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Purpose: Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The inhibitor of differentiation 3 (Id3) gene , a member of the Id gene family, governs the expression and progression of transforming growth factor beta (TGFβ)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal insults, corneal keratocytes differentiate into myofibroblasts for wound repair. Excessive development or persistence or both of myofibroblasts after wound repair results in corneal haze that compromises corneal clarity and visual function. The objective of this study was to investigate whether Id3 overexpression in human corneal stromal fibroblasts governs TGFβ-driven cellular differentiation and inhibits keratocyte to myofibroblast transformation., Methods: Primary human corneal stromal fibroblast (h-CSF) cultures were generated from donor human corneas. Human corneal myofibroblasts (h-CMFs) were produced by growing h-CSF in the presence of TGFβ1 under serum-free conditions. The Id3 gene was cloned into a mammalian expression vector (pcDNA3 mCherry LIC cloning vector), and the nucleotide sequence of the vector constructs was confirmed with sequencing as well as through restriction enzyme analysis. The Id3 mammalian overexpression vector was introduced into h-CSFs using a lipofectamine transfection kit. The expression of Id3 in selected clones was characterized with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and western blotting. Phase contrast microscopy and trypan blue exclusion assays were used to evaluate the effects of the transfer of the Id3 gene on the hCSF phenotype and viability, respectively. To analyze the inhibitory effects of the Id3 gene transfer on TGFβ-induced formation of h-CMFs, expression of the mRNA and protein of the myofibroblast marker alpha smooth muscle actin (α-SMA) was examined with qRT-PCR, western blotting, and immunocytochemistry. Student t test, analysis of variance (ANOVA), and Bonferroni adjustment for repeated measures were used for statistical analysis., Results: The results indicate that Id3 overexpression does not alter the cellular phenotype or viability of h-CSFs. Overexpression of the Id3 gene in h-CSF cells grown in the presence of TGFβ1 under serum-free conditions showed a statistically significant decrease (76.3±4.3%) in α-SMA expression (p<0.01) compared to the naked-vector transfected or non-transfected h-CSF cells. Id3-transfected, naked-vector transfected, and non-transfected h-CSF cells grown in the absence of TGFβ1 showed the expected low expression of α-SMA (0-5%). Furthermore, Id3 overexpression statistically significantly decreased TGFβ-induced mRNA levels of profibrogenic genes such as fibronectin , collagen type I , and collagen type IV (1.80±0.26-, 1.70±0.35- and 1.70±0.36-fold, respectively; p<0.05) that a play role in stromal matrix modulation and corneal wound healing. Results of the protein analysis with western blotting indicated that Id3 overexpression in h-CSF cells effectively slows TGFβ-driven differentiation and formation of h-CMFs. Results for subsequent overexpression studies showed that this process occurs through the regulation of E2A, a TATA box protein., Conclusions: Id3 regulates TGFβ-driven differentiation of h-CSFs and formation of h-CMFs in vitro. Targeted Id3 gene delivery has potential to treat corneal fibrosis and reestablish corneal clarity in vivo., (Copyright © 2020 Molecular Vision.)

Purpose: Previous research in our laboratory indicated that prothrombin and other coagulation enzymes required to activate prothrombin to thrombin are synthesized by the cornea and that apoptotic human corneal stromal cells can provide a surface for prothrombin activation through the intrinsic and extrinsic coagulation pathways. The purpose of the work reported here is to study the role of thrombin activity in the regulation of matricellular protein Cyr61 (CCN1) produced by wounded phenotype human corneal stromal fibroblasts and myofibroblasts., Methods: Stromal cells from human donor corneas were converted to defined wounded phenotype fibroblasts and myofibroblasts with fetal bovine serum, followed by basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGFβ-1), respectively, and stimulated with varying concentrations (0-10.0 units (U)/ml) of thrombin from 1-7 h. Cyr61 transcript levels were determined using reverse transcriptase-PCR (RT-PCR) and quantitative PCR (qPCR) while protein forms were analyzed using western blot data. Protease activities were characterized via protease class-specific inhibitors and western blot analysis. Thrombin activity was quantified using the fluorogenic peptide Phe-Pro-Arg-AFC. Protease-activated receptor (PAR) agonist peptides-1 and -4 were used to determine whether cells increased Cyr61 through PAR signaling pathways. The PAR-1 antagonist SCH 79797 was used to block the thrombin cleavage of the receptor. PCR data were analyzed using MxPro software and western blot data were analyzed using Image Lab™ and Image J software. Student t test and one- and two-way ANOVA (with or without ranking, depending on sample distribution), together with Dunnett's test or Tukey comparison tests for post-hoc analysis, were used to determine statistical significance., Results: Full-length Cyr61 is expressed by human corneal stromal fibroblasts and myofibroblasts and is significantly upregulated by active thrombin stimulation at the message (p<0.03) and protein (p<0.03) levels for fibroblasts and myofibroblasts. Inhibition by the allosteric thrombin-specific inhibitor hirudin prevented the thrombin-associated increase in the Cyr61 protein expression, indicating that the proteolytic activity of thrombin is required for the increase of the Cyr61 protein level. PAR-1 agonist stimulation of fibroblasts and myofibroblasts significantly increased cell-associated Cyr61 protein levels (p<0.04), and PAR-1 antagonist SCH 79797 significantly inhibited the thrombin stimulated increase of Cyr61 in fibroblasts but not in myofibroblasts. In the fibroblast and myofibroblast conditioned media, Cyr61 was detected as the full-length 40 kDa protein in the absence of thrombin, and mainly at 24 kDa in the presence of thrombin at ≥0.5 U/ml, using an antibody directed toward the internal linker region between the von Willebrand factor type C and thrombospondin type-1 domains. Although known to undergo alternative splicing, Cyr61 that is synthesized by corneal fibroblasts and myofibroblasts is not alternatively spliced in response to thrombin stimulation nor is Cyr61 directly cleaved by thrombin to generate its 24 kDa form; instead, Cyr61 is proteolytically processed into 24 kDa N- and 16 kDa C-terminal fragments by a thrombin activated leupeptin-sensitive protease present in conditioned media with activity distinct from the proteolytic activity of thrombin., Conclusions: In cultured human corneal stromal fibroblasts and myofibroblasts, thrombin regulates Cyr61 through two mechanisms: 1) thrombin increases the Cyr61 expression at the message and protein levels, and 2) thrombin increases the activation of a leupeptin-sensitive protease that stimulates the cleavage of Cyr61 into N- and C-terminal domain populations in or near the thrombospondin type-1 domain. Generation of Cyr61 peptides during corneal injury stimulation may reveal additional functions of the protein, which modulate corneal wound healing activities or decrease activities of the full-length Cyr61 form., (Copyright © 2020 Molecular Vision.)

