Abstract-Endothelial cell (EC) proliferation and migration are important for reendothelialization and angiogenesis. We have demonstrated that reactive oxygen species (ROS) derived from the small GTPase Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)-mediated endothelial responses mainly through the VEGF type2 receptor (VEGFR2). Little is known about the underlying molecular mechanisms. IQGAP1 is a scaffolding protein that controls cellular motility and morphogenesis by interacting directly with cytoskeletal, cell adhesion, and small G proteins, including Rac1. In this study, we show that IQGAP1 is robustly expressed in ECs and binds to the VEGFR2. A pulldown assay using purified proteins demonstrates that IQGAP1 directly interacts with active VEGFR2. In cultured ECs, VEGF stimulation rapidly promotes recruitment of Rac1 to IQGAP1, which inducibly binds to VEGFR2 and which, in turn, is associated with tyrosine phosphorylation of IQGAP1. Endogenous IQGAP1 knockdown by siRNA shows that IQGAP1 is involved in VEGF-stimulated ROS production, Akt phosphorylation, endothelial migration, and proliferation. Wound assays reveal that IQGAP1 and phosphorylated VEGFR2 accumulate and colocalize at the leading edge in actively migrating ECs. Moreover, we found that IQGAP1 expression is dramatically increased in the VEGFR2-positive regenerating EC layer in balloon-injured rat carotid artery. These results suggest that IQGAP1 functions as a VEGFR2-associated scaffold protein to organize ROS-dependent VEGF signaling, thereby promoting EC migration and proliferation, which may contribute to repair and maintenance of the functional integrity of established blood vessels. Regeneration of the endothelium after vascular damage is important in limiting atherogenesis. 1 EC activation, migration, and proliferation are important for endothelial wound repair and neovascularization, a process by which new blood vessels are formed from preexisting vessels. 2 The underlying molecular mechanisms are incompletely understood.Vascular endothelial growth factor (VEGF) stimulates EC migration and proliferation primarily through the VEGF type 2 receptor (VEGFR2, KDR/Flk-1), thereby contributing to angiogenesis in vivo. 3 In ECs, VEGF binding initiates dimerization and transphosphorylation (autophosphorylation) of tyrosine residues in the cytoplasmic kinase domain of VEGFR2, which is followed by activation of key signaling enzymes involved in angiogenesis/neovascularization including mitogen activated proteins (MAP) kinases and Akt. 4 VEGF also promotes mobilization and recruitment of endothelial progenitor cells into ischemic sites, which contribute to neovascularization. 5,6 Moreover, VEGF is upregulated and promotes regeneration of ECs in balloon-injured arteries. 7,8 We and others demonstrated that VEGF stimulates an increase in reactive oxygen species (ROS) generation via activation of the small GTPase Rac1-dependent NAD(P)H oxidase and that ROS participate in VEGFR2-mediated signaling, EC migration...

Background-Neovascularization is potentially important for the treatment of ischemic heart and limb disease. We reported that reactive oxygen species (ROS) derived from gp91 phox (Nox2)-containing NAD(P)H oxidase are involved in angiogenesis in mouse sponge models as well as in vascular endothelial growth factor (VEGF) signaling in cultured endothelial cells. The role of gp91 phox -derived ROS in neovascularization in response to tissue ischemia is unknown, however. Methods and Results-Here, we show that neovascularization in the ischemic hindlimb is significantly impaired in gp91 phoxϪ/Ϫ mice as compared with wild-type (WT) mice as evaluated by laser Doppler flow, capillary density, and microsphere measurements. In WT mice, inflammatory cell infiltration in the ischemic hindlimb was maximal at 3 days, whereas capillary formation was prominent at 7 days when inflammatory cells were no longer detectable. Increased O 2 ⅐Ϫ production and gp91 phox expression were present at both time points. The dihydroethidium staining of ischemic tissues indicates that O 2 ⅐Ϫ is mainly produced from inflammatory cells at 3 days and from neovasculature at 7 days after operation. Relative to WT mice, ischemia-induced ROS production in gp91 phoxϪ/Ϫ mice at both 3 and 7 days was diminished, whereas VEGF expression was enhanced and the inflammatory response was unchanged. Infusion of the antioxidant ebselen into WT mice also significantly blocked the increase in blood flow recovery and capillary density after ischemia. Conclusions-gp91phox -derived ROS play an important role in mediating neovascularization in response to tissue ischemia. NAD(P)H oxidases and their products are potential therapeutic targets for regulating angiogenesis in vivo.

No abstract

Abstract-Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenicrelated responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6 (T27N)

Six months of DAPT was not inferior to 18 months of DAPT following implantation of a DES with a biodegradable abluminal coating. However, this result needs to be interpreted with caution given the open-label design and wide noninferiority margin of the present study. (Nobori Dual Antiplatelet Therapy as Appropriate Duration [NIPPON]; NCT01514227).

