Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.     

uPA, tPA and PAI-1 mRNA expression in periretinal membranes

PURPOSE: Formation of periretinal membranes occurs in proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) and includes cell migration, proliferation, extracellular matrix formation and tissue contraction, processes in which plasminogen activation (PA) system is involved. METHODS: Twenty PVR, PDR or pucker membranes were examined to identify the cells with cell specific markers and to detect the expression of urokinase (uPA), tissue-type plasminogen activator (tPA) or plasminogen activator inhibitor-1 (PAI-1) by in situ hybridization and by immunohistochemistry. RESULTS: In PVR, uPA, tPA and PAI-1 were expressed by retinal pigment epithelial (RPE) cells, macrophages or retinal glial cells. In PDR, PA components were also expressed by endothelial cells. Semiquantitative analysis in in situ hybridization and immunohistochemistry results demonstrated no notable differences in uPA, tPA or PAI-1 expression between PDR and PVR membranes. CONCLUSIONS: We conclude that local proteolytic activation is involved in extracellular matrix production both in diabetic and non-diabetic membranes.     

Pathogenesis of corneal epithelial defects: role of plasminogen activator

Previous studies have suggested that the plasminogen activator (PA)/plasmin system has important roles in the pathogenesis of epithelial defects and stromal ulceration. The current studies were performed to localize PA species and identify them as tissue-type PA (tPA) or urokinase-like PA (uPA) as the two have distinct regulatory properties potentially related to the mechanisms of defect formation and ulceration. To determine the locations and types of PA species, antibodies to tPA or to uPA or the drug amiloride (a drug that inhibits uPA but not tPA) were incorporated into fibrin/fibronectin (Fn) clots overlying frozen sections to block regional fibrinolysis. Normal rabbit eyes showed tPA activity in association with corneal epithelium, corneal endothelium, and ciliary body/iris. After epithelial scrape or alkali burn, corneal tPA activity was detected initially in the defect zone colinear with fibrin/Fn and was symmetrical to resurfacing epithelium. The observation that initial fibrinolysis occurs in the defect zone, known to contain fibrin/Fn, suggests that tPA from blood (limbal vascular endothelium) and/or from corneal epithelium has become bound to (and activated on) the fibrin/Fn. PA activity was also associated with the leading edges of migrating epithelium post-scrape and post-burn and was not inhibited by antibodies to either tPA or uPA but was inhibited by amiloride. After complete closure of the primary defect post-scrape, only tPA appeared to be associated with the epithelium in that all PA activity was inhibited by antibodies to tPA. The observation that leading edge activity post-burn, in correlation with the formation of secondary defects, continues to be inhibitable by amiloride but not by antibodies to tPA suggests that uPA remains abnormally on the leading edge, and that sustained uPA activity in that location results in inappropriate degradation of subepithelial fibrin/Fn to result in a defect. Successful regulation of uPA activity at the leading edge of corneal epithelium post-burn would be expected to be useful therapeutically in the healing of epithelial defects and the prevention of stromal ulceration.     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

Tissue type plasminogen activator facilitates NMDA-receptor-mediated retinal apoptosis through an independent fibrinolytic cascade

PURPOSE: To investigate the association between apoptosis and the fibrinolytic system in retinal cell damage. METHODS: Tissue type plasminogen activator-deficient (tPA(-/-)), urokinase type plasminogen activator-deficient (uPA(-/-)), plasminogen activator inhibitor-1-deficient (PAI-1(-/-)), alpha2 antiplasmin-deficient (alpha2 AP(-/-)) mice, and their wild-type counterparts were used. Retinal cell damage was induced by intravitreal injection of the excitotoxin N-methyl-d-aspartate (NMDA). The TdT-dUTP terminal nick-end labeling (TUNEL) method was used to examine retinal cell damage. RESULTS: tPA(-/-) mice were resistant to retinal cell damage caused by administration of NMDA, and PAI-1(-/-) mice were more injured than their wild-type. No significant difference was observed between uPA(-/-) or alpha2 AP(-/-) and their wild-type mice. CONCLUSIONS: The results strongly suggest that endogenous tPA, but not uPA acts as a facilitator in NMDA-induced retinal cell damage, and that its mechanism may not be associated with cleavage of plasminogen into plasmin in the fibrinolytic cascade.     

