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Smooth Muscle Apoptosis in Pulmonary Hypertension Is Regulated by the Sodium-Hydrogen Exchanger
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A2068 - Smooth Muscle Apoptosis in Pulmonary Hypertension Is Regulated by the Sodium-Hydrogen Exchanger
Author Block: J. Huetsch1, X. Yun1, H. Jiang1, L. A. Shimoda2; 1Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, MD, United States, 2John Hopkins Univ, Baltimore, MD, United States.
Objective: A key component of the remodeling of the pulmonary vasculature in pulmonary hypertension (PH) is resistance to apoptosis by pulmonary arterial smooth muscle cells (PASMCs). 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.
Methods: PH 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.
Results: ER stress, which often induces 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 mediates apoptosis, 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 increased both basal and H2O2-induced CHOP expression and apoptosis in SuHx PASMCs.
Conclusions: Decreased CHOP expression in PH PASMCs prevents ER stress-induced apoptosis. Increasing CHOP expression via NHE-inhibition restores stress-induced apoptosis, suggesting a novel mechanism to control vascular remodeling in PH.
Author Block: J. Huetsch1, X. Yun1, H. Jiang1, L. A. Shimoda2; 1Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, MD, United States, 2John Hopkins Univ, Baltimore, MD, United States.
Objective: A key component of the remodeling of the pulmonary vasculature in pulmonary hypertension (PH) is resistance to apoptosis by pulmonary arterial smooth muscle cells (PASMCs). 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.
Methods: PH 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.
Results: ER stress, which often induces 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 mediates apoptosis, 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 increased both basal and H2O2-induced CHOP expression and apoptosis in SuHx PASMCs.
Conclusions: Decreased CHOP expression in PH PASMCs prevents ER stress-induced apoptosis. Increasing CHOP expression via NHE-inhibition restores stress-induced apoptosis, suggesting a novel mechanism to control vascular remodeling in PH.
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