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Nutrition, Metabolism & Cardiovascular Diseases (2006) 16, S39eS45

Endothelial dysfunction in type 2

diabetes mellitus
Angelo Avogaro*, Gian Paolo Fadini, Alessandra Gallo,
Elisa Pagnin, Saula de Kreutzenberg

Department of Clinical and Experimental Medicine, Division of Metabolic Diseases,

University of Padova, School of Medicine, Via Giustiniani 2, 35128 Padova, Italy

Received 18 October 2005; received in revised form 20 October 2005; accepted 29 October 2005

KEYWORDS Abstract Aims: Vascular endothelial cells play a major role in maintaining cardio-
Endothelium; vascular homeostasis in health. Diabetes mellitus (DM) substantially impairs the
Diabetes; vasodilating properties of the endothelium and leads to endothelial dysfunction,
Protein kinase C; which can thus be considered the first step in the progression of cardiovascular dis-
Diabetic ease. The aim of the present study is to illustrate possible mechanisms responsible
complications; for endothelial dysfunction in DM.
Cardiovascular disease Data synthesis: We have shown that NADPH oxidase gene expression is increased
in circulating lymphomonocytes from patients with DM, and that this increased
gene expression is dependent upon metabolic control. Hyperglycemia can mediate
its adverse effects through the activation of protein kinase C. We have shown an
increase in membrane-associated PKC beta 2 activity in monocytes from patients
with DM. This activity was reduced by 40% in the euglycemic condition. Finally,
we show a reduction of the circulating endothelial progenitor cells, a subset of
bone marrow-derived endothelial-oriented stem cells, which can give rise to
mature endothelial cells.
Conclusion: Endothelial dysfunction, the initial step of the atherosclerotic process,
is reversible. Thus, major efforts should be made to control not only hyperglycemia
but also the other risk factors for cardiovascular disease, in order to prevent the
onset of all these processes that eventually leads the diabetic patient to premature
ª 2005 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: þ39 049 8212178; fax: þ39 049 8754179.
E-mail address: (A. Avogaro).

0939-4753/$ - see front matter ª 2005 Elsevier B.V. All rights reserved.
S40 A. Avogaro et al.

Introduction (NO-sensitive GC), which is the most important re-

ceptor for the signalling molecule NO. This leads to
Vascular endothelial cells play a major role in the conversion of GTP to cyclic guanosine mono-
maintaining cardiovascular homeostasis in health. phosphate (cGMP) [4]. The NO/cGMP signalling
In addition to providing a physical barrier between cascade is crucial in the cardiovascular system,
the vessel wall and lumen, the endothelium where it controls smooth muscle relaxation, and
secretes a number of mediators that regulate inhibition of platelet aggregation; cyclic nucleo-
platelet aggregation, coagulation, fibrinolysis and tide phosphodiesterases hydrolyze cGMP and thus
vascular tone. The term endothelial dysfunction terminate their action. NO has several important
refers to a condition in which the endothelium effects on the vasculature: first, it maintains basal
loses its physiological properties: the tendency to tone by relaxing vascular smooth muscle cells; it
promote vasodilation, fibrinolysis and antiaggrega- also inhibits platelet adhesion, activation, secre-
tion. Endothelial cells secrete several mediators tion, and aggregation and promotes platelet disag-
that can alternatively mediate either vasoconstric- gregation, in part through a cGMP-dependent
tion, such as endothelin-1 and thromboxane A2, or mechanism. In addition to these effects, endothe-
vasodilation such nitric oxide (NO), prostacyclin lial-derived NO inhibits leukocyte adhesion to the
and endothelium-derived hyperpolarizing factor endothelium and smooth muscle cell migration
(EDHF) [1]. NO is the major contributor to endo- and proliferation [2]: therefore NO is a powerful
thelium-dependent relaxation in conduit arteries inhibitor of the earlier phases of atherosclerosis
whereas the contribution of EDHF predominates and all the mechanisms that lead to neointimal
in smaller resistance vessels. Diabetes mellitus proliferation after vascular injury.
(DM) substantially impairs the vasodilating proper-
ties of the endothelium and leads to endothelial
dysfunction, which can thus be considered the first
Molecular mechanisms underlying
step in the progression of cardiovascular disease
(CVD) [2]. endothelial dysfunction in
diabetes mellitus

