BS65 Plasma membrane calcium atpase 4 regulates pro-inflammatory signalling in the pathophysiology of pulmonary artery endothelial cells

IntroductionPulmonary Arterial Hypertension (PAH) is a chronic and life-threatening human disease. It is characterised by sustained vasoconstriction, and progressive narrowing and occlusion of small pulmonary arteries, leading to increased pulmonary resistance, right ventricular hypertrophy, and heart failure. There is no cure for PAH. Inflammation is widely associated with pathological remodelling of pulmonary arteries in PAH, with patients presenting high serum levels of pro-inflammatory cytokines TNFα, IL1β and IL-6. In this study, we have analysed the effects of pro-inflammatory stimuli on the expression of Plasma Membrane Calcium ATPase 4 (PMCA4) in Pulmonary Artery Endothelial Cells (PAEC) and analysed its role in apoptosis regulation.MethodsHuman primary PAEC were stimulated for different times with pro-inflammatory stimuli. Expression of PMCA4 RNA and protein was determined by qPCR and western blot. PMCA4 expression was silenced using siRNA specific for human PMCA4. Quantification of apoptotic cells was performed by TUNNEL assay.ResultsExpression of PMCA4 decreased significantly in PAEC by stimulation with TNFα. RNA decay experiments where transcription was blocked using actinomycin D, indicated that TNFα has a negative effect on PMCA4 RNA stability. To determine the functional consequences of PMCA4 reduction in PAEC, we have silenced PMCA4 expression using siRNA technology. We are characterising the molecular pathway alteration in PMCA4-silenced cells that are responsible for PAEC being sensitized to TNFα-induced apoptosis. On analysing different possibilities, BMP9 has been reported to protect PAEC against TNFα/CHX apoptosis by activating the smad1/5/8 pathway. Analysis of smad1/5/8 phosphorylation (activation) in PMCA4-lacking PAEC did not reveal any differences with respect to activation of the pathway in control cells expressing PMCA4 expressed cells. This result suggests that BMP9 mediated protection of apoptosis is not altered by PMCA4 suppression.ConclusionPMCA4 protects PAEC from apoptosis induced by pro-inflammatory cytokines. TNFα-induced downregulation of PMCA4 might be implicated in the apoptotic loss of endothelial cells observed in the pulmonary arterioles of patients with PAH.Conflict of InterestNone