Localization Of Plasminogen Activator Research Articles

Localization of plasminogen activators and plasminogen-activator inhibitors in human gingival tissues demonstrated by immunohistochemistry and in situ hybridization

The plasminogen-activating system plays an important part in tissue proteolysis in physiological as well as pathological processes. Plasminogen activators u-PA (urokinase) and t-PA (tissue) as well as the inhibitors PAI-1 and PAI-2 are present in gingival crevicular fluid in concentrations significantly greater than in plasma. This fact, and the finding that the concentrations of t-PA and PAI-2 are higher in areas with gingival inflammation, indicate local production of these components. The present study describes, by means of in situ hybridization and immunohistochemistry, the localization of the plasminogen activators and their inhibitors in gingival tissues from patients undergoing periodontal surgery. t-PA mRNA and t-PA antigen were primarily found in the epithelial tissues, predominantly in the sulcular and junctional regions, although occasionally in the oral epithelium and in blood vessels of the connective tissue. u-PA and u-PA-receptor signals were seen in single cells within the junctional and sulcular epithelia and adjacent to blood vessels close to the junctional epithelium, but rarely in the oral epithelium. Similar to t-PA, the predominant location of PAI-2 mRNA was the gingival epithelia. In the junctional and sulcular epithelia, PAI-2 mRNA was seen throughout the thickness, while in the oral epithelium the strongest signals were seen in stratum granulosum and stratum spinosum. PAI-1 mRNA was invariably found in the connective tissue associated with blood vessels. The present study confirms earlier indications of local production of plasminogen activators and their inhibitors in gingival tissues. In addition, the results demonstrate that t-PA and PAI-2 in these patients are produced predominantly in the epithelial tissues. Furthermore, the presence of t-PA and PAI-2 seems to be most pronounced in the areas likely to be subjected to bacterial assault.

Localization of plasminogen activator and inhibitor, LH and androgen receptors and inhibin subunits in monkey epididymis.

Epididymis is a site of sperm maturation and storage. Limited and directed-proteolysis regulated by plasminogen activator (PA), plasminogen activator inhibitor type-1 (PAI-1) and other related factors may play an essential role in these processes. Our previous studies have demonstrated that rat epididymis expressed luteinizing hormone receptor (LHR), tissue type (t) and urokinase type (u)PA, mRNAs, and tPA activity was stimulated in vitro by human chorionic gonoadotrophin (HCG). In the present study we further examined localization of mRNAs for tPA, uPA, LHR, androgen receptor (AR), as well as inhibin subunits alpha, betaA and betaB in rhesus monkey epididymis. Using in-situ hybridization with digoxygenin-labelled cRNA probes, we have demonstrated that tPA and PAI-1 mRNAs were localized in epithelial cells of adult monkey epididymis. uPA mRNA was localized in the same areas, but to a much smaller extent. tPA, uPA and PAI-1 mRNAs were greatly expressed in the caput and corpus of adult epididymis than in other regions. In-vitro experiments showed that both tPA and uPA activities in epididymal cells were dramatically stimulated by HCG, but not by follicle stimulating hormone (FSH). LHR (but not FSH receptor) and AR mRNAs were localized in the epithelial cells of the epididymis. However, LHR mRNA was detected in both adult and immature infant monkeys, whereas AR was found only in the adult. Inhibin alpha, betaA and betaB mRNAs were also detected in this organ, betaA mRNA being more strongly expressed in the caput than in other regions of the epididymis. We suggest that LH and androgen may be the key hormones in coordination with the PA-PAI-1 system in regulating epididymal differentiation and sperm maturation.

Localization of plasminogen activators and their inhibitor in squamous cell carcinomas of the head and neck.

Plasminogen activators (PAs) and their inhibitors are thought to play an important role in tumor invasion and metastasis. However, there have been few reports in which histologic localization of PAs has been demonstrated in head and neck tumors. We examined the patterns of expression of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), and vitronectin in head and neck squamous cell carcinomas using immunohistochemical techniques. We also studied the correlation between the immunohistologic expression of these fibrinolytic proteins and the clinical staging of the tumor. Of 28 tumor specimens, 15 (54%) showed immunoreactivity for u-PA; 8 (36%), for t-PA; and 23 ( 82%), for PAI-1. There was a significant correlation between PAI-1 expression and the extent of the primary tumor. The present study demonstrates the existence and possible pathophysiologic significance of u-PA and PAI-1 in squamous cell carcinomas of the head and neck.

Localization of plasminogen activators and their inhibitor in squamous cell carcinomas of the head and neck

Background Plasminogen activators (PAs) and their inhibitors are thought to play an important role in tumor invasion and metastasis. However, there have been few reports in which histologic localization of PAs has been demonstrated in head and neck tumors. Methods We examined the patterns of expression of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), and vitronectin in head and neck squamous cell carcinomas using immunohistochemical techniques. We also studied the correlation between the immunohistologic expression of these fibrinolytic proteins and the clinical staging of the tumor. Results Of 28 tumor specimens, 15 (54%) showed immunoreactivity for u-PA; 8 (36%), for t-PA; and 23 ( 82%), for PAI-1. There was a significant correlation between PAI-1 expression and the extent of the primary tumor. Conclusions The present study demonstrates the existence and possible pathophysiologic significance of u-PA and PAI-1 in squamous cell carcinomas of the head and neck. © 1997 John Wiley & Sons, Inc. Head Neck 19: 611–616, 1997.

