Acute Corneal Edema in a Middle-Aged Patient.

A middle-aged patient who had bilateral penetrating keratoplasty 20 years ago for keratoconus presented with pain and blurriness of the right eye for 2 days. Despite prompt corticosteroid therapy, they had no vision improvement and reported occasional pain. What would you do next?

Netarsudil-associated reticular corneal epithelial edema.

PURPOSE: To describe 8 cases of reversible reticular corneal epithelial edema of the cornea that developed after use of the topical Rho-kinase inhibitor netarsudil. METHODS: This is a retrospective chart review case series of 8 patients treated with netarsudil at an academic medical center. OBSERVATIONS: Patients had predisposing corneal conditions including penetrating keratoplasty, corneal decompensation after trabeculectomy-associated endophthalmitis, congenital glaucoma with Haab striae, aphakic bullous keratopathy, history of Ahmed valve and silicone oil, and Fuchs endothelial corneal dystrophy undergoing Descemet stripping only. One patient did not have clear predisposing corneal disease other than low endothelial cell density and a history of trabeculectomy. All patients developed reticular corneal epithelial edema, which appeared as collections of moderate sized superficial epithelial bullae arranged in a reticular pattern resembling a honeycomb. Most developed these changes within weeks of initiating netarsudil, but unique to this series are 2 cases in which netarsudil was tolerated by the cornea for months before developing reticular corneal epithelial edema after diode laser cyclophotocoagulation. In cases which underwent anterior segment optical coherence tomography, the imaging demonstrated that the corneal stroma was not edematous, and the reticular corneal epithelial edema involved both host and donor corneal epithelium in cases of penetrating keratoplasty. This fully resolved in all cases upon cessation of netarsudil, and this series is the first to document resolution via a pattern in which the individual bullae become smaller and more widely spaced apart. CONCLUSION: Netarsudil can cause a reversible reticular corneal epithelial edema.

FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro.

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-ß1 and -ß3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-ß1, TGF-ß3, or TGF-ß1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-ß1 or TGF-ß3 impart distinct effects on genes involved in wound healing and fibrosis-ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-ß1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-ß3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-ß1 + FAKi attenuated TGF-ß1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-ß1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

Initiation of fibrosis in the integrin Αvß6 knockout mice.

We previously demonstrated that ß6 knockout mice showed impaired wound repair in corneal debridement and keratectomy wounds. In the current investigation, we continued our examination of integrin αvß6 in order to determine if it was required for the initiation of wound healing in a corneal wound model that normally heals in a fibrotic manner. A full-thickness corneal incision was made in C57BL/6 J wild type (WT) and C57BL/6-Itgb6 KO (ß6-/-) mice. The mice were observed at 3, 7, 14, and 28 days post-incision. The morphology of corneal restoration was observed in tissue sections stained with hemotoxilin and eosin (H&E). In addition, indirect-immunofluorescence (IF) was performed on sections and/or whole mounts to evaluate the immunolocalization of α-smooth muscle actin (SMA) and thrombospondin-1 (TSP-1). H&E staining revealed that the corneas in ß6-/- mice healed slower than those in WT mice, with an obvious delay in the restoration of the stromal matrix and epithelium. In sections at 3 and 7 days, SMA and TSP-1 were greatly reduced in the ß6-/- mice as compared to WT, but peaked at 28 days after incision. Whole mount SMA IF results were consistent with those from sections. Therefore, the initiation of fibrosis was inhibited by the lack of αvß6; however, there appeared to be an alternate mechanism that initiated fibrosis 7-14 days later. Localization of TSP-1 correlated with expression of SMA whether wound healing was delayed or initiated immediately after wounding.

Inhibition of Human Corneal Myofibroblast Formation.

