Protein kinase C epsilon mediates angiotensin II-induced activation of beta1-integrins in cardiac fibroblasts.

OBJECTIVE: Angiotensin II (AII) promotes cardiac fibrosis by increased extracellular matrix production and enhanced interaction of matrix proteins with their cellular receptors, including integrins. AII and other growth factors augment beta(1)-integrin-dependent adhesion and spreading by "inside-out signaling" without affecting the total number of integrin receptors. "Inside-out signaling" involves specific signaling pathways, including protein kinase C (PKC), leading to activation of beta1-integrins. In the present study we investigated the mechanisms involved in AII-increased adhesion of adult rat cardiac fibroblasts (CFBs), obtained from Sprague-Dawley rats, to collagen I mediated by beta1-integrin. METHODS AND RESULTS: Treatment of CFBs with AII induced a concentration-dependent increase in adhesion to collagen I (2.2-fold, p<0.01) within 3-6 h of treatment. This effect was mediated by beta1-integrin via the angiotensin AT1 receptor and was significantly reduced by protein kinase C inhibition. AII significantly induced phosphorylation of PKC epsilon (PKCepsilon), which is involved in beta1-integrin-dependent adhesion and motility, and phosphorylation of the cytoplasmatic tail of beta1-integrin at threonine 788/789, required for adhesion. Phosphorylation of beta1-integrin and an increase in adhesion was also induced by l-alpha-phosphatidylinositol-3,4,5-triphosphate (l-alpha-PIP3), an activator of endogenous PKCepsilon. AII failed to increase adhesion in myofibroblasts obtained from PKCepsilon (-/-) mice, but not in cells obtained from control mice. Co-immunoprecipitation and double immunofluorescence demonstrated that AII induced a close association of PKCepsilon with beta1-integrin in CFBs. CONCLUSION: The present study demonstrates that AII increased beta1-integrin-dependent adhesion to collagen I in cardiac fibroblasts by inside-out signaling via PKCepsilon and phosphorylation of the cytoplasmatic tail of the beta1-integrin.

Chronic CRP-exposure inhibits VEGF-induced endothelial cell migration.

UNLABELLED: C-reactive protein (CRP) is a pluripotent mediator of inflammation and is present at sites of vascular injury and in atherosclerotic lesions. CRP stimulates endothelial cell adhesion molecule expression and monocyte migration, thereby contributing to the development and progression of vascular lesion formation. In addition, chronic exposure to CRP is known to inhibit angiogenesis and endothelial cell (EC) proliferation. AIM: Whether CRP also affects EC migration, however, has yet to be determined. The present study investigates how long-term exposure to CRP interacts with vascular endothelial growth factor (VEGF) -induced EC migration. METHODS AND RESULTS: Using a Transwell chamber migration assay, VEGF (20 ng/mL, 5 h incubation)-induced migration of human umbilical vein EC was significantly inhibited in cells pretreated with CRP (10 microg/mL) for 24 h by more than 75%. EC migration in response to VEGF is known to require activation of the protein kinase B (Akt)/endothelial NO synthase (eNOS)- and the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway. We therefore investigated the long-term effects of CRP on these signalling events. Immunoblotting with phosphospecific antibodies revealed rapid and transient activation/phosphorylation of the protein kinase Akt within 20 minutes after stimulation with VEGF, which was inhibited by 86% in EC pretreated with CRP (10 microg/mL, 24 h, p<0.05). Subsequent VEGF-induced phosphorylation of eNOS downstream of Akt was completely inhibited in CRP-treated EC. In contrast, CRP-pretreatment did not affect VEGF-induced phosphorylation of ERK1/2. Interestingly, stimulation of EC with CRP for 16-24 h induced marked expression of the phosphatase and tensin homolog (PTEN), which functions as a negative regulator of phosphatidylinositol 3 kinase (PI3K) -->Akt signalling. CONCLUSION: The observed time course for CRP-mediated PTEN upregulation corresponds to the exposure time needed for inhibition of Akt phosphorylation and migration and may therefore constitute a potential mechanism by which CRP inhibits inducible Akt phosphorylation and EC migration.

Leptin induces endothelial cell migration through Akt, which is inhibited by PPARgamma-ligands.

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-gamma (PPARgamma). This study investigates the role of leptin in EC migration, the chemotactic signaling pathways involved, and the effects of the TZD-PPARgamma 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)-->Akt-->eNOS 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-PPARgamma-ligands TRO and CIG significantly inhibited the chemotactic response toward leptin. Both PPARgamma-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 PPARgamma-ligand-mediated upregulation of PTEN, a phosphatase that functions as a negative regulator of PI3K-->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 PPARgamma-ligands TRO and CIG through inhibition of Akt underscores their potential in the prevention of diabetes-associated complications.