ABSTRACT
Transglutaminase 2 (TG2) is the most abundant crosslinking enzyme in murine and human cornea, while retinoids are well-known inducers of TG2 expression. This study aims to determine if the retinoic acid supplementation can increase corneal stiffness by crosslinking through upregulating the corneal TG2 expression. The right eyes of C57BL/6 mice were treated with 2 × 10-2M retinol palmitate (VApal) eyedrops or control eyedrops and hold for 30 min, once a day for 28 consecutive days. The WB and qPCR results showed increased expression of TG2 in murine cornea with the prolongation of VApal eyedrop application. After 28 days of VApal eyedrop treatment, the increased TG2 were found catalytically active and distributed in corneal epithelium and stroma as detected by 5-(biotinamido) pentylamine (5-BP) incorporation method and immunofluorescence staining. The transmission electron microscope image revealed that VApal treated cornea manifested with increased collagen density in anterior and middle layer of stroma. The higher elastic module was found among VApal treated cornea by nano-indentation test. In cultured corneal epithelial cells and keratocytes, all-trans retinoid acid (ATRA) treatment increased the content of TG2 in cell lysis and in culture medium. These results indicate that retinoic acid induce the reinforcement of the cornea by TG2 mediated crosslinking via increasing the TG2 expression in corneal epithelium and keratocyte. As TG2 was found to be less in the cornea of keratoconus patients in several RNA-sequencing studies, retinoic acid could serve as a non-invasive prevention method for keratoconus progression.
Subject(s)
Antineoplastic Agents/administration & dosage , Cornea/drug effects , Gene Expression Regulation, Enzymologic/physiology , Protein Glutamine gamma Glutamyltransferase 2/genetics , Tretinoin/administration & dosage , Administration, Ophthalmic , Animals , Blotting, Western , Cells, Cultured , Cornea/enzymology , Cornea/physiopathology , Corneal Keratocytes/drug effects , Corneal Keratocytes/enzymology , Cross-Linking Reagents , Electrophoresis, Polyacrylamide Gel , Epithelium, Corneal/drug effects , Epithelium, Corneal/enzymology , Female , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Ophthalmic Solutions , Up-RegulationABSTRACT
PURPOSE: This study aimed to analyze the association of tear matrix metalloproteinase 9 (MMP-9) immunoassay with the severity of dry eye (DE) signs and symptoms through qualitative, semiquantitative, and quantitative evaluations of immunoassay band. MATERIALS AND METHODS: This cross-sectional study enrolled 320 eyes of 320 patients. The clinical signs of DE were assessed using the Ocular Surface Disorder Index (OSDI) score, visual analogue scale (VAS), tear breakup time (tBUT), tear volume evaluation by tear meniscometry, and staining scores of the cornea and conjunctiva by the Oxford grading scheme. The tear MMP-9 immunoassay results were interpreted using qualitative (positive or negative), semi-quantitative (reagent band density on a four-point scale: 0 = negative; 1 = weakly positive; 2 = moderately positive; 3 = strongly positive), and quantitative (ratio of reagent band density to control band density) indicators. RESULTS: Positive MMP-9 immunoassay results were significantly related to shorter tBUT, tBUT ≤3 seconds, higher corneal staining score, corneal staining score ≥2, and conjunctival staining score ≥2. The semi-quantitative results of the MMP-9 immunoassay were positively correlated with higher corneal staining score (r = 0.122, p = 0.029) and negatively correlated with tBUT (r = -0.125, p = 0.025). However, in the quantitative analysis, none of the DE signs or symptoms were correlated to the band density of the MMP-9 immunoassay. CONCLUSIONS: The positive MMP-9 immunoassay results were related to the severity of ocular signs of DE. However, using quantitative measures of the MMP-9 immunoassay to assess the clinical severity of DE requires further investigation.
Subject(s)
Dry Eye Syndromes/enzymology , Immunoassay , Matrix Metalloproteinase 9/metabolism , Qualitative Research , Tears/enzymology , Adolescent , Adult , Aged , Aged, 80 and over , Conjunctiva/enzymology , Conjunctiva/pathology , Cornea/enzymology , Cornea/pathology , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Point-of-Care Testing , Young AdultABSTRACT
Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes) have mutations in the gene encoding ß1,3-glucosyltransferase (B3GLCT). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients.
