mRNA analysis of several components of the plasminogen activator and matrix metalloproteinase systems in endometriosis using a real-time quantitative RT-PCR assay

BACKGROUND: The plasminogen activator (PA) and matrix metalloproteinase (MMP) systems are implicated in the establishment of endometriosis. The mechanisms by which these systems are involved in the pathogenesis of this disease are not well defined and controversial results have been published. The aim of this study was to analyse mRNA and protein levels of several components of the PA and MMP systems in endometriotic tissue and endometrium from women with and without endometriosis. METHODS and RESULTS: Real-time quantitative RT-PCR assays were developed to quantify mRNA levels of these components in 57 women with endometriosis and 32 controls. Endometrium of women with endometriosis showed higher mRNA and antigenic levels of urokinase type-PA (uPA) and MMP-3 than endometrium from controls. In these patients, ovarian endometriotic tissue had higher mRNA and antigenic levels of PA inhibitor type 1 (PAI-1) and MMP inhibitor type 1 (TIMP-1) than endometrium. CONCLUSIONS: The increase in mRNA and protein levels of uPA and MMP-3 observed in endometrium of women with endometriosis may facilitate the attachment of endometrial tissue to the peritoneum and ovarian surface, as well as the invasion of the extracellular matrix. This process would lead to the formation of early endometriotic lesions. Once the ovarian endometriotic cyst is developed, PAI-1 and TIMP-1 would increase which could explain the frequent clinical finding of an endometrioma without invasion of the adjacent ovarian tissue.     

Differential localization and expression of urokinase plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1) mRNA and protein in endometrial tissue during the menstrual cycle

Normal endometrium is a highly dynamic tissue, which responds to ovarian steroids with cyclic proliferation, differentiation (secretion), and degradation (menstruation). The urokinase plasminogen activator (uPA)-dependent proteolytic cascade as well as ligand activation of the uPA receptor (uPAR) is critically involved in physiological as well as pathophysiological aspects of tissue expansion and remodelling. Cyclic variation and distribution of uPA, uPAR and plasminogen activator inhibitor 1 (PAI-1) mRNA were examined by in situ hybridization, real-time PCR and northern blot in normal endometrium. Their corresponding proteins were localized with immunohistochemistry. uPA mRNA is exclusively expressed by stromal cells, whereas uPA protein is present in both epithelial and stromal cells. Immunostaining for uPA protein is reduced or undetectable at midcycle, thus coinciding with peak concentration of uPA in the uterine fluid. uPAR mRNA is expressed by epithelial cells in the proliferative phase and by stromal cells in the secretory phase. However, epithelial cells stain for uPAR protein throughout the cycle, suggesting that uPAR may detach from stromal cells and then bind to epithelial cells in the secretory phase. PAI-1 mRNA is located in vessel walls. The late secretory phase has greatly increased expression of all three mRNA and their proteins, mainly in pre-decidual cells in the superficial stroma. Discordant localization of the mRNA and proteins suggest that uPA is produced by stromal cells, released and bound to epithelial cells in both the proliferative and secretory phases, whereas uPAR is released from the stroma and bound to epithelial cells in the secretory phase. Also, the present data together with earlier reports suggest that uPA is released from the epithelial cells to the uterine fluid.     

Expression of several components of the plasminogen activator and matrix metalloproteinase systems in endometriosis

BACKGROUND: Endometriosis is considered a benign disease that has the ability to invade normal tissue. As in neoplastic growth, local extracellular proteolysis may take place. The aim of this study is to analyse several components of the plasminogen activator (PA) pathway and the matrix metalloproteinase (MMP) system in endometriotic tissue, endometrium and peritoneal fluid from women with and without endometriosis (controls). METHODS AND RESULTS: Thirty-nine women with endometriosis and 35 controls were studied. In eutopic endometrium of women with endometriosis, the antigenic levels of urokinase-type PA (uPA) and MMP-3 were elevated when compared with endometrium from controls. Ovarian endometriotic tissues had higher antigenic levels of PA inhibitor type 1 (PAI-1) and tissue inhibitor of metalloproteinases type 1 (TIMP-1) than endometrium. The peritoneal fluid from women with endometriosis showed a significant increase in uPA levels compared with controls. CONCLUSIONS: The increase in antigenic levels of uPA and MMP-3 in endometrium of women with endometriosis might contribute to the invasive potential of endometrial cells. Once the ovarian endometriotic cyst is developed, an increase in PAI-1 and TIMP-1 is detected and significant proteolytic activity is no longer observed. This increase in inhibitors and decrease in proteolytic activity could explain the frequent clinical finding of isolated endometriotic cyst without invasion of the surrounding ovarian tissue.     

