The effects of gonadotropin-releasing hormone (GnRH) I and GnRH II on the urokinase-type plasminogen activator/plasminogen activator inhibitor system in human extravillous cytotrophoblasts in vitro

The regulated expression of the urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) is believed to modulate the invasive capacity of human trophoblastic cells in vitro and in vivo. To date, the factors capable of regulating the expression of uPA and PAI-1 in these cells remain poorly characterized. In these studies, we have examined the ability of the classical mammalian GnRH I and the second form of GnRH (GnRH II) to regulate uPA and PAI-1 mRNA and protein expression levels in primary cultures of human extravillous cytotrophoblasts using quantitative competitive PCR and ELISA, respectively. Both GnRH I and II increased uPA and concomitantly decreased PAI-1 mRNA and protein expression levels in our extravillous cytotrophoblast cultures in a dose- and time-dependent manner. Cetrorelix, a peptide GnRH antagonist specific for the GnRH I receptor, was capable of inhibiting the regulatory effects of GnRH I, but not GnRH II on uPA and PAI-1 expression levels in primary cell cultures. Taken together, these observations suggest that GnRH I and GnRH II may facilitate trophoblast invasion by increasing the ratio of uPA/PAI-1 expression via interactions with two distinct GnRH receptors.     

Differential regulation of tissue factor and plasminogen activator inhibitor by human mononuclear cells

Fibrin is a hallmark of immune-mediated tissue lesions. The presence of fibrin in such lesions implies both the formation of fibrin via coagulation and the accompanying restriction of fibrinolysis, allowing fibrin to persist. Previous work has shown that human monocytes exposed to an inflammatory stimulus such as lipopolysaccharide (LPS) produce both tissue factor (TF) and plasminogen activator inhibitor--type 2 (PAI-2). These two proteins favor fibrin deposition, and evidence implies that cellular production of these two molecules may be linked. Another proinflammatory process pertinent to immune-mediated tissue damage and fibrin deposition is the response to alloantigen. Peripheral- blood mononuclear cells (PBM), consisting of lymphocytes and monocytes together, responded to alloantigen stimulation with differential expression of TF and PAI-2. PBM exposed to alloantigen developed high levels of TF activity, with no concomitant increase in PAI-2 activity or antigen. Alloantigen-stimulated PBM did not accumulate intracellular PAI-2, nor did they degrade PAI-2 added to cultures. This lack of PAI-2 production was not due to inadequate stimulation, as tritiated thymidine uptake and TF production demonstrated recognition of, and a vigorous reaction to, alloantigen. The divergent TF and PAI-2 responses of PBM exposed to alloantigen was maintained over 5 days and was reflected by mRNA profiles. These results imply that under specific physiologically relevant conditions, the procoagulant and antifibrinolytic effectors of inflammatory mononuclear cells can be independently regulated. This would imply more flexibility to monocyte mechanisms that favor fibrin deposition than previously thought.     

Prourokinase activation on the surface of human rhabdomyosarcoma cells: localization and inactivation of newly formed urokinase-type plasminogen activator by recombinant class 2 plasminogen activator inhibitor.

Recombinant class 2 plasminogen activator inhibitor (PAI-2) was used in an approach to probe the formation and location of enzymatically active urokinase-type plasminogen activator (u-PA) sites on the surface of cultured human rhabdomyosarcoma cells (RD cells). Activation of pro-u-PA on the cell surface and consequent binding of PAI-2 was dependent on the addition of native plasminogen to serum cultures of the cells. Inhibition of the enzyme activity of surface-bound u-PA by the added PAI-2 resulted in a 79% reduction in the capacity of the RD cells to generate cell surface-associated plasmin activity from bound plasminogen. Under these conditions, the PAI-2 probe was localized at focal adhesions of RD cells, where it colocalized with both extracellular u-PA and intracellular vinculin antigens in double immunofluorescence labeling. Specificity of the probe's interaction with cell surface-bound u-PA was confirmed by blocking with a monoclonal antibody to human u-PA, which could also inhibit the formation of bound plasmin activity. These results showed the assembly of the plasmin-generating system at focal adhesions and the accessibility of bound u-PA on which it depends to added PAI-2. Therefore, PAI-2 has the potential both to localize at sites of tumor expression of functionally active u-PA and simultaneously to inhibit cell surface plasminogen activation.     