Plasminogen Activators and Inhibitors in the Corneas of Mice Infected with Pseudomonas aeruginosa

PURPOSE: To characterize the presence of plasminogen activators and their inhibitors in the corneas during the inflammatory response in na?ve and immunized mice intracorneally infected with Pseudomonas aeruginosa. METHODS: RT-PCR was used to detect gene expression for plasminogen activators and their inhibitors in na?ve and immunized mice. Immunoblot analysis, zymography, and ELISA were used to demonstrate the syntheses of these proteins. RESULTS: Na?ve mice intracorneally infected with P. aeruginosa showed a temporally enhanced expression of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2), over a several-day holding period. Immunized mice demonstrated a lower and shorter expression of these factors over the same period. Expression of these factors at the mRNA and protein levels may have been due to enzymes and inhibitors present in inflammatory cells and in resident corneal cells. CONCLUSIONS: These results show a correlation between the overexpression of the PA system in infected na?ve mice as part of the inflammatory response, with eventual ocular destruction. Immunized mice exhibit a more balanced and shorter expression of the PA system, which may contribute to the restoration of corneal clarity.     

Mechanisms regulating plasminogen activators in transformed retinal ganglion cells

Irreversible loss of retinal ganglion cells (RGCs) is a major clinical issue in glaucoma, but the mechanisms that lead to RGC death are currently unclear. We have previously reported that elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) cause the death of RGCs in vivo and transformed retinal ganglion cells (RGC-5) in vitro. Yet, it is unclear how secreted proteases such as tPA and uPA directly cause RGCs' death. In this study, by employing RGC-5 cells, we report that tPA and uPA elicit their direct effect through the low-density lipoprotein-related receptor-1 (LRP-1). We also show that blockade of protease-LRP-1 interaction leads to a complete reduction in autocrine synthesis of tPA and uPA, and prevents protease-mediated death of RGC-5 cells. RGC-5 cells were cultured in serum-free medium and treated with 2.0 microM Staurosporine to induce their differentiation. Neurite outgrowth was observed by a phase contrast microscope and quantified by NeuroJ imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by MTT assays. Compared to untreated RGC-5 cells, cells treated with Staurosporine differentiated, synthesized and secreted elevated levels of tPA and uPA, and underwent cell death. In contrast, when RGC-5 cells were treated with Staurosporine along with the receptor associated protein (RAP), proteolytic activities of both tPA and uPA were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived and showed increased neurite outgrowth. These results indicate that LRP-1 regulates autocrine synthesis of tPA and uPA in RGC-5 cells and suggest that the use of RAP to antagonize the effect of proteases may be a way to prevent RGC death in glaucoma.     

Maspin: synthesis by human cornea and regulation of in vitro stromal cell adhesion to extracellular matrix.

PURPOSE: Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules. METHODS: Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. RESULTS: Maspin mRNA and micromolar amounts of the protein were found in all three layers of the human cornea in situ, including the stroma. Maspin was also detected in primary and first-passage corneal stromal cells, but its expression was downregulated in subsequent passages. Late-passage stromal cells, which did not produce maspin, responded to exogenous recombinant maspin as measured by increased cell adhesion not only to fibronectin, similar to mammary gland tumor epithelial cells, but also to type I collagen, type IV collagen, and laminin. CONCLUSIONS: The corneal stromal cell is the first nonepithelial cell type shown to synthesize maspin. Loss of maspin expression in late-passage corneal stromal cells in culture and their biological response to exogenous maspin suggests a role for maspin on the stromal cells in the cornea. Maspin may function within the cornea to regulate cell adhesion to extracellular matrix molecules and perhaps to regulate the migration of activated fibroblasts during corneal stromal wound healing.     

