Deflazacort modulates the fibrinolytic pattern and reduces uPA-dependent chemioinvasion and proliferation in rheumatoid arthritis synoviocytes

OBJECTIVE: Extracellular fibrinolysis, controlled by the cell-associated fibrinolytic system (urokinase plasminogen activator, uPA; uPA receptor, uPAR; plasminogen activator inhibitor type-1, PAI-1), is involved in cartilage damage generation and in rheumatoid arthritis (RA) synovitis. Since steroids reduce the rate of radiological progression of RA, we planned to evaluate in healthy and RA synoviocytes the effects of the steroid deflazacort on uPA, uPAR and PAI-1 expression, and subsequent phenotypic modifications in terms of uPA/uPAR-dependent invasion and proliferation. METHODS: uPA, uPAR and PAI-1 levels were studied by ELISA, RT-PCR (uPAR) and zymography (uPA) in synoviocytes from four RA patients and four healthy controls. Chemoinvasion was assessed by the Boyden chamber invasion assay, using Matrigel as the invasion substrate. Proliferation was evaluated by cell counting. Both invasion and proliferation were measured upon treatment with deflazacort 5 muM with or without parallel stimulation with uPA 500 ng/ml or in the presence of monoclonal anti-uPA and anti-uPAR antibodies. RESULTS: Invasion and proliferation of RA synoviocytes require a proper functional balance of the fibrinolytic system. Both deflazacort and monoclonal antibodies against uPA and uPAR reduced expression and activity of the system, thus inhibiting invasion and proliferation. In RA synoviocytes, deflazacort induced higher PAI-1 and lower uPA and uPAR levels, as well as a decrease in uPA enzymatic activity. The levels of uPAR mRNA were concomitantly reduced, as was uPA-induced chemoinvasion. All these effects were also shown in controls, though to a lesser extent. CONCLUSIONS: Deflazacort might control RA synovial proliferation and invasion by differential modulation of single members of the fibrinolytic system.     

Plasminogen activation in synovial tissues: differences between normal, osteoarthritis, and rheumatoid arthritis joints

OBJECTIVE—To analyse the functional activity of the plasminogen activators urokinase (uPA) and tissue type plasminogen activator (tPA) in human synovial membrane, and to compare the pattern of expression between normal, osteoarthritic, and rheumatoid synovium. The molecular mechanisms underlying differences in PA activities between normal and pathological synovial tissues have been further examined.
METHODS—Synovial membranes from seven normal (N) subjects, 14 osteoarthritis (OA), and 10 rheumatoid arthritis (RA) patients were analysed for plasminogen activator activity by conventional zymography and in situ zymography on tissue sections. The tissue distribution of uPA, tPA, uPA receptor (uPAR), and plasminogen activator inhibitor type-1 (PAI-1) was studied by immunohistochemistry. uPA, tPA, uPAR, and PAI-1 mRNA values and mRNA distribution were assessed by northern blot and in situ hybridisations respectively.
RESULTS—All normal and most OA synovial tissues expressed predominantly tPA catalysed proteolytic activity mainly associated to the synovial vasculature. In some OA, tPA activity was expressed together with variable amounts of uPA mediated activity. By contrast, most RA synovial tissues exhibited considerably increased uPA activity over the proliferative lining areas, while tPA activity was reduced when compared with N and OA synovial tissues. This increase in uPA activity was associated with increased levels of uPA antigen and its corresponding mRNA, which were localised over the synovial proliferative lining areas. In addition, in RA tissues, expression of the specific uPA receptor (uPAR) and of the plasminogen activator inhibitor-type 1 (PAI-1) were also increased.
CONCLUSION—Taken together, these results show an alteration of the PA/plasmin system in RA synovial tissues, resulting in increased uPA catalytic activity that may play a part in tissue destruction in RA.