尿激酶型纤溶酶原激活物途径在大鼠酒精性肝纤维化形成中的作用

目的 通过观察尿激酶型纤溶酶原激活物(uPA)及其抑制物(PAI-1)在大鼠酒精性肝纤维化形成中的动态变化,探讨uPA纤溶途径在酒精性肝纤维化形成中的作用.方法 雄性SD大鼠随机分为空白组、四氯化碳(CCl4)组和造模组.采用56度二锅头酒、玉米油、吡唑混合物灌胃联合腹腔注射CCl4橄榄油溶液(CCl4∶橄榄油=1∶3...     

人尿激酶型纤溶酶原激活剂重组腺病毒的构建及其在肝星形细胞中的表达

我们利用AdEasy系统在细菌内构建携带非分泌型尿激酶型纤溶酶原激活剂（urokinase-type plasminogen acti-vator,uPA）和绿色荧光蛋白（greenfluorescent protein,GFP）cDNA的复制缺陷型腺病毒,并观察其在肝星形细胞（hepatic stellate cell,HSC）中的表达。     

Differential regulation by troglitazone of plasminogen activator inhibitor type 1 in human hepatic and vascular cells

Troglitazone, a novel oral insulin sensitizer, normalizes increased plasma activity of plasminogen activator inhibitor type 1 (PAI-1) in hyperinsulinemic patients such as women with polycystic ovary syndrome and patients with type 2 diabetes mellitus. However, underlying mechanisms have not yet been fully elucidated. Human hepatic and vascular cells, the main sources of circulating PAI-1, were studied in cell culture. In human hepatic cells, PAI-1 accumulated in conditioned medium by 23% within 24 h after exposure to 3 microg/mL troglitazone (P = 0.001). The accumulation depended on the concentration of troglitazone, but not that of insulin (known to stimulate PAI-1 synthesis). By contrast, in human aortic smooth muscle cells, 3 microg/mL troglitazone decreased basal PAI-1 expression by 23% (P = 0.037) and decreased transforming growth factor-beta-induced expression by 34% (P = 0.026). Concomitant insulin had no effect. Tissue-type plasminogen activator was decreased by 38% (P = 0.002). In human endothelial cells, PAI-1 was diminished by 32% (P < 0.001), whereas tissue-type plasminogen activator was unaffected. The results suggest that the reduction in plasma activity of PAI-1 induced by troglitazone in patients may reflect both directly mediated diminution of its elaboration from vessel walls and indirectly mediated reduction of its hepatic synthesis secondary to attenuation of hyperinsulinemia (known to increase the hepatic synthesis of PAI-1).     

Tumor necrosis factor induces the production of urokinase-type plasminogen activator by human endothelial cells

