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.     

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.     

Regulation by reactive oxygen species of matrix metalloproteinase-1, 3 and tissue inhibitor of matrix metalloproteinase-1 in smooth muscle cells

AIM: To understand whether reactive oxygen species promote the rupture of atherosclerotic plaques by regulating the balance of matrix metalloproteinase-1, 3 (MMP-1, 3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in smooth muscle cells. METHODS: Aortic smooth muscle cells from 4-6months-healthy abortive fetuses were incubated for 24 hours with xanthine (100 μmol/L) and xanthine oxidase (5 U/L) in vitro . MMP-1, 3 and TIMP-1 in the concentrated culture media were measured by Western blotting ( n =3 independent experiments). RESULTS: Incubation with xanthine/xanthine oxdiase decreased the amount of MMP-1 in the aortic smooth muscle cells (21.2%±5.5% of the control group), and pro-MMP-1 was activated completely. Reactive oxygen species (ROS) also activated pro-MMP-3, and increased the production of MMP-3 in the aortic smooth muscle cells. On the other hand, ROS inhibited the production of TIMP-1 in the aortic smooth muscle cells. CONCLUSION: It is complicated that ROS regulates the balance of MMPs and TIMPs. ROS may contribute to matrix degradation and the rupture in the atherosclerotic plaques.     

Recent advances in lectin-like oxidized low-density lipoprotein receptor-1 and atherosclerosis

Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is a type-Ⅱ membrane protein belonging to the C-type lectin family molecules, which acts as a cell surface endocytosis receptor for atherogenic oxidized LDL (Ox-LDL). LOX-1 supports the binding internalization and proteolytic degradation of oxidized LDL, but not of significant amounts of acetylated LDL. LOX-1 is initially synthesized as a 40 kD precursor protein with N-linked high mannose-type carbohydrate, which is further glycosylated and processed into a 48-kD mature form. In vivo, endothelial cells that cover early therosclerotic lesions, intimal macrophages and smooth muscle cells in advanced atherosclerotic plaques express LOX-1. LOX-1 is cleaved at membrane proximal extracellular domain and released from the cell surface. Measurement of soluble LOX-1 in vivo may provide novel diagnostic strategy for the evaluation and prediction of atherosclerosis and vascular diseases.     

Native and oxidized low density and very low density lipoprotein enhance the expression of MIP-1α mRNA in aortic smooth muscle cells

AIM: To understand whether native and oxidized low density and very low density lipoprotein (n-LDL, n-VLDL, ox-LDL, ox-VLDL) enhance the expression of macrophage inflammatory protein (MIP)1α mRNA in cultured aortic smooth muscle cells (SMCs). METHODS: Native low density and very low density lipoprotein were isolated from normal blood donors by density gradient ultracentrifugation, and were oxidatively modified by adding CuCl2. After a 24 h-exposure of the cultured SMCs to n-LDL， n-VLDL， ox-LDL and ox-VLDL, respectively, the expression of MIP-1α mRNA was determined by in situ hybridization and RT-PCR. RESULTS: Cultured aortic SMCs expressed MIP-1α mRNA at low level. N-LDL， n-VLDL， ox-LDL and ox-VLDL enhanced the expression of MIP-1α mRNA in SMCs, ox-LDL and ox-VLDL showed stronger effect than n-LDL and n-VLDL, respectively. The effect of ox-VLDL was most striking. There was a significant difference between groups (P<0.01). CONCLUSION: N-LDL, n-VLDL, especially ox-LDL and ox-VLDL, may play an important role in the formation of early atherosclerotic lesion by inducing SMCs to express MIP-1α.     

Effects of sodium ferulate on cholesterol and triglyceride metabolism in atherosclerosis with hyperlipidemia

AIM:To investigate the effects of sodium ferulate on cholesterol and triglyceride metabolism in atherosclerosis with hyperlipidemia. METHODS:The rabbit model of atherosclerosis was produced by feeding high lipid forages. RAW264.7 foam cell and HepG2 injured cell models were established by incubation with oxidized low density lipoprotein (ox-LDL). The atherosclerotic plaque area was measured, and serum lipids were detected. The cellular lipid accumulation was examined by oil red O staining. The cellular contents of total cholesterol and cholesterol ester were quantified by high performance liquid chromatography. The hepatic lipase (HL) mRNA expression was determined by RT-PCR. RESULTS:(1) Compared with hyperlipid group, the aorta atherosclerosis plaque area and the serum triglyceride level were significantly decreased in sodium ferulate-treated rabbits, but the serum cholesterol level showed little change. (2) Compared with ox-LDL group, the HL mRNA expression in HepG2 cells was enhanced significantly in sodium ferulate-treated group, but the cellular contents of total cholesterol and cholesterol ester in RAW264.7 foam cells showed little change. CONCLUSION:Sodium ferulate inhibits the formation of atherosclerotic plaque in high-cholesterol-fed rabbits aorta. This antiatherosclerotic function may reduce serum triglyceride level through enhancing the expression of HL mRNA without influencing serum cholesterol level and foam cell formation.     

Sodium Ferulate protects human aortic smooth muscle cells against oxidized Lipoprotein(a)

AIM: To investigate the influences of native and oxidized lipoprotein(a) on human arterial smooth muscle cell (SMC) proliferation, change of intracellular free calcium concentration ( [Ca²⁺]_i) and the protective effect of sodium ferulate(SF). METHODS: Lp(a) was oxidized by Cu²⁺ and the extent of oxidation was assessed by the MDA content.Human SMC were incubated in culture media with SF for 12 h, then exposed to Lp(a) and oxidized-Lp(a) , respectively. MTT colorimetry and flow cytometry were used to evaluated the proliferation of SMC and flurorescent indicator Fura-2/AM was used to determined [Ca²⁺]_i. RESULTS: ox-Lp(a) significantly promoted proliferation of SMC and increased [Ca²⁺]_i compared with Lp(a). SF(40,80 mg/L) remarkedly inhibited SMC proliferation and decreased the rising of [Ca²⁺]_i induced by ox-Lp(a) in a dose-dependent manner, but no effect on SMC proliferation and the increase in [Ca²⁺]_i induced by Lp(a).CONCLUSION: ox-Lp(a) induces the strong growth-promoting effect in SMC through increasing in [Ca²⁺]_i, which might be one of the cellular mechanisms responsible for the higher atherogenic potential of ox-Lp(a) compared with Lp(a), and this process can be prevented by inhibiting of oxidation by SF.     

ox-LDL increases endothelial lipase in RAW264.7 macrophages

AIM: To investigate the effect of oxidized low-density lipoprotein (ox-LDL) on the expression of endothelial lipase (EL) in murine RAW264.7 macrophages. METHODS: RAW264.7 cells were incubated with ox-LDL at concentrations of 0~100 mg/L for 24 h.On the other hand, the cells were incubated with or without PDTC (NF-κB inhibitor) for 30 min and then with ox-LDL (50 mg/L) for 24 h. The expression of EL and p65 was detected by Western blotting. RESULTS: The level of EL was significantly increased after ox-LDL incubation in RAW264.7 cells (P<0.05). NF-κB was activated by ox-LDL at concentration of 50 mg/L for 15~30 min in RAW264.7 cells. The increase in EL induced by ox-LDL was markedly inhibited by a NF-κB inhibitor PDTC (P<0.05). CONCLUSION: ox-LDL significantly increases the expression of EL in RAW264.7 macrophages, which is possibly related to NF-κB activation.     

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.     

Effects of oxidized HDL on the levels of MCP-1, ICAM-1 and free calcium in cultured human umbilical venous endothelial cells

AIM:To study the effects of oxidized high-density lipoprotein (oxHDL) on the expression of monocyte chemoattractant protein-1(MCP-1) and intercellular adhesion molecule-1(ICAM-1) and intracellular free calcium concentration ([Ca²⁺]i) level in cultured human umbilical venous endothelial cells(HUVECs). METHODS:The MCP-1 protein content in the medium of conditioned HUVEC was measured by ELISA, and the ICAM-1 on HUVECs was detected by indirect immunofluorescence, and [Ca²⁺]i was determined by Fluo-3/AM, the injury of cells was observed by scanning electron microscopy (SEM).RESULTS:oxHDL could induce the expression of MCP-1 and ICAM-1 in HUVECs. In oxHDL group (HUVECs were incubated with 100 mg protein/L oxHDL for 24 h), the levels of MCP-1, ICAM-1 and [Ca²⁺]i increased by 160%, 60% and 70% respectively compared with the control group (P＜0.01). When HUVECs were incubated with 300 mg protein/L oxHDL for 24 h, cells were injured obviously. CONCLUSION:By inducing the expression of ICAM-1 and MCP-1 in endothelial cells, oxHDL may promote monocyte-endothelium adhesion and monocyte migration to intima, it may promote atherosclerosis as oxidized low-density lipoprotein (oxLDL).     

Autophagy protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting C/EBP homologous protein expression

AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: The RAW264.7 macrophages were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid (PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The activities of lactate dehydrogenase (LDH) in the medium and caspase-3 in the cells were determined by detection kits. The protein levels of beclin-1 (a molecular marker of autophagy), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein (CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis) were examined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope.RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability, and dramatic elevation in LDH leakage, cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autophagy inducer). ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap. Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL. Moreover, PBA (endoplasmic reticulum stress inhibitor) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granulation of LC3. CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages, and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression.     

Lectin-like oxidized low density lipoprotein receptor-1 mediates expression of MCP-1 induced by ox-LDL in cultured human vascular endothelial cells

AIM: To investigate the effects of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) on the expression of MCP-1 in the cultured human unbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs was incubated with ox-LDL, or preincubated with carrageenan and polyinosinic acid. LOX-1 and MCP-1 mRNA and protein were determined by RT-PCR and Western blot. RESULTS: Incubation of HUVECs with ox-LDL (from 0-100 mg/L) for 24 h markedly increased the expression of LOX-1 and MCP-1 (mRNA and protien) in a concentration-dependent fashion. Preincubation of HUVECs with carrageenan and polyinosinic acid, the chemical inhibitors of LOX-1, for 2 h, ox-LDL-mediated upregulation of LOX-1 and MCP-1 was suppressed (P<0.05). CONCLUSION: These findings indicate that ox-LDL upregulates MCP-1 and its own endothelial receptor, and ox-LDL-induced MCP-1 is mediated by the action of LOX-1. LOX-1 plays a critical role in the pathogenesis of atherosclerosis.     

Preventive effect of 3,4-dihydroxyacetophenone on atherosclerosis and role of visfatin expression

AIM: To observe the preventive effect of 3,4-dihydroxyacetophenone (DHAP) on atherosclerosis (AS) and the role of visfatin expression in ApoE(-/-) mice.METHODS: Eight-week-old normal mice were used in normal control group (n=8). Eight-week-old male ApoE (-/-) mice were randomly divided into 3 groups: AS group (n= 8, im. NS), DHAP treatment group (n=8, im. DHAP 10 mg·kg^-1·d^-1) and simvastatin treatment group (n=8, im. simvastatin 10 mg·kg^-1·d^-1). All mice were fed with Western diet (21% fat, 0.15% cholesterol) for 12 weeks. The blood samples were collected and the concentrations of blood lipids and visfatin were detected. The frozen sections of aortic root were stained with oil red O. The visfatin in atherosclerotic plaques at aortic roots was examined by Western blotting. The structures of smooth muscle cells and endothelial cells were observed under electron microscope. RESULTS: In DHAP-treated mice, the concentrations of visfatin, TG and TC were decreased, the formation of AS plaque was reduced, the injuries of smooth muscle cells and endothelia cells were attenuated. Visfatin was also decreased at atherosclerosis plaque in DHAP-treated mice.CONCLUSION: DHAP effectively prevents and treats AS by inhibiting the production of visfatin and reducing lipid.     

Role of Toll-like receptor 4/MAPKs pathway on monocyte chemoattractant protein-1 secretion induced by oxidized low density lipoprotein in vascular smooth muscle cells

AIM: To investigate the role of Toll-like receptor 4/MAPKs pathway on the secretion of monocyte chemoattractant protein-1 (MCP-1) induced by oxidized low density lipoprotein (ox-LDL) in the vascular smooth muscle cells (VSMCs). METHODS: mRNA and protein expressions of MCP-1 in VSMCs stimulated with oxidized low density lipoprotein were determined by RT-PCR and ELISA, respectively. The phosphorylated forms of ERK1/2 and p38MAPK were determined by Western blotting. TLR4 neutralizing antibodies (a specific TLR4 inhibitor), PD98059 (ERK1/2 specific inhibitor), SB23015 (p38MAPK specific inhibitor) and SP600125 (JNK specific inhibitor) were used to investigate the underlying mechanisms. RESULTS: The mRNA and protein expressions of MCP-1 in VSMCs were up-regulated by ox-LDL (P<0.05), while those were inhibited by TLR4 neutralizing antibodies, PD98059 or SB23015 (P<0.05), but not by SP600125 (P>0.05). TLR4 had regulatory effect on the phosphorylation of ERK1/2 and p38MAPK. CONCLUSION: ox-LDL is an endogenous ligand of TLR4. The secretion of MCP-1 induced by ox-LDL in VSMCs is at least in part via TLR4/ERK1/2 and TLR4/p38MAPKs pathways.     

Low density lipoprotein from patients of diabetes mellitus induces apoptosis of murine macrophages via activating caspase-12 pathway

