Oxidized low-density lipoprotein and atherosclerosis implications in antioxidant therapy

Low-density lipoprotein (LDL)-cholesterol is important for cellular function, but in high concentrations, it can lead to atheroma formation. Over the past several decades, it has become abundantly evident that the oxidized form of LDL-cholesterol (ox-LDL) is more important in the genesis and progression of atherosclerosis than native unmodified LDL-cholesterol. Ox-LDL leads to endothelial dysfunction, an initial step in the formation of an atheroma. Ox-LDL acts via binding to a number of scavenger receptors (SR), such as SR-A1, SR-A2 and lectin-like oxidized low-density lipoprotein receptor (LOX-1). Ox-LDL can upregulate expression of its own receptor LOX-1 on endothelial cells and activate these cells. In addition, ox-LDL promotes the growth and migration of smooth muscle cells, monocytes/macrophages and fibroblasts. Ox-LDL also leads to the generation of reactive oxygen species that in physiologic concentrations combat invasion of the body by noxious agents, but when in excess, can lead to a state of oxidative stress. There is evidence for the presence of oxidative stress in a host of conditions such as atherosclerosis and aging. In this review, we discuss the role of oxidative stress, ox-LDL and LOX-1 in atherogenesis and the reasons why the traditional approaches to limit oxidant stress have not been successful.     

LOX-1在动脉粥样硬化中的作用研究新进展

动脉粥样硬化(AS)是一种血管慢性炎症性病变,其中内皮细胞功能异常、单核细胞的黏附和迁移、平滑肌细胞的凋亡、泡沫细胞的形成和血小板的活化是AS形成的关键环节,最终结果是形成大、中动脉内膜下的粥样硬化斑块,造成管腔狭窄,远端组织器官供血不足甚至栓塞。低密度脂蛋白（LDL） 氧化形成的氧化型LDL（ox-LDL）在AS发生、发展过程中起着重要作用。目前在与AS发生、发展相关的细胞（如血管内皮细胞、血管平滑肌细胞、单核细胞、巨噬细胞以及泡沫细胞）上已经发现和鉴定了多种oxLDL受体,其中瘦素样氧化型低密度脂蛋白受体(LOX)-1表达于血管内皮细胞、巨噬细胞、血小板上,是ox-LDL的主要受体［1］,在AS的发生、发展中起着重要作用,本文将着重阐述近年来LOX-1影响AS发生发展相关效应与机制的新进展。     

Roles of oxidized low-density lipoprotein and its receptors in the pathogenesis of atherosclerotic diseases

In elderly populations, atherosclerotic diseases, including ischemic heart disease and stroke, frequently impair quality of life and affect mortality. Hypercholesterolemia, especially increased plasma low-density lipoprotein (LDL), is one of the strongest risk factors for atheroscletorotic diseases. Oxidative modification of LDL appears to convert LDL particles to more atherogenic forms. Scavenger receptor class A (SR-A) and CD36 have been identified and well-characerized as receptors for Ox-LDL in macrophages. In addition to these molecules, lectin-like oxidized LDL receptor (LOX)-1 and scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX) are type II and I membrane glycoproteins, respectively, both of which can act as cell-surface endocytosis receptors for atherogenic oxidized LDL (Ox-LDL). LOX-1 expression can dynamically be induced by pro-inflammatory stimuli, and is detectable in cultured macrophages and activated vascular smooth muscle cells (VSMC), in addition to endothelial cells. LOX-1-dependent uptake of Ox-LDL induces apoptosis of cultured VSMC. In vivo , endothelial cells that cover early atherosclerotic lesions, and intimal macrophages and VSMC in advanced atherosclerotic plaques dominantly express LOX-1. LOX-1 expressed on the cell-surface can be cleaved in part and released as soluble molecules, suggesting the diagnostic value of soluble LOX-1. SR-PSOX is a newly identified receptor for Ox-LDL, which appears to be identical to CXCL16, a novel membrane-anchored chemokine directed to CXCR6-positive lymphocytes. In contrast to LOX-1, which is expressed by a variety of cell types, SR-PSOX expression appeared relatively confined to macrophages in atherogenesis. Taken together, oxidized LDL receptors, including LOX-1 and SR-PSOX, may play important roles in atherogenesis and atherosclerotic plaque rupture.     

