Aquaporin-1-facilitated keratocyte migration in cell culture and in vivo corneal wound healing models

Aquaporin-1 (AQP1) water channels are expressed in corneal keratocytes, which become activated and migrate following corneal wounding. The purpose of this study was to investigate the role of AQP1 in keratocyte migration. Keratocyte primary cell cultures from wildtype and AQP1-null mice were compared, as well as keratocyte cultures from pig cornea in which AQP1 expression was modulated by RNAi knockdown and adenovirus-mediated overexpression. AQP1 expression was found in a plasma membrane pattern in corneal stromal and cultured keratocytes. Osmotic water permeability, as measured by calcein fluorescence quenching, was AQP1-dependent in cultured keratocytes, as was keratocyte migration following a scratch wound. Keratocyte migration in vivo was compared in wildtype and AQP1 knockout mice by histology and immunofluorescence of corneal sections at different times after partial-thickness corneal stromal debridement. AQP1 expression in keratocytes was increased by 24 h after corneal debridement. Wound healing and keratocyte appearance near the wound margin were significantly reduced in AQP1 knockout mice, and the number of neutrophils was increased. These results implicate AQP1 water permeability as a new determinant of keratocyte migration in cornea.     

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.     

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.     

Comparison of stromal remodeling and keratocyte response after corneal incision and photorefractive keratectomy]

PURPOSE: We investigated the keratocyte response and stromal remodeling after corneal incision and photorefractive keratectomy, respectively to learn the difference between the two surgeries histophysiologically and immunohistochemically. METHODS: We performed corneal incision or photorefractive keratectomy in rabbits or rats, and then we chronologically observed the histological changes and the changes in localization of extracellular matrix proteins. RESULTS: In both types of surgery, the keratocyte population in the damaged stroma became sparse, and the cells were undergoing apoptosis immediately after the procedures. After that, activated keratocytes migrated into the acellular zone, and the cells formed multiple layers at the resurfaced subepithelial regions. Deposition of amorphous substances was observed between migrated keratocytes, and stromal remodeling began. Three months after the surgery, corneal structure had recovered to near normal condition in the corneal incisions. In photorefractive keratectomy, however, strong immunoreactivity of extracellular matrix proteins was observed in the subepithelial regions. CONCLUSIONS: These results suggested that stromal wound healing processes were similar in both corneal incision and photorefractive keratectomy. Corneal incision may induce transient keratocyte response during the stromal remodeling, but photorefractive keratectomy may induce sustained keratocyte response.     

Comparison of stromal remodeling and keratocyte response after corneal incision and photorefractive keratectomy

PURPOSE: We investigated the keratocyte response and stromal remodeling after corneal incision and photorefractive keratectomy to understand the histophysiological and immunohistochemical differences between these two types of surgery. METHODS: Corneal incision or photorefractive keratectomy was performed in rabbits or rats. Then we chronologically observed the histological changes and the changes in the localization of extracellular matrix proteins. RESULTS: In both types of surgery, the keratocyte population at the damaged stroma became sparse, and the cells began undergoing apoptosis immediately after the surgical procedure. Subsequently, activated keratocytes migrated into the acellular zone, and the cells formed multiple layers at the resurfaced subepithelial regions. We observed deposition of amorphous substances between keratocytes that had migrated, and stromal remodeling appeared to start. Three months after surgery, the corneal structure had recovered to a near-normal condition at the corneal incision. After photorefractive keratectomy, however, extracellular matrix proteins were strongly immunoreactive at the subepithelial regions. CONCLUSIONS: These results suggest that the stromal wound-healing processes are similar after corneal incision and photorefractive keratectomy. A corneal incision may induce a transient keratocyte response during stromal remodeling, but photorefractive keratectomy may induce a sustained keratocyte response.     

