Perindopril, a centrally active angiotensin-converting enzyme inhibitor, prevents cognitive impairment in mouse models of Alzheimer's disease.

The purpose of this work was to test whether brain-penetrating angiotensin-converting enzyme (ACE) inhibitors (e.g., perindopril), as opposed to non-brain-penetrating ACE inhibitors (e.g., enalapril and imidapril), may reduce the cognitive decline and brain injury in Alzheimer's disease (AD). We first compared the effect of perindopril, enalapril, and imidapril on cognitive impairment and brain injury in a mouse model of AD induced by intracerebroventricular (i.c.v.) injection of amyloid-ß (Aß)1₋40. Perindopril, with significant inhibition of hippocampal ACE, significantly prevented cognitive impairment in this AD mouse model. This beneficial effect was attributed to the suppression of microglia/astrocyte activation and the attenuation of oxidative stress caused by iNOS induction and extracellular superoxide dismutase down-regulation. In contrast, neither enalapril nor imidapril prevented cognitive impairment and brain injury in this AD mouse. We next examined the protective effects of perindopril on cognitive impairment in PS2APP-transgenic mice overexpressing Aß in the brain. Perindopril, without affecting brain Aß deposition, significantly suppressed the increase in hippocampal ACE activity and improved cognition in PS2APP-transgenic mice, being associated with the suppression of hippocampal astrocyte activation and attenuation of superoxide. Our data demonstrated that the brain-penetrating ACE inhibitor perindopril, as compared to non-brain-penetrating ACE inhibitors, protected against cognitive impairment and brain injury in experimental AD models.

Telmisartan protects against diabetic vascular complications in a mouse model of obesity and type 2 diabetes, partially through peroxisome proliferator activated receptor-γ-dependent activity.

Experimental and clinical data support the notion that peroxisome proliferator-activated receptor γ (PPARγ) activation is associated with anti-atherosclerosis as well as anti-diabetic effect. Telmisartan, an angiotensin receptor blocker (ARB), acts as a partial PPARγ agonist. We hypothesized that telmisartan protects against diabetic vascular complications, through PPARγ activation. We compared the effects of telmisartan, telmisartan combined with GW9662 (a PPARγ antagonist), and losartan with no PPARγ activity on vascular injury in obese type 2 diabetic db/db mice. Compared to losartan, telmisartan significantly ameliorated vascular endothelial dysfunction, downregulation of phospho-eNOS, and coronary arterial remodeling in db/db mice. More vascular protective effects of telmisartan than losartan were associated with greater anti-inflammatory effects of telmisartan, as shown by attenuation of vascular nuclear factor kappa B (NFκB) activation and tumor necrosis factor α. Coadministration of GW9662 with telmisartan abolished the above mentioned greater protective effects of telmisartan against vascular injury than losartan in db/db mice. Thus, PPARγ activity appears to be involved in the vascular protective effects of telmisartan in db/db mice. Moreover, telmisartan, but not losartan, prevented the downregulation of vascular PPARγ in db/db mice and this effect of telmisartan was cancelled by the coadministration of GW9662. Our data provided the first evidence indicating that PPARγ activity of telmisartan contributed to the protective effects of telmisartan against diabetic vascular complication. PPARγ activity of telmisartan was involved in the normalization of vascular PPARγ downregulation in diabetic mice. Thus, telmisartan seems to exert vascular protective effects in hypertensive patients with diabetes.

Beneficial effects of combination of valsartan and amlodipine on salt-induced brain injury in hypertensive rats.

