Abstract 20829: Galectin-3 Promoter Hypomethylation Drives Gene Expression in Hypertensive PASMC

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Abstract

Introduction: Pulmonary Arterial Hypertension (PAH) is a progressive disease of the lung vasculature that is accompanied by excessive proliferation of pulmonary artery smooth muscle cells (PASMC). We have identified galectin-3 (Gal-3) as a pro-proliferative, pro-fibrotic and anti-apoptotic gene that is not only upregulated in PASMC from several models of PAH as well as in human PAH, but is functionally important as determined by the ability of Gal-3 inhibitors as well as a Gal-3 knockout rat to ameliorate altered hemodynamics and right ventricle remodeling in multiple models of PAH.

Hypothesis: However, how Gal-3 is upregulated remains poorly understood.

Results: PASMC isolated from PAH rats exhibited an enduring capacity for increased proliferation and expressed higher levels of Gal-3 suggesting an epigenetic mechanism of regulation. Treatment of PASMC with DNA methylation inhibitors robustly increased Gal-3 expression in both control human and rat PASMC but not in MCT-derived PASMC. Pyrosequencing analysis of DNA isolated from PA revealed hypomethylation of the Gal-3 proximal promoter. Analysis of DNA methyltransferase expression in PA revealed a significant decrease in Dnmt3A expression in hypertensive pulmonary arteries. Targeted methylation Gal-3 promoter using a CRISPR dCas9-DNMT3A fusion construct effectively reduced Gal-3 expression in SMC isolated from MCT rat PA and reversed the excessive proliferation. Regions on interest on the Gal-3 promoter were explored using a range of gRNAs that revealing location specific influences of DNA methylation. Bromodomain (BET) inhibitors had no significant effect on Gal-3 expression.

Conclusions: These results advance an important role of DNA methylation-dependent mechanisms in the regulation of Gal-3 expression during PAH.

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