CD40系统与动脉粥样硬化研究的新进展

越来越多的研究证实动脉粥样硬化是一种炎症发应,CD40系统的相互作用是炎症信号的重要传导通路,还参与粥样斑块内重要细胞如血管内皮细胞、血管平滑肌细胞及巨噬细胞等的炎症反应调节。阻断CD40CD40配体之间的相互作用可以抑制动脉粥样斑块的发生发展。最进,动脉粥样硬化(Atherosclerosis,AS)发病机制的研究有了很大进展,以前AS仅仅被认为是脂质堆积造成显著管腔狭窄或闭合。现在许多研究都证实AS是一种多种免疫因素参与的慢性炎症性疾病[1]。炎症发应贯穿于AS的发生、发展、变化的全过程,并在很大程度上决定着动脉粥样斑块的稳定性[2…     

复合羟丁基壳聚糖纳米载体携载CD133＋抗体温敏膜对猪髂动脉内皮细胞生长的影响

目的：观察复合羟丁基壳聚糖纳米载体携载CD133＋抗体温敏膜对培养的猪髂动脉内皮细胞生长的影响。方法：将猪髂动脉内皮细胞接种于裸钴铬合金片膜（裸金属支架组）、雷帕霉素药物涂层钴铬合金片膜（雷帕霉素支架组）、复合羟丁基壳聚糖纳米载体携载CD133＋抗体温敏膜钴铬合金片膜（抗温敏膜组）,培养24h。在光镜下观察细胞形态,以活细胞计数器（CCK-8）测定细胞增殖;以酶联免疫吸附试验（ELISA）试剂盒测定各组血管内皮生长因子（VEGF）、血小板衍化生长因子（PDGF）的水平。结果：动脉内皮细胞在抗温敏膜上生长良好,形态正常。培养24h,与裸金属支架组、雷帕霉素支架组比较,在抗温敏膜上存活的内皮细胞[光密度值：（0.812±0.003）、（0.783±0.005）比（0.903±0.003）],VEGF水平[（11.583±1.281）ng/L、（9.145±1.065）ng/L比（19.731±2.773）ng/L]显著升高,PDGF水平显著降低[（113.627±17.369）ng/μl、（91.251±17.097）ng/μl比（66.278±18.045）ng/μl],P均〈0.05。结论：复合羟丁基壳聚糖纳米载体携载CD133＋抗体温敏膜有助于血管内皮细胞的增殖。     

靶向USPIO分子探针联合MR监测动脉粥样硬化细胞间黏附分子-1变化

目的制备细胞间黏附分子-1(ICAM-1)靶向超微型超顺磁性氧化铁颗粒(USPIO)分子探针并联合磁共振(MR)研究动脉粥样硬化中ICAM-1的变化。方法通过水浴法将聚乙二醇(PEG)包被的USPIO和ICAM-1抗体合成ICAM-1靶向USPIO分子探针。通过透射电镜、动态光散射、酶联免疫吸附间接法检测分子探针的理化特性。使用ApoE基因敲除雄性小鼠建立动脉粥样硬化斑块模型后进行常规MR及增强MR扫描。分别注射ICAM-1靶向USPIO、非特异性IgG-USPIO分子探针,注药前及注药后0.5 h、2 h进行MR横断位扫描。MR扫描完成后进行病理学检查、免疫组织化学染色及电镜检查。结果磁共振扫描显示:靶向探针组增强后,粥样硬化斑块处信号明显减低。靶向探针组及非特异性IgG-USPIO组增强前、增强后0.5 h、2 h平均相对信号强度(rSI)有统计学意义(F=55.988,P 0.01);非特异性IgG-USPIO探针组增强后斑块信号轻度降低,增强后0.5 h、2 h rSI与靶向分子探针组比较均有统计学意义(t=-2.403、-3.147,P 0.05)。电镜检查显示:靶向探针组增强后铁颗粒在动脉粥样硬化斑块处较多沉积,非特异性IgG-USPIO探针组增强后铁颗粒在内皮下组织内有较多沉积。结论 ICAM-1在粥样硬化进程中具有空间效应,结合MR可作为动脉粥样硬化发生和发展的指标。     

