Tear metabolite changes in keratoconus.

While efforts have been made over the years, the exact cause of keratoconus (KC) remains unknown. The aim of this study was to identify alterations in endogenous metabolites in the tears of KC patients compared with age-matched healthy subjects. Three groups were tested: 1) Age-matched controls with no eye disease (N = 15), 2) KC - patients wearing Rigid Gas permeable lenses (N = 16), and 3) KC - No Correction (N = 14). All samples were processed for metabolomics analysis using LC-MS/MS. We identified a total of 296 different metabolites of which >40 were significantly regulated between groups. Glycolysis and gluconeogenesis had significant changes, such as 3-phosphoglycerate and 1,3 diphosphateglycerate. As a result the citric acid cycle (TCA) was also affected with notable changes in Isocitrate, aconitate, malate, and acetylphosphate, up regulated in Group 2 and/or 3. Urea cycle was also affected, especially in Group 3 where ornithine and aspartate were up-regulated by at least 3 fold. The oxidation state was also severely affected. Groups 2 and 3 were under severe oxidative stress causing multiple metabolites to be regulated when compared to Group 1. Group 2 and 3, both showed significant down regulation in GSH-to-GSSG ratio when compared to Group 1. Another indicator of oxidative stress, the ratio of lactate - pyruvate was also affected with Groups 2 and 3 showing at least a 2-fold up regulation. Overall, our data indicate that levels of metabolites related to urea cycle, TCA cycle and oxidative stress are highly altered in KC patients.

Reversal of fibrosis by TGF-ß3 in a 3D in vitro model.

Corneal scarring following moderate to severe injury is inevitable. Despite significant advancements in the field, current treatments following these types of injuries are limited, and often, the visual recovery is poor. One of the problems and limitations is that corneal wound healing is a complex process, involving corneal cells, extracellular matrix components and growth factors. Therefore, further understanding is required, along with new treatments and techniques to reduce or prevent corneal scarring following injury. Two isoforms of transforming growth factor-beta (TGF-ß), TGF-ß1 and -ß3 (T1 and T3, respectively), are associated with corneal wound healing. T1 has been shown to drive the corneal keratocytes to differentiate into myofibroblasts; whereas, T3 has been found to inhibit fibrotic markers. In the current study, we examined whether the fibrotic characteristics expressed by human corneal fibroblasts (HCF) in our 3-dimensional (3D) construct following T1 stimulation could be reversed by introducing T3 to the in vitro system. To do this, HCF were isolated and cultured in 10% serum, and when they reached confluence, the cells were stimulated with a stable Vitamin C (VitC) derivative for 4 weeks, which allowed them to secrete a self-assembled matrix. Three conditions were tested: (1) CONTROL: 10% serum (S) only, (2) T1: 10%S + T1, or (3) Rescue: 10%S + T1 for two weeks and then switched to 10%S + T3 for another two weeks. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and transmission electron microscopy (TEM). Different collagens that are normally present in healthy corneas in vivo, such as Type I and V, as well as Type III, which is a fibrotic indicator, were examined. In addition, we examined smooth muscle actin (SMA), a marker of myofibroblasts, and thrombospondin-1 (TSP-1), a multifunctional matrix protein known to activate the latent complex of TGF-ß and appear upon wounding in vivo. Our data showed high expression of collagens type I and V under all conditions throughout the 3D constructs; however, type III and SMA expression were higher in the constructs that were stimulated with T1 and reduced to almost nothing in the Rescue samples. A similar pattern was seen with TSP-1, where TSP-1 expression following "rescue" was decreased considerably. Overall, this data is in agreement with our previous observations that T3 has a significant non-fibrotic effect on HCFs, and presents a novel model for the "rescue" of both cellular and matrix fibrotic components with a single growth factor.

In vitro model suggests oxidative stress involved in keratoconus disease.

Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-ß3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.

Transforming growth factor-ß3 regulates assembly of a non-fibrotic matrix in a 3D corneal model.

