantly improved this. Inhibition of cyclo-oxygenase (COX) and nitric oxide synthase (NOS) abolished the endotheliumdependent relaxation almost totally in all studies. This is in line with findings from other studies reporting a marked attenuation in the endothelium-derived hyperpolarizing factor (EDHF)-mediated component in SHR (Bssemaker et al. 2003, Michel et al. 2008) and also in GK rats (Oniki et al. 2006, Matsumoto et al. 2010). Despite the inhibition of COX and NOS, there was still some residual relaxation left in the aortic rings of SHR that had received test product +sterols (study II) and mesenteric artery rings of GK rats treated with test product (study III), indicating that tripeptides affected the EDHF-mediated component of endothelium-dependent relaxation to some extent. The effect of Ile-Pro-Pro on EDHF was also demonstrated in the in vitro study, where mesenteric artery rings incubated with Ile-Pro-Pro for 24 h showed increased relaxation compared to control arteries in the presence of COX- and NOS-inhibitors. To be strict, the contribution of NO on the relaxation responses should have been assessed also by inhibiting COX and EDHF but not NOS leaving NO as the sole mediator of relaxation. Endothelial dysfunction has been demonstrated both in resistance and conduit arteries of several animal models of hypertension. The presence and severity of endothelial dysfunction is highly dependent on the age of the rat and the vascular bed studied (for review, see Bernatova et al. 2009). Mesenteric arteries of SHR used in the present studies did not show endothelial dysfunction almost at all, as endothelium-dependent relaxations were nearly 100 %. However, there are also other reports that endothelium-dependent relaxation has either remained at normal level (Behr-Roussel et al. 2005) or even increased in SHR (Bernatova et al. 2007). In contrast, mesenteric arteries of GK rats (study III) showed greatly impaired endothelium-dependent relaxation, despite the animals were still quite young (15-16 weeks of age). This was most likely due to the high-salt feeding, which, in addition to its hypertensive effect, has been shown to deteriorate endothelial function in experimental animals (Cheng et al. 2001, Oniki et al. 2006) as well as in humans (Bragulat et al. 2001). Several pharmacological antihypertensive agents, such as ACE-inhibitors, reverse the endothelial dysfunction in experimental rat models (Hutri-Khnen et al. 1997, Khnen et al. 1999, Schfer et al. 2007) and in hypertensive patients (for review, see Taddei et al. 2002). In rats, improvements in both endothelium-dependent and -independent relaxations have been observed. In the present studies, no effects on endothelium-independent relaxations were seen by the treatment with tripeptides. Augmented function of K+ channels in smooth muscle has been suggested to explain the 102

enhanced endothelium-independent relaxation in SHR by ACE-inhibitors (Hutri-Khnen et al. 1997). As the improvements in vascular function by tripeptides were endothelium-dependent and EDHF seemed to be involved, the effect may be attributed to the small and intermediate conductance Ca2+-activated K+ channels (SKCa and IKCa, respectively), which are located at the endothelial layer of vascular wall, and not the large conductance Ca2+-activated K+ channels (BKCa), which reside in the smooth muscle layer and probably account for the improvements in endothelium-independent relaxations observed with ACE-inhibitors, for example. However, the role of different Ca2+-activated K+ channels in vascular function after long-term tripeptide treatment should be properly assessed by specific inhibition of one K+ channel at a time. Some concern has arisen about the possible harmful effects of plant sterol consumption in the pathogenesis of atherosclerosis and deterioration of vascular function due to high levels of circulating plant sterols. Women with elevated levels of plant sterols were shown to have enhanced risk of coronary artery disease (Rajaratnam et al. 2000). Furthermore, treatment of C57/Bl6 mice with diet containing 2% plant sterols for 4 weeks impaired aortic endothelium-dependent relaxation and increased cerebral lesion size after middle cerebral artery occlusion (Weingrtner et al. 2008). Even so, large population-based studies have indicated otherwise (for review, see Calpe-Berdiel et al. 2009). The potential risk may be counteracted by the notable reduction in plasma cholesterol. In the present studies, no consistent association with plant sterols and impaired vascular function was observed. However, to properly address this, the effect of plant sterols on vascular function should be investigated by feeding them alone and not combined with any other active components (as with tripeptides in the present studies). It appears that in experimental studies like the present studies, the animals should be enough old and enough diseased to thoroughly characterize the effects of a treatment on vascular function. Namely, if the animals do not possess endothelial dysfunction, one may not see any beneficial effects by the treatment, at least on relaxation responses. Based on the present findings, it can be concluded that hypertension precedes endothelial dysfunction in SHR and endothelial dysfunction is more like consequence than cause of increased blood pressure. In GK rats, high-salt feeding induced a severe endothelial dysfunction, which was paralleled by an increase in blood pressure and which was slightly but significantly attenuated by the treatment with fermented milk products containing Ile-Pro-Pro and Val-Pro-Pro.

