Human Corneal Fibroblasts Research Articles

Upregulation of Tight-Junctional Proteins in Corneal Epithelial Cells by Corneal Fibroblasts in Collagen Vitrigel Cultures

To investigate the effects of corneal fibroblasts on the differentiation of corneal epithelial cells in a coculture system based on a collagen vitrigel membrane. Simian virus 40-transformed human corneal epithelial (HCE) cells and human corneal fibroblasts were cultured on opposite sides of a collagen vitrigel membrane. The distribution of HCE cells and corneal fibroblasts on the collagen membrane was determined by immunofluorescence staining and immunoblot analysis of marker proteins. Expression of the tight-junctional proteins ZO-1, occludin, and claudin and of the adherens-junctional proteins E- and N-cadherin in HCE cells was determined at the mRNA and protein levels by reverse transcription-polymerase chain reaction analysis and immunoblot analysis, respectively. The abundance of ZO-1, occludin, and claudin mRNA and proteins in HCE cells was markedly increased by coculture with corneal fibroblasts. The expression of E- or N-cadherin did not differ between HCE cells cultured with corneal fibroblasts and those cultured without them. PD98059, a specific inhibitor of signaling by extracellular signal regulated kinase (ERK), prevented the upregulation of tight-junctional proteins in HCE cells by corneal fibroblasts. Human corneal fibroblasts regulated the expression of tight-junctional proteins in HCE cells, suggesting that corneal fibroblasts may play an important role in the differentiation of corneal epithelial cells.

Non-destructive mechanical characterisation of UVA/riboflavin crosslinked collagen hydrogels

Aims:To establish a non-destructive method of characterising the mechanical properties of collagen hydrogels to model corneal tissue and to examine the effect of photochemical crosslinking on their mechanical properties.Methods:Collagen hydrogels...

Expression of Indoleamine 2,3-Dioxygenase in Human Corneal Cells as a Local Immunosuppressive Factor

To identify the localization of indoleamine 2,3-dioxygenase (IDO) in human corneal cells and to evaluate its functional ability as a local immunosuppressive factor. The expression profile of IDO was identified in primary cultures of human corneal cells (fibroblasts, epithelial cells, endothelial cells) by RT-PCR and Western blot analysis. The immunosuppressive function of IDO was assessed by culturing human CD4(+) T cells with conditioned medium derived from the three types of human corneal cells, and changes in proliferation and apoptosis were determined. IDO expression and its apoptotic effects on CD4(+) T cells were also investigated after IFN-gamma treatment. Among the three types of human corneal cells, IDO mRNA and protein expression were observed in human corneal fibroblasts and epithelial cells, with higher levels in the human corneal fibroblasts. Human CD4(+) T cells cultivated in conditioned medium derived from human corneal fibroblasts showed decreased cell proliferation and increased apoptosis. IFN-gamma treatment significantly induced IDO expression and showed apoptotic effects on immune cells. These results suggest that human corneal fibroblasts are relatively immunoresistant and that the IDO expression can act as one of the factors for the maintenance of immune privilege in the cornea.

Morphologic Characterization of Organized Extracellular Matrix Deposition by Ascorbic Acid–Stimulated Human Corneal Fibroblasts

To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures. Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to 5 weeks. Constructs were assessed by light (phase-contrast and differential interference-contrast) and transmission (standard and quick freeze/deep etch) microscopy. Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27-51 nm) that often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct, whereas quick-freeze, deep-etch micrographs showed the details of the matrix interaction with fibroblasts through arrays of membrane surface structures. Human keratocytes, cultured in a stable vitamin C derivative, are capable of assembling extracellular matrix, which comprises parallel arrays of ECM fibrils. The resultant constructs, which are highly cellular, are morphologically similar to the developing mammalian stroma, where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggests a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes that govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue engineering a biomimetic stroma.

Role of 3- O-sulfated heparan sulfate in virus-induced polykaryocyte formation

One way herpes simplex virus type-1 (HSV-1) spreads in vivo is by polykaryocytes formation. Here we demonstrate that polykaryocyte production during HSV-1 spread in cultured human corneal fibroblasts (CF) required heparan sulfate (HS) and more specifically 3- O sulfated HS (3- OS HS). The polykaryocyte formation heavily depended on the expression of HS on target (CF) cells but not on glycoprotein expressing effector cells. Furthermore, we provide the first visual evidence of 3- OS HS and HSV-1 gD colocalization during the membrane fusion process. Taken together our results provide novel insight into the significance of HS in polykaryocyte formation.