The optimum antihypertensive treatment for prevention of hypertensive stroke has yet to be elucidated. This study was undertaken to examine the benefit of a combination of valsartan, an angiotensin II type 1 (AT1) receptor blocker, and amlodipine, a calcium channel blocker, in prevention of high-salt-induced brain injury in hypertensive rats. High-salt-loaded stroke-prone spontaneously hypertensive rats (SHRSPs) were given 1) vehicle, 2) valsartan (2 mg/kg/day), 3) amlodipine (2 mg/kg/day), or 4) a combination of valsartan and amlodipine for 4 weeks. The effects on brain injury were compared between all groups. High-salt loading in SHRSPs caused the reduction of cerebral blood flow (CBF), cerebral hypoxia, white matter lesions, glial activation, AT1 receptor up-regulation, endothelial nitric-oxide synthase (eNOS) uncoupling, inducible nitric-oxide synthase induction, and nitroxidative stress. Valsartan, independently of blood pressure, enhanced the protective effects of amlodipine against brain injury, white matter lesions, and glial activation in salt-loaded SHRSPs. These beneficial effects of valsartan added to amlodipine were associated with an additive improvement in CBF and brain hypoxia because of an additive improvement in cerebral arteriolar remodeling and vascular endothelial dysfunction. Furthermore, valsartan added to amlodipine enhanced the attenuation of cerebral nitroxidative stress through an additive suppression of eNOS uncoupling. Valsartan, independently of blood pressure, augmented the protective effects of amlodipine against brain injury in salt-loaded hypertensive rats through an improvement in brain circulation attributed to nitroxidative stress. Our results suggest that the combination of valsartan and amlodipine may be a promising strategy for the prevention of salt-related brain injury in hypertensive patients.

Nifedipine prevents vascular endothelial dysfunction in a mouse model of obesity and type 2 diabetes, by improving eNOS dysfunction and dephosphorylation.

The effect of calcium channel blockers (CCBs) on type 2 diabetes is still unclear. The present study was undertaken to examine the efficacy of nifedipine, a dihydropyridine CCB, on obesity, glucose intolerance and vascular endothelial dysfunction in db/db mice (a mouse model of obesity and type 2 diabetes). db/db mice, fed high-fat diet (HFD) were treated with vehicle, nifedipine (10 mg kg(-1) day(-1)) or hydralazine (5 mg kg(-1) day(-1)) for 4 weeks, and the protective effects were compared. Although nifedipine and hydralazine exerted similar blood pressure lowering in db/db mice, neither affected body weight, fat weight, and glucose intolerance of db/db mice. However, nifedipine, but not hydralazine, significantly improved vascular endothelial function in db/db mice, being accompanied by more attenuation of vascular superoxide by nifedipine than hydralazine. These protective effects of nifedipine were attributed to the attenuation of eNOS uncoupling as shown by the prevention of vascular endothelial nitric oxide synthase (eNOS) dimer disruption, and the prevention of dihydrofolate reductase (DHFR) downregulation, the key enzyme responsible for eNOS uncoupling. Moreover, nifedipine, but not hydralazine, significantly prevented the decreases in phosphorylation of vascular akt and eNOS in db/db mice. Our work provided the first evidence that nifedipine prevents vascular endothelial dysfunction, through the inhibition of eNOS uncoupling and the enhancement of eNOS phosphorylation, independently of blood pressure-lowering effect. We propose that nifedipine may be a promising therapeutic agent for cardiovascular complications in type 2 diabetes.

Ezetimibe ameliorates cardiovascular complications and hepatic steatosis in obese and type 2 diabetic db/db mice.

Type 2 diabetes plays a major role in the development of cardiovascular diseases. The present study was undertaken to investigate the effect of ezetimibe, a potent cholesterol absorption inhibitor, on cardiovascular injury of obese and type 2 diabetic db/db mice. Diabetic db/db mice fed a Western diet were given ezetimibe for 9 weeks, and the effects on cardiovascular injury and hepatic steatosis were examined. Ezetimibe treatment of db/db mice significantly improved vascular endothelial function, which was associated with the restoration of the decreased phospho-Akt and phospho-endothelial nitric-oxide synthase (eNOS). Moreover, ezetimibe also reduced vascular superoxide levels in db/db mice, accompanied by the attenuation of NADPH oxidase subunit gp91(phox) and Nox4 and the prevention of down-regulation of Cu/Zn-superoxide dismutase (SOD) and extracellular SOD. Thus, the improvement of vascular endothelial function by ezetimibe in diabetic mice seems to be attributed to the improvement of eNOS function and the attenuation of oxidative stress. Ezetimibe treatment also significantly attenuated cardiac interstitial fibrosis and coronary arterial thickening of diabetic mice and ameliorated cardiac macrophage infiltration. This improvement of cardiac injury was also related to the attenuation of NADPH oxidase-mediated oxidative stress. Furthermore, ezetimibe significantly prevented hepatic steatosis, inflammation, and oxidative stress in diabetic mice. Our work provides the first evidence that ezetimibe prevented cardiovascular injury and hepatic steatosis in diabetic mice. These beneficial effects were attributed to the attenuation of oxidative stress and inflammation and the improvement of eNOS function. Therefore, we propose that ezetimibe may be a promising therapeutic drug for obese and type 2 diabetes.