Purpose: Patients with a heterozygous mutation in the gene encoding the transcription factor, PAX6, have a degenerative corneal opacity associated with failure of normal radial epithelial cell migration across the corneal surface and a reported wound healing defect. This study investigated the guidance mechanisms that drive the directed migration of corneal epithelial cells., Methods: In vivo corneal epithelial wounding was performed in adult wild-type and Pax6(+/-) mice, and the healing migration rates were compared. To investigate the control of the cell migration direction, primary corneal epithelial cells from wild-type and Pax6(+/-) mice were plated on grooved quartz substrates, and alignment relative to the grooves was assayed. A reconstructed corneal culture system was developed in which dissociated wild-type and genetically mutant corneal epithelial cells could be cultured on a de-epithelialized corneal stroma or basement membrane and their migration assayed with time-lapse microscopy., Results: The Pax6(+/-) cells efficiently re-epithelialized corneal wounds in vivo but had mild slowing of healing migration compared to the wild-type. Cells aligned parallel to quartz grooves in vitro, but the Pax6(+/-) cells were less robustly oriented than the wild-type. In the reconstructed corneal culture system, corneal epithelial cells continued to migrate radially, showing that the cells are guided by contact-mediated cues from the basement membrane. Recombining wild-type and Pax6 mutant corneal epithelial cells with wild-type and Pax6 mutant corneal stroma showed that normal Pax6 dosage was required autonomously in the epithelial cells for directed migration. Integrin-mediated attachment to the substrate, and intracellular PI3Kγ activity, were required for migration. Pharmacological inhibition of cAMP signaling randomized migration tracks in reconstructed corneas., Conclusions: Striking patterns of centripetal migration of corneal epithelial cells observed in vivo are driven by contact-mediated cues operating through an intracellular cAMP pathway, and failure to read these cues underlies the migration defects that accompany corneal degeneration in patients with mutations in PAX6.

Purpose: The purpose of this study was to examine the expression of corneal epithelial basement membrane (EBM) components in different corneal stromal cell types. In vitro model systems were used to explore the expression of EBM components nidogen-1, nidogen-2, and perlecan that are the primary components in the lamina lucida and the lamina densa that defectively regenerate in corneas with stromal opacity after in -9.0 D photorefractive keratectomy (PRK)., Methods: Primary rabbit corneal stromal cells were cultured using varying serum concentrations and exogenous growth factors, including fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-β1, to optimize the growth of each cell type of interest. The expression of the keratocyte-specific marker keratocan and the myofibroblast-specific marker α-smooth muscle actin (α-SMA) were analyzed with real-time PCR, western blot, and immunocytochemical staining to evaluate the specificity of the cell types and select optimal conditions (high keratocan and low α-SMA for keratocytes; low keratocan and high α-SMA for myofibroblasts; low keratocan and low α-SMA for corneal fibroblasts). The expression of the EBM components nidogen-1, nidogen-2, and perlecan was evaluated in each corneal cell type using real-time PCR, immunostaining, and western blotting. In agreement with previous studies, serum-free DMEM was found to be optimal for keratocytes, DMEM with 10% serum and 40 ng/ml FGF-2 yielded the best marker profile for corneal fibroblasts, and DMEM with 1% serum and 2 ng/ml TGF-β1 was found to be optimal for myofibroblasts., Results: Nidogen-1 and nidogen-2 mRNAs were highly expressed in keratocytes, whereas perlecan was highly expressed in myofibroblasts. In keratocytes, nidogen-2 and perlecan proteins were expressed predominantly in intracellular compartments, whereas in myofibroblasts expression of both EBM components was observed diffusely throughout the cell. Although the perlecan mRNA levels were high in the myofibroblasts, the qualitative protein expression was different from that of the keratocytes. Corneal fibroblasts produced a low amount of each EBM component., Conclusions: We have demonstrated qualitative and quantitative differences in the expression of nidogen-1, nidogen-2, and perlecan by keratocytes compared to myofibroblasts that may contribute to defective regeneration of the lamina lucida and the lamina densa of the EBM associated with late stromal haze after high-correction PRK.

Purpose: In an earlier study, we showed that human antigen R (HuR) and β-actin expression levels were downregulated in fibroblasts isolated from human keratoconus stroma compared to normal corneal stroma. To further extend the finding, we determined whether HuR expression affects β-actin gene expression and in turn affects corneal fibroblast migration and wound healing., Methods: Stromal keratocytes from normal human corneas were cultured in the presence of serum. Cells were transfected with siRNA specific for β-actin or HuR. SiRNAs specific for GAPDH or a scrambled sequence were used as positive and negative controls (siCTR) for transfection, respectively. The effects of gene silencing were analyzed at the transcriptional and translational levels. Specific proteins were immunohistochemically localized using confocal imaging. The effects of gene silencing on cell migration and cell proliferation were analyzed using a modified Boyden chamber and with a wound healing assay, respectively., Results: Reverse-transcription PCR (RT-PCR) and western blot analyses showed that when the HuR gene was silenced, β-actin expression was significantly downregulated. This was further confirmed at the translational level with immunohistochemical-confocal analysis. However, when the β-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression. When the β-actin or HuR gene was individually silenced, the motility and proliferation of corneal fibroblasts were significantly reduced., Conclusions: The results show that downregulation of the HuR gene results in decreased β-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts. We conclude that decreased β-actin expression in normal corneal stroma clearly disrupts the cytoskeletal structure and functions, including keratocyte motility and wound healing.

