Endothelial diaphragms are protein barriers associated with vascular endothelial subcellular structures, such as fenestrae, transendothelial channels (TEC), caveolae and vesiculo-vacuolar organelles implicated in vascular permeability and immune response. Fenestrae are circular transcellular pores in vascular endothelial cells playing critical roles in the maintenance of basal permeability. Recent data in humans and mice demonstrate that fenestrae and their diaphragms play critical roles in the maintenance of normal endothelial barrier function, blood homeostasis and ultimately survival. Our group demonstrated that Plasmalemma Vesicle Associated Protein (PLVAP or PV1) is critical for diaphragm assembly, and that absence of PV1 in mice and humans causes abnormal fenestrae and perinatal lethality due to multiple vascular defects. PV1 is also critical in the immune response, and therapeutic strategies based on PV1 biology are being developed for myocardial infarction, immune diseases and cancer. While there is emerging interest in diaphragms and fenestrae in the clinic, there is a significant knowledge gap in our understanding of the components and regulatory pathways governing fenestrae and diaphragm morphogenesis, addressed in this proposal. Critically for this proposal, PV1 is the only known marker for diaphragms, and we will exploit this property in our investigation of regulation diaphragm morphogenesis. Based on our preliminary data and literature, our central hypothesis is that multiple signaling pathways synergize in vascular bed specific manner for diaphragm and fenestrae formation. Specifically, crosstalk between growth factors and laminin binding integrin receptors are critical for PV1 expression and fenestrae and diaphragm morphogenesis. This hypothesis is based on our key new observations including: 1) differential segmental and spatial control of fenestrae in different vascular beds; 2) Gene expression screens for factors involved in PV1 regulation, identified SCF, FGF-5, VEGF-A and specific laminins signaling via their respective c-KIT, FGFR1, VEGFR2, and ITGB1 receptors as necessary for PV1 expression in ERK1/2 dependent manner; and 3) the combination of laminin with SCF, FGF-5 and VEGF- A results in robust PV1 expression and de novo fenestrae formation We will test our hypothesis using a combination of functionally validated endothelial cell culture systems and genetically engineered animal models using loss of function, gain of function and reconstitution approaches. Our Aims will define the role and downstream signaling pathways of newly identified growth factors and laminins and their integrin receptors regulating PV1 expression and fenestrae morphogenesis. We are uniquely positioned both in terms of key and necessary expertise to complete this work. These investigations will define regulatory factors and pathways controlling the assembly for an intricate and physiologically relevant cellular structure, providing valuable insight...