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.

Intraoperative Performance and Early Postoperative Outcomes Following Phacoemulsification With Three Fluidic Systems: A Randomized Trial.

PURPOSE: To compare intraoperative performance and early postoperative outcomes following phacoemulsification with two systems using active fluidics and one using gravity-based fluidics. METHODS: In this prospective randomized trial, 200 eyes were randomized to the traditional and Active Sentry groups (n = 80 eyes each) where the Centurion Vision System was used with traditional or Active Sentry (Alcon Laboratories, Inc) hand-pieces, respectively, or the Infinit group (n = 40 eyes) where the Infiniti Vision System (Alcon Laboratories, Inc) was used. Within the traditional and Active Sentry groups, there were two subgroups with low (30 mm Hg) or high (55 mm Hg) intraocular pressure (IOP) used. Outcome measures compared were: cumulative dissipated energy (CDE), percentage change in central corneal thickness (CCT) at 1 day, 1 week, and 1 month, anterior chamber cells at 1 day and 1 week, rate of rise and fall of IOP following occlusion break, corneal endothelial cell density (ECD), and macular thickness 6 months postoperatively. RESULTS: CDE was significantly lower in group II compared to the traditional group (2.96 ± 1.4 vs 4.14 ± 2.2, P = .001). With 30 mm Hg IOP, the Active Sentry group had significantly less percentage change in CCT at 1 week postoperatively compared to the traditional handpiece group (0.01% vs 0.02%, P = .008). Incidence of anterior chamber cells less than grade 2 on day 1 was significantly higher in the Active Sentry group (82.9% vs 52%, P = .03). Percentage change in ECD was significantly lower in the Active Sentry group (-0.957 vs -0.98%, P = .005). Significantly faster rise of IOP to baseline following occlusion break was seen in the Active Sentry group. CONCLUSIONS: The use of Active Sentry handpiece was associated with lower CDE, less postoperative increase in CCT, fewer anterior chamber cells, and faster rise of IOP following occlusion break. [J Refract Surg. 2024;40(5):e304-e312.].

Senescent characteristics of human corneal endothelial cells upon ultraviolet-A exposure.

PURPOSE: The objective of this study was to investigate the senescent phenotypes of human corneal endothelial cells (hCEnCs) upon treatment with ultraviolet (UV)-A. METHODS: We assessed cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation and expression of senescence markers (p16 and p21) in hCEnCs exposed to UV-A radiation, and senescent hCEnCs induced by ionizing radiation (IR) were used as positive controls. We performed RNA sequencing and proteomics analyses to compare gene and protein expression profiles between UV-A- and IR-induced senescent hCEnCs, and we also compared the results to non-senescent hCEnCs. RESULTS: Cells exposed to 5 J/cm2 of UV-A or to IR exhibited typical senescent phenotypes, including enlargement, increased SA-ß-gal activity, decreased cell proliferation and elevated expression of p16 and p21. RNA-Seq analysis revealed that 83.9% of the genes significantly upregulated and 82.6% of the genes significantly downregulated in UV-A-induced senescent hCEnCs overlapped with the genes regulated in IR-induced senescent hCEnCs. Proteomics also revealed that 93.8% of the proteins significantly upregulated in UV-A-induced senescent hCEnCs overlapped with those induced by IR. In proteomics analyses, senescent hCEnCs induced by UV-A exhibited elevated expression levels of several factors part of the senescence-associated secretory phenotype. CONCLUSIONS: In this study, where senescence was induced by UV-A, a more physiological stress for hCEnCs compared to IR, we determined that UV-A modulated the expression of many genes and proteins typically altered upon IR treatment, a more conventional method of senescence induction, even though UV-A also modulated specific pathways unrelated to IR.

Evaluation of the results of contact lens assisted corneal cross-linking treatment in keratoconus patients with thin corneas.