Purpose: The peripheral cornea contains mature and immature resident dendritic cells (DCs) while the central cornea is exclusively equipped with immature DCs. There must be some factors that cause immature DCs. This study investigated whether corneal stroma cells (CSCs) inhibit DC maturation by secreting cytokines., Methods: The messenger ribonucleic acid (mRNA) and protein level of transforming growth factor beta 2 (TGF-β(2)) was analyzed using reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Immature DCs were induced to mature in the presence of lipopolysaccharide (LPS) and with concentrations of CSC culture supernatant (containing and not containing neutralizing TGF-β(2) antibodies). Then, the DC phenotypic and functional maturation were analyzed., Results: CSCs exhibited positive expressions of TGF-β(2) mRNA and secreted high concentrations of TGF-β(2) protein. In the presence of LPS, DCs, which were treated with a CSC culture supernatant, displayed reduced expressions of cluster of differentiation 80 (CD80), CD86, and major histocompatibility complex II (MHC II) in a dose-dependent manner. Moreover, treated DCs showed lower T-cell stimulation capacity and a higher endocytosis function. However, these phenotypic and functional modifications were partially reversed after the application of neutralizing TGF-β(2) antibodies., Conclusions: This study demonstrates that CSCs can partially inhibit LPS-induced DC maturation through TGF-β(2) secretion in vitro.

Purpose: Previously, the authors demonstrated that the lack of the P2X(7) receptor impairs epithelial wound healing and stromal collagen organization in the cornea. The goal here is to characterize specific effects of the P2X(7) receptor on components of the corneal stroma extracellular matrix., Methods: Unwounded corneas from P2X(7) knockout mice (P2X(7) (-/-)) and C57BL/6J wild type mice (WT) were fixed and prepared for quantitative and qualitative analysis of protein expression and localization using Real Time PCR and immunohistochemistry. Corneas were stained also with Cuprolinic blue for electron microscopy to quantify proteoglycan sulfation in the stroma., Results: P2X(7) (-/-) mice showed decreased mRNA expression in the major components of the corneal stroma: collagen types I and V and small leucine-rich proteoglycans decorin, keratocan, and lumican. In contrast P2X(7) (-/-) mice showed increased mRNA expression in lysyl oxidase and biglycan. Additionally, we observed increases in syndecan 1, perlecan, and type III collagen. There was a loss of perlecan along the basement membrane and enhanced expression throughout the stroma, in contrast with the decreased localization of other proteoglycans throughout the stroma. In the absence of lyase digestion there was a significantly smaller number of proteoglycan units per 100 nm of collagen fibrils in the P2X(7) (-/-) compared to WT mice. While digestion was more pronounced in the WT group, double digestion with Keratanase I and Chondroitinase ABC removed 88% of the GAG filaments in the WT, compared to 72% of those in the P2X(7) (-/-) mice, indicating that there are more heparan sulfate proteoglycans in the latter., Conclusions: Our results indicate that loss of P2X(7) alters both the expression of proteins and the sulfation of proteoglycans in the corneal stroma.

Purpose: Corneal fibroblasts contribute to collagen remodeling in the corneal stroma in part by mediating collagen degradation. Given that corneal structure is influenced by sex hormone status, we examined the effects of sex hormones on collagen degradation by corneal fibroblasts., Methods: Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels with or without sex hormones including 17β-estradiol, progesterone, testosterone, and dehydroepiandrosterone (DHEA). Collagen degradation was determined by measurement of hydroxyproline after acid hydrolysis. The expression and activity of matrix metalloproteinases (MMPs) were evaluated by immunoblot analysis and gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappa B (NF-κB) inhibitor NF kappa B Inhibitor-alpha (IκB-α) in corneal fibroblasts was examined by immunoblot analysis. Cell proliferation and viability were evaluated by measurement of bromodeoxyuridine incorporation and the release of lactate dehydrogenase, respectively., Results: 17β-Estradiol and progesterone each inhibited interleukin (IL)-1β-induced collagen degradation by corneal fibroblasts in a concentration-dependent manner, whereas testosterone and DHEA had no such effect. MMP expression and activation in corneal fibroblasts exposed to IL-1β were also inhibited by 17β-estradiol and progesterone. These female sex hormones did not affect cell proliferation or viability. Both 17β-estradiol and progesterone inhibited the IL-1β-induced phosphorylation of p38 MAPK without affecting that of the MAPKs extracellular Signal-regulated Kinase (ERK) or c-jun N-terminal kinase (JNK). 17β-Estradiol also inhibited the IL-1β-induced phosphorylation of IκB-α., Conclusions: 17β-Estradiol and progesterone inhibited MMP expression and activity in IL-1β-stimulated corneal fibroblasts and thereby suppressed collagen degradation by these cells.

Purpose: Adipose-derived stem cells (ADSC) are an abundant population of adult stem cells with the potential to differentiate into several specialized tissue types, including neural and neural crest-derived cells. This study sought to determine if ADSC express keratocyte-specific phenotypic markers when cultured under conditions inducing differentiation of corneal stromal stem cells to keratocytes., Methods: Human subcutaneous adipose tissue was obtained by lipoaspiration. ADSC were isolated by collagenase digestion and differential centrifugation. Side population cells in ADSC were demonstrated using fluorescence-activated cell sorting after staining with Hoechst 33342. Differentiation to keratocyte phenotype was induced in fibrin gels or as pellet cultures with serum-free or reduced-serum media containing ascorbate. Keratocyte-specific gene expression was characterized using western blotting, quantitative RT-PCR, and immunostaining., Results: ADSC contained a side population and exhibited differentiation to adipocytes and chondrocytes indicating adult stem-cell potential. Culture of ADSC in fibrin gels or as pellets in reduced-serum medium with ascorbate and insulin induced expression of keratocan, keratan sulfate, and aldehyde dehydrogenase 3 family, member A1 (ALDH3A1), products highly expressed by differentiated keratocytes. Expression of differentiation markers was quantitatively similar to corneal stromal stem cells and occurred in both serum-free and serum containing media., Conclusions: ADSC cultured under keratocyte-differentiation conditions express corneal-specific matrix components. Expression of these unique keratocyte products suggests that ADSC can adopt a keratocyte phenotype and therefore have potential for use in corneal cell therapy and tissue engineering.

