Hiroshi Sakonjo

Eplerenone Inhibits Atherosclerosis in Nonhuman Primates

Aldosterone may be involved in the pathogenesis of atherosclerosis. We investigated the effect of eplerenone, a selective mineralocorticoid receptor blocker, on atherosclerosis in monkeys fed a high-cholesterol diet. Monkeys fed a high-cholesterol diet for 9 months were divided into 3 groups: those treated with a low dose of eplerenone (30 mg/kg per day); those treated with a high dose of eplerenone (60 mg/kg per day); and the placebo-treated group. The normal group consisted of monkeys fed a normal diet. There were no significant differences in blood pressure and cholesterol levels between the placebo- and eplerenone-treated groups. On the other hand, monocyte chemoattractant protein-1 and malondialdehyde-modified LDL were significantly higher in the placebo-treated group than in the normal group, whereas they were suppressed in the eplerenone-treated groups. The ratio of intimal volume to total volume by intravascular ultrasound analysis imaging of the aortas was dose-dependently lower in the eplerenone-treated groups than in the placebo-treated group. Acetylcholine-induced vasorelaxation was significantly weaker in the placebo-treated group than in the normal group, but the vasorelaxation was strengthened in the eplerenone-treated groups. A significant upregulation of angiotensin-converting enzyme activity was observed in the placebo-treated group, but the activity was suppressed in the eplerenone-treated groups. In conclusion, eplerenone may strengthen the endothelium-dependent relaxation and suppress angiotensin-converting enzyme activity in the vasculature, thus preventing the development of atherosclerosis in nonhuman primates.

Activation of two angiotensin-generating systems in the balloon-injured artery

Participation of angiotensin II in the myointimal proliferation following a vascular injury was postulated. This study assessed the potential involvement of the local angiotensin II‐forming enzymes in injured arteries of dogs. The potential angiotensin II‐forming enzymes are angiotensin‐converting enzyme (ACE) and chymostatin‐sensitive angiotensin II‐generating enzyme (CAGE) which is highly homologous to or could be identical to the mast cell chymase. Both ACE and CAGE catalyze the conversion of angiotensin I to angiotensin II. We found that the enzymatic activities of ACE and CAGE, and the mRNA levels of ACE and chymase were increased in the injury‐induced hypertrophied vessels. The results suggest that ACE and CAGE participate in the hypertrophy through the production of angiotensin II which is a growth promoter for vascular smooth muscle cells.

Anti-atherosclerotic effects of an angiotensin converting enzyme inhibitor and an angiotensin II antagonist in Cynomolgus monkeys fed a high-cholesterol diet

We investigated the relationship between angiotensin II formation and the development of atherosclerotic lesions in the aorta of monkeys (Macaca fascicularis) fed a high‐cholesterol (4% cholesterol and 6% corn oil) diet for 6 months, and studied the effects of an angiotensin converting enzyme (ACE) inhibitor, trandolapril (10 mg kg−1 per day, p.o.), and an angiotensin II type 1 receptor antagonist, 2‐butyl‐4‐(methylthio)‐1‐[[2′[[[(propylamino)carbonyl]amino]sulfonyl](1,1′‐biphenyl)‐4‐yl]methyl]‐1H‐imidazole‐5‐carboxylate (HR 720; 20 mg kg−1 per day, p.o.). The level of low‐density lipoprotein was significantly increased by the cholesterol diet, whereas that of high‐density lipoprotein was significantly decreased. The relative areas of the atherosclerotic lesions in the thoracic aorta in the normal and cholesterol‐diet groups were 1.3±0.3 and 64±10%, respectively. Plasma renin and ACE activities showed no differences between the normal and cholesterol‐diet groups. ACE activity and the concentration of angiotensin II were significantly increased in the aorta of the cholesterol‐fed monkeys. Trandolapril and HR 720 decreased significantly the area of the atherosclerotic lesions in the thoracic aorta of cholesterol‐fed monkeys, but not the mean blood pressure and the levels of low‐density and high‐density lipoproteins. In plasma and aorta, trandolapril, but not HR 720, decreased significantly the ACE activities in the cholesterol‐fed monkeys, while both of these drugs decreased significantly the angiotensin II levels. In conclusion, blockade of angiotensin II function in vascular tissues by trandolapril or HR 720 may play an important role in preventing the development of atherosclerotic lesions.

