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Introduction:Pulmonary hypertension (PH) is associated with significant morbidity and mortality. RASA3, a GTPase-activating protein, is integral to processes highly relevant to PH. We explore the association of RASA3 genetic variation with PH risk in patients with sickle cell disease (SCD)-associated PH and pulmonary arterial hypertension (PAH).Methods:Using peripheral blood mononuclear cells (PBMC) from 3 cohorts of subjects with SCD, cis-expression quantitative trait loci (eQTL) were queried for RASA3. Genome-wide single nucleotide polymorphisms (SNP) near or in the RASA3gene that associate with lung RASA3 expression were identified using Genotype Tissue Expression project data, reduced to 9 tagging SNPs for RASA3and associated with PH markers. Associations between the top RASA3SNP and PAH severity were tested using whole genome genotype data from PAH Biobank subjects. Using RNAseq samples aligned with SNP arrays from lung tissue from the Pulmonary Hypertension Breakthrough Initiative (PHBI), we evaluated the frequency of the risk allele at our SNP of interest.Results:PBMC RASA3expression was significantly lower in patients with SCD-associated PH using elevated TRV≥2.5m/s and RHC data and associated with significantly higher mortality. One eQTL for RASA3 was identified, rs9525228, with the risk allele correlating with PH risk and severity in patients with SCD-associated PH. Analyzing this SNP in PAH patients based on either European or African ancestry (EA, AA), we found that rs9525228 associated with markers of PH in EA but not AA individuals (Table 1). There was a trend towards decreased survival in EA individuals carrying rs9525228 (p=0.08) that was not seen among AA individuals. Using PHBI samples, individuals homozygous for the risk allele had lower RASA3 expression (p=0.05).Conclusions:RASA3is a novel candidate gene in SCD-associated PH and PAH and appears to be protective. Further studies are ongoing to delineate the role of RASA3 in PH.

Introduction:Despite improvements in quality of life and functional status, pulmonary hypertension (PH) remains a highly morbid disease with poor life expectancy with no targeted treatments available. RASA3 is a ubiquitously expressed GTPase activating protein that is integral to angiogenesis and barrier function in endothelial cells (EC). In this study, we sought to explore the differential expression of RASA3 in response to PH-relevant stimuli and downstream markers of EC health when RASA3 expression is lost.Methods:Human pulmonary artery cells (PAECs) from Lonza were grown in culture and treated with 1% hypoxia, increasing concentrations of TNF-α for 24 hours or IL-1β for 48 hours and then harvested. Cells were transfected with siRNA directed against RASA3 or non-targeting control and harvested to evaluate downstream markers of EC health, or plated on Matrigel to evaluate angiogenic capabilities. Non-contact co-culture experiments were performed using human pulmonary artery smooth muscle cells (PASMC) with PAECs that were transfected with siRNA against RASA3 or non-targeting control.Results:We found that when untreated PAECs were exposed to 1% hypoxia for up to 96 hours, RASA3 expression was significantly decreased (p=0.046). When treated with increasing concentrations of TNF-α for 24 hours, RASA3 expression decreased (p=0.0002). When PAECs were treated with IL-1β 10ng/mL for 48 hours, RASA3 expression significantly increased (p=0.001). Silenced RASA3 PAECs had significantly less PCNA (p=0.0474), a trend towards increased HIF-2α expression (p=0.08) and increased VEGFR2 expression (p=0.0878). Cells that had silenced RASA3 expression when plated on Matrigel were significantly less able to form rings compared to non-targeting control (p=0.0152). PASMCs that had been co-cultured with silenced RASA3 had significantly increased PCNA compared to PASMCs co-cultured with non-targeting control PAECs (p=0.0467).Conclusions:RASA3 is essential to PAEC homeostasis, and loss of RASA3 affects endothelial cell health as well as affects its local environment. Further studies are ongoing to determine how loss of RASA3 contributes to the development of PH.

Introduction:Sickle cell disease (SCD)-associated PH is associated with increased mortality when tricuspid regurgitant jet velocity (TRV) is ≥2.5m/s and mPAP is ≥25mmHg on right heart catheterization. RASA3, a ubiquitously expressed GTPase-activating protein, is integral to angiogenesis and maintenance of endothelial barrier function.Hypothesis:Loss of RASA3 leads to the development of PH in patients with SCD-associated PH and PAH.Methods:Cis-eQTLs were queried for RASA3using whole genome genotype arrays and gene expression profiles from PBMCs of subjects with SCD from 3 cohorts. Genome-wide SNPs near or in the RASA3gene that may associate with lung RASA3 expression were identified using data from the Genotype-Tissue Expression project, reduced to 9 tagging SNPs for RASA3 and associated with TRV≥2.5m/s and RHC hemodynamics. Associations between the top RASA3 SNP and RHC hemodynamics were tested using whole genome genotype data from subjects in the PAH Biobank. PAECs collected from explanted lungs from patients with IPAH or control lungs were cultured with RASA3 expression evaluated on Western blot.Results:PBMC-derived RASA3 expression was significantly lower in patients with SCD-associated PH using TRV≥2.5m/s and RHC hemodynamics, with significantly higher mortality (Figure 1). The risk allele (T) at rs9525228, an eQTL for RASA3, correlated with PH risk, higher TRV and higher PVR among PBMCs associated with decreased RASA3 expression in patients with SCD-associated PH. The T allele at rs9525228 was significantly associated with increased mPAP (p=0.014), PVR (p=0.041) and mean right atrial pressure (p=0.04) in patients from the PAH Biobank. RASA3 expression was significantly lower in PAECs from patients with IPAH compared to control.Conclusions:RASA3is a novel candidate gene in SCD-associated PH and PAH, with RASA3 expression appearing to be protective. Further studies are ongoing to delineate the role of RASA3 in PH.