| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
CLINICAL STUDIES |
From the Division of Cardiology, Taipei Medical University-Wan Fang Hospital (Dr Yang, Dr Kao, Dr Chan); Faculty of Chinese Medicine, Macau University of Science and Technology, Macau (Dr Chan); the Faculty of Medicine, the Chinese University of Hong Kong (Dr Tomlinson); and the Division of Cardiology, Cathay General Hospital, Taipei, Taiwan (Dr Ko).
Address for reprints: Address for correspondence: Paul Chan, MD, PhD, Division of Cardiology, Taipei Medical University-Wan Fang Hospital, No 111, Hsing-Lung Road, Sec. 3, Wen-Shan District, Taipei City 116, Taiwan; e-mail: chanpaul{at}wanfang.gov.tw.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
Key Words: gene • hypertension • reactive oxygen species • superoxide dismutase • valsartan
Many clinical and laboratory studies have suggested that the renin-angiotensin system is involved in the pathogenesis of cardiovascular diseases and that this has changed the traditional view on the role of angiotensin (Ang) II. It is now generally accepted that locally formed Ang II regulates the expression of many substances, including growth factors, cytokines, chemokines, and adhesion molecules, which are involved in cell growth/apoptosis, fibrosis, and inflammation.3-7 The production of Ang II within the arterial wall is important in the normal regulation of arterial tone and is clearly involved in the pathogenesis of atherosclerosis. Ang II regulates many processes implicated in vascular pathophysiology, including cell growth/apoptosis of vascular cells, migration of vascular smooth muscle cells, inflammatory responses, and extracellular matrix remodeling.4-6,8 Drugs that reduce the effects of Ang II actions, such as angiotensinconverting enzyme (ACE) inhibitors or angiotensin receptor antagonists, are currently employed in the treatment of hypertension, heart failure, and diabetic nephropathy and appear to be useful in atherosclerosis and other cardiovascular diseases.4-6
Cellular protection mechanisms against oxidants include both enzymatic and nonenzymatic defense systems. Among the most important antioxidant enzymes are superoxide dismutase (SOD), catalase, cytochrome c peroxidase, glutathione peroxidase, and glutathione reductase. Superoxide dismutase is a ubiquitous enzyme in aerobic organisms with 3 isoenzymes: Fe-SOD, Mn-SOD, and Cu/Zn-SOD. The Fe-SOD is present in prokaryotic organisms, plant chloroplasts, and some protozoa.9 Mammalian cells produce 2 different types of Cu/Zn-SOD enzymes, 1 intracellular form and 1 extracellular form (ECSOD), which catalyze the dismutation reaction with similar efficiencies.9
Valsartan is an angiotensin II type 1 (AT1) receptor antagonist, which has aromatic rings in its chemical structure.10 The presence of either a phenolic ring in the chemical structure or conjugated double bonds (as in vitamin E) seems to be necessary for effective scavenging of oxygen free radicals.11 This study was undertaken to evaluate whether valsartan has effects on the antioxidant enzyme SOD in patients with stage I or II essential hypertension, which might imply antioxidant activity.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
Patient Population
Eligible patients of both sexes (aged 20 to 75 years) with newly diagnosed mild (stage I) to moderate (stage II) essential hypertension (meeting Joint National Committee [JNC] VI criteria), having a sitting systolic blood pressure (SBP) between 140 and 179 mm Hg and a sitting diastolic blood pressure (DBP) between 90 and 109 mm Hg,12 were enrolled in the study. Otherwise, the patients had to be apparently healthy and free of target organ damage caused by hypertension, secondary causes of hypertension, other cardiac disease, malignancies, significant renal impairment (serum creatinine level >2.0 mg/dL), or hepatic dysfunction. Patients were asked to avoid consuming any antioxidants, including tea, vitamin E, vitamin C, and beta-carotene, during the course of the study. A normal control group of 20 people was also included for measurement of SOD activity.