Corneal tissue engineering has attracted the attention of many researchers over the years, in part due to the cornea's avascularity and relatively straightforward structure. However, the highly organized and structured nature of this optically clear tissue has presented a great challenge. We have previously developed a model in which human corneal fibroblasts (HCFs) are stimulated by a stable vitamin C (VitC) derivative to self-assemble an extracellular matrix (ECM). Addition of TGFß1 enhanced the assembly of ECM; however, it was accompanied by the upregulation of specific fibrotic markers. In this study, we tested the effects of all three TGFß isoforms (-ß1, -ß2 and -ß3) on ECM production, as well as expression of fibrotic markers. HCFs were grown in four media conditions for 4 weeks: control, VitC only; T1, VitC + TGFß1; T2, VitC + TGFß2; and T3, VitC + TGFß3. The cultures were analysed with western blots, TEM and indirect immunofluorescence (IF). Compared to controls, all TGFß isoforms stimulated matrix production by about three-fold. IF showed the presence of type III collagen and smooth muscle actin (SMA) in T1 and T2; however, T3 showed little to no expression. In western blots, T3 stimulated a lower type III:type I collagen ratio when compared to the other conditions. In addition, TEM indicated that T3 stimulated a higher level of matrix alignment and organization. HCFs stimulated by VitC and TGFß3 appear to generate a matrix that mimics the normal adult or developing human cornea, whereas TGF-ß1 and -ß2 drive the constructs towards a more fibrotic path.

Human primary corneal fibroblasts synthesize and deposit proteoglycans in long-term 3-D cultures.

Our goal was to develop a 3-D multi-cellular construct using primary human corneal fibroblasts cultured on a disorganized collagen substrate in a scaffold-free environment and to use it to determine the regulation of proteoglycans over an extended period of time (11 weeks). Electron micrographs revealed multi-layered constructs with cells present in between alternating parallel and perpendicular arrays of fibrils. Type I collagen increased 2-4-fold. Stromal proteoglycans including lumican, syndecan4, decorin, biglycan, mimecan, and perlecan were expressed. The presence of glycosaminoglycan chains was demonstrated for a subset of the core proteins (lumican, biglycan, and decorin) using lyase digestion. Cuprolinic blue-stained cultures showed that sulfated proteoglycans were present throughout the construct and most prominent in its mid-region. The size of the Cuprolinic-positive filaments resembled those previously reported in a human corneal stroma. Under the current culture conditions, the cells mimic a development or nonfibrotic repair phenotype.

Extracellular matrix and wound healing.

Over the years, most researchers have approached corneal epithelial and stromal wound healing as separate events. However, it is becoming increasingly clear that even the simplest epithelial debridement wound results in keratocyte death and a subsequent stromal response to regenerate the affected area. Thus, the interaction between stromal and epithelial healing must be considered to fully understand corneal wound healing. Although wound healing has been an active area of research for many years, the advent of refractive surgery has stimulated research into the regulation of wound repair and provided important insights into the molecular components involved in repair. Epithelial and stromal wound healing are influenced by extracellular matrix components. The purpose of the current article is to review progress in the year 2000 toward understanding mechanisms involved in corneal wound healing and how extracellular matrix affects the healing processes.

A role for MAP kinase in regulating ectodomain shedding of APLP2 in corneal epithelial cells.

We previously reported an increased secretion of amyloid precursor-like protein 2 (APLP2) in the healing corneal epithelium. The present study sought to investigate signal transduction pathways involved in APLP2 shedding in vitro. APLP2 was constitutively shed and released into culture medium in SV40-immortalized human corneal epithelial cells as assessed by Western blotting, flow cytometry, and indirect immunofluorescence. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) caused significant increases in APLP2 shedding. This was inhibited by staurosporine and a PKC-epsilon-specific, N-myristoylated peptide inhibitor. Epidermal growth factor (EGF) also induced APLP2 accumulation in culture medium. Basal APLP2 shedding as well as that induced by PMA and EGF was blocked by a mitogen-activated protein kinase (MAPK) kinase inhibitor, U-0126. Our results suggest that MAPK activity accounts for basal as well as PKC- and EGF-induced APLP2 shedding. In addition, PKC-epsilon may be involved in the induction of APLP2 shedding in corneal epithelial cells.

