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Thrombin and angiotensin II (angII) have trophic properties as mediators of vascular remodeling. Focal adhesions and actin cytoskeleton are involved in cell growth, shape, and movement and may be important in vascular remodeling. To characterize mechanisms by which thrombin and angII modulate vessel structure, we studied the effects of these G protein-coupled receptor ligands on focal adhesions in vascular smooth muscle cells (VSMCs). Both thrombin and angII stimulated bundling of actin filaments to form stress fibers, assembly of focal adhesions, and protein tyrosine phosphorylation at focal adhesions, such as p130Cas, paxillin, and tensin. To test whether c-Src plays a critical role in focal adhesion rearrangement, we analyzed cells with altered c-Src activity by retroviral transduction of wild-type (WT) and kinase-inactive (KI) c-Src into rat VSMCs, and by use of VSMCs from WT (src +/+ ) and Src-deficient (src -/-) mice. Tyrosine phosphorylation of Cas, paxillin, and tensin were markedly decreased in VSMCs expressing KI-Src and in src -/-VSMCs. Expression of KI-Src did not inhibit stress fiber formation by thrombin. Surprisingly, actin bundling was markedly decreased in VSMCs from src -/-mice both basally and after thrombin stimulation, compared with src +/+ mice. We also studied the effect of KI-Src and WT-Src on VSMC spreading. Expression of KI-Src reduced the rate of VSMC spreading on collagen, whereas WT-Src enhanced cell spreading. In conclusion, c-Src plays a critical role in agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in VSMCs. c-Src kinase activity is required for the cytoskeletal turnover that occurs in cell spreading, whereas c-Src appears to regulate actin bundling via a kinase-independent mechanism.

Shc⅐PDGF␤-R complex was inhibited by antioxidants such as N-acetylcysteine and Tiron, but not by calcium chelation. However, transactivation of PDGF␤-R by AngII (measured by PDGF␤-R tyrosine phosphorylation) differed significantly from PDGF-BB. Evidence to support different mechanisms of PDGF␤-R phosphorylation includes differences in the time course of PDGF␤-R phosphorylation, differing effects of inhibitors of the endogenous PDGF␤-R tyrosine kinase and Src family tyrosine kinases, differing results when the PDGF␤-R was directly immunoprecipitated (PDGF␤-R-antibody) versus coimmunoprecipitated (Shc-antibody), and cell fractionation studies that suggested that the Shc⅐PDGF␤-R complexes phosphorylated by AngII and PDGF-BB were located in separate subcellular compartments. These studies are the first to suggest that transactivation of tyrosine kinase receptors by G protein-coupled receptors involves a unique pathway that regulates a population of tyrosine kinase receptors different from the endogenous tyrosine kinase ligand.

The angiotensin II type-1 (AT1) receptor, a G protein-coupled receptor, lacks intrinsic kinase activity. However, recent data show that angiotensin II (Ang II) stimulates tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), Stat91 (one of the signal transducers and activators of transcription), and paxillin in vascular smooth muscle cells. The tyrosine kinases responsible for these phosphorylation events are unknown. Src family kinases have been shown to phosphorylate PLC-gamma 1 and to be activated by G protein-coupled receptors. We hypothesized that pp60c-src associates with the AT1 receptor and is activated after Ang II stimulation of smooth muscle cells. We immunoprecipitated pp60c-src from Ang II-stimulated vascular smooth muscle cells and measured pp60c-src activity by autophosphorylation and by phosphorylation of enolase. Both assays demonstrated an approximately threefold increase in pp60c-src activity within 1 minute. A similar increase in Ang II-stimulated pp60c-src activity was observed in Chinese hamster ovary cells transfected with the AT1 receptor but not in untransfected cells. These data are the first to show that pp60c-src is activated by Ang II. To determine if pp60c-src associated with the AT1 receptor, the AT1 receptor was immunoprecipitated (with two different antibodies), and Western blots were performed with two different anti-pp60c-src antibodies. No pp60c-src was detected. In addition, direct interaction between the AT1 receptor and pp60c-src could not be demonstrated by using a glutathione S-transferase (GST)-AT1 fusion protein to bind proteins from cell lysates stimulated by Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

Among the angiotensin II (Ang II)-mediated signal events likely to be important in vascular smooth muscle cells (VSMCs) is activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). The upstream mediators by which Ang II activates ERK1/2 remain poorly defined. Recently, we showed that Ang II activated c-Src, a nonreceptor kinase, which is a candidate to mediate Ang II signal events. To determine whether c-Src is required for ERK1/2 activation by Ang II, we studied the effects of Src family-selective tyrosine kinase inhibitors on ERK1/2 activation and also studied Ang II-mediated signal events in Src-deficient and Src-overexpressing VSMCs. The tyrosine kinase inhibitors, genistein and CP-188,556, blocked Ang II-mediated ERK1/2 activation in rat VSMCs (rVSMCs). We derived Src-deficient VSMCs from the aortas of c-Src knockout mice (Src-/- mVSMCs). Basal ERK1/2 activity was lower, and activation of ERK1/2 by Ang II was significantly decreased in Src-/- mVSMCs compared with wild-type mVSMCs, whereas ERK1/2 protein expression and ERK1/2 activation by phorbol 12-myristate 13-acetate were similar. To examine the role of c-Src further, we overexpressed wild-type or dominant-negative c-Src in rVSMCs using retroviral vectors. ERK1/2 activation by Ang II was significantly increased in rVSMCs that overexpressed c-Src, whereas ERK1/2 activation by Ang II was significantly inhibited in rVSMCs that overexpressed dominant-negative c-Src compared with control rVSMCs. These findings demonstrate that c-Src activation is required for Ang II stimulation of ERK1/2 in VSMCs and suggest an important role for c-Src in Ang II-mediated signal transduction.

