Corneal Endothelial Wound Healing Research Articles

Transcription factor 4 promotes increased corneal endothelial cellular migration by altering microtubules in Fuchs endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD) is a complex corneal disease characterized by the progressive decline and morphological changes of corneal endothelial cells (CECs) that leads to corneal edema and vision loss. The most common mutation in FECD is an intronic CTG repeat expansion in transcription factor 4 (TCF4) that leads to its altered expression. Corneal endothelial wound healing occurs primarily through cell enlargement and migration, and FECD CECs have been shown to display increased migration speeds. In this study, we aim to determine whether TCF4 can promote cellular migration in FECD CECs. We generated stable CEC lines derived from FECD patients that overexpressed different TCF4 isoforms and investigated epithelial-to-mesenchymal (EMT) expression, morphological analysis and cellular migration speeds. We found that full length TCF4-B isoform overexpression promotes cellular migration in FECD CECs in an EMT-independent manner. RNA-sequencing identified several pathways including the negative regulation of microtubules, with TUBB4A (tubulin beta 4A class IVa) as the top upregulated gene. TUBB4A expression was increased in FECD ex vivo specimens, and there was altered expression of cytoskeleton proteins, tubulin and actin, compared to normal healthy donor ex vivo specimens. Additionally, there was increased acetylation and detyrosination of microtubules in FECD supporting that microtubule stability is altered in FECD and could promote cellular migration. Future studies could be aimed at investigating if targeting the cytoskeleton and microtubules would have therapeutic potential for FECD by promoting cellular migration and regeneration.

ROCK Inhibitors as an Alternative Therapy for Corneal Grafting: A Systematic Review.

Purpose: Currently, corneal blindness is affecting >10 million individuals worldwide, and there is a significant unmet medical need because only 1.5% of transplantation needs are met globally due to a lack of high-quality grafts. In light of this global health disaster, researchers are developing corneal substitutes that can resemble the human cornea in vivo and replace human donor tissue. Thus, this review examines ROCK (Rho-associated coiled-coil containing protein kinases) inhibitors as a potential corneal wound-healing (CWH) therapy by reviewing the existing clinical and nonclinical findings. Methods: The systematic review was done from PubMed, Scopus, Web of Science, and Google Scholar for CWH, corneal injury, corneal endothelial wound healing, ROCK inhibitors, Fasudil, Netarsudil, Ripasudil, Y-27632, clinical trial, clinical study, case series, case reports, preclinical study, in vivo, and in vitro studies. After removing duplicates, all downloaded articles were examined. The literature search included the data till January 2023. Results: This review summarized the results of ROCK inhibitors in clinical and preclinical trials. In a clinical trial, various ROCK inhibitors improved CWH in individuals with open-angle glaucoma, cataract, iris cyst, ocular hypertension, and other ocular diseases. ROCK inhibitors also improved ocular wound healing by increasing cell adhesion, migration, and proliferation in vitro and in vivo. Conclusions: ROCK inhibitors have antifibrotic, antiangiogenic, anti-inflammatory, and antiapoptotic characteristics in CWH, according to the existing research. ROCK inhibitors were effective topical treatments for corneal infections. Ripasudil, Y-27632, H-1152, Y-39983, and AMA0526 are a few new ROCK inhibitors that may help CWH and replace human donor tissue.

Mice Deficient in TAZ (Wwtr1) Demonstrate Clinical Features of Late-Onset Fuchs' Endothelial Corneal Dystrophy.

