Experimental Study On Calcification Of Vascular Smooth Muscle Cells Induced By High Phosphoru Activates Of TLR4/NF-?B Signaling Pathway

The morbidity and mortality of chronic kidney disease(CKD)are still increasing even though numerous progresses have been made in modern medicine and public health all over the world.Many CKD patients are not effectively controlled,and their diseases eventually deteriorate into the end-stage renal disease(ESRD),which needs dialysis or kidney transplantation.It is noteworthy that the complications of the CDK can affect many other organs besides the kidney.Cardiovascular disease(CVD),which accounts for more than 50% of the total CKD mortality,is the most prevalent and important factor among all the complications in CKD patients.Thus,CKD has attracts great attentions.Notably,vascular calcification(VC)is not only an important pathological change in CKD patients,but also an independent risk factor of the morbidity and mortality in cardiovascular diseases.Therefore,it is quite important to prevent the development of vascular calcification(VC)in clinical treatments.Vascular calcification often occurs with the aging of our body,but also occurs in some diseases,such as chronic kidney disease,diabetes,chronic heart failure and so on.Among them,CKD–induced vascular calcification often occurs in the medial layer,which is thought to be a simple deposition of calcium-phosphate in vascular smooth muscle cells(VSMCs)and extracellular matrix,suggested by previous studies.However,the current studies show that the vascular calcification in the medial layer is a process that is active and gene-controlled.The trans-differentiation of vascular smooth muscle cells into osteoblasts is considered to be the key step of vascular calcification in the medial layer.Therefore,it is very important to explore the reasons and mechanisms of the trans-differentiation of VSMCs into osteoblast,which will provide new ideas and new targets for the clinical prevention of vascular calcification.Hyperphosphatemia and chronic inflammation are not only the important pathological features of chronic kidney disease,but also play important roles in its development.Although hyperphosphatemia often occurs in phase 4 or 5 of CKD,the increase in serum phosphorus begins at the earlier stages.Under this condition,hormones such as FGF23 can be adaptively increased to maintain the normal level of serum phosphorus,but the damage caused by the increase of serum phosphorus is quietly developed.Now it is believed that hyperphosphatemia is one of the major causes of vascular calcification,however,the mechanisms of high phosphorus-induced VCMCs differentiation into osteoblasts have not been completely elucidated.Studies have shown that inflammatory responses and inflammatory factors(tumor necrosis factor-alpha,etc.)can accelerate the differentiation of VSMCs into osteoblasts in vitro,which suggests that the inflammatory response and vascular calcification are causally-related.However,their relationship is not yet clear in vivo.NF-?B plays an important role in chronic inflammation.The transcription of interleukin-1?(IL-1?),interleukin-6(IL-6),tumor necrosis factor-? and other inflammatory factors can activated by the translocation of NF-?B into the nucleus,while the TOLL-like receptor 4(TLR4)is the major signaling molecule that activates NF-?B signaling pathway.TLR4 can activate NF-?B classical pathway through mediator-like protein 88(My D88),which facilitates the dissociation,release and activation of phosphorylated NF-?B by I?B? and leads to the translocation NF-?B into the nucleus,resulting in the activation and transcription of various inflammatory factors.These studies suggest that hyperphosphatemia and chronic inflammation(TLR4 / NF-?B signaling pathway)are closely related to the trans-differentiation of VSMCs into osteoblasts.However,the relationship between hyperphosphatemia and chronic inflammation is still unknown.Furthermore,it remains unclear whether phosphate(Pi)can activate TLR4 / NF-?B pathway directly to induce the calcification of vascular smooth muscle cell or not.Therefore,in this study,by using mouse vascular smooth muscle cells as an in vitro model,we aims to verify the key role of high phosphorus in vascular calcification,and clarify whether high phosphorus could induce the trans-differentiation of VSMCs into osteoblasts through the NF-?B activation,and finally investigate whether high-phosphorus could induce the trans-differentiation VSMCs