Hyperphosphorus-induced Cholesterol-sensing SCAP Dysfunction Promotes Foaming Of Vsmcs

Part one:High phosphorus induces cholesterol accumulation in VSMCsObjective:Atherosclerosis(AS)disease is a major complication and cause of death in patients with chronic kidney disease(CKD).Recent studies have shown that serum phosphorus and cholesterol may have similar atherogenic effects.Vascular smooth muscle foaming is the basic pathological process of atherosclerosis.In order to investigate whether high phosphorus interferes with the development of atherosclerosis through cholesterol metabolism,we observed the accumulation of cholesterol in the cells after inducing primary human Vascular Smooth Muscle Cells(VSMCs)with high phosphorus.Methods:1 A high phosphorus VSMCs model was established:6-8 generations of primary human VSMCs were starved for 24 hours in 2%FBS M199 medium,and incubated with gradient phosphorus concentrations of 1.0 mmol/L,2.0mmol/L,3.0mmol/L,4.0mmol/L,and 5.0mmol/L,respectively,for 24 hours.The total protein of VSMCs was routinely extracted and the expression of SCAP and HMGCR protein were detected by western blotting.2 After the normal starvation,the 6-8 generations of primary human VSMCs were divided into control group and high phosphorus group.In control group,VSMCs were cultured with 2%FBS M199 containing phosphorus at a concentration of 1.0mmol/L,and the concentration of phosphorus is 3.0mmol/L in high-phosphorus group.After 24 hours of treatment mentioned above,neutral lipid accumulation in VSMCs was observed by oil red O staining.Cholesterol content in VSMCs was determined by enzymatic quantification.The expression of SCAP,HMGCR and N-SREBP2 were measured by western blotting.Result:After VSMCs were intervened by different phosphorus concentration gradients,western blotting results showed that phosphorus up-regulated the expression of SCAP and HMGCR protein in VSMCs in a dose-dependent manner.2.The results of oil red O staining showed that in the high phosphorus group,the neutral lipid particles in VSMCs were significantly increased compared with the control group.Determination of cholesterol content in VSMCs by enzymatic method showed that the total cholesterol and free cholesterol in VSMCs were significantly increased in the high-phosphorus group,comparing with the control group(P<0.05).Western blotting results showed that the levels of SCAP,HMGCR and N-SREBP2 in the high-phosphorus group were significantly higher than those in the control group.3.Real time PCR results showed that the mRNA expression of HMGCR(P<0.05),LDLr(P<0.05),and SREBP2(P<0.01)increased significantly in the high-phosphorus group.Conclusion:Phosphorus can up-regulate the expression of SCAP and HMGCR protein in VSMCs in a dose-dependent manner.Phosphorus ion concentration of 3.0mmol/L was closer to the hyperphosphatemia state in vivo,so 3.0mmol/L of phosphorus was selected to establish a high-phosphorus cell model in vitro.High phosphorus(3.0mmol/L)can cause cholesterol accumulation in VSMCs,enhance the expression of SCAP,HMGCR,and N-SREBP2 proteins and enhances transcription of HMGCR,SREBP2,and LDLr genes.The SCAP and N-SREBP2 proteins are key proteins in the regulation of intracellular cholesterol homeostasis(LDLr negative feedback regulation),and HMGCR is a key protein in intracellular cholesterol metabolism.These phenomena suggest that once CKD patients are complicated by hyperphosphatemia,high phosphorus induces foaming of vascular smooth muscle cells through a mechanism that induces cholesterol metabolism,which triggers atherosclerotic disease.However,the molecular mechanism of the interference of high phosphorus on cholesterol metabolism needs further exploration.Part two:Hyperpnospnorus-inauced cnolesterol-sensing SCAP dysfunction promotes foaming of VSMCsObjective:It has been demonstrated that cholesterol accumulation in VSMCs caused by high phosphorus may be closely related to the negative feedback regulation of LDLr in previous work.Under normal physiological conditions,the balance of intracellular cholesterol homeostasis is mainly controlled by the LDLr negative feedback regulatory system through the interaction of Insig-SCAP-SREBPs,in which the translocation of SCAP from the endoplasmic reticulum to the Golgi apparatus is a crucial step.Studies have shown that after induction of SCAP upregulation,inflammation caused a large accumulation of cholesterol in peripheral cells.Does high phosphorus also disrupt cholesterol metabolism through the same pathway?Therefore,specific pitl/2 channel blocker(PFA)was used to intervene in the uptake of VSMCs and Betulin(a specific inhibition of SCAP from endoplasmic reticulum to Golgi translocation compound)was used to intervene in the level of SCAP translocation in order to investigate whether the high phosphorus leads to foaming of VSMCs by inducing SCAP dysfunction in cells.Methods:After the normal starvation,VSMCs were divided into 6 groups:control group(pbs),high-phosphorus group(phosphorus 3.0mmol/L),PFA group(PFA 1.mmol/L),PFA + high-phosphorus group(PFA 1.0mmol/L + Phosphorus 3.0mmol/L),Betulin group(Betulin 3.0μg/mL),and Betulin + high-phosphorus group(Betulin 3.0μg/mL+3.0mmol/L).After VSMCs were cultured with 2%FBS M199 for 24h,the accumulation of neutral lipid particles in VSMCs was detected by Oil red O staining.The cholesterol content in VSMCs was detected by Enzyme assay.Results:1 The results of oil red O showed that there was no significant difference in neutral lipid particles in VSMCs between the PFA group and the control group,whereas in the PFA+ high phosphate group,the neutral lipid particles in VSMCs were significantly reduced compared to the high phosphate group.The degree of neutral lipid particle aggregation in VSMCs in the Betulin group was similar to that in the control group.The neutral lipid particles in VSMCs in the Betulin + high-phosphorus group were significantly less than those in the high-phosphorus group.2 Enzymatic quantitative results showed that there was no significant difference in the cholesterol content between the PFA group and the control group.Comparing with the high phosphorus group,the intracellular cholesterol was significantly decreased in the PFA + high phosphorus group(total cholesterol(P<0.05)and free cholesterol(P<0.05)).The cholesterol content in the Betulin group was significantly lower than that in the control group(total cholesterol(P<0.05)and free cholesterol(P<0.01)),and the intracellular cholesterol was also significantly reduced in the Betulin + high phosphorus group compared with the control group(total cholesterol(P<0.05)and free cholesterol(P<0.05)).3 The results of Real time PCR showed that the expression of HMGCR,LDLr,and SREBP2 mRNA in high phosphorus + PFA group was significantly lower than those in high phosphate group(P<0.05).The expression of HMGCR and LDLr mRNA in Betulin group was significantly lower than those in control group(P<0.05).The expression of HMGCR,LDLr,and SREBP2 mRNA in the P + Betulin group was significantly lower than those in the high-phosphorus group(HMGCR and LDLr(P<0.01),SREBP2(P<0.05)).There was no significant changes in the expression of SCAP mRNA under PFA or Betulin.Conclusion:In the high-phosphorus state,PFA blocks the entry of phosphorus into the cells,and the intracellular cholesterol is significantly reduced,indicating that in the high-phosphorus state,phosphorus needs to enter the cells to achieve interference with cholesterol metabolism.After the SCAP translocation process was inhibited,the massive accumulation of intracellular lipids caused by high phosphorus was significantly reduced.This phenomenon indicates that high phosphorus may cause foaming of VSMCs by interfering with the function of SCAP.