The presence of lysyl oxidase-like enzymes in human control and keratoconic corneas.

PURPOSE: Lysyl oxidases, a family comprising lysyl oxidase (LOX) and four LOX-like enzymes (LOXL1-4), catalyse the cross-linking of elastin and collagen fibrils. Keratoconus (KC) is characterized by progressive thinning leading to irregular astigmatism, resulting in significant visual impairment. Although the pathogenesis of KC remains unclear, one of the current hypotheses is based on alterations in the organization and structure of collagen fibrils. To extend existing general knowledge about cross-linking enzymes in the human cornea, in the present study we have focused on the detection of LOXL enzymes. METHOD: The localization and distribution of LOXL1-4 were assessed in cryosections of 7 control donors (three males and three females; 25-68 years; mean age 46±17.6 years) and 8 KC corneas (5 males and 3 females; 25-46 years; mean age 31.3±7.5 years) using indirect fluorescent immunohistochemistry (IHC). The specimens were examined using an Olympus BX51 microscope (Olympus Co., Tokyo, Japan) at a magnification of 200-1000x. Western blot analysis of 4 control and 4 KC corneas was performed for all tested enzymes. RESULTS: All four LOX-like enzymes were present in all layers of control corneas as well as in the limbus and conjunctiva. Almost no differences between control and pathological specimens were found for LOXL1. A lower staining intensity of LOXL2 was found using IHC and Western blot analysis in KC specimens. Decreases of the signal and small irregularities in the staining were found in the epithelium, keratocytes and extracellular matrix, where a gradual anterior-posterior weakening of the signal was observed. LOXL3 IHC staining was lower in the corneal stromal extracellular matrix and keratocytes of KC samples. No prominent differences were detected using IHC for LOXL4, but a slight decrease was observed in KC corneas using Western blot analysis. CONCLUSION: We presume that the decrease of LOXL2 in KC corneas is more likely a consequence of the associated pathological processes (activation of stromal cells due to tissue weakening and consequent structural changes) than a direct cause leading to KC development. At this time, we are unable to provide a coherent explanation for the observed decrease of LOXL3 and LOXL4 in KC corneas.

Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas.

Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per µg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic samples (2.60 ± 2.23 nM H(2)O(2)/µg of total protein) was more than 2.5-fold lower than in control tissue (6.83 ± 2.53 nM H(2)O(2)/µg of total protein), and the decrease was statistically significant (p = 0.0178). The location of lysyl oxidase in the healthy cornea, limbus and perilimbal conjunctiva was described. We hypothesize that the restricted lysyl oxidase distribution in keratoconic corneas, and particularly the decrease of total lysyl oxidase activity in cultured keratoconic fibroblasts, is one potential reason for the inadequate collagen cross-linking that is a hallmark of this disease.

PI3-K/Akt/JNK/NF-κB is essential for MMP-9 expression and outgrowth in human limbal epithelial cells on intact amniotic membrane.

Matrix metalloproteinase-9 (MMP-9) plays an important role in the outgrowth of expanded human limbal epithelial cells on intact amniotic membranes (AM). The mechanisms of MMP-9 expression and cell outgrowth remain unknown. Here, we demonstrated that MMP-9 is preferentially expressed at the leading edge of limbal epithelial outgrowth. Treatment with the inhibitors of PI3-K (LY294002), Akt (SH-5), MEK1/2 (U0126), and JNK1/2 (SP600125) attenuated the outgrowth area, indicating that PI3-K/Akt, p42/p44 MAPK, and JNK1/2 are involved in the outgrowth of intact AM-expanded limbal epithelial cells. However, MMP-9 expression at both transcriptional and translational levels was attenuated by treatment with SP600125, LY294002, or SH-5, not by U0126 and SB202190, suggesting that JNK1/2 and PI3-K/Akt participate in MMP-9 expression. Moreover, NF-κB phosphorylation and nuclear translocation was especially noted at the leading edge, which was attenuated by treatment with SP600125 or LY294002. Helenalin, a selective NF-κB inhibitor, reduced both the limbal epithelial outgrowth and MMP-9 expression. Finally, the data reveal that PI3-K/Akt is an upstream component of the JNK1/2 pathway in MMP-9 expression. Thus, both MAPKs and PI3-K/Akt are required for limbal epithelial outgrowth on intact AM, only the PI3-K/Akt/JNK is essential for MMP-9 expression mediated through activation of transcriptional factor NF-κB in this model.