PURPOSE: We aimed to compare the efficacy and safety of accelerated contact lens-assisted cross-linking (CA-CXL) with Lotrafilcon B and Comfilcon A lenses in keratoconus (KC) patients with thin corneas. STUDY DESIGN: Retrospective, single-center study. MATERIALS AND METHODS: We retrospectively included 51 eyes of 39 KC patients with corneal thickness <400µm after epithelial scraping (Epi-off), who underwent accelerated CA-CXL treatment with Lotrafilcon B (n=20) and Comfilcon A (n=31). Uncorrected and corrected distance visual acuity (UDVA and CDVA), manifest refraction values, corneal topographic data and endothelial cell density were recorded at preoperative and postoperative 1st, 3rd and 6th month controls. RESULTS: CDVA in the Comfilcon A group was higher than CDVA before surgery at 6 months postoperatively (p<0.001). When the two lenses were compared, CDVA was found to be significantly higher in the Lotrafilcon B group in the preoperative, postoperative 1st month and 3rd month values, but there was no significant difference between the postoperative 6th month values (p=0.028, p=0.018, p=0.044, p=0.181, respectively). The maximum keratometry (Kmax) value at the 6th month after surgery in the Comfilcon A group was significantly lower than in the Lotrafilcon B group (p=0,009). There was no significant difference between the endothelial cell density values between the groups (p=0.623, p=0.609, p=0.794, p=0.458, respectively). There was no significant difference between the progression, regression, and stability rates of the two groups (p=0.714). CONCLUSIONS: Accelerated CA-CXL with Lotrafilcon B and Comfilcon A silicone hydrogel lenses is a safe and effective method to stop progression in patients with thin corneas.

Comparison of keratoplasty outcomes at the scar versus edema stages of keratoconus.

PURPOSE: To assess the outcomes of deep anterior lamellar keratoplasty or penetrating keratoplasty at the scar and the edema stages. METHODS: Forty-five patients (45 eyes) with keratoconus scar stage (scar group, n=26; penetrating keratoplasty a subgroup, n=7; deep anterior lamellar keratoplasty b subgroup, n=19) and keratoconus edema stage (edema group, n=19; penetrating keratoplasty c subgroup, n=12; deep anterior lamellar keratoplasty d group, n=7) who received penetrating keratoplasty or deep anterior lamellar keratoplasty from 2000 to 2022 were retrospectively studied. At 1, 6, and 12 months after surgery, the best-corrected visual acuity, astigmatism, spherical equivalent, corneal endothelial cell density, and complications were analyzed. RESULTS: The best-corrected visual acuity and average corneal endothelial cell loss rate were not significantly different between the scar and edema groups (p>0.05). At 6 and 12 months after surgery, the astigmatism and spherical equivalent in the scar group were significantly lower than those in the edema group (p<0.05). The spherical equivalent of the deep anterior lamellar keratoplasty b subgroup was lower than that of the penetrating keratoplasty a subgroup in the scar group 6 months after surgery (p<0.05). In the edema group, there was no significant difference in spherical equivalent between subgroups (p>0.05). There were no significant differences in best-corrected visual acuity and astigmatism between subgroups within the two groups (p>0.05). In comparison to the scar group, the edema group experienced more complications. According to a survival analysis, there was no statistically significant difference between the scar group and the edema group regarding the progression of vision. CONCLUSIONS: In terms of the outcomes and prognosis for vision after keratoplasty with edema stage and scar stage, deep anterior lamellar keratoplasty may be as effective as penetrating keratoplasty.

Long-term dynamic changes and influencing factors of corneal morphology after multiple intravitreal injections of anti-VEGF drugs.

