Abstract-Endothelial cell (EC) migration is required for angiogenesis, neovascularization, and reendothelialization.Integrins, known as ␣␤-heterodimeric cell-surface receptors, regulate cell migration and are essential for mechanotransduction of hemodynamic forces. Therefore, we investigated the effect of shear stress on EC migration and the contribution of the integrins and integrin-dependent signaling pathways in a scratched-wound assay. Laminar shear stress-induced EC migration was significantly reduced by integrin-receptor blocking with RGD peptides or with neutralizing antibodies against integrin subunits ␣ 5 and ␤ 1 , whereas antibodies against ␣ v ␤ 3 or ␣ 2 ␤ 1 had no effect. Cell-surface levels of the integrin ␣ 5 and ␤ 1 were specifically upregulated in migrating ECs at the wound edges. Consistent with the important role of integrins for shear stress-increased cell migration, blockade of the integrinassociated adapter protein Shc by overexpression of dominant negative construct inhibited shear stress-stimulated EC migration. Moreover, pharmacological inhibition of the integrin downstream effector signaling molecules ERK1/2 or phosphatidyl-inositol-3-kinase prevented shear stress-induced EC migration. In contrast, inhibition of the NO synthase had no effect. Taken ntegrins are ␣␤-heterodimeric cell-surface receptors that recognize a large number of extracellular ligands. 1-3 Currently, 18 ␣-and 8 ␤-subunits have been identified, which could form more than 24 heterodimeric receptors. 3,4 Ligand binding by integrins regulates such processes as cell adhesion, growth, differentiation, and migration. 1,5 Cell adhesion and migration are required for angiogenesis, which was thought to depend primarily on ␣ v -integrins, for example, ␣ v ␤ 3 . 6 Studies in ␣ v -knockout mice, however, indicate that other receptors and their ligands are also involved in vasculogenesis and angiogenesis. 7 Consistent with these findings, recent knockout studies indicate a more important role of other integrins, such as ␤ 1 or ␣ 5 . 4 ␣ 5 -Knockout mice have severe defects in vascular development, 8 and ␣ 1 ␤ 1 -and ␣ 2 ␤ 1 -integrins seems to contribute to angiogenesis. 9,10 Integrins play a further important role in mediating mechanical stress-induced signals. Specifically, integrins like ␣ 5 ␤ 1 and ␣ v ␤ 3 are essential for the mechanotransduction of hemodynamic forces into biochemical signals. [11][12][13][14][15] Hemodynamic forces are important regulators of blood vessel function and structure. Of these forces, the laminar blood flow (shear stress) is a potent endogenous mediator of atheroprotective signals in endothelial cells (ECs). 14 Several studies have shown that the beneficial effects of shear stress, such as activation of the prosurvival kinases Akt and ERK1/2, require an intact integrin signaling. 12,13,16 We have previously demonstrated that laminar shear stress upregulates the fibronectin receptor subunits ␣ 5 and ␤ 1 in ECs and enhances EC adhesion and survival. 17 In addition, integrins are important for cell m...

Endostatin is an anti-angiogenic factor that inhibits endothelial cell (EC) migration and induces EC apoptosis. Because nitric oxide (NO) plays a key role in vascular endothelial growth factor (VEGF)-induced angiogenesis, we hypothesized that endostatin interferes with the activation of the endothelial NO synthase (eNOS). Human recombinant endostatin significantly reduced VEGF-induced NO-release, which suggests that endostatin inhibits eNOS activation. Because the activation of eNOS by VEGF is associated with the Akt-dependent phosphorylation of eNOS at Ser1177, we investigated whether endostatin interferes with phosphorylation of eNOS. Endostatin reduced VEGF-induced phosphorylation of eNOS at Ser1177, whereas Akt phosphorylation was not affected. Coinciding with the inhibition of eNOS phosphorylation, endostatin completely blocked VEGF-induced EC migration. The NO-donor SNAP reversed the inhibitory effect of endostatin on EC migration. In addition, endostatin significantly inhibited VEGF-induced tube formation, whereas endostatin did not affect tube formation induced by NO. Finally, a non-dephosphorylatable constitutive active eNOS construct (S1177D), but not constitutive active Akt, abolished the inhibitory effect of endostatin on EC migration. Endostatin activated PP2A, which is known to directly dephosphorylate eNOS at Ser1177. Inhibition of PP2A prevented the inhibitory effect of endostatin. Thus, endostatin inhibits VEGF-induced EC migration and angiogenesis upstream of NO-synthesis via dephosphorylation of eNOS at Ser1177.

Abstract-Tyrosine kinase cascades may play a role in the hypoxic regulation of hypoxia-inducible factor (HIF)-1. We investigated the role of tyrosine kinase phosphorylation and of the Shc/Ras cascade on hypoxic HIF-1 stabilization. Exposure of human umbilical vein endothelial cells to hypoxia results in HIF protein stabilization as early as 10 minutes, with a maximum at 3 hours, and also in Shc tyrosine phosphorylation, with a maximum at 10 minutes. Key Words: hypoxia Ⅲ hypoxia-inducible factor-1 Ⅲ Shc Ⅲ Ras Ⅲ endothelial cells H ypoxia is an important regulatory stimulus for a variety of different biological processes, such as vascular tone and structure, angiogenesis, and erythropoiesis. These events are mediated by a variety of hypoxia-sensitive cellular proteins, which modulate cell-cell interaction and cell proliferation and differentiation. Many of these proteins, such as erythropoietin, vascular endothelial growth factor, the inducible NO synthase gene, or glycolytic enzymes, 1-4 are under the control of a master regulator of O 2 homeostasis, hypoxia-inducible factor (HIF)-1, a transcription factor. This DNA binding protein is a heterodimer composed of 2 subunits, HIF-1␣ and HIF-1␤. HIF-1␤ can dimerize with several different basic helix-loop-helix-PAS proteins, whereas HIF-1␣ is the O 2 -regulated subunit that determines its biological activity. 5,6 The regulation of HIF is complex and involves not only changes on the transcriptional level but also posttranscriptional and posttranslational alterations in response to hypoxia. 6 -9 Perhaps most striking is HIF protein stabilization under hypoxic conditions and its rapid degradation on reoxygenation. Under normoxic conditions, HIF is subject to ubiquitination and proteasomal degradation. 6,9,10 However, the regulatory step in hypoxia-mediated HIF regulation has not been determined. The von Hippel-Lindau tumor suppressor protein, a putative ubiquitin-protein ligase, 11 appears to play a role in HIF protein stabilization, inasmuch as loss of the von Hippel-Lindau protein results in constitutive expression of the HIF protein. 12 In addition, there is evidence that multiple domains in the HIF protein regulate its expression and that they do so by altering the ubiquitination of HIF-1␣ under nonhypoxic conditions. 13 In addition to changes in the redox state affecting hypoxic signaling, changes in phosphorylation may influence HIF-1 expression and, eventually, the transactivation of genes. Indeed, hypoxic induction of HIF-1 is inhibited by pretreating cells with the tyrosine kinase inhibitor genistein or the serine/threonine phosphatase inhibitor sodium fluoride. 14 The adaptor protein Shc is an immediate substrate of tyrosine kinase and may play an important role in linking activated tyrosine kinase receptors to downstream hypoxic signaling, involving HIF protein stabilization. 15,16 The Shc family of adaptor proteins consists of multiple protein-protein interaction domains: an amino-terminal phosphotyrosine binding domain, a central collagen homology domain, and...