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Clinical study In the present study, single administration of tripeptide- and plant sterol-containing fermented milk product did not affect arterial stiffness assessed by pulse-wave analysis (PWA) and evaluated by pulse-wave velocity (PWV), aortic augmentation index (AIx), brachial AIx, pulse pressure and aortic SBP. In the previous studies, beneficial effects of long-term treatment with fermented milk products containing tripeptides have been observed. Daily consumption of 50 mg Ile-Pro-Pro and Val-Pro-Pro for 10 weeks significantly decreased aortic arterial stiffness index (AASI) calculated from 24 h ambulatory blood pressure recordings (Jauhiainen et al. 2007b). In another long-term study, 50 mg Ile-Pro-Pro and Val-Pro-Pro daily in a fermented milk product decreased AIx (Jauhiainen et al. 2010b). However, no improvements on arterial stiffness were observed with a daily intake of 5 mg tripeptides consumed as spread (Turpeinen et al. 2009). In the present study, the tripeptide dose was 21.5 mg. It seems that the antihypertensive effects of tripeptides are evident at lower doses as the effects on arterial stiffness. Therefore, to improve vascular function, higher doses should be used. Also the beneficial effects on vascular function may not be achieved by a single administration, which would parallel the findings from the present in vitro study on the time-dependent effects of tripeptides.

To summarize, protective effect of tripeptides Ile-Pro-Pro and Val-Pro-Pro and fermented milk products containing them on vascular function was demonstrated in in vitro studies and long-term experimental studies with GK rats. The effect was shown to be endothelium-dependent and possibly involving endothelium-derived hyperpolarizing factor (EDHF). In the clinical study, single administration of tripeptide-containing fermented milk product did not affect measures of arterial stiffness. Based on experimental and clinical findings, beneficial effects of tripeptides on vascular function seem not to be acute of a kind.

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6.4. MECHANISTIC INSIGHTS INTO THE BENEFICIAL EFFECTS OF ILE-PRO-PRO AND VAL-PRO-PRO Findings from the present experimental studies suggest that long-term treatment with tripeptides or fermented milk products containing them has an influence on the renin-angiotensin system (RAS) as well as L-arginine-NO pathway. In contrast, no effects on the components of RAS were observed in the clinical study. Angiotensin-converting enzyme (ACE) activity (study II) or the amount of ACE (study III) was decreased in serum of SHR and GK, respectively, which had received tripeptides for 8 weeks. This is in line with the study of Nakamura et al. (1996), who showed that long-term feeding with diet containing 2.5% lyophilized sour milk decreased ACE-activity in the SHR aorta. Also in another study a single administration of fermented milk containing Ile-Pro-Pro and Val-Pro-Pro decreased ACEactivity in the aorta of SHR (Masuda et al. 1996). The findings suggest the involvement of ACEinhibition in the attenuation of blood pressure development by the tripeptides. Also the present in vitro study (study I) supports the concept of ACE-inhibition. However, it is not known why statistically significantly lower ACE levels were seen only in the group receiving both tripeptides and plant sterols in the present long-term studies. Furthermore, these groups of rats did not show any superiority in blood pressure decrease compared to the other tripeptide-treated groups. ACE - plant sterol interaction has not been studied much. Hagiwara et al. (1986) showed that plant sterol fucosterol decreased ACE levels in cultured bovine carotid endothelial cells indirectly by inhibiting the synthesis of glucocorticoid receptors involved in the regulation of ACE level. The potential effect of plant sterols on ACE clearly deserves further attention. RAS controls extracellular fluid volume via regulation of aldosterone secretion (for review, see Epstein 2001). Angiotensin II (Ang II) stimulates aldosterone release from the adrenal cortex. Treatment with tripeptides decreased serum aldosterone levels in GK rats in the present study. Interestingly, chronic treatment with aldosterone is known to induce endothelial dysfunction in experimental animals (Blanco-Rivero et al. 2005, Xavier et al. 2008). Consistent with this, the groups of rats that had the lowest aldosterone levels exhibited also less endothelial dysfunction in the present study. ACE-inhibitors have been shown to decrease plasma or serum aldosterone levels (Haddad et al. 2007, Nakamura et al. 2009b). Thus, the concept of simultaneous ACE-inhibition and aldosterone suppression is consistent and demonstrates that the long-term treatment with Ile-ProPro and Val-Pro-Pro has inhibitory effects on RAS in hypertensive rats. Furthermore, as chronic exposure to Ang II has been shown to attenuate EDHF-mediated relaxations in rat mesenteric arteries (Dal-Ros et al. 2009), decreased levels of Ang II due to ACE-inhibition by tripeptides could

