摘要
背景纤溶系统不仅参与纤维蛋白溶解，而且对细胞粘附、增殖、迁移、基质破坏、不稳定斑块的形成和破裂以及动脉瘤的形成等均起到了关键作用。在动脉粥样硬化病程中，纤溶系统与冠心病严重并发症如急性心肌梗死和急性再闭塞密切相关，实际上它与动脉粥样硬化的整个过程都不可分割，其主要成分包括纤溶酶原及纤溶酶，后者是多种重要的生理病理过程，如细胞迁移、组织重构和肿瘤细胞转移的关键蛋白酶。纤溶酶原的激活需要纤溶酶原激活物，分为组织型(tPA)和尿激酶型(uPA)两种。目前认为tPA激活纤溶酶原后可以溶解循环中纤维蛋白；uPA与其细胞受体(uPAR)结合后激活纤溶酶原，可以将蛋白酶解限制在细胞周围。uPAR位于多种细胞表面，近年对uPAR的研究多集中在肿瘤转移方面，认为uPA和uPAR的高表达与肿瘤的远处转移正相关，是预后的重要指标。并非偶然的是，有人对动脉粥样硬化斑块中的uPA和uPAR表达进行了检测，发现粥样硬化斑块中两者都高表达。
目的本研究旨在明确人单核／巨噬细胞表达uPAR的促动脉粥样硬化作用：首先在临床水平，检测uPAR在冠心病高危患者外周血单核细胞上是否已经高表达，分析其表达水平与冠心病危险因素分层的相关性。同时在体外细胞水平，探讨粥样硬化过程中的炎症因子如γ干扰素(INF-γ)、氧化低密度脂蛋白(oxLDL)和单核细胞趋化蛋白-1(MCP-1)对巨噬细胞生成uPAR的影响，并且了解阿托伐他汀对这些因子的干预作用。在细胞功能水平，通过Transwell和细胞分选方法，将uPA、uPA抗体、纤溶酶原激活物抑制剂-1(PAI-1)、抗uPAR等因子与单核细胞共同孵育，探讨uPA和uPAR在单核细胞趋化过程中的作用及机制。最后在人粥样斑块的组织水平，检测斑块中uPAR表达的情况，以及在不同病变部位表达的差异。
方法与结果
1、收集临床上稳定和不稳定冠心病、初发糖尿病以及具有不同冠心病危险因素的病人，检测血浆中tPA，uPA，PAI-1等纤溶相关指标的水平；应用流式细胞仪测量外周血单核细胞上uPAR表达的比例，结果显示：初发糖尿病患者处于高凝和纤溶亢进状态；稳定冠心病患者和ACS患者纤溶活性均明显下降，以ACS患者最为严重。初发糖尿病、ACS和代谢综合征患者外周血单核细胞上uPAR表达确实已经显著升高，这种升高受到代谢异常的调节，尤其是高脂血症能显著增加单核细胞表达uPAR。血脂和血糖异常同时存在可能是一对影响uPAR表达的关键因素。
2、表达uPAR的单核细胞在相差显微镜下表现为颗粒感和立体感明显增强，细胞周围有光晕，与不表达uPAR的细胞明显不同，而且这种形态学的改变和uPAR的表达呈一一对应关系。
3、本研究采用密度梯度离心和粘附法分离、纯化单核细胞，用细胞原位ELISA法检测uPAR抗原，底物发色法检测uPA活性和RT-PCR检测uPAR mRNA水平。我们发现在体外实验中，巨噬细胞粘附和分化本身可以诱导产生uPA，但不诱导uPAR。INF-γ和MCP-1对于巨噬细胞产生uPA的活性是一种抑制作用，而高浓度oxLDL则是刺激作用。INF-γ、MCP-1和oxLDL三种因素对巨噬细胞生成uPAR的影响是不同的：INF-γ抑制uPAR表达，oxLDL和MCP-1能够增强uPAR的表达，oxLDL是慢刺激，MCP-1则是快刺激。中浓度和高浓度oxLDL刺激巨噬细胞表达uPAR的效应可以被阿托伐他汀显著抑制，这是通过降低mRNA水平实现的。
4、利用流式细胞分选的方法，将外周血单核细胞分选为uPAR阳性和uPAR阴性两组，给予uPA抗体、uPAR抗体、uPA和PAI-1等因子作用观测单核细胞迁移情况：PAI-1和抗uPA抗体对单核细胞迁移的影响不大，而uPA的氨基末端(ATF)则显著抑制单核细胞的迁移，说明uPA与uPAR结合后的相互作用在迁移中起关键作用。随着抗uPAR抗体浓度的递增，单核细胞的迁移呈递减。ATF对于uPAR的阻断作用强于抗uPAR抗体，对细胞迁移的抑制作用也更明显。
5、从临床上收集动脉粥样硬化斑块剥脱标本，通过免疫组化等方法，观察uPAR在斑块中的表达部位；与巨噬细胞、平滑肌细胞的关系以及半定量检测斑块不同部位uPAR表达量的差异。研究结果显示，uPAR在正常乳内动脉的内膜和中膜无表达；而在动脉粥样硬化斑块的内膜中uPAR广泛表达，与巨噬细胞和平滑肌细胞分布的部位一致；uPAR在脂质池附近表达量最高，在斑块肩部和破裂斑块的部位也有大量表达，呈团块状。斑块内膜处uPAR的表达比中膜处显著升高。
