Regulation of gelatinase B production in corneal cells is independent of autocrine IL-1alpha. (1/1253)

PURPOSE: The matrix metalloproteinase gelatinase B is synthesized by cells at the leading edge of the corneal epithelium migrating to heal a wound. Recent data from the authors' laboratory suggest that excessive synthesis contributes to repair defects. The goal of the study reported here was to investigate mechanisms controlling gelatinase B production by corneal epithelial cells. METHODS: Freshly isolated cultures of corneal epithelial cells and early passage stromal fibroblasts from rabbit were used for these studies. RESULTS: In a previous study, it was found that the cytokine interleukin (IL)-1alpha is released into the culture medium of corneal epithelial cells more efficiently when they are plated at low density with limited cell-cell contact than when plated at high density. In this study, we show that production of gelatinase B by these cells is similarly affected by cell plating density. However, it is further demonstrated that these two events are not dependent on one another but occur in parallel: IL-1alpha does not regulate gelatinase B production (synthesis), nor was there evidence that any other secreted autocrine cytokine acts as mediator. Instead, our data suggest that gelatinase B production is downregulated directly by high cell density and indicate a connection to the level of protein kinase C activity. Nevertheless, the anticancer agent suramin, which blocks collagenase synthesis by interfering with autocrine cytokine-receptor interactions, still inhibits synthesis of gelatinase B. CONCLUSIONS: Unlike collagenase synthesis by corneal stromal fibroblasts, production (synthesis) of gelatinase B does not appear to be controlled by secreted autocrine cytokines but can still be inhibited by suramin. Suramin may make an effective therapeutic agent for controlling pathologic overproduction of gelatinase B in corneal ulcers.  (+info)

PAF binding to a single receptor in corneal epithelium plasma membrane. (2/1253)

PURPOSE: To study the binding characteristics and the expression of platelet-activating factor receptors (PAF-R) in corneal epithelium to elucidate the site of action of PAF. METHODS: Binding of [3H]PAF was investigated in subcellular fractions of the epithelia of bovine corneas and in membranes from cultured rabbit corneal epithelial cells. Dose-response inhibition curves of [3H]PAF-specific binding were generated using increasing concentrations of several PAF-R antagonists. RNA from rabbit corneal epithelial cells was probed for PAF-R expression by reverse transcription-polymerase chain reaction (RT-PCR) with specifically designed degenerated primers. RESULTS: Scatchard analysis showed a high-affinity binding site in bovine and rabbit corneal epithelium. The dissociation constant (Kd) and the maximum binding sites (Bmax) in a bovine membrane preparation and similar rabbit fraction were 0.77+/-0.03 nM and 180+/-21 femtomoles/mg protein and 4.3 nM and 1.3 picomoles/mg protein, respectively. Specific PAF-binding sites were found in bovine preparations enriched in plasma membranes with a Kd = 69.6 pM and Bmax = 80 femtomoles/mg protein; no specific binding was found in nuclei or microsomal fractions. RT-PCR of rabbit corneal epithelium generated a single product of the predicted size (478 bp). The deduced amino acid sequence of the purified PCR product was 87% homologous to human PAF-R. The hetrazepines BN 50727 and BN 50730 and the PAF structural analogues CV 3988 and CV 6209 competitively inhibited [3H]PAF binding to corneal epithelium with similar potency. WEB 2086 BS was two orders of magnitude less active in antagonizing PAF binding. CONCLUSIONS: Corneal epithelium contains a single population of receptors localized in the plasma membrane. PAF antagonists exert their actions by blocking this PAF-R. The partial sequence deduced in rabbit corneal PAF-R show a higher homology to the human PAF-R.  (+info)

Zyxin and vinculin distribution at the cell-extracellular matrix attachment complex (CMAX) in corneal epithelial tissue are actin dependent. (3/1253)