We sought to define the role of Wwtr1 in murine ocular structure and function and determine the role of mechanotransduction in Fuchs' endothelial corneal dystrophy (FECD), with emphasis on interactions between corneal endothelial cells (CEnCs) and Descemet's membrane (DM). A Wwtr1 deficient mouse colony was established, and advanced ocular imaging, atomic force microscope (AFM), and histology/immunofluorescence were performed. Corneal endothelial wound healing was assessed using cryoinjury and phototherapeutic keratectomy in Wwtr1 deficient mice. Expression of WWTR1/TAZ was determined in the corneal endothelium from normal and FECD-affected patients; WWTR1 was screened for coding sequence variants in this FECD cohort. Mice deficient in Wwtr1 had reduced CEnC density, abnormal CEnC morphology, softer DM, and thinner corneas versus wildtype controls by 2 months of age. Additionally, CEnCs had altered expression and localization of Na/K-ATPase and ZO-1. Further, Wwtr1 deficient mice had impaired CEnC wound healing. The WWTR1 transcript was highly expressed in healthy human CEnCs comparable to other genes implicated in FECD pathogenesis. Although WWTR1 mRNA expression was comparable between healthy and FECD-affected patients, WWTR1/TAZ protein concentrations were higher and localized to the nucleus surrounding guttae. No genetic associations were found in WWTR1 and FECD in a patient cohort compared to controls. There are common phenotypic abnormalities seen between Wwtr1 deficient and FECD-affected patients, suggesting that Wwtr1 deficient mice could function as a murine model of late-onset FECD. Despite the lack of a genetic association between FECD and WWTR1, aberrant WWTR1/TAZ protein subcellular localization and degradation may play critical roles in the pathogenesis of FECD.

Rock inhibitors and antisense oligonucleotides for Fuchs corneal endothelial dystrophy

AbstractFuchs corneal dystrophy (FECD) is the most common primary cause of corneal endothelial dystrophy. FECD is a complex disease where interaction between multiple genetic and environmental factors results in endothelial cells apoptosis and atypical extracellular matrix deposition. Due to the shortage of donor tissue, and high costs of transplants, a topical therapy for treating corneal endothelial dysfunction is urgently required. The topical treatment of several agents has been investigated as therapies for FECD, by the promotion of endothelial cell proliferation and migration, in experimental models of the disorder. ROCK1 and ROCK2 inhibition promotes cell adhesion, prevents apoptosis, and enhances the proliferation of and human corneal endothelial cells. These therapies have involved the knockdown of connexin by siRNA, the release of cell–cell contact inhibition with EDTA, and inhibition of Rho‐associated protein kinase (ROCK) with Y‐27632. Recently several papers showed that Ripasudil, a rho kinase inhibitor, has been shown to promote corneal endothelial wound healing in animal models and in clinical trials. An antisense oligonucleotide (ASO) has also been developed to target the repetitive RNA. This has been shown to decrease foci formation and reversal of misplacing in ex‐vivo FECD specimens. Antisense oligonucleotides (ASO) reduced the RNA aggregates and their toxic downstream events in patient cells. ASO has to be designed to target a particular genetic mutation. This new medical‐based regenerative therapy may provide a less invasive and more effective method for treating patients with FECD.

Corneal endothelial wound healing after Descemet tear with a rho kinase inhibitor

Introduction: To the authors’ knowledge, this is the first report of Descemet tear postcataract surgery that was successfully treated with ripasudil hydrochloride hydrate. Patient and Clinical Findings: An 85-year-old pseudophakic man presented with blurry vision and mild photophobia in the left eye. Slitlamp biomicroscopy showed moderate corneal edema and a central Descemet tear from cataract surgery 7 years earlier. Diagnosis, Intervention, and Outcomes: The patient was treated for 1 month with prednisolone acetate 1% and sodium chloride 5% hyperosmotic ophthalmic solution with no clinical improvement. He declined surgical intervention and opted for medical management with ripasudil 0.4% ophthalmic solution, a rho kinase inhibitor. He applied it topically 4 times daily for 3 months, with improvement of corneal edema and corrected distance visual acuity. After discontinuation of ripasudil, his edema and vision worsened and then improved again after retreatment, showing a strong clinical response. 2 years after starting ripasudil therapy, the patient had continued to use ripasudil 3 times daily and reported worsened vision at lower doses. Conclusions: The use of topical ripasudil may be a viable medical intervention for corneal edema due to Descemet tear instead of surgery, although chronic use may be required.

