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 ^phox−/− 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 ^·− production and gp91 ^phox expression were present at both time points. The dihydroethidium staining of ischemic tissues indicates that O ₂ ^·− 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 ^phox−/− 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— gp91 ^phox -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.

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.

Although gait speed and six-minute walk distance are used to assess functional capacity in older patients with cardiovascular disease, their prognostic capabilities have not been directly compared. The study population was identified from the Kitasato University Cardiac Rehabilitation Database and consisted of 1474 patients ≥60 years old with a mean age of 72.2 ± 7.1 years that underwent evaluation of both usual gait speed and six-minute walk distance in routine geriatric assessment between 1 June 2008–30 September 2015. Both gait speed and six-minute walk distance were determined on the same day at hospital discharge. Mean gait speed and six-minute walk distance in the whole population were 1.04 m/s and 381 m, respectively, and were strongly positively correlated (r = 0.80, p < 0.001). A total of 180 deaths occurred during a follow-up of 2.3 ± 1.9 years. After adjusting for confounding factors, both gait speed (adjusted hazard ratio per 0.1 m/s increase: 0.87, 95% confidence interval: 0.81–0.93, p < 0.001) and six-minute walk distance (adjusted hazard ratio per 10-metre increase: 0.96, 95% confidence interval: 0.94–0.97, p < 0.001) were independent predictors of all-cause mortality. There was no significant difference in prognostic capability between gait speed and six-minute walk distance (c-index: 0.64 (95% confidence interval: 0.60–0.69) and 0.66 (95% confidence interval: 0.61–0.70), respectively, p = 0.357). Gait speed and six-minute walk distance showed similar prognostic predictive ability for all-cause mortality in older cardiovascular disease patients, indicating the potential utility of gait speed as a simple risk stratification tool in older cardiovascular disease patients.

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.

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 β-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/β-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.