Formation And Mechanism Of Vascular Smooth Muscle Cells Derived Foam Cell Induced By OxLDL

Background and Objectives:Atherosclerosis(AS)is the primarycause of death worldwide through its manifestations including ischemic heart disease,stroke and peripheral vascular diseases.The formation of fat-laden foam cells,contributing to the fatty streaks of the plaques of atheroma,is the critical early process in AS.Within vascular cells,lipids accumulate as cytoplasmic droplets of cholesterol esters and triglycerides;cells with an abundance of these droplets are termed foam cells.Macrophages are considered the primary source of foam cells in AS.Besides,atherosclerotic lesions also contain abundant foam cells with vascular smooth muscle cell(VSMC)identity,especially in advanced atherosclerotic lesions.Oxidized low density lipoprotein(oxidized low-density,oxLDL)is generally considered to be the strongest factor causing AS,and in vivo and in vitro studies have confirmed that VSMC can promote the formation of foam cell.Herein,the purpose of this study was to investigate the role of macropinocytosis in the uptake of lipids and the role of SIRT1 in the process of oxLDL induced VSMC formation.Macropinocytosis has been shown to play an important role in the formation of macrophage derived FC.A number of studies have demonstrated that macrophages can uptake a large amount of extracellular modified or native LDL in the form of macrpinocytosis,and form foam cells.However,the role of macrpinocytosis in VSMC derived FC is less unknown.Previous reports confirmed that PDGF can stimulate VSMC to intaking drug particles and VSMC uptake serum form LDL in foam cell formationboth through macropinocytosis,so this research in the application of PDGF tostimulated VSMC,and to explore whether there will be macropinocytosis and oxLDL can enter the cell through this form.SIRT1(Sirtuin1)in mammals,the closest homolog of yeast Silent information regulator 2(Sir2)protein,is a member of the highly conserved sirtuin family of nicotinamide adenine dinucleotide(NAD+)-dependent histone deacetylases.SIRT1 can target numerous proteins including peroxisome proliferator-activated receptor(PPAR)-?,PPAR-? coactivator(PGC)-1?,uncoupling protein 2(UCP2),liver X receptors(LXRs)and nuclear factor(NF)-?B and thus regulate various pathological and physiological processes.For instance,deacetylation of LXR by SIRT1 up-regulates its activity and promotes reverse cholesterol transport to remove cholesterol from cells,ultimately inhibits the foam cell formation.SIRT1 has also been found to exert salutary actions against endothelial dysfunction through inhibiting premature senescence.Upregulating endothelial nitric oxide synthase(eNOS)expression by SIRT1 rescued endothelium dependent vasorelaxation in vivo impaired by high fat diet.Besides,the positive effects of SIRT1 on stabilizing plaques,decreasing cholesterol uptake,inhibiting macrophage foam cell formation and relieving inflammatory raise the prospect of SIRT1 as a promising therapeutic target for AS treatment.However,there are few reports on the role of SIRT1 in the process of VSMC foam cell formation.Therefore,this study focuses on the role of SIRT1 in VSMC foam cell formation and its underlying mechanism.Acyl coenzyme A: cholesterol acyltransferase 1(A-cholesterol acyltransferase,ACAT1)is a key enzyme in the synthesis of cholesterol esterswhich catalyzethe free cholesterol and fatty acid in cells.Previous studies have shown ACAT1 plays a key role in macrophage and VSMC derivedfoam cell formation,so whether SIRT1 can affect the expression of ACAT1 in VSMCderivedfoam cellisstillnotclear.Before SIRT1 has been reported to function directly with PPAR?,while PPAR? in macrophages and VSMC can directly regulate ACAT1 function,in this experiment we also detected the expression of PPAR? in FC formation.Materials and Methods:1.VSMCs were isolated from the thoracic aorta of C57BL/6Jwild-type(WT)mice using the explant technique.The cultured VSMC were verified through immunofluorescence and flow cytometry to test ?-smooth muscle actin(?-SMA).2.VSMC-derived foam cells by oxLDL were verified by Oil Red O staining.3.VSMC was stimulated byPDGF-BB,and the cell endocytosis tracer Lucifer Yellow(LY)as well as fluorescently labeled(Di I)LDL and oxLDL was detected using the flow cytometry.4.PDGF-BB stimulated VSMC with Di I-oxLDL,using inhibitor including actin polymerization inhibitor cytochalasin D,PI3-K inhibitor LY 294002,Na+/H+ inhibitor amiloride,dynamin inhibitor dynasore and clathrin dependent endocytosis inhibitors(hypertonic sucrose).Di I-oxLDL uptake was observed by FACS.5.SIRT1 agonist SRT1720(SRT)and antagonist nicotinamide(Nic)were used to modulate the expression of SIRT1 pharmacologically.6.PPAR? agonist Rosiglitazone(RSG)and antagonist GW9662 were used to influence the expression of PPAR?.Results:1.PDGF-BB can stimulate VSMC to increase the uptake of LY,LDL and oxLDL.PDGF stimulated VSMC significantly increased the percentage of cells containing LY.With the increase of PDGF concentration gradient,the uptake of LDL by VSMC could be increased.Similarly,the use of PDGF can stimulate VSMC to increase the uptake of oxLDL.2.The inhibitors of macropinocytosis and the inhibitors of receptor mediated endocytosisboth inhibited the uptake of oxLDL partially by VSMC.The use of actin polymerization inhibitor cytochalasin D,PI3-K pathway inhibitor LY294002 can significantly inhibit the uptake of oxLDL;and dynamin inhibitor dynasore also can significantly inhibit the uptake of oxLDL.3.SIRT1 inhibitedthe ox-LDL induced VSMC foam cell formation.In this study,80 ?g/m L oxLDL stimulated VSMC 48 hours,oil red O staining indicated that oxLDL promoted the accumulation of red lipid dropletsin cultured VSMC cells,the oxLDL can induce VSMC derived foam cell formation;at the same time,stimulated by SRT,can significantly reduce the foam cells formation and red lipid droplets decreased significantly;and this effect was blocked by SIRT1 antagonist Nic.The results of Western blot showed that the expression of SIRT1 was significantly decreased after 24 hours of stimulation with oxLDL in VSMC.4.SIRT1 inhibited FC formation by decreasing ACAT1 and increasing the expression of PPAR?.In this study,the expression of ACAT1 protein was significantly increased after oxLDL stimulation for 24 hours with the decrease of PPAR?.The expression of SIRT1 protein was regulated by SRT and Nic.SRT could downregulate the expression of PPAR? and increase the expression of ACAT1,while Nic reversed the expression of ACAT1 and PPAR by SRT.The next step is to regulate the expression of PPAR? protein by PPAR?activator Rosiglitazone(RSG)and inhibitor GW9662,RSG can significantly inhibit the expression of ACAT1,and GW9662 can reverse this effect.Conclusions:In conclusion,PDGF-BB can significantly stimulate the increase of oxLDL in VSMC,and through micropinocytosis partially.We showed that SIRT1 could inhibit the formation of VSMC derived foam cell induced byoxLDL and decrease the expression of SIRT1.Activation of SIRT1 inhibits the formation of FC,accompanied by the decrease of ACAT1 expression and the increase of PPAR? expression.This study suggests that SIRT1 is a potential target for improving VSMC function and enriches the way of oxLDL entry into cells.