Oxidative reactions are crucial in all the events

Nitric oxide synthesis and that lead to atherogenesis, including endothelial
biological effects dysfunction. In endothelium exposed to agents
that damage the vasculature there is stimulation
NO, which is the most powerful vasodilating of several enzymes that can produce reactive
compound, derives from L-arginine: the reaction oxygen species (ROS): the enzymes of the mito-
is catalyzed by a family of enzymes, called the chondrial electron transport chain, xanthine
NO synthases (NOS). Three NOS isoforms have oxidase, cyclooxygenases, lipoxygenases, myelo-
been identified: endothelial NOS (eNOS), neuronal peroxidases, cytochrome P450 monooxygenase,
NOS (nNOS) and inducible or inflammatory NOS uncoupled NOS, heme oxygenases, peroxidases,
(iNOS). In endothelial cells, eNOS generates NO and NAD(P)H oxidases. In turn, ROS can be pro-
only if one essential cofactor such as tetrahydro- duced intracellularly, extracellularly, or in specific
biopterin (BH4) is optimally present within the intracellular compartments. Among these en-
cells. Enzyme activity can be regulated by post- zymes, nicotinamide adenine dinucleotide/NADPH
translational modifications: these modifications oxidase is relevant because it is a major vascular
occur through the phosphorylation of Ser1179, source of ROS [5]. There is evidence that strong
which increases the activity of the enzyme [1]. correlations exist among NADPH oxidase activity,
Several kinases can phosphorylate this site, includ- atherosclerotic risk factors, and endothelial
ing protein kinase A, protein kinase C, and serine/ dysfunction. We have recently shown that NADPH
threonine kinase Akt/PKB. On the contrary eNOS oxidase gene expression is increased in circulating
activity can be downregulated by other proteins, lymphomonocytes from patients with diabetes
including caveolin-1, the major coating protein of mellitus (DM), and that this increased gene expres-
caveolae, vesicles found throughout endothelial sion is dependent upon metabolic control [6]. In-
cells, which are implicated not only in signal trans- terestingly, NADPH activity is increased not only
duction but also in endothelial barrier function and by factors that damage vascular endothelium,
mechanotransduction [3]. In order to induce but possibly by insulin itself [7,8]. ROS generation
vasodilation in the vascular smooth muscle cells, is enhanced in the blood vessels of hypertensive
NO must activate NO-sensitive guanylyl cyclase animal models and in atherosclerotic lesions in
Endothelium and diabetes S41