Soluble urokinase-type plasminogen activator receptor is over-expressed in uterine endometrium from women with endometriosis

Extracellular matrix degradation by secreted proteases, e.g. plasmin, is essential for endometrial functions such as blastocyst implantation and menstruation. We investigated whether the expression of plasmin(ogen) activating or inhibiting factors in endometrial cells from women with endometriosis was different from women without the disease. Endometrial biopsies were obtained from 10 patients with and 16 women without endometriosis. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with diethylstilboestrol (10(-10) M) alone or combined with promegestone (5 x 10(-8) or 5 x 10(-6) M). Urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI)-1 and -2, and soluble uPA receptor (suPA-R) concentrations were assayed by enzyme-linked immunosorbent assay (ELISA) in the conditioned media. uPA and PAI-2 concentrations were not influenced by steroid treatment and did not differ between women with and without endometriosis, whereas PAI-1 was significantly up-regulated by promegestone in both groups. In contrast, suPA-R expression was not influenced by steroid treatment but was significantly higher in cells from endometriosis patients. This is the first report on suPA-R secretion in endometrial cells and the results indicate an altered activation of plasmin(ogen) in endometrium from women with endometriosis that could lead to a higher proteolytic potential of retrogradely menstruated endometrial fragments with consecutive development of endometriotic foci.      

Expression of angiogenic factors in endometriosis: relationship to fibrinolytic and metalloproteinase systems

BACKGROUND: Endometriosis is a highly prevalent, benign disease in which the angiogenic, fibrinolytic and metalloproteinase (MMP) systems may be implicated. The objective of this study is to analyse mRNA expression and protein levels of several angiogenic factors and to correlate them with several components of the fibrinolytic and MMP systems in samples from 71 women with endometriosis and 50 controls. METHODS AND RESULTS: Eutopic endometrium showed higher mRNA expression of vascular endothelial growth factor (VEGF) in patients than in controls. However, ovarian endometrioma had lower VEGF mRNA levels than did the eutopic endometrium of patients. Similar results were obtained for VEGF protein levels. On the other hand, a significant increase in thrombospondin-1 (TSP-1) levels was observed in ovarian endometrioma than in eutopic endometrium. The peritoneal fluid from women with endometriosis showed a significant increase in VEGF, urokinase-type plasminogen activator (uPA) and MMP-3 levels than that of controls. A significant correlation was observed between the levels of VEGF and uPA in endometrium and in peritoneal fluid. CONCLUSIONS: Endometrium and peritoneal fluid from women with endometriosis have increased levels of VEGF, uPA and MMP-3 levels. Therefore, the development of endometriotic implants at ectopic sites may be facilitated, promoting the progress of the endometriosis.     

Differential expression and localization of de-novo synthesized endometriotic haptoglobin in endometrium and endometriotic lesions

