Stanislava Vrankova

Continuous light and L-NAME-induced left ventricular remodelling: different protection with melatonin and captopril.

Objective Blood pressure enhancement induced by continuous light exposure represents an attractive but rarely investigated model of experimental hypertension. Design and methods The aim of this study was to show whether the combination of continuous light (24 h/day) exposure and chronic NG-nitro-L-arginine-methyl ester (L-NAME) treatment induces remodelling of the left ventricle and whether captopril or melatonin can modify these potential alterations. Six groups of 3-month-old Wistar rats (nine per group) were treated for 6 weeks: control (untreated), L-NAME (40 mg/kg per day), exposed to continuous light, L-NAME treated and exposed to continuous light (L24), L24 rats treated with either captopril 100 mg/kg per day, or melatonin (10 mg/kg/24 h). Systolic blood pressure (SBP), relative weights of the left ventricle, endothelial nitric oxide synthase (eNOS) and angiotensin-converting enzyme (ACE) expression in tissues, malondialdehyde and advanced oxidation protein product concentrations in the plasma and hydroxyproline levels in collagenous protein fractions were measured. Results The continuous light and L-NAME treatment led to hypertension, left ventricular hypertrophy (LVH) and fibrosis. An increase in SBP was completely prevented by captopril and partly by melatonin in the L24 group. Both drugs reduced oxidative damage and attenuated enhanced expression of ACE in the myocardium. Neither of the drugs prevented the attenuation of eNOS expression in the combined hypertensive model. Only captopril reduced LVH development in L24, whereas captopril and melatonin reduced left ventricular hydroxyproline concentrations in soluble and insoluble collagen, respectively. The total hydroxyproline concentration was reduced only by melatonin. Conclusion In hypertension induced by a combination of continuous light and L-NAME treatment, melatonin and captopril protect the heart against pathological left ventricular remodelling differently.

Effect of chronic apocynin treatment on nitric oxide and reactive oxygen species production in borderline and spontaneous hypertension

The purpose of this study was to investigate the effect of NAD(P)H oxidase inhibitor - apocynin (4-hydroxy-3-methoxyacetophenone) on the increase of systolic blood pressure (SBP) in borderline (BHR) and spontaneously hypertensive rats (SHR). Young 6-week-old male BHR (offspring of SHR dams and Wistar Kyoto sires) and SHR were treated with apocynin (30 mg/kg/day) for six weeks. SBP was measured by tail-cuff plethysmography. Nitric oxide synthase (NOS) activity was determined in the left ventricle and aorta. Protein expression of nuclear factor kappa B (NF-kappaB) and NAD(P)H oxidase subunits p67phox and p22phox as well as concentration of cGMP were determined for the left ventricle. Apocynin significantly decreased SBP in all groups investigated. Administration of apocynin had no effect on NOS activity in either tissue studied. However, apocynin decreased protein expression of NF-kappaB (p65) and NAD(P)H oxidase subunit p22phox in both hypertensive groups and p67phox subunit in the SHR group. Moreover, apocynin was able to prevent a decrease in cGMP concentration in the left ventricle of both hypertensive groups. In conclusion, our study demonstrated that apocynin treatment partially prevented SBP rise in borderline and spontaneously hypertensive rats, yet without increasing activity of NOS in the left ventricle and aorta. However, apocynin was able to decrease production of reactive oxygen species in hypertensive rats; thus preventing the decrease in cGMP formation.

The effects of new Alibernet red wine extract on nitric oxide and reactive oxygen species production in spontaneously hypertensive rats.

We aimed to perform a chemical analysis of both Alibernet red wine and an alcohol-free Alibernet red wine extract (AWE) and to investigate the effects of AWE on nitric oxide and reactive oxygen species production as well as blood pressure development in normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHRs). Total antioxidant capacity together with total phenolic and selected mineral content was measured in wine and AWE. Young 6-week-old male WKY and SHR were treated with AWE (24,2 mg/kg/day) for 3 weeks. Total NOS and SOD activities, eNOS and SOD1 protein expressions, and superoxide production were determined in the tissues. Both antioxidant capacity and phenolic content were significantly higher in AWE compared to wine. The AWE increased NOS activity in the left ventricle, aorta, and kidney of SHR, while it did not change NOS activity in WKY rats. Similarly, increased SOD activity in the plasma and left ventricle was observed in SHR only. There were no changes in eNOS and SOD1 expressions. In conclusion, phenolics and minerals included in AWE may contribute directly to increased NOS and SOD activities of SHR. Nevertheless, 3 weeks of AWE treatment failed to affect blood pressure of SHR.

