BMP2 Regulates SMAD And MicroRNA Mediated Vascular Calcification In Chronic Renal Failure

Research backgroundIn recent years,the incidence of patients with chronic renal failure(CRF)is rapidly increasing in China.Although renal replacement therapy,represented by blood purification technology,has been able to rid patients with CRF of uremia,the mortality rate due to cardiovascular events in patients with CRF is still high compared with the general population.The main independent risk factor for this process is vascular calcification(VC),so it is of great significance to investigate the mechanism of vascular calcification in CRF patients.Vascular calcification induced by CRF is a type of vascular calcification that is different from atherosclerosis.It starts from the osteogenic differentiation of vascular smooth muscle cells(VSMC)and mainly involves the deposition of hydroxyapatite crystals in the vascular wall,which is closely related to the nephro-vascular-bone axis.The Wnt/β-catemin pathway plays an important role in promoting osteoblast differentiation and biomineralization of extracellular matrix secreted by osteoblasts.However,control of hyperphosphonemia alone and use of Wnt pathway inhibitors cannot completely prevent or significantly reduce the incidence of vascular calcification in uremia.In recent years,the BMP/Smad pathway,another signaling pathway associated with changes in calcium and phosphorus metabolism,has been paid more attention.BMP is an acidic glycoprotein widely existing in bone matrix.It is the most widely involved family in the transforming growth factor-β(TGF-β)superfamily.It can up-regulate the expression of 66 genes including Smad6 and Smad7 transcription factors.Studies suggest that BMP2 in the BMP family plays an important role in atherosclerosis,vascular and valve calcification,and other processes.BMP2 activates Smads and induces osteoblast differentiation by activating the expression of runt-related transcription factor 2(Runx2)via both distal and proximal promoters.However,it has not been determined which smad BMP2 regulates Runx2 expression and which molecule this smad causes calcification.In this study,rat and VSMC calcification models were established.By means of immunohistochemistry,immunofluorescence,PCR and other experimental methods,the role of BMP2 in calcification was clarified,and the regulatory mechanism of BMP2 on downstream Smad family proteins was studied.In addition,this study further explored the potential role of microRNA in this process and looked for microRNA types that might be involved in regulating calcification.Objective1.The role of clear BMP2 in chronic renal failure VC;2.Clear smad proteins family role in the VC;3.Explore smad family involved in the mechanism of VC BMP2 regulation;4.Explore the role of microRNA in VC and search for microRNA types involved in the regulation of VC.Methods1.The first part:VSMC cells were transferred to 6-well plates for culture after 5 passages,and the planting density was 1×105 个/mL.Phosphorus concentration was 5 mM,and intervention time was 0,3,5,7 and 9 days,respectively.ALP concentration and cell calcium content were detected at different intervention time,and the optimal intervention time was determined.Then VSMC divided into control group,high phosphorus group(Pi:5mm),intervention group(Pi:5mm+BMP-2 inhibitor LDN-193189 250nM),each group of each intervention on the above terms and conditions for five days;2.The second part:Twenty Wistar rats were rando mLy divided into control group(n=5),CRF group(n=5)and intervention group(n=10),the control group was routinely fed,the CRF group and the intervention group were fed with 0.75%adenine high calcium and high phosphorus diet for 4 weeks to establish the chronic renal failure model,after the modeling success,the CRF group was maintained fed with high calcium and high phosphorus diet.On the basis of CRF group,the intervention group was intraperitoneally injected with LDN-193189 at 2.5mg/kg/d,and the control group and CRF group were simultaneously injected with normal saline as the control.A total of 2 weeks of treatment.After the success of the two parts of calcification model building,Von Kossa staining,RT-QPCR,immunofluorescence,immunohistochemistry and Western blot were used to analyze differences in calcification,gene and protein expressions of Runx2,BMP2 and Smad family,as well as microRNA levels between cell and animal groups.ResultsThe first Part:The mechanism of high phosphorus induced BMP2 expression promoting VSMC calcification in rats.1.High phosphorus stimulation can increase expression of BMP2,promoting VSMC transdifferentiation sample for osteogenesis cells,promote calcium deposits.2.The inhibition of BMP2 can significantly inhibit VSMC transdifferentiation and calcium deposits;3.BMP2 by raising Runx2 and p-smad 1/5/8 expression levels play a role of promote calcification;4.VSMC calcification,miR-30b、miR-30c expression significantly lowered,P<0.05,the inhibition of BMP2,increased significantly,P<0.05,confirmed thatmiR-30b、miR-30c is regulated by the BMP2,may play a negative regulatory role in the process of VC.The second part Mechanism of BMP2 promoting vascular calcification in CRF rats.1.The CRF rats significantly vascular calcification,high expression of BMP2,Runx2;2.The inhibition of BMP2 can significantly inhibit calcification,cut Runx2 expression,prove that BMP2 by promoting Runx2 express promote calcification;3.The CRF vascular calcification,smad158 phosphorylation levels,cut smad6 expression,inhibition of BMP2 physiological function can significantly reduce the phosphorylation level,improve the level of smad6,P<0.05,proving that BMP2 by promoting smad158 phosphorylation,reduce smad6 express promote calcification,Smad6 may play a role in inhibiting calcification.4.CRF calcification,miR-30b,miR-30c expression significantly lowered,P<0.05,the inhibition of BMP2,increased significantly,P<0.05,confirmed that miR-30b and miR-30c are regulated by the BMP2,may play a negative regulatory role in the process of VC.Research conclusionsBMP2 rely on phosphorylation of smad 1/5/8 promote the expression of Runx2,in turn,promote the CRF vascular calcification,smad6,miR-30b and miR-30c play a negative regulatory role in the process,application of BMP-2 inhibitor can significantly reduce vascular calcification,can be used as potential therapeutic targets.