Effects of flow on LOX-1 and oxidized low-density lipoprotein interactions in brain endothelial cell cultures

Fluid shear stress and uptake of oxidized low-density lipoprotein (ox-LDL) into the vessel wall both contribute to atherosclerosis, but the relationship between shear stress and ox-LDL uptake is unclear. We examined the effects of flow, induced by orbital rotation of bEnd.3 brain endothelial cell cultures for 1 wk, on ox-LDL receptor (LOX-1) protein expression, ox-LDL uptake and ox-LDL toxicity. Orbitally rotated cultures showed no changes in LOX-1 protein expression, ox-LDL uptake or ox-LDL toxicity, compared to stationary cultures. Flow alone does not modify ox-LDL/LOX-1 signaling in bEnd.3 brain endothelial cells in vitro, suggesting that susceptibility of atheroprone vascular sites to lipid accumulation is not due solely to effects of altered flow on endothelium.     

Protamine may have anti-atherogenic potential by inhibiting the binding of oxidized-low density lipoprotein to LOX-1

Oxidized low-density lipoprotein (ox-LDL) leads to atherosclerosis via lectin-like oxidized lipoprotein receptor-1 (LOX-1), one of the major receptor for ox-LDL. Inhibition of the binding of ox-LDL to LOX-1 decreases the proinflammatory and atherosclerotic events. The aim of the present study was to investigate whether protamine, a polybasic nuclear protein, interferes the binding of ox-LDL to LOX-1. Using sandwich ELISA with newly generated antibody, we measured the blocking effect of protamine on the binding of ox-LDL to LOX-1. Protamine dose-dependently inhibited the binding of ox-LDL to LOX-1. DiI-labeled ox-LDL uptake assay in two types of cultured human endothelial cells was performed with fluorescence microplate reader. Activation of extracellular-signal-regulated kinase (ERK)1/2 by ox-LDL was analyzed by immunoblotting. We found that protamine suppressed uptake of ox-LDL in endothelial cells and inhibited ERK1/2 activation by ox-LDL. These results suggest that protamine may possess anti-atherogenic potential by inhibiting ox-LDL binding to LOX-1 through electrostatic interactions.     

Modulation of oxidized-LDL receptor-1 (LOX1) contributes to the antiatherosclerosis effect of oleanolic acid

Oleanolic acid (OA) is a bioactive pentacyclic triterpenoid. The current work studied the effects and possible mechanisms of OA in atherosclerosis. Quails (Coturnix coturnix) were treated with high fat diet with or without OA. Atherosclerosis was assessed by examining lipid profile, antioxidant status and histology in serum and aorta. Human umbilical vein endothelial cells (HUVECs) were exposed to 200 μg/mL ox-LDL for 24 h, then cell viability was assessed with MTT assay; reactive oxygen species (ROS) was assessed with DCFDA staining. Expression levels of LOX-1, NADPH oxidase subunits, nrf2 and ho-1 were measured with real time PCR and western blotting. Furthermore, LOX-1 was silenced with lentivirus and the expression levels assessment was repeated. OA treatment improved the lipid profile and antioxidant status in quails fed with high fat diet. Histology showed decreased atherosclerosis in OA treated animals. Ox-LDL exposure decreased viability and induced ROS generation in HUVECs, and this progression was alleviated by OA pretreatment. Moreover, elevated expression of LOX-1, NADPH oxidase subunits, nrf2 and ho-1 were observed in ox-LDL exposed HUVECs. OA pretreatment prevented ox-LDL induced increase of LOX-1 and NADPH oxidase subunits expression, while further increased nrf2 and ho-1 expression. Silencing of LOX-1 abolished ox-LDL induced effects in cell viability, ROS generation and gene expression. OA could alleviate high fat diet induced atherosclerosis in quail and ox-LDL induced cytotoxicity in HUVECs; the potential mechanism involves modulation of LOX-1 activity, including inhibition of expression of NADPH oxidase subunits and increase of the expression of nrf2 and ho-1.     