Effect of nuclear factor kappa B inhibition on L-NAME-induced hypertension and cardiovascular remodelling.

Objective We aimed to analyze effects of nuclear factor-kappa B (NF-κB) inhibition on blood pressure (BP) regulation and cardiovascular remodelling. Design Adult 12-week-old male Wistar Kyoto rats (WKY) were treated with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg/day) for seven weeks. From the fourth week of L-NAME treatment, the NF-κB inhibitor lactacystin (1 μg/kg) was applied once a week. Furthermore, age-matched WKY received L-NAME or lactacystin alone for 7 or 3 weeks, respectively. Methods Total NOS activity was determined in the left ventricle (LV) and aorta. The concentration of conjugated dienes, fibrosis, and collagen I and III levels were determined in the LV. The cross-sectional area (CSA) and wall thickness to internal diameter ratio (WT/ID) were measured in the aorta. Results L-NAME treatment increased BP significantly (145 ± 2 mmHg vs. 110 ± 1 mmHg in controls). The addition of lactacystin resulted in further significant increase in BP (161 ± 3 mmHg). Similarly, lactacystin potentiated the increased conjugated dienes concentration induced by L-NAME. Whereas L-NAME alone did not affect NOS activity, the addition of lactacystin decreased it in both tissues investigated. The addition of lactacystin did not affect LV hypertrophy, fibrosis, and collagen I and III, already increased by L-NAME; however, it further amplified CSA in the aorta increased by L-NAME alone. WT/ID increased significantly only after the addition of lactacystin. Conclusion Decreased NOS activity along with increased oxidative load may be responsible for decreased NO bio-availability and further BP increase after NF-κB inhibition in L-NAME-induced hypertension. Increased CSA and WT/ID could contribute to this hypertensive process.

Lactacystin-Induced Model of Hypertension in Rats: Effects of Melatonin and Captopril

Lactacystin is a proteasome inhibitor that interferes with several factors involved in heart remodelling. The aim of this study was to investigate whether the chronic administration of lactacystin induces hypertension and heart remodelling and whether these changes can be modified by captopril or melatonin. In addition, the lactacystin-model was compared with NG-nitro-l-arginine-methyl ester (L-NAME)- and continuous light-induced hypertension. Six groups of three-month-old male Wistar rats (11 per group) were treated for six weeks as follows: control (vehicle), L-NAME (40 mg/kg/day), continuous light (24 h/day), lactacystin (5 mg/kg/day) alone, and lactacystin with captopril (100 mg/kg/day), or melatonin (10 mg/kg/day). Lactacystin treatment increased systolic blood pressure (SBP) and induced fibrosis of the left ventricle (LV), as observed in L-NAME-hypertension and continuous light-hypertension. LV weight and the cross-sectional area of the aorta were increased only in L-NAME-induced hypertension. The level of oxidative load was preserved or reduced in all three models of hypertension. Nitric oxide synthase (NOS) activity in the LV and kidney was unchanged in the lactacystin group. Nuclear factor-kappa B (NF-κB) protein expression in the LV was increased in all treated groups in the cytoplasm, however, in neither group in the nucleus. Although melatonin had no effect on SBP, only this indolamine (but not captopril) reduced the concentration of insoluble and total collagen in the LV and stimulated the NO-pathway in the lactacystin group. We conclude that chronic administration of lactacystin represents a novel model of hypertension with collagenous rebuilding of the LV, convenient for testing antihypertensive drugs or agents exerting a cardiovascular benefit beyond blood pressure reduction.

