Endothelial Rap1 restricts inflammation in the retina - PROJECT SUMMARY Endothelial-leukocyte interactions are pivotal in Diabetic Retinopathy (DR), a major cause of global vision loss. Proinflammatory factors like tumor necrosis factor alpha (TNF-α) drive hyperactivity in retinal endothelial cells (RECs), leading to inflammation, increased leukocyte adhesion, and ultimately pathological neovascularization. Understanding REC-leukocyte dynamics is crucial for early intervention. Vascular Endothelial Growth Factor (VEGF), is a key player in DR influencing REC behavior and serving as therapy target. However, it also maintains cellular homeostasis during inflammation. Deciphering retinal mechanisms balancing proinflammatory and homeostatic responses is crucial for optimizing DR treatments. REC responsiveness hinges on lipid rafts and specialized plasma membranes domains, caveolae. Understanding how homeostatic and proinflammatory signals are balanced in these structures remains a critical research goal. Our published data indicate that endothelial Rap1B (Ras association proximate 1B), a signaling molecule in ECs, enhances nitric oxide release and limits proinflammatory signaling in ECs, and is essential for VEGFR2 activation and signaling. In the retina, Rap1B is critical for physiological retinal development yet endothelial Rap1B knockout (KO) reduces VEGF-driven pathological hyper-permeability in streptozotocin-induced diabetes. Our preliminary data indicate Rap1B localizes to cholesterol-rich lipid rafts to orchestrate VEGFR2 signaling. Restoring cholesterol in Rap1B-deficient ECs reinstates deficient VEGF-triggered NO signaling. These results led to our hypothesis: Rap1B governs the equilibrium between homeostasis and inflammation in RECs during DR progression by modulating the composition and organization of lipid rafts. To investigate the regulatory role of Rap1B in RECs in progression of DR, we propose to: (Aim1) Assess Rap1B's role in regulating VEGF- VEGFR2 lipid rafts. Employ a two-tier genetic strategy (Rap1B KO and functional mutants in RECs), complemented by advanced microscopy, omics, and signal transduction analyses, to elucidate Rap1B's influence on VEGFR2 lipid raft structure, composition, and signaling. (Aim 2) Examine how Rap1B controls the balance between VEGFR2 and TNFR1 signaling. Apply methods from Aim 1 to discern Rap1B's effect on TNFR1 lipid raft organization, composition, and redox signaling. Evaluate Rap1B's role in ROS and NO-mediated leukostasis and explore hyperglycemia's impact on Rap1B signaling. (Aim 3) Establish the impact of EC Rap1B on EC-leukocyte dynamics in vivo. Utilize advanced microscopy and transcriptomics to determine Rap1B's role in Rap1B on leukocyte-dependent vascular obliteration, permeability and leukostasis in DR and using a retinopathy of prematurity model, on neovascularization. These studies will define molecular roles of Rap1 in RECs and provide critical insights into the mechanisms controlling the balance of REC responses to VEGF and proinflammatory stimuli in DR, information critical to improve therapeutic strategies for retinal ischemic diseases.
