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1.  Ethyl Pyruvate Ameliorates Endotoxin-Induced Corneal Inflammation 
The purpose of this study was to evaluate the anti-inflammatory effect of ethyl pyruvate (EP) in a mouse model of lipopolysaccharide (LPS)-induced corneal inflammation.
LPS was injected intrastromally into the corneas of C57BL/6 mice followed by treatment with a solution of 2.5% EP in 0.2% hydroxypropyl methylcellulose (HPMC) every 90 minutes during the course of 12 hours. Prednisolone acetate 1% solution (PRED FORTE) was used as a positive control. Mice were sacrificed after 3 days, and corneas were examined by in vivo confocal microscopy and analyzed for infiltrated cells by flow cytometry. Gr-1, TNF-α, and pNF-κB-p65 were detected immunohistochemically, and TNF-α, IL-6, and IL-1β levels were quantified by ELISA.
LPS-induced haze in mice corneas was decreased by 2-fold upon EP treatment; however, it was not changed upon PRED FORTE treatment. Flow cytometry and immunohistochemistry showed infiltration of leukocytes in the LPS-treated corneas; among the infiltrated cells, neutrophils (Gr-1+ and CD11b+) and macrophages (F4/80+ and CD11b+) were 3403.4- and 4.5-fold higher in number, respectively, than in vehicle-treated control corneas. EP or PRED FORTE treatment of LPS-injected corneas decreased the number of neutrophils 7.5- and 7.2-fold and macrophages by 5.6- and 3.5-fold, respectively. Both EP and PRED FORTE decreased TNF-α and IL-6 expression considerably, and to a lesser extent IL-1β expression, in the LPS-treated corneas.
The present study demonstrated that EP reduces LPS-induced inflammation in the cornea and thus may have a potential therapeutic application in the inhibition of corneal inflammation.
The anti-inflammatory effect of ethyl pyruvate (EP) in a mouse model of lipopolysaccharide (LPS)-induced corneal inflammation was evaluated. EP reduced LPS-induced infiltration of inflammatory cells and cytokine levels; thus it has a potential therapeutic use in the inhibition of corneal inflammation. The present study demonstrated that EP reduces LPS-induced inflammation in the cornea in mice as effectively as prednisolone (PRED FORTE). Therefore, EP may have a potential therapeutic application as a nonsteroidal anti-inflammatory chemotherapeutic agent.
PMCID: PMC3460388  PMID: 22918642
2.  Optical Coherence Tomography as a Rapid, Accurate, Noncontact Method of Visualizing the Palisades of Vogt 
OCT is able to safely, rapidly, and effectively image the palisades of Vogt without direct contact to the eye. This ability will greatly enhance our understanding of this stem cell niche and will allow development of new clinical and research techniques.
This study explored the efficacy of optical coherence tomography (OCT) as a high-resolution, noncontact method for imaging the palisades of Vogt by correlating OCT and confocal microscopy images.
Human limbal rims were acquired and imaged with OCT and confocal microscopy. The area of the epithelial basement membrane in each of these sets was digitally reconstructed, and the models were compared.
OCT identified the palisades within the limbus and exhibited excellent structural correlation with immunostained tissue imaged by confocal microscopy.
OCT successfully identified the limbal palisades of Vogt that constitute the corneal epithelial stem cell niche. These findings offer the exciting potential to characterize the architecture of the palisades in vivo, to harvest stem cells for transplantation more accurately, to track palisade structure for better diagnosis, follow-up and staging of treatment, and to assess and intervene in the progression of stem cell depletion by monitoring changes in the structure of the palisades.
PMCID: PMC3339911  PMID: 22266521
3.  FGF-2– and TGF-β1–Induced Downregulation of Lumican and Keratocan in Activated Corneal Keratocytes by JNK Signaling Pathway 
Keratan sulfate proteoglycans (KSPGs) are important for corneal stromal transparency. This study shows that JNK signaling downregulates KSPGs in TGF-β1– and FGF-2–activated corneal keratocytes, which is known to occur during corneal wound healing.
Downregulation of lumican and keratocan expression is an undesirable phenotypic change that occurs during corneal wound healing. The present study was intended to determine whether the activation of Jun N-terminal kinase (JNK)-signaling pathway is involved in their downregulation in TGF-β1– and FGF-2–activated keratocytes.