Overexpression of matrix metalloproteinase-1 (MMP-1) and MMP-3 in superior limbic keratoconjunctivitis.

PURPOSE: To explore the presentations of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in superior limbic keratoconjunctivitis (SLK) with typical redundant superior conjunctiva. METHODS: Eight surgical specimens from medically refractory SLK patients were examined. Another nine conjunctival specimens from patients who underwent cataract and retinal surgery served as controls. Expression of RNA and proteins of MMPs and TIMPs in conjunctival specimens and cultured conjunctival fibroblasts were determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). RESULTS: The expression of mRNA of MMP-1 and -3 was detected in six and seven SLK patients, respectively, but was not detected in any case in the control group. IHC showed more prominent immunostaining of MMP-1 and -3 in the subepithelial stroma of the SLK patients than in the controls. After culturing, conjunctival fibroblasts of the SLK patients also shown apparent overexpression of MMP-1 and -3 compared with that in the controls. MMP-9 was not detected in both groups. MMP-2 and TIMP-1 and -2 were detected in some cases in both groups without a statistically significant difference between groups. CONCLUSIONS: Overexpression of MMP-1 and -3 was found in surgical specimens and cultured conjunctival fibroblasts from SLK patients. MMP imbalance may contribute to SLK pathogenesis. (ClinicalTrials.gov number, NCT00167050.).

CD38 and CD157 ectoenzymes mark cell subsets in the human corneal limbus.

Nicotinamide adenine dinucleotide (NAD(+)), a precursor of molecules involved in cell regulatory processes, is released in extra-cellular compartments after stress or inflammation.This study investigates the expression in the human cornea of CD38 and CD157, two NAD(+)-consuming ectoenzymes and surface receptors. The analysis in corneal epithelial and stromal cells was performed by means of multiple approaches, which included immunofluorescence, reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, and confocal microscopy. The presence of enzymatically active NAD(+)-consumers in intact corneal cells was analyzed by high performance liquid chromatography (HPLC)-based assays. The results obtained show that CD38 and CD157 are expressed constitutively by corneal cells: CD38 appears as a 45-kDa monomer, while CD157 is a 42- to 45-kDa doublet. The molecules are enzymatically active, with features reminiscent of those observed in human leukocytes. CD38 is expressed by cells of the suprabasal limbal epithelium, whereas it is not detectable in central corneal epithelium and stroma. CD157 is expressed by basal limbal clusters, a p63(+)/cytokeratin 19(+) cell subset reported to contain corneal stem cells, and by stromal cells. The results of the work indicates that the human cornea is equipped with molecular tools capable of consuming extracellular NAD(+), and that CD157 is a potential marker of corneal limbal cells in the stem cell niche. The presence and characteristics of these ectoenzymes may be exploited to design drugs for wound repair or for applications in tissue transplantation.

The Cdk5 inhibitor olomoucine promotes corneal debridement wound closure in vivo.

PURPOSE: To investigate the effect of the Cdk5 inhibitor olomoucine on corneal debridement wound healing in vivo. METHODS: Corneal debridement wounds of 1.5 mm were made on the ocular surface of CD-1 mice. A 20 microl drop of 15 microM olomoucine in 1% DMSO was applied to the wound area immediately after wounding and again after 6 h. Control mice received identical applications of 1% DMSO. Mice were euthanized after 18 h, two weeks, and three weeks for evaluation of wound healing and restratification. Corneas were stained with Richardson's dye, photographed, and processed for histology and immunofluorescence as whole mounts or paraffin sections. The remaining wound area at 18 h was measured by image analysis. Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro. MMP-2 and MMP-9 were detected by immunofluorescence and immunoblotting. RESULTS: Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05). The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced. Olomoucine treatment of scratch wounded HCLE cells produced similar changes in MMP-9 and MMP-2 expression. The examination of treated corneas two and three weeks after wounding showed normal epithelial restratification with no evidence of inflammation or stromal disorganization. CONCLUSIONS: Topical application of olomoucine in 1% DMSO significantly enhances closure of small epithelial debridement wounds without increasing inflammation or impairing reepithelialization.