     

类风湿关节炎患者滑液和血浆中尿激酶型纤溶酶原激活物及其受体测定的临床意义

目的 检测尿激酶型纤溶酶原激活物（uPA）及其受体uPAR在类风湿关节炎（RA）患者滑液和血浆中的含量,并结合临床和实验室资料分析其实际意义。方法 运用酶联免疫吸附法（ELISA）双抗体夹心法分别测定46例RA、8例骨关节炎（OA）和12名健康对照个体的血浆以及15例RA患者滑液中的uPA和uPAR含量,同时将类风湿因子阳性（RF^ ）和阴性（RF^-）组之间uPA和uPAR含量进行比较,并采用直线相关与回归方法分析血浆和滑膜中uPA、uPAR的含量与相评价RA病情活动指标之间的相互关系。结果 RA患者滑液中uPA、uPAR的含量均显著高于其自身血浆（P＜0．001,P＜0．01）;RA血浆中uPA、uPAR的含量又明显高于OA组（P＜0．05,P＜0．0001）和正常对照组（P＜0．0001,P＜0．001);RF^ 和RF^-组之间uPA、uPAR在滑液和血浆中的含量差异均无显著性（P＞0．05）。RA滑液和血浆中uPA、uPAR在滑液和血浆中的含量差异均无显著性（P＞0．05）。RA滑液和血浆中uPA、uPAR的含量还与反映RA病情活动的指标C反应蛋白（CRP）和类风湿因子（RF）之间均呈正相关;RA滑液中uPA、uPAR的含量还与关节肿胀个正相关。结论 RA患者滑液和血浆中uPA和uPAR的含量是反映RA病情活动的有用指标,提示uPA和uPAR基因可能通过降解细胞外基质的蛋白裂解作用参与RA的免疫作用机制并发挥重要作用。     

Increased expression of plasminogen activator and plasminogen activator inhibitor during liver fibrogenesis of rats: role of stellate cells.

BACKGROUND/AIMS: Plasminogen activators and plasminogen activator inhibitors are important regulators of the balance between the proteolytic and antiproteolytic activities that determine extracellular matrix turnover. We examined the expression of plasminogen activator-plasmin system components in experimental liver fibrosis of rats. METHODS: Liver fibrosis was produced in rats by injecting carbon tetrachloride for 6 to 12 weeks. Gene expression for plasminogen activator inhibitor-1 (PAI-1), urokinase and tissue plasminogen activators (uPA and tPA), urokinase plasminogen activator receptor (uPAR), and transforming growth factor-beta1 (TGF-beta1) was examined by Northern analysis. Western analysis was performed to detect protein expression of PAI-1, uPA and uPAR. An immunohistochemical study was performed to detect the localization of PAI-1. Additionally, primary cultured liver cells were examined by Northern and Western analyses for this protein with or without prior incubation with TGF-beta1. RESULTS: At 6 weeks, when fibrosis had occurred, uPA and uPAR mRNAs had increased 2.8-fold and 1.8-fold, respectively; PAI-1 and tPA mRNA levels were unchanged. At the cirrhotic stage (9 to 12 weeks), mRNA levels for PAI-1, uPA, uPAR and tPA were all increased. Western analysis also showed increased uPA and uPAR expressions in fibrotic liver, and increased PAI-1, uPA and uPAR expressions in cirrhotic liver. PAI-1 protein was also demonstrated immunohistochemically along sinusoids, vessels, and bile duct cells of normal and fibrotic liver. In liver cell cultures, Kupffer cells, hepatocytes, and especially stellate cells, expressed PAI-1. Expression was enhanced in stellate cells cultured from fibrotic or cirrhotic liver or stimulated in vitro with TGF-beta1. CONCLUSION: Though increased uPA and uPAR may act on matrix degradation in fibrotic liver, increased PAI-1 together with uPA, uPAR and tPA are associated with overall inhibition of matrix degradation in cirrhotic liver. Hepatic stellate cells are an important source of PAI-1 during liver fibrosis.     

Plasminogen activators and their inhibitors in synovial fluids from normal, osteoarthritis, and rheumatoid arthritis knees.