Dynamic protrusive cell behaviour generates force and drives early matrix contraction by fibroblasts

We investigated the cellular mechanisms underlying force generation and matrix contraction, using human corneal, Tenon's and scleral fibroblasts in a standard collagen matrix. We used timelapse light and confocal reflection microscopy to analyse concomitantly cell behaviour and matrix remodeling during contraction and devised a novel index to quantify dynamic cell behaviour in 3D. Based on the previously described culture force monitor, a novel simultaneous imaging and micro-culture force monitor system (SIM–CFM) was developed to measure the mechanical strain generated during matrix contraction whilst simultaneously recording cell and matrix behaviour. Ocular fibroblasts show marked differences in macroscopic matrix contraction profiles, with corneal fibroblasts inducing the strongest, and scleral the weakest, contraction. We identified four factors that determine the early matrix contraction profile: 1) cell size, 2) intrinsic cellular force, 3) dynamic cell protrusive activity and 4) net pericellular matrix displacement. Intrinsic cellular force and dynamic activity appear to be independent unique characteristics of each cell type and might serve as predictors of matrix contraction. The identification of these factors raises the fundamental new possibilities of predicting the ability of tissues to contract and scar and of modulating tissue contraction by targeting intracellular pathways linked to protrusive activity and force generation.

Herpes simplex virus type 2 entry into cultured human corneal fibroblasts is mediated by herpesvirus entry mediator

Herpes simplex virus type 2 (HSV-2) infections in the eye are becoming increasingly common in adults. The most likely point of entry for HSV-2 into the eye is through the cornea. By using primary cultures of human corneal fibroblasts (CFs), a natural target-cell type for infection, it was demonstrated that CFs are highly susceptible to HSV-2 entry and replication. RT-PCR and flow-cytometry analyses demonstrated expression of herpesvirus entry mediator (HVEM), a known mediator for HSV-2 entry into cells. Blocking of virus entry into CFs by anti-HVEM antibody implicated HVEM as a potential receptor for HSV-2 infection. These results indicate that HVEM may play a crucial role in HSV-2-induced corneal infections.

Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

The purpose of this study was to investigate the role of microtubules in regulating corneal fibroblast structure and mechanical behavior using static (3-D) and dynamic (4-D) imaging of both cells and their surrounding matrix. Human corneal fibroblasts transfected to express GFP-zyxin (to label focal adhesions) or GFP-tubulin (to label microtubules) were plated at low density inside 100 μm thick type I collagen matrices. After 24 h, the effects of nocodazole (to depolymerize microtubules), cytochalasin D (to disrupt f-actin), and/or Y-27632 (to block Rho-kinase) were evaluated using 3-D and 4-D imaging of both cells and ECM. After 24 h of incubation, cells had well organized microtubules and prominent focal adhesions, and significant cell-induced matrix compaction was observed. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and additional cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process. Following 24 h exposure to nocodazole, there was an even greater increase in both the number of stress fibers and the amount of matrix compaction and alignment at the ends of cells. When Rho-kinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/Rho-kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture.

Bioreactor design for cornea tissue engineering: Material–cell interactions

Several materials were evaluated for potential use in a bioreactor system for a tissue-engineered cornea. Two types of cytotoxicity tests were performed using human corneal stromal fibroblasts: a 24 h cytotoxicity test based on the ASTM standard F813-01 and a 7 days growth inhibition test. It was determined that culture configuration, autoclaving and materials surface preparation were all important factors influencing cell viability. Poly(etheretherketone) and titanium–6Al–4V were found to be the most appropriate materials for use in a corneal bioreactor system. Furthermore, poly(oxymethylene) copolymer and poly(tetrafluoroethlylene) are not safe for use with human corneal fibroblasts after autoclaving.