Purpose: Superoxide (O(2) (.-)) may function as a second messenger or regulator of signal transduction when produced at low concentrations in the proper locations within cells. The purpose of these studies was to determine whether human corneal stromal (HCS) fibroblasts are capable of producing O(2) (.-) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, a family of protein complexes believed to be responsible for the localized and limited production of O(2) (.-) with regulatory activity., Methods: HCS cells, grown as primary and low-passage cultures of fibroblasts, were used as the sources of RNA for reverse transcriptase PCR, with primers specific for mRNAs encoding the proteins that comprise NADPH oxidases. Small interfering (si)RNAs were used to knockdown specific NOX mRNAs. Proteins composing the NADPH oxidase complexes were identified using western blots. The production of O(2) (.-) by whole cells and cell-free preparations was assessed by measurement of NADPH-dependent superoxide dismutase-inhibitable cytochrome c reduction., Results: Whole cells and cell-free extracts of corneal stromal fibroblasts produced O(2) (.-) in an NADPH-dependent manner. These fibroblasts constitutively produced mRNAs encoding eight proteins known to comprise NADPH oxidase complexes. mRNAs encoding NOX1, NOX4, NOX5, p22 phox, p47 phox, p67 phox, and p40 phox as well as Rac were expressed. Treatment of HCS fibroblasts with siRNA pools specific for each of these three NOXs significantly reduced the steady state levels of the respective mRNAs. Western blots confirmed the existence of all the proteins required for O(2) (.-) production. Rac 1, a regulator of the activity of some forms of NADPH complexes was present in membranous cell fractions containing the oxidase proteins., Conclusions: HCS fibroblasts produced O(2) (.-) in a NADPH-dependent manner via at least three isoforms of NADPH oxidase. These cells expressed NOX1, NOX4, NOX5, p22 phox, p47 phox, p67 phox, and p40 phox as well as Rac. SiRNAs directed against each of the three putative isoforms of NOX significantly reduced the steady state levels of the appropriate NOX mRNA pools, thus confirming the existence of the three isoforms. The O(2) (.-) produced by the NADPH oxidases in HCS fibroblasts is a potential contributor to signal transduction pathways and a regulator of gene expression as well as a potential participant in processes that occur during inflammation.

Purpose: To evaluate the stability and biocompatibility of artificial corneal stroma that was prepared by using ultrahigh hydrostatic pressurization treatment to decellularize corneas., Methods: The porcine cornea was decellularized by two methods, a detergent method and an ultrahigh hydrostatic pressure (UHP) method. Either 1% w/v Triton X-100 or sodium dodecyl sulfate (SDS) was used for the detergent method, and 10,000 atmospheres (atm; 7.6x10(6) mmHg) was applied to the cornea for 10 min at 10 degrees C by a high-pressure machine for the UHP method. Hematoxylin-eosin staining was performed to confirm the removal of the corneal cells, and then decellularized porcine corneal stroma was implanted into rabbit corneal pockets. After eight weeks, the rabbit eyes were enucleated to examine the tissue compatibility of the implanted stroma., Results: Complete decellularization was confirmed only in corneas treated by the UHP method, and little inflammation was seen when they were implanted into the rabbit corneal pockets., Conclusions: Porcine corneal stroma completely decellularized by the UHP method has extremely high biocompatibility and is a possible corneal scaffold for an artificial cornea.

Purpose: Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1alpha (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor)., Methods: Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1alpha, and HIF-1alpha siRNA to test if HIF-1alpha activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1alpha and VEGF in transcriptional and translational levels., Results: During hypoxia (0.5% O2), 5 muM cadmium chloride completely inhibited HIF-1alpha expression and reversed the protection by hypoxia preconditioning. HIF-1alpha siRNA (15 nM) reduced HIF-1alpha expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1alpha dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning. VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning. However, the transcription and translation of VEGF were paradoxically increased by siHIF-1alpha, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1alpha., Conclusions: These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1alpha, but that VEGF is not a component of the protection.

Purpose: To examine the efficacy of a novel method of decellularizing porcine corneal stroma using N(2) gas from liquid N(2) and the feasibility of using decellularized porcine corneal stroma in a corneal transplantation model in rabbits., Methods: Porcine corneas were placed in a tube, and N(2) gas from liquid N(2) was poured into the tube to freeze the corneas and make the inside of the tube hypoxic. After fastening the cap firmly, the tube was kept at room temperature for seven days, and the porcine corneas were examined histologically. A porcine corneal stromal disk treated with the aforementioned method was inserted into a pocket of rabbit corneal stroma and observed for six months., Results: Hoechst 33342 and hematoxylin and eosin staining both showed few cellular nuclei in the porcine corneal stroma incubated in N(2) gas for one week. A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay showed many positively stained nuclei in the porcine corneal stroma incubated in N(2) gas for three days. The porcine corneal stroma that was decellularized and transplanted into a rabbit corneal stromal pocket remained clear for six months after transplantation., Conclusions: This method using N(2) gas decellularizes corneal stroma without reducing corneal transparency.