To observe alterations in corneal morphology caused by repeated intravitreal injections of anti-vascular endothelial growth factor (VEGF). Prospective cohort study. Seventy-seven eyes were treated with intravitreal injection of anti-VEGF from June 2021 to March 2023. There were 25 eyes of neovascular age-related macular degeneration (nAMD), 24 eyes of diabetic macular edema (DME), and 28 eyes of retinal vein occlusion (RVO). Aflibercept was used in 37 eyes and Ranibizumab was used in 40 eyes. 3 + PRN was used. Corneal endothelium and corneal thickness were measured using a corneal endothelial microscope. The data related to central corneal thickness, corneal endothelial cell density (ECD), average cell size, coefficient of variation (CV), proportion of hexagonal cells (Hex%) was collected. A comparison was also made between baseline and the dynamic changes of all indexes 1 year following the last injection. It was observed that in comparison to baseline, ECD and Hex% decreased significantly after the 3rd injection of Aflibercept and Ranibizumab. However, ECD did not decrease further and remained at the same level as after the last injection. Hex% and average cell size increased to a certain extent in comparison to the last injection. All the changes were found to be statistically significant (P < .01). After 3 injections, ECD in DME group was markedly lower than that in nAMD and RVO group, but the CV in DME group was higher than that in nAMD as well as RVO groups, and all the differences were statistically significant (P < .05). Following intravitreal anti-VEGF therapy, DME is more likely than other disorders to result in a decrease in ECD. Repeated intravitreal injections of anti-VEGF drugs can reduce the Hex% and ECD to a certain extent. After the last injection, Hex% can progressively recover, and ECD can remain stable without further declining. After injections, ECD in DME group was found to be significantly lower than that in nAMD and RVO groups, but CV in DME group was significantly higher in comparison to the other 2 groups. In patients with macular edema, repeated intravitreal injections of anti-VEGF may have certain effects on corneal morphology. Patients with diabetes mellitus in particular should pay special attention to corneal safety following repeated intravitreal injections if they have significantly reduced ECD at baseline.

Determination of progressive endothelial cell loss after posterior chamber phakic intraocular lens implantation.

Comments about endothelial cell loss after posterior chamber phakic intraocular lens are provided, with a particular emphasis on the importance of determining progressive postoperative cell density reduction, excluding that related to surgical trauma.

A Shape Memory Polymeric Shield for Protecting Corneal Endothelium During Phacoemulsification.

Background: The purpose of this study was to explore the protective effect of a shape memory polymeric shield on corneal endothelium during phacoemulsification in rabbits. Methods: Poly-(glycerol dodecanedioate) (PGD) with a transition temperature of 24.416°C was prepared to make a shape memory shield with a thickness of 100 µm, an arc length of 14 mm, and a radius of curvature of 8.8 mm. In the control group, a phaco-tip with bevel-down was used to simulate injury to the corneal endothelium by phacoemulsification in rabbits. In the experimental group, the pre-cooled and curled shape memory shield was injected into and removed from the anterior chamber before and after phaco-power release. Anterior segment optical coherence tomography (AS-OCT), confocal microscope, trypan blue/alizarin red staining, and scanning electron microscope were performed to measure endothelial damage after surgery. Results: One day postoperatively, the lost cell ratio of the control group and the experimental group were 28.08 ± 5.21% and 3.50 ± 1.43%, respectively (P < 0.0001), the damaged cell ratios were 11.83 ± 2.30% and 2.55 ± 0.52%, respectively (P < 0.0001), and the central corneal thicknesses (CCT) were 406.75 ± 16.74 µm and 340. 5 ±13.48 µm, respectively (P < 0.0001). Seven days postoperatively, the endothelial cell density (ECD) of the control group and the experimental group were 1674 ± 285/mm2 and 2561 ± 554/mm2, respectively (P < 0.05). The above differences were all statistically significant. Conclusions: This PGD based shape memory shield has a protective effect on corneal endothelium during phacoemulsification. It reduces postoperative corneal edema and ECD decrease in the short term after surgery. Translational Relevance: The shape memory PGD "shield" in this study may have a use in certain human patients with vulnerable corneas of low endothelial cell count or shallow anterior chambers.

Nicotinamide promotes the differentiation of functional corneal endothelial cells from human embryonic stem cells.

