ER Stress Induced Apoptosis of Smooth Muscle in Pulmonary Hypertension is Regulated by the Sodium‐Hydrogen Exchanger

Abstract

ObjectiveAltering the apoptosis‐resistant phenotype of pulmonary arterial smooth muscle cells (PASMCs) may be key to reversing pathologic pulmonary vascular remodeling in pulmonary hypertension (PH). Endoplasmic reticulum (ER) stress and sodium‐hydrogen exchanger (NHE) activity have each been implicated in vascular remodeling in PH. We studied the effect of NHE activity upon ER stress‐induced apoptosis in PASMCs isolated from the SU5416‐hypoxia (SuHx) rat model of PH.MethodsPH was induced in male Wistar rats via exposure to the vascular endothelial growth factor receptor inhibitor SU5416 plus 3 wk of 10% oxygen followed by 2 wk of normoxia (SuHx model), while control rats were exposed to vehicle and 5 wk of normoxia. PASMCs were isolated from SuHx and control rats and expanded in culture. Apoptosis was measured using Hoechst stain. Thioflavin fluorescence was quantified as a measure of ER stress. Protein levels of the ER stress‐induced transcription factor, CCAAT/enhancer‐binding protein homologous protein (CHOP), were measured via immunoblot. To measure NHE activity, PASMCs were incubated with pH‐sensitive fluorescent dye and the Na+‐dependent recovery of intracellular pH following acidification by ammonium pulse challenge was measured via fluorescence microscopy. Cell stress was induced with 250 μM H2O2. NHE was pharmacologically inhibited with 10 μM ethyl‐isopropyl amiloride.ResultsER stress, a known stimulus of apoptosis, was increased by H2O2 in control PASMCs and was elevated at baseline in SuHx PASMCs. H2O2 exposure increased apoptosis in control PASMCs. Despite increased ER stress, SuHx PASMCs exhibited low levels of apoptosis both at baseline and in response to H2O2. CHOP protein expression, which is induced by ER stress and triggers the apoptotic cascade, was increased by H2O2 in control cells, but remained low in H2O2‐treated SuHx PASMCs. NHE activity was increased in PASMCs from SuHx rats, and pharmacologic NHE inhibition in SuHx PASMCs increased both basal and H2O2‐induced CHOP expression, and also increased apoptosis.ConclusionsPH PASMCs are resistant to ER‐stress induced apoptosis due to decreased CHOP expression. NHE inhibition increases CHOP expression in PH PASMCs and restores ER‐stress induced apoptosis, suggesting a novel mechanism to control vascular remodeling in PH.Support or Funding InformationNIH K08HL133475 & R01HL073859This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.