Role of afadin in vascular endothelial growth factor- and sphingosine 1-phosphate-induced angiogenesis.

Angiogenesis contributes to physiological and pathological conditions, including atherosclerosis. The Rap1 small G protein regulates vascular integrity and angiogenesis. However, little is known about the effectors of Rap1 involved in angiogenesis. It is not known whether afadin, an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1, plays a role in angiogenesis. We investigated the role of endothelial afadin in angiogenesis and attempted to clarify the underlying molecular mechanism. Treatment of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) induced the activation of Rap1. Activated Rap1 regulated intracellular localization of afadin. Knockdown of Rap1 or afadin by small interfering RNA inhibited the VEGF- and S1P-induced capillary-like network formation, migration, and proliferation, and increased the serum deprivation-induced apoptosis of HUVECs. Knockdown of Rap1 or afadin decreased the accumulation of adherens and tight junction proteins to the cell-cell contact sites. Rap1 regulated the interaction between afadin and phosphatidylinositol 3-kinase (PI3K), recruitment of the afadin-PI3K complex to the leading edge, and the activation of Akt, indicating the involvement of Rap1 and afadin in the PI3K-Akt signaling pathway. Binding of afadin to Rap1 regulated the activity of Rap1 in a positive-feedback manner. In vivo, conditional deletion of afadin in mouse vascular endothelium using a Cre-loxP system impaired the VEGF- and S1P-induced angiogenesis. These results demonstrate a novel molecular mechanism by which Rap1 and afadin regulate the VEGF- and S1P-induced angiogenesis.

Tawa H ，Rikitake Y ，Takahashi M ，Amano H ，Miyata M ，Satomi-Kobayashi S ，Kinugasa M ，Nagamatsu Y ，Majima T ，Ogita H ，Miyoshi J ，Hirata K ，Takai Y ... -  《-》

Necl-5/poliovirus receptor interacts with VEGFR2 and regulates VEGF-induced angiogenesis.

Vascular endothelial growth factor (VEGF), a major proangiogenic agent, exerts its proangiogenic action by binding to VEGF receptor 2 (VEGFR2), the activity of which is regulated by direct interactions with other cell surface proteins, including integrin α(V)β(3). However, how the interaction between VEGFR2 and integrin α(V)β(3) is regulated is not clear. To investigate whether Necl-5/poliovirus receptor, an immunoglobulin-like molecule that is known to bind integrin α(V)β(3), regulates the interaction between VEGFR2 and integrin α(V)β(3), and to clarify the role of Necl-5 in the VEGF-induced angiogenesis. Necl-5-knockout mice displayed no obvious defect in vascular development; however, recovery of blood flow after hindlimb ischemia and the VEGF-induced neovascularization in implanted Matrigel plugs were impaired in Necl-5-knockout mice. To clarify the mechanism of the regulation of angiogenesis by Necl-5, we investigated the roles of Necl-5 in the VEGF-induced angiogenic responses in vitro. Knockdown of Necl-5 by siRNAs in human umbilical vein endothelial cells (HUVECs) inhibited the VEGF-induced capillary-like network formation on Matrigel, migration, and proliferation, and conversely, enhanced apoptosis. Coimmunoprecipitation assays showed the interaction of Necl-5 with VEGFR2, and knockdown of Necl-5 prevented the VEGF-induced interaction of integrin α(V)β(3) with VEGFR2. Knockdown of Necl-5 suppressed the VEGFR2-mediated activation of downstream proangiogenic and survival signals, including Rap1, Akt, and endothelial nitric oxide synthase. These results demonstrate the critical role of Necl-5 in angiogenesis and suggest that Necl-5 may regulate the VEGF-induced angiogenesis by controlling the interaction of VEGFR2 with integrin α(v)β(3), and the VEGFR2-mediated Rap1-Akt signaling pathway.

