Effects of anthocyanin on cholesterol efflux from mouse peritoneal macrophage foam cells

AIM: To explore the effect of anthocyanin on cholesterol efflux and elucidate its possible molecular mechanism. METHODS: Mouse Peritoneal macrophages were loaded with 50 mg/L AcLDL to induce macrophage-derived foam cells. Cholesterol efflux from macrophage-derived foam cells induced by anthocyanin was determined by enzymatic methods. PPAR-γ mRNA and protein expression in macrophage-derived foam cells was assayed by quantitative PCR and western blotting. RESULTS: Anthocyanins had the capacity of promoting cholesterol efflux from mouse peritoneal macrophage-derived foam cells and increased PPAR-γ mRNA and protein expression. CONCLUSION: Anthocyain-induced cholesterol efflux may be related to its enhancing PPAR-γ mRNA and protein expression.     

Action of ATP binding cassette transporter A 1 on cholesterol efflux in THP-1 macrophage-derived foam cells

AIM:To study the action of ATP binding cassette transporter(ABC) A 1 on cholesterol efflux in THP-1 macrophage-derived foam cells.METHODS:After exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) at different concentration for 24 hours, cholesterol efflux and ABCA1 mRNA level were determined by FJ-2107P type liquid scintillator and reverse trancriptase-polymerase chaim reaction(RT-PCR), respectively.RESULTS:Oxidized LDL promoted cholesterol efflux in THP-1 macrophages and 22(R)-hydroxycholesterol increased cholesterol efflux in THP-1 macrophage-derived foam cells in a dose-dependent manner and DIDS inhibited cholesterol efflux in THP-1 macrophage-derived foam cells in a dose-dependent manner. Exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and DIDS at different concentration for 24 hours, resulted in increase and decrease in the expression of ABCA1 mRNA in THP-1 macrophage-derived foam cells in a dose-dependent manner, respectively.CONCLUSION:ABCA1 playes an important role in cholesterol efflux in THP-1 macrophage-derived foam cells.     

Mast cells change cellular cholesterol content and inhibit cholesterol efflux from THP-1 macrophage-derived foam cells

AIM:Mast cells (MC) are present in the arterial intima,the site of atherogenesis. The present studies explore the effect of MC on cholesterol content,distribution and efflux in THP-1 macrophage-derived foam cells (THP-1FCs）. METHODS:THP-1FCs were incubated with high-density lipoproteins 3 (HDL₃) in the absence or presence of mast cell granules (MCGs) harvested from compound 48/80-stimulated rat peritoneal MC. The intracellular cholesterol level,cholesterol effluxing capacity,ATP-binding cassette transporter A1(ABCA1) mRNA and HDL₃ treated with MCGs were detected to characterize the role of MC on intracellular cholesterol. RESULTS:MCGs had high levels of cellular total cholesterol(TC),free cholesterol(FC) but not esterifed cholesterol(EC) compared to control group where the TC concentrations ranged from 527.3 mg/g to 917.9 mg/g cellular protein with EC accounting for 7.6% of the cholesterol. Cholesterol efflux was 14% less in MCGs group compared to control group. ABCA1 mRNA expression in MCG-treated THP-1FCs remained unchanged in 20 hours. In contrast,treatment of HDL₃ with MCGs resulted in rapid degradation of the main HDL₃ apoliproteins,apoA-Ⅰ. SDS-PAGE revealed that a minor polypeptide band with about 26 kD molecular mass appeared below the apoA-Ⅰband. Densitometric analysis of the gel demonstrated that ≈ 28% of apoA-Ⅰhad been degraded by the MCGs. CONCLUSION:These results indicate that MC decreases cholesterol efflux,increases cellular accumulation in TC and FC by depleting HDL₃ and apoA-Ⅰ,but not by inhibiting ABCA1 mRNA expression.     