Current Concepts of the Role of Oxidized LDL Receptors in Atherosclerosis

Atherosclerosis is characterized by accumulation of lipids and inflammatory cells in the arterial wall. Oxidized low-density lipoprotein (ox-LDL) plays important role in the genesis and progression of atheromatous plaque. Various scavenger receptors have been recognized in the past two decades that mediate uptake of ox-LDL leading to formation of foam cells. Inhibition of scavenger receptor A and CD36 has been shown to affect progression of atherosclerosis by decreasing foam cell formation. Lectin-type oxidized LDL receptor 1 (LOX-1) participates at various steps involved in the pathogenesis of atherosclerosis, and in experimental studies its blockade has been shown to affect the progression of atherosclerosis at multiple levels. In this review, we summarize the role of ox-LDL and scavenger receptors in the formation of atheroma with emphasis on effects of LOX-1 blockade.     

New oxidized LDL receptors and their functions in atherogenesis

Oxidized low density lipoprotein (Ox-LDL) appears to play key roles in atherosclerotic progression and plaque rupture. Biological effects of Ox-LDL on vascular cells may, at least in part, be mediated by cell surface receptors for Ox-LDL. Lectin-like oxidized LDL receptor (LOX)-1 and scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX) are type II and I membrane glycoprtoeins, respectively, both of which can act as cell-surface endocytosis receptors for atherogenic oxidized LDL (Ox-LDL). LOX-1 expression can dynamically be induced by proinflammatory stimuli, and is detectable in cultured macrophages and activated vascular smooth muscle cells (VSMC), in addition to endothelial cells. LOX-1-dependent uptake of Ox-LDL induced apoptosis of cultured VSMC. In vivo, endothelial cells that cover early atherosclerotic lesions, and intimal macrophages and VSMC in advanced atherosclerotic plaques dominantly express LOX-1. LOX-1 expressed on the cellsurface can be cleaved, in part, and released as soluble molecules, suggesting the diagnostic significance of plasma soluble LOX-1 levels. SR-PSOX appeared to be identical to CXCL16, a novel membrane-anchored chemokine directed to CXCR6-positive lymphocytes, suggesting another role of SR-PSOX as T-cell chemoattractant. In contrast to LOX-1 expressed by a variety of cell types. SR-PSOX expression appeared relatively confined to macrophages in atherogenesis. Taken together, LOX-1 and SR-PSOX may play important roles in atherogenesis and athrosclerotic plaque rupture.     

Upregulation of endothelial receptor for oxidized LDL (LOX-1) by oxidized LDL and implications in apoptosis of human coronary artery endothelial cells: evidence from use of antisense LOX-1 mRNA and chemical inhibitors

A specific lectin-like endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL-induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 microg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL-mediated upregulation of LOX-1 was suppressed (P<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL-mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL-mediated apoptosis of HCAECs. Nuclear factor (NF)-kappaB was markedly activated in ox-LDL-treated HCAECs. The critical role of NF-kappaB activation became evident in experiments with antisense LOX-1, which abolished ox-LDL-mediated NF-kappaB activation. In this process, an NF-kappaB inhibitor, caffeic acid phenethyl ester, also inhibited ox-LDL-mediated apoptosis of HCAECs. These findings indicate that ox-LDL upregulates its own endothelial receptor. Ox-LDL-induced apoptosis is mediated by the action of LOX-1. In this process, NF-kappaB activation may play an important role as a signal transduction mechanism.     

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in atherogenesis.

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 can act as a cell surface endocytosis receptor for atherogenic oxidized LDL (Ox-LDL). LOX-1 is synthesized as a 40 kDa precursor protein with N-linked high mannose-type carbohydrate, which is further glycosylated and processed into a 50 kDa mature form. LOX-1 expression is not constitutive but can be induced by proinflammatory, oxidative, and mechanical stimuli. In addition to endothelial cells, macrophages and activated vascular smooth muscle cells express LOX-1. In vivo, endothelial cells covering early atherosclerotic lesions and macrophages and smooth muscle cells accumulated in the intima of advanced atherosclerotic plaques express LOX-1. LOX-1 is cleaved at membrane proximal extracellular domain by some protease activities 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.     

Adhesion molecule expression in fibroblasts: alteration in fibroblast biology after transfection with LOX-1 plasmids

The endothelial lectinlike, oxidatively (ox-) modified LDL receptor LOX-1 is a critical player in the pathogenesis of atherosclerosis and myocardial ischemia. Ox-LDL binding of LOX-1 results in the expression of various adhesion molecules, which attract monocytes to endothelial cells, an initial step in atherogenesis. We wished to examine the role of the ox-LDL/LOX-1 signaling pathway in fibroblasts, which naturally express low levels of LOX-1. Rat cardiac fibroblasts were transfected with either cytomegalovirus (CMV)-LOX-1wt (amino acids [aa] 1 to 273) or CMV-LOX-1(1-261) (an ox-LDL-binding negative mutant, aa 1 to 261) plasmid. Western blots showed that LOX-1 protein expression was increased significantly in cells transfected with CMV-LOX-1wt or CMV-LOX-1(1-261) plasmid (P<0.01 vs control). Fibroblasts transfected with CMV-LOX-1wt showed ox-LDL binding, whereas fibroblasts without transfection and those transfected with CMV-LOX-1(1-261) did not bind ox-LDL. Compared with untransfected cells, ox-LDL treatment (50 microg/mL, 24 hours) markedly induced the expression of the leukocyte adhesion molecules intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM)-1 as well as matrix metalloproteinase (MMP)-1 in cells transfected with CMV-LOX-1wt (P<0.05) but not in cells transfected with CMV-LOX-1(1-261). Concurrently, ox-LDL treatment enhanced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) (P<0.05 vs control) in CMV-LOX-1wt-transfected cells. These data suggest that in cardiac fibroblasts, ox-LDL binds to LOX-1 and activates p38 MAPK, followed by the expression of ICAM-1, VCAM-1, and MMP-1. Thus, fibroblasts transform into an endothelial phenotype on transfection with CMV-LOX-1wt and subsequent exposure to ox-LDL. This study provides a useful model system (plasmid-transfected fibroblasts) to study the molecular biology of LOX-1.     

Synergically increased expression of CD36, CLA-1 and CD68, but not of SR-A and LOX-1, with the progression to foam cells from macrophages

Several species of scavenger receptors have so far been identified. However, it remains unclear which receptors are more crucial for the foam cell formation and progression. In the present study, we compared five major scavenger receptors (SR-A, CD36, CLA-1, CD68, and LOX-1) in their levels of expression at the different stages of foam cells derived from THP-1 cells. The expression of all scavenger receptors examined was up-regulated by the stimulation with TPA for 48 hours, despite the expressions of SR-A, CD36 and LOX-1 being very low before the treatment with TPA. Four to 7 days after the removal of TPA, the levels of CD36, CLA-1 and CD68 were increased significantly. In contrast, the expression of SR-A was suppressed significantly, and no change was observed in that of LOX-1. Furthermore, when the transformed macrophages were incubated with oxidized LDL, in which the uptake of [3H] cholesteryl oleoyl ether-labeled OxLDL was linear up to 7 days after the addition of OxLDL, the expression of CD36, CLA-1 and CD68 were greatly enhanced. This enhancement was more prominent than that without oxidized LDL, and the enhancement was sustained throughout the experimental period. On the other hand, SR-A was not up-regulated, and LOX-1 was down-regulated. We thus propose that CD36, CLA-1 and CD68, but not SR-A and LOX-1, may play crucial roles in the progression of macrophages to foam cells, which is a key step for the initiation of atherosclerosis.     

LOX-1: A Critical Player in the Genesis and Progression of Myocardial Ischemia

Myocardial ischemia is the most common cause of mortality and morbidity in the developed countries and rapidly becoming a common malady in the developing countries. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a fundamental role in the genesis and progression of atherosclerosis and its complications. LOX-1 has been identified as a major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, cardiomyocytes and fibroblast. In vitro and in vivo studies show that LOX-1 is upregulated during acute myocardial ischemia, and continues to be upregulated during chronic ischemia. Further, LOX-1 inhibition reduces ischemic myocardial injury and limits cardiac remodeling. LOX-1 inhibition decreases oxidative stress and inflammatory response to injury resulting in limitation of ischemic injury. Molecular studies show that LOX-1 inhibition reduces release of pro-inflammatory cytokines and expression of angiotensin II type 1 receptor via inhibition of redox-sensitive pathways. These alterations limit cardiomyocyte hypertrophy and collagen accumulation in the ischemic regions. These alterations in molecular signaling and physical alterations can result in improved cardiac function and better survival after ischemic myocardial injury.     