肥大细胞类胰蛋白酶促进动脉粥样硬化斑块内出血

目的体内外实验研究类胰蛋白酶在动脉粥样硬化斑块内出血中的作用。方法体外通过慢病毒载体干扰肥大细胞P815类胰蛋白酶表达,取培液上清作为条件培养基孵育小鼠微血管内皮细胞(bEnd.3),观察细胞增殖、迁移、管腔形成情况,Western blot检测PAI、tPA表达改变。体内实验利用ApoE-/-小鼠,右侧颈总动脉套环建立斑块内出血模型,随机分为6组:①肥大细胞脱颗粒剂;②肥大细胞稳定剂;③肥大细胞脱颗粒剂+类胰蛋白酶病毒载体;④肥大细胞稳定剂+对照病毒载体;⑤肥大细胞稳定剂+对照siRNA病毒载体;⑥肥大细胞稳定剂+siRNA病毒载体。小鼠处死后取颈动脉斑块,冷冻切片,荧光显微镜观察斑块内荧光表达,HE染色观察出血及血管新生状况,免疫组化染色检测PAI、tPA、CD31表达。结果过表达类胰蛋白酶P815条件培养基促进bEnd3细胞增殖、迁移能力以及管腔形成能力;而且促进bEnd3细胞tPA表达,同时抑制PAI表达。ApoE-/-小鼠体内过表达类胰蛋白酶后,未套环侧颈总动脉斑块面积增大,狭窄程度加重;套环侧斑块内出血加重,斑块内CD31表达增加;同时PAI表达降低、tPA表达升高。结论类胰蛋白酶通过促进血管新生...     

靶向活化血小板的MRI显影可以在体内查出血栓形成和监察溶栓效果

血小板是血栓形成的关键，在动脉粥样硬化形成中起一定作用。作者评估用一种MRI显影剂[包括氧化铁微颗粒（MPIOs）和一种活化PⅡb／Ⅲa配体-引发结合部位（LIBS）的单链抗体]作颈动脉血栓和动脉粥样斑块影像。     

动脉斑块内骨桥蛋白靶向的新型超声对比剂的制备及体外实验研究

目的骨桥蛋白(OPN)在动脉易损斑块中高表达,本研究通过构建偶联骨桥蛋白(OPN)抗体的聚乳酸(PLA)纳米颗粒,实现OPN的超声纳米分子成像。方法通过乳化溶剂挥发法制备包裹液态氟碳的PLA纳米颗粒,经碳二亚胺法进行聚乙二醇(PEG)亲水性修饰和OPN抗体偶联,得到OPN靶向的超声成像纳米探针。对探针进行表征,CCK-8法检测探针毒性,激光共聚焦显微镜评估细胞摄取,超声诊断仪进行体外超声成像。结果 PLA纳米颗粒呈球形或类球形,水合粒径为(267.60±82.40)nm;CCK-8毒性实验显示,经不同浓度探针(0.00~0.50 mg/ml)的孵育后,各实验组与空白对照组比较细胞活性差异无统计学意义(P0.05);靶向OPN的纳米颗粒在泡沫细胞内摄取明显增多(P0.05);乳胶管超声成像见纳米探针在反向脉冲谐波成像(PIHI)模式下显示为点状密集高回声影像。结论本实验构建的靶向OPN纳米探针生物相容性好,能够在体外与OPN高表达的泡沫细胞模型特异性结合,超声成像效果良好。     