Endothelial cells play an important role in the regulation of fibrinolysis by the production of several key regulatory proteins. The cytokines tumor necrosis factor (TNF), lymphotoxin, and interleukin-1 (IL-1), but not interleukin-6, increase the production of plasminogen activator inhibitor-1 (PAI-1) by endothelial cells, whereas they have no stimulatory effect on the production of tissue-type plasminogen activator (t-PA). Primary cultures of human endothelial cells release very little urokinase-type plasminogen activator (u-PA). We report here that TNF and lymphotoxin induce, in a concentration-dependent way, the production of both cellular and secreted u-PA antigen in primary and subcultured human endothelial cells. The TNF-induced increase was accompanied by a more than 10-fold increase in u-PA mRNA. Upon stimulation of early passage umbilical vein endothelial cells by TNF, u-PA was predominantly secreted at the basolateral side, whereas PAI activity and t-PA were found in more equal amounts at the apical and basolateral sides of the cell monolayers. TNF-stimulated u-PA secretion by subcultured human aorta endothelial cells showed only a marginal polarity. The u-PA antigen was present in a plasmin-activatable form (single chain u-PA) and in a nonactivatable form (probably u-PA: PAI-1 complex). During the induction of u-PA by TNF, the ratio between plasmin-activatable u-PA and total u-PA decreased markedly. This may indicate that TNF also increases the degree of u-PA activation. The parallel induction of the synthesis and secretion of both u-PA and PAI-1 by endothelial cells adds a new aspect to the alterations of the fibrinolytic system caused by inflammatory mediators. This aspect may be significant for the regulation of cell-associated and interstitial plasminogen activator activity.     

Demonstration of single chain urokinase-type plasminogen activator on human platelet membrane

Fibrinolytic activity was found to be associated with sonicated platelet membranes after separation from cytosol by differential centrifugation. This fibrinolytic activity was attributed to the presence of a plasminogen activator, which was immunochemically identified as urinary-type plasminogen activator (uPA) by antibody neutralization assay, immunoblotting, and immunofluorescence. The molecular weight (mol wt) of this uPA was 54,000 and was present as the single chain form, although a small amount was detected in a higher mol wt complex indicative of a uPA-inhibitor complex. Treatment of membrane preparations with Triton X-100, 3 mol/L KCl, and 0.1 mol/L glycine, (pH 2.3), but not 10 mmol/L ethylenediamine tetraacetic acid (EDTA), removed the uPA from the membrane. This suggests that uPA is a peripheral membrane protein and that metal ions do not mediate protein- membrane association. Immunofluorescent staining revealed the presence of uPA on the outer surface of the platelet in preparations of intact unstimulated platelets. Thus, uPA is associated with the outer leaflet of the platelet membrane and may be involved with the acceleration of thrombus degradation observed with platelet-rich thrombi.     

Hyperglycemia is associated with lower levels of urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor in wound fluid

AimsWounds in patients with hyperglycemia show impaired healing. Plasminogen activation is crucial in several overlapping phases of wound healing process. In this study, we aimed i) to compare acute wound fluid in patients with hyperglycemia and normoglycemia, ii) to focus on the elements of plasminogen activation in the wound fluid, and iii) to determine if the acute wound fluid characteristics are associated with surgical site infections.MethodsIn a cohort of 54 patients, a closed suction drain was placed in the wound above the anterior abdominal wall fascia under the skin in order to collect postoperative acute wound fluid samples for 3 following days after colorectal surgery. Patients were classified as normoglycemic (n = 25) or hyperglycemic (n = 29; 17 with type 2 diabetes and 12 with stress induced hyperglycemia). Surgical site infection was defined according to the Centers for Disease Control criteria. The levels of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAr), plasminogen activator inhibitor-1 (PAI-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and fibroblast growth factor-1 (FGF-1) were measured in the wound fluid.ResultsCompared to normoglycemic subjects, patients with hyperglycemia had significantly lower levels of uPA and uPAr in the wound fluid despite similar or even higher circulating levels. There was no significant difference in IL-1β, TNF-α, PAI-1 and FGF-1 levels. In the whole study population, the wound fluid levels of uPA and uPAr were negatively correlated with circulating glucose levels. No difference was detected in the wound fluid characteristics of patients with and without surgical site infection.ConclusionPatients with hyperglycemia exhibit decreased levels of uPA and uPAr in the wound fluid, suggesting a local failure in plasminogen activation at the wound site.     