Plasminogen activator activity in vitamin A-deficient rat corneas

Plasminogen activator (PA) activity during epithelial wound healing in vitamin A-deficient rats was investigated to determine whether a relationship exists between corneal defect formation and PA activity. Uniform, central 3 mm diameter corneal epithelial wounds were made by scalpel debridement in vitamin A-deficient and in pair-fed control rats. Cryostat sections of such corneas, taken at various times post-scrape, were overlaid with fibrin films containing plasminogen to examine the distribution of PA activity; and antibodies to tissue plasminogen activator (tPA) or to urokinase-like activator (uPA) were incorporated into the films so that the immunochemical natures of detected PA activities could be determined. Corneas from pair-fed controls showed tPA-dependent lysis in association with the regenerating epithelium as well as in the defect region during epithelial wound healing. Corneas from vitamin A-deficient rats also demonstrated tPA activity in association with corneal epithelium post-scrape but showed no detectable tPA activity in the defect region. Histological examination of the vitamin A-deficient corneas demonstrated that a pseudomembrane composed of PMNs, cell debris, and fibrinous exudate had formed on the scrape-debrided stromal surface. The migrating edge of regenerating epithelium overlaid this membrane and was, therefore, not in contact with the stromal surface. The formation of the pseudomembrane, which delays reepithelialization, might have resulted from the absence of PA activity in the defect region. Both excessive and inadequate levels of PA activity may result in impaired epithelial wound healing.     

Promotion of glaucoma filter bleb with tissue plasminogen activator after sclerectomy under a clot

Since 1987 we have performed, with good results, glaucoma filtering surgery in which the limbo-scleral fistula was closed by an autogenic full blood clot (sclerectomy under a clot). In 8 patients with advanced stages of glaucoma simplex, 1–3 days after sclerectomy under a clot with rise IOP, 25g of tissue plasminogen activator (tPA) was injected subconjunctivally. Two weeks after surgery normalization of IOP below 15 mmHg and normal outflow facility in all tPA treated eyes were obtained. No increase in early postoperative complications, such as hemorrhage or corneal haze, was attributable to tPA use. In early period after sclerectomy under a clot, when blockage of outflow appears, the use of tPA can cause the re-creation of filtering tract.     

Urokinase binding to bovine corneal endothelial cells

Previously, we showed that the clotting factor thrombin binds to bovine corneal endothelial cells forming a covalent complex with a protein released by these cells into the culture media. We report that the plasminogen activator, urokinase, an enzyme involved in dissolution of blood clots, binds specifically to bovine corneal endothelial cells. [125I]-urokinase is rapidly bound by corneal endothelial cells. The serine active site of urokinase is required for binding since [125I]-urokinase inactivated with diisoprophylfluorophosphate does not bind to the cells. Pre-incubation of corneal cells with unlabeled urokinase for 20 hr results in increased release of binding sites for [125I]-urokinase into the culture media and at least a 3.5-fold increase in binding by the cells. [125I]-urokinase forms a 73 300 dalton sodium dodecyl sulfate complex with a corneal endothelial cell protein. Although thrombin competes with urokinase for binding to corneal endothelial cells, urokinase has at least a 10-fold higher affinity for binding to the cells. Furthermore, these cells make a plasminogen activator identical in molecular weight to urokinase. Thus, under physiological conditions, urokinase rather than thrombin binds to corneal endothelial cells. Binding may serve to regulate endogenous urokinase activity in the anterior chamber of the eye by limiting its extracellular proteolytic activity.     

