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.

Abstract-Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo. (Circ Res. 2008;102:1182-1191.)Key Words: protein tyrosine phosphatase 1B Ⅲ vascular endothelial growth factor Ⅲ endothelial cell Ⅲ cell-cell adhesions Ⅲ angiogenesis

Forkhead transcription factor FoxO1 and the NADThe yeast Sir2 (silent information regulator 2) and its mammalian homologue SIRT1, class III histone deacetylases of the sirtuin family, modulate aging, oxidative stress resistance, cell metabolism, energy homeostasis, insulin resistance, and angiogenesis in several species (1, 2). In the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans, increased expression of Sir2 promotes longevity under calorie restriction conditions (3-5). In mammals, SIRT1 also promotes cell survival during calorie restriction (6 -8). SIRT1 plays diverse roles in a number of cellular processes through deacetylation of histones, transcription factors, and transcriptional cofactors (9). SIRT1 mediates stress resistance in mammalian cells by deacetylating stress response mediators, such as FoxO (forkhead box O) transcription factors (10) and p53 (11). Reduced expression or activity of SIRT1 contributes to insulin resistance and its related diseases, such as type II diabetes mellitus (12, 13). In contrast, SIRT1 up-regulates adiponectin expression by deacetylating FoxO1 and thus protects against insulin resistance (14 -16). SIRT1 transgenic mice display improved glucose tolerance and increased metabolic efficiency and have reduced aging-induced diabetes when fed a normal diet (17). Evidence from genetically engineered mouse models (18) shows a key role of SIRT1 in controlling endothelial angiogenic functions during vascular growth. These observations suggest that activation or increased expression of SIRT1 might have a broad spectrum of beneficial effects in metabolic diseases, including diabetes and obesity, as well as in aging-associated cardiovascular diseases, such as atherosclerosis.Expression and activity of SIRT1 are tightly regulated at multiple levels. Apoptosis transcriptional regulator E2F1 induces SIRT1 transcriptional expression in response to the stress of DNA damage (19). Furthermore, deacetylation of E2F1 by SIRT1 inhibits its activity by a negative feedback mechanism (19). SIRT1 transcription is also negatively regulated by HIC1 (hypermethylated in cancer 1), a tumor suppressor gene, which binds the enhancer elements of the SIRT1 promoter and represses SIRT1 expression (20,21). Interestingly, a concomitant induction of FoxO3a and SIRT1 expression was observed in response to acute nutritional stress (22). FoxO3a interacts with p53 and binds to the p53 response elements within the mouse Sirt1 promoter, thereby up-regulating SIRT1 transcription (22). HUR, an RNA-binding protein, associates with the 3Ј-untranslated region of SIRT1 mRNA and increases SIRT1 expression by stabilizing the SIRT1 mRNA (23). Oxidative stress reduces SIRT1 mRNA and protein levels, and a concomitant reduction of HUR and SIRT1 expression appears during the replicative senescence of human diploid fibroblasts (23). AROS (active regulator of SIRT1) (24) and DBC1 (deleted in breast cancer 1) (25) were recently identified as positive and negative regulators of SIRT1 enzymatic activity, respecti...

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)

Objective-Endothelial cell (EC) migration is a key event for repair process after vascular injury and angiogenesis. EC migration is regulated by reorganization of the actin cytoskeleton at the leading edge and localized production of reactive oxygen species (ROS) at the site of injury. However, underlying mechanisms are unclear. We reported that IQGAP1, an actin binding scaffold protein, mediates VEGF-induced activation of gp91phox (Nox2)-dependent NAD(P)H oxidase and EC migration. We thus hypothesized that Nox2 and IQGAP1 may play important roles in ROS-dependent EC migration in response to injury. Methods and Results-Using a monolayer scratch assay with confluent ECs, we show that ROS production is increased at the margin of scratch area and Nox2 translocates to the leading edge, where it colocalizes and associates with both actin and IQGAP1 in migrating ECs. Knockdown of IQGAP1 using siRNA and inhibition of the actin cytoskeleton blocked scratch injury-induced H 2 O 2 production, Nox2 translocation and its interaction with actin, and EC migration toward the injured site. Conclusions-These suggest that IQGAP1 may function to link Nox2 to actin at the leading edge, thereby facilitating ROS production at the site of injury, which may contribute to EC migration. (Arterioscler Thromb Vasc Biol. 2005;25:2295-2300.)Key Words: actin cytoskeleton Ⅲ endothelial cell migration Ⅲ IQGAP1 Ⅲ NAD(P)H oxidase Ⅲ reactive oxygen species E ndothelial migration is a key event during the repair of damaged vessels after vascular injury and angiogenesis, and this may contribute to limiting the development of atherogenesis. 1,2 Cell migration is regulated by the dynamic reorganization of the actin cytoskeleton, protrusion at the front of the cell, and retraction at the rear. It is a highly localized event, involving the generation of spatially and temporally restricted signaling molecules, including the small GTPase Rac1 3 and phosphatidylinositol 3,4,5 trisphosphate [PI(3,4,5)P 3 ], 4 the product of PI 3-kinase, at the site of the new leading edge. Although excess amounts of reactive oxygen species (ROS) are toxic, physiological levels of ROS serve as signaling molecules to regulate many growth and migratory responses. 5,6 ROS are also necessary for reparative angiogenesis in the ischemic heart 7 and hindlimb 8 as well as wound-healing in vivo. 9 The PI 3kinase-Rac pathway is also involved in ROS production. 10 In endothelial cells (ECs), endogenous H 2 O 2 accumulates in actively migrating cells at the site of injury, which is required for cytoskeletal reorganization and cell migration. 11 However, underlying regulatory mechanisms are unclear.In ECs, NAD(P)H oxidase is a major source of ROS. 12 ECs express NAD(P)H oxidase subunits that are identical to those found in phagocytes, including the membrane-bound gp91 phox (now known as Nox2) and p22 phox , the cytosolic components p47 phox and p67 phox , and Rac1. 12 On stimulation, cytosolic components translocate to the membrane to form a multimeric protein complex, leading to product...