Protein S-guanylation by the biological signal 8-nitroguanosine 3',5'-cyclic monophosphate.

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.

Excess salt causes cerebral neuronal apoptosis and inflammation in stroke-prone hypertensive rats through angiotensin II-induced NADPH oxidase activation.

BACKGROUND AND PURPOSE: The precise mechanism of salt-induced brain injury is unclear. We examined the detailed causative role of angiotensin II and NADPH oxidase in salt-accelerated brain injury of stroke-prone spontaneously hypertensive rats (SHRSP). METHODS: We examined the effect of salt loading on brain reactive oxygen species (ROS), inflammation, and apoptosis in SHRSP. Salt-loaded SHRSP were given vehicle, valsartan (an angiotensin AT1 receptor blocker), or hydralazine to compare their efficacy on brain injury. We also examined the efficacy of apocynin (a NADPH oxidase inhibitor) on brain injury of salt-loaded SHRSP. RESULTS: Cerebral NADPH oxidase activity and ROS in SHRSP were already increased at 1 week after salt loading followed by the significant increase in ED-1-positive cells and neuronal apoptosis. Thus, cerebral NADPH oxidase activation preceded cerebral inflammation and neuronal apoptosis. Despite comparable hypotensive effects between valsartan and hydralazine in salt-loaded SHRSP, valsartan reduced cerebral NADPH oxidase activity and ROS more than hydralazine being accompanied by more prevention of stroke by valsartan than hydralazine. Valsartan, but not hydralazine, prevented neuronal apoptosis, being associated with the suppression of apoptosis signal-regulating kinase 1 activation by valsartan. Moreover, cerebral inflammation was also prevented by valsartan more than hydralazine, being associated with more suppression of monocyte chemotactic protein-1 and tumor necrosis factor-alpha expressions by valsartan. Thus, angiotensin II was directly involved in salt-induced neuronal NADPH oxidase activation, ROS, apoptosis, and inflammation in SHRSP. Apocynin attenuated the enhancement of ROS, cerebral inflammation, neuronal apoptosis, and apoptosis signal-regulating kinase 1 activation and prevented stroke in salt-loaded SHRSP, indicating the causative role of cerebral NADPH oxidase in salt-induced brain injury. CONCLUSIONS: We obtained the evidence that excess salt, through ROS produced by angiotensin II-activated NADPH oxidase, caused cerebral neuronal apoptosis and inflammation as well as stroke in SHRSP.

Pravastatin enhances beneficial effects of olmesartan on vascular injury of salt-sensitive hypertensive rats, via pleiotropic effects.

OBJECTIVE: This work was undertaken to investigate comparative effect of AT1 receptor blocker (ARB), 3-hydroxy-3-methylglutaryl (HMG) coenzymeA (CoA) reductase inhibitor (statin), and their combination on vascular injury of salt-sensitive hypertension. METHODS AND RESULTS: Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats) were treated with (1) vehicle, (2) hydralazine (5 mg/kg/d), (3) olmesartan (0.5 mg/kg/d), (4) pravastatin (100 mg/kg/d), and (5) combined olmesartan and pravastatin for 4 weeks. Olmesartan or pravastatin significantly and comparably improved vascular endothelium-dependent relaxation to acetylcholine, coronary arterial remodeling, and eNOS activity of DS rats. Olmesartan prevented vascular eNOS dimer disruption or the downregulation of dihydrofolate reductase (DHFR) more than pravastatin, whereas Akt phosphorylation was enhanced by pravastatin but not olmesartan, indicating differential pleiotropic effects between olmesartan and pravastatin. Add-on pravastatin significantly enhanced the improvement of vascular endothelial dysfunction and remodeling by olmesartan in DS rats. Moreover, pravastatin enhanced the increase in eNOS activity by olmesartan, being associated with additive effects of pravastatin on phosphorylation of Akt and eNOS. CONCLUSIONS: Olmesartan and pravastatin exerted beneficial vascular effects in salt-sensitive hypertension, via differential pleiotropic effects. Pravastatin enhanced vascular protective effects of olmesartan. Thus, the combination of ARB with statin may be the potential therapeutic strategy for vascular diseases of salt-sensitive hypertension.

