To report a novel mutation within the CHST6 gene, as well as describe light and electron microscopic features of a case of macular corneal dystrophy. A 59-year old woman with macular corneal dystrophy in both eyes who had decreased visual acuity underwent penetrating keratoplasty. Further studies including light and electron microscopy, as well as DNA analysis were performed. Light microscopy of the cornea revealed glycosaminoglycan deposits in the keratocytes and endothelial cells, as well as extracellularly within the stroma. All samples stained positively with alcian blue, colloidal iron, and periodic acid-Schiff. Electron microscopy showed keratocytes distended by membrane-bound intracytoplasmic vacuoles containing electron-dense fibrillogranular material. These vacuoles were present in the endothelial cells and between stromal lamellae. Some of the vacuoles contained dense osmophilic whorls. A novel homozygous mutation (c.613 C>T [p.Arg205Trp]) was identified within the whole coding region of CHST6. A novel CHST6 mutation was detected in a Korean macular corneal dystrophy patient.
Corneal Dystrophies, Hereditary/diagnosis/*genetics/metabolism ; Corneal Keratocytes/ultrastructure ; DNA/*genetics ; DNA Mutational Analysis ; Female ; Humans ; Microscopy, Electron ; Middle Aged ; *Mutation, Missense ; Pedigree ; Polymerase Chain Reaction ; Republic of Korea ; Sulfotransferases/*genetics/metabolism

PURPOSE: To investigate the biologic effect of mitomycin C, dexamethasone and cyclosporine A 0.05% on cultured human keratocytes in vitro. METHODS: Human corneal keratocytes were exposed to a concentration of mitomycin C (0.05%), dexamethasone (0.05%) and cyclosporine A (0.05%) for a period of 3, 5, and 10 minutes. MTT-based colorimetric assay was performed to assess the metabolic activity of cellular proliferation and the concentration of type I procollagen COOH-terminal peptide (PIP) and laminin were measured. Cell damage was determined by using the lactate dehydrogenase (LDH) assay. Apoptotic response was evaluated utilizing flow cytometric analysis with Annexin V and propiodium iodide. RESULTS: The inhibitory effect of cellular proliferation and cytotoxicity in cultured human keratocytes showed a time-dependent response in all drugs. The production of PIP and laminin showed a time-dependent response in cultured cells. Apoptosis was observed in flow cytometry after being treated with mitomycin C, dexamethasone and cyclosporine A. Cyclosporin A resulted in less apoptosis of keratocytes than mitomycin C and dexamethasone. CONCLUSIONS: The apoptotic response of mitomycin C, dexamethasone and cyclosporine A is associated with the inhibitory effect of human corneal keratocyte proliferation. To decrease corneal opacity, mitomycin C and dexamethasone were more effective than cyclosporine A in the present study. Additionally, a high concentration of cyclosporine A greater than 0.05% is necessary to lower corneal opacity.
Annexin A5 ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Collagen Type I ; Corneal Keratocytes ; Corneal Opacity ; Cyclosporine ; Dexamethasone ; Flow Cytometry ; Humans ; L-Lactate Dehydrogenase ; Laminin ; Mitomycin

BACKGROUNDTransforming growth factor β (TGFβ) is one of the most important growth factors in the development of fibrosis and scarring on cornea. Smad7, an inhibitory Smad, can inhibit TGFβ signal transduction. In recent years, effects of lentiviral-mediated Smad7 on inhibition of fibrosis on some organs have been studied, while little is known about the effects on cornea. This study aimed to determine the effects of lentiviral-mediated Smad7 gene expression on keratocyte proliferation and fibrosis induced by TGF β2 in vitro.

METHODSKeratocytes were cultured from corneal tissue isolated from Sprague-Dawley (SD) rats and transfected with Smad7 expressing lentiviral vector (Lv-Smad7) or non-functioning control vector (Lv-blank). Following the exposure to TGFβ2, keratocytes were processed for immunoblotting to assess the phosphorylation of Smad2 as down-stream event of TGFβ/Smad signaling. Expression of fibrotic markers α-smooth muscle actin (α-SMA), type III collagen (collagen III) were measured by Western blotting and quantitative real time polymerase chain reaction (PCR). Overall cell proliferation was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression of cell cycle-related marker Ki67 at both mRNA and protein levels.