Purpose: Keratocytes, mesenchymal cells populating the corneal stroma, secrete the unique transparent connective tissue of the cornea as well as opaque scar tissue after injury. Previous studies identified factors mediating keratocyte phenotype in vitro, particularly the expression of the keratan sulfate proteoglycans, which are essential for vision. Whereas earlier work emphasized effects of cytokines, the current study examines the effects of substratum attachment on keratocyte phenotype., Methods: Primary keratocytes from collagenase digestion of bovine corneas were cultured on tissue-culture plastic or on poly (2-hydroxyethylmethacrylate)(polyHEMA)-coated, non-adhesive surfaces. Secreted proteoglycans from culture media and cell-associated proteins were characterized using western blotting or isotopic labeling. Gene expression was characterized with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Secreted matrix was examined with immunostaining., Results: We observed that virtually all primary keratocytes participate in the formation of spheroidal aggregates, remaining viable for at least four weeks in vitro. Spheroid keratocytes secrete more keratan sulfate and keratocan than attached cells in the same culture medium. In spheroids, keratocytes accumulate substantial matrix in intercellular spaces, including keratan sulfate, lumican, keratocan, and collagens V and VI. The unattached cells undergo limited cell division and do not differentiate into myofibroblasts in response to transforming growth factor beta (TGFbeta), which is based on the expression of extra domain A (EDA) fibronectin and alpha-smooth muscle actin. Similarly, the platelet derived growth factor, a cytokine initiating the fibroblastic phenotype in attached keratocytes, had a limited effect on the spheroid-associated keratocytes. Ascorbate-2-phosphate was the only agent stimulating keratan sulfate secretion in the spheroid keratocytes., Conclusions: These results provide a new paradigm for understanding signals that regulate extracellular matrix secretion. For primary keratocytes, the alteration of the cellular environment in terms of cell-cell and cell-matrix interactions mediates and can override signals from soluble cytokines in influencing matrix expression and also in adopting other aspects of the fibroblastic and myofibroblastic phenotypes found in healing wounds.

Purpose: To isolate multipotent precursors from the rabbit corneal stroma and to compare the distribution and proliferative capacity of keratocyte precursors obtained from the central and peripheral regions of the corneal stroma., Methods: The rabbit corneal stroma was divided into a peripheral region (6.0-10.0 mm in diameter) and a central region (6.0 mm in diameter). A sphere-forming assay was then performed to isolate precursors from the stroma of each region. To promote differentiation, isolated sphere colonies were plated in wells with a medium containing fetal bovine serum. Expression of various markers by the sphere colonies and their progeny was examined using immunocytochemistry and/or reverse-transcription polymerase chain reaction (RT-PCR)., Results: The rate of primary sphere formation by cells from the peripheral stroma (51.4+/-10.1/10,000 cells) was significantly higher than by cells from the central stroma (35.9+/-3.0/10,000 cells; p=0.00021). Secondary sphere formation rate was significantly higher in the peripheral stroma (45.6+/-6.4/10,000 cells) than in the central stroma (33.4+/-2.1/10,000 cells; p=0.00002). Cells from the spheres were positive for CD34 and nestin. Their progeny showed a keratocyte-like spindle shape and expressed vimentin, alpha-smooth muscle actin, and two neural differentiation markers (microtubule-associated protein-2 and neuron-specific enolase). Expression of nestin and vimentin was confirmed by RT-PCR., Conclusions: Our findings demonstrate that both the peripheral and central regions of the corneal stroma contain a significant number of precursors, but the peripheral stroma has more precursors with a stronger proliferative capacity than that of cells from the central stroma.

Purpose: To isolate fibroblast precursors from rabbit corneal stroma using a sphere-forming assay, to engineer corneal stroma with the precursors and gelatin, and to establish the therapeutic application of precursors in a rabbit corneal stroma., Methods: In the in vitro study, a sphere-forming assay was performed to produce precursors from rabbit corneal stroma. Corneal stroma was engineered by cultivating precursors in porous gelatin for one week. In the in vivo study, the engineered corneal stromal sheet with precursors (precursor/gelatin group) or with fibroblasts (fibroblast /gelatin group) or without cells (gelatin group) was transplanted to a pocket of rabbit corneal stroma. Gene expression and extracellular matrix production were examined immunohistochemically in each group one week and four weeks after surgery., Results: In the in vitro study, cells in the spheres were BrdU-positive, and their progeny were keratocan-positive. The study also showed that the corneas transplanted with a porous gelatin sheet did not show any opacity four weeks after transplantation in any group. In the gelatin sheet of the precursor/gelatin group, a more intense expression of type I collagen was observed relative to the other two groups four weeks after the surgery., Conclusions: Our findings demonstrate that the transplantation of fibroblast precursors combined with gelatin hydrogel into the corneal stroma is a possible treatment strategy for corneal stromal regeneration.

Purpose: Corneal myofibroblasts differentiated from activated corneal stromal cells are the major cellular sources of extracellular matrix synthesis for the repair of corneal injury. In this study, the effects of histone deacetylase (HDAC) inhibitors on the activation, proliferation, migration and senescence of corneal stromal cells were evaluated., Methods: Primary human and mouse corneal stromal cells were harvested by sequential digestion with dispase and collagenase, and cultured in DMEM/F-12 media under serum-free (keratocytes), serum- (corneal fibroblasts) and TGFbeta1-supplemented (corneal myofibroblasts) conditions. The responses of corneal stromal cells to HDAC inhibitors were characterized by cDNA microarray, real time PCR, immunocytochemistry and western blot analysis. The effects of HDAC inhibitors on corneal fibroblast proliferation, cell cycle distribution, migration and senescence were also assessed in vitro., Results: Fetal bovine serum and TGFbeta1 activated the transdifferentiation of corneal stromal cells into fibroblasts and myofibroblasts, indicated by cell spreading, renewed assembly of actin filaments and enhanced expression of extracellular matrix components, all of which were suppressed by the addition of HDAC inhibitors. HDAC inhibitors inhibited the proliferation of corneal fibroblasts by decreasing the proportion in the S-phase and increasing the proportion in the G0/G1 and G2/M cell cycle checkpoints. HDAC inhibitors showed a dose-dependent inhibitory effects on the migration of corneal fibroblasts. In addition, HDAC inhibitors induced the senescence of corneal myofibroblasts as shown by enhanced staining of beta-galactosidase and upregulated expression of p16(ink4a)., Conclusions: HDAC inhibitors may affect corneal stromal cells by inhibiting myofibroblastic differentiation, cell proliferation, migration and by inducing cell senescence. Thus, this has implications for future studies in the development of promising drugs in the prevention or treatment of corneal haze and scar formation.

