原代狒狒股动脉内皮细胞的炎症损伤机制和干预研究
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摘要
背景
动脉粥样硬化(Atherosclerosis, AS)是一个严重危害人类健康的疾病,其发病机制一直以来成为研究者关注的焦点。目前研究常见的动物模型有鼠、家兔、禽类、猪、食肉类(犬,猫)和非人灵长类等AS动物模型。狒狒是灵长类动物之一,其基因学,解剖学和病理生理学与人类相近,是研究AS较为理想的实验动物。内皮细胞损伤是AS发生发展中的起始和关键环节。本实验选用原代狒狒股动脉内皮细胞(Baboon Femoral Arterial Endothelial Cell, BAEC)进行体外实验,模拟炎症因子对体内大血管早期损伤的机制。
目前许多研究证实炎症因子对内皮细胞毒性损伤在心血管疾病的发病机制中发挥重要的作用。在病理条件下,炎症介质可以损伤内皮的屏障功能,导致通透性增高,表面粘附因子表达增加,单核细胞粘附聚集和泡沫细胞的形成,从而造成血管功能障碍,进而导致大血管动脉粥样硬化。肿瘤坏死因子-α (TNF-α)是一个明确的心血管风险因子和炎症因子,可通过损伤血管内皮细胞,促进单核细胞对内皮细胞的粘附,参与AS发生、发展过程。临床研究发现它富集于动脉粥样硬化的病变处,不存在于正常的组织中,成为其参与动脉粥样硬化炎症反应的直接证据。
白藜芦醇(Resveratrol, RSV)是一种生物性较强的天然多酚类物质,又称芪三酚。天然RSV广泛存在于花生、葡萄和虎杖等植物中。流行病学研究显示高脂饮食的法国人群冠心病发病率较低,归因于红酒中的白藜芦醇成分,具有抗氧化,抗炎,调脂等心血管保护作用和防治动脉粥样硬化的作用,但具体作用机制尚需进一步探索。目前研究显示RSV对人动静脉内皮细胞具有抑制或促进生长的作用,存在明显争议。本研究选择了不同剂量的白藜芦醇,研究其对炎症损伤状态下的原代BAEC的作用特点,进一步探讨RSV保护血管的可能机制。
材料和方法
1.细胞培养
消化狒狒股动脉,获得原代股内皮细胞(BAEC),悬浮于20%FCS-F-12K培养基,接种于铺有明胶的6孔板上。每三天全量换液,细胞长至85-95%可传代或冻存。
2.CD31+免疫磁珠标记内皮细胞
选用CD31+免疫磁珠标记内皮细胞,从而筛选、纯化原代分离的股动脉内皮细胞。
3.内皮细胞功能和细胞骨架测定
免疫荧光染色法简述如下：固定,封闭,一抗孵育,冲洗,二抗孵育,DAPI染色,封片,荧光显微镜下观察。Dil-LDL/UEA-1、vWF和VE-cadherin特异性标记内皮细胞,F-actin和β-tubulin非特异性染色细胞骨架,α-actin为肌纤维细胞的相对特异性染色。
4.内皮细胞胞增殖实验
选用MTT法测定内皮细胞的增殖。观察不同剂量的白藜芦醇(RSV,0.1-100μmol/L)和或TNF-α (lOng/ml)对内皮细胞增殖的影响。
5.流式细胞技术测定内皮细胞表面粘附因子
本实验选用适合于狒狒的抗体为：抗人E-选择素-FITC(R&D Systems公司,#1C0025),抗人VCAM-1-PE (US Biological公司,#400122),抗人ICAM-1-APC (BD Biosciences公司,#555749)。观察TNF-α (10ng/ml)和或不同剂量的白藜芦醇(RSV,0.1-100μmol/L)对内皮细胞表面粘附因子表达的影响。
6.细胞因子测定
ELISA法测定内皮细胞分泌的单核细胞趋化蛋白-1(MCP-1)和白介素-1β(IL-1β)因子。
7.内皮细胞炎症损伤测定
划痕实验法测定内皮细胞迁移愈合能力。管腔形成实验测定内皮细胞血管形成的能力
结果
1.狒狒股动脉内皮细胞形态和功能鉴定
原代狒狒股动脉内皮细胞(baboon femoral arterial endothelial cells, BAECs),早期(Figl. A, day3)呈小多角、球形、呈团状,48-72h生长最快,逐渐生长成梭形,有些细胞排列呈鱼贯状相连,间有旋祸状排列。7-10天胞体呈多角形,相互嵌合,为单层呈铺路石状排列(Figl.B,day7)。培养基为20%FCS-F-12K,每三天全量换液,约7-10天,可传代。狒狒肱动脉纤维细胞呈长梭形,生长迅速,与内皮细胞形态差别显著(Figl. C)。镜下观察收集的内皮细胞纯度通常>85%(Figl. B),有时收集到纯度<50%的内皮细胞(Figl. D),可见混杂的纤维细胞。
VE-Cadherin (Fig2.A)和vWF (Fig2. B)为狒狒股动脉内皮细胞特异性染色。F-肌动蛋白(F-actin, Fig2. C)和β-微管蛋白(β-tubulin, Fig2. D)为BAEC非特异性骨架染色,可见内皮细胞骨架结构完整。
α-肌动蛋白(α-actin, Fig2. E)和p-微管蛋白(β-tubulin,Fig2. F)为纤维细胞骨架染色,其中α-肌动蛋白不能染色内皮细胞。
CD31+免疫磁珠标记内皮细胞(Fig3.A,B),金黄色磁珠附着于内皮细胞表面,随传代逐渐脱落,密度减少,不影响内皮细胞生长和功能。
内皮细胞Dil-LDL/UEA染色双阳性。
2.肿瘤坏死因子-α (TNF-α, lOng/ml)激活内皮细胞
TNF-α激活的内皮细胞高表达ICAM-1, VCAM-1, E-selectin和分泌MCP-1和IL-1β。同时TNF-α能抑制内皮细胞的增殖,延缓内皮细胞划痕愈合速率。
3.白藜芦醇对内皮细胞的保护作用
小剂量(0.1,1μmol/L)白藜芦醇对内皮细胞生长无明显影响。10-100μmol/L白藜芦醇能明显促进狒狒股动脉内皮细胞的增殖,并且能减轻肿瘤坏死因子-α(TNF-α, lOng/ml)对内皮细胞的损伤作用,但不能逆转炎症损伤。
白藜芦醇10μmol/L能明显促进内皮细胞的划痕愈合速率。白藜芦醇(1,10,50μmol/L)能明显减轻肿瘤坏死因子-β (TNF-α,10ng/ml)对内皮细胞划痕愈合速率的损伤,但不能逆转损伤。
白藜芦醇(10,50μmol/L)和TNF-a (lOng/ml)共孵育24小时后,能明显抑制VCAM-1和ICAM-1的表达,可能是通过抑制NF-KB信号通路实现的。
结论
1.狒狒原代股动脉内皮细胞具有结构完整,高纯度和高生物活性特点,且狒狒便于活检取大血管,为深入研究动脉粥样硬化机制提供了理想的动物模型。
2.肿瘤坏死因子-α (TNF-α)损伤狒狒股动脉内皮细胞,促进动脉粥样硬化的发生发展。
3.白藜芦醇(RSV)能抑制TNF-α对内皮细胞的损伤,具有明显的抗炎和抗动脉粥样硬化作用。
背景
近30年来发现,低密度脂蛋白的氧化修饰在动脉粥样硬化(AS)发生机制中起着重要的作用(1,2)。有学者发现人、兔动脉粥样硬化斑块处分离出的LDL的特性与Ox-LDL相似,包括电泳速度加快、ApoB降解等,而正常动脉壁处LDL无此特性。Ox-LDL刺激内皮细胞分泌粘附分子(E-selectin、ICAM-1、VCAM-1),诱导单核细胞分化为巨噬细胞并分泌特异的针对单核细胞的趋化剂(MCP-1)。进而,巨噬细胞通过清道夫受体聚积OxLDL,转变为泡沫细胞。目前低密度脂蛋白颗粒浓度和脂肪酸组成成分都不能被证实具有直接促动脉粥样硬化作用,并且不同的研究者得出了大量的LDL促动脉粥样硬化和抗动脉粥样硬化的结论(6),因此低密度脂蛋白在动脉粥样硬化发生发展中的具体作用机制尚存在争议。
目前研究常见的动物模型有鼠、家兔、禽类、猪和食肉类(犬,猫)等AS动物模型。因不同AS动物模型对高脂饮食敏感性不一,血脂基线水平不同,发病时间、发病部位及斑块特点与人类存在不同程度的差异性,研究结论各有差异,因此选用合理的动物模型显得尤为重要。狒狒是灵长类动物之一,其基因学,解剖学和病理生理学与人类相近,是研究AS较为理想的实验动物。特别重要的是,狒狒和人的低密度脂蛋白极其相似(18),因为蛋白和脂肪在形成氧化低密度脂蛋白时经历了复杂的氧化过程(6,16,17)。狒狒为我们提供了极为宝贵的机会深入研究低密度脂蛋白在AS形成发展中的机制。
AS主要侵犯大动脉和中等动脉,如主动脉、冠状动脉和脑动脉。血管内皮损伤为AS发生发展中始动环节。有些研究显示,低密度脂蛋白或氧化的低密度脂蛋白能造成内皮细胞凋亡(11,12),也有研究显示这些物质具有促生长作用(13,14)。本研究选用了狒狒原代股动脉内皮细胞,同时改进了低密度脂蛋白的制备,研究低密度脂蛋白单独以及与单核细胞共培养是否损伤大血管内皮细胞及可能机制。
材料和方法
1.1低密度脂蛋白的分离、氧化和特点
血浆库来源于7只狒狒的新鲜血液。超速离心技术分离出血中的低密度脂蛋白(LDL)。加入0.1mM抗氧化剂二丁基羟基甲苯,可获得天然LDL(Na-LDL)。加入新配置的5μM的CuSO4,37℃孵育LDL6小时,制备过氧化低密度脂蛋白(ox-LDL)。加入无CuSO4的PBS液,37℃孵育LDL6小时,制备微氧化低密度脂蛋白(mm-LDL)。
Lowry法根据总蛋白含量衡量分离的LDL的浓度。TBARS蛋白浓度衡量LDL的氧化程度。2%琼脂糖凝胶电泳相对迁移率测定不同氧化程度的LDL相对电泳迁移率(REM)的变化。相对电泳迁移率=氧化的LDL/未氧化的LDL。
1.2狒狒股动脉内皮细胞和单核细胞的分离及培养
狒狒股动脉内皮细胞分离和培养方法同第一部分。
Ficoll (1.077)密度梯度离心法获取外周血单核细胞(PBMNCs),孵育过夜,去上清,贴壁细胞即为单核细胞。抗CD14-FITC和抗CD36-FITC孵育流式细胞仪鉴定单核细胞,DIL-LDL和UEA-1染色来测定单核细胞的功能。
1.3不同氧化程度的LDL对内皮细胞的作用特点
1.3.1内皮细胞生长和凋亡的测定
浓度为50μg/mL和100μg/mL的天然LDL、微氧化LDL和过氧化LDL单独孵育股动脉内皮细胞24h。Lowry法和Annexin V染色法观察内皮细胞的生长和凋亡。
1.3.2内皮细胞表面粘附因子的测定
100μg/mL天然LDL、微氧化LDL和过氧化LDL单独孵育股动脉内皮细胞4h和24h。或天然LDL (50μg/mL和100μg/mL)与单核细胞共孵育内皮细胞4小时。流式细胞技术测定内皮细胞表面粘附因子的表达。
1.4单核细胞的活化
浓度为50和100μg/ml的天然LDL分别刺激单核细胞0,0.5,1,4,8,12,24小时,取细胞上清液。ELISA法测TNF-α和IL-1p的含量。500ng/ml的LPS作为阳性对照。
1.5天然LDL和单核细胞的共培养体系
共培养小室(8.0μm,德国)置于12孔板上,小室上层为单核细胞,培养基为单核细胞培养液。12孔板内为BAECs (0.5-1X106/孔),培养基为0.5m12%FCS-F-12K溶液。小室上层加入50μg/ml或100μg/ml的天然LDL,等量PBS和TNF-α作为空白对照和阳性对照。
天然LDL (50μg/mL)单独或和单核细胞共培养24小时。流式细胞技术测定内皮细胞表面粘附因子的表达
1.6抗体中和试验测定内皮细胞表面粘附因子