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contribute to the improvement of EDHF-mediated relaxation in the resistance arteries of hypertensive animals. Although the findings from the present experimental studies and previous in vitro studies (Nakamura et al. 1995b, Lehtinen et al. 2010) suggest the involvement of ACE-inhibition in the antihypertensive mechanisms of Ile-Pro-Pro and Val-Pro-Pro, the present clinical study does not support this. Also previous clinical studies have failed to show the ACE-inhibitory effect consistently (Jauhiainen et al. 2005b, Engberink et al. 2008, Usinger et al. 2010). Nonetheless, significant decreases in blood pressure have been observed, as was also in the present study. The reason for this discrepancy may be that the doses used in humans are too low to cause ACE-inhibition or that ACE is inhibited at the tissue level (local RAS) and circulating ACE is not affected, at least with these doses. As was observed in a kinetic study with rats (Jauhiainen et al. 2007a), tripeptides seem to accumulate into tissues (e.g. aorta, kidney). Urinary excretion of cyclic GMP (cGMP) was shown to be increased after long-term treatment of SHR with tripeptide powders (study IV) as well as in the clinical study (study V) by the single administration of the tripeptide-containing milk product. cGMP is a second messenger of nitric oxide (NO), and its accumulation in the vascular smooth muscle causes relaxation of the blood vessel (Archer et al. 1994). Also a tendency towards increased urinary NOX (NO metabolites) was observed in the clinical study. In accordance with these findings, plasma citrulline levels were increased in the SHR that had received tripeptide powders. Citrulline is produced as a byproduct in NO synthesis from L-arginine (Frstermann and Mnzel 2006). Altogether these findings (increased urinary cGMP and NOX excretion, increased plasma citrulline concentration) would suggest increased NO production and would possibly be reflected to improved vascular function. However, in the present studies, these treatment groups did not differ from controls as regards endothelial function and arterial stiffness. There are no previous reports on the effects of tripeptides Ile-Pro-Pro and Val-ProPro on these variables, but increases in plasma or serum cGMP and NOX levels have been observed after treatment with ACE-inhibitors (Takase et al. 2000, Yamanari et al. 2004). Concerning the present clinical study, a single administration of the tripeptide-containing milk product may not have been enough to induce changes down to the vascular function despite the observed increase in cGMP levels. Long-term treatment of SHR with tripeptide powders also strongly decreased urinary excretion of albumin. The same has been observed in humans with antihypertensive agents such as ACEinhibitors. Reduction of urinary albumin excretion is important, as the elevated level of albumin is considered to indicate hypertension-induced target organ damage and renal dysfunction (Mancia et 106

al. 2007). This finding thus demonstrates that in the present study, attenuating effect of tripeptides on blood pressure was paralleled with renal protection.

Taken together, long-term treatment with fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro inhibited angiotensin-converting enzyme (ACE) and decreased aldosterone levels thus showing beneficial effects on the renin-angiotensin system (RAS) in experimental models of hypertension. No changes in the components of RAS were observed by the single administration of the same product in mildly hypertensive subjects. Increased levels of cGMP, NOX and citrulline suggest increased nitric oxide (NO) production by the tripeptides. However, no direct relationship between these markers of NO production and improved vascular function could be shown.

6.5. CLINICAL RELEVANCE AND OPEN QUESTIONS Hypertension and related disorders cause a global burden increasing cardiovascular morbidity and mortality. Prevention and early treatment of hypertension should receive high attention. Any reductions in blood pressure, however small, are meaningful; systolic blood pressure (SBP) reduction of 9 mmHg and diastolic blood pressure (DBP) reduction of 5 mmHg reduces the risk of stroke by 3540% and coronary heart disease by 20-25% (Mensink et al. 2003). However, even these reductions may be difficult to achieve; two meta-analyses on the antihypertensive effects of the two most used drug classes, ACE-inhibitors and AT1-antagonists, showed that both drug classes decrease SBP and DBP by 8 and 5 mmHg, respectively (Heran et al. 2008a, Heran et al. 2008b). It should be noted that blood pressure levels may be effectively affected also by nutrition. For example, a modest reduction in salt intake for 4 weeks or more reduces SBP and DBP by 5.0 and 2.7 mmHg, respectively, in hypertensive patients (He et al. 2004). According to the two recent meta-analyses on the antihypertensive effects of casein-derived Ile-ProPro and Val-Pro-Pro or fermented milk products containing them, long-term treatment has decreased SBP and DBP by approximately 5 and 2 mmHg, respectively (Pripp 2008, Xu et al. 2008). In the present clinical study, fermented milk product containing the two tripeptides and plant sterols decreased SBP and DBP by 2.1 and 1.6 mmHg, respectively, over a period of 8 hours after a single administration. Although the antihypertensive effect of tripeptide-containing products is lesser than 107