结论本研究证实动脉粥样斑块中uPAR大量表达，而且首次发现在冠心病高危因素的患者外周血单核细胞上uPAR的表达就已经显著增高，这种升高和代谢异常明确相关，就将uPAR在致粥样硬化中的作用追溯到了粥样硬化形成的早期，并为临床利用uPAR来评价冠心病稳定性、干预uPAR表达或功能来治疗和预防冠心病提供了一个可能的方向。本研究还探讨了体外INF-γ、MCP-1和oxLDL对巨噬细胞表达uPAR的影响和特点；发现阿托伐他汀可以抑制oxLDL对巨噬细胞表达uPAR的刺激作用，这是通过转录水平来实现的，这些结果为进一步深入研究uPAR在粥样硬化形成中的作用和机制打下了基础。
Background Fibrinolytic system participates in the degradation of fibrin and many other physiopathological process, as cell adhesion, proliferation and migration. It breaks down extra cellular matrix, causes unstable atherosclerotic plaque and rupture incidents, and moreover, accelerates the progression of aneurysm. Fibrinolytic system is not only involved in severe coronary artery complications as acute myocardial infarction or acute coronary restenosis, but actually through the whole atherosclerosis. Fibrinolytic system is composed mainly of plasminogen and its active form called plasmin. Plasmin is one of the crucial proteases that enables cell migration, tissue remodeling and cancer metastasis. The activation of plasminogen depends on two member activators: tissue type (tPA) and urokinase type plasminogen activator (uPA). Now it is recognized tPA associated plasminogen activation can dissolve fibrin in circulation, while uPA related activation can limit proteinase activities at certain sites of the cell surfaces, and the latter requires uPA to bind to its receptor(uPAR). uPAR is located on membranes of various cells. It has been reported in recent years that uPAR plays an important role in cancer metastasis. The high level expression of uPA and uPAR is corresponding to the remote metastasis of malignant tumors, and is a strong signal for adverse prognosis. Interestingly, when researchers detected human atherotic artery samples, they have found high level expression of uPA and uPAR in atherosclerotic lesions likewise.
