Takahiro Taniguchi

Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells

Involvement of Akt/Protein kinase B (PKB), a serine/threonine kinase with a pleckstrin-homology domain, in angiotensin II (ANG II)-induced signal transduction was investigated in cultured vascular smooth muscle cells (VSMC). Stimulation of the cells with ANG II led to a marked increase in the kinase activity of Akt/PKB, which coincided with Ser-473 phosphorylation. ANG II-stimulated Akt/PKB activation was rapid, concentration dependent, and inhibited by the AT1-receptor antagonist CV-11974, but not by pertussis toxin. Akt/PKB activity was stimulated by the Ca2+ ionophore ionomycin, suggesting the possible involvement of Ca2+ in ANG II-stimulated Akt/PKB activation. However, blockade of Ca2+ mobilization by BAPTA-AM only partially inhibited ANG II-stimulated Akt/PKB activation. ANG II-stimulated Akt/PKB activation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A and by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002. These results indicate that ANG II stimulates Akt/PKB activity via AT1 receptors in VSMC and that the activities of tyrosine kinase and PI3K are required for this activation.Involvement of Akt/Protein kinase B (PKB), a serine/threonine kinase with a pleckstrin-homology domain, in angiotensin II (ANG II)-induced signal transduction was investigated in cultured vascular smooth muscle cells (VSMC). Stimulation of the cells with ANG II led to a marked increase in the kinase activity of Akt/PKB, which coincided with Ser-473 phosphorylation. ANG II-stimulated Akt/PKB activation was rapid, concentration dependent, and inhibited by the AT1-receptor antagonist CV-11974, but not by pertussis toxin. Akt/PKB activity was stimulated by the Ca2+ ionophore ionomycin, suggesting the possible involvement of Ca2+ in ANG II-stimulated Akt/PKB activation. However, blockade of Ca2+ mobilization by BAPTA-AM only partially inhibited ANG II-stimulated Akt/PKB activation. ANG II-stimulated Akt/PKB activation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A and by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002. These results indicate that ANG II stimulates Akt/PKB activity via AT1 receptors in VSMC and that the activities of tyrosine kinase and PI3K are required for this activation.

High glucose accelerates MCP-1 production via p38 MAPK in vascular endothelial cells

In diabetes mellitus (DM), hyperglycemia causes cardiovascular lesions through endothelial dysfunction. Monocyte chemoattractant protein-1 (MCP-1) is implicated in the pathogenesis of cardiovascular lesions. By using human umbilical vein endothelial cells, we investigated the effect of hyperglycemia on MCP-1 production and its signaling pathways. Chronic incubation with high glucose increased mRNA expression and production rate of MCP-1 in a time (1-7 days)- and concentration (10-35 mM)-dependent manner. Chronic exposure to high glucose resulted in enhancement of generation of reactive oxygen species (ROS), as determined by increasing level of 2,7-dichlorofluorescein (DCF), and subsequent activation of p38 mitogen-activated protein kinase (MAPK). Neither c-Jun NH(2)-terminal kinase nor extracellular signal-regulated kinase1/2 was affected. SB203580 or FR167653, p38 MAPK specific inhibitors, completely suppressed MCP-1 expression. Catalase suppressed p38 MAPK phosphorylation and MCP-1 expression. These results indicate that hyperglycemia can accelerate MCP-1 production through the mechanism involving p38 MAPK, ROS-sensitive signaling pathway, in vascular endothelial cells.

Tranilast Inhibits Vascular Smooth Muscle Cell Growth and Intimal Hyperplasia by Induction of p21waf1/cip1/sdi1 and p53

