The Study Of The Molecular Mechanism Under The Atherogenic Role Of Hyperphosphatemia By Inducing SCAP Dysfunction

Part one: Hyperphosphatemia promote atherosclerosis formation in ApoE-/-mice by inducing dysfunction of SREBP2 cleavage activation protein(SCAP)Objects: Hyperphosphatemia is one of the most important risk factors for coronary artery atherosclerotic disease in patients with chronic renal disease,but its atherogenic mechanism remains unclear.Sterol regulatory element binding Protein 2(SREBP2)and its cleavage activating protein(SCAP)act as the sensor of cholesterol in mammal cells,playing crucial roles in intracellular homeostasis of cholesterol.Many researches have revealed that the dysfunction of SCAP modulates intracellular cholesterol accumulation in varous pathological state.By establishing hyperphosphatemia models in ApoE-/-mice,this study was aimed to investigate the atherogenic role of hyperphosphate and its underlying mechanism.Methods: Male ApoE-/-mice aged 8 weeks were divided into four groups randomly: control group(chow,phosphate 0.6%,n=8),high phosphate feeding group(h P,phosphate 1.6%,n=8),high phosphate plus sevelamer group(h PS,phosphate 1.6% + 3% sevelamer,n=12)and hyperphosphatemia sevelamer treatment group(h PST,phosphate 1.6% for 8 weeks,then phosphate 1.6% + 3% sevelamer for 8 weeks,n=8).The mice were feed in SPF environment for 16 weeks before sacrifice,serum concentration of biochemical indexes such as ionic phosphate,calcium,urea nitrogen,blood lipids et al were measured.The atherosclerosis burden was determined by oil red staining of aortic sinus frozen sections,the expression of sterol regulatory element binding protein(SREBP)and its cleavage activating protein(SCAP),low density lipoprotein receptor(LDLr)and 3-hydroxy-3-methyl-glutaryl coenzyme A reductase(HMGCo AR)levels were measured by Immunohistochemical staining and Western blotting with aorta.Results: The serum phosphate concentration of ApoE-/-mice in high phosphate treatment group was higher than control group after 8 weeks of preliminary experiment.After 16 weeks of different diet feeding,the serum concentration of phosphate in h P group were significantly increased when compared with control group(P<0.05),and the level of serum phosphate of h PS group were significantly lower than that in h P group(P<0.05),whitch in h PST group showed an increasing trend but did not reach the significance when comparied with control group(P>0.05),While other biochemical indexes such as calcium,BUN,LDL and Tcho did not change significantly among groups.The quantitative analysis of oil red staining of artic sinus continuous frozen section demonstrated that,the relative areas of plaque(atheroma area/ cross area of aortic sinus)of mice in h P group were larger than that in control group(P<0.05),but not changed significantly when compared with h PST group.The Immunohistochemical staining and Western blotting examination showed that the protein expression of N-SREBP2,HMGCo AR and LDLr were obviously higher in the aorta of mice in h P group when compared with control group and h PS grou;however,the operation of sevelamer treatment after establishment of hyperphosphatemia seems noneffective on the expression of these proteins.Conclusion: Hyperphosphatemia promoted the development of atherosclerosis in ApoE-/-mice,which might via upregulating the SCAP protein level in vascular tissue cells.This enhance the translocation of SREBP2 from endoplasmic reticulum to Golgi apparatus and release the active fragment N-SREBP2 by proteolytic cleavages,promoting the transcription of target genes such as LDLr and MGCo AR.As a consequence,the extracellular LDL uptake and intracellular cholesterol de novo synthesis were increased,finally promoted atherosclerosis.The promotion of atherosclerosis by hyperphosphatemia induced high-phosphate intake could be delayed effectively by giving oral phosphate binder sevelamer in ApoE-/-mice.But in the mice which already have hyperphosphatemia,the treatments of sevslamer do not react effectually,which might because of the compensatory response of PTH,FGF 23 induced by sustained hyperphosphatemia.Part two: Phosphate Promotes Cholesterol Accumulation by Inducing Cholesterol Sensor SCAP Dysfunction in MacrophagesObjective:To investigate the effect of phosphate on the homeostasis of cholesterol in human macrophages and its underlying mechanism.Methods: The THP-1 macrophages were divided into control group(Pi 1.0 mmol/L),high phosphate group(Pi 3.0 mmol/L),phosphonoformic acid(PFA)treatment group(PFA 1.0 mmol/L)and high phosphate plus PFA treatment group.After treated for 24 h,the content of intracellular neutral lipids was observed by Oil red o staining,the total cholesterol(TC),cholesterol ester(CE)and free cholesterol(FC)in macrophages were measured by quantitative assay.The relative m RNA expression of sterol regulatory element binding protein(SREBP)cleavage activating protein(SCAP),low density lipoprotein receptor(LDLr)and 3-hydroxy-3-methyl-glutaryl coenzyme A reductase(HMGCo AR)were examined by Real-time PCR,the protein expression of SCAP,LDLr,HMGCo AR,and nuclear SREBP 2(N-SREBP2)were assessed by Western-blotting.Results: High phosphate exposure increased the accumulation of intracellular cholesterol ester(CE)and total cholesterol(FC)but not free cholesterol(FC)in THP-1 macrophages when compared with the control(P<0.05),and these alterations can be reconciled effectively by the blocker of the sodium dependent phosphate transporter Pit-1 on the cell membrane(high phosphate plus PFA treatment group)(P<0.05).The m RNA and protein expression of LDLr and HMGCo AR were obviously higher in high phosphate group than that in control(P<0.01),the elevation of which can be dismayed by addition of PFA.Meanwhile,we found an elevated protein level of N-SREBP2 in macrophage nucleus which is the active transcription factor for LDLr and HMGCo AR gene.Further,high phosphate treatment increased the protein level but not the m RNA expression of SCAP which is the key chaperon molecular for SREBP2 in eukaryocyte,and this up-regulatory effect on SCAP protein can be significantly suppressed by PFA.Conclusion: When cultured in high phosphate environment,the inorganic phosphorus entered the macrophages via the sodium dependent phosphate transporters Pit1 on the cell surface,then the elevated intracellular phosphorus induced the dysfunction of SCAP by increasing its protein level in an post transcriptional pathway,which facilitated the transport of SREBP2 from endoplasmic reticulum to Golgi apparatus,leading to more N-SREBP2 production in the nucleus,and therefore upregulated LDLr and HMGCo AR gene transcription,which increased the uptake of native LDL and the de novo synthesis of endogenous cholesterol,thereby accelerated macrophages derived foam cell formation.