The role of electrical signals in murine corneal wound re-epithelialization

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.     

Knockdown of heme oxygenase-2 impairs corneal epithelial cell wound healing

Heme oxygenase (HO) represents an intrinsic cytoprotective system based on its anti‐oxidative and anti‐inflammatory properties mediated via its products biliverdin/bilirubin and carbon monoxide (CO). We showed that deletion of HO‐2 results in impaired corneal wound healing with associated chronic inflammatory complications. This study was undertaken to examine the role of HO activity and the contribution of HO‐1 and HO‐2 to corneal wound healing in an in vitro epithelial scratch injury model. A scratch wound model was established using human corneal epithelial (HCE) cells. These cells expressed both HO‐1 and HO‐2 proteins. Injury elicited a rapid and transient increase in HO‐1 and HO activity; HO‐2 expression was unchanged. Treatment with biliverdin or CORM‐A1, a CO donor, accelerated wound closure by 10% at 24 h. Inhibition of HO activity impaired wound closure by more than 50%. However, addition of biliverdin or CORM‐A1 reversed the effect of HO inhibition on wound healing. Moreover, knockdown of HO‐2 expression, but not HO‐1, significantly impaired wound healing. These results indicate that HO activity is required for corneal epithelial cell migration. Inhibition of HO activity impairs wound healing while amplification of its activity restores and accelerates healing. Importantly, HO‐2, which is highly expressed in the corneal epithelium, appears to be critical for the wound healing process in the cornea. The mechanisms by which it contributes to cell migration in response to injury may reside in the cytoprotective properties of CO and biliverdin. J. Cell. Physiol. 226: 1732–1740, 2011. © 2010 Wiley‐Liss, Inc.     

Advanced glycation end products delay corneal epithelial wound healing through reactive oxygen species generation

Delayed healing of corneal epithelial wounds is a serious complication in diabetes. Advanced glycation end products (AGEs) are intimately associated with the diabetic complications and are deleterious to the wound healing process. However, the effect of AGEs on corneal epithelial wound healing has not yet been evaluated. In the present study, we investigated the effect of AGE-modified bovine serum albumin (BSA) on corneal epithelial wound healing and its underlying mechanisms. Our data showed that AGE-BSA significantly increased the generation of intracellular ROS in telomerase-immortalized human corneal epithelial cells. However, the generation of intracellular ROS was completely inhibited by antioxidant N-acetylcysteine (NAC), anti-receptor of AGEs (RAGE) antibodies, or the inhibitor of NADPH oxidase. Moreover, AGE-BSA increased NADPH oxidase activity and protein expression of NADPH oxidase subunits, p22phox and Nox4, but anti-RAGE antibodies eliminated these effects. Furthermore, prevention of intracellular ROS generation using NAC or anti-RAGE antibodies rescued AGE-BSA-delayed epithelial wound healing in porcine corneal organ culture. In conclusion, our results demonstrated that AGE-BSA impaired corneal epithelial wound healing ex vivo. AGE-BSA increased intracellular ROS generation through NADPH oxidase activation, which accounted for the delayed corneal epithelial wound healing. These results may provide better insights for understanding the mechanism of delayed healing of corneal epithelial wounds in diabetes.     

Rho kinases regulate corneal epithelial wound healing

We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and beta-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.     

Role of lumican in the corneal epithelium during wound healing

Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e. g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum(-/-) mice was significantly delayed compared with Lum(+/-) mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.     

Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.     

Effects of Topically Applied Vitamin D during Corneal Wound Healing

Vitamin D is an important regulator of immune function and largely acts to dampen chronic inflammatory events in a variety of tissues. There is also accumulating evidence that vitamin D acts to enhance initial inflammation, beneficial during both infection and wound healing, and then promotes resolution and prevention of chronic, damaging inflammation. The current study examines the effect of topical vitamin D in a mouse of model of corneal epithelial wound healing, where acute inflammation is necessary for efficient wound closure. At 12 and 18 hours post-wounding, vitamin D treatment significantly delayed wound closure by ~17% and increased infiltration of neutrophils into the central cornea. Basal epithelial cell division, corneal nerve density, and levels of VEGF, TGFβ, IL-1β, and TNFα were unchanged. However, vitamin D increased the production of the anti-microbial peptide CRAMP 12 hours after wounding. These data suggest a possible role for vitamin D in modulating corneal wound healing and have important implications for therapeutic use of vitamin D at the ocular surface.     

