Vascular smooth muscle cell (VSMC) proliferation and migration are responses to arterial injury that are highly important to the processes of restenosis and atherosclerosis. In the arterial balloon injury model in the rat, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced in the vessel wall and regulate these VSMC activities. Novel insulin sensitizing agents, thiazolidinediones, have been demonstrated to inhibit insulin and epidermal growth factor-induced growth of VSMCs. We hypothesized that these agents might also inhibit the effect of PDGF and bFGF on cultured

Angiotensin II (A II ) is a critical factor in cardiac remodeling which involves hypertrophy, fibroblast proliferation, and extracellular matrix production. However, little is known about the mechanism by which A II accelerates these responses. Osteopontin is an acidic phosphoprotein with RGD (arginine-glycine-aspartate) sequences that are involved in the vascular smooth muscle cell remodeling process. We identified the presence of osteopontin mRNA and protein in cultured rat cardiac fibroblasts and its prominent regulation by A II (10 Ϫ 11 M). Osteopontin message levels were increased fourfold ( P Ͻ 0.01) and protein fivefold ( P Ͻ 0.05) at 24 h after addition of A II (10 Ϫ 7 M). This response was inhibited by the AT 1 receptor blocker, losartan. Osteopontin mRNA levels were increased in hypertrophied ventricles from animals with renovascular hypertension (1.6-fold, P Ͻ 0.05) and aortic banding (2.9-fold, P Ͻ 0.05). To examine the function of osteopontin, we determined its effects on ( a ) the ability of cardiac fibroblasts to contract three-dimensional collagen gels and ( b ) cardiac fibroblast growth. A monoclonal antibody against osteopontin partially blocked A II -induced three-dimensional collagen gel contraction by cardiac fibroblasts (64 Ϯ 4 vs. 86 Ϯ 5% in the presence of antibody, P Ͻ 0.05), while osteopontin itself promoted contraction of the gels by fibroblasts (71 Ϯ 5%, P Ͻ 0.05 compared with control). Either a monoclonal antibody against ␤ 3 integrin which is a ligand for osteopontin or the RGD peptide blocked both A II and osteopontin-induced collagen gel contraction. Thus, the osteopontin RGD sequence binds to ␤ 3 integrins on the fibroblast to promote fibroblast binding to collagen. A II induced a threefold increase in DNA synthesis of cardiac fibroblasts, which was completely blocked by antibodies against osteopontin and ␤ 3 integrin, or by RGD peptide, but not by controls. Thus, A II -induced growth of cardiac fibroblasts also requires osteopontin engagement of the ␤ 3 integrin. Taken together, these results provide the first evidence that osteopontin is a potentially important mediator of A II regulation of cardiac fibroblast behavior in the cardiac remodeling process. ( J. Clin. Invest. 1996. 98:2218-2227.)

mice expressed less CD68, C-C-chemokine receptor 2, and VCAM-1. In response to intraperitoneal thioglycollate, recruitment of leukocytes in OPN -/-mice was impaired, and OPN -/-leukocytes exhibited decreased basal and MCP-1-directed migration. Furthermore, macrophage viability in atherosclerotic lesions from Ang II-infused ApoE -/-OPN -/-mice was decreased. Finally, Ang II-induced abdominal aortic aneurysm formation in ApoE -/-OPN -/-mice was reduced and associated with decreased MMP-2 and MMP-9 activity. These data suggest an important role for leukocytederived OPN in mediating Ang II-accelerated atherosclerosis and aneurysm formation.

Background-This study is the first to examine the effect of direct angiotensin II type 2 (AT 2 ) receptor stimulation on postinfarct cardiac function with the use of the novel nonpeptide AT 2 receptor agonist compound 21 (C21). Methods and Results-Myocardial infarction (MI) was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with C21 (0.01, 0.03, 0.3 mg/kg per day IP) was started 24 hours after MI and was continued until euthanasia (7 days after MI). Infarct size was assessed by magnetic resonance imaging, and hemodynamic measurements were performed via transthoracic Doppler echocardiography and intracardiac Millar catheter. Cardiac tissues were analyzed for inflammation and apoptosis markers with immunoblotting and real-time reverse transcription polymerase chain reaction. C21 significantly improved systolic and diastolic ventricular function. Scar size was smallest in the C21-treated rats. In regard to underlying mechanisms, C21 diminished MI-induced Fas-ligand and caspase-3 expression in the peri-infarct zone, indicating an antiapoptotic effect. Phosphorylation of the p44/42 and p38 mitogen-activated protein kinases, both involved in the regulation of cell survival, was strongly reduced after MI but almost completely rescued by C21 treatment. Furthermore, C21 decreased MI-induced serum monocyte chemoattractant protein-1 and myeloperoxidase as well as cardiac interleukin-6, interleukin-1␤, and interleukin-2 expression, suggesting an antiinflammatory effect. Conclusions-Direct

The present study provides the first evidence that cardiomyocytes are a prominent source of OP in vivo and suggests that induction of OP expression is strongly associated with ventricular hypertrophy.

