Ryo Takeda

AMP-Activated Protein Kinase Inhibits Angiotensin II–Stimulated Vascular Smooth Muscle Cell Proliferation

Background—AMP-activated protein kinase (AMPK) is a stress-activated protein kinase that works as a metabolic sensor of cellular ATP levels. Here, we investigated whether AMPK signaling has a role in the regulation of the angiotensin II (Ang II)–induced proliferation signal in rat vascular smooth muscle cells (VSMCs). Methods and Results—Aminoimidazole-4-carboxamide-1-&bgr;-ribofuranoside (AICAR) activated AMPK in rat VSMCs and inhibited Ang II–induced extracellular signal–regulated kinase 1/2 phosphorylation but not that of p38 MAPK or Akt/PKB. Although Ang II activated AMPK, this activation was significantly inhibited by catalase, N-acetylcysteine, and diphenyleneiodonium chloride, an NADPH oxidase inhibitor. Moreover, the observation that AMPK was activated by H2O2 suggests that AMPK is redox sensitive. The Ang II type 1 receptor antagonist valsartan but not the Ang II type 2 receptor antagonist PD123319 significantly inhibited Ang II–induced AMPK activation, suggesting that Ang II–induced AMPK activation was Ang II type 1 receptor dependent. Whereas 3H-thymidine incorporation by VSMCs treated with Ang II was significantly inhibited when the cells were pretreated with 1 mmol/L AICAR, the inhibition of AMPK by dominant-negative AMPK overexpression augmented Ang II–induced cell proliferation. Subcutaneous injection of AICAR (1 mg/g body weight per day) for 2 weeks suppressed neointimal formation after transluminal mechanical injury of the rat femoral artery. Conclusions—Our findings indicate that Ang II–induced AMPK activation is synchronized with extracellular signal-regulated kinase signaling and that AMPK works as an inhibitor of the Ang II proliferative pathway. AMPK signaling might serve as a new therapeutic target of vascular remodeling in cardiovascular diseases.

Angiotensin II and tumor necrosis factor-α synergistically promote monocyte chemoattractant protein-1 expression: roles of NF-κB, p38, and reactive oxygen species

We examined whether ANG II and TNF-alpha cooperatively induce vascular inflammation using the expression of monocyte chemoattractant protein (MCP)-1 as a marker of vascular inflammation. ANG II and TNF-alpha stimulated MCP-1 expression in a synergistic manner in vascular smooth muscle cells. ANG II-induced MCP-1 expression was potently inhibited to a nonstimulated basal level by blockade of the p38-dependent pathway but only partially inhibited by blockade of the NF-kappaB-dependent pathway. In contrast, TNF-alpha-induced MCP-1 expression was potently suppressed by blockade of NF-kappaB activation but only modestly suppressed by blockade of p38 activation. ANG II- and TNF-alpha-induced activation of NF-kappaB- and p38-dependent pathways was partially inhibited by pharmacological inhibitors of ROS production. Furthermore, ANG II- and TNF-alpha-stimulated MCP-1 expression was partially suppressed by ROS inhibitors. We also examined whether endogenous ANG II and TNF-alpha cooperatively promote vascular inflammation in vivo using a wire injury model of the rat femoral artery. Blockade of both ANG II and TNF-alpha further suppressed neointimal formation, macrophage infiltration, and MCP-1 expression in an additive manner compared with blockade of ANG II or TNF-alpha alone. These results suggested that ANG II and TNF-alpha synergistically stimulate MCP-1 expression via the utilization of distinct intracellular signaling pathways (p38- and NFkappaB-dependent pathways) and that these pathways are activated in ROS-dependent and -independent manners. These results also suggest that ANG II and TNF-alpha cooperatively stimulate vascular inflammation in vivo as well as in vitro.

Blockade of Endogenous Cytokines Mitigates Neointimal Formation in Obese Zucker Rats

Background—It is well known that diabetes mellitus is a major risk factor for vascular diseases such as atherosclerosis and restenosis after angioplasty. It has become clear that advanced glycation end products (AGE) and their receptor (RAGE) are implicated in vascular diseases, especially in diabetes mellitus. Nevertheless, the mechanisms by which diabetes mellitus is often associated with vascular diseases remain unclear. Methods and Results—To study the role of endogenous cytokines such as tumor necrosis factor-&agr; (TNF-&agr;) and interleukin-6 in the development of vascular diseases and in the expression of RAGE, we used semapimod, a pharmacological inhibitor of cytokine production, and examined its effect on neointimal formation in the femoral artery of obese Zucker (OZ) rats. We also used an adenovirus construct expressing a dominant negative mutant of the receptor for TNF-&agr; (AdTNFR&Dgr;C) to block the action of endogenous TNF-&agr;. Semapimod significantly suppressed neointimal formation and RAGE expression in OZ rats compared with untreated OZ rats. This inhibitory effect of semapimod on neointimal formation was overcome by infection of an adenovirus expressing RAGE into the femoral artery of OZ rats. Furthermore, AdTNFR&Dgr;C infection significantly suppressed neointimal formation and RAGE expression in the femoral artery of OZ rats. Conclusions—These results suggest that endogenous cytokines, especially TNF-&agr;, were implicated in neointimal formation in OZ rats and that RAGE was a mediator of the effect of these cytokines on neointimal formation.

