Molecular mechanisms regulating wound repair: Evidence for paracrine signaling from corneal epithelial cells to fibroblasts and immune cells following transient epithelial cell treatment with Mitomycin C.

In this paper, we use RNAseq to identify senescence and phagocytosis as key factors to understanding how mitomyin C (MMC) stimulates regenerative wound repair. We use conditioned media (CM) from untreated (CMC) and MMC treated (CMM) human and mouse corneal epithelial cells to show that corneal epithelial cells indirectly exposed to MMC secrete elevated levels of immunomodulatory proteins including IL-1α and TGFß1 compared to cells exposed to CMC. These factors increase epithelial and macrophage phagocytosis and promote ECM turnover. IL-1α supplementation can increase phagocytosis in control epithelial cells and attenuate TGFß1 induced αSMA expression by corneal fibroblasts. Yet, we show that epithelial cell CM contains factors besides IL-1α that regulate phagocytosis and αSMA expression by fibroblasts. Exposure to CMM also impacts the activation of bone marrow derived dendritic cells and their ability to present antigen. These in vitro studies show how a brief exposure to MMC induces corneal epithelial cells to release proteins and other factors that function in a paracrine way to enhance debris removal and enlist resident epithelial and immune cells as well as stromal fibroblasts to support regenerative and not fibrotic wound healing.

Identification of presumed corneal neuromas and microneuromas using laser-scanning in vivo confocal microscopy: a systematic review.

BACKGROUND/AIMS: This systematic review critically evaluated peer-reviewed publications describing morphological features consistent with, or using terms related to, a 'neuroma' or 'microneuroma' in the human cornea using laser-scanning in vivo confocal microscopy (IVCM). METHODS: The review was prospectively registered on PROSPERO (CRD42020160038). Comprehensive literature searches were performed in Ovid MEDLINE, Ovid Embase and the Cochrane Library in November 2019. The review included primary research studies and reviews that described laser-scanning IVCM for examining human corneal nerves. Papers had to include at least one of a pre-specified set of keyword stems, broadly related to neuromas and microneuromas, to describe a corneal nerve feature. RESULTS: Twenty-five papers (20 original studies; 5 reviews) were eligible. Three original studies evaluated corneal nerve features in healthy eyes. Most papers assessed corneal nerves in ocular and systemic conditions; seven studies did not include a control/comparator group. There was overlap in terminology used to describe nerve features in healthy and diseased corneas (eg, bulb-like/bulbous, penetration, end/s/ing). Inspection of IVCM images within the papers revealed that features termed 'neuromas' and 'microneuromas' could potentially be physiological corneal stromal-epithelial nerve penetration sites. We identified inconsistent definitions for terms, and limitations in IVCM image acquisition, sampling and/or reporting that may introduce bias and lead to inaccurate representation of physiological nerve characteristics as pathological. CONCLUSION: These findings identify a need for consistent nomenclature and definitions, and rigorous IVCM scanning and analysis protocols to clarify the prevalence of physiological, as opposed to pathological, corneal nerve features.

Uveitis-mediated immune cell invasion through the extracellular matrix of the lens capsule.

While the eye is considered an immune privileged site, its privilege is abrogated when immune cells are recruited from the surrounding vasculature in response to trauma, infection, aging, and autoimmune diseases like uveitis. Here, we investigate whether in uveitis immune cells become associated with the lens capsule and compromise its privilege in studies of C57BL/6J mice with experimental autoimmune uveitis. These studies show that at D14, the peak of uveitis in these mice, T cells, macrophages, and Ly6G/Ly6C+ immune cells associate with the lens basement membrane capsule, burrow into the capsule matrix, and remain integrated with the capsule as immune resolution is occurring at D26. 3D surface rendering image analytics of confocal z-stacks and scanning electron microscopy imaging of the lens surface show the degradation of the lens capsule as these lens-associated immune cells integrate with and invade the lens capsule, with a subset infiltrating both epithelial and fiber cell regions of lens tissue, abrogating its immune privilege. Those immune cells that remain on the surface often become entwined with a fibrillar net-like structure. Immune cell invasion of the lens capsule in uveitis has not been described previously and may play a role in induction of lens and other eye pathologies associated with autoimmunity.

