Yasuhiro Izumiya

Role of JNK, p38, and ERK in Platelet-Derived Growth Factor–Induced Vascular Proliferation, Migration, and Gene Expression

Objective—We investigated the comparative roles of mitogen-activated protein (MAP) kinases, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38, in vascular smooth muscle cell (VSMC) proliferation, migration, and gene expression. Methods and Results—VSMCs were infected with recombinant adenovirus containing dominant-negative mutants of ERK, p38, and JNK (Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK, respectively) to specifically inhibit the respective MAP kinases and then stimulated with platelet-derived growth factor (PDGF)-BB. Ad-DN-ERK attenuated PDGF-BB–induced VSMC proliferation more potently than Ad-DN-p38 or Ad-DN-JNK, indicating the dominant role of ERK in VSMC proliferation. Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK similarly inhibited PDGF-induced VSMC migration. Ad-DN-ERK and Ad-DN-JNK suppressed PDGF-BB–induced downregulation of cyclin-dependent kinase inhibitor p27Kip1, whereas Ad-DN-p38 decreased PDGF-BB–induced upregulation of p21Cip1. Ad-DN-ERK inhibited PDGF-BB–induced plasminogen activator inhibitor type-1 (PAI-1), monocyte chemoattractant protein-1, and transforming growth factor-&bgr;1 expressions, Ad-DN-p38 blocked monocyte chemoattractant protein-1 and transforming growth factor-&bgr;1 expression but not PAI-1, whereas Ad-DN-JNK suppressed only PAI-1 expression. Moreover, in vivo gene transfer of Ad-DN-p38 to rat carotid artery caused the inhibition of intimal hyperplasia by balloon injury, indicating the involvement of p38 in vascular remodeling in vivo. Conclusions—We propose that these 3 MAP kinases participate in vascular diseases via differential molecular mechanisms and are new therapeutic targets for treatment of vascular diseases.

Apoptosis Signal-Regulating Kinase 1 Plays a Pivotal Role in Angiotensin II–Induced Cardiac Hypertrophy and Remodeling

Abstract— Multiple lines of evidence establish that angiotensin II (Ang II) induces not only hypertension but also directly contributes to cardiac diseases. Apoptosis signal-regulating kinase 1 (ASK1), one of mitogen-activated protein kinase kinase kinases, plays a key role in stress-induced cellular responses. However, nothing is known about the role of ASK1 in cardiac hypertrophy and remodeling in vivo. In this study, by using mice deficient in ASK1 (ASK1−/− mice), we investigated the role of ASK1 in cardiac hypertrophy and remodeling induced by Ang II. Left ventricular (LV) ASK1 was activated by Ang II infusion in wild-type mice, which was mediated by angiotensin II type 1 receptor and superoxide. Although Ang II-induced hypertensive effect was comparable to wild-type and ASK1−/− mice, LV ASK1 activation by Ang II was not detectable in ASK1−/− mice, and p38 and c-Jun N-terminal kinase (JNK) activation was lesser in ASK−/− mice than in wild-type mice. Elevation of blood pressure by continuous Ang II infusion was comparable between ASK1−/− and wild-type mice. However, Ang II–induced cardiac hypertrophy and remodeling, including cardiomyocyte hypertrophy, cardiac hypertrophy–related mRNA upregulation, cardiomyocyte apoptosis, interstitial fibrosis, coronary arterial remodeling, and collagen gene upregulation, was significantly attenuated in ASK1−/− mice compared with wild-type mice. These results provided the first in vivo evidence that ASK1 is the critical signaling molecule for Ang II–induced cardiac hypertrophy and remodeling. Thus, ASK1 is proposed to be a potential therapeutic target for cardiac diseases.

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.

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.