单核细胞培养基中加入100μg/mL天然LDL,24小时后收集培养基为条件培养液。并将其分为两组：中和组加入10μg/mL山羊抗人TNF-α和10μg/mL抗人IL-1βIgG,对照组加入等量的非特异性山羊IgG,置于C02培养箱中37℃培养4小时,将两组液体孵育内皮细胞4小时。流式细胞技术测定E-选择素,ICAM-1和VCAM-1的表达。
结果
1.低密度脂蛋白制备特点
天然低密度脂蛋白中TBARS浓度为0-2nmol/mg蛋白,过氧化低密度脂蛋白(ox-LDL) TBARS浓度为160-200nmol/mg蛋白。与微氧化(mm-LDL)和天然低密度脂蛋白(Na-LDL)相比,过氧化低密度脂蛋白有较高的电泳迁移率。微氧化低密度脂蛋白显示出弥散的电泳带。
2.低密度脂蛋白对狒狒内皮细胞生长和凋亡的作用
Lowry法结果显示,低浓度(50μg/mL) Na-LDL对人HAEC和三只狒狒的股动脉内皮细胞生长无影响,高浓度(100μg/mL)孵育24小时可以造成编号为1x8000的狒狒来源的股动脉内皮细胞的明显损伤,对人HAEC和另外两只狒狒的BAEC无影响。浓度为50和100μg/ml的mm-LDL,对BAEC和HAEC24小时无直接损伤。100μg/ml ox-LDL能显著损伤人HAEC细胞生长,且实验组7只狒狒中,5只狒狒的股动脉内皮细胞有明显的损伤。
细胞形态学变化及Annexin V测定也证实,ox-LDL具有明显的内皮细胞损伤作用,Na-LDL和mm-LDL对股动脉内皮细胞无明显的直接损伤作用。
3.LDL对内皮细胞粘附因子表达的影响
与TNF-α相比,不同氧化程度的LDL单独孵育不能有效的刺激BAECs表面粘附分子(CAM)的表达。Ox-LDL可轻度升高部分狒狒的CAM,可能存在基因敏感差异性。
4.天然LDL对单核细胞的活化
Na-LDL增加单核细胞分泌TNF-α和IL-1β,呈剂量和时间依赖性,并与单核细胞结合,明显降低单核细胞CD36的表达水平。
5.LDL和单核细胞共培养对内皮细胞粘附因子表达的影响
天然LDL (50μg/mL,100μg/mL)和单核细胞共培养孵育内皮细胞4小时,可强烈刺激内皮细胞表达E-选择素,细胞间粘附分子-1(VCAM-1)和血管细胞粘附分子-1(ICAM-1)。
5.抗体中和试验测定内皮细胞表面粘附因子
Na-LDL与单核细胞共培养可强烈刺激内皮细胞CAM,而这种协同激活作用能够被人抗TNF-α和IL-1βIgG中和抵消。
结论
1.首次制备了同一来源、不同氧化程度的LDL,各具特点和活性。
2. Na-LDL和mm-LDL对人和狒狒大血管内皮细胞均无直接的损伤作用。研究提示LDL对内皮细胞的损伤取决于其氧化程度,不是激活内皮细胞表达CAM的有效刺激物。
3. Na-LDL能够激活单核细胞分泌炎症因子TNF-α和IL-1β,共培养介导内皮细胞损伤。抗TNF-α和IL-1β抗体可抑制此协同作用。
4.狒狒来源的股动脉内皮细胞对炎症刺激反应存在差异性,其基因差异性发挥重要的作用。
Background
It is well recognized that endothelial cell cytotoxicity induced by inflammatory factors plays a key role in the pathogenesis of cardiovascular disease. TNF-alpha, an endothelial cell-derived cytokine commonly found in atherosclerotic lesions, can promote apoptosis and inflammation[1], which subsequently contribute to endothelial cell injury and cellular dysfunction[3], So attenuating TNF-alpha induced endothelial cell cytotoxicity is important in preventing cardiovascular disease.
Epidemiological studies have shown that Mediteranean diets are associated with reduced risk of cardiovasular diseases [4,5]. Resveratrol (RSV), a polyphenol found in grapes and red wine, is the most important constituent involved in mediteranean dietary. Resveratrol has been shown to have direct cardiovascular protective effect by improving myocardial perfusion, enhancing angiogenesis, reducing oxidant stress or inhibiting platelet aggregation [6-9]. However, the precise mechanism of its action is not completely understood.
In the present study, we examined whether resveratrol could attenuate endothelial cell injury generated by TNF-alpha in baboon femoral arterial endothelial cells (BAECs) and investigated the molecular mechanisms involved in the process.
Methods
1. Cell culture
Baboon Femoral Arterial Endothelial Cells (BAEC) were isolated from baboon femoral arteries. Baboons were immobilized and a2-cm segment of femoral artery in the upper part of the thigh was obtained under sterile surgical procedure. The artery was digested with0.1%collagenase I at37℃for15min. The released cells were seeded immediately on1.0%gelatin-coated culture plates. Primary BAEC were cultured in F-12K growth medium supplemented with20%FCS, and treated with tumor necrosis factor (TNF lOng/mL) with and without resveratrol at various concentrations and for the different time periods indicated in the text.
2. CD31+magnetic beads selection
BAECs were labeled by CD31+immunomagnetic beads for endothelial selection, purification and cell culture.
3. Endothelial function and cytoskeleton determination
Immunocytochemistry method was simplified as fixiation, blocking, primary antibody incubation, washing, secondary antibody incubation, DAPI staining, mounted with slowfade reagents and observed under fluorescence microscopy. Dil-LDL/UEA-1, vWF and VE-cadherin are specific markers of endothelial cells, F-actin and β-tubulin are non-specific cytoskeleton staining of endothelial cells, a-actin is relatively specific staining of the fiberblast cells.
4. Endothelial proliferation assay
MTT assay was used to test proliferation of BAECs with different treatments by resveratrol (RSV,0.1-100μmol/L) and/or TNF-α(10ng/mL). According to the protocol of MTT assay, BAECs were incubated with20μl (1mg/ml) MTT for4hours at37℃to allow MTT to form formazan crystals and disolved by150μl of DMSO. The absorbance of each well was measured by a microplate reader at490nm wavelength (A490).
5. Endothelial cellular adhension moleculars expression
Flowcytometry was used to test the expression of endothelial cellular adhension moleculars (CAMs) under different treatment with resveratrol (RSV,0.1-100μmol/L) and/or TNF-α (10ng/mL).
Following antibodies were used in this study:anti-human CD62E (clone BBIG-E5; R&D Systems, Inc), anti-human CD54(clone HA58; BD Biosciences, San Jose, CA), and anti-human CD106(clone5K267; US Biological, Swampscott, MA).
6. Cytokine expression
ELISA was used to quantification of, test the expression of cytokine production induced by TNF-α(10ng/mL).