Novel mechanism and role of angiotensin II induced vascular endothelial injury in hypertensive diastolic heart failure.

OBJECTIVE: The mechanism and role of angiotensin II-induced vascular endothelial injury is unclear. We examined the molecular mechanism of angiotensin (AII)-induced vascular endothelial injury and its significance for hypertensive diastolic heart failure. METHODS AND RESULTS: We compared the effect of valsartan and amlodipine on Dahl salt-sensitive hypertensive rats (DS rats). Valsartan improved vascular endothelial dysfunction of DS rats more than amlodipine, by inhibiting endothelial apoptosis and eNOS uncoupling more. Moreover, valsartan inhibited vascular apoptosis signal-regulating kinase 1 (ASK1) more than amlodipine. Thus, AT1 receptor contributed to vascular endothelial apoptosis, eNOS uncoupling, and ASK1 activation of DS rats. Using ASK1(-/-) mice, we examined the causative role of ASK1 in endothelial apoptosis and eNOS uncoupling. AII infusion in wild-type mice markedly caused vascular endothelial apoptosis and eNOS uncoupling accompanied by vascular endothelial dysfunction, whereas these effects of AII were absent in ASK1(-/-) mice. Therefore, ASK1 participated in AII-induced vascular endothelial apoptosis and eNOS uncoupling. Using tetrahydrobiopterin, we found that eNOS uncoupling was involved in vascular endothelial dysfunction in DS rats with established diastolic heart failure. CONCLUSIONS: AII-induced vascular endothelial apoptosis and eNOS uncoupling were mediated by ASK1 and contributed to vascular injury in diastolic heart failure of salt-sensitive hypertension.

Pioglitazone exerts protective effects against stroke in stroke-prone spontaneously hypertensive rats, independently of blood pressure.

BACKGROUND AND PURPOSE: Very recent subgroup analysis from the PROspective pioglitAzone Clinical Trial In macroVascular Events has shown that pioglitazone reduces the risk of recurrent stroke in type 2 diabetic patients. However, the underlying mechanism of stroke prevention by pioglitazone is unknown. Our aim was to examine the effect of pioglitazone on hypertension-based stroke in rats. METHODS: Pioglitazone (1 mg x kg(-1) x d(-1)) was orally administered to stroke-prone spontaneously hypertensive rats (SHRSP) to examine the effect on incidental stroke, cerebrovascular injury, brain inflammation, oxidative stress, and vascular endothelial dysfunction induced by hypertension. RESULTS: Treatment of SHRSP with pioglitazone for 4 weeks, without affecting blood pressure and blood glucose values, improved vascular endothelial dysfunction (P<0.05), suppressed remodeling of the middle cerebral artery (P<0.05) and brain microvessels (P<0.05), and inhibited brain macrophage infiltration (P<0.05) and the upregulation of brain monocyte chemoattractant protein-1 and tumor necrosis factor-alpha expression (P<0.01). Furthermore, pioglitazone treatment significantly delayed the onset of stroke signs and death in SHRSP (P<0.05). These beneficial effects of pioglitazone on cerebrovascular injury and stroke in SHRSP were associated with a reduction of brain and vascular superoxide via the inhibition of NADPH oxidase activity. CONCLUSIONS: Our work provides the first evidence that pioglitazone significantly protects against hypertension-induced cerebrovascular injury and stroke by improving vascular endothelial dysfunction, inhibiting brain inflammation, and reducing oxidative stress. These beneficial effects of pioglitazone were independent of blood pressure or blood sugar values. Thus, pioglitazone appears to be a potential therapeutic agent for stroke in type 2 diabetes with hypertension.