Mulberry leaf aqueous fractions inhibit TNF-alpha-induced nuclear factor kappaB (NF-kappaB) activation and lectin-like oxidized LDL receptor-1 (LOX-1) expression in vascular endothelial cells

Mulberry (Morus Alba L., family Moraceae) leaf extracts have various biological effects including inhibition of oxidative modification of low-density lipoprotein (LDL), which is the major cause of atherosclerosis. Endothelial dysfunction elicited by oxidized LDL (Ox-LDL) has been implicated in atherogenesis. Lectin-like Ox-LDL receptor-1 (LOX-1), a cell-surface receptor for atherogenic Ox-LDL, appears to mediate Ox-LDL-induced inflammation, which may be crucial in atherogenesis. Previous studies revealed that expression of LOX-1 is highly inducible by proinflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and transforming growth factor-beta (TGF-beta). Therefore, we examined whether mulberry leaf aqueous fractions inhibit LOX-1 expression induced by proinflammatory stimuli. Pretreatment of cultured bovine aortic endothelial cells (BAECs) with mulberry leaf aqueous fractions inhibited TNF-alpha- and LPS-induced expression of LOX-1 at both protein and mRNA levels in a time- and concentration-dependent manner. In contrast, mulberry leaf aqueous fractions did not affect TGF-beta-induced LOX-1 expression. Furthermore, mulberry leaf aqueous fractions inhibited TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) and phosphorylation of inhibitory factor of NF-kappaB-alpha (IkappaB-alpha) in a time- and concentration-dependent fashion. Thus, mulberry leaf aqueous fractions suppress TNF-alpha- and LPS-induced LOX-1 gene expression, by inhibiting NF-kappaB activation.     

Lectin-like oxidized low-density lipoprotein receptor 1 mediates leukocyte infiltration and articular cartilage destruction in rat zymosan-induced arthritis

OBJECTIVE: The relationship between rheumatoid arthritis and atherosclerosis has been recognized for >20 years. This study aimed to elucidate the roles of oxidized low-density lipoprotein (ox-LDL; one of the main pathogenic factors of atherosclerosis) and its endothelial receptor, lectin-like ox-LDL receptor 1 (LOX-1), in arthritic joints using a rat zymosan-induced arthritis (ZIA) model. METHODS: LOX-1 expression and ox-LDL accumulation in arthritic joints were detected by immunohistochemistry using specific mouse anti-LOX-1 and anti-ox-LDL monoclonal antibodies, respectively. To elucidate the effects of the expressed LOX-1 on arthritis, ZIA rats were treated with anti-LOX-1 antibody or normal mouse IgG. The severity of arthritis was analyzed by joint swelling. Cell infiltration, synovial hyperplasia, and proteoglycan losses were also determined by histologic scoring. Proinflammatory cytokine and nitrite levels in serum and joint fluid were also measured. RESULTS: Immunohistochemical study of ZIA demonstrated LOX-1 expression on synovial endothelium and postcapillary venules at 6 hours after the induction of inflammation, with maximum expression detected at 24 hours. LOX-1 was also expressed weakly on both joint cartilage and synovium. Ox-LDL, a ligand of LOX-1, was also detected in articular chondrocytes. Administration of anti-LOX-1 antibody, which blocks LOX-1 activity, suppressed joint swelling (by 33.5%), leukocyte infiltration, and joint nitrite accumulation at 24 hours, as well as cartilage destruction at 7 days, compared with control rats. CONCLUSION: LOX-1 induction in arthritic joints might play a role in promoting joint inflammation and cartilage destruction by mediating leukocyte infiltration into the arthritic joints of ZIA rats.     

Role of caspases in Ox-LDL-induced apoptotic cascade in human coronary artery endothelial cells