TGF-beta receptor types I and II are differentially expressed during corneal epithelial wound repair.

PURPOSE: It has been demonstrated that cells migrating to cover an epithelial débridement wound exit the cell cycle and that the cell-cycle inhibitor p15(INK4b) is upregulated in these cells. TGF-beta signaling has been implicated in both of these processes, and this study was conducted to determine whether the expression and localization of TGF-beta receptor (TbetaR)-I and -II are altered during corneal epithelial wound repair. METHODS: Three-millimeter superficial keratectomy wounds and 3-mm débridement wounds were made in central rat cornea and allowed to heal in vivo for 1 to 48 hours. Immunofluorescence microscopy and Western blot analysis were used to determine the localization and expression of TbetaR-I and -II. Unwounded rat corneas served as control samples. To determine the effect of epidermal growth factor (EGF) and TGF-beta1 on p15(INK4b) and TbetaR-I and -II expression, human corneal epithelial cells were grown in culture to 50% to 60% confluence, and EGF (5 ng/ml) and/or TGF-beta1 (2 ng/ml) were added for 6 hours. Cells were harvested and p15(INK4b) and TBR-I and -II levels were assayed by using Western blot analysis. RESULTS: In unwounded corneas, TbetaR-I and TbetaR-II were present at low levels across the cornea, with higher levels in limbal epithelium. Both TbetaR-I and -II were upregulated after wounding. However, levels of TbetaR-II appeared to increase in the epithelial cells that had migrated to cover the wound area, whereas TbetaR-I was upregulated in the entire corneal epithelium. Western blot analysis indicated that both TbetaR-I and -II were upregulated threefold after wounding. In cultured cells, EGF and TGF-beta1 stimulated TbetaR-II; however, neither one stimulated TbetaR-I expression. TGF-beta1 stimulated p15(INK4b) protein levels threefold. CONCLUSIONS: After wounding, TbetaR-I and TbetaR-II were both expressed at high levels in cells migrating to cover a corneal wound, suggesting that TGF-beta signaling is involved in blocking migrating cells from progressing through the cell cycle. This blockage, at least in part, involves the inhibitor p15(INK4b). In addition, although both TbetaR-I and TbetaR-II are upregulated during wound repair, they appear to be differentially regulated.

Kinetics of keratocyte proliferation in response to epithelial debridement.

Over the past 40 years, several groups have shown that epithelial debridement results in the death of keratocytes subjacent to the wound area. More recently this cell death has been shown to involve apoptosis. The purpose of this project was to examine the proliferative response of the normally quiescent keratocytes to repopulate the apoptotic area. Three mm wounds were made in the central cornea of adult rats and allowed to heal 4 hr to 14 days. Cryostat sections were stained with propidium iodide to mark the nuclei of all cells. Actively proliferating cells were identified with anti-Ki67, a marker of the late G1-M phase of the cell cycle. Anti-alpha-smooth muscle actin was used to determine if myofibroblasts were present. In unwounded corneas, keratocytes were uniformly spread throughout the stroma, and less than one proliferating cell per mm was observed. By 4 hr after wounding, the anterior one-half to three-fourths of the stroma subjacent to the wound was devoid of cells. No increase in Ki67-expressing cells was observed in the stroma until 24 hr after wounding (3.9 +/- 0.5 and 6. 3 +/- 0.5 mm(-1)in the wound center and edge, respectively). The number of Ki67-expressing cells steadily increased, peaking 44 hr after debridement (41.2 +/- 1.7 and 39.6 +/- 1.0). These cells were confined to a narrow zone adjacent to the area of cell death. No change in the number of cells expressing Ki67 was observed in the keratocytes distal to the original debridement. Ki67 levels did not return to control levels until 7 days after wounding. No alpha-smooth muscle actin was detected at any time point. This study indicates that epithelial debridement stimulates a synchronous increase in keratocyte proliferation. This stimulation is specific for cells immediately adjacent to the area of cell death. This activation does not involve the transformation of the stromal cells to a myofibroblast phenotype.

Regulation of conjunctival goblet cell secretion by Ca(2+)and protein kinase C.