Postnatal corneal transparency, keratocyte cell cycle exit and expression of ALDH1A1

PURPOSE: Recent studies have shown that rabbit corneal keratocytes abundantly express two water-soluble proteins, transketolase (TKT) and aldehyde dehydrogenase class 1A1 (ALDH1A1), in vivo and that these proteins may contribute to corneal transparency at the cellular level. The purpose of this study was to determine the relationship between the expression of these proteins and the development of postnatal corneal transparency. METHODS: Rabbits 1 day to 42 days of postnatal age were evaluated by in vivo confocal microscopy (CM) to measure corneal epithelial thickness, stromal thickness, and corneal haze. Selected corneas were then processed for immunocytochemistry and Western and Northern blot analyses, to determine stromal cell density, cell cycle entry, and expression of ALDH1A1 and TKT. RESULTS: Quantitative measurement of corneal haze showed that the postnatal cornea was hazy after birth and became transparent during the first weeks after eyelid opening. Development of transparency was associated with decreased cytoplasmic light-scattering from postnatal corneal stromal cells, with the appearance of nuclear light-scattering after eyelid opening. Four days after birth, stromal cell density decreased rapidly, and the cells became quiescent, showing decreased staining by Ki67, a cell cycle marker. Whereas expression of TKT showed a gradual increase after birth, ALDH1A1 showed a marked increase after eyelid opening, and the combined expression significantly correlated with the reduction in light-scattering by postnatal stromal cells. CONCLUSIONS: The data suggest that development of postnatal corneal transparency is associated with decreased keratocyte density and quiescence and the expression of TKT/ALDH1A1.     

IGF-II and collagen expression by keratocytes during postnatal development

Keratocytes produce the extensive stromal matrix of the cornea during the late embryonic and neonatal time periods. We propose to test the hypothesis that their biosynthetic activity declines during this process. Keratocytes were isolated from corneas of 6-8-week-old rabbits and corneas of 1-2-year-old cows and their ability to proliferate and synthesize collagen in serum-free media was determined. Rabbit keratocyte cultures increased 38% in DNA content after one week and deposited collagen type I and IGF-II in the media. Bovine keratocyte cultures, in contrast, did not increase in DNA or produce detectable collagen and IGF-II. Bovine keratocytes cultured in media previously conditioned by rabbit keratocytes, however, increased 56% in DNA content, and deposited collagen type I into the media. Microarray analysis of mRNA from neonatal and adult mouse keratocytes was used to confirm these differences. Compared to adult mouse keratocytes, neonatal keratocytes showed high expression levels of IGF-I, IGF-II and collagen types III and V. Since previous studies showed that IGFs stimulate bovine keratocytes to proliferate and to synthesize procollagen type I, we therefore propose that the results of this study suggests that the IGFs may play an important role in regulating early corneal growth in vivo.     

Hepatocyte growth factor and keratinocyte growth factor regulation of epithelial and stromal corneal wound healing

PURPOSE: To investigate the effects of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) on early wound healing in the corneal epithelium and stroma. SETTING: Cell and Molecular Biology Unit, Department of Optometry and Vision Sciences, Cardiff University, and the Cardiff Institute of Tissue Engineering and Repair, Cardiff, United Kingdom. METHODS: Corneal keratocyte cell cultures and wounded corneal organ cultures (both maintained in serum-free conditions) were treated with 0.1 to 100 ng/mL of HGF or KGF for up to 5 days. Cell cultures were assessed for proliferation, migration, and differentiation into myofibroblasts. Organ cultures were used to evaluate the effect of HGF and KGF on reepithelialization following a wound, epithelial morphology and stratification, keratocyte numbers directly beneath the wounded area, and differentiation into myofibroblasts. RESULTS: The 2 growth factors had opposite effects on the rate of reepithelialization, with HGF delaying and KGF accelerating epithelial coverage of the wound. Morphologic assessment showed that both growth factors affected the stratification and differentiation of the epithelium. Both factors stimulated proliferation of keratocytes in serum-free cell culture, although neither induced the appearance of myofibroblasts. This was in contrast to wounded organ cultures treated with 100 ng/mL HGF, in which large numbers of myofibroblasts were observed under the wound. Control corneas and those receiving KGF contained very few myofibroblasts. Keratocyte repopulation of the denuded area under the wound was enhanced in the presence of HGF but decreased in response to KGF. CONCLUSIONS: Hepatocyte growth factor and KGF appeared to have potent and often opposite effects on epithelial and stromal cells following a wound. Hepatocyte growth factor was more detrimental than KGF, resulting in an aberrant epithelium and mass differentiation of keratocytes into myofibroblasts. Inhibition of HGF may be an appropriate therapeutic intervention in the case of persistent epithelial defects and to prevent fibrosis following a corneal stromal wound such as can occur after refractive surgery.     

