Role of VLDL Receptor in the Process of Foam Cell Formation

Journal of Huazhong University of Science and Technology [Med Sci]24 (1); 1-4, 20041华中科技大学学报[医学(英德文)版了Role of VLDL Receptor in the Process of Foam Cell Formation "qu Shen(屈伸), WUFan(昊凡), TIAN Jun(田俊). LI Yinghong (李映红), WANG Yan(王燕)，WANG Yuzhe (王宇哲), ZONG Yiqiang (宗义强)Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences. Tomgji Medical College.Hua zhong University of Science and Technology. Wuhan 430030Summary: The role of very low density lipoprotein reeptor (LVLDR) in the process of foam cellformation was investigated. After the primary cultured mouse peritoneal macrophages were incuba-ted with VLDL, βVLDL or low density lipoprotein (LDL), respectively for 24 h and 48 h. foamcells formation was identified by oil red O staining and cellular contents of triglyceride (TG) and to-tal cholesterol (TC) were detrmined. The mRNA levels of LDLR, LDLR related protein (LRP)and VLDLR were detected by semi- quantitative RRT-PCR. The rsults demonstrated that VLDL. PVLDL and LDL could increase the contents of TG and TC in macrophages. Cells treated withVLDL or 9-VLDL showed markedly increased expression of VLDLR and decreased expression ofLDLR，whereas LRP was up- regulated slightly. For identifying the effect of VLDL receptor on cel-lular lipid aceumulation, IdI-A7-VR cells, which expresses VLDLR and trace amount of LRP with-out functional LDLR, was used to incubate with lipoproteins for further examination. The resultselucidated that the uptake of triglyceride rich lipoprotein mediated by VLDLR plays an importantrole in accumulation of lipid and the formation of foam cells.Key words: very low density lipoprotein receptor; triglyceride rich lipoprotein; expression regula-tion; foam cellCholesterol has been showed to play an impor-DNA polymerase, DNA marker, dNTPs were pur-tant role in development of atherosclerosis (AS).chased from Promega (USA). Trizol reagent wasRecent researches further demonstrated that plas-from Shanghai Huashun Company. Test kit forma triglyceride levels are positively correlated withlipid mensuration was purchased from Beijingthe coronary artery disease. Increasing clinical evi-Zhongsheng Company.Hepes, RPM11640 culturedence and epidemiological studies indicate that tri-medium, fetal bovine serum (FBS) were purchasedglyceride- rich lipoprotein (TRL) is an independentfrom Gibco (USA). ldI-A7 cell was gifted byatherogenic factor 0. Hiltunen et al reported thatProf. Monty Krieger and THP-1 cell was purthe expression of very low density lipoprotein re-chased from ATCC. ldl-A7-VR cell that steadilyceptor (VLDLR) in smooth muscle cells ( SMC)expresses VLDLR was constructed by Prof. Qu etand macrophages in the aorta wall of New Zealandal间. PCR primers were synthesized by Shanghairabbit was increased significantly after 3-week up-Shenggong Company. J apanese white rabbit andtake of cholesterol. 