Gene transfer to trabecular meshwork endothelium via direct injection into the Schlemm canal and in vivo toxicity study.

PURPOSE: Our aim was to investigate the efficiency of adenoviral gene transfer via direct injection into the Schlemm canal ex vivo in human donor eyes and to examine the effect of human MMP-3 transgene expression in a rat model in vivo. METHODS: A viscocanalostomy-like operation was performed and adenoviral vector encoding for MMP-3 and green fluorescent protein was injected into human Schlemm canal or rat anterior chamber. RESULTS: Transgene expression was high in trabecular meshwork endothelium in human donor eyes. In vivo, adenovirus caused dose-dependent inflammation. CONCLUSIONS: Direct injection of adenoviral vectors into the Schlemm canal has potential in glaucoma treatment.

[Experimental research of the expression of telomerase in corneal limbal epithelial cells].

OBJECTIVE: To explore the possibility of using telomerase as a marker of corneal limbal stem cells. METHODS: Corneal limbal tissues and central corneal epithelial tissues from 8 rabbits were examined for telomerase activity qualitatively by telomere repeat amplification protocol (TRAP) and quantitatively by detecting the light value with bioluminescent technique. 5-Fu (20 mg) was injected subconjunctivally in 4 rabbits and the other 4 rabbits were not injected. RESULTS: Telomerase activity was positive in all corneal limbal tissues and negative in all central corneal tissues. Telomerase activity of corneal limbal cells (light value 165,575) was significantly higher than that of central corneal epithelial cells (light value 34,912) by bioluminescent technique (P = 0.001). 5-Fu injection group showed higher telomerase activity than that of the group without injection (light value 145,754) (P = 0.02). CONCLUSIONS: Positive telomerase activity is detected in the corneal limbal tissues. It suggests that there are cells with high proliferative ability in the corneal limbus. Telomerase activity may be used as a marker of corneal limbal stem cells.

Role of matrix metalloproteinase-9 in ex vivo expansion of human limbal epithelial cells cultured on human amniotic membrane.

PURPOSE: To investigate the expression and pivotal role of matrix metalloproteinase (MMP)-9 in the ex vivo expansion of human limbal explants with or without amniotic membrane (AM). METHODS: Corneoscleral buttons were cultured on intact, denuded AM or plastic dishes for 3 weeks. To determine the role of MMP-9 in cell migration, either the MMP inhibitor GM6001 or an MMP-9 antibody was used. Expression of MMP-9 was determined by gelatin zymography, reverse transcription-polymerase chain reaction, and immunohistochemical staining. RESULTS: The expression of MMP-9 in all culture conditions increased in a time-dependent manner. However, the active form of MMP-9 emerged only in cultures on both intact and denuded AM from the second week. The averaged corrected ratio of MMP-9 expression in cultures on intact AM versus those on denuded AM or plastic dishes was 2.76 +/- 0.69- or 4.25 +/- 0.30-fold, respectively, when total RNA was used as an internal control. MMP-9 transcripts were upregulated in cultures on intact AM compared with the other two culture conditions. Immunohistochemical staining demonstrated that the MMP-9 protein was located on the limbal epithelial cells. Upregulation of MMP-9 associated with cell migration was significantly attenuated by both GM6001 and MMP-9 antibody, consistent with the inhibition of MMP-9 activity, as determined by gelatin zymography. In contrast, the sizes of limbal outgrowth were not different between the control and MMP-9 antibody-treated plastic dishes. CONCLUSIONS: These results demonstrated that MMP-9 not only was upregulated, it was also involved in the outgrowth of limbal epithelial cells. These results suggest that cell-cell matrix interaction is involved in the expansion of limbal epithelial cells on intact AM, and MMP-9 may be a key element.

A Rho-associated protein kinase: differentially distributed in limbal and corneal epithelia.