Endometriosis protein-I (ENDO-I) mRNA expression and protein localization were evaluated using in-situ hybridization and immunohistochemistry in endometriotic lesions and eutopic endometrium from women with endometriosis, and in eutopic endometrium from women without endometriosis (controls). When present, ENDO-I mRNA and protein were observed in the functionalis zone of endometrial stroma and the stroma of endometriotic lesions. Expression and localization differences were scored and statistically analysed. During the secretory stage, ENDO-I mRNA expression by endometriotic lesions and eutopic endometrium from women with disease was significantly greater than ENDO-I mRNA expression by proliferative stage eutopic endometrium from women with disease or eutopic endometrium from controls, regardless of cycle stage (P < 0.001). More ENDO-I protein was localized in endometriotic lesions and eutopic endometrium from women with disease than in eutopic endometrium from controls, regardless of cycle stage (P < 0.001). Differential expression and localization of ENDO-I may help develop minimally invasive diagnostic strategies for endometriosis. Further, as ENDO-I shares nucleotide sequence and amino acid sequence with hepatic haptoglobin-which in certain disease states is immunosuppressive and angiogenic-differences in ENDO-I expression and localization in the peritoneal cavity may contribute to the pathogenesis of endometriosis and/or facilitate development of unprecedented diagnostic or therapeutic approaches for management of this enigmatic disease.     

Plasminogen activator system, vascular endothelial growth factor, and colorectal cancer progression.

AIMS: To plasminogen activator system (PAS) consists of the plasminogen activators (urokinase (uPA) and tissue-type (tPA) plasminogen activators), the uPA receptor (uPAR), and the plasminogen activator inhibitors (PAI-1 and PAI-2). Plasminogen activators activate plasminogen to plasmin, which can break down extracellular matrix (ECM) components. Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and is involved in angiogenesis. VEGF has been shown to upregulate uPA and this may facilitate tumour angiogenesis further. METHODS: PAS components and VEGF were determined by enzyme linked immunosorbent assay (ELISA) in paired colorectal tumour and normal tissue (n = 50) and correlated with pathological staging. RESULTS: uPA, uPAR, PAI-1, and VEGF values were significantly higher in tumour tissue (for example, tumour uPA: median, 2.3 (range, 0.1-6.7) ng/mg protein v normal uPA: median, 0.2 (range, 0-2.6) ng/mg protein). tPA was significantly higher in normal mucosa and there was no difference in PAI-2. uPA, uPAR, PAI-1, and VEGF values significantly correlated with each other and with Dukes's staging (uPA in adenomas: median, 0.42 (range, 0.1-1.2) ng/mg protein; upA in Dukes's B tumours: median, 2.1 (range, 0.4-4.3) ng/mg protein; and uPA in Dukes's D tumours: median, 4.0 (range, 3.7-4.2) ng/mg protein) and lymphatic invasion. In addition PAI-1 also correlated with tumour size and differentiation. CONCLUSION: The involvement of the PAS and VEGF in colorectal cancer appears to be complex. uPA, uPAR, PAI-1, and VEGF were upregulated in tumour tissue and this correlated with Dukes's staging and lymphatic invasion.     

Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma

Adult stem cells are thought to be responsible for the high regenerative capacity of the human endometrium, and have been implicated in the pathology of endometriosis and endometrial carcinoma. The RNA-binding protein Musashi-1 is associated with maintenance and asymmetric cell division of neural and epithelial progenitor cells. We investigated expression and localization of Musashi-1 in endometrial, endometriotic and endometrial carcinoma tissue specimens of 46 patients. qPCR revealed significantly increased Musashi-1 mRNA expression in the endometrium compared to the myometrium. Musashi-1 protein expression presented as nuclear or cytoplasmic immunohistochemical staining of single cells in endometrial glands, and of single cells and cell groups in the endometrial stroma. Immunofluorescence microscopy revealed colocalization of Musashi-1 with its molecular target Notch-1 and telomerase. In proliferative endometrium, the proportion of Musashi-1-positive cells in the basalis layer was significantly increased 1.5-fold in the stroma, and three-fold in endometrial glands compared to the functionalis. The number of Musashi-1 expressing cell groups was significantly increased (four-fold) in proliferative compared to secretory endometrium. Musashi-1 expressing stromal cell and cell group numbers were significantly increased (five-fold) in both endometriotic and endometrial carcinoma tissue compared to secretory endometrium. A weak to moderate, diffuse cytoplasmic glandular staining was observed in 50% of the endometriosis cases and in 75% of the endometrioid carcinomas compared to complete absence in normal endometrial samples. Our results emphasize the role of Musashi-1-expressing endometrial progenitor cells in proliferating endometrium, endometriosis and endometrioid uterine carcinoma, and support the concept of a stem cell origin of endometriosis and endometrial carcinoma.     

