Vascular integrity is essential for the prevention of vascular diseases. Barrier function of the endothelium is tightly regulated to ensure proper homeostasis of various components of the blood vessel wall. Sustained endothelial activation and loss of barrier function significantly contributes to tissue edema, perpetuates the inflammatory response, and exacerbates tissue injury ultimately resulting in organ failure. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid found in high concentration in the blood and lymph. S1P signals through five G protein-coupled receptors, S1PR1-S1PR5. In endothelial cells, activation of S1PR1 induces vasodilation, reduces inflammation and decreases endothelial permeability. Aberrant S1P signaling is observed in atherosclerosis, hypertension and stroke. The endocytosis of S1PR1 is a key regulatory mechanism to control its signaling and function. However, multiple factors can influence the endocytosis of S1PR1, and specific factors present on endothelial cells modulating S1PR1 endocytosis are poorly understood. Hence, I hypothesize that novel regulatory factors of S1PR1 endocytosis will affect its cell surface expression and modify its function in the microvasculature. These modifications will affect vessel morphology, endothelial integrity and blood flow. In my first aim, I will identify novel regulatory molecules of S1PR1 endocytosis by a whole genome RNAi screen in U2OS cells stably expressing S1P1-GFP, and confirm putative hits, stimulators or inhibitors of S1PR1 endocytosis, in a secondary screen in endothelial cells (HUVEC). I will characterize factors in vitro by the knock down of their expression with a CRISPR/Cas9 method in HUVEC. I will assess their regulatory role on S1PR1 endocytosis, signaling, barrier function and proliferation. My second aim will focus on the characterization of these regulating factors of S1PR1 in vivo. I will use a Cre inducible CRISPR/Cas9 mouse strain bred with a VE-Cadherin Cre mouse. I will deliver the gRNAs targeting the protein with an adeno-associated virus. As a readout for S1PR1 function on vessel morphology and integrity, I will use Matrigel based tumor-induced angiogenesis. Tumor growth, vessel morphology and leakage will be evaluated in the role of these regulatory factors on S1PR1 to comprehend the mechanism of action of the regulatory factors in the activation pathway of S1PR1, which is part of the endocytosis process and represent a potential pharmacological target.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||07/01/2018 → 06/30/2020|