TGFbeta induced myofibroblast differentiation of rabbit keratocytes requires synergistic TGFbeta,PDGF and integrin signaling

There is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes which undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast. Both in vivo and in vitro studies have shown this transition to be dependent, in part, on transforming growth factor beta (TGFbeta). In many fibroblastic cells autocrine production of platelet derived growth factor (PDGF) is known to mediate the growth up-regulation by TGFbeta. In this study, blocking antibodies to PDGF significantly reduced by 80% (P<0.025) the TGFbeta1 stimulated cell cycle entry of serum-free cultured rabbit corneal keratocytes. AntiPDGF treatment also markedly reduced the TGFbeta1-induced intracellular actin filament re-organization, fibronectin fibril assembly, and focal contact formation as well as reducing by 80% the expression of alpha-smooth muscle (alpha-SM) specific isoform of actin characteristic of myofibroblast differentiation. Although PDGF treatment of quiescent keratocytes produced an activated, fibroblastic cell type, PDGF stimulated keratocytes exhibited the same temporal, myofibroblastic differentiation response to TGFbeta1 as did quiescent keratocytes. Furthermore, blocking TGFbeta1 induction of myofibroblast differentiation with the Arg-Gly-Asp containing peptide, GRGDdSP, for 3 days followed by allowing progression of myofibroblast differentiation by removing GRGDdSP did not change the temporal response or tyrosine phosphorylation cascade (2-72 hr) leading to myofibroblast differentiation. Nor did PDGF treatment of keratocytes reverse the RGD blockade of TGFbeta1 induced myofibroblast differentiation. Overall these cumulative findings indicate that myofibroblast differentiation in the rabbit corneal keratocyte requires synergistic growth factor/integrin signaling involving TGFbeta, PDGF, and the fibronectin receptor. Additionally, the similar TGFbeta1 temporal response of PDGF-stimulated compared to nai;ve keratocytes suggests that myofibroblast differentiation does not require transition through a fibroblast phenotype.     

Transforming growth factor(beta)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly.

PURPOSE: Recent studies indicate that transforming growth factor (TGF)beta is a potent inducer of corneal myofibroblast differentiation and expression of smooth muscle-specific, alpha-actin (alpha-SMA). Although TGFbeta is known to enhance synthesis of extracellular matrix proteins and receptors, little is known about how it modulates the expression of smooth muscle proteins in nonmuscle cells. The purpose of this study was to identify the role of Arg-Gly-Asp (RGD)-dependent tyrosine phosphorylation in regulating alpha-SMA gene expression and ultimately myofibroblast development. METHODS: Because cell culture in serum-containing media mimics myofibroblast transformation, all experiments were performed on freshly isolated rabbit keratocytes plated in defined, serum-free media. Cells were exposed to TGFbeta (1 ng/ml), Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP, 50 microM), Gly-Arg-AL-Asp-Ser-Pro (GRADSP; 100 microM), or herbimycin A (0.1-10 nM) at 24 hours (sparse) or 7 days (confluent). Cells were evaluated by immunocytochemistry and proteins and RNA collected for western and northern blot analyses using antibodies specific for alpha-SMA, fibronectin, focal adhesion proteins, and phosphotyrosine (clones 4G10 and PY20); and probes directed against rabbit alpha-SMA. All experiments were repeated at least three times. RESULTS: Keratocytes exposed to TGFbeta showed expression of alpha-SMA that coincided with the intracellular reorganization of the actin cytoskeleton and the extracellular assembly of fibronectin fibrils. Addition of RGD containing but not control peptides blocked the organization of intracellular actin, extracellular fibronectin, and alpha-SMA protein and mRNA. Immunoprecipitation of cell proteins with 4G10 or PY20 identified the TGFbeta-associated tyrosine phosphorylation of paxillin, pp125fak, p130, PLCgamma, and tensin, which was blocked by addition of GRGDdSP. Addition of herbimycin A to keratocytes exposed to TGFbeta showed a dose-dependent loss of alpha-SMA protein and mRNA which correlated with loss of tyrosine phosphorylation, absence of actin reorganization, and fibronectin assembly. CONCLUSIONS: The data suggest that TGFbeta-mediated alpha-SMA gene expression leading to myofibroblast transformation may involve an RGD-dependent phosphotyrosine signal transduction pathway.     

Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines control in vitro contractility and extracellular matrix contraction

The purpose of this study was to evaluate specific keratocyte phenotypes (keratocyte, fibroblast, myofibroblast) for cell contractility and ability to contract extracellular matrix. Rabbit keratocyte phenotype was modulated by exposure to optimal proliferative doses of IGF-I, IL-1alpha, FGF2, PDGF-AB, and TGFbeta(1). Cells were then evaluated by immunocytochemistry, western blot, collagen gel contraction and LPA stimulation to measure: (1) focal adhesion (FA), fibronectin (FN) and f-actin assembly; (2) expression of alpha-smooth muscle actin (alpha-SMA); (3) ability to contract extracellular matrix and (4) determine contractile ability, respectively. Untreated keratocytes showed no ability to contract collagen matrix. IGF-I and IL-1alpha increased cell proliferation (70.2 and 74.3%, respectively) but did not alter keratocyte phenotype or ability to contract matrix. FGF2 and PDGF induced fibroblast differentiation with FA and FN assembly and significant (p<0.05) extracellular matrix contraction. TGFbeta(1) induced myofibroblast differentiation with prominent FA and FN assembly, expression of alpha-SMA and significantly greater (p<0.05) matrix contraction. Addition of LPA induced actin filament assembly in growth factor starved fibroblasts and myofibroblasts but had no effect on the cultured keratocyte phenotype. We report for the first time that the keratocyte phenotype is non-contractile and that cell quiescence is not a defining characteristic. We further establish that changes in environmental conditions modulate the keratocyte phenotype resulting in physiologically functional differences regarding cell contractility and capacity to contract extracellular matrix.     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes

Transparency is essential for normal corneal function. Recent studies suggest that corneal cells express high levels of so-called corneal crystallins, such as aldehyde dehydrogenase (ALDH) and transketolase (TKT) that contribute to maintaining cellular transparency. Stromal injury leads to the appearance of repair phenotype keratocytes, the corneal fibroblast and myofibroblast. Previous studies on keratocytes from species such as bovine and rabbit indicate that the transformation from the normal to repair phenotype is accompanied by a loss of corneal crystallin expression, which may be associated with loss of cellular transparency. Here we investigated if a similar loss occurs with human keratocyte repair phenotypes. Human corneal epithelial cells were collected by scraping and keratocytes were isolated by collagenase digestion from cadaveric corneas. The cells were either processed immediately (freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. RT-PCR, western blotting and immunolabeling were used to detect mRNA and protein expression of ALDH isozymes and TKT. ALDH enzyme activity was also quantitated and immunolabeling was performed to determine the expression of ALDH3A1 in human corneal tissue sections from normal and diseased corneas. Human corneal keratocytes isolated from three donors expressed ALDH1A1 and ALDH3A1 mRNA, and one donor also expressed ALDH2 and TKT. Corneal epithelial cells expressed ALDH1A1, ALDH2, ALDH3A1 and TKT. Compared to normal keratocytes, corneal fibroblast expression of ALDH3A1 mRNA was reduced by 27% (n=5). ALDH3A1 protein expression as detected by western blotting was markedly reduced in passage zero fibroblasts and undetectable in higher passages (n=3). TKT protein expression was reduced in fibroblasts compared to keratocytes (n=2). ALDH3A1 enzyme activity was not detectable in corneal fibroblasts (n=6) but was readily detected in corneal epithelial cells (0.29+/-0.1U/mg protein, n=4) and keratocytes (0.05+/-0.009U/mg protein, n=7). ALDH3A1 expression was also reduced in corneal fibroblasts and myofibroblasts as determined by immunolabeling of the cells in culture (n=3) and in diseased corneal tissues in situ (n=2). We conclude that expression of the crystallin ALDH3A1 is decreased in repair phenotype human keratocytes, compared to normal human keratocytes. Extrapolating from studies of bovine and rabbit, the reduced expression of ALDH3A1 may contribute to the loss of corneal transparency experienced by human patients after injury and refractive surgeries.     