Keratocytes, isolated from rabbit corneal stroma, and cultured in a serum-free medium, pretreated or not treated with JNK inhibitor (SP600125), were activated with FGF-2/heparin sulfate (HS) or TGF-β1 in the presence or absence of SP600125. In another set of experiments, keratocytes were transfected with JNK1/2 Dicer-substrate RNA (DsiRNA) and then activated with TGF-β1 or FGF-2/HS. Specific phenotypic changes were analyzed immunocytochemically and correlated with Western blot analyses. The relative levels of specific mRNAs were estimated by quantitative RT-PCR using specific reagents.
The FGF-2/HS– or TGF-β–induced activation of corneal stromal keratocytes to fibroblast- or myofibroblast-phenotype, respectively, resulted in marked decreases in cell surfaceassociated and secreted keratan sulfate proteoglycans (KSPGs). Both keratocan and lumican proteins and their mRNAs were downregulated in the activated keratocytes. However, JNK inhibition during the activation of keratocytes, pretreated with the JNK inhibitor, suppressed the reduction in the cell–surface associated and secreted KSPGs (lumican and keratocan), and their mRNA transcripts. Downregulation of total KSPGs and their mRNAs was also inhibited by decreasing JNK1 and JNK2 levels via JNK1/2 DsiRNA transfection of keratocytes before their activation.
Extrapolating from the present study, FGF-2– and TGF-β1–activation of JNK signaling pathway may be partly responsible for the downregulation of keratocan and lumican expression in activated corneal keratocytes observed during corneal stromal wound healing.
PMCID: PMC3231797  PMID: 22025571
4.  Initial In Vitro Investigation of the Human Immune Response to Corneal Cells from Genetically Engineered Pigs 
This study is the first to demonstrate the humoral and cellular immune response in humans to corneal endothelial cells from genetically engineered pigs. The results suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.
To compare the in vitro human humoral and cellular immune responses to wild-type (WT) pig corneal endothelial cells (pCECs) with those to pig aortic endothelial cells (pAECs). These responses were further compared with CECs from genetically engineered pigs (α1,3-galactosyltransferase gene-knockout [GTKO] pigs and pigs expressing a human complement-regulatory protein [CD46]) and human donors.
The expression of Galα1,3Gal (Gal), swine leukocyte antigen (SLA) class I and class II on pCECs and pAECs, with or without activation by porcine IFN-γ, was tested by flow cytometry. Pooled human serum was used to measure IgM/IgG binding to and complement-dependent cytotoxicity (CDC) to cells from WT, GTKO, and GTKO/CD46 pigs. The human CD4+ T-cell response to cells from WT, GTKO, GTKO/CD46 pigs and human was tested by mixed lymphocyte reaction (MLR).
There was a lower level of expression of the Gal antigen and of SLA class I and II on the WT pCECs than on the WT pAECs, resulting in less antibody binding and reduced human CD4+ T-cell proliferation. However, lysis of the WT pCECs was equivalent to that of the pAECs, suggesting more susceptibility to injury. There were significantly weaker humoral and cellular responses to the pCECs from GTKO/CD46 pigs compared with the WT pCECs, although the cellular response to the GTKO/CD46 pCECs was greater than to the human CECs.
These data provide the first report of in vitro investigations of CECs from genetically engineered pigs and suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.
PMCID: PMC3176056  PMID: 21596821
5.  Responses of Cultured Human Keratocytes and Myofibroblasts to Ethyl Pyruvate: A Microarray Analysis of Gene Expression 
During corneal stromal wound healing, quiescent stromal keratocytes are activated to myofibroblasts with changed phenotypic characteristics, which are responsible for the formation of nontransparent scar tissue. The present study demonstrates that ethyl pyruvate (EP) modulates phenotypic changes during TGF-β1–induced activation of keratocytes to myofibroblasts in vitro, and EP may be a potential therapeutic agent in corneal wound healing.
Ethyl pyruvate (EP) has pharmacologic effects that remediate cellular stress. In the organ-cultured murine lens, EP ameliorates oxidative stress, and in a rat cataract model, it attenuates cataract formation. However, corneal responses to EP have not been elucidated. In this study, the potential of EP as a therapeutic agent in corneal wound healing was determined by examining its effects on the transition of quiescent corneal stromal keratocytes into contractile myofibroblasts.
Three independent preparations of cultured human keratocytes were treated with TGF-β1, to elicit a phenotypic transition to myofibroblasts in the presence or absence of 10 or 15 mM EP. Gene expression profiles of the 12 samples (keratocytes ± EP ± TGF-β1 for three preparations) were produced by using gene microarrays.