RESULTSThe Smad7 gene transfer suppressed TGFβ/Smad signaling in keratocytes by down-regulating phosphorylation of Smad2. Markers of cell proliferation and fibrosis including Ki67, α-SMA, collagen III were inhibited by introduction of Smad 7 into TGFβ exposed keratocytes. Consequently, the rate of cell proliferation was attenuated.

CONCLUSIONSmad7 gene transfer inhibited fibrogenic responses of keratocytes to TGFβ2.

Actins ; genetics ; metabolism ; Animals ; Blotting, Western ; Cell Proliferation ; Cells, Cultured ; Collagen Type III ; genetics ; metabolism ; Corneal Keratocytes ; cytology ; drug effects ; metabolism ; Genetic Vectors ; genetics ; Ki-67 Antigen ; genetics ; metabolism ; Lentivirus ; genetics ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; genetics ; Smad7 Protein ; genetics ; metabolism ; pharmacology ; Transforming Growth Factor beta2 ; pharmacology

PURPOSE: To investigate the characteristics of cultured rabbit corneal keratocytes in vitro and evaluate the possibility of differentiation of mesenchymal stem cells to keratocytes using the keratocyte conditioned medium (KCM). METHODS: Isolated keratocytes were seeded on the stromal side of amniotic membranes (AM) or plastic dishes, and morphologic changes were evaluated. Rabbit mesenchymal stem cells were cultured on AM with alpha-MEM (minimum essential medium alpha) and KCM. The gene expression patterns of specific keratocyte markers (keratocan, lumican, and aldehyde dehydrogenase family, member A1 (ALDH1A1)) of cultured cells were evaluated by RT-PCR. RESULTS: Keratocytes on AM showed dendritic morphology with slow proliferation in contrast, cells on dishes were stellate in shape with fast proliferation. Cultured keratocytes on AM maintained the expression of keratocan, lumican and ALDH1A1 while keratocytes on plastic dishes steadily lost their keratocyte marker gene expression. Additionally, mesenchymal stem cells cultured with KCM on AM induced expression of keratocan and ALDH1A1. CONCLUSIONS: Keratocytes cultured on AM stromal matrix maintained their characteristic morphology and marker gene expression. Morphology changes and marker gene expressions of mesenchymal stem cells suggest an ability to differentiate into keratocytes when grown on AM with KCM.
Aldehyde Dehydrogenase ; Amnion ; Cells, Cultured ; Chondroitin Sulfate Proteoglycans ; Corneal Keratocytes ; Culture Media, Conditioned ; Gene Expression ; Humans ; Keratan Sulfate ; Mesenchymal Stromal Cells ; Organic Chemicals ; Plastics ; Seeds

PURPOSE: To evaluate the biological effects and cytotoxicity of gel-type artificial tears on human corneal keratocytes and conjunctival cells in vitro. METHODS: Human corneal keratocytes and conjunctival epithelial cells were exposed to Soothe(R) and Systane(R) at variable concentrations. Evaluations were conducted through an MTT-based calorimetric assay to measure the metabolic activity and through a lactate dehydrogenase (LDH) assay to assess cellular damage. Apoptotic response was examined using fluorescent microscopy and flow cytometric analysis, and cellular morphologic results were evaluated with a transmission electron microscope. RESULTS: The inhibitory effects of corneal keratocyte and conjunctival cell proliferations increased at higher concentrations and longer exposure times to Soothe(R) and Systane(R). The LDH titers increased after Soothe(R) exposure, but showed no significant difference after Systane(R) exposure. Soothe(R) and Systane(R) treatments both produced fluorescence, representing apoptotic cells. In flow cytometry, the maximal apoptotic response was observed for both types of artificial tears, although Systane(R) showed less edema, as well as reduced cytoplasmic and nuclear cell degeneration compared to those of Soothe(R). CONCLUSIONS: The apoptotic responses of Soothe(R) and Systane(R) are associated with inhibitory effects of human corneal keratocyte and conjunctival epithelial cell proliferations. To inhibit the cellular proliferation of human corneal keratocytes and conjunctival epithelial cells, Systane(R) may be less severe than Soothe(R) at higher concentrations and longer exposure times.
Apoptosis ; Cell Proliferation ; Corneal Keratocytes ; Cytoplasm ; Edema ; Electrons ; Epithelial Cells ; Flow Cytometry ; Fluorescence ; Humans ; L-Lactate Dehydrogenase ; Microscopy ; Ophthalmic Solutions