Involvement of S100A4 in stromal fibroblasts of the regenerating cornea

PURPOSE. S100A4 is a member of the S100 family of calcium-binding proteins. Members of the S100 family have been implicated in a variety of cellular events, including growth, signaling, differentiation, and motility. It has been suggested that S100A4 modulates cell shape and motility by interacting with components of the cytoskeleton. In the present study, the distribution patterns of S100A4 were investigated in normal and regenerating mouse corneas. METHODS. Rabbit cDNA libraries were prepared from cultures of corneal fibroblasts. S100A4 was identified as the most abundant message present. Expression of S100A4 in the cornea was determined using Northern blot analysis, in situ hybridization, and immunohistochemistry. Distribution patterns of S100A4 in primary corneal fibroblast cultures treated with either FGF-2/heparin or TGFbeta1 were analyzed by immunofluorescence. RESULTS. S100A4 mRNA was rarely detected in keratocytes or epithelial cells of the normal rabbit cornea. Likewise, S100A4 antigen was not found in normal mouse corneas. However, after removal of the corneal epithelium, fibroblasts are activated and had readily detectable S100A4 expression 6 days after wounding. In the in vitro equivalent of activated keratocytes, cultured rabbit corneal fibroblasts, S100A4 was restricted to the cytoplasm. In contrast, in cultures treated with TGFbeta1, which induces a myofibroblast phenotype, more than 90% of the cells showed a nuclear localization of S100A4. CONCLUSIONS. The findings show that S100A4 is expressed in the keratocyte phenotypes that appear in stromal tissue of corneas recovering from damage, the fibroblasts, and myofibroblasts. Its expression and distinct subcellular redistribution patterns suggest that S100A4 may be involved in the interconversions that occur between keratocytes, fibroblasts, and myofibroblasts during corneal wound healing.     

CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-beta1

PURPOSE: To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-beta1-induced myofibroblast differentiation. METHODS: Collagenase-isolated keratocytes were seeded and subcultured on plastic or amniotic membrane matrix (AM) in DMEM, with or without 10% FBS, in serum-free DMEM containing insulin-transferrin-sodium selenite (ITS) with 10, 100, and 1000 pg/mL TGF-beta1 or in DMEM with 1% FBS and 10 ng/mL TGF-beta1. Protein expression of CD34 and alpha-smooth muscle actin (alpha-SMA) was measured by Western blot and immunostaining. RESULTS: Keratocytes, expressing CD34 in normal human corneas, continued to express CD34 when cultured on AM in serum-containing medium and on plastic in serum-free medium, but expression was lost on plastic in serum-containing medium. In serum-containing medium, expression of CD34, but not alpha-SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed alpha-SMA without CD34 when continuously passaged on plastic. Expression of alpha-SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas alpha-SMA expression was upregulated when cells were subcultured on plastic. In serum-free medium, CD34 expression was maintained by cells treated with 10 pg/mL TGF-beta1, but was lost when treated with a higher concentration on plastic for 5 days. In 1% FBS, AM-expanded keratocytes rapidly became alpha-SMA-expressing myofibroblasts if subpassaged on plastic and treated with 10 ng/mL TGF-beta1, but failed to do so if cultured on AM, even for 7 days. CONCLUSIONS: These findings indicate that CD34 is expressed by human keratocytes in vivo and in vitro. Myofibroblast differentiation promoted by TGF-beta1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-beta signaling.     

