Evaluating the effect of pulse energy on femtosecond laser trabeculotomy (FLT) outflow channels for glaucoma treatment in human cadaver eyes.

BACKGROUND AND OBJECTIVES: Femtosecond laser trabeculotomy (FLT) creates aqueous humor outflow channels through the trabecular meshwork (TM) and is an emerging noninvasive treatment for open-angle glaucoma. The purpose of this study is to investigate the effect of pulse energy on outflow channel creation during FLT. MATERIALS AND METHODS: An FLT laser (ViaLase Inc.) was used to create outflow channels through the TM (500 µm wide by 200 µm high) in human cadaver eyes using pulse energies of 10, 15, and 20 µJ. Following treatment, tissues were fixed in 4% paraformaldehyde. The channels were imaged using optical coherence tomography (OCT) and assessed as full thickness, partial thickness, or not observable. RESULTS: Pulse energies of 15 and 20 µJ had a 100% success rate in creating full-thickness FLT channels as imaged by OCT. A pulse energy of 10 µJ resulted in no channels (n = 6), a partial-thickness channel (n = 2), and a full-thickness FLT channel (n = 2). There was a statistically significant difference in cutting widths between the 10 and 15 µJ groups (p < 0.0001), as well as between the 10 and 20 µJ groups (p < 0.0001). However, there was no statistically significant difference between the 15 and 20 µJ groups (p = 0.416). CONCLUSIONS: Fifteen microjoules is an adequate pulse energy to reliably create aqueous humor outflow channels during FLT in human cadaver eyes. OCT is a valuable tool when evaluating FLT.

Nonlinear optical crosslinking (NLO CXL) for correcting refractive errors.

Ultraviolet A (UVA) light-based photoactivation of riboflavin (Rf) to induce corneal crosslinking (CXL) and mechanical stiffening is now a well-known treatment for corneal ectasia and Keratoconus that is being used in a topographically guided photorefractive intrastromal CXL (PiXL) procedure to treat low degrees of refractive errors. Alternative approaches for non-invasive treatment of refractive errors have also been proposed that use femtosecond lasers (FS) that provide much faster, more precise, and safer results than UVA CXL. One such treatment, nonlinear optical crosslinking (NLO CXL), has been able to replicate the effects of UVA CXL, while producing a smaller area of cellular damage and requiring a shorter procedure time. Unlike UVA CXL, the treatment volume of NLO CXL only occurs within the focal volume of the laser, which can be placed at any depth and scanned into any pattern for true topographically guided refractive correction. This review presents our experience with using FS lasers to photoactivate Rf and perform highly controlled corneal CXL that leads to mechanical stiffening and changes in corneal shape.

Sensory nerve supports epithelial stem cell function in healing of corneal epithelium in mice: the role of trigeminal nerve transient receptor potential vanilloid 4.

In order to understand the pathobiology of neurotrophic keratopathy, we established a mouse model by coagulating the first branch of the trigeminal nerve (V1 nerve). In our model, the sensory nerve in the central cornea disappeared and remaining fibers were sparse in the peripheral limbal region. Impaired corneal epithelial healing in the mouse model was associated with suppression of both cell proliferation and expression of stem cell markers in peripheral/limbal epithelium as well as a reduction of transient receptor potential vanilloid 4 (TRPV4) expression in tissue. TRPV4 gene knockout also suppressed epithelial repair in mouse cornea, although it did not seem to directly modulate migration of epithelium. In a co-culture experiment, TRPV4-introduced KO trigeminal ganglion upregulated nerve growth factor (NGF) in cultured corneal epithelial cells, but ganglion with a control vector did not. TRPV4 gene introduction into a damaged V1 nerve rescues the impairment of epithelial healing in association with partial recovery of the stem/progenitor cell markers and upregulation of cell proliferation and of NGF expression in the peripheral/limbal epithelium. Gene transfer of TRPV4 did not accelerate the regeneration of nerve fibers. Sensory nerve TRPV4 is critical to maintain stemness of peripheral/limbal basal cells, and is one of the major mechanisms of homeostasis maintenance of corneal epithelium.

Cell-independent matrix configuration in early corneal development.

Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3.

Collagen fiber crimping following in vivo UVA-induced corneal crosslinking.