Commercial enzyme-linked immunosorbent assay kits from R&D Systems were used for the measurement of MCP-1and Human Cytokine Lincoplex kit (LINCO Research Inc, MO) for the measurement of measurement of IL-1β.
7. Determination of inflammatory injury of endothelial cells
Wound-Healing Assay was used to test endothelial migration with treatments by resveratrol (RSV,0.1-100μmol/L) and/or TNF-α (10ng/mL) incubation. Tube formation was used to test in vitro angiogenesis ability of BAECs with RSV and TNF-α (10ng/mL) incubation.
Results
1. Isolation and Identification of BAECs
Baboon arterial endothelial cells have a typical cobblestone shape (Fig.l A, B), take3-7days to reach confluence according to cell seeding density and passages. We identified BAEC by staining the cells with VE-Cadherin, vWF, CD31+magnetic beads selection and Dil-LDL/UEA uptake and expression of cellular adhension molecules.
2. BAECs can be activated by TNF-α(10ng/ml)
BAEC can be activated by TNF-α and highly express ICAM-1, VCAM-1and E-selectin, by41.38,3-5,12.2folds when compared with normal control。As well as increased MCP-1and IL-1β secretion。 Proliferation and migration of BAEC can be impared by TNF-α(10ng/ml).
3. RSV can attenuate impairment of BAECs induced by TNF-alpha
RSV can improve proliferation and migration ability of BAECs impaired by TNF-alpha, especially at10,50μmol/L. RSV (10,50μmol/L) can inhibit TNF-alpha induced expression of VCAM (CD106) and ICAM (CD54) in BAECs, when BAECs were incubated by RSV and TNF-a together. While RSV pre-treatment and after-treatment has no effects on activated BAECs. This anti-inflammation effect can be attributed to the inhibition of NF-K B singnal pathway activated by TNF-a
Conclusions
1. Primary femeral arterial endothelial cells which can be constantly abtained from baboon with typical cobblestone morphology and was identified by vWF/VE-Cadherin immunocytochemistry staining and CD31+magnetic beads selection.
2. RSV improves BAEC proliferation and attenuates BAECs migration impaired by TNF-alpha.
3. RSV can inhibit TNF alpha induced expression of VCAM (CD106) and ICAM (CD54) in BAECs, which may be attributed to inhibition of NF-K B activated by TNF-alpha in BAECs, while no effects can be seen in pretreatment and aftertreatment incubation.
Background
Since its discovery30years ago, oxidative modification of LDL is considered an important mechanism in atherogenesis. Results from animal models and human subjects suggest that LDL and its derivatives are involved in endothelial dysfunction creating a procoagulatory and proinflammatory local environment, which is a key step in the genesis of both stable and unstable atherosclerosis. However, the mechanisms by which LDL mediates these effects have been debated for decades; oxidized LDL preparations by individual investigators have shown numerous pro-and antiatherogenic properties. While neither LDL particle concentration nor the fatty acid composition of the particles has been proven to promote atherosclerosis directly, a strong relationship between LDL and risk of coronary vascular disease has been well documented.
The endothelium, a monolayer tissue lining on the inner surface of blood vessels, is the first target of circulating LDL. However, assessment of the effect of LDL on endothelial cells has produced conflicting results. For example, while some have reported that LDL or oxidized LDL causes endothelial apoptosis, others have claimed that these substances have growth-promoting effects. Because oxidative LDL preparations have neither been well defined nor thoroughly characterized, the heterogeneous nature of the preparations due to their sources, methods of preparation and treatment, and conditions of storage and use has been attributed to be responsible for the variable pathophysiological effects of LDL on the endothelium. In addition, there are few data demonstrating whether LDL damages the endothelium directly by itself or indirectly in concert with other cell types. To better understand LDL-mediated effects, we investigated both the effects of defined LDL preparations on endothelial cells and the potential involvement of monocytes in mediating the impact of LDL on endothelial cells.
This study was conducted using an Old World nonhuman primate, the baboon. There is increasing awareness that animal models play a critical role in providing relevant information about mechanistic questions. Different animal models have provided insights into mechanisms of atherogenesis, but large animals, especially nonhuman primates, are better suited for translation to humans. This is particularly important in regard to investigations involving LDL because the oxidation of LDL is a complex process during which both the proteins and the lipids undergo oxidative changes and form complex products. Since baboon and human LDL are remarkably similar, the baboon provides us a unique opportunity to address this question. At the Southwest National Primate Research Center, we are able to obtain a large and steady supply of blood and arteries that are from healthy baboons for experimental use and that give consistent and reproducible results. Our results have a direct impact on understanding the relationship of LDL to atherosclerosis in human beings.
Methods
Isolation and oxidation of LDL
Plasma was ultracentrifuged at105,000x g for24h at4℃. LDL in the density range of1.019-1.063g/mL was collected.0.1mM antioxidant butylated hydroxytoluene (BHT) was added to the plasma that was used for obtaining native LDL. The LDL preparation was then sterilized by passing through a0.2-μM filter. Extensively oxidized LDL was prepared by adding freshly prepared CuSO4solution at the final concentration of5μM at37℃for6hours. Minimally oxidized LDL was obtained by incubation of freshly isolated LDL at37℃for6hours without CuSO4.
Endothelial cell and monocyte isolation and culture
Baboons were anesthetized under10mg/kg ketamine hydrochloride, and a2-4cm segment of femoral artery in the upper part of the thigh was obtained under sterile surgical procedure. The artery was gently digested with0.1%collagenase at37℃for20min. The released cells were seeded immediately on1.0%gelatin-coated culture plates. Cells were allowed to reach70-90%confluence until treatments.
Peripheral blood mononuclear cells (PBMNCs) were obtained from buffy coats using a Ficoll gradient and centrifuged for10min at1300rpm to pellet cells. And incubated overnight at37℃in5%CO2. Attached cells were detached by add-ing10mL PBS/0.53mM EDTA and incubated for10min at37℃in a5%CO2incubator. The cells were identified by flowcytometry with anti-CD14-FITC and anti-CD36-FITC incubation. And monocyte functionalities were determined by measuring uptake of Dil-LDL and UEA-1using fluorescence microscopy.
Monocyte activation by LDL
We exposed cells to50and100μg/ml native LDL for0,0.5,1,4,8,12and24hours in replicate wells.500ng/mL LPS was used as a positive control. We measured TNF-a, IL-1β and MCP-1with ELISA kits.
Endothelial activation by LDL and monocyte co-culture
We added native LDL, minimally oxidized LDL, and extensively oxidized LDL at indicated concentrations for24hours. In regard to co-culture, monocytes were seeded into the co-culture insert (8.0μm pore) on top of the endothelial layer at a density of0.5-1.0×106/well in the12-well plates in0.5mL F-12K medium with2%FCS. We added50μg/ml or100μg/ml native LDL in2%FCS medium for24hours; equal volume of PBS and10ng/mL TNF-a were added as vehicle and positive controls. Triplicate wells were evaluated for each treatment. Endothelial activation was determined by examining either cellular adhesion molecule expression or MCP-1release.
Roles of TNF-a and IL-1β in monocyte conditioned media on endothelial activation
We added100μg/mL native LDL to monocyte cultures and collected conditioned media24hours later and divided the conditioned media into two parts; we added10μg/mL goat anti-human TNF-a and10μg/mL anti-human IL-1β IgG to the neutralization groups and an equal amount of nonspecific goat IgG to the control groups. After incubation for4hours at37℃in a humidified CO2incubator, the control and neutralized groups were separately added to endothelial cell cultures and incubated for an additional4hours. Endothelial activation was monitored by expression of E-selectin, ICAM-1and VCAM-1using flow cytometry.
Results
Characteristics of LDL preparations
Native LDL had TBARS content ranging from zero to2nmol/mg protein, and extensively oxidized LDL had TBARS content from160to200nmol/mg protein. Extensively oxidized LDL had a higher electrophoretic mobility than minimally oxidized LDL and native LDL. Minimally oxidized LDL often showed a diffuse band of electrophoresis. Native LDL prepared using the current protocol retained maximally the chemical and physical features of freshly isolated LDL.
Effects of LDL preparations on endothelial cell growth
Native LDL at a lower dosage (50μg/mL) had no effect on cell growth, but a higher dosage of100μg/ml led to cell loss after24hours of incubation.
Minimally oxidized LDL at concentrations of50and100μg/mL had no significant changes in baboon endothelial cells or human arterial endothelial cells (HAEC) after24hours of incubation.
Comprised to native LDL and minimally oxidized LDL. Extensively oxidized LDL caused significant cell loss, which can be confirmed by EC morphology and by Annexin V detection, whereas native or minimally oxidized LDL caused minimal damage.
Cytokine expression
Compared with TNF-a, various LDL preparations were not potent stimuli for endothelial CAM expression, even at24h incubation.
Extensively oxidized LDL tremendously decreased MCP-1expression, while native LDL and minimally oxidized LDL tends to maintain endothelial cells and activate them to secrete chemokine MCP-1.
Native LDL increased the expression of TNF-a and IL-1β in dosage-and time-dependent manners, and significant decreased CD36levels of monocyte.
Interactive effects of LDL and monocytes on endothelial cells
Native LDL greately increased E-Selectin, VCAM and ICAM expression of endothelial cells when co-cultured with monocyte, which can be inhibited by neutralized media with anti-humans TNF-a and10μg/mL anti-human IL-1β IgG.
Conclusions
1. We set up standard method to define three LDL preparations with varying extents of oxidation from the same source for the first time.