Immune responses to injury and their links to eye disease.

The eye is regarded as an immune privileged site. Since the presence of a vasculature would impair vision, the vasculature of the eye is located outside of the central light path. As a result, many regions of the eye evolved mechanisms to deliver immune cells to sites of dysgenesis, injury, or in response to the many age-related pathologies. While the purpose of these immune responses is reparative or protective, cytokines released by immune cells compromise visual acuity by inducing inflammation and fibrosis. The response to traumatic or pathological injury is distinct in different regions of the eye. Age-related diseases impact both the anterior and posterior segment and lead to reduced quality of life and blindness. Here we focus attention on the role that inflammation and fibrosis play in the progression of age-related pathologies of the cornea and the lens as well as in glaucoma, the formation of epiretinal membranes, and in proliferative vitreoretinopathy.

Combination therapy of cold atmospheric plasma (CAP) with temozolomide in the treatment of U87MG glioblastoma cells.

Cold atmospheric plasma (CAP) technology, a relatively novel technique mainly investigated as a stand-alone cancer treatment method in vivo and in vitro, is being proposed for application in conjunction with chemotherapy. In this study, we explore whether CAP, an ionized gas produced in laboratory settings and that operates at near room temperature, can enhance Temozolomide (TMZ) cytotoxicity on a glioblastoma cell line (U87MG). Temozolomide is the first line of treatment for glioblastoma, one of the most aggressive brain tumors that remains incurable despite advancements with treatment modalities. The cellular response to a single CAP treatment followed by three treatments with TMZ was monitored with a cell viability assay. According to the cell viability results, CAP treatment successfully augmented the effect of a cytotoxic TMZ dose (50 µM) and further restored the effect of a non-cytotoxic TMZ dose (10 µM). Application of CAP in conjunction TMZ increased DNA damage measured by the phosphorylation of H2AX and induced G2/M cell cycle arrest. These findings were supported by additional data indicating reduced cell migration and increased αvß3 and αvß5 cell surface integrin expression as a result of combined CAP-TMZ treatment. The data presented in this study serve as evidence that CAP technology can be a suitable candidate for combination therapy with existing chemotherapeutic drugs. CAP can also be investigated in future studies for sensitizing glioblastoma cells to TMZ and other drugs available in the market.

Corneal Epithelial "Neuromas": A Case of Mistaken Identity?

Laser scanning in vivo confocal microscopy is a useful clinical tool to assess the corneal nerves in human and laboratory animals. With this new technology, the use of terms such as "neuromas" and "microneuromas" is becoming popular to describe nerve structures seen in humans. Here, we point out that the sites where stromal nerves enter the corneal epithelium are often hyperreflective and can appear dysmorphic when imaged using in vivo confocal microscopy. Furthermore, we clarify what is known anatomically about how the nerves enter the corneal epithelium from the stroma, and we urge colleagues to differentiate between hyperreflective foci at the corneal stromal-epithelial nerve penetration sites and alterations in nerve morphology secondary to injury or disease.

Transient Mitomycin C-treatment of human corneal epithelial cells and fibroblasts alters cell migration, cytokine secretion, and matrix accumulation.

A single application of Mitomycin C (MMC) is used clinically in ophthalmology to reduce scarring and enhance wound resolution after surgery. Here we show in vitro that a 3-hour MMC treatment of primary and telomerase immortalized human corneal limbal epithelial (HCLE) cells impacts their migration and adhesion. Transient MMC treatment induces HCLE expression of senescence associated secretory factors, cytokine secretion, and deposition of laminin 332 for several days. Transient MMC treatment also reduces migration and deposition of transforming growth factor-ß1 (TGFß1)-stimulated collagen by corneal fibroblasts. Using conditioned media from control and MMC treated cells, we demonstrate that factors secreted by MMC-treated corneal epithelial cells attenuate collagen deposition by HCFs whereas those secreted by MMC-treated HCFs do not. These studies are the first to probe the roles played by corneal epithelial cells in reducing collagen deposition by corneal fibroblasts in response to MMC.

Reduced Corneal Innervation in the CD25 Null Model of Sjögren Syndrome.

