Ethyl pyruvate ameliorates endotoxin-induced corneal inflammation.

Purpose: 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.

Methods: LPS was injected intrastromally into the corneas of C57BL/6 mice followed by treatment with a solution of 2.5% EP in 0.

Optical coherence tomography as a rapid, accurate, noncontact method of visualizing the palisades of Vogt.

Purpose: 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.

Methods: 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.

FGF-2- and TGF-β1-induced downregulation of lumican and keratocan in activated corneal keratocytes by JNK signaling pathway.

Purpose: 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.

Methods: 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.

Initial in vitro investigation of the human immune response to corneal cells from genetically engineered pigs.

Purpose: 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.

Methods: 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.

Responses of cultured human keratocytes and myofibroblasts to ethyl pyruvate: a microarray analysis of gene expression.

Purpose: 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.

Rho-mediated regulation of TGF-beta1- and FGF-2-induced activation of corneal stromal keratocytes.

Purpose: To investigate the role of Rho GTPase signaling in FGF-2- and TGF-beta1-induced activation of corneal keratocytes.

Methods: Keratocytes isolated from rabbit corneal stroma and plated in a serum-free medium were treated with FGF-2/heparin or TGF-beta1 in the presence or absence of Rho inhibitor (C3 exoenzyme) or ROCK (Rho kinase) inhibitor (Y27632). Specific phenotypic changes were analyzed by immunocytochemistry and Western blot analysis, and the relative abundance of specific mRNAs was estimated by quantitative RT-PCR.

Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.

Purpose: The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression.

Methods: Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium.

Rho GTPase and cAMP/protein kinase A signaling mediates myocilin-induced alterations in cultured human trabecular meshwork cells.

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 in TM cells, and an altered myocilin level may be the culprit in conditions such as corticosteroid glaucoma.

Secretion and organization of a cornea-like tissue in vitro by stem cells from human corneal stroma.

Purpose: 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.

Methods: 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.

Compositional differences between infant and adult human corneal basement membranes.

Purpose: 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.

Methods: Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.

Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation.

Purpose: To determine whether changes in the expression of type IV alpha1, alpha2, or alpha3 collagen isoforms are stringently associated with corneal stromal cell activation.

Methods: Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-beta1-, 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.

Alpha-smooth muscle actin expression enhances cell traction force.

Using an established corneal stromal cell differentiation model, we manipulated alpha-smooth muscle actin (alpha-SMA) protein expression levels in fibroblasts by treating them with TGF-beta1, bFGF, TGF-beta type I receptor inhibitor (SB-431542), and siRNA against alpha-SMA. The corresponding cell traction forces (CTFs) were determined by cell traction force microscopy. With all these treatments, we found that alpha-SMA is not required for CTF induction, but its expression upregulates CTF.

Multipotent stem cells in human corneal stroma.

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.

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.

Rho-mediated assembly of stress fibers is differentially regulated in corneal fibroblasts and myofibroblasts.

Corneal keratocytes (stromal cells) are activated to fibroblasts and myofibroblasts during wound healing. Myofibroblast transdifferentiation is accompanied by the expression of alpha-smooth muscle actin (alpha-SMA) and the assembly of a robust stress fiber network and larger focal adhesions (FAs). The regulation of the assembly of stress fibers was evaluated in cultured corneal fibroblast and myofibroblast phenotypes.

Downstream effects of ROCK signaling in cultured human corneal stromal cells: microarray analysis of gene expression.

Purpose: Rho-associated coiled-coil-containing protein kinase (ROCK) is a downstream target of Rho GTPase signaling and regulates the assembly of stress fibers. Previous reports indicate that Rho/ROCK signaling is involved in the regulation of several cellular processes, some of which may be cell-type specific and are probably critical to corneal stromal cell activation. The present study identified ROCK-regulated gene expression in corneal stromal cells.

Wounding induces motility in sheets of corneal epithelial cells through loss of spatial constraints: role of heparin-binding epidermal growth factor-like growth factor signaling.

Cellular responses to wounding have often been studied at a molecular level after disrupting cell layers by mechanical means. This invariably results in damage to cells at the edges of the wounds, which has been suggested to be instrumental for initiating wound healing. To test this, we devised an alternative procedure to introduce gaps in layers of corneal epithelial cells by casting agarose strips on tissue culture plates.

Rho and Rho-kinase (ROCK) signaling in adherens and gap junction assembly in corneal epithelium.

Purpose: To examine whether Rho and its downstream target, a Rho-associated kinase (ROCK), are involved in the regulation of the assembly of cadherin-mediated cell adhesion and connexin 43 (Cx43) gap junctions in corneal epithelium.

Methods: Rho and ROCK activities in rabbit corneal epithelial cells in culture were inhibited by microinjection of a Clostridium botulinum ADP-ribosyltransferase (C3) and treatment with a ROCK specific inhibitor (Y-27632), respectively. Immunocytochemical and Western blot techniques were used to study the distribution and relative concentrations of E-cadherin and Cx43.