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》

A phosphodiesterase-5 inhibitor vardenafil enhances angiogenesis through a protein kinase G-dependent hypoxia-inducible factor-1/vascular endothelial growth factor pathway.

We examined whether phosphodiesterase-5 (PDE5) inhibition can promote ischemia-induced angiogenesis. Unilateral hindlimb ischemia was generated by resecting right femoral artery in wild-type C3H/He mice, treated with either vehicle or a PDE5 inhibitor vardenafil (10 mg/kg per day). Four weeks after surgery, vardenafil significantly enhanced blood flow recovery and augmented capillary collateral formation in ischemic muscle (blood flow ratios of ischemic/nonischemic leg: 0.52+/-0.17 [vehicle] versus 0.92+/-0.09 [vardenafil], P<0.01). Vardenafil upregulated protein expression of vascular endothelial growth factor and hypoxia-inducible factor (HIF)-1 alpha in ischemic muscle and enhanced mobilization of Sca-1/Flk-1-positive endothelial progenitor cells (EPCs) in peripheral blood and bone marrow, contributing to neovascularization. Vardenafil also promoted capillary-like tube formation of human umbilical vein endothelial cells and increased the number of human blood mononuclear cell-derived EPCs in vitro. Furthermore, reporter assays showed that vardenafil and cGMP activated the transactivation activity of HIF-1 under hypoxia. These effects of vardenafil were markedly inhibited by genetic ablation of endothelial nitric oxide synthase, a soluble guanylate cyclase inhibitor, and a protein kinase G inhibitor, respectively. Our results suggest that PDE5 inhibition enhances ischemia-induced angiogenesis with mobilization of EPCs through a protein kinase G-dependent HIF-1/vascular endothelial growth factor pathway. PDE5 inhibition may have a therapeutic potential to treat ischemic cardiovascular diseases.

Sahara M ，Sata M ，Morita T ，Nakajima T ，Hirata Y ，Nagai R ... -  《-》

Fractalkine, a CX3C chemokine, as a mediator of ocular angiogenesis.

Fractalkine (FKN) is a chemoattractant and adhesion molecule for leukocytes. Angiogenic effect of FKN also has been reported. This study was an investigation of FKN-mediated angiogenesis in vitro and in vivo to determine its role in ocular angiogenic disorders. FKN effects on cultured human umbilical vein endothelial cells (HUVECs) and bovine retinal capillary endothelial cells (BRECs) were evaluated with chemotaxis assay and a synthetic matrix capillary tube formation assay in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were used to detect mRNA and protein expression of FKN and its receptor, CX3CR1, in HUVECs and BRECs. A rabbit corneal neovascularization assay and an oxygen-induced retinopathy (OIR) model of mice were used to test the angiogenic property of FKN in vivo. FKN levels of vitreous samples from patients with proliferative diabetic retinopathy were measured by enzyme-linked immunosorbent assay (ELISA). Immunodepletion of FKN in PDR vitreous samples by anti-FKN polyclonal antibody was observed in endothelial cell chemotaxis assays. FKN significantly induced migration of HUVECs and BRECs. FKN induced formation of endothelial cell capillary tubes on synthetic matrix. Expression of FKN and CX3CR1 was detected in HUVECs and BRECs by RT-PCR and Western blot analysis. FKN significantly induced more blood vessel growth than did the control in the rabbit corneal pocket neovascularization assay. Intravitreal injection of anti-mouse FKN antibody decreased retinal angiogenesis in the OIR model. The vitreous level of FKN was elevated in patients with PDR compared with control subjects. Immunodepletion of soluble FKN from PDR vitreous samples caused 36.6% less migration of BRECs. FKN is an angiogenic mediator in vitro and in vivo. The vitreous level of FKN was elevated in patients with PDR. FKN may play an important role in ocular angiogenic disorders such as PDR.

You JJ ，Yang CH ，Huang JS ，Chen MS ，Yang CM ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

HIF-1alpha and HIF-2alpha play a central role in stretch-induced but not shear-stress-induced angiogenesis in rat skeletal muscle.

Angiogenesis, which is essential for the physiological adaptation of skeletal muscle to exercise, occurs in response to the mechanical forces of elevated capillary shear stress and cell stretch. Increased production of VEGF is a characteristic of endothelial cells undergoing either stretch- or shear-stress-induced angiogenesis. Because VEGF production is regulated by hypoxia inducible factors (HIFs), we examined whether HIFs play a significant role in the angiogenic process initiated by these mechanical forces. Rat extensor digitorum longus (EDL) muscles were overloaded to induce stretch, or exposed to the dilator prazosin to elevate capillary shear stress, and capillaries from these muscles were isolated by laser capture microdissection for RNA analysis. HIF-1alpha and HIF-2alpha transcript levels increased after 4 and 7 days of stretch, whereas a transient early induction of HIF-1alpha and HIF-2alpha transcripts was detected in capillaries from prazosin-treated muscles. Skeletal muscle microvascular endothelial cells exposed to 10% stretch in vitro showed an elevation in HIF-1alpha and HIF-2alpha mRNA, which was preceded by increases in HIF-binding activity. Conversely, HIF-1alpha and HIF-2alpha mRNA were reduced significantly, and HIF-alpha proteins were undetectable, after 24 h exposure to elevated shear stress (16 dyn cm(-2) (16 x10(-5) N cm(-2)). Given the disparate regulation of HIFs in response to these mechanical stimuli, we tested the requirement of HIF-alpha proteins in stretch- and shear-stress-induced angiogenesis by impeding HIF accumulation through use of the geldanamycin derivative 17-DMAG. Treatment with 17-DMAG significantly impaired stretch-induced, but not shear-stress-induced, angiogenesis. Together, these results illustrate that activation of HIF-1alpha and HIF-2alpha contributes significantly to stretch- but not to shear-stress-induced capillary growth.

Milkiewicz M ，Doyle JL ，Fudalewski T ，Ispanovic E ，Aghasi M ，Haas TL ... -  《-》

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 ... -  《-》