Objective-Angiotensin II (Ang II) is a potent mediator of vascular hypertrophy in vascular smooth muscle cells (VSMCs).These effects are mediated through the Ang II type 1 receptor (AT 1 R) and require its trafficking through caveolin-1 (Cav1)-enriched lipid rafts and reactive oxygen species (ROS) derived from Rac1-dependent NAD(P)H oxidase. The specific role(s) of Cav1 in AT 1 R signaling is incompletely understood. Methods and Results-Knockdown of Cav1 protein by small interfering RNA (siRNA) inhibits Ang II-stimulated Rac1 activation and membrane translocation, H 2 O 2 production, ROS-dependent epidermal growth factor receptor (EGF-R) transactivation, and subsequent phosphorylation of Akt without affecting ROS-independent extracellular signalregulated kinase 1/2 phosphorylation. Ang II stimulates tyrosine phosphorylation of Sos-1, a Rac-guanine nucleotide exchange factor, which is inhibited by Cav1 siRNA, demonstrating involvement of Cav1 in Rac1 activation. Detergent-free fractionation showed that EGF-Rs are found basally in Cav1-enriched lipid raft membranes and associate with Cav1. Ang II stimulates AT 1 R movement into these microdomains contemporaneously with the egress of EGF-R. Both aspects of this bidirectional receptor trafficking are inhibited by Cav1 siRNA. Moreover, Cav1 siRNA inhibits Ang II-induced vascular hypertrophy. Key Words: angiotensin II Ⅲ reactive oxygen species Ⅲ caveolin Ⅲ caveolae Ⅲ vascular hypertrophy Ⅲ vascular smooth muscle A ngiotensin II (Ang II), the major effector peptide of the renin-angiotensin system, is a pleuripotent hormone in vascular smooth muscle cells (VSMCs) and stimulates arterial hypertrophy, a hallmark of remodeling in hypertension. These effects are mediated primarily through the G-proteincoupled Ang II type1 receptor (AT 1 R). Major outputs of the AT 1 R are dependent on the transactivation (tyrosine phosphorylation) of the epidermal growth factor receptor (EGF-R), leading to activation of downstream targets mitogen-activated protein kinases and Akt, which are important for the expression of the full hypertrophy-related Ang II signaling repertoire in VSMCs. 1,2 Many of these processes are mediated via generation of reactive oxygen species (ROS), which act as signaling molecules. [3][4][5][6] Emerging evidence indicates that NAD(P)H oxidase is a major source of vascular ROS in VSMCs. 1 Caveolae/lipid rafts are cholesterol-enriched, specialized membrane microdomains in which multimolecular complexes of signaling molecules such as EGF-R and Src are compartmentalized via interacting with caveolin-1 (Cav1). 7,8 The pathophysiological importance of Cav1 is reflected in the cardiovascular phenotype of Cav1 Ϫ/Ϫ mice. 8,9 We showed that Ang II promotes association of AT 1 R with Cav1 and AT 1 R trafficking into caveolin-enriched/lipid rafts in VSMCs. 10,11 This event is associated with egress of EGF-R from these specialized microdomains, where they are basally located. 11 Using cholesterol-binding agents such as ␤-cyclodextrin, we reported previously that cholesterol...

Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells.