Glaucoma is associated with ocular hypertension, and lowering intraocular pressure is the primary objective of current therapies. Recent studies have established a key role for Schlemm’s canal endothelium in this pressure increase and have shown that it has a unique, lymphatic-like hybrid phenotype characterized by expression of the lymphatic transcription factor PROX1. However, the functional importance of this hybrid phenotype in the adult canal remains unclear, as long-term studies have been limited by systemic requirements for lymphatic gene expression and a lack of Schlemm’s canal–specific animal models. Here, we designed and validated a51 strategy using 4OH-tamoxifen-loaded nanocarriers to generate targeted, Schlemm’s canal-52 specific Prox1 knockout mice that specifically lacked lymphatic characteristics in the canal53 endothelium. Within 4 weeks, intraocular pressure was significantly elevated, and ocular hypertension was maintained for at least 24 weeks. Unlike lymphatic vessels, which degenerate following Prox1 deletion, Schlemm’s canal persisted but reverted to a less functional vein-like phenotype with no change in size or morphology. Together, these findings demonstrate the utility of nanocarrier-mediated tamoxifen delivery and establish the importance of the Schlemm’s canal lymphatic-like phenotype in intraocular pressure regulation, providing targets for future glaucoma therapies and a mouse model of adult-onset ocular hypertension.

BACKGROUND: Systemic hypertension is a risk factor for glaucoma, a leading cause of irreversible blindness, yet the mechanistic basis connecting the 2 conditions remains elusive. METHODS: A longitudinal assessment of blood pressure, intraocular pressure, retinal function, structure, and perfusion was conducted in spontaneously hypertensive rats and Wistar-Kyoto rats. Transcriptomic analyses and immunofluorescence staining were performed to investigate hypertension-associated retinal biochemical changes. AAV-mediated knockdown of endothelin 2 was used to investigate its mechanistic role. RESULTS: Hypertensive rats develop accelerated retinal degeneration marked by retinal thinning, functional impairment, reduced perfusion, and progressive neuroinflammation. Critically, these changes occur despite persistently low intraocular pressure, indicating an intraocular pressure-independent mechanism underlying the pathology. Biochemical analyses identified robust activation of the retinal endothelin system, with endothelin 2 the most significantly upregulated component. AAV-mediated retinal endothelin 2 knockdown substantially restored retinal function, ameliorated neuroinflammation, and enhanced retinal blood flow in hypertensive rats. Pharmacological blockade of endothelin receptor type A, but not type B, replicated these protective effects without altering intraocular pressure. CONCLUSIONS: Our findings establish endothelin 2 as an important, intraocular pressure-independent contributing factor to hypertension-associated retinal neurodegeneration and identify the endothelin-endothelin receptor type A axis as a potential therapeutic target to prevent vision loss in patients with hypertension.

Glaucoma is associated with ocular hypertension and lowering intraocular pressure is a key objective of glaucoma therapies. Recent studies have established a role for the Schlemm's canal endothelium in this pressure increase and have shown it to have a unique, lymphatic-like, hybrid phenotype. However, the role of these lymphatic phenotypes in the adult canal remains uncertain. Long-term functional studies have been limited by systemic importance of lymphatic genes and lack of Schlemm's canal-specific animal models. Here, we designed and validated a strategy using 4OH-tamoxifen-loaded nanocarriers to generate targeted, Schlemm's canal specific knockout mice lacking lymphatic phenotypes. Using this system, we selectively deleted Prox1, the master transcription factor governing lymphatic fate. Within four weeks, intraocular pressure significantly increased, and ocular hypertension was maintained for at least 24 weeks. Unlike lymphatic vessels, which degenerate following Prox1 deletion, Schlemm's canal reverted to a less functional vein-like phenotype with no change in size or morphology. These results highlight the utility of nanocarriers for tissue-specific genetic recombination and demonstrate that changes in lymphatic phenotypes alter intraocular pressure, providing new targets for glaucoma therapy. Moreover, as we found that PROX1 was downregulated with age in human Schlemm's canal, these canal-specific conditional Prox1 knockout mice are a valuable new adult-onset model of ocular hypertension that captures key features of age-related human disease.

Rho-associated protein kinase (ROCK) inhibitors reduce cytoskeletal tension in trabecular meshwork (TM) cells, but the mechanisms linking this acute response to sustained extracellular matrix remodelling remain incompletely understood. In this study, we investigated whether the ROCK inhibitor Y39983 regulates thrombospondin-1 (THBS1) and DNA methylation programs, elements known to modulate extracellular matrix (ECM) remodelling, in primary human trabecular meshwork (hTM) cells. Primary hTM cells were treated with Y39983, 5-azacytidine (5-Aza, a DNA methyltransferase inhibitor), or both in combination. Gene expression of THBS1, DNA methyltransferase, ECM, and cytoskeletal molecules was assessed using RT-qPCR. THBS1 promoter methylation was examined through methylation-specific PCR, while genome-wide methylation remodelling was profiled using whole-genome bisulphite sequencing (WGBS) followed by pathway enrichment analysis. 5-Azacytidine abolished Y39983-dependent effects on DNMT1 and DNMT3A and attenuated THBS1 regulation. Methylation-specific PCR suggested altered THBS1 promoter methylation after Y39983 treatment, while whole-genome bisulphite sequencing identified 8845 differentially methylated genes, including 5608 hypomethylated and 3237 hypermethylated genes, enriched in ErbB signalling, focal adhesion, and regulation of the actin cytoskeleton. Independently, Y39983 reduced the basal and stimulus-induced expression of THBS1, as well as ECM- and cytoskeleton-associated genes, including COL4A1, COL6A1, and ACTG1. These findings demonstrate that ROCK inhibition is associated with changes in DNMT-related gene expression and DNA methylation patterns in hTM cells. However, because 5-azacytidine has effects beyond selective DNMT inhibition, further studies using orthogonal approaches are required to establish whether DNA methylation directly mediates transcriptional responses to Y-39983.