Activation of the Na /H exchanger isoform-1 (NHE-1) by angiotensin II is an early signal transduction event that may regulate vascular smooth muscle cell (VSMC) growth and migration. Many signal transduction events stimulated by angiotensin II are mediated by the mitogen-activated protein (MAP) kinases. To define their roles in angiotensin II-mediated NHE-1 activity, VSMCs were treated with angiotensin II and the activities of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured. Angiotensin II rapidly (peak, 5 minutes) activated p38 and ERK1/2, whereas JNK was activated more slowly (peak, 30 minutes). Because angiotensin II stimulated Na /H exchange within 5 minutes, the effects of p38 and ERK1/2 antagonists on Na /H exchange were studied. The MEK-1 inhibitor PD98059 decreased ERK1/2 activity and Na /H exchange stimulated by angiotensin II. In contrast, the specific p38 antagonist SKF-86002 increased Na /H exchange. Two mechanisms were identified that may mediate the effects of p38 and SKF-86002 on angiotensin II-stimulated Na /H exchange. First, angiotensin II activation of ERK1/2 was increased 1.5-to 2.5-fold (depending on assay technique) in the presence of SKF-86002, demonstrating that p38 negatively regulates ERK1/2. Second, the ability of angiotensin II-stimulated MAP kinases to phosphorylate a glutathione S-transferase fusion protein containing amino acids 625 to 747 of NHE-1 in vitro was analyzed. The relative activities of endogenous immunoprecipitated p38, ERK1/2, and JNK were 1.0, 2.0, and 0.05 versus control, respectively suggesting that p38 and ERK1/2, but not JNK, may phosphorylate NHE-1 in VSMC. These data indicate important roles for p38 and ERK1/2 in angiotensin II-mediated regulation of the Na /H exchanger in VSMC. (Circ Res. 1998;83:824-831.) Key Words: mitogen-activated protein kinase Na /H exchange angiotensin II vascular smooth muscle A ngiotensin II is a multifunctional agonist for vascular smooth muscle cells (VSMC), stimulating ion fluxes, protein phosphorylation, contractility, gene expression, and cell growth. Our laboratory has characterized the signal transduction events stimulated by angiotensin II in VSMC to gain insight into its mechanisms of action. Many of the signal transduction events stimulated by angiotensin II are mediated by members of mitogen-activated protein (MAP) kinase family of protein kinases. 1-4 Characterization of substrates for the MAP kinases should provide important insights into the mechanisms by which angiotensin II regulates VSMC function. The Na /H exchanger isoform-1 (NHE-1) is an important membrane protein whose activity may be regulated by protein kinases activated by angiotensin II. Stimulation of Na /H exchange by angiotensin II is an early event that is required for VSMC growth, migration, and contraction. 2,5-7 In fact, inhibiting Na /H exchange with specific amiloride-derivative antagonists decreases neointimal proliferation in the rat carotid injury model. 7 The Na /H exchanger is a phospho...

We clearly showed that the expression of the MMP-9 increased in fibrillating atrial tissue, which may have contributed to the atrial structural remodeling and atrial dilatation during AF.

ObjectiveOmega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), likely prevent cardiovascular disease, however their mechanisms remain unclear. Recently, the role of DNA damage in atherogenesis has been receiving considerable attention. Here, we investigated the effects of EPA and DHA on DNA damage in vascular endothelial cells to clarify their antiatherogenic mechanisms.Methods and resultsWe determined the effect of EPA and DHA on H2O2-induced DNA damage response in human aortic endothelial cells. Immunofluorescence staining showed that γ-H2AX foci formation, a prominent marker of DNA damage, was significantly reduced in the cells treated with EPA and DHA (by 47% and 48%, respectively). H2O2-induced activation of ATM, a major kinase orchestrating DNA damage response, was significantly reduced with EPA and DHA treatment (by 31% and 33%, respectively). These results indicated EPA and DHA attenuated DNA damage independently of the DNA damage response. Thus the effects of EPA and DHA on a source of DNA damage were examined. EPA and DHA significantly reduced intracellular reactive oxygen species under both basal condition and H2O2 stimulation. In addition, the mRNA levels of antioxidant molecules, such as heme oxygenase-1, thioredoxin reductase 1, ferritin light chain, ferritin heavy chain and manganese superoxide dismutase, were significantly increased with EPA and DHA. Silencing nuclear factor erythroid 2-related factor 2 (NRF2) remarkably abrogated the increases in mRNA levels of antioxidant molecules and the decrease in intracellular reactive oxygen species. Furthermore, EPA and DHA significantly reduced H2O2-induced senescence-associated β-galactosidase activity in the cells (by 31% and 22%, respectively), which was revoked by NRF2 silencing.ConclusionsOur results suggested that EPA and DHA attenuate oxidative stress-induced DNA damage in vascular endothelial cells through upregulation of NRF2-mediated antioxidant response. Therefore omega-3 fatty acids likely help prevent cardiovascular disease, at least in part, by their genome protective properties.