The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures

PURPOSE: Early phase keratoconic corneas and their cultured keratocytes abnormally produce the Mr 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are involved in the regulation of MMP activity and tissue inhibitor of metalloproteinase (TIMP) production in non-ocular tissues. The purpose of this enquiry was to determine whether these growth factors also play a role in the activity and/or production of corneal MMP-2 and TIMP, and whether their activity could account for the existence of the Mr 62,000 form of MMP-2 in keratoconic corneas. METHODS: Confluent cultures of normal and early-phase keratoconic corneal keratocytes were established and incubated in serum-free media in the presence or absence of PDGF and TGF-beta. The proteins secreted by these cells over periods of 7 days were harvested for analysis. The total protein produced was determined spectrophotometrically. MMP-2 was visualised by SDS-gelatin polyacrylamide gel electrophoresis and assayed using radiolabelled type IV collagen as substrate. The enzyme inhibitors, TIMP-1 and TIMP-2, were quantified by dot blot immunoassay. RESULTS: The addition of PDGF or TGF-beta to the culture medium of keratoconic corneal keratocytes had no significant effect on overall protein production, MMP-2 activity or on the amounts of TIMP- 1 and TIMP-2 secreted. These observations also applied to normal corneal keratocytes, with the exception that PDGF induced expression of the Mr 62,000 species of MMP-2. CONCLUSIONS: PDGF may be involved in the production of the Mr 62,000 species of MMP-2 that is abnormally produced by early-phase keratoconic corneal keratocytes.     

Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6+/- mouse cornea

PURPOSE: Corneal wound healing involves a cascade of interactions between the epithelium and stroma. Pax6 is upregulated, and early events include epithelial cell migration and apoptosis of superficial keratocytes. The mouse heterozygous Pax6 (Pax6+/-) corneal phenotype mimics human aniridia-related keratopathy (ARK), and some aspects of wound healing have been shown to be abnormal, including matrix metalloproteinase (MMP)-9 expression. The purpose of this study was to test whether the Pax6+/- genotype affects corneal wound-healing responses, including stromal cell apoptosis, epithelial cell migration rate, and MMP secretion in culture. METHOD: Pax6+/- and wild-type (Pax6+/+) mice were killed and their corneas wounded by epithelial debridement. Whole eyes were cultured in organ culture and corneal epithelial healing rates and keratocyte apoptosis were quantified by topical fluorescein staining and TUNEL, respectively. Dissociated corneal epithelial cells from Pax6+/- and wild-type mice were cultured, and the activities of secreted MMP-9 were determined by zymography. RESULTS: Wound-healing rates during the first 6 hours were significantly faster for larger wounds and for Pax6+/- corneas. Compared with wild-type, wounded Pax6+/- eyes showed significantly more stromal cell apoptosis, and cultured Pax6+/- corneal epithelial cells produced lower MMP-9 activity. CONCLUSIONS: The cumulative effect of abnormal wound-healing responses, characterized by increased stromal cell apoptosis and reduced levels of MMP-9 secretion may contribute to the corneal changes in the Pax6+/- mice. Possible contributions of elevated stromal cell apoptosis and other abnormal wound-healing responses to ARK are discussed.     

Temporal and spatial expression of matrix metalloproteinases during wound healing of human corneal tissue

Our understanding of MMP expression during corneal repair has previously relied upon animal models, isolated human biopsy specimens and cell culture studies. The aim of this study was to determine the temporal and spatial expression of matrix metalloproteinases following wounding of cultured human corneal tissue. Human corneas were cultured and cut into six pieces. The epithelium was removed with a corneal brush. The tissue was then re-cultured and tissue pieces were fixed up to 7 days post-wounding. Matrix metalloproteinases were detected by in situ hybridisation and immunohistochemistry. Intracellular laminin-5, a marker of migratory epithelial cells, was located immunohistologically. In the time scale studied tissue series from nine corneas achieved coverage of the stroma with epithelial cells and partial repair of damaged basement membrane, demonstrated by the Periodic acid-Schiff reaction and haematoxylin and eosin counter-staining. By day 3, migrating epithelial cells and stromal cells beneath the wounded area expressed collagenase-1 (MMP-1). Stromelysin-1 (MMP-3) was expressed only by fibroblast-like stromal cells. Stromelysin-2 (MMP-10) was detected in migrating epithelial cells and remained when the stroma was surrounded by a monolayer of epithelial cells. By day 7, development of multi-layered epithelium around the tissue coincided with cessation of MMP expression in both epithelial and stromal cells, except for MMP-9, which remained in epithelial basal cells. Tissue inhibitor of matrix metalloproteinase-1 was mainly associated with stromal cells and was reduced upon formation of a multi-layered epithelium. This study demonstrates matrix metalloproteinase expression in epithelial and fibroblast-like cells following wounding of human corneal tissue in culture where the cells remain in contact with their natural matrices.     

