The Role And Mechanism Of Hypoxia Induced Mitogenic Factor As A Nonclassical Ligand For Extracellular Calcium-sensitive Receptor In Hypoxic Pulmonary Hypertension

Objictive:To study the role and mechanism of hypoxia induced mitogenic factor(HIMF)as a nonclassical ligand for extracellular calcium-sensing receptor(CaSR)in the proliferation of pulmonary artery smooth muscle cells(PASMCs)and pulmonary hypertension induced by chronic hypoxia.Methods:(1)Screened of G protein-coupled receptors interaction with HIMF in chronic hypoxic pulmonary hypertension by immunoprecipitation;(2)Primary culture of PASMCs;(3)Detected the interaction and co-localization between HIMF and CaSR in PASMCs and PA by immunoprecipitation and immunofluorescence;(4)Yeast two-hybrid technique was used to screen the amino acid sites of CaSR which binds to HIMF;(5)Treatment of-PASMCs with different concentrations of extracellular calcium([Ca2+]o)and detection of cytosolic Ca2+([Ca2+]i)by calcium ion fluorescence probe Fura-2/AM and calcium ion fluorescence imaging system;(6)PASMCs transfected with CaSR shRNA vector and CaSR mutation vector(vector of specifically mutant the four amino acid sites of CaSR binding to HIMF)to down-regulated and mutated CaSR expression,PASMCs transfected.with HIMF shRNA vector and HIMF overexpression vector to down-regulated and up-regulated HIMF expression;(7)Synthesised the specific cell-penetrating peptide to compete the HIMF and CaSR binding and prevent the binding of HIMF and CaSR;(8)Used CCK8 kit to detect the proliferation of PASMCs;(9)Calhex231 and specific cell-penetrating peptide was injected to SD rats and lentivirus was used to infect SD rats;(10)western and ELLISA was used to detect the effect of lentivirus;(11)The power lab four conductivity meters to detect pulmonary artery pressure、systemic pressure and cardiac output;(12)Western blotting was used to detect whether HIMF binds to CaSR affects the dimer formation of CaSR;(13)It is confirmed by calcium imaging technique whether the specific peptide that inhibits the interaction between HIMF and CaSR affects the normal function of CaSR under normal conditions.Results:(1)The GPCR binds to HIMF is CaSR in chronic hypoxic hypertension;(2)HIMF and CaSR can interact with each other and co-located in pulmonary artery(PA)and PASMCs detected by immunoprecipitation technique and immunofluorescence technique;(3)The amino acid sites of CaSR interacted with HIMF screened by yeast two-hybrid technique is 885-888aa;(4)Chronic hypoxia can lead to a significant increase in CaSR sensitivity and the proliferation of PASMCs.which is significantly inhibited by CaSR-specific inhibitor Calhex231、CaSR shRNA、CaSR mutation、specific cell-penetrating peptide and HIMF shRNA;(5)Under normoxia condition overexpression of HIMF can a significant increase in CaSR sensitivity and the proliferation of PASMCs,and this effect can also be inhibited by CaSR inhibitor Calhex231 and CaSR shRNA;(6)The lentivirus packaging of CaSR and HIMF specific interferential effects plasmids、CaSR mutation plasmid and HIMF overexpression plasmid were injected into SD rats from the airway,after 72 hours,we began to build animal model.In vivo experiments,chronic hypoxia can increase the mean pressure of pulmonary artery(Ppa)、pulmonary vascular resistance(PVR)、and right ventricle hypertrophy(right ventricular weight/left ventricular+septal weight,Rv/LV+S)、hypertrophy of pulmonary arteries of 50-100 μm diamter and neo-muscularization of small vessels(<30μm)(medial thickness%),the effect can be inhibited by CaSR-specific inhibitors Calhex231、CaSR shRNA、CaSR mutation and HIMF shRNA and specific cell-penetrating peptide;(7)Overexpression of HIMF can lead to an increase in the mean pressure of pulmonary artery(Ppa)、pulmonary vascular resistance(PVR)、and right ventricle hypertrophy(right ventricular weight/left ventricular+septal weight,Rv/LV+S)、and hypertrophy of pulmonary arteries of 50-100 μm diameter and neo-muscularization of small vessels(<30 μm)(medial thickness%)、this effect can also be inhibited by CaSR-specific inhibitors Calhex231 and CaSR shRNA;(8)ELISA assay detect that HIMF shRNA can interfere the HIMF expression and HIMF overexpression lentivirus can increase the expression of HIMF gene in normoxia.Western blot to detect the CaSR shRNA causes decreased expression of CaSR and CaSR mutation increased expression of CaSR;(9)HIMF binds to CaSR increased the sensitivity of CaSR by increasing the dimer formation of CaSR;(10)Under normal conditions,the specific cell-penetrating peptide only blocked the interaction between CaSR and HIMF,but did not affect the physiological function of CaSR.Conclusion:Chronic hypoxia induced an increase in HIMF expression,enhanced the interaction between HIMF and CaSR,increased the dimer formation of CaSR and enhanced the sensitivity of CaSR,it can be activated by 2.5 mM[Ca2+]o.which caused the[Ca2+]o influx,PASMCs[Ca2+]i increased,PASMCs proliferation and pulmonary hypertension.