Subject(s)
Cleft Lip/enzymology , Cleft Lip/genetics , Cornea/abnormalities , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Growth Disorders/enzymology , Growth Disorders/genetics , Limb Deformities, Congenital/enzymology , Limb Deformities, Congenital/genetics , Mutation/genetics , ADAMTS Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Biocatalysis , Cornea/enzymology , Enzyme Stability , Fucose/metabolism , Galactosyltransferases/chemistry , Glucose/metabolism , Glucosyltransferases/chemistry , HEK293 Cells , Humans , Kinetics , Models, Molecular , Protein Domains , Repetitive Sequences, Amino Acid , Structural Homology, ProteinABSTRACT
Lecithin:cholesterol acyltransferase (LCAT) is an enzyme secreted by the liver and circulates with high-density lipoprotein (HDL) in the blood. The enzyme esterifies plasma cholesterol and increases the capacity of HDL to carry and potentially remove cholesterol from tissues. Cholesterol accumulates within the extracellular connective tissue matrix of the cornea stroma in individuals with genetic deficiency of LCAT. LCAT can be activated by apolipoproteins (Apo) including ApoD and ApoA1. ApoA1 also mediates cellular synthesis of HDL. This study examined the expression of LCAT by epithelial cells, keratocytes, and endothelial cells, the cell types that comprise from anterior to posterior the three layers of the cornea. LCAT and ApoD were immunolocalized to all three cell types within the cornea, while ApoA1 was immunolocalized to keratocytes and endothelium but not epithelium. In situ hybridization was used to detect LCAT, ApoD, and ApoA1 mRNA to learn what cell types within the cornea synthesize these proteins. No corneal cells showed mRNA for ApoA1. Keratocytes and endothelium both showed ApoD mRNA, but epithelium did not. Epithelium and endothelium both showed LCAT mRNA, but despite the presence of LCAT protein in keratocytes, keratocytes did not show LCAT mRNA. RNA sequencing analysis of serum-cultured dedifferentiated keratocytes (commonly referred to as corneal stromal fibroblasts) revealed the presence of both LCAT and ApoD (but not ApoA1) mRNA, which was accompanied by their respective proteins detected by immunolabeling of the cultured keratocytes and Western blot analysis of keratocyte lysates. The results indicate that keratocytes in vivo show both ApoA1 and LCAT proteins, but do not synthesize these proteins. Rather, keratocytes in vivo must take up ApoA1 and LCAT from the corneal interstitial tissue fluid.
Subject(s)
Apolipoprotein A-I/metabolism , Apolipoproteins D/metabolism , Cholesterol/metabolism , Cornea/metabolism , Phosphatidylcholine-Sterol O-Acyltransferase/metabolism , Aged , Apolipoprotein A-I/blood , Apolipoprotein A-I/genetics , Apolipoproteins D/blood , Apolipoproteins D/genetics , Cornea/enzymology , Cornea/pathology , Cornea/ultrastructure , Corneal Dystrophies, Hereditary/genetics , Corneal Dystrophies, Hereditary/metabolism , Coronary Artery Disease/genetics , Coronary Artery Disease/metabolism , Endothelial Cells/metabolism , Epithelial Cells/metabolism , Female , Fibroblasts/metabolism , Humans , Keratinocytes/metabolism , Lecithin Cholesterol Acyltransferase Deficiency/genetics , Lecithin Cholesterol Acyltransferase Deficiency/metabolism , Lipoproteins, HDL/blood , Male , Microscopy, Electron , Middle Aged , Phosphatidylcholine-Sterol O-Acyltransferase/blood , Phosphatidylcholine-Sterol O-Acyltransferase/genetics , Phospholipids/metabolism , RNA-Seq , Tangier Disease/genetics , Tangier Disease/metabolismABSTRACT
Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the ß3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose ß1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.