Activation of Apoptosis Signal-Regulating Kinase 1 in Injured Artery and Its Critical Role in Neointimal Hyperplasia

Background—Apoptosis signal-regulating kinase 1 (ASK1), recently identified as one of the mitogen-activated protein kinase kinase kinases, is activated by various extracellular stimuli and involved in a variety of cellular function. Therefore, we first examined the role of ASK1 in vascular remodeling. Methods and Results—We used rat balloon injury model and cultured vascular smooth muscle cells (VSMCs). Arterial ASK1 activity was rapidly and dramatically increased after balloon injury. To specifically inhibit endogenous ASK1 activation, dominant-negative mutant of ASK1 (DN-ASK1) was transfected into rat carotid artery before balloon injury. Gene transfer of DN-ASK1 significantly prevented neointimal formation at 14 days after injury. Bromodeoxyuridine labeling index at 7 days after injury showed that DN-ASK1 remarkably suppressed VSMC proliferation in both the intima and the media. We also examined the role of ASK1 in cultured rat VSMCs. Infection with DN-ASK1 significantly attenuated serum-induced VSMC proliferation and migration. We also compared neointimal formation after cuff placement around the femoral artery between mice deficient in ASK1 (ASK1−/− mice) and wild-type (WT) mice. Neointimal formation at 28 days after cuff injury in ASK1−/− mice was significantly attenuated compared with WT mice. Furthermore, we compared the proliferation and migration of VSMCs isolated from ASK1−/− mice with WT mice. Both proliferation and migration of VSMCs from ASK1−/− mice were significantly attenuated compared with VSMCs from WT mice. Conclusions—ASK1 activation plays the key role in vascular intimal hyperplasia. ASK1 may provide the basis for the development of new therapeutic strategy for vascular diseases.

Beneficial Effects of Combined Blockade of ACE and AT1 Receptor on Intimal Hyperplasia in Balloon-Injured Rat Artery

Objective—The present study was undertaken to elucidate the effect of the ACE inhibitor and the angiotensin II type 1 (AT1) receptor antagonist in combination on neointimal hyperplasia after balloon injury. Methods and Results—Temocapril (an ACE inhibitor), CS-866 (an AT1 receptor antagonist), or their combination was given orally to rats, and their effects were compared on vascular hyperplasia induced by balloon injury. The maximal preventive effect of temocapril and CS-866 alone on neointimal thickening after balloon injury was obtained at a dose of 20 and 10 mg/kg per day, respectively. However, compared with either agent alone, combined temocapril and CS-866 (20 and 10 mg/kg per day, respectively) prevented intimal thickening to a larger extent. Furthermore, compared with either agent alone, combined temocapril and CS-866 prevented vascular smooth muscle cell proliferation in the intima more potently. The increase in platelet-derived growth factor receptor tyrosyl phosphorylation was reduced more potently by the combination of both agents compared with either agent alone. The nonpeptide bradykinin B2 receptor antagonist or the NO synthase inhibitor reduced the prevention of intimal thickening by combined temocapril and CS-866. Conclusions—Compared with either agent alone, the combination of an ACE inhibitor and an AT1 receptor antagonist is more effective in the prevention of vascular hyperplasia due to bradykinin or NO.

Dominant-negative c-Jun inhibits rat cardiac hypertrophy induced by angiotensin II and hypertension

Cardiac activator protein-1 (AP-1), composed of c-Jun, is significantly activated by hypertension or angiotensin II (AngII). This study was undertaken to elucidate whether c-Jun could be the potential target for treatment of cardiac hypertrophy. We constructed recombinant adenovirus carrying dominant-negative mutant of c-Jun (Ad.DN-c-Jun). Using catheter-based technique of adenoviral gene transfer, we achieved global myocardial transduction of DN-c-Jun in rats, to specifically inhibit cardiac AP-1. (1) AngII (200u2009ng/kg/min) infusion in rats caused cardiac hypertrophy, increased cardiac p70S6 kinase activity by 1.3-fold (P<0.05) and enhanced the gene expression of cardiac hypertrophic markers. Ad.DN-c-Jun, which was transferred to the heart 2 days before AngII infusion, prevented cardiac hypertrophy (P<0.01), decreased p70S6 kinase phosphorylation (P<0.05), and suppressed cardiac gene expression of brain natriuretic peptide, collagen I, III, and IV, monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) (P<0.01). (2) In genetically hypertensive rats with cardiac hypertrophy, cardiac gene transfer of Ad.DN-c-Jun, without affecting hypertension, regressed cardiac hypertrophy (P<0.05), and suppressed p70S6 kinase phosphorylation by 20% (P<0.05) and suppressed the enhanced expression of collagen I, III, and IV, MCP-1 and PAI-1. These results provided the first evidence that in vivo blockade of cardiac c-Jun inhibits pathologic cardiac hypertrophy.