Expression of urokinase plasminogen activator and the urokinase plasminogen activator receptor in myeloma cells

Binding of urokinase (uPA) to its receptor (uPAR; CD87) focuses proteolytic activity on the cell surface and this system is of importance in malignant matrix degradation and tumour invasion. By immunocytochemistry and flow cytometry, we found that primary myeloma cells and myeloma cell lines expressed uPA and uPAR. Soluble uPA was present in cell line supernatants and lysates in low concentrations. In cell lines, uPA and uPAR were located both on the cell surface and intracellularly, but the expression of both proteins was low. Higher levels of uPAR was detected on the cell surface of primary myeloma cells. When primary myeloma cells were gated by CD45 expression, stronger expression was found on immature CD45+ cells than on mature CD45-/dim cells. Finally, both myeloma cell lines and primary cells were able to cleave a uPA-specific substrate showing that the uPA system is functionally active. We conclude that myeloma cells are able to produce uPA and uPAR. This opens up a possible role of the uPA system in myeloma cell invasion and in the proteolytic digestion of bone matrix.     

Elevated soluble urokinase plasminogen activator receptor in plasma from patients with idiopathic myelofibrosis or polycythaemia vera

Extracellular proteolytic enzymes of the urokinase-type plasminogen activator (uPA) system and the family of metalloproteases play a crucial role in the matrix degradation and tissue remodelling processes characteristic of malignant disorders. The receptor for urokinase plasminogen activator (uPAR) serves to localise and intensify the action of uPA and is expressed on the surface of malignant as well as tumour stromal cells including fibroblasts. A soluble form of uPAR (suPAR) cleaved from its glycosyl-phosphatidylinositol anchor is detected in plasma from healthy individuals and increased levels of suPAR have been found in advanced malignancy, suggesting that suPAR may be a marker of extensive tissue remodelling. In an attempt to clarify whether suPAR might be a marker for bone marrow tissue remodelling we measured plasma suPAR levels in a patient cohort comprising 17 with myelofibrosis (MF), 17 with polycythaemia vera (PV), 15 with essential thrombocythaemia (ET), one with a transitional myeloproliferative disorder evolving from PV and 30 controls. Compared with controls suPAR levels were significantly higher in the patients (P < 0.0001) (median 3.35 vs. 2.32 microg L(-1)). Moreover, in subgroup analyses including patients with MF, PV, and ET, respectively, suPAR levels differed significantly with the highest levels found in patients with MF and PV (MF vs. PV vs. ET; P = 0.0003). When comparing suPAR levels of the individual patient subgroups with controls, only suPAR levels of PV and MF patients were significantly increased (P < 0.0001). Sixty-five percent of patients with PV and MF (22/34) had suPAR plasma values that were above the mean +2 standard deviations (SD) of controls. The concentration of suPAR was significantly correlated to plasma lactate dehydrogenase, thrombomodulin, and complex of tPA:PAI-1 in the patients. There was no difference between patients and controls when comparing plasma uPA levels. Increased plasma suPAR levels in patients with chronic myeloproliferative disorders may reflect increased uPAR production in the bone marrow, leading to enhanced bone marrow remodelling.     

