This study successfully generated an Angpt2-miR-145 mouse model carrying the rs76020419 (G>T) mutation, confirming that this mutation causes elevated ANGPT2 expression and Schlemm's canal area enlargement, suggesting its potential role in glaucoma protective mechanisms. This model provides a critical tool for future investigations of ANGPT2's functions in intraocular pressure regulation and retinal neurodegeneration.
Literature Overview
This article 'A Glaucoma-Protective Human SNP in the Angpt2 Locus', published in the journal Arteriosclerosis, Thrombosis, and Vascular Biology, reviews functional validation studies of rs76020419 (G>T), a human single nucleotide polymorphism located in the miR-145 binding site within the ANGPT2 3'UTR region. This mutation has been associated with reduced intraocular pressure and lower risk of primary open-angle glaucoma in genome-wide association studies, and functions as an expression quantitative trait locus (eQTL) in the GTEx database showing correlation with ANGPT2 upregulation. The research team employed CRISPR/Cas9 technology to construct a mouse model carrying this SNP and evaluated ANGPT2 expression levels and Schlemm's canal morphological changes in ocular tissues and multiple systemic organs.
Background Knowledge
Glaucoma represents a leading cause of blindness globally, affecting approximately 60 million individuals. Maintaining aqueous humor production and drainage equilibrium is critical for intraocular pressure (IOP) regulation, where fluid primarily drains through the trabecular meshwork (TM) and Schlemm's canal (SC). Previous studies demonstrated that ANGPT1 and TEK gene functional deficiencies associate with congenital glaucoma, while ANGPT2 can partially compensate for ANGPT1 loss. miR-145 is a known ANGPT2 suppressor; the SNP rs76020419 (G>T) at its binding site disrupts this inhibition, leading to elevated ANGPT2 expression that may increase SC caliber and reduce IOP. Prior to this work, no mouse model existed for in vivo validation of this SNP, making this study a significant contribution to understanding ANGPT2's protective role in SC homeostasis and glaucoma prevention.
Research Methods and Experiments
The research team introduced the rs76020419 (G>T) mutation into C57BL/6 mice using CRISPR/Cas9 technology to generate the Angpt2-miR-145 mouse model. ANGPT2 protein expression levels in plasma and tissues (ocular, pulmonary, renal) were quantified through qPCR and ELISA assays. Immunofluorescence staining combined with confocal microscopy analyzed SC area changes, while intraocular pressure was measured using a Tonolab rebound tonometer. Fluorescent angiography assessed vascular leakage to confirm model stability.
Key Conclusions and Perspectives
Research Significance and Prospects
The findings establish a functional relationship between ANGPT2 upregulation and SC structural modifications, supporting its potential as a genetic protective factor against glaucoma. Future studies should integrate multiple ocular hypertension models to evaluate this mutation's protective effects against elevated IOP and retinal neuron degeneration, establishing foundational evidence for targeting ANGPT2 or miR-145 in glaucoma therapeutics.
Conclusion
This study represents the first validation in a mouse model of human ANGPT2 gene rs76020419 (G>T) mutation's effects on intraocular pressure and Schlemm's canal morphology. Using CRISPR/Cas9 gene editing, the research team successfully generated mutant mice demonstrating ANGPT2 protein upregulation across multiple tissues. The observed SC area expansion and reduced IOP in mutant animals provide in vivo evidence for miR-145's regulatory role on ANGPT2 expression. This model establishes a critical platform for investigating ANGPT2-mediated protective mechanisms in glaucoma and related neurodegenerative diseases, while identifying novel molecular targets for genetic therapies.
