The Mechanism Of LPS Regulating BMP4 On Osteogenic Differentiation Of VICs Through P65

Objective: To explore the effect and mechanism of inflammation mimic LPS on osteogenic differentiation of porcine aortic VICs(valve interstitial cells)by upregulating BMP4(bone morphogenetic protein 4),which could provide a theoretical basis for the intervention and treatment of CAVD(calcific aortic valve disease).Methods: Calcium salt deposition and the expressions of Runx2(runt-related transcription factor2),OPN(osteopontin)and BMP4 in non-CAVD group(n=1)and CAVD group(n=3)were detected by Alizarin red tissue staining and immunohistochemistry.The expressions of osteogenic maker Runx2,OPN,OPG(osteoprotegerin),myofibroblast phenotypic markers α-SMA and collagen I,apoptosis maker Bax,anti-apoptotic maker Bcl2 and BMP4 were measured by Western blot in non-CAVD group(n=1)and CAVD group(n=4).Then,the porcine VICs were isolated by collagenase(type I)after the digestion of the valve,and the phenotype was identified by immunofluorescence staining.The early and late osteogenic differentiation ability of VICs were detected by ALP staining and Alizarin red S staining respectively induced by LPS.The expressions of Runx2,OPN and BMP4 were detected by q RT-PCR and Western blot.And the expressions of OPG,Bax and Bcl2 were measured by Western blot by LPS.After VICs was infected with Ad-BMP4,the early and late osteogenic differentiation ability of VICs were detected by ALP staining and Alizarin red S staining respectively.The expressions of Runx2,OPN and BMP4 were detected by q RT-PCR and Western blot.And the expression of OPG was measured by Western blot.Next,VICs were infected with Ad-si BMP4 induced by LPS,the early and late osteogenic differentiation ability of VICs were detected by ALP staining and Alizarin red S staining respectively.The expressions of Runx2,OPN and BMP4 were detected by q RT-PCR and Western blot.And the expression of OPG was measured by Western blot.After VICs were induced by LPS,the changes of p65,Smad1/5/8,Smad4 and Smad7 signaling pathways were found by Western blot.The effect of p65 inhibitor Bay 11-7082 on the osteoblastic differentiation of VICs and the expression of BMP4 were measured by Western blot.After VICs were infected with Ad-si BMP4 induced by LPS,the changes of p65,Smad1/5/8,Smad4 and Smad7 signaling pathways were detected by Western blot.Results: Compared with non-CAVD group,BMP4 was significantly increased in CAVD group,and Runx2,OPN,OPG,Collagen I and Bax were significantly increased in CAVD group.Bcl2 was decreased in CAVD group and there was no significant changes in α-SMA.The porcine primary VICs were successfully isolated.The staining of α-SMA and vimentin were positive,while the staining of CD31 was negative.The early and late osteogenic differentiation ability of VICs were both enhanced by LPS.The expressions of BMP4,Runx2,OPN,OPG and Bax were increased while the expression of Bcl2 was significantly inhibited by LPS.The early and late osteogenic differentiation ability of VICs were both enhanced by Ad-BMP4.The expressions of BMP4,Runx2,OPN and OPG were significantly increased compared with the control group.After VICs were treated with LPS,it was infected with Ad-si BMP4.Compared with the control group,the early and late osteogenic differentiation ability of VICs were both decreased and the expressions of BMP4,Runx2,OPN and OPG were mostly inhibited.The expressions of p-p65 and p-Smad1/5/8of VICs were significantly increased,and Smad4 and Smad7 signaling pathways were activated by LPS.The activation of p65 signaling pathway and the expressions of BMP4 and Runx2 were inhibited by Bay11-7082.The expressions of p-Smad1/5/8 was decreased and the activation of Smad4 and Smad7 signaling pathways were significantly inhibited after VICs were infected with Ad-si BMP4 induced by LPS.However,the expression of p-p65 was not changed.Conclusion: LPS can promote osteoblastic differentiation of aortic valve interstitial cells by regulating BMP4 through p65.The Smad1/5/8,Smad4 and Smad7 signaling pathways may play an important role in this process.