S42 Characterizing ATP13A3 loss of function in pulmonary arterial hypertension (PAH)

Abstract

Introduction Idiopathic pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vascular occlusion and eventually right heart failure. Causal genetic mutations are implicated in both familial (>80%) and sporadic (30%) forms of idiopathic PAH. From a recent whole genome sequencing study of 1038 PAH patients, we identified ATP13A3 mutations in a subset of patients. ATP13A3 is a poorly characterized P-type ATPase. The aim of this study is to understand the impact of ATP13A3 loss of function on pulmonary vascular cell function and provide insights into the pathogenesis of PAH in these patients. Method ATP13A3 mRNA expression was compared in human pulmonary vascular cells using qPCR. ATP13A3 knockdown was achieved using siRNA transfection. Pulmonary artery endothelial cell (PAEC) proliferation was determined by cell counting. PAEC Apoptosis was assessed by Annexin V/PI staining and Caspase-Glo 3/7 assay. Endothelial permeability was assayed by measuring the transit of horseradish peroxidase across PAEC monolayers in the presence or absence of thrombin. Results ATP13A3 mRNA expression was similar between human PAECs, pulmonary artery smooth muscle cells and blood outgrowth endothelial cells, although immunohistochemistry in human lung indicated a predominant endothelial expression pattern. ATP13A3 was expressed at higher levels in PAECs than other P-type ATPase family members. Knockdown of ATP13A3 in PAECs resulted in reduced cell proliferation, associated with reduced expression of cyclins. Loss of ATP13A3 in PAECs under low serum conditions (0.1%FBS) predisposed cells to early apoptosis, confirmed by both Annexin V/PI staining and increased caspase3/7 activity. ATP13A3 deficiency in human PAECs also resulted in an increase of endothelial monolayer permeability and an enhanced response to thrombin. Conclusion ATP13A3 is expressed in PAECs and plays a role in cell proliferation, apoptosis and endothelial permeability. These data provide initial insights into the pathogenicity of ATP13A3 mutations in patients with PAH. This work is produced by Prof. Nick Morrell’s Group on behalf of the UK PAH Cohort Study.