Purpose: The purpose of this investigation was to elucidate the regulation of corneal extracellular superoxide dismutase (SOD3) synthesis in keratoconus. We compared the basal and cytokine-regulated SOD3 synthesis in cultured human stromal cells from keratoconus corneas to stromal cells from normal and bullous keratopathy corneas., Methods: Keratocyte cultures were obtained from patients undergoing corneal transplantation for keratoconus and bullous keratopathy, and from healthy donor corneas. The cell lines obtained were cultured until near confluence and interleukin-1alpha, interleukin-6, transforming growth factor beta, or platelet derived growth factor were added to the media. The phenotypes of the cultured cells were assessed by immunocytochemical expression of alpha-smooth muscle actin and CD34. SOD3 protein contents were determined in the culture media with ELISA after 24, 48, 72, and 96 h., Results: Interleukin-1alpha had an inhibitory effect on SOD3 synthesis exclusively in the keratoconus cultures (p<0.01). Platelet derived growth factor induced a reduction in SOD3 synthesis in all groups (p<0.05)., Conclusions: Here, we demonstrate that cultured keratoconus stromal cells respond with a reduced SOD3 synthesis to interleukin-1alpha, which is not the case in corresponding normal or bullous keratopathy cells. Since interleukin-1alpha is upregulated in corneal trauma and inflammation, keratoconus corneas may muster an insufficient oxidative defense under such conditions.

Purpose: To study the phenotypes, distribution, and morphologies of different antigen-presenting cells (APCs) in the murine cornea., Methods: Intravitreal injection of fluorescently tagged ovalbumin (OVA) or antibodies to MHC-II (I-A(d)), F4/80, CD11c, B7-1, and B7-2 was performed to label cells in the murine cornea. Light and transmission electron microscopy were used to examine corneal histology. Intravital microscopy, epifluorescence microscopy, and confocal microscopy were used to evaluate the labeled cells. In vitro staining was performed to validate the in vivo staining and localize the labeled cells. Three-dimensional rotatable images were taken to evaluate relationships between two differently labeled cells., Results: Histological examination revealed no observable change in the cornea following intravitreal injection. In vivo staining showed that OVA+ cells and cells positive for MHC-II, F4/80, CD11c, B7-1, or B7-2 were noted throughout the cornea with a decreasing density from limbus toward the central cornea. Two populations with distinct morphological features were identified among these APCs. Labeled cells were found beneath the epithelium or in the shallow stroma in the central and paracentral cornea, but in all layers in the peripheral cornea. A number of F4/80+ and CD11c+ cells were also positive for OVA, MHC-II, B7-1, or B7-2. Rotatable images showed a close contact between two differently labeled cells., Conclusions: Intravitreal injection of labeled antibodies can be adapted to visualize labeled cells in the cornea. APCs with distinct morphologies, phenotypes, and distribution may contribute to the immunologically privileged feature of the cornea.

Purpose: Maintenance of avascularity of the normal cornea and control of neovascularization during wound healing depend on a balance of angiogenic and antiangiogenic factors. The purpose of this paper is to determine the ability of corneal cells to convert plasminogen to angiostatins and to compare these products with those made by intact corneas., Methods: RT-PCR was performed using plasminogen specific primers and the generated cDNA was sequenced. The proteins in corneal extracts, cornea conditioned medium, and medium from corneal epithelial cells, stromal fibroblasts, and myofibroblasts incubated with plasminogen were separated by SDS-PAGE and electroblotted. Western blots used monoclonal antibodies to kringles 1-3 to detect plasminogen and angiostatins. Angiostatins were isolated and tested for activity in a vascular endothelial cell proliferation inhibition assay., Results: Plasminogen, its mRNA and angiostatins were found in human corneal tissue extracts from the epithelial, stromal, and endothelial layers and from cornea conditioned medium, but not in medium from cultured epithelial cells, stromal fibroblasts, or myofibroblasts. However, cultures of corneal epithelial cells and stromal fibroblasts were able to convert exogenously added plasminogen to angiostatins, whereas cultured myofibroblasts did not. Angiostatins of 38 and 34 kDa were found under all angiostatin generating conditions; however other angiostatins differed in size. Further, the angiostatins isolated from fibroblast culture supernatants inhibited vascular endothelial cell proliferation., Conclusions: Conversion of plasminogen to angiostatin is cell-type dependent. Because corneal cells generate angiostatins, use of human angiostatins may be a means of treating abnormal corneal neovascularization without the risk of side effects.

Purpose: Stromal vascularization is a frequent occurrence in herpes simplex keratitis (HSK) and carries a poor prognosis for penetrating keratoplasty. The pathogenesis may involve disruption of the normal equilibrium between angiogenic and anti-angiogenic factors in and around the cornea. Thrombospondin (TSP) 1 and 2 are multifunctional matricellular glycoproteins with potent anti-angiogenic properties and are expressed by human keratocytes in a stromal wound repair model. We hypothesize that the synthesis of these anti-angiogenic proteins by keratocytes is inhibited by HSV1 and that such a mechanism may contribute to stromal vascularization in HSK., Methods: Nonconfluent monolayers of human keratocytes were infected with HSV1 at a multiplicity of infection of 5 virus particles/keratocyte. Expression of TSP1 and TSP2 was determined by immunohistochemistry and SDS-polyacrylamide gel electrophoresis at 0, 2, 4, 6, 8, 24, 48, and 72 h after infection (ai). Expression of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) served as a control. Expression of immediate early and late viral proteins was also determined. Protein expression was quantified by densitometric analysis of the immunoblot bands., Results: Human keratocytes supported the growth of HSV1 at all times ai. TSP1 and TSP2 were downregulated as early as 4 h ai to a 50% reduction by 8 h (p<0.002), and were absent from 24 h ai (p<0.001). There was no change in the level of expression of GAPDH throughout the duration of the experiment. Immediate early viral proteins (HSV1:ICP27) could be detected from 6 h ai reaching maximum intensity 24 h ai and late proteins (HSV:1gD) were expressed from 24 h., Conclusions: The synthesis of TSP1 and TSP2 is selectively downregulated by HSV1 infection in human keratocytes. Addition of these proteins or their angio-active peptides in early stage HSK therapy may be an important adjuvant in controlling HSV1 induced corneal vascularization.