Induction of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs correlates with outcome of acute experimental pseudomonal keratitis

This study aimed to investigate expressions and sources of matrix metalloproteinases (MMP)-2 and MMP-9, and of tissue inhibitors of MMP (TIMP)-1 and TIMP-2 in experimental Pseudomonas aeruginosa keratitis in rabbits. Pseudomonal keratitis was induced in New Zealand white rabbits, and macroscopic and microscopic examinations were performed at appropriate time points (3, 9, 12, 18, 24, 72 h). Expressions and sources of MMP-2, 9, and TIMP-1, 2 were determined using immunohistochemistry, gelatin zymography, ELISA, and RT-PCR. A typical corneal ulcer with a ring abscess was observed 12-72 h post-infection (p.i.) with P. aeruginosa. In microscopic examinations, massive inflammatory cell (mostly polymorphonuclear leukocytes, PMNs) infiltration and liquefactive necrosis were characteristic features. MMP-2 was constitutively expressed in keratocytes, and its expression was not apparently enhanced after pseudomonal infection as evidenced by zymography, immunostaining, and RT-PCR. However, MMP-9 and its activated form were induced, and were significantly enhanced 12-24 h p.i. MMP-9 appeared to derive from PMNs rather than from resident corneal cells. TIMP-1 was expressed in PMNs, macrophages, and keratocytes, and its expression was enhanced 72 h p.i. Although TIMP-2 was constitutively expressed as seen by immunostaining and RT-PCR, its concentration was below detection limits during the experiments. We demonstrated that MMP-9 was one of the important factors for corneal tissue destruction, because it was induced and significantly expressed in keratocytes and inflammatory cells after pseudomonal infection. Although TIMP-1 was expressed in later stages of infection, enhancement and activation of MMP-9 were much faster and stronger than those of TIMP-1, thereby facilitating tissue destruction leading to corneal ulceration.     

Transforming growth factor-beta and interleukin-1 modulate metalloproteinase expression by corneal stromal cells.

The enzyme collagenase participates in remodeling the extracellular matrix of corneal stroma during normal wound healing and mediates the degradation of extracellular matrix that occurs in many corneal pathologic states. Because this enzyme is synthesized and secreted by corneal cells, therapy of degradative disorders might be geared toward control of enzyme expression. The effects of two cytokines, transforming growth factor-beta (TGF-beta) and interleukin-1 (IL-1), on the expression of collagenase by cultured corneal stromal cells are reported. In addition, the concomitant effects of these cytokines on expression of three additional members of the matrix metalloproteinase (MMP) family--stromelysin, 72-kilodalton (kD) gelatinase, and 92-kD gelatinase--were investigated. When stromal cells are situated in the normal corneal stroma, they produce only a single MMP, 72-kD gelatinase. This pattern of expression was reproduced by stromal cells freshly plated in primary culture. However after passage in culture, the cells also began to secrete collagenase and stromelysin. Treatment of primary cultures with recombinant human IL-1 also induced collagenase and stromelysin expression. In addition, 92-kD gelatinase expression was induced and 72-kD gelatinase expression was increased further by IL-1 treatment. Treatment of passaged cultures or IL-1-treated primary cultures with recombinant human TGF-beta reverted the pattern of enzyme expression toward that exhibited by primary, untreated cultures, ie, expression of collagenase and stromelysin was repressed while expression of 72-kD gelatinase was increased. These results suggest that TGF-beta and IL-1 may be important agents for controlling MMP expression in healthy and diseased corneas.     