Mitomycin C Upregulates IL-8 and MCP-1 Chemokine Expression via Mitogen-Activated Protein Kinases in Corneal Fibroblasts

To investigate the expression of chemokines and their signaling pathways after application of mitomycin C (MMC) to corneal fibroblasts. Primary porcine and human corneal fibroblasts from passages 3 to 6 were treated with MMC at concentrations of 0.05, 0.1, or 0.2 mg/mL for 1, 2, 5, or 10 minutes. The relative expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were investigated with reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The effects of MMC on the activation of kinases were analyzed by Western blot analysis with specific antiphosphokinase antibodies. The signaling pathways by which MMC regulates the expression of IL-8 and MCP-1 were evaluated by pharmacological kinase-specific inhibitors. The expression of IL-8 and MCP-1 were upregulated after MMC treatment in a time- and concentration-dependent manner. Furthermore, the upregulated expression of IL-8 and MCP-1 increased with longer incubation time. MMC treatment enhanced the phosphorylation of p38, JNK, and ERK at different time points. The MMC-related IL-8 and MCP-1 expression was inhibited by both a p38 inhibitor (SB203580) and an ERK inhibitor (PD98059). A JNK inhibitor (SP600125) reduced the expression of MMC-induced MCP-1 but not of IL-8. MMC treatment upregulated the expression of IL-8 and MCP-1 mRNA and protein secretion by the activation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts.

003 Génération de fibroblastes “like” cornéens issus de la différenciation de cellules souches mésenchymales humaines in vitro

Aim Tissue engineering (TE) offers considerable promise in damaged tissues repair or replacement. Corneal Fibroblasts (CF) result from mesodermal cell extension into optic cap. They participate in the genesis of corneal stroma during the 5th week of embryonic development. We used MSC to generate CF phenotype cells for corneal TE and clinical applications. Materials and Methods Genotype CF, obtained from 8 corneal grafts, were evaluated by genomic profiling of total RNA using microarray analysis (1300 genes). The MSC were isolated from 12 human bone marrows with the magnetic beads separation method. These cells were cultured in specific media. Three weeks later, capacity of CD34+ cells to differentiate into fibroblast like cells was analyzed. The resulting cells were renamed as mesenchymal stem cell “derivated corneal fibroblast-like” (DCFL) cells. Their phenotypes were analysed by imunocytochemistry (ICC) using specific antibodies against CD34, Von Willbrand factor (VWF), 3G5 (CF ganglioside being used as corneal keratocytes marker), stromal derived factor SDF-1 and its receptor CXCR4, VEGF receptors, Aqp 1, and α-SMA. These phenotypes were compared to CF microarray results. Results Microarray indicates corneal fibroblasts expression of SDF1, CXCR4, VEGF C, Flt1 and Flt4 mRNAs. Corresponding proteins were highlighted in corneal fibroblasts and DCFL cells by ICC. Indeed Aquaporin-1, SDF1alpha, CXCR4, VEGF and Flt1 and Flt4 were detected. 3G5 expression was detected on 0,2% DCFL. Both corneal fibroblasts and endothelial cells were stained with anti-alphaSMA antibody whereas only endothelial cells were positive for anti VWF staining. Interestingly, no corneal fibroblasts analysed by gene array (n = 2) and immunohistochemistry (n = 8) expressed CD34 + . Discussion 3G5 antigen is constitutively expressed by cultured human corneal fibroblast and is found in CD34 + BMDF cells which suggests the potential for bone marrow to generate CF like cells. Conclusion These cells, characterized by surface expression of CXCR4 + , SDF1- alpha + , Flt-4 + , VEGF-C + and 3G5 + should be considered as candidates for corneal tissue engineering.

Rho plays a central role in regulating local cell‐matrix mechanical interactions in 3D culture

The purpose of this study was to assess quantitatively the role of the small GTPase Rho on cell morphology, f-actin organization, and cell-induced matrix remodeling in 3D culture. Human corneal fibroblasts (HTK) were infected with adenoviruses that express green fluorescent protein (GFP) or GFP-N19Rho (dominant negative Rho). One day later cells were plated inside collagen matrices and allowed to spread for 24 h. Cells were fixed and stained for f-actin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) images were acquired using confocal microscopy. Fourier transform analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. The decrease in matrix height was used as an indicator of global matrix contraction. HTK and HTK-GFP cells induced significant global matrix contraction; this was inhibited by N19Rho. HTK and HTK-GFP fibroblasts generally had a bipolar morphology and occasional intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. In contrast, HTK-GFPN19 cells were elongated, and had a more cortical f-actin distribution. Numerous small extensions were also observed along the cell body. In addition, both local collagen fibril density and alignment were significantly reduced. Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3D culture. Overall, the data suggest that Rho-kinase dependent cell contractility contributes to global and local matrix remodeling, whereas Rho dependent activation of mDia and/or other downstream effectors regulates the structure and number of cell processes.