Effect of puerarin on cholesterol influx and efflux in RAW264.7-treated foam cells

AIM: To study the protective effect of puerarin on the atherosclerosis of RAW264.7-derived foam cells. METHODS: The model of foam cells was established by incubating the RAW264.7 cells with ox-LDL. The cholesterol uptake was evaluated by a DiI-ox-LDL binding assay. The ability of cholesterol efflux of the RAW264.7-derived foam cells was detected by cholesterol efflux assay. The protein levels of LC3Ⅱ, P62, CD36, ABCA1, LAL and p-AMPK were determined by Western blot. RESULTS: Puerarin treatment reduced the cholesterol uptake capacity and enhanced the cholesterol efflux rate. The protein levels of LC3Ⅱ, ABCA1 and LAL in puerarin group were higher than that in ox-LDL group, while the protein levels of P62 and CD36 were obviously decreased, and those in rapamycin treatment group had the same change as puerarin group. The protein levels of LC3Ⅱ, ABCA1 and LAL were obviously decreased and the protein level of p-AMPK was increased after co-treated with 3-MA. CONCLUSION: Puerarin promotes LAL and ABCA1-mediated cholesterol efflux in ox-LDL-treated RAW264.7 macrophages, which might enhance autophagy through AMPK-dependent pathway for cholesterol efflux regulation, and reduce the uptake of lipids by CD36 negative regulation.     

Proliferation and apoptosis of human umbilical vein endothelial cells induced by oxidized low density lipoprotein

AIM:To investigate proliferation and apoptosis of cultured endothelial cell (ECV-304 cell line) induced by varied concentrations of oxidized low density lipoprotein (ox-LDL). METHODS:Cell morphology, Typan blue test, MTT test, LDH release test, flow cytometry and micro-molecular weight DNA fragment gel electrophoresis of apoptosis were used for the detection of the cytotoxic effects of ox-LDL on ECV-304 cell line.RESULTS:0.1, 1, 10 mg/L ox-LDL could promote proliferation of ECV-304 cells. When the concentration of ox-LDL reached up to 100 mg/L and above, the distinct cytotoxic effect appeared. Further study showed that the apoptosis rate of endothelial cells, induced by ox-LDL of 150 mg/L and 200 mg/L for 12 hours, are 15.86% and 21.89%, respectively. 18 h and above hours after incubation, the apoptosis rate began to decrease and rate of necrosis increased. CONCLUSION:ox-LDL has strong cytotoxic effects on endothelial cells and could give rise to different pathologic process, such as proliferation, apoptosis prophase, apoptosis and necrosis.     

Lipophagy inhibits foam cell formation by reducing cholesterol content

AIM To observe the changes of lipophagy during foam cells formation, and to determine the effect of lipophagy on the lipid content and cholesterol outflow of foam cells. METHODS Human THP-1 monocytes were induced by phorbol-12-myristate-13-acetate for 48 h to differentiate into macrophages, and then were incubated with 50 mg/L oxidized low-density lipoprotein （oxLDL） to form foam cells. Lipids in foam cell were stained by oil red O, and the lipid content was determined. The total cholesterol （TC） and free cholesterol （FC） levels in foam cells were measured by cholesterol testing kit. Cholesteryl ester （CE） and CE/TC ratio were calculated. The cholesterol efflux rate was detected by cholesterol efflux assay kit. The expression of autophagy-related proteins, including autophagy-related protein 5 （Atg5）, microtubule-associated protein 1 light chain 3 （LC3） and P62, were detected by Western blot. The colocalization of lipid droplets （LD） and LC3 was detected by immunofluorescence staining. The autophagy inducer rapamycin （Rap） or blocker 3-methyladenine （3MA） was used to intervene foam cells, and the expression of Atg5, LC3 and P62, the co-expression of LD and LC3, the cholesterol content and the cholesterol efflux rate were determined. RESULTS Formation of foam cells was observed at 24 h after stimulation with oxLDL at 50 mg/L, as indicated by intracellular CE/TC ratio exceeding 50%.Cholesterol efflux assay revealed that the cholesterol efflux rate increased within 24 h during foam cell formation but decreased after 48 h （P<0.05）. Western blot results displayed that the expression of Atg5 and LC3-II/LC3-I ratio were increased within 24 h of foam cell formation, but was deceased after 48 h （P<0.05）. The expression of P62 was decreased within 24 h but was increased at 48 h （P<0.05）. The colocalization of LD and LC3 was increased at 24 h but was decreased at 48 h after oxLDL stimulation. Treatment with Rap up-regulated the expression of Atg5 and LC3-II/LC3-I ratio, reduced the level of P62, increased the colocalization of LD and LC3, promoted the cholesterol efflux, anf reduced cholesterol content in foam cells （P<0.05）. On the contrary, 3MA inhibited the expression of Atg5, reduced LC3-II/LC3-I ratio, elevated the level of P62, decreased the colocalization of LD and LC3, reduced the outflow of cholesterol, increased the content of TC and CE, and elevated CE/TC ratio in foam cells （P<0.05）. CONCLUSION Lipophagy is enhanced at 24 h but decreased at 48 h during foam cell formation. Lipophagy inhibited foam cell formation by reducing cholesterol content and increasing cholesterol efflux.     

