Coordinated reassembly of the basement membrane and junctional proteins during corneal epithelial wound healing

PURPOSE: To characterize changes in the localizations of the basement membrane protein laminin-1 and of adhesion proteins of intercellular junctions during wound healing after epithelial ablation in the rat cornea. METHODS: Epithelial ablation was performed with an excimer laser. Rats were killed immediately, 12 hours, 24 hours, 3 days, or 4 weeks after ablation, and corneal cryosections were subjected to two-color immunofluorescence staining with antibodies to laminin-1 and antibodies to connexin43 for gap junctions, desmoglein 1 or 2 (desmoglein 1 + 2) for desmosomes, or E-cadherin for adherens junctions. Sections were also stained with antibodies to occludin for examination of tight junctions. RESULTS: Laminin-1 was detected in the basement membrane, connexin43 in the basal cell layer, desmoglein 1 + 2 in the wing cell layer, E-cadherin in all cell layers, and occludin in the wing and superficial cell layers of the intact corneal epithelium. Laminin-1 immunostaining was not detected at the leading edge of migrating epithelial cells until 24 hours after ablation. Expression of connexin43 and desmoglein 1 + 2 coincided with the reappearance of laminin-1, whereas that of E-cadherin and occludin was apparent regardless of the absence or presence of laminin-1. Epithelial remodeling was complete after 4 weeks. The basement membrane was re-established, and the expression patterns for all the adhesion proteins were identical with those characteristic of the intact cornea. CONCLUSIONS: Actively migrating epithelial cells no longer manifested gap junctions and desmosomes in the wounded area with no basement membrane. Re-establishment of the basement membrane coincided with reassembly of these intercellular junctions, suggesting that the presence of the basement membrane may be required for their reformation in the rat cornea.     

Removal of the basement membrane enhances corneal wound healing

Recurrent corneal erosions are painful and put patients’ vision at risk. Treatment typically begins with debridement of the area around the erosion site followed by more aggressive treatments. An in vivo mouse model has been developed that reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously within two weeks after a single 1.5 mm corneal debridement wound created using a dulled-blade. This study was conducted to determine whether 1) inhibiting MMP9 function during healing after dulled-blade wounding impacts erosion development and 2) wounds made with a rotating-burr heal without erosions. Oral or topical inhibition of MMPs after dulled-blade wounding does not improve healing. Wounds made by rotating-burr heal with significantly fewer erosions than dulled-blade wounds. The localization of MMP9, β4 integrin and basement membrane proteins (LN332 and type VII collagen), immune cell influx, and reinnervation of the corneal nerves were compared after both wound types. Rotating-burr wounds remove the anterior basement membrane centrally but not at the periphery near the wound margin, induce more apoptosis of corneal stromal cells, and damage more stromal nerve fibers. Despite the fact that rotating-burr wounds do more damage to the cornea, fewer immune cells are recruited and significantly more wounds resolve completely.     

Human amniotic membrane, like corneal epithelial basement membrane, manifests the alpha5 chain of type IV collagen

PURPOSE: To reexamine whether the alpha5 chain of type IV (alpha5[IV]) collagen, thought to be absent, is in fact present in human amniotic membrane. METHODS: Cryosections of human amniotic membrane obtained at Cesarean section were immunohistochemically examined for the presence of alpha5(IV), with or without inclusion of the denaturing step. Amniotic membrane was digested with collagenase to release the noncollagenous NC1 domain from the alpha-chain. The NC1 domain of alpha5(IV) was then assayed on Western blot analysis. Identical experiments were performed with human corneas and conjunctivae obtained from an American eye bank. RESULTS: The basement membrane of denatured samples of amniotic membrane and cornea stained positive for alpha5(IV). Without the denaturing step, only corneal samples were positive. With or without denaturing, conjunctival epithelium did not stain. Western blot analysis detected NC1 domains of alpha5(IV) in amniotic membrane and corneal samples. CONCLUSIONS: The basement membrane of amniotic membrane resembles that of corneal epithelium but not conjunctiva. Amniotic membrane may be an excellent substrate for corneal epithelial cells.     

Protein synthesis during corneal epithelial wound healing

Previous investigations have shown that corneal epithelium, migrating to cover a wound, synthesizes protein and glycoprotein at a faster rate than does normal stratified epithelium. The authors have found that the maximal rate of synthesis, as indicated by the incorporation of leucine and glucosamine, occurs 16 hr after wounding, 6 hr before wound closure. A comparison of total protein and protein synthesized during migration indicates that the increased synthesis is the result of the enhanced synthesis of many of the proteins present in unwounded epithelia. However, one protein band with a molecular weight of 110 K daltons was present to a much greater extent in migrating tissue than in normal epithelium. A time course analysis indicates that this band is apparent during migration and is not present either before wounding or 24 hr after wound closure.     