2. We demonstrated in this study that LDL-mediated effects were largely dependent on the extent of LDL oxidation.
3. We explored the synergistic effects of monocytes and native LDL in endothelial cell activation under co-culture system, which can be blocked by anti-humans TNF-a and10μg/mL anti-human IL-1β IgG.
4. Our results also suggest that genetic variation controlling endothelial and monocyte response to circulating LDL may have an important role in determining risk of atherosclerosis.
动脉粥样硬化(Atherosclerosis, AS)是一个严重危害人类健康的疾病,其发病机制一直以来成为研究者关注的焦点。目前研究常见的动物模型有鼠、家兔、禽类、猪、食肉类(犬,猫)和非人灵长类等AS动物模型。狒狒是灵长类动物之一,其基因学,解剖学和病理生理学与人类相近,是研究AS较为理想的实验动物。内皮细胞损伤是AS发生发展中的起始和关键环节。本实验选用原代狒狒股动脉内皮细胞(Baboon Femoral Arterial Endothelial Cell, BAEC)进行体外实验,模拟炎症因子对体内大血管早期损伤的机制。
目前许多研究证实炎症因子对内皮细胞毒性损伤在心血管疾病的发病机制中发挥重要的作用。在病理条件下,炎症介质可以损伤内皮的屏障功能,导致通透性增高,表面粘附因子表达增加,单核细胞粘附聚集和泡沫细胞的形成,从而造成血管功能障碍,进而导致大血管动脉粥样硬化。肿瘤坏死因子-α (TNF-α)是一个明确的心血管风险因子和炎症因子,可通过损伤血管内皮细胞,促进单核细胞对内皮细胞的粘附,参与AS发生、发展过程。临床研究发现它富集于动脉粥样硬化的病变处,不存在于正常的组织中,成为其参与动脉粥样硬化炎症反应的直接证据。
白藜芦醇(Resveratrol, RSV)是一种生物性较强的天然多酚类物质,又称芪三酚。天然RSV广泛存在于花生、葡萄和虎杖等植物中。流行病学研究显示高脂饮食的法国人群冠心病发病率较低,归因于红酒中的白藜芦醇成分,具有抗氧化,抗炎,调脂等心血管保护作用和防治动脉粥样硬化的作用,但具体作用机制尚需进一步探索。目前研究显示RSV对人动静脉内皮细胞具有抑制或促进生长的作用,存在明显争议。本研究选择了不同剂量的白藜芦醇,研究其对炎症损伤状态下的原代BAEC的作用特点,进一步探讨RSV保护血管的可能机制。
材料和方法
1.细胞培养
消化狒狒股动脉,获得原代股内皮细胞(BAEC),悬浮于20%FCS-F-12K培养基,接种于铺有明胶的6孔板上。每三天全量换液,细胞长至85-95%可传代或冻存。
2.CD31+免疫磁珠标记内皮细胞
选用CD31+免疫磁珠标记内皮细胞,从而筛选、纯化原代分离的股动脉内皮细胞。
3.内皮细胞功能和细胞骨架测定
免疫荧光染色法简述如下：固定,封闭,一抗孵育,冲洗,二抗孵育,DAPI染色,封片,荧光显微镜下观察。Dil-LDL/UEA-1、vWF和VE-cadherin特异性标记内皮细胞,F-actin和β-tubulin非特异性染色细胞骨架,α-actin为肌纤维细胞的相对特异性染色。
4.内皮细胞胞增殖实验
选用MTT法测定内皮细胞的增殖。观察不同剂量的白藜芦醇(RSV,0.1-100μmol/L)和或TNF-α (lOng/ml)对内皮细胞增殖的影响。
5.流式细胞技术测定内皮细胞表面粘附因子
本实验选用适合于狒狒的抗体为：抗人E-选择素-FITC(R&D Systems公司,#1C0025),抗人VCAM-1-PE (US Biological公司,#400122),抗人ICAM-1-APC (BD Biosciences公司,#555749)。观察TNF-α (10ng/ml)和或不同剂量的白藜芦醇(RSV,0.1-100μmol/L)对内皮细胞表面粘附因子表达的影响。
6.细胞因子测定
ELISA法测定内皮细胞分泌的单核细胞趋化蛋白-1(MCP-1)和白介素-1β(IL-1β)因子。
7.内皮细胞炎症损伤测定
划痕实验法测定内皮细胞迁移愈合能力。管腔形成实验测定内皮细胞血管形成的能力
结果
1.狒狒股动脉内皮细胞形态和功能鉴定
原代狒狒股动脉内皮细胞(baboon femoral arterial endothelial cells, BAECs),早期(Figl. A, day3)呈小多角、球形、呈团状,48-72h生长最快,逐渐生长成梭形,有些细胞排列呈鱼贯状相连,间有旋祸状排列。7-10天胞体呈多角形,相互嵌合,为单层呈铺路石状排列(Figl.B,day7)。培养基为20%FCS-F-12K,每三天全量换液,约7-10天,可传代。狒狒肱动脉纤维细胞呈长梭形,生长迅速,与内皮细胞形态差别显著(Figl. C)。镜下观察收集的内皮细胞纯度通常>85%(Figl. B),有时收集到纯度<50%的内皮细胞(Figl. D),可见混杂的纤维细胞。
VE-Cadherin (Fig2.A)和vWF (Fig2. B)为狒狒股动脉内皮细胞特异性染色。F-肌动蛋白(F-actin, Fig2. C)和β-微管蛋白(β-tubulin, Fig2. D)为BAEC非特异性骨架染色,可见内皮细胞骨架结构完整。
α-肌动蛋白(α-actin, Fig2. E)和p-微管蛋白(β-tubulin,Fig2. F)为纤维细胞骨架染色,其中α-肌动蛋白不能染色内皮细胞。
CD31+免疫磁珠标记内皮细胞(Fig3.A,B),金黄色磁珠附着于内皮细胞表面,随传代逐渐脱落,密度减少,不影响内皮细胞生长和功能。
内皮细胞Dil-LDL/UEA染色双阳性。
2.肿瘤坏死因子-α (TNF-α, lOng/ml)激活内皮细胞
TNF-α激活的内皮细胞高表达ICAM-1, VCAM-1, E-selectin和分泌MCP-1和IL-1β。同时TNF-α能抑制内皮细胞的增殖,延缓内皮细胞划痕愈合速率。
3.白藜芦醇对内皮细胞的保护作用
小剂量(0.1,1μmol/L)白藜芦醇对内皮细胞生长无明显影响。10-100μmol/L白藜芦醇能明显促进狒狒股动脉内皮细胞的增殖,并且能减轻肿瘤坏死因子-α(TNF-α, lOng/ml)对内皮细胞的损伤作用,但不能逆转炎症损伤。
白藜芦醇10μmol/L能明显促进内皮细胞的划痕愈合速率。白藜芦醇(1,10,50μmol/L)能明显减轻肿瘤坏死因子-β (TNF-α,10ng/ml)对内皮细胞划痕愈合速率的损伤,但不能逆转损伤。
白藜芦醇(10,50μmol/L)和TNF-a (lOng/ml)共孵育24小时后,能明显抑制VCAM-1和ICAM-1的表达,可能是通过抑制NF-KB信号通路实现的。
结论
1.狒狒原代股动脉内皮细胞具有结构完整,高纯度和高生物活性特点,且狒狒便于活检取大血管,为深入研究动脉粥样硬化机制提供了理想的动物模型。
2.肿瘤坏死因子-α (TNF-α)损伤狒狒股动脉内皮细胞,促进动脉粥样硬化的发生发展。
3.白藜芦醇(RSV)能抑制TNF-α对内皮细胞的损伤,具有明显的抗炎和抗动脉粥样硬化作用。
背景
近30年来发现,低密度脂蛋白的氧化修饰在动脉粥样硬化(AS)发生机制中起着重要的作用(1,2)。有学者发现人、兔动脉粥样硬化斑块处分离出的LDL的特性与Ox-LDL相似,包括电泳速度加快、ApoB降解等,而正常动脉壁处LDL无此特性。Ox-LDL刺激内皮细胞分泌粘附分子(E-selectin、ICAM-1、VCAM-1),诱导单核细胞分化为巨噬细胞并分泌特异的针对单核细胞的趋化剂(MCP-1)。进而,巨噬细胞通过清道夫受体聚积OxLDL,转变为泡沫细胞。目前低密度脂蛋白颗粒浓度和脂肪酸组成成分都不能被证实具有直接促动脉粥样硬化作用,并且不同的研究者得出了大量的LDL促动脉粥样硬化和抗动脉粥样硬化的结论(6),因此低密度脂蛋白在动脉粥样硬化发生发展中的具体作用机制尚存在争议。
目前研究常见的动物模型有鼠、家兔、禽类、猪和食肉类(犬,猫)等AS动物模型。因不同AS动物模型对高脂饮食敏感性不一,血脂基线水平不同,发病时间、发病部位及斑块特点与人类存在不同程度的差异性,研究结论各有差异,因此选用合理的动物模型显得尤为重要。狒狒是灵长类动物之一,其基因学,解剖学和病理生理学与人类相近,是研究AS较为理想的实验动物。特别重要的是,狒狒和人的低密度脂蛋白极其相似(18),因为蛋白和脂肪在形成氧化低密度脂蛋白时经历了复杂的氧化过程(6,16,17)。狒狒为我们提供了极为宝贵的机会深入研究低密度脂蛋白在AS形成发展中的机制。
AS主要侵犯大动脉和中等动脉,如主动脉、冠状动脉和脑动脉。血管内皮损伤为AS发生发展中始动环节。有些研究显示,低密度脂蛋白或氧化的低密度脂蛋白能造成内皮细胞凋亡(11,12),也有研究显示这些物质具有促生长作用(13,14)。本研究选用了狒狒原代股动脉内皮细胞,同时改进了低密度脂蛋白的制备,研究低密度脂蛋白单独以及与单核细胞共培养是否损伤大血管内皮细胞及可能机制。
材料和方法
1.1低密度脂蛋白的分离、氧化和特点
血浆库来源于7只狒狒的新鲜血液。超速离心技术分离出血中的低密度脂蛋白(LDL)。加入0.1mM抗氧化剂二丁基羟基甲苯,可获得天然LDL(Na-LDL)。加入新配置的5μM的CuSO4,37℃孵育LDL6小时,制备过氧化低密度脂蛋白(ox-LDL)。加入无CuSO4的PBS液,37℃孵育LDL6小时,制备微氧化低密度脂蛋白(mm-LDL)。
Lowry法根据总蛋白含量衡量分离的LDL的浓度。TBARS蛋白浓度衡量LDL的氧化程度。2%琼脂糖凝胶电泳相对迁移率测定不同氧化程度的LDL相对电泳迁移率(REM)的变化。相对电泳迁移率=氧化的LDL/未氧化的LDL。
1.2狒狒股动脉内皮细胞和单核细胞的分离及培养
狒狒股动脉内皮细胞分离和培养方法同第一部分。
Ficoll (1.077)密度梯度离心法获取外周血单核细胞(PBMNCs),孵育过夜,去上清,贴壁细胞即为单核细胞。抗CD14-FITC和抗CD36-FITC孵育流式细胞仪鉴定单核细胞,DIL-LDL和UEA-1染色来测定单核细胞的功能。
1.3不同氧化程度的LDL对内皮细胞的作用特点
1.3.1内皮细胞生长和凋亡的测定
浓度为50μg/mL和100μg/mL的天然LDL、微氧化LDL和过氧化LDL单独孵育股动脉内皮细胞24h。Lowry法和Annexin V染色法观察内皮细胞的生长和凋亡。
1.3.2内皮细胞表面粘附因子的测定
100μg/mL天然LDL、微氧化LDL和过氧化LDL单独孵育股动脉内皮细胞4h和24h。或天然LDL (50μg/mL和100μg/mL)与单核细胞共孵育内皮细胞4小时。流式细胞技术测定内皮细胞表面粘附因子的表达。
1.4单核细胞的活化
