Genomics of corneal wound healing: a review of the literature

Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.     

Stromal-epithelial interactions in the cornea

Stromal-epithelial interactions are key determinants of corneal function. Bi-directional communications occur in a highly coordinated manner between these corneal tissues during normal development, homeostasis, and wound healing. The best characterized stromal to epithelial interactions in the cornea are mediated by the classical paracrine mediators hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF). HGF and KGF are produced by the keratocytes to regulate proliferation, motility, differentiation, and possibly other functions, of epithelial cells. Other cytokines produced by keratocytes may also contribute to these interactions. Epithelial to stromal interactions are mediated by cytokines, such as interleukin-1 (IL-1) and soluble Fas ligand, that are released by corneal epithelial cells in response to injury. Other, yet to be identified, cytokine systems may be released from the unwounded corneal epithelium to regulate keratocyte viability and function. IL-1 appears to be a master regulator of corneal wound healing that modulates functions such as matrix metalloproteinase production, HGF and KGF production, and apoptosis of keratocyte cells following injury. The Fas/Fas ligand system has been shown to contribute to the immune privileged status of the cornea. However, this cytokine-receptor system probably also modulates corneal cell apoptosis following infection by viruses such as herpes simplex and wounding. Pharmacologic control of stromal-epithelial interactions appears to offer the potential to regulate corneal wound healing and, possibly, treat corneal diseases in which these interactions have a central role.     

Progress in corneal wound healing

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β (TGF-β) system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells.     

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.     

Corneal cells: chatty in development,homeostasis, wound healing,and disease

PURPOSE: To provide an overview of cell-cell interactions in the cornea that have a critical role in corneal development, homeostasis, wound healing, and disease. DESIGN: Review. METHODS: Review of the literature. RESULTS; Cell-cell interactions make critical contributions to development, homeostasis, and wound healing in the cornea. Many of these interactions are mediated by cytokines, growth factors, and chemokines. The best characterized are stromal-epithelial interactions between epithelial cells and stromal cells such as keratocytes, keratoblasts, and myofibroblasts. However, interactions also occur between corneal nerves and epithelial cells and between corneal cells (epithelial cells and stromal cells) and corneal immune cells. Although investigations are limited, it is likely that there are interactions between corneal endothelial cells and keratocytes in the posterior stroma. CONCLUSIONS: Cellular communications in the cornea are critical during development, homeostasis, and wound healing. Disorders of cellular communication likely contribute to many corneal diseases.     

Corneal alkali burn in the rabbit. Waterbalance, healing and transparency.

Healing following a standardized central corneal alkali wound was studied morphologically in New Zealand white rabbits up to one year after the initial wound. Clinical examination, light and transmission electron microscopy was performed. The study was focused on how permanent scar tissue formed. Following the penetrating alkali injury, all cells (epithelium, keratocytes and endothelium) in the wound area disappeared. The fibroblasts/keratocytes repopulated an extensively swollen central corneal stroma. Cells and extracellular matrix filled stromal lacunae in an irregular fashion and upon deswelling the lacunae remained as irregularities in the stroma, reducing the transparency. In the periphery of the wound repopulation occurred in a less swollen stroma and normal cytoarchitecture and transparency resumed. It appears that the degree of swelling decides the degree of scar tissue formation in the corneal stroma following alkali wound healing.     

Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor

PURPOSE: To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing. METHODS: A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture. RESULTS: Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator. CONCLUSIONS: The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.     

Corneal femtosecond laser keratotomy results in isolated stromal injury and favorable wound-healing response

PURPOSE: To examine the corneal repair response after intrastromal femtosecond (fs) laser keratotomy. METHODS: Twelve rabbits underwent monocular intrastromal keratotomy performed with an fs laser at a preoperatively determined corneal depth of 160 to 200 microm. The fs laser-induced corneal repair response was compared with that of nonoperated control eyes and eyes treated with photorefractive keratectomy (PRK). Follow-up examinations were performed 1, 3, 7, and 28 days after surgery. Corneas were evaluated using slit lamp, in vivo confocal microscopy, and light microscopy. The extracellular matrix components fibronectin and tenascin were located using immunofluorescence staining. Anti-Thy-1 and anti-alpha-SMA antibodies and phalloidin were used to identify repair fibroblasts. Cell proliferation and nuclear DNA fragmentation were detected using an anti-Ki-67 antibody and the TUNEL assay, respectively. RESULTS: Intrastromal fs keratotomy resulted in a hypocellular stromal scar discernible as a narrow band of increased reflectivity on slit lamp examination. Deposition of fibronectin and tenascin as well as death and subsequent proliferation of keratocytes were observed. No differentiation of keratocytes into Thy-1- or alpha-SMA-positive fibroblasts could be detected. In contrast, after PRK, which causes epithelial and stromal wounding, all markers for repair fibroblasts were found in subepithelial stromal layers. On slit lamp examination, a fibrotic scar and a corneal haze were revealed. CONCLUSIONS: Isolated stromal injury using an fs laser avoids epithelial injury and is associated with a favorable wound-healing response preserving corneal transparency. Thus, fs laser keratotomy is a highly selective laser treatment that can be useful for the treatment of refractive errors.     

