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Intraocular pressure (IOP) follows a circadian rhythm. In both humans and mice, IOP is normally slightly elevated at night during the dark phase of the light cycle. In studying a strain of mice for possible indices of glaucoma, we incidentally discovered that C57BL/6J mice homozygous for the beige-J mutation of the Lyst gene lack a circadian fluctuation in IOP. Instead of having an elevated dark phase IOP, homozygotes exhibit a uniform IOP characteristic for light period values of C57BL/6J mice. The beige-J mutation results from deletion of a single isoleucine amino acid in the LYST WD40 motif likely to influence protein-protein interactions. Based on the literature, we hypothesized that CSNK2B (casein kinase 2, beta polypeptide) might be a relevant interacting protein, which we confirmed with a pulldown assay as a binding partner of wild-type, but not beige-J encoding, LYST protein. Treating wild-type mice with 4,5,6,7-tetrabromobenzotriazole (TBB), a casein kinase 2 inhibitor, recapitulated the beige-J mutant phenotype in preventing a rise in IOP during the dark period. Together, these results identify Lyst beige-J mice as a new strain for studying circadian IOP regulation and point to casein kinase 2 as a key molecule of interest., Competing Interests: Declaration of competing interest CMM, None; LD, None; ST, None; MR, None; MGA is a paid consultant for Santen Pharmaceutical., (Published by Elsevier Ltd.)

Objective: To describe our multidisciplinary bronchopulmonary dysplasia (BPD) consult team's systematic approach to BPD associated pulmonary hypertension (PH), to report our center outcomes, and to evaluate clinical associations with outcomes., Study Design: Retrospective cohort of 60 patients with BPD-PH who were referred to the Seattle Children's Hospital BPD team from 2018 to 2020. Patients with critical congenital heart disease were excluded. Demographics, comorbidities, treatments, closure of hemodynamically relevant intracardiac shunts, and clinical outcomes including time to BPD-PH resolution were reviewed., Results: Median gestational age of the 60 patients was 25 weeks (IQR: 24-26). 20% were small for gestational age (SGA), 65% were male, and 25% received a tracheostomy. With aggressive cardiopulmonary management including respiratory support optimization, patent ductus arteriosus (PDA) and atrial septal defect (ASD) closure (40% PDA, 5% ASD, 3% both), and limited use of pulmonary vasodilators (8%), all infants demonstrated resolution of PH during the follow-up period, including three (5%) who later died from non-BPD-PH morbidities. Neither SGA status nor the timing of PH diagnosis (<36 vs. ≥36 weeks PMA) impacted the time to BPD-PH resolution in our cohort [median 72 days (IQR 30.5-166.5)]., Conclusion: Our multidisciplinary, systematic approach to BPD-PH management was associated with complete resolution of PH with lower mortality despite less sildenafil use than reported in comparable cohorts. Unique features of our approach included aggressive PDA and ASD device closure and rare initiation of sildenafil only after lack of BPD-PH improvement with respiratory support optimization and diagnostic confirmation by cardiac catheterization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SB declared a shared affiliation with the author AB to the handling editor at the time of review., (© 2023 Yung, Jackson, Blumenfeld, Redding, DiGeronimo, McGuire, Riker, Tressel, Berkelhamer and Eldredge.)

The endothelium responds to numerous chemical and mechanical factors in regulating vascular tone, blood pressure, and blood flow. The endothelial volume-regulated anion channel (VRAC) has been proposed to be mechanosensitive and thereby sense fluid flow and hydrostatic pressure to regulate vascular function. Here, we show that the leucine-rich repeat-containing protein 8a, LRRC8A (SWELL1), is required for VRAC in human umbilical vein endothelial cells (HUVECs). Endothelial LRRC8A regulates AKT-endothelial nitric oxide synthase (eNOS) signaling under basal, stretch, and shear-flow stimulation, forms a GRB2-Cav1-eNOS signaling complex, and is required for endothelial cell alignment to laminar shear flow. Endothelium-restricted Lrrc8a KO mice develop hypertension in response to chronic angiotensin-II infusion and exhibit impaired retinal blood flow with both diffuse and focal blood vessel narrowing in the setting of type 2 diabetes (T2D). These data demonstrate that LRRC8A regulates AKT-eNOS in endothelium and is required for maintaining vascular function, particularly in the setting of T2D., Competing Interests: AA, JA, JM, AK, CT, SG, UF, CK, LX, OA, MR, ME, RM, CG, RM, AS, RS No competing interests declared, (© 2021, Alghanem et al.)