CHIR99021 balance TGFβ1 induced human corneal endothelial-to-mesenchymal transition to favor corneal endothelial cell proliferation

Corneal endothelial cells (CECs) play a major role in the maintenance of stromal hydration via the barrier and pump function for clear vision. Adult CECs cannot regenerate after injury. CECs cultured in vitro can undergo mitosis but may undergo corneal endothelial-to-mesenchymal transition (EnMT) and lose their endothelial characteristics. In this study, we examined the effects of CHIR99021 on transforming growth factor beta-1(TGFβ1)-induced EnMT in human CECs (hCECs) lines. CHIR99021 kept hCECs in the hexagonal shape and could downregulate the EnMT markers alpha-smooth muscle actin (α-SMA) and fibronectin (FN1), meanwhile maintained the hCECs function markers Na+/K+-ATPase and zonula occludens-1 (ZO-1) at levels comparable to those in the normal control. Interestingly, we found that the combination of CHIR99021 and TGFβ1 at appropriate concentrations would significantly promote the proliferation and migration of hCECs. These effects may be related to the inhibition of RhoA or Rac1, as well as the activation of Wnt and Erk pathway, with a calcium homeostasis. Our findings indicate that CHIR99021 inhibit EnMT and that the combination of CHIR99021 and TGFβ1 may provide new ideas for corneal endothelial regeneration and wound healing.

Multiple roles of FGF10 in the regulation of corneal endothelial wound healing

Corneal endothelial dysfunction usually induces corneal haze and oedema, which seriously affect visual function. The main therapeutic strategy for this condition is corneal transplantation, but the use of this strategy is limited by the shortage of healthy donor corneas. Compared with corneal transplantation, drug intervention is less invasive and more accessible; thus, finding an effective pharmaceutical alternative for cornea transplantation is critical for the treatment of corneal endothelial dysfunction. In this study, we established a rabbit scratch model to investigate the effect of fibroblast growth factor 10 (FGF10) on corneal endothelial wound healing. Results showed that FGF10 injection accelerated the recovery of corneal transparency and increased the protein expression levels of ZO1, Na+/K+-ATPase and AQP-1. Moreover, FGF10 significantly inhibited the expression levels of endothelial-to-mesenchymal transition proteins and reduced the expression levels of the proinflammatory factors IL-1β and TNF-α in the anterior chamber aqueous humour. FGF10 also enhanced the Na+/K+-ATPase activity by enhancing mitochondrial function as a result of its direct interaction with its conjugate receptor. Thus, FGF10 could be a new pharmaceutical preparation as treatment for corneal endothelial dysfunction.

Rho kinase (ROCK) inhibitors in the management of corneal endothelial disease.

Rho kinase (ROCK) inhibitors are growing increasingly relevant in ophthalmology, and the goal of this review is to summarize their mechanisms of action and potential applications in the subspecialties of glaucoma, retina, and cornea. We will focus specifically on corneal endothelial wound healing, for which ROCK inhibition demonstrates particular promise. ROCK inhibition has been shown to promote corneal endothelial cell proliferation, increase intercellular adhesion, and suppress apoptosis. Topical ROCK inhibitor treatment has exhibited potential use in Fuchs endothelial dystrophy, corneal edema from acute surgical trauma and other etiologies, and tissue engineering therapy for the endothelial disease. Ripasudil and netarsudil, the two ROCK inhibitors available for ophthalmic use, are generally very well tolerated with mild and transient local side effects. ROCK inhibitors are revolutionizing the subspecialty of cornea, and further research is needed to compare long-term outcomes of ROCK inhibitor therapy to those of conventional endothelial keratoplasty, including visual acuity and endothelial cell density. Other possible avenues include the use of ROCK inhibitors to prolong corneal graft survival, and early data appears promising.

Y-27632 Promotes the Repair Effect of Umbilical Cord Blood-Derived Endothelial Progenitor Cells on Corneal Endothelial Wound Healing.

To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits. UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds. Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own. Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.

PTEN Inhibition Accelerates Corneal Endothelial Wound Healing through Increased Endothelial Cell Division and Migration.