The macrophage stimulating protein/RON system: a potential novel target for prevention and treatment of endometriosis

Our recent DNA microarray analysis using tissue obtained by laser capture microdissection (LCM) identified up-regulation of RON (a tyrosine kinase receptor) during the late secretory phase in eutopic endometrial epithelial cells from patients with deep endometriosis compared with control endometrium from women with macroscopically normal pelvic cavities. In the present study, we further investigated mRNA expression of RON and its ligand, macrophage stimulating protein (MSP), in deep endometriotic lesions, eutopic endometrium from patients with deep endometriosis and control endometrium by using LCM and quantitative real-time RT-PCR. MSP mRNA expression in endometrial epithelial cells was significantly up-regulated in endometriosis patients during the late secretory phase compared with expression in controls. Furthermore, we detected up-regulation of MSP mRNA in ectopic endometrial epithelial cells compared with matched eutopic endometrial epithelial cells within the same patients regardless of the menstrual phase. MSP has an intrinsically dual functional nature through its receptor RON-it is a trophic cytokine preventing apoptosis and a scatter factor promoting invasion, both of which may be necessary for the initial development and growth of endometriosis. The present findings suggest that the MSP/RON system may be involved in the pathophysiology of endometriosis.     

The plasminogen activator inhibitor "paradox" in cancer

Proteolysis in general and specifically the plasminogen activating system regulated by urokinase (uPA) its specific receptor, the GPI membrane anchored urokinase receptor (uPAR) and the specific plasminogen activator inhibitor 1 (PAI-1) plays a major role in tumorigenesis, tumor progression, tumor invasion and metastasis formation. This is exemplified by a body of published work showing a positive correlation between the expression of uPA or uPAR in several tumors and their malignancy. It is generally assumed that such a "pro-malignant" effect of the uPA-uPAR system is mediated by increased local proteolysis thus favoring tumor invasion, by a pro-angiogenic effect of this system and also by uPA-uPAR signaling towards the tumor thereby shifting the tumor phenotype to a more "malignant" one. However, when tumor patients are analyzed for long term survival, those with high levels of the inhibitor of the system, PAI-1 have a much worse prognosis than those with lower PAI-1 levels. This indicates that increased overall proteolysis alone cannot be made responsible for the adverse effects of the plasminogen activating system in tumors. Moreover, it becomes increasingly evident that components of the fibrinolytic system secreted by the tumor cells themselves are not solely responsible for a correlation between the plasminogen activating system and tumor malignancy; components of the plasminogen activating system secreted by stroma cells or cells of the immune system such as macrophages contribute also to the impact of fibrinolysis on malignancy. This review summarizes the evidence for the role of plasminogen activator inhibitor-1 in mediating the malignant phenotype and possible mechanism thereby trying to explain the "PAI-1 paradox in cancer" on a molecular level.     

Induction of the plasminogen activator system by mechanical stimulation of human bronchial epithelial cells

Mechanical stimulation of the airway epithelium, as would occur during bronchoconstriction, is a potent stimulus and can activate profibrotic pathways. We used DNA microarray technology to examine gene expression in compressed normal human bronchial epithelial cells (NHBE). Compressive stress applied continuously over an 8-h period to NHBE cells led to the upregulation of several families of genes, including a family of plasminogen-related genes that were previously not known to be regulated in this system. Real-time PCR demonstrated a peak increase in gene expression of 8.0-fold for urokinase plasminogen activator (uPA), 16.2-fold for urokinase plasminogen activator receptor (uPAR), 4.2-fold for plasminogen activator inhibitor-1 (PAI-1), and 3.9-fold for tissue plasminogen activator (tPA). Compressive stress also increased uPA protein levels in the cell lysates (112.0 versus 82.0 ng/ml, P = 0.0004), and increased uPA (4.7 versus 3.3 ng/ml, P = 0.02), uPAR (1.3 versus 0.86 ng/ml, P = 0.007), and PAI-1 (50 versus 36 ng/ml, P = 0.006) protein levels in cell culture media. Functional studies demonstrated increased urokinase-dependent plasmin generation in compression-stimulated cells (0.0090 versus 0.0033 OD/min, P = 0.03). In addition, compression led to increased activation of matrix metalloproteinase (MMP)-9 and MMP-2 in a urokinase-dependent manner. In postmortem human lung tissue, we observed an increase in epithelial uPA and uPAR immunostaining in the airways of two patients who died in status asthmaticus compared with minimal immunoreactivity noted in airways from seven lung donors without asthma. Together these observations suggest an integrated response of airway epithelial cells to mechanical stimulation, acting through the plasminogen-activating system to modify the airway microenvironment.     