14-week later, VLDLR mRNAKunming mice were from Experimental Animallevel was increased to 100 folds compared to con-Center of Tongji Medical College. All other chemi-trol2. Additionally, markedly elevated level ofcals were of domestic analytical grade.VLDLR was also observed in human atherosclerot-1.2 Lipoprotein Separationic plaque. These data indicate that the increasedVLDL and LDL were separated from healthyVL.DLR expression on the surface of SMC andhuman blood plasma by ultracentrifugationl4. βmacrophages in atherosclerotic plaque may play anVLDL was isolated from rabbit blood plasma. Theimportant role in foam cell formation.rabbits had been fed with cholesterol for oneThe progress of AS is affected by a variety ofmonth.factors through different mechanisms. In this stud-1.3 Cell Culturey, the regulation of VLDLR expression by three1.3.1 Isolation and Culture of Mouse Peritonealtypes of lipoproteins in mouse peritoneal macro-MacrophagesMouse peritoneal macrophagesphages was investigated and found that up-regula-from female Kunming Mice were cultured in RP-tion of VLDLR had an independent effect on theMI1640 culture medium supplemented with 10 %accumulation of cellular lipids and the formation ofFBS, 20 mmol/L HEPES and 125 mg/ml L-gluta-foam cell.mine, at 37 C，5 % CO2. For experiment, thecells were plated at the density of 5X105 in 100 ml1 MATERIALS AND METHODSflask and used after 24 h preincubation in serum-free medium. Then cells were incubated with three1.1 Materialskinds of lipoproteins (50 mg/ ml) respectively for 0Reverse- Transcriptase and Oligo dT， Taqh,24h,48h.1.3.2: Cultureldl中国煤化工red in F12, con-QU Shen, male, born in 1954. Professor" This project was supported by a grant from National Natu-taininHEPES and 125ral Sciences Foundation of China (No. 39970307).mg/YHCNMHGoCo2. Porexperiment, the cells were plated at the density of 12Journal of Huazhonug Univesity of Science and Tchnology [Med Ssi] 24 (1): 1-4. 2004X10° in 100 ml flask and used after 24 h preincu-medium was discarded. and the cells were washedbation in (MEM. Then, cells were incubated withand fixed, the cells were stained with 0.3 % oilthree kinds of lipoproteins (8 mg/ ml) respectivelyred () and hematoxylin. Then the foam cells werefor 2 days, 4 days, 6 days.photographed by cell scan analysis system (CIS-1.4 Lipid Extraction and Measurement1000)Lipid was extracted by Folch methods and de-1.6 RT-PCR Detecting the Expression of VLDL-R,tected according to Test Kit manual. The cellularLRP, LDL-R mRNAprotein content was measured by Lowery methods.Total RNA was isolated by the single stepThe cellular contents of TG or TC were calculatedmethod using the Trizol reagent. First-strand cD-by the following formula:NA was prepared from 3. 5 μg RNA using Super-TG (TC) = Value of lipid/ Volume of extrac-SeriptTPreamplification System ( Promega,ting solution/Protein concentration (μg/ mg)USA). PCR was performed in a 50 μl final volume1.5 Oil Red 0 Staining for Foam Cellscontaining 4 μl reverse transcribed DNA from each5X 10' macrophages or 1X 10' ldl-A7, ldI-A7-reaction. Sequence of the primers, expected lengthVR cells were plated in 24-well plates, incubatedof PCR products and PCR conditions were listed inwith lipoproteins for definite time. After culturetable 1.Table 1 PCR primers sequence and PCR conditionsFragmentAnnealingNumber ofSequencessize .temperaturecyclesVILDL-R Sense5-GGTCAGACTGGGGCGAGCCA-3'451bp58 C35Antisense5'-GCTGGCAGGCAGAGATATTC -3'L.RPSense5'-GTATCTCAAAGGGCTGGCGGTG -3'619bp56C.375'-TGCACCCAGCATACGTCTC -3'LDL-R .5'-GAAGACTCATGCAGCAGGAACG-3'468bp56 C5'-CTCATCGGAGCCGTCAACACAG -3'pβ-Actin5'-TGAGACCTTCAACACCCAG -3'316bp60C275'-GCCATCTCTTGCTCGAAGTC -3'The PCR products were analyzed by electro-phoresis on 2 % agarose gels containing 0.5 mg/Lethidium bromide. Amplificates were visualizedand photographed under ultraviolet light, scannedand analyzed by GIS system. The mRNA levels forLDLR，VLDLR and LRP were normalized to β-ac-tin mRNA.2 RESULTS2.1 mRNA Level of the Lipoprotein Receptors inFig. 