Independent regulation of plasminogen activator inhibitor 2 and plasminogen activator inhibitor 1 in human synovial fibroblasts

Objective. To study the plasminogen activator inhibitor(s) (PAI) produced in vitro by human synovial fibroblast-like cells. Methods. Human synovial cell explant cultures were established using cells from nonrheumatoid donors. PAI-2 and PAI-1 antigens were measured by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) levels were determined by Northern blot. Results. The synovial fibroblasts produced both PAI-2 and PAI-1. Interleukin-1 (IL-1) increased PAI-2 but decreased PAI-1 formation, both at the protein and the mRNA levels. Using cyclooxygenase inhibitors, evidence was obtained that an endogenous cyclooxygenase product(S) in the IL-1—treated cultures inhibited formation of both PAIs; exogenous prostaglandin E₂ (10⁻⁷M) reversed the effect of cyclooxygenase inhibition. The glucocorticoid dexamethasone (10⁻⁶ to 10⁻⁷M) inhibited IL-1—stimulated PAI-2 formation but reversed the suppressive effect of IL-1 on PAI-1 production. Conclusion. PAI-2 formation and PAI-1 formation can be regulated independently in human synoviocytes, illustrating the complexity of the modulation of the net PA activity expressed by these cells.     

导入外源人尿激酶型纤溶酶原激活剂基因对肝星状细胞胶原沉积的影响

目的　利用重组复制缺陷型腺病毒载体将外源非分泌型人尿激酶型纤溶酶原激活剂(uPA)基因导入活化的肝星状细胞 (HSC)内 ,研究uPA表达对HSC胶原沉积的影响。方法　细菌内同源重组构建表达非分泌型uPA的复制缺陷型重组腺病毒AduPA。体外感染肝星状细胞株HSC T6 ,Northern印迹法和免疫细胞化学法检测uPA在HSC中的表达。酶联免疫吸附实验测定培养上清基质金属蛋白酶 2 (MMP 2 )分泌水平。免疫细胞荧光法测定外源uPA基因导入对HSC胞质内Ⅰ、Ⅲ型胶原含量变化的影响。结果　同源重组最终获得约 5× 1 0 1 1 efu/mlAduPA。AduPA体外感染HSC 3d后 ,Northern印迹法和免疫细胞化学法显示uPAmRNA及蛋白表达明显增加 ,细胞上清液中MMP 2分泌水平达 (2 74 .4 5± 7.6 3) pg/ml,明显高于对照组的 (1 4 5 .85± 6 .5 8)pg/ml(P <0 .0 1 )。免疫细胞荧光法提示uPA基因导入HSC后 ,胞质内Ⅰ、Ⅲ型胶原含量明显减少。结论　通过重组腺病毒载体将外源uPA基因导入HSC内可维持uPA高效表达并上调MMP 2分泌水平 ,减少细胞外基质沉积 ,是基因治疗肝纤维化的有效方法     

Plasminogen activator inhibitor 2 and urokinase-type plasminogen activator in plasma and leucocytes in patients with severe sepsis

Proteins influencing plasminogen activation to plasmin, namely plasminogen activators tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and their principal inhibitors, plasminogen activator inhibitor 1 (PAI-1) and PAI-2, were measured in the plasma, the polymorph and mononuclear cell fractions taken from patients with major sepsis who were entering a general intensive care unit. The purpose of this study was to elucidate the factors favouring the persistence of fibrin in the microvasculature and thus contributing to multiple organ failure. Levels of u-PA antigen in plasma rose in sepsis and u-PA activity, not detectable in normal plasma, appeared. Levels of u-PA antigen in the cell fractions fell concomitantly. t-PA antigen in plasma and in the mononuclear cell fraction rose in sepsis, but t-PA activity was not detectable. Plasma PAI-1 antigen levels were strikingly raised in sepsis, presumably accounting for the complete neutralization of t-PA activity. PAI-2 antigen, not normally detected in plasma, appeared in the plasma of some patients, whereas it disappeared from the cellular fractions. Appearance of PAI-2 in plasma was associated with non-survival of the patient. The observations indicate that all the agents involved in plasminogen activation are released into the plasma in major sepsis. The levels of PAI-1 reached were quantitatively sufficient to suppress all activity of the released t-PA, but the inhibitors did not prevent expression of u-PA activity in the circulation. Circulating active u-PA and PAI-2 in the plasma of patients with severe sepsis may represent material originating from leucocytes. Leucocyte release of these agents within fibrin deposits may influence the persistence of fibrin and thus the development of multiple organ failure.     

