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Introduction Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive vasoconstriction, vascular remodelling, and occlusion of small pulmonary arteries. It leads to increased pulmonary resistance and finally right ventricular failure. The disorder has no cure and current therapies only target vasoconstriction but have little effect on vessel remodelling. Increased activity of pro-inflammatory cytokines is linked to PAH pathogenesis. In this study, we analysed the effect of TNF-alpha and IL-1Betaβon the expression of Plasma Membrane Calcium ATPase 4 (PMCA4) in pulmonary artery endothelial cells (PAEC). Methods PAEC were cultured for different times and with different doses of TNF-alpha or IL-1Beta. Expression of PMCA4 RNA and protein was determined by qPCR and western blot respectively. PMCA4 expression was silenced using siRNA specific for human PMCA4. Quantification of apoptotic cells was performed by flow cytometry and TUNNEL. Results Treatment of PAEC with TNF-alpha or IL-1Beta induced a time- and dose-dependent decrease in the levels of RNA for PMCA4. Analysis of PMCA4 RNA levels in the lungs of mice with overexpression of ectopic TNF-alpha confirmed the in vivo relevance of these observations. RNA decay experiments performed by blocking cellular transcription with Actinomycin D indicate that the downregulation of PMCA4 RNA levels mediated by pro-inflammatory stimuli in PAEC is the result of a decrease in RNA stability. In agreement with the reduction observed in RNA levels, PMCA4 protein expression was strongly decreased by treating PAEC with TNF-alpha or IL-1Beta. Silencing PMCA4 gene expression sensitised PAEC to apoptosis, suggesting that PMCA4 protects PAEC to apoptosis induced by pro-inflammatory cytokines. Conclusion The pro-inflammatory cytokines TNF-alpha and IL-1Beta significantly downregulate the expression of the PMCA4 gene in PAEC at the RNA and protein level. Decrease in PMCA4 expression sensitised PAEC to apoptosis. This indicates that the PMCA 4 gene might play an important role in the apoptotic loss of endothelial cells observed in the pulmonary arterioles of patients with PAH. Conflict of interest None

Pulmonary arterial hypertension (PAH) is a chronic and life-threatening disease characterised by a progressive narrowing and occlusion of small pulmonary arteries leading to increased pulmonary resistance, right ventricular hypertrophy, and, finally, right ventricular failure. Several studies have demonstrated that proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) play a pivotal role in the vascular remodelling characteristic of PAH. Levels of cytoplasmic calcium are an important determinant of PASMC proliferation and migration. The Plasma Membrane Calcium ATPase (PMCA) proteins extrude calcium from the cytosol to the extracellular medium. Here, we have investigated whether inducers of PAH trigger any changes in the expression of PMCA genes in PASMCs. Treatment of PASMCs with the PAH inducers Platelet Derived Growth Factor (PDGF) or TNF-alpha induced a significant increase in the RNA levels of PMCA1. PMCA1 RNA levels are also elevated in lungs of rats with monocrotaline-induced PAH. In silico analysis of the PMCA1 gene promoter region has shown putative binding sites for the transcription factors NFAT and NFkB that could mediate the transcriptional upregulation triggered by PDGF and TNF-alpha respectively. However, we show here that upregulated PMCA1 gene expression is not mediated by binding of these transcription factors to the proximal promoter region. No changes were observed in the RNA levels of PMCA4, the other major PMCA isoform expressed in PASMCs. Our results suggest an important role for PMCA1 in PASMC deregulation during PAH, although a full understanding of the role of PMCA1 on the onset and progression of PAH requires further investigation.

Pulmonary arterial hypertension (PAH) is a chronic and life-threatening disease with high morbidity and mortality in adult and paediatric patients. PAH is characterised by a progressive narrowing and occlusion of small pulmonary arteries leading to increased pulmonary resistance, right ventricular hypertrophy, and, finally, right ventricular failure. A large body of data has shown that proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) represent key events in the vascular remodelling of pulmonary arteries that occurs during PAH. Levels of cytoplasmic calcium are an important determinant of PASMC proliferation and migration, and failure in maintaining appropriate levels of intracellular calcium are associated with PAH. The plasma membrane calcium ATPase (PMCA) proteins extrude calcium from the cytosol to the extracellular medium, and in doing so, play a critical role in the modulation of intracellular calcium levels. In this work, we have investigated whether inducers of PAH trigger any changes in the expression of PMCA proteins in PASMCs. Analysis of RNA expression levels for PMCA genes has revealed that treatment of PASMCs with PDGF results in a significant increase in the level of the RNA encoding for the protein PMCA1. Interestingly, PMCA1 RNA levels were also elevated in lungs of rats with monocrotaline-induced PAH. No changes were observed in the RNA levels for PMCA4, the other major PMCA isoform expressed in PASMCs. Although previous studies on the regulation of PMCA1 gene expression have identified functional binding sites for the transcription factors NFAT in the PMCA1 promoter region, we show here that PDGF-mediated upregulation of PMCA1 transcriptional expression is independent of activation of the calcineurin/NFAT signalling pathway. Our results suggest the involvement of PMCA1 in PASMC deregulation during PAH, although determination of the link between increased expression of PMCA1 and PAH requires further investigation.