Oxidized low-density lipoprotein (ox-LDL) induces apoptosis in endothelial cells. However, steps leading to ox-LDL-induced apoptosis remain unclear. We examined the role of ox-LDL and its newly described receptor LOX-1 in the expression of intracellular pro- and antiapoptotic proteins and caspase pathways in human coronary artery endothelial cells (HCAECs). Cells were cultured and treated with different concentrations (10 to 80 microg/mL) of ox-LDL for different times (2 to 24 hours). Ox-LDL induced apoptosis in HCAECs in a concentration- and time-dependent manner. Ox-LDL also activated caspase-9 and caspase-3, but not caspase-8. After ox-LDL treatment, there was a significant release of activators of caspase-9, including cytochrome c and Smac from mitochondria to cytoplasmic compartment, and their release was not affected by treatment of cells with inhibitors of either caspase-8 or caspase-9. Ox-LDL also decreased expression of antiapoptotic proteins Bcl-2 and c-IAP (inhibitory apoptotic protein)-1, which are involved in the release of cytochrome c and Smac and activation of caspase-9, in a concentration- and time-dependent manner. On the other hand, ox-LDL did not change the expression of Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP) and proapoptotic protein Fas, which are required for the activation of caspase-8. Further, ox-LDL did not cause the truncation of Bid, which implies the activation of caspase-8. In other experiments, pretreatment of HCAECs with the caspase-9 inhibitor z-LEHD-fmk, but not the caspase-8 inhibitor z-IETD-fmk, blocked ox-LDL-induced activation of caspase-3 and apoptosis. As expected, pretreatment with the caspase-3 inhibitor DEVD-CHO inhibited ox-LDL-induced activation of caspase-3 and resultant apoptosis. The proapoptotic effects of ox-LDL were mediated by its receptor LOX-1, because pretreatment of HCAECs with antisense-LOX-1, but not sense-LOX-1, blocked these effects of ox-LDL. These findings suggest that ox-LDL through its receptor LOX-1 decreases the expression of antiapoptotic proteins Bcl-2 and c-IAP-1. This is followed by activation of apoptotic signaling pathway, involving release of cytochrome c and Smac and activation of caspase-9 and then caspase-3.     

Lectin-like oxidized low-density lipoprotein receptor 1 mediates matrix metalloproteinase 3 synthesis enhanced by oxidized low-density lipoprotein in rheumatoid arthritis cartilage

OBJECTIVE: To investigate for the presence of oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor 1 (LOX-1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox-LDL with LOX-1 can induce matrix metalloproteinase 3 (MMP-3) in articular cartilage explant culture. METHODS: Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX-1 and ox-LDL by confocal fluorescence microscopy. The association between ox-LDL and LOX-1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox-LDL, with or without preincubation with neutralizing anti-LOX-1 antibody. MMP-3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme-linked immunosorbent assay. RESULTS: The majority of the RA chondrocytes stained positively with both anti-LOX-1 and anti-ox-LDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti-LOX-1 antibody suppressed the binding of DiI-labeled ox-LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX-1. In contrast to native LDL, ox-LDL induced MMP-3 synthesis by articular chondrocytes in association with the induction of LOX-1, which resulted in enhanced secretion of MMP-3 into the culture medium. Anti-LOX-1 antibody reversed ox-LDL-stimulated MMP-3 synthesis to control levels. CONCLUSION: Ox-LDL, principally mediated by LOX-1, enhanced MMP-3 production in articular chondrocytes. Increased accumulation of ox-LDL with elevated expression of LOX-1 in RA cartilage indicates a specific role of the receptor-ligand interaction in cartilage pathology in RA.     

Lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) expression in human articular chondrocytes

OBJECTIVE: To investigate the involvement of oxidized low density lipoprotein (ox-LDL) and the expression of its receptor lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) in osteoarthritis, by determining the ox-LDL in synovial fluid and the expression of LOX-1 mRNA and protein in osteoarthritic as well as normal cartilage. In addition, the effect of ox-LDL on chondrocyte viability and the effect of ascorbic acid (a well-known anti-oxidant) on LOX-1 expression were studied. METHODS: Fifteen patients were included in the study. Osteoarthritic articular cartilage was obtained from two distinct locations in the knee (n = 10) and hip (n = 5), specifically from weight-bearing and non-weight-bearing areas of the same joints. Five individuals were used as controls. mRNA and protein expression were studied by RT-PCR and immunofluorescence, respectively. Ox-LDL was measured in the synovial fluid and in paired serum samples from the patients using the ELISA method. RESULTS: Ox-LDL was detected in the synovial fluid and its receptor LOX-1 was detected in cartilage from both weight-bearing and non-weight-bearing areas, whereas no LOX-1 expression was found in normal cartilage. Ox-LDL reduced chondrocyte viability in cell cultures, while the addition of ascorbic acid to osteoarthritic chondrocytes resulted in a decrease in LOX-1 mRNA expression. CONCLUSION: The detection of LOX-1 mRNA and protein expression in osteoarthritic cartilage drawn from both weight-bearing and non-weight-bearing regions of the same patients suggest that LOX-1 may be involved in the progression and pathogenesis of osteoarthritis.     