肝硬化患者血浆中尿激酶型纤溶酶激活物的检测及其意义

目的 探讨肝硬化患者血浆尿激酶型纤溶酶激活物(uPA)、尿激酶型纤溶酶激活物受体(uPAR)、纤溶酶原激活物抑制剂-1(PAI-1)的变化及其意义。 方法 确诊的72例乙型肝炎后肝硬化患者,Child-pugh分级A级23例(A组),B级29例(B组),C级20例(C组)。6例健康志愿献血者为正常对照组。酶联免疫吸附实验测定血浆uPA、uPAR、PAI-1的变化。并同时检测血透明质酸(HA)、Ⅳ型胶原(C Ⅳ)、Ⅲ型前胶原(PC Ⅲ)、血浆白蛋白、胆红素、凝血酶原时间及其活动度改变。 结果 随着肝硬化的进展,血浆uPA、uPAR、PAI-1逐渐增加,HA、PC Ⅲ也明显增加。Child C组患者血浆uPA、uPAR、PAI-1水平(μg/L)分别为1.88±0.64、4.82±2.02和52.60±16.87,A组分别为1.36±0.43、3.03±1.48和24.09±7.14,B组分别为1.79±0.62、4.80±2.22和41.40±17.52,C组与A、B组比较,t值为2.81～7.38,P值均<0.01。A组血浆uPA与PC Ⅲ呈负相关(r=-0.4785,P<0.05);C组PAI-1与HA呈正相关(r=0.5447,P<0.01)。 结论 肝硬化晚期,虽然血浆uPA、PAI-1增加,但总的效应表现为uPA相对不足,肝基质纤维降解受抑制,血浆uPA、PAI-1与肝硬化发展密切相关。     

Circulating soluble urokinase plasminogen activator receptor predicts cancer,cardiovascular disease,diabetes and mortality in the general population

Abstract. Eugen‐Olsen J, Andersen O, Linneberg A, Ladelund S, Hansen TW, Langkilde A, Petersen J, Pielak T, Møller LN, Jeppesen J, Lyngbæk S, Fenger M, Olsen MH, Hildebrandt PR, Borch‐Johnsen K, Jørgensen T, Haugaard SB (Copenhagen University, Hvidovre Hospital, Hvidovre; Copenhagen University Hospital, Glostrup; Copenhagen University Hospital, Copenhagen; Copenhagen University Hospital, Glostrup; Copenhagen University, Hvidovre Hospital, Hvidovre; Steno Diabetes Center, Gentofte; University of Aarhus, Aarhus; University of Copenhagen, Copenhagen; Copenhagen University, Hvidovre Hospital, Hvidovre, Denmark). Circulating soluble urokinase plasminogen activator receptor predicts cancer, cardiovascular disease, diabetes and mortality in the general population. J Intern Med 2010; 268 : 296–308. Background. Low‐grade inflammation is thought to contribute to the development of cardiovascular disease (CVD), type‐2 diabetes mellitus (T2D), cancer and mortality. Biomarkers of inflammation may aid in risk prediction and enable early intervention and prevention of disease. Objective. The aim of this study was to investigate whether plasma levels of the inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) are predictive of disease and mortality in the general population. Design. This was an observational prospective cohort study. Cohort participants were included from June 1993 to December 1994 and followed until the end of 2006. Setting. General adult Caucasian population. Participants. The MONICA10 study, a population‐based cohort recruited from Copenhagen, Denmark, included 2602 individuals aged 41, 51, 61 or 71 years. Measurements. Blood samples were analysed for suPAR levels using a commercially available enzyme‐linked immunosorbent assay. Risk of cancer (n = 308), CVD (n = 301), T2D (n = 59) and mortality (n = 411) was assessed with a multivariate proportional hazards model using Cox regression. Results. Elevated baseline suPAR level was associated with an increased risk of cancer, CVD, T2D and mortality during follow‐up. suPAR was more strongly associated with cancer, CVD and mortality in men than in women, and in younger compared with older individuals. suPAR remained significantly associated with the risk of negative outcome after adjustment for a number of relevant risk factors including C‐reactive protein levels. Limitation. Further validation in ethnic populations other than Caucasians is needed. Conclusion. The stable plasma protein suPAR may be a promising biomarker because of its independent association with incident cancer, CVD, T2D and mortality in the general population.     

Serum levels of soluble urokinase plasminogen activator receptor is associated with parasitemia in children with acute Plasmodium falciparum malaria infection