Conjunctival goblet cells secrete mucus in response to cholinergic (muscarinic) agonists, but the underlying signaling pathways activated in this tissue are not well understood. Cholinergic agonists usually activate phospholipase C to produce inositol 1,4,5 trisphosphate and diacylglycerol. Inositol 1,4,5 trisphosphate increases the intracellular Ca(2+)concentration ([Ca2(+)](i)) while diacylglycerol activates protein kinase C (PKC). PKC and Ca(2+), either by itself or with calmodulin, activate cellular functions. Goblet cell glycoprotein secretion, our index of mucin secretion, was measured from pieces of rat conjunctiva with an enzyme-linked lectin assay using the lectin Ulex europaeus agglutinin I (UEA-I). UEA-I selectively recognizes high molecular weight glycoproteins secreted by the goblet cells. Increasing the [Ca(+)](i)with the Ca(2+)ionophore ionomycin stimulated glycoprotein secretion from conjunctival goblet cells. Cholinergic agonist-induced secretion was completely blocked by chelation of extracellular Ca(2+)and by the Ca(2+)/calmodulin-dependent protein kinase inhibitors KN93 and W7 as well as their inactive analogs KN92 and W5. Activation of classical and novel PKC isozymes by phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate stimulated goblet cell glycoprotein secretion. When ionomycin and PMA were added simultaneously, secretion was additive. PKC isozymes were identified by Western blotting analyses with antibodies specific to nine of the 11 PKC isozymes (PKCgamma and zeta were not tested). All nine PKC isozymes were identified in the conjunctival epithelium. The cellular location of the PKC isozymes was determined by immunofluorescence microscopy. Goblet cells contained the classical PKC isozymes PKCalpha, -betaI and -betaII, the novel PKC isozymes PKCepsilon, -theta;, and - mu, and the atypical PKC isozyme PKCzeta. We were unable to determine if PKC activation is required for cholinergic-agonist induced secretion because the PKC inhibitors chelerythrine and staurosporine alone greatly increased secretion. We conclude that Ca(2+)plays a major role in cholinergic agonist-induced conjunctival goblet cell secretion, but this agonist appears not to use Ca(2+)/calmodulin-dependent protein kinases. We also conclude that activated PKC can stimulate goblet cell secretion and that seven different PKC isoforms are present in the goblet cells.

Glucose transporter 1 expression in corneal wound repair under high serum glucose level.

PURPOSE: To determine glucose transporter (GLUT) 1 mRNA and protein expression during corneal epithelial wound healing in diabetic rat. METHODS: Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. At 10 days after injection, unilateral 3-mm epithelial debridement was carried out in the central cornea. At 2, 4, 6, and 24 hours after wounding, whole corneal epithelium was collected and GLUT1 protein and mRNA levels were determined by Western blotting and reverse transcription-polymerase chain reaction, respectively. Sugar content in collected samples was measured by the Anthrone reaction. Normal rats were used as controls. RESULTS: Glucose transporter 1 protein and mRNA levels in unwounded cornea were similarly low in the diabetic and control groups. Healing of corneal wounds was slower in diabetic rats than in controls. After wounding, GLUT1 mRNA and protein expression in both groups were similarly enhanced compared to unwounded epithelium. Sugar content at all time points did not show significant alteration in any group, although in diabetic rats it was significantly higher than in controls throughout the time course. CONCLUSION: Glucose transporter 1 expression in diabetic rat cornea showed little difference from that in normal rat cornea, suggesting minimal influence of GLUT1 on the delayed healing of diabetic corneal wounds.

Development of conjunctival goblet cells and their neuroreceptor subtype expression.