Oxidation of Prx2 and phosphorylation of GRP58 by angiotensin II in human coronary smooth muscle cells identified by 2D-DIGE analysis.

To study early changes in angiotensin II (Ang II)-induced signaling with post-translational modifications, we analyzed proteins from cultured human coronary smooth muscle cells stimulated with Ang II, using two-dimensional difference gel electrophoresis (2D-DIGE) combined with ProQ Diamond and SYPRO Ruby staining, followed by mass spectrometry or Western blotting. Among 40 proteins identified, peroxiredoxin 2 (Prx2) was oxidized and 58 kDa glucose-regulated protein (GRP58) was phosphorylated after 5 min of Ang II (1 microM) stimulation. Valsartan, a selective Ang II type 1 (AT1) receptor blocker, and N-acetylcysteine, an antioxidant, inhibited both of these modifications, indicating the contribution of AT1 receptor and reactive oxygen species to oxidation of Prx2 and phosphorylation of GRP58 by Ang II.

Enhancement of cardiac oxidative stress by tachycardia and its critical role in cardiac hypertrophy and fibrosis.

OBJECTIVE: To examine the mechanism and significance of tachycardia-induced cardiac damage, using azelnidipine, a relatively new dihydropyridine calcium channel blocker which does not increase heart rate. METHODS: Comparing azelnidipine and amlodipine, we examined the cardiac effects and the direct effects on a sinus node/atrial preparation in stroke-prone spontaneously hypertensive rats (spSHRs). By pacing the right atrium, we examined the effect of tachycardia per se on cardiac oxidative stress. Using apocynin, a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, we investigated the role of oxidative stress in cardiac remodelling. RESULTS: Azelnidipine suppressed cardiac hypertrophy, fibrosis, NADPH oxidase and superoxide in spSHRs more potently than amlodipine, and was associated with lower heart rates than amlodipine. Azelnidipine caused a greater reduction than amlodipine in the beat rate of the sinus node/atrial preparation of spSHRs. A 10 or 20% increase in heart rate, independent of blood pressure or sympathetic nerve activity, significantly enhanced cardiac NADPH oxidase activity, superoxide and activated mitogen-activated protein kinases. Reduction of cardiac oxidative stress by apocynin led to the suppression of cardiac hypertrophy, inflammation and fibrosis in spSHRs, beyond its hypotensive effect. CONCLUSIONS: Our work provided evidence that the increase in heart rate per se, independent of sympathetic nerve activity, enhances cardiac oxidative stress and activates mitogen-activated protein kinases, which seem to be responsible for cardiac remodelling. Azelnidipine, without causing an increase in heart rate, has the potential to be useful for the treatment of cardiac remodelling.

Effects of alpha(1)-acid glycoprotein on isometric tension of mouse aorta.

We examined the effects of human alpha(1)-acid glycoprotein on isometric tension of mouse aortic rings. alpha(1)-Acid glycoprotein (7.5-75 microM) produced a transient, concentration-dependent relaxation of the phenylephrine-precontracted preparation. Although N(G)-nitro-L-arginine methyl ester or removal of endothelium rarely affected the alpha(1)-acid glycoprotein-induced relaxation, extracellular heparin inhibited the alpha(1)-acid glycoprotein-induced relaxation. In 10 mM Ca(2+)-containing external solutions, the alpha(1)-acid glycoprotein-induced relaxation was significantly potentiated. In the 60 mM KCl-precontracted preparation, alpha(1)-acid glycoprotein produced weaker relaxation than in the phenylephrine-precontracted preparation. These results suggest that the vasorelaxant effect of alpha(1)-acid glycoprotein is mainly achieved by block of Ca(2+) entry in the vascular smooth muscle cells. The interaction between alpha(1)-acid glycoprotein molecules and plasmalemmal Ca(2+) entry channels may be modified by extracellular Ca(2+) and heparin.

Apoptosis signal-regulating kinase 1 deficiency eliminates cardiovascular injuries induced by high-salt diet.