Sphere formation from corneal keratocytes and phenotype specific markers

The keratocytes are specialized mesenchymal cells that produce and maintain the extracellular matrix of the corneal stroma. With a typical dendritic and flattened appearance, these cells can morph into fibroblasts and myofibroblasts upon injury, and produce abnormal or fibrotic extracellular matrices detrimental to corneal transparency. Insights into mechanisms that regulate these phenotypic switches and optimal culture conditions that preserve the keratocyte phenotype are important for tissue engineering of the corneal stroma. Like other cell types with self-renewing capacity, keratocytes can form spheres in culture. Here we investigated human and bovine keratocytes with respect to their sphere forming capabilities, and sought to identify potentially distinguishing markers for the keratocyte and fibroblast phenotypes. Keratocytes, isolated from bovine and human corneas, cultured in serum-free medium supplemented with insulin, selenium and transferrin, assumed typical keratocyte morphology, converted to fibroblasts in serum-containing medium and reverted to keratocytes after serum-deprivation. The bovine keratocytes produced spheres under adherent or low attachment conditions, while the human keratocytes produced spheres under low attachment conditions only. The primary keratocytes and fibroblasts expressed vimentin, confirming their mesenchymal origin. Keratocan, considered to be a marker for keratocytes, was also detected in early passage bovine fibroblasts. BMP3 was expressed in keratocytes and keratocyte-derived spheres, while cadherin 5 in keratocytes only, suggesting these as potential keratocyte markers.     

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.     

Maspin increases extracellular plasminogen activator activity associated with corneal fibroblasts and myofibroblasts

Maspin, an inhibitor of cell migration and a stimulator of adhesion of cells to the ECM, is synthesized and released by corneal keratocytes into the extracellular matrix. When the cornea is wounded, the quiescent stromal keratocytes underlying the wound undergo apoptosis and cells adjacent to this apoptotic area convert to fibroblasts or myofibroblasts. This study explores the effect of extracellular maspin on the plasminogen–plasminogen activator system of corneal stromal cells following wounding. Treatment of corneal fibroblasts and myofibroblasts with r-maspin increased extracellular but not cell-associated tissue-type plasminogen activator (tPA), urinary-type plasminogen activator (uPA) or plasminogen activator inhibitor-1 (PAI-1). Despite the extracellular increase in PAI-1, the net effect of maspin treatment was an increase in plasminogen activation. At physiological levels, maspin did not alter uPA or tPA mRNA levels, in these cells. The increase in pro and active uPA was due to decreased clearance in the presence of maspin for myofibroblasts but not for fibroblasts. The clearance of pro and active tPA was normal in fibroblasts indicating different mechanisms for the increase of these homologous enzymes in the two cell types. Increased generation of plasmin by maspin treated corneal stromal fibroblasts and myofibroblasts led to conversion of plasminogen to active plasmin degradation products and angiostatin-like molecules. This study suggests that extracellular maspin increased pro and active uPA and tPA released by corneal fibroblasts and myofibroblasts on the short time scale of 1–4 h, but by 24 h there was no increase over the levels produced without maspin. This augmentation of plasminogen activator activity increases plasmin activation and angiostatin generation. It further indicates that the effect of maspin on uPA and tPA levels is cell type dependent.     