Serum levels of soluble urokinase plasminogen activator receptor (suPAR) are significantly elevated and of prognostic value in patients suffering from serious infectious diseases such as HIV and tuberculosis. Our objective was to investigate suPAR levels during symptomatic malaria infection and 7 days after treatment. Children younger than 6 years who presented with fever or other symptoms compatible with malaria were enrolled. Blood films and samples were collected on day 0 and day 7. Twenty-five children were allocated to each of three groups according to the amount of Plasmodium falciparum detected in their initial blood film. Children in group 1 had parasite densities in excess of 20 parasites per 200 leucocytes. The median plasma suPAR level was 6.49 ng/mL (interquartile range [IQR]: 4.90-7.61) and correlated to parasitemia (Spearman 0.43, P < 0.0001). Blood was obtained from 20 children in group 1 after 7 days of treatment. All became malaria negative in their blood slides and all decreased in suPAR level to median 3.48 ng/mL (IQR: 3.08-3.91) (P < 0.0001). Group 2 consisted of 25 children with 1-20 parasites in their blood slide. The suPAR level was median 2.91 ng/mL (IQR: 2.27-4.40) and decreased with median 0.5 ng/mL following treatment (P = 0.0002). Group 3 showed to be negative in their blood slides and most received antibiotic treatment. suPAR decreased from median 3.26 ng/mL (IQR: 2.77-4.46) to median 2.47 ng/mL (IQR: 2.01-3.75), on day 7 (P = 0.006). This study demonstrates an important association between suPAR and acute malaria infection in humans.     

Interleukin-1beta regulates urokinase plasminogen activator (u-PA), u-PA receptor, soluble u-PA receptor, and plasminogen activator inhibitor-1 messenger ribonucleic acid expression in cultured human endometrial stromal cells

The interleukin-1 (IL-1) system plays an integral role in local intercellular interactions during implantation. In addition, the plasminogen activator system, especially urokinase plasminogen activator (u-PA), plasminogen activator inhibitor (PAI-1), and u-PA receptor (u-PAR), are crucial during embryo implantation. Decidualization and implantation are complex processes dependent upon several proteases, including u-PA, and IL-1 is known to affect PA activity in several cell types. We investigated the role of IL-1beta in regulating u-PA, PAI-1, u-PAR, and soluble u-PAR messenger ribonucleic acid (mRNA) expression in cultured human endometrial stromal cells using quantitative competitive PCR. For confirmation of the mRNA data, we measured PAI-1 and u-PAR protein by enzyme-linked immunosorbent assay. Confluent stromal cell cultures treated with progesterone and estradiol for 9 days were stimulated with IL-1beta, and IL-1beta plus IL-1beta antibody for an additional 24 h. Total RNA was extracted, reverse transcribed, and coamplified using quantitative and competitive PCR with internal standards. IL-1beta increased PAI-1, u-PAR, and soluble u-PAR expression in a dose-dependent manner, and this result was reversed by anti-IL-1beta antibody treatment. u-PA mRNA expression was not dependent on IL-1beta. These results suggest that IL-1 may be important in regulating PAI-1 and u-PAR during stromal cell decidualization before implantation.     

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.     

Soluble urokinase plasminogen activator receptor as a prognostic marker in men participating in prostate cancer screening

