‘Send Orders for Reprints to reprints@benthamscience.ae Current Vascular Pharmacology, 2015, 13, 801-808 801 Effect of Micronized Purified Flavonoid Fraction Thera i ; i py on Endothelin-1 and TNF-a Levels in Relation to Antioxidant Enzyme Balance in the Peripheral Blood of Women with Varicose Veins Agata Pietrzycka'”, Mariusz Kézka">, Tomasz Urbanek‘, Marek Stepniewski! and Marek Kucharzewski* ‘Cytobiology Department of Pharmacobiology Chair, Jagiellonian University, Medical College, Pharmacy Faculty, 9 Medyczna st, 30-688 Krakow, Poland; ‘Department of General Surgery, Jagiellonian University Medical College, 21 Kopernika st, 31-50] Krakow, Poland; ‘Department of General and Vascular Surgery, 5th Military Hospital, 1-3 Wroclawska st, 31-901 Krakow, Poland; ‘Department of General and Yascutar Surgery Medical University of Silesia, 45/47 Ziolowa st, 40-635 Katowice, Poland: “School of Medicine with the Division of Dentistry in Zabrze, Chair and Department of Descriptive and Topographic Anatomy, Medical University of Silesia ul Jordana 19, 41-808 Zabrze, Poland [Absieact: Objective: The aetiology of varicose veins involves various factors and pathomchanims including endothelial call activation or dysfunction, venous hypertension, vein wall hypo bear sess distances, inlmmatory reaction Setivatio or fee radical production To improve our understanding ofthe mechanisms of potential pharmacological i- terventons for ehroni venous disease, we evalusied the influence of micronized purified flavonoid faction (MPFF) on the relationship between antioxidant enzyme balance, endothelin-1 (ET-1) aad tumour neroisfactor-o (INF-o) levels. Material and Methods: Blood samples were obtained from 89 women wih primary vaticose veins, 34 were treated with MPEF and 35 did not receive any phlcbotopic dr treatment. For the evaaation of th blood antioxidant enzyre bal- ance, catalase (CAT) and superoxide dismutase (SOD) activity was asssted and the CAT/SOD rato was calculated. Re- sulls: Patient taking MPF had significantly lower ET-1 levels than tose not teking MPFF [median (255th quail) 242 (22,30-2787) vs 37.62 (249-4458) pg.m' p <0.05}. Ia those taking MPFF, a higher CAT/SOD ratio (39.8 (24.7-72.6) 4¥e.28.8 (163-57), 0.08) anda lower TNF-a concentration [682 (442-1339) vs 12.94 (601-2738) pgm p<0.05] was also observed. In women not taking MPFF, ET-1 levels increased with the CAT/SOD ratio. In those taking MPFF, the ET-1 level was stable at approximately 25.0 pg mt" up to a CAT/SOD ratio of 100. TNF-a level increased continuously svth an increasing CATSOD ati; however, the highest levels of TNPaa were observed in women not taking MPFF. Conclusions We demonstrate the ability of MPFF to effectively lover the levels of ET-1 and TNF-a in patents with Shon venous disease. Further investigations are nested to defi the therapeutic potential of MPFF inlusing the pten- fal eneet on chonie sbclnieainlanmaton, antioxidant imbalance and vascular dysfunction during the development of chronic venous dissat. Keywords: Endothelin, micronized purified flavonoid fraction, tumour necrosis factor-a, varicose veins, superoxide dismutase, catalase. INTRODUCTION Chronic venous ificant proportion of the Westen world populations with higher prevalence among the elderly and females [1-4]. Despite the number of studies performed, the aetiology of this disease remains un- clear [5-7]. Vein valve failure was suggested to play a role [5, 8], Currently, primary vein wall injury and resultant structure degeneration have been emphasized (3, 5]. Various stirmuli were initially considered, including endothelial cell activation or dysfunction, venous hypertension, vein wall hypoxia, shear stress disturbances, inflammatory reaction activation or the production of free radicals [3, 5, 6, -12] eee TAdivess concapondence to this author atthe Cyrobiology Department of Pharmacobiology Chait, Jagiellonian University, Medical