The purpose of this study was to measure collagen fiber crimping (CFC) using nonlinear optical imaging of second harmonic generated (SHG) signals to determine the effects of UVA-riboflavin induced corneal collagen crosslinking (UVA CXL) on collagen structure. Two groups, four rabbits each, were treated in the right eye with standard UVA CXL. In vivo confocal microscopy was performed at 1, 2, and 4 weeks after treatment for the first group and up to three months for the second group to measure epithelial/stromal thickness and corneal haze during recovery. Rabbits were sacrificed at one and three months, respectively, and their corneas fixed under pressure. Regions of crosslinking were identified by the presence of collagen autofluorescence (CAF) and then collagen structure was imaged using SHG microscopy. The degree of CFC was determined by measuring the percentage difference between the length of the collagen fiber and the linear distance traveled. CFC was measured in the central anterior and posterior CXL region, the peripheral non-crosslinked region in the same cornea, and the central cornea of the non-crosslinked contralateral eye. No change in corneal thickness was detected after one month, however the stromal thickness surpassed its original baseline thickness at three months by 25.9 µm. Corneal haze peaked at one month and then began to clear. Increased CAF was detected in all CXL corneas, localized to the anterior stroma and extending to 42.4 ±â€¯3.4% and 47.7 ±â€¯7.6% of the corneal thickness at one and three months. There was a significant (P < 0.05) reduction in CFC in the CAF region in all eyes averaging 1.007 ±â€¯0.006 and 1.009 ±â€¯0.005 in one and three month samples compared to 1.017 ±â€¯0.04 and 1.016 ±â€¯0.06 for controls. These results indicate that there is a significant reduction in collagen crimping following UVA CXL of approximately 1%. One possible explanation for this loss of crimping could be shortening of the collagen fibers over the CXL region.

Axial mechanical and structural characterization of keratoconus corneas.

PURPOSE: Previous studies indicate that there is an axial gradient of collagen lamellar branching and anastomosing leading to regional differences in corneal tissue stiffness that may control corneal shape. To further test this hypothesis we have measured the axial material stiffness and quantified the collagen lamellar complexity in ectatic and mechanically weakened keratoconus corneas (KC). METHODS: Acoustic radiation force elastic microscopy (ARFEM) was used to probe the axial mechanical properties of the cone region of three donor KC buttons. 3 Dimensional second harmonic generation microscopy (3D-SHG) was used to qualitatively evaluate lamellar organization in 3 kC buttons and quantitatively measure lamellar branching point density (BPD) in a separate KC button that had been treated with epikeratophakia (Epi-KP). RESULTS: The mean elastic modulus for the KC corneas was 1.67 ±â€¯0.44 kPa anteriorly and 0.970 ±â€¯0.30 kPa posteriorly, substantially below that previously measured for normal human cornea. 3D-SHG of KC buttons showed a simplified collagen lamellar structure lacking noticeable angled lamellae in the region of the cone. BPD in the anterior, posterior, central and paracentral regions of the KC cornea were significantly lower than in the overlying Epi-KP lenticule. Additionally, BPD in the cone region was significantly lower than the adjacent paracentral region in the KC button. CONCLUSIONS: The KC cornea exhibits an axial gradient of mechanical stiffness and a BPD that appears substantially lower in the cone region compared to normal cornea. The findings reinforce the hypothesis that collagen architecture may control corneal mechanical stiffness and hence corneal shape.

Meibocyte differentiation and renewal: Insights into novel mechanisms of meibomian gland dysfunction (MGD).

This paper reviews our current understanding of age-related meibomian gland dysfunction (MGD) and the role of the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ), in the regulation of meibomian gland function, meibocyte differentiation and lipid synthesis. The studies suggest that PPARγ is a master regulator of meibocyte differentiation and function, whose expression and nuclear signaling coupled with meibocyte renewal is altered during aging, potentially leading to atrophy of the meibomian gland as seen in clinical MGD. Study of meibomian gland stem cells also suggest that there is a limited number of precursor meibocytes that provide progeny to the acini, that may be susceptible to exhaustion as occurs during aging and other environmental factors. Further study of pathways regulating PPARγ expression and function as well as meibocyte stem cell maintenance may provide clues to establishing cellular and molecular mechanisms underlying MGD and the development of novel therapeutic strategies to treating this disease.

Ocular surface inflammation impairs structure and function of meibomian gland.