人源化抗肝癌双链抗体纳米颗粒对荷人肝癌裸鼠放射免疫显像研究

目的：观察99mTc 标记的人源化抗肝癌双链抗体 BDM3及双链抗体纳米颗粒在荷肝癌动物体内的靶向分布及放射免疫显像,探讨其对肝癌的靶向诊断和治疗的价值。方法使用99mTc 对人源化抗肝癌双链抗体 BDM3及双链抗体纳米颗粒进行标记,并测定标记率,将标记好的抗体及抗体纳米颗粒经腹腔注入荷瘤裸鼠体内,在注射后不同时间点进行放射免疫显像。取各脏器及肿瘤组织,计算肿瘤/非肿瘤比值。结果99mTc 标记的双链抗体BDM3和双链抗体纳米颗粒标记率为91%和92%,显像率均达100%。注射后1 h 肿瘤/血比值为（1.63±0.17）和（3.63±0.21）,4 h 肿瘤/血比值为（3.82±0.24）和（5.63±0.26）,8 h 肿瘤/血比值为（0.48±0.21）和（1.41±0.32）,两组比较,差异均有统计学意义（P〈0.01,P〈0.001和 P〈0.01）,表明同位素标记的双链抗体和双链抗体纳米颗粒均能在肿瘤组织中浓聚,而在非肿瘤组织中无浓聚现象,因而具有明确的靶向性,而抗体纳米颗粒因具有双重靶向作用,其浓聚现象较抗体本身更显著,能进一步增加显像效果。结论99mTc 标记的双链抗体 BDM3及其纳米颗粒对肝癌组织具有很好的亲和力,可作为肝癌诊断和治疗的靶向载体。     

动脉粥样硬化病变组织中PAI—1和uPA抗原表达的变化

目的了解动脉粥样硬化（ＡＳ）病变组织中Ｉ型组织型纤溶酶原激活剂抑制物（ＰＡＩ－１）与尿激酶型纤溶酶原激活剂（ｕＰＡ）抗原表达的变化。方法采用免疫组织化学染色检测了８例ＡＳ病变动脉，并以３例非血管性疾病死亡的新鲜尸检动脉为对照。结果对照动脉中，ＰＡＩ－１在内皮细胞和中膜平滑肌细胞表达；ＡＳ病变中，ＰＡＩ－１在病变表面的内皮细胞中表达增强，粥样斑块中的泡沫细胞呈阳性，病灶的细胞外基质中有广泛的ＰＡＩ－１分布，陈旧性病灶形成的肉芽组织中的毛细血管壁ＰＡＩ－１呈阳性反应。对照动脉内皮细胞中ｕＰＡ呈弱表达；ＡＳ病变中，纤维斑块病变的结缔组织帽下可见数层泡沫细胞ｕＰＡ表达呈强阳性，粥样斑块中新生毛细血管壁呈阳性，新生毛细血管周围有散在的阳性反应细胞。结论ＡＳ病变血管组织中，ＰＡＩ－１和ｕＰＡ抗原的分布和表达强度不一致，可能与它们参与ＡＳ过程的作用机理有关。     

抗血小板药物与阻断CD40信号对动脉粥样硬化的影响

目的探讨抗血小板药物、抗CD40L(CD40配体)抗体及合用对动脉粥样硬化的影响.方法雄性载脂蛋白E基因敲除小鼠随机分为模型对照组(n=10)、抗血小板药物组(n=10,阿司匹林 氯吡格雷)、抗CD40L抗体组(n=8,抗CD40L抗体)、合用组(n=9,阿司匹林 氯吡格雷 抗CD40L抗体).并取与载脂蛋白E基因敲除小鼠有相同遗传背景的健康雄性近交系C57BL/6小鼠6只作为正常对照组.测血脂、可溶性血管细胞黏附分子-1(sVCAM-1)和可溶性细胞间黏附分子-1(sICAM-1)浓度.观察主动脉组织病理形态学改变,免疫组织化学法测定斑块部位巨噬细胞、平滑肌细胞百分率.Western杂交分析测定基质金属蛋白酶-9的蛋白表达.结果抗血小板药物及抗CD40L抗体治疗可明显降低sVCAM-1和sICAM-1浓度(P＜0.01),减轻动脉粥样硬化病变,减少斑块部位巨噬细胞,增加平滑肌细胞数量(P＜0.01),对血脂无影响(P＞0.05),合用则作用增强(P＜0.05).抗CD40L抗体可降低基质金属蛋白酶-9的表达(P＜0.01),抗血小板治疗无此作用.结论抗血小板治疗及阻断CD40信号可抑制炎症反应,减轻动脉粥样硬化病变,稳定斑块,对血脂无影响,联合治疗有协同作用.     

