Anthony J. Aldave

Purpose: To report the visually significant recurrence of the transforming growth factor beta-induced gene ( TGFBI ) dystrophy associated with p.Met619Lys mutation in the posterior stroma after deep anterior lamellar keratoplasty (DALK), necessitating penetrating keratoplasty (PK). Methods: Slit-lamp and anterior segment optical coherence tomography imaging were performed for 2 individuals with a variant TGFBI corneal dystrophy associated with the p.Met619Lys mutation. Histopathologic examination of the corneal buttons excised at the time of PK from each individual was performed. Results: An unrelated 58-year-old woman (case 1) and a 61-year-old man (case 2) with a variant TGFBI corneal dystrophy associated with p.Met619Lys mutation underwent manual (case 1) and big-bubble (case 2) DALK for visually significant dystrophic stromal deposits. Both individuals developed decreased vision approximately 5 years after DALK, attributed to recurrence of dystrophic deposits in the posterior stroma, necessitating PK in both individuals. Histopathologic examination of the excised residual posterior host stroma (case 1) and the excised posterior donor stroma (case 2) revealed dystrophic deposits that demonstrated staining with Masson trichrome and red-green dichroism with Congo red stain. Conclusions: The TGFBI dystrophy secondary to the p.Met619Lys mutation is associated with recurrence of visually significant dystrophic deposits in the posterior stroma after DALK. Given the need for PK only 5 years after DALK in the cases that we report, we recommend genetic testing in individuals in whom a variant TGFBI corneal dystrophy is suspected, and the conversion to PK instead of DALK for such individuals if intraoperative baring of Descemet membrane cannot be achieved.

Purpose: To describe the clinical, histopathological, and genetic characteristics of a posterior polymorphous corneal dystrophy-4 pedigree spanning 3 generations with a novel GRHL2 variant and to highlight intrafamilial clinical variability. Methods: Comprehensive ophthalmic examinations were performed on affected family members. Histopathological evaluation of proband's corneal tissue excised during penetrating keratoplasty was conducted using hematoxylin & eosin, periodic acid–Schiff, and cytokeratin stains. Genomic DNA was analyzed using Sanger sequencing of the promoter and coding regions of ZEB1, GRHL2 , and OVOL2 . Results: The proband and her sibling exhibited steep corneas with variable ground-glass haze, vesicular, and band-like endothelial changes. The mother and maternal grandfather showed milder endothelial vesicles and subtle posterior stromal haze, with the grandfather least affected. Histopathology showed posterior stromal thickening with edema, Descemet membrane reduplication, and thinning along with endothelial loss. Genetic analysis identified a novel heterozygous intronic GRHL2 variant (c.20 + 544G > C) segregating in all affected members, absent in 80 controls, and located in a known regulatory hotspot associated with ectopic GRHL2 expression. Conclusions: Intronic regulatory variants of GRHL2 can cause familial posterior polymorphous corneal dystrophy (PPCD) with marked phenotypic variability. This is the first report of the c.20 + 544G > C variant and the second implicating the same intronic position in PPCD, underscoring the diagnostic importance of this hotspot and the need to include regulatory regions in targeted genetic testing for PPCD.

Purpose: To determine the outcomes of Descemet stripping endothelial keratoplasty (DSEK) in eyes with anterior chamber intraocular lenses (ACIOLs). Methods: Retrospective chart review of DSEK procedures in eyes with ACIOLs from May 2006 to December 2024 and posterior chamber intraocular lenses (PCIOLs) from May 2006 to July 2020 (control group). Cox analysis of risk factors for secondary graft failure (SGF) was performed. Results: A total of 132 and 608 DSEK procedures were performed in ACIOL and PCIOL groups with a median follow-up of 21.7 and 36.8 months, respectively ( P < 0.01). There were no significant differences between the groups in the mean VA improvement, or incidence of primary graft failure, graft dislocation, endothelial rejection, or postop intraocular pressure elevation, although glaucoma surgery was more common in the ACIOL group (14.4% vs. 6.7%, P < 0.01). The incidences of SGF in the ACIOL and PCIOL groups were 0.14 and 0.13 per eye-year ( P = 0.47) and 0.11 and 0.01 per eye-year ( P < 0.01) in eyes with and without glaucoma, respectively. An ACIOL increased the risk of SGF in eyes without glaucoma [hazard ratio (HR): 6.5 (2–21.4)], with a trend toward significance in eyes with medically treated glaucoma [HR: 1.9 (0.8–4.7)] but not in eyes with prior glaucoma surgery. Prior graft failure [HR: 1.8 (1.2–2.7)] and endothelial rejection [HR: 2.2 (1.2–3.3)] also increased the risk of SGF. Conclusions: An ACIOL increases the risk of SGF after DSEK in eyes without glaucoma but not in eyes with prior glaucoma surgery. Therefore, IOL exchange should be considered before DSEK in eyes with an ACIOL and without prior glaucoma surgery.

