Epithelial tissue-type plasminogen activator expression, unlike that of urokinase, its receptor, and plasminogen activator inhibitor-1, is increased in chronic venous ulcers.

BACKGROUND: The plasminogen activation system represents a potent mechanism of extracellular proteolysis and is an essential component of normal wound healing. It has also been implicated in the pathogenesis of chronic, nonhealing ulcers. Traditionally, urokinase-type plasminogen activator (uPA) has been associated with pericellular proteolytic activity involved in tissue remodelling processes, and tissue-type plasminogen activator (tPA) mainly with intravascular fibrinolysis. OBJECTIVES: The present study was conducted to characterize the spatial distribution of the various plasminogen activation system components in chronic ulcers and acute, well-granulating wounds. METHODS: The expression of uPA, tPA, urokinase receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and vitronectin was investigated by immunohistochemical staining, in addition to uPA, tPA and PAI-1 expression by in-situ hybridization, in samples from eight chronic venous ulcers, five decubitus ulcers, five well-granulating acute wounds and five normal skin samples. RESULTS: In chronic venous leg ulcers tPA mRNA was detected in basal and suprabasal keratinocytes at the leading wound edge, while in well-granulating wounds and in decubitus ulcers tPA mRNA was expressed only in a few keratinocytes. However, tPA was widely expressed in fibroblast- and macrophage-like cells in the stroma of well-granulating wounds, while less tPA was detected in the granulation tissue of chronic ulcers. tPA mRNA and protein were localized in the superficial granular layers in normal skin. Although no qualitative differences in expression of uPA, PAI-1 or uPAR in the wound edge keratinocytes in chronic ulcers vs. normally granulating wounds were found, their expressions were more pronounced in the granulation tissue of well-granulating wounds. CONCLUSIONS: These results suggest that in poorly healing venous leg ulcers, the pattern of tPA expression is altered in keratinocytes at the leading edge of the wound, and the patterns of tPA, uPA and PAI-1 expression are altered in the granulation tissue.

Plasminogen activation in neurofibromatosis 2-associated and sporadic schwannomas.

BACKGROUND: Schwannomas are usually benign tumours which occur sporadically or in association with neurofibromatosis 2 (NF2), an autosomal dominant disorder. Invasiveness and higher proliferative potential compared to sporadic tumours are features of NF2-associated schwannomas. METHOD: We studied urokinase (uPA), tissue-type plasminogen activator (tPA), urokinase receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) expression by in situ hybridization and by immunohistochemistry in 14 NF2 and 15 sporadic patients with 34 schwannomas. uPAR and vitronectin immunohistochemistry were also studied. Three sural nerve specimens were included as Schwann cell controls. FINDINGS: Both schwannoma groups expressed prominent levels of uPA and tPA. Semiquantitative analysis of the in situ hybridization and immunoreactivity demonstrated that NF2 schwannomas expressed less PAI-1 at the mRNA level than sporadic schwannomas (score 1.63+/-0.41 vs. 2.05+/-0.75) and less total PAI-1 at the antigen level (score 1.55+/-0.66 vs. 2.07+/-0.56). PAI-1 was mostly in a free form in NF2 schwannomas compared to the sporadic counterparts (score 1.85+/-0.73 vs. 1.46+/-0.58), whereas there was less uPAR antigen in NF2 schwannomas than in the sporadic counterparts (score 1.18+/-0.49 vs. 1.68+/-0.56). Sural nerve Schwann cells did not express detectable level of PAI-1 and at the most a minor amount of tPA. CONCLUSIONS: Schwann cells of tumour cell origin, both in sporadic and NF2 schwannomas, expressed elevated levels of plasminogen activators and PAI-1 compared to normal suralic nerve Schwann cells. Furthermore, there seemed to be an imbalance in the PA-PAI-1 system in NF2-associated schwannomas. Although our methods are more descriptive than quantitative, we suggest that the somewhat more aggressive behavior of NF2-associated schwannomas compared to sporadic schwannomas may be based on the local proteolytic activity.

Differential effects of acute and chronic wound fluids on urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and tissue-type plasminogen activator in cultured human keratinocytes and fibroblasts.