Estrogen negatively regulates epithelial wound healing and protective lipid mediator circuits in the cornea

Estrogen receptors (ERs) are expressed in leukocytes and in every ocular tissue. However, sex-specific differences and the role of estradiol in ocular inflammatory-reparative responses are not well understood. We found that female mice exhibited delayed corneal epithelial wound closure and attenuated polymorphonuclear (PMN) leukocyte responses, a phenotype recapitulated by estradiol treatment both in vivo (topically in male mice) and in vitro (corneal epithelial cell wound healing). The cornea expresses 15-lipoxygenase (15-LOX) and receptors for lipoxin A(4) (LXA(4)), which have been implicated in an intrinsic lipid circuit that regulates corneal inflammation and wound healing. Delayed epithelial wound healing correlated with lower expression of 15-LOX in the regenerated epithelium of female mice. Estradiol in vitro and in vivo down-regulated epithelial 15-LOX expression and LXA(4) formation, while estradiol abrogation of epithelial wound healing was completely reversed by treatment with LXA(4). More important, ERβ and ERα selectively regulated epithelial wound healing, PMN cell recruitment, and activity of the intrinsic 15-LOX/LXA(4) circuit. Our results demonstrate for the first time a sex-specific difference in the corneal reparative response, which is mediated by ERβ and ERα selective regulation of the epithelial and PMN 15-LOX/LXA(4) circuit. These findings may provide novel insights into the etiology of sex-specific ocular inflammatory diseases.     

垂体腺苷酸环化酶激活肽衍生多肽RHMP的重组制备及促角膜损伤修复的初步研究

利用基因工程技术高效制备具有促进角膜损伤修复功能的垂体腺苷酸环化酶激活肽(PACAP)衍生多肽RHMP,并在体外研究其生物学效应,为角膜疾病的治疗提供了新思路。采用基因重组技术表达重组肽RHMP,经Chitin-Beads柱纯化、HPLC及SDS-PAGE、质谱鉴定后,研究其对小鼠角膜损伤修复的影响。实验结果表明:利用基因重组技术制备的PACAP衍生多肽RHMP的分子量为3.4kDa,纯度为96%;将重组肽作用于创伤后的小鼠角膜,12h、24h、36h、48h后角膜修复率分别为(49.58±1.74)%、(93.66±3.39)%、(99.6±0.43)%、(99.8±0.14)%,而对照组修复率分别为(9.76±1.58)%、(29.02±1.63)%、(55.10±1.49)%、(78.59±2.52)%,P<0.001,差异具有统计学意义,重组肽RHMP可显著促进小鼠角膜损伤修复。因此,利用以上确立的表达、纯化策略,可实现新型基因重组PACAP衍生多肽RHMP的高效制备,重组多肽RHMP可快速有效地促进损伤角膜的修复,从而有望成为一种新型角膜损伤治疗药物;同时,建立的小鼠角膜上皮损伤模型可用于相关药物的生物学效应研究。     

Matrix metalloproteinase activity is necessary for thymosin beta 4 promotion of epithelial cell migration

Studies from our laboratory provide substantial evidence that thymosin beta 4, (Tbeta(4)), an actin-sequestering protein, promotes corneal wound healing through its ability to stimulate epithelial cell migration. Matrix metalloproteinases (MMPs), which are expressed in a wide variety of tissues including the cornea, also play a key role in epithelial cell migration and wound healing. In this study we investigated the role of MMPs in Tbeta(4)-stimulated corneal epithelial cell migration. In Boyden chamber assays, XG076, an inhibitor of the conversion of pro- to active MMPs, had no effect on epithelial cell migration stimulated by exogenous activated MMP-1. However, in in vitro migration assays where the activation of pro-MMPs was blocked, XG076 significantly inhibited cell migration and wound healing in the presence or absence of Tbeta(4). GM6001, a broad-spectrum inhibitor of active MMPs and selective MMP inhibitors, also suppressed Tbeta(4)-stimulated cell migration. Tbeta(4) upregulated MMP-1 gene and protein expression in primary human corneal epithelial cells and in transformed human corneal epithelial cells following scrape wounding. From these results we conclude that MMP catalytic activity is necessary for Tbeta(4) promotion of epithelial cell migration. These novel findings are the first to demonstrate a functional link between the two.     

Intestinal trefoil factor/TFF3 promotes re-epithelialization of corneal wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3(+)(/)(+)) and Tff3-deficient (Tff3(-)(/)(-)) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3(-/-) mice is significantly prolonged in comparison to Tff3(+/+) mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3(+/+) and Tff3(-/-) mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.     

Keratinocyte growth factor signalling: a mathematical model of dermal-epidermal interaction in epidermal wound healing

A wealth of growth factors are known to regulate the various cell functions involved in the repair process. An understanding of their therapeutic value is essential to achieve improved wound healing. Keratinocyte growth factor (KGF) seems to have a unique role as a mediator of mesenchymal-epithelial interactions: it originates from mesenchymal cells, yet acts exclusively on epithelial cells. In this paper, we study KGF's role in epidermal wound healing, since its production is substantially up-regulated after injury. We begin by modelling the dermal-epidermal signalling mechanism of KGF to investigate how this extra production affects the signal range. We then incorporate the effect of KGF on cell proliferation, and using travelling wave analysis we obtain an approximation for the rate of healing. Our modelling shows that the large up-regulation of KGF post-wounding extends the KGF signal range but is above optimal for the rate of wound closure. We predict that other functions of KGF may be more important than its role as a mitogen for the healing process.     

