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Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

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The Molecular Mechanisms and Therapeutic Potentials of Engrailed-1 in Fibrotic Scar Formation Post Glaucoma

Yi Lin¹, Wangdu Luo¹, Xiaomin Zhu¹

¹Department of Ophthalmology, The Third Affiliated Hospital of Chongqing Medical University, Chongqing, China.

Investigative Ophthalmology & Visual Science

|February 5, 2026

Abstract

Purpose:

Conjunctival scarring is a major cause of glaucoma filtration surgery (GFS) failure, necessitating effective antifibrotic strategies. This study investigated the role of engrailed-1 (EN1) in conjunctival fibrosis and evaluated the therapeutic potential of EN1-targeted biomaterials for antiscarring in post-GFS patients.

Methods:

Fibrotic models were developed using TGF-β2-induced human Tenon fibroblasts (HTFs) and a rat GFS model. EN1 was identified as a key therapeutic target using RNA interference, transcriptome sequencing, chromatin immunoprecipitation-qPCR, and dual-luciferase assays. Exosomes (Exos) loaded with EN1 small interfering RNA were encapsulated in a GelMA hydrogel to form the Gel-Exo-siEN1 composite material.

Results:

EN1 expression was significantly elevated in TGF-β2-stimulated HTFs and conjunctival scar tissue post GFS. Inhibition of EN1 reduced TGF-β2-induced proliferation and migration in HTFs and decreased fibrosis-related gene expression. These effects may be mediated through the Yes-related protein/transcriptional co-activator PDZ-binding motif and SMAD3 pathways. Assays confirmed that EN1 inhibition suppressed proliferation and migration and downregulated fibrosis markers like fibronectin, collagen I, and α-smooth muscle actin. Western blot analysis showed increased Yes-related protein/transcriptional co-activator PDZ-binding motif expression after TGF-β2 induction, which was reduced by verteporfin. Chromatin immunoprecipitation-PCR confirmed that SMAD3 binds to the EN1 promoter, regulating its expression. Exo analysis showed Exo-siEN1 maintained stability and effectively delivered siEN1, leading to significant EN1 knockdown and reduced fibrosