SUMMARY/ABSTRACT: Venous stenosis (VS) and neointimal hyperplasia (VNH) often occur in arteriovenous fistulas (AVFs) after percutaneous transluminal angioplasty (PTA), leading to reduced AVF blood flow for hemodialysis (HD) in end- stage renal disease (ESRD) patients. This renewal's long-term goals are to: 1. Define mechanisms by which VS and VNH occur in AVFs after PTA; and 2. Develop new pharmacologic therapies to attenuate VS/VNH in AVFs after PTA in rodent/porcine models for a future clinical trial. In 2016, 726,331 US patients (~2M globally) were diagnosed with ESRD, with an annual 3% rising incidence. Roughly 87% of ESRD patients undergo renal replacement therapy with HD. Optimal long-term HD necessitates vascular access via AVFs or grafts. One year AVF patency rate is only 62%, with frequently repeated angioplasty maintenance at >$3B/yr. The mechanisms underpinning AVF VS/VNH after angioplasty are unknown. Therapies preventing VS/VNH, the major cause of AVF failure after PTA, will reduce morbidity and healthcare costs, of huge benefit to patients with ESRD. In the prior award (with 3 years and reduced funding), we studied the efficacy of adventitial delivery of 1,25(OH)2D3- coated nanoparticles (1,25 NP) to the AVF outflow vein to reduce VNH in mice and pigs with chronic kidney disease (CKD). We showed treatment of AVFs with 1,25 NP reduces VNH and VS of AVFs, the number of macrophages, smooth muscle cells (SMC) and fibroblasts, immediate early response gene (Iex-1/Ier3) expression and cellular proliferation, leads to less outflow vein fibrosis. This proposal examines AVF failure pathophysiology following PTA for which no effective therapies exist. Recent trials using paclitaxel coated technologies yielded mixed results— clinical practice adoption has been diminished due to increased all-cause mortality in patients treated with these devices for peripheral arterial disease symptoms. New therapies based on mechanistic observations are needed to prevent VS/VNH after PTA. In response, we developed murine and porcine models of CKD with stenotic AVFs treated with PTA and obtained data establishing the central role of Ier3/Iex-1 and monocyte chemoattractant protein-1 (Mcp-1/Ccl2) driven cellular infiltration in the pathogenesis of AVF VS. Adventitial delivery of 1,25 NP to the stenotic vein after PTA reduced VS and VNH in AVFs in our models. This application examines the mechanism of VS/VNH following PTA, testing 1,25NP efficacy in both murine and porcine models for a future clinical trial. Central Hypothesis: Treatment of stenotic outflow veins after angioplasty with adventitial 1,25NP will reduce SMC Ier3 and subsequent Mcp-1/Ccl2 expression and vessel wall infiltration by monocytes and macrophages resulting in less VS and VNH. To test this hypothesis, we propose two aims: Aim 1: Assess the role(s) of SMC Ier3 deletion and Mcp-1/Ccl2 knockdown on VS/VNH after PTA of murine AVFs. Aim 2: Determine the mechanism of action and effect of 1,25 NP on...