骨髓间充质干细胞移植对损伤血管的内皮修复作用

目的探讨兔颈动脉粥样硬化狭窄血管成形术后,骨髓间充质干细胞(BMSC)移植对损伤血管内皮修复的影响及可能机制。方法制作兔颈动脉粥样硬化狭窄模型48只,随机分成BMSC移植组(n=24)和对照组(n=24)。体外培养兔BMSC,流式细胞仪鉴定BMSC表面标志,DAPI标记后备用。球囊损伤颈动脉的即刻,BMSC移植组以107/kg的细胞数经颈外动脉移植到损伤动脉局部,对照组注射等量的PBS液。术前及细胞移植后3、7、14及28天采集外周血用ELISA法测定血管内皮生长因子水平。移植后7天取材观察DAPI标记BMSC的归巢。移植后14天免疫组织化学染色检测损伤血管组织的血小板-内皮细胞黏附分子(CD31)的表达;移植后28天HE染色后测定损伤血管新生内膜面积、新生内膜面积/中膜面积及再狭窄率。结果 BMSC移植组在移植后各时间点血管内皮生长因子水平均显著高于对照组(P<0.01)。移植后7天BMSC移植组损伤血管的内膜表面见DAPI标记的BMSC。移植后14天血管内膜有CD31的连续性表达,对照组没有表达。与对照组比较,移植后28天BMSC移植组血管新生内膜面积(0.092±0.009比0.189±0.007,P<...     

Specific induction of tie1 promoter by disturbed flow in atherosclerosis-prone vascular niches and flow-obstructing pathologies

Nonlaminar flow is a major predisposing factor to atherosclerosis. Yet little is known regarding hemodynamic gene regulation in disease-prone areas of the vascular tree in vivo. We have determined spatial patterns of expression of endothelial cell receptors in the arterial tree and of reporter gene constructs in transgenic animals. In this study we show that the endothelial cell-specific receptor Tie1 is induced by disturbed flow in atherogenic vascular niches. Specifically, tie1 expression in the adult is upregulated in vascular bifurcations and branching points along the arterial tree. It is often confined to a single ring of endothelial cells functioning as sphincters and hence experiencing the steepest gradient in shear stress. In aortic valves, tie1 is asymmetrically induced only in endothelial cells encountering changes in flow direction. Disturbance of laminar flow by a surgical interposition of a vein into an artery led to induction of tie1, specifically in the region where the differently sized vessels adjoin. In pathological settings, tie1 expression is specifically induced in areas of disturbed flow because of the emergence of aneurysms and, importantly, in endothelial cells precisely overlying atherosclerotic plaques. Hemodynamic features of atherosclerotic lesion-prone regions, recreated in vitro with the aid of a flow chamber with a built-in step, corroborated an upregulated tie1 promoter activity only in cells residing where flow separation and recirculation take place. These defined promoter elements might be harnessed for targeting gene expression to atherosclerotic lesions.     

Impact of glycoprotein VI and platelet adhesion on atherosclerosis—A possible role of fibronectin

Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE−/− mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE−/− mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE−/− mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly via fibronectin-could relevantly contribute to platelet-triggered atheroprogression.     