The effect of wound fluids collected from acute well-healing wounds and chronic nonhealing venous leg ulcers on the plasminogen activation system of keratinocyte and fibroblast cell cultures was studied in a simplified wound-healing model. Acute wound fluid was collected from donor sites of split skin grafts at different time points representing the progressive healing of the wound. Urokinase-type plasminogen activator, tissue-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor 1 expression were studied. The methods used were immunocapture assay and immunocytochemistry. The results indicated that the later the acute wound fluid was collected, the greater the urokinase-type plasminogen activator and the lower the plasminogen inhibitor-1 level in treated cells. In contrast, the level of urokinase-type plasminogen activator receptor remained stable irrespective of wound fluid treatment. Immunostaining for urokinase-type plasminogen activator of acute wound fluid-treated cells showed a disseminated punctate pattern over the cell surface, but with chronic wound fluid, urokinase-type plasminogen activator was localized to focal contacts. Our findings support the view that in the acute wound environment the plasminogen activator system is proteolytically active and that in chronic leg ulcers urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor may also be organized for cell adhesion and migration.

Tissue plasminogen activator gene expression in multiple sclerosis brain tissue.

Recent studies have implicated tissue-type plasminogen activator (tPA) in neurodegeneration. We studied multiple sclerosis (MS) brain tissue for tPA gene and protein expression in comparison with reference tissue, by in situ hybridisation and immunohistochemistry. MS is characterised by demyelination in the central nervous system. In this study, neuronal cell bodies in MS brain showed high expression of tPA mRNA and protein, while in reference brains, staining for protein and mRNA expression were very low in neurons and mostly restricted to blood vessel walls. In MS, there was an additional staining of mononuclear cells within perivascular cuffs and foamy macrophages within demyelinating plaques. In view of evidence that the final process of demyelination in MS is thought to be enzyme-mediated, our work suggests the involvement of tPA and by inference plasmin, in the demyelinating process. Blocking tPA or plasmin activity may be a potentially beneficial therapeutic approach in MS.

Transforming growth factor beta induces urokinase receptor expression in cultured retinal pigment epithelial cells.

The effect of transforming growth factor-beta1 (TGF-beta1) and interferon-gamma (IFN-gamma) was studied on urokinase receptor (uPAR) expression of cultured human retinal pigment epithelial (RPE) cells. Human RPE cells were incubated with 1, 5 or 10 ng/ml of TGF-beta1 or with 10, 100 or 1,000 IU/ml of IFN-gamma to measure total cellular uPAR protein and released uPAR by enzyme immunoassay. uPAR at cell surface was measured by flow cytometric analysis at 8, 12, 24 and 48 h. uPAR mRNA levels were assayed by Northern blotting at 2, 6, 12 and 24 h. The increase in uPAR gene expression in RPE cells exposed to TGF-beta1 paralleled enhanced uPAR level at the cell surface and in conditioned medium. TGF-beta appeared to induce also membrane-bound uPA activity and the release of active plasminogen activator inhibitor-1, indicating that TGF-beta has the potential to regulate plasminogen activation at the RPE cell surface. The increase in uPAR gene expression by IFN-gamma did not seem to translate into the protein level. We conclude that TGF-beta regulates the pericellular proteolysis in RPE cells by increasing uPAR expression.

Biodegradable tube implants in experimental glaucoma surgery in the rabbit.

Although ocular drainage implants are manufactured from biocombatible materials to reduce foreign-body reaction, the formation of excessive scar tissue around the implant is a common cause for implant failure. In this study, the suitability of poly(D, L-lactide-co-glycolide) copolymer, impregnated with an antiproliferative agent retinoic acid, was evaluated as a material for biodegradable tubular implants, as well as the duration and magnitude of the intraocular pressure reduction obtained with the prototype implant. Subconjunctivally placed retinoid-impregnated polymer particles caused a milder inflammatory reaction than plain polymer, and the layer of connective tissue around the material was thinner after the follow-up period of 60 d. In the anterior chamber, the inflammatory response elicited by the material was milder than subconjunctivally. The plain polymer caused a transiently stronger reaction than the retinoid-impregnated polymer, but after 60 d no difference was evident between the two materials. In all operated eyes with the tubular implant, the intraocular pressure was statistically significantly lower (p<0.05) than in control eyes for 9 wk after the operation. The intraocular pressure of the eyes with the retinoid-impregnated implant was statistically significantly lower (p<0.05) than in eyes with a plain polymer implant for up to 7 wk post-operatively. However, the use of retinoid did not prolong the effective functioning time of the implants.

Interferons and retinoids enhance and dexamethasone suppresses urokinase-mediated plasminogen activation in promyelocytic leukemia cells.