Dysfunction of the meibomian glands alters secreted meibum quantitatively and qualitatively that can lead to damage to the ocular surface epithelium. In response to an unstable tear film cause by meibomian gland dysfunction, ocular surface epithelium is damaged and expresses inflammatory cytokines leading to secondary ocular inflammation. In turn, inflammatory disorders of the palpebral conjunctiva and lid margin may affect the structure and function of meibomian gland. The disorders include allergic conjunctivitis, long-term usage of contact lenses, dermatological diseases that affect conjunctival homeostasis, Stevens-Johnson's syndrome or chemical burning of the ocular surface and lid margin.

Transcriptome analysis of aging mouse meibomian glands.

PURPOSE: Dry eye disease is a common condition associated with age-related meibomian gland dysfunction (ARMGD). We have previously shown that ARMGD occurs in old mice, similar to that observed in human patients with MGD. To begin to understand the mechanism underlying ARMGD, we generated transcriptome profiles of eyelids excised from young and old mice of both sexes. METHODS: Male and female C57BL/6 mice were euthanized at ages of 3 months or 2 years and their lower eyelids removed, the conjunctival epithelium scrapped off, and the tarsal plate, containing the meibomian glands, dissected from the overlying muscle and lid epidermis. RNA was isolated, enriched, and transcribed into cDNA and processed to generate four non-stranded libraries with distinct bar codes on each adaptor. The libraries were then sequenced and mapped to the mm10 reference genome, and expression results were gathered as reads per length of transcript in kilobases per million mapped reads (RPKM) values. Differential gene expression analyses were performed using CyberT. RESULTS: Approximately 55 million reads were generated from each library. Expression data indicated that about 15,000 genes were expressed in these tissues. Of the genes that showed more than twofold significant differences in either young or old tissue, 698 were identified as differentially expressed. According to the Gene Ontology (GO) analysis, the cellular, developmental, and metabolic processes were found to be highly represented with Wnt function noted to be altered in the aging mouse. CONCLUSIONS: The RNA sequencing data identified several signaling pathways, including fibroblast growth factor (FGF) and Wnt that were altered in the meibomian glands of aging mice.

Synergistic Cysteamine Delivery Nanowafer as an Efficacious Treatment Modality for Corneal Cystinosis.

A synergy between the polymer biomaterial and drug plays an important role in enhancing the therapeutic efficacy, improving the drug stability, and minimizing the local immune responses in the development of drug delivery systems. Particularly, in the case of ocular drug delivery, the need for the development of synergistic drug delivery system becomes more pronounced because of the wet ocular mucosal surface and highly innervated cornea, which elicit a strong inflammatory response to the instilled drug formulations. This article presents the development of a synergistic cysteamine delivery nanowafer to treat corneal cystinosis. Corneal cystinosis is a rare metabolic disease that causes the accumulation of cystine crystals in the cornea resulting in corneal opacity and loss of vision. It is treated with topical cysteamine (Cys) eye drops that need to be instilled 6-12 times a day throughout the patient's life, which causes side effects such as eye pain, redness, and ocular inflammation. As a result, compliance and treatment outcomes are severely compromised. To surmount these issues, we have developed a clinically translatable Cys nanowafer (Cys-NW) that can be simply applied on the eye with a fingertip. During the course of the drug release, Cys-NW slowly dissolves and fades away. The in vivo studies in cystinosin knockout mice demonstrated twice the therapeutic efficacy of Cys-NW containing 10 µg of Cys administered once a day, compared to 44 µg of Cys as topical eye drops administered twice a day. Furthermore, Cys-NW stabilizes Cys for up to four months at room temperature compared to topical Cys eye drops that need to be frozen or refrigerated and still remain active for only 1 week. The Cys-NW, because of its enhanced therapeutic efficacy, safety profile, and extended drug stability at room temperature, can be rapidly translated to the clinic for human trials.

From nano to macro: studying the hierarchical structure of the corneal extracellular matrix.

In this review, we discuss current methods for studying ocular extracellular matrix (ECM) assembly from the 'nano' to the 'macro' levels of hierarchical organization. Since collagen is the major structural protein in the eye, providing mechanical strength and controlling ocular shape, the methods presented focus on understanding the molecular assembly of collagen at the nanometre level using X-ray scattering through to the millimetre to centimetre level using non-linear optical (NLO) imaging of second harmonic generated (SHG) signals. Three-dimensional analysis of ECM structure is also discussed, including electron tomography, serial block face scanning electron microscopy (SBF-SEM) and digital image reconstruction. Techniques to detect non-collagenous structural components of the ECM are also presented, and these include immunoelectron microscopy and staining with cationic dyes. Together, these various approaches are providing new insights into the structural blueprint of the ocular ECM, and in particular that of the cornea, which impacts upon our current understanding of the control of corneal shape, pathogenic mechanisms underlying ectatic disorders of the cornea and the potential for corneal tissue engineering.