College, Phannacy Fooully, 9 Medycra st, 30-688 Krakow, Poland; Tel: +48126205711; Fax: 448126205405, E-mail apietizy @em-uj.krakowpl 1s7O-1611/1S $58.004.00 Excessive oxidative stress has been reported in patients with lower varicose veins [7, 9, 13-17]. The generation of radical oxygen species (ROS) in cells is controlled by anti oxidant defence enzymes. One of the most important mecha- nisms is the balance between superoxide dismutase (SOD), ‘which catalyses the dismutation of superoxide to hydrogen peroxide, and catalase (CAT). Together with glutathione peroxidase, SOD and CAT convert hydrogen peroxide to water and oxygen [18-20]. Depletion or inactivation of these enzymes can lead to the overproduction of superoxide ani ons, hydroxyl radicals or hydrogen peroxide (11, 14] ‘The balance between CAT and SOD may be expressed as the CATISOD ratio [21] If SOD is depleted or inactivated (due to an increase in superoxide anions) and/or CAT is activated (resulting from increasing amounts of hydrogen peroxide), the CAT/SOD ratio increases. Alternatively, an increase in SOD activity (© 2015 Bentham Science Publishers802 Current Vascular Pharmacology, 2015, Vol 13, No.6 and/or inhibition of CAT could lead to a decrease in the CATISOD ratio. An imbalance between CAT and SOD re- sults in the overproduction of ROS, which in turn may lead fo a destruction of lipid membranes, proteins and other molecules in the endothelium, ROS can also stimulate the release of proinflammatory cytokines from activated or dam- aged endothelial cells including tumur necrosis. factor-a (TNF-a) and endothelin-1 (ET-1) (3, 5, 20, 22]. These destructive effects may be attenuated by activated leukocytes ‘tapped in the venous microcireulation. Activated neutrophils play an important role in the mechanisms leading to tissue injury, especially through reactive oxygen metabolites, re- lease of hydrolytic enzymes and the activation of inflamma- tory reactions (3, 5, 23, 24] The potential role of antioxidant therapy must be consid- fered when new targets for the pharmacological treatment of vascular diseases are considered [25-27]. Among the strong antioxidants, are the polyphenolic flavonoid compounds [28- 32]. The micronized purified flavonoid fraction (MPFF) has been suggested to exert a positive effect on regulating oxida- tive stress and a protective effect on endothelial cells [26, 33, 34]. However, the precise mechanisms linking MPEF anti- oxidant activity with the endothelium and maintenance of vein wall homeostasis still need to be investigated. In a study examining an ischemic and reperfusion model, MPFF was suggested to exert a protective effect through a dose- dependent reduction of the amount of hydrogen peroxide released from activated leukocytes [29]. Another study showed that MPFF may also affect the hydrogen peroxide- hypochlorite-myeloperoxidase system [25]. According to research performed on venous hypertension models and in cohorts of chronic venous disease patients, MPFF improves venous tonus and lymphatic drainage, reduces swelling and capillary permeability and increases capillary resistance [26, 35]. MPFF also acts as a potential anti-inflammatory drug by inhibiting enzymes involved in activating pro-inflammatory signalling cascades and preventing the expression of specific adhesion molecules involved in leucocyte recruitment [35- 40]. Its therapeutic benefit might also be associated with the capacity to inhibit platelet aggregation [28] as well as pros taglandin synthesis [37] To improve the understanding of the potential mecha- nisms of possible pharmacological interventions for patients with chronic venous disease, this study evaluated the influ- ence of MPFF on the relationship between antioxidant en- zyme balance, ET-1 and TNF-a levels. White blood cell (WBC) counts and plasma C-reactive protein (CRP) concen- trations were also measured. MATERIAL AND METHODS Peripheral blood samples were