Ghrelin Improves Renal Function in Mice with Ischemic Acute Renal Failure

Growth hormone and IGF-1 have been suggested to have tissue-protective effects. Ghrelin is a stomach-derived growth hormone secretagogue. The effects of ghrelin on ischemia/reperfusion-induced renal failure in mice were examined. Ischemic acute renal failure was induced by bilateral renal artery clamping for 45 min and reperfusion for 24 h. Ghrelin (100 microg/kg mouse) or vehicle was injected subcutaneously six times before surgery and three times after surgery every 8 h. Twenty-four hours after reperfusion, the right kidney was isolated and perfused. Acetylcholine (ACh)- and adrenomedullin-induced endothelium-dependent vasorelaxation of renal vessels significantly improved in ghrelin-pretreated mice (%Delta renal perfusion pressure by 10(-7) M ACh -63.5 +/- 3.7 versus -41.2 +/- 5.5%; P < 0.05). This change was associated with significant increases of nitric oxide release in the kidneys of ghrelin-treated mice (10(-7) M ACh 35.5 +/- 5.8 versus 16.9 +/- 3.5 fmol/g kidney per min; P < 0.05). Serum concentration of urea nitrogen (53 +/- 7 versus 87 +/- 15 mg/dl; P < 0.05) and renal injury score were significantly lower in the ghrelin group (2.5 +/- 0.8 versus 5.3 +/- 1.5; P < 0.01). Tubular apoptotic index was significantly lower in the ghrelin group (5 +/- 5 versus 28 +/- 4; P < 0.05). Furthermore, the survival rate after the 60-min ischemic period was higher in the ghrelin group (80 versus 20%; P < 0.05). Ghrelin treatment significantly increased the serum level of IGF-1. However, such renal protective effects of ghrelin on ischemia/reperfusion injury were not observed in insulin receptor substrate-2 knockout mice. These results suggest that ghrelin may protect the kidneys from ischemia/reperfusion injury and that this effect is related to an improvement of endothelial function through an IGF-1-mediated pathway.

Calcineurin Promotes the Expression of Monocyte Chemoattractant Protein-1 in Vascular Myocytes and Mediates Vascular Inflammation

Abstract— Although the role of the calcineurin-dependent pathway in the development of cardiac hypertrophy has been intensively studied, little is known of its role in vascular inflammatory diseases such as atherosclerosis and restenosis after angioplasty. To help elucidate the role of calcineurin in vascular inflammation, we infected cultured vascular smooth muscle cells (VSMCs) with an adenovirus construct expressing a constitutively active mutant of calcineurin, and examined its effect on the expression of monocyte chemoattractant protein-1 (MCP-1). We also examined the role of calcineurin in vivo using a transluminal wire injury model of the rat femoral artery. Forced activation of calcineurin significantly increased the expression of MCP-1 both at the transcriptional and protein levels. Angiotensin II (Ang II) also significantly stimulated MCP-1 expression, and this increase was significantly inhibited by cyclosporin A (CyA). Constitutive activation of calcineurin stabilized MCP-1 mRNA without enhancing MCP-1 promoter activity. In accordance with the results, Ang II–induced increase of MCP-1 promoter activity was not suppressed by CyA. Ang II stabilized MCP-1 mRNA, and this effect of Ang II was diminished by CyA. CyA suppressed MCP-1 expression in the femoral artery after the transluminal mechanical injury. CyA also inhibited macrophage infiltration and neointimal formation in the wire-injured femoral arteries. These results suggested that calcineurin mediates vascular inflammation via stimulation of MCP-1 expression in VSMCs and macrophage infiltration.

Myocyte Enhancer Factor 2 Mediates Vascular Inflammation via the p38-Dependent Pathway

Although it has been established that myocyte enhancer factor 2 (MEF2) plays pivotal roles in the development of the cardiovascular system as well as skeletal muscle cells, little is known of its role in vascular inflammatory diseases such as atherosclerosis and restenosis after angioplasty. To investigate the role of MEF2 in vascular inflammation and that of p38 in the activation of MEF2, we infected cultured rat vascular smooth muscle cells (VSMCs) with an adenovirus construct expressing a dominant-negative mutant of MEF2A (MEF2ASA) or mitogen-activated protein kinase kinase 6 (MEK6AA), and examined their effects on the expression of monocyte chemoattractant protein-1 (MCP-1), which is known to play important roles in vascular inflammation. We also examined the role of MEF2 in vivo using a rat model of transluminal wire-induced injury of the femoral artery. Angiotensin II (Ang II)–induced expression of MCP-1 mRNA was significantly inhibited by infection with adenoviruses encoding MEF2ASA (AdMEF2ASA) or MEK6AA. Ang II–induced increase of MCP-1 promoter activity was also significantly suppressed by overexpression of MEF2ASA or MEK6AA. Ang II stimulated the transactivating function of MEF2A and this activation was inhibited by overexpression of MEK6AA. Infection with AdMEF2ASA suppressed MCP-1 expression in the femoral artery after the transluminal mechanical injury. AdMEF2ASA infection also inhibited macrophages infiltration and neointimal formation in the wire-injured femoral arteries. These results suggested that MEF2 activation via the p38-dependent pathway mediates vascular inflammation via stimulation of MCP-1 expression in VSMCs and macrophages infiltration.

