Monocrotaline Induces Endothelial Injury And Pulmonary Hypertension By Targeting The Extracellular Calcium-sensing Receptor

Pulmonary hypertension(PH)is a progressive,complex and often fetal disease,its pathogenesis is second only to hypertension and coronary heart disease,ranking third in cardiovascular disease.The disease is very high degree of malignancy,about 75%of patients died within 5 years after diagnosis,and the average survival after the onset of symptoms is just 1.9 years,so it is also called "cancer" of cardiovascular diseases.However,because of its etiology and pathogenesis diversity,the patient's clinical manifestations,histological features.severity,response to treatment and prognosis are very different.Therefore,extensive and in-depth research is needed to overcome the disease.An important part of the research is the establishment of an animal model of pulmonary hypertension.The model establishment has a variety of methods,including chronic hypoxia-induced pulmonary hypertension model,nonocrotaline induction pulmonary hypertension model,monocrotaline plus lung resection induced pulmonary hypertension model,Sugen 5416 plus chronic hypoxia induced pulmonary hypertension model and so on.The MCT model is one of the most widely used models of pulmonary hypertension because of its simple operation(single subcutaneous or intraperitoneal injection),short time(2 weeks can form pulmonary hypertension),good stability and low cost.But the molecular mechanism of the model has been unclear.Monocrotaline is an 11-me,mbered macrocyclic alkaloid that is predo,minantly present in leguminous plants.After being taken into the liver.MCT is oxidized by cytochrome P450 into monocrotaline pyrrole(MCTP),which is active form of MCT.By reviewing the previous study on MCT,it was found that the binding of MCTP to protein,DNA and other nucleophilic macromolecules was considered to be an important mechanism of MCT toxicity.MCTP is mainly associated with the protein thiol,including actin,galectin,protein disulfide isomerase,cytoskeleton myosin,ERp57,etc.Studies have shown that MCTP has a high affinity for this motif Cys-XXa-Xaa-Cys(CXXC).In addition,it has been found that MCTP can also form a covalent bonds with histidine and tryptophan,such as reticulocalbin 1,3 and ATP synthase[1-4].In recent years,more and more papers report that CaSR play an important role in the development and progression of pulmonary hypertension[5-7],and CaSR extracellular domain(ECD)contains 19 cysteines,13 histidine,10 tryptophan and 3 CXXC motifs.In summary,the structural characteristics of CaSR may make it an important target for MCT.Therefore,this study uses CaSR as a starting point to explore the molecular mechanism of MCT-induced pulmonary hypertension,in order to provide more clues to the mechanism of pulmonary hypertension and provide a potential target for the treatment of pulmonary hypertension.PurposeMonocrotaline has been widely used to establish an animal mode of pulmonary hypertension.The molecular target underlying monocrotaline-induced pulmonary artery endothelial injury and pulmonary hypertension still remains unknown.The extracellular calcium-sensing receptor is a member of G protein-couple receptor C family,and its particularly extracellular domain hold the potential structural basis for monocrotaline to bind.This study aims to reveal whether monocrotaline induces pulmonary hypertension by targeting the extracellular calcium-sensing receptor.Methods1.Expression and purification of recombinant CaSR extracellular domain protein,the interaction between CaSR extracellular domain protein and MCT was detected by WaterLOGSY(Water-ligand observed via gradient spectroscopy)and STD-NMR2.Mark the MCT with FITC.then the distribution of MCT and the distribution of CaSR protein in cultured PAECs were observed by confocal.3.The changes of[Ca2+]i in pulmonary artery endothelial cells were detected by the calcium fluorescence probe Fura-2/AM.4.PAEC survival was detected by the CCK-8 kit.cleaved caspase 3 expression level was detected by Western Blot.5.Construction of CFP-CaSR and YFP-CaSR plasmid,detect the interaction between proteins by FRET.6.The model of pulmonary hypertension was established by subcutaneous injection of MCT,and the pulmonary artery pressure,body circulation pressure,cardiac output and vascular wall thickness were measured.Results1.CaSR extracellular domain protein binds to MCT was demonstrated by WaterLOGSY and STD-NMR.2.The distribution of MCT on the cell membrane is more abundant than the cytoplasm and co-localization with CaSR in cultured PAECs3.MCT caused[Ca2+]i of PAECs significantly increased.While this effect is inhibited by Callhex231(CaSR inhibitor)or CaSR shRNA.4.MCT induced the survival rate of PAECs significantly decreased.While this effect is inhibited by Calhex 231(CaSR inhibitor)or CaSR shRNA.MCT induced the expression of cleaved caspase 3 in PAECs significantly increased.While this effect is also inhibited by Calhex231(CaSR inhibitor)or CaSR shRNA.5.MCT leads to the energy transfer from CFP-CaSR to YFP-CaSR significantly increased.6.After two weeks of MCT injection,the four indexes of pulmonary artery pressure.pulmonary vascular resistance,right heart hypertrophy index and vessel wall thickness were significantly increased.And this effect was significantly attenuated or abolished by general or lung knockdown or knockout of extracellular calcium-sensing receptor.ConclusionMCT can bind to CaSR and cause CaSR polymerization to activate CaSR,which caused pulmonary artery endothelial cell cytoplasmic calcium concentration increased,leading to pulmonary artery endothelial cell apoptosis,and then cause pulmonary artery endothelial injury,eventually induces pulmonary hypertension.