into osteoblast-like cells or chondrocytes by activating TLR4/NF-?B signaling pathways that lead to VC.The main findings are list below:1,High phosphorus induced the trans-differentiation of VSMCs into osteoblasts that leads to calcificationVascular smooth muscle cells were cultured using normal DMEM high glucose medium or DMEM high glucose medium containing 2.6 mmol/L phosphate for 5 days in vitro,respectively.After 5 days culture,VSMC cells were stained with alizarin red S to observe the calcification.Western blot was applied to analyze the protein levels of trans-differentiational markers.Alizarin red S staining showed that VSMCs in high phosphorus group had obvious calcification in high phosphorus environment,compared with that in the normal group.The protein expression of Bone morphogenetic protein 2(BMP2)and Runt-related transcription factor 2(Runx2)in VSMCs was significantly higher,while VSMC-specific marker SM22? was down-regulated.These results indicate that hyperphosphatemia can induce calcification and trans-differentiation of vascular smooth muscle cells.2,High phosphorus induced the trans-differentiation of VSMCs and VC by activation of NF-?BIn order to observe the effect of high phosphorus on NF-?B,we also cultured VSMCs for 5 days using conventional DMEM medium and medium that containing 2.6 mmol / L phosphate.The expression of NF-?B was detected by Western blot after 5 days of treatment.The results showed that the protein level of phosphorylated NF-?B(P65)in high-phosphorus group was significantly higher than that in normal control group,indicating that high-phosphorus could activate NF-?B and play a role in translocation into the nucleus.In order to further elucidate whether hyperphosphorones induce calcification of VSMCs by NF-?B activation,we use NF-?B inhibitor(PDTC)to incubate with VSMCs in normal medium and medium containing 2.6 mmol / L phosphate for 5 days.5 days later,Western blot was used to detect intracellular protein expression.Alizarin red S staining was used to observe the cell calcification.The results showed that the protein level of phosphorylated NF-?B was significantly inhibited by PDTC,compared with that in high-phosphorus group.At the same time,the protein levels of other markers such as BMP2 and Runx2 were also significantly downregulated,while smooth muscle markers SM22? was significantly increased,as the activation of NF-?B was inhibited by PDTC.Alizarin red S result also demonstrated that PDTC inhibited the calcification of VSMCs,compared with that in high phosphorus group.3,High phosphorus activated TLR4 / NF-?B signaling pathway to induce VSMCs calcificationTo further observe whether TLR4 / NF-?B signaling pathway can be activated during calcification of VSMCs in high phosphate medium,we first cultured VSMCs in normal medium or medium containing 2.6 mmol / L phosphate for 5 days,then the expression of TLR4 protein was then detected by Western blot.Moreover,we used TLR4 si RNA to interfere with the expression of TLR4.Similarly,Alizarin red S staining was used to observe the cell calcification.Western blot showed that the protein level of TLR4 was significantly induced by high phosphate medium,which suggested that TLR4 was involved in VC.To analyze the role of TLR4 in VC,the protein expression of TLR4 was knockdown by TLR4 si RNA,and the result showed its efficiency was very well,as judged by Realtime PCR and Western blot.Next,we detected the effects of TLR4 si RNA on NF-Kb and other protein markers.The results showed that the phosphorylation of NF-?B was significantly inhibited by TLR4 si RNA,while the expression of SM22? was significantly increased and osteoblast associated protein expression was decreased significantly.Alizarin red S showed that the degree of cell calcification was significantly reduced while TLR4 was knockdown by TLR4 si RNA.All these data clearly indicated high phosphorus can activate TLR4 / NF-?B signaling pathway to induce VSMCs calcification.In summary,our study found that under the condition of CKD,high phosphorus may up-regulate TLR4 expression and activating TLR4 / NF-?B signaling pathway,which give rise to the initiation of trans-differentiation of VSMCs into osteoblast and / or chondrocyte,and thus induce vascular calcification subsequently,which may be an important mechanism of vascular calcification in CKD patients.Moreover,how to inhibit the activation of TLR4 / NF-?B signaling pathway may be a key intervention to prevent vascular calcification for CKD patients.