LOX-1在氧化型低密度脂蛋白诱导U937细胞凋亡中的作用和卡托普利的干预作用

目的探讨ox-LDL受体LOX-1在ox-LDL诱导单核/巨噬细胞凋亡中的角色和卡托普利的干预作用。方法应用流式细胞术检测细胞凋亡,采用免疫组织化学技术和Western Blot测定Caspase3、8、9的表达。结果ox-LDL可诱导U937细胞的凋亡峰出现(12.773±1.413),应用卡托普利和LOX-1的阻断剂角叉菜胶、PIA后凋亡峰明显减少,其百分比分别为(1.02±0.166)(4.94±0.47)(2.62±0.656),同时U937细胞在用ox-LDL培养后代表线粒体通路的Cas-pase9,3和代表死亡受体通路caspases8,3表达都增加,应用卡托普利和LOX-1的阻断剂角叉菜胶、PIA后caspase3、8、9的表达均减少。结论ox-LDL是通过与其受体LOX-1结合发挥损伤作用的。卡托普利可以抑制ox-LDL对U937细胞凋亡的诱导作用,从而对单核巨噬细胞起到保护作用。     

白细胞介素10对人THP-1源巨噬细胞泡沫化过程中SR-A表达的影响

目的:本研究旨在观察白细胞介素-10(IL-10)对人THP-1源巨噬细胞泡沫化过程中清道夫受体A(SR-A)表达的影响,探讨在动脉粥样硬化形成中IL-10对氧化型低密度脂蛋白(ox-LDL)诱导巨噬细胞泡沫化的干预作用。方法:体外建立泡沫细胞培养体系,将细胞分为5组即THP-1单核细胞组,巨噬细胞组,IL-10刺激巨噬细胞组,ox-LDL刺激巨噬细胞组(泡沫细胞组),ox-LDL和IL-10联合刺激巨噬细胞组。采用RT-PCR和Westernbloting分别检测SR-A的mRNA和蛋白表达变化,脂质油红O化学染色方法检测各组细胞脂质摄取量的情况。结果:当THP-1分化为巨噬细胞时,SR-A开始大量表达;IL-10刺激可显著抑制巨噬细胞组SR-A的表达,其mRNA和蛋白分别下降为未刺激前的0.6倍和0.7倍,泡沫细胞形成率也下降为未刺激前的0.6倍。巨噬细胞经ox-LDL刺激形成泡沫细胞时,SR-A的表达进一步升高,其mRNA和蛋白分别增加为巨噬细胞组的1.5倍和1.4倍,泡沫细胞形成率提高为巨噬细胞组的1.7倍。在此过程中加入IL-10联合刺激,观察到SR-A的表达量有显著降低,其mRNA和蛋白均下降为单用ox-LDL刺激巨噬细胞组的0.4倍,ox-LDL的摄取量也下降为单用ox-LDL刺激巨噬细胞组的0.3倍。结论:IL-10抑制巨噬细胞SR-A表达,IL-10对ox-LDL诱导巨噬细胞SR-A的表达具明显干预作用。IL-10通过抑制SR-A表达可能在抗动脉粥样硬化的发生中发挥重要作用。     

Heparin-binding EGF-like growth factor induces expression of lectin-like oxidized LDL receptor-1 in vascular smooth muscle cells

Receptor-mediated endocytosis of oxidized LDL (Ox-LDL) has been implicated in lipid accumulation and vascular cell dysfunction. Lectin-like Ox-LDL receptor-1 (LOX-1) is highly inducible by proinflammatory cytokines, as well as angiotensin II and Ox-LDL in vitro. LOX-1 is expressed in macrophages and smooth muscle cells accumulated in the intima of advanced atherosclerotic plaques in vivo. Here we show that heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells, induces LOX-1 expression in cultured bovine aortic smooth muscle cells. HB-EGF (1-100 ng/ml) induced LOX-1 expression, which was peaked between 8 and 16 h after HB-EGF stimulation. HB-EGF-induced expression of LOX-1 was suppressed by ZD1839, an inhibitor of EGF receptor phosphorylation. Both MEK and p38 mitogen-activated protein kinase (MAPK) inhibitors significantly blocked LOX-1 upregulation induced by HB-EGF. Phosphatidylinositol 3-kinase (PI3K) inhibitors also blocked HB-EGF-induced LOX-1 expression. HB-EGF induced phosphorylation of ERK, p38 MAPK and Akt, which were suppressed by ZD1839. Upregulated expression of LOX-1 was associated with enhanced uptake of DiI-labeled Ox-LDL in smooth muscle cells. Taken together, HB-EGF can also act as an inducer of LOX-1 expression and play an integral role in foam cell transformation, cellular dysfunction, and proliferation of smooth muscle cells in atherogenesis.     

