Mechanistic Roles of Transforming Growth Factor-Beta and Receptor Tyrosine Kinase Cross-Talks in Sprouting Angiogenesis

Abstract

Vascular sprouting is characterized by a series of morphological and functional transformations of endothelial cells (ECs) that result in the formation of an interconnected network of blood vessels. This complex phenomenon inevitably involves multiple signal transduction pathways and requires their precise synchronization in order to ensure accurate spatio-temporal coordination of sprouting angiogenesis. Through this dissertation, I have shed light on molecular cross-talks between transforming growth factor beta (TGF) and two receptor tyrosine kinases (RTKs), vascular endothelial growth factor receptor 2 (VEGFR2) and insulin receptor (IR) involved in angiogenesis. Despite considerable effort on its activation, little is known about the turnover or degradation of VEGFR2, the primary driver of sprouting angiogenesis. I report BMP9, a TGF family cytokine, as a crucial regulator of VEGFR2 in endothelial cells. My work shows that BMP9 enhances VEGFR2 degradation by involving a cytoskeletal protein complex of IV-spectrin/CamKII and critically inhibits vascular sprouting through a signaling cascade separate from the hallmark ID1/3 and Notch signaling pathways in determining tip/stalk cell selection during angiogenesis. The inhibitory roles of BMP9 on vascular sprouting were validated both in time and space though multiple in vivo tissue based studies. Insulin receptor or IR is another important RTK involved in angiogenesis. Although insulin is known to be a pro-angiogenic factor while in circulation and impaired insulin signaling is implicated in many common vascular disorders, the exact mechanism of insulin/IR controlling angiogenesis remain unclear. I report a distinct crosstalk between IR and endoglin/ALK1, an endothelial cell specific TGFβ receptor. While endoglin–ALK1, normally binds to TGFβ or BMP9 to promote Smad1/5 activation, I showed that insulin rapidly activates Smad 1/5 in ECs. This non-canonical smad1/5 activation in response to insulin involves a direct physical interaction of IR and endoglin–ALK1 complex at the endothelial cell surface. Additionally, I have identified EPDR1 as a major insulin responsive Smad1/5 gene target involved in angiogenesis. Together, this study indicated that insulin can signal through pathways other than the established PI3K/Akt and ERK pathways during angiogenesis and suggests important therapeutic implications for EPDR1 and the TGFβ pathways in pathologic angiogenesis during hyperinsulinemia and insulin resistance