Abstract 18643: Role for RUNX2 in Pulmonary Arterial Hypertension

Abstract

Introduction: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced pulmonary artery smooth muscle cells (PASMC) proliferation, calcification and suppressed apoptosis. Numerous biological pathways have been implicated in the sustainability of this phenotype, including P53/P21, Bcl-2 along with the STAT3/NFAT axis. Several studies have demonstrated that miR-204 is downregulated in PAH. Interestingly, miR-204 directly regulates the expression of the transcription factor RUNX2, which is implicated in many features characterizing PAH, including modulation of the BMPR2, P53/P21, and Bcl2 pathways. Hypothesis: Thus, we hypothesized that a miR-204 dependent upregulation of RUNX2 in PAH promotes PASMC proliferation, resistance to apoptosis and calcification. Methods/Results: Using human explanted lungs, fresh distal PAs and freshly isolated PASMC from both control and PAH patients (n=5 to 12), we demonstrated a significant (p<0.05) upregulation of RUNX2 (RT-qPCR, western blot and immunofluorescence) in PAH compared to controls. Gain and loss functions experiments in PASMC showed that downregulation of miR-204 in control PASMC increases RUNX2 expression, while increases in miR-204 in PAH-PASMC decreases it (n=3 to 5 p<0.05). Increased RUNX2 expression was associated with a downregulation of P53/P21 and an upregulation of Bcl2 (n=3 to 8 p<0.05). More importantly, restoring miR-204 or decreasing RUNX2 expression decreased calcification (red alizarin), proliferation (Ki67) and resistance to apoptosis (TUNEL) in human PAH-PASMC (n=3 p<0.05), while upregulating RUNX2 in control PASMC using an adenovirus mimicked PAH phenotype (n=3 p<0.05). Finally, in Sugen/hypoxia rat model of PAH, nebulization of RUNX2 siRNA (n=15) decreased mean PA pressure, pulmonary vascular resistance, and increased exercise tolerance and cardiac output (p<0.05). Conclusions: Taken together, our study uncovers a new miR-204-RUNX2 axis contributing to a P53/P21-dependent pro-proliferative and Bcl2-dependent anti-apoptotic phenotype in PAH-PASMC.