PURPOSE: To investigate expression of muscarinic, cholinergic, and adrenergic receptors on developing conjunctival goblet cells. METHODS: Eyes were removed from rats 9 to 60 days old, fixed, and used for microscopy. For glycoconjugate expression, sections were stained with Alcian blue/periodic acid-Schiffs reagent (AB/PAS) and with the lectins Ulex europeus agglutinin I (UEA-I) and Helix pomatia agglutinin (HPA). Goblet cell bodies were identified using anti-cytokeratin 7 (CK7). Nerve fibers were localized using anti-protein gene product 9.5. Location of muscarinic and adrenergic receptors was investigated using anti-muscarinic and beta-adrenergic receptors. RESULTS: At days 9 and 13, single apical cells in conjunctival epithelium stained with AB/PAS, UEA-I, and CK7. At days 17 and 60, increasing numbers of goblet cells were identified by AB/PAS, UEA-I, HPA, and CK7. Nerve fibers were localized around stratified squamous cells and at the epithelial base at days 9 and 13, and around goblet cells and at the epithelial base at days 17 and 60. At days 9 and 13, M2- and M3-muscarinic and beta2-adrenergic receptors were found in stratified squamous cells, but M1-muscarinic and beta1-adrenergic receptors were not detected. At days 17 and 60, M2- and M3-muscarinic receptors were found in goblet cells, whereas M1-muscarinic receptors were in stratified squamous cells. Beta1- and beta2-adrenergic receptors were found on both cell types. Beta3-adrenergic receptors were not detected. CONCLUSIONS: In conjunctiva, nerves, M2- and M3-muscarinic, and beta1- and beta2-adrenergic receptors are present on developing goblet cells and could regulate secretion as eyelids open.

Activation of epidermal growth factor receptor during corneal epithelial migration.

PURPOSE: Epidermal growth factor (EGF) and related growth factors: transforming growth factor (TGF)-alpha, heprin-ding (HB)-EGF, and amphiregulin (AR), have been shown to stimulate events associated with epithelial wound repair. These growth factors function by binding to a common EGF receptor (EGFR), tyrosine kinase. We have used in vivo and organ culture wound-healing models to examine the kinetics and extent of EGFR activation during corneal epithelial wound repair and whether the epithelium itself produces EGFR ligands capable of stimulating the healing process. METHODS: In the in vivo model, 3-mm debridement wounds were made in rat corneas and allowed to heal in situ. Activation of EGFR was analyzed by 1) indirect immunofluorescence microscopy, 2) immunoprecipitation using anti-EGFR and anti-phosphotyrosine (anti-PT), and 3) binding-site localization using EGF-fluorescein isothiocyanate (FITC). Relative levels of mRNA for EGF, TGF-alpha, HB-EGF, and AR were determined using reverse transcription-polymerase chain reaction. To determine whether inhibiting EGFR activation slows epithelial migration, wounded corneas were allowed to heal in organ culture in the presence of tyrphostin AG1478 (0-50 microM), a specific inhibitor of EGFR kinase activity. RESULTS: In unwounded corneas, EGFR was localized in basal cells and appeared to be membranous. Within 1 hour after wounding, EGFR was no longer immunolocalized in the membranes of cells migrating into the wound area. EGF-FITC-binding assays indicated that EGFR ligands could penetrate all the way to the limbus. Immunoprecipitation showed that EGFR was phosphorylated on tyrosine residues within 30 minutes after wounding and that phosphorylation levels increased after wounding. Levels of mRNA for TGF-alpha, HBEGF, and AR all appeared to increase after wounding. In organ culture experiments, tyrphostin AG1478 inhibited migration rates in a dose-dependent manner. CONCLUSIONS: These data indicate that EGFR was activated during corneal epithelial wound healing in vivo. Furthermore, this activation appears to be a necessary component of the process, because inhibition of the EGFR signaling cascade significantly slowed migration rates.

Expression of cyclin-dependent kinase inhibitors during corneal wound repair.

During corneal epithelial wound repair, cells migrating to cover the wound area exhibit a drastic reduction in proliferative activity. In contrast, cells distal to the original wound exhibit a greatly enhanced level of proliferative activity. At least 90% of the basal cells in limbal and peripheral corneal epithelia synchronously progress through the cell cycle. The question addressed in this article is whether cyclin-dependent kinase inhibitors play a role in the alterations in proliferative activity seen during corneal wound repair. These inhibitors specifically block cells in the G1-phase of the cell cycle. Two families of cyclin-dependent kinase inhibitors have been identified. The CIP/KIP family includes p21, p27, and p57, while the INK4 family consists of p16. p15. p18. and pI9. At least five of these inhibitors are present in the corneal epithelium. The expression of two of these, p15 and p27. is dramatically altered during wound repair, suggesting that they may be involved in the changes in cell proliferation observed during corneal wound healing.