Purpose: Keratoconus is a non-inflammatory thinning disorder of the corneal stroma. Recently, we showed that these corneas contain inducible nitric oxide synthase and an accumulation of nitrotyrosine, representing oxidative damage from peroxynitrite. Previously, we suggested that keratoconus corneas and their cell cultures have alterations in a gelatinase system with increased matrix metalloproteinase-2 (MMP-2) activity and decreased tissue inhibitor of metalloproteinase-1 (TIMP-1). This study examines whether a peroxynitrite donor (3-morpholinosydomine N-ethylcarbamide, SIN-1) or nitric oxide donor (S-nitroso-N-acetylpenicillamine, SNAP) could alter TIMP-1 and/or MMP-2 in vitro., Methods: Normal stromal fibroblasts were cultured in the presence or absence of either SIN-1 or SNAP for varying times. These cultures were analyzed by western and northern blot analyses, gelatin zymography, and a quantitative gelatinase/MMP assay., Results: In vitro, SIN-1 treatment led to protein nitration, increased RNA levels of TIMP-1 and MMP-2, and loss of TIMP-1 immunostaining, but did not diminish gelatinase activity. SNAP treatment led to activation of MMP-2 and significantly increased gelatinase/MMP activity, without a change in TIMP-1 levels., Conclusions: Our data show that peroxynitrite or nitric oxide can decrease TIMP-1 and increase gelatinase activity, respectively. This demonstrates a relationship between elements of oxidative stress and tissue degradation in human corneal fibroblasts. This effect may play a significant role in the stromal thinning that occurs in keratoconus.

Purpose: The human mimecan/osteoglycin promoter contains multiple interferon-stimulated response elements (ISRE) and interferon-gamma-activation sites (GAS). ISRE and GAS motifs are present in a variety of interferon (IFN)-inducible genes. The purpose of this study was to investigate whether IFN-gamma affects mimecan gene expression and, if so, to determine the cis-elements and transcription factors that mediate its action., Methods: Electrophoretic mobility shift assay (EMSA) was used to investigate whether nuclear proteins from IFN-gamma-treated cells bind to regions of the human mimecan promoter containing ISRE sites. Incubation of nuclear extracts with specific antibodies was used to identify transcription factors that bind to these sites. Transcriptional activity of the promoter was evaluated by transient transfections of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Co-transfection experiments were used to study the role of transcription factors that bind ISRE elements in the promoter and mediate the IFN-gamma response. Expression of mRNAs was analyzed by reverse transcription-polymerase chain reaction., Results: Using probes that correspond to two ISRE sites located in the first intron of the human mimecan gene, we detected specific DNA-protein complexes with nuclear extracts from IFN-gamma-treated cells. Formation of DNA-protein complexes was abrogated by competition with unlabeled probe and one of the complexes was supershifted by the anti-interferon regulatory factor-1 (IRF-1) antibody. Interestingly, when probe that corresponds to a conserved E-box (CACATG) in the proximal promoter and nuclear extracts from IFN-gamma-treated cells were used in EMSA, increased binding of upstream stimulatory factor-1 (USF-1) was observed. Co-transfection of a mimecan promoter construct that contained the entire first intron with IRF-1, or with both IRF-1 and USF-1 expression plasmids, suppressed luciferase activity of the promoter in corneal keratocytes and T-47D cells. In contrast, co-transfection experiments with IRF-2, or with both IRF-2 and USF-1, led to increased luciferase activity of the same promoter construct. RT-PCR analyses demonstrate that IFN-gamma rapidly and transiently suppresses mimecan expression and induces IRF-1 and IRF-2 mRNAs in bovine corneal keratocytes., Conclusions: IRF-1 binds to ISRE sites, located in the first intron of the human mimecan gene, and negatively regulates mimecan expression at the level of transcription. Consistent with these observations, an inverse correlation between the expression of mimecan and IRF-1 in bovine corneal keratocytes and non-corneal cells was demonstrated. IRF-2 positively regulates mimecan transcription in corneal keratocytes. Because the intact E-box in the proximal promoter was required for IRF-1 and IRF-2 effects on mimecan transcription, potential direct or indirect interactions between USF-1 and IRF-1 and IRF-2 are likely.

Purpose: To determine whether human keratocytes synthesize thrombospondins 2 and 3 (TSP-2, TSP-3) in a collagen matrix and the effect of addition of antibodies to TSP-2 and TSP-3 on keratocyte-populated collagen matrix contraction., Methods: Keratocyte-populated collagen matrices were evaluated for TSP-2 and TSP-3 mRNA. Sections of matrices were stained by immunohistochemistry for TSP-2 and TSP-3. Keratocyte populated collagen matrices were treated with antibodies specific for TSP-2 and TSP-3 and these preparations were evaluated for contraction and keratocyte morphology., Results: Keratocyte derived fibroblasts in collagen matrices contained TSP-2 and TSP-3 mRNA, and the cells were immunoreactive for both proteins. Compared to controls, an antibody specific to the N-terminal domain of TSP-2 significantly inhibited matrix contraction for up to 10 days at a concentration of 20 microg/ml antibody. At 2 microg/ml TSP-2 antibody concentration significant inhibition occurred up to 3 days. Removal of the antibody from the media reversed the inhibitory effect. Cultured keratocytes in TSP-2 treated collagen matrices appeared more rounded than keratocytes in control matrices. An antibody specific to TSP-3 had no effect on matrix contraction or keratocyte morphology., Conclusions: Keratocyte derived fibroblasts synthesize TSP-2 and TSP-3 when seeded in collagen matrices. Antibody specific to TSP-2 reversibly inhibits matrix contraction. TSP-2 may play a key role in keratocyte/collagen matrix interactions, as may occur during corneal stromal repair.