OBJECTIVES: To establish baseline concentrations of plasminogen activators and their inhibitors in normal knee synovial fluids, and to compare them with well characterised osteoarthritis (OA) and rheumatoid arthritis (RA) knee fluids. METHODS: A total of 26 normal subjects, 71 patients with OA, and 17 patients with RA underwent knee aspiration. Patients with OA were subclassified according to presence of nodal generalised OA (NGOA) and synovial fluid calcium pyrophosphate crystals. Clinical assessment of inflammation (graded 0-6) was undertaken in OA and RA patients. Plasminogen activator (PA), plasminogen activator inhibitor (PAI), and urokinase-type PA receptor (uPAR) antigen concentrations were determined by enzyme linked immunosorbent assay. The species of PAs present were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS: Concentrations of all antigens (uPA, tissue-type PA (tPA), uPAR, and PAI-1), were significantly greater in RA than OA; those in OA were significantly greater than normal. The concentrations showed no direct association with clinically assessed inflammation of the knee. In normal fluids, no associations with age were observed. Antigen concentrations (uPA, tPA, and uPAR) in NGOA differed from those in other subclasses of OA, but the species of PA present did not appear to vary between disease groups. The predominant PA appeared to have identity with uPA. CONCLUSION: Because of the greater concentrations of these antigens in OA compared with normal fluids, OA cannot be used as a surrogate normal control in studies of the PA/PAI system. Alteration of the PA/PAI system was confirmed in RA and OA knee fluids, with greater changes evident in RA. The finding of different concentrations of PA antigens in NGOA compared with other OA fluids further supports a different pathogenic mechanism in this subset.     

Plasminogen activator inhibitor type-1 deficiency does not influence the outcome of murine pneumococcal pneumonia

Urokinase-type plasminogen activator (uPA) and its receptor uPAR are components of the fibrinolytic system and are important for an adequate immune response to respiratory tract infection, in part through their role in the migration of inflammatory cells. PA inhibitor-1 (PAI-1) is the predominant inhibitor of soluble and receptor-bound uPA. To determine the role of PAI-1 in host defense against pneumococcal pneumonia, the following studies were performed: (1) Patients with unilateral community-acquired pneumonia demonstrated elevated PAI-1 concentrations together with decreased PA activity in bronchoalveolar lavage fluid (BALF) obtained from the infected, but not from the contralateral, site. (2) Mice with Streptococcus pneumoniae pneumonia displayed elevated PAI-1 protein and mRNA levels in their lungs. (3) PAI-1 gene-deficient mice, however, had an unaltered immune response to pneumococcal pneumonia, as measured by cell recruitment into lungs, bacterial outgrowth, and survival. Furthermore, plasminogen-gene-deficient mice also had an unremarkable defense against pneumococcal pneumonia. These data indicate that pneumonia is associated with inhibition of the fibrinolytic system at the site of the infection secondary to increased production of PAI-1; an intact fibrinolytic response is not required for an adequate host response to respiratory tract infection, however, suggesting that the previously described role of uPA and uPAR are restricted to their function in cell migration.     

The urokinase plasminogen activator system in cancer: Implications for tumor angiogenesis and metastasis

Substantial evidence exists which implicates the urokinase plasminogen activator system [urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1)] in the neo-vascularization, invasion and metastasis of many solid tumors. Clinical studies have demonstrated an association between high levels of expression of the components of this system in tumors and poor patient prognosis and outcome. Components of the uPA/uPAR system are differentially expressed or activated on motile cells including invading tumor cells and leukocytes, and migrating endothelial cells. In contrast, there is little or no expression on most normal, quiescent cells. Studies performed in vitro have demonstrated the regulation of the expression of uPA and uPAR by growth and differentiation factors as well as by oncogenes. In this review, we summarize recent findings on the role of the components of the uPA/uPAR system in angiogenesis, invasiveness and tumor metastasis. The activities of this system in endothelial and leukocyte cell biology and the relevance of these activities to angiogenesis and tumor metastasis will be considered. Recent experimental evidence obtained using inhibitors of uPA and uPAR has validated this system as a therapeutic target for the development of anti-angiogenic and anti-metastatic therapeutic agents. These studies, as well as additional therapeutic and diagnostic implications for uPAR targeting, will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plasminogen activation in human acute leukaemias