Granulocyte Macrophage Colony–Stimulating Factor Expression in Human Herpetic Stromal Keratitis: Implications for the Role of Neutrophils in HSK

Granulocyte macrophage colony-stimulating factor (GM-CSF) is thought to play a key role in chronic inflammatory diseases by governing the survival and function of infiltrating neutrophils. The objective of this study was to determine the putative role of GM-CSF in the pathogenesis of human herpetic stromal keratitis (HSK). Primary human corneal fibroblast (HCF) cultures and a telomerase-immortalized human corneal epithelial (HCE) cell line representative of native HCE were stimulated with the known HSK-inducing cytokines interferon (IFN)-gamma, interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha. Alternatively, the T-cell cytokine IL-17 was added solely or simultaneously. Human neutrophils were incubated with conditioned medium (CM) of the HCF and HCE stimulated with the aforementioned cytokines, or recombinant GM-CSF, and their viability or activation status was determined by flow cytometry. GM-CSF and IL-8 secretion levels in the CM were determined by ELISA. The antibody-dependent cellular cytotoxicity (ADCC) of neutrophils toward herpes simplex virus (HSV)-infected HCFs was determined by flow cytometry. The expression of GM-CSF was determined in HSK and control corneal buttons by real-time RT-PCR and immunohistology. Compared with IFN-gamma, CM of either cell type stimulated with IL-1beta, or in the case of HCE cells, stimulated with TNF-alpha or IL-17, delayed neutrophil apoptosis significantly. Only in HCFs did IL-17 exhibit a synergistic effect with TNF-alpha. The antiapoptotic activity was attributable in part to the GM-CSF secreted by the activated HCFs and HCE cells. GM-CSF stimulation of neutrophils induced their activation and the secretion of IL-8. GM-CSF did not increase significantly the ADCC reaction of neutrophils toward HSV-infected HCFs. Finally, GM-CSF was expressed in corneas of the patients with HSK but not in control subjects. The data suggest that GM-CSF, expressed by cornea-resident cells such as HCFs and HCE cells, may play a role in the immunopathogenesis of HSK by prolonging the survival and modulating the effector function of corneal infiltrating neutrophils.

Expression of Extracellular Matrix Proteins Fibulin-1 and Fibulin-2 by Human Corneal Fibroblasts

Purpose: The fibulins are a family of extracellular matrix (ECM) molecules that regulate the organ shape along with other growth factors and stromal cells. We report here the in vitro expression of ECM proteins fibulin-1 and fibulin-2 by human corneal fibroblasts. The ability of fibulin-1 to modulate cell motility was investigated. Methods: Fibulin-1 and fibulin-2 mRNA and proteins expression were analyzed in primary and immortalized human corneal fibroblasts (CHN) respectively by gene array, RT-PCR, and immunocytochemistry. The motility and adhesion of the cells transfected with fibulin-1 siRNA were analyzed on tissue culture polystyrene coated with Matrigel or ECM secreted by those fibroblasts. Results: (1) The microarray analysis shows the expression of fibulin-1, fibulin-2, and their binding partners (i.e., fibronectin, nidogen-1, aggrecan, fibrilin-1, endostatin, and laminin alpha-2 chain). Interestingly, a matrix metalloprotease, ADAMTS-1, for which fibulin-1 acts as a cofactor, was also detected in CHN. (2) The synthesis by CHN of fibulin-1 and 2 mRNA and proteins was confirmed respectively by RT-PCR and immunocytochemistry. (3) Transfection of CHN by fibulin-1 siRNA has no effect on cell adhesion but increases cell migration compared with that of the control cells. This observation suggests an important role of fibulin-1 on cell motility. Conclusions: The expression of fibulins and that of their binding partners by human corneal fibroblasts indicate the important role of these proteins in the organization of supramolecular ECM structures of cornea. The variation of their expression and the structural changes of fibulins remain to be determined in corneal pathology.

