Hiroshi Iwao

Critical Role of Angiotensin II in Excess Salt-Induced Brain Oxidative Stress of Stroke-Prone Spontaneously Hypertensive Rats

Background and Purpose— The detailed role of angiotensin II in salt-exacerbated stroke is unclear. We examined the role of angiotensin II in salt-accelerated stroke of stroke-prone spontaneously hypertensive rats (SHRSP). Methods— Salt-loaded SHRSP were orally given the angiotensin II type 1 (AT1) receptor blocker candesartan (0.3 to 3 mg/kg per day) and calcium channel blocker amlodipine (1 mg/kg per day), and the effects on stroke (n=61) and brain superoxide were compared between them. We also examined the effect of angiotensin II infusion (200 ng/kg per min) on brain superoxide production and blood-brain barrier. Results— Despite the comparable hypotensive effect between candesartan and amlodipine, candesartan prolonged survival of salt-loaded SHRSP much more than amlodipine (P<0.01), being associated with more improvement of cerebral arteriolar thickening, cerebral arteriolar cell proliferation, and hippocampal CA1 neuronal cell reduction (1024.9±20.6 versus 724.9±22.8 cells/mm2; P<0.01; n=7 to 10 in each group) in SHRSP by candesartan (P<0.05) than amlodipine. Salt loading increased superoxide and NADPH oxidase activity in brain cortex and hippocampus of SHRSP, and this increase was prevented by candesartan (P<0.01) but not amlodipine. Angiotensin II infusion, via AT1 receptor, directly increased brain superoxide by 1.8-fold (P<0.05; n=6 to 7 in each group) and impaired blood-brain barrier in salt-loaded SHRSP by 1.7-fold (P<0.05), and this increase in brain superoxide and blood-brain barrier impairment was prevented by tempol as well as candesartan. Conclusion— Excess salt, via oxidative stress, accelerates stroke, and angiotensin II, via AT1 receptor, plays a pivotal role in brain superoxide production of SHRSP by excess salt.

AP-1 and colorectal cancer

Activator protein-1 (AP-1) is a transcription factor that consists of either a Jun-Jun homodimer or a Jun-Fos heterodimer. AP-1 regulates the expression of multiple genes essential for cell proliferation, differentiation and apoptosis. Numerous reports suggest that AP-1 plays an important role in various human diseases. Among them, the roles relating to human cancers have been strongly suggested for a long time. In human cancers, colorectal cancer is still a leading cause of morbidity and mortality in the world. Since there are some reports about the role of AP-1 in colorectal cancer response to a number of stimuli, such as cytokines and growth factors, and oncogenic transformation, therapeutic inhibition of AP-1 activity has attracted considerable interest. Here, we demonstrate the biological properties of AP-1 and its role in colorectal cancer, and discuss a possibility of an AP-1 inhibitor, an adenovirus dominant-negative mutant of c-Jun, as a therapeutic a gent for gene therapy.

Excess aldosterone under normal salt diet induces cardiac hypertrophy and infiltration via oxidative stress.

Aldosterone is known to play a role in the pathophysiology of some cardiovascular diseases. However, previous studies on aldosterone infusion have been mostly performed in animals receiving sodium loading and uninephrectomy, and thus the cardiac action of aldosterone alone remains to be fully clarified. The present study was undertaken to investigate the direct cardiac action of aldosterone infusion alone in rats not subjected to salt loading and uninephrectomy. Aldosterone (0.75 μg/h) was subcutaneously infused into rats via an osmotic minipump for 14 days. Aldosterone infusion, under a normal salt diet, induced only a slight increase in the blood pressure of normal rats throughout the infusion. However, aldosterone significantly induced cardiac hypertrophy, as shown by echocardiography and measurement of cardiomyocyte cross-sectional area. Furthermore, aldosterone caused not only cardiac interstitial macrophage infiltration but also cardiac focal inflammatory lesions, which were associated with an increase in cardiac monocyte chemoattractant protein-1 (MCP-1) and osteopontin mRNA. The slight elevation of blood pressure by aldosterone infusion was completely prevented by tempol, the superoxide dismutase mimetic. However, tempol failed to suppress cardiac hypertrophy, the formation of inflammatory lesions, and upregulation of cardiac MCP-1 and osteopontin by aldosterone, while N-acetylcysteine could inhibit all of them. Our data provide evidence that aldosterone alone can induce cardiac hypertrophy and severe inflammatory response in the heart, independently of blood pressure, even in the absence of salt loading or nephrectomy. Aldosterone seems to induce cardiac inflammation and gene expression via oxidative stress that is inhibited by N-acetylcysteine but not by tempol.