obtained from 89 con- secutive women with primary varicose veins who qualified for great saphenous vein and varicose vein surgical removal as well as approved and signed the patient informed consent. All patients had diagnosed chronic venous disease in clinical stages C2 or C3 according to the CEAP (Clinical, Etiologi- cal, Anatomical and Pathological) classification [41]. Of these, 62 patients had symptomatic varicose veins in stage C2 (heaviness of the legs and crural or varicose vein Piearsycha eat pain when standing or sitting) and 27 patients had varicose veins and leg edema (C3). In all the patients a duplex Doppler ultrasound of the deep and superficial vein system was performed. Patients with deep or superficial vein thrombosis or post-thrombotic changes in the lower extremity vein system were excluded. Other exclusion criteria were: diabetes, peripheral arterial disease, systemic or local inflammation, previous surgery within 6 months, need for anticoagulant or antiplatelet treat- ‘ment, being pregnant or post-partum, hormonal or lipid low- ering therapy, renal insufficiency, recent trauma or imnmobi- lization, In the 89 patients, 34 were treated with MPFF (Detralex, Servier, France) orally administered at a dose of 500 mg twice daily for at least 12 weeks. The remaining 55 patients were not treated with any phlebotropic drug. In all patients, class two compression stockings were prescribed 23 months before study recruitment. Previous MPFF. treatment was su- pervised by the patients” physicians and the decision to ad minister MPFF was not related to study recruitment. Com- parisons between the 2 groups (patients taking or not taking MPFF) for patient age, symptoms and stage of the chronic venous diseases and presence of concomitant diseases are presented in Table 1. In order to monitor the response to treatment, the 34 patients taking MPFF were divided into 2 subgroups: treatment for 12 ‘months [median - 80 weeks (min. 56 weeks, max 140 ‘weeks)] - (14 women). Informed consent was obtained from all paticnts. The study protocol was approved by the Bioeth- ies Committee, Medical College, Jagiellonian University. Laboratory Study Blood samples were collected before the routine varicose vein surgical procedures after a I2h overnight fast and before anesthesia and surgery (to exclude the potential influence of the surgery on the laboratory test results). Blood from an upper extremity peripheral vein was drawn into Ky-EDTA~ containing tubes (S-Monovettel, Sarstedt AG, Sevelen, Switzerland) to determine red blood cell count, hemoglobin, platelet count, white blood cell count (WBCs) count and hematocrit, Plasma samples were stored st -30°C until analy- sis of ET-1, TNF-a and CRP concentrations. ET-1 and TNF- a levels (pg,m{") in plasma samples were measured using immunoassay kits (Cayman Chemical, USA). High sensitiv ity C-reactive protein (hsCRP) was measured by immu- nonephelometry using the reagent kit CardioPhase (Dade Behring). For the evaluation of the CAT/SOD ratio, the red blood cell supernatant was washed 4 times with 150 mM NaCl, lysed in 4.0 ml of ice-cold double distilled water and centrifuged. The activity of catalase was determined accord- ing to Aebi on the basis of the decomposition of 10 mMol hydrogen peroxide in a sodium, potassium phosphate buffer pH = 7 (the absorbance was measured at a wavelength of 240 nm using UV- Vis spectrophotometer) [42]. SOD activ- ity was assessed according to the Misra and Fridovich method that is based on monitoring the auto-oxidation rate of epinephrine. The percentage of auto oxidation inhibition is proportional to SOD activity [43]Antioxidant Enzyme Balance in Varicose Vein Patiens Statistical Analysis Statistical analysis was performed with STATISTICA ENG, software, version 10.0 (Statsoft, POLAND). Due to the distribution of all tested variables being significantly different from the normal distribution, test and