Plasminogen activation is implicated in solid tumour growth, invasion and metatastic spread. However, little is known about its role in leukaemia. We investigated the production by leukaemic cells of plasminogen activators [urokinase (uPA) and tissue-type PA (tPA)], cell surface receptor for uPA (uPAR) and PA inhibitors (PAI-1 and PAI-2). Leukaemic cells from 37 patients [26 with acute myeloid leukaemia (AML) and 11 with acute lymphoid leukaemia (ALL)] were analysed for mRNA content and enzymatic activities. High levels of uPA mRNA were found in M1, M2, M3 and M4-M5 AMLs, whereas tPA mRNA was not detected in any of the analysed cases. uPAR mRNA was confined to subtypes M4-M5. PAI-1 mRNA was detected in M3 and M4-M5. PAI-2 mRNA was found predominantly in M2 and M4-M5. SDS-PAGE/zymography analyses of cell extracts and supernatants after 24 and 48 h of culture confirmed the production of active uPA by AML cells (mainly M4-M5). but not by ALL. The finding of uPA, uPAR, PAI-1 and PAI-2 synthesized by leukaemic cells suggests that plasminogen activation may contribute to the invasive behaviour of these cells, the fibrinolytic imbalance observed in leukaemic patients and the differentiation and proliferation of M4-M5 by interaction of uPA with uPAR.     

类风湿关节炎中尿激酶型纤溶酶原激活物及其受体蛋白和基因的表达

目的　检测尿激酶型纤溶酶原激活物 (uPA)及其受体 (uPAR)蛋白和mRNA在类风湿关节炎 (RA)的表达 ,探讨uPA、uPAR基因在RA细胞外基质降解中的作用。方法　采用免疫组化和cDNA mRNA原位分子杂交技术分别检测了 2 4例RA、18例骨关节炎 (OA)和 6例正常滑膜组织中uPA、uPAR蛋白和mRNA的分布及表达情况。结果　 2 4例RA滑膜组织均呈uPA、uPAR蛋白和mRNA的阳性表达 ,uPA、uPAR蛋白的强阳性率高于mRNA。uPA、uPAR蛋白和mRNA阳性信号主要分布在RA滑膜衬里细胞、滑膜下层单核细胞、巨噬细胞样细胞及血管内皮细胞 ;18例OA滑膜组织中 ,uPA、uPAR蛋白和mRNA的表达部位类似于RA ,但阳性率、阳性程度及分布范围均明显低于RA滑膜组织 ,两组之间蛋白和mRNA表达的差异均有显著性 (P <0 0 1或P <0 0 0 1)。 6例正常滑膜组织呈阴性反应。结论　RA滑膜组织存在高水平uPA、uPAR蛋白和mRNA的表达 ,提示在RA的发生发展过程中 ,uPA和uPAR基因起着重要作用 ;RA和OA中uPA、uPAR基因表达水平的差异 ,可能与这两种疾病软骨和骨基质降解的程度及进程等临床表现密切相关     

尿激酶型纤溶酶原激活物对糖尿病大鼠肾脏系膜基质的影响

目的 探讨尿激酶型纤溶酶原激活物（uPA）对糖尿病大鼠肾脏系膜基质表达的影响及机制.方法 8周龄健康雄性SD大鼠（体质量150～200 g）20只尾静脉注射链脲佐菌素,成功建立糖尿病大鼠模型后采用数字表法随机分为糖尿病组（n=10）和uPA组（n=10;尾静脉注射2500 U·kg^-1·d^-1 uPA,共4周）.另以10只正常大鼠为对照组.29d后处死大鼠,心脏取血检测血糖、血肌酐水平.过碘酸六胺银染色测定肾小球平均面积、肾小球平均容积和肾小球系膜区面积.免疫组织化学法检测肾脏尿激酶型纤溶酶原激活物受体（uPAR）、纤溶酶原激活物抑制剂-1（PAI-1）和Ⅳ型胶原表达水平.采用方差分析和q检验进行数据统计.结果 与正常对照组比较,糖尿病组大鼠明显出现尿蛋白[（25.4±4.3）mg/24 h vs（5.5±2.1）mg/24 h,q=4.27,P＜0.01],肾小球体积及系膜基质显著增加,肾小球系膜uPAR、PAI-1、Ⅳ型胶原表达显著增加（q值分别为3.63、3.97、4.21,均P＜0.05）.糖尿病大鼠注射uPA后,尿蛋白明显减少[（12.6±5.4）mg/24 h,q=3.45,P＜0.05],肾小球体积、系膜基质异常有所改善（q值分别为4.34、4.27,均P＜0.01）,肾小球系膜PAI-1、Ⅳ型胶原表达明显减少（q值分别为3.98、4.17,均P＜0.05）.结论注射uPA可明显降低糖尿病大鼠肾小球PAI-1蛋白表达,对uPAR表达的影响不大,提示uPA可能通过与uPAR结合、摄取PAI-1并加速其降解,从而调节肾小球系膜细胞及其基质表达,改善糖尿病系膜基质病变.     

