Fellowship for Bradshaw: EG-VEGF Mediated Angiogenesis

Hypoxia-induced angiogenesis is a compensatory mechanism to supply blood to ischemic tissues. In the peripheral vasculature, ischemia-initiated angiogenesis is controlled by a variety of growth factors, which allow for endothelial permeability, proliferation, and migration. Endocrine gland derived vascular endothelial growth factor (EG-VEGF) is a hypoxia-inducible mitogen. EG-VEGF causes proliferation, migration, and fenestration of the endothelium. EG-VEGF acts as an angiogenic factor on endothelial cells causing massive vascularization. EG-VEGF does not belong to the classical VEGF family, rather it has a unique in gene sequence, protein structure, and receptor subtype. To date, prokineticin receptor (EG-VEGF receptor) localization within the plasma membrane and resulting protein-protein interactions have not been elucidated. Also, EG-VEGF-mediated downstream molecular mechanisms of nitric oxide production and angiogenesis have not been fully assessed. Caveolae are cholesterol and sphingolipid rich invaginations of the plasma membrane, which are characterized by caveolin-1. Caveolae are recognized as centers for signal transduction, and caveolins modulate cellular signaling cascades. Caveolin-1 acts as a negative regulator to local molecules including receptors and endothelial nitric oxide synthase (eNOS). We have confocal data that shows prokineticin receptor-1 and -2 both colocalize with caveolin-1 and eNOS in SW-13 cells as well as transfected Chinese hamster ovary (CHO) cells, suggesting that these prokineticin receptors localize in caveolae. As a complementary approach, we employed subcellular fractionation which shows that prokineticin receptor-1 and -2 are enriched in caveolae membrane fraction along with caveolin-1. Furthermore, immunoprecipitation studies show that prokineticin receptor-1 and -2 bind caveolin- directly in transfected CHO cells as well as murine testes lysate. Upon binding of EG-VEGF, prokineticin receptor activates Akt, which is known to activate eNOS. Akt phosphorylates eNOS at ser1177, which causes increased and prolonged production of nitric oxide. Activation of eNOS has been linked to endothelial cell proliferation and increased endothelial cell permeability. It is important to elucidate EG-VEGF receptor localization and its molecular mechanism of angiogenesis because angiogenic molecules are the focus of many therapeutic efforts to promote new vessel development in ischemic tissue.