Abstract 17192: ALDH1A3 Induces NFY to Coordinate Transcription of Cell Cycle and Metabolic Genes Necessary for Smooth Muscle Proliferation in Pulmonary Hypertension

Abstract

Background: In developing disease modifying therapies for pulmonary arterial hypertension (PAH), it is important to understand the molecular basis for hyperproliferation of pulmonary arterial smooth muscle cells (PASMC) that progressively occlude the vessel lumen. Previously we presented studies using H3K27 ChIP Seq and RNA Seq in PAH vs. control PASMC, and showed that an increase in ALDH1A3 is responsible for both heightened proliferation and H3K27ac enhancer marks in cell cycle and metabolic enzyme genes. Methods and Results: We now present molecular and metabolic studies and experiments in transgenic mice that delineate the mechanism by which ALDH1A3 plays a critical role in PAH PASMC proliferation. Promoter binding motif analysis of sites that are ALDH1A3 dependent in PAH PA SMC identified NFY family members as putative transcription factors. We confirmed their role in increasing three energy producing metabolic enzymes PKM2, DLD and IDH1 and two cell cycle genes, CCNA2 and CDC20. We verified that their mRNA transcripts and protein levels are elevated in PAH PASMC and dependent on increased ALDH1A3 and NFY(n=3, p<0.05) The accumulation of acetyl-CoA is increased in PAH PASMC with siControl vs. siALDH1A3. The nuclear localization of increased PAH PASMC ALDH1A3 indicates that generation of acetyl CoA from acetylaldehde could acetylate histones at H3K27 target sites marked by NFY binding. From non-targeted metabolomic analyses by LC/MS, we found ALDH1A3 dependent metabolites in the glycolytic and pentose phosphate pathways in PAH PASMC, consistent with the functions of PKM2, DLD and IDH1. We then showed that ALDH1A3 is induced by hypoxia, and required for SMC proliferation and pulmonary hypertension in mice exposed to three weeks of chronic hypoxia (10% oxygen). Gender matched transgenic mice with SMC deletion of Aldh1a3 had reduced pulmonary hypertension when compared to littermate controls, assessed by right ventricular systolic pressure (n=8, p<0.05). Conclusions: Our studies reveal an ALDH1A3-dependent coordination of histone acetylation at NFY transcription factor binding sites of key metabolic enzyme and cell cycle genes in highly proliferative PAH PASMC from PAH patients, that is critical in the development of pulmonary hypertension.