Winston W.-Y. Kao

Smad3 Signaling Is Required for Epithelial-Mesenchymal Transition of Lens Epithelium after Injury

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

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.

A role for MEK kinase 1 in TGF‐β/activin‐induced epithelium movement and embryonic eyelid closure

MEKK1‐deficient mice show an eye open at birth phenotype caused by impairment in embryonic eyelid closure. MEK kinase 1 (MEKK1) is highly expressed in the growing tip of the eyelid epithelium, which displays loose cell–cell contacts and prominent F‐actin fibers in wild‐type mice, but compact cell contacts, lack of polymerized actin and a concomitant impairment in c‐Jun N‐terminal phosphorylation in MEKK1‐deficient mice. In cultured keratinocytes, MEKK1 is essential for JNK activation by TGF‐β and activin, but not by TGF‐α. MEKK1‐driven JNK activation is required for actin stress fiber formation, c‐Jun phosphorylation and cell migration. However, MEKK1 ablation does not impair other TGF‐β/activin functions, such as nuclear translocation of Smad4. These results establish a specific role for the MEKK1–JNK cascade in transmission of TGF‐β and activin signals that control epithelial cell movement, providing the mechanistic basis for the regulation of eyelid closure by MEKK1. This study also suggests that the signaling mechanisms that control eyelid closure in mammals and dorsal closure in Drosophila are evolutionarily conserved.

Roles of lumican and keratocan on corneal transparency

Lumican and keratocan are members of the small leucine-rich proteoglycan (SLRP) family, and are the major keratan sulfate (KS) proteoglycans in corneal stroma. Both lumican and keratocan are essential for normal cornea morphogenesis during embryonic development and maintenance of corneal topography in adults. This is attributed to their bi-functional characteristic (protein moiety binding collagen fibrils to regulate collagen fibril diameters, and highly charged glycosaminoglycan (GAG) chains extending out to regulate interfibrillar spacings) that contributes to their regulatory role in extracellular matrix assembly. The absence of lumican leads to formation of cloudy corneas in homozygous knockout mice due to altered collagenous matrix characterized by larger fibril diameters and disorganized fibril spacing. In contrast, keratocan knockout mice exhibit thin but clear cornea with insignificant alteration of stromal collaegenous matrix. Mutations of keratocan cause cornea plana in human, which is often associated with glaucoma. These observations suggest that lumican and keratocan have different roles in regulating formation of stromal extracellular matrix. Experimental evidence indicates that lumican may have additional biological functions, such as modulation of cell migration and epithelium-mesenchyme transition in wound healing and tumorgenesis, besides regulating collagen fibrillogenesis. Published in 2003.

Stem Cell Therapy Restores Transparency to Defective Murine Corneas

Corneal scarring from trauma and inflammation disrupts vision for millions worldwide, but corneal transplantation, the primary therapy for corneal blindness, is unavailable to many affected individuals. In this study, stem cells isolated from adult human corneal stroma were examined for the ability to correct stromal opacity in a murine model by direct injection of cells into the corneal stroma. In wild‐type mice, injected human stem cells remained viable for months without fusing with host cells or eliciting an immune T‐cell response. Human corneal‐specific extracellular matrix, including the proteoglycans lumican and keratocan, accumulated in the treated corneas. Lumican‐null mice have corneal opacity similar to that of scar tissue as a result of disruption of stromal collagen organization. After injection with human stromal stem cells, stromal thickness and collagen fibril defects in these mice were restored to that of normal mice. Corneal transparency in the treated mice was indistinguishable from that of wild‐type mice. These results support the immune privilege of adult stem cells and the ability of stem cell therapy to regenerate tissue in a manner analogous to organogenesis and clearly different from that of normal wound healing. The results suggest that cell‐based therapy can be an effective approach to treatment of human corneal blindness. STEM CELLS 2009;27:1635–1642

Apoptosis down-regulates inflammation under the advancing epithelial wound edge: Delayed patterns in diabetes and improvement with topical growth factors

BACKGROUND Wound healing is involved in many aspects of care, ranging from anastomoses and skin incisions to foot ulcers and decubitus. Clinical healing failures are a major challenge to the physician and cause significant morbidity and mortality in select patient populations. As we are starting to understand more fully the mechanisms of impaired wound healing, the diabetic mouse (C57BL/KsJ-db/db) has been a good model for research. The diabetic wound exhibits significant delays in healing, previously identified as impaired cellular infiltration and granulation tissue formation. Apoptosis, or programmed cell death, is intimately involved in the regulation of inflammation and ultimately should play a role in the inflammatory phase of wound healing. METHODS To examine its role in wound healing, patterns of apoptosis in large, full-thickness cutaneous wounds were compared between groups of diabetic and nondiabetic mice. RESULTS Initially apoptosis was mainly limited to the wound edge and followed the advancing epithelial edge toward the center of the wound as healing progressed. Significant delays in the appearance of the apoptotic pattern were noted in the diabetic mice. Wounds in diabetic mice were then treated with topical application of growth factors. The delay in apoptotic pattern was reversed after treatment with the combination of insulin-like growth factor-II and platelet-derived growth factor, approaching levels in nondiabetic animals. CONCLUSIONS Apoptosis appears concurrently with reepithelialization of the wound and may signal the end of the inflammatory phase of healing at that site in the wound. One can speculate that a signal for apoptosis and down-regulation of inflammation in the wound is derived from the epithelium.

CHARACTERIZATION AND EXPRESSION OF THE MOUSE LUMICAN GENE

Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3′ noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5′-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency.

ABCB5 is a limbal stem cell gene required for corneal development and repair

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali.

Damage to the cornea from chemical burns is a serious clinical problem that often leads to permanent visual impairment. Because transforming growth factor (TGF)-beta has been implicated in the response to corneal injury, we evaluated the effects of altered TGF-beta signaling in a corneal alkali burn model using mice treated topically with an adenovirus (Ad) expressing inhibitory Smad7 and mice with a targeted deletion of the TGF-beta/activin signaling mediator Smad3. Expression of exogenous Smad7 in burned corneal tissue resulted in reduced activation of Smad signaling and nuclear factor-kappaB signaling via RelA/p65. Resurfacing of the burned cornea by conjunctival epithelium and its differentiation to cornea-like epithelium were both accelerated in Smad7-Ad-treated corneas with suppressed stromal ulceration, opacification, and neovascularization 20 days after injury. Introduction of the Smad7 gene suppressed invasion of monocytes/macrophages and expression of monocyte/macrophage chemotactic protein-1, TGF-beta1, TGF-beta2, vascular endothelial growth factor, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-2 and abolished the generation of myofibroblasts. Although acceleration of healing of the burned cornea was also observed in mice lacking Smad3, the effects on epithelial and stromal healing were less pronounced than those in corneas treated with Smad7. Together these data suggest that overexpression of Smad7 may have effects beyond those of simply blocking Smad3/TGF-beta signaling and may represent an effective new strategy for treatment of ocular burns.

Therapeutic Effect of Topical Administration of SN50, an Inhibitor of Nuclear Factor-κB, in Treatment of Corneal Alkali Burns in Mice

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 microg/microl) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semi-quantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-kappaB activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-alpha in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-alpha, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-alpha-/- mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-alpha/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.