Abstract 14210: Impaired Mitophagy and Reduced Parkin Expression in Human Pulmonary Arterial Smooth Muscle Cells (PASMCs) in Pulmonary Arterial Hypertension (PAH)

Abstract

Introduction: Mitophagy is the key mitochondrial quality control process of selective removal of damaged mitochondria which is necessary for maintaining cellular homeostasis. While studies have demonstrated that abnormalities in mitochondrial metabolism drive a hyperproliferative phenotype of pulmonary vasculature in PAH, there is limited understanding of mitophagy dysregulation in PAH. Recent studies have shown that hypoxia induced Parkin/Pink1-mediated mitophagy promotes vascular remodeling, however another report suggests that loss of Pink1 and reduced mitophagy in hypoxia promotes PASMC proliferation. The role and mechanisms of altered mitochondrial dynamics/mitophagy dysregulation in driving pulmonary vascular remodeling in PAH remain incompletely understood. Hypothesis: We hypothesized that impaired mitophagy and reduced Parkin expression may drive the apoptosis-resistant, hyperproliferative phenotype in PAH PASMCs. Methods: Mitophagy was assessed in human PASMCs from control donors and PAH patients stably expressing mtKeima protein and in lungs from hypoxia-exposed mtKeima transgenic mice. Parkin expression was assessed by Western blotting and immunocytochemistry. Immunofluorescence staining for Parkin was performed in lungs from mice exposed to chronic hypoxia. Human PASMCs from control donors were transfected with siControl and siParkin and assessed for proliferation and apoptosis by Ki67 and Annexin V/PI flow cytometry and PCNA expression. Results: PASMCs from PAH patients showed reduced KeimaRed vs. KeimaGreen ratio compared to control donors indicating reduced mitophagy i.e. further confirmed by decreased Parkin in PAH PASMCs. Parkin deficient PASMCs demonstrated more Ki67 and less Annexin V/PI positive cells and increased PCNA. Interestingly, lungs from hypoxia-exposed mtKeima transgenic mice showed increased KeimaRed vs. KeimaGreen ratio and increased Parkin in lungs of hypoxia-exposed mice. Conclusions: PAH PASMCs demonstrate impaired mitophagy and reduced Parkin expression; Parkin deficiency promotes proliferation and inhibits apoptosis in PASMCs. Our findings suggest that Parkin may regulate remodeling phenotypes in PASMCs and thus may represent a potential therapeutic target in PAH.