control groups were reported as medians and interquartile ranges. Between-subject differences were analyzed using two-sided Mann-Whitney U tests; p < 0.05 was considered signifi- Current Vascular Pharmacology, 2015, Vol 13, No. 6 803 RESULTS There were no statistically significant differences be- tween the MPFF user and MPFF non-user groups for the stages of chronic venous disease, patient age, body mass indexes, concomitant diseases and daily activity (Table 1). In both groups a similar rate of smokers was reported (50% in the group with MPFF therapy and 48% in the group without MPFF). There were also no significant differences in cant, fibrinogen, WBC and CRP between the groups (Table 2). Tables, Group characteris Grout women ig Group of women not taking MPFF | sedan 5s quite Men 5 grt , | re Age Gear) «7 0555) “sa4si) [xs | Sota ww (eee 2 ® 1s sane Connrng AP | eee 120525%) san [os sage Ch aecag a CEAP eo ie fein si 100 isan ss sug vee) se) ss sine suing | 10% va ws The prec ae ven ny 2A 26m 8 in een 13.08) ry ¥ Tet akg 176%) 266 8 Conpresionaacing I 34 100%) 55000) NS Ver cama pitd deel es oe Table2, Charcerites of rups prtpatingin the sud pat). Gopeimeme ung | Grey efremen tig ee vr ezan sven quinn | Melon 575 geri a a Fibrinogen gl 2.69 (2.24-3.1), 3.0 (2.45-3.54) NS* ‘White blood ceil count x 10° ul"! 7 6.02 (3.6-6.8) 6.2 (3.8-17.1) ‘NS Cesc pon na 2815630) 220548) ss CATISOD mio Bai canny AB U63I5II) pans ET-1 pgml* 24.2 22.30-27: 51) 37.62 (24.9-44.58) P<0.05 Trapani" en earin3s 129 039) rans ‘dotlia-I; MPFF-misonied pari flavonoid action; NS on sigan (CAT ~ catalase CATISOD - the balance bcween catalase and superoxide dismutase: ET S0D-superoniedirnuase; TNF-o amour necrosis factor {SL103X01 1 1y supplied and printed for onal Library, University of Debracer sito eV. censed cus riversity an804 Current Vascular Pharmacology, 2015, Vol 13,No. 6 Table 3. ‘and longer. Pletryha etal ‘The differences in chosen laboratory parameters between women with varicose veins taking MPEF for less than one year Patients taking MPFF <1 year (a= 20) Patients taking MPFF >t yenr (n= 14) Median | P Level of significance ae ET-1 pg.mt" 22.11 a 22.80 0.007 CCAT ~ Catause; CATISOD - he balance between eatate and superoxide dismutase; ET-1 - Endoielin-I; MPFF- Micronized Puifled Flavonoid Fraction; SOD ~ Superxile Dismutase, TNF-a Tumour Necrosis astra, Additionally, no differences in the hemoglobin, haematocrit and platelet count were seen (data not shown). Patients taking MPFF had significantly lower ET-I levels, than those not taking MPFF [median (25-75th quartile): 24.2 (22.30-27.87vs 37.62 (24.9-44.58) pg:mt"; p<0.05]. Patients in the MPFF group also had a significantly higher CAT/SOD ratio than the group of patients not taking MPFF [39.8 (24.7- 72.6) vs 28.8 (16:3-57.7); p<0.05}. Simultaneously in the patients treated with MPFF, a significantly lower TNF-a concentration was observed (Table 2). Furthermore, women taking MPFF for a longer duration (>1 year) had a higher CAT/SOD ratio and lower ET-I level than women on shorter term therapy. No differences in TNF-a were noted in relation to MPFF treatment duration (Table 3), To assess the relationship between ET-1 levels and oxi- dative enzyme balance, ET-1 levels correlated with the CATISOD ratio in the untreated group as well as in those on MPFF therapy (shorter and longer than 12 months). In the aforementioned 3 therapeutic subgroups (not taking MPFF, taking MPFF less than | year or longer than 1 yeat), (Fig. 1) shows that ET-1 levels increased with the CAT/SOD ratio. In women not taking MPFF, ET-I rose markedly when the CATISOD ratio increased above 100. In women taking MPFF, ET-1 was stable at approximately 25.0 pg.ml" up to a CATISOD ratio of 100. Above a CAT/SOD ratio of 100, the lowest ET-1 was observed in women taking MPFF for >1 year, ‘The relationship between the CAT/SOD ratio and TNF-a is presented in Fig, (2). The TNF-a