OBJECTIVES: High-salt diet is closely associated with the increase in cardiovascular events. However, the mechanism of high-salt-induced cardiovascular injury is unknown. The present study was undertaken to test our hypothesis that apoptosis signal-regulating kinase (ASK) 1 may be involved in salt-induced cardiovascular injury. METHODS: Wild-type and ASK1-/- mice were fed a low-salt or a high-salt diet for 10 weeks and the effects of high-salt diet on food intake, urinary volume and electrolyte excretion, and cardiovascular injury were compared between both groups of mice. RESULTS: High-salt diet in wild-type and ASK1-/- mice similarly increased food intake, water intake, urine volume, and urinary sodium excretion, and comparably decreased plasma renin activity and aldosterone. Thus, ASK1 appears to play a minor role in the increase in natriuresis and the decrease in plasma renin, and aldosterone caused by high-salt diet. High-salt diet enhanced the phosphorylation of cardiovascular ASK1 in wild-type mice. High-salt diet in wild-type mice enhanced cardiac transforming growth factor-ß1, interstitial fibrosis, coronary perivascular fibrosis, and inflammatory cell infiltration, and these changes were associated with the increase in cardiac superoxide and Nox2. ASK1 deficiency abolished the above-mentioned high-salt-induced cardiac injury. High-salt diet also caused the impairment of vascular endothelium-dependent relaxation by acetylcholine and increased vascular superoxide, and Nox2 in wild-type mice, whereas it did not cause vascular injury in ASK1-/- mice. CONCLUSION: ASK1 is implicated in cardiac inflammation and fibrosis, and vascular endothelial dysfunction caused by high-salt diet, through the enhancement of oxidative stress.

Novel mechanism of salt-induced glomerular injury: critical role of eNOS and angiotensin II.

OBJECTIVES: The present study was undertaken to examine the role of endothelial nitric oxide synthase (eNOS) in salt-sensitive renal injury. METHODS: The effects of high-salt diet on renal injury were compared between wild-type and eNOS-/- mice. To examine the role of glomerular angiotensin II and oxidative stress, high-salt fed eNOS-/- mice were given irbesartan, an angiotensin receptor blocker, or tempol, an antioxidant. RESULTS: Four weeks of high-salt diet in wild-type mice, which rapidly caused glomerular eNOS activation and subsequent increase in nitric oxide, did not at all induce renal injury, indicating that wild-type mice are salt-resistant. On the contrary, high-salt diet in eNOS-/- mice, which little increased nitric oxide, rapidly increased urinary albumin excretion, followed by glomerular macrophage infiltration and glomerular sclerosis. Thus, eNOS deficiency caused salt-sensitive glomerular injury. Salt-induced glomerular injury in eNOS-/- mice was preceded by rapid enhancement of glomerular superoxide followed by enhancement of glomerular endothelial angiotensinogen and angiotensin II. Irbesartan and tempol, independently of blood pressure, markedly prevented salt-induced glomerular injury in eNOS-/- mice, and these protective effects were attributed to the attenuation of glomerular oxidative stress and glomerular angiotensinogen-derived angiotensin II. CONCLUSION: We propose that eNOS dysfunction plays a causative role in salt-induced glomerular injury, through augmentation of glomerular oxidative stress-induced angiotensinogen.

Vascular responses to 8-nitro-cyclic GMP in non-diabetic and diabetic mice.