Decreased corneal innervation is frequent in patients with Sjögren Syndrome (SS). To investigate the density and morphology of the intraepithelial corneal nerves (ICNs), corneal sensitivity, epithelial cell proliferation, and changes in mRNA expression of genes that are involved in autophagy and axon targeting and extension were assessed using the IL-2 receptor alpha chain (CD25 null) model of SS. ICN density and thickness in male and female wt and CD25 null corneas were assessed at 4, 6, 8, and 10/11 wk of age. Cell proliferation was assessed using ki67. Mechanical corneal sensitivity was measured. Quantitative PCR was performed to quantify expression of beclin 1, LC3, Lamp-1, Lamp-2, CXCL-1, BDNF, NTN1, DCC, Unc5b1, Efna4, Efna5, Rgma, and p21 in corneal epithelial mRNA. A significant reduction in corneal axon density and mechanical sensitivity were observed, which negatively correlate with epithelial cell proliferation. CD25 null mice have increased expression of genes regulating autophagy (beclin-1, LC3, LAMP-1, LAMP-2, CXCL1, and BDNF) and no change was observed in genes that were related to axonal targeting and extension. Decreased anatomic corneal innervation in the CD25 null SS model is accompanied by reduced corneal sensitivity, increased corneal epithelial cell proliferation, and increased expression of genes regulating phagocytosis and autophagy.

Molecular basis of Mitomycin C enhanced corneal sensory nerve repair after debridement wounding.

The ocular surface is covered by stratified squamous corneal epithelial cells that are in cell:cell contact with the axonal membranes of a dense collection of sensory nerve fibers that act as sentinels to detect chemical and mechanical injuries which could lead to blindness. The sheerness of the cornea makes it susceptible to superficial abrasions and recurrent erosions which demand continuous regrowth of the axons throughout life. We showed previously that topical application of the antibiotic and anticancer drug Mitomycin C (MMC) enhances reinnervation of the corneal nerves and reduces recurrent erosions in mice via an unknown mechanism. Here we show using RNA-seq and confocal imaging that wounding the corneal epithelium by debridement upregulates proteases and protease inhibitors within the epithelium and leads to stromal nerve disruption. MMC attenuates these effects after debridement injury by increasing serpine1 gene and protein expression preserving L1CAM on axon surfaces of reinnervating sensory nerves. These data demonstrate at the molecular level that gene expression changes in the corneal epithelium and stroma modulate sensory axon integrity. By preserving the ability of axons to adhere to corneal epithelial cells, MMC enhances sensory nerve recovery after mechanical debridement injury.

Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells.

Cold atmospheric plasma (CAP), an ionized gas operated at near-ambient temperatures, has been introduced as a new therapeutic opportunity for treating cancers. The effectiveness of the therapy has been linked to CAP-generated reactive oxygen and nitrogen species such as hydrogen peroxide and nitrite. In this study, we monitor in real-time cancer cell response to CAP over the course of 48 h. The results demonstrate a correlation between cell viability, exposure time (30, 60, 90, and 180 s), and discharge voltage (3.16 and 3.71 kV), while stressing the likely therapeutic role of plasma-generated reactive species. A 30-60 s increase in CAP exposure time and/or a discharge voltage adjustment from 3.16 to 3.71 kV is consistently accompanied by a significant reduction in cell viability. Comparably, levels of hydrogen peroxide and nitrite vary as a function of voltage with elevated levels detected at the highest tested voltage condition of 3.71 kV. CAP ultimately initiates a reduction in cell viability and triggers apoptosis via damage to the mitochondrial membrane, while also deregulating protein synthesis. The findings presented in this study are discussed in the context of facilitating the development of an adaptive CAP platform which could improve treatment outcomes.

EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling.