Abstract. Kjellman A, Akre O, Gustafsson O, Høyer‐Hansen G, Lilja H, Norming U, Piironen T, Törnblom M (Department of Clinical Science, Intervention, and Technology; Clinical Epidemiology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Copenhagen, Denmark; Departments of Clinical Laboratories, Urology and Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Department of Clinical Science and Education, Södersjukhuset, Karolinska Institutet, Stockholm, Sweden; and Syrinx Bioanalytics Oy, Turku, Finland; formerly at Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Copenhagen, Denmark). Soluble urokinase plasminogen activator receptor as a prognostic marker in men participating in prostate cancer screening. J Intern Med 2010; 269 : 299–305. Background. The urokinase plasminogen activator (uPA) system is involved in tissue remodelling processes and is up‐regulated in many types of malignancies. We investigated whether serum levels of different forms of soluble uPA receptor (suPAR) are associated with survival and in particular with prostate cancer and cardiovascular disease mortality. Methods. Using time‐resolved fluorescence immunoassays, we measured intact suPAR [suPAR(I‐III)] and intact plus cleaved suPAR [suPAR(I‐III) + suPAR(II‐III)] and thus calculated the amount of suPAR(II‐III) in serum samples from 375 men participating in a prostate cancer screening trial. A total of 312 men were free of prostate cancer and 63 men had prostate cancer diagnosed at the time of screening. The cohort was followed for 15 years. We assessed survival using Kaplan–Meier estimation and Cox proportional hazards regression. Results. The mean age at blood sampling was 64 years. In total, 152 men died during follow‐up. SuPAR(I‐III) and suPAR(II‐III) were significantly positively associated with mortality (P = 0.001 and P < 0.0001, respectively). In a Cox regression model adjusting for age and prostate cancer status, an increase in suPAR(I‐III) and suPAR(II‐III) by 1‐unit (ln‐scale) was associated with a hazard ratio (HR) of 2.26 [95% confidence interval (CI) 1.17–4.35] and 2.53 (95% CI 1.56–4.10), respectively. There was a trend towards an increased risk of death from prostate cancer in screening‐detected prostate cancer patients with increased values of either suPAR form. However, this difference was not significant and the association disappeared after adjusting for age, tumour stage, tumour grade and prostate‐specific antigen. Being in the highest quartile of any of the suPAR forms was associated with a highly significant increased risk of cardiovascular death, with HR adjusted for age of 3.27 (95% CI 1.38–7.73) for suPAR(I‐III) quartile 4 versus quartile 1. Conclusions. High concentrations of serum suPAR(I‐III) and suPAR(II‐III) were associated with poor overall survival. The association was particularly strong for death from cardiovascular disease. No similar association was found for prostate cancer after adjustment for other prognostic factors.     

Elevation of serum urokinase plasminogen activator receptor and liver stiffness in postoperative biliary atresia

AIM To investigate serum urokinase-type plasminogen activator receptor(u PAR) and liver stiffness in biliary atresia(BA) and examine the correlation of circulating u PAR, liver stiffness, and clinical outcomes in postoperative BA children.METHODS Eighty-five post Kasai BA children and 24 control subjects were registered. Circulating u PAR was measured using enzyme-linked immunosorbent essay. Liver stiffness was analyzed using transient elastography.RESULTS BA children had significantly greater circulating u PAR andliver stiffness scores than control subjects(P 0.001). Circulating u PAR and liver stiffness were substantially higher in jaundiced BA children than non-jaundiced BA children(P 0.001). In addition, circulating u PAR was positively associated with serum aspartate aminotransferase(r = 0.507, P 0.001), alanine aminotransferase(r = 0.364, P 0.001), total bilirubin(r = 0.559, P 0.001), alkaline phosphatase(r = 0.325, P 0.001), and liver stiffness scores(r = 0.508, P 0.001).CONCLUSION Circulating u PAR and liver stiffness values were greater in BA children than healthy controls. The increased circulating u PAR was associated with liver dysfunction in BA. As a consequence, serum u PAR and liver stiffness may be used as noninvasive biomarkers indicating the progression of liver fibrosis in post Kasai BA.     

Soluble urokinase plasminogen activator receptor suPAR as a marker for inflammation in pediatric inflammatory bowel disease

Abstract Objective. In inflammatory bowel disease (IBD), more means to monitor early therapeutic response are needed. In pediatric IBD, blood inflammatory markers erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) may be low in 10 to 20% of patients with severe disease. Recently, soluble urokinase plasminogen activator receptor (suPAR) was described as a potential blood inflammatory marker in adult IBD. Methods. We tested the performance of suPAR by the start of therapy with glucocorticoids (n = 19) or TNF-α-antagonist (n = 16) in pediatric IBD (Crohn's disease n = 19, ulcerative colitis (UC) n = 16). Results. The levels of suPAR were low in both patient groups studied. There was no difference in the values regarding the presence of Crohn's disease or ulcerative colitis. Thus, all analyses were performed on the entire sample set. Glucocorticoid therapy, however, resulted in a significant decline in suPAR levels from a median of 3.06 to 2.54 ng/ml (p < 0.01). In contrast, TNF-α-antagonist had no effect. The suPAR levels did not associate with ESR or CRP or fecal calprotectin (FC). Conclusions. In pediatric IBD, the suPAR levels in blood are low and do not reflect the level of intestinal inflammation assessed with FC. The introduction of corticoids, however, results in a decline of suPAR levels in blood but not reflect therapeutic response to TNF-α-antagonist. Thus, suPAR is of limited value in assessing systemic inflammatory responses in pediatric IBD.     