Subject(s)
ADAMTS Proteins/metabolism , ADAMTS9 Protein/metabolism , Cleft Lip/metabolism , Cornea/abnormalities , Glycosyltransferases/deficiency , Growth Disorders/metabolism , Limb Deformities, Congenital/metabolism , Alleles , Animals , Cleft Lip/enzymology , Cleft Lip/genetics , Cornea/enzymology , Cornea/metabolism , Disease Models, Animal , Female , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Glycogen Debranching Enzyme System/metabolism , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Growth Disorders/enzymology , Growth Disorders/genetics , Limb Deformities, Congenital/enzymology , Limb Deformities, Congenital/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Organogenesis/geneticsABSTRACT
To observe the expression of MMP-2 and MMP-9 and of the FAK/PI3K/Akt signaling pathway in HSK. Fifty BALB/c mice were infected to establish the model and killed on days 0, 2, 7, 14, and 28. The cornea samples were prepared, respectively. Slit lamp examination, immunofluorescence staining, reverse transcription PCR, and Western blot were used to detect the index. After HSV-1 infection, different degrees of epithelial or stromal damage and corneal opacity were observed. Immunofluorescence staining showed that the expressions of MMP-2 and MMP-9 at different levels of corneal tissue were observed on the 0d, 2d, 7d, 14d, and 28d. Compared with 0d, the relative expression levels of MMP-2 and MMP-9 mRNA at 2d, 7d, 14d, and 28d were significantly increased (all P< 0.05). Compared with 14d, the relative expression of MMP-2 and MMP-9 mRNA decreased on the 2d, 7d, and 28d (all P< 0.05). Western blot showed that the protein expressions of p-FAK, p-PI3K, p-Akt, MMP-2, and MMP-9 at 2d, 14d, and 28d were all significantly higher than 0d (all P< 0.05). At 14 d, the expressions of p-FAK, p-PI3K, p-Akt, and MMP-2 were significantly higher than those at 2d, 7d, and 28d (all P< 0.05). The protein expression of FAK, PI3K, and Akt in corneal of mice in each time period had no significant (all P> 0.05). These data suggest that FAK/PI3K/Akt signaling pathway and MMP-2 and MMP-9 may be involved in the development of HSK.
Subject(s)
Focal Adhesion Kinase 1/metabolism , Gene Expression Regulation, Enzymologic , Keratitis, Herpetic/enzymology , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Animals , Cornea/enzymology , Cornea/pathology , Keratitis, Herpetic/pathology , Male , Mice , Mice, Inbred BALB CABSTRACT
Purpose: Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases involved in wound healing processes, including neovascularization and fibrosis. We assessed MMP protein expression levels in diseased corneas of patients requiring penetrating and deep anterior lamellar keratoplasty. The purpose of this study was to test the hypothesis that upregulation of MMPs in diseased corneas is positively associated with clinical levels of corneal neovascularization and fibrosis. Methods: Protein expression levels of nine individual MMPs were quantified simultaneously in human corneal lysates by using the Bio-Plex Pro Human MMP 9-Plex Panel and the MAGPIX technology. Measurements of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, and MMP13 were performed on diseased specimens from 21 patients undergoing corneal transplantation (17 for penetrating keratoplasty and 4 for deep anterior lamellar keratoplasty) and 6 normal control corneas. Results: Luminex-based expression analysis revealed a significant overexpression of four of the nine MMPs tested (MMP2, MMP8, MMP12, and MMP13) in patient samples compared to control. Significant overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 was observed in diseased corneas with neovascularization compared with diseased corneas without neovascularization. Overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 also corresponded with the levels of corneal fibrosis. Finally, reduced expression of MMP3 was detected in keratoconus patients. Conclusions: Multiple MMPs are expressed in the corneas of patients with chronic disease requiring keratoplasty even when the pathologic process appears to be clinically inactive. In particular, the expression of several MMPs (MMP2, MMP8, MMP12, and MMP13) is positively associated with increased levels corneal fibrosis and neovascularization.