The serpin PAI-1 inhibits cell migration by blocking integrin alpha V beta 3 binding to vitronectin.

During wound healing, migrating cells increase expression of both the vitronectin receptor (VNR) integrins and plasminogen activators. Here we report that vitronectin significantly enhances the migration of smooth muscle cells (SMCs), and that the specific VNR alpha V beta 3 is required for cell motility. We also show that the alpha V beta 3 attachment site on vitronectin overlaps with the binding site for plasminogen activator inhibitor (PAI)-1, and that the active conformation of PAI-1 blocks SMC migration. This effect requires high-affinity binding to vitronectin, and is not dependent on the ability of PAI-1 to inhibit plasminogen activators. Formation of a complex between PAI-1 and plasminogen activators results in loss of PAI-1 affinity for vitronectin and restores cell migration. These data demonstrate a direct link between plasminogen activators and integrin-mediated cell migration, and show that PAI-1 can control cell-matrix interactions by regulating the accessibility of specific cell-attachment sites. This indicates that the localization of plasminogen activators at sites of focal contact does not initiate a proteolytic cascade leading to generalized matrix destruction, but instead is required to expose cryptic cell-attachment sites necessary for SMC migration.

A Quantitative Receptor Assay Using TRITON X-114 for Plasminogen Activator Binding Proteins in Solubilized Membranes from Human Liver and Placenta

Cell surface binding proteins play an important role in the localization of plasminogen activator (PA) activity at the cell surface or in the clearance of PAs. We describe a rapid and quantitative receptor assay applicable to the quantification and affinity determination of binding proteins for tissue-type PA and urokinase-type PA in solubilized membranes obtained from human liver, human placenta, or human monocyte-like cells. The method is based on the ability of a solution of the nonionic detergent Triton X-114 to phase separate at temperatures above 20°C. After incubation of integral membrane proteins with radiolabeled ligand, a solution of Triton X-114 is added at 4°C and warmed to 37°C to allow phase partitioning. Radiolabeled ligand bound to membrane protein is recovered in the detergent-rich lower phase which is separated by centrifugation from the detergent-poor upper phase containing free radiolabeled ligand.

Localization of plasminogen activator inhibitor‐1 production in inflamed appendix by in situ mRNA hybridization

The peritoneum has been shown to possess fibrinolytic activity which is thought to play a role in the prevention of intra-abdominal adhesion formation. Recently inflamed peritoneal tissue has been shown to have reduced fibrinolytic activity secondary to increased levels of plasminogen activator inhibitor-1 (PAI-1). The aim of this study was to localize the production of PAI-1 in appendix tissue using in situ mRNA hybridization. Sections of normal and inflamed appendix were hybridized with a digoxigenin-labelled cDNA probe. PAI-1 production was localized to both mesothelium and serosal blood vessel endothelium in all inflamed appendix samples. Cell identities were confirmed using immunohistochemistry directed against mesothelial and endothelial cell markers. Staining was not seen on 1 probe following ribonuclease digestion). The identification of the cells expressing the PAI-1 gene in peritoneum increases our understanding of the pathophysiological changes in fibrinolytic activity which occur in inflammation and may lead to adhesion formation.

Regulation and Localization of Plasminogen Activators in the Rat Seminiferous Epithelium

Regulation and Localization of Plasminogen Activators in the Rat Seminiferous Epithelium

Immunohistochemical localization of plasminogen activators in human saphenous veins

Human saphenous veins were analyzed for presence of plasminogen activators (t-PA) in the endothelium of the vessel walls by immunohistochemical techniques. We used a polyclonal rabbit antihuman t-PA antibody, a monoclonal mouse-antihuman t-PA and a goat antibody to low molecular weight urokinase. To demonstrate presence of antigen we used FITC conjugated anti-IgG or the Biotin-Avidin technique. Both monoclonal and polyclonal anti-t-PA demonstrated positive fluorescence in the endothelial layer. Tests for urokinase were negative. The present study thus demonstrated that t-PA was present in the vascular endothelium. This is in accordance with data from earlier histochemical studies for detection of PA activity.

