Masaya Fukuda

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-&agr; 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. Conclusion— 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.

Aliskiren Enhances the Protective Effects of Valsartan Against Cardiovascular and Renal Injury in Endothelial Nitric Oxide Synthase–Deficient Mice

The protective effect of aliskiren, a direct renin inhibitor, against hypertensive cardiovascular and renal injury remains to be defined. This study was undertaken to examine the protective effects of the combination of aliskiren and valsartan, an angiotensin receptor blocker, against cardiovascular and renal injury. Endothelial NO synthase–deficient mice, subjected to cuff injury of femoral artery, were divided into 5 groups and were treated with the following: (1) vehicle; (2) aliskiren (25 mg/kg per day); (3) valsartan (8 mg/kg per day); (4) combined aliskiren (12.5 mg/kg per day) and valsartan (4 mg/kg per day); and (5) hydralazine (10 mg/kg per day) for 4 weeks. Aliskiren and valsartan alone markedly and similarly suppressed cardiac hypertrophy, inflammation and fibrosis, and coronary remodeling; prevented cuff injury–induced arterial intimal thickening; and reduced urinary albumin excretion, glomerular inflammation, and glomerulosclerosis in endothelial NO synthase–deficient mice. These beneficial effects of aliskiren and valsartan were associated with the significant attenuation of oxidative stress in these tissues. Hence, aliskiren and valsartan markedly exert the protective effects against cardiovascular and renal injury through the reduction of oxidative stress. Furthermore, compared with monotherapy with aliskiren or valsartan, the combination of a half dose of these drugs more greatly improved the above-mentioned cardiovascular and renal injuries of endothelial NO synthase–deficient mice, which were associated with greater attenuation of tissue oxidative stress by the combination therapy. Thus, the combination of aliskiren and valsartan exerts the synergistic organ-protective effects through synergistic attenuation of oxidative stress. The combination of aliskiren and valsartan seems to be a promising therapeutic strategy for hypertensive organ injury caused by endothelial NO synthase dysfunction.

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-&bgr;1 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.

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.

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 gp91phox 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.

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-β 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 p22phox-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.

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.

Benidipine, a dihydropyridine L-type/T-type calcium channel blocker, affords additive benefits for prevention of cardiorenal injury in hypertensive rats.

Objectives Benidipine is a dihydropyridine calcium channel blocker inhibiting not only L-type but also T-type calcium channels. To elucidate potential additive benefit of benidipine for prevention of cardiorenal injury, we compared the cardiac and renal protective effects of equihypotensive doses of benidipine and cilnidipine in stroke-prone spontaneously hypertensive rats (SHRSP). Methods SHRSP were divided into five groups, and were given vehicle, benidipine at 1 or 3 mg/kg per day, or cilnidipine at 1 or 3 mg/kg per day for 7 weeks, and the protective effects against cardiorenal injury were compared among each group. Results Benidipine and cilnidipine at the same doses exerted comparable hypotensive effects on SHRSP throughout the treatment. Despite equihypotensive effects between both drugs, benidipine prevented cardiac hypertrophy, fibrosis, and inflammation to a greater extent than cilnidipine. Moreover, benidipine prevented glomerulosclerosis, tubulointerstitial injury, and renal inflammation more than cilnidipine. To elucidate the underlying mechanism of more beneficial effects of benidipine than cilnidipine, we compared the effects of these drugs on cardiac and renal oxidative stress, and aldosterone in SHRSP. Benidipine reduced both cardiac and renal NADPH oxidase activities in SHRSP more than cilnidipine, being associated with more attenuation of cardiac and renal superoxide by benidipine. Furthermore, serum aldosterone was significantly reduced by benidipine but not by cilnidipine. Conclusion Benidipine exerted more protective effects against cardiorenal injury of hypertensive rats than cilnidipine, through more attenuation of oxidative stress than cilnidipine, and the reduction of aldosterone. Benidipine, via blockade of T-type calcium channels, seems to elicit additive benefits for prevention of hypertensive cardiorenal injury.

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.

A Q312X mutation in the hemojuvelin gene is associated with cardiomyopathy due to juvenile haemochromatosis

Juvenile haemochromatosis (JH) is an autosomal recessive iron disorder characterized by the early onset of secondary cardiomyopathy. The candidate modifier genes are hemojuvelin (HJV) and hepcidin antimicrobial peptide (HAMP). In the Japanese population, the prevalence of JH is quite low. The influence of HJV mutation on the JH phenotype is still unclear.