Involvement of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in corneal fibroblasts during corneal wound healing

PURPOSE: The involvement of downstream messengers of transforming growth factor (TGF)-beta in the differentiation of corneal fibroblasts into myofibroblasts was investigated. The effects of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 upregulated by TGF-beta were examined in human corneal fibroblasts, and the possible involvement of IGF axis components in corneal wound healing was assessed in a mouse model. METHODS: Human corneal fibroblasts were incubated with TGF-beta2 or IGF-I, to investigate IGF-I, IGF-II, IGFBP-3, type I collagen, and alpha-smooth muscle actin (alpha-SMA) mRNA, as well as IGFBP-3 protein expression, during myofibroblast differentiation. DNA synthesis was evaluated with a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. IGFBP-3 mRNA expression, protein expression, and immunolocalization were investigated in mouse corneas after photorefractive keratectomy (PRK). RESULTS: TGF-beta2 treatment induced expression of IGF-I and IGFBP-3 mRNA and of IGFBP-3 protein in human corneal fibroblasts. TGF-beta2 and IGF-I both stimulated expression of type I collagen. TGF-beta2 but not IGF-I potently stimulated alpha-SMA mRNA expression. IGF-I potently stimulated basal DNA synthesis, whereas IGFBP-3 inhibited it. IGF-I potently stimulated proliferation of TGF-beta2-activated myofibroblasts without reversing the activated fibrogenic phenotype, whereas IGFBP-3 suppressed IGF-I-induced proliferation of corneal fibroblasts. IGFBP-3 mRNA and protein increased in mouse corneas soon after PRK, when in vivo immunostaining of the corneas showed expression of IGFBP-3 in the deep layer of the corneal stroma. CONCLUSIONS: These results suggest that during corneal wound healing, TGF-beta stimulates IGF axis components, whereas IGFBP-3 may modulate IGF-I-induced myofibroblast proliferation to suppress corneal mesenchymal overgrowth.     

Electrophilic PPARγ ligands inhibit corneal fibroblast to myofibroblast differentiation in vitro: a potentially novel therapy for corneal scarring

A critical component of corneal scarring is the TGFβ-induced differentiation of corneal keratocytes into myofibroblasts. Inhibitors of this differentiation are potentially therapeutic for corneal scarring. In this?study, we tested the relative effectiveness and mechanisms of action of two electrophilic peroxisome proliferator-activated receptor gamma (PPARγ) ligands: cyano-3,12-dioxolean-1,9-dien-28-oic acid-methyl ester (CDDO-Me) and 15-deoxy-Δ(-12,14)-prostaglandin J(2) (15d-PGJ(2)) for inhibiting TGFβ-induced myofibroblast differentiation in?vitro. TGFβ was used to induce myofibroblast differentiation in cultured, primary human corneal fibroblasts. CDDO-Me and 15d-PGJ(2) were added to cultures to test their ability to inhibit this process. Myofibroblast differentiation was assessed by measuring the expression of myofibroblast-specific proteins (αSMA, collagen I, and fibronectin) and mRNA (αSMA and collagen III). The role of PPARγ in the inhibition of myofibroblast differentiation by these agents was tested in genetically and pharmacologically manipulated cells. Finally, we assayed the importance of electrophilicity in the actions of these agents on TGFβ-induced αSMA expression via Western blotting and immunofluorescence. Both electrophilic PPARγ ligands (CDDO-Me and 15d-PGJ(2)) potently inhibited TGFβ-induced myofibroblast differentiation, but PPARγ was only partially required for inhibition of myofibroblast differentiation by either agent. Electrophilic PPARγ ligands were able to inhibit myofibroblast differentiation more potently than non-electrophilic PPARγ ligands, suggesting an important role of electrophilicity in this process. CDDO-Me and 15d-PGJ(2) are strong inhibitors of TGFβ-induced corneal fibroblast to myofibroblast differentiation in?vitro, suggesting this class of agents as potential novel therapies for corneal scarring warranting further study in pre-clinical animal models.     

Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing

PURPOSE: To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. METHODS: The expression of VSX1 was evaluated through semiquantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation. RESULTS: Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1. CONCLUSIONS: The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.     

Fibroblast growth factor reversal of the corneal myofibroblast phenotype

PURPOSE: Keratocytes give rise to fibroblasts and myofibroblasts in wounded cornea. It is well established that treatment of fibroblasts with transforming growth factor (TGF) beta will induce myofibroblast differentiation. We investigated whether this differentiation could be reversed by the administration of fibroblast growth factor (FGF). METHODS: Cultured corneal myofibroblasts were plated at 200 cells/mm(2), and cells were grown in DMEM/F12 containing (1) 10% FBS or (2) 10% FBS with FGF and heparin or (3) 1% FBS or (4) 1% FBS with TGF-beta. As distinguished from the fibroblast phenotype, the myofibroblast phenotype was identified by the assembly of alpha-smooth muscle (SM) actin protein into the stress fiber cytoskeleton. To further characterize growth factor regulation of the two phenotypes, the phenotypic expression of TGF-beta receptor types I and II, cadherins, and connexin 43 by immunocytochemistry, Western blot analysis, and immunoprecipitation and of alpha-SM actin mRNA in Northern blot analysis were evaluated. RESULTS: Corneal myofibroblasts replated and grown in the presence of FGF-1 or FGF-2 (20 ng/ml) plus heparin (5 microg/ml) in 10% FBS medium had decreased expression of alpha-SM actin protein, TGF-beta receptors, and cadherins. Thus, FGF-heparin decreased the myofibroblast phenotype and promoted the fibroblast phenotype. Administration of either 20 ng/ml FGF or 5 microg/ml heparin alone was not effective. Addition of TGF-beta further enhanced the expression of alpha-SM actin mRNA and protein and cell surface expression of TGF-beta receptors in myofibroblast cultures. CONCLUSIONS: FGF-1 or -2 and heparin promoted the fibroblast phenotype and reversed the myofibroblast phenotype. This finding supports the idea that corneal myofibroblasts and fibroblasts are alternative phenotypes rather than terminally differentiated cell types.     

Uncoupling keratocyte loss of corneal crystallin from markers of fibrotic repair

PURPOSE: Corneal crystallins are lost from resident cells of the corneal stroma during wound repair, and this is associated with a loss of cell transparency. The goal of this study was to identify factors inducing loss of the corneal crystallin transketolase (TKT). METHODS: A cell culture model of freshly isolated rabbit corneal keratocytes was used. Fibrotic markers included cell proliferation, adoption of a "fibroblastic" spindle-shaped morphology associated with cytoskeletal rearrangement, loss of TKT, and expression of alpha-smooth muscle actin (alpha-sm actin), a marker for the myofibroblast. RESULTS: When freshly isolated keratocytes were cultured in the continuous presence of 10% calf serum, the high level of intracellular TKT protein was reduced dramatically within 24 to 48 hours. In contrast, TKT protein was retained in cells maintained in the absence of serum. When cells were prevented from proliferating by exposure to serum for <24 hours or by continuously exposing to serum at a contact-inhibiting plating density, TKT loss was inhibited. TKT loss was induced by treatment of serum-free cultures with the serum cytokines platelet-derived growth factor or basic fibroblast growth factor, both of which also stimulated keratocyte proliferation, although not other changes associated with fibrosis. However, TKT loss was not induced by treatment of serum-free cultures with a third serum cytokine, transforming growth factor- (TGF)-beta, even though TGF-beta stimulated cell proliferation at low doses and induced the fibroblastic spindle-shape and express alpha-sm actin at high doses. CONCLUSIONS: TKT loss in corneal keratocytes can be induced by PDGF or bFGF and this loss can be uncoupled from other fibrotic markers. Targeting these cytokines or the signaling pathways that they activate could enable retention of corneal crystallin in stromal cells during repair, a more regenerative outcome. The result would be enhanced clarity of the cornea.     