Transplantation of amniotic membrane in murine herpes stromal keratitis modulates matrix metalloproteinases in the cornea

PURPOSE: To study matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the corneas from mice with ulcerative herpes stromal keratitis (HSK) treated with amniotic membrane transplantation (AMT). METHODS: The corneas from BALB/c mice were infected with HSV-1. Mice with ulcerative HSK on postinfection (PI) day 14 were used for the experiments. In one group of mice, the corneas were treated with transplantation of amniotic membrane (AMT) that was secured with a tarsorrhaphy, and a control group underwent tarsorrhaphy alone. After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically. Corneal sections were studied by immunohistochemistry for the expression of MMP-2, -8, and -9 and TIMP-1 and -2. MMP activity in the corneas was investigated by zymography, and the expression of the enzymes was measured by the Western blot technique. RESULTS: At day 14 PI, the ulcers stained intensely positive for MMP-2, -8, and -9 and TIMP-1 and -2. Ulceration (P < 0.001), stromal inflammation (P < 0.01) and inflammatory cell infiltration (P < 0.001) markedly improved by day 2 after AMT. This was associated with reduced expression (P < 0.01) and activity of MMP-8, and -9 and increased localization of TIMP-1 (P < 0.01), whereas TIMP-2 was not affected. In contrast, high levels of expression of MMP-8 and -9 remained in the cornea after tarsorrhaphy, and the TIMP-1 expression was only slightly upregulated. CONCLUSIONS: Rapid improvement of HSV-1-induced ulcerative keratitis is noted after amniotic membrane transplantation. This may be caused by reduced expression and activity of MMP-8 and -9, increased expression of TIMP-1, and sustained expression of TIMP-2.     

Apoptosis in the endothelium of human corneas for transplantation

PURPOSE: To determine whether endothelial cell loss of human corneas stored in organ culture before transplantation is due to apoptosis. METHODS: The corneal endothelium of human corneas, stored in organ culture at 34 degrees C for varying periods of time, were analyzed for the presence of apoptotic cells using the TdT-mediated dUTP nick-end labeling (TUNEL) technique. Corneal endothelial cell apoptosis was confirmed by Hoechst staining and immunolabeling with anti-caspase 3 active antibody. RESULTS: Apoptotic cells were identified in the corneal endothelium of human organ cultured corneas: their number and distribution demonstrated a close correlation with corneal folding and overall quality of the corneal endothelium. TUNEL-positive labeling of cells was confirmed as apoptotic by the presence of morphologic nuclear alterations identified by Hoechst staining and the presence of immunostaining for caspase-3 activity. Corneal endothelial cell apoptosis was independent of cause of donor death, death to enucleation time, and death to culture times. CONCLUSIONS: Corneal endothelial cell apoptosis appears to determine the suitability of a cornea for transplantation.     

Expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinase 1 and 2 in inflammation-induced corneal neovascularization.

PURPOSE: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMP-2, MMP-9 and TIMPs in the corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9 and TIMPs during the course of cauterization-induced corneal neovascularization in a rat model. METHODS: Neovascularization of rat corneas was induced by silver nitrate cauterization. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 was examined by immunohistochemistry and RT-PCR. The protein activities of MMPs and TIMPs were compared in pre- and postcauterization corneas by gelatin zymography and reverse zymography, respectively. RESULTS: MMP-2, TIMP-1 and TIMP-2 immunoreactivities were expressed in normal corneas, predominantly in the corneal epithelium. After injury, immunoreactivities of both MMPs and TIMPs were increased, notably in the healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts and ingrowing vascular endothelial cells. The increase in gross MMP-2 enzymatic activity paralleled the maximal vascular ingrowth on day 4, while the gross MMP-9 enzymatic activity rose immediately on day 1, then decreased steadily, which paralleled the magnitude of inflammatory cell infiltration. The immunoreactivity of MMPs/TIMPs decreased significantly 2 weeks after cauterization. On day 35, MMP-2, TIMP-1 and TIMP-2 staining was seen only in corneal epithelium and vascular endothelial cells. Both the RT-PCR and reverse zymography results revealed a more constant expression of TIMP-2, while the TIMP-1 expression appeared to be more inducible. CONCLUSION: MMPs as well as TIMPs were upregulated in cauterization-induced corneal neovascularization, suggesting that both may participate in extracellular matrix remodeling in the corneal wound healing, inflammation and neovascularization processes.     