Corneal Opacity: Cell Biological Determinants of the Transition From Transparency to Transient Haze to Scarring Fibrosis,and Resolution,After Injury

PurposeTo highlight the cellular, matrix, and hydration changes associated with opacity that occurs in the corneal stroma after injury.MethodsReview of the literature.ResultsThe regulated transition of keratocytes to corneal fibroblasts and myofibroblasts, and of bone marrow-derived fibrocytes to myofibroblasts, is in large part modulated by transforming growth factor beta (TGFβ) entry into the stroma after injury to the epithelial basement membrane (EBM) and/or Descemet''s membrane. The composition, stoichiometry, and organization of the stromal extracellular matrix components and water is altered by corneal fibroblast and myofibroblast production of large amounts of collagen type I and other extracellular matrix components—resulting in varying levels of stromal opacity, depending on the intensity of the healing response. Regeneration of EBM and/or Descemet''s membrane, and stromal cell production of non-EBM collagen type IV, reestablishes control of TGFβ entry and activity, and triggers TGFβ-dependent myofibroblast apoptosis. Eventually, corneal fibroblasts also disappear, and repopulating keratocytes reorganize the disordered extracellular matrix to reestablish transparency.ConclusionsInjuries to the cornea produce varying amounts of corneal opacity depending on the magnitude of cellular and molecular responses to injury. The EBM and Descemet''s membrane are key regulators of stromal cellularity through their modulation of TGFβ. After injury to the cornea, depending on the severity of the insult, and possibly genetic factors, trace opacity to severe scarring fibrosis develops. Stromal cellularity, and the functions of different cell types, are the major determinants of the level of the stromal opacity.     

Plasminogen activator inhibitor-1 (PAI-1) stimulates human corneal epithelial cell adhesion and migration in vitro

In addition to its role as an inhibitor of urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) is hypothesized to regulate epithelial cell adhesion and migration. We have previously reported that PAI-1 may be an important regulatory factor of the uPA system in cornea. The purpose of this study was to extend those observations by determining the effect of exogenous PAI-1 on the migration and adhesion of human corneal epithelial cells (HCEC) in vitro. The expression of PAI-1 in non-transformed early passage HCEC was confirmed by immunofluorescence microscopy and Western blot analysis. Colorimetric assays coupled with function-inhibiting antibody studies using the matrix assembled in situ by cultured cells demonstrate that immobilized PAI-1 serves as an efficient substrate for HCEC adhesion. HCEC attachment to PAI-1 is comparable to that of laminin-10, a known strong adhesion protein for epithelial cells. In addition to serving as an adhesion substrate, PAI-1 also functions as a chemotactic agent for corneal epithelium. Additionally it promotes the random migration of HCEC, from an initial cell cluster, along a culture substrate. Our results in corneal epithelium are consistent with reports from other investigators showing that PAI-1 facilitates both epithelial adhesion and migration. From our studies we conclude that PAI-1 may play a dual role in corneal wound healing. Initially PAI-1 may function to stimulate migration and facilitate the reepithelialization of the wound bed. Post-reepithelization, PAI-1 may ensure corneal epithelial cell adhesion to matrix to promote successful wound healing.     

Evidence for a role of the plasminogen activator--plasmin system in corneal ulceration