PURPOSE: The authors have developed monoclonal antibodies (mAbs) to characterize the sequential biochemical changes in corneal epithelial cells after they differentiate from stem cells, located in the limbus, and migrate centripetally to follow the pathway of terminal differentiation. The purpose of this study was to identify a protein (recognized by mAb HE1/11F) with increased expression associated with the transition of the limbal epithelium to corneal epithelium. METHODS: The distribution and identification of the protein(s) were performed using an indirect immunohistochemical staining technique and a western blot analysis, respectively. A rabbit corneal epithelial cDNA library, constructed in the Uni-Zap XR vector, was screened with mAb HE1/11F to select cDNA clones expressing polypeptide(s) recognized by this mAb. Additional overlapping cDNA clones were obtained from a primer extension cDNA library to determine the sequence of the complete open reading frame encoding the protein recognized by mAb HE1/11F. RESULTS: Rabbit corneal epithelium exhibited strong immunostaining with mAb HE1/11F, however, the limbal epithelial cells stained weakly. HE1/11F recognized 160-kDa (HEBM1) and 100-kDa (HEBM2) polypeptides in the corneal epithelial extracts. The amino acid sequence of the protein deduced from the nucleotide sequence of the cDNA exhibited a close homology to that of a RhoA (Ras-related small GTPase)-associated serine-threonine kinase (ROCK-I or Rho-associated coiled-coil kinase). A 160-kDa RhoA-binding polypeptide with a molecular mass similar to that of HEBM1 and ROCK-I was detected in the corneal epithelial extracts. These findings strongly suggested that HEBM1 was rabbit ROCK-I. The identity of HEBM1 was further confirmed from the reactivity of mAb HE1/11F with ROCK-I immunoprecipitated from rabbit corneal epithelial extracts using anti-ROCK-I antibodies. CONCLUSIONS: The increased expression of a protein identified as ROCK-I from cDNA analyses is associated with rabbit corneal epithelial differentiation and transition from the limbal to corneal surface. Therefore, a RhoA signaling pathway is likely to be associated with corneal epithelial differentiation (maturation). A close homology among the cDNA sequences of rabbit, mouse, rat, and human ROCK-I indicates that this RhoA-associated kinase is a well-conserved protein.

Alpha-2,3 sialylation differentiate the limbal and corneal epithelial cell phenotypes.

PURPOSE: The initial differentiation event for the corneal epithelial cell lineage occurs as the limbally localized stem cells yield, through mitosis, the highly proliferative, transiently amplifying corneal peripheral cells. This differentiation is characterized by the expression of tissue-specific cytokeratins, as well as the loss of alpha-enolase and pigmentation. All these are intracellular events. The aim of this study was to identify and characterize, through lectin analysis, changes in cell surface properties associated with differentiation. METHODS: Cryostat sections of the limbo-corneal area from freshly dissected pigmented rabbit corneas were stained with fluorescent lectins. RESULTS: Peanut lectin (PNA; binds to Ser/Threo-GalNAc-beta-1,3-Gal, if the Gal residue is not sialylated) stained the plasma membrane of all layers of the conjunctiva and limbus but was excluded from corneal cell membranes. Maakia amurensis agglutinin (MAA; binds to sialic acid attached to galactose through alpha-2,3 bonds in either N-glycans or O-glycans) stained exclusively corneal cell plasma membrane. After complete tissue desialylation, all corneal plasma membranes became PNA positive with equal stain intensity across both sides of the limbo-corneal margin. The binding of the agglutinins from Limax flavus (binds unselectively to sialic acid) and Sambucus nigra (binds to sialic acid attached through alpha-2,6 bonds) to the basement membrane displayed a large increase at the corneal side of limbo-corneal demarcation. CONCLUSIONS: Limbal (stem) cells express on the cell surface unsialylated galactose residues that are recognized by PNA and that lack any sialic acid bound through alpha-2,3 bonds. The initial differentiation involves sialylation of these residues and the concurrent appearance of alpha-2,3 sialic acid residues, suggesting expression or activation of alpha-2-3 sialytransferase. Changes in basement membrane composition, charge, or both may underpin this expression.