Objective Our study was aiming to evaluate the atherogenic role of uPAR expressed on human monocytes or macrophages. First, the percentage of peripheral monocytes expressing uPAR was measured to observe whether uPAR have been highly expressed in patients at high risk to coronary artery diseases (CAD), and if it has, to find an association between this high level expression and risk factors. Second, interferon-γ(INF-γ), oxLOL and monocyte chemotactic protein-1 (MCP-1) were utilized to investigate their effect on uPAR expression by macrophages, and the response to atorvastatin. Third, after incubated with uPA, uPA antibody, PAI-1, uPAR antibody, human monocytes were put into upper chambers oftranswell to prove the role and mechanism played by uPA and uPAR in MCP-1 induced migration. Last, human atherosclerotic plaque were tested to detect the expression of uPAR within, and the expression difference at various locations. Methods and results
1、Clinically stable and unstable patients with CAD, patients with initially diagnosed diabetes, or those with risk factors for CAD were enrolled in the study. Serum levels of tPA, uPA and PAI-1 were measured and the proportion of peripheral monocytes expressing uPAR was surveyed by flow cytometer. Patients with initial diabetes were in a situation of hypercoagulation and hyperfibrinolysis. Fibrinolytic activity was significantly impaired in patients with stable CAD and acute coronary syndrome (ACS). The expression of uPAR on peripheral monocytes had been notably raised in patients with initial diabetes, ACS or metabolic syndrome. It was also found the expression of uPAR was modulated by metabolic abnormalities, and hyperlipidemia strikingly stimulated the expression of uPAR on monocytes. Hyperlipidemia and pathoglycemia was probably a critical pair of factors activating uPAR expression.
2、Monocytes expressing uPAR appeared grainier and more three-dimensional under contrast phase microscope. A halation could be observed around uPAR positive cells, which obviously differed from non-uPAR expressing cells, and moreover, the morphological changes were corresponding to the expression of uPAR precisely.
3、Monocytes were separated and purified by density gradient centrifugation and adhering assay. ELISA was used for detecting antigen, chromogenic substrate assay for uPA activity and RT-PCR for mRNA. The results indicated that in vitro, adhesion and differentiation of macrophages could induce the production of uPA, but not that of uPAR. INF-γand MCP-1 inhibited uPA activity, while high concentration of oxLDL stimulated it. As for uPAR, INF-γ、MCP-1 and oxLDL had distinct effect on its expression on macrophages: INF-γsuppressed it, oxLDL and MCP-1 stimulated it. Interestingly, oxLDL stimulation was in a slow style, but MCP-1 in a quick one. The stimulating effect at moderate and high concentration of oxLDL could be inhibited by adding atorvastatin, which was achieved at the level ofmRNA.
4、Using cell sorting, peripheral monocytes were divided into uPAR positive and negative groups. After incubated with uPA antibody、uPAR antibody、uPA and PAI-1, the number of monocytes having migrated through transwell were calculated. PAI-1 and uPA antibody had no effect on monocyte migration, but the amino terminal fragment of uPA (ATF) suppressed the migration significantly. With the increasing uPAR antibody concentration, the number of migrated monocytes decreased. ATF had a stronger inhibitory effect on uPAR than uPAR antibody had, and suppressed migration more thoroughly than the antibody.
5、Stripping samples were collected from human peripheral artery atheroma, and immunohistochemistry was employed to detect the location of uPAR protein as well as its association with macrophages and SMC in plaque. No uPAR was detected in intima or tunica media of normal internal mammary arteries; however it was extensively expressed in the intima of atherosclerotic lesions and coexisted with macrophages and SMC in the plaque. Maximal uPAR expression was observed in lipid pool, and fairly massive expression at plaque shoulders and rupture sites. The expression of uPAR in intima was significantly higher than in tunica media.
Conclusions Our study confirmed a significant expression of uPAR in atheroma, and for the first time we found the express of uPAR on peripheral monocytes had been notably raised in patients with risk factors of CAD. The high level expression was clearly related to metabolic abnormalities, thus the role of uPAR in atherogenesis could be traced back to the early stage of atherosclerosis, and made it possible to use uPAR as a parameter to evaluate the stability of CAD or to treat and prevent CAD by interfering uPAR expression or its function at early stage. We also verified that INF-γ, MCP-1 and oxLDL had distinct impact on uPAR synthesis by macrophages; oxLDL associated uPAR expression was inhibited by atorvastatin, which was achieved at a transcriptional level. Our results provided new knowledge of monocyte/macrophage's atherogenic role by uPAR, and further investigation was required to elucidate its mechanism and the possibility of clinical application.