浓度为50和100μg/ml的天然LDL分别刺激单核细胞0,0.5,1,4,8,12,24小时,取细胞上清液。ELISA法测TNF-α和IL-1p的含量。500ng/ml的LPS作为阳性对照。
1.5天然LDL和单核细胞的共培养体系
共培养小室(8.0μm,德国)置于12孔板上,小室上层为单核细胞,培养基为单核细胞培养液。12孔板内为BAECs (0.5-1X106/孔),培养基为0.5m12%FCS-F-12K溶液。小室上层加入50μg/ml或100μg/ml的天然LDL,等量PBS和TNF-α作为空白对照和阳性对照。
天然LDL (50μg/mL)单独或和单核细胞共培养24小时。流式细胞技术测定内皮细胞表面粘附因子的表达
1.6抗体中和试验测定内皮细胞表面粘附因子
单核细胞培养基中加入100μg/mL天然LDL,24小时后收集培养基为条件培养液。并将其分为两组：中和组加入10μg/mL山羊抗人TNF-α和10μg/mL抗人IL-1βIgG,对照组加入等量的非特异性山羊IgG,置于C02培养箱中37℃培养4小时,将两组液体孵育内皮细胞4小时。流式细胞技术测定E-选择素,ICAM-1和VCAM-1的表达。
结果
1.低密度脂蛋白制备特点
天然低密度脂蛋白中TBARS浓度为0-2nmol/mg蛋白,过氧化低密度脂蛋白(ox-LDL) TBARS浓度为160-200nmol/mg蛋白。与微氧化(mm-LDL)和天然低密度脂蛋白(Na-LDL)相比,过氧化低密度脂蛋白有较高的电泳迁移率。微氧化低密度脂蛋白显示出弥散的电泳带。
2.低密度脂蛋白对狒狒内皮细胞生长和凋亡的作用
Lowry法结果显示,低浓度(50μg/mL) Na-LDL对人HAEC和三只狒狒的股动脉内皮细胞生长无影响,高浓度(100μg/mL)孵育24小时可以造成编号为1x8000的狒狒来源的股动脉内皮细胞的明显损伤,对人HAEC和另外两只狒狒的BAEC无影响。浓度为50和100μg/ml的mm-LDL,对BAEC和HAEC24小时无直接损伤。100μg/ml ox-LDL能显著损伤人HAEC细胞生长,且实验组7只狒狒中,5只狒狒的股动脉内皮细胞有明显的损伤。
细胞形态学变化及Annexin V测定也证实,ox-LDL具有明显的内皮细胞损伤作用,Na-LDL和mm-LDL对股动脉内皮细胞无明显的直接损伤作用。
3.LDL对内皮细胞粘附因子表达的影响
与TNF-α相比,不同氧化程度的LDL单独孵育不能有效的刺激BAECs表面粘附分子(CAM)的表达。Ox-LDL可轻度升高部分狒狒的CAM,可能存在基因敏感差异性。
4.天然LDL对单核细胞的活化
Na-LDL增加单核细胞分泌TNF-α和IL-1β,呈剂量和时间依赖性,并与单核细胞结合,明显降低单核细胞CD36的表达水平。
5.LDL和单核细胞共培养对内皮细胞粘附因子表达的影响
天然LDL (50μg/mL,100μg/mL)和单核细胞共培养孵育内皮细胞4小时,可强烈刺激内皮细胞表达E-选择素,细胞间粘附分子-1(VCAM-1)和血管细胞粘附分子-1(ICAM-1)。
5.抗体中和试验测定内皮细胞表面粘附因子
Na-LDL与单核细胞共培养可强烈刺激内皮细胞CAM,而这种协同激活作用能够被人抗TNF-α和IL-1βIgG中和抵消。
结论
1.首次制备了同一来源、不同氧化程度的LDL,各具特点和活性。
2. Na-LDL和mm-LDL对人和狒狒大血管内皮细胞均无直接的损伤作用。研究提示LDL对内皮细胞的损伤取决于其氧化程度,不是激活内皮细胞表达CAM的有效刺激物。
3. Na-LDL能够激活单核细胞分泌炎症因子TNF-α和IL-1β,共培养介导内皮细胞损伤。抗TNF-α和IL-1β抗体可抑制此协同作用。
4.狒狒来源的股动脉内皮细胞对炎症刺激反应存在差异性,其基因差异性发挥重要的作用。
Background
It is well recognized that endothelial cell cytotoxicity induced by inflammatory factors plays a key role in the pathogenesis of cardiovascular disease. TNF-alpha, an endothelial cell-derived cytokine commonly found in atherosclerotic lesions, can promote apoptosis and inflammation[1], which subsequently contribute to endothelial cell injury and cellular dysfunction[3], So attenuating TNF-alpha induced endothelial cell cytotoxicity is important in preventing cardiovascular disease.
Epidemiological studies have shown that Mediteranean diets are associated with reduced risk of cardiovasular diseases [4,5]. Resveratrol (RSV), a polyphenol found in grapes and red wine, is the most important constituent involved in mediteranean dietary. Resveratrol has been shown to have direct cardiovascular protective effect by improving myocardial perfusion, enhancing angiogenesis, reducing oxidant stress or inhibiting platelet aggregation [6-9]. However, the precise mechanism of its action is not completely understood.
In the present study, we examined whether resveratrol could attenuate endothelial cell injury generated by TNF-alpha in baboon femoral arterial endothelial cells (BAECs) and investigated the molecular mechanisms involved in the process.
Methods
1. Cell culture
Baboon Femoral Arterial Endothelial Cells (BAEC) were isolated from baboon femoral arteries. Baboons were immobilized and a2-cm segment of femoral artery in the upper part of the thigh was obtained under sterile surgical procedure. The artery was digested with0.1%collagenase I at37℃for15min. The released cells were seeded immediately on1.0%gelatin-coated culture plates. Primary BAEC were cultured in F-12K growth medium supplemented with20%FCS, and treated with tumor necrosis factor (TNF lOng/mL) with and without resveratrol at various concentrations and for the different time periods indicated in the text.
2. CD31+magnetic beads selection
BAECs were labeled by CD31+immunomagnetic beads for endothelial selection, purification and cell culture.
3. Endothelial function and cytoskeleton determination
Immunocytochemistry method was simplified as fixiation, blocking, primary antibody incubation, washing, secondary antibody incubation, DAPI staining, mounted with slowfade reagents and observed under fluorescence microscopy. Dil-LDL/UEA-1, vWF and VE-cadherin are specific markers of endothelial cells, F-actin and β-tubulin are non-specific cytoskeleton staining of endothelial cells, a-actin is relatively specific staining of the fiberblast cells.
4. Endothelial proliferation assay
MTT assay was used to test proliferation of BAECs with different treatments by resveratrol (RSV,0.1-100μmol/L) and/or TNF-α(10ng/mL). According to the protocol of MTT assay, BAECs were incubated with20μl (1mg/ml) MTT for4hours at37℃to allow MTT to form formazan crystals and disolved by150μl of DMSO. The absorbance of each well was measured by a microplate reader at490nm wavelength (A490).
5. Endothelial cellular adhension moleculars expression
Flowcytometry was used to test the expression of endothelial cellular adhension moleculars (CAMs) under different treatment with resveratrol (RSV,0.1-100μmol/L) and/or TNF-α (10ng/mL).