Design of a novel LOX-1 receptor antagonist mimicking the natural substrate

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is overexpressed in atherosclerotic lesions. LOX-1 specific inhibitors, urgently necessary to reduce the rate of atherosclerotic and inflammation processes, are not yet available. We have designed and synthesized a new modified oxidized phospholipid, named PLAzPC, which plays to small scale the ligand-receptor recognition scheme. Molecular docking simulations confirm that PLAzPC disables the hydrophobic component of the ox-LDL recognition domain and allows the interaction of the l-lysine backbone charged groups with the solvent and with the charged/polar residues located around the edges of the LOX-1 hydrophobic tunnel. Binding assays, in a cell model system expressing human LOX-1 receptors, confirm that PLAzPC markedly inhibits ox-LDL binding to LOX-1 with higher efficacy compared to previously identified inhibitors.     

Blockade of NEAT1 represses inflammation response and lipid uptake via modulating miR-342-3p in human macrophages THP-1 cells

Atherosclerosis has been recognized as a chronic inflammation process induced by lipid of the vessel wall. Oxidized low-density lipoprotein (ox-LDL) can drive atherosclerosis progression involving macrophages. Recently, long noncoding RNAs (lncRNAs) have been reported to play critical roles in atherosclerosis development. In our current study, we focused on the biological roles of lncRNA NEAT1 in atherosclerosis progress. Here, we found that ox-LDL was able to trigger human macrophages THP-1 cells, a human monocytic cell line, apoptosis in a dose-dependent and time-dependent course. In addition, we observed that NEAT1 was significantly increased in THP-1 cells incubated with ox-LDL and meanwhile miR-342-3p was greatly decreased. Then, NEAT1 was silenced by transfection of small interfering RNA (siRNA) of NEAT1 into THP-1 cells. As exhibited, CD36, oil-red staining levels, total cholesterol (TC), total cholesterol (TG) levels and THP-1 cell apoptosis were obviously repressed by knockdown of NEAT1. Furthermore, inhibition of NEAT1 contributed to the repression of inflammation in vitro. Interleukin 6 (IL-6), IL-1β, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-α) protein levels were remarkably depressed by NEAT1 siRNA in THP-1 cells. By using bioinformatics analysis, miR-342-3p was predicted as a downstream target of NEAT1 and the correlation between them was confirmed in our study. Moreover, overexpression of miR-342-3p could also greatly suppress inflammation response and lipid uptake in THP-1 cells. Knockdown of NEAT1 and miR-342-3p mimics inhibited lipid uptake in THP-1 cells. In conclusion, we implied that blockade of NEAT1 repressed inflammation response through modulating miR-342-3p in human macrophages THP-1 cells and NEAT1 may offer a promising strategy to treat atherosclerotic cardiovascular diseases.     

Oxidized LDL through LOX-1 modulates LDL-receptor expression in human coronary artery endothelial cells

Experimental studies have shown that oxidized low-density lipoprotein (ox-LDL) up-regulates its receptor LOX-1. Both ox-LDL and LOX-1 are expressed in atherosclerotic plaques. Native LDL concentrations are elevated in atherosclerosis, suggesting a reduction in LDL-receptors. We hypothesized that ox-LDL via LOX-1 could influence the expression of LDL-receptors. This study was designed to examine the interaction between ox-LDL, LOX-1, and LDL-receptors in human coronary artery endothelial cells (HCAECs). HCAECs were incubated with ox-LDL (10-80 microg/ml) for 3-24h. Ox-LDL decreased the expression of LDL-receptor in a concentration- and time-dependent fashion. The effects of ox-LDL were mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92, 10 microg/ml) prevented the effect of ox-LDL on LDL-receptor expression. The role of LOX-1 was further confirmed by the use of an antisense to LOX-1 mRNA, which also blocked the effect of ox-LDL in LDL-receptor expression. In other experiments, ox-LDL as expected induced superoxide anion generation; and pretreatment of HCAECs with the anti-oxidants trolox and alpha-tocopherol (each 10 microM) inhibited the formation of superoxide anions as well as the down-regulation of LDL-receptor in response to ox-LDL. These studies provide the first evidence that ox-LDL via LOX-1 modulates LDL-receptor expression in HCAECs. The generation of free radicals elicited by ox-LDL may be a key step in this process.     