Corneal stromal wound healing: Major regulators and therapeutic targets

Corneal stromal wound healing is a complex event that occurs to restore the transparency of an injured cornea. It involves immediate apoptosis of keratocytes followed by their activation, proliferation, migration, and trans-differentiation to myofibroblasts. Myofibroblasts contract to close the wound and secrete extracellular matrix and proteinases to remodel it. Released proteinases may degenerate the basement membrane allowing an influx of cytokines from overlying epithelium. Immune cells infiltrate the wound to clear cellular debris and prevent infections. Gradually basement membrane regenerates, myofibroblasts and immune cells disappear, abnormal matrix is resorbed, and transparency of the cornea is restored. Often this cascade deregulates and corneal opacity results. Factors that prevent corneal opacity after an injury have always intrigued the researchers. They hold clinical relevance as they can guide the outcomes of corneal surgeries. Studies in the past have shed light on the role of various factors in stromal healing. TGFβ (transforming growth factor-beta) signaling is the central player guiding stromal responses. Other major regulators include myofibroblasts, basement membrane, collagen fibrils, small leucine-rich proteoglycans, biophysical cues, proteins derived from extracellular matrix, and membrane channels. The knowledge about their roles helped to develop novel therapies to prevent corneal opacity. This article reviews the role of major regulators that determine the outcome of stromal healing. It also discusses emerging therapies that modulate the role of these regulators to prevent stromal opacity.     

Corneal keratocytes: phenotypic and species differences in abundant protein expression and in vitro light-scattering

PURPOSE: Previous studies suggest that corneal haze after injury involves changes in the light-scattering properties of keratocytes that are possibly linked to the abundant expression of water-soluble proteins. The purpose of this study was to determine the protein expression pattern of keratocytes from different species and different cultured rabbit keratocyte phenotypes and to assess differences in light-scattering in vitro. METHODS: Water-soluble proteins were isolated from corneal epithelial cells and keratocytes of several species, including human (Hu), mouse (Mo), rabbit (Ra), chicken (Ch), and pig (P) and different cultured rabbit keratocyte phenotypes. Proteins were then characterized by SDS-PAGE, tryptic peptide sequence analysis, and Western blot analysis. Light-scattering and actin organization from cultured cells were determined with confocal reflectance and fluorescence microscopy, respectively. RESULTS: Protein expression patterns varied substantially between species and cell types, with five new abundantly expressed proteins identified including, LDH (Ra, Ch), G3PDH (Hu, Ch), pyruvate kinase (Ch), Annexin II (Ch), and protein disulfide isomerase (Ch). Different rabbit keratocyte phenotypes also showed different levels of expression of ALDH1A1 and TKT, with myofibroblasts showing the greatest reduction. Myofibroblasts showed significantly greater (P < 0.05) light-scattering but also showed the greatest organization of actin filaments. CONCLUSIONS: Abundant protein expression is a characteristic feature of corneal keratocytes that is lost when cells are phenotypically modulated in culture. Greater light-scattering by myofibroblasts also provides support for a link between cellular transparency and haze after injury that is possibly related to loss of protein expression or development of prominent actin filament bundles.     

Immunohistochemical study of subepithelial haze after phototherapeutic keratectomy

PURPOSE: Subepithelial haze is a frequent complication and is often the cause of regression after photorefractive keratectomy (PRK). The lack of understanding of this undesirable complication following PRK is in part due to the limited availability of suitable tissues for pathological studies. METHODS: We examined the expression of various extracellular components in the cornea of a 46-year-old man who underwent phototherapeutic keratectomy (PTK) to remove a central corneal scar secondary to trauma. The patient subsequently underwent penetrating keratoplasty. A scar-free region containing an area of slight subepithelial haze adjacent to normal cornea was used for immunohistochemical staining with antibodies directed against cytoskeletal proteins, ie, vimentin, desmin and smooth muscle actin, and the extracellular components, laminin, heparan sulfate, keratan sulfate, and collagen types III, IV, V, and VII. RESULTS: Immunohistochemistry revealed that basal epithelial cells expressed components of basement membrane. The stromal fibroblasts within the haze tissue were labeled by anti-smooth muscle actin antibodies, a characteristic of myofibroblasts, which synthesized and secreted extracellular matrix components that contributed to the formation of the disorganized collagenous matrix and may account for subepithelial haze. CONCLUSIONS: The expression patterns for the cytoskeletal proteins and extracellular components indicated that the formation of subepithelial haze is a process of tissue remodeling, involving both corneal basal epithelial cells and keratocytes during wound repair.     