角膜基质修复的机制和调控因素研究进展

角膜基质是维持角膜透明度的重要结构。外伤、感染、手术等可造成角膜基质损伤,引起修复的过程包括基质细胞表型改变、细胞外基质重塑、免疫细胞迁移。当基质严重受损,肌成纤维细胞增多和细胞外基质沉积发生基质纤维化反应,形成角膜瘢痕,是全球致盲的主要原因。目前治疗方式主要是角膜移植手术,因角膜供体资源短缺、手术技巧要求和术后移植排斥风险等治疗效果不佳。近年来,各种分子、细胞和组织对角膜基质损伤修复的调控机制取得一定研究进展。本文就角膜基质损伤修复的机制和角膜损伤原因、角膜结构、分子因素对角膜基质损伤修复的调控进行综述,为探索促进角膜基质修复和再生的途径提供新思路,希望帮助临床预防角膜瘢痕的发生。     

Extracellular matrix and growth factors in corneal wound healing

The crystal clear cornea has been challenged by refractive surgeries. The surgical outcome depends on the healing responses of the cornea. The factors responsible for the corneal wound healing have been characterized. The orchestrated action of extracellular matrix proteins, growth factors, cytokines, and their receptors have been investigated extensively over the past decade. The clinical results with refractive surgeries provide us various important information with regard to the physiology and pathology of the cornea. The role of basement membrane or Bowman's membrane is now challenged for the maintenance and repair of the epithelium. Furthermore, the interactions between epithelium and stroma is another field to be investigated. The regulatory mechanisms of the maintenance of stromal collagen by keratocytes is also studied. This review discusses the current advancement in the healing responses of the cornea to various injuries and refractive surgeries.     

Extracellular matrix and wound healing

Over the years, most researchers have approached corneal epithelial and stromal wound healing as separate events. However, it is becoming increasingly clear that even the simplest epithelial debridement wound results in keratocyte death and a subsequent stromal response to regenerate the affected area. Thus, the interaction between stromal and epithelial healing must be considered to fully understand corneal wound healing. Although wound healing has been an active area of research for many years, the advent of refractive surgery has stimulated research into the regulation of wound repair and provided important insights into the molecular components involved in repair. Epithelial and stromal wound healing are influenced by extracellular matrix components. The purpose of the current article is to review progress in the year 2000 toward understanding mechanisms involved in corneal wound healing and how extracellular matrix affects the healing processes.     

Direct epithelial–stromal interaction in corneal wound healing: Role of EMMPRIN/CD147 in MMPs induction and beyond

In the cornea, the epithelium and the underlying stroma are separated by the basement membrane and Bowman's layer. The disruption of these anatomical barriers during wound healing represents a key step which initiates tissue remodeling through the modification of the epithelial–stromal interactions (ESI). Diffusible cytokines are generally viewed as central modulators in the bidirectional communication between these epithelial and stromal compartments and their implication in all stages of the wound healing process has been an active area of research for many years. Our studies which aimed to explore mechanisms of matrix degradation in pathological corneal wound healing have shown that EMMPRIN, a glycoprotein expressed on corneal epithelial cell surface, can induce matrix metalloproteinase (MMP) production and myofibroblasts differentiation after direct interaction with corneal fibroblasts. EMMPRIN appears therefore as a potential mediator of ESI by direct cell–cell contact which represents a new mechanism for dysregulated MMPs' induction observed in corneal ulcerations. These direct epithelial–stromal interactions (direct-ESI) can occur when delayed epithelial healing prevents regeneration of the basement membrane and allows the two cell types to come into close proximity. We propose that prevention of these interactions through inhibition of EMMPRIN may represent a promising therapeutic strategy in the inhibition of MMP induction in ulceration.     

The molecular basis of corneal transparency

The cornea consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells. The stroma consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25-30 nm fibrils that are regularly packed within lamellae and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sufate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-β would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.     

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.     

RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal wound healing

PURPOSE: To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ. METHODS: Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry. RESULTS: A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury. CONCLUSIONS: Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.     

Interleukin-1 and Transforming Growth Factor Beta: Commonly Opposing,but Sometimes Supporting,Master Regulators of the Corneal Wound Healing Response to Injury