Subject(s)
Corneal Diseases/enzymology , Corneal Diseases/surgery , Keratoplasty, Penetrating , Matrix Metalloproteinases/metabolism , Adult , Aged , Aged, 80 and over , Cornea/enzymology , Cornea/pathology , Corneal Neovascularization/enzymology , Corneal Transplantation , Female , Fibrosis/enzymology , Humans , Immunoassay/methods , Male , Middle Aged , Young AdultABSTRACT
Optimal ex vivo corneal storage in eye banks is crucial to increase both the number of corneas suitable for graft and their intrinsic quality, mainly the number of viable endothelial cells, which dictates graft survival in recipients. With both passive storage methods used worldwide (short-term cold storage in the United States, long-term organ culture in Europe), significant endothelial cell loss is inevitable. Here we show that, with an active storage machine, also called a bioreactor, which restores 2 fundamental physiological parameters, intraocular pressure and medium renewal, endothelial cell survival is improved by 23% compared with organ culture after 4 weeks' storage. Also observed in the bioreactor is a 4-fold higher expression of Na+ /K+ ATPase, which supports one of the major endothelial cell pumping functions. In addition, corneas remain thin and transparent, so they are suitable for surgery at any time. This new active eye banking method may help to reduce the severe global scarcity of donor corneas.
Subject(s)
Cornea , Corneal Transplantation , Eye Banks , Organ Preservation/instrumentation , Bioreactors , Cell Survival , Cornea/cytology , Cornea/enzymology , Electron Transport Complex IV/metabolism , Endothelial Cells/cytology , Endothelial Cells/enzymology , Equipment Design , Humans , In Vitro Techniques , Organ Culture Techniques/instrumentation , Prospective Studies , Sodium-Potassium-Exchanging ATPase/metabolism , Time FactorsABSTRACT
OBJECTIVE To characterize the distribution and intensity of cyclooxygenase (COX)-2 expression in the eyes of cats with and without uveitis and to determine whether COX-2 expression is correlated with severity of inflammation. SAMPLES Archived ocular tissue specimens from 51 cats with and 10 cats without ocular disease. PROCEDURES Specimens from only 1 eye were evaluated for each cat. Specimens were stained with H&E stain or immunohistochemical stain for detection of COX-2 and reviewed. For each eye, the type, severity, and distribution of inflammation and the distribution and intensity of COX-2 expression were determined for the uvea and other ocular tissues. Correlation between COX-2 expression and inflammation severity was also assessed. RESULTS COX-2 was not expressed in any nondiseased eye. Of the 51 diseased eyes, 20 had histologic evidence of lymphocytic-plasmacytic uveitis, 13 had neutrophilic uveitis, 11 had diffuse iris melanoma with uveitis, and 7 had diffuse iris melanoma without uveitis. Of the 44 eyes with uveitis, COX-2 was detected in the uvea of 16, including 11 eyes with lymphocytic-plasmacytic uveitis, 4 with neutrophilic uveitis, and 1 with diffuse iris melanoma-induced uveitis. Inflammation was severe, moderate, or mild in 10, 5, and 1 of those eyes, respectively. Cyclooxygenase-2 was detected in the cornea of 21 eyes with uveitis and 1 eye with diffuse iris melanoma without uveitis. Uveitis severity was positively correlated with COX-2 expression in both the uvea and cornea. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that COX-2 is an inflammatory mediator in feline uveitis but not diffuse iris melanoma.
Subject(s)
Cyclooxygenase 2/biosynthesis , Eye/enzymology , Uveitis/veterinary , Animals , Cats , Cornea/enzymology , Cyclooxygenase 2/metabolism , Female , Male , Melanoma/enzymology , Melanoma/veterinary , Uvea/enzymology , Uveal Neoplasms/enzymology , Uveal Neoplasms/veterinary , Uveitis/enzymologyABSTRACT
PURPOSE: Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a compound of significant biological activity. The aim of this study is to investigate the presence and distribution of KAT immunoreactivity in the healthy human cornea. METHODS: Data on gene expression in human eye structures were extracted from public microarray experiments using Genevestigator software. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, II, and III on sections of eight enucleated eyes from patients with choroidal melanoma. RESULTS: Bioinformatics analysis showed that all four KAT isoforms were actively transcribed in the cornea and the conjunctiva. Immunohistochemical analysis revealed the presence of KAT I, II, and III in all examined corneal sections. The corneal endothelium showed the strongest reactivity for all three KAT isoforms. There was a slight positive staining of the corneal stroma for KAT I and II. KAT III immunoreactivity was found only in the stroma of the limbal region. In the corneal epithelium, the expression of all three KAT isoforms showed a specific pattern of the stain with fine squatter granules throughout the cytoplasm. This reactivity was more pronounced in the basal cell layers. The intermediate cell layers showed only faint immunoreactivity, and occasionally, there was no staining. KAT I, II, and III were also present in the adjacent limbal conjunctiva. CONCLUSIONS: The results indicate that kynurenine can be metabolized to KYNA in the corneal epithelium, stroma, and endothelium.
Subject(s)
Computational Biology , Cornea/enzymology , Gene Expression Regulation, Enzymologic , Transaminases/genetics , Adult , Aged , Aged, 80 and over , Conjunctiva/enzymology , Female , Humans , Immunohistochemistry , Kynurenine , Male , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , Retina/enzymology , Transaminases/metabolismABSTRACT
PURPOSE: Keratoconus (KC) is thinning of the central cornea. Its etiology is unknown, but it may result from degrading of collagen type IV. The major protein in the cornea is collagen. Matrix metalloproteinase-9 (MMP-9) is able to degrade collagen type IV from the basement membrane and extracellular matrix (ECM). MMP-9 enzymatic activity is inhibited by the tissue inhibitor of metalloproteinase-1 (TIMP-1). In the present study, we sought to investigate and evaluate the effects of single nucleotide polymorphisms in COL4A3, MMP-9, and TIMP-1 on the risk of KC in an Iranian population sample. METHODS: This case-control study was performed on 140 KC patients and 150 healthy controls. Genotyping of the COL4A3 rs55703767, MMP-9 rs17576, and TIMP-1 rs6609533 polymorphisms was done using amplification refractory mutation system polymerase chain reaction (ARMS-PCR). RESULTS: Our findings showed that the rs55703767G/T polymorphism decreased the risk of KC (OR = 0.26, 95% CI = 0.08-0.82, P = 0.022). rs17576A/G, associated with KC and the A allele, was significantly overrepresented in healthy individuals. rs6609533A/G (X-chromosome) increased the risk of KC in females (OR = 2.27, 95% CI = 1.06-4.76, P = 0.036). In males, the allele frequency was not associated with KC risk/protection. CONCLUSIONS: This study indicates that in our population, the COL4A3 rs55703767 polymorphism decreased the risk of KC. However, the TIMP-1 rs6609533 polymorphism was associated with an increased risk of KC.
Subject(s)
Autoantigens/genetics , Collagen Type IV/genetics , Cornea/enzymology , DNA/genetics , Keratoconus/genetics , Matrix Metalloproteinase 9/genetics , Polymorphism, Genetic , Tissue Inhibitor of Metalloproteinase-1/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Autoantigens/metabolism , Child , Collagen Type IV/metabolism , Cornea/pathology , Female , Genotype , Humans , Incidence , Iran/epidemiology , Keratoconus/enzymology , Keratoconus/epidemiology , Male , Matrix Metalloproteinase 9/metabolism , Middle Aged , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction , Tissue Inhibitor of Metalloproteinase-1/metabolism , Young AdultABSTRACT
The enzyme soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate (HCO3-) to produce the signaling molecule cyclic adenosine monophosphate (cAMP). Because sAC and sAC-related enzymes are found throughout phyla from cyanobacteria to mammals and they regulate cell physiology in response to internal and external changes in pH, CO2, and HCO3-, sAC is deemed an evolutionarily conserved acid-base sensor. Previously, sAC has been reported in dogfish shark and round ray gill cells, where they sense and counteract blood alkalosis by regulating the activity of V-type H+- ATPase. Here, we report the presence of sAC protein in gill, rectal gland, cornea, intestine, white muscle, and heart of leopard shark Triakis semifasciata Co-expression of sAC with transmembrane adenylyl cyclases supports the presence of cAMP signaling microdomains. Furthermore, immunohistochemistry on tissue sections, and western blots and cAMP-activity assays on nucleus-enriched fractions demonstrate the presence of sAC protein in and around nuclei. These results suggest that sAC modulates multiple physiological processes in shark cells, including nuclear functions.