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8. Florea Lupu, Dominik A. Heim, Fedor Bachmann et al. Plasminogen Activator Expression in Human Atherosclerotic Lesions. Arterioscler Thromb Vasc Biol. 1995; 15:1444-1455.
9. Joachim Kienast, Teresa Padro, Martin Steins et al. Relation of Urokinase-type Plasminogen Activator Expression to Presence and Severity of Atherosclerotic Lesions in Human Coronary Artery. Thromb Haemost 1998; 79:579-86.
10. Martin B. Steins, Teresa Padro, Carsten Schwaenen et al. Overexpression of Urokinase Receptor and Cell surface Urokinase-type Plasminogen Activator in the Human Vessel Wall Different Types of Atherosclerotic Lesions. Blood Coagulation and Fibrinolysis 2004.15:383-391.
11. M.Y.Salame, N.J.Samani, I. Masood et al. Expression of the plasminogen activator system in the human vascular wall. Atherosclerosis 2000; 152:19-28.
12. Teresa Padro, Jef J. Emeis, Martin Steins et al. Quantification of Plasminogen Activator and Their Inhibitors in the Aortic Vessel Wall in Relation to the Presence and Severity of Atherosclerotic Disease. Arterioscler Thromb Vasc Biol. 1995; 15:893-902.
13. Aernout Luttun, Florea Lupu, Erik Storkebaum et al. Lack of Plasminogen Activator Inhibitor-1 Promotes Growth and Abnormal Matrix Remodeling of Advanced Atherosclerotic Plaques in Apolipoprotein E-Deficiant Mice. Arterioscler Thromb Vasc Biol, 2002; 22:499-505.
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15. P.N. Raghunath, John E. Tomaszewski, Stephen T. Brady et al. Plasminogen Activator System in Human Coronary Atherosclerosis. Arterioscler Thromb Vasc Biol 1995;15:1432-1443.
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18. Jean Marc Herbert, Isabelle Lamarche, Valerie Prabonnaud et al, Tissue-type Plasminogen Activator is a Potent Mitogen for Human Aortic Smooth Muscle Cells. The Journal of Biological Chemistry. 1994;269:3076-3080.
19. Sandip M. Kanse, Omar Benzakour, Chryso Kanthou et al. Induction of Vascular SMC Proliferation by Urokinase Indicates a Novel Mechanism of Action in Vasoproliferative Disorders. Arteriosclerosis, Thrombosis & Vascular Biology. 1997,17(11):2848-2854.
20. Teresa Padro, Rolf M. Mesters, Berno Dankbar et al. The Catalytic Domain of Endogenous urokinase-type Plasminogen Activator is Requred for the Mitogenic Activity of Platelet-Derived and basic Fibroblast growth Factors in Human Vascular Smooth Muscle Cells. Journal of Cell Science 2002; 115:1961-1971.
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26. Margaret R. Gyetko, Robert F. Todd III, Camille C. Wilkinson et al. The Urokinase Receptor is Required for Human Monocyte Chemotaxis in vitro. The Journal of Clinical Investigation. 1994, 93:1380-1387.
27. Nathalie Busso, Sandra K.Masur, David Lazega et al. Induction of Cell Migration by Pro-urokinase Binding to Its Receptor:Possible Mechanism for Signal Transduction in Human Epithelial Cells. The Journal of Cell Biology.1994;126:259-270.
28. Sandip M. Kanse, Triantafyllos Chavakis, Nadia AI-Fakhri et al. Reciprocal Regulation of Urokinase Receptor(CD87)-Mediated Cell Adhesion by Plasminogen Activator Inhibitor-1 and Protease Nexin-1. Journal of Cell Science 2004,117, 477-485.