BACKGROUND AND PURPOSE: 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), formed nitric oxide (NO)-dependently, is a physiological second messenger, yet little is known about its role in the pathophysiology of vascular diseases. To study the pharmacological activity of 8-nitro-cGMP in diabetic mice, we compared its effects on vascular reactivity of aortas from non-diabetic and diabetic mice. EXPERIMENTAL APPROACH: Vascular tension recording was performed in thoracic aortic rings from wild-type (C57BL/6), non-diabetic db/+ and obese/diabetic db/db mice. Endothelial NO synthase (eNOS) uncoupling and superoxide were tested by Western blot and dihydroethidium fluorescence respectively. KEY RESULTS: 8-Nitro-cGMP, at concentrations up to 10 µM, enhanced phenylephrine-induced contractions in aortas from C57BL/6 and db/+ mice, but not from db/db mice. This enhancement was not observed with 8-bromo-cGMP. Pretreatment of aortas from C57BL/6 and db/+ mice with l-NAME (100 µM), superoxide dismutase (100 U·mL(-1) ) or tiron (1 mM), abolished 8-nitro-cGMP-induced enhancement of the phenylephrine contraction. In 8-nitro-cGMP (10 µM)-treated C57BL/6 aortas, eNOS dimer/monomer ratio was significantly decreased and vascular superoxide production increased, suggesting that 8-nitro-cGMP-induced superoxide production via eNOS uncoupling may mediate the enhancement of the phenylephrine contraction. At higher concentrations (>10 µM), 8-nitro-cGMP produced relaxation of the phenylephrine-contracted aortas from C57BL/6, db/+ and db/db mice. The 8-nitro-cGMP-induced relaxation in db/db mouse aortas was found to be resistant to a phosphodiesterase 5 inhibitor, zaprinast (1 µM). CONCLUSIONS AND IMPLICATIONS: The vasodilator effect of 8-nitro-cGMP may contribute to amelioration of the vascular endothelial dysfunction in diabetic mice, representing a novel pharmacological approach to prevent the complications associated with diabetes.

Eplerenone potentiates protective effects of amlodipine against cardiovascular injury in salt-sensitive hypertensive rats.

The clinical value of the combination of amlodipine and eplerenone is unclear. This study was undertaken to test whether eplerenone potentiates the protective effects of amlodipine against hypertensive cardiovascular injury. Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats) were given (1) vehicle, (2) an antihypertensive dose of amlodipine, (3) a non-antihypertensive dose of eplerenone or (4) combined amlodipine and eplerenone for 6 weeks, and the effects on cardiovascular injuries were compared. There was no significant difference among the four groups regarding plasma aldosterone, urine volume or urinary electrolytes. A subpressor dose of eplerenone markedly ameliorated vascular endothelial dysfunction, cardiac inflammation and fibrosis in DS rats to a similar degree as an antihypertensive dose of amlodipine. Addition of eplerenone to amlodipine, without affecting blood pressure, enhanced the improvement by amlodipine of vascular endothelial function, cardiac inflammation, fibrosis and diastolic dysfunction in DS rats. Additive beneficial effects of eplerenone were attributed to additive potentiation of eNOS and Akt phosphorylation and additive reduction of oxidative stress. Eplerenone significantly attenuated cardiovascular NADPH oxidase activity by reducing gp91(phox) upregulation and attenuated the upregulation of cardiovascular AT1 receptor, but amlodipine failed to affect them. Thus, the normalization by eplerenone of gp91(phox) and AT1 receptor upregulation seems to be at least partially responsible for the additive benefits of eplerenone in the prevention of hypertensive cardiovascular injury. The combination of amlodipine and eplerenone may be a promising therapeutic strategy for cardiovascular disease in salt-sensitive hypertension.

Potentiation by candesartan of protective effects of pioglitazone against type 2 diabetic cardiovascular and renal complications in obese mice.

OBJECTIVES: The efficacy of renin-angiotensin system (RAS) blockers on type 2 diabetes and its complications remains to be defined. This study was undertaken to test the hypothesis that candesartan may enhance the protective effects of pioglitazone against type 2 diabetes. METHODS: We compared the effects of pioglitazone, candesartan, and their combination on cardiorenal and vascular injury, diabetes, and tissue oxidative stress in obese and type 2 diabetic db/db mice, and also examined the effects of tempol, a superoxide dismutase (SOD) mimetic, on db/db mice to define the role of oxidative stress. RESULTS: The addition of candesartan to pioglitazone significantly potentiated the suppressive effects of pioglitazone on cardiac macrophage infiltration and interstitial fibrosis, and glomerular macrophage infiltration and sclerosis in db/db mice. These benefits of the combination of pioglitazone and candesartan in db/db mice were attributed to additive attenuation of cardiorenal oxidative stress, through the attenuation of NADPH oxidase or the restoration of Cu/Zn-SOD and EC-SOD. The combination of these drugs reversed vascular endothelial dysfunction in db/db mice more than either monotherapy, by causing more phosphorylation of eNOS. Candesartan slightly augmented the improvement of glucose tolerance by pioglitazone in db/db mice, and this additive effect was mediated by more attenuation of oxidative stress. CONCLUSIONS: Our work demonstrated that candesartan significantly potentiated the protective effects of pioglitazone against cardiorenal and vascular injury, and diabetes in obese type 2 diabetic mice. Thus, the combination of pioglitazone with candesartan is potentially a promising therapeutic strategy for type 2 diabetes.