Subject(s)
Adenylyl Cyclases/isolation & purification , Bicarbonates/metabolism , Cell Nucleus/enzymology , Cytoplasm/enzymology , Sharks/metabolism , Animals , Cornea/enzymology , Intestines/enzymology , Myocardium/enzymology , Salt Gland/enzymologyABSTRACT
BACKGROUND: Keratoconus has classically been defined as a noninflammatory disorder, although recent studies show elevated levels of inflammatory markers suggesting that keratoconus could be, at least in part, an inflammatory condition. Heparanase upregulation has been described in multiple inflammatory disorders. In this article, we study the differential expression of heparanase in cornea and tears from keratoconus patients and healthy controls. METHODS: A transcriptomic approach was used employing quantitative polymerase chain reaction to analyze the expression of heparanase and heparanase 2 in stromal and epithelial corneal cells. The protein expression was analyzed by immunohistochemistry in corneal sections. Enzymatic activity in tears was measured using [3H]-labeled heparan sulfate as substrate. RESULTS: Heparanase transcription was detected in stromal and epithelial cells and appeared upregulated in keratoconus. Overexpression of the enzyme was also detected by immunohistochemistry. Corneal expression of heparanase 2 was detected in some cases. Heparanase catalytic activity was found in tears and displayed a positive correlation with the degree of keratoconus. CONCLUSIONS: Heparanase overexpresses in keratoconic corneas, possibly reinforcing the inflammatory condition of the pathology. The presence of heparanase activity in tears allows us to propose its use as a biomarker for the diagnosis of the disorder.
Subject(s)
Glucuronidase/metabolism , Keratoconus/metabolism , Tears/metabolism , Biomarkers/metabolism , Cells, Cultured , Cornea/enzymology , Cornea/metabolism , Glucuronidase/genetics , Heparitin Sulfate/metabolism , Humans , Keratoconus/enzymology , Keratoconus/pathology , Tears/enzymology , Up-RegulationABSTRACT
OBJECTIVE To compare the anticollagenase efficacy of fresh feline, canine, and equine serum and plasma on in vitro corneal degradation. SAMPLE Grossly normal corneas from recently euthanized dogs, cats, and horses and fresh serum and plasma from healthy dogs, cats, and horses. PROCEDURES Serum and plasma were pooled by species and used for in vitro experiments. Corneas were collected and stored at -80°C. Sections of cornea were dried, weighed, and incubated in saline (0.9% NaCl) solution with clostridial collagenase and homologous fresh serum or plasma. Corneal degradation was assessed as the percentage of corneal weight loss and hydroxyproline concentration, compared with results for positive and negative control samples. RESULTS Homologous fresh serum and plasma significantly reduced the percentage of corneal weight loss, compared with results for positive control samples. No significant difference was found in percentage of corneal weight loss between incubation with serum or plasma for feline, canine, and equine corneas. Canine serum and plasma significantly reduced hydroxyproline concentrations, whereas inclusion of feline and equine serum or plasma did not, compared with results for positive control samples. Hydroxyproline concentrations were moderately correlated with percentage of corneal weight loss for feline samples and weakly correlated for equine samples, but they were not correlated for canine samples. CONCLUSIONS AND CLINICAL RELEVANCE In this study, the anticollagenase efficacy of fresh feline, canine, and equine serum was not different from that of plasma. Plasma should be an acceptable substitute for serum in the topical treatment of keratomalacia.
Subject(s)
Cats , Cornea/enzymology , Dogs , Horses , Plasma , Serum , Animals , Collagenases/metabolism , Hydroxyproline , Matrix Metalloproteinase Inhibitors/pharmacologyABSTRACT
The pharmacokinetic behaviors of novel ophthalmic drugs are often preliminarily investigated in preclinical studies using ex vivo animal cornea or corneal cell culture models. During transcorneal passage, topically applied drugs may be affected by drug metabolizing enzymes. The knowledge regarding the functional expression of metabolic enzymes in corneal tissue is marginal; thus, the aim of this study was to investigate cytochrome P450 activity in an organotypic three-dimensional human cornea construct and to compare it with porcine and rabbit corneas, which are commonly used ex vivo cornea models. The total cytochrome P450 activity was determined by measuring the transformation of 7-ethoxycoumarin. Furthermore, the expression of the cytochrome P450 enzyme 2D6 (CYP2D6) was investigated at the protein level using immunohistochemistry and western blotting. CYP2D6 activity measurements were performed using a d-luciferin-based assay. In summary, similar levels of the total cytochrome P450 activity were identified in all 3 cornea models. The protein expression of CYP2D6 was confirmed in the human cornea construct and porcine cornea, whereas the signals in the rabbit cornea were weak. The analysis of the CYP2D6 activity indicated similar values for the human cornea construct and porcine cornea; however, a distinctly lower activity was observed in the rabbit cornea.