Matrix metalloproteinase activity is enhanced during corneal wound repair in high glucose condition.

PURPOSE: (1) To investigate the effect of elevated extracellular glucose on migration, proliferation, and the activity of matrix metalloproteinases (MMPs) of SV40-transformed human corneal epithelial cells (HCEC). (2) To examine MMP activity in wounded corneal epithelium in diabetic rats. METHODS: HCEC were cultured in media containing 5.5 mM or 31.2 mM D-glucose, or in a combination of 5.5 mM D-glucose and 25.7 mM D-mannitol on fibronectin/collagen I-coated 48-well plates. After reaching confluence (day 0), cells in the central part of the plate were wounded and the residual cells were cultured for 3 days. Migration and proliferation were evaluated by assessing the increasing amount of area covered by cells, and the day-3 to day-0 ratio of DNA levels, respectively. To determine MMP activity, cells were reacted with synthetic fluorogenic substrates specific to MMPs 1, 2, 3, 7, 9, and MMP activity was determined by a fluorometric kinetic assay. Diabetic rats were induced by streptozotocin injection. Corneal epithelium was scraped from limbus-to-limbus and allowed to heal. Normal rats were treated similarly to serve as controls. Healing epithelium was collected 24 hours later, and gelatin zymography was performed. RESULTS: In the cell culture study, migration in 31.2 mM glucose was significantly slower than that in 5.5 mM, but proliferation in each concentration was similar. The osmotic effect of D-mannitol did not alter migration or proliferation. MMP activity in 31.2 mM was significantly higher than that in 5.5 mM. Zymography revealed enhanced activity of pro and active MMP-9 in healing corneal epithelium in diabetic rats. CONCLUSIONS: MMP activity was enhanced in healing corneal epithelium, both in in vitro and in vivo diabetic models, suggesting its involvement in diabetic keratopathy.

Expression patterns of retinoblastoma and E2F family proteins during corneal development.

PURPOSE: To determine the expression patterns of the retinoblastoma protein and the E2F transcription factor families in limbal and corneal epithelia and in corneal keratocytes in situ during corneal development and differentiation. METHODS: Retinoblastoma protein (pRb) and its family members p107 and p130; E2F-1, -2, and -4, members of the E2F family of transcription factors; and Ki67, a marker of actively cycling cells, were localized by indirect immunofluorescence microscopy, in corneas of neonatal, juvenile, and adult rats. Presence of mRNA for pRb, p107, p130, and E2F types 1 to 5 in adult corneal epithelium was determined by reverse transcription-polymerase chain reaction. RESULTS: mRNA for all members of pRb and E2F families was present in adult corneal epithelium. The greatest number of Ki67-positive corneal and limbal epithelial cells were present at days 13 to 19, and Ki67-positive stromal keratocytes at day 2. pRb and E2F-2 were localized to all cells in neonatal, juvenile, and adult corneas. With age, p130 localization became more intense and nuclear in stromal keratocytes and suprabasal cells of corneal and limbal epithelia; p107, initially nuclear in limbal and corneal epithelia, became increasingly cytoplasmic in corneal epithelium. E2F-1 was initially nuclear in keratocytes and diminished after day 10. E2F-1 was localized in the basal cell layer of limbal and corneal epithelia after day 10. E2F4 was always nuclear in limbal epithelium and cytoplasmic in corneal epithelium. CONCLUSIONS: Expression patterns of pRb and E2F family proteins vary with corneal cell differentiation, but are most apparent with p130 and p107. Nuclear localization of p130 appears to correlate with terminal differentiation in epithelium and entrance into a quiescent state by keratocytes. In contrast, p107 is nuclear in the undifferentiated limbal basal cells and is cytoplasmic in the remainder of the corneal epithelial cells.

Synchronization of the G1/S transition in response to corneal debridement.

PURPOSE: This study's intention was to examine the progression of ocular surface epithelium through the G1/S transition of the cell cycle after corneal epithelial debridement. METHODS: Three-millimeter debridements were made in central rat cornea and allowed to heal 4 to 48 hours in vivo. Unwounded contralateral eyes served as controls. Two hours before the animals were killed, 5-bromo-2-deoxyuridine (BrdU) was injected to detect S-phase cells. Incorporated BrdU was visualized by indirect immunofluorescence microscopy, and expression of G1 cell-cycle markers cyclins D and E was examined by indirect immunofluorescence and immunoblotting. RESULTS: The number of BrdU-labeled cells in conjunctival, limbal, and peripheral epithelium peaked at 28 hours after wounding (3.9-, 4.5-, and 3.2-fold increases, respectively). In unwounded eyes, cyclin D showed diffuse cytoplasmic localization with occasional basal cells exhibiting a nuclear localization, while anti-cyclin E showed intense localization in limbal and conjunctival basal cells but only minimal labeling in corneal epithelium. Within 8 to 12 hours after wounding, the nuclei of most corneal basal cells outside the wound area were bound intensely by anti-cyclins D and E. Immunoblotting revealed that cyclin D and E protein levels increased 4.5- and 12.1-fold after wounding, respectively. Epithelium migrating into the wound area did not incorporate BrdU and did not exhibit nuclear localization of cyclins D and E. CONCLUSIONS: Corneal epithelial debridement stimulates basal cells outside the wound area to synchronously enter the cell cycle. However, cells migrating to cover the wound area do not progress through the cell cycle. These data suggest a compartmentalization of the proliferative and migratory phases of wound repair.

Immunolocalization of muscarinic and VIP receptor subtypes and their role in stimulating goblet cell secretion.

PURPOSE: To determine the subtypes of cholinergic muscarinic receptors and receptors for vasoactive intestinal peptide (VIP) present in rat conjunctival goblet cells and whether cholinergic agonists and VIP stimulate goblet cell secretion. METHODS: Immunofluorescence studies were performed using antibodies against the m1, m2, and m3 muscarinic receptor subtypes and VIP receptors 1 and 2 (VIPR1 and VIPR2). The lectin Ulex europeus agglutinin I was used to measure glycoconjugate secretion, the index of secretion, from goblet cells in an enzyme-linked lectin assay. In this assay, pieces of conjunctiva were placed on filter paper and incubated for 15 to 120 minutes, with or without increasing concentrations of the cholinergic agonist carbachol or VIP. The muscarinic antagonist atropine and the muscarinic receptor-subtype-selective antagonists pirenzepine (M1), gallamine (M2), and 4-4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard; M3) were incubated with carbachol to determine specificity of receptor activation. RESULTS: Immunoreactivity to M2 and M3 receptors was found on goblet cell membranes subjacent to the secretory granules. Immunoreactivity to M1 receptor was not on goblet cells but was on the stratitfied squamous cells. Immunoreactivity to VIPR2 was found on goblet cells with a localization similar to that of the M2 and M3 receptors. VIPR1 was not found on goblet cells or on the stratified squamous cells. Carbachol and VIP induced a time- and concentration-dependent stimulation of glycoconjugate secretion. Carbachol, at 10(-4) M, induced a threefold increase in glycoconjugate secretion, which was completely inhibited by atropine (10(-5) M). Carbachol-induced secretion was inhibited 54% +/- 8% by pirenzepine (10(-5) M), 69% +/- 14% by gallamine (10(-5) M), and 72% +/- 11% by 4-DAMP mustard (10(-5) M). A twofold increase in glycoconjugate secretion was obtained with VIP at 10(-8) M. CONCLUSIONS: Cholinergic agonists, through M2 and/or M3 muscarinic receptors, and VIP, through VIPR2, regulate conjunctival goblet cell secretion, suggesting that goblet cell secretion in vivo is under the control of parasympathetic nerves.

Matrix metalloproteinases in epithelia from human recurrent corneal erosion.