Kinugasa M ，Amano H ，Satomi-Kobayashi S ，Nakayama K ，Miyata M ，Kubo Y ，Nagamatsu Y ，Kurogane Y ，Kureha F ，Yamana S ，Hirata K ，Miyoshi J ，Takai Y ，Rikitake Y ... -  《-》

FHL-2 suppresses VEGF-induced phosphatidylinositol 3-kinase/Akt activation via interaction with sphingosine kinase-1.

In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs). Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers. FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

Hayashi H ，Nakagami H ，Takami Y ，Koriyama H ，Mori M ，Tamai K ，Sun J ，Nagao K ，Morishita R ，Kaneda Y ... -  《-》

Activation of fractalkine/CX3CR1 by vascular endothelial cells induces angiogenesis through VEGF-A/KDR and reverses hindlimb ischaemia.

The present study investigated the detailed mechanism by which fractalkine (Fkn), a CX3C chemokine, induces angiogenesis and its functional implication in alleviating ischaemia in vivo. Fkn induced new vessel formation on the excised rat aorta and chick chorioallantoic membrane (CAM) through CX3CR1 activation. Immunoblotting analysis, promoter assay and electrophoretic mobility shift assay showed that Fkn upregulated hypoxia-inducible factor-1 alpha (HIF-1alpha) by cultured human aortic endothelial cells (ECs), which in turn induced mRNA and protein levels of vascular endothelial growth factor (VEGF)-A through a p42/44 mitogen-activated protein kinase pathway. In vivo Fkn-induced angiogenesis on CAM was completely blocked by functional inhibition of VEGF receptor 2 kinase insert domain-containing receptor (KDR) and Rho GTPase. C57/BL6 mice with CX3CR1(-/-) bone marrow-derived cells developed angiogenesis in the implanted Fkn-mixed Matrigel plug, suggesting CX3CR1 activation in vascular ECs is sufficient for Fkn-induced angiogenesis in vivo. The condition of rat hindlimb ischaemia, which rapidly stimulated mRNA expression of both Fkn and VEGF-A, was significantly alleviated by the injection of whole-length Fkn protein. Fkn-induced activation of CX3CR1 by ECs leads to in vivo angiogenesis through two sequential steps: the induction of HIF-1alpha and VEGF-A gene expression by CX3CR1 activation and the subsequent VEGF-A/KDR-induced angiogenesis. The potent induction of angiogenesis by Fkn can be used as a therapeutic strategy for alleviating peripheral ischaemia.

Ryu J ，Lee CW ，Hong KH ，Shin JA ，Lim SH ，Park CS ，Shim J ，Nam KB ，Choi KJ ，Kim YH ，Han KH ... -  《CARDIOVASCULAR RESEARCH》

Involvement of the nectin-afadin complex in PDGF-induced cell survival.

The nectin-afadin complex is involved in the formation of cell-cell junctions, such as adherens junctions (AJs) and tight junctions (TJs). Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules, whereas afadin is an intracellular nectin-binding protein that connects nectins to the cadherin-catenin system at AJs and to the claudin-zona-occludens (ZO) protein system at TJs. Afadin(-/-) mice show embryonic lethality, resulting from impaired migration and improper differentiation of cells due to disorganization of cell-cell junctions during gastrulation. However, it remains to be elucidated whether disruption of afadin affects apoptosis. In the present study, we first found that embryoid bodies derived from afadin-knockout embryonic stem (ES) cells contained many more apoptotic cells than those derived from wild-type ES cells. We also revealed that apoptosis induced by serum starvation or Fas-ligand stimulation was increased in cultured NIH3T3 cells when afadin or nectin-3 was knocked down. The nectin-afadin complex was involved in the platelet-derived growth factor (PDGF)-induced activation of phosphatidylinositol 3-kinase (PI3K)-Akt signaling for cell survival. This complex was associated with PDGF receptor on the plasma membrane at cell-cell adhesion sites. Thus, the nectin-afadin complex is involved in PDGF-induced cell survival, at least through the PI3K-Akt signaling pathway.

Kanzaki N ，Ogita H ，Komura H ，Ozaki M ，Sakamoto Y ，Majima T ，Ijuin T ，Takenawa T ，Takai Y ... -  《JOURNAL OF CELL SCIENCE》