Following antibodies were used in this study:anti-human CD62E (clone BBIG-E5; R&D Systems, Inc), anti-human CD54(clone HA58; BD Biosciences, San Jose, CA), and anti-human CD106(clone5K267; US Biological, Swampscott, MA).
6. Cytokine expression
ELISA was used to quantification of, test the expression of cytokine production induced by TNF-α(10ng/mL).
Commercial enzyme-linked immunosorbent assay kits from R&D Systems were used for the measurement of MCP-1and Human Cytokine Lincoplex kit (LINCO Research Inc, MO) for the measurement of measurement of IL-1β.
7. Determination of inflammatory injury of endothelial cells
Wound-Healing Assay was used to test endothelial migration with treatments by resveratrol (RSV,0.1-100μmol/L) and/or TNF-α (10ng/mL) incubation. Tube formation was used to test in vitro angiogenesis ability of BAECs with RSV and TNF-α (10ng/mL) incubation.
Results
1. Isolation and Identification of BAECs
Baboon arterial endothelial cells have a typical cobblestone shape (Fig.l A, B), take3-7days to reach confluence according to cell seeding density and passages. We identified BAEC by staining the cells with VE-Cadherin, vWF, CD31+magnetic beads selection and Dil-LDL/UEA uptake and expression of cellular adhension molecules.
2. BAECs can be activated by TNF-α(10ng/ml)
BAEC can be activated by TNF-α and highly express ICAM-1, VCAM-1and E-selectin, by41.38,3-5,12.2folds when compared with normal control。As well as increased MCP-1and IL-1β secretion。 Proliferation and migration of BAEC can be impared by TNF-α(10ng/ml).
3. RSV can attenuate impairment of BAECs induced by TNF-alpha
RSV can improve proliferation and migration ability of BAECs impaired by TNF-alpha, especially at10,50μmol/L. RSV (10,50μmol/L) can inhibit TNF-alpha induced expression of VCAM (CD106) and ICAM (CD54) in BAECs, when BAECs were incubated by RSV and TNF-a together. While RSV pre-treatment and after-treatment has no effects on activated BAECs. This anti-inflammation effect can be attributed to the inhibition of NF-K B singnal pathway activated by TNF-a
Conclusions
1. Primary femeral arterial endothelial cells which can be constantly abtained from baboon with typical cobblestone morphology and was identified by vWF/VE-Cadherin immunocytochemistry staining and CD31+magnetic beads selection.
2. RSV improves BAEC proliferation and attenuates BAECs migration impaired by TNF-alpha.
3. RSV can inhibit TNF alpha induced expression of VCAM (CD106) and ICAM (CD54) in BAECs, which may be attributed to inhibition of NF-K B activated by TNF-alpha in BAECs, while no effects can be seen in pretreatment and aftertreatment incubation.
Background
Since its discovery30years ago, oxidative modification of LDL is considered an important mechanism in atherogenesis. Results from animal models and human subjects suggest that LDL and its derivatives are involved in endothelial dysfunction creating a procoagulatory and proinflammatory local environment, which is a key step in the genesis of both stable and unstable atherosclerosis. However, the mechanisms by which LDL mediates these effects have been debated for decades; oxidized LDL preparations by individual investigators have shown numerous pro-and antiatherogenic properties. While neither LDL particle concentration nor the fatty acid composition of the particles has been proven to promote atherosclerosis directly, a strong relationship between LDL and risk of coronary vascular disease has been well documented.
The endothelium, a monolayer tissue lining on the inner surface of blood vessels, is the first target of circulating LDL. However, assessment of the effect of LDL on endothelial cells has produced conflicting results. For example, while some have reported that LDL or oxidized LDL causes endothelial apoptosis, others have claimed that these substances have growth-promoting effects. Because oxidative LDL preparations have neither been well defined nor thoroughly characterized, the heterogeneous nature of the preparations due to their sources, methods of preparation and treatment, and conditions of storage and use has been attributed to be responsible for the variable pathophysiological effects of LDL on the endothelium. In addition, there are few data demonstrating whether LDL damages the endothelium directly by itself or indirectly in concert with other cell types. To better understand LDL-mediated effects, we investigated both the effects of defined LDL preparations on endothelial cells and the potential involvement of monocytes in mediating the impact of LDL on endothelial cells.
This study was conducted using an Old World nonhuman primate, the baboon. There is increasing awareness that animal models play a critical role in providing relevant information about mechanistic questions. Different animal models have provided insights into mechanisms of atherogenesis, but large animals, especially nonhuman primates, are better suited for translation to humans. This is particularly important in regard to investigations involving LDL because the oxidation of LDL is a complex process during which both the proteins and the lipids undergo oxidative changes and form complex products. Since baboon and human LDL are remarkably similar, the baboon provides us a unique opportunity to address this question. At the Southwest National Primate Research Center, we are able to obtain a large and steady supply of blood and arteries that are from healthy baboons for experimental use and that give consistent and reproducible results. Our results have a direct impact on understanding the relationship of LDL to atherosclerosis in human beings.
Methods
Isolation and oxidation of LDL
Plasma was ultracentrifuged at105,000x g for24h at4℃. LDL in the density range of1.019-1.063g/mL was collected.0.1mM antioxidant butylated hydroxytoluene (BHT) was added to the plasma that was used for obtaining native LDL. The LDL preparation was then sterilized by passing through a0.2-μM filter. Extensively oxidized LDL was prepared by adding freshly prepared CuSO4solution at the final concentration of5μM at37℃for6hours. Minimally oxidized LDL was obtained by incubation of freshly isolated LDL at37℃for6hours without CuSO4.
Endothelial cell and monocyte isolation and culture
Baboons were anesthetized under10mg/kg ketamine hydrochloride, and a2-4cm segment of femoral artery in the upper part of the thigh was obtained under sterile surgical procedure. The artery was gently digested with0.1%collagenase at37℃for20min. The released cells were seeded immediately on1.0%gelatin-coated culture plates. Cells were allowed to reach70-90%confluence until treatments.
Peripheral blood mononuclear cells (PBMNCs) were obtained from buffy coats using a Ficoll gradient and centrifuged for10min at1300rpm to pellet cells. And incubated overnight at37℃in5%CO2. Attached cells were detached by add-ing10mL PBS/0.53mM EDTA and incubated for10min at37℃in a5%CO2incubator. The cells were identified by flowcytometry with anti-CD14-FITC and anti-CD36-FITC incubation. And monocyte functionalities were determined by measuring uptake of Dil-LDL and UEA-1using fluorescence microscopy.
Monocyte activation by LDL
We exposed cells to50and100μg/ml native LDL for0,0.5,1,4,8,12and24hours in replicate wells.500ng/mL LPS was used as a positive control. We measured TNF-a, IL-1β and MCP-1with ELISA kits.
Endothelial activation by LDL and monocyte co-culture
We added native LDL, minimally oxidized LDL, and extensively oxidized LDL at indicated concentrations for24hours. In regard to co-culture, monocytes were seeded into the co-culture insert (8.0μm pore) on top of the endothelial layer at a density of0.5-1.0×106/well in the12-well plates in0.5mL F-12K medium with2%FCS. We added50μg/ml or100μg/ml native LDL in2%FCS medium for24hours; equal volume of PBS and10ng/mL TNF-a were added as vehicle and positive controls. Triplicate wells were evaluated for each treatment. Endothelial activation was determined by examining either cellular adhesion molecule expression or MCP-1release.
Roles of TNF-a and IL-1β in monocyte conditioned media on endothelial activation
We added100μg/mL native LDL to monocyte cultures and collected conditioned media24hours later and divided the conditioned media into two parts; we added10μg/mL goat anti-human TNF-a and10μg/mL anti-human IL-1β IgG to the neutralization groups and an equal amount of nonspecific goat IgG to the control groups. After incubation for4hours at37℃in a humidified CO2incubator, the control and neutralized groups were separately added to endothelial cell cultures and incubated for an additional4hours. Endothelial activation was monitored by expression of E-selectin, ICAM-1and VCAM-1using flow cytometry.
Results
Characteristics of LDL preparations
Native LDL had TBARS content ranging from zero to2nmol/mg protein, and extensively oxidized LDL had TBARS content from160to200nmol/mg protein. Extensively oxidized LDL had a higher electrophoretic mobility than minimally oxidized LDL and native LDL. Minimally oxidized LDL often showed a diffuse band of electrophoresis. Native LDL prepared using the current protocol retained maximally the chemical and physical features of freshly isolated LDL.
Effects of LDL preparations on endothelial cell growth
Native LDL at a lower dosage (50μg/mL) had no effect on cell growth, but a higher dosage of100μg/ml led to cell loss after24hours of incubation.
Minimally oxidized LDL at concentrations of50and100μg/mL had no significant changes in baboon endothelial cells or human arterial endothelial cells (HAEC) after24hours of incubation.
Comprised to native LDL and minimally oxidized LDL. Extensively oxidized LDL caused significant cell loss, which can be confirmed by EC morphology and by Annexin V detection, whereas native or minimally oxidized LDL caused minimal damage.
Cytokine expression
Compared with TNF-a, various LDL preparations were not potent stimuli for endothelial CAM expression, even at24h incubation.
Extensively oxidized LDL tremendously decreased MCP-1expression, while native LDL and minimally oxidized LDL tends to maintain endothelial cells and activate them to secrete chemokine MCP-1.
Native LDL increased the expression of TNF-a and IL-1β in dosage-and time-dependent manners, and significant decreased CD36levels of monocyte.
Interactive effects of LDL and monocytes on endothelial cells
Native LDL greately increased E-Selectin, VCAM and ICAM expression of endothelial cells when co-cultured with monocyte, which can be inhibited by neutralized media with anti-humans TNF-a and10μg/mL anti-human IL-1β IgG.