that of the most effective antihypertensive drugs, they may suit to people with high normal blood pressure before pharmacological therapy is required. Functional foods, consumed as a part of the normal diet, may reduce the threshold of commitment to the treatment of hypertension. ACE-inhibitory drugs, such as captopril and enalapril, possess some common adverse effects (cough, angioedema), which could theoretically concern also milk-derived antihypertensive peptides because of their ACE-inhibitory activity. However, no treatment-related safety concerns have appeared in clinical studies with different kinds of antihypertensive peptide products. Also neither in vitro genotoxicity tests, 90-day repeated-dose toxicity studies in rats nor a pre-natal development study in rabbits show any adverse effects related to tripeptides or tripeptide-containing casein hydrolysates (Dent et al. 2007, Ponstein-Simarro Doorten et al. 2009). Therefore, consumption of tripeptide-containing products as a part of normal diet should not involve any safety issues. Although the present studies provided more evidence on the mechanisms of the tripeptides Ile-ProPro and Val-Pro-Pro, several questions remain. The effects on the main components of reninangiotensin (RAS) system have been characterized, but still there are several possible targets to which the effects of tripeptides have not been studied. Besides RAS, kallikrein-kinin system participates in blood pressure regulation. ACE-inhibitors are known to inhibit the degradation of bradykinin, which has multiple effects related to vascular function. Thus, the effects of tripeptides on bradykinin responses deserve further attention. Furthermore, to better characterize the antihypertensive effect of tripeptides in experimental models of hypertension, older animals with high blood pressure could be used in long-term studies in order to see if tripeptides decrease already established hypertension. In addition, the effects of plant sterols on vascular function is an interesting subject; in the present studies, treatment with a fermented milk product containing both tripeptides and plant sterols differed to some extent from the other, only tripeptide-containing products. The research concerning these questions has already started. Many food-derived peptides have been shown to possess antioxidative properties (for review, see Mller et al. 2008). As regards Ile-Pro-Pro and Val-Pro-Pro, no studies addressing this question have been published. Preliminary data from our group does not support the concept of antioxidative action of Ile-Pro-Pro and Val-Pro-Pro, but the question is not yet fully answered. Furthermore, concerning the protective effects of Ile-Pro-Pro and Val-Pro-Pro on vascular function observed in the present studies, morphological examination of the arteries of rat treated with tripeptides would possibly provide more information on the mechanisms underneath.

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7. SUMMARY AND CONCLUSIONS

The present series of studies investigated the effects of casein-derived tripeptides Ile-Pro-Pro and Val-Pro-Pro and fermented milk products containing them on vascular function and blood pressure in experimental models of hypertension and in humans as well as the mechanisms behind them. The potential synergistic effect of plant sterols and influence of different fermentation processes was also studied. The main findings of the present studies are as follows: x Long-term treatment with fermented milk products containing casein-derived tripeptides Ile-Pro-Pro and Val-Pro-Pro attenuates the development of hypertension in young spontaneously hypertensive rats (SHR) and type 2 diabetic Goto-Kakizaki (GK) rats. Plant sterols do not enhance this effect. Differently produced tripeptide powders produce a similar attenuating effect on systolic blood pressure (SBP) of SHR.

A single administration of fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro and plant sterols acutely lowers brachial systolic and diastolic blood pressure in mildly hypertensive subjects.

Based on in vitro studies and long-term experimental studies, tripeptides Ile-Pro-Pro and ValPro-Pro and fermented milk products containing them have a protective effect on arterial function. The effect is shown to be endothelium-dependent and possibly involving endothelium-derived hyperpolarizing factor (EDHF).

Arterial stiffness is not affected by a single administration of tripeptide-containing fermented milk product in humans. Despite this, increased levels of markers of nitric oxide (NO) production suggest that tripeptides may have beneficial effects on endothelial function also after single administration.