Purpose: Transforming growth factor-beta (TGF-ß) isoform 1 (T1) is involved in corneal fibrotic wound healing by stimulating myofibroblast transformation and altering fibrotic gene expression. In this study, two specific inhibitors were used to dissect the relationship between myofibroblast generation and the TGF-ß/Smad- or TGF-ß/p38-signaling pathway in human corneal fibroblasts (HCF). Methods: In HCF, Trx-SARA (Smad-pathway inhibitor) was used to block the TGF-ß/Smad-signaling pathway, and the p38 inhibitor (p38inh, SB202190) was used to inhibit p38MAPK, thus blocking the TGF-ß/p38-signaling pathway. HCF ± Trx-SARA or Trx-GA (SARA control) were serum starved overnight in Eagle's minimum essential medium (EMEM) ± p38inh, grown in EMEM ± T1 ± p38inh for 24 hours, and then processed for indirect-immunofluorescence, Western blot, or quantitative real-time polymerase chain reaction to examine α-smooth muscle actin (αSMA) and other fibrotic genes, such as fibronectin, thrombospondin1, and type III collagen. In addition, the morphology and the effect of p38inh on myofibroblast phenotype after myofibroblast formation were examined. Results: We observed that Trx-SARA had little effect on αSMA expression, indicating that blocking the Smad pathway did not significantly inhibit myofibroblast formation. However, p38inh did significantly inhibit αSMA and other fibrotic genes, thus efficiently preventing the transition of HCFs to myofibroblasts. In addition, morphology changed and αSMA decreased in myofibroblasts exposed to p38inh medium, as compared with controls. Conclusions: HCF transition to myofibroblasts was mainly through the p38 pathway. Therefore, blocking the p38 pathway may be a potential therapeutic tool for human corneal fibrosis prevention/treatment, because it controls myofibroblast formation in human corneal cells, while leaving other functions of T1 unaffected.

Peripheral Endothelial Cell Count Is a Predictor of Disease Severity in Advanced Fuchs Endothelial Corneal Dystrophy.

PURPOSE: In advanced Fuchs endothelial corneal dystrophy (FECD), central endothelial changes do not correlate with disease severity. The peripheral endothelial cell count (ECC) has not been studied as a marker of FECD severity. The goal of this study was to determine the relationship between the peripheral ECC and known clinical markers of FECD in advanced cases. METHODS: Patients with FECD examined between January 1, 2013, and September 1, 2016, by 1 cornea specialist were identified. Medical records from all previous visits were reviewed to include eyes with high-quality central and peripheral in vivo confocal microscopy images performed on the same day as a clinical evaluation. Endothelial photographs were used to perform manual cell counts centrally and peripherally. Clinical grading of FECD from 1 to 4 was performed at the slit-lamp. RESULTS: We identified 154 eyes of 126 patients that met criteria for inclusion. With higher disease grades, central ECC and peripheral ECC decreased, visual acuity worsened, and central corneal thickness (CCT) increased (all P < 0.05). In patients with advanced disease (defined as either grade 3 or 4, CCT >700, or central ECC <350), the peripheral ECC was the best predictor of disease severity and had the highest number of statistically significant correlations with other clinical markers compared with competing variables. CONCLUSIONS: In advanced FECD, severity is best determined by the peripheral ECC compared with the central ECC, visual acuity, clinical disease grade, and CCT. The peripheral ECC should be added to the clinical parameters used to evaluate FECD severity.

Development of wound healing models to study TGFß3's effect on SMA.

The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFß3) in reducing α-smooth muscle actin (SMA) expression in two models-an ex vivo organ culture and an in vitro 3D cell construct-both of which closely mimic an in vivo environment. For the ex vivo organ culture system, a central 6.0 mm corneal keratectomy was performed on freshly excised rabbit globes The corneas were then excised, segregated into groups treated with 1.0 ng/ml TGFß1 or ß3 (T1 or T3, respectively), and cultured for 2 weeks. The corneas were assessed for levels of haze and analyzed for SMA mRNA levels. For the 3D in vitro model, rabbit corneal fibroblasts (RbCFs) were cultured for 4 weeks on poly-transwell membranes in Eagle's minimum essential media (EMEM) + 10% FBS + 0.5 mM vitamin C ± 0.1 ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and RT-qPCR. The RT-qPCR data showed that SMA mRNA expression in T3 samples for both models was significantly lower (p < 0.05) than T1 treatment (around 3-fold in ex vivo and 2-fold in constructs). T3 also reduced the amount of scarring in ex vivo corneas as compared with the T1 samples. IF data from RbCF constructs confirmed that T3-treated samples had up to 4-fold (p < 0.05) lower levels of SMA protein expression than samples treated with T1. These results show that T3 when compared to T1 decreases the expression of SMA in both ex vivo organ culture and in vitro 3D cell construct models. Understanding the mechanism of T3's action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti-scarring therapy.

TGF-ß-target genes are differentially regulated in corneal epithelial cells and fibroblasts.