All-trans retinoic acid (RA) has been successfully used in the treatment of patients with acute promyelocytic leukemia (APL). It induces differentiation of APL cells and reduces the bleeding tendency in APL patients. It has been proposed that plasminogen activation could affect the fibrinolytic balance in patients with leukemia. In our earlier study we found that treatment of APL cells with RA results in changes in urokinase (uPA) production. As interferons (IFNs) and dexamethasone can be used together with RA in the treatment of patients with APL, we have now studied the effects of RA together with IFNs and dexamethasone on the plasminogen activation cascade of these cells, including measurement of plasmin generation and uPA receptor (uPAR), using enzyme immunoassays, fluorescence-activated cell sorter analysis and RNA extraction with Northern blotting. Our main results were: (1) plasmin was formed on the surface of APL cells; (2) RA stimulated transiently plasmin generation and increased uPAR mRNA level; (3) IFNs alpha and gamma potentiated RA in its effects on uPA and plasmin activities and on uPAR level; (4) dexamethasone suppressed totally the effect of RA on uPA induction and plasminogen activation; and (5) IFNs and dexamethasone alone did not have potent effects on plasminogen activation. These results may assist in the design of therapy for APL patients.

Plasminogen activator inhibitor-1 in neointima of vein grafts: its role in reduced fibrinolytic potential and graft failure.

BACKGROUND: Intimal smooth muscle cell proliferation is an underlying pathogenetic mechanism for neointimal hyperplasia and consequent vein graft failure. This study characterizes the expression of tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor-1 (PAI-1) in hyperplastic vein grafts and normal venous tissue. METHODS AND RESULTS: Failing graft and control vein specimens from 14 donors were homogenized, and TPA and PAI-1 were quantified with ELISA. The amount of PAI-1 was seven times higher (4.2+/-2.1 versus 0.6+/-0.6 ng/mg protein, P<.005), but the TPA antigen content was markedly lower (3.1+/-2.1 versus 8.1+/-3.7 ng/mg protein, P<.005) in the stenosed grafts compared with the control veins. Strong immunohistochemical PAI-1 reactivity and in situ hybridization signals for PAI-1 and UPA mRNA were associated with the smooth muscle cells of the thickened intima of the grafts. Functional assays of the graft specimens showed an increased UPA/TPA ratio and a decreased total fibrinolytic activity in comparison with normal veins. CONCLUSIONS: Upregulation of PAI-1 mRNA expression and markedly increased amounts of PAI-1 antigen were detected in the vein grafts after the development of neointima. Furthermore, augmented UPA activity was found in the graft wall, but TPA was clearly depleted. Altogether, our findings imply decreased fibrinolytic potential in the stenosed graft, which may contribute to the graft occlusion.

Plasminogen activation in epiretinal membranes.

BACKGROUND: Formation of epiretinal membranes occurs in proliferative vitreoretinopathy, macular pucker and after penetrating trauma. Epiretinal membrane formation includes cell migration and proliferation, extracellular matrix formation and tissue contraction. Generally in scar tissue formation, the production of new extracellular matrix occurs concomitantly with its proteolytic degradation, resulting in continuous tissue remodelling. The plasminogen activator-mediated proteolytic cascade is an important mechanism for pericellular degradation of the extracellular matrix. Therefore we wanted to study the presence of the plasminogen activator-mediated proteolytic cascade in epiretinal membranes. METHODS: Specimens of 18 epiretinal and 3 subretinal membranes were obtained during vitreous surgery for retinal detachment with proliferative vitreoretinopathy or macular pucker. Plasminogen activators and plasmin were characterized in frozen sections of epiretinal membranes by in situ zymography and in membrane lysates by zymography. Indirect immunofluorescence staining was performed to localize urokinase in epiretinal membranes. RESULTS: Urokinase was present in 17/21 and tissue-type plasminogen activator in 12/21 of the membranes studied. Active plasmin was not detected in the frozen sections of epiretinal membranes. Immunofluorescence staining localized urokinase predominantly in the areas invaded by macrophages and cells of retinal pigment epithelial origin. CONCLUSION: Our results demonstrate the presence of proteolytic activity in periretinal scar tissue. Urokinase was more consistently present, but smaller amounts of tissue-type plasminogen activator were also found in the specimens. These results indicate that continuous tissue remodelling with simultaneous extracellular matrix production and breakdown regulates the growth of epiretinal membranes.

Cerebrospinal fluid activity of tissue plasminogen activator in patients with neurological diseases.