Meibomian gland dysfunction: hyperkeratinization or atrophy?

Meibomian gland dysfunction (MGD) is the major cause of evaporative dry eye disease (EDED) and dysfunction is widely thought to mechanistically involve ductal hyperkeratinization, plugging and obstruction. This review re-evaluates the role of hyperkeratinization in MGD based on more recent findings from mouse models. In these studies, eyelids from normal young and old mice or mice exposed to desiccating stress were evaluated by immunofluorescent tomography and 3-dimensional reconstruction to evaluate gland volume, expression of hyperkeratinization markers and cell proliferation or stimulated Raman scattering (SRS) microscopy to assess lipid quality. Results indicate that aging mice show dropout of meibomian glands with loss of gland volume and a forward migration of the mucocutaneous junction anterior to the gland orifice; similar age-related changes that are detected in human subjects. Atrophic glands also showed evidence of epithelial plugging of the orifice without the presence of hyperkeratinization. Mice exposed to desiccating stress showed hyperproliferation of the meibomian gland and ductal dilation suggesting a marked increase in lipid synthesis. Lipid quality was also affected in EDED mice with an increase in the protein content of lipid within the duct of the gland. Overall, age-related changes in the mouse show similar structural and functional correlates with that observed in clinical MGD without evidence of hyperkeratinization suggesting that gland atrophy may be a major cause of EDED. The response of the meibomian gland to desiccating stress also suggest that environmental conditions may accelerate or potentiate age-related changes.

TRPA1 is required for TGF-ß signaling and its loss blocks inflammatory fibrosis in mouse corneal stroma.

We examined whether the loss of transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing ion channel, or TRPA1 antagonist treatment affects the severity inflammation and scarring during tissue wound healing in a mouse cornea injury model. In addition, the effects of the absence of TRPA1 on transforming growth factor ß1 (TGF-ß1)-signaling activation were studied in cell culture. The lack of TRPA1 in cultured ocular fibroblasts attenuated expression of TGF-ß1, interleukin-6, and α-smooth muscle actin, a myofibroblast the marker, but suppressed the activation of Smad3, p38 MAPK, ERK, and JNK. Stroma of the healing corneas of TRPA1(-/-) knockout (KO) mice appeared more transparent compared with those of wild-type mice post-alkali burn. Eye globe diameters were measured from photographs. An examination of the corneal surface and eye globes suggested the loss of TRPA1 suppressed post-alkali burn inflammation and fibrosis/scarring, which was confirmed by histology, immunohistochemistry, and gene expression analysis. Reciprocal bone marrow transplantation between mice showed that KO corneal tissue resident cells, but not KO bone marrow-derived cells, are responsible for KO mouse wound healing with reduced inflammation and fibrosis. Systemic TRPA1 antagonists reproduced the KO phenotype of healing. In conclusion, a loss or blocking of TRPA1 in mice reduces inflammation and fibrosis/scarring in the corneal stroma during wound healing following an alkali burn. The responsible mechanism may include the inhibition of TGF-ß1-signaling cascades in fibroblasts by attenuated TRPA1 signaling. Inflammatory cells are considered to have a minimum involvement in the exhibition of the KO phenotype after injury.

A microfabricated, optically accessible device to study the effects of mechanical cues on collagen fiber organization.

As the primary structural protein of our bodies, fibrillar collagen and its organizational patterns determine the biomechanics and shape of tissues. While the molecular assembly of individual fibrils is well understood, the mechanisms determining the arrangement of fibers and thus the shape and form of tissues remain largely unknown. We have developed a cell culture model that successfully recapitulates early tissue development and the de novo deposition of collagen fibers to investigate the role of mechanical cues on collagen fiber alignment. The devices used a thin, collagen-coated deformable PDMS membrane inside a tissue culture well built on microscope-grade coverslips. Deformations and strains in the PDMS membrane were quantified by tracking fluorescent bead displacement and through the use of a COMSOL model. Cyclical strains were applied to serum-cultured rabbit corneal cells at 0.5 Hz for 24-48 h and showed a preferred alignment after 36 h of cyclical loading. Cells cultured with ascorbic acid under methylcellulose serum-free conditions deposited a collagenous matrix that was visible under live second harmonic generation microscopy at week 4. Our microfabricated tissue culture system allows for the controllable application of a wide range of stress profiles to cells, and for the observation and quantification of cells and de novo collagen formation in vitro. Future studies will involve the fabrication of models to study the formation and organization of collagen in ocular diseases.