PURPOSE: Transforming growth factor-beta (TGF-ß) activates the canonical Smad pathway, which includes the Smad family of proteins and SARA (Smad Anchor for Receptor Activation) and other less understood pathways, including one involving p38MAPK. The goal of the current research was to determine if corneal epithelial cells and fibroblasts used the classical or alternative TGF-ß-signaling pathways. To examine this question, we made use of Trx-SARA, which inhibits native SARA, thus blocking the Smad pathway. METHODS: A human corneal epithelial cell line (HCE-TJ), and stromal fibroblasts (HCF) were infected with retroviruses (RTV) containing either Trx-SARA or Trx-GA (a control plasmid). The effect of Trx-SARA on thrombospondin-1 (TSP-1) expression in both cell types, p15ink4b expression in HCE-TJ, and cellular fibronectin (cFN) expression in HCF was determined. In addition, the effect of p38MAPK inhibitor on TSP-1 and p15ink4b were examined. RESULTS: In HCE-TJ with TGF-ß1, TSP-1-protein levels increased and peaked at 24 hours. Trx-SARA reduced TSP-1 expression in HCE-TJ, but had no effect on p15ink4b. With HCF, Trx-SARA failed to reduce TSP-1 expression; however, cFN expression decreased and proliferation was inhibited. By blocking the p38MAPK pathway, TSP-1 expression was reduced in HCF and p15ink4b expression was decreased in HCE-TJ. CONCLUSIONS: Surprisingly, TSP-1 was regulated through the Smad pathway in HCE-TJ and the p38MAPK pathway in HCF. The p38MAPK pathway also induced p15ink4b in HCE-TJ. Our results indicate that not all TGF-ß-target proteins require the Smad pathway, and it may be possible to block certain TGF-ß-target proteins without blocking the expression of all the TGF-ß-target proteins.

PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts.

Purpose: The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor ß3 (TGF-ß3) and TGF-ß1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-ß3's response to wounding. Methods: A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-ß1 or -ß3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. Results: In HCF, TGF-ß3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-ß1, while in HCF-P, both TGF-ß1 and -ß3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-ß3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. Conclusions: These results show that TGF-ß3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-ß3 treatment. Understanding the role of PDGFRα in TGF-ß3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-ß1 and TGF-ß3 in corneal wound healing.

Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization.

Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions.

Defective Phagocytic Corpse Processing Results in Neurodegeneration and Can Be Rescued by TORC1 Activation.

The removal of apoptotic cell corpses is important for maintaining homeostasis. Previously, defects in apoptotic cell clearance have been linked to neurodegeneration. However, the mechanisms underlying this are still poorly understood. In this study, we report that the absence of the phagocytic receptor Draper in glia leads to a pronounced accumulation of apoptotic neurons in the brain of Drosophila melanogaster. These dead cells persist in the brain throughout the lifespan of the organism and are associated with age-dependent neurodegeneration. Our data indicate that corpses persist because of defective phagosome maturation, rather than recognition defects. TORC1 activation, or inhibition of Atg1, in glia is sufficient to rescue corpse accumulation as well as neurodegeneration. These results suggest that phagocytosis of apoptotic neurons by glia during development is essential for brain homeostasis in adult flies. Furthermore, it suggests that TORC1 regulates Draper-mediated phagosome maturation. SIGNIFICANCE STATEMENT: Previously, defects in dead cell clearance were linked to neurodegeneration, but the exact mechanisms are not well understood. In this study, we report that the absence of an engulfment receptor leads to a pronounced accumulation of dead neurons in the brain of the fruit fly Drosophila melanogaster. These dead cells persist in the brain throughout the lifespan of the organism and are associated with age-dependent neurodegeneration. Our data indicate that corpses persist because of defective degradation of cells rather than recognition of dead cells.

Cornea As a Model for Testing CTGF-Based Antiscarring Drugs.

Scarring remains a serious complication of the wound healing process that can lead to the formation of excessive fibrous connective tissue in an organ or tissue leading to pain and loss of function. This process is mainly regulated by Transforming growth factor ß1 (TGF-ß1), which binds to receptors and induces its downstream mediator, Connective tissue growth factor (CTGF). The number of drugs targeting CTGF for treating scars has been on the rise in the past few years. The purpose of this article is to suggest the possibility of using cornea as a model for testing anti-CTGF therapies for scarring.