Avian embryonic corneal epithelia are two cell layers thick. If isolated without (-) basal lamina, the basal cells have unorganized actin and project cytoplasmic protrusions termed blebs. The actin-based cytoskeleton at the cell-extracellular matrix junction (termed the actin cortical mat) is disrupted. These epithelia respond to soluble extracellular matrix molecules by reorganizing the actin cortical mat. Sheets of epithelia were isolated + or -basal lamina. Epithelia isolated -basal lamina were cultured +/- laminin-1 and/or +/- cytochalasin D (CD). The intracellular localization of zyxin, vinculin, paxillin, focal adhesion kinase, and tensin was determined using indirect immunohistochemistry. Protein levels were determined by Western blot analysis. Zyxin and vinculin were concentrated in two areas of the tissue. The interface between the upper cell layer (periderm) and the basal cells. The second area of concentration was at the inferior 1-4 microns of the basal cells in an area with multiple actin bundles termed the actin cortical mat. The actin bundles align toward zyxin and vinculin that were located near basal lateral membranes. Zyxin was displaced from the basal compartment of blebbing basal cells. In contrast tensin, vinculin and focal adhesion kinase were found diffusely throughout the blebs. Zyxin and vinculin redistributed to the basal-lateral membranes as actin bundles reorganized in laminin-stimulated epithelia. In contrast to the altered protein distribution, extractable protein levels were similar in blebbing and laminin-stimulated epithelia. Zyxin, vinculin, and other associated proteins were disrupted in the CD-treated tissues and do not colocalize with each other or CD-induced actin aggregates. The intracellular localization of zyxin and vinculin were concentrated in distinct areas along the inferior basolateral membranes of basal cells termed the cell-extracellular matrix attachment complex (CMAX). The distribution of CMAX proteins was dependent upon actin bundle organization.  (+info)

ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent. (4/1253)

Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75-fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15-30 min. Focal adhesion kinase, p190 Rho-GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk-1, erk-2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase activation.  (+info)

The ultrastructure of corneal epithelium after co-cultivation with herpes simplex virus. (5/1253)

To elucidate the ultrastructural change of corneal epithelium co-cultured with herpes simplex virus (HSV), the corneal epithelium of 3 rabbits was excised and cultivated in culture media. After 7 days, the Kos strain of herpes simplex virus was inoculated in the cultured cornea epithelium until cytopathic effect was occurred. It was fixed in the solution of 3% glutaraldehyde and examined with electronmicroscope. In co-cultured cells, the intercellular spaces had increased and microvilli were seen prominently. The virus particles that initiated the infection by fusing the viral envelope with the plasma membrane were also seen. The nuclear degeneration in an infected cell was prominent. The nuclear membrane was folded markedly, and the chromatin was degraded, condensed and displaced toward the nuclear membrane. Numerous viral particles and inclusion bodies were present in the nuclei. These findings suggest that the infectious process of herpes simplex virus in the human corneal epithelium may occur in a similar way. This result would be helpful in understanding the pathogenesis of herpes simplex epithelial keratitis.  (+info)

Epithelial ingrowth of anterior chamber and anterior surface of vitreous. (6/1253)

This study is a case report of the histopathologic findings of the anterior chamber epithelial ingrowth in a patient who had penetrating injury in the right eye from an arrow approximately 20 years ago. The patient underwent the enucleation in the right eye due to pthisis bulbi and was fitted with a prosthetic eye. Specimens were prepared from the enucleated right eye for histopathologic observation using hematoxyllin-eosin to be observed under light microscopy. Epithelial ingrowth in the anterior chamber was noted in one layer or multi-layered epithelial cell growth. The ingrowth had spread to the posterior surface of the cornea to the anterior chamber angle, to the iris surface, and to the anterior surface of the vitreous. The finding suggests that epithelial ingrowth could invade even through a perforation site and spread wherever the cells could reach.  (+info)

Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin. (7/1253)

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.  (+info)

Conjunctival-limbal autografts for primary and recurrent pterygia: technique and results. (8/1253)

Our technique of pterygium excision with conjunctival-limbal autografting is described and the safety and efficacy of the procedure in India is analysed. Case records of 51 consecutive patients (53 eyes) who underwent surgery at our institute between November 1992 and September 1994 were retrospectively analysed. Recurrence was defined as fibrovascular tissue crossing the corneoscleral limbus onto clear cornea in the area of previous pterygium excision. 2 (3.8%) of the 53 pterygia (primary 36; recurrent 17) recurred, after a mean follow up of 18.9 +/- 12.1 months (range: 1.5-43 months). Both recurrences occurred within a year of follow up, in patients who were < or = 40 years of age. No major operative or postoperative complications were encountered. The inclusion of limbal tissue in conjunctival autografts following pterygium excision appears to be essential to ensure low recurrence rates. The technique is safe, simple and inexpensive and is recommended for the management of both primary and recurrent pterygia in Indian eyes.  (+info)