level increased continu- ously with an increasing CAT/SOD ratio. However, the highest levels of TNF-a were observed in women not taking MPFF. No significant differences were observed between ‘women taking MPFF for 1 year (Fig. 2) Conceming the potential influence of MPFF on the in- flammatory reaction compounds, the white blood cell count was the highest (above 10,000 ut) when the CAT/SOD ratio was above 100 and lowest when the CAT/SOD ratio was exactly 100 in women not taking MPFF. In women treated with MPF, the WBC count was stable (approximately 6,000 ul"), independent of the CAT/SOD ratio. The ET-1 level increased together with the leukocyte count in peripheral blood only in samples obtained from women not taking MPFF. In patients taking MPFF, ET-I was observed to be at 90.0 80,0 70.0 60.0 50,0 40.0 ET-1 pg* mi 30,0 20.0 10,0 0,0-50,0 100,1-150,0 50,1-100,0 150,1-200,0 CATISOD Ratio Fig. (1), Endothelin-1 (ET-1) level as a function of antioxidant balance expressed as the CAT/SOD ratio: patients not taking MPF (black line), patients taking MPFF for short term therapy (erey line) and taking MPFF for more than 1 year (dashed line). Points represent median values, and whiskers represent the 25" - 75" quaciles. CAT/SOD ratio - the balance between catalase (CAT) and superoxide dismutase (SOD); MPFE- micronized puri- fied flavonoid fraction. ‘The scrum CRP levels did not differ between the patients ‘aking and not taking MPEF (median (25-75th quartile: 2.8 (1.56-3.0) vs 2.8 (2.05-4.05) mg 1“!; p>0.05]. However, the ET-I levels correlated with hsCRP concentration, especially in the range of hsCRP 4.1-6.0 mg 1 in chronic venous dis- cease patients not treated with MPFF. This relationship was not observed in the blood of women taking MPFF shorter than 12 months and >1 year (Fig. 4), DISCUSSION ‘Vascular endothelial dysfunction may be an important factor leading to chronic venous disease and varicose vein development [3, 5, [1, 39, 40]. Venous hypertension, hy- poxia and shear stress changes alongside leucocyte and inflammatory reaction activation can lead to endothelial x sonstan level of22-25 pest independent of he duration Gyan, eytkine rlewse and metallopro‘ias?Antioxidant Encyme Balance in Varicose Vein Patients 429 42.9 0,0-50,0 100,1-150,0 50,1-100,0 150,1-200,0. CATISOD Ratio Fig. @). Tumour necrosis factor-a (TNF-a) level as a function of antioxidant balance expressed as the CAT/SOD ratio as follows: patients not taking MPFF (black line), patients taking MPFF shorter than 12 months (grey line) and patients taking MPFF longer than 1 year (dashed line). Points represent median values, whiskers repre- ‘ent the 25% - 75" quartiles. CAT/SOD ratio - the balance between catalase (CAT) and superoxide dismutase (SOD), MPFF- mi- cronized purified flavonoid fraction, 90,0 7,6-10,0 10,1-15,0 051-75 WBCs 10° * lt Fig. (3). Relationship between endothelin-1 (ET-1) level and white blood cell count (WBCs) in the peripheral blood of ‘women with varicose veins as follows: not taking MPFF (black lines), taking MPFF shorter than 12 months (grey lines) and taking MPFF longer than 1 year (black and dotted lines). Points represent ‘median values, and whiskers in the picture represent the 25° - 75 quartiles. MPFF- micronized purified flavonoid fraction. Current Vascular Pharmacology, 2018, Vol 13, No.6 805 90.0 800 700 600 50,0 ET-I pe* mi 40,0 300 200 10.0 0.0-2,0 2,130 31-40 4,1-5,0 5,1-6,0 CRP mg * del * Fig. (4). Relationship between the endothelin-1 (ET-1) level and C-reactive protein (CRP) in the peripheral blood of women ‘with varicose veins as follows: patients not taking drugs (black lines); patients taking MPFF shorter than 12 months (grey lines), and taking MPFF longer than I year (black and dotted lines), Points represent median values, and whiskers in the picture represent the 25-75 quartiles. MPFF- micronized purified flavonoid fraction expression and activation [5, 8, 38, 