Corneal transplantation represents the primary therapeutic approach for managing corneal endothelial dysfunction, but corneal donors remain scarce. Anterior chamber cell injection emerges as a highly promising alternative strategy for corneal transplantation, with pluripotent stem cells (PSC) demonstrating considerable potential as an optimal cell source. Nevertheless, only a few studies have explored the differentiation of functional corneal endothelial-like cells originating from PSC. In this investigation, a chemical-defined protocol was successfully developed for the differentiation of functional corneal endothelial-like cells derived from human embryonic stem cells (hESC). The application of nicotinamide (NAM) exhibited a remarkable capability in suppressing the fibrotic phenotype, leading to the generation of more homogeneous and well-distinctive differentiated cells. Furthermore, NAM effectively suppressed the expression of genes implicated in endothelial cell migration and extracellular matrix synthesis. Notably, NAM also facilitated the upregulation of surface marker genes specific to functional corneal endothelial cells (CEC), including CD26 (-) CD44 (-∼+-) CD105 (-) CD133 (-) CD166 (+) CD200 (-). Moreover, in vitro functional assays were performed, revealing intact barrier properties and Na+/K+-ATP pump functionality in the differentiated cells treated with NAM. Consequently, our findings provide robust evidence supporting the capacity of NAM to enhance the differentiation of functional CEC originating from hESC, offering potential seed cells for therapeutic interventions of corneal endothelial dysfunction.

Microstructure of the corneal endothelial transition zone in different laboratory animals.

Purpose: To compare the microstructure of the corneal endothelial transition zone in different laboratory animals. Methods: Flat-mount corneas of rabbits, rats, and mice were stained with Alizarin Red S (ARS) and observed using scanning electron microscopy (SEM). The progenitor cell markers p75 neurotrophin receptor (p75NTR), SRY-box transcription factor 9 (SOX9), leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), telomerase reverse transcriptase (TERT), and proliferation marker Ki-67 were examined in the flat-mounted corneas of three laboratory animals using immunofluorescence microscopy. Results: On flat mounts, proximity to the trabecular meshwork correlated with weaker ARS staining and greater polymorphism of endothelial cells in the transition zone in all animals. On SEM, distinct and smooth structures of the transition zone were negligibly detected in all animals. The endothelial cells in the transition zone had irregular shapes, with less dense, less wavy intercellular junctions, especially in murine corneas, exhibiting unique intercellular cystic spaces. In the transition zone of the rabbit cornea, progenitor cell markers p75NTR, SOX9, Lgr5, TERT, and proliferation marker Ki-67 were expressed, in contrast to those in other murine corneas. Conclusions: Although the transition zone was not identified clearly, irregular cell morphology and loss of cell-cell contact were observed in all animal corneal endothelial cells. The proliferative capacity and the presence of progenitor cells were confirmed in the transition zone, especially in the rabbit cornea.

Corneal endothelial changes seven years after phacoemulsification cataract surgery.

PURPOSE: To evaluate long-term postoperative corneal changes after phacoemulsification cataract surgery. METHODS: Twenty patients who participated in a previous study regarding corneal endothelial changes after phacoemulsification cataract surgery were examined after 7 years. The patients were divided in three groups based on their initial increase in central corneal thickness day one after the surgery: < 5% increase, 6-20% increase and ≥ 20% increase. The primary outcome measures were corneal endothelial cell loss (ECL), endothelial cell count (ECC) and endothelial morphology. RESULTS: After 7 years, a difference in cell loss between the groups was observed, except for groups 1 and 2. Endothelial cell count (ECC) differed significantly between groups 1 and 3 at 3 months. At 7 years, there was no difference in ECC between the three groups. Cell loss was found exclusively in group 1 between 3 months and 7 years. Endothelial cell morphology showed a converging pattern between 3 months and 7 years. CONCLUSION: After phacoemulsification cataract surgery, long-term ECC and morphology appear to converge towards a comparable steady state regardless of initial corneal swelling and endothelial cell loss.