Expression of Local Immunosuppressive Factor, Indoleamine 2,3-dixygenase, in Human Coreal Cells

Purpose: To identify the localization of indoleamine 2,3-dioxygenase (IDO) in human corneal cells and to evaluate its ability to act as a local immunosuppressive factor. Methods: The expression profile of IDO was obtained with RT-PCR and Western blot of in a primary culture of human corneal cells (fibroblasts, epithelial cells and endothelial cells). In order to investigate the immunosuppressive function of IDO, immune cells were cultured in a human corneal cell-conditioned medium, and their prolifleration was identified by the MTT assay. Moreover, apoptotic effects of IDO in immune cells treated with IFN-γ were also investigated with apoptosis ELISA. Results: Among the three different types of human corneal cells analyzed, mRNA and protein expression of IDO was observed only in human corneal fibroblasts. Immune cells cultured in a human corneal fibroblast-conditioned medium showed inhibited proliferation. Moreover, IFN-γ-induced expression of IDO significantly enhanced apoptotic ability in a dose-depandant manner. Conclusions: Our results suggest that human corneal fibroblasts are relatively immuno-resistant and that expression of IDO may be one of the factors involved in the immune tolerance observed in corneal grafts.

Inhibition by Triptolide of Chemokine, Proinflammatory Cytokine, and Adhesion Molecule Expression Induced by Lipopolysaccharide in Corneal Fibroblasts

The production of proinflammatory cytokines and chemokines as well as the surface expression of intercellular adhesion molecule (ICAM)-1 by corneal fibroblasts contribute to corneal inflammation. The effects of triptolide on the expression of these proteins induced by lipopolysaccharide (LPS) in human corneal fibroblasts were examined in comparison with those of dexamethasone. The release of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1beta, and IL-8 from cultured corneal fibroblasts was measured with assay kits. Surface expression of ICAM-1 on the cultured cells was measured with a whole-cell enzyme-linked immunosorbent assay. Lipopolysaccharide (LPS) induced the release of the proinflammatory cytokine IL-6 and that of the chemokines G-CSF, MCP-1, MIP-1beta, and IL-8 as well as surface expression of ICAM-1 by corneal fibroblasts, whereas IL-1beta, TNF-alpha, and GM-CSF were not detected in the culture supernatants of cells incubated with or without LPS. Triptolide and dexamethasone each inhibited in a concentration-dependent manner the LPS-induced release of IL-6, G-CSF, MCP-1, and IL-8 by corneal fibroblasts. Whereas the inhibitory effect of dexamethasone on LPS-induced IL-6 release was greater than that of triptolide, the inhibitory effect of triptolide on LPS-induced G-CSF release was more pronounced than was that of dexamethasone. Dexamethasone also inhibited LPS-induced MIP-1beta release, whereas triptolide did not. Both compounds inhibited the LPS-induced surface expression of ICAM-1. Triptolide inhibits the LPS-induced expression of IL-6, chemokines (G-CSF, MCP-1, IL-8), and ICAM-1 in cultured human corneal fibroblasts. This compound might thus be expected to limit the infiltration of immune cells into the cornea.

Role for 3-O-sulfated heparan sulfate as the receptor for herpes simplex virus type 1 entry into primary human corneal fibroblasts.

Herpes simplex virus type 1 (HSV-1) infection of the corneal stroma remains a major cause of blindness. Primary cultures of corneal fibroblasts (CF) were tested and found susceptible to HSV-1 entry, which was confirmed by deconvolution imaging of infected cells. Plaque assay and real-time PCR demonstrated viral replication and hence a productive infection of CF by HSV-1. A role for glycoprotein D (gD) receptors in cultured CF was determined by gD interference assay. Reverse transcription-PCR analysis indicated expression of herpesvirus entry mediator and 3-O-sulfated (3-OS) heparan sulfate (HS)-generating enzyme 3-O sulfotransferase 3 (3-OST-3) but not nectin-1 or nectin-2. Subsequently, HS isolated from these cells was found to contain two distinct disaccharides (IdoUA2S-AnMan3S and IdoUA2S-AnMan3S6S) that are representative of 3-OST-3 activity. The following lines of evidence supported the important role of 3-OS HS as the mediator of HSV-1 entry into CF. (i) Blockage of entry was observed in CF treated with heparinases. The same enzymes had significantly less effect on HeLa cells that use nectin-1 as the entry receptor. (ii) Enzymatic removal of cell surface HS also removed the major gD-binding receptor, as evident from the reduced binding of gD to cells. (iii) Spinoculation assay demonstrated that entry blockage by heparinase treatment included the membrane fusion step. (iv) HSV-1 glycoprotein-induced cell-to-cell fusion was inhibited by either prior treatment of cells with heparinases or by HS preparations enriched in 3-OS HS. Taken together, the data in this report provide novel information on the role of 3-OS HS in mediating infection of CF, a natural target cell type.