LOX-1在D-葡萄糖诱导人肾小球系膜细胞表达TGF-β1中的作用

目的探讨血凝素样氧化低密度脂蛋白受体1（LOX-1）在D-葡萄糖诱导人肾小球系膜细胞表达转化生长因子β1（TGF-β1）中的作用。方法在体外培养人肾小球系膜细胞,在不同时间加入不同浓度的D-葡萄及LOX-1特异性阻滞剂JTX92,用半定量RT-PCR法检测LOX-1和TGF-β1基因表达的相对含量,用Western blot法检测p38 MAPK蛋白质的相对含量,用酶联免疫吸附法（ELISA）检测细胞培养液中TGF-β1浓度。结果D-葡萄糖以时间和浓度依赖的方式增加细胞内LOX-1和TGF-β1 mRNA表达和培养液中TGF-β1浓度,同时也以时间和浓度依赖的方式增加p38 MAPK的表达,JTX92可以明显抑制LOX-1、TGF-β1和p38 MAPK的表达。结论高浓度D-葡萄糖可能通过上调LOX-1的表达,激活细胞内的p38 MAPK信号传递途径,促使人肾小球系膜细胞合成并分泌大量TGF-β1,参与糖尿病肾病的发生发展。     

Chlamydia pneumoniae binds to the lectin-like oxidized LDL receptor for infection of endothelial cells

The association of Chlamydia pneumoniae and atherosclerosis has been well documented. Recently, it has been demonstrated that C. pneumoniae up-regulates expression of the lectin-like ox-LDL receptor (LOX-1) in endothelial cells. Many of the pro-atherogenic effects of ox-LDL occur through its activation and uptake by LOX-1. This class E scavenger receptor contains a carbohydrate-recognition domain common to the C type lectin family. Previously, we have demonstrated that the major outer membrane protein of the chlamydiae is glycosylated and glycan removal abrogates infectivity of C. pneumoniae for endothelial cells. In this study, we investigated whether C. pneumoniae binds to LOX-1. The results show that 1) infection of endothelial cells by C. pneumoniae is inhibited by ligands that bind to the LOX-1 receptor, but not by ligands binding to other scavenger receptors; 2) anti-LOX-1 antibody inhibits C. pneumoniae infectivity, while antibodies against other scavenger receptors do not; 3) anti-LOX-1 antibody inhibits attachment of C. pneumoniae to endothelial cells; and 4) C. pneumoniae co-localizes with LOX-1. These effects were not observed for Chlamydia trachomatis. In conclusion, C. pneumoniae binds to the LOX-1 receptor, which is known to promote atherosclerosis.     

NEAT1 contributes to ox-LDL-induced inflammation and oxidative stress in macrophages through inhibiting miR-128

Long noncoding RNAs (lncRNA) have been recognized as significant regulators in the progression of atherosclerosis (AS). Oxidized low-density lipoprotein (ox-LDL) can induce macrophage inflammation and oxidative stress, that serves important roles in AS. However, the exact function of lncRNA NEAT1 and its possible molecular mechanism in AS remain unclear. Here, we concentrated on the roles and molecular mechanisms of NEAT1 in AS development. In our current study, we observed that NEAT1 was elevated by ox-LDL in a dose-dependent and time-dependent manner. RAW264.7 cell survival was greatly enhanced, and cell apoptosis was significantly inhibited by LV-shNEAT1 transfection. In addition, knockdown of NEAT1 in RAW264.7 cells repressed CD36 expression and foam cell formation while NEAT1 overexpression shown an opposite process. Moreover, NEAT1 downregulation inhibited inflammation molecules including IL-6, IL-1β, and TNF-α. Meanwhile, silencing of NEAT1 can also suppress reactive oxygen species (ROS) and malondialdehyde (MDA) levels with an enhancement of superoxide dismutase (SOD) activity in RAW264.7 cells. MicroRNAs are some short RNAs, and they can regulate multiple biological functions in many diseases including AS. Here, we found that miR-128 expression was remarkably decreased in ox-LDL-incubated RAW264.7 cells. Interestingly, miR-128 mimics was able to reverse AS-correlated events induced by overexpression of NEAT1. By using bioinformatics analysis, miR-128 was predicted as a target of NEAT1 and the correlation between them was validated in our study. Taken these together, it was implied that NEAT1 participated in ox-LDL-induced inflammation and oxidative stress in AS development through sponging miR-128.     

