Abstract Body (Do not enter title and authors here): BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by obliteration of distal pulmonary arteries (PA) in association with endothelial cell (EC) dysfunction, leading to smooth muscle proliferation. SOX17 is a transcription factor (TF) expressed in arterial EC that is critical in vascular development. Deleterious variants causing reduced expression of SOX17 are linked to PAH, particularly in congenital heart defects (CHD) that cause increased PA flow and high shear stress (HSS). HYPOTHESIS: SOX17 deficiency is additive to HSS in compromising PAEC homeostasis and in promoting severe PAH. METHODS: SOX17 was reduced (>70%) by siRNA in primary human PAEC cultured in chamber slides in the IBIDI perfusion system. Computational modeling of distal PA indicated pathological HSS of 100 dyn/cm² in CHD with PAH whereas physiological laminar shear stress (LSS) is 15 dyn/cm². EC were preconditioned under LSS for 24h, followed by HSS or LSS for 24h. RESULTS: SOX17 expression was increased under LSS versus static condition, as were known SOX17 target genes (e.g., GJA5, GJA4, CGNL1, JAG1, and CTNNB1). SOX17 siRNA and HSS similarly reduced SOX17 target genes and when combining SOX17 siRNA with HSS they were further decreased owing to an interaction between SOX17 and ERG, a TF reduced by HSS. Indeed, SOX17 and ERG motifs marked most enhancer and promoter H3K27acetyl marks that were reduced under HSS. We then carried out RNAseq of PAEC to find genes co-regulated by SOX17 and ERG (reduced by siRNA for either TF under LSS), decreased under HSS and more-so with HSS + SOX17 siRNA. Those downregulated included SOX17 targets (e.g., CGNL1 and GJA5) and others not previously described, with links to BMPR2 signaling, YAP1 (inducer of BMP ligands and suppressor of NF-kB), SETBP1 (inducer of BMPR2 co-receptor BMPR1b), and TMEM100 and SULT1B1 important in NOTCH signaling and EC specification. We also found novel extracellular matrix target genes, e.g., elastin (ELN, top DEG), HMCN1, TMTC1 and chromatin remodeler (TOX). Among genes upregulated, were NAMPT, FAS, LYN, HPSE related to NF-kB activation and/or inflammation. CONCLUSION: HSS plus SOX17 deficiency profoundly compromises EC homeostatic genes, among which are those affecting BMPR2 and NOTCH pathways, ELN fiber assembly, and those promoting inflammation. This can explain why SOX17 mutations are associated with severe PAH in HSS-related congenital heart defects.