Purpose: A major environmental stress encountered by humans is solar UV light, which can cause a spectrum of eye diseases, such as photokeratitis, cataract, pterygia, and ocular neoplasms. Mammalian defense mechanisms in response to adverse effects of UV light result in induction of a number of genes. Studies on the transcriptional regulation of genes that are expressed in the eye will increase understanding of both the physiological functions of these genes in the mammalian UV response, and the molecular bases for abnormalities associated with the above diseases. Mimecan is an extracellular matrix proteoglycan that is abundantly expressed in the cornea. The purpose of this study was to determine and characterize the UV responsive regulatory elements of the human mimecan promoter., Methods: Transcriptional activity of the promoter was evaluated, before and after UV irradiation, using transient transfection of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Site directed mutagenesis and corresponding functional assays were used to determine the contribution of UV responsive regions to human mimecan transcription. Co-transfection experiments were used to investigate the role of transcription factors that bind these elements in the promoter and mediate the UV response. mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR)., Results: The shortest promoter construct that was strongly activated following UV irradiation contained three initiator elements, an E-box element that is conserved between species, and the entire first intron of the human mimecan gene. Deletion of the intronic p53 binding site from this construct considerably diminished transcription and the UV response of the promoter. Surprisingly, deletion of the E-box sequence from this construct completely abolished both transcription and UV response of the promoter. These results demonstrated that the E-box is essential to transcription of the human mimecan gene and also is required for activation by p53. The role of the E-box, and the E-box binding protein, USF-1, in transcription and UV responses of the human mimecan promoter were confirmed by co-transfection experiments using dominant negative transcription factor, A-USF. In addition to these positive regulators, we demonstrate that the region between nucleotides -1314 and -1907 contains a transcriptional repressor site that is active in a time dependent manner following UV irradiation. Finally, we show that UV irradiation results in changes in mimecan mRNA levels in bovine corneal keratocytes in a time-dependent manner., Conclusions: The human mimecan promoter contains several UV responsive regulatory elements that are conserved between human and bovine species and include the intronic p53 DNA binding site, the E-box in the proximal promoter, and the region between nucleotides -1314 and -1907. The E-box plays an important role in transcription and UV response of the human mimecan promoter. UV irradiation modulates expression of mimecan mRNA in bovine corneal keratocytes and non-corneal cells.

Purpose: Procollagen I is synthesized and intracellularly degraded in corneal endothelial cells (CEC), whereas type IV and VIII collagens are secreted into Descemet's membrane. In our previous study, we demonstrated that procollagen I synthesized by CEC is improperly folded and that the molecule was largely colocalized with protein disulfide isomerase (PDI) within the endoplasmic reticulum (ER). In the present study, we further investigated whether the alpha-subunit of prolyl 4-hydroxylase (P4Halpha) and glucose regulatory protein/immunoglobulin heavy chain binding protein (Grp78/BiP) were also involved in ER retention of procollagen I in CEC., Methods: Immunocytochemical analysis was performed to determine the colocalization of procollagen I with molecular chaprones. Protein synthesis was measured by immunoblot analysis and the association between proteins was determined by coimmunoprecipitation followed by immunoblot analysis. mRNA was quantitated using RT-PCR., Results: To study the interaction of procollagen I with certain molecular chaperones involved in the collagen biosynthetic pathway, we determined whether procollagen I colocalized with P4Halpha and Grp78/Bip, and then compared this molecular chaperone colocalization with their association with type IV collagen. Procollagen I was colocalized with either P4Halpha or Grp78/Bip to a much lesser degree than type IV collagen was colocalized with these same ER proteins. Colocalization between the molecular chaperones demonstrated that P4Halpha and Grp78/Bip were largely colocalized in the peripheral region of the ER, whereas colocalization of P4Halpha and PDI was mostly limited to a small region of the ER. When cells were treated with alpha,alpha-dipyridyl, the inhibitor did not affect the colocalization profiles of collagens with the molecular chaperones. However, the inhibitor markedly increased colocalization of P4Halpha and PDI, but it significantly decreased colocalization between P4Halpha and Grp78/Bip. When synthesis of the molecular chaperones was compared between CEC and corneal stromal fibroblasts (CSF), more Grp78/Bip and PDI were produced by CEC than by CSF. On the other hand, expression of Hsp47 was lower in CEC than it was in CSF. Coimmunoprecipitation was used to compare the association of P4Halpha or Grp78/Bip with collagens in CEC and CSF. The association of collagens (regardless of type) with P4Halpha or Grp78/Bip was much higher in CEC than in CSF. When the association of collagen molecules with respective molecular chaperones was compared in CEC, the degree of association between Grp78/Bip and procollagen I was similar to that between the molecular chaperone and type IV collagen. On the other hand, the degree of association between P4Halpha and type IV collagen was much higher than that between P4Halpha and procollagen I., Conclusions: These data suggest that procollagen I and type IV collagen may use different molecular chaperones in the ER, thus targeting their distinctive destinations.

Purpose: Immortalized cell lines representing fibroblast cells from corneal stroma would facilitate studies of corneal cell biology and injury response., Methods: Primary cultures of cells derived from mouse corneal stroma were transfected with a human telomerase reverse transcriptase (hTERT) expression construct to maximize chances of cellular immortalization. A resulting cell line was analyzed for telomerase activity, cell growth characteristics, senescence and gene expression patterns. Specific responses to transforming growth factor beta (TGF-beta) were also analyzed., Results: An immortalized cell line was derived and was named MK/T-1. MK/T-1 cells show no signs of cellular senescence or transformation at over 100 passages. Telomerase activity was significantly higher in MK/T-1 cells as compared to the parental cell cultures. However, relative telomere length (RTL) in the MK/T-1 and parental cells was not significantly different. Senescence associated beta-galactosidase (SA-beta-Gal) activity was not detected in late passage MK/T-1 cells while the parental cells had already upregulated SA-beta-Gal at high levels by passage 9. The MK/T-1 cells express vimentin, tubulin, lumican, mimecan, decorin and collagen I, but not keratocan. Exposure of the MK/T-1 cells to TGF-beta induces the expression of smooth muscle alpha-actin (ASMA), the activation of MAP Kinase (p38-MAPK) and morphological changes consistent with cytoskeletal reorganization., Conclusions: MK/T-1 cells represent an immortalized fibroblast cell line derived using cultures from corneal stroma cell preparations. Expression of hTERT may contribute to immortalization of the MK/T-1 cells by a mechanism other than increases in RTL. MK/T-1 cells may be a useful model in which to study the responses of corneal fibroblast cells to cytokines and other diverse environmental factors in vitro.