Abstract Introduction Pulmonary arterial hypertension (PAH) is a fatal disease characterized by progressive pulmonary vascular remodeling, which results in right heart failure and premature death. Pulmonary vascular remodeling is known to occur due to excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) caused by abnormal activation of growth factor signals. Prostaglandin E2 receptor 4 (EP4), a member of the prostanoid receptor family, is known to have cAMP-mediated vasodilative effect and exert anti-proliferative effects by suppressing extracellular signal-regulated kinase (ERK) in cancer cells. However, its impact on PAH remains unknown. Hypothesis: A selective oral EP4 agonist, KAG-308, reduces proliferation of PASMCs isolated from human PAH, and ameliorates pulmonary vascular remodeling and pulmonary hypertension (PH) in an experimental rat model with PAH. Methods and Results We investigated the effects of KAG-308 on proliferation and related signals in PASMCs of patients with PAH (PAH-PASMCs). Compared to controls, phosphorylation levels of Mitogen-Activated Protein Kinase Kinase (MEK) and ERK were increased with higher mRNA expression of Ki67 (Figure A-B). Treatment with KAG-308 increased cAMP in a concentration-dependent manner (Figure C). KAG-308 suppressed MEK-ERK activation as well as gene expression of Ki67 (Figure D-E). ERK inhibition by U0126 attenuated Ki67 expression to similar level. These data suggest that KAG-308 attenuates PAH-PASMC proliferation by enhancement of cAMP and suppression of ERK. Next, we investigated the impact of KAG-308 in a monocrotaline (MCT)-exposed PH rat model. Compared to normal rats, MCT-exposed rats at day21 had a higher right ventricular systolic pressure (RVSP), total pulmonary vascular resistance index (TPRI) and RV hypertrophy (RVH) (Figure F). Immunofluorescent analyses in a rat lung demonstrated the co-expression of EP4 and αSMA. Immunoblotting showed increased MEK-ERK activation in the lungs (Figure G). Chronic oral administration of KAG-308 (1mg/kg, twice daily, day0 to 21) lowered RVSP (53.1±9.3 vs 81.0±18.5 mmHg, p0.01), TPRI (112.0±16.6 vs 161.4±37.0 mmHg/mL/min/g, p0.05) and RVH (0.47±0.04 vs 0.57±0.10, p0.05) (Figure F). Histological analysis revealed that KAG-308 attenuated medial wall thickening (Figure H). Furthermore, KAG-308 reduced ERK activation as well as pro-inflammatory cytokines such as Il6 and Il1β in the whole lungs with reducing the numbers of Ki67-positive cells in the medial layer of small vessels (Figure I). In addition, although single administration of KAG-308 in MCT-exposed rats increased the cAMP levels in lungs, RVSP did not reduce (Figure J). These data suggest that the TPRI is due to anti-proliferative effect by KAG-308 chronic administration. Conclusions KAG-308 reduced PAH-PASMCs proliferation and ameliorated pulmonary vascular remodeling and PH in MCT-exposed rats. Activation of EP4 could be a potential therapeutic agent for the treatment of PAH.Figures
Kimuro et al. (Sat,) studied this question.