Comparison of the effects of indapamide and captopril on the development of spontaneous hypertension

Objectives The effects of indapamide, a thiazide-like diuretic, and captopril, an angiotensin-converting enzyme inhibitor, on spontaneous hypertension and the development of left ventricular hypertrophy (LVH), nitric oxide generation and oxidant status were investigated. Methods Six-week-old male spontaneously hypertensive rats (SHR) were treated with indapamide (1 mg/kg per day) or captopril (10 mg/kg per day) or a combination of indapamide plus captopril. After the 6-week treatment, nitric oxide synthase (NOS) activity, the expression of NOS isoform proteins, conjugated dienes concentration and relaxation responses of the femoral artery were analyzed. Results Indapamide and captopril partly prevented a blood pressure increase in young SHR. Captopril in contrast to indapamide reduced LVH. The effect of the combined indapamide and captopril treatment on the prevention of hypertension was additive. Combined indapamide and captopril treatment increased NOS activity and endothelial NOS protein expression in the aorta and decreased conjugated dienes concentration in the kidney compared with the indapamide monotherapy group. Indapamide and indapamide and captopril treatment increased acetylcholine-induced relaxation of the femoral artery. Conclusion Whereas captopril reduced LVH, indapamide enhanced NOS activity and decreased oxidative damage in the case of the combined treatment. It is concluded that the complex protective effects of the combined indapamide plus captopril treatment on hypertension may be exerted via its effects on blood pressure, hypertrophy and vasorelaxation.

The Regulatory Role of Nuclear Factor Kappa B in the Heart of Hereditary Hypertriglyceridemic Rat

Activation of nuclear factor-κB (NF-κB) by increased production of reactive oxygen species (ROS) might induce transcription and expression of different antioxidant enzymes and also of nitric oxide synthase (NOS) isoforms. Thus, we aimed at studying the effect of NF-κB inhibition, caused by JSH-23 (4-methyl-N 1-(3-phenyl-propyl)-benzene-1,2-diamine) injection, on ROS and NO generation in hereditary hypertriglyceridemic (HTG) rats. 12-week-old, male Wistar and HTG rats were treated with JSH-23 (bolus, 10 μmol, i.v.). After one week, blood pressure (BP), superoxide dismutase (SOD) activity, SOD1, endothelial NOS (eNOS), and NF-κB (p65) protein expressions were higher in the heart of HTG rats compared to control rats. On the other hand, NOS activity was decreased. In HTG rats, JSH-23 treatment increased BP and heart conjugated dienes (CD) concentration (measured as the marker of tissue oxidative damage). Concomitantly, SOD activity together with SOD1 expression was decreased, while NOS activity and eNOS protein expression were increased significantly. In conclusion, NF-κB inhibition in HTG rats led to decreased ROS degradation by SOD followed by increased oxidative damage in the heart and BP elevation. In these conditions, increased NO generation may represent rather a counterregulatory mechanism activated by ROS. Nevertheless, this mechanism was not sufficient enough to compensate BP increase in HTG rats.

604 EFFECT OF MELATONIN ON BLOOD PRESSURE AND FIBROSIS ENLARGEMENT IN THE HEART AND AORTA IN EXPERIMENTAL METABOLIC SYNDROME

Objectives: Melatonin is involved in many signaling processes via receptor-mediated or receptor-independent mechanisms. We aimed to find out whether melatonin can modify blood pressure (BP) and morphological parameters of heart and aorta in rats with metabolic syndrome. Methods: Males, 6-week-old rats were divided into four groups: Wistar Kyoto rats; spontaneously hypertensive rats (SHR); rats with metabolic syndrome (SHR-cp); and SHR-cp treated with melatonin (10 mg/kg/day) for 3 weeks. BP was measured by telemetry. Tissue sections embedded in paraffin were stained with hematoxylin–eosin and picrosirius red. Heart and aorta collagen levels, cross section area and wall thickness of the aorta were analyzed. Melatonin MT(1) and MT(2) receptors were determined by immunohistochemical and Western blot methods. Results: BP increase in SHR-cp was comparable to that of SHR. Melatonin treatment reduced BP increase of SHR-cp by 12%. Both heart and aorta collagen levels in SHR-cp were increased significantly comparing to SHR. Melatonin failed to affect fibrosis enlargement in the heart. On the other hand, it was able to reduce fibrosis enlargement in the aorta. Comparing to SHR, expression of MT(1) receptors was elevated in SHR-cp with higher density in the aorta than in the heart. Thus, melatonin reduced fibrosis enlargement in the aorta – the tissue where expression of MT(1) receptors was shown to be higher. In Conclusion: Decrease of fibrosis enlargement in the aorta after melatonin treatment may contribute to BP reduction in rats with metabolic syndrome. Moreover, contribution of MT(1) receptor – mediated effect of melatonin is suggested. (APVV-0538-07, APVV-0742-10, VEGA-2/0190/11, VEGA-2/0178/09).