Conduct of Study
After randomization, patients were requested to attend follow-up every 4 weeks for 12 weeks during active treatment to assess side effects and to measure blood pressure. The active treatment group patients were given valsartan capsules (Novartis Pharma, Basel, Switzerland) 80 to 160 mg once or twice a day, and the control group patients were given a matching placebo. An electrocardiogram and laboratory examinations (including activity and mRNA level of SOD) were performed at the beginning and then monthly during double-blind treatment. Blood pressure was measured using a mercury sphygmomanometer.13 Each trough value was the mean of 3 readings taken 10 minutes apart. Venous blood was drawn between 8 and 10 AM after subjects had fasted overnight. The participants were asked to abstain from heavy meals for 48 hours before their visit. Blood was collected for determination of glucose, lipids, renal and liver function, and electrolytes and antioxidant parameters. Glucose, cholesterol, triglycerides, uric acid, blood urea nitrogen, alanine aminotransferase, asparate aminotransferase, and creatinine levels were measured with a Monarch Autoanalyzer System (Instrumentation Laboratories, Tex).
Preparation of Polymorphonuclear Leukocytes
Blood was obtained from the subjects of each group. Polymorphonuclear leukocytes (PMNs) were separated out using the method of Williams and Cole,14 with slight modification. Blood with heparin (10 U/mL) was added to half of its volume of monopolyresolving medium (Ficoll-Hypaque, density 1.114 g/mL, Flow Laboratory, Meckenheim, Germany). After centrifuging at 1600 rpm for 30 minutes at 4°C, the PMN layers were collected and treated with 0.17 Tris-ammonium chloride. The cells were washed with Hank's balanced salt solution. The PMNs were suspended in culture medium (Life Technologies, Inc, New York) (4 x 105 PMNs/mL) and kept in silicone glass containers until use. Luminol (Sigma, St Louis, Mo) was dissolved in dimethyl sulphoxide (Sigma) at a concentration of 2 x 10-3 M. Prior to use, this stock solution was further diluted in medium to the required concentration.
Assay of SOD Activity
The activity of SOD was measured using a commercial assay kit (Wako, Osaka, Japan). Erythrocytes were isolated and homogenized in 1 mL of 0.9% NaCl.15 The crude homogenate was centrifuged at 10 000 g for 1 hour to get the supernatant (cytosolic) and pellet (particulate). For assay of Mn-SOD activity in the pellet, 1 mmol/L potassium cyanide was added to the incubation mixture to inhibit Cu/Zn-SOD activity. The activity of Cu/Zn-SOD was derived by the subtraction of Mn-SOD activity from total SOD activity in the pellet.15 The units of SOD activity were derived from bovine erythrocytes (S-2515; Sigma). Results were expressed as unit/mg of protein determined.16
Northern Blot Analysis
Cells (PMNs) for RNA isolation were frozen in liquid nitrogen immediately after removal and then stored at -80°C. Total RNA was isolated as described previously.17 RNA was then transferred to Hybond N nylon membranes (Amersham, Manchester, UK) overnight in 2 volumes of saline-sodium citrate. The transfer was controlled on an ultraviolet transilluminator and also by staining the blot membrane with 0.05% methylene blue.18 The filters were rapidly prehybridized at 65°C in hybridization solution (Quikhyb, Stratagene, La Jolla, Calif). The cDNA probes were also prepared. Plasmids containing cDNA of SOD were supplied by Dr Y. S. Ho, and plasmids containing cDNAs of catalase and glutathione peroxidase were obtained from Dr T. S. Chiou. Transformation in Escherichia coli, plasmid preparation, and cDNA purification were performed according to standard methods.19 Radioactive probes (P32) were prepared using the multiprime DNA labeling system (Amersham). The prepared cDNA inserts and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes were added directly into the prehybridization solution (Quikhyb, Stratagene) at a radioactivity of 1 x 106 ct/(min·mL). Hybridization was performed at 68°C for 70 minutes. After washing, the wet blot membranes were sealed in plastic foil and exposed to medium-sensitive medical x-ray film (Fuji, Tokyo, Japan) at -70°C using intensifier screens. Exposure times were 2 to 3 days for Northern blots. Hybridization intensity of autoradiographic signals was measured using 2-dimensional densitometry. The obtained density (optical units) was calculated versus the value of slot blot for GAPDH to serve as the internal control for quantification of mRNA.