Purpose: Progenitor cells of the limbal epithelium reside in a discrete area peripheral to the more differentiated corneal epithelium and maintain tissue homeostasis. What regulates the limbal-corneal epithelial boundary is a major unanswered question. Ephrin-A1 ligand is enriched in the limbal epithelium, whereas EphA2 receptor is concentrated in the corneal epithelium. This reciprocal pattern led us to assess the role of ephrin-A1 and EphA2 in limbal-corneal epithelial boundary organization. Methods: EphA2-expressing corneal epithelial cells engineered to express ephrin-A1 were used to study boundary formation in vitro in a manner that mimicked the relative abundance of these juxtamembrane signaling proteins in the limbal and corneal epithelium in vivo. Interaction of these two distinct cell populations following initial seeding into discrete culture compartments was assessed by live cell imaging. Immunofluoresence and immunoblotting was used to evaluate the contribution of downstream growth factor signaling and cell-cell adhesion systems to boundary formation at sites of heterotypic contact between ephrin-A1 and EphA2 expressing cells. Results: Ephrin-A1-expressing cells impeded and reversed the migration of EphA2-expressing corneal epithelial cells upon heterotypic contact formation leading to coordinated migration of the two cell populations in the direction of an ephrin-A1-expressing leading front. Genetic silencing and pharmacologic inhibitor studies demonstrated that the ability of ephrin-A1 to direct migration of EphA2-expressing cells depended on an a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and epidermal growth factor receptor (EGFR) signaling pathway that limited E-cadherin-mediated adhesion at heterotypic boundaries. Conclusions: Ephrin-A1/EphA2 signaling complexes play a key role in limbal-corneal epithelial compartmentalization and the response of these tissues to injury.

Topical Mitomycin-C enhances subbasal nerve regeneration and reduces erosion frequency in the debridement wounded mouse cornea.

Corneal epithelial basement membrane dystrophies and superficial injuries caused by scratches can lead to recurrent corneal erosion syndrome (RCES). Patients and animals with reduced corneal sensory nerve innervation can also develop recurrent erosions. Multiple wild-type mouse strains will spontaneously develop recurrent corneal erosions after single 1.5 mm debridement wounds. Here we show that this wound is accompanied by an increase in corneal epithelial cell proliferation after wound closure but without a commensurate increase in corneal epithelial thickness. We investigated whether excess corneal epithelial cell proliferation contributes to erosion formation. We found that topical application of Mitomycin C (MMC), a drug used clinically to improve healing after glaucoma and refractive surgery, reduces erosion frequency, enhances subbasal axon density to levels seen in unwounded corneas, and prevents excess epithelial cell proliferation after debridement wounding. These results suggest that topically applied MMC, which successfully reduces corneal haze and scarring after PRK, may also function to enhance subbasal nerve regeneration and epithelial adhesion when used to treat RCES.

Partial denervation of sub-basal axons persists following debridement wounds to the mouse cornea.

Although sensory reinnervation occurs after injury in the peripheral nervous system, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify sub-basal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of sub-basal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7 days after superficial trephination, sub-basal axon density returns to control levels; by 28 days the vortex reforms. Although axon density is similar to control 14 days after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14 days, axons retract from the center leaving the sub-basal axon density reduced by 37.2 and 36.8% at 28 days after dulled blade and rotating burr wounding, respectively, compared with control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration-associated genes involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7 days after injury and by 14 and 28 days after wounding, many of these basal cells undergo apoptosis and die. Although sub-basal axons are restored to their normal density and morphology after superficial trephination, sub-basal axon recovery is partial after debridement wounds. The increase in corneal epithelial basal cell apoptosis at the apex observed at 14 days after corneal debridement may destabilize newly reinnervated sub-basal axons and lead to their retraction toward the periphery.

Syndecan-1 and Its Expanding List of Contacts.

Significance: The binding of cytokines and growth factors to heparan sulfate (HS) chains on proteoglycans generates gradients that control development and regulate wound healing. Syndecan-1 (sdc1) is an integral membrane HS proteoglycan. Its structure allows it to bind with cytosolic, transmembrane, and extracellular matrix (ECM) proteins. It plays important roles in mediating key events during wound healing because it regulates a number of important processes, including cell adhesion, cell migration, endocytosis, exosome formation, and fibrosis. Recent Advances: Recent studies reveal that sdc1 regulates wound healing by altering integrin activation. Differences in integrin activation lead to cell-type-specific changes in the rate of cell migration and ECM assembly. Sdc1 also regulates endocytosis and the formation and release of exosomes. Critical Issues: Understanding how sdc1 facilitates wound healing and resolution will improve treatment options for elderly and diabetic patients with delayed wound healing. Studies showing that sdc1 function is altered in cancer are relevant to those interested in controlling fibrosis and scarring. Future Directions: The key to understanding the various functions ascribed to sdc1 is resolving how it interacts with its numerous binding partners. The role played by chondroitin sulfate glycosaminoglycan (GAG) chains on the ability of sdc1 to associate with its ligands needs further investigation. At wound sites heparanase can cleave the HS GAG chains of sdc1, alter its ability to bind cytokines, and induce shedding of the ectodomain. This review will discuss how the unique structure of sdc1 allows it to play key roles in cell signaling, ECM assembly, and wound healing.