Background: Sex differences have been described in several corneal diseases such as Fuchs endothelial corneal dystrophy and keratoconus, with estrogens implicated in the induction of these differences. Here, we report the identification of sex differences in a cohort of 177 individuals with Corneal Hereditary Endothelial Dystrophy (CHED), a rare corneal endothelial dystrophy associated with biallelic SLC4A11 gene mutations, and in a Slc4a11 ^−/− mouse model of CHED. Methods: Central corneal thickness (CCT) was measured in individuals with CHED and in Slc4a11 ^−/− and Slc4a11 ^+/+ mice to identify a correlation between sex and the degree of corneal edema. To investigate potential causes of such a correlation, RNAseq analysis and mitochondrial superoxide measurement were performed on corneal endothelium from male and female Slc4a11 ^−/− and Slc4a11 ^+/+ mice, for which body composition analysis was also performed. Gonadectomy or sham surgery was performed in Slc4a11 ^−/− and Slc4a11 ^+/+ mice at 4 weeks of age with subsequent longitudinal CCT and body weight monitoring, followed by an analysis of the interaction effect of surgery type, sex and genotype on CCT. Results: Male sex is associated with increased CCT, and thus more severe corneal edema, the characteristic clinical feature of CHED, in affected individuals and Slc4a11 ^−/− mice. The corneal endothelium in male Slc4a11 ^−/− mice demonstrates increased levels of oxidative stress compared to Slc4a11 ^−/− female mice, as evidenced by higher levels of glucose- and glutamine-derived mitochondrial superoxide, controlling for age. Removal of gonadal hormones in Slc4a11 ^−/− mice increases corneal edema in female mice, suggesting a protective role for ovarian hormones. Transcriptomic analysis of corneal endothelium and body composition analysis in Slc4a11 ^+/+ and Slc4a11 ^−/− mice suggest that estrogens play a role in promoting corneal endothelial utilization of lipids via β-oxidation as an alternative energy source in the absence of SLC4A11-mediated NH ₃ :H transport function, thereby reducing oxidative stress from glucose and glutamine metabolism. Conclusions: Male sex is associated with a more severe corneal phenotype in individuals with CHED and a Slc4a11 ^−/− mouse model of the disease. Increased corneal edema in female Slc4a11 ^−/− mice following gonadectomy suggests ovarian hormones play a protective role in maintaining corneal deturgescence in the setting of loss of SLC4A11 function.

Purpose: To evaluate the efficacy of cenegermin 0.002% in neurotrophic keratopathy (NK). Methods: This is a single-center retrospective case series. Data including demographic information, clinical characteristics, and staging of NK at baseline and 8 weeks were collected and analyzed. The primary outcome was improvement in NK 8 weeks after treatment, defined as a reduction in corneal epithelial defect size, or decreased corneal staining score in those without epithelial defect. Secondary outcomes include recurrence and the need for additional intervention. Results: Fifty-two eyes of 49 patients (mean age 66.0 ± 18.0 years) were included. The mean follow-up was 26.0 ± 16.0 (range 4–51) months. Twenty eyes (38.5%) were classified as Mackie stage 1, 24 (46.2%) as stage 2, and 8 (15.4%) as stage 3 NK. Overall, 67.3% of patients showed improvement by week 8, with resolution of corneal epithelial defect in 62.5% of patients with stage 2 and 3 NK, and a significant decrease in staining score among those with stage 1 disease (2.57 ± 1.07–2.15 ± 1.27, P = 0.002). Multiple logistic regression showed a significant association between Mackie staging and treatment success with cenegermin (OR: 4.79, 95% CI: 1.13–20.26, P = 0.03). Recurrence of NK after cenegermin treatment was observed in 37.5% of eyes within 4 months of follow-up. In patients who received a second course subsequently, 58.3% showed treatment success. Conclusions: Cenegermin is an effective treatment of NK in the clinical setting, although a notable proportion of patients experience disease recurrence subsequently. Mackie staging is a significant prognostic factor for treatment success.

Purpose: To report a case of delayed onset infectious crystalline keratopathy after corneal cross-linking (CXL). Methods: Slit-lamp examination, confocal microscopy, anterior segment optical coherence tomography imaging, and corneal culture were performed to characterize the corneal opacification. Results: A 49-year-old man presented with progressively worsening vision in the left eye 8 months after uncomplicated epithelium-off CXL for keratoconus. Examination demonstrated focal central corneal stromal thinning with surrounding stromal opacification. Confocal microscopy did not demonstrate evidence of parasitic or fungal infection. Two months later, gray-white crystalline stromal opacities were observed surrounding the area of central stromal thinning, consistent with infectious crystalline keratopathy. Corneal scrapings of the left eye were obtained, which were positive for Enterococcus faecalis. After cessation of topical loteprednol and initiation of topical vancomycin and linezolid, the stromal infiltrate increased in size and a hypopyon developed but had resolved before the discontinuation of 7 weeks of topical antibiotic therapy. A deep anterior lamellar keratoplasty was performed 6 weeks later, with no recurrence of infection 10 months after surgery. Conclusions: Infectious crystalline keratopathy can present more than 6 months after CXL. Progressive corneal stromal thinning associated with stromal opacification should raise suspicion of this rare late onset complication of CXL and prompt corneal scraping for stains and cultures to rule out an indolent infectious process.

