Abstract 427: Epigenetic Regulation Of Endothelial Anoctamin1 In Pulmonary Arterial Hypertension

Introduction/Hypothesis: Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH), by formation of occlusive plexiform lesions. We previously reported that Anoctamin-1 (ANO1), a calcium-activated chloride channel, expression is increased in lung ECs from patients with PAH and is associated with increased EC proliferation. However, the regulatory mechanism underlying ANO1 expression in PAH remains unclear. DNA methylation has been recognized as an important mechanism regulating gene expression. In this study, we test the hypothesis that ANO1 expression in PAH is epigenetically regulated via DNA methylation. Methods/Results: DNA was extracted from lung ECs of patients with idiopathic PAH (n=7) and controls (n=6), followed by Bisulfite Next-Generation Sequencing (EpigenDx, MA) to determine DNA methylation profile in human ANO1 gene (Ensembl Gene ID: ENSG00000131620). We used 19 assays to cover 97 CpG sites in the human ANO1 gene, including regions of 5’-upstream, 5’-UTR, and different introns. Our data show that the methylation of 29 of 97 (30%) CpG sites in human ANO1 gene is significantly altered in lung ECs from PAH patients than that from control subjects. Among the significantly altered 29 CpG sites, the methylation at 22 CpG sites (76%) is significantly decreased. We developed a CRISPR-based approach to target four differentially methylated CpG sites to determine the specific contribution of their methylation in regulation of human ANO1 gene. We demonstrate that demethylation of one of the sites, CpG#-73, in human embryonic kidney cells resulted in an increase in ANO1 mRNA expression compared to control, suggesting that decreased DNA methylation is sufficient to upregulate human ANO1 expression. Conclusions: Our results demonstrate that DNA methylation in human ANO1 gene is significantly altered in PAH and reduction in DNA methylation may serve as a regulatory mechanism for ANO1 upregulation in settings of PAH.