Atherosclerosis Induced Arterial Urokinase-type Plasminogen Activator Receptor (uPAR) Role And Mechanism

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 results1,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.