Monocyte chemoattractant protein-1 induces scavenger receptor expression and monocyte differentiation into foam cells

Accumulation of monocytes and the entrapment of oxidized-low-density lipoprotein (ox-LDL) in monocytes are important in the differentiation into "foam" macrophages and the pathogenesis of atherosclerosis. We investigated the role of monocyte chemoattractant protein-1 (MCP-1) in the expression of scavenger receptor (SCR) by using resting monocytes prepared by counterflow centrifugal elutriation. Our results showed that: (1) MCP-1 increased the expression of CD36 SCR by flow cytometric analysis. (2) MCP-1 increased incorporation of 125I-labeled ox-LDL and oil red O staining. (3) MCP-1 and ox-LDL enhanced in vitro transendothelial monocyte migration. (4) These functions were mediated at least in part via extracellular signal-regulated kinase (ERK) pathway. (5) MCP-1 and ox-LDL did not induce monocyte proliferation. Our results imply that MCP-1 is involved in the inflammatory process of atherosclerosis through the induction of SCR expression via the ERK pathway and differentiation of monocytes into foam macrophages, as well as induction of monocyte migration.     

LncRNA UCA1 sponges miR-206 to exacerbate oxidative stress and apoptosis induced by ox-LDL in human macrophages

Long noncoding RNA UCA1 has exerted a significant effect in cardiovascular disease. The biological role of UCA1 in atherosclerosis is unclear. Our study was to identify the potential mechanisms in the progression of atherosclerosis. Here, we observed that ox-LDL increased UCA1 expression greatly in THP-1 cells. Knockdown of UCA1 greatly inhibited CD36 expression, a crucial biomarker in atherosclerosis. Meanwhile, 20 μg/ml ox-LDL induced foam cell formation, which can be reversed by downregulation of UCA1. In addition, TC and TG levels induced by ox-LDL was rescued by UCA1 small interfering RNA. Accumulating studies have indicated that oxidative stress contributes to atherosclerosis progression. Here, we also found that reactive oxygen species, MDA, and THP-1 cell apoptosis were restrained by decreased of UCA1 with an increase of the superoxide dismutase activity. Moreover, miR-206 was predicted as a target of UCA1 and knockdown of UCA1 was able to repress miR-206 expression. Furthermore, overexpression of miR-206 inhibited oxidative stress process and it was reversed by UCA1 upregulation in vitro. In conclusion, we indicated that UCA1 sponged miR-206 to exacerbate atherosclerosis events induced by ox-LDL in THP-1 cells.     

LOX-1特异性小发夹RNA 表达载体的构建及其对巨噬细胞源性泡沫细胞形成的影响

[摘　要]　目的：靶向血凝素样氧化型低密度脂蛋白受体-1基因的发卡样siRNA(shRNA)表达载体及其对巨噬细胞源性泡沫细胞形成的影响。方法：(1)采用DNA重组技术,将LOX-1 shRNA双链与线性化pGenesil-1质粒表达载体连接,脂质体法转染小鼠单核巨噬细胞（RAW264.7）,半定量逆转录聚合酶链反应法检测LOX-1 mRNA的表达,Western blot法检测LOX-1蛋白的表达。(2) Ox-LDL诱导巨噬细胞建立泡沫细胞模型, LOX-1-shRNA进行干预,干预组使用脂质体法进行细胞转染,转染24小时后,再加入Ox-LDL作用24小时,用油红O染色法及细胞内游离胆固醇及总胆固醇测定法观察对泡沫细胞形成的影响,倒置荧光显微镜观察转染LOX-1 shRNA后RAW264.7细胞对Dil-ox-LDL的摄取率。结果：测序鉴定发现插入的发卡样序列正确,成功合成了发卡样LOX-1基因RNA干扰表达载体;靶向LOX-1基因的发卡样shRNA表达载体转染RAW264.7细胞后,其LOX-1基因和蛋白表达显著下调, 同时可抑制巨噬细胞源性泡沫细胞形成及对Dil-ox-LDL的摄取。结论：成功构建了能有效抑制LOX-1 mRNA表达的发卡样LOX-1基因RNA干扰表达载体,并在一定程度上能抑制巨噬细胞源性泡沫细胞的形成,为进一步利用RNA干扰技术防治动脉粥样硬化提供理论基础。     

Inhibition of LOX-1 by statins may relate to upregulation of eNOS.