Conclusions
1. We set up standard method to define three LDL preparations with varying extents of oxidation from the same source for the first time.
2. We demonstrated in this study that LDL-mediated effects were largely dependent on the extent of LDL oxidation.
3. We explored the synergistic effects of monocytes and native LDL in endothelial cell activation under co-culture system, which can be blocked by anti-humans TNF-a and10μg/mL anti-human IL-1β IgG.
4. Our results also suggest that genetic variation controlling endothelial and monocyte response to circulating LDL may have an important role in determining risk of atherosclerosis.
引文
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[29]Dimayuga PC, Chyu KY, Cercek B. Immune responses regulating the response to vascular injury. Curr Opin Lipidol 2010; 21:416-421.
[30]Mestas J, Ley K. Monocyte-endothelial cell interactions in the development of atherosclerosis. Trends Cardiovasc Med 2008; 18:228-232.
[31]Elkind MS. Impact of innate inflammation in population studies. Ann N Y Acad Sci 2010; 1207:97-106.
[32]Shi Q, Cox LA, Glenn J, Tejero ME, Hondara V, Vandeberg JL, Wang XL. Molecular pathways mediating differential responses to lipopolysaccharide between human and baboon arterial endothelial cells. Clin Exp Pharmacol Physiol 2010; 37:178-84.
[1]Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. CircRes1995;77:510-518.
[2]Steinberg D, Witztum JL. Oxidized low-density lipoprotein and atherosclerosis. Arterioscler Thromb Vase Biol 2010; 30:2311-2316.
[3]Libby P. Molecular bases of the acute coronary syndromes. Circulation 1995; 91: 2844-2850.
[4]Hansson GK. Inflammatory mechanisms in atherosclerosis. J Thromb Haemost 2009; Suppl 1:328-331.
[5]Levitan I, Volkov S, Subbaiah PV. Oxidized LDL:diversity, patterns of recognition, and pathophysiology. Antioxid Redox Signal 2010; 13:39-75.
[6]Parthasarathy S, Raghavamenon A, Garelnabi MO, Santanam N. Oxidized low-density lipoprotein. Methods Mol Biol 2010; 610:403-417.
[7]Lada AT, Rudel LL. Associations of low density lipoprotein particle composition with atherogenicity. Curr Opin Lipidol 2004; 15:19-24.
[8]Krauss RM. Lipoprotein subfractions and cardiovascular disease risk. Curr Opin Lipidol 2010; 21:305-311.
[9]Hazen SL, Chisolm GM. Oxidized phosphatidylcholines:pattern recognition ligands for multiple pathways of the innate immune response. Proc Natl Acad Sci USA 2002; 99:12515-12517.
[10]Vanhoutte PM. Endothelial dysfunction:the first step toward coronary arteriosclerosis. Circ J 2009; 73:595-601.
[11]Napoli C. Oxidation of LDL, atherogenesis, and apoptosis. Ann N Y Acad Sci 2003; 1010:698-709.
[12]Takahashi M, Okazaki H, Ogata Y, Takeuchi K, Ikeda U, Shimada K. Lysophosphatidylcholine induces apoptosis in human endothelial cells through a p38-mitogen-activated protein kinase-dependent mechanism. Atherosclerosis 2002; 161:387-394.
[13]Zettler ME, Prociuk MA, Austria JA, Massaeli H, Zhong G, Pierce GN. OxLDL stimulates cell proliferation through a general induction of cell cycle proteins. Am J Physiol Heart Circ Physiol 2003; 284:H644-53.
[14]Galle J, Hansen-Hagge T, Wanner C, Seibold S. Impact of oxidized low density lipoprotein on vascular cells. Atherosclerosis 2006; 185:219-226.
[15]Vilahur G, Padro T, Badimon L. Atherosclerosis and thrombosis:insights from large animal models. J Biomed Biotechnol 2011; 2011:907575.
[16]Barter PJ, Gooden JM, Rajaram OV. Species differences in the activity of a serum triglyceride transferring factor. Atherosclerosis 1979; 33:165-169.
[17]Groener JE, Bax W, Stuani C, Pagani F. Difference in substrate specificity between human and mouse lysosomal acid lipase:low affinity for cholesteryl ester in mouse lysosomal acid lipase. Biochim Biophys Acta 2000; 1487:155-162.
[18]Chapman MJ, Goldstein S. Comparison of the serum low density lipoprotein and of its apoprotein in the pig, rhesus monkey and baboon with that in man. Atherosclerosis 1976; 25:267-291.
[19]Shi Q, Vandeberg JF, Jett C, Rice K, Leland MM, Talley L, Kushwaha RS, Rainwater DL, Vandeberg JL, Wang XL. Arterial endothelial dysfunction in baboons fed a high-cholesterol, high-fat diet. Am J Clin Nutr 2005; 82:751-759.
[20]Patel RP, Darley-Usmar VM. Molecular mechanisms of the copper dependent oxidation of low-density lipoprotein. Free Radic Res 1999; 30:1-9.
[21]Shi Q, Wang J, Wang XL, VandeBerg JL. Com-parative analysis of vascular endothelial cell activation by TNF-α and LPS in humans and baboons. Cell Biochem Biophys 2004; 40:289-303.
[22]Rainwater DL, Shi Q, Mahaney MC, Hodara V, Vandeberg JL, Wang XL. Genetic regulation of endothelial inflammatory response in baboons. Arterioscler Thromb Vasc Biol 2010; 30:1628-1633.
[23]Amar S, Oyaisu K, Li L, Van Dyke T. Moesin:a potential LPS receptor on human monocytes. J Endotoxin Res 2001; 7:281-286.
[24]Rader DJ, Daugherty A. Translating molecular discoveries into new therapies for atherosclerosis. Nature 2008; 451:904-913.
[25]Adams MR, Kinlay S, Blake GJ, Orford JL, Ganz P, Selwyn AP. Atherogenic lipids and endothelial dysfunction:mechanisms in the genesis of ischemic syndromes. Annu Rev Med 2000; 51:149-167.
[26]Batt KV, Avella M, Moore EH, Jackson B, Suck-ling KE, Botham KM. Differential effects of low-density lipoprotein and chylomicron remnants on lipid accumulation in human macrophages. Exp Biol Med (Maywood) 2004; 229:528-537.
[27]Henriksen T, Evensen SA, Carlander B. Injury to human endothelial cells in culture induced by low density lipoproteins. Scand J Clin Lab Invest 1979; 39: 361-368.
[28]Coffey MD, Cole RA, Colles SM, Chisolm GM. In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals. J Clin Invest 1995; 96:1866-1873.
[29]Dimayuga PC, Chyu KY, Cercek B. Immune responses regulating the response to vascular injury. Curr Opin Lipidol 2010; 21:416-421.
[30]Mestas J, Ley K. Monocyte-endothelial cell interactions in the development of atherosclerosis. Trends Cardiovasc Med 2008; 18:228-232.
[31]Elkind MS. Impact of innate inflammation in population studies. Ann N Y Acad Sci 2010; 1207:97-106.
[32]Shi Q, Cox LA, Glenn J, Tejero ME, Hondara V, Vandeberg JL, Wang XL. Molecular pathways mediating differential responses to lipopolysaccharide between human and baboon arterial endothelial cells. Clin Exp Pharmacol Physiol 2010; 37:178-84.
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[I]Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. CircRes1995;77:510-518.
[2]Steinberg D, Witztum JL. Oxidized low-density lipoprotein and atherosclerosis. Arterioscler Thromb Vasc Biol 2010; 30:2311-2316.
[3]Libby P. Molecular bases of the acute coronary syndromes. Circulation 1995; 91: 2844-2850.
[4]Hansson GK. Inflammatory mechanisms in atherosclerosis. J Thromb Haemost 2009; Suppl 1:328-331.
[5]Levitan I, Volkov S, Subbaiah PV. Oxidized LDL:diversity, patterns of recognition, and pathophysiology. Antioxid Redox Signal 2010; 13:39-75.
[6]Parthasarathy S, Raghavamenon A, Garelnabi MO, Santanam N. Oxidized low-density lipoprotein. Methods Mol Biol 2010; 610:403-417.
[7]Lada AT, Rudel LL. Associations of low density lipoprotein particle composition with atherogenicity. Curr Opin Lipidol 2004; 15:19-24.
[8]Krauss RM. Lipoprotein subtractions and cardiovascular disease risk. Curr Opin Lipidol 2010; 21:305-311.
[9]Hazen SL, Chisolm GM. Oxidized phosphatidylcholines:pattern recognition ligands for multiple pathways of the innate immune response. Proc Natl Acad Sci USA 2002; 99:12515-12517.
[10]Vanhoutte PM. Endothelial dysfunction:the first step toward coronary arteriosclerosis. Circ J 2009; 73:595-601.
[11]Napoli C. Oxidation of LDL, atherogenesis, and apoptosis. Ann N Y Acad Sci 2003; 1010:698-709.
[12]Takahashi M, Okazaki H, Ogata Y, Takeuchi K, Ikeda U, Shimada K. Lysophosphatidylcholine induces apoptosis in human endothelial cells through a p38-mitogen-activated protein kinase-dependent mechanism. Atherosclerosis 2002; 161:387-394.
[13]Zettler ME, Prociuk MA, Austria JA, Massaeli H, Zhong G, Pierce GN. OxLDL stimulates cell proliferation through a general induction of cell cycle proteins. Am J Physiol Heart Circ Physiol 2003; 284:H644-53.
[14]Galle J, Hansen-Hagge T, Wanner C, Seibold S. Impact of oxidized low density lipoprotein on vascular cells. Atherosclerosis 2006; 185:219-226.