40, 44-46), Vein wall structure, qualitative and quantitative changes can be ob- served as sequelae of this process, resulting in vein wall de- generation and weakening (3, 5]. One of the potential key points in this process can be excessive generation of ROS and depletion of antioxidant defence [8, 9, 13, 14, 27]. In previous studies, ROS generation was reported to be en- hanced in varicose vein walls. According to the study by Kozka et al. the presence of chronic venous ciated with an increased oxidative stress measured by ‘malonyldialdehyde (MDA) level [9]. In another study by the same group, SOD activity was observed in the wall of the insufficient ‘and varicose veins, and the total antioxidant power was lower and inversely proportional to MDA con- centration and levels of SOD enzyme and its substrate, su- peroxide radicals, in the varicose vein walls (14. According to their results, a higher superoxide radical concentration was noticed in the distal part of the incompetent saphenous vein wall compared with proximal specimens. Simultancously, the mean SOD level in the distal calf varicosities was sig- nificantly higher compared with the mid-thigh great saphe- nous vein (14]. The higher activity level of SOD in varicose vein specimens was also documented in another study that also confirmed the role of free radicals in chronic venous disease development [15]. Condezo-Hoyos ef al. evaluated the presence of the oxidative stress in the plasma of varicose vein patients [16]. They calculated the individual global in- dex of oxidative stress, documenting decreased catalase ac- tivity and thiol levels and increased levels of MDA-bound protein and protein carbonyls in varicose vein patient plasina [16]. Searching for the mechanisms responsible for the nd Nations806 Current Vascular Pharmace 1S, Tak 13, No.6 increased superoxide production in varicose veins, Guzik ef al. reported a higher superoxide production in varicose veins compared with control human saphenous veins, espe- cially in the distal, erural part of veins [17]. According to this study, the major sources of superoxide were nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and un- coupled nitrie oxide synthase (NOS). The authors also sug gested a Link between oxidative stress, endothelial dysfune~ tion, inflammation and immune activation and the develop- ment of the chronic venous dysfunction (17]. The potential role of the oxidative stress in the mecha- nisms linking inflammation, free radical production, as well as cytokine expression in varicose vein patients was also documented in our study. One of the important markers of endothelial activation and/or dysfunction is ETI, acting as a potent vasoconstrictor as well as a pro-inflammatory peptide (46. 47]. The biological effects of ET-1 are transduced by two receptor subtypes ET, and ET» [10, 47, 48]. According to another study, plasma ET-1 concentrations were higher in varicose veins than in normal saphenous veins [46]. The higher ET-1 concentrations in the varicose vein wall were also confirmed by Lowell er al. [48]. According to some studies, higher ET-1 content docs not only contribute to counterbalancing varicose vein relaxation but can also be reactive to endothelin receptor abnormalities. In patients with chronic venous disease, down-expression of ET=1 re- ceptors (ET and ETg) is observed along with decreases in the vein wall contraction mediated by ET-I [10]. ET-I re- ceptor down-regulation in varicose veins was also suggested by others as a response to the increased ET-1 production [49]. Concerning the potential role of ET-1 in the mechanism of other cardiovascular disease development. except for the influence of ET-1 on vessel vasoconstriction, the effect of ET-1 on the platelet function should also be mentioned (50, Si}. Based on previous studies as well as our results, other potential mechanisms related to ET-1 release and activity should be