Endoglin, a TGF-beta receptor-associated protein, is expressed by smooth muscle cells in human atherosclerotic plaques

Endoglin is a transmembrane protein that is found in association with transforming growth factor-beta (TGF-beta) superfamily receptor complexes and has an expression pattern that appears to be restricted primarily to endothelial cells, activated macrophages, trophoblasts, and fibroblasts. Since mutations in endoglin have been shown to be linked to hereditary hemorrhagic telangiectasia type 1, a disease manifested as vascular malformations characterized by excessive layers of vascular smooth muscle cells (VSMC), the expression of endoglin was investigated in VSMC. In vivo, the majority of SMC in human atherosclerotic plaques expressed high levels of endoglin, while endoglin was not detected in SMC from samples of the normal arterial wall. In vitro studies demonstrate that human aortic smooth muscle cells (HASMC) express the L-isoform of endoglin. Like endothelial cells, HASMC express endoglin protein as a dimer on the cell surface that binds TGF-beta1. In vitro, endoglin expression by HASMC is upregulated in response to TGF-beta1, suggesting that the presence of this factor in the atherosclerotic plaque might be responsible for the increased expression of endoglin. The demonstration of increased levels of endoglin in VSMC in human atherosclerotic plaques suggests a role for SMC endoglin in the maintenance of vascular integrity and in the response of the vessel wall to injury.     

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.     

Periodicity in tumor vasculature targeting kinetics of ligand-functionalized nanoparticles studied by dynamic contrast enhanced magnetic resonance imaging and intravital microscopy

In the past two decades advances in the development of targeted nanoparticles have facilitated their application as molecular imaging agents and targeted drug delivery vehicles. Nanoparticle-enhanced molecular imaging of the angiogenic tumor vasculature has been of particular interest. Not only because angiogenesis plays an important role in various pathologies, but also since endothelial cell surface receptors are directly accessible for relatively large circulating nanoparticles. Typically, nanoparticle targeting towards these receptors is studied by analyzing the contrast distribution on tumor images acquired before and at set time points after administration. Although several exciting proof-of-concept studies demonstrated qualitative assessment of relative target concentration and distribution, these studies did not provide quantitative information on the nanoparticle targeting kinetics. These kinetics will not only depend on nanoparticle characteristics, but also on receptor binding and recycling. In this study, we monitored the in vivo targeting kinetics of α_vβ₃-integrin specific nanoparticles with intravital microscopy and dynamic contrast enhanced magnetic resonance imaging, and using compartment modeling we were able to quantify nanoparticle targeting rates. As such, this approach can facilitate optimization of targeted nanoparticle design and it holds promise for providing more quantitative information on in vivo receptor levels. Interestingly, we also observed a periodicity in the accumulation kinetics of α_vβ₃-integrin targeted nanoparticles and hypothesize that this periodicity is caused by receptor binding, internalization and recycling dynamics. Taken together, this demonstrates that our experimental approach provides new insights in in vivo nanoparticle targeting, which may proof useful for vascular targeting in general.     

Leucocyte recruitment in rupture prone regions of lipid-rich plaques: a prominent role for neovascularization?

OBJECTIVE: Microvessels in atherosclerotic plaques provide an alternative pathway for the recruitment of leucocytes in the lesions. The present study was designed to investigate the potential role of these microvessels in creating vulnerable sites in atherosclerotic plaques. METHODS: Thirty-four atherosclerotic plaques were obtained from 25 patients undergoing carotid endartherectomy (n = 16), femoral endartherectomy (n = 6) and aortic surgery (n = 12). Plaques were histologically classified as either lipid-rich (rupture prone, n = 21) or fibrous (stable, n = 13). Serial cryostat sections were immunohistochemically investigated using monoclonal antibodies against endothelial cells (ULEX-E and F-VIII), vascular endothelial growth factor (VEGF), endothelial adhesion molecules (ICAM-1, VCAM-1, E-Selectin, CD40) and inflammatory cells (macrophages (CD68) and T lymphocytes (CD3). RESULTS: The microvessel density in lipid-rich plaques was significantly increased as compared to fibrous plaques. Most of these vessels were located in the shoulder-region of the plaque and at the base of the atheroma. Microvessels in lipid-rich plaques also expressed increased levels of ICAM-1, VCAM-1, E-Selectin and CD40. Moreover, inflammation was most abundantly present in the proximity of microvessels. VEGF was only observed on vessels and mononuclear cells in lipid-rich plaques, suggesting that this factor may play a role in microvessels formation. CONCLUSIONS: Neovascularisation and expression of adhesion molecules by microvessels at sites of vulnerable lipid-rich plaques may sustain the influx of inflammatory cells and hence, could contribute to plaque destabilization.     