PurposeTo investigate the role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in the regulation of corneal endothelial cell (CECs) focusing on proliferation and migration, and to further evaluate the application of PTEN inhibitors in the treatment of corneal endothelial dysfunction in a rat model.MethodsExpression of PTEN in human and rat corneal endothelium was determined by immunocytochemistry, western blotting, and ELISA. A small molecular inhibitor of PTEN, bpV(pic), was applied in the culture of human CEC cell line B4G12 and organ-cultured rat cornea in the presence of transforming growth factor beta 2 (TGF-β2). Cell cycle status was detected by flow cytometry and BrdU staining. Subcellular localization for endogenous p27Kip1 was detected by immunocytochemistry and western blotting. Moreover, exogenous transfected YFP-p27Kip1 was observed under a fluorescent microscope. Cell migration was examined with a wound scratch model and transwell invasion assay. Finally, bpV(pic) was intracamerally injected in a rat corneal endothelial injury model. The wound healing process was evaluated by slit lamp biomicroscopy, optical coherence tomography, histological and scanning electron microscope examination.ResultsThe expression of PTEN in human corneal endothelium was higher compared with rat, which we speculate was mostly responsible for the relatively less proliferation capacity of human CEC than rat. PTEN inhibition by bpV(pic) could reverse TGF-β2-induced CEC G1-arrest by alleviating p27Kip1 nuclear accumulation and decreasing total p27Kip1 expression. In addition, bpV(pic) promoted CEC migration, which acted synergistically with TGF-β2. Finally, intracameral injection of bpV(pic) could promote corneal endothelial wound healing in a rat model.ConclusionsOur study provided experimental basis for the development of therapeutic agent targeting on PTEN for the treatment of corneal endothelial dysfunction.

Transcription Factor 4 Regulates the Regeneration of Corneal Endothelial Cells.

PurposeHuman corneal endothelial cells (hCECs) have limited regenerative capacity in vivo. Reduced hCEC density results in bullous keratopathy requiring corneal transplantation. This study reveals the role of transcription factor 4 (TCF4) in hCEC diseases and suggests that TCF4 may be a molecular target for hCEC regeneration.MethodsCell shape, cell proliferation rates, and proliferation-associated proteins were evaluated in normal or senescent hCECs. TCF4 was blocked by siRNA (si-TCF4) or activated using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 activation systems (pl-TCF4). The corneal endothelium of six-week-old Sprague-Dawley (SD) rats was transfected by electroporation followed by cryoinjury.ResultsCell proliferation rates and TCF4 levels were reduced in senescent cells. TCF4 CRISPR activation enhanced corneal endothelial wound healing. TCF4 regulated mitochondrial functions including mitochondrial membrane potential, mitochondrial superoxide levels, and energy production. The percentage of cells in the S-phase was reduced with si-TCF4 and increased with pl-TCF4. Cell proliferation and cell cycle-associated proteins were regulated by TCF4. Autophagy was induced by si-TCF4. In vivo transfection of CRISPR/dCas9 activation systems (a-TCF4) induced regeneration of corneal endothelium.ConclusionsCorneal endothelial diseases are associated with TCF4 reduction; TCF4 may be a potential target for hCEC diseases. Gene therapy using TCF4 CRISPR/dCas9 may be an effective treatment for hCEC diseases.

Establish an In Vitro Cell Model to Explore the Impacts of UVA on Human Corneal Endothelial Wound Healing

ABSTRACT Purpose To provide scientific data for clinical practice in making strategies for accelerating corneal endothelial wound healing, we investigated the impact of UVA on the corneal endothelial wound healing process and the underlying mechanism using an in vitro cell model. Materials and methods An in vitro cell model for corneal endothelial wound healing was established by scratching the in vitro cultured human corneal endothelial cell (HCEnC) confluent layer. Then, we investigated the impacts of UVA irradiation and Ascorbic acid-2-phosphate (Asc-2p) on the wound healing process of the in vitro HCEnC model by examining wound-healing index, F-actin+ rate, Ki-67+ rate, and ROS production. Results After scratching, the Ki-67+ and F-actin+ HCEnCs occupied the scratching gap. Furthermore, the F-actin+ rates were significantly higher than Ki-67+ rates in the wound closure area. After irradiated with UVA, the wound-healing indexes, Ki-67+ rates and F-actin+ rates of the wound-healing model significantly reduced, whereas the ROS production significantly increased in a dose-dependent manner. Pretreatment with Asc-2p significantly reduced the ROS production as well as increased the wound-healing indexes, Ki-67+rates and F-actin+ rates of the UVA irradiated wound-healing model. Conclusion The migration of HCEnC plays a major role in the wound healing process of the established cell model, which is like the wound healing process in vivo. UVA decreases the wound closure of the in vitro HCEnC model dose-dependently, while antioxidant Asc-2p can attenuate the damage to UVA to HCEnCs probably via reducing ROS to improve their migration.