Spontaneous skin erosions and reduced skin and corneal wound healing characterize CLIC4(NULL) mice.

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-ß (TGF-ß) signaling, and mice lacking components of the TGF-ß signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-ß pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of ß4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-ß was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-ß. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-ß pathway.

αVß6 integrin promotes corneal wound healing.

PURPOSE: To appreciate the role of the integrin αvß6 in corneal wound repair, corneal debridement and keratectomy in ß6 knockout (ß6(-/-)) mice were examined. METHODS: Either a 2-mm debridement or keratectomy was made in 129SVE wild type mice (WT) and ß6(-/-) mice and allowed to heal for up to 4 months. The pattern of corneal restoration was studied "in vivo" by slit lamp and in tissue sections by means of both light and electron microscopy. In addition, αvß6, α6ß4, laminin, and fibronectin were evaluated by indirect immunofluorescence microscopy and/or Western blot analysis. RESULTS: αvß6 expression was upregulated in migrating corneal epithelium after a keratectomy. Healing rates were unaffected in debridement wounds, but were significantly slowed in keratectomy wounds. Most dramatically, mice lacking αvß6 had a severe defect in basement membrane zone (BMZ) regeneration. Levels of laminin were greatly reduced and no BMZ reformation was observed in transmission electron microscopy (TEM). In addition, hemidesmosome reformation was also impaired in the ß6(-/-) mice. Analysis of the hemidesmosome component α6ß4 indicated that normal amounts of this integrin were synthesized, suggesting that the defect was in reassembly of the hemidesmosomes. Finally, fibronectin persisted in the BMZ for as long as 4 months after keratectomy in the ß6(-/-) mice. CONCLUSIONS: It is hypothesized that the lack of αvß6 leads to reduced laminin production during wound repair. This lack of laminin prevents reassembly of the BMZ and mature hemidesmosomes after keratectomy in ß6(-/-) mice.

Loss of syndecan-1 is associated with malignant conversion in skin carcinogenesis.

Syndecan-1 (sdc-1) is a cell surface proteoglycan that mediates the interaction of cells with their matrix, influencing attachment, migration, and response to growth factors. In keratinocytes, loss of sdc-1 delays wound healing, reduces migration, and increases Transforming growth factor beta (TGFbeta) 1 expression. In this study we show that sdc-1 expression is significantly reduced in basal cell, squamous cell, and metastatic human skin cancers compared to normal human skin. In experimental mouse skin tumor induction, compared to wildtype (wt) BALB/c mice, papilloma formation in sdc-1 null mice was reduced by 50% and the percent of papillomas converting to squamous cell carcinoma (SCC) was enhanced. sdc-1 expression on wt mouse papillomas decreased as they converted to SCC. Furthermore, papillomas forming on sdc-1 null mice expressed suprabasal alpha3 and beta4 integrins; suprabasal beta4 integrin is a marker of a high risk for progression. While the proliferative response to phorbol-12-myristate-13-acetate (TPA) did not differ among the genotypes, sdc-1 null mice had an enhanced inflammatory response and retained higher levels of total TGFbeta1 within their skin after TPA treatment. sdc-1 null keratinocytes, transduced in vitro by oncogenic ras(Ha), expressed higher levels of beta4 integrin and had enhanced pSmad2 signaling and reduced senescence when compared to wt ras(Ha)-transduced keratinocytes. When ras(Ha)-transduced cells of both genotypes were grafted onto nude mice, null tumors converted to SCC with higher frequency confirming the skin painting experiments. These data indicate that sdc-1 is important both early in the development of skin tumors and in progression of skin cancers suggesting that reduced expression of sdc-1 could be a useful marker for progression in neoplastic skin lesions.