牛眼球の硝子体および房水におけるプラスミノーゲン・アクチベーターの活性および局在について

We studied the fibrinolytic activity, amount and localization of plasminogen activator (PA) in bovine vitreous extract (VE) and aqueous humor (AH). Fibrinolytic activity of VE and AH was quenched by adding anti-human t-PA IgG, but not by anti-human HMW u-PA IgG. After SDS electrophoretic analysis, enzymographic distributions of PA in VE and AH were detected at about Mr. 66K and 110K. These findings suggested the fact that the main PA in VE and AH was t-PA. Estimating fibrinolytic activity of VE and AH by amidolytic assay, activity was 0.12±0. 10IU/ml (n=10) in VE and 0.04±0.07IU/ml (n=10) in AH. Measuring the amount of t-PA by RIA, t-PA was 4.1±0.9ng/ml (n=10) in VE, and 3.3±0.8ng/ml in AH. Investigating the PA activity by Todd's fibrinolysis autography, clear lytic areas could be seen on the small vessels of the iris, and the subepithelial stroma and along the endothelium of the cornea. In immunohistochemical studies using anti-human t-PA IgG, vascular endothelial cells and corneal epithelial cells showed the positive reactivity.

Localization of plasminogen activator(s) in primary and secondary rat adenocarcinoma cells.

Localization of plasminogen activator(s) in primary and secondary rat adenocarcinoma cells.

Modulation in the levels and localization of plasminogen activator in differentiating neuroblastoma cells

Variations in the specific activity and localization of plasminogen activator (PA) were examined prior to and during the differentiation of mouse neuroblastoma and rat glioma cells in culture, in an attempt to reveal whether PA is actively involved in neurite extension and development in these cells. Following differentiation induced by dibutyryl cyclic AMP, the level of PA increased by 10 to 20-fold in neuroblastoma N18TG-2 cells and in the hybrid cell line NG108-15, obtained by fusion of mouse neuroblastoma N18TG-2 and rat glioma C6BU-1 cells, but was very low in the glioma parental cells throughout differentiation. Plasminogen-dependent proteolysis was found not to be an obligatory requirement for the differentiation of the hybrid cells in culture, since the extension of neurites, the appearance of toxin-activated Rb +-efflux and the eletrical properties were largely unaffected by eliminating and blocking this activity. Confluent cells from several clones derived from mouse neuroblastoma C1300 cells displayed significant intracellular and secreted PA activities, and the axon-minus neuroblastoma NIA-103 cells were found to secrete the highest amounts of PA. Upon gel electrophoresis, all mouse neuroblastoma cell lines examined displayed a major molecular form of active PA with an apparent molecular weight of 80,000 and a minor one of 50,000. Only the larger enzyme form was expressed by the rat glioma or the hybrid cells, and the electrophoretic migration pattern of the active enzyme species remained unchanged upon differentiation of the various cell lines. Distribution of PA activity on the surface of differentiating neuroblastoma N1E-115 cells and hybrid NG108-15 cells was visualized by high resolution autoradiography of [ 125I]fibrin degradation products, cross-linked to the cell surface. Particularly intense labeling was observed along membranes of cell bodies in both developing and mature cells, around branching zones, along individual neurite extensions and in part, but not all, of the growth cones. These findings suggest that PA plays a role in the metabolism of mature cells of neuronal origin, and imply its involvement in the development and/or the functions of specific neuronal processes and growth cones.

Plasminogen activator activity and plasmin inhibition in nerves.

Histochemical studies of the distribution of fibrinolytic activity in the vagus and sciatic nerves of rat, guinea pig, and rabbit showed large areas of diffuse lysis related to the fascicles of the nerve. Foci of lysis were also located in the perineurium, in the peripheral epineurium, and particularly in the epineurium between the fascicles of the nerve. The fibrinolytic activity was caused by a plasminogen activator. The activity was highest in the rat, lowest in the rabbit and intermediate in the guinea pig. Plasmin inhibitors were located histochemically by the fibrin slide 'sandwich' technique. In all three species plasmin inhibition was most pronounced in relation to the nerve fascicles, with the highest degree of inhibition in the guinea pig and less in the rat and rabbit. The discrete localization of plasminogen activator and plasmin inhibitor might be of importance in the control of fibrin deposition and tissue repair following nerve injury or in inflammatory demyelinating diseases.

The localization of fibrinolytic enzyme in ovulation of rat and swine follicles

The localization of plasminogen activator in ovulation was investigated using the fibrin slide method. In regular cycle rats, plasminogen activator activity appeared in the external area of the follicle wall (stigma) from 12 hours before ovulation. No activity was noticed in the follicular cavity at this time. A peak of activity was seen at 2 hours before ovulation. After ovulation, the activity decreased markedly. The activity was completely inhibited on fibrin slides to which 10-2M t-AMCHA had been added. No activity was apparent on plasminogen-free fibrin slides.The plasminogen activator activities on the follicular wall and in the follicular fluid of swine were also measured using the fibrin plate method. The activity on the follicular wall increased from the diestrous to the estrous phase. No activity was apparent in the follicular fluid at any phase.

Fibrinolysis of the lung: An experimental study on the localization of plasminogen activator in the rat lung

The localization of tissue fibrinolysis in the rat lung was studied with a histochemical method. To identify the vessels a latex paint, most of which did not pass the capillary network, was injected into either the pulmonary veins, arteries or aorta. Fibrinolytic activity in the rat lung was found to be based on a plasminogen activator mainly localized to the cells of the pulmonary and bronchial arterial endothelium. No fibrinolytic activity was found in the pulmonary veins.