Characteristics of fibrinolytic disorders in acute promyelocytic leukemia

ABSTRACT

Objectives: Catastrophic hemorrhage remains the main cause of acute promyelocytic leukemia (APL) treatment failure. This study was aimed to study the pathogenesis of coagulopathy in patients with APL.

Methods: Multiple procoagulant and profibrinolytic parameters in plasma and peripheral leukocytes from 24 patients with newly diagnosed APL accompanied by coagulopathy before and after arsenic trioxide (ATO) treatment were evaluated.

Results: Prior to the treatment, the patients had elevated D-dimer and decreased fibrinogen levels. Plasma urokinase-type plasminogen activator receptor (uPAR) and plasmin–ɑ₂ antiplasmin complexes (PAP) levels, plasmin (Pn) activity, and cell surface levels of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) were significantly higher; plasma plasminogen activator inhibitor-1 (PAI-1) levels and plasminogen (Pg) activity were significantly decreased; plasma plasminogen activator (PA) activity, uPA and tPA levels; and cell surface levels of uPAR and annexin II were not significantly different from levels in the control group. During ATO treatment, both patients’ plasma PA activity and uPAR on leukocytes gradually increased, annexin II on leukocytes increased initially and decreased afterwards, and tPA and uPA on leukocytes remained consistently higher in the patients than in the controls. Other parameters gradually tended toward normal values.

Conclusions: In APL, activated coagulation system activated fibrinolytic system, and increased uPAR levels could contribute to the hyperfibrinolysis. Annexin II might not be involved in the coagulopathy.     

Fibrinolytic characteristics and their significance in malignant, tuberculous and cirrhotic pleural and ascitic fluids

The fibrinolytic characteristics and clinical pathological significance of pleural and ascitic fluid were studied in patients with malignant tumour, tuberculosis or liver cirrhosis. Urokinase plasminogen activator (uPA) and urokinase plaminogen activator receptor (uPAR) levels were measured by enzyme-linked immunoadsorbent assay and tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), plasminogen (Plg), plasmin (Pl) and alpha(2) plasmin inhibitor (alpha(2)PI) by colorimetric assay. uPA and uPAR levels were higher in malignant tumour and tuberculosis compared with liver cirrhosis, whereas tPA levels were significantly higher in liver cirrhosis and malignant tumour than in tuberculosis patients. Tuberculosis patients showed statistically higher PAI-1, Plg and Pl concentrations than malignant tumour patients, which, in turn, were higher than those in liver cirrhosis patients. alpha(2)PI levels were markedly higher in malignant tumour and liver cirrhosis than in tuberculosis. In the malignant tumour group, only uPA level was significantly different between the samples that contained cancer cells and those that did not. We found significant differences between the fibrinolytic characteristics in pleural and ascitic fluid in patients with malignant tumour, tuberculosis and liver cirrhosis. The analysis of several fibrinolytic parameters could help to determine the quality of pleural and ascitic fluid, and also to further understand the pathological processes of these diseases.     