Tranilast Suppresses Vascular Chymase Expression and Neointima Formation in Balloon-Injured Dog Carotid Artery

BACKGROUND Activation of vascular chymase plays a major role in myointimal hypertrophy after vascular injury by augmenting the production of angiotensin (ANG) II. Because chymase is synthesized mainly in mast cells, we assumed that the chymase-dependent ANG II formation could be downregulated by tranilast, a mast cell-stabilizing antiallergic agent. We have assessed inhibitory effects of tranilast on neointima formation after balloon injury in the carotid artery of dogs, which share a similar ANG II-forming chymase with humans, and further explored the pathophysiological significance of vascular chymase. METHODS AND RESULTS Either tranilast (50 mg/kg BID) or vehicle was orally administered to beagles for 2 weeks before and 4 weeks after balloon injury. Four weeks after the injury, remarkable neointima was formed in the carotid arteries of vehicle-treated dogs. Chymase mRNA levels and chymaselike activity of vehicle-treated injured arteries were increased 10.2- and 4.8-fold, respectively, those of uninjured arteries. Angiotensin-converting enzyme (ACE) activity was slightly increased in the injured arteries, whereas ACE mRNA levels were not. Tranilast treatment completely prevented the increase in chymaselike activity, reduced the chymase mRNA levels by 43%, and decreased the carotid intima/media ratio by 63%. In vehicle-treated injured arteries, mast cell count in the adventitia showed a great increase, which was completely prevented by the tranilast treatment. Vascular ACE activity and mRNA levels were unaffected by tranilast. CONCLUSIONS Tranilast suppressed chymase gene expression, which was specifically activated in the injured arteries, and prevented neointima formation. Suppression of the chymase-dependent ANG II-forming pathway may contribute to the beneficial effects of tranilast.

Involvement of Angiotensin II-Dependent Vascular Endothelial Growth Factor Gene Expression via NADPH Oxidase in the Retina in a Type 2 Diabetic Rat Model

Purpose: To clarify the involvement of angiotensin II-dependent vascular endothelial growth factor (VEGF) via NADPH oxidase in the retina in spontaneously diabetic Torii (SDT) rats, a type 2 diabetic rat model. In SDT rats, the plasma glucose level and angiotensin-converting enzyme (ACE) levels were measured, and effects of angiotensin II receptor blocker (ARB) and angiotensin II were also studied. Materials and Methods: We evaluated the age-dependent changes in the peripheral and ocular angiotensin II-forming systems in SDT rats at 15 (n = 8), 20 (n = 8), 30 (n = 7), and 50 weeks of age (n = 8). We also evaluated the effect of an ARB (2.5 mg/kg/day candesartan) or angiotensin II (500 ng/kg/min) on retinal gene expressions of VEGF and p22phox, a subunit of NADPH oxidase. Results: The plasma glucose level was significantly increased from 20 weeks of age. No significant changes in ACE activities in the plasma, aorta, and eye were observed until 30 weeks of age. At 50 weeks, ACE activity in the eyes was significantly increased, whereas ACE activities in the plasma and aorta were not. At 50 weeks, significant increases in VEGF and p22phox, an NADPH oxidase subunit, were significantly reduced by candesartan. Angiotensin II infusion resulted in significant increases in VEGF and p22phox levels. Conclusions: Angiotensin II is involved in the gene expression of VEGF via NADPH oxidase in the retina of SDT rats.

Mechanisms of angiotensin II type 1 receptor blocker for anti-atherosclerotic effect in monkeys fed a high-cholesterol diet.