Enhanced expression of a transmembrane phosphotyrosine phosphatase (LAR) in keratoconus cultures and corneas

The purpose of the study was to identify genes that are differentially expressed in normal versus keratoconus corneas. Total RNA isolated from corneal stromal cell cultures was reverse-transcribed and then amplified by the polymerase chain reaction (PCR) using defined, arbitrary primers. The products were displayed on polyacrylamide gels and bands that were differentially expressed were excised, re-amplified and subcloned. The resulting clones were sequenced and utilized as probes for Northern blots with cultured cell RNA or Southern blots of corneal cDNA. One of the products that appeared to be more highly expressed in keratoconus cultures and corneas displayed 100% homology with leukocyte common antigen related protein (LAR), a transmembrane phosphotyrosine phosphatase. Western analyses and immunohistochemistry with monoclonal and/or polyclonal antibodies to LAR were used to examine keratocyte cultures and fresh frozen normal, keratoconus and pseudophakic bullous corneas. We identified a gene product with 100% homology to LAR that is expressed at the RNA level in keratoconus corneas and cell cultures but is found only at low or undetectable levels in normal cultures and normal and pseudophakic bullous keratopathy (PBK) corneas. By Western blotting and immunofluorescence with specific LAR antibodies, the protein was identified in keratoconus stromal cell cultures but not in normal cultures. When fresh frozen tissue was examined, LAR protein was localized to numerous stromal cells throughout central keratoconus corneas, while no central staining was seen in normal or bullous keratopathy corneas. LAR, a transmembrane phosphotyrosine phosphatase, is more highly expressed in keratoconus corneas and stromal cell cultures as demonstrated by differential display, Northern analyses, immunohistochemistry and Western blotting.     

Reduced expression of the gap junction protein Connexin 43 in keratoconus

PURPOSE: The purpose of the present study was to examine whether keratoconus, which is a bilateral noninflammatory corneal ectasia with multifactorial aetiology, shows altered expression of Connexin (Cx43). Cx43 is an important gap junction protein that contributes crucially to epithelial and stromal integrity of cornea. METHODS: Eight keratoconic human corneal buttons were examined with immunohistochemistry and Western blotting and compared with eight normal human corneal buttons, to unravel changes in Cx43 expression. RESULTS: All normal corneas exhibited similar epithelial Cx43 expression patterns, with the protein located in the basal epithelial layer. In contrast, some keratoconic corneas showed an altered pattern of immunostaining and Western blotting confirmed a decreased expression of Cx43 in keratoconic corneas. CONCLUSIONS: Our results indicate that a decrease in Cx43 amount together with functional alteration of the protein is associated with keratoconus pathophysiology However, these changes apply only to some of the corneas examined and may not generally account for the development of keratoconus.     

Adenovirus mediated gene delivery of tissue inhibitor of metalloproteinases-3 induces death in retinal pigment epithelial cells.

BACKGROUND: Sorsby's fundus dystrophy (SFD) and age related macular degeneration (ARMD) are retinal diseases associated with a high level of accumulation of mutant and wild type TIMP-3, respectively, in Bruch's membrane. The pathogenic role of TIMP-3 in these diseases is uncertain, but causative mutations have been identified in the TIMP-3 gene of patients with SFD. Recent reports that TIMP-3 causes apoptosis in certain cell types and not in others prompted the authors to investigate whether TIMP-3 causes apoptosis in cultured retinal pigment epithelium (RPE) cells. METHODS: RPE and MCF-7 cells (as a positive control) were initially infected with replication deficient adenovirus, to overexpress beta-galactosidase (RAdLacZ) or TIMP-3 (RAdTIMP-3). TIMP-3 was detected by western blotting and ELISA. Cell viability was defined by cell counts. ISEL was used to investigate the mechanism of cell death. RESULTS: Cultured RPE cells produced small quantities of endogenous TIMP-3 and remained viable. However, overexpression of TIMP-3 caused a dose related death of RPE cells. The mechanism of cell death was apoptosis. CONCLUSION: The previously unreported finding of TIMP-3 induced apoptosis of RPE cells may account for some of the early features seen in SFD and ARMD.     