AIM: To study cerebrospinal fluid (CSF) activity of tissue plasminogen activator (tPA) in patients with neurological diseases. METHODS: CSF tPA and urokinase (uPA) activities were studied using an immunocapture assay and zymography in 44 patients with neurological disease and 20 reference subjects. The patient group comprised three patients with meningitis, 21 with encephalitis, nine with acute lymphoblastic (n = 7) and myeloid (n = 2) leukaemia, seven with multiple sclerosis, three with facial paresis, and one with polyradiculitis. RESULTS: Raised tPA activities were observed in patients with multiple sclerosis, leukaemia and encephalitis. In contrast, there were no differences in the mean activities of tPA in patients with meningitis or other diseases compared with the reference subjects. The highest tPA activities were found in patients with multiple sclerosis. The mean activity in patients with leukaemia was higher than in those with meningitis and polyradiculitis, but not encephalitis and facial paresis. Although the CSF tPA activity correlated positively with age in reference subjects, no correlation was observed in patients. Samples were qualitatively screened for both tPA and uPA activity by zymography and positive samples were quantitated. Some of the samples had quantifiable levels of uPA activity: three of seven multiple sclerosis samples, 10 of 21 samples from patients with encephalitis and five of nine leukaemic samples. The highest activities were recorded in patients with leukaemia. uPA was not detected in the CSF of the patients with meningitis, facial paresis or polyradiculitis. CONCLUSIONS: Plasminogen activator activity can be measured reliably in CSF and the assessment of tPA activity may be useful for studying the pathogenesis of neurological diseases.

Keratinocyte growth factor enhances urokinase-type plasminogen activator activity in HPV16 DNA-immortalized human uterine exocervical epithelial cells.

We recently demonstrated that keratinocyte growth factor (KGF), a fibroblast-derived growth factor, induced anchorage-independent growth of HPV16 DNA-immortalized human uterine exocervical epithelial cells (HCE16/3 cell line) in soft agarose assay. We have now studied whether KGF might also regulate the plasminogen activator (PA) system, another transformation parameter related to cell invasiveness. HCE16/3 cells were found to produce urokinase-type PA (uPA) and small amounts of tissue-type PA (tPA) as determined by immunocapture assay. Secretion of both uPA and tPA was increased when HCE16/3 cells were exposed to KGF for 4 h and continued to increase during the next 24 h. The early increase following KGF treatment was presumably mediated by binding of KGF to its receptors. Enhanced secretion of uPA in HCE16/3 cells by KGF was also seen by zymographic analysis and immunofluorescence. According to Northern blotting KGF upregulated the transcription of uPA gene in HCE16/3 cells; the upregulation occurred only after 12 h of KGF treatment, suggesting that stimulation of uPA production by KGF is a biphasic event. As proteolysis is a prerequisite for tumor cell invasion, taken together with our previous results, the present findings suggest that KGF may play an important role in the transition of immortalized uterine cervical epithelial cells from in situ to invasive carcinoma.

Proteinases in subretinal fluid.

BACKGROUND: Degradation of the extracellular matrix by secreted proteases is connected to cell migration and proliferation in invasive growth and in scar tissue formation. In retinal detachment, retinal pigment epithelium (RPE) cells loosened from their monolayer are often seen in the subretinal fluid (SRF) and the vitreous, where they may participate in the scar tissue formation of proliferative vitreoretinopathy. To evaluate the role of SRF constituents on the release of RPE cells, we analyzed SRF in patients with retinal detachment for the presence of enzymes able to degrade extracellular matrix. METHODS: SRF was collected altogether from 16 patients undergoing retinal reattachment surgery and analyzed for activities against some of the key enzymes in extracellular proteolysis, namely collagenases, gelatinases, elastase and cathepsin G. RESULTS: Seventy-two-kilodalton gelatinase was found in all SRF samples studied, whereas the neutrophil-type 92-kDa gelatinase could not be detected. Low collagenase, elastase and cathepsin G activities could also be detected in some samples. CONCLUSIONS: The predominant type of matrix metalloproteinase present in SRF is the 72-kDa MMP-2. The proteolytic activity in SRF may be connected to the release of RPE cells into SRF and to degradation of components of the vitreous exposed to SRF.

Secretion of plasminogen activators by cells cultured from subretinal fluid.

Secretion of plasminogen activators was examined in 11 cells cultures from subretinal fluid of patients with retinal detachment. The plasminogen activator production was compared to 4 retinal pigment epithelial cell cultures from post-mortem donors with no known retinal pathology. Plasminogen activator activity was evaluated by zymography and was corrected for the total cell protein in the cultures. Cells from subretinal fluid had more proteolytic activity and secreted more active urokinase-type plasminogen activator than retinal pigment epithelial cells of donor origin. It is possible that cells from subretinal fluid are modulated by the conditions in subretinal fluid to secrete more urokinase-type plasminogen activator, generally considered to be a sign of invasive growth or that the subretinal fluid cultures contain other cell types than solely retinal pigment epithelial cells, such as pigmented macrophages, with an active plasminogen activator production. Alternatively, retinal pigment epithelial cells detached into the subretinal fluid may represent a selected population of retinal pigment epithelial cells with a more active extracellular proteolysis.