Cell-matrix and cell-cell interactions during corneal epithelial wound healing

The corneal epithelium serves as a barrier and contributes to the maintenance of corneal transparency and rigidity. In most instances, corneal epithelial defects caused by simple injury are resurfaced promptly. However, in individuals with certain clinical conditions, such as herpes simplex virus infection, neurotrophic keratopathy or diabetic keratopathy, corneal epithelial defects persist and do not respond to conventional treatment regimens because of delayed epithelial wound healing. After the corneal epithelium is removed by injury, the remaining epithelial cells migrate over the denuded surface of the cornea in a manner that is dependent both on the interaction of the cells with the underlying substrate and on cell-cell adhesion. In this review, we describe the specific roles of cell-matrix and cell-cell interactions during the course of corneal epithelial wound healing. The clinical implications of the basic research findings are also discussed.     

Effect of a collagen shield on cat corneal epithelial wound healing

We have previously demonstrated that a corneal bandage lens made from porcine scleral collagen may be useful in treating various ocular surface problems. In order to determine whether the collagen shield would accelerate epithelial healing, a 7 mm diameter circular area in the center of the left cornea of ten domestic cats was mechanically deepithelialized. In five of the cats, a 14.5 mm non-cross-linked collagen shield was then placed on the cornea covering the wound. Another shield was applied 24 hr after surgery. The wound size was determined immediately after surgery and at 8-hr intervals until wound closure. Using analysis of variance for experiments with repeated observations, there was a significantly greater healing response in the treated group than in the control group. There was, however, no significant difference in slope between the two groups, suggesting that the shield did not increase the speed of epithelial cell migration. Rather, the effect of the shield was most pronounced during the first 8 hr after wounding. In contrast to that of the treated group, the mean defect radius of the control group was larger at t = 8 hr than at t = 0 hr. The earlier wound closure exhibited by the treated group, which may be due to protection and lubrication of the epithelial cells at the margins of the fresh wound, suggests that the collagen shield may be useful in treating corneal surface conditions of which deepithelialization is a component.     

Alteration of epithelial paracellular permeability during corneal epithelial wound healing.

We studied the paracellular permeability to mannitol of corneas with epithelium of corneal, limbal, or conjunctival origin. Corneas with epithelial defects reepithelialized by corneal or limbal epithelium were nonvascularized; the corneal permeability was initially increased and returned to normal 3 days later. When epithelial defects extended beyond the limbus, they were healed by conjunctival epithelium. If corneas remained avascular or minimally vascularized, the conjunctiva-derived epithelium underwent a transdifferentiation process into a cornealike morphology in which the corneal permeability was initially increased upon complete reepithelialization, and gradually decreased to a level similar to that of normal cornea, 4 weeks after healing. However, when corneas became vascularized, the conjunctiva-derived epithelium retained its original phenotype, and corneal permeability remained increased throughout the 8-month period of study. The deranged barrier functions noted in the above vascularized cornea were demonstrated further by horseradish peroxidase tracer, which was found in the intercellular spaces of conjunctiva-derived epithelium of vascularized corneas but not in the avascular corneas with epithelia of corneal or limbal origin, or transdifferentiated conjunctival epithelium. To study further the effect of subsequent ocular surface trauma, conjunctival biopsy was performed on transdifferentiated avascular corneas 3 months after initial wounding. The biopsy resulted in extensive vascularization in three of eight previously nonvascularized corneas. Two weeks later, the corneal permeability was increased to a level similar to that of conjunctiva. These results indicate that corneal epithelial paracellular permeability correlates well with the status of the epithelial phenotype.     

