Recent studies suggest that statins can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. We show here that statins rapidly activate the protein kinase Akt/PKB in endothelial cells. Accordingly, simvastatin enhanced phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS), inhibited apoptosis and accelerated vascular structure formation in vitro in an Akt-dependent manner. Similar to vascular endothelial growth factor (VEGF) treatment, both simvastatin administration and enhanced Akt signaling in the endothelium promoted angiogenesis in ischemic limbs of normocholesterolemic rabbits. Therefore, activation of Akt represents a mechanism that can account for some of the beneficial side effects of statins, including the promotion of new blood vessel growth.

A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serinethreonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of antiapoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.During angiogenesis, endothelial cells receive cues from growth factors to initiate mitosis, migration, and organization of endothelial cells into primitive angiotubes and patent vascular networks (1, 2). These processes critically depend on preservation of endothelial cell viability. Disruption of endothelial cell-matrix contacts or interference with extracellular survival signals is sufficient to initiate caspase-dependent apoptosis in endothelium, culminating with rapid involution of vascular structures (3, 4). Unlike most angiogenic regulators, including fibroblast growth factor or vascular endothelial growth factor (VEGF), 1 angiopoietin-1 (Ang-1) does not stimulate endothelial cell growth but rather promotes stabilization of vascular networks and branching morphogenesis in vivo and in vitro (5-8). Little is known about the signaling requirements of these responses, and the mechanism(s) of Ang-1-induced cytoprotection are unknown (7, 9). The major goal of this paper was to elucidate a potential link between endothelial cell viability and maintenance of angiogenesis by examining the ability of Ang-1 to activate the antiapoptotic serine-threonine kinase, Akt (or protein kinase B). Moreover, we examined the relationship between Ang-1, Akt activation, and the expression of the anti-apoptotic genes, bcl-2 and survivin, in cultured microvascular endothelial cells (MVEC). MATERIALS AND METHODSCell Culture and Reagents-Bovine MVEC (Vec Technologies, Rensselaer, NY) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, L-glutamine, and antibiotics (penicillin and streptomycin). Cells (up to passage 12) were used for the experiments. In experiments examining endogenous survivin expression, human umbilical vein endothelial cells (HUVEC) were used, because the survivin antibody recognized human survivin better than bovine survivin. HUVEC were cultured on gelatin-coated tissue culture flasks in M199 medium containing 20% fetal bovine serum, 50 g/ml endothelial cell growth supplement (a commercial preparation that contains main...

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.

Abstract-17␤-Estradiol (E 2 ) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E 2 -stimulated endothelial NO release can occur without an increase in cytosolic Ca 2ϩ . Here we demonstrate for the first time, to our knowledge, that E 2 rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E 2 treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182,780. E 2 stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E 2 -activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca 2ϩ levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E 2 -stimulated NO release. The membrane-impermeant E 2 BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca 2ϩ fluxes and describes an important pathway relevant to cardiovascular pathophysiology. Key Words: estrogen Ⅲ endothelial nitric oxide synthase Ⅲ Akt Ⅲ membrane receptor E ndogenous and exogenous estrogen in premenopausal and postmenopausal women, respectively, is protective against the development of atherosclerotic cardiovascular disease. 1,2 The relevant biological effects of estrogen are numerous and include improvements in lipid and lipoprotein profiles as well as endothelial-dependent vasodilation stimulated by estrogen administered at physiological concentrations. Reports have described significant estrogen-stimulated increases in bioavailable NO. [3][4][5] Because the antiatherogenic properties of NO are emerging, it has been proposed that the cardiovascular protective effect of estrogen is mediated through augmentation of endothelial NO production. Using a human endothelial cell (EC) in vitro model, we have previously shown that 17␤-estradiol (E 2 ) induces endothelial NO release within minutes, is estrogen receptor (ER)-dependent and gene transcription-independent, and is the result of activation of endothelial nitric oxide synthase (eNOS). 6 The regulation of eNOS activity is multifaceted. This includes regulated palmitoylation and myristoylation, which are required for eNOS partitioning into membrane caveolae and consequent function. 7-10 A variety of cofactors are required for enzymatic function, including Ca 2ϩ , calmodulin, and tetrahydrobiopterin. 11,12 Recently, heat shock p...

Abstract-Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation. Key Words: nitric oxide Ⅲ signaling Ⅲ scaffold Ⅲ hsp90 Ⅲ Akt E ndothelial nitric oxide synthase (eNOS) continually produces low levels of nitric oxide (NO) to regulate several aspects of cardiovascular homeostasis. In endothelial cells and cells transfected with the eNOS cDNA, eNOS behaves as a peripheral membrane protein that is regulated by the allosteric activator, calmodulin (CaM). In vitro, the addition of CaM to recombinant eNOS markedly accelerates NOS catalytic function and NO synthesis. 1 However, in vivo, additional regulatory mechanisms other than CaM participate in eNOS activation/inactivation. This concept is supported by studies demonstrating that mislocalization of eNOS secondary to mutations that block its membrane association do not influence its catalytic function or calcium dependency in vitro; however, agonist-stimulated NO release from cells is markedly diminished. 2-4 These studies imply that spatial and temporal regulation of membrane associated eNOS function must involve other protein-protein or protein-lipid interactions that impact on its activation state.In the past several years, many protein partners that interact with eNOS have been described, including caveolins-1 and -3, 5,6 heat shock protein 90 (hsp90), 7 dynamin-2, 8 G protein-coupled receptors, 9 and certain kinases including Akt and mitogen-activated protein kinase family members. 10,11 All these proteins have been show to interact with eNOS in conventional in vitro assays, including coprecipitations, affinity chromatography, and yeast 2-hybrid analysis. In vivo, there is compelling evidence supporting the importance of caveolin-1, hsp90, and Akt in regulating NO release because overe...