Abstract 17806: Novel Synthetic Prostacyclin Agonist, Ono1301, Inhibits Proliferation of Pulmonary Arterial Smooth Muscle Cells by Suppressing Raf Kinase Signal Pathway
Objectives Proliferation of smooth muscle cells (SMCs), which is associated with activation of Raf kinase pathway, is one of the integral features of the pathological processes of pulmonary arterial hypertension (PAH). Synthetic prostacyclin agonist, ONO1301 may have a possibility of alleviation of PAH by inhibition of SMC proliferation. We hypothesize ONO1301 inhibits proliferation of pulmonary arterial SMCs via inhibition of Raf signal pathway, leading to prevent pulmonary vascular remodeling in rat PAH model.
Methods In vitro study, human pulmonary arterial SMCs were isolated from recipient lung of idiopathic PAH and cultured in absence or presence of ONO1301. PCNA assay and western blotting were performed to qualify Raf-1 and ERK1/2 phosphorylation. In vivo study, Monocrotaline (60mg/kg) was subcutaneously injected to male Wistar rats to induce PH. Slow releasing form of ONO-1301 micro-sphere (1mg/kg) was administered intravenously (O group, n=15), and PLGA vehicle (1mg/kg) as control group(C group, n=15). Survival rate in 42 days, RV/LV pressure ratio, and RV/LV+septum weight ratio were evaluated. Medial wall thickness of pulmonary arteries and SMC proliferation were assessed by immunofluorescent staining. Medial layer of pulmonary artery was selectively micro-dissected and assessed by western blotting to evaluate phosphorylation of Raf-1 and ERK1/2.
Results In vitro study, western blotting revealed phosphorylation of Raf-1 and ERK1/2 was significantly suppressed in the presence of ONO1301. PCNA assay showed the number of PCNA positive SMCs was decreased in presence of ONO1301. Survival rate was significantly improved in O group. Catheterization analysis demonstrated RV/LV pressure ratio was significantly decreased (0.49±0.03) in O group compared to C group (0.80±0.12, p<0.05). RV/LV+S weight ratio was significantly decreased and thickening of medial layer was also inhibited in O group. The number of PCNA positive SMCs was considerably reduced after treatment. Phosphorylation of Raf-1 and ERK1/2 in medial layer was significantly suppressed in O group.
Conclusion ONO-1301 inhibits proliferation of pulmonary arterial SMCs via suppressing Raf signal pathway, warranting therapeutic potential in clinical scenario.
- © 2012 by American Heart Association, Inc.