Effects Of High-density Lipoprotein 1 On The Formation Of Foam Cells From Human Monocytes-derived Macrophages

BackgroundIt is confirmed that serum high density lipoprotein(HDL) possess properties of atherosclerosis(AS) resistance and heart protection.HDL is composed of different subclasses with different particle characteristics and physiological functions. Different HDL subclasses play different roles in the AS resisting functions of HDL, which is of great significance to the prevention and treatment of coronary heart disease(CHD).However,understandings on the composition and particle characteristics of HDL subclasses are limited,and no obvious therapeutic effects can be obtained by the regulation of serum HDL subclasses in clinic.The compositions of HDL are complex,and its constitutions are special.How to detect HDL subclasses in serum so as to help the status analysis of reverse cholesterol transport(RCT),and make further studies on the physical and chemical structures and physical functions of HDL,especially its new types,are hot and hard at present.In our prophase studies,13 zones of lipoproteins could be clearly seen in the isolated gel from sudan black B(SBB) pre-dyed serum after sectile density polyacrylamide gel electrophoresis (sd-PAGE),among which 2 were free fatty acids(FFA_1-2),5 were HDL subclasses(HDL_1-5,among which HDL₁ and HDL₅ were new types),3 were phenotypic LDL,intermediated-density lipoprotein(IDL),2 were very low density lipoprptein(VLDL),and chylomicra(CM),which stagnated in the starting point of gel. Sd-PAGE new methods could not only isolated LDL,HDL,VLDL,CM,IDL and other lipoproteins in gel from human serum clearly,but also could make a further subclass isolation of them,of which 5 types of HDL subclasses were especially prominent.In our prophase studies we also found that HDL₁ appeared alone in isolated gels in all sd-PAGE experiments,which indicating that HDL₁ is a new type of HDL subclass that is relative isolated.This is a new phenomenon which is deserved to be further studied and which will have important academic values and clinical significance.ObjectivesThe purpose of present study was to isolate and prepare a special subclass of HDL(HDL₁ from human serum by sd-PAGE,and to investigate its effects on the formation of foam cells from human peripheral blood monocyte-derived macrophages, so as to explore its functions in resisting AS,and to offer clues for further studies of it from molecular level and receptor level.Methods1.Preparation and oxidized of low density lipoprotein(LDL):LDL was isolated from human plasma by one time density gradient centrifugation.Agarose gel electrophoresis was used to identify the purity of LDL,and modified Lowry protein assay was applied to detect the concentration of LDL.10 umol/L Cu²⁺ was used to induce the oxidation of LDL(ox-LDL),and thiobarbituric acid reactive substance (TBARS) value was detected so as to estimate the oxidation degree of LDL in 532 nm. 2.Isolation and preparation of serum HDL₁:Serum was pre-dyed with SBB,and sectile density polyacrylamide gel electrophoresis(sd-PAGE) was applied to isolate serum LDL,HDL as well as their subclasses,and other serum lipoproteins simultaneously.The isolated HDL₁ was prepared by self-made eluting system and condensing system.Modified Lowry protein assay was used to quantified HDL₁ concentration,biochemistry enzymic method and turbidimetric method were applied to detect apoA-I concentration in HDL₁ solution.3.Isolation of monocytes from human peripheral blood:Monocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation and plastic adsorptive process.Trypan blue staining and flow cytometry were applied to access the survival rate and purity of isolated cells.4.Establishment of foam cell model:4.1 Transformation of monocytes to macrophages:The isolated cells were induced and stimulated by 50 nmol/L phorbol 12-myristate 13-acetate(PMA) for 48 h so as to make them transfer to macrophages.Morphological changes of the cells were observed under light microscope.4.2 Transformation of macrophages to foam cells:The successfully transfer cells were then coincubated with 80 mg/L oxidized low density lipoprotein(ox-LDL) for 24 h,morphological changes of macrophages were observed through transmission electron microscope(TEM),and by red oil O staining under light microscope.Total cholesterol(TC),free cholesterol(FC),cholesteryl ester(CE),and protein