Oxidized low density lipoprotein induces the expression of macrophage migration inhibitory factor

AIM: To examine expression of macrophage migration inhibitroy factor (MIF) gene and protein in macrophages induced by oxidized low density lipoprotein (ox-LDL). METHODS: Macrophages were incubated with ox-LDL at the concentration of 150 mg/L for time course (0-36 h) and with ox-LDL at the different concentrations (0-300 mg/L) for 24 h, expression of MIF mRNA and protein were detected by RT-PCR and ELISA. RESULTS: The results showed that ox-LDL increased MIF gene and protein expression in macrophages in a dose and time-dependent manner. After the exposure of macrophage to ox-LDL, the expression of MIF mRNA level increased consistently with protein. CONCLUSION: MIF may play an important role in atherosclerosis.     

Study on scavenger receptor A from mouse peritoneal macrophages

AIM: To study the role of scavenger receptor A(SR A) in the uptake of oxidized low density lipoprotein(OxLDL) in mouse peritoneal macrophages(MPM). METHODS: Comparing the difference of the uptake of OxLDL in SR A-deficient and wild-type MPM. RESULTS: The results showed that the binding of OxLDL wasn't apparently reduced in SR A-deficient MPM. The association of OxLDL was reduced by 35.8% and degradation of OxLDL was reduced by 42% in SR A-deficient MPM compared with those in wild-type MPM. CONCLUSION: Studies showed that SR A didn't play an important role in the uptake of OxLDL in MPM. Approximately 70% of the uptake of OxLDL in macrophages was attributable to non-SR A receptor.     

Effect of 17β-estradiol on the levels of plasma oxidized low density lipoprotein in ovariectomized rabbits

AIM: To observe the changes of plasma oxidized low density lipoprotein(oxLDL) levels of ovariectomized cholesterol-fed rabbits with or without 17β-estradiol(E₂) replacement therapy.METHODS: All rabbits were ovariectomized and fed standard chow supplemented with 1.5% cholesterol for 14 weeks. Two weeks after operation, the rabbits were randomly assigned to four groups. Three groups were treated with E₂ 1 mg, 2 mg, 4 mg respectively, the other group served as control. Serum superoxide dismutase (SOD) levels and plasma oxLDL levels were measured at 0, 3, 8, 12 weeks after hormone replacement therapy. The aortic atherosclerotic lesion areas were determined by computer. RESULTS: We found that there were striking increase of serum SOD levels ( P<0.05 ) and significant decrease in both the plasma oxLDL levels and the aortic atherosclerotic lesion areas ( P<0.01 respectively). Moreover, a positive correlation was found between plasma oxLDL levels and the areas of atherosclerotic plaque in all rabbits. CONCLUSIONS: Estrogen attenuates atherogenesis in cholesterol-fed ovariectomized rabbits. And this beneficial effect of E₂ may be duo to its lowering of plasma oxLDL level.     

XBP-01 accelerated apoptosis induced by oxidized low density lipoprotein in vascular smooth muscle cells

AIM: Previous studies performed with XBP-01 in vitro indicated that XBP-01 could inhibit vascular smooth muscle cells from being transformed into foam cell and could eliminate the atherosclerotic plaque in C57BL/6J mouse. This experiment is to investigate its mechanism of eliminating plaques in vitro. METHODS: The cultured porcine artery smooth muscle cells incubated with XBP-01 of 0.1 mg/L for 24 h after preincubated with oxidized low density lipoprotein of 15 mg/L for 72 h in vitro. The samples were analyzed by fluorescence microscope, confocal microscope system and flow cytometry. RESULTS: Apoptosis was triggered by being incubated with oxidized low density lipoprotein and this process was accelerated additionally by being incubated with XBP-01. CONCLUSION: XBP-01 can be effective in eliminating atherosclerotic plaque by accelerating the process in which oxidized low density lipoprotein induced smooth muscle cell apoptosis.     

