Characterization, enrichment, and computational modeling of cross-linked actin networks in trabecular meshwork cells.

Purpose: Cross-linked actin networks (CLANs) are prevalent in the glaucomatous trabecular meshwork (TM), yet their role in ocular hypertension remains unclear. We used a human TM cell line that spontaneously forms fluorescently-labeled CLANs (GTM3L) to explore the origin of CLANs, developed techniques to increase CLAN incidence in GMT3L cells, and computationally studied the biomechanical properties of CLAN-containing cells. Methods: GTM3L cells were fluorescently sorted for viral copy number analysis. CLAN incidence was increased by (i) differential sorting of cells by adhesion, (ii) cell deswelling, and (iii) cell selection based on cell stiffness. GTM3L cells were also cultured on glass or soft hydrogel to determine substrate stiffness effects on CLAN incidence. Computational models were constructed to mimic and study the biomechanical properties of CLANs. Results: All GTM3L cells had an average of 1 viral copy per cell. LifeAct-GFP expression level did not affect CLAN incidence rate, but CLAN rate was increased from ~0.28% to ~50% by a combination of adhesion selection, cell deswelling, and cell stiffness-based sorting. Further, GTM3L cells formed more CLANs on a stiff vs. a soft substrate. Computational modeling predicted that CLANs contribute to higher cell stiffness, including increased resistance of the nucleus to tensile stress when CLANs are physically linked to the nucleus. Conclusions: It is possible to greatly enhance CLAN incidence in GTM3L cells. CLANs are mechanosensitive structures that affect cell biomechanical properties. Further research is needed to determine the effect of CLANs on TM biomechanics and mechanobiology as well as the etiology of CLAN formation in the TM.

GSK3ß Inhibitors Inhibit TGFß Signaling in the Human Trabecular Meshwork.

Purpose: Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFß and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFß2-induced TM changes and ocular hypertension (OHT). Methods: Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFß signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP. Results: We found that the small-molecule Wnt activators (GSK3ß inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFß signaling as well as TGFß2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and ß-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFß and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFß2-induced OHT in perfusion-cultured human eyes. Conclusions: Our results showed that small-molecule Wnt activators have the potential for treating TGFß signaling-induced OHT in patients with POAG.

Biomechanical and Vascular Metrics Between Eyes of Patients With Asymmetric Glaucoma and Symmetric Glaucoma.

PRCIS: Corneal hysteresis (CH) and pulsatile ocular blood volume (POBV) were significantly lower in the eye with greater damage in asymmetric glaucoma, without a difference in intraocular pressure (IOP) or central corneal thickness (CCT), and no difference in elastic parameters. OBJECTIVE: To compare biomechanical and vascular metrics between the eyes of patients with asymmetric glaucoma (ASYMM) and those with symmetric glaucoma (SYMM). PATIENTS AND METHODS: Forty-five patients were prospectively recruited and divided into ASYMM, defined as cup-to-disc (C/D) ratio difference >0.1 between eyes and SYMM, with C/D difference ≤0.1. For ASYMM, the smaller C/D was defined as the best eye ("best") and the fellow eye was defined as the worst eye ("worse"). All metrics were subtracted as "worse" minus "best," including the viscoelastic parameter CH, and elastic parameters from the Corvis ST, including stiffness parameter at first applanation, stiffness parameter at highest concavity, integrated inverse radius, deformation amplitude ratio, IOP, CCT, mean deviation (MD), ganglion cell complex (GCC), and POBV were included. Paired t tests were performed between eyes in both groups. Statistical analyses were performed with SAS using a significance threshold of P <0.05. RESULTS: For ASYMM (16 patients), "worse" showed significantly lower CH (-0.76 ± 1.22), POBV (-0.38 ± 0.305), MD (-3.66 ± 6.55), and GCC (-7.9 ± 12.2) compared with "best." No other parameters were significantly different. For SYMM (29 patients), there were no significantly different metrics between eyes. CONCLUSIONS: Lower CH, POBV, GCC, and worse MD were associated with greater glaucomatous damage in asymmetric glaucoma without a difference in IOP or CCT. Lower CH and GCC are consistent with previous studies. POBV, a new clinical parameter that may indicate reduced blood flow, is also associated with greater damage.

Assessment of Pupil Size and Angle Kappa in Refractive Surgery: A Population-Based Epidemiological Study in Predominantly American Caucasians.

Purpose This retrospective study aims to establish normative values for pupil size, angle kappa, higher-order aberration, and astigmatism type in a largely Caucasian population in Utah, United States, utilizing the NIDEK OPD-Scan III system (Gamagori, Japan). Methods This study included 716 patients (1432 eyes) grouped based on spherical equivalence and age. Measurements were conducted under mesopic and photopic conditions. Statistical analysis involved Pearson's correlation and linear regression using the generalized estimating equation. NIDEK OPD-Scan III measured mesopic and photopic pupil size and angle kappa. The subjects were then grouped based on their spherical equivalence in diopters (D) and age in decades. The spherical equivalence groups were defined: >-6 D, -5.99 to -3 D, -2.99 to -0.25 D, -0.24 to 0.24 D, and >0.25 D (range 0.25-5.75 D). The higher-order aberration groups were based on the reason for the visit: laser-assisted in situ keratomileusis, photorefractive keratectomy, and small incision lenticule extraction as one group; cataract evaluation; and keratoconus. Astigmatism measurements were grouped into with-the-rule (WRT), against-the-rule (ATR), and oblique astigmatism, with further subgrouping into a young cohort (20-40 years) and an old cohort (>65 years).  Results Among 716 participants, 49.2% were men; the mean age was 42.1±15.5 (range 7-88 years). The average spherical equivalence for myopia eyes was -3.28±2.34 D, and 1.51±1.46 D for hyperopia eyes. The mean mesopic pupil size was 5.68 ± 1.09 mm; the photopic pupil size was 4.65±1.09 mm. Pearson's correlation coefficient for mesopic pupil size versus age was -0.551, and -0.42 for photopic pupil (p < 0.001); sphere vs mesopic pupil size was -0.200, and -0.173 for photopic pupil (p < 0.001). The regression analysis for mesopic pupil size versus age revealed a 0.39 mm decrease in average pupil size per decade increase in age, and 0.25 mm decrease per decade for photopic pupil. The regression analysis for mesopic pupil size versus sphere revealed a 0.22 mm decrease in average pupil size per 3D increase in sphere, and a 0.16 mm decrease 3 D increase in sphere for the photopic pupil. The mean mesopic angle kappa was 0.33 ± 0.15 mm; photopic angle kappa was 0.31±0.15 mm. Pearson's correlation coefficient for mesopic angle kappa vs spherical equivalence was 0.32, and 0.296 for photopic angle kappa (p <0.001 for both). Regression analysis for mesopic angle kappa vs spherical equivalence demonstrated a 0.051 mm increase in angle kappa per 3 D increase in spherical equivalence, and a 0.048 mm increase for photopic angle kappa (p < 0.001 for both). Among the higher-order aberration groups, the keratoconus group exhibited the highest levels. In terms of astigmatism type, WRT astigmatism was the most common in the young cohort, while ATR astigmatism was most prevalent in the older cohort. Conclusions The results of this study reveal significant associations between pupil size and increasing age, as well as between pupil size and increasingly positive refractive errors. These findings hold particular clinical relevance to older patients and individuals with hyperopia, as they undergo photoablative corneal refractive surgery or multifocal intraocular lens implantation. Understanding the established normative values for pupil size, angle kappa, higher-order aberration, and astigmatism type can aid clinicians in making more informed decisions and improving patient outcomes.