PurposeInterleukin (IL)-1α/IL-1β and transforming growth factor (TGF)β1/TGFβ2 have both been promoted as “master regulators” of the corneal wound healing response due to the large number of processes each regulates after injury or infection. The purpose of this review is to highlight the interactions between these systems in regulating corneal wound healing.MethodsWe conducted a systematic review of the literature.ResultsBoth regulator pairs bind to receptors expressed on keratocytes, corneal fibroblasts, and myofibroblasts, as well as bone marrow-derived cells that include fibrocytes. IL-1α and IL-1β modulate healing functions, such as keratocyte apoptosis, chemokine production by corneal fibroblasts, hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) production by keratocytes and corneal fibroblasts, expression of metalloproteinases and collagenases by corneal fibroblasts, and myofibroblast apoptosis. TGFβ1 and TGFβ2 stimulate the development of myofibroblasts from keratocyte and fibrocyte progenitor cells, and adequate stromal levels are requisite for the persistence of myofibroblasts. Conversely, TGFβ3, although it functions via the same TGF beta I and II receptors, may, at least in some circumstances, play a more antifibrotic role—although it also upregulates the expression of many profibrotic genes.ConclusionsThe overall effects of these two growth factor-cytokine-receptor systems in controlling the corneal wound healing response must be coordinated during the wound healing response to injury or infection. The activities of both systems must be downregulated in coordinated fashion to terminate the response to injury and eliminate fibrosis.Translational RelevanceA better standing of the IL-1 and TGFβ systems will likely lead to better approaches to control the excessive healing response to infections and injuries leading to scarring corneal fibrosis.     

Regulation of gelatinase B production in corneal cells is independent of autocrine IL-1alpha

PURPOSE: The matrix metalloproteinase gelatinase B is synthesized by cells at the leading edge of the corneal epithelium migrating to heal a wound. Recent data from the authors' laboratory suggest that excessive synthesis contributes to repair defects. The goal of the study reported here was to investigate mechanisms controlling gelatinase B production by corneal epithelial cells. METHODS: Freshly isolated cultures of corneal epithelial cells and early passage stromal fibroblasts from rabbit were used for these studies. RESULTS: In a previous study, it was found that the cytokine interleukin (IL)-1alpha is released into the culture medium of corneal epithelial cells more efficiently when they are plated at low density with limited cell-cell contact than when plated at high density. In this study, we show that production of gelatinase B by these cells is similarly affected by cell plating density. However, it is further demonstrated that these two events are not dependent on one another but occur in parallel: IL-1alpha does not regulate gelatinase B production (synthesis), nor was there evidence that any other secreted autocrine cytokine acts as mediator. Instead, our data suggest that gelatinase B production is downregulated directly by high cell density and indicate a connection to the level of protein kinase C activity. Nevertheless, the anticancer agent suramin, which blocks collagenase synthesis by interfering with autocrine cytokine-receptor interactions, still inhibits synthesis of gelatinase B. CONCLUSIONS: Unlike collagenase synthesis by corneal stromal fibroblasts, production (synthesis) of gelatinase B does not appear to be controlled by secreted autocrine cytokines but can still be inhibited by suramin. Suramin may make an effective therapeutic agent for controlling pathologic overproduction of gelatinase B in corneal ulcers.     

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.     

Corneal wound healing

PURPOSE: To examine the latest theories in understanding wound healing, primarily as it pertains to refractive surgery but also, briefly, incisional surgery and nonsurgical corneal conditions (iatrogenic injury and noninfectious corneal ulcers). RECENT FINDINGS: Corneal wound healing involves transformation of fibroblasts; intercellular signaling-for example between epithelial and stromal cells-from cytokines, neuropeptides, growth factors, and chemokines; action of matrix metalloproteinases; and protection of tissue from free radical damage. There may be a common wound healing pathway after different types of surgery and injury to the cornea-for example, the wound healing cascades after laser in situ keratomileusis and photorefractive keratectomy may be similar, and yet the effects on the cornea differ because of the extent of disruption to the basement membrane-but the outcomes may depend on small differences in the cascade. SUMMARY: Interest in the study of corneal wound healing has increased with the proliferation of keratorefractive surgery. Important contributions come from intercellular signaling, fibroblast transformation, remodeling of the extracellular matrix, and free radical scavengers. Despite these advances, a formalized, unified vision of the wound healing cascade remains elusive.     

Loss of fenamate-activated K+ current from epithelial cells during corneal wound healing.

PURPOSE: The corneal epithelium provides a barrier between the external environment and the cornea. It also serves as an ion transporting epithelium. Because of its proximity with the external environment, the corneal epithelium is frequently injured through physical or chemical insult. The purpose of this study was to determine whether corneal epithelial cell whole-cell currents change during corneal wound healing as the author of the present study has previously reported for corneal keratocytes and endothelial cells. METHODS: Rabbit corneal epithelial cells were injured by scraping, heptanol exposure, or freezing. The epithelium was allowed to heal for 12 to 74 hours. Cells were dissociated from corneas, and whole-cell currents were examined using the amphotericin-perforated-patch technique. RESULTS: Cells from the wounded corneal groups had significantly increased capacitance values, indicating increased surface area compared with that of control cells. As previously reported, the primary control whole-cell current was a fenamate-activated K+ current. An inwardly rectifying K+ current and a Cl- current were also observed. In epithelial cells from heptanol-wounded corneas, these conductances were generally unchanged. In cells from scrape- and freeze-wounded corneas, however, the fenamate-activated current was absent or significantly attenuated. CONCLUSIONS: As they do in corneal keratocytes and endothelial cells, K+ channels disappear during some models of corneal epithelial wound healing. In addition, cell capacitance, a measurement of cell surface area, increases. These results suggest that substantial K+ channel activity is not required for in vivo epithelial cell proliferation during corneal wound healing.