contents in cells were detected,so as help to make a biological identification of the established model.4.3 Detection of cell survival rates:Trypan blue staining was applied to access the survival rates and cytoactive of cultured cells in different stages of experiment,so as to make certain that weather HDL₁ at different concentrations,or HDL1 at certain concentration but functioned for different times,would exert adverse effects on cultured cells.5.Detection of cholesterol and protein in cells:Enzymic cholesteryl ester assay was applied to detect the contents of TC,FC in cells,and CE was expressed as TC-FC.Modified Lowry protein assay was applied to detect the contents of protein in cells,the ratio of CE to TC and TC to Pro were calculated finally.6.Effects of HDL₁ on contents of cholesterol and protein in macrophages:The monocyte-derived macrophages were allocated to control groups and HDL₁ groups. Control groups were further divided into blank control(RPMI-1640 group),positive control(ox-LDL group),and standard control(ox-LDL+apoA-I group).The cells were firstly coincubated with 80 mg/L ox-LDL and HDL₁ at different concentration(0～10.0 mg/L) for 24 h,respectively.Oil red O dyeing experiment and transmission electron microscope(TEM) were performed to identify the formation of foam cells, and TC,FC and protein in cultured cells were quantitatively analyzed by enzymic cholesteryl ester assay and modified Lowry protein assay,respectively.The dose-effect of HDL₁ to the ratio of TC to Pro in cultured cells was observed.When the best control concentration of HDL₁ was confirmed,the cells were then coincubated with 80 mg/L ox-LDL and HDL₁ of the best concentration for 0,6,12 and 24 h,respectively,and the time-effect relationship of HDL₁ to the ratio of TC to Protein in cells were statistically and graphically observed.7.Statistical treatment:All measurement data was indicated by Mean±Standard deviation((?)±S).One-way ANOVA analysis followed by LSD analysis(SPSS10.0) were used for the analysis of difference between groups,andα=0.05 indicates significant difference.Results1.Isolation effect of serum lipoprotein:13 zones of lipoproteins could be clearly seen in the isolated gel from SBB pre-dyed serum after sd-PAGE,among which 2 were free fatty acids(FFA_1-2),5 were HDL subclasses(HDL_1-5,among which HDL₁ and HDL₅ were new types),3 were phenotypic LDL,intermediated-density lipoprotein(IDL),2 were very low density lipoprptein(VLDL),and chylomicra(CM), which stagnated in the starting point of gel.2.Establishment of foam cell model2.1 Transformation of monocytes to macrophages:After induced by 50 nmol/L PMA,most of the cells got polygon from round,and aggregated,adherence easily, under light microscopes,which indicated the successful transformation of monocytes to macrophages.2.2 Transformation of macrophages to foam cells:When macrophages were coincubated with 80 mg/L ox-LDL,the ratio of CE/TC in cells was beyond 60%by cholesterol assay,and there were also a great deal of lipid droplets and lipid vacuole in ox-LDL treated cells both under TEM and light microscope,which indicated the formation of foam cells.3.Effects of HDL₁ on the formation of macrophages to foam cells:HDL₁ could decreased TC/Pro ratio in macrophages in a concentration-dependent manner(0～10.0 mg/L)(P＜0.01).At the concentration of 10.0 mg/L,HDL₁ decreased TC/Pro ratio in cells more than 70%.HDL₁(10.0 mg/L) could also decreased TC/Pro ratio in cells in a time-dependent manner,which differed significantly to that of control groups(P＜0.01).Conclusions1.Sd-PAGE new methods could not only isolated LDL,HDL,VLDL,CM,IDL and other lipoproteins in gel from human serum clearly,but also could make a further subclass isolation of them,of which 5 types of HDL subclasses were especially prominent.Compared with many of other methods concerning the isolation of serum lipoprotein subclasses,the introduced method has evident superiority.2.In the formation of macrophages to foam cells,treatment of HDL₁ at different concentrations and for different times have obvious dose-effect and time-effect with the ratio of TC/Pro in cells,which indicates that HDL₁ can by means of decreasing TC in cells so as to decrease TC/Pro ratio and CE/TCratio in cells.The obtained results suggest that HDL₁ is capable of inhibiting or relieving the formation of foam cells by decreasing of TC in cells,so as playing a role in resisting AS of HDL.