Monoclonal antibody (3G5)-defined ganglioside: cell surface marker of corneal keratocytes

PURPOSE: To evaluate the anti-ganglioside monoclonal antibody 3G5 as a marker of corneal keratocytes. METHODS: 3G5 expression on keratocytes was investigated by immunofluorescence microscopy. Studies were performed on frozen sections of normal human, bovine, porcine, rabbit, rat, and mouse corneas and on repairing rabbit cornea. In vitro studies were performed on tissue-cultured human, bovine, porcine, mouse, and rabbit keratocytes. RESULTS: 3G5 stained frozen sections of human, bovine, porcine, rat, and rabbit cornea but not mouse cornea and the staining pattern followed the distribution of stromal keratocytes but did not stain epithelium or endothelium. Subconfluent human and bovine keratocyte cultures were 3G5 negative. Almost 100% of the human and bovine cells that were maintained at confluence without replacement of serum-containing culture medium for 2 weeks became 3G5 positive. The 3G5 antigen was constitutively expressed on cultured rabbit and porcine keratocytes under all conditions examined. Mouse keratocyte cultures did not express 3G5. The 3G5 antigen was not present on myofibroblastic cells in the repairing area of a full-thickness wound in rabbit cornea that had been healing for 20 days. The area surrounding the healing wound expressed 3G5 antigen in an altered distribution, whereas 3G5 antigen was distributed in the expected pattern in areas that were distant from the wound. When rabbit keratocytes were induced to express the myofibroblast marker alpha-smooth muscle actin by treatment with TGFbeta1 in vitro, the pattern of 3G5 staining was altered. CONCLUSIONS: The 3G5 antigen is a useful marker for the identification of corneal keratocytes and for documenting their response to environmental stimuli associated with wound repair.     

Corneal stromal wound healing in refractive surgery: the role of myofibroblasts

While laser and incisional refractive surgery offer the promise to correct visual refractive errors permanently and predictably, variability and complications continue to hinder wide-spread acceptance. To explain variations, recent studies have focused on the role of corneal wound healing in modulating refractive outcomes. As our understanding of the corneal response to refractive surgery broadens, it has become apparent that the response of one cell, the corneal stromal keratocyte, plays a pivotal role in defining the results of refractive surgery. Studies reviewed herein demonstrate that injury-induced activation and transformation of keratocytes to myofibroblasts control the deposition and organization of extracellular matrix in corneal wounds. Myofibroblasts establish an interconnected meshwork of cells and extracellular matrix that deposits new matrix and contracts wounds using a novel and unexpected "shoe-string-like" mechanism. Transformation of keratocytes to myofibroblasts is induced in culture by transforming growth factor beta (TGFbeta) and blocked in vivo by antibodies to TGFbeta. Overall, myofibroblast appearance in corneal wounds is associated with wound contraction and regression following incisional keratotomy and the development of "haze" or increased scattered light following laser photorefractive keratectomy (PRK). By contrast, absence of myofibroblasts is associated with continued widening of wound gape and progressive corneal flattening after incisional procedures. Based on these studies, we have arrived at the inescapable conclusion that a better understanding of the cellular and molecular biology of this one cell is required if refractive surgery is ever to achieve predictable and safe refractive results.     

Localization of thrombospondin-1 and myofibroblasts during corneal wound repair

Thrombospondin-1 (TSP-1) is a multifunctional matrix protein that has recently been examined in various wound processes, primarily for its ability to activate the latent complex of transforming growth factor-beta (TGF-β). TGF-β has been shown to play a major role in stimulating mesenchymal cells to synthesize extracellular matrix. After injury, corneal keratocytes become activated and transform into fibroblasts and myofibroblasts. Our hypothesis is that TSP-1 regulates the transformation of keratocytes into myofibroblasts (MF) via TGF-β. In the current study, we examined the expression of TSP-1 and α-smooth muscle actin (SMA), a marker of MF, during rat corneal wound healing. Three-mm keratectomy or debridement wounds were made in the central rat cornea and allowed to heal from 8 hours to 8 weeks in vivo. Unwounded rat corneas served as controls. Expression of TSP-1, SMA and Ki67, a marker of proliferating cells, were examined by indirect-immunofluorescence microscopy. In unwounded corneas, TSP-1 expression was observed primarily in the endothelium. No expression was seen in the stroma, and only low levels were detected in the epithelium. Ki67 was localized in the epithelial basal cells and no SMA was present in the central cornea of unwounded eyes. After keratectomy wounds, TSP-1 expression was seen 24 h after wounding in the stroma immediately subjacent to the wound-healing epithelium. The expression of TSP-1 increased daily and peaked 7–8 days after wounding. SMA expression, however, was not observed until 3–4 days after wounding. Interestingly, SMA-positive cells were almost exclusively seen in the stromal zone expressing TSP-1. Peak expression of SMA-positive cells was observed 7–8 days after wounding. Ki67-expressing cells were seen both in the area expressing TSP-1 and the adjacent area. In the debridement wounds, no SMA expressing cells were observed at any time point. TSP-1 was localized in the basement membrane zone from 2 to 5 days after wounding, and the localization did not appear to penetrate into the stroma. These data are in agreement with our hypothesis that TSP-1 localization in the stromal matrix is involved in the transformation of keratocytes into myofibroblasts.     

