Pulmonary hypertension (PH) is a complication of sickle cell disease (SCD) that causes significant morbidity and mortality. Novel therapeutic strategies that target multiple mediators in SCD-PH have the potential to reduce this devastating complication of SCD. Recent findings indicate that activation of the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARγ), attenuates PH and several mediators of hypoxia-induced PH, including NOX4 and ET-1, and favorably modulates microRNAs (miRNAs or miR) by reducing miR-27a (which reduces PPARγ) and increasing miR-98 (which reduces ET-1). miRNAs are differentially expressed during development and disease, but pharmacological targeting of miRNAs has not been defined in SCD-PH. Based on our preliminary and published studies, we will explore the hypothesis that heme released during intravascular hypoxia/hemolysis in SCD induces miRNAs in the vascular wall that reduce PPARγ levels and increase NF-κB levels thereby contributing to endothelial dysfunction and PH pathogenesis. New preliminary data support the feasibility of using chronic hypoxia to induce more severe PH, right ventricle hypertrophy (RVH), and pulmonary vascular remodeling in SCD to test novel therapeutic approaches. Aim 1 will examine regulation of pathogenic miRNAs in SCD-PH. Preliminary data indicate that hemin activates v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1) to increase miR-27a thereby reducing PPARγ and enabling miR-98 upregulation in the lung in SCD-PH and in hemin-treated human pulmonary artery endothelial cells (HPAECs). The role of altered endothelial PPARγ in SCD-PH will be further examined using novel chimeric mouse models generated by transplanting SCD bone marrow into mice with endothelial-targeted PPARγ gain (SSePPARγOX) or loss (SSePPARγKO) of function. These models will permit direct analysis of the ETS1/miR-27a/PPARγ/miR-98 signaling axis in SCD-PH. Aim 2 is based on evidence that PPARγ increases levels of the E3 ubiquitin ligase, HUWE1 (which also constitutes the host gene of miR-98) to induce ubiquitination of NF-κB and termination of NF-κB activation in SCD-PH. Aim 2 will explore the role of reduced PPARγ in NF-κB activation and endothelial dysfunction in SCD-PH. Aim 2 will determine the impact of PPARγ on HUWE1 and NF-κB using PPARγ gain or loss of function approaches or using PPARγ activation with pharmacological ligands. PPARγ regulation of HUWE1 will be confirmed with in vitro studies using luciferase reporter and ChIP assays. Pull-down and ubiquitination assays will determine if HUWE1 acts as an E3 ligase for promoting NF-κB ubiquitination in vitro, and endothelial-targeted HUWE1 KO mice will be used for in vivo studies. The overall goal of this proposal is to define novel molecular pathways involved in SCD-PH pathogenesis that can be therapeutically targeted. The results of these pre-clinical studies can inform future clinical trials examining PPARγ ligands as a novel t...