Effects of metoprolol on epinephrine-induced takotsubo-like left ventricular dysfunction in non-human primates

Takotsubo cardiomyopathy, alternatively known as stress cardiomyopathy, is an increasingly recognized clinical syndrome characterized by acute reversible apical ventricular dysfunction. To elucidate the mechanism, we tried to make a new model of takotsubo-like cardiomyopathy in non-human primates. Echocardiography revealed that repeated intravenous infusion of epinephrine overdose in cynomolgus monkeys induced takotsubo-like cardiomyopathy, which is characterized by progressive left ventricle and depressed systolic function with severe hypokinesis in apical regions and hyperkinesis in the basal region. Although this cardiac dysfunction almost normalized after a month even without any treatment, metoprolol, a β-blocker, improved the decreased ejection fraction earlier than in the control. Luxol fast blue staining, which is useful for estimating myocytolysis, showed that increased myocytolysis was observed in the apical ventricle of the epinephrine-infused heart. Metoprolol diminished epinephrine-induced cardiomyocytolysis. To explain the mechanism of takotsubo myopathy and the effect of metoprolol, gene expressions in apical or basal ventricle were compared. Heart failure-related genes, such as brain natriuretic peptide, connective tissue growth factor and osteopontin; calcium signaling-related genes, such as ryanodine receptor 2, sarcoendoplasmic reticulum Ca2+-ATPase 2A2 and adenylate cyclase 7; renin–angiotensin system-related genes, such as angiotensinogen, angiotensin II receptor, type 1 and type 2; and mitochondria-related genes, such as peroxisome proliferator-activated receptor-γ co-activator-1α, cytochrome c and transcription factor A mitochondrial, were significantly changed at the apical ventricle rather than at the basal ventricle. The changes of some genes improved with metoprolol treatment. These results indicate that this model is valuable in understanding the pathogenesis of takotsubo cardiomyopathy and the effectivity of β-blockers.

Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation

Protein phosphatase 4 catalytic subunit (PP4c) is a PP2A-related protein serine/threonine phosphatase with important functions in a variety of cellular processes, including microtubule (MT) growth/organization, apoptosis, and tumor necrosis factor signaling. In this study, we report that NDEL1 is a substrate of PP4c, and PP4c selectively dephosphorylates NDEL1 at Cdk1 sites. We also demonstrate that PP4c negatively regulates Cdk1 activity at the centrosome. Targeted disruption of PP4c reveals disorganization of MTs and disorganized MT array. Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1. In addition, abnormal NDEL1 phosphorylation facilitates excessive recruitment of katanin p60 to the centrosome, suggesting that MT defects may be attributed to katanin p60 in excess. Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition. Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.

Heat Shock Cognate Protein 70 Is Essential for Akt Signaling in Endothelial Function

Objective—Heat shock protein 70s (Hsp70s) are molecular chaperones that protect cells from damage in response to various stress stimuli. However, the functions and mechanisms in endothelial cells (ECs) have not been examined. Herein, we investigate the role of Hsp70s, including heat shock cognate protein 70 (Hsc70), which is constitutively expressed in nonstressed cells (ie, ECs). Methods and Results—The Hsp70 inhibitor, KNK437, significantly decreased vascular endothelial growth factor (VEGF)–induced cell migration and tube formation in vitro. KNK437 inhibited the phosphorylation of VEGF-induced Akt and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells. In a mouse hind limb model of vascular insufficiency, intramuscular inhibition of Hsp70s attenuated collateral and capillary vessel formation. Silencing the Hsc70 gene by short interfering RNA abolished VEGF-induced Akt phosphorylation and VEGF-stimulated human umbilical vein endothelial cell migration and tube formation. As the molecular mechanisms, Hsc70 knockdown reduced the expression of phosphatidylinositol 3-kinase. Conclusion—Collectively, Hsc70 plays a significant role in ECs via the phosphatidylinositol 3-kinase/Akt pathway. Hsc70 may provide the basis for the development of new therapeutic strategies for angiogenesis.

Important Role of Apoptosis Signal-Regulating Kinase 1 in Ischemia-Induced Angiogenesis

Objective—We first examined the role of apoptosis signal-regulating kinase 1 (ASK1), one of mitogen-activated protein kinase kinase kinases, in ischemia-induced angiogenesis. Methods and Results—Unilateral hindlimb ischemia was induced surgically in C57BL/6J wild-type (WT) mice or mice deficient in ASK1 (ASK1−/−). ASK1 activity in WT mouse hindlimb was increased dramatically after ischemia. By laser Doppler analysis, well-developed collateral vessels and angiogenesis were observed in WT mice in response to hindlimb ischemia, whereas these responses were reduced in ASK1−/− mice. Immunostaining revealed that infiltration of macrophages and T lymphocytes was suppressed in the ischemic tissues of ASK1−/− mice compared with WT mice. The expression of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) proteins in ischemic tissues was weaker in ASK1−/− mice compared with WT mice. In vitro study on endothelial cells indicated that dominant-negative ASK1 significantly attenuated hydrogen peroxide–induced VEGF and MCP-1 production. Furthermore, in vivo blockade of MCP-1 by its neutralizing antibody suppressed the recovery of the blood flow and capillary formation after ischemia. Conclusions—ASK1 pathway promotes early angiogenesis by inducing inflammatory cell infiltration and VEGF and MCP-1 expression. ASK1 may provide the basis for the development of new therapeutic strategy for angiogenesis.