Platelet GPVI binds to collagenous structures in the core region of human atheromatous plaque and is critical for atheroprogression in vivo

Platelet adhesion to the atherosclerotic vascular wall induces thrombosis and boosters vascular inflammation and atheroprogression. In the present study we studied the binding of the platelet collagen receptor glycoprotein (GP) VI to human atherosclerotic plaques (AP) and the role of GPVI-mediated platelet adhesion for atheroprogression. Soluble GPVI-Fc fusion protein bound to immobilized collagen type I, collagen type III, and predominantly to the core region of human carotid atheromatous plaques. The pattern of GPVI-Fc binding was similar to the immunostaining pattern of collagen type III and differed from the immunostaining of collagen type I, which was more intense in the cap than in the core. Plaque-induced platelet aggregation in stirred blood and platelet adhesion/aggregate formation under flow were inhibited by the anti-GPVI monoclonal antibody 5C4 or by pretreatment of plaques with anti-collagen type I and anti-collagen type III antibody, or GPVI-Fc. However, there was no correlation between GPVI-Fc binding and platelet aggregating activity of individual plaques. GPVI bound also to atherosclerotic arteries of ApoE-deficient mice in vivo as assessed by small animal positron emission tomography (PET). Prolonged administration of soluble GPVI attenuated atheroprogression in ApoE-deficient mice. In humans, GPVI binding to collagenous type I and type III structures of the plaque core region mediates plaque-induced platelet adhesion and aggregation, but GPVI binding is not the sole platelet-activating determinant of plaques. In mice, GPVI-mediated platelet adhesion to the atherosclerotic vascular wall is involved in atheroprogression in vivo. Taken together, our data suggests that GPVI is a relevant target to prevent atherothrombotic events and atheroprogression.     

High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents

A cell delivery strategy was investigated that was hypothesized to enable magnetic targeting of endothelial cells to the steel surfaces of intraarterial stents because of the following mechanisms: (i) preloading cells with biodegradable polymeric superparamagnetic nanoparticles (MNPs), thereby rendering the cells magnetically responsive; and (ii) the induction of both magnetic field gradients around the wires of a steel stent and magnetic moments within MNPs because of a uniform external magnetic field, thereby targeting MNP-laden cells to the stent wires. In vitro studies demonstrated that MNP-loaded bovine aortic endothelial cells (BAECs) could be magnetically targeted to steel stent wires. In vivo MNP-loaded BAECs transduced with adenoviruses expressing luciferase (Luc) were targeted to stents deployed in rat carotid arteries in the presence of a uniform magnetic field with significantly greater Luc expression, detected by in vivo optical imaging, than nonmagnetic controls.     

Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance

Time-dependent thrombolytic resistance is a critical problem in thrombolytic therapy for acute myocardial infarction. Platelets have been regarded as the main source of plasminogen activator inhibitor-1 (PAI-1) found in occlusive platelet-rich clots. However, endothelial cells are also known to influence the fibrinolytic capacity of blood vessels, but their ability to actively mediate time-dependent thrombolytic resistance has not been fully established. We will show that, in vitro, tumor necrosis factor-alpha-stimulated endothelial cells secrete large amounts of PAI-1 over a period of hours, which then binds to fibrin and protects the clot from tissue plasminogen activator- induced fibrinolysis. In vivo, endothelial cells covering atherosclerotic plaques are influenced by cytokines synthesized by plaque cells. Therefore, we propose that continuous activation of endothelial cells in atherosclerotic blood vessels, followed by elevated PAI-1 secretion and storage of active PAI-1 in the fibrin matrix, leads to clot stabilization. This scenario makes endothelial cells a major factor in time-dependent thrombolytic resistance.     

Oxidized phospholipids stimulate angiogenesis via autocrine mechanisms, implicating a novel role for lipid oxidation in the evolution of atherosclerotic lesions

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2-generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.