IL-33-induced Differential Phenotipic Transition Of Valvular Interstitial Cells Promotes Calcific Aortic Valve Disease Progression

ObjectiveIL-33 is a mediator which has been proven to be involved in the pathogenesis of several inflammation-associated diseases.ST2 is the receptor for IL-33,which has been reported to be overexpressed in CAVD.Our present study compared the expression of ?-SMA,OPN and ST2 between specimens from fibrotic and calcific stages of CAVD and explored the effects and mechanisms of phenotypic transition of porcine VICs in the presence of IL-33.Methods49 and 53 CAVD patients and healthy adults were included into this study.The mRNA and protein level of members of IL-1 family were quantified by Real Time RT-qPCR and ELISA.Immunohistochemistry and immunofluorescence were used to detect the expression of ?-SMA,OPN and ST2 in fibrotic and calcific stage of CAVD speciemens.Porcine VICs were isolated by collagenase digestion.Real Time RT-qPCR and Western Blot were used to quantify the mRNA and protein level of ?-SMA,Runx2,OPN and ST2 in porcine VICs which were stimulated by IL-33,IL-33+SC75741 or IL-33+SB203580.Phosphorylation of NF-?B and p38 MAPK signaling pathways were examined by Western Blot.siRNA lentivirus infection was used to knockdown the expression of ST2.Immunofluorescence was used to detect the co-expression of ?-SMA,OPN and ST2.ResultsThe results showed that the mRNA and protein levels of IL-33 and sST2 in CAVD patients were higher than those in healthy adults.Immunohistochemistry and immunofluorescence demonstrated higher expression of ?-SMA,OPN and ST2 in the calcific stage of CAVD speciemens than that in the fibrotic stage.Co-expressed ?-SMA/ ST2 or OPN/ST2 was found only in the calcific stage,while co-expression of ?-SMA,OPN and ST2 was scarce in either stage.Phosphorylation of NF-?B was associated with IL-33-induced porcine VICs differentiation into myofibroblasts,while IL-33-mediated porcine VICs acquisition of an osteoblastic phenotype and enhanced ALP activity were dependent on activation of the p38 MAPK signaling pathway.The overexpression of ?-SMA and OPN could be abolished by treatment with the NF-?B signaling pathway inhibitor SC75741 and p38 MAPK signaling pathway inhibitor SB203580,respectively.Stimulation with IL-33 promoted the co-expression of OPN/ST2 or ?-SMA/OPN/ST2.All these effects could be diminished by ST2 knockdown.Conclusion: IL-33 might serve as a potential biomarker for CAVD.After stimulation with IL-33,porcine VICs exhibited differential phenotypic transition and differentiation into myofibroblasts and osteoblasts via activation of the NF-?B and p38 MAPK signaling pathways,respectively.