A common indication for corneal transplantation, which is the most transplanted tissue, is a dysfunctional corneal endothelium due to Fuchs' endothelial dystrophy (FED). FED is diagnosed by the presence of in vivo pathological microtopography on the Descemet membrane, which is called corneal guttata. Minimally invasive corneal endothelial cell regenerative procedures such as endothelial cell injection therapy and Rho kinase inhibitor pharmacotherapy have been proposed as alternatives to conventional corneal transplantation for FED patients. However, the effect of guttata on monolayer reformation following such therapies is unknown and there is no equivalent in vitro or animal model to study monolayer reformation. Using a synthetic guttata FED disease model, the formation of the monolayer is investigated to evaluate the efficacy of both therapies. Results obtained suggest that guttata dimensions, density, and spacing greatly affect the fate of corneal endothelial cells in terms of migratory behavior and monolayer reformation. Densely packed synthetic guttata mimicking late-stage FED hinders monolayer reformation, while synthetic guttata of lower height and density show improved monolayer formation. These results suggest that severity of the FED, as determined by height and density of existing guttata, can potentially attenuate corneal endothelial monolayer formation of corneal cell injection therapy and pharmacotherapy.

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Background: Some patients with neovascular age-related macular degeneration (nAMD) respond insufficiently to anti-VEGF treatment despite maximal monthly intravitreal injections. Their short-term response between injections was investigated for extent and visual prognosis. Subjects/Methods: Monocentric retrospective observational study. 45 eyes from 41 patients with refractory nAMD, evaluated by optical coherence tomography (OCT) in between monthly anti-VEGF injections. The fluid profile on OCT was evaluated before, 1 week after, and 1 month after an intravitreal injection, using central retinal thickness (CRT), manual measurements, and fluid specific volumetric measurements performed by an automated algorithm based on artificial intelligence. Results: A significant improvement was found at week 1 in terms of CRT (p<0.0001), intraretinal (IRF) (p=0.007), subretinal fluid (SRF) (p<0.0001), and pigment epithelium detachment (PED) volume (p<0.0001). Volumetric fluid measures revealed a >50% reduction at week 1 for both IRF and SRF for approximately two-thirds of eyes. Poorer short-term response was associated with larger exudative fluid amounts (IRF+SRF) (p=0.003), larger PED (p=0.007), lower visual acuity (p=0.004) and less anatomic changes at treatment initiation (p<0.0001). Univariate and multivariate analysis revealed that visual outcomes 4 and 5 years later was significantly worse with weaker short-term responsiveness (p=0.005), with the presence of atrophy (p=0.01) and larger PED volumes (p=0.002). Conclusions: Incomplete responders to anti-VEGF showed a significant short-term response, identifiable at 1 week after injection, with rapid recurrence at 1 month. Weaker short-term responsiveness at 1 week was associated with poorer long term visual prognosis. These patients may need adjuvant treatment to improve their prognosis.

(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds—AR-13324 (Netarsudil) and its active metabolite, AR-13503—and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM; * p < 0.05); (ii) untreated vs. AR-13503 (10 μM; *** p < 0.001); (iii) Y-27632 vs. AR-13503 (10 μM; ** p < 0.005); (iv) AR-13324 (1 μM) vs. AR-13503 (10 μM; ** p < 0.005); and (v) AR-13324 (0.1 μM) vs. AR-13503 (10 μM; * p < 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.

Micro‐structural changes in the corneal endothelial basement membrane cause Fuchs dystrophy, which leads to corneal blindness. On page 2896, Muhammad Rizwan and co‐workers demonstrate a micro‐fabricated in vitro disease model of Fuchs dystrophy by recapitulating the topographical details of diseased basement membrane. This model is employed to elucidate complex interaction of corneal cells with the disease‐like surface to study corneal endothelial monolayer reformation for the development of corneal cell regenerative therapies.