PURPOSE: To assay for the presence of matrix metalloproteinases (MMPs) in human corneal epithelium affected by recurrent erosion compared with that in normal corneal epithelium. METHODS: Corneal epithelial debridement samples were obtained from 13 patients with recurrent epithelial erosion. For control specimens, epithelia were obtained from healthy patients undergoing photorefractive keratectomy. Zymography was performed on all samples to identify MMPs. Immunolocalization of MMP-2, laminin, and collagen type VII was determined in two samples with human recurrent epithelial erosion and compared with that in control epithelium. RESULTS: Twelve of 13 erosion samples showed MMP-2 enzymatic activity; one of the 12 also showed MMP-9 activity. Only one erosion sample showed no MMP enzymatic activity. All normal control specimens were negative for MMP. Immunohistochemical analysis of two recurrent erosion samples showed MMP-2 presence in basal cells, whereas, in normal epithelium it was not detected. One sample with epithelial erosion showed laminin localization in basal epithelial cells and basal lamina. Type VII collagen localized in basal epithelial cells only in this sample. A second erosion sample showed localization of laminin and type VII collagen in basal epithelial cells only. Normal corneal epithelium showed presence of laminin and type VII collagen in basal epithelium and basal lamina. CONCLUSIONS: Matrix metalloproteinase-2 expression is upregulated in human epithelia affected by recurrent erosion compared with that in normal control samples. Immunolocalization studies suggest that this enzyme is concentrated in basal epithelial cells where it may play an important role in degradation of the epithelial anchoring system and the recurrent epithelial slippage and erosion observed in these patients.

Mitotic inhibition of corneal endothelium in neonatal rats.

PURPOSE: Corneal endothelium in humans does not divide to any significant extent after birth; therefore, with age there is a gradual loss of cells. When cell density is reduced to a critical level, the endothelium cannot function to maintain corneal clarity, and the cornea becomes permanently cloudy. Currently, the blindness that results can be treated only by corneal transplantation. The long-term goal is to find methods to stimulate corneal endothelial proliferation in a clinically relevant manner. The first step toward achieving this goal is to identify mechanisms responsible for the induction and maintenance of mitotic inhibition of the corneal endothelium in vivo. During corneal development, the endothelium is formed by migration and proliferation of mesenchymal cells from the ocular periphery. Soon after the monolayer is formed, proliferation ceases. In tissue culture, many cell types cease proliferating upon formation of stable cell-cell and cell-substrate attachments. The goal of the present studies was to determine whether establishment of stable contacts correlates with cessation of endothelial proliferation during corneal development in vivo. METHODS: Corneas from neonatal (days 1, 3, 7, 10, 13, 14, 17, 21, 28, and 42) and adult rats were used for immunolocalization of the following: bromodeoxyuridine (BrdU), an S-phase marker; p27kip1 and p21cip1, G1-phase inhibitors; connexin-43 and ZO-1, proteins associated with gap and tight junctions, respectively; Na+/K+-ATPase and beta3-integrin, markers of plasma membrane polarity; and fibronectin and collagen type IV, constituents of Descemet's membrane. Nuclei staining positively for BrdU were counted to determine the relative number of S-phase cells at various times after birth. Marker protein expression and localization were determined by conventional fluorescence microscopy and by confocal microscopy. RESULTS: The number of endothelial cells staining positively for BrdU gradually decreased between postnatal days 1 and 13. After postnatal day 13, positive BrdU staining was no longer detectable. During the first postnatal week, cells stained positively for the G1-phase inhibitor p27kip1 but not for p21cip1. Connexin-43 achieved its mature location by postnatal day 1. ZO-1, Na+/K+-ATPase, beta3-integrin, fibronectin, and collagen type IV achieved their mature localization patterns between postnatal days 14 and 21. CONCLUSIONS: In neonatal rat, corneal endothelial cells are still entering the cell cycle at birth, but cell cycle entry gradually decreases, so that by postnatal day 13 cells are no longer entering the S-phase. The G1-phase inhibitor p27kip1, but not p21cip1, may help mediate this inhibition. Stable cell-cell and cell-substrate contacts gradually form, and monolayer maturation is complete between postnatal days 14 and 21. The results lead to the hypothesis that, in developing rat cornea in vivo, the establishment of stable cell-cell and cell-substrate contacts initiates a cascade of events, mediated by p27kip1, which induces mitotic inhibition in the endothelial monolayer.