AIM:To explore the effect of low density lipoprotein from the patients of diabetes mellitus (DM-LDL) on the activation of caspase-12 an important molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, in the murine macrophages, and to clarify the underlying molecular mechanisms of apoptosis. METHODS:Murine macrophage RAW264.7 was exposed to DM-LDL (25, 50 and 100 mg/L), normal low density lipoprotein (n-LDL, 50 mg/L), or tunicamycin (TM, 4 mg/L) for 24 h. Additionally, RAW264.7 macrophages were precultured with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then exposed to DM-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were detected by MTT assay and flow cytometry with Annexin V-FITC/propidium iodide staining, respectively. Lactate dehydrogenase (LDH) activity in the media was measured by assay kit. The protein level of caspase-12 was determined by Western blot. RESULTS:Similar to TM (an ERS inducer), treatment with DM-LDL caused significantly decrease in the viability and increase in LDH activity in the media and apoptotic rate of the RAW264.7 macrophages (P<0.05). Additionally, DM-LDL induced activation of caspase-12 especially at the dose of 50 and 100 mg/L (P<0.01). However, the ERS inhibitor PBA protected RAW264.7 macrophages from DM-LDL-induced decrease in viability and increase in LDH activity and apoptosis (P<0.05). Furthermore, PBA attenuated DM-LDL-induced activation of caspase-12 (P<0.05). CONCLUSION:DM-LDL may induce apoptosis in RAW264.7 macrophages, and the mechanism may be related to the activation of caspase-12.     

Effect of pioglitazone on balance of regulatory and effector T cells of uremic apolipoprotein E knockout mice in vitro

AIM: To investigate the effects of pioglitazone on the quantity and function-related factors of regulatory and effector T cells (Treg and Teff ) of uremic apolipoprotein E knockout mice in vitro with or without the stimulation of atherosclerotic plaque-specific antigen oxidized low-density lipoprotein (oxLDL). METHODS: Uremic apolipoprotein E knockout mouse model was established by 2-step surgical procedure. After intervention with different concentrations (2 μmol/L and 20 μmol/L) of pioglitazone and PPARγ antagonist GW9662 (5 μmol/L) on splenocytes of uremic mice for 12 h in the presence or absence of oxLDL (2 mg/L), the levels of CD4⁺ CD25⁺ Foxp3⁺ Treg and IFNγ⁺ CD4⁺ Teff were determined by flow cytometry. The mRNA expressions of Foxp3 and IFNγ was detected by real-time fluorescent quantitative PCR. RESULTS: In vitro, oxLDL induced a Treg/Teff imbalance in splenocytes from the uremic mice. Pioglitazone upregulated the level of Treg and mRNA expression of Foxp3 in the presence of oxLDL, which was not antagonized by GW9662. Meanwhile, pioglitazone downregulated the level of Teff and mRNA expression of IFNγ in the presence or absence of oxLDL, which was reversed by GW9662. CONCLUSION: oxLDL induces a Treg/Teff imbalance in uremic apolipoprotein E knockout mice. Pioglitazone modulates the Treg/Teff imbalance probably through PPARγ-independent and-dependent mechanisms.     

Effects of several herbal extractives with the effect of promoting blood flow and detoxication on atherosclerotic plaque stability in aorta of ApoE-gene knockout mice

AIM: To observe the effects of saponins of panax pseudo-ginseng, coptis chinensis extractive, giant knotweed rhizome and rhubarb extractives on the stability of vulnerable atherosclerotic plaques in the aortic roots of fat-fed ApoE knockout mice. METHODS: Eighty ApoE knockout mice were fed high-fat diet for 26 weeks. Thereafter, they were randomized to one of seven groups and treated with these herbal extractives for 13 weeks. The morphology and composition of atherosclerotic plaques in aortic roots were examined in tissue sections. Four sections were chosen and stained with Movat and HE staining, respectively, and the protein expressions of smooth muscle in plaque were determined by immunohistochemistry. The percentages of the plaque composition of foam cells, extracellular lipids, smooth muscle cells and collagen in each area were counted in the whole plaque. The number of buried caps and the ratio of the length of fibrous cap/plaque surface were also counted. The effect of the drugs on plaque stability was evaluated by using the vulnerability index 〔(foam cells＋extracellular lipids)/(collagens+smooth muscle cells)〕 and the number of buried fibrous caps. RESULTS: The vulnerability indexes in all drug treatment groups were decreased (P<0.05, P<0.01). The giant knotweed rhizome and rhubarb extractives, which had both the effect of promoting blood flow and detoxication, inhibited it the most significantly among them (P<0.01). The significant differences between the promoting blood flow group (saponins of panax pseudo-ginseng) and detoxication group (coptis chinensis extractive) were observed. Furthermore, the mean number of buried caps in plaque of giant knotweed rhizome extractive group and coptis chinensis extractive group were significantly decreased compared with that of control(P<0.01, P<0.05). CONCLUSION: Saponins of panax pseudo-ginseng, coptis chinensis extractive, giant knotweed rhizome and rhubarb extractives stabilize vulnerable plaque by changing plaque composition in clinical commended doses. Among them the herbal extractives with both the effects of promoting blood flow and detoxication (giant knotweed rhizome and rhubarb extractives) have the best effect on stabilizing vulnerable atherosclerotic plaque. Our investigation indicates that this kind of herbal extractives may be have similar effectiveness on stabilizing vulnerable atherosclerotic plaque for decreasing acute cardiovascular events, which is superior to herbs that has the effect of promoting blood flow only or detoxication only.