Objective To clarify the mechanism of the anti-atherosclerotic effect of angiotensin II type 1 receptor blocker (ARB) in primates, we investigated whether an ARB (CS-866) affects the serum markers of inflammation and growth factors, and the endothelial function in monkeys fed a high-cholesterol diet. Design Monkeys fed a high-cholesterol diet for 6 months were divided into two groups: one group was given an ARB, CS-866 (10 mg/kg per day), and the other group was not. The control group was fed a normal diet. Results Blood pressure and the plasma cholesterol level were not affected by CS-866. Plasma levels of angiotensin II, renin, angiotensin converting enzyme and chymase were not changed by the high-cholesterol diet, whereas vascular angiotensin converting enzyme, but not chymase, was significantly increased. Serum levels of macrophage-colony stimulating factor, transforming growth factor-β1 and intracellular adhesion molecule-1 were significantly increased in monkeys fed a high-cholesterol diet but they were suppressed by CS-866. The relaxation response of isolated carotid arteries to acetylcholine was suppressed in the high-cholesterol group, whereas it was improved by CS-866. Conclusions CS-866 reduced lipid deposition along with the suppression of serum macrophage-colony stimulating factor, transforming growth factor-β1 and intracellular adhesion molecule-1, and the improvement of vascular functions, suggesting that ARB has multiple mechanisms for reducing lipid deposition in primates.

Inhibition of Vascular Angiotensin-Converting Enzyme by Telmisartan via the Peroxisome Proliferator-Activated Receptor γ Agonistic Property in Rats

The angiotensin receptor blocker (ARB) telmisartan is a partial agonist of peroxisome proliferator–activated receptor γ (PPARγ). Typical PPARγ agonists suppress the gene expression of angiotensin-converting enzyme (ACE) in vascular tissues. However, it remains unclear whether or not PPARγ activation by telmisartan can inhibit vascular ACE activity. We compared the effects of PPARγ agonistic telmisartan and non-agonistic valsartan on ACE, vascular function and oxidative stress in stroke-prone spontaneously hypertensive rats (SHR-SP) and in sodium (1% NaCl)-loaded SHR-SP. SHR-SP and sodium-loaded SHR-SP received placebo, 1 mg/kg telmisartan, or 10 mg/kg valsartan for 2 weeks. Systolic blood pressure (SBP) was equally reduced in SHR-SP given either telmisartan or valsartan compared with SHR-SP given placebo. However, neither telmisartan nor valsartan suppressed SBP in sodium-loaded SHR-SP. Acetylcholine induced significantly less vasorelaxation in SHR-SP than in Wistar-Kyoto rats, but telmisartan and valsartan each significantly prevented such vasorelaxation. However, telmisartan significantly attenuated acetylcholine-induced vasorelaxation in sodium-loaded SHR-SP, whereas valsartan did not. Telmisartan significantly attenuated NADPH oxidase subunit p22phox gene expression in both SHR-SP and sodium-loaded SHR-SP, whereas valsartan did not. Likewise, telmisartan also significantly attenuated the significantly increased vascular ACE activity in sodium-loaded SHR-SP, whereas valsartan did not. In conclusion, the partial PPARγ agonist telmisartan might inhibit vascular ACE activity, and result in the prevention of oxidative stress and endothelial dysfunction more effectively than non-agonistic valsartan.

Significance of angiotensin II receptor blockers with high affinity to angiotensin II type 1 receptors for vascular protection in rats.

Angiotensin II receptor blockers (ARBs) vary in their binding affinities to angiotensin II type 1 (AT1) receptors in in vitro experiments. We compared a high-affinity ARB, olmesartan, and a low-affinity ARB, valsartan, in terms of their vascular protective effects in stroke-prone spontaneously hypertensive rats (SHR-SP). Blood pressure was equally reduced by placebo, olmesartan (1 mg kg−1) and valsartan (3 mg kg−1) daily for 2 weeks. In another experiment, 12-week-old SHR-SP were fed 8% salt, and olmesartan (1 mg kg−1), valsartan (3 mg kg−1) or placebo were administered daily until a survival rate of 60% was reached. In the experiment using SHR-SP, the reduction of acetylcholine-induced vascular relaxation and the increase of p22phox expression in the placebo-treated group were significantly attenuated by olmesartan and valsartan, but this attenuation was significantly greater for olmesartan. In immunohistological analysis, all areas positive for angiotensin II, p22phox and 4-hydroxy-2-nonenal were significantly reduced by olmesartan and valsartan, but again this reduction was significantly greater for olmesartan. In salt-loaded SHR-SP, the number of days to reach a 60% survival rate was 25 and 42 in placebo and valsartan-treated rats, respectively, and this represented a significant difference. The survival rate in olmesartan-treated rats was 95% at day 42, when valsartan-treated rats reached 60% survival, and this difference was also significant. In the surviving rats, olmesartan, but not valsartan, augmented acetylcholine-induced vascular relaxation and attenuated vascular p22phox expression. Thus, heterogeneity in binding affinity to AT1 receptors among ARBs may result in different degrees of vascular protection and lifespan extension.