Soluble urokinase plasminogen activator receptor suPAR as a marker for inflammation in pediatric inflammatory bowel disease

Abstract

Objective. In inflammatory bowel disease (IBD), more means to monitor early therapeutic response are needed. In pediatric IBD, blood inflammatory markers erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) may be low in 10 to 20% of patients with severe disease. Recently, soluble urokinase plasminogen activator receptor (suPAR) was described as a potential blood inflammatory marker in adult IBD. Methods. We tested the performance of suPAR by the start of therapy with glucocorticoids (n = 19) or TNF-α-antagonist (n = 16) in pediatric IBD (Crohn's disease n = 19, ulcerative colitis (UC) n = 16). Results. The levels of suPAR were low in both patient groups studied. There was no difference in the values regarding the presence of Crohn's disease or ulcerative colitis. Thus, all analyses were performed on the entire sample set. Glucocorticoid therapy, however, resulted in a significant decline in suPAR levels from a median of 3.06 to 2.54 ng/ml (p < 0.01). In contrast, TNF-α-antagonist had no effect. The suPAR levels did not associate with ESR or CRP or fecal calprotectin (FC). Conclusions. In pediatric IBD, the suPAR levels in blood are low and do not reflect the level of intestinal inflammation assessed with FC. The introduction of corticoids, however, results in a decline of suPAR levels in blood but not reflect therapeutic response to TNF-α-antagonist. Thus, suPAR is of limited value in assessing systemic inflammatory responses in pediatric IBD.     

Collagen metabolism and enzymes of the urokinase plasminogen activator system in chronic myeloproliferative disorders: correlation between plasma-soluble urokinase plasminogen activator receptor and serum markers for collagen metabolism

Extracellular proteolytic enzymes of the urokinase-type plasminogen activator (uPA) system and the family of metalloproteinases (MMPs) catalyse the matrix degradation and remodelling processes characteristic of invasive malignant disorders. In a cohort of 50 patients with chronic myeloproliferative disorders (MPD) serum markers for collagen metabolism were compared to plasma levels of enzymes of the uPA and MMP system. Serum aminoterminal propeptide of type III procollagen (S-PIIINP) (P < 0.0001) concentration was significantly higher in the patients (median 3.7 micro g/L vs. 2.5 micro g/L) compared with controls. In a subgroup analysis comprising patients with myelofibrosis (MF), polycythaemia vera (PV) and essential thrombocythaemia (ET), respectively, S-PIIINP levels differed significantly with the highest values found in patients with MF (MF vs. PV vs. ET; P = 0.0027). Serum concentration of carboxyterminal telopeptide of type I collagen (S-ICTP) (P = 0.0006), reflecting type I collagen degradation, was significantly higher in patients compared with controls (median 4.0 micro g/L vs. 2.7 micro g/L). When comparing S-ICTP measurements between patient subgroups and controls there were only significantly higher values among MF and PV patients (MF vs. controls; P < 0.0001, PV vs. controls; P = 0.0016). A significant correlation between the marker for collagen synthesis (S-PIIINP) and degradation (S-ICTP) (r = 0.59; P < 0.0001) was demonstrated. A correlation analysis between serum markers for bone marrow remodelling processes (S-PIIINP, S-ICTP and S-hyaluronan) and plasma-soluble urokinase plasminogen receptor (suPAR) disclosed a significant relationship between suPAR and S-PIIINP (r = 0.48; P = 0.0009), S-hyaluronan (r = 0.56; P < 0.0001) and S-ICTP (r = 0.47; P = 0.0013), respectively. Plasma levels of MMP-2 and -9 were not correlated to serum markers for collagen metabolism. These findings suggest that enzymes of the uPA system might participate in the bone marrow remodelling processes characteristic of MPD.