humans and animals: this process appears to pro- interface of blood and tissue, which maintains
mote vascular proliferation, hypertrophy and a balance between these two compartments.
remodelling, and is involved in the progression of With disruption of this balance, mediated by in-
atherosclerosis. Insulin resistance, which most flammation, the vascular wall becomes susceptible
often precedes the onset of type 2 diabetes, and to atheroma formation [16]. In response to DM,
increased FFA levels cause oxidative stress due blood leukocytes attach to the endothelium where
to increased mitochondrial uncoupling. Moreover, there is an exaggerated production of ROS [17]. In
oxidative stress plays a key role in the pathogene- their turn, ROS induce several inflammatory cyto-
sis of late diabetic complications. Hyperglycemia kines such that systemic oxidative stress also
leads to mitochondrial dysfunction and activation means systemic inflammation. Accumulating evi-
of stress pathways. Both in vitro and in vivo studies dence suggests a major link between C reactive
reveal that oxidative stress due to hyperglycemia protein (CRP) and atherosclerosis. CRP, which is
occurs before late complications become clinically produced in the liver in response to pro-inflamma-
evident. This indicates that oxidative stress plays tory states, potently downregulates eNOS tran-
a crucial role in the pathogenesis of late diabetic scription and destabilizes eNOS mRNA, with
complications [9]. resultant decreases in both basal and stimulated
Hyperglycemia also leads to generation of ad- NO release [18]. CRP also stimulates endothelin-1
vanced glycation endproducts (AGE), the products and interleukin-6 release, upregulates adhesion
of non-enzymatic glycation of proteins and lipids molecules, and stimulates MCP-1. Thus, CRP is
that accumulate in the vessel wall. They are signal not only a marker of endothelial dysfunction/
transduction ligands for the receptor for AGE cardiovascular risk [19,20] but also takes part in
(RAGE) [10]. AGEs accumulate in the vessel wall its pathogenesis [21]. The inflammatory response
exposed to hyperglycemia and alter the structural involved in the initiation and progression of ath-
integrity of the vessel wall and underlying base- erosclerotic lesions involves many mediators that
ment membrane and are able to quench nitric activate NF-kB, which has specific target sequen-
oxide. This contributes substantially to negatively ces on many immune and inflammatory gene
affect endothelial function [11]. promoters [22].
Hyperglycemia is one of the major causal There is general agreement that hyperglycemia
factors in the development of diabetic vascular impairs endothelium-dependent vasodilation in
complications and can mediate their adverse vivo, in humans [23e27]. Interestingly, even in
effects through multiple pathways. One of those normoglycemic subjects who are prone to develop
mechanisms is the activation of PKC by hypergly- DM and insulin resistance syndrome, impaired en-
cemia-induced increases in diacylglycerol (DAG) dothelial function has been observed during an
level, partly due to de novo synthesis [12]. There is oral glucose tolerance test [28]. This has led to
increasing evidence that PKC activation is impor- the hypothesis that endothelial dysfunction may
tant in diabetes-related endothelial dysfunction; precede the development of overt DM, and that
impaired NO-vasodilation and increased ET-1 re- a prolonged and repeated exposure to postprandial
lease involved PKC-mediated inhibition of eNOS hyperglycemia may play an important role in the
[11]. We have shown an increase in membrane- development of atherosclerosis, even in those
associated PKC beta 2 activity in monocytes from who have normal fasting plasma glucose levels.
patients with DM; this activity was reduced by With endothelial function being defined as the
40% in the euglycemic condition [13]. PKC activa- blood flow response to acetylcholine, insulin resis-
tion also mediates the overexpression of adhesion tance itself is characterized by endothelial dys-
molecules such as ICAM, VCAM and E-selectin. PKC function [29]. In DM, endothelial dysfunction
also play a crucial role in mediating vascular appears a consistent although not widespread
smooth muscle cell contractility thus mediating finding; our previous finding of a normal vascular
vascular vasoconstriction in response to risk fac- response to acetylcholine in DM without other
tors such as hyperglycemia. known risk factors for CHD suggests that the endo-
Atherosclerosis is also regarded as a progressive thelial dysfunction in DM cannot be attributed to
disease arising from the combination of endothe- a single, but possibly, to a variety of factors such
lial dysfunction and inflammation. Indeed, inflam- as the diameter of LDL particles, total cholesterol,
mation has been recognized as a keypoint in the leukocyte count, von Willebrand factor and serum
cluster of the insulin resistance syndrome, includ- AGEs [30]. However, there is general agreement
ing diabetes, obesity, hypertension and hyperlipe- that hyperglycemia and DM lead to an impairment
mia [14,15]. Much of the inflammatory reactions of NO production and activity. We have recently
are mediated by the endothelium, located at the shown that the fraction of L-arginine converted
S42 A. Avogaro et al.