PURPOSE: To identify the effects of microenvironmental changes caused by human corneal epithelial damages to characteristics or differentiation of human mesenchymal stem cells (hMSCs). METHODS: Artificial corneal damage was induced onto a cultured monolayer of human corneal epithelial cells. hMSCs were then co-cultured with damaged human corneal epithelial cells (dIHCE). Morphological changes in the co-cultured hMSCs were observed. To elucidate the differentiation of hMSCs into corneal keratocytes or epithelial cells, the expressions of alpha-smooth muscle actin, keratin-3/-12, and E-cadherin were confirmed by immunofluorescence. RESULTS: hMSCs co-cultured with dIHCE showed enhanced adherence in the neighborhood of dIHCE and morphological change into dendritic shapes at 6 days post-seeding. Although the expression of alpha-smooth muscle actin, known as hMSCs marker, significantly decreased at the dIHCE-contacted site of hMSCs; there were no expressional changes on keratin-3/-12 and E-cadherin, the markers of corneal epithelial cells. Interestingly, positive expression of corneal epithelial marker keratin-3/-12 was observed in dIHCE co-cultured hMSCs. hMSCs co-cultured with normal human corneal epithelial cells (nIHCE) were unable to attach, and showed no change in the expression of alpha-smooth muscle actin. CONCLUSIONS: It is proposed that dIHCE causes a morphological change in hMSCs, and decreased expression of alpha-smooth muscle actin. These results suggest that dIHCE can affect a change in the characteristics and differentiation of hMSCs.
Actins ; Cadherins ; Coculture Techniques ; Corneal Keratocytes ; Epithelial Cells* ; Fluorescent Antibody Technique ; Humans* ; Mesenchymal Stromal Cells* ; Residence Characteristics

PURPOSE: To evaluate the effects of intrastromal air injection and intrastromal balanced salt solution (BSS) injection on corneal keratocyte apoptosis. METHODS: Twelve right eyes of New Zealand White rabbits were divided into an air-injected group (n=6) and a hydro-injected group (n=6). Contralateral eyes served as a control. Air or Balanced salt solution (BSS(R), Alcon, USA) was injected into the deep corneal stroma at the paracentral area to propagate into nearly the entire cornea. To reduce the intraocular pressure, anterior chamber paracentesis was performed. The animals were sacrificed 4 hours (n=6) and 24 hours (n=6) after surgery. Central cornea buttons were retrieved to stain with Hematoxylin & Eosin and TUNEL (Apoptag(R), Chemicon). The mean number of apoptotic keratocytes was counted in 24.67+/-4.04 consecutive high power field (HPF). RESULTS: The mean number of TUNEL-positive cells at 4 hours was 12.85+/-7.25/HPF and 0.25+/-0.44/HPF in air-injected and hydro-injected eyes, respectively. It was reduced to 6.25+/-4.02/HPF and 0.15+/-0.37/HPF in air-injected and hydro-injected eyes after 24 hours. The air-injected group showed significantly more TUNEL-positive cells compared with the hydro-injected or control group until 24 hours (p=0.001, p=0.001, Mann-Whitney U test). CONCLUSIONS: Intrastromal air injection induces significant apoptosis of keratocytes suggesting some damages in the peripheral cornea when used in deep lamellar keratoplasty.
Animals ; Anterior Chamber ; Apoptosis* ; Cornea ; Corneal Keratocytes ; Corneal Stroma ; Corneal Transplantation ; Eosine Yellowish-(YS) ; Hematoxylin ; In Situ Nick-End Labeling ; Intraocular Pressure ; Paracentesis ; Rabbits

