The Influence Of PTGIS Mutation On The Function Of Pulmonary Endothelia Cells

Background:Pulmonary arterial hypertension(PAH)is a severe,rare disease with high clinical heterogeneity.Bone morphogenetic protein receptor 2(BMPR2),which is main causative gene,could only partially explain the genetic etiology of PAH.Identification of novel PAH susceptibility loci remains challenge.Aim:We tend to explore the role of PTGIS gene in the pathogenesis of pulmonary vascular remolding and provide novel insights into the molecular genetic basis of PAH.Method:According to our previous experiments,whole genome sequencing found two mutations of PTGIS(Prostaglandin I2 synthase),which will increase the risk of PAH.Based on the wild type plasmid,two mutant plasmids(R252Q and A447T)were constructed by site-directed mutagenesis using the QuickChange mutagenesis kit.Human pulmonary microvascular endothelial cell(HPAEC)were transfected with 4 different plasmids,including the empty vector control,wild-type plasmid,and two mutant plasmids(R252Q,A447T).The mutation effects on enzymatic activity and protein function were tested on HPMEC.Results:Compared with the wild type plasmid,transfection of HPMEC with R252Q and A447T resulted in 43%(P<0.001)and 50%(P<0.001)of decrement of the prostacyclin metabolites at baseline,and 33%(P<0.001)and 34%(P<0.001)at hypoxia condition,respectively.CCK-8 assay was used to examine cell viability of HPMEC exposed to normal or hypoxia stimulation.Overexpression of PTGIS enhanced 20-30%cell viability either at the baseline or at the hypoxia condition.The mutant R252Q has no influence on the cell viability.Whereas the mutant A447T decreased approximate 18%and 10%cell viability both in baseline and hypoxia condition,respectively.Caspase3/7 activity assay was adopted to investigate the effects of overexpressing mutant PTGIS on apoptosis of HPMEC.Overexpression of PTGIS had an effect of pro-apoptosis.However,the mutant R252Q and A447T exhibited a pro-apoptosis effect at baseline,while the mutant R252Q and A447T exhibited an anti-apoptosis effect in hypoxia condition.Conclusion:The mutant PTGIS would impair the protective function of PTGIS in HPMEC.Both R252Q and A447T decreased the metabolic concentration of prostacyclin and induced apoptosis resistant phenotype.The A447T also decreased the cell variability of HPMEC both in baseline and hypoxia condition.