CD40/CD40L Regulates Intracellular Calcium Concentration To Regulate Smooth Muscle Cell Dedifferentiation And Participate In Pulmonary Vascular Remodeling

Objective:pulmonary artery smooth muscle cells(PASMC)undergo phenotypic transformation and dedifferentiation during pulmonary arterial hypertension(PAH).Inflammatory cytokines play an important role in the dedifferentiation of smooth muscle cells(SMC),and the CD40/CD40L axis is the central link mediating inflammatory regulation.This study focused on the mechanism of CD40/CD40L axis regulation of the dedifferentiation of SMC which involving in PAH vascular remodeling.Methods:(1)PASMCs were treated with normal culture,sCD40L,and hypoxia combined with sCD40L to determine the effects of sCD40L and hypoxia on the biological function and molecular expression of PASMCs.Cell proliferation ability was detected by CCK8,and the changes in the expression of contraction and secretion marker molecules were determined by qRT-PCR and Western Blot.(2)PASMCs were treated with normal culture,sCD40L,hypoxia,and hypoxia combined with sCD40L to determine the effects of sCD40L and hypoxia on the cAMP/PKA signaling pathway activity and cytoplasmic Ca2+concentration.The concentration of cAMP and the activity of PKA were detected by ELISA,the expression of CaV1.2(Ca2+in-flow channel protein)and RYR2(Ca2+out-flow channel protein)were determined by Western Blot,and the cytoplasmic Ca2+concentration was determined by Fura-2/AM fluorescence probe method.(3)Digoxin was used to reduce the cytoplasmic Ca2+concentration and research the relationship between the cytoplasmic Ca2+concentration and the cellular biological function and molecular expression of PASMCs.(4)Agonist of cAMP was used to excite the cAMP/PKA pathway to research the effects of cAMP/PKA pathway on cytoplasmic Ca2+concentration,biological function and molecular expression of PASMCs.Conclusion:By inhibiting the activity of cAMP/PKA signaling pathway,sCD40L causes increased expression of CaV1.2 and decreased expression of RYR2,leading to the rise in cytoplasmic Ca2+concentration,thereby promoting the proliferation and dedifferentiation of PASMCs.