Apoptosis in human non-necrotizing stromal herpes simplex keratitis

BACKGROUND: Herpes simplex virus (HSV-1) stromal keratitis is an immune-mediated disease. Recently, it has been shown that infection of cells with HSV-1 induces apoptosis. In this study we investigated the presence of apoptotic cell death in keratectomy specimens from patients with HSV-1 stromal keratitis. PATIENTS AND METHODS: Keratectomy specimens from six patients with HSV-1 non-necrotizing stromal keratitis were chosen and compared to healthy corneas. Paraffin sections were analyzed histologically and cryosections were studied by the immunoperoxidase technique for the presence of HSV-1, Fas and FasLigand (FasL) antigens. Apoptosis was assessed by TUNEL assay (TdT-mediated dUTP nick-end labeling). RESULTS: In healthy corneas, Fas was detected in the epithelium, keratocytes and endothelium; FasL was present in the epithelium and endothelium; TUNEL-positive cells were only found in the superficial epithelial cells. In contrast, inflammatory cells and scars were found in all HSV-diseased corneas; HSV-1 antigen was detected in only one specimen. Cells within inflammatory infiltrations and epithelium were apoptotic. Fas was detected in all corneal cell layers and inflammatory cells. FasL was restricted to areas of inflammation. CONCLUSIONS: The data suggest that apoptosis plays a role in the pathogenesis of HSV keratitis. The Fas-FasL system appears to be involved in the induction of apoptosis. Apoptosis could be involved in the depletion of inflammatory cells in the cornea and may limit the extension of viral replication to the eye.     

Epithelial cell, keratocyte, and endothelial cell apoptosis in Fuchs' dystrophy and in pseudophakic bullous keratopathy

PURPOSE: To elucidate the pathomechanism of Fuchs' dystrophy and pseudophakic bullous keratopathy (PBK) by examining cell apoptosis in different corneal layers. METHODS: The authors studied corneal buttons obtained from 21 eyes following central penetrating keratoplasty: 14 corneal buttons (13 patients, age 70.8+/-10.0 years) with Fuchs' dystrophy, and 7 buttons (7 patients, age 69.6+/-10.2 years) with PBK. Four buttons from enucleated eyes with choroidal melanoma served as controls. Histologic changes were examined using light microscopy with hematoxylin-eosin (HE) staining. The average numbers of apoptotic cells per field of view (125x magnification) in separate samples of the epithelial, stromal, and endothelial layers were determined using the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling) assay. RESULTS: In 11 of the Fuchs' dystrophy corneas and 2 of the PBK corneas, apoptotic activity was detected. In the control corneas no apoptotic activity was found. Compared to the controls there was a statistically significant difference in the mean (normalized) apoptotic cell numbers for all three layers (p=0.01 in each case) in the Fuchs' dystrophy corneas, and for the stromal layer (p<0.01) in PBK corneas. The apoptotic cell numbers for the epithelial and endothelial layers of the latter were higher, but the difference was not statistically significant (p=0.07, 0.07). CONCLUSIONS: Apoptosis may play a role in the pathomechanism of Fuchs' dystrophy and in keratocyte death in corneas with PBK.     

Keratocyte activation and apoptosis in transplanted human corneas in a xenograft model