Increases in collagen type IV and laminin in galactose-induced retinal capillary basement membrane thickening--prevention by an aldose reductase inhibitor

Biochemical alterations in the composition of retinal capillary basement membrane components were investigated in galactosemic rats, an animal model that develops basement membrane lesions comparable to those of diabetic retinopathy. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for 9 months developed significant thickening of retinal capillary basement membranes by comparison with animals fed a control test diet (P less than 0.001), or animals on a diet containing 30% galactose and 250 mg kg-1 of the aldose reductase inhibitor sorbinil (P less than 0.001). A quantitative electron microscopic immunogold technique applied on ultrathin sections of the retinas of these animals showed that the labeling densities of collagen type IV and laminin per unit cross-sectional area (which is presumably proportional to the concentrations of these molecules) were significantly increased in the retinal capillary basement membranes of galactose-fed rats, compared with animals on the control test diet. Increases in these two components of basement membranes were prevented by addition of sorbinil to the diet. However, there was no significant change in the labeling density of heparan sulfate proteoglycan (HSPG) core protein in the basement membranes of galactose-fed rats in comparison to animals on either the control diet or galactose-sorbinil diet. Two types of striated fibrillar materials were frequently found in areas of focal thickening of basement membranes of galactose fed rats only. Thinner fibrils reacted strongly with collagen type III antibody, whereas thicker fibrils reacted weakly with collagen type I antibody. Our results indicate that there is an increase in labeling densities of collagen type IV and laminin in thickened basement membranes of retinal capillaries of galactosemic rats along with the expression of interstitial collagens like collagen type III and an abnormal collagen that weakly cross-reacts with antibody to collagen type I, and these effects of galactosemia on the basement membranes are preventable by an aldose reductase inhibitor.     

The conjunctiva in corneal epithelial wound healing

BACKGROUND/AIMS—During the healing of corneal epithelial wounds with limbal involvement, conjunctival epithelium often migrates across the denuded limbus to cover the corneal surface. It is believed that, over a period of time, conjunctival epithelium covering the cornea assumes characteristics of corneal epithelium by a process referred to as conjunctival transdifferentiation. The purpose of this study was to examine, clinically, the fate of conjunctival epithelial cells covering the cornea and to assess the healing of corneal epithelial wounds when the conjunctival epithelium was removed or actively prevented from crossing the limbus and extending onto the cornea.
METHODS—10 patients with conjunctivalisation of the cornea were followed for an average of 7.5 months. Five patients in this group had their conjunctival epithelium removed from the corneal surface and allowed to heal from the remaining intact corneal epithelium. In another four patients with corneal epithelial defects, the conjunctival epithelium was actively prevented from crossing the limbus by mechanically scraping it off.
RESULTS—The area of cornea covered by conjunctival epithelium appeared thin, irregular, attracted new vessels and was prone to recurrent erosions. Conjunctivalisation of the visual axis affected vision. Removal of conjunctival epithelium from the cornea allowed cells of corneal epithelial phenotype to cover the denuded area with alleviation of symptoms and improvement of vision. It was also established that migration of conjunctival epithelium onto corneal surface could be anticipated by close monitoring of the healing of corneal epithelial wounds, and prevented by scraping off conjunctival epithelium before it reached the limbus.
CONCLUSION—This study shows that there is little clinical evidence to support the concept that conjunctival transdifferentiation per se, occurs in humans. "Replacement" of conjunctival epithelium by corneal epithelial cells may be an important mechanism by which conjunctival "transdifferentiation" may occur. In patients with partial stem cell deficiency this approach can be a useful and effective alternative to partial limbal transplantation, as is currently practised.

Keywords: corneal epithelium; conjunctiva; stem cells; transdifferentiation     

Phosphorylation of MAP kinase in corneal epithelial cells during wound healing

PURPOSE: To investigate the role of mitogen-activated protein kinase (MAPK), such as p44/42 MAPK, p38 MAPK and stress-activated protein kinase (SAPK), in corneal epithelial cells during the wound healing process. METHODS: A single non-penetrating incision was produced on rat cornea. Then the corneal wound healing process was observed with an immunocytochemical technique using specific antibodies reacting only with phosphorylated p44/42 MAPK, p38 MAPK or SAPK. Cell lysates of corneal epithelial cells in rabbits stimulated with keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were processed for Western blot using antibodies to phosphorylated p44/42 MAPK. RESULTS: Maximum activation of p44/42 MAPK was observed in wing and basal cells at wounded regions in rat cornea at 1 hour after the incision. Activation of p44/42 MAPK was still detected in all basal and wing cells at wounded regions at up to 24 hours when the incisions were completely closed, and then receded to normal intensity after 7 days. Neither p38 MAPK nor SAPK were activated during the wound healing process. Western blot analysis of cultured corneal epithelial cells in rabbits showed phosphorylation of p44/42 MAPK after 30 minutes in response to KGF and HGF, whereas non-activated p44/42 MAPK was ordinarily detected even at the absence of KGF or HGF. CONCLUSIONS: These results demonstrate that p44/42 MAPK is activated during the corneal wound healing process and suggest that KGF and HGF play an important role in initiation of cell migration and proliferation in the initial wound healing process by activating p44/42 MAPK.     