Expression and Impact of Fibronectin, Tenascin-C, Osteopontin, and Type XIV Collagen in Fuchs Endothelial Corneal Dystrophy.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by Descemet's membrane (DM) abnormalities, namely an increased thickness and a progressive appearance of guttae and fibrillar membranes. The goal of this study was to identify abnormal extracellular matrix (ECM) proteins expressed in FECD DMs and to evaluate their impact on cell adhesion and migration. Methods: Gene expression profiles from in vitro (GSE112039) and ex vivo (GSE74123) healthy and FECD corneal endothelial cells were analyzed to identify deregulated matrisome genes. Healthy and end-stage FECD DMs were fixed and analyzed for guttae size and height. Immunostaining of fibronectin, tenascin-C, osteopontin, and type XIV collagen was performed on ex vivo specimens, as well as on tissue-engineered corneal endothelium reconstructed using healthy and FECD cells. An analysis of ECM protein expression according to guttae and fibrillar membrane was performed using immunofluorescent staining and phase contrast microscopy. Finally, cell adhesion was evaluated on fibronectin, tenascin-C, and osteopontin, and cell migration was studied on fibronectin and tenascin-C. Results: SPP1 (osteopontin), FN1 (fibronectin), and TNC (tenascin-C) genes were upregulated in FECD ex vivo cells, and SSP1 was upregulated in both in vitro and ex vivo FECD conditions. Osteopontin, fibronectin, tenascin-C, and type XIV collagen were expressed in FECD specimens, with differences in their location. Corneal endothelial cell adhesion was not significantly affected by fibronectin or tenascin-C but was decreased by osteopontin. The combination of fibronectin and tenascin-C significantly increased cell migration. Conclusions: This study highlights new abnormal ECM components in FECD, suggests a certain chronology in their deposition, and demonstrates their impact on cell behavior.

Narrative review after post-hoc trial analysis of factors that predict corneal endothelial cell loss after phacoemulsification: Tips for improving cataract surgery research.

PURPOSE: Identifying pre/perioperative factors that predict corneal endothelial-cell loss (ECL) after phacoemulsification may reveal ways to reduce ECL. Our literature analysis showed that 37 studies have investigated one or several such factors but all have significant limitations. Therefore, the data of a large randomized controlled trial (PERCEPOLIS) were subjected to post-hoc multivariate analysis determining the ability of nine pre/perioperative variables to predict ECL. METHODS: PERCEPOLIS was conducted in 2015-2016 to compare two phacoemulsification techniques (subluxation and divide-and-conquer) in terms of 3-month ECL. Non-inferiority between the techniques was found. In the present study, post-hoc univariate and multivariate analyses were conducted to determine associations between ECL and age, sex, cataract density, preoperative endothelial-cell density, phacoemulsification technique, effective phaco time (EPT), and 2-hour central-corneal thickness. The data are presented in the context of a narrative review of the literature. RESULTS: Three-month data were available for 275 patients (94% of the randomized cohort; mean age, 74 years; 58% women). Mean LOCSIII cataract grade was 3.2. Mean EPT was 6 seconds. Mean ECL was 13%. Only an older age (beta = 0.2%, p = 0.049) and higher EPT (beta = 1.2%, p = 0.0002) predicted 3-month ECL. Cataract density was significant on univariate (p = 0.04) but not multivariate analysis. The other variables did not associate with ECL. CONCLUSIONS: Older age may amplify ECL due to increased endothelial cell fragility. EPT may promote ECL via cataract density-dependent and -independent mechanisms that should be considered in future phacoemulsification research aiming to reduce ECL. Our literature analysis showed that the average ECL for relatively unselected consecutively-sampled cohorts is 12%.

Epithelial ingrowth in descemet membrane endothelial keratoplasty associated with vitreous loss.