PURPOSE: To study keratocyte activation and cellular apoptosis in transplanted human corneas during the early postoperative period. METHODS: Ten human donor corneas preserved for 6 days at 4 degrees C were transplanted into the eyes of 10 adult cats. After confocal and specular microscopy in vivo 1 week after keratoplasty, the cats were killed, and the fixed corneas were examined by TUNEL assay and by scanning (SEM) and transmission electron microscopy (TEM). RESULTS: Abnormal keratocytes, in which portions of cell bodies and processes as well as nuclei were visible, were present in all corneas and occupied the anterior 16 to 562 microm of the stroma. By TEM in the same corneas, these abnormalities represented keratocytes that were activated to a repair phenotype. Only 0% to 1% of all corneal cells were apoptotic by TUNEL assay, except for the donor keratocytes near the wound, where 7% were apoptotic. The midstromal keratocyte density was decreased at 13,936 +/- 5,910 cells/mm(3) (mean +/- SD), and the endothelial cell density was 2,298 +/- 688 cells/mm(2), representing an endothelial cell loss of 7% +/- 16%. CONCLUSIONS: Substantial keratocyte activation and low levels of cellular apoptosis occur 1 week after human corneal transplantation. The human-to-cat xenograft model of corneal transplantation demonstrated endothelial cell loss and other clinical findings similar to human allografts. The model will be useful for preclinical testing of new methods of long-term corneal preservation and of donor endothelial cell augmentation, as well as the study of human corneal wound healing and keratocyte replacement during the early postoperative period.     

Immunohistochemical Expression of Fas Ligand (FasL) and Neprilysin (Neutral endopeptidase/CD10) in Keratoconus

Recent evidence demonstrated a correlation between apoptosis and neprilysin expression. The aim of this study was to investigate the immunohistochemical expression of Fas ligand (FasL) and neprilysin in keratoconic corneas in comparison to normal cadaver corneas to evaluate if such molecules play a role in the pathogenesis of keratoconus. We studied the expression of FasL and neprilysin in corneal specimens removed during penetrating keratoplasty in 15 cases with keratoconus and compared them with 5 normal cadaver corneas. In keratoconus, FasL was expressed in epithelium, endothelium and sub-Bowman’s stroma only, while neprilysin was expressed in epithelium, endothelium and all stromal layers. All normal corneas showed weak expression of both markers in basal epithelial layer only. In keratoconus, corneal epithelium with higher expression of FasL may evoke apoptosis in keratocytes, while neprilysin could prevent possible rescue of keratocytes from apoptosis.     

Effects of riboflavin/UVA corneal cross‐linking on keratocytes and collagen fibres in human cornea

Purpose: To evaluate the effects of corneal cross‐linking on keratocytes and collagen fibres in human corneas. Methods: Fifteen corneal buttons were examined. Ten were from patients with keratoconus submitted to penetrating keratoplasty and five of them were treated with cross‐linking 6 months before penetrating keratoplasty. Five normal corneal buttons from healthy donors were used as controls. All samples were prepared for TUNEL assay and Western blot analysis for the detection of keratocyte apoptosis and immunohistochemical analysis for the morphological evaluation of keratocytes and collagen fibre diameter. Results: Normal corneas exhibited no TUNEL‐positive keratocytes and keratoconic and cross‐linked corneas showed moderate apoptotic cells mainly in the anterior part of the stroma. This apoptotic trend was confirmed by the cleavage of poly (ADP‐ribose) polymerase assessed using Western blot. The Ki‐67 staining showed a significant increase in the keratocyte proliferation in cross‐linked corneas compared with normal and keratoconus. In cross‐linked corneas CD34‐positive keratocytes were regularly distributed throughout the whole corneal stroma as in the control, and keratoconus was associated with patchy loss of immunoreactivity. The immunohistochemical analysis of collagen type I showed a significant increase in fibre diameter of cross‐linked corneas compared with control and keratoconus. Conclusion: Corneal cross‐linking leads to keratocyte damage; after 6 months a repopulation by proliferating cells, a distribution of CD34‐positive keratocytes as in control and an increase in collagen fibre diameter were observed. These modifications are the morphological correlate of the process leading to an increase in biomechanical stability.     