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.     

Localization of fibronectin and actin in cultured rabbit keratocytes

Migration of activated keratocytes toward the corneal stromal wound is one of the most important processes of successful healing. To understand the motility of keratocytes and the interaction of fibronectin and intracellular actin filaments, we cultured rabbit corneal keratocytes and studied dynamic movements of the cells by time-lapse cinematography. We also examined the changes in the localization of fibronectin and actin using double staining immunofluorescent microscopy. The cultured keratocytes first attached to the substratum in round globular shape and then spread with many extending processes. In the early stage of the cultivation, fibronectin was observed inside the cells. Later, fibronectin was observed outside the cells, suggesting formation of the extracellular matrix. When keratocytes spread, actin was observed as a stress fiber inside the cells. At the edge of the cellular processes, a close interaction between fibronectin and actin was observed. The present results demonstrated that cultured keratocytes had active motility and that there were close interactions between the extracellular fibronectin and intracellular actin filaments. The organization of fibrillar actin filaments (F-actin) might be affected by the binding of extracellular fibronectin to the cell surface receptor for fibronectin.     

In vivo and in vitro demonstration of epithelial cell-induced myofibroblast differentiation of keratocytes and an inhibitory effect by amniotic membrane

PURPOSE: To examine the role of epithelial cells in inducing the differentiation of keratocytes into myofibroblasts and to determine whether this effect may be inhibited by amniotic membrane matrix. METHODS: In vivo, a 9-mm diameter, partial-thickness corneal flap was created in 12 rabbit eyes (6 rabbits), which were equally subdivided into three groups. The first group was implanted with one layer of a 6-mm diameter human amniotic membrane, from which the epithelium had been removed by dispase. The second group received an implantation of dispase-treated amniotic membrane with cultured rabbit corneal epithelial cells. The third group received the same implantation as the second group except that the cultured corneal epithelial cells were sandwiched between two layers of membrane. All corneas were removed 2 weeks later and were subjected to Masson trichrome staining and immunofluorescence staining with monoclonal antibodies to alpha-smooth muscle (alpha-SM) actin for myofibroblasts and cytokeratins for epithelial cells. In vitro collagen gels impregnated with different types of human ocular surface fibroblasts were seeded with or without rabbit corneal epithelial cells before testing for gel contraction. RESULTS: Positive staining of alpha-SM actin was noted only in keratocytes adjacent to corneal epithelial cells at the incision site and those grown on the basement membrane side of the amniotic membrane. Negative staining was noted when epithelial cells were removed by dispase or when cultured corneal epithelial cells were sandwiched between two layers of membrane. Gel contraction by fibroblasts was significantly promoted when epithelial cells were seeded on the gel. In the latter situation, positive staining of alpha-SM actin was noted in fibroblasts subjacent to epithelial cells but not in those impregnated in the gel. CONCLUSION: Epithelial cells are capable of inducing the differentiation of adjacent fibroblasts into myofibroblasts; such an induction requires a close epithelial-mesenchymal contact. Amniotic membrane alone does not induce this effect and can help block such induction by epithelial cells.     

Increased SPARC accumulation during corneal repair

Keratocytes can become fibroblasts and myofibroblasts during corneal injury and wound healing. We used the in vitro bovine keratocyte repair model system, which involves culturing collagenase-isolated keratocytes in serum-free media and then adding serum or serum plus TGF-beta to the culture media to induce the fibroblast and myofibroblast phenotypes, respectively, to evaluate the synthesis of secreted products by the cells. Serum and serum plus TGF-beta rapidly induced the fibroblast morphology and alpha smooth muscle actin, a marker of myofibroblasts. Keratocytes cultured in serum and serum plus TGF-beta also increased the synthesis of several high molecular weight products (approximately 100kD and larger) and the accumulation of a 43kD protein shown to be osteonectin/SPARC by both sequencing tryptic peptides from the protein and by reaction with antisera to osteonectin/SPARC. Immunohistochemical staining of mouse corneas with antisera to SPARC seven days post-wounding also demonstrated an increased accumulation of SPARC in the regions undergoing repair. These results indicate SPARC accumulation is a marker for stromal repair.     

