PROJECT SUMMARY/ABSTRACT Heart failure with preserved ejection fraction (HFpEF) affects more than 3 million adults of the United States and has no effective therapy. The promising effect of general strategies for improving endothelial cell (EC) function in HFpEF patients has highlighted the crucial roles of EC dysfunction in the disease pathogenesis. However, the molecular mechanism of EC dysfunction and how it contributes to the pathogenesis of HFpEF are still poorly understood. A recent patient study identified serum neuropilin-1 (NRP1) as a prognostic marker in HFpEF patients. Our preliminary data indicate that endothelial NRP1 is increased in the cardiac tissues of murine HFpEF models and mice deficient of endothelial NRP1 show improved EC and cardiac diastolic functions in these models. Tumor necrosis factor-α (TNFα) is a major inducer of EC dysfunction and associates with the disease progression of HFpEF. Our preliminary data suggest that knockdown of NRP1 reduces TNFα-induced expression of adhesion molecules but enhances the levels of endothelial nitric oxide synthase (eNOS) in ECs. Computational docking model and protein binding assay collectively indicate the direct interaction between NRP1 and TNFα. Based on these results, this project will test the central hypothesis that endothelial NRP1, acting as a novel co-receptor with TNFR, leads to the pathogenesis of HFpEF by increasing cardiac inflammation and impairing myocardial nitric oxide bioavailability. Two Specific Aims are proposed: Aim 1: Delineate the crosstalk between NRP1 and TNFα/TNFR in EC dysfunction. We will characterize the interaction between NRP1 and TNFα/TNFR using microplate-based binding assay and cultured ECs. The role of NRP1 in TNFα-induced EC dysfunction will be examined in both mouse and zebrafish endothelial cell specific NRP1 knockout models. Aim 2: Reveal the role of endothelial NRP1 in the pathogenesis of HFpEF. We will use mouse HFpEF models to investigate how endothelial NRP1 cooperates with TNFR1 to control the disease initiation and progression of HFpEF.