Sloughing of corneal epithelium and wound healing complications associated with laser in situ keratomileusis in patients with epithelial basement membrane dystrophy

PURPOSE: To report sloughing of corneal epithelium during laser in situ keratomileusis and subsequent wound healing complications in patients with epithelial basement membrane dystrophy. METHODS: In a retrospective study, the surgical procedures, postoperative course, and visual acuities of 16 eyes of nine patients with epithelial basement membrane dystrophy who underwent laser in situ keratomileusis complicated with epithelial sloughing at three centers were reviewed. The mean follow-up period was 23 weeks (range, 4 to 52 weeks). RESULTS: In 13 (81%) of 16 eyes with epithelial basement membrane dystrophy, epithelial sloughing occurred during laser in situ keratomileusis. In eight of the 13 eyes, epithelial growth beneath the flap was observed. The flap was lifted and the interface epithelium scraped in six eyes. Flap melt or keratolysis occurred in four eyes. At the last follow-up visit, 13 of 16 eyes had an uncorrected visual acuity of 20/30 or better, and all eyes had a best-corrected visual acuity of 20/30 or better. CONCLUSIONS: Patients with epithelial basement membrane dystrophy have poorly adherent corneal epithelium and are predisposed to epithelial sloughing during the microkeratome pass of laser in situ keratomileusis. This may lead to flap distortion, interface epithelial growth, flap keratolysis, and corneal scarring. It is not recommended that laser in situ keratomileusis be performed in patients with classic, symptomatic epithelial basement membrane dystrophy. In patients who present with mild and asymptomatic epithelial basement membrane dystrophy, laser in situ keratomileusis should be performed with caution, or photorefractive keratectomy may be the preferred refractive procedure.     

Clinical patterns of corneal epithelial wound healing

We studied the reepithelialization of corneal abrasions in 21 patients. All abrasions, irrespective of the nature of injury, followed a consistent pattern during reepithelialization. Three to six convex leading fronts of migrating epithelial sheets developed along the circumference of the defect and progressed toward the center. Neighboring fronts met along their sides, resulting in the formation of various geometric shapes. In the final stage of the healing process a contact line shaped like a "Y" or two "Y's" placed with their long axes end to end was seen where the advancing fronts of migrating epithelial sheets met. The rate of healing of the abrasions was determined by measuring the area of the abrasions at daily intervals from serial photographs. The area of the epithelial defects decreased exponentially with time, indicating a constant rate of epithelial cell migration.     

角膜上皮细胞基底膜的研究进展

角膜上皮细胞基底膜是一层很薄的组织,它的结构与其他部位的基底膜既有相似之处,也有独特的地方,在角膜创伤修复中有着重要的作用.基底膜组织的破坏会导致一系列棘手的角膜病变,而近来对角膜上皮细胞基底膜的研究使我们在这些病变的治疗方面有了新的人手点.就近年来角膜上皮细胞基底膜的研究进展做一综述.     

整合素在角膜上皮创伤愈合中的研究进展

整合素作为一类重要的细胞黏附分子,通过影响细胞的形态,介导细胞的黏附、迁移和增生,在角膜上皮创伤愈合中发挥了重要的作用。讨论α2β1、α3β1、α5β1、αvβ3、α6β4、α9β1和αvβ6这7种整合素在角膜上皮创伤愈合中的研究进展及其临床意义。α6β4整合素为半桥粒的主要组成部分,介导角膜上皮细胞在细胞外基质上的静态黏附,损伤后该黏附就转变为α2β1、α3β1整合素介导的动态黏附,细胞在黏附-去黏附的过程中实现迁移,从而修复创面。α6β4、α3β1整合素相互协调作用,实现上皮细胞的板层状运动。研究还发现α6β4、α3β1整合素的活化还能促进细胞的增生。损伤后上皮细胞表面α5β1、αvβ3整合素的表达上调,二者与黏着斑的形成密切相关。α9β1和αvβ6为近年来新发现的与角膜上皮创伤愈合有关的整合素,其具体作用尚有待进一步的研究。     

Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing

PURPOSE: To purify and characterize the glycoprotein lumican, isolated from human amniotic membrane (AM), and to examine its efficacy in treating corneal epithelium debridement. METHODS: An affinity-purified, anti-human lumican antibody-conjugated protein A Sepharose column was used to purify soluble lumican protein from human AM. The purified AM lumican was characterized by two-dimensional and SDS gel electrophoresis, plus Western blot analysis with anti-lumican antibody. The effects of lumican on corneal epithelial wound healing were examined in an organ culture mouse eye model. RESULTS: Lumican was found to be abundantly present in the stroma of human AM. It was extracted from the AM by isotonic, 1 M NaCl, and 4 M guanidine HCl solutions, suggesting that it is present in both the soluble and matrix-bound states. In two-dimensional gel electrophoresis, the 50-kDa human amniotic lumican purified by antibody-conjugated affinity chromatography migrated in a smear between pH 3.0 and 6.0. After endo-beta-galactosidase digestion, it existed as a single core protein at pH 6.0, suggesting that native human amniotic lumican is a glycoprotein with short sugar moiety. Addition of purified human AM lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. In lumican-knockout (lum(-/-)) mice, the effect of human lumican on promoting corneal epithelial wound healing was even more dramatic than in wild-type mice. CONCLUSIONS: The diversified functions of lumican include modulation of epithelial cells in wound healing and serving as an extracellular matrix component. Administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.     

白细胞介素-6对糖尿病小鼠角膜缘干细胞活化的促进作用和角膜上皮愈合的加速作用

背景 白细胞介素(IL)-6既介导炎症反应过程又在损伤修复中发挥重要作用,但其具体机制及其在糖尿病角膜组织损伤修复中的作用值得探讨. 目的 探讨IL-6在正常和糖尿病小鼠角膜缘干细胞活化和角膜上皮修复过程中的作用及其机制.方法 正常6～8周龄C57B L/6小鼠52只,采用随机数字表法随机分为正常对照鼠32只和糖尿病模型鼠20只,采用50 mg/kg链脲佐菌素连续腹腔内注射5d的方法诱导建立小鼠糖尿病模型.正常对照小鼠和糖尿病模型小鼠均行角膜上皮刮除术,然后分别于刮除后即刻和48 h结膜下注射IL-6或等容量PBS,采用荧光素染色法评价随时间延长的角膜上皮的愈合情况.体外培养小鼠角膜上皮干/祖细胞(TKE2细胞系),采用结晶紫染色法评估不同质量浓度IL-6处理后细胞克隆率(CFE),并与空白对照组进行比较;采用免疫荧光检测法和Western blot法检测细胞和小鼠再生上皮中干细胞标志物△NP63、Ki67的表达以及关键转录因子STAT3磷酸化水平;采用实时荧光定量PCR法和ELISA法检测小鼠角膜再生上皮中IL-6的mRNA及蛋白水平.结果 角膜荧光素染色检查显示,正常对照小鼠和糖尿病模型小鼠PBS注射组与IL-6注射组注射后24、48和72 h残留角膜上皮缺损面积占原始缺损面积的百分比随角膜上皮损伤后时间延长均明显缩小,同时间点IL-6注射组角膜上皮缺损面积均明显小于PBS注射组,组间总体差异均有统计学意义(正常对照组:F分组=19.982,P＜0.01;F时间=589.350,P＜0.01;糖尿病组:F分组=25.411,P＜0.01;F时间=334.807,P＜0.01).空白对照组及10、20、50、100 ng/ml IL-6处理组CFE分别为(13.23±1.12)％、(15.87±1.30)％、(21.69±1.62)％、(25.33±1.28)％和(18.67±1.54)％,随着IL-6质量浓度的增加CFE逐渐增加,总体比较差异有统计学意义(F=35.547,P＜0.01).50 ng/ml IL-6处理5、10、15、30和60 min细胞中△NP63、Ki67和p-STAT3蛋白的相对表达量随着处理时间的延长均逐渐增加,各时间点细胞中△NP63、Ki67和p-STAT3蛋白的相对表达量均明显高于空白对照组,差异均有统计学意义(均P＜0.05).糖尿病组和正常对照组小鼠角膜上皮刮除后24 h的再生上皮中IL-6 mRNA相对表达量分别为0.45±0.21和1.00±0.16,糖尿病组小鼠角膜再生上皮中IL-6 mRNA相对表达量较正常对照组小鼠下降56％,差异有统计学意义(t=3.42,P=0.03),糖尿病小鼠角膜上皮刮除后48 h的再生上皮中IL-6蛋白质量浓度为(257±12)ng/μl,明显低于正常对照组小鼠的(323±17) ng/μl,差异有统计学意义(t=5.60,P＜0.01). 结论 IL-6能够通过激活STAT3信号通路促进正常和糖尿病小鼠角膜缘干细胞的活化和增生,进而促进角膜上皮修复,而封闭内源性IL-6则会延迟小鼠角膜上皮的修复.