Tranilast, which is an antiallergic drug, has a potent effect on preventing postangioplasty restenosis. To elucidate this mechanism, we studied the effect of tranilast on the proliferation of vascular smooth muscle cells (SMCs) in vitro and in vivo. Tranilast decreased the growth rate of SMCs stimulated by either 10% FBS or platelet-derived growth factor. The IC50 value, evaluated as cell number, was 100 micromol/L. These inhibitory effects were associated with inhibition of the retinoblastoma gene product (pRb) phosphorylation. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDK), we investigated CDK2 and CDK4 activities and the expression of CDK inhibitor p21(waf1/cip1/sdi1) (p21). When SMCs were stimulated by 10% FBS or platelet-derived growth factor, CDK2 and CDK4 activities reached a maximum near the G1/S transition. Tranilast suppressed their activities by >80% without reduction of CDK2/cyclin E and CDK4/cyclin D1 protein levels. These inhibitory effects were associated with enhanced expression of p21 and elevated complexing of p21 with CDK2/CDK4. Next, rat balloon-injured carotid artery was analyzed for intimal thickening and p21 expression. Tranilast-treated rats had a 70% (P<0.001) smaller neointima/media area ratio at 14 days after balloon injury compared with the controls. Immunohistochemical staining demonstrated that, in tranilast-treated rats, p21 was already present in the neointima at day 7 and strongly expressed throughout the neointima at day 14. In control rats, p21 was not observed in the neointima at day 7 but was sparsely expressed at day 14. These data demonstrate that inhibition of CDK2/CDK4 activities by the increased expression of p21 may be one mechanism by which tranilast inhibits SMC proliferation and prevents postangioplasty restenosis.

The structures of the asparagine-linked sugar chains of human apolipoprotein B-100

The asparagine-linked sugar chains of human apolipoprotein B-100 were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Their structures were elucidated by sequential exoglycosidase digestion in combination with methylation analysis after fractionation by paper electrophoresis and gel permeation chromatography. One neutral and two acidic fractions were obtained by paper electrophoresis in a molar ratio of 7:8:5. The neutral fraction contained high-mannose type oligosaccharides consisting of Man5GlcNAc2 to Man9GlcNAc2. The acidic fractions contained monosialylated and disialylated biantennary complex type oligosaccharides. As minor components in the monosialylated fraction, biantennary complex-type oligosaccharides which were absent one terminal galactose residue, monoantennary complex type, and hybrid type oligosaccharides were detected. Apolipoprotein B-100 was calculated to contain 5-6 mol of high-mannose type and 8-10 mol of complex type oligosaccharides per mole protein.

Chylomicron remnant induces apoptosis in vascular endothelial cells.

Chylomicron remnant (CR) is a cholesteroland apoE-enriched particle derived from the lipolytic processing of intestinal chylomicron, and has been regarded as an atherogenic lipoprotein in postprandial hyperlipidemia.1,2 However, little is known about the mechanisms through which CR promotes atherosclerosis. Recent studies suggest that remnant lipoproteins induce vascular endothelial dysfunction assessed by measuring endothelium-dependent vasorelaxation.3 Endothelial injury and dysfunction induced by atherogenic lipoprotein are believed to play pivotal roles in atherogenesis. Apoptosis in endothelial cell is considered to be involved in this process.4,5 This hypothesis is supported by the findings that proatherosclerotic factors, such as oxidized low density lipoprotein (LDL), reactive oxygen species, and inflammatory cytokines, have all been shown to induce apoptosis of vascular endothelial cells.6,7 We postulated that CR may promote atherosclerosis by inducing apoptosis in vascular endothelial cells. To examine our hypothesis, we isolated CR and investigated whether CR induces apoptosis in vascular endothelial cells in vitro.

Mouse Genetic Evidence That Tranilast Reduces Smooth Muscle Cell Hyperplasia via a p21waf1-Dependent Pathway