Modeling meibum secretion: Alternatives for obstructive Meibomian Gland Dysfunction (MGD).

PURPOSE: While changes in meibum quality are correlated with severity of meibomian gland dysfunction (MGD) and dry eye disease, little is known regarding the mechanics of meibum secretion. The purpose of this study was to develop a finite element model of meibum secretion and evaluate the effect of various factors that might impact meibum delivery to the ocular surface. METHODS: A finite element analysis in COMSOL 6.0 was used to simulate the flow of meibum within the gland's terminal excretory duct. Historical normal human meibum rheology data taken over the meibum melting range from fluid (35-40 °C) to solid (25-30 °C) were then used to calculate the minimum yield stress and plastic viscosity of meibum. The effects of meibum melting state, eyelid pressure and terminal duct diameter on meibum flow rates were then systematically investigated. RESULTS: The melting state of meibum from liquid to solid was associated with an increase in the minimum yield stress and plastic viscosity that caused an exponential decrease in meibum flow. Modeling also established that there was a linear correlation between meibum flow rate and eyelid pressure needed to express meibum and the 4th power of the terminal duct radius. CONCLUSIONS: Our results suggest that changes in the melting state of meibum from fluid to solid, as well as changes in the radius of the terminal excretory duct and the force exerted by the eyelid can lead to dramatic decreases in the flow of meibum. Together these findings suggest alternative mechanisms for meibomian gland obstruction.

The effects of age and dysfunction on meibomian gland population dynamics.

PURPOSE: While meibomian gland dysfunction (MGD) is widely recognized as a major cause of evaporative dry eye disease, little is known about normal gland differentiation and lipid synthesis or the mechanism underlying gland atrophy and abnormal lipid secretion. The purpose of this study was to use single-cell and spatial transcriptomics to probe changes in cell composition, differentiation, and gene expression associated with two murine models of MGD: age-related gland atrophy in wild-type mice and altered meibum quality in acyl-CoA wax alcohol acyltransferase 2 (Awat2) knockout (KO) mice. METHODS: Young (6 month) and old (22 month) wild type, C57Bl/6 mice and young (3 month) and old (13 month) Awat2 KO mice were used in these studies. For single-cell analysis, the tarsal plate was dissected from the upper and lower eyelids, and single cells isolated and submitted to the UCI Genomic Core, while for the spatial analysis frozen tissue sections were shipped to Resolve Biosciences on dry ice and sections probed in duplicate using a meibomian gland specific, 100 gene Molecular Chartography panel. RESULTS: Analysis of gene expression patterns identified the stratified expression of lipogenic genes during meibocyte differentiation, which may control the progressive synthesis of meibum lipids; an age-related decrease in meibocytes; and increased immune cell infiltration. Additionally, we detected unique immune cell populations in the Awat2 KO mouse suggesting activation of psoriasis-like, inflammatory pathways perhaps caused by ductal dilation and hyperplasia. CONCLUSION: Together these findings support novel mechanism controlling gland function and dysfunction.

The Yin and Yang of non-immune and immune responses in meibomian gland dysfunction.

Meibomian gland dysfunction (MGD) is a leading cause of dry eye disease and one of the most common ophthalmic conditions encountered in eye clinics worldwide. These holocrine glands are situated in the eyelid, where they produce specialized lipids, or meibum, needed to lubricate the eye surface and slow tear film evaporation - functions which are critical to preserving high-resolution vision. MGD results in tear instability, rapid tear evaporation, changes in local microflora, and dry eye disease, amongst other pathological entities. While studies identifying the mechanisms of MGD have generally focused on gland obstruction, we now know that age is a major risk factor for MGD that is associated with abnormal cell differentiation and renewal. It is also now appreciated that immune-inflammatory disorders, such as certain autoimmune diseases and atopy, may trigger MGD, as demonstrated through a T cell-driven neutrophil response. Here, we independently discuss the underlying roles of gland and immune related factors in MGD, as well as the integration of these two distinct mechanisms into a unified perspective that may aid future studies. From this unique standpoint, we propose a revised model in which glandular dysfunction and immunopathogenic pathways are not primary versus secondary contributors in MGD, but are fluid, interactive, and dynamic, which we likened to the Yin and Yang of MGD.

