The Effect And Mechanism Of TLR4 Activation Regulating BMP7 On Osteoblastic Differentiation Of Aortic Valve Interstitial Cells

Objective:To investigate the effect and mechanism of TLR4inflammatory receptor activation regulating bone morphogenetic protein7?BMP7?on the osteoblastic differentiation of aortic valve interstitial cells,and to provide a theoretical basis for the intervention and treatment of calcific aortic valve disease?CAVD?.Methods:The non-CAVD valve tissues?non-CAVD group?were taken from patients with surgical treatment of aortic dissection,the CAVD valve tissues?CAVD group?were taken from patients undergoing aortic valve replacement because of calcified aortic valve stenosis.The expression levels of TLR4,BMP7,Runx2 and?-smooth muscle actin??-SMA?in the non-CAVD group and CAVD group were tested by immunohistochemistry or Western blot.Healthy domestic pigs were sacrificed and the aortic valve leaflets were aseptically removed immediately.The porcine aortic valve interstitial cells?VICs?were isolated by continuous collagenase digestion,its morphological characteristics were observed and the phenotypes were identified by immunofluorescence staining.LPS was used to activate the inflammatory receptor TLR4 in the VICs,then Alkaline phosphatase?ALP?staining and Alizarin red S staining was used to evaluate the cell early and late osteogenic differentiation abilities.The mRNA and protein levels of BMP7 and osteogenesis factors Runx2,OPN was detected by qPCR and Western blot.In addition,the conditioned medium induced osteogenic differentiation of porcine VICs for the establishment of calcification model of aortic valve interstitial cells in vitro,then VICs were transfected with Ad-BMP7,ALP staining and Alizarin red S staining was used to evaluate the cell early and late osteogenic differentiation abilities.The mRNA and protein levels of inflammatory cytokines IL-6,IL-8 and osteogenesis factors Runx2,OPN were determined by qPCR and Western blot,respectively.After LPS activates the TLR4 inflammatory receptor,VICs were transfected with BMP7-siRNA by liposome method.ALP staining and Alizarin red S staining was used to evaluate the cell early and late osteogenic differentiation abilities.The protein levels of TLR4,BMP7,Runx2 and OPN was determined by Western blot.The pathway of AKT,ERK1/2 and Smad1/5/8 were measured also by Western blot.In vivo,ApoE^-/-mice were given a high-fat diet to construct CAVD calcification mouse model.Echocardiography and HE staining were used to verify whether the model was successfully constructed.The expression levels of TLR4,BMP7 and Runx2 in heart valve tissues were tested by immunohistochemistry.Results:In human valve tissue,the expression of TLR4,BMP7,Runx2 and?-SMA in CAVD group was significantly higher than that in non-CAVD group.The primary porcine aortic valve interstitial cells were successfully isolated and the staining of?-SMA and Vimentin were positive,the staining of von Willebrand factor?vWF?was negative,the cells were identified as porcine aortic valve interstitial cells.After LPS activates the inflammatory receptor TLR4,the expression of BMP7 at mRNA and protein levels in the porcine VICs was increased,the cell early and late osteogenic differentiation abilities were significantly increased and the mRNA and protein levels of osteogenesis factors Runx2and OPN were also significantly increased.The protein levels of p-AKT,p-ERK1/2 and p-Smad1/5/8 were up-regulated.After porcine VICs were transfected with Ad-BMP7,the cell early and late osteogenic differentiation abilities were significantly increased,the inflammatory response and the expression of osteogenesis factors Runx2 and OPN significantly increased.After LPS activates the TLR4,VICs transfected with BMP7-siRNA,the cell early and late osteogenic differentiation abilities were significantly decreased,the expression of Runx2 and OPN at protein levels were also significantly decreased,but there was no influence on TLR4 expression.Only the protein levels of p-Smad1/5/8 in the VICs transfected with BMP7-siRNA was significantly decreased,while the p-AKT and p-ERK1/2 protein levels were not significantly changed.The CAVD calcified mouse model was successfully constructed,and the calcified mouse model showed higher expression of TLR4,BMP7,and Runx2 in heart valve tissues.Conclusion:Activation of TLR4 inflammatory receptor promotes osteogenic differentiation of aortic valve interstitial cells by up-regulation of BMP7.The BMP7/Smads signaling pathways may play an important role in these processes.