BACKGROUND: Epithelial ingrowth is a rare but potentially sight-threatening complication caused by the invasion of corneal or conjunctival epithelial cells into the eye during ocular surgeries. DMEK is emerging as a widely used surgery for endothelial keratoplasty with its improved safety profile. We describe a case of epithelial ingrowth in the graft-host interface after uneventful DMEK associated with vitreous prolapse in the anterior chamber. CASE PRESENTATION: An 81-year-old female with Fuchs endothelial dystrophy underwent DMEK for corneal decompensation following cataract surgery. During the DMEK procedure, vitreous prolapse was observed around the intraocular lens (IOL). Her early postoperative course was unremarkable, but a dense paracentral interface opacity was observed during the 3-month follow-up. The area of epithelial ingrowth was imaged with optical coherence tomography (OCT) as a uniform nodule with a discrete increase in interface hyperreflectivity. A low-energy YAG laser was applied to remove the opacity. She maintained good vision and clear cornea without reoccurrence after treatment. CONCLUSIONS: We propose that, in addition to the introduction of epithelial cells during surgery, vitreous retention in the anterior chamber may be a risk factor by providing a scaffold that potentially aggravates epithelial ingrowth in DMEK. Our case demonstrated that early YAG intervention may disrupt interface epithelial cell growth, and the transmitted laser energy may fragment the scaffold vitreous noninvasively.

Plastic vs. glass: the effect of the synthetic material in contact with DMEK tissue during staining on endothelial cell loss.

PURPOSE: Endothelial cell loss (ECL) during Descemet membrane endothelial keratoplasty (DMEK) graft preparation has been shown to affect graft survival and the need for re-grafting. The purpose of this study was to quantitatively assess the impact of the plastic and glass mediums in contact with DMEK donor tissue during intra-operative graft staining on ECL. METHODS: Retrospective study that included patients who underwent DMEK surgery between January 2019 and June 2021 at Hôpital Maisonneuve-Rosemont and the Jewish General Hospital in Montreal, Canada. DMEK grafts were stained with 0.06% Trypan blue ophthalmic solution (VisionBlue®, Dutch Ophthalmic, USA, Exeter, NH) for 120 s in either a plastic or glass medium prior to delivery into the recipient's eye. The ECL was compared between the two groups 12-30 months post-operatively. RESULTS: ECL at 12-30 months was significantly less in the eyes that had received grafts stained in a plastic medium compared to those stained in a glass medium. Graft survival and re-bubbling was higher in the glass group however this difference was not statistically significant. CONCLUSION: Staining of the DMEK graft in a plastic medium caused less ECL compared to the glass medium.

"Keep on ROCKIn": Repurposed ROCK inhibitors to boost corneal endothelial regeneration.

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.

Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

Purpose: To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation. Methods: CECs were divided into central zone (8 mm trephined boundary) and peripheral zone (8 mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscopy. Compressed type I collagen gels with different stiffness were constructed as an in vitro model system to test the role of stiffness on phenotype using cultured rabbit CECs. Cell morphology, expression and intracellular distribution of Yes-associated protein (YAP), differentiation (ZO-1, Na+/K+-ATPase), stemness (FOXD3, CD34, Sox2, Oct3/4), and endothelial-mesenchymal transition (EnMT) markers were analyzed by immunofluorescence, quantitative RT-PCR, and Western blot. Results: The results showed that the peripheral area of rabbit and human DM is softer than the central area ex vivo. Using the biomimetic extracellular matrix collagen gels in vitro model, we then demonstrated that soft substrate weakens the differentiation and EnMT in the culture of CECs. It was further proved by the inhibitor experiment that soft substrate enhances stemness maintenance via inhibition of paxillin-YAP signaling, which was activated on a stiff substrate. Conclusions: Our findings confirm that substrate stiffness modulates the stemness maintenance and differentiation of CECs and suggest a potential strategy for CEC-based corneal tissue engineering.