Aliskiren enhances protective effects of valsartan against type 2 diabetic nephropathy in mice.

OBJECTIVES: Addition of aliskiren, a direct renin inhibitor, to losartan provides additive reduction of urinary albumin excretion in type 2 diabetic patients. However, the detailed effect of aliskiren on type 2 diabetic nephropathy is still unknown. This study was undertaken to examine the efficacy of aliskiren and the combination of aliskiren with valsartan on type 2 diabetic nephropathy. METHODS: db/db mice were treated with aliskiren (3 mg/kg per day), valsartan (5 or 10 mg/kg per day), combined aliskiren (3 mg/kg per day) and valsartan (5 mg/kg per day), and hydralazine (80 mg/kg per day), for 6 weeks, and the protective effects against diabetic nephropathy were compared among each group. RESULTS: Aliskiren significantly attenuated albuminuria and glomerular mesangial matrix expansion in db/db mice, which was associated with the improvement of the increased glomerular transforming growth factor-beta and type IV collagen expressions, the increased macrophage infiltration, and the decreased glomerular nephrin expression of db/db mice. These protective effects of aliskiren in db/db mice were attributed to the attenuation of p22(phox)-related nicotinamide adenine dinucleotide phosphate oxidase-induced superoxide. Addition of aliskiren to valsartan treatment provided more beneficial effects on all the above-mentioned parameters than valsartan monotherapy. CONCLUSION: Aliskiren protected against type 2 diabetic nephropathy, through pleiotropic effects, and significantly enhanced the protective effects of valsartan against diabetic nephropathy in db/db mice.

Critical role of apoptosis signal-regulating kinase 1 in aldosterone/salt-induced cardiac inflammation and fibrosis.

The molecular mechanism underlying aldosterone/salt-induced cardiovascular injury remains to be defined. This work was undertaken to determine the role of apoptosis signal-regulating kinase 1 (ASK1) in the mechanism underlying aldosterone-induced cardiac injury in vivo. We compared the in vivo effects of 4 weeks of aldosterone/salt treatment on wild-type and ASK1-deficient mice. Aldosterone infusion plus high salt intake in wild-type mice significantly increased blood pressure and urinary albumin excretion and decreased plasma potassium concentrations, and these effects of aldosterone/salt were not affected by ASK1 deficiency. Thus, ASK1 seems to play a minor role in aldosterone-induced hypertension and renal injury. ASK1 deficiency also failed to affect aldosterone-induced cardiac hypertrophy. However, ASK1 deficiency markedly ameliorated aldosterone-induced cardiac injury, eg, the enhancement of cardiac macrophage infiltration, monocyte chemotactic protein 1 expression, interstitial fibrosis, perivascular fibrosis, and transforming growth factor-beta1 and collagen type I expressions. Thus, ASK1 participates in aldosterone-induced cardiac inflammation and fibrosis. Furthermore, the enhancement of NADPH oxidase-mediated cardiac oxidative stress caused by aldosterone infusion was markedly lessened by ASK1 deficiency, which was associated with the significant amelioration by ASK1 deficiency of aldosterone-induced cardiac Nox2 upregulation. Furthermore, aldosterone/salt treatment significantly enhanced cardiac expression of the angiotensin-converting enzyme and angiotensin II type 1 receptor in wild-type mice, whereas the enhancement of these proteins by aldosterone/salt was abolished by ASK1 deficiency. Our results demonstrate that ASK1 is implicated in aldosterone/salt-induced cardiac inflammation and fibrosis through the enhancement of NADPH oxidase-mediated oxidative stress and the upregulation of the cardiac renin-angiotensin system.