Increased expression of syndecan-1 protects against cardiac dilatation and dysfunction after myocardial infarction.

BACKGROUND: The cell-associated proteoglycan syndecan-1 (Synd1) closely regulates inflammation and cell-matrix interactions during wound healing and tumorigenesis. The present study investigated whether Synd1 may also regulate cardiac inflammation, matrix remodeling, and function after myocardial infarction (MI). METHODS AND RESULTS: First, we showed increased protein and mRNA expression of Synd1 from 24 hours on, reaching its maximum at 7 days after MI and declining thereafter. Targeted deletion of Synd1 resulted in increased inflammation and accelerated, yet functionally adverse, infarct healing after MI. In concordance, adenoviral gene expression of Synd1 protected against exaggerated inflammation after MI, mainly by reducing transendothelial adhesion and migration of leukocytes, as shown in vitro. Increased inflammation in the absence of Synd1 resulted in increased monocyte chemoattractant protein-1 expression, increased activity of matrix metalloproteinase-2 and -9, and decreased activity of tissue transglutaminase, associated with increased collagen fragmentation and disorganization. Exaggerated inflammation and adverse matrix remodeling in the absence of Synd1 increased cardiac dilatation and impaired systolic function, whereas gene overexpression of Synd1 reduced inflammation and protected against cardiac dilatation and failure. CONCLUSIONS: Increased expression of Synd1 in the infarct protects against exaggerated inflammation and adverse infarct healing, thereby reducing cardiac dilatation and dysfunction after MI in mice.

Corneal integrins and their functions.

Integrins were first described just over 20 years ago and have been studied in the cornea by many groups interested in how the cornea functions in health and disease. There are a minimum of 12 different integrin heterodimers reported to be expressed by the major resident cells of the cornea: the corneal and limbal epithelial cells, keratocytes/fibroblasts, and corneal endothelial cells. These different integrin heterodimers play important and varied roles in maintaining the cornea and organizing how its cells interact with their surrounding extracellular matrix to maintain corneal clarity. In this review, an overview of the discovery and functions of integrins is provided along with a description of the current state of our knowledge of this large family of important proteins. While we have learned a lot about corneal integrins over the past 20 years, there is still much to learn. Areas where gaps in our knowledge of integrin functions in the cornea are slowing our progress in understanding corneal diseases and dystrophies at a molecular level are highlighted.

Effect of wound type on Smad 2 and 4 translocation.

PURPOSE: In a prior study, it was reported that both TGF-beta receptors type-I and -II are upregulated after wounding, suggesting that TGF-beta signaling may play a role in corneal epithelial repair. The Smad proteins, which translocate into the nucleus after activation of the TGF-beta receptors, are key factors in the major TGF-beta signaling pathway. The present study was undertaken to examine whether Smads 2 and 4 translocate into the nucleus during wound repair and whether the wound type affects the extent of translocation. METHODS: Either a 3-mm superficial keratectomy or epithelial debridement was performed on adult Sprague-Dawley rats. The eyes were allowed to heal from 4 hours to 2 weeks. Indirect immunofluorescence was performed with anti-Smads 2 and 4, anti-laminin, a marker of basement membrane, and anti-alphavbeta6 integrin, which has been implicated in TGF-beta activation. In addition, the effect of the p38MAPK inhibitor SB202190 on healing rates of debridement and keratectomy wounds was determined in organ culture. RESULTS: In unwounded tissue, Smad 2 was cytoplasmic. By 4 hours after keratectomy, nuclear localization was visible in a few epithelial basal cells at the leading edge of the wound. The number of basal cells expressing nuclear Smad 2 in the wound area increased with time, peaking at 48 hours (95%). However, in the debridement model, Smad 2 localization remained primarily cytoplasmic. Smad 4 showed similar localization. In both wound models, p38MAPK inhibitor slowed epithelial migration, and alphavbeta6 integrin appeared to be upregulated with localization primarily observed in the basal cells migrating over the wound area. CONCLUSIONS: The presence of the basement membrane appears to have an effect on the extent and duration of translocation of the Smad 2 and 4 proteins during corneal epithelial wound repair. The Smad pathway does not appear to be essential for migration; rather, it may play a role in resynthesis of the basement membrane.