Plasminogen activator inhibitor-1 as a measure of vascular remodelling in breast cancer

The generation of urokinase plasminogen activator (uPA) by tumours is an important pathway for neoplastic cell invasion and metastasis. Indeed in several tumour types, elevated levels of uPA, its receptor (uPAR) or its inhibitor plasminogen activator inhibitor-1 (PAI-1) is associated with a poorer prognosis. Since endothelial cells also use this proteolytic system to remodel the extracellular matrix during angiogenesis and since angiogenesis, as assessed by microvessel density, is also a predictor of patient survival, this study was designed to investigate the relationship between angiogenesis and the urokinase system in breast tumours. The aims were to assess whether the uPA, uPAR and/or PAI-1 correlates with angiogenic activity and could therefore be a useful objective clinical measure of tumour neovascularization; and to clarify whether the poor outcome associated with high levels of the urokinase system is due to its association with angiogenesis. The study also sought to examine the relationship between the uPA system and vessel remodelling using loss of a basement membrane epitope (LH39) normally associated with established capillaries. The cytosolic levels of uPA, PAI-1 and uPAR were therefore measured by enzyme linked immunoabsorbent assay, together with tumour vascularity, in 136 well-characterized invasive breast carcinomas. There were significant relationships between uPA and uPAR (Spearman r=0.37, p<0.0001), uPA and PAI-1 (Spearman r=0.19, p=0.03) and between uPAR and PAI-1 (Spearman r=0.23 p=0.01). A significant correlation was also observed between PAI-1 and vessel remodelling (Spearman r=0.34, p=0.04), patient age (p=0.01), nodal status (p=0.047) and tumour grade (p=0.04), but no association between tumour vascularity and PAI (p=0.96), uPA (p=0.69) or uPAR (p=0.81) was present. No significant association was seen between any of the urokinase variables and expression of the angiogenic factor thymidine phosphorylase. Furthermore, no significant associations were found between any of the studied parameters and overall survival in a univariate analysis of the cancer patients. A multivariate Cox proportional hazard model of overall survival showed that uPA (p=0.15), but not uPAR (p=0.52) or PAI-1 (p=0.61), gave no additional prognostic information. These findings show that uPA may work via an independent pathway to angiogenesis and therefore combined blockade of uPA and angiogenesis may have additional therapeutic benefits. It also shows, as recently demonstrated in animal models, that PAI-1 may be a key regulator of vascular remodelling in human cancer.     