Alpha- and gamma-interferon inhibit plasminogen activator inhibitor-1 gene expression in human retinal pigment epithelial cells.

The effect of interferons IFN-alpha and IFN-gamma on the production of plasminogen activators (PAs) and plasminogen activator inhibitors (PAIs) was examined in human retinal pigment epithelial (RPE) cells in culture. Cultures of human RPE cells were incubated with either of the interferons for 48-96 h. The cell cultures were assayed using mRNA analysis and solid-phase immunocapture assay. Both interferons caused a marked decrease in PAI-1 mRNA expression after 48 h with no change in urokinase-type plasminogen activator (u-PA) mRNA expression. A marked decrease in PAI-1 activity and concomitant increase in u-PA activity in the culture medium appeared only after 72-96 h. We conclude that IFN-alpha and IFN-gamma stimulate plasminogen-mediated pericellular proteolysis of human RPE cells in culture by inhibiting PAI-1 gene expression.

Retinoids increase urokinase-type plasminogen activator production by human retinal pigment epithelial cells in culture.

PURPOSE: To examine the effect of two retinoids, all-trans-retinoic acid (tretinoin) and all-trans-9-(4-methoxy-2,3,6- trimethylphenyl)-3,7-dimethyl- 2,4,6,8-nonatetraenoic acid (acitretin) on the production of plasminogen activators and plasminogen activator inhibitors by human retinal pigment epithelial cells in culture. METHODS: Cultures of human retinal pigment epithelial cells were incubated with either of the retinoids at a concentration of 1 microM for 24-72 hours. The media were assayed using solid-phase immunocapture assays and zymography. RESULTS: Both retinoids caused a twofold to sevenfold increase in urokinase-type plasminogen activator in the medium. The effect was seen after 24 hours in culture and was further augmented up to 72 hours. No significant amounts of tissue-type plasminogen activator were detected. The plasminogen activator inhibitor activity was unaffected by the retinoids. Proliferation and morphology of retinal pigment epithelial cells were also unaffected by the retinoids in incubations for up to 72 hours. CONCLUSIONS: Retinoids profoundly affect the extracellular proteolysis of retinal pigment epithelial cells in culture. This effect may be related to the differentiation-inducing activity of retinoids seen in other cell types, often connected with changes in extracellular proteolysis. It is possible that retinoids may modulate dedifferentiation, proliferation, and migration of retinal pigment epithelial cells seen in vitro, as well as in the pathogenesis of retinal disease.

Retinal pigment epithelial cells secrete urokinase-type plasminogen activator and its inhibitor PAI-1.

Secretion of plasminogen activators and their inhibitors was examined in cultures of human retinal pigment epithelial (RPE) cells. The methods employed were zymography and reverse zymography, solid-phase immunocapture assay, metabolic labeling followed by immunoprecipitation, and immunofluorescence. The results showed that these cells produce urokinase-type plasminogen activator (u-PA) and a plasminogen activator inhibitor (PAI) which is immunologically and biochemically similar to PAI-1. Tissue-type plasminogen activator activity (t-PA) was not detected, but we detected small amounts of t-PA in an inactive complex with inhibitor in RPE cell-conditioned media. We conclude that RPE cells have the potential to utilize u-PA-catalyzed plasminogen activation which is subject to regulation by PAI-1. These results may have a bearing on the pathogenesis of proliferative retinal diseases.

Culture of retinal pigment epithelial cells from subretinal fluid.

Culture of cells from subretinal fluid (SRF) was performed using 29 SRF samples obtained at retinal reattachment surgery. Proliferating cells were found in 58.6% of the samples studied. The cells were of retinal pigment epithelial (RPE) origin, as evidenced by their brown pigmentation in primary culture and their positive immunostaining for cytokeratins 8/18. The age of the patients did not affect the proliferative capacity of the cells. Proliferating cells were present in all samples from eyes with proliferative vitreoretinopathy (PVR) of grade C1 or more. In primary culture the cells had a fibroblast-like morphology, resembling that of ordinary RPE cells exposed to the vitreous. We conclude that the SRF of many patients with PVR contains viable proliferating RPE cells and that SRF offers a new source of RPE cells for studies on the pathogenesis of PVR.