TGF-β1–driven twofold changes in at least two of three experiments defined a group of 1961 genes. Genes showing twofold modulation by EP in at least two experiments appeared exclusively in myofibroblasts (857 genes), exclusively in keratocytes (409 genes), or in both phenotypes (252 genes). Analysis of these three EP-modulated groups showed that EP (1) inhibited myofibroblast proliferation with concomitant modulation of some cell cycle genes, (2) augmented the NRF2-mediated antioxidant response in both keratocytes and myofibroblasts, and (3) modified the TGF-β1–driven transition of keratocytes to myofibroblasts by inhibiting the upregulation of a subset of profibrotic genes.
These EP-induced phenotypic changes in myofibroblasts indicate the potential of EP as a therapeutic agent in corneal wound healing.
PMCID: PMC2891457  PMID: 20053976
6.  PAX6 expression identifies progenitor cells for corneal keratocytes 
Keratocytes of the corneal stroma produce a transparent extracellular matrix required for vision. During wound-healing and in vitro, keratocytes proliferate, becoming fibroblastic, and lose biosynthesis of unique corneal matrix components. This study sought identification of cells in the corneal stroma capable of assuming a keratocyte phenotype after extensive proliferation. About 3% of freshly isolated bovine stromal cells exhibited clonal growth. In low-mitogen media, selected clonal cultures displayed dendritic morphology and expressed high levels of keratan sulfate, aldehyde dehydrogenase 3A1, and keratocan, molecular markers of keratocyte phenotype. In protein-free media, both primary keratocytes and selected clonal cells aggregated to form attachment-independent spheroids expressing elevated levels of those marker molecules. The selected clonal cells exhibited normal karyotype and underwent replicative senescence after 65–70 population doublings; however, they continued expression of keratocyte phenotypic markers throughout their replicative life span. The progenitor cells expressed elevated mRNA for several genes characteristic of stem cells and also for genes expressed during ocular development PAX6, Six2, and Six3. PAX6 protein was detected in the cultured progenitor cells and a small number of stromal cells in intact tissue but was absent in cultured keratocytes and fibroblasts. Cytometry demonstrated PAX6 protein in 4% of freshly isolated stromal cells. These results demonstrate the presence of a previously unrecognized population of PAX6-positive cells in adult corneal stroma that maintain the potential to assume a keratocyte phenotype even after extensive replication. The presence of such progenitor cells has implications for corneal biology and for cell-based therapies targeting corneal scarring.
PMCID: PMC2876310  PMID: 15901670
cornea; keratan sulfate; stem cell
7.  Secretion and Organization of a Cornea-like Tissue In Vitro by Stem Cells from Human Corneal Stroma 
To investigate the potential of human corneal stromal stem cells to assume a keratocyte phenotype and to organize extracellular matrix (ECM) in vitro similar to corneal stromal tissue.
Human corneal stromal stem cells (hCSSC) were isolated as side population cells by flow cytometry. Cloned hCSSC were cultured as free-floating pellets in serum-free media for 3 weeks. Gene expression was examined using gene array, quantitative RT-PCR, immunostaining, and immunoblotting. Transmission electron microscopy showed collagen fibril size and alignment.
Pellet cultures of hCSSC in serum-free media upregulated the expression of keratocyte-specific genes and secreted substantial ECM containing characteristic stromal components: keratocan, keratan sulfate, collagen I, collagen V, and collagen VI. Abundant connexin 43 and cadherin 11 in pellets demonstrated cell-cell junctions typical of keratocytes in vivo. Electron microscopy of the pellet cultures revealed abundant fibrillar collagen, some of which was aligned in parallel arrays similar to those of stromal lamellae. Gene array identified expression in pellets of several genes highly expressed by keratocytes. Transcripts for these keratocyte genes—FLJ30046, KERA, ALDH3A1, CXADR, PTGDS, PDK4, MTAC2D1, F13A1—were increased by as much as 100-fold in pellets compared with hCSSC. Simultaneously, expression of stem cell genes BMI1, KIT, NOTCH1, SIX2, PAX6, ABCG2, SPAG10, and OSIL was reduced by a similar factor in pellets compared with hCSSC.
Scaffolding-free pellet culture of hCSSC induces keratocyte gene expression patterns in these cells and secretion of an organized stroma-like ECM. These cells offer a novel potential for corneal bioengineering.