Roles of wound geometry, wound size, and extracellular matrix in the healing response of bovine corneal endothelial cells in culture

It has classically been accepted that the healing of narrow wounds in epithelia occurs by the formation of a contractile actin cable, while wide wounds are resurfaced by lamellipodia-dependent migration of border cells into the denuded area. To further investigate the general validity of this idea, we performed systematic experiments of the roles of wound geometry, wound size, and extracellular matrix (ECM) in wound healing in monolayers of bovine corneal endothelial cells, a system shown here to predominantly display any of the two healing mechanisms according to the experimental conditions. We found that, in this system, it is the absence or presence of the ECM on the wound surface that determines the specific healing mode. Our observations demonstrate that, independent of their size and geometry, wounds created maintaining the ECM heal by migration of cells into the wound area, while ECM removal from the wound surface determines the predominant formation of an actin cable. While the latter mechanism is slower, the actin cable permits the maintainance of the epithelial phenotype to a larger extent during the healing process, as also confirmed by our finding of a more conserved localization of cadherin and vinculin. We also introduce a model that simulates experimental findings about the dynamics of healing mechanisms, both for the maintenance or removal of the ECM on the wound surface. The findings of this study may contribute to the understanding of physiological and pathological aspects of epithelial wound healing and to the design of therapeutic strategies.     

The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration

Diabetic keratopathy (DK) is an important diabetic complication at the ocular surface. Chronic low-grade inflammation mediated by the NLRP3 inflammasome promotes pathogenesis of diabetes and its complications. However, the effect of the NLRP3 inflammasome on DK pathogenesis remains elusive. Wild-type (WT) and Nlrp3 knockout (KO) C57BL/6 mice were used to establish a type I diabetes model by intraperitoneal injection of streptozotocin. The effect of the NLRP3 inflammasome on diabetic corneal wound healing and never regeneration was examined by a corneal epithelial abrasion model. Western blot, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and pharmacological treatment were performed to investigate the regulatory mechanism of advanced glycation end products (AGEs) on NLRP3 inflammasome activation and corneal wound healing in vivo. The cultured mouse corneal epithelial cells (TKE2) were used to evaluate the effect and mechanism of AGEs on NLRP3 inflammasome activation in vitro. We revealed that NLRP3 inflammasome-mediated inflammation and pyroptosis contributed to DK pathogenesis. Under physiological conditions, the NLRP3 inflammasome was required for corneal wound healing and nerve regeneration. However, under a diabetic scenario, sustained activation of the NLRP3 inflammasome resulted in postponed corneal wound healing and impaired nerve regeneration. Mechanistically, the accumulated AGEs promoted hyperactivation of the NLRP3 inflammasome through ROS production. Moreover, genetically and pharmacologically blocking the AGEs/ROS/NLRP3 inflammasome axis significantly expedited diabetic corneal epithelial wound closure and nerve regeneration. Our results revealed that AGEs-induced hyperactivation of the NLRP3 inflammasome resulted in delayed diabetic corneal wound healing and impaired nerve regeneration, which further highlighted the NLRP3 inflammasome as a promising target for DK treatment.     

The immunoreactivity of a chimeric multi-epitope DNA vaccine against IBV in chickens

Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins α2 and β1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin β1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.     

Continuum model of fibroblast-driven wound contraction: inflammation-mediation.

We propose a mathematical model to aid the understanding of how events in wound healing are orchestrated to result in wound contraction. Ultimately, a validated model could provide a predictive means for enhancing or mitigating contraction as is appropriate for managing a particular wound. The complex nature of wound healing and the lack of a modeling framework which can account for both the relevant cell biology and biomechanics are major reasons for the absence of models to date. Here we adapt a model originally proposed by Murray and co-workers to show how cell traction forces can result in spatial patterns of cell aggregates since it offers a framework for understanding how traction exerted by wound fibroblasts drives wound contraction. Since it is a continuum model based on conservation laws which reflect assumed cell and tissue properties, it is readily extended to account for emerging understanding of the cell biology of wound healing and its relationship to inflammation. We consider various sets of assumed properties, based on current knowledge, within a base model of dermal wound healing and compare predictions of the rate and extent of wound contraction to published experimental results.     

New insights in wound response and repair of epithelium

Epithelial wounds usually heal relatively quickly, but repair may be impaired by environmental stressors, such as hypoxic or diabetic states, rendering patients vulnerable to a number of corneal pathologies. Though this response appears simple, at first, years of research have uncovered the complicated biochemical pathways coordinating the wound healing response. Here, we investigate signaling cascades and individual proteins involved in the corneal epithelium's self‐repair. We will explore how an epithelial cell migrates across the wound bed and attaches itself to its new post‐injury surroundings, including its neighboring cells and the basement membrane, through focal adhesions and hemidesmosomes. We will also discuss how the cell coordinates this motion physiologically, through calcium signaling and protein phosphorylation, focusing on the communication through purinergic, glutamatergic, and growth factor receptors. Many of these aspects reflect and can be extended to similar epithelial surfaces, and can be used to facilitate wound healing in patients with various underlying pathologies. The collective library of laboratory and clinical research done around the world has demonstrated how important precise regulation of these processes is in order for the injured corneal epithelium to properly heal. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.