The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Mutations in myocilin ( MYOC) may cause either juvenile open angle glaucoma (JOAG) or adult-onset primary open angle glaucoma (POAG). MYOC encodes a glycoprotein that is normally secreted from trabecular meshwork cells that regulate intraocular pressure. Prior in vitro, transgenic rodent, and organ culture experiments have suggested that abnormal accumulation of MYOC protein within trabecular meshwork cells is a key step in glaucoma pathophysiology. We investigated the pathogenesis of MYOC glaucoma by examining a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation., Design: Case-control, immunohistochemical study of a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation and age-matched control donor eyes., Subjects: An eye from a 59-year-old male with JOAG caused by a Tyr437His MYOC mutation and eyes from five donors (ages 51-66) with no known ocular disease were examined., Methods: Frozen fixed sections of the iridocorneal angle were prepared from the donor eyes of the MYOC glaucoma patient and control eyes. We used antibodies directed against MYOC, collagen IV, and BiP/GRP78 as well as wheat germ agglutinin and concanavalin A lectins to localize MYOC protein in the trabecular meshwork., Main Outcome Measure: Qualitative comparison of MYOC protein labeling and localization in the trabecular meshwork of donor eyes from a glaucoma patient with a MYOC mutation and from control subjects., Results: Using immunohistochemistry, we detected more abundant MYOC protein within the trabecular meshwork of the MYOC glaucoma patient's eye than in control eyes. We further localized MYOC protein within the trabecular meshwork cells of the MYOC glaucoma patient's eye by co-labeling with the endoplasmic reticulum (ER) marker GRP78 (BiP). Little to no MYOC was identified within the trabecular meshwork cells of control eyes. Minimal extracellular MYOC was detected in both MYOC glaucoma eyes and control eyes., Conclusions: This is the first histopathological analysis of an eye from a glaucoma patient with a MYOC mutation. Furthermore, this analysis supports our model of MYOC-associated glaucoma, in which MYOC mutations cause abnormal intracellular retention of MYOC within the ER of trabecular meshwork cells as a key step towards development of glaucoma.

Purpose: The gene that causes normal tension glaucoma (NTG) in a large pedigree was recently mapped to a region of chromosome 12q14 (GLC1P) that contains the genes TBK1, XPOT, RASSF3, and GNS. We sought to investigate the structure of the chromosome 12q14 duplication and explore the ocular expression of GLC1P locus genes., Methods: The location of the chromosome 12q14 duplication in this pedigree was examined with fluorescent in situ hybridization (FISH) using probes for TBK1 and GNS. The expression pattern of XPOT, TBK1, RASSF3, and GNS was investigated with immunohistochemistry of human eyes., Results: The karyotype of an NTG patient from pedigree GGO-414 was normal and FISH studies demonstrated that the duplicated DNA is organized as a tandem repeat on chromosome 12q14. Of the genes in or near the chromosome 12q14 duplication, TBK1 showed expression in the retina that is specific to the retinal ganglion cells and the retinal nerve fiber layer. Expression of RASSF3 and XPOT was relatively uniform throughout the retina, while GNS expression was expressed in a pattern consistent with Müller cells., Conclusions: Previous studies demonstrated that chromosome 12q14 duplications are associated with NTG inherited as an autosomal dominant trait. FISH studies now demonstrate that the duplicated segments are tandemly organized on chromosome 12q14 in close proximity. The specific expression of TBK1 in human retinal ganglion cells compared to the widespread pattern of expression of neighboring genes provides additional evidence that TBK1 is the glaucoma gene in the chromosome 12q14 duplication within the GLC1P locus.