Subject(s)
Cornea/enzymology , Cytochrome P-450 Enzyme System/metabolism , 7-Alkoxycoumarin O-Dealkylase/metabolism , Administration, Ophthalmic , Animals , Cell Line , Cells, Cultured , Cytochrome P-450 CYP2D6/metabolism , Drug Delivery Systems , Humans , Immunohistochemistry , Models, Biological , Rabbits , Species Specificity , SwineABSTRACT
Complex sphingolipids are strongly expressed in neuronal tissue and contain ceramides in their backbone. Ceramides are synthesized by six ceramide synthases (CerS1-6). Although it is known that each tissue has a unique profile of ceramide synthase expression and ceramide synthases are implicated in several neurodegenerative disorders, the expression of ceramide synthase isoforms has not been investigated in the retina. Here we demonstrate CerS1, CerS2 and CerS4 expression in mouse retina and cornea, with CerS4 ubiquitously expressed in all retinal neurons and Müller cells. To test whether ceramide synthase deficiency affects retinal function, we compared electroretinograms and retina morphology between wild-type and CerS1-, CerS2- and CerS4-deficient mice. Electroretinograms were strongly reduced in amplitude in ceramide synthase-deficient mice, suggesting that signalling in the outer retina is affected. However, the number of photoreceptors and cone outer segment length were unaltered and no changes in retinal layer thickness or synaptic structures were found. Mass spectrometric analyses of ceramides, hexosyl-ceramides and sphingomyelins showed that C20 to C24 acyl-containing species were decreased whereas C16-containing species were increased in the retina of ceramide synthase-deficient mice. Similar but smaller changes were also found in the cornea. Thus, we hypothesize that the replacement of very long-chain fatty acyl residues by shorter C16 residues may affect the electrical properties of retina and cornea, and alter receptor-mediated signal transduction, vesicle-mediated synaptic transmission or corneal light transmission. Future studies need to identify the molecular targets of ceramides or derived sphingolipids in light signal transduction and transmission in the eye.
Subject(s)
Cornea/metabolism , Light Signal Transduction , Oxidoreductases/metabolism , Retina/metabolism , Sphingolipids/metabolism , Animals , Ceramides/metabolism , Cornea/enzymology , Electroretinography , Mice , Oxidoreductases/genetics , Retina/enzymology , Retina/physiologyABSTRACT
Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas.