Meibomian gland stem/progenitor cells: The hunt for gland renewal.

Meibomian glands (MGs) secrete lipid (meibum) onto the ocular surface to form the outermost layer of the tear film. Proper meibum secretion is essential for stabilizing the tear film, reducing aqueous tear evaporation, and maintaining the homeostasis of the ocular surface. Atrophy of MG as occurs with aging, leads to reduction of meibum secretion, loss of ocular surface homeostasis and evaporative dry eye disease (EDED). Since MGs are holocrine glands, secretion of meibum requires continuous self-renewal of lipid-secreting acinar meibocytes by stem/progenitor cells, whose proliferative potential is dramatically reduced with age leading to MG atrophy and an age-related meibomian gland dysfunction (ARMGD). Understanding the cellular and molecular mechanisms regulating meibocyte stem/progenitor cell maintenance and renewal may provide novel approaches to regenerating MG and treating EDED. Towards that end, recent label retaining cell and lineage-tracing experiments as well as knock-out transgenic mouse studies have begun to identify the location and identities of meibocyte progenitor cells and potential growth and transcription factors that may regulate meibocyte renewal. In addition, recent reports have shown that ARMGD may be reversed by novel therapeutics in mice. Herein, we discuss our current understanding of meibocyte stem/progenitor cells and the hunt for gland renewal.

Aberrant expression of a beta-catenin gain-of-function mutant induces hyperplastic transformation in the mouse cornea.

Beta-catenin signaling has been shown to play a fundamental role in embryonic development and tumorigenesis. In this study, we investigated the role of beta-catenin (Ctnnb1) in corneal homeostasis and tumorigenesis. Conditional expression of a murine Ctnnb1 gain-of-function mutation alone caused corneal neoplasia and neovascularization, resembling human ocular surface squamous neoplasia (OSSN). These corneas displayed an upregulation of cell proliferative markers (PCNA and p63), while presenting downregulation of both the Pax-6 transcription factor and the corneal differentiation marker cytokeratin 12. In addition, the expression of limbal-type keratin 15 ectopically extended to cornea, but the pattern of conjunctival keratin 4 and epidermal keratin 10 were unchanged. Moreover, epithelial E-cadherin and laminins decreased concomitantly with elevated levels of MMP-7. We also noticed a dramatic upregulation of pro-angiogenic factors (Vegf-A, Vegfr1) and angiopoietins in these corneas. Interestingly, all human OSSN specimens examined revealed nuclear beta-catenin immunoreactivity. Taken together, these results argue that beta-catenin activation is a crucial step during OSSN pathogenesis. Thus, inhibition of beta-catenin might be beneficial for treating this disease.

Quiescent keratocytes fail to repair MMC induced DNA damage leading to the long-term inhibition of myofibroblast differentiation and wound healing.

PURPOSE: The purpose of this study was to determine the acute and long-term effects of mitomycin C (MMC) on quiescent rabbit corneal keratocytes regarding cell proliferation, myofibroblast differentiation and DNA repair. METHODS: Quiescent keratocytes cultured in serum-free media were exposed to various concentrations of MMC and then treated with transforming growth factor-ß (TGFß). DNA damage was evaluated in both cultured keratocytes and live rabbit eyes following treatment with MMC. The long-term ability of quiescent keratocytes to repair MMC induced damage in vivo was evaluated in rabbits treated with MMC 2 months before 100 µm deep lamellar keratectomy (LK) injury. RESULTS: MMC significantly blocked TGFß-induced cell proliferation and myofibroblast differentiation in cultured quiescent keratocytes and altered the transcriptional regulation of macrophage chemotactic protein-1 (MCP-1) and alpha smooth muscle actin (αSMA). MMC also induced phosphorylation of the nuclear histone marker of DNA damage, γH2AX (a member of the H2A histone family), without induction of cell cycle entry or immediate DNA repair measured by Comet assay. In live rabbits, 0.2 mg/ml MMC significantly induced γH2AX nuclear immunostaining (p<0.05) throughout the cornea and corneas receiving 0.2 mg/ml MMC treatment 2 months before LK injury showed complete absence of any corneal scarring. CONCLUSIONS: MMC induces DNA damage to quiescent corneal keratocytes, which remains unrepaired, resulting in abnormal cell replication and gene transcription that leads to long-term effects on corneal repair. Overall these findings suggest that there may be long-term and perhaps permanent consequences to the application of MMC as an anti-fibrotic therapy.