Core Protein of Hepatitis C Virus Induces Cardiomyopathy

Hepatitis C virus (HCV) has been reported to be associated with cardiomyopathy. However, the mechanism of cardiomyopathy in chronic HCV infection is still unclear. Therefore, we investigate the development of cardiomyopathy in mice transgenic for the HCV-core gene. After the age of 12 months, mice developed cardiomyopathy that appeared as left ventricular dilatation, and systolic and diastolic dysfunction assessed by Doppler echocardiography. Histologically, hypertrophy of cardiomyocytes, cardiac fibrosis, disarray and scarcity of myofibrils, vacuolization and deformity of nuclei, myofibrillar lysis, streaming of Z-bands, and an increased number of bizarre-shaped mitochondria were found in HCV-core transgenic mice. These histological changes are just consistent with cardiomyopathy. In conclusion, the HCV-core protein directly plays an important role in the development of cardiomyopathy.

Pravastatin accelerates ischemia-induced angiogenesis through AMP-activated protein kinase.

Statins exert pleiotropic effects on the cardiovascular system, in part through an increase in nitric oxide (NO) bioavailability. In this study, we examined the role of pravastatin in ischemia-induced angiogenesis. Unilateral hindlimb ischemia was surgically induced in C57BL/6J mice. Phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC) and endothelial NO synthase (eNOS) was increased in ischemic tissues. Furthermore, mice treated with pravastatin showed higher increases in phosphorylation than did untreated mice. Laser Doppler analysis has shown that pravastatin treatment accelerates the development of collateral vessels and angiogenesis in response to hindlimb ischemia. Capillary density in the ischemic hindlimb was also increased by pravastatin treatment. An in vitro study on human umbilical vein endothelial cells (HUVECs) revealed that pravastatin increased the phosphorylation of AMPK. Pravastatin-induced phosphorylation of eNOS, one of the downstreams of AMPK, was inhibited by compound C, an AMPK antagonist. The increased migration and tube formation of HUVECs by pravastatin were significantly blocked by compound C treatment. The accelerated angiogenesis by pravastatin after hindlimb ischemia was significantly reduced after treatment with compound C. Thus, ischemia induced AMPK phosphorylation in vivo. Furthermore, pravastatin could also activate AMPK in vivo and in vitro. Such phosphorylation results in eNOS activation and angiogenesis, which provide a novel explanation for one of the pleiotropic effects of statins that is beneficial for angiogenesis.

Effects of eplerenone on transcriptional factors and mRNA expression related to cardiac remodelling after myocardial infarction

Objective: To examine the effects of eplerenone, a selective aldosterone blocker, on cardiac function after myocardial infarction (MI) and myocardial remodelling related transcriptional factors and mRNA expression in non-infarcted myocardium. Methods: MI was induced by ligation of the coronary artery in Wistar rats. Rats were randomly assigned to a vehicle treated group or an eplerenone treated group (100 mg/kg/day). Results: At four weeks after MI, left ventricular (LV) end diastolic pressure, LV weight, and LV end diastolic dimension were increased in MI rats. Eplerenone significantly reduced the increase in LV end diastolic pressure, LV weight, and LV end diastolic dimension. In the MI rats the decreased ejection fraction indicated systolic dysfunction and the increased E wave to A wave ratio and E deceleration rate indicated diastolic dysfunction. Eplerenone significantly attenuated this systolic and diastolic dysfunction. Myocardial interstitial fibrosis, transcriptional activities of activator protein 1 and nuclear factor κB, and mRNA expression of monocyte chemoattractant protein 1, plasminogen activator inhibitor 1, atrial natriuretic peptide, brain natriuretic peptide, and collagen types I and III were significantly increased at four weeks after MI. Eplerenone significantly attenuated interstitial fibrosis and suppressed transcriptional activity and mRNA expression of these genes. Conclusions: When administered after MI, eplerenone prevents cardiac remodelling accompanied by systolic and diastolic dysfunction and inhibits abnormal myocardial transcriptional activities and gene expression.