Independent regulation of plasminogen activator inhibitor 2 and plasminogen activator inhibitor 1 in human synovial fibroblasts.

OBJECTIVE. To study the plasminogen activator inhibitor(s) (PAI) produced in vitro by human synovial fibroblast-like cells. METHODS. Human synovial cell explant cultures were established using cells from nonrheumatoid donors. PAI-2 and PAI-1 antigens were measured by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) levels were determined by Northern blot. RESULTS. The synovial fibroblasts produced both PAI-2 and PAI-1. Interleukin-1 (IL-1) increased PAI-2 but decreased PAI-1 formation, both at the protein and the mRNA levels. Using cyclooxygenase inhibitors, evidence was obtained that an endogenous cyclooxygenase product(s) in the IL-1-treated cultures inhibited formation of both PAIs; exogenous prostaglandin E2 (10(-7) M) reversed the effect of cyclooxygenase inhibition. The glucocorticoid dexamethasone (10(-6) to 10(-7) M) inhibited IL-1-stimulated PAI-2 formation but reversed the suppressive effect of IL-1 on PAI-1 production. CONCLUSION. PAI-2 formation and PAI-1 formation can be regulated independently in human synoviocytes, illustrating the complexity of the modulation of the net PA activity expressed by these cells.     

Differential effects of nebivolol and metoprolol on insulin sensitivity and plasminogen activator inhibitor in the metabolic syndrome

Early-generation β-blockers lower blood pressure and reduce cardiovascular morality in coronary artery disease and congestive heart failure but worsen glucose homeostasis and fibrinolytic balance. Nebivolol is a third-generation β-blocker that increases the bioavailability of nitric oxide. We compared the effect of nebivolol (5 mg/d) and the β(1)-selective antagonist metoprolol (100 mg/d) on glucose homeostasis and markers of fibrinolysis in 46 subjects with metabolic syndrome. Subjects underwent a frequently sampled IV glucose tolerance test after 3-week washout and placebo treatment and after randomized treatment with study drug. After 12-week treatment, nebivolol and metoprolol equivalently decreased systolic blood pressure, diastolic blood pressure, and heart rate. Neither drug affected β-cell function, disposition index, or acute insulin response to glucose. Metoprolol significantly decreased the insulin sensitivity index. In contrast, nebivolol did not affect insulin sensitivity, and the decrease in sensitivity was significantly greater after metoprolol than after nebivolol (-1.5±2.5×10(-4)×min(-1) per milliunit per liter versus 0.04±2.19×10(-4)×min(-1) per milliunit per liter after nebivolol; P=0.03). Circulating plasminogen activator inhibitor also increased after treatment with metoprolol (from 9.8±6.8 to 12.3±7.8 ng/mL) but not nebivolol (from 10.8±7.8 to 10.5±6.2 ng/mL; P=0.05 versus metoprolol). Metoprolol, but not nebivolol, increased F(2)-isoprostane concentrations. In summary, treatment with metoprolol decreased insulin sensitivity and increased oxidative stress and the antifibrinolytic plasminogen activator inhibitor 1 in patients with metabolic syndrome, whereas nebivolol lacked detrimental metabolic effects. Large clinical trials are needed to compare effects of nebivolol and the β(1) receptor antagonist metoprolol on clinical outcomes in patients with hypertension and the metabolic syndrome.     

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.