884 Effect of nuclear factor-kappa B inhibition on heart remodelling in experimental hypertension

Objectives: We aimed to analyze effects of NF-&kgr;B inhibition on blood pressure (BP) and heart remodelling in NG-nitro-L-arginine methyl ester (L-NAME)-hypertensive animals. Methods: Males, 12-week-old Wistar Kyoto rats (WKY) were treated with nitric oxide synthase (NOS) inhibitor L-NAME (40 mg/kg/day) for seven weeks. From the fourth week of treatment, NF-&kgr;B inhibitor lactacystin (1 mg/kg) was applied once a week. Furthermore, age-matched WKY received L-NAME or lactacystin alone for 7 or 3 weeks, respectively. NOS activity, expressions of eNOS mRNA and protein, concentration of conjugated dienes and collagen I and III levels were determined in the heart. NF-&kgr;B (p65) protein expression was measured by immunohistochemical and Western blot methods. Results: L-NAME treatment increased BP significantly (145 ± 2 vs. 110 ± 3 mmHg in controls). Addition of lactacystin resulted in further significant BP increase (161 ± 3 mmHg). L-NAME led to increased NF-&kgr;B (p65) expression followed by elevation of both eNOS mRNA and protein expressions. Addition of lactacystin blocked, however, elevated eNOS expression followed by decreased NOS activity. Furthermore, lactacystin potentiated increased conjugated dienes concentration and fibrotic process induced by L-NAME treatment. Addition of lactacystin, however, did not affect heart hypertrophy and collagen I and III already increased by L-NAME. Conclusion: Decreased NOS activity along with increased oxidative load may be responsible for decreased NO bioavailability and further BP increase after NF-&kgr;B inhibition in L-NAME-hypertension. Decreased level of NO may also contribute to fibrosis enlargement in the heart after lactacystin treatment. Thus, NO could represent a regulatory factor responsible for different NF-&kgr;B-dependent growth responses. (APVV-0538-07, APVV-0742-10, VEGA-2/0190/11, VEGA-2/0178/09).

THE ROLE OF NUCLEAR FACTOR KAPPA B IN L-NAME-INDUCED HYPERTENSION: PP.29.139

Objective: Recently we have demonstrated involvement of NF-κB in the upregulation of endothelial nitric oxide synthase (eNOS) in hypertension induced by NG-nitro-L-arginine methyl ester (L-NAME). Thus, the goal of our study was to analyze an effect of NF-κB inhibitor, lactacystin, in L-NAME-induced hypertension. Methods and Design: Adult 12-week-old male Wistar rats were subjected to treatment with L-NAME (40 mg/kg/day) for seven weeks (n = 14). Half of the rats received lactacystin together with L-NAME for last three weeks. Next 16-week-old male Wistar rats received lactacystin only for 3 weeks (n = 7). Blood pressure was measured by tail-cuff plethysmography every week. Total NOS activity was determined by measuring the formation of L-[3H] citrulline from L-[3H] arginine. Endothelial NOS and NF-κB (p65) protein expressions were determined immunohistochemically and by Western blot analysis. Results: Lactacystin treatment did not affect the blood pressure (103 ± 6 mmHg), while 7-week-L-NAME treatment increased blood pressure (141 ± 3 mmHg) by 38% comparing the age-matched untreated animals (104 ± 5 mmHg). Addition of lactacystin to the L-NAME increased blood pressure significantly (159 ± 4 mmHg) by 54% comparing the untreated control group and by 12% comparing the L-NAME group. L-NAME treatment led to increased NF-κB expression followed by elevation of both eNOS protein expression and total NOS activity in the aorta, heart and kidney. Addition of lactacystin blocked, however, elevated eNOS protein expression in all tissues investigated. Conclusion: We hypothesized that NF-κB is responsible for upregulation of eNOS protein expression which may represent one of the counterregulatory mechanisms activated to compensate decreased NO production and increased blood pressure after long-term L-NAME treatment.