Oxidized-LDL through LOX-1 increases the expression of angiotensin converting enzyme in human coronary artery endothelial cells

BACKGROUND AND OBJECTIVES: Our previous studies have shown that oxidized low-density lipoprotein (ox-LDL) and angiotensin II (Ang II) influence each other's action in endothelial cells. This study was designed to examine the regulation by ox-LDL of the expression of angiotensin converting enzyme (ACE) gene in human coronary artery endothelial cells (HCAECs). In addition, we studied the effect of the HMG CoA reductase inhibitor simvastatin on this interaction. METHODS AND RESULTS: Cultured HCAECs were incubated with ox-LDL (10-80 microg/ml) for 1-24 h. Ox-LDL increased the expression of ACE in a concentration- and time-dependent fashion. The upregulation of ACE expression in response to ox-LDL was mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 prevented the expression of ACE (P<0.01). Native-LDL had no significant effect on ACE expression. In this process, ox-LDL-induced activation of mitogen-activated protein kinase (MAPK p42/44) played an important role, since pretreatment of HCAECs with the MAPK p42/44 inhibitor (PD98059, 10 microM) inhibited MAPK activation and subsequently attenuated the expression of ACE (P<0.01 vs. ox-LDL alone). In other experiments, we pretreated HCAECs with simvastatin (10 microM) and then exposed the cells to ox-LDL. Simvastatin markedly attenuated ox-LDL-induced MAPK activation, and concurrently reduced ACE expression (P<0.01 vs. ox-LDL alone). CONCLUSIONS: Our observations provide direct evidence that ox-LDL via LOX-1 activation induces ACE gene expression in HCAECs, and MAPK activation plays a signal transduction role in this process. Simvastatin, which inhibits MAPK activation, also blocks ox-LDL-mediated upregulation of ACE.     

8-iso-prostaglandin F(2alpha) increases expression of LOX-1 in JAR cells

Lectinlike oxidized LDL receptor-1 (LOX-1), a cell-surface receptor for oxidized LDL (ox-LDL), is proposed to be involved in endothelial dysfunction and in the pathogenesis of atherosclerosis. Preeclampsia is a pregnancy complication diagnosed by hypertension and proteinuria, characterized by endothelial dysfunction, and supposedly caused by compounds from hypoxic uteroplacental tissues. A feature of preeclampsia is formation of foam cells in maternal arterial walls of gestational tissue ("acute atherosis"). Oxidative stress is believed to play a role in the pathophysiology of preeclampsia. 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) is a marker of oxidative stress in vivo, is biologically active in vitro, and is elevated in preeclamptic plasma and gestational tissue. In the present article, we hypothesized that 8-iso-PGF(2alpha) could induce the expression of LOX-1 in trophoblastic cells (JAR). We demonstrated augmented cellular uptake of (125)I-tyraminylcellobiose ox-LDL in JAR cells incubated with 8-iso-PGF(2alpha) (10 micromol/L) versus control cells. Ligand blots revealed an increased binding of ox-LDL to LOX-1 in JAR cells incubated with 8-iso-PGF(2alpha) (10 micromol/L). Incubation with 8-iso-PGF(2alpha) (10 micromol/L) also resulted in augmented LOX-1 protein levels (Western blots) and mRNA levels (Northern blots). JAR cells transfected with 3 copies of a nuclear factor-kappaB binding site demonstrated dose-dependent activation of the reporter gene luciferase after incubation with 8-iso-PGF(2alpha) (0 to 10 micromol/L). We also demonstrated increased accumulation of neutral fats in JAR cells incubated with 8-iso-PGF(2alpha) (10 micromol/L) and ox-LDL compared with controls by oil red O staining. We speculate a potential role of isoprostanes and LOX-1 in preeclampsia in the development of "acute atherosis" of gestational spiral arteries.