To assess the impact of MitoQ, a mitochondria-targeted antioxidant, on viability of human corneal endothelial cell (hCEnC) lines expressing SLC4A11 mutations associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy type 4 (FECD4). SLC4A11 wildtype (SLC4A11^WT) and mutant (SLC4A11^MU) hCEnC lines were created to express either SLC4A11 variant 2 (V2) or variant 3 (V3) by stable transduction of SLC4A11^-/- hCEnC-21T with lentiviruses containing either SLC4A11^WT or one of the following mutations: V2 (V3) mutants c.374 G>A (c.326 G>A) (CHED), c.1813C>T (c.1765C>T) (CHED), c.2263C>T (c.2215C>T) (CHED), or c.2224 G>A (c.2176 G>A) (FECD4). A SLC4A11^{-/- empty} hCEnC line was created by stable transduction of SLC4A11^-/- hCEnC-21T with an empty lentiviral plasmid. Cell viability was measured by exposing MitoQ treated and untreated cells to oxidative stress agent tert-butyl hydroperoxide (tBH) followed by performing XTT assays and spectrophotometry. SLC4A11^{-/- empty}, SLC4A11 V2^WT, and SLC4A11 V3^WT hCEnC exposed to ≤0.01 μM MitoQ retained over 90% of the viability of untreated SLC4A11^{-/- empty} hCEnC. When treated with MitoQ, SLC4A11^{-/- empty} was able to demonstrate partial restoration of cell viability. All CHED-associated mutant hCEnC lines treated with 0.01 μM MitoQ demonstrated increased viability compared to untreated following exposure to tBH. The FECD4-associated mutant hCEnC line treated with 0.01 μM MitoQ showed no significant increase in cell viability compared to untreated following exposure to tBH. Media supplementation with antioxidant MitoQ has beneficial effects on cell viability in hCEnC harboring CHED-associated SLC4A11 mutations following exposure to tBH-induced oxidative stress.

Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11^WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11^MU). SLC4A11^WT and SLC4A11^MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2^WT and V2^MU) or variant 3 (V3^WT and V3^MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH₃-induced membrane conductance. We demonstrate SLC4A11^-/- and SLC4A11^MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11^WT hCEnC. Additionally, SLC4A11^-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11^WT hCEnC. Induction with 10mM NH₄Cl led SLC4A11^WT hCEnC to depolarize; conversely, SLC4A11^-/- hCEnC hyperpolarized and the majority of SLC4A11^MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH₄Cl induction. Immunostaining of primary hCEnC and SLC4A11^WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.

Purpose: The purpose of this study was to characterize the oxidative stress and inflammatory responses in ex vivo corneal endothelial cells (evCEnC) during expansion and assess the impact of SkQ1, an antioxidant and anti-inflammatory compound, on measures of oxidative stress and inflammation. Methods: A CEnC line (HCEnC-21T) was cultured in media supplemented with varying SkQ1 concentrations to determine the optimal SkQ1 dose range of toxicity and protective effect on CEnC exposed to acute oxidative stress. The impact of SkQ1 treatment on intracellular free radical (IFR) levels, NRF2-mediated oxidative stress response, and NFkB-mediated inflammatory response was determined at each passage of evCEnC isolated from donor corneas and cultured in SkQ1-supplemented and untreated media. Results: HCEnC-21T cultured in media supplemented with ≤250 nM SkQ1 retained over 95% cell viability compared with untreated cells. SkQ1 provided oxidative stress protection to HCEnC-21T in a dose-dependent manner up to 500 nM. In evCEnC, 50 nM and 250 nM SkQ1 supplementation significantly reduced IFR levels across passages 0 to 3 (P-values of 0.015 and 0.023, respectively) and 50 nM SkQ1 supplementation led to decreased levels of active NRF2 in evCEnC at passage 2. However, media supplementation with SkQ1 (50 nM and 250 nM) did not alter NFkB activation at any passage. Conclusions: SkQ1 media supplementation provides oxidative stress protection to HCEnC-21T in a dose-dependent manner and decreases IFR levels and NRF2 activation in evCEnC during expansion at concentrations that do not negatively affect CEnC viability. These findings indicate that SkQ1 supplementation may increase the expansion potential of evCEnC.