Objective-Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating reactive oxygen species (ROS) production primarily through the VEGF receptor-2 (VEGFR2). One of the initial responses in established vessels to stimulate angiogenesis is loss of vascular endothelial (VE)-cadherin-based cell-cell adhesions; however, little is known about the underlying mechanisms. IQGAP1 is a novel VEGFR2 binding protein, and it interacts directly with actin, cadherin, and ␤-catenin, thereby regulating cell motility and morphogenesis. Methods and Results-Confocal microscopy analysis shows that IQGAP1 colocalizes with VE-cadherin at cell-cell contacts in unstimulated human endothelial cells (ECs). VEGF stimulation reduces staining of IQGAP1 and VE-cadherin at the adherens junction without affecting interaction of these proteins. Knockdown of IQGAP1 using siRNA inhibits localization of VE-cadherin at cell-cell contacts, VEGF-stimulated recruitment of VEGFR2 to the VE-cadherin/␤-catenin complex, ROS-dependent tyrosine phosphorylation of VE-cadherin, which is required for loss of cell-cell contacts and capillary tube formation. IQGAP1 expression is increased in a mouse hindlimb ischemia model of angiogenesis. Conclusions-IQGAP1 is required for establishment of cell-cell contacts in quiescent ECs. To induce angiogenesis, it may function to link VEGFR2 to the VE-cadherin containing adherens junctions, thereby promoting VEGF-stimulated, ROS-dependent tyrosine phosphorylation of VE-cadherin and loss of cell-cell contacts. (Arterioscler Thromb Vasc

Abstract-Important output signals of the angiotensin subtype 1 receptor (AT 1 R) in vascular smooth muscle cells (VSMCs) are mediated by angiotensin II (Ang II)-stimulated transactivation of the epidermal growth factor receptor (EGF-R), which is critical for vascular hypertrophy. Ang II-induced EGF-R transactivation is mediated through cSrc, a proximal target of reactive oxygen species (ROS) derived from NAD(P)H oxidase (NOX) and is dependent on AT 1 R trafficking through caveolin1 (Cav1)-enriched lipid rafts. Underlying molecular mechanisms are incompletely understood. The nonreceptor tyrosine kinase, proto-oncogene cAbl is a substrate of Src and is a major mediator for ROS-dependent tyrosine phosphorylation of Cav1. We thus hypothesized that cAbl is important for ROS-, cSrc-, and Cav1-dependent growth-related AT 1 R signal transduction. Here we show that Ang II induces tyrosine phosphorylation of cAbl in rat VSMCs and mouse aorta, and that Ang II promotes association of cAbl with AT 1 R, both of which are Src-dependent. Pretreatment of rat VSMCs with the NOX inhibitor diphenylene iodonium or the antioxidants N-acetylcysteine or ebselen significantly inhibited Ang II-induced cAbl phosphorylation. Cell fractionation shows that both EGF-Rs and cAbl are found basally in Cav1-enriched membrane fractions. Knockdown of cAbl protein using small interference RNA inhibits Ang II-stimulated: (1) trafficking of AT 1 R into, and EGF-R out of, Cav1-enriched lipid rafts; (2) EGF-R transactivation; (3) appearance of the transactivated EGF-R and phospho-Cav1 at focal adhesions; and (4) vascular hypertrophy. These studies provide a novel role of cAbl in the spatial and temporal organization of growth-related AT 1 R signaling in VSMCs and suggest that cAbl may be generally important in signaling of G-protein coupled receptors. Key Words: cAbl Ⅲ caveolin-1 Ⅲ NAD(P)H oxidase Ⅲ angiotensin II Ⅲ vascular smooth muscle A ngiotensin II (Ang II) is important in mediating vascular remodeling and hypertrophy in hypertension. These effects are mediated, in large part, through the G-protein coupled angiotensin subtype 1 receptors (AT 1 Rs). The growth-related outputs of the AT 1 R are dependent on the transactivation (tyrosine phosphorylation) of the epidermal growth factor receptor (EGF-R) and its activation of mitogenactivated protein kinases and Akt, which are involved in vascular smooth muscle cell (VSMC) hypertrophy. 1,2 Many effects of Ang II are dependent on the AT 1 R stimulation of reactive oxygen species (ROS) production by NAD(P)H oxidase. 2-5 EGF-R transactivation in VSMCs is mediated through the activation of cSrc, a proximal ROS-dependent event. 5 Caveolae/lipid rafts are specialized membrane microdomains where multimolecular signaling molecule complexes are compartmentalized, in part, via interacting with caveolin1 (Cav1). We showed that in VSMCs, Ang II stimulation promotes AT 1 R association with Cav1 and its trafficking into Cav1-enriched lipid rafts, events which in turn are associated contemporaneously with egress o...

BackgroundGlucagon-like peptide-1 (GLP-1) is an incretin hormone that has a wide range of effects on glucose metabolism and cardiovascular function (e.g., improving insulin sensitivity, reduction in appetite, modulation of heart rate, blood pressure and myocardial contractility). Metabolic syndrome (MetS) is associated with an increased risk of developing atherosclerotic cardiovascular diseases. Novel glycemic control drugs, the dipeptidyl-peptidase-4 (DPP-4) inhibitors, work by inhibiting the inactivation of incretin hormones, GLP-1 and glucose-dependent insulinotropic polypeptide (GIP). In spite of good effects of these drugs in diabetic patients, circulating levels of incretins and their role in MetS are largely unknown.MethodsTo examine relationships between incretin hormones and MetS risk factors, we measured circulating levels of incretins in obese high-risk patients for cardiovascular disease. Fasting serum GLP-1 and GIP levels were measured by ELISA. We performed a cross-sectional analysis of metabolic variables in the fasting state in two subject groups: with MetS (n = 60) and pre-MetS (n = 37).ResultsFasting levels of Serum GLP -1 in the peripheral circulation were significantly increased correlated with the accumulation of MetS risk factors components (r = 0. 470, P < 0.001). There was a significant interaction between circulating GLP-1 and GIP, serum high-density lipoprotein cholesterol, triglyceride, and serum uric acid concentrations but not waist circumference, fasting glucose, HbA1c, or presence of diabetes.ConclusionCirculating levels of GLP-1 in relation to the accumulation in MetS factors suggested that MetS patients with elevated levels of GLP-1 are high-risk patients for cardiovascular disease, independent with the presence of diabetes.