considered beyond vasoconstriction (52, 53]. Ac- cording to the results of our study, a link between oxidative stress level and ET-1 in the blood plasma of the varicose vein patients can be seen, ET-I levels correlated with the CAT/SOD ratio in varicose vein patients from our study group that confirm some of the previous observation and the Potential role of ET-1 and oxidative stress induction. In other studies ET-1 increases superoxide production in the rat aorta ‘and stimulates NADPH oxidase-derived superoxide produc- tion in hypertensive animals (53, 54]. The stimulation of the superoxide production in endothelial cells via the ET recep- tor in the human vascular endothelial cells was also sug gested [55]. Increased oxidative stress provoked by ET=1 in the vessel wall is a potential stimulus responsible for endo- thelial cell dysfunetion [47]. Some studies conducted in hu- ‘mans are also confirm the association between ET-1 expres- sion and the presence of oxidative stress and endothelial dys- function; in these studies, ET-1 was shown to induce super- ‘exide production in human venous bypass in diabetic pa- tients [52, 53]. Considered together, ET-I may have a sig- nificant influence on the increase in oxidative stress by in- ducing ROS, However, very little data concerning this poten- tial mechanism in chronic venous discase patients or labora tory models has been available until now and the presented Pletrayeka etal study indicates the new potential goals for the further the research concerning an effective pharmacological therapy of chronic venous disease patients. Looking for the new targets for the pharmacological treatment in pationts with chronic venous disease, for the first time, we found that MPFF ther- apy can potentially maintain constant ET-1 levels during an imbalance between catalase and superoxide dismutase (CATISOD ratio). This protective MPFF effect could possi- bly be used to regulate oxidative stress and stabilize ET-1 levels and could be used in further studies examining poten- tial efficacy of phlebotropic drugs in this setting. Notably, the stabilization of ET-1 levels was greater with prolonged MPFF therapy (compared with short term treatment). Another interesting observation concerns the relationship between the CAT/SOD ratio and some inflammatory reac- tion markers in patients treated and not treated with MPFF. Except for its vasoconstrictor effect and free radical genera- tion stimulation, ET-1 is also an important pro-inflammatory stimulus [47]. As previously documented, ET-1 has been demonstrated to activate macrophages to release the proin- flammatory mediators such as interleukin-6, interleukin-8 and TNF-a [56, 57]. Simultaneously, ET-1 also increases the expression of adhesion molecules in TNF-a stimulated vas- cular endothelial cells (47]. In our study, the ET-1 level in- creased together with the leukocyte count ih peripheral blood obtained from the subjects not taking MPFF. Simultanc- ously, the ET-1 levels correlated with CRP concentration in the patients not treated with MPFF. ‘According to animal studies performed on venous hyper- tension models, inflammation plays a crucial role in the vein wall and vein valve injury (38, 39]. MPFF can potentially inhibit the pro-inflammatory ‘cascade, prevent adhesion molecule expression as well as leucocyte recruitment and activation [28, 34, 37, 58-60]. In our study additional protec- tive MPFF action based on the oxidative stress control as well as ET-1 expression decrease was documented Considering the important role of the leukocytes and in- flammatory reaction in chronic venous disease occurrence and progression, we did not find significant differences in the WBC counts in both groups. However, when analysing the levels of oxidative stress markers (CAT/SOD ratio), the highest WBC counts were noticed in patients with a high CATISOD ratio who were not taking MPFF. In the patients ‘weated