Plasminogen is present in the cornea andcan be activated to plasmin by plasminogen activator. Plasmin is able, in turn, to activate latent collagenase. This system could initiate and perpetuate the collagen degradation of corneal ulceration. This report details evidence for such a system in the cornea. Plasmin has been found to activate latent collagenase from organ cultures of ulcerating rabbit corneas and from fibroblast cultures derived from such corneas. As in the case of activation by trypsin, activation by plasmin results in the conversion of the 40,000 MW latent form to an active species of 23,000 MW. Explants of normal or alkali-burned, ulcerating corneas demonstrated plasminogen-dependent lysis of fibrin clots; frozen sections of such corneas demonstrated that lysis begins in the superficial stroma near the periphery of the cornea. Multiply freeze-thawed ulcerating corneas, but not normal corneas, showed initial lysis, not peripherally but at the ulcer region containing polymorphonuclear leukocytes. The fact that the peripheral lytic pattern existed in corneas that were obtained from eyes prefrozen in liquid nitrogen before excision of the corneas would suggest that plasminogen activator is normally contained in cells in vivo and is not made only in response to tissue injury. There was no correlation between the location of blood vessels or the presence of the corneal endothelium and the plasminogen-dependent lysis. Plasminogen activator from the ulcerating cornea and from fibroblasts was characterized by sodium dodecyl sulfate--gel electrophoresis of its cleavage products of plasminogen. The activator cleaves plasminogen into heavy- and light-chain fragments similar to those produced from plasminogen by urokinase. Plasminogen activator activity was quantitated by a new assay that restricts diffusion of the enzyme to one dimension into a narrow bore tube. The addition of plasminogen daily to cultures of ulcerating corneas resulted in earlier rises of plasminogen activator, collagenase, and collagen degradation fragments in the culture media. Although total plasminogen activator levels were not increased by the addition of plasminogen to culture, levels of both collagenase and solubilized collagen were approximately doubled. It is concluded that the plasminogen activator--plasmin system might play an important role in the destruction of stromal matrix in corneal ulceration.     

Changes of Plasma tPA and PAI Activities in Patients with Diabetic Retinopathy

Purpose:To investigate the relationship between the diabetic retinopathy (DR) and the activity of plasma tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI).Methods :tPA and PAI activities were measured by chromatogenous substrate assay in plasma samples obtained from patients with and without diabetic retinopathy (n = 42). Retinopathy was determined by stereoscopic color fundus photographs graded according to a modification of Chinese National Fundus Disease Academic Meeting. And 21 sex-age matched normal people were as controls. This study was in a masked fashion.Results:①tPA activity was lower and PAI activity was higher in all of diabetic patients than those in controls (P<0. 001); ②tPA activity was lower and PAI activity was higher in proliferative DR (PDR) subgroup than those in non-DR (NDR) and background-DR (BDR) subgroups (P<0.01,respectively); ③there was no significant difference between BDR and NDR subgroups (P>0. 05); and ④the results also suggested that the seve     

The molecular basis of corneal transparency

The cornea consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells. The stroma consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25-30 nm fibrils that are regularly packed within lamellae and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sufate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-β would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.     

Microarray studies reveal macrophage-like function of stromal keratocytes in the cornea

PURPOSE: To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea. METHODS: Murine Genome_U74Av2 arrays (Affymetrix Inc., Santa Clara, CA) were used to elucidate gene expression patterns of adult and postnatal day-10 corneal stroma, keratocytes, fibroblasts, and myofibroblasts. RESULTS: Mobilization of stromal cells by culturing led to a wound-healing cascade in which specific extracellular matrix and cornea-transparency-related genes were turned off, and a repertoire of macrophage genes were switched on. Thus, novel transparency-related crystallins detected in the corneal gene expression patterns were downregulated in culture, whereas macrophage genes, mannose receptor type-1, Cd68, serum amyloid-A3, chemokine ligands (Ccl2, Ccl7, Ccl9), lipocalin-2, and matrix metalloproteinase-3 and -12 of innate immunity were induced in primary keratocyte cultures. Fibroblasts expressed the growth-related genes lymphocyte antigen 6 complex locus-A and preprokephalin-1, and myofibroblasts expressed annexin-A8, WNT1-inducible signaling pathway protein-1, arginosuccinate synthetase-1, and procollagen XI of late-stage wound healing. CONCLUSIONS: The emergent biological process suggests a dual role for resident stromal keratocytes in the avascular cornea: one of cornea maintenance, which involves synthesis of proteins related to the extracellular matrix and corneal transparency, and a second of barrier protection macrophage functions, which are switched on during corneal infection and injury.     