to NO is lower in DM patients than in normal sub- amputation. Thus, emerging evidence indicating
jects using a stable isotope approach [31]. It has that bone marrow-derived EPCs participate in
also been shown that an inappropriate production postnatal neovascularization led to the hypothesis
of asymmetric dimethylarginine (ADMA) can alter that alterations in EPC number or function could
endothelial function in patients with DM: this be involved in the pathogenesis of vascular com-
endogenous competitor for NOS increases after plications in DM. Lambiase et al. have shown that
a fatty meal in DM and its circulating levels corre- poor coronary collateral development, which is
late with a reduction of vascular function [32]. typical of DM, is related to low levels of circulating
EPCs [37].
A possible role for EPCs in diabetic vascular
disease was first investigated in diabetic mice with
The role of circulating endothelial experimental hindlimb ischemia, in which infusion
progenitor cells in DM of human CD34-positive leukocytes was able to
accelerate blood flow restoration [38]. Tepper
Circulating endothelial progenitor cells (EPCs) are et al. showed that PBMC-derived EPCs isolated
considered a subset of bone marrow-derived en- from patients with DM displayed a proliferation
dothelial-oriented stem cells, expressing both rate in culture decreased by 48% compared to con-
hematopoietic immature surface markers, such trol subjects, a weaker adherence to activated
as CD34 and CD133, and endothelial lineage human umbilical vein endothelial cells (HUVEC)
markers [33]. EPCs give rise to mature endothelial and a 2.5-fold reduced incorporation into vascular
cells, as demonstrated in pioneer in vitro studies structures in vitro [39]. The rate of EPC prolifera-
and recently confirmed in vivo. EPCs originally tion from plated PBMCs in patients with DM was
reside in the bone marrow and are mobilized to inversely correlated with the levels of glycated
peripheral blood by a number of stimuli, including hemoglobin, suggesting a possible relation be-
tissue ischemia, physical exercise, cytokines and tween glucose control and EPC function. Almost
certain drugs provided with pleiotropic cardiovas- identical results have been reported by Loomans
cular effects [34]. Currently, it is suggested that in type 1 diabetic patients [40]. EPC reduction in
increase in EPCs may be one of the mechanisms diabetes has been hailed as a novel concept in
by which these stimuli would ameliorate vascular the pathogenesis of macrovascular complications.
function: once in the bloodstream, EPCs could We have recently demonstrated that patients
help to maintain endothelial integrity and stimu- with DM and peripheral arterial disease have a pro-
late angiogenesis in ischemic organs. found reduction of circulating EPCs in comparison
On the contrary, reduction in circulating EPCs with controls, with patients with vascular disease
has been demonstrated in the presence of risk but without DM and with patients with DM but
factors for atherosclerosis, endothelial dysfunction, without vascular disease, thus strengthening the
hypercholesterolemia, aging, smoking, chronic re- hypothesis that EPC reduction may have a role in
nal failure, coronary artery disease, cerebral the pathogenesis of vascular disease in DM. We
vascular disease and DM [35]. Low levels of circu- also confirmed the negative relation between
lating EPCs may be associated with both endothe- EPC levels and the number of risk factors and
lial dysfunction and poor collateral development. showed a strong positive correlation between
Indeed, the concept that EPCs are involved both EPCs from patients with limb ischemia and the
in early and advanced events of the development ankle-brachial index [41].
of cardiovascular diseases is of crucial importance
for diabetology since DM is widely associated with
endothelial dysfunction as well as with poor new
vessel generation [36]. Antidiabetic therapy improves
In diabetes, atherosclerotic vascular disease is endothelial function
characterized by high prevalence, early develop-
ment and rapid progression. The severity of macro- Glycemic control remains the major intervention
vascular complications in DM seems to be due to for prevention of both micro- and macrovascular
profound impaired collateralization of vascular disease. However, in the UKPDS study, the 12%
ischemic beds, but mechanisms that hinder reduction in all diabetes-related endpoints
ischemia-induced neovascularization remain elu- achieved by intensive insulin treatment was mainly
sive. In DM collateralization results are insufficient due to a 25% decrease in microvascular complica-
to overcome the loss of blood flow through occluded tions, while myocardial infarction showed a non-
arteries, leading to ischemia, and often to limb statistical 16% reduction [42]. Stronger predictors
Endothelium and diabetes S43

of macrovascular complications were factors such On the contrary, insulin has also been shown to
as LDL cholesterol and blood pressure, that may exert pro-atherogenic effects. In fact, chronically
be related more to insulin resistance than to glyce- elevated insulin levels, such those reached in the
mic control. setting of insulin resistance and when the hormone
Nonetheless, the correction of both hypergly- is chronically administered, force insulin to act as
cemia and insulin resistance improves endothelial a growth factor through the MAP kinase pathway,
function. Although weight loss ameliorates insulin thus promoting the atherogenetic process [52].
sensitivity, a parallel sharp improvement in endo-
thelial function has not been observed. However,
weight loss decreases serum levels of inflammatory
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