LOX-1, a receptor for oxidized low-density lipoprotein (ox-LDL), plays a critical role in endothelial dysfunction and atherosclerosis; both of these conditions are associated with diminished expression of constitutive endothelial nitric oxide synthase (eNOS). Recent studies show that HMG CoA reductase inhibitors (statins) exert cardioprotective effect. We examined the role of LOX-1 in eNOS expression and modulation of this relationship by two different statins, simvastatin and atorvastatin in human coronary artery endothelial cells (HCAECs). Ox-LDL (40 microg/ml) upregulated the expression of LOX-1; simultaneously, there was a reduction in eNOS expression. Pretreatment of HCAECs with simvastatin or atorvastatin (1 and 10 microM) reduced ox-LDL-induced upregulation of LOX-1 and downregulation of eNOS (both P < 0.05). High concentration of statins (10 microM) was more potent than the low concentration (1 microM) (P < 0.05). Both statins also attenuated ox-LDL-mediated activation of MAP kinase. These observations indicate that statins attenuate the effect of ox-LDL on eNOS expression. Inhibitory effect on LOX-1 and subsequently MAP kinase activity provides a potential mechanism of beneficial effects of statins beyond lowering cholesterol.     

The hydrophobic tunnel present in LOX-1 is essential for oxidized LDL recognition and binding

Lectin-like oxidized LDL (ox-LDL) receptor-1 (LOX-1) is a type-II transmembrane protein that belongs to the C-type lectin family of molecules. LOX-1 acts as a cell surface endocytosis receptor and mediates the recognition and internalization of ox-LDL by vascular endothelial cells. Internalization of ox-LDL by LOX-1 results in a number of pro-atherogenic cellular responses implicated in the development and progression of atherosclerosis. In an effort to elucidate the functional domains responsible for the binding of ox-LDL to the receptor, a series of site-directed mutants were designed using computer modeling and X-ray crystallography to study the functional role of the hydrophobic tunnel present in the LOX-1 receptor. The isoleucine residue (I(149)) sitting at the gate of the channel was replaced by phenylalanine, tyrosine, or glutamic acid to occlude the channel opening and restrict the docking of ligands to test its functional role in the binding of ox-LDL. The synthesis, intracellular processing, and cellular distribution of all mutants were identical to those of wild type, whereas there was a marked decrease in the ability of the mutants to bind ox-LDL. These studies suggest that the central hydrophobic tunnel that extends through the entire LOX-1 molecule is a key functional domain of the receptor and is critical for the recognition of modified LDL.     

Paeoniflorin attenuates oxidized low-density lipoprotein-induced apoptosis and adhesion molecule expression by autophagy enhancement in human umbilical vein endothelial cells

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction is recognized as a driving force in the development of atherosclerosis (AS). Paeoniflorin (Pae), a typical traditional herbal medicine, possesses anti-inflammatory, antioxidative, antihyperglycaemic, and antiapoptotic properties. Our study aimed to investigate the effects of Pae on ox-LDL-induced injury of the human umbilical vein endothelial cells (HUVECs) and to explore its molecular mechanism. We found that ox-LDL stimulation inhibited cell viability, activated autophagy, and induced apoptosis and adhesion molecule expression in HUVECs. Pae rescued ox-LDL-induced viability reduction and enhanced the ox-LDL-induced autophagy activation in HUVECs. Pae inhibited ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement in HUVECs. In addition, inhibition of SIRT1 by EX-527 abolished the promoting effect of Pae on autophagy and restored the inhibitory effect of Pae on apoptosis and adhesion molecule expression in the presence of ox-LDL. In conclusion, Pae attenuated ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement via upregulation of SIRT1 in HUVECs, shedding light on the mechanism underlying the protective effect of Pae on ox-LDL-induced injury of HUVECs.     

Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-gamma

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.