PURPOSE: To evaluate the effect of polyhexamethylene biguanide (PHMB) and chlorhexidine on Acanthamoeba cysts and cultured human keratocytes. METHODS: Each well of two-fold diluted PHMB and chlorhexidine were treated on the Acanthamoeba cyst suspension of 5 x 10(4) cysts/ml for 8, 24, and 48 hours to measure the minimal cysticidal concentration (MCC) of each disinfectant and was exposed to the human corneal keratocytes of 5 x 10(4) cells/ml for same hours to measure the survival rate of keratocytes. Inverted phase-contrast micrograph and electron microscopy for observing the morphologic changes were evaluated. RESULTS: MCC of PHMB was 9.42 microgram/ml, 5.62 microgram/ml, and 2.37 microgram/ml, and chlorhexidine was 24.32 microgram/ml, 10.02 microgram/ml, and 7.02 microgram/ml respecitvely in 8, 24, and 48 hours. The survival rate of keratocytes at MCC was 91.7%, 64.6%, and 49.7% in PHMB of which significant decreases were found at 24 and 48 hours, and 95.7%, 90.6%, and 78.1% in chlorhexidine of which significant decrease was only found at 48 hours. The higher the concentration of disinfectants, cysts and keratocytes demonstrated more damaged appearance. CONCLUSIONS: The amoebicidal efficacy of PHMB and chlorhexidine was similar. However, in consideration of toxic effect on keratocytes by disinfectants, chlorhexidine is suggested to be more clinically useful than PHMB.
Acanthamoeba ; Chlorhexidine ; Corneal Keratocytes* ; Disinfectants ; Humans* ; Microscopy, Electron ; Survival Rate

PURPOSE: This study was to identified differentiated genes concerned with apoptosis that are up-regulated or down-regulated in OLETF corneal keratocytes stimulated with interleukin-1alpha. METHODS: Otsuka Long Evans Tokushima Fatty (OLETF) corneal keratocytes were primarily cultured and treated with 20 ng/ml of IL-1a for 6 hours. Hybridization was carried out using Oligonucleotide microarray. A sample of normal keratocytes was labeled with Cy 3, and a diabetic keratocytes sample was labeled with Cy 5. RESULTS Diabetes showed 20 down-regulated genes and 24 up-regulated genes compared with normal keratocytes. Also, IL-1alpha-treated diabetic keratocytes expressed 31 down-regulated and 33 up-regulated genes. Compared with diabetic keratocytes, the newly expressed genes in OLETF treated with IL-1alpha were Bcl, Lam, Timp, Mmp, Socs, Bmp, Ccl, Lcn, C7, etc. CONCLUSIONS: There were some genes related with apoptosis that expressed differently between normal and diabetic keratocytes of OLETF. Also, IL-1alpha may stimulate differing effects of apoptosis on diabetic corneal keratocytes. Studies analyzing the apoptotic significance of these differences identified in diabetic keratocytes are needed.
Animals ; Apoptosis* ; Corneal Keratocytes ; Diabetes Mellitus ; Interleukin-1alpha ; Oligonucleotide Array Sequence Analysis ; Rats

PURPOSE: This study was to identified differentiated genes concerned with apoptosis that are up-regulated or down-regulated in OLETF corneal keratocytes stimulated with interleukin-1alpha. METHODS: Otsuka Long Evans Tokushima Fatty (OLETF) corneal keratocytes were primarily cultured and treated with 20 ng/ml of IL-1a for 6 hours. Hybridization was carried out using Oligonucleotide microarray. A sample of normal keratocytes was labeled with Cy 3, and a diabetic keratocytes sample was labeled with Cy 5. RESULTS Diabetes showed 20 down-regulated genes and 24 up-regulated genes compared with normal keratocytes. Also, IL-1alpha-treated diabetic keratocytes expressed 31 down-regulated and 33 up-regulated genes. Compared with diabetic keratocytes, the newly expressed genes in OLETF treated with IL-1alpha were Bcl, Lam, Timp, Mmp, Socs, Bmp, Ccl, Lcn, C7, etc. CONCLUSIONS: There were some genes related with apoptosis that expressed differently between normal and diabetic keratocytes of OLETF. Also, IL-1alpha may stimulate differing effects of apoptosis on diabetic corneal keratocytes. Studies analyzing the apoptotic significance of these differences identified in diabetic keratocytes are needed.
Animals ; Apoptosis* ; Corneal Keratocytes ; Diabetes Mellitus ; Interleukin-1alpha ; Oligonucleotide Array Sequence Analysis ; Rats