Mechanisms of Basement Membrane Regulation During Angiogenesis

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PROJECT SUMMARY During angiogenesis, endothelial cells secrete a variety of proteins that compose a planar protein network that encapsulate blood vessels, collectively termed basement membrane. Missteps in this crucial process can manifest as a life-threatening heritable form of intracerebral hemorrhage as well as contributing to stroke and aneurysm. This association with disease highlights the importance of the vascular basement membrane as an indispensable macromolecular scaffold to withstand the continuous mechanical strain of blood circulation. To date, the biomedical community knows extraordinarily little about how basement membrane proteins are regulated by the vascular endothelium during angiogenesis. Our preliminary investigations suggest a novel scenario in which a Rab10 endosome-mediator directly regulates secretion and storage of multiple basement membrane proteins during blood vessel formation. Our results indicate that Rab10 may be a never-before recognized common pipeline for basement membrane proteins, easily switching from polarized secretion to degradation, depending on the stimulus. Our general working hypothesis is that endothelial cells predominately use a Rab10 trafficking pathway to regulate basement membrane deposition, contributing directly to maintenance of vascular stability. We will test this general hypothesis in three aims. First, we will mechanistically explore how Rab10 orchestrates the secretion of multiple, critical basement membrane proteins in endothelial cells. In this in vitro aim, using biochemical, 2-dimensional and 3-dimensional static and live-imaging assays, we will thoroughly explore how Rab10 and its related effectors influence endothelial basement membrane deposition. Second, we will used transgenic zebrafish to evaluate Rab10's role in angiogenesis and vascular basement membrane regulation, levering zebrafish's innate live-imaging capabilities. Third, we will employ a gold-standard transgenic mouse model to conditionally ablate Rab10 in the vasculature to probe how Rab10 affects blood vessel morphogenesis as well as adult blood vessel basement membrane integrity. We believe an understanding of this vascular basement membrane biology would constitute an important first-step toward determining how disease states disrupt this process and manifest into life-threatening vascular disorders, as well as a significant advancement of our understanding of the angiogenic process as a whole.