PMCID: PMC2874676  PMID: 17962455
8.  Rho GTPase and cAMP/Protein Kinase A Signaling Mediates Myocilin-induced Alterations in Cultured Human Trabecular Meshwork Cells* 
The Journal of biological chemistry  2007;283(1):603-612.
Myocilin is a gene linked to the most common form of glaucoma, a major blinding disease. The trabecular meshwork (TM), a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The myocilin expression is known to be up-regulated by glucocorticoids inTMcells, and an altered myocilin level may be the culprit in conditions such as corticosteroid glaucoma. Wild type myocilin, when transfected into cultured human TM cells, induced a dramatic loss of actin stress fibers and focal adhesions. Myocilin transfectants displayed a heightened sensitivity to trypsin. Adhesion to fibronectin, collagens, and vitronectin was compromised. The fibronectin deposition and the levels of fibronectin protein and mRNA were also reduced in myocilin transfectants. The fibronectin deposition could be restored by treatment with lysophosphatidic acid, a Rho stimulator. Assays further revealed that upon myocilin overexpression, the activity of RhoA was diminished, whereas the cAMP level and the protein kinase A (PKA) activity were augmented. Myocilin protein did not affect actin polymerization. The collapse of actin stress fibers and increased trypsin sensitivity from myocilin transfection could be reverted by co-expression of constitutively active RhoA or by treatment with PKA inhibitor H-89. The PKA activity, however, was not modified by co-expression of either constitutively active or dominant negative RhoA. These results demonstrate that myocilin has a deadhesive activity and triggers signaling events. cAMP/PKA activation and the downstream Rho inhibition are possible mechanisms by which myocilin in overabundance may lead to TM cell or tissue damage.
PMCID: PMC2729092  PMID: 17984096
9.  Loss of Alpha3(IV) Collagen Expression Associated with Corneal Keratocyte Activation 
To determine whether changes in the expression of type IV α1, α2, or α3 collagen isoforms are stringently associated with corneal stromal cell activation.
Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-β1-, or fetal bovine serum (FBS)-supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents.
Immunohistochemical analysis indicated that type IV α1, α2, and α3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, α3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-β1. α3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-β1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels.
The loss in the expression of α3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-β1, and it accompanies the downregulation of keratocan expression.
PMCID: PMC2714546  PMID: 17251459
10.  Compositional Differences between Infant and Adult Human Corneal Basement Membranes 
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. In the infant DM, type IV collagen α1-α6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
The distribution of laminin γ3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
PMCID: PMC2151758  PMID: 17962449
11.  Multipotent Stem Cells in Human Corneal Stroma 
Stem cells (Dayton, Ohio)  2005;23(9):1266-1275.
Keratocytes of the corneal stroma secrete a specialized extracellular matrix essential for vision. These quiescent cells exhibit limited capacity for self-renewal and after cell division become fibroblastic, secreting nontransparent tissue. This study sought to identify progenitor cells for human keratocytes. Near the corneal limbus, stromal cells expressed ABCG2, a protein present in many adult stem cells. The ABCG2-expressing cell population was isolated as a side population (SP) by cell sorting after exposure to Hoechst 33342 dye. The SP cells exhibited clonal growth and continued to express ABCG2 and also PAX6, product of a homeobox gene not expressed in adult keratocytes. Cloned SP cells cultured in medium with fibroblast growth factor-2 lost ABCG2 and PAX6 expression and upregulated several molecular markers of keratocytes, including keratocan, aldehyde dehydrogenase 3A1, and keratan sulfate. Cloned corneal SP cells under chondrogenic conditions produced matrix staining with toluidine blue and expressed cartilage-specific markers: collagen II, cartilage oligomatrix protein, and aggrecan. Exposure of cloned SP cells to neurogenic culture medium upregulated mRNA and protein for glial fibrillary acidic protein, neuro filament protein, and beta-tubulin II. These results demonstrate the presence of a population of cells in the human corneal stroma expressing stem cell markers and exhibiting multipotent differentiation potential. These appear to be the first human cells identified with keratocyte progenitor potential. Further analysis of these cells will aid elucidation of molecular mechanisms of corneal development, differentiation, and wound healing. These cells may be a resource for bioengineering of corneal stroma and for cell-based therapeutics.
PMCID: PMC1941788  PMID: 16051989
Cornea; Keratocyte; Keratocan; Keratan sulfate; ABCG2; PAX6; Side population; Adult stem cells; Progenitor cells; Chondrogenesis

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