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Dissection of the Role of PPP2CA in the Pathogenesis of Pulmonary Arterial Hypertension

Revathi Rajkumar


JC Sembrat

Ferhaan Ahmad


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Conference: 2010 International PHA Conference and Scientific Sessions

Release Date: 06.24.2010

Presentation Type: Abstracts

Rajkumar R, Sembrat J, Ahmad F.
Cardiovascular Institute, Department of Medicine, University of Pittsburgh, PA, USA

BACKGROUNDPulmonary arterial hypertension (PAH) is a life threatening disease characterized by vascular remodeling resulting in increased mean pulmonary artery pressure (≥30 mm Hg during exercise) followed by subsequent right ventricular hypertrophy and failure. Lung vascular lesions in PAH comprise of enlarged, apoptosis resistant, proliferating pulmonary artery endothelial and smooth muscle cells (PAECs and PASMCs). However, the mechanisms leading to the proliferative and antiapoptotic environment in the vascular wall are poorly understood.

OBJECTIVE: As the first stage of a two-stage study designed to understand the pathogenesis of PAH, we have previously carried out an unbiased, genome-wide RNA expression profiling of lung tissues from 18 PAH subjects, 8 idiopathic pulmonary fibrosis subjects with secondary PH, and 13 normal subjects.

The present second stage in vitro study aims to validate the pathogenetic role of differentially expressed genes identified from PAH RNA expression profiling. This study, in particular, aims to determine the proliferative role of protein phosphatase 2, catalytic subunit, alpha isoform (PPP2CA) in developing PAH like characteristics in normal human PASMCs. The enzyme PPP2CA is one of four major protein phosphatases that plays a role in the regulation of most major metabolic pathways and is involved in negative regulation of cell growth.

METHODS AND RESULTS: RNA microarray analysis showed a 4-fold downregulation of PPP2CA in PAH lungs as compared to normal controls and PH secondary to IPF, which was confirmed by QPCR. siRNA targeting PPP2CA(20nM) was used to selectively knock down this gene in human PASMCs. Scrambled siRNA conjugated with Cy3 at the 5' end was used for monitoring transfection efficiency and also acted as negative control. QPCR confirmed 90% knockdown of PPP2CA mRNA 72 hours post transfection (Figure A), and immunoblots confirmed protein knockdown 4 days post transfection (Figure B).

Dissection of the Role of PPP2CA in the Pathogenesis of Pulmonary Arterial Hypertension

CONCLUSION: We anticipate that proliferation analysis and assessment of downstream expression changes (e.g., SOX18, CBP, Axin 2, beta-catenin, PPARG3) following PPP2CA knockdown in PASMCs will establish the role ofPPP2CA in PAH.