[15]Vilahur G, Padro T, Badimon L. Atherosclerosis and thrombosis:insights from large animal models. J Biomed Biotechnol 2011; 2011:907575.
[16]Barter PJ, Gooden JM, Rajaram OV. Species differences in the activity of a serum triglyceride transferring factor. Atherosclerosis 1979; 33:165-169.
[17]Groener JE, Bax W, Stuani C, Pagani F. Difference in substrate specificity between human and mouse lysosomal acid lipase:low affinity for cholesteryl ester in mouse lysosomal acid lipase. Biochim Biophys Acta 2000; 1487:155-162.
[18]Chapman MJ, Goldstein S. Comparison of the serum low density lipoprotein and of its apoprotein in the pig, rhesus monkey and baboon with that in man. Atherosclerosis 1976; 25:267-291.
[19]Shi Q, Vandeberg JF, Jett C, Rice K, Leland MM, Talley L, Kushwaha RS, Rainwater DL, Vandeberg JL, Wang XL. Arterial endothelial dysfunction in baboons fed a high-cholesterol, high-fat diet. Am J Clin Nutr 2005; 82:751-759.
[20]Patel RP, Darley-Usmar VM. Molecular mechanisms of the copper dependent oxidation of low-density lipoprotein. Free Radic Res 1999; 30:1-9.
[21]Shi Q, Wang J, Wang XL, VandeBerg JL. Com-parative analysis of vascular endothelial cell activation by TNF-a and LPS in humans and baboons. Cell Biochem Biophys 2004; 40:289-303.
[22]Rainwater DL, Shi Q, Mahaney MC, Hodara V, Vandeberg JL, Wang XL. Genetic regulation of endothelial inflammatory response in baboons. Arterioscler Thromb Vasc Biol 2010; 30:1628-1633.
[23]Amar S, Oyaisu K, Li L, Van Dyke T. Moesin:a potential LPS receptor on human monocytes. J Endotoxin Res 2001; 7:281-286.
[24]Rader DJ, Daugherty A. Translating molecular discoveries into new therapies for atherosclerosis. Nature 2008; 451:904-913.
[25]Adams MR, Kinlay S, Blake GJ, Orford JL, Ganz P, Selwyn AP. Atherogenic lipids and endothelial dysfunction:mechanisms in the genesis of ischemic syndromes. Annu Rev Med 2000; 51:149-167.
[26]Batt KV, Avella M, Moore EH, Jackson B, Suck-ling KE, Botham KM. Differential effects of low-density lipoprotein and chylomicron remnants on lipid accumulation in human macrophages. Exp Biol Med (Maywood) 2004; 229:528-537.
[27]Henriksen T, Evensen SA, Carlander B. Injury to human endothelial cells in culture induced by low density lipoproteins. Scand J Clin Lab Invest 1979; 39: 361-368.
[28]Coffey MD, Cole RA, Colles SM, Chisolm GM. In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals. J Clin Invest 1995; 96:1866-1873.
[29]Dimayuga PC, Chyu KY, Cercek B. Immune responses regulating the response to vascular injury. Curr Opin Lipidol 2010; 21:416-421.
[30]Mestas J, Ley K. Monocyte-endothelial cell interactions in the development of atherosclerosis. Trends Cardiovasc Med 2008; 18:228-232.
[31]Elkind MS. Impact of innate inflammation in population studies. Ann N Y Acad Sci 2010; 1207:97-106.
[32]Shi Q, Cox LA, Glenn J, Tejero ME, Hondara V, Vandeberg JL, Wang XL. Molecular pathways mediating differential responses to lipopolysaccharide between human and baboon arterial endothelial cells. Clin Exp Pharmacol Physiol 2010; 37:178-84.
[1]Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. CircRes1995;77:510-518.
[2]Steinberg D, Witztum JL. Oxidized low-density lipoprotein and atherosclerosis. Arterioscler Thromb Vase Biol 2010; 30:2311-2316.
[3]Libby P. Molecular bases of the acute coronary syndromes. Circulation 1995; 91: 2844-2850.
[4]Hansson GK. Inflammatory mechanisms in atherosclerosis. J Thromb Haemost 2009; Suppl 1:328-331.
[5]Levitan I, Volkov S, Subbaiah PV. Oxidized LDL:diversity, patterns of recognition, and pathophysiology. Antioxid Redox Signal 2010; 13:39-75.
[6]Parthasarathy S, Raghavamenon A, Garelnabi MO, Santanam N. Oxidized low-density lipoprotein. Methods Mol Biol 2010; 610:403-417.
[7]Lada AT, Rudel LL. Associations of low density lipoprotein particle composition with atherogenicity. Curr Opin Lipidol 2004; 15:19-24.
[8]Krauss RM. Lipoprotein subfractions and cardiovascular disease risk. Curr Opin Lipidol 2010; 21:305-311.
[9]Hazen SL, Chisolm GM. Oxidized phosphatidylcholines:pattern recognition ligands for multiple pathways of the innate immune response. Proc Natl Acad Sci USA 2002; 99:12515-12517.
[10]Vanhoutte PM. Endothelial dysfunction:the first step toward coronary arteriosclerosis. Circ J 2009; 73:595-601.
[11]Napoli C. Oxidation of LDL, atherogenesis, and apoptosis. Ann N Y Acad Sci 2003; 1010:698-709.
[12]Takahashi M, Okazaki H, Ogata Y, Takeuchi K, Ikeda U, Shimada K. Lysophosphatidylcholine induces apoptosis in human endothelial cells through a p38-mitogen-activated protein kinase-dependent mechanism. Atherosclerosis 2002; 161:387-394.
[13]Zettler ME, Prociuk MA, Austria JA, Massaeli H, Zhong G, Pierce GN. OxLDL stimulates cell proliferation through a general induction of cell cycle proteins. Am J Physiol Heart Circ Physiol 2003; 284:H644-53.
[14]Galle J, Hansen-Hagge T, Wanner C, Seibold S. Impact of oxidized low density lipoprotein on vascular cells. Atherosclerosis 2006; 185:219-226.
[15]Vilahur G, Padro T, Badimon L. Atherosclerosis and thrombosis:insights from large animal models. J Biomed Biotechnol 2011; 2011:907575.
[16]Barter PJ, Gooden JM, Rajaram OV. Species differences in the activity of a serum triglyceride transferring factor. Atherosclerosis 1979; 33:165-169.
[17]Groener JE, Bax W, Stuani C, Pagani F. Difference in substrate specificity between human and mouse lysosomal acid lipase:low affinity for cholesteryl ester in mouse lysosomal acid lipase. Biochim Biophys Acta 2000; 1487:155-162.
[18]Chapman MJ, Goldstein S. Comparison of the serum low density lipoprotein and of its apoprotein in the pig, rhesus monkey and baboon with that in man. Atherosclerosis 1976; 25:267-291.
[19]Shi Q, Vandeberg JF, Jett C, Rice K, Leland MM, Talley L, Kushwaha RS, Rainwater DL, Vandeberg JL, Wang XL. Arterial endothelial dysfunction in baboons fed a high-cholesterol, high-fat diet. Am J Clin Nutr 2005; 82:751-759.
[20]Patel RP, Darley-Usmar VM. Molecular mechanisms of the copper dependent oxidation of low-density lipoprotein. Free Radic Res 1999; 30:1-9.
[21]Shi Q, Wang J, Wang XL, VandeBerg JL. Com-parative analysis of vascular endothelial cell activation by TNF-α and LPS in humans and baboons. Cell Biochem Biophys 2004; 40:289-303.
[22]Rainwater DL, Shi Q, Mahaney MC, Hodara V, Vandeberg JL, Wang XL. Genetic regulation of endothelial inflammatory response in baboons. Arterioscler Thromb Vasc Biol 2010; 30:1628-1633.
[23]Amar S, Oyaisu K, Li L, Van Dyke T. Moesin:a potential LPS receptor on human monocytes. J Endotoxin Res 2001; 7:281-286.
[24]Rader DJ, Daugherty A. Translating molecular discoveries into new therapies for atherosclerosis. Nature 2008; 451:904-913.
[25]Adams MR, Kinlay S, Blake GJ, Orford JL, Ganz P, Selwyn AP. Atherogenic lipids and endothelial dysfunction:mechanisms in the genesis of ischemic syndromes. Annu Rev Med 2000; 51:149-167.
[26]Batt KV, Avella M, Moore EH, Jackson B, Suck-ling KE, Botham KM. Differential effects of low-density lipoprotein and chylomicron remnants on lipid accumulation in human macrophages. Exp Biol Med (Maywood) 2004; 229:528-537.
[27]Henriksen T, Evensen SA, Carlander B. Injury to human endothelial cells in culture induced by low density lipoproteins. Scand J Clin Lab Invest 1979; 39: 361-368.
[28]Coffey MD, Cole RA, Colles SM, Chisolm GM. In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals. J Clin Invest 1995; 96:1866-1873.
[29]Dimayuga PC, Chyu KY, Cercek B. Immune responses regulating the response to vascular injury. Curr Opin Lipidol 2010; 21:416-421.
[30]Mestas J, Ley K. Monocyte-endothelial cell interactions in the development of atherosclerosis. Trends Cardiovasc Med 2008; 18:228-232.
[31]Elkind MS. Impact of innate inflammation in population studies. Ann N Y Acad Sci 2010; 1207:97-106.
[32]Shi Q, Cox LA, Glenn J, Tejero ME, Hondara V, Vandeberg JL, Wang XL. Molecular pathways mediating differential responses to lipopolysaccharide between human and baboon arterial endothelial cells. Clin Exp Pharmacol Physiol 2010; 37:178-84.
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