Mtgratton of vascular smooth muscle cells (VSMCs) 1s a crucial response to vascular mjury resulting m neomtima formatton and atherosclerosis Platelet-derived growth factor (PDGF-BB) functions as a potent chemoattractant for VSMCs and enhances these pathologies m the vasculature. However, little is known about the mtracellular pathways that mediate VSMC mtgratton In the present study, we mvestigated the role of mltogen-achvated protein kmase (MAPK) activatton m this function, since PDGF-BB as well as other growth factors activate this pathway Usmg an m-gel kmase assay, we observed that PD 98059, an mhlbttor of MEK that activates MAP kmase, inhibited PDGF-BB-induced activation of ERK-1 and ERK-2 m cultured rat aorttc smooth muscle cells m a concentration-dependent manner In contrast, PDGF-mediated acttvation of mtracellular calcmm release was not affected by PD 98059 The chemotacttc response of both rat aorttc smooth muscle cells (RASMCs) and human umbilical vem smooth muscle cells (HUSMCs) toward PDGF-BB (10 ng/mL) was stgmticantly reduced by PD 98059 (10 pmol/L) to 41 7?7 1% m RASMCs (P<.Ol) and to 47 2 (1) increased mtracellular calcmm and the activation of calcmm/calmodulm-dependent protein kmase II', (2) mcreased phosphattdylinosttol turnover linked to the acttvatton of phosphohpase Cy or phospholipase Cp3, (3) actrvatron of phosphatidylmosuol3-kmase4; and (4) activation of RAS and RAF 8 The mrtogenic stgnalmg pathway for PDGF involves the activation of RAS, which activates the serme threonme kmase RAF, which triggers a protein kmase cascade.9 The signal for prohferatton 1s transmitted into the nucleus though the actrvatton of MAPK, which phosphorylates transcription factors that induce expresston of c-fos and other early growth response genes. The involvement of the RAS-RAF-MAPK cascade m cell migration 1s controverstal In fibroblasts expressing a dominant negative RAS, directed migration toward PDGF was suppressed,* suggesting this pathway contributed to cell migration In contrast, IGF-I and PDGF-BB were shown to equally induce VSMC migration, but IGF-1 only weakly mduced MAPK m contrast to PDGF 3 The authors concluded that actrvatron of MAPK may not required for VSMC migration The avatlabrlity of the MEK mhtbrtor PD 98059, which is widely used as a pharmacologrcal mhibttor of the MAPK pathway, and antisense ODN against MAPK mRNA now provides cnttcal tools to determine the specific role of MAPK activation m cell functions such as migration

mice expressed less CD68, C-C-chemokine receptor 2, and VCAM-1. In response to intraperitoneal thioglycollate, recruitment of leukocytes in OPN -/-mice was impaired, and OPN -/-leukocytes exhibited decreased basal and MCP-1-directed migration. Furthermore, macrophage viability in atherosclerotic lesions from Ang II-infused ApoE -/-OPN -/-mice was decreased. Finally, Ang II-induced abdominal aortic aneurysm formation in ApoE -/-OPN -/-mice was reduced and associated with decreased MMP-2 and MMP-9 activity. These data suggest an important role for leukocytederived OPN in mediating Ang II-accelerated atherosclerosis and aneurysm formation.

Abstract-Migration of endothelial cells (EC) is a key event in angiogenesis that contributes to neovascularization in diabetic vasculopathy. Leptin induces angiogenesis and is elevated in obesity and hyperinsulinemia. The antidiabetic thiazolidinediones (TZD) inhibit leptin gene expression and vascular smooth muscle cell migration through activation of the peroxisome proliferator-activated receptor-␥ (PPAR␥). This study investigates the role of leptin in EC migration, the chemotactic signaling pathways involved, and the effects of the TZD-PPAR␥ ligands troglitazone (TRO) and ciglitazone (CIG) on EC migration. We demonstrate that leptin induces EC migration. Because activation of two signaling pathways, the phosphatidylinositol-3 kinase (PI3K)3 Akt3eNOS and the ERK1/2 MAPK pathway, is known to be involved in cell migration, we used the pharmacological inhibitors wortmannin and PD98059 to determine if chemotactic signaling by leptin involves Akt or ERK1/2, respectively. Both wortmannin and PD98059 significantly inhibited leptin-induced migration. Treatment with the TZD-PPAR␥-ligands TRO and CIG significantly inhibited the chemotactic response toward leptin. Both PPAR␥-ligands inhibited leptin-stimulated Akt and eNOS phosphorylation, but neither attenuated ERK 1/2 activation in response to leptin. The inhibition of Akt-phosphorylation was accompanied by a PPAR␥-ligand-mediated upregulation of PTEN, a phosphatase that functions as a negative regulator of PI3K3 Akt signaling. These experiments provide the first evidence that activation of Akt and ERK 1/2 are crucial events in leptin-mediated signal transduction leading to EC migration. Moreover, inhibition of leptin-directed migration by the PPAR␥-ligands TRO and CIG through inhibition of Akt underscores their potential in the prevention of diabetesassociated complications.