Angiotensin-converting enzyme (ACE) inhibition and aldosterone suppression suggest the involvement of renin-angiotensin system (RAS) in the attenuating effect of tripeptides on blood pressure increase in experimental models of hypertension. In the present clinical study, no effects on circulating RAS were detected.

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8. ACKNOWLEDGEMENTS

This study was carried out in 2007-2010 at the Institute of Biomedicine, Pharmacology, University of Helsinki within the Valio Ltd. milk protein project and funded by the Finnish Funding Agency for Technology and Innovation (Tekes). I owe my deepest gratitude to my supervisors Professor (emeritus) Heikki Vapaatalo, MD, PhD and Professor Riitta Korpela, PhD. They have opened the door to me into the world of science. I wish to express my sincerest gratefulness to Heikki Vapaatalo for believing in me right from the beginning. His exceptional enthusiasm towards science and experience in the field of cardiovascular research has encouraged me to go further, better, faster. I cannot thank enough for Heikkis endless support in every step of the work. It has been a privilege to be supervised also by Riitta Korpela. Her neverending inspiration and vast knowledge in nutrition has encouraged me to go into this side of science too. I am most grateful to Riitta for her heartfelt encouragement and guidance throughout this work. Professor Esa Korpi, the Head of the Institute of Biomedicine, is sincerely acknowledged for the opportunity to work at the Institute of Biomedicine, Pharmacology. I also owe my sincerest appreciation to Professor Tiina Mattila-Sandholm, Executive Vice President of Valio R&D, for the opportunity to work in collaboration with Valio R&D. Official reviewers of this thesis, Professor Hannu J. Korhonen and Docent Risto Kerkel, are respectfully thanked for the fluent review process and their contribution to improve my thesis. My co-authors are greatly acknowledged for their expertise and collaboration. Docent Anu Turpeinen at Valio R&D is warmly thanked for her invaluable support and guidance throughout the work. Tiina Jauhiainen PhD and Kirsi Rajakari DI at Valio R&D are acknowledged for their expertise in the studies. Docent Frank Wagner and his group at Charit Research Organization, Berlin, Germany is thanked for the excellent cooperation in the clinical study. I also would like to thank Anne Kivimki BSc, Enni Pere BM and Risto Lehtinen BSc for their valuable help and cooperation in the laboratory work and biochemical analyses for this thesis. I owe my warmest thanks to Professor Valrie B. Schini-Kerth, Cyril Auger PhD and Eric Anselm PhD at the Faculty of Pharmacy, Strasbourg, France, for introducing me to the fascinating world of blood vessels. Their encouragement to vascular function studies has been one of the cornerstones for my inspiration for the whole thesis project. I am truly thankful to my co-workers at the Institute of 111

Biomedicine for all their advice and support during the studies. Professor Eero Mervaala, Agnieszka Biala MSc, Anne Kivimki BSc, Marjut Louhelainen PhD, Saara Merasto MSc and all the other PhD students and co-workers are warmly thanked for the memorable moments inside and outside the lab. Ms Anneli von Behr and Ms Nada Bechara-Hirvonen are greatly acknowledged for their valuable guidance in the vascular laboratory and Ms Sari Laakkonen for the help with the animal care. I am most thankful to Taru Pilvi PhD and Riina Kekkonen PhD at Valio R&D for the fruitful cooperation within the milk protein project. I also thank Suvi Honkaniemi, Outi Kerojoki, Elina Lausvaara, Pivi Perjoki and Petri Silfverberg at Valio R&D for their collaboration in providing and analyzing the study products. Hannu Kautiainen BA and Salme Jrvenp MSc at Medcare Ltd. are thanked for their advice in statistics. All my co-workers and collaborators expertise has been valuable throughout the thesis. My friends outside the academic world, Tiia and Lauri, Annukka and Tero, Marianne and Kristian, pharmacy fellows, Rockettes girls and others thank you for supporting me and reminding me of the other aspects of life as well. The joyful moments with you have given me energy to go through the project. Last, I owe my deepest gratefulness to my parents, mother Raija and father Markku. You have raised me to believe in myself, to be myself and to work hard for the aims. I am most grateful for your endless support and encouragement in my life. I warmly thank my brother Juho for supporting me and reminding me that sometimes life is not that serious, after all. I wish to express my special thanks also to my godparents Riitta and Reijo for always showing interest in my life and work. Finally, my warmest gratitude belongs to my dear husband Henkka. Thank you for your support, patience and love. Thank you for being by my side.

Espoo, August 2010

Pauliina Ehlers (ne Jkl)

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