Statistics
All values are presented as the mean ± standard deviation (SD) from each group. Statistical significance was evaluated using 1-way analysis of variance (ANOVA) with Kruskal-Wallis's test when multiple groups were compared; when only 2 groups were compared, Student t test was used. A value of P < .05 was considered to be statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
Efficacy of Valsartan
The baseline and endpoint blood pressure (BP) results are summarized in Table I. The BP levels in the placebo and active treatment groups were not different at baseline, but after 12 weeks, both SBP and DBP values for the active treatment group were significantly different from the placebo group. The mean reduction in SBP was 10 mm Hg, and DBP was 6 mm Hg (Table I). The mean dosage of valsartan used was 128.6 mg daily.
Safety
The drug was well tolerated. Only 4 patients (2 from the placebo group, 2 from the valsartan group) were withdrawn before the last scheduled study visit for the following reasons: lost to follow-up (2 patients) and side effects (2 patients, 1 from active treatment group and 1 from placebo group) due to dizziness and skin rash. These patients were not included in the statistical analysis. At the outset, 3 patients from the active treatment group and 3 from the placebo group experienced abdominal fullness, headache, nausea, and asthenia, but all of the symptoms disappeared after continued intake of drugs for 1 week. Laboratory tests, either at baseline or during double-blind treatment, showed no significant difference between the 2 groups. All remaining subjects followed the prescribed treatment schedule during the entire 12-week treatment period. Subject compliance was evaluated by capsule counting, which showed a similar degree of compliance in the 2 treatment groups throughout the entire course of randomized treatment. Capsule intake in the placebo group averaged 95% ± 4% of the planned number of capsules at randomization and 92% ± 3% during double-blind treatment. In the valsartan group, the intake averaged 96% ± 3% at randomization and 93% ± 3% during double-blind treatment. There were no cardiovascular events or any deaths in either group during the study.
Superoxide Dismutase Activity
The mean ± SD baseline SOD activity of hypertensive subjects was significantly higher than that in normal controls (240.4 ± 43.3 vs 96.2 ± 26.0 U/mg protein). After treatment for 3 months, the SOD activity was reduced in the valsartan group, although the mean level was still higher than the normal control group (96.2 ± 26.0 µ/mg protein; Table I). However, the SOD activity of the placebo group showed no significant change after 3 months.
SOD-mRNA After Valsartan Treatment
Both Cu/Zn-SOD and Mn-SOD mRNA levels (Figure 1) were reduced significantly and progressively over the 3-month period of treatment with valsartan (Table II). However, for the patients in the placebo group, the SOD-mRNA levels did not show any change. The baseline value of the SOD-mRNA of the control group was 100 ± 0; the value at 3 months was 99.2 ± 0.5. These changes were not significant.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
The role of oxidative stress in the pathogenesis of vascular disease has been well recognized.20 Ang II stimulates the production of ROS by inducing vascular NADH/NADPH oxidase. Ang II promotes atherosclerosis by 2 redox mechanisms: (1) by increasing the levels of lipid-oxidizing ROS, which promotes the loading of lipid into foam cells, and (2) by inducing the expression of redox-sensitive gene products, such as vascular cell adhesion molecule-1 monocyte chemoattractant protein-1 (MCP-1).21 As the role of oxidative stress in the pathogenesis of hypertension-related disease becomes more appreciated, our impression of hypertension as a rather indolent, solely hemodynamic process is being revised. The ROS are the end result of the univalent reduction of oxygen, resulting in the production of superoxide anion (
), hydrogen peroxide (H2O2), and water (H2O). The ROS influence both normal and abnormal cellular processes, including cellular growth, hypertrophy, remodeling, lipid oxidation, modulation of vascular tone, and inflammation.22 In vascular smooth muscle cells, the induction of MCP-1 and interleukin-6 (IL-6) by Ang II is dependent on the activation of NADPH oxidase.23,24 Both Ang II and IL-6 are localized to the macrophages present in the shoulder region of the atherosclerotic plaques, the region suggested to be prone to plaque rupture in acute coronary syndromes.25 Ang II is also associated with other sources of oxidative stress, such as oxidized low-density lipoprotein (LDL) and nitric oxide, which can participate in inflammation and apoptosis.26
Drugs that block the actions of Ang II, such as ACE inhibitors or angiotensin receptor antagonists, are currently employed in the treatment of hypertension and heart failure and appear useful for atherosclerosis and other cardiovascular disease.3-8 A recent study showed that valsartan could inhibit neointimal proliferation in patients who received percutaneous coronary intervention.27 We speculate that valsartan could attain this result via ROS scavenging.