with MPFF, the WBC was noted to be stable and independent of the CAT/SOD ratio. A similar influence of MPFF therapy was also observed on WBC count, CRP and ET-1 in patients on this therapy, constant ET-I levels and CRP levels were reported, which suggest a positive influence ‘of MPFF on inflammatory reactions inkibition. CRP is one ‘of the acute-phase proteins whose production during in- flammation or tissue injury is stimulated by the proinflam- matory cytokines IL-1 and TNF-ot [47, 58, 61, 62]. Similar to ET-I levels, TNF-a levels increased with increased anti- ‘oxidant enzyme imbalance (CAT/SOD ratio) in varicose vein patients not on MPFF - in patients treated with MPFF, lower ‘TNF-a levels were seen. Of course for the better assessment of the potential influence of MPFF on the inflammatory cas- ‘cade, except above mentioned, other inflammatory reaction ‘compounds, including interleukins as well as adhesive mole- ‘cules, should be evaluated in future research. National Library, University of Debrecen (SL1D3X01 1286Antioxidant Enzyme Balance in Varicose Vein Patienss Some other limitations of the study should also be taken into consideration. Despite the inclusion and exclusion crite- ria, the potential heterogeneity of the investigated group of chronic venous disease patients should be taken into account (concerning local disease advancement as well as vein wall degenerative changes). In the study, ET-I levels were inves- tigated, however, for the better explanation of the role of ET- 1 in varicose vein development endothelin receptors should be assessed in further studies. Except for the CAT/SOD rae tio, other oxidative stress markers have to be taken into ac- count for the better and more precise assessment of the por tential anti-oxidant effect of MPFF. To summarize, according to our results, the possibility of a pharmacological intervention focused on the new targets ‘was confirmed, which could be based on the previously sug- gested potential anti-oxidant and antiinflammatory effects of MPFF [25-27, 29, 35, 36, 63-65]. CONCLUSION ‘These results demonstrate the ability of MPFF to effec- tively lower ET-1 and TNF-a levels in patients with chronic ‘venous disease. This effect may result in antioxidant capacity and the stabilization of inflammatory processes in chronic ‘venous disease Further investigations are needed to define the therapeutic potential of MPFF including the potential effect on chronic subclinical inflammation, antioxidant im- balance and vascular dysfunction during the development of chronic venous disease LIST OF ABBREVIATIONS CAT — = Catalase CATISOD = The balance between Catalase and Superox- ide Dismutase ET-1 = Endothelin-1 it = Intesteukin MPFF = =‘ Micronized Purified Flavonoid Fraction SOD —-=_ Superoxide Dismutase TNFa «= Tumour Necrosis Factor a WBCs = White Blood Cell Count (CONFLICT OF INTEREST The authors confirm that this anticle conde hus 90 ca fice of itexest. ACKNOWLEDGEMENTS “Te tectmical avsisiamce thom Wancke Kabtaiske fone the Radiligad Depavinicut of Phamnacsbiology Departament, Ingiclloniae Univessity Medial College. and Faculty of Phormcy to cary out the meauxemerts of eptikines andl antionidas enczyrnes activites és gneatly appprociviet. This ork owas supported fiom the Jagietloniine Uinives sity Programs No. 50): P:205F sand Ne K-ZDSIMUOSHS, printed for U REFERENCES: wu ry BI 4) Is) (6 am os o 110) on 102) wos eal re) 16} my fey tsp ey em rT eal eal ps iwersity and National Library, University of Ds Current Vaveutar Phurnaculogs, 2018, Vik 14, Noe 6 807 Partch H. Varicose veins und tone vets isucienny, Vasa 2009; 34: 23.301 BesbesDinmer It, Pree deniology a eh Upidemy 200818: 178-4 nares L, Schmid-Schonbein CW, Puhowensh of inary cvonie vetoun dan Inge tom sina models at Yenout yperennion, J Vane Surg 2008; 47. 1812 Melsmer Mil, Glove Bergin se Primary ec venue inorder. 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