1 The mRNA level of receptors in macropha-Mouse Peritoneal Macrophagesges_1: Marker; 2: VLDL-R; 3: LRP; 4: L.DL-The mRNA levels of VLDL-R，LDL-R andR; 5: F-ActinLRP which were rela:ed to lipoprotein metabolismin mice peritoneal macrophages were detected.、250The results showed that the correspondingOLDLstrips of VLDI-R, LDL-R, LRP and β-actin were，20■VLDLall amplified in mice macrophages, suggesting thatDβ-VUDLTthese three receptors could be all expressed in。15mouse macrophages (fig. 1).2.2 Effect of Lipoprotein in vitro on the Contentsi 10of TG and TC in Mouse Peritoneal Macrophages andEffect on the Formation of Foam CellsE5The cellular contents of TG and TC were de-termined after the macrophages were incubated re-spectively with three kinds of lipoproteins for 24 h0h4h8hand 48 h. The results were shown in fig. 2 andfig.3.中国煤化工The intracellular lipid content was increasedCon triglycerideafter the macrophages were incubated with threeMHCNMHGphagecellipoproteins respectively for 24 h, and reached itspeakat 48 h. The effects of VLDL, 3-VLDL. on3QU Shen et al. VLDL. Receptor and Foam Cell Formationfor 24 h, and the configuration of foam cells was30more clear after incubating for 48 h. But it changed25slightly when incubating with LDL ( Data notshown. )20.3 Efect of Lipoprotein on the mRNA Level ofLDLR, VLDLR and LRP in Mice Peritoneal Macro-phagesAfter incubation with three lipoproteins for 24h and 48 h respectively, total RNA of the miceperitoneal macrophages was extracted with Trizol-reagent guanidine isothiocyanate centrifugationmethod. Semi-quantitation RT-PCR was per-OH24h48hformed to detect the expression of each receptor byTime (h)using β-actin as contrast.Fig. 3 Effeet of lipoprotein on TC accumulation inFrom fig. 4- 6, it was concluded that VLDImacrophage cellor β-VLDL could increase the expression of VLDLTG accumulation were more remarkable than thatR in macrophages, and decrease that of LDL-R,of L.DL. Otherwise, the effect of LDL on accumu-but had lttle effect on that of LRP. While thelation of cholesterol was outstanding.macrophages were incubated with LDL， LDL-RThe result of oil red () staining suggested thatmRNA level was decreased, LRP mRNA level wasthe formation of foam cells could be observed whenincreased, and that of VLDL-R had no significantmacrophages were incubated with VLDL, β-VLDL .23456712345671234 5 67一LDLR:1-aCLDLH acu .BDFig. 4 Elfect of p-VLDL. on the mRNA levels of LDL-R(A), LRP(B), VLDL-R(C) in macrophagesA: mRNA level of I.DL-R 1: DNA marker; 2, 5, 7; mRNA level of f-actin after incubated with βVLDLfor0h. 24 h, 48 h;3, 4, 6; mRNA level of LDL-R after incubated with β-VLDL forOh, 24 h, 48 h.B: mRNA level of LRP 1: DNA marker; 2. 3, 4; mRNA level of β actin after incubated with β VLDL for 05. 6.7: mRNA level of LRP after incubated with β-VLDL for0 h, 24 h, 48 hC: mRNA level of VLDL-R 1: DNA marker; 2, 4, 6; mRNA level of p-actin after incubated with β-VLDLfor0h, 24h. 48 h;，3. 5. 7: mRNA level of VLDL R afer incubated with p-VLDL for0h, 24 h, 48 hD: Quntified results of electrophoresis strapof A. B and c2.4 Effect of Lipoprotein Incubation on the Con-both cells .tents of TG and TC in ldI-A7 Cells and ldl-A7-VRCells and Effect on the Formation of Foam Cell3 DISCUSSIONldI-A7 cells and ldl-A7 cells expressing VLDL-R steadily