Clinical studies have shown the occurrence of increased ROS production in humans with essential hypertension.28,29 In physiological conditions, levels are modulated by endogenous scavenging systems, such as SOD. It seems that in essential hypertension, there may be an imbalance between an enhanced generation and a decreased antioxidant activity. Superoxide dismutase activity is a major cellular defense mechanism that combats oxygen toxicity.30 The dismutation of superoxide anions () to H2O2 and O2 by SOD is often called the primary defense.31 H2O2 is a comparatively less reactive oxidizing agent than , and glutathione-peroxidase and catalase rapidly convert it to water and O2. The phenomenon of down-regulation of SOD-mRNA was clearly demonstrated in this study. This is compatible with previous reports, which suggested that there is increased ROS production in human essential hypertension, and these increases in production of ROS may be secondary to increased Ang II secretion.28,29 If valsartan is able to reduce ROS production, less SOD will be required, and this could result in down-regulation of SOD expression. However, there are still 3 limitations of this study: (1) the sample size is relatively small and will not apply to other studies; (2) the methodology of measuring SOD is not standardized in biomedical research, but the method we used is commonly employed; (3) direct measurement of the antioxidant activity of valsartan (enhanced chemiluminescence) was not performed systematically, but there have been reports showing the antioxidant effect of valsartan.10,27
Berry et al32 have demonstrated that NAD(P)H oxidase is a source of basal production in human internal mammary arteries and saphenous veins. The same authors have reported that Ang II increases in human arteries. This effect is mediated by NAD(P)H oxidase and is completely inhibited by the AT1 receptor antagonist losartan. Higher basal concentration in arteries, compared with that in veins, was maintained after endothelial denudation by rubbing, suggesting that vascular smooth muscle cells might be an important source of generation in the human arterial wall.
In conclusion, this study shows that hypertensive patients have higher levels of oxygen free radicals and that treatment with valsartan results in down-regulation of SOD-mRNA and SOD activity in these hypertensive patients, which probably implies that valsartan effectively reduces ROS production. This phenomenon signifies that this AT1 receptor antagonist may have benefits in hypertensive patients in addition to blood pressure reduction.
|
ACKNOWLEDGEMENTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
1. MacMahon SW, Cutler JA, Neaton JD, et al. The effects of drug treatment for hypertension on morbidity and mortality from cardiovascular disease: a review of randomized controlled trials. Prog Cardiovasc Dis. 1986;29: 99-118.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
2. Prabha PS, Das UN, Koratkar R, Sagar PS, Ramesh G. Free radical generation, lipid peroxidation and essential fatty acids in uncontrolled essential hypertension. Prostaglandins Leukot Essent Fatty Acids. 1990;41: 27-33.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
3. Egido J. Vasoactive hormones and renal sclerosis. Kidney Int. 1996;49: 578-597.[Web of Science][Medline] [Order article via Infotrieve]
4. Matsubara H. Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal disease. Circ Res. 1998; 83: 1182-1191.
5. Mezzano SA, Ruiz-Ortega M, Egido J. Angiotensin II and renal fibrosis. Hypertension. 2001;38: 635-638.
6. Sadoshima J. Cytokine actions of angiotensin II. Circ Res. 2000;86: 1187-1189.
7. Ruiz-Ortega M, Lorenzo O, Suzuki Y, Ruperez M, Egido J. Proinflammatory actions of angiotensins. Curr Opin Nephrol Hypertens. 2001;10: 321-329.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
8. Tu
on J, Ruiz-Ortega M, Egido J. Regulation of matrix proteins and impact on vascular structure. Curr Hypertens Res. 2000; 2: 106-113.