Nicotinamide inhibits corneal endothelial mesenchymal transition and accelerates wound healing

Corneal endothelial cells (CECs) maintain the clarity of the cornea through the barrier and pump function. Ex vivo culture or injury may cause corneal endothelial-mesenchymal transition (EnMT) and lead to loss of function. In this study, we explored the effects of nicotinamide (NIC) on the wound healing of rabbit corneal endothelium and the proliferation, migration, and EnMT of cultured human CEC lines. The animal results showed that corneal clarity was rapidly recovered within seven days through topical application of NIC in the rabbits with mechanical injury of the corneal endothelium, while the control corneas remained edematous and cloudy. Whole-mounted corneal staining found the expressions of Na+/K+-ATPase, aquaporin-1, and zonula occludens-1 were mainly localized to the boundaries of regenerated endothelium in NIC-treated eyes, in contrast to the scattered staining in vehicle-treated eyes. Interestingly, we found that NIC application inhibited the expression of typical EnMT marker alpha-smooth muscle actin, which appeared in the rabbit corneal endothelial wound healing. In vitro, NIC promoted the proliferation, but not the migration, of cultured human CECs. Moreover, NIC effectively inhibited transforming growth factor beta-1-induced corneal EnMT and decreased the levels of EnMT regulators snail and slug. Therefore, our study indicates that NIC enhances corneal endothelial wound healing through the promotion of proliferation and the inhibition of EnMT, which may provide a potential pharmaceutical agent for treating corneal endothelial dysfunction.

Topical Rho-Associated Kinase Inhibitor, Y27632, Accelerates Corneal Endothelial Regeneration in a Canine Cryoinjury Model.

Corneal endothelial cell regeneration varies by species, with nonhuman primates (NHPs) and rabbits displaying low and high proliferative capacities, respectively. Recent studies report that topical application of rho-associated kinase (ROCK) inhibitors accelerates corneal endothelial wound healing in animal models and human patients with endothelial dysfunction. This study determines the regenerative capacity of canine corneal endothelial cells in vivo and their response to a topical ROCK inhibitor, Y27632, after transcorneal freezing. Right eyes of 6 beagles underwent transcorneal freezing; 10 mM ROCK inhibitor Y27632 or vehicle control was applied topically to both eyes at least 4 times daily for 56 days. Endothelial cell density was evaluated by in vivo confocal microscopy, and corneal thickness was measured by Fourier-domain optical coherence tomography (FD-OCT) and ultrasound pachymetry. Transcorneal freezing induced severe central corneal edema in dogs, with restoration of transparency occurring within 4 weeks. Y27632 significantly decreased corneal thickness by FD-OCT and ultrasound pachymetry in the acute phase and significantly increased endothelial cell density at days 28 and 42 post-cryoinjury, suggesting faster restoration of endothelial cell recovery. Canine corneal endothelial function recovers at a similar rate as NHPs but more slowly than rabbits after cryoinjury. Faster corneal endothelial wound healing was observed by in vivo confocal microscopy and FD-OCT in dogs treated with Y27632 versus vehicle controls. Thus, a canine cryoinjury model may be a useful alternative to NHPs in detecting a response to therapies directed at endothelial regeneration.

Myosin phosphatase accelerates cutaneous wound healing by regulating migration and differentiation of epidermal keratinocytes via Akt signaling pathway in human and murine skin

Wound healing is a complex sequence of cellular and molecular processes such as inflammation, cell migration, proliferation and differentiation. ROCK is a widely investigated Ser/Thr kinase with important roles in rearranging the actomyosin cytoskeleton. ROCK inhibitors have already been approved to improve corneal endothelial wound healing. The purpose of this study was to investigate the functions of myosin phosphatase (MP or PPP1CB), a type-1 phospho-Ser/Thr-specific protein phosphatase (PP1), one of the counter enzymes of ROCK, in skin homeostasis and wound healing. To confirm our hypotheses, we applied tautomycin (TM), a selective PP1 inhibitor, on murine skin that caused the arrest of wound closure. TM suppressed scratch closure of HaCaT human keratinocytes without having influence on the survival of the cells. Silencing of, the regulatory subunit of MP (MYPT1 or PPP1R12A), had a negative impact on the migration of keratinocytes and it influenced the cell-cell adhesion properties by decreasing the impedance of HaCaT cells. We assume that MP differentially activates migration and differentiation of keratinocytes and plays a key role in the downregulation of transglutaminase-1 in lower layers of skin where no differentiation is required. MAPK Proteome Profiler analysis on human ex vivo biopsies with MYPT1-silencing indicated that MP contributes to the mediation of wound healing by regulating the Akt signaling pathway. Our findings suggest that MP plays a role in the maintenance of normal homeostasis of skin and the process of wound healing.