Spitz naevi may express components of the plasminogen activation system

The plasminogen activation (PA) system is involved in the process of invasion and metastasis. Its major components are urokinase (uPA) and tissue-type plasminogen activator (tPA), plasminogen activation inhibitor type 1 and 2 (PAI-1 and PAI-2) and a receptor for urokinase (uPAR). In this study, the expression of plasminogen activation components in Spitz naevi was compared with that in common and dysplastic naevi on the one hand and primary cutaneous melanomas on the other. Spitz naevi had melanocytic positivity for uPA in 0% (0/36), tPA in 30% (6/20), PAI-1 in 10% (3/35), PAI-2 in 40% (8/21) and uPAR in 60% (13/21) of cases. This far exceeded the expression found in common (n = 25) and dysplastic (n = 15) naevi, which only showed melanocytic positivity for PAI-2 (20% and 15% respectively) and in one dysplastic naevus also for uPAR. This was much (for most components significantly) less than the proportion of primary melanomas with tumour cell positivity, which was 30% (11/38) for uPA, 80% (19/24) for tPA, 75% (28/38) for PAI-1, 80% (19/24) for PAI-2 and 80% (19/24) for uPAR. The main findings of this study are that Spitz naevi, firstly, may express plasminogen activator (tPA), inhibitors and the receptor of the PA system, but in a much smaller proportion than cutaneous melanomas; and secondly, do not express urokinase, whereas some of the melanomas do. uPA positivity may therefore be suggestive of melanoma. However, overlapping staining results imply that the PA system has limited value in the differential diagnosis between Spitz naevus and primary melanoma. As serine protease components are expressed, Spitz naevi may use this proteolytic machinery to accomplish matrix degradation, although in a more restricted, possibly transient manner than melanomas.     

Vitamin D3 modulation of plasminogen activator inhibitor type-1 in human breast carcinomas under organ culture

Urokinase plasminogen activator (uPA), its cell-bound receptor (uPAR) and its main inhibitor plasminogen activator type 1 (PAI-1) are present primarily in stromal cells in invasive breast carcinoma. The purpose of this study was to investigate the regulation by 1,25 dihydroxyvitamin-D3 (VD3) of these invasion-associated markers expressed in breast cancer tumors under organ culture, which preserves the interacting network of tumor and stromal cells. Breast carcinoma slices (30 cases), obtained using the Krumdieck tissue slicer, cultured for 48 h in the presence or absence of 100 nM vitamin D3, were embedded in formalin-fixed paraffin. uPA, uPAR, PAI-1 and VD3 receptor (VDR) were analyzed by immunohistochemistry, and their expression, detected in tumor cells and fibroblasts of the specimens, was not statistically changed by culture conditions. The proportion of cases expressing uPA, uPAR and PAI-1 was not affected by VD3 in epithelial cells, but the fraction of cases displaying strong PAI-1 reactivity in fibroblasts was reduced (P=0.016) compared with control slices. Fibroblasts isolated from invasive ductal carcinomas and from normal breast tissues expressed higher VDR mRNA levels than epithelial cells. In cultured tumor fibroblasts, PAI-1 immunostaining and mRNA levels were reduced by VD3-limiting fibroblast contribution to invasion.     

Expression of urokinase-type plasminogen activator,its receptor,and its inhibitor in gastric adenocarcinoma tissues.

The plasminogen and plasmin system, which is mainly regulated by urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1), is generally believed to play a role in cancer invasion and metastasis. This study was conducted to investigate the role of uPA, uPAR and PAI-1 in the invasion and metastasis of gastric adenocarcinoma. The expression of mRNAs for uPA and PAI-1 was determined by Northern blot analysis in nine primary gastric cancer tissues, nine paired metastatic lymph nodes and normal gastric mucosa. The mRNA of uPA was not or faintly detected in normal mucosa, while the expression was increased in both primary gastric cancer tissues and metastatic lymph nodes to a similar degree. The mRNA expression for PAI-1 in the gastric cancer tissues was not different from that in the paired metastatic lymph nodes and normal mucosae. uPAR was determined by immunohistochemical staining, demonstrating that five (56%) and six (67%) out of nine primary gastric cancer tissues and nine paired metastatic lymph nodes were positive, respectively and the intensity was stronger in metastatic lymph nodes. The results support the concept that most gastric cancer cells may have an innately moderate level of uPA and uPAR, and that increase of uPAR expression can be considered to be closely associated with cancer invasion and metastasis.     

Could the Defects in the Endometrial Extracellular Matrix During the Implantation Be a Cause for Impaired fertility?