Stress fiber formation is required for matrix reorganization in a corneal myofibroblast cell line

Corneal wound healing fibroblasts (myofibroblasts) develop a muscle-like contractile apparatus composed of prominent microfilament bundles (stress fibers) and express alpha-smooth muscle actin (alpha-SMA). In this study, gelsolin, an actin filament-severing protein, was overexpressed in a alpha-SMA-expressing corneal myofibroblast cell line (TRK43) to assess whether intact stress fibers are required for in vitro matrix organization and wound contraction. Stably integrated gelsolin was introduced by electroporation of an expression construct (pREPCG8) into cultured cells. Thirty-seven clones were isolated with half of the clones showing a fibroblastic phenotype while the remaining half appeared epithelioid. One fibroblastic clone, GS56, and one epithelioid clone, GS44, were selected for detailed characterization. The GS56 cells appeared highly elongated and spindle-shaped and had prominent stress fibers and focal adhesions. GS44 cells showed disruption of stress fibers and a cortical f-actin organization as well as the down regulation of alpha-SMA expression by immunocytochemistry and Western blotting. Both phenotypes showed enhanced gelsolin expression; however, fractionation of cell extracts demonstrated differences in the subcellular distribution of gelsolin with GS44 cells having markedly reduced and GS56 cells having markedly increased cytoskeletal gelsolin. In an in vitro wound contraction assay, epithelioid GS44 cells showed a significantly impaired ability to contract a collagen matrix compared to that of TRK43 cells, CT9 or GS56 transfectants. Loss of stress fibers in GS44 cells also correlated with enhanced cell motility. Together, these results demonstrate that the ability to form microfilament bundles or stress fibers is required for matrix organization and contraction by corneal myofibroblasts. Although no clear explanation is available, we suspect that differences in gene insertion of the gelsolin overexpression vector may have led to differential intercellular localization of gelsolin and its effect on stress fiber formation in the two cell lines.     

Serum-free spheroid culture of mouse corneal keratocytes

PURPOSE: To develop a serum-free mass culture system for mouse keratocytes. METHODS: Corneas of C57BL6/J mice were enzyme digested after the epithelium and endothelium were removed. Stromal cells were cultured in serum-free DMEM/F12 (1:1) containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and B27 supplement. Primary spheres were dissociated by trypsin and subcultured as suspended secondary spheres. Cells from postnatal day (P)6 to P10 spheres were subcultured onto plastic dishes or type I collagen gels for phenotype analysis. The expression of the keratocyte markers keratocan, aldehyde dehydrogenase (Aldh), and CD34, were analyzed by RT-PCR, and vimentin and alpha-smooth muscle actin (alpha-SMA) were examined by immunocytochemistry. RESULTS: Primary keratocytes formed spheres, which were cultured for over 12 passages. Suspended sphere cells expressed vimentin, keratocan, CD34, and lumican, but were negative for cytokeratin K12 (K12) and Pax6. Sphere cells subcultured on plastic exhibited a dendritic morphology characteristic of keratocytes, and maintained keratocan, Aldh, and CD34 expression in serum-free medium. Sphere cells subcultured with 10% serum became fibroblastic, and expressed alpha-SMA when stimulated by transforming growth factor (TGF)-beta. alpha-SMA-positive cells demonstrated contractile properties on collagen gels, compatible with the myofibroblast phenotype. CONCLUSIONS: The phenotype of mouse keratocytes can be maintained in vitro for more than 12 passages by the serum-free sphere culturing technique.