Promotion by fibronectin of collagen gel contraction mediated by human corneal fibroblasts

Collagen contraction mediated by corneal fibroblasts (CFs) is implicated in the maintenance of corneal shape. Given that fibronectin is expressed at sites of corneal stromal wounding, we investigated the effect of fibronectin on CF-mediated collagen gel contraction. Human CFs were cultured in a three-dimensional gel of type I collagen in the absence or presence of various extracellular matrix (ECM) components. The contraction of collagen gels mediated by CFs was evaluated by measurement of changes in gel diameter. The formation of stress fibers and focal adhesions in CFs was examined by fluorescence microscopy. The abundance of paxillin, phosphorylated paxillin, integrins α5, β1, and α2, and α-smooth muscle actin in CFs was examined by immunoblot analysis. Fibronectin promoted CF-mediated collagen gel contraction in a concentration- and time-dependent manner. Other ECM proteins or glycosaminoglycans did not exhibit such an effect. Fibronectin also induced cell spreading, the formation of stress fibers, and the establishment of focal adhesions containing paxillin in CFs cultured in three-dimensional collagen gels. In addition, it increased the amounts of paxillin, phosphorylated paxillin, and integrins α5 and β1 in these cells. The expression of integrin α2 and α-smooth muscle actin was not affected by fibronectin, however. Furthermore, the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the stimulatory effect of fibronectin on CF-mediated collagen gel contraction. These results suggest that fibronectin promoted CF-mediated collagen gel contraction in a manner dependent on the formation of stress fibers and focal adhesions, the activation of paxillin, and the up-regulation of integrin α5β1. Fibronectin may therefore contribute to the maintenance of corneal shape by CFs during the healing of stromal wounds.

Quantitative assessment of local collagen matrix remodeling in 3-D Culture: The role of Rho kinase

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-μm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase ( p < 0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced ( p < 0.01). Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.

Herpes simplex virus 1 (HSV-1) DNA and immune complex (HSV-1–human IgG) elicit vigorous interleukin 6 release from infected corneal cells via Toll-like receptors

Toll-like receptor 3 (TLR-3) and TLR-9 gene expression and interleukin 6 (IL-6) secretion were studied in corneal cells with components of herpes simplex virus (HSV). Human corneal epithelial cells (HCEs) and primary human corneal fibroblasts (HCRFs) were infected with live HSV or UV-inactivated HSV (UV-HSV), transfected with HSV DNA or treated with HSV-anti-HSV IgG immune complexes. Gene expression of TLR-3 and -9 was analysed by real-time PCR. Supernatants were assayed for IL-6 by ELISA. Incubation of HCEs and HCRFs with live HSV-1, UV-HSV and HSV DNA resulted in augmented TLR-3 and -9 gene expression and IL-6 release. Moreover, infected or transfected HCRFs released greater amounts of IL-6 than did HCEs. As virus is frequently in the form of neutralized virus immune complexes, the ability of these immune complexes to interact with TLRs and trigger IL-6 production was evaluated. Here, it is shown that HSV-anti-HSV IgG complexes were as potent as HSV DNA in their ability to induce IL-6. Treatment of HCRFs transfected with HSV DNA with the TLR-9-inhibitory oligomer iODN, anti-TLR-3 antibody or phosphatidylinositol 3-kinase inhibitor indicated that IL-6 release from HCRFs was mediated by TLR-3 and -9 gene expression. These results demonstrated that neutralized HSV immune complexes were as potent as HSV DNA in enhancing IL-6 release from corneal fibroblasts. These phenomena were mediated via augmented TLR-3 and -9 gene expression.