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...

Nitric oxide (NO) production by endothelial cell nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. We sought to understand the mechanism underlying defective eNOS function. Phosphorylation of the serine-threonine kinase Akt, which activates eNOS, was substantially reduced in sinusoidal endothelial cells from injured livers. Overexpression of Akt in vivo restored phosphorylation of Akt and production of NO and reduced portal pressure in portal hypertensive rats. We found that Akt physically interacts with G-protein-coupled receptor kinase-2 (GRK2), and that this interaction inhibits Akt activity. Furthermore, GRK2 expression increased in sinusoidal endothelial cells from portal hypertensive rats and knockdown of GRK2 restored Akt phosphorylation and NO production, and normalized portal pressure. Finally, after liver injury, GRK2-deficient mice developed less severe portal hypertension than control mice. Thus, an important mechanism underlying impaired activity of eNOS in injured sinusoidal endothelial cells is defective phosphorylation of Akt caused by overexpression of GRK2 after injury.

Despite substantial evidence that nitric oxide (NO) and/or endogenous S-nitrosothiols (SNOs) exert protective effects in a variety of cardiovascular diseases, the molecular details are largely unknown. Here we show that following left coronary artery ligation, mice with a targeted deletion of the S-nitrosoglutathione reductase gene (GSNOR ؊/؊ ) have reduced myocardial infarct size, preserved ventricular systolic and diastolic function, and maintained tissue oxygenation. These profound physiological effects are associated with increases in myocardial capillary density and S-nitrosylation of the transcription factor hypoxia inducible factor-1␣ (HIF-1␣) under normoxic conditions. We further show that S-nitrosylated HIF-1␣ binds to the vascular endothelial growth factor (VEGF) gene, thus identifying a role for GSNO in angiogenesis and myocardial protection. These results suggest innovative approaches to modulate angiogenesis and preserve cardiac function.angiogenesis ͉ HIF-1␣ ͉ myocardial infarction ͉ nitric oxide ͉ S-nitrosylation

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P < 0.0001) in an in vitro angiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P < 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.

Rationale: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis (IPF). Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents. Objective: To show that myofibroblast differentiation is regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity in vivo, and to identify a potential mechanism by which this occurs. Methods: We used tissue sections of surgical lung biopsies from patients with IPF to localize expression of PTEN and ␣-smooth muscle actin (␣-SMA). We used cell culture of pten ؊/؊ and wildtype fibroblasts, as well as adenoviral strategies and pharmacologic inhibitors, to determine the mechanism by which PTEN inhibits ␣-SMA, fibroblast proliferation, and collagen production.

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Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd induces neuronal apoptosis in part through activation of the mammalian target of rapamycin (mTOR) pathway. However, the underlying mechanism is unknown. Here we show that Cd induced generation of reactive oxygen species (ROS) by upregulating expression of NADPH oxidase 2 (NOX2) and its regulatory proteins (p22 phox , p67 phox , p40 phox , p47 phox and Rac1) in PC12 and SH-SY5Y cells. Cd induction of ROS contributed to activation of mTOR signaling, as pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, prevented this event. Further studies reveal that Cd induction of ROS increased phosphorylation of type I insulin-like growth factor receptor β subunit (IGFRβ), which was abrogated by NAC. Wortmannin, a phosphoinositide 3′-kinase (PI3K) inhibitor, partially attenuated Cd-induced phosphorylation of Akt, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), as well as apoptosis of the neuronal cells. In addition, overexpression of wild-type phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or pretreatment with aminoimidazole carboxamide ribonucleotide (AICAR), an AMPactivated protein kinase (AMPK) activator, partially prevented Cd-induced ROS and activation of mTOR pathway, as well as cell death. The results indicate that Cd induction of ROS activates mTOR signaling, leading to neuronal cell death, in part by activating the positive regulators IGFR/ PI3K, and by inhibiting the negative regulators PTEN/AMPK. The findings suggest that the inhibitors of PI3K and mTOR, activators of AMPK, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.

Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.Tie2 (also called Tek) is a member of a novel family of receptor tyrosine kinases (RTKs) (16,17,37,42,72) that are expressed predominantly on endothelial cells or their embryonic precursors (14,16,17,37,42) and that are required for normal vascular development (15,52,55). Functional disruption of Tie2 in transgenic mice results in embryonic lethality by day E9.5 to 10.5 with effects on the microvasculature resulting in reduced numbers of endothelial cells and abnormalities of vascular morphogenesis (15,55). Knockout of the activating Tie2 ligand, angiopoietin-1 (Ang1), or overexpression of a related, inhibitory ligand, angiopoietin-2 (Ang2), resulted in phenotypes similar to the Tie2 knockout (43, 64). Taken together, these findings suggest a role for Tie2 in endothelial cell maintenance, survival, and/or vascular morphogenesis (24).In addition to a role in embryonic vascular development, data from our laboratory suggest that Tie2 plays an important role in the adult vasculature. For example, Tie2 expression is increased in the vasculature of malignant breast tumors (49), and a soluble extracellular domain of Tie2 inhibits tumor angiogenesis and growth (39). Tie2 is also broadly expressed and tyrosine phosphorylated in a variety of adult tissues in which the endothelium is normally quiescent (69). These findings are consistent with a dual role for Tie2 in both the growth and the maintenance of the adult vasculature.To better understand the role of Tie2 in vascular growth and maintenance, it will be important to identify the signal transduction pathways responsible for these functions. Currently, little is known about the specific signaling proteins and pathways utilized by Tie2. We demonstrated previously that Tie2 a...