Expression of urokinase-type plasminogen activator receptor and plasminogen activator inhibitor-1 in gastric cancer

In gastric cancer, the urokinase-type plasminogen activator (uPA) system plays important roles in invasion and metastasis, processes which entail proteolysis and adhesion. Both the urokinasetype plasminogen activator receptor (uPAR) and the plasminogen activator inhibitor-1 (PAI-1) are thought to be important factors in this system. To clarify the relationship between these two factors and gastric cancer invasiveness, we evaluated the expression of uPAR and PAI-1 in 91 cases of gastric cancer by immunohistochemistry and in situ hybridization. Urokinase-type plasminogen activator receptor-mRNA, PAI-1-mRNA, uPAR and PAI-1 protein were diffusely distributed in the cytoplasm of the cancer cells and concentrated at invasive foci. Urokinase-type plasminogen activator receptor protein expression correlated with lymphatic, venous invasion (P<.01) and lymph node metastasis (P<0.05); uPAR-mRNA expression correlated with lymphatic, venous invasion and lymph node metastasis (P<0.05). Plasminogen activator inhibitor-1 protein expression correlated with lymphatic, venous invasion, lymph node metastasis and depth of invasion (P<0.01); PAI-1-mRNA expression was linked to lymphatic, venous invasion (P<0.01), lymph node metastasis and depth of invasion (P<0.05). This suggests that the proteolytic activity of uPAR and the cellular motility of PAI-1 in gastric cancer cells may determine penetration of lymphatic and blood vessels, whereby lymph node metastasis may be promoted and that the promotion of cellular motility by PAI-1 may influence the depth of cancer invasion.     

Urokinase-type plasminogen activator receptor regulates leukocyte recruitment during experimental pneumococcal meningitis

Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) have been suggested to play an important role in inflammatory diseases. Increased levels of tPA, uPA, uPA receptor (uPAR), and their inhibitor, plasminogen activator inhibitor (PAI)-1, have been found in the cerebrospinal fluid (CSF) of patients with bacterial meningitis. Here, we show that expression of tPA, uPA, uPAR, PAI-1, and PAI-2 is up-regulated during experimental pneumococcal meningitis. In uPAR-deficient mice, CSF pleocytosis was significantly attenuated 24 h after infection, compared with that in infected wild-type (wt) mice. Lack of uPAR did not influence blood-brain barrier permeability, intracranial pressure, expression of chemokines (keratinocyte-derived cytokine and macrophage inflammatory protein-2), bacterial killing, or clinical outcome. No differences in pathophysiological alterations were observed in tPA-deficient mice, compared with those in infected wt mice. These results indicate that uPAR participates in the recruitment of leukocytes to the CSF space during pneumoccal meningitis.     

类风湿关节炎患者滑膜、滑液和血浆中PAI的检测及其临床意义

目的 检测Ⅰ型和Ⅱ型纤深酶原激活物抑制剂（ＰＡＩ－１和ＰＡＩ－２）在类风湿关节炎（ＲＡ）患者滑膜组织中的定位和表达,同时测定ＲＡ患者滑液和血浆中ＰＡＩ－１的含量和活性,并分析其临床意义。方法 运用免疫组织化学方法检测了２４例ＲＡ、１８例骨关节炎（ＯＡ）和６例正常滑膜组织中ＰＡＩ－１和ＰＡＩ－２的定位及其表达;采用ＥＬＩＳＡ双抗体夹心法测定４６例ＲＡ和８例ＯＡ患者血浆、１４例ＲＡ滑液和１２例正常对照     

Chromosomal localization of the human urokinase plasminogen activator receptor and plasminogen activator inhibitor type-2 genes: Implications in colorectal cancer

Abstract Activation of the proenzyme of urokinase (uPA) on the surface of cancer cells has been implicated in the initiation of focal proteolytic mechanisms that permit invasion and metastasis by colon cancers. The activity of uPA on the cell surface appears to be a function of the number of uPA-specific receptors (uPAR) and the extent of inhibition of uPA by plasminogen activator inhibitors (PAI). The mapping of the genes coding for uPAR, and for PAI-2, was performed to determine whether their chromosomal localization suggested their involvement in the genetic alterations associated with cancer cell DNA.
This study confirms the localization of the human urokinase plasminogen activator receptor gene to chromosome 19q and, using in situ hybridization, provides a precise localization to chromosome 19q13.2. In addition, our results confirm the previous allocation of the human plasminogen activator inhibitor-2 gene to a location 18q21.3 → 18q21.1, a location that corresponds to the commonest (>70%) somatic deletions found in colorectal carcinomas. The mapping of the uPAR and PAI-2 genes enables the elucidation of their possible involvement in the genetic alterations that determine the invasive and metastatic phenotypes in colorectal cancer.