Allogeneic Descemet's Membrane Transplantation Enhances Corneal Endothelial Monolayer Formation and Restores Functional Integrity Following Descemet's Stripping.

To characterize the differences in corneal endothelial wound healing in the presence or absence of Descemet's membrane (DM), in vivo. New-Zealand white rabbits were subjected to 7-mm endothelial wound either by scraping (n = 8; DM intact), peeling (n = 6; DM removed), or a combinatory scrape/peel wound (n = 6). In a second experiment, rabbits underwent peel wound with immediate transplantation of pure decellularized human DM (n = 4). In vivo endothelial migration was assessed via trypan blue staining. Recovery of corneal clarity and thickness was performed by using slit-lamp biomicroscopy and optical coherence tomography. Cell proliferation, phenotype, and morphology were assessed by using immunofluorescence and scanning electron microscopy. In vivo wound closure was faster in the presence of DM; 25.4% ± 1.4%/d versus 5.5% ± 0.6%/d (P < 0.0001). At day 8, complete wound closure was seen in all of the scrape samples but none of the peel group, with wound closure preceding clinical recovery by approximately 6 days in the scrape group. Endothelial cells in the scraped areas reformed functional monolayers capable of restoring corneal thickness and transparency whilst those in the peeled area underwent mesenchymal-like transformation resulting in scar formation. Transplanting decellularized DM in animals receiving a peel wound resulted in clarity and thickness comparable to the scrape group. Endothelial proliferation (Ki67 +ve cells) was higher in scraped versus peeled areas: 54.7% ± 3.5% vs. 8.8% ± 0.7%, (P < 0.01). The presence of DM promoted endothelial wound healing, proliferation, and maintenance of a normal phenotype. DM transplantation recovered the abnormal peel phenotype back to that observed after endothelial scraping.

Descemet\u2019s Membrane Supports Corneal Endothelial Cell Regeneration in Rabbits

Descemet’s membrane (DM) helps maintain phenotype and function of corneal endothelial cells under physiological conditions, while little is known about the function of DM in corneal endothelial wound healing process. In the current study, we performed in vivo rabbit corneal endothelial cell (CEC) injury via CEC scraping, in which DM remained intact after CECs removal, or via DM stripping, in which DM was removed together with CECs. We found rabbit corneas in the CEC scraping group healed with transparency restoration, while there was posterior fibrosis tissue formation in the corneas after DM stripping on day 14. Following CEC scraping on day 3, cells that had migrated toward the central cornea underwent a transient fibrotic endothelial-mesenchymal transition (EMT) which was reversed back to an endothelial phenotype on day 14. However, in the corneas injured via DM stripping, most of the cells in the posterior fibrosis tissue did not originate from the corneal endothelium, and they maintained fibroblastic phenotype on day 14. We concluded that corneal endothelial wound healing in rabbits has different outcomes depending upon the presence or absence of Descemet’s membrane. Descemet’s membrane supports corneal endothelial cell regeneration in rabbits after endothelial injury.

Ribonuclease 5 facilitates corneal endothelial wound healing via activation of PI3-kinase/Akt pathway