Blockade of Endogenous Proinflammatory Cytokines Ameliorates Endothelial Dysfunction in Obese Zucker Rats

To study the role of endogenous proinflammatory cytokines in endothelial dysfunction in diabetes, we administered semapimod, an inhibitor of proinflammatory cytokine production, to obese Zucker (OZ) rats, and examined its effect on endothelium-dependent vasorelaxation. Endothelium-dependent vasorelaxation induced by acetylcholine and adrenomedullin (AM) was significantly reduced in OZ rats compared to a control group of lean Zucker rats. Semapimod significantly restored endothelium-dependent vasorelaxation in OZ rats. This effect of semapimod was well correlated with the reduction in the serum concentrations of tumor necrosis factor-α (TNF-α), interleukin-6, and C-reactive protein, as well as with the recovery of AM-induced Akt phosphorylation and cGMP production. Furthermore, acute administration of TNF-α significantly suppressed endothelium-dependent vasorelaxation and AM-induced cGMP production. These results implicate endogenous proinflammatory cytokines, especially TNF-α, in endothelial dysfunction in diabetes, and indicate that blockade of these cytokines will be a promising strategy for inhibiting the progression of vascular inflammation.

Clinical usefulness of drug-eluting stents in the treatment of dialysis patients with coronary artery disease

AIMS To investigate the clinical outcomes of paclitaxel-eluting stents (PES) and sirolimus-eluting stents (SES) in patients on dialysis. METHODS AND RESULTS Between May 2004 and December 2008, 95 patients on dialysis with 124 lesions were treated with PES alone, and were compared to 184 patients on dialysis with 244 lesions treated with SES alone, retrospectively. One-year major adverse cardiac event (MACE) including stent thrombosis, target lesion revascularisation (TLR), myocardial infarction (MI) and cardiac death were compared. Baseline characteristics were similar except for previous CABG (p = 0.02) and reference vessel diameter (p = 0.04). During hospitalisation, all cause death was more frequently observed in the PES group (p = 0.004). In-hospital MACE was not significantly different (p = 0.8). The incidence of 1-year MACE in the PES group was lower than that in the SES group (14.7%, 28.3%, p = 0.04), mainly due to the reduction of TLR (11.6%, 25.0%, p = 0.03). Rates of stent thrombosis (0%, 2.7%, p = 0.1), MI (1.1%, 3.8%, p = 0.2), and cardiac death (3.2%, 4.4%, p = 0.6) were not significantly different. CONCLUSIONS PES appears to be more efficient in reducing angiographic and clinical restenosis in dialysis patients compared with SES.

Effects of adrenomedullin on acute ischaemia-induced collateral development and mobilization of bone-marrow-derived cells

Adrenomedullin exerts not only vasodilatory effects, but also angiogenic effects. In the present study, we investigated the effects of adrenomedullin on collateral formation and circulating bone-marrow-derived cells after acute tissue ischaemia. Bone marrow of 8-10-week-old female C57BL/6J mice was replaced with that from GFP (green fluorescent protein) transgenic mice (GFP mice). At 8 weeks after transplantation, hindlimb ischaemia was induced by resecting the right femoral artery and a plasmid expressing human adrenomedullin (50 mug) was injected into the ischaemic muscle, followed by in vivo electroporation on a weekly basis. Overexpression of adrenomedullin significantly enhanced the blood flow recovery compared with controls (blood flow ratio, 1.0+/-0.2 compared with 0.6+/-0.3 respectively, at week 4; P<0.05) and increased capillary density in the ischaemic leg as determined by anti-CD31 immunostaining of the ischaemic muscle (567+/-40 compared with 338+/-65 capillaries/mm(2) respectively, at week 5; P<0.05). There were more GFP-positive cells in the thigh muscle of the mice injected with adrenomedullin than in that of the control mice (29.6+/-4.5 compared with 16.5+/-3.3 capillaries/mm(2) respectively, at week 5; P<0.05). We repeated the same experiments using LacZ-knock-in mice instead of GFP mice, and obtained similar results. These findings suggest that adrenomedullin may augment ischaemia-induced collateral formation with some effects on circulating bone-marrow-derived cells.

A case of acute coronary syndrome caused by extrinsic compression of the left main coronary artery due to pulmonary hypertension

Stenosis of the left main coronary artery (LMCA) due to extrinsic compression, producing symptoms of myocardial ischemia, is called left main compression syndrome. We report on a 43-year-old male with acute coronary syndrome who developed left main compression syndrome while waiting for a lung transplantation secondary to interstitial pneumonia, but underwent successful LMCA stenting as emergent treatment. Coronary angiography 3 months after the operation showed good stent patency in the LMCA, and the clinical course was favorable.