Effects of tranilast on cultured rabbit corneal keratocytes and corneal haze after photorefractive keratectomy

PURPOSE: In vitro and in vivo studies were performed to elucidate the effects of tranilast on cellular proliferation and collagen synthesis. METHODS: Subculturing was carried out using keratocytes from rabbits that underwent photorefractive keratectomy (PRK) and developed corneal haze, and keratocytes from normal rabbit cornea. RESULTS: Tranilast suppressed proliferation in cultured keratocytes from the corneal haze region at doses of 30 and 300 micromol/L and collagen synthesis at doses of 3, 30, and 300 micromol/L. Normal corneal cultures showed suppression of keratocyte proliferation and collagen synthesis only at a high dose of tranilast (300 micromol/L). Betamethasone suppressed proliferation of keratocytes in both haze and normal cornea at a dose of 10 micromol/L, as well as collagen synthesis at respective doses of 1 and 10 micromol/L. Diclofenac sodium suppressed collagen synthesis of keratocytes in haze cornea at a high dose of 100 micromol/L, and in keratocytes in normal cornea, at doses of 10 and 100 micromol/L. In an in vivo study, either 0.5% tranilast, 0.1% betamethasone phosphate eye drops, or a tranilast base solution (control) was instilled four times daily to rabbits that had undergone PRK. Weekly evaluation of the inhibitory effect of these drugs on the development of haze was performed 2 weeks after surgery. Tranilast suppressed haze 6-13 weeks after PRK, but betamethasone phosphate showed no effect. CONCLUSION: These results indicate that tranilast is potentially effective for inhibiting the corneal haze that occurs after PRK.     

Corneal Opacity: Cell Biological Determinants of the Transition From Transparency to Transient Haze to Scarring Fibrosis,and Resolution,After Injury

PurposeTo highlight the cellular, matrix, and hydration changes associated with opacity that occurs in the corneal stroma after injury.MethodsReview of the literature.ResultsThe regulated transition of keratocytes to corneal fibroblasts and myofibroblasts, and of bone marrow-derived fibrocytes to myofibroblasts, is in large part modulated by transforming growth factor beta (TGFβ) entry into the stroma after injury to the epithelial basement membrane (EBM) and/or Descemet''s membrane. The composition, stoichiometry, and organization of the stromal extracellular matrix components and water is altered by corneal fibroblast and myofibroblast production of large amounts of collagen type I and other extracellular matrix components—resulting in varying levels of stromal opacity, depending on the intensity of the healing response. Regeneration of EBM and/or Descemet''s membrane, and stromal cell production of non-EBM collagen type IV, reestablishes control of TGFβ entry and activity, and triggers TGFβ-dependent myofibroblast apoptosis. Eventually, corneal fibroblasts also disappear, and repopulating keratocytes reorganize the disordered extracellular matrix to reestablish transparency.ConclusionsInjuries to the cornea produce varying amounts of corneal opacity depending on the magnitude of cellular and molecular responses to injury. The EBM and Descemet''s membrane are key regulators of stromal cellularity through their modulation of TGFβ. After injury to the cornea, depending on the severity of the insult, and possibly genetic factors, trace opacity to severe scarring fibrosis develops. Stromal cellularity, and the functions of different cell types, are the major determinants of the level of the stromal opacity.