I008 MRP4 inhibition prevents human pulmonary artery smooth muscle cells proliferation and hypoxia-induced pulmonary arterial hypertension in mice

Abstract

Inhibition of the active efflux pump Multidrug Resistance Associated Protein 4 (MRP4) can increase intracellular levels of cyclic nucleotides cAMP and cGMP in smooth muscle cells, thereby limiting their proliferation. MRP4 inhibition thus appears as a promising therapeutic target in vasculoproliferative disorders such as pulmonary arterial hypertension (PAH). We investigate whether MRP4 inhibition can exert beneficial effects on pulmonary vascular remodeling in PAH. MRP4-knock out (KO) and wild-type (WT) mice were placed for 4 weeks in hypoxic (10 % O2) or normoxic conditions (21 % O2). After this period, the right ventricular systolic pressure (RSVP) was gauged. The Fulton Index (ratio of right ventricle to left ventricle plus septum weights), a marker of right ventricular hypertrophy, was calculated. Complementary in vitro studies were conducted in isolated human pulmonary artery smooth muscle cells (hPASMC). hPASMC proliferation was measured by BrdU incorporation quantification after transfection with scrambled or MRP4 small interfering RNAs (siRNA). Whereas there were no differences between WT and MRP4-KO mice in normoxic conditions (23.8±0.3 vs 23.0±0.6 mmHg, NS), chronic hypoxia resulted in pulmonary vascular remodeling and increased RVSP in WT but not in MRP4-KO mice (35.0±1.8 vs 25.8±0.7 mmHg, p<0.001). The right ventricular hypertrophy was strongly decreased in MRP4-KO compared to WT mice (0.27±0.01 vs 0.37±0.01 respectively, p<0.001). In vitro specific inhibition of MRP4 by siRNA caused an increase of the intracellular/extracellular ratio for both cAMP and cGMP (185±18 % and 162±22 %, relative to scrambled siRNA, p<0.05) and was associated with a significant decrease in proliferation compared to scrambled siRNA (219±13 % vs 432±44 %, relative to control in 0.1 % growth factors, p< 0.001). This antiproliferative effect was abolished by blocking PKA or PKG activity: 225±9 % with PKA inhibitor PKI and 194±11 % with PKG inhibitor KT5823 vs 160±7 % for MRP4 siRNA alone (p<0.05), compared to 238±16 % for siRNA scrambled. MRP4 inhibition reduces hPASMC pathological proliferation by controlling PKA and PKG pathways and prevents PAH development in vivo. MRP4 inhibition appears as a new target for therapeutic intervention in PAH.