9. Bannister JV, Bannister WH, Rotilio G. Aspects of the structure, function, and applications of superoxide dismutase. Crit Rev Biochem. 1987;22: 111-180.[Web of Science][Medline] [Order article via Infotrieve]
10. Seeger H, Mueck AO, Lippert TH. Effects of valsartan and 17ß-estradiol on the oxidation of low-density lipoprotein in vitro. Coron Artery Dis. 2000;11: 347-349.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
11. Chan P, Cheng JT, Tsao CW, Niu CS, Hong CY. The in vitro antioxidant activity of trilinolein and other lipid-related natural substances as measured by enhanced chemiluminescence. Life Sci. 1996;59: 2067-2073.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
12. Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure. The sixth report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC VI). Arch Intern Med. 1997;157: 2413-2446.
13. Perloff D, Grim C, Flack J, et al. Human blood pressure determination by sphygmomanometry. Circulation. 1993;88: 2460-2470.
14. Williams AJ, Cole PJ. Human bronchoalveolar lavage cells and luminol-dependent chemiluminescence. J Clin Pathol. 1981;34: 167-171.
15. Chan PH, Chen SF, Yu AC. Induction of intracellular superoxide radical formation by arachidonic acid and by polyunsaturated fatty acids in primary astrocytic cultures. J Neurochem. 1988;50: 1185-1193.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72: 248-254.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
17. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162: 156-159.[Web of Science][Medline] [Order article via Infotrieve]
18. Herrin DL, Schmidt GW. Rapid, reversible staining of Northern blots prior to hybridization. Biotechnique. 1988;6: 196-197, 199-200.[Web of Science][Medline] [Order article via Infotrieve]
19. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
20. Irani K. Angiotensin II-stimulated vascular remodeling: the search for the culprit oxidase. Circ Res. 2001;88: 858-860.
21. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994;74: 1141-1148.
22. Alexander RW. Theodore Cooper Memorial Lecture. Hypertension and the pathogenesis of atherosclerosis. Oxidative stress and the mediation of arterial inflammatory response: a new perspective. Hypertension. 1995;25: 155-161.
23. Chen XL, Tummala PE, Olbrych MT, Alexander RW, Medford RM. Angiotensin II induces monocyte chemoattractant protein-1 gene expression in rat vascular smooth muscle cells. Circ Res. 1998;83: 952-959.
24. Kranzhofer R, Schmidt J, Pfeiffer CA, Hagl S, Libby P, Kubler W. Angiotensin induces inflammatory activation of human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1999; 19: 1623-1629.
25. Schieffer B, Schieffer E, Hilfiker-Kleiner D, et al. Expression of angiotensin II and interleukin 6 in human coronary atherosclerotic plaques: potential implications for inflammation and plaque instability. Circulation. 2000;101: 1372-1378.
26. Weiss D, Sorescu D, Taylor WR. Angiotensin and atherosclerosis. Am J Cardiol. 2001;87(suppl): 25C-32C.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
27. Peters S, Gotting B, Trummel M, Rust H, Brattstrom A. Valsartan for prevention of restenosis after stenting of type B2/C lesions: the Val-PREST trial. J Invasive Cardiol. 2001;13: 93-97.[Medline] [Order article via Infotrieve]
28. Mehta JL, Lopez LM, Chen L, Cox OE. Alterations in nitric oxide synthase activity, superoxide anion generation, and platelet aggregation in systemic hypertension, and effects of celiprolol. Am J Cardiol. 1994;74: 901-905.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
29. Lacy F, O'Connor DT, Schmid-Schonbein GW. Plasma hydrogen peroxide production in hypertensive and normotensive subjects at genetic risk of hypertension. J Hypertens. 1998;16: 291-303.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
30. McCord JM. Human disease, free radicals, and the oxidant/antioxidant balance. Clin Biochem. 1993;26: 351-357.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
31. Kehrer JP. Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol. 1993;23: 21-48.[Web of Science][Medline] [Order article via Infotrieve]
32. Berry C, Hamilton CA, Brosnan MJ, et al. Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries. Circulation. 2000;101: 2206-2212.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