Urokinase-type plasminogen activator in human aqueous humor.

In the anterior segment of the eye, fibrin clots must be rapidly resorbed to prevent further fibrosis and scarring. The aqueous humor of patients undergoing cataract surgery was analyzed for the presence of components of the fibrinolytic cascade. In 30 patients, aqueous humor and plasma were compared for their content of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen activators inhibitors (PAIs), plasminogen, and total proteins. With gel electrophoresis and zymographic assays of serial dilutions of plasma and aqueous humor, all these components were found to be present at lower concentrations in aqueous humor than in plasma. For total proteins, the aqueous/plasma ratio was approximately 0.003, and for plasminogen it was 0.001. Interestingly, the aqueous/plasma ratio for uPA was not as low and varied from 0.01 to 0.03. A significant proportion of the uPA in aqueous humor was present in the two-chain active form. In addition to uPA, aqueous humor contained lower levels of tPA, but no detectable levels of reactive plasminogen activators inhibitors (PAIs). The presence of a relatively high concentration of active uPA shows that the proteolytic balance of the aqueous humor in the anterior chamber of the eye is shifted toward fibrinolysis.     

Latent and active plasminogen activator in corneal ulceration

Previous studies of alkali burns have provided evidence for an important role of the plasminogen activator (PA)/plasmin system in corneal ulceration. Current studies have utilized a sensitive, plasminogen-dependent fluorescent assay to demonstrate that PA is present mostly in a latent (trypsin- or plasmin-activatable) form (proactivator) in cultures of rabbit corneal epithelial cells or normal corneas. Cultures of ulcerating corneas demonstrate only active PA early in organ culture, whereas, latent PA levels increase later in culture. Thus, ulceration is correlated with the apparent conversion of latent to active PA. Moreover, profiles of proactivator and latent collagenase and of active PA and active collagenase in vitro, respectively, are similar, suggesting that activator and collagenase are under coordinate control. Cultures of normal epithelial cells and nonulcerating corneas contain PA molecular weight species of 72,000 and 46,000 MW, and ulcer corneas, species of 72,000, 46,000, and 35,000 MW. Double-diffusion analysis indicates that rabbit epithelial cells, fibroblasts, and ulcer corneas produce urokinase (UK)-like PA; and human cornea extracts and tears also contain PA immunoreactive with anti-UK antibodies. The existence of PA in a latent form identifies another level of regulation in the cascades that lead to stromal ulceration.     

Galardin inhibits collagen degradation by rabbit keratocytes by inhibiting the activation of pro-matrix metalloproteinases.

We investigated the inhibitory action of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase (MMP), Galardin (GM6001), on collagen degradation by rabbit corneal stromal fibroblasts (keratocytes) cultured three-dimensionally in the type I collagen gel with medium containing interleukin 1alpha (IL-1alpha) and/or plasminogen. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline, and activation of MMPs was also analyzed by gelatin zymography and Western blotting. Plasmin activity was measured using a synthetic substrate. In the absence of plasminogen, treatment of the cells with IL-1alpha in collagen gel greatly enhanced the production of proMMP-1, -3 and -9, but no significant degradation of collagen was detected. In the presence of plasminogen, IL-1alpha stimulated collagen degradation by keratocytes in a dose-dependent manner. This resulted from the plasminogen activator-plasmin system-dependent activation of proMMP-1, -3 and -9. Galardin inhibited the collagen degradation in a dose-dependent fashion in the presence of plasminogen, whether IL-1alpha was present or not. Galardin inhibited the activation of proMMP-3, and also prevented the activation of proMMP-9 and the conversion of MMP-1 intermediates to the fully active MMP-1. Galardin did not affect plasmin activity. The present results suggest that Galardin inhibits IL-1alpha-stimulated collagen degradation in the presence of plasminogen, resulting from not only inhibiting active MMPs but also preventing the conversion of proMMPs to active MMPs.