Objective—N-(3′4′-dimethoxycinnamoyl)-anthranilic acid (tranilast) is a drug that has been shown to reduce the incidence of restenosis after angioplasty in middle-scale clinical trials. Despite clinical interest in this drug, the pharmacological actions of tranilast remain relatively unexplored at a molecular level. Methods and Results—We evaluated the effects of tranilast on vascular smooth muscle cell (VSMC) proliferation in wild-type mice and in mice lacking a cyclin-dependent kinase inhibitor, p21WAF1 (p21). Tranilast potently inhibited the proliferation of VSMC cultures derived from wild-type mice, but VSMCs derived from p21-deficient (p21−/−) mice were unaffected by this treatment. In a mouse femoral artery model of vascular injury, tranilast administration to wild-type mice led to an upregulation of p21 expression and a decrease in the number of proliferating VSMCs, as determined by immunostaining for proliferating cell nuclear antigen. In contrast, tranilast had no effect on the number of proliferating cell nuclear antigen–positive cells in the injured arteries of p21−/− mice. Administration of tranilast significantly reduced the neointimal VSMC hyperplasia in wild-type mice at 4 weeks but had no effect on lesion formation in p21−/− mice. Conclusions—Our findings provide genetic evidence that tranilast inhibits intimal hyperplasia via a p21-dependent pathway, an activity that may contribute to its efficacy in the prophylactic treatment of postangioplasty restenosis.

Participation of reactive oxygen intermediates in the angiotensin II-activated signaling pathways in vascular smooth muscle cells.

Angiotensin II (Ang II), the main peptide hormone of the renin-angiotensin system, has been known to play an important role in the development of various cardiovascular diseases in addition to its key regulatory role in the regulation of blood pressure and circulating volume.1 Although Ang II is a potent hypertrophic factor for vascular smooth muscle cells (VSMC),1 molecular mechanisms responsible for a growth-promoting action of Ang II have not been fully understood. Recent studies suggest that reactive oxygen intermediates (ROI) may function as second messengers in the intracellular signaling pathways that mediate cellular responses induced by growth factors and cytokines.2 Therefore, we examined whether ROI were involved in Ang II–activated signaling pathways in cultured VSMC.

Papillary fibroelastoma of the left atrial appendage: echocardiographic findings

Papillary fibroelastoma is a small and rare benign intracardiac tumor that most frequently arises from the valvular endocardium. We report a patient with acute myocardial infarction in whom a papillary fibroelastoma in the left atrial appendage was detected by a transesophageal echocardiography during evaluation of the myocardial infarction. The roles of transesophageal echocardiography and surgical intervention are discussed.

Effects of lipoprotein(a) and low density lipoprotein on growth of mitogen-stimulated human umbilical vein endothelial cells

We investigated the effects of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) on proliferation of human umbilical vein endothelial cells (HUVECs). Both Lp(a) and LDL stimulated the growth of HUVECs synergistically with basic fibroblast growth factor and insulin in a dose-dependent manner. The potency of Lp(a) to promote the cell proliferation was 40% less than that of LDL. Addition of anti-transforming growth factor-beta 1 neutralizing antibody into the medium could not diminish the difference of HUVECs proliferation by Lp(a) and LDL. However, addition of anti-LDL receptor antibody suppressed HUVECs proliferation to the same level and sequestered the difference by the two lipoproteins. Moreover, cholesteryl ester content incubated with Lp(a) was 50% less than that with LDL. These results suggest that Lp(a) has less effect on HUVECs proliferation and cholesterol delivery to the cells than LDL. Therefore, Lp(a) may play a role as an atherogenic lipoprotein by delaying the repair of endothelium after injury.

Effects of hypoxia on cholesterol metabolism in human monocyte-derived macrophages

We assessed the metabolism of low density lipoprotein (LDL) of human monocyte-derived macrophages under hypoxia. The specific binding and association of 125I-labeled LDL (125I-LDL) were not changed under hypoxia compared to normoxia. However, the degradation of 125I-LDL under hypoxia decreased to 60%. The rate of cholesterol esterification under hypoxia was 2-fold greater on incubation with LDL or 25-hydroxycholesterol. The cellular cholesteryl ester content was also greater under hypoxia on incubation with LDL. Secretion of apolipoprotein E into the medium was not altered under hypoxia, suggesting that apolipoprotein E independent cholesterol efflux may be reduced under hypoxia. Thus, hypoxia affects the intracellular metabolism of LDL, stimulates cholesterol esterification, and enhances cholesteryl ester accumulation in macrophages. Hypoxia is one of the important factors modifying the cellular lipid metabolism in arterial wall.