To maintain corneal transparency, corneal endothelial cells (CECs) exert a pump function against aqueous inflow. However, human CECs are arrested in the G1-phase and non-proliferative in vivo. Thus, treatment of corneal endothelial decompensation is limited to corneal transplantation, and grafts are vulnerable to immune rejection. Here, we show that ribonuclease (RNase) 5 is more highly expressed in normal human CECs compared to decompensated tissues. Furthermore, RNase 5 up-regulated survival of CECs and accelerated corneal endothelial wound healing in an in vitro wound of human CECs and an in vivo cryo-damaged rabbit model. RNase 5 treatment rapidly induced accumulation of cytoplasmic RNase 5 into the nucleus, and activated PI3-kinase/Akt pathway in human CECs. Moreover, inhibition of nuclear translocation of RNase 5 using neomycin reversed RNase 5-induced Akt activation. As a potential strategy for proliferation enhancement, RNase 5 increased the population of 5-bromo-2′-deoxyuridine (BrdU)-incorporated proliferating CECs with concomitant PI3-kinase/Akt activation, especially in CECs deprived of contact-inhibition. Specifically, RNase 5 suppressed p27 and up-regulated cyclin D1, D3, and E by activating PI3-kinase/Akt in CECs to initiate cell cycle progression. Together, our data indicate that RNase 5 facilitates corneal endothelial wound healing, and identify RNase 5 as a novel target for therapeutic exploitation.

Effect of the Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) on Corneal Endothelial Wound Healing.

Ripasudil (Glanatec), a selective rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, was approved as a glaucoma and ocular hypertension treatment in Japan in 2014. The purpose of this study was to investigate the feasibility of using ripasudil eye drops to treat corneal endothelial injuries. Cultured human corneal endothelial cells (HCECs) were treated with ripasudil, and 5-bromo-2'-deoxyuridine (BrdU) incorporation was evaluated by ELISA. A rabbit corneal endothelial damage model was also created by mechanically scraping the corneal endothelium, followed by topical ripasudil eye drop application for 2 weeks. The anterior segment was evaluated by slit-lamp microscopy, and central corneal thickness was measured by ultrasound pachymetry. Corneal specimens were evaluated by phalloidin staining and immunohistochemical analysis using antibodies against Ki67, N-cadherin, and Na+/K+-ATPase. Many more BrdU-positive cells were observed among the HCECs treated with ripasudil (0.3-30 μM) than among the control HCECs. Ripasudil-treated eyes in a rabbit model showed 91.5 ± 2.0% Ki67-positive cells after 48 hours, whereas control eyes showed 52.6 ± 1.3%. Five of six corneas became transparent in ripasudil-treated eyes, whereas zero of six corneas became transparent in the control eyes. Regenerated cell densities were higher in the eyes treated with ripasudil than in eyes treated with vehicle. Eyes treated with ripasudil expressed N-cadherin and Na+/K+-ATPase in almost all CECs, whereas this expression was decreased in control eyes. Ripasudil promoted corneal endothelial wound healing, supporting its development as eye drops for treating acute corneal endothelial damage due to eye surgeries, especially cataract surgery.

Effect of the Rho Kinase Inhibitor Y-27632 on Corneal Endothelial Wound Healing.

The purpose of this study was to investigate the feasibility of using Rho-associated kinase (ROCK) inhibitor eye drops for treating severe corneal endothelial damage due to surgical invasion. A rabbit corneal endothelial damage model was created by mechanically scraping half the area of the corneal endothelium of eighteen eyes of Japanese white rabbits. A selective ROCK inhibitor, Y-27632 (10 mM), was applied topically for 2 weeks, and then the anterior segment was evaluated by slitlamp microscopy. The corneal endothelium was evaluated by phalloidin staining and immunohistochemical analysis. We then conducted pilot clinical research and applied Y-27632 eye drops topically to three patients who exhibited severe corneal edema due to corneal endothelial damage. In the corneal endothelial damage rabbit model, more Ki67-positive cells were detected in Y-27632-treated eyes than in control eyes. Five of six corneas became transparent in Y-27632-treated eyes, whereas zero of six corneas became transparent in the control eyes (P < 0.01). Actin fibers were distributed at the cell cortex in the eyes treated with Y-27632, whereas actin distribution was partially disrupted, and stress fibers were observed in control eyes. N-cadherin and Na+/K+-ATPase were expressed in almost all cells in Y-27632-treated eyes, but expression decreased in control eyes. Preliminary human cases confirmed that ROCK inhibitor eye drops were considerably effective for treatment of corneal edema associated with cataract surgery. ROCK inhibitor may be developed as an eye drop for treating acute corneal endothelial damage to prevent progression of bullous keratopathy. (University Hospital Medical Information Network Clinical Trial Registry no. UMIN000003625; www.umin.ac.jp/ctr).