In vivo corneal confocal microscopy in keratoconus

PURPOSE: To evaluate the corneas of keratoconic subjects using in vivo confocal microscopy. METHODS: Slit scanning confocal microscopy was used to evaluate the central cornea of one eye of each of 29 keratoconic subjects (mean age 31 +/- 10 years; range 16-49 years). Quantitative aspects of corneal morphology were compared against data from control subjects. RESULTS: Compared with normal control corneas, epithelial wing cell nuclei were larger (p < 0.0001) and epithelial basal cell diameter was larger (p < 0.05) in the keratoconic cornea. Many of the keratoconic corneas investigated showed increased levels of stromal haze and reflectivity, which appeared to be related to the presence of apical scarring on slit lamp examination. A grading scale was devised to quantify the levels of haze. This scale was shown to provide a measure of the level of scarring present. The anterior keratocyte density (AKD) and posterior keratocyte density were 19% lower (p < 0.0001) and 10% lower (p = 0.004) than in controls, respectively. The reduction in AKD was significantly associated with three factors: a history of atopy, eye rubbing and the presence of corneal staining. The mean endothelial cell density in keratoconus was 6% greater than that of normal controls (p = 0.05). The level of endothelial polymegethism was shown not to be different between keratoconic subjects and matched controls (paired t-test: t = 1.82, p = 0.08). CONCLUSIONS: Confocal microscopy demonstrates significant quantitative alterations of corneal morphology in keratoconus.     

Effect of ectopic epithelial tissue within the stroma on keratocyte apoptosis,mitosis, and myofibroblast transformation

The purpose of this study was to examine the effects of the epithelium on processes involved in stromal wound healing. Lamellar epithelial-stromal flaps were produced in rabbit corneas with a microkeratome. Peripheral corneal epithelial tissue, central corneal epithelial tissue, or no epithelial tissue (control) was introduced beneath the flap. Corneas were removed at time points from 4 hr to 1 month after surgery. Tissue sections were analyzed with immunocytochemistry for Keratin 3 (K3) to detect epithelial antigen, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell proliferation, and immunocytochemistry for alpha-smooth muscle actin (SMA) to detect myofibroblasts. K3 was detected at the level of the interface from 4 hr to 1 month after surgery in corneas in which epithelial tissue was introduced, but not control corneas, with the exception of one that developed epithelial in growth. Keratocyte apoptosis was significantly higher at 4 hr after flap formation in both groups in which corneal epithelial tissue was introduced beneath the flap compared with controls. Keratocyte proliferation was significantly greater at 72 hr in corneas in which epithelial tissue was introduced beneath the flap compared to the controls. Corneas in which epithelial tissue was introduced into the interface, but not control corneas, had stromal cells expressing alpha-SMA in the stroma anterior and posterior to the interface at 1 week and 1 month after surgery. This was also noted in the control cornea in which there was epithelial ingrowth. Signals derived from the corneal epithelium promote keratocyte apoptosis. Keratocyte proliferation is higher in corneas that have lamellar surgery when epithelial tissue is introduced into the interface. Epithelium-derived signals also participate in the generation and/or maintenance of myofibroblasts in the corneal stroma.     

Matrix metalloproteinase 2: involvement in keratoconus

PURPOSE: The activation of matrix metalloproteinase-2 (MMP-2) is postulated to be a crucial pathogenic factor behind progressive and chronic diseases in which basement membranes are disrupted. An ocular example is keratoconus. The purpose of the present enquiry was therefore to investigate and compare the activities of the MMP-2 secreted by keratocytes of normal and keratoconic corneas. METHODS: The spectrum of MMP-2 activities secreted by cultures of keratocytes derived from normal and keratoconic corneas was analysed by zymography. Subsequently, selected preparations were assayed for peptidase activity, using Type I, Type III, Type IV and Type V collagen as substrate, under native conditions and after treatment with a variety of putative activating reagents. RESULTS: Although MMP-2 of Mr 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocytes of early-phase keratoconic corneas secrete an additional zymographic activity of Mr 61,000. From their N-terminal amino acid sequences, both these proteins were shown to be conformers of proMMP-2. Treatment with SDS followed by protein fractionation was required to achieve in vitro activation of the MMP-2 secreted by normal corneal keratocytes. Treatment with SDS alone partially activated the enzyme produced by early-phase keratoconic corneal keratocytes. This procedure and autocatalysis, yielded an enzyme of Mr 43,000 that selectively hydrolysed Type IV and denatured Type 1 collagen. CONCLUSIONS: The zymographic gelatinase activities of apparent Mr 65,000 and 61,000 are conformers of corneal proMMP-2. Activated enzyme, of Mr 43,000, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparations of the culture media of normal corneal keratocytes.