The role of chlamydia pneumoniae in the course of transformation of C57 BL/6J mouse peritoneal macrophages into foam cells

AIM: In order to understand the role of chlamydia pneumoniae in the course of macrophages transformation into foam cells experiments with chlamydia pneumoniae standard strain AR-39 wese made. METHOD: C57 BL/6J Mouse peritoneal macrophages C57 BL/6J wese incubated 24 h, and they were divided into 6 groups to be incubated continually: A₁~DMEM; A₂~DMEM+10 IFUs/L AR-39; B₁~DMEM+10mg/L LDL; B₂~DMEM+10mg/L LDL+10 IFUs/L AR-39; C₁~DMEM+10mg/L OxLDL;C₂~DMEM+10mg/L OxLDL+10 IFUs/L AR-39. 72 h later, morphological changes of the cells were observed and of intracellular cholesterol of content was detected. RESULTS: 1、Morphological studies: there were no lipid particles in A₁, A₂ and B₁ groups, but the lipid particles could be found in B₂、C₁ and C₂ group, Among six groups, the most lipid particles were seen in C₂ groups. 2、Biochemical detection:The ratio of cholesterol ester to total cholesterol was much higher than 50% in B₂、C₁ and C₂ groups, but was less than 50% in A₁、A₂ and B₁ groups. CONCLUSION: Chlamydia pneumoniae may have played a role in promoting C57 BL/6J mouse peritoneal macrophages into foam cells.     

Effects of oxidized low density lipoprotein and vitamin E on the levels of IL-6,IL-8 and TNF-α in cultured human umbilical vein endothelial cells

AIM:To investigate effects of OX-LDL and VitE on the levels of IL-6,IL-8 and TNF-α in human umbilical vein endothelial cells(HUVEC).METHODS: Human umbilical vein endothelial cells were obtained by in vitro culture. HUVEC treated with or without Vit E was incubated with OX-LDL, and the levels of IL-6, IL-8 and TNF-α were determined by enzyme-linked immunosorbent assy technique. RESULTS:50 μg/L,100 μg/L, 200 μg/L OX-LDL induced the release of IL-6,IL-8 and TNF-α by HUVEC in a dose-dependent manner. Compared with the control group , the levels of IL-6 and IL-8 were significantly increased at 6-12 h of stimulation with OX-LDL . Maximal levels of IL-6 and IL-8 occurred after 24-36 h, reaching a plateau maintained for at least 48 h. TNF-α rose after 2-6 h in HUVEC, and reached a maximum after 12 h. In contrast to IL-6 and IL-8, TNF-α declined after 48 h. However, when VitE (50 mg/L,100 mg/L,200 mg/L)was added, it can significant inhibited the release of IL-6, IL-8 and TNF-α in a dose-dependent manner, and after 48 h these cytokines have no diference between OX-LDL+VitE groups and OX-LDL groups. CONCLUSION: OX-LDL can obviously stimulate the production of IL-6,IL-8 and TNF-α in vascular endothelial cells, which can significantly be inhibited by VitE in a short time.     

IGF-1R,p-ERK is involved in LDLs-induced mouse peritoneal macrophage survival

AIM: To study the effects of low density lipoproteins (LDLs) on insulin-like growth factor-1 receptor (IGF-1R), phosphorylated extracellular signal-regulated kinase (p-ERK), Bcl-2 and Bax protein expression in mouse peritoneal macrophages (MPMs). METHODS: Using the methods of immunocytochemistry and Western blotting, the expression of IGF-1R, p-ERK, Bcl-2 and Bax protein in MPMs treated with LDLs, anti-IGF-1R antibody (α-IR-3) or ERK inhibitor (PD98059) were detected. RESULTS: oxLDL significantly increased the IGF-1R protein expression in a dose and time-dependent manner. P-ERK protein expression induced by oxLDL peaked at 5 min. Moreover, oxLDL could induced ERK translocation from cytoplasm to nuclear. When given α-IR-3, p-ERK protein expression was significantly inhibited, and ERK translocation disappeared. oxLDL increased Bcl-2 protein expression and reduced Bax expression in a dose and time-dependent manner. When given PD98059, Bcl-2 and Bax protein expression induced by oxLDL altered significantly. Native LDL had no significant effect on the expression of these four proteins. CONCLUSIONS: oxLDL may promote cultured MPMs survival at least by enhancing IGF-1R expression and ERK phosphorylation, and there may be many pathways involved in MPMs survival induced by oxLDL, Whereas native LDL had no effects on culture MPMs.     

Oxidative modification of low density lipoprotein induced byhyperhomocysteinaemia following application of methionine

AIM:To observe the oxidative modification of low density lipoprotein iinduced by hyper homocysteinaemia following application of methionine. RESULTS:Following application of methionine, the levels of hyperhomocysteine, ox-LDL and TBARS in group M and group M+Ch were significntly higher than those of group C and group Ch (P＜0.01). The oxidative modification of low density lipoprotein induced by homocysteine in vitro were significantly increased with the concentrations of LDL and homocysteine (P＜0.01).     