Candesartan and amlodipine combination therapy provides powerful vascular protection in stroke-prone spontaneously hypertensive rats

The vascular protective effects of placebo, candesartan (1 mg kg−1 per day) monotherapy, candesartan (1 mg kg−1 per day) and amlodipine (1 mg kg−1 per day) combination therapy, and candesartan (1 mg kg−1 per day) and hydrochlorothiazide (HCTZ) (10 mg kg−1 per day) combination therapy for 2 weeks were compared in stroke-prone, spontaneously hypertensive rats. Candesartan monotherapy significantly reduced blood pressure, and both combination therapies were equally and significantly lower than the monotherapy. Acetylcholine-induced vascular relaxation was significantly stronger in all therapeutic groups than in the placebo-treated group. Furthermore, the relaxation was significantly stronger in the candesartan plus amlodipine-treated group than in the candesartan-treated group; however, there was no significant difference between the candesartan- and candesartan plus HCTZ-treated groups. Vascular gene expressions of the NADPH oxidase subunits p22phox, gp91phox, NOX1 and NOX4 were significantly attenuated in all therapeutic groups compared with the placebo-treated group, and there were no significant differences among those groups. However, a significant augmentation of vascular superoxide dismutase activity was observed in the candesartan plus amlodipine-treated group, but not in other groups. Malondialdehyde levels in the vascular tissues were significantly attenuated in all therapeutic groups. Compared with the candesartan-treated group, significant attenuation was observed in the candesartan plus amlodipine-treated group, but not in the candesartan plus HCTZ-treated group. Immunohistological analysis showed that areas positive for 4-hydroxy-2-nonenal were significantly reduced in all therapeutic groups, but this reduction was significantly greater for the candesartan plus amlodipine-treated group than for the candesartan-treated group. Thus, candesartan and amlodipine combination therapy could have a powerful protective effect in vascular tissues via the reduction of oxidative stress.

Combination therapy with irbesartan and efonidipine for attenuation of proteinuria in Dahl salt-sensitive rats

Angiotensin receptor blockers (ARBs) or T- and L-type calcium channel blockers (CCBs) are useful for glomerular protection; however, the protective effects of combination therapy remain unclear. In this study, Dahl salt-sensitive rats were fed a high-salt diet and were treated daily with placebo, irbesartan (60 mg kg−1), efonidipine (30 mg kg−1), irbesartan (60 mg kg−1)+efonidipine (30 mg kg−1), amlodipine (3 mg kg−1), or irbesartan (60 mg kg−1)+amlodipine (3 mg kg−1) for 4 weeks. Significant reductions in systolic blood pressure were seen in the irbesartan-, efonidipine- and amlodipine-treated groups compared with the placebo-treated group; a further significant reduction was seen in the irbesartan+efonidipine-treated group compared with the irbesartan-treated group. Compared with the placebo-treated group, proteinuria was significantly lower in the irbesartan- and efonidipine-treated groups, but not in the amlodipine-treated group. Furthermore, a significant attenuation of proteinuria in the irbesartan+efonidipine-treated group compared with the irbesartan-treated group was observed; this effect was not observed in the irbesartan+amlodipine-treated group. The glomerulosclerosis index was significantly attenuated by all active treatments except amlodipine. The glomerulosclerosis index in the irbesartan+efonidipine-treated group, but not in the irbesartan+amlodipine-treated group, was significantly lower than that in the irbesartan-treated group. Significant attenuations of gene expressions of p22phox, transforming growth factor-β, monocyte chemoattractant protein-1 and collegen I were observed in the irbesartan- and efonidipine-treated groups, but not in the amlodipine-treated group. Values for these parameters were reduced to control levels in the irbesartan+efonidipine-treated group. Combination therapy with ARB and T- and L-type CCB might produce a powerful renal protective effect.