PROBLEM: Assessment of concentration of the metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinases 1 (TIMP1), urokinase plasminogen activator (uPA) and its receptor (uPAR) and leukaemia inhibitory factor (LIF) in the uterine fluid in women with idiopathic infertility and unknown cause for recurrent miscarriage. METHOD OF STUDY: Uterine fluid obtained from patients with idiopathic infertility (n = 13), patients with unknown cause of recurrent miscarriages (n = 16) and healthy fertile women (n = 14). The assessment of MMP9, TIMP1, uPA, uPAR and LIF were performed with enzyme-linked immunosorbent assay. RESULTS: Statistically lower expression of MMP9 and TIMP1 in uterine fluid during implantation window in women with infertility and recurrent miscarriage of unknown cause. CONCLUSION: Changed turnover of the extracellular matrix might be a cause of improper endometrial transition and in effect cause faulty implantation.     

Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes

The expression and localization of mRNA's for tissue plasminogen activator (tPA), urokinase PA (uPA), uPA receptor (uPAR) and inhibin subunits, alpha, beta A and beta B in monkey testes was investigated. Using in-situ hybridization with digoxigenin-labelled cRNA probes (dig- cRNA), we demonstrated that tPA and plasminogen activator inhibitor type 1 (PAI-1) were expressed in testes of both immature and mature rhesus monkeys. tPA mRNA was localized predominantly in Sertoli cells. Expression level was low in immature testis, increased dramatically in the adult and varied with seminiferous cycle. PAI-1 mRNA was localized mainly in germ cells except late spermatids. uPA mRNA was expressed stage-specifically in Sertoli cells of adult testis. uPA receptor mRNA was localized in germ cells of mature testis but not in spermatogonia or late spermatids. Assayed by fibrin overlay technique, PA activity in conditioned media of purified Sertoli cells (Sc) was negligible, PA activity in media obtained from co-cultured Sertoli and Leydig cells (LS), however, was significantly increased, although Leydig cells alone were not capable of producing any PA activity. Addition of follicle stimulating hormone (FSH) to the incubation medium remarkably increased PA secretion in both Sc and LS cultures. Human chronic gonadotrophin (HCG) had no significant effect on PA activity in the Sc culture but dramatically stimulated PA activity in the co-culture system. Dihydrotestosterone (DHT) did not mimic the effect of HCG. PAI-1 activity was secreted mainly by germ cells and did not differ between the two culture systems. FSH and forskolin inhibited PAI-1 secretion. Inhibin alpha, beta A and beta B subunit mRNAs were localized in Sertoli cells of adult monkey testes, with no obvious difference in the expression levels. These data suggest that PA/PAI-1 and other related factors are expressed in rhesus monkey testis under the control of various hormones, seminiferous cycle and cell-cell interactions through paracrine or autocrine regulation. Locally generated fibrinolysis may play an important role in the process of spermatogenesis.      

Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor-1 and -2 in hepatocellular carcinoma

It has become more and more clear in recent decades that the plasminogen activation system, which includes urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), plasminogen activator inhibitor (PAI)-1 and PAI-2, plays a very important role in the aggressiveness of cancer. Using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), the expression of these four components of the uPA system was analyzed in 19 cases of hepatocellular carcinoma (HCC) and 18 cases of the adjacent non-cancer tissues which all had chronic active hepatitis with liver fibrosis or liver cirrhosis. Four cases of normal liver tissues, as controls for immunohistochemical stains, were obtained from the hepatectomized liver of patients with metastatic cancer in the liver. The positive rates of uPA, uPAR, PAI-1 and PAI-2 for immunohistochemical stains in cancer tissues were 78.9, 68.4, 57.9 and 31.6%, respectively. Positive signals were mainly distributed in the cytoplasm of the cancer and in stromal cells. Moreover, the strong stains were chiefly located in the invasive front of the cancer cells. No specific stain was detected in four cases of normal liver tissues. In ELISA, there were significant differences between cancer and non-cancer tissues in concentration of uPA, uPAR and PAI-1 (P < 0.0003, 0.0024 and 0.01, respectively), but there was no significant difference in that of PAI-2 (P = 0.37). These results suggest that uPA, uPAR and PAI-1 are related to invasion of HCC.