Role of receptors for oxidized low-density lipoprotein in pathogenesis of atherosclerosis

The formation of atherosclerosis （AS） is an evolutionary process. It undergoes lipid deposition, focal fibrosis in the intima of the arteries and atherosclerotic plaque formation, resulting in hardening of the vessel wall and stenosis of the lumen, which eventually leads to ischemic changes in the corresponding organs. In these processes, oxidized low-density lipoprotein （oxLDL）, an oxidative product of low-density lipoprotein, is considered to be a key trigger for AS development. In this paper, the role of the receptors for oxLDL in the development of AS is reviewed based on the recent studies.     

Effect of mininally modified low density lipoprotein on the adhesive function of vascular endothelial cell and its mechanism

AIM: To observe the effect of MM-LDL (minimally modified LDL) on the interaction between human umbilical vein endothelial cell (HUVEC) and U937 monocyte-like cell line and the exporession of vascular cell adhesion-1 (VCAM-1), intercellular adhesion molecule-1(ICAM-1), P-selectin.METHODS:The adhesive percentage between HUVEC treated with MM-LDL and U937 was determined by counting and the expression of VCAM-1, ICAM-1, P-selectin were examined by ELISA. RESULTS: Treatment of HUVEC with MM-LDL (75 mg/L) for 4 hours significantly increased adhesion of U937 to HUVEC ( P <0.01) and did not induce the surface expression of VCAM-1, ICAM-1, P-selectin . Recombination tumor necrosis factor-alpha (rTNF_α) 5.0 μg/L, as a positive control, induced the expression of these adhesion molecules ( P <0.05). Prolonged (18 h) exposure to MM-LDL resulted in the expression of P-selectin, but not VCAM-1.CONCLUSION: the adhesion of monocytes to endothelial cells induced by MM-LDL is not mediated by VCAM-1, ICAM-1. P-selectin induction may be partly involved in the process.     

Effect of oxidized LDL on expression of connexin43 in cultured human umbilical vein endothelial cells

AIM: To investigate the effect of oxidized LDL (ox-LDL) on the expression of gap junction protein connexin43 in cultured human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Human umbilical vein endothelial cells cultured in normal condition were divided into blank control group, 50 mg/L,100 mg/L and 200 mg/L ox-LDL intervention groups. The mRNA expression of connexin43 in cultured HUVECs was detected with RT-PCR method; while the protein level of connexin43 was determined by the method of immunocytochemistry in the control and 100 mg/L ox-LDL intervention groups 24 h after ox-LDL was given. RESULTS: Different concentrations (50 mg/L, 100 mg/L, 200 mg/L) of ox-LDL up-regulated mRNA expression of connexin43 in cultured HUVECs after 24 h intervention (P<0.01). The protein level of connexin43 in cultured HUVECs intervened with 100 mg/L Ox-LDL for 24 h was up-regulated as compared to the control cells (P<0.01).CONCLUSION: Ox-LDL may up-regulate the expression of connexin43 at mRNA and protein levels in cultured human umbilical vein endothelial cells within short time, indicating that connexin43 plays an important role in the pro-atherosclerotic effect of Ox-LDL.     

Insulin induced LPL mRNA expression in THP-1 macrophage and acceleration of transferring the macrophage to foam cell

AIM:To investigate the effect of insulin on ox-LDL transferring the THP-1 cells to foam cells and influencing the LPL mRNA expression in THP-1 cells.METHODS:THP-1 cells were incubated with 50 mg/Lox-LDL and insulin at concentrations of 10 mU/L, 100 mU/L, 1 000 mU/L and 10 000 mU/L, respectively. The expression of LPL mRNA in cells was detected by RT-PCR. Lipoprotein lipase of THP-1 cells was presented by no-specific lipase staining. THP-1 cells were stained with oil red O. Accumulation of total cholesterol (TC) in THP-1 cells was determined with oxidase assay.RESULTS:In 100 mU/L、1 000 mU/L、10 000 mU/L insulin groups, LPL mRNA expression increased 2 times, the average cell perilength was longer, the percentage of positive oil red O staining cells was significant higher, the content of cholesterol in THP-1 cells was higher than in ox-LDL control (P＜0.05).CONCLUSION:Insulin accelerates transferring of THP-1 cells to foam cell with exposed to ox-LDL because LPL mRNA expression increased in the cells.