Subject(s)
Cornea/enzymology , Epithelium, Corneal/enzymology , Glutathione S-Transferase pi/metabolism , Glutathione Transferase/metabolism , Animals , Blotting, Western , Cell Line , Ethacrynic Acid/metabolism , Fluorescent Antibody Technique , Glutathione/analogs & derivatives , Glutathione/metabolism , Humans , Rabbits , Species Specificity , SwineABSTRACT
Our previous studies have shown that Dexamethasone (Dex) reduced the expression of matrix-metalloproteinases (MMPs -1,-3,-9,-13), IL-1ß and IL-6, while it significantly increased MMP-8 mRNA transcripts in a concomitant dry eye and corneal alkali burn murine model (CM). To investigate if MMP-8 induction is responsible for some of the protective effects of Dex in CM, MMP-8 knock out mice (MMP-8KO) were subjected to the CM for 2 or 5 days and topically treated either with 2 µl of 0.1% Dexamethasone (Dex), or saline QID. A separate group of C57BL/6 mice were topically treated with Dex or BSS and received either 100 nM CAM12 (MMP-8 inhibitor) or vehicle IP, QD. Here we demonstrate that topical Dex treated MMP-8KO mice subjected to CM showed reduced corneal clarity, increased expression of inflammatory mediators (IL-6, CXCL1, and MMP-1 mRNA) and increased neutrophil infiltration at 2D and 5D compared to Dex treated WT mice. C57BL/6 mice topically treated with Dex and CAM12 IP recapitulated findings seen with MMP-8KO mice. These results suggest that some of the anti-inflammatory effects of Dex are mediated through increased MMP-8 expression. J. Cell. Physiol. 231: 2506-2516, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Burns, Chemical/drug therapy , Burns, Chemical/enzymology , Cornea/pathology , Dexamethasone/therapeutic use , Dry Eye Syndromes/complications , Eye Burns/drug therapy , Eye Burns/enzymology , Matrix Metalloproteinase 8/metabolism , Alkalies , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Burns, Chemical/complications , Cornea/enzymology , Desiccation , Dexamethasone/pharmacology , Disease Models, Animal , Dry Eye Syndromes/enzymology , Eye Burns/complications , Female , Matrix Metalloproteinase 8/deficiency , Matrix Metalloproteinase 8/genetics , Matrix Metalloproteinase Inhibitors/pharmacology , Matrix Metalloproteinase Inhibitors/therapeutic use , Mice, Inbred C57BL , Mice, Knockout , Neutrophil Infiltration/drug effects , Neutrophils/drug effects , Neutrophils/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Stress, PhysiologicalABSTRACT
Caused by fungus, fungal keratitis is a kind of infections corneal disease with high rate of blindness, which patients are mainly farmers in developing countries. Interleukin, as important proinflammatory cytokines, involve in immune defense process against fungal infection of cornea. The expression of interleukin in the pathogenesis of fungal keratitis, especially the main source of its cells, is not clear and the cell signaling pathways which regulate the synthesis and modification of interleukin is still unknown. Caspase-11 was obtained and cultured. And the ELISA and Western-blot methods were used to explore the regulatory effect of Caspse-11 on Interleukin-1ß in the fungal keratitis. neutrophils were the main cell lineage of IL-1ß to take part in the innate anti-fungi immunity in the cornea; IL-1ß generation induced by fungal infection might not be through the pre-excitation in the classical signal pathway; TLR4/TRIF pathway was not involved in pro-IL-1ß generation; while Dectin-1/syk pathway was involved in IL-1ß generation in the fungal keratitis; Caspase-l participated in the modification of IL-1ß to change from the precursor into the mature body; but NLRP3 inflammasome and ASC inflammasome were not involved in IL-1ß generation; Caspase-11 was involved in IL-1ß generation through regulating the modified process of Caspase-l to turning from precursor into mature body. TLR4/TRIF pathway and NLRP3 inflammasome and ASC inflammasome are not involved in the pro-IL-1ß generation, while Caspase-l, Caspase-11 and Dectin-1/syk pathway are involved in the IL-1ß generation.
Subject(s)
Aspergillosis/enzymology , Aspergillus fumigatus/pathogenicity , Caspase 1/metabolism , Caspases/metabolism , Cornea/enzymology , Eye Infections, Fungal/enzymology , Interleukin-1beta/metabolism , Keratitis/enzymology , Neutrophils/enzymology , Adaptor Proteins, Vesicular Transport/deficiency , Adaptor Proteins, Vesicular Transport/genetics , Animals , Aspergillosis/genetics , Aspergillosis/immunology , Aspergillosis/microbiology , Aspergillus fumigatus/immunology , Caspase 1/genetics , Caspases, Initiator , Cells, Cultured , Cornea/immunology , Cornea/microbiology , Disease Models, Animal , Eye Infections, Fungal/genetics , Eye Infections, Fungal/immunology , Eye Infections, Fungal/microbiology , Female , Genotype , Host-Pathogen Interactions , Keratitis/genetics , Keratitis/immunology , Keratitis/microbiology , Lectins, C-Type/deficiency , Lectins, C-Type/genetics , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/immunology , Neutrophils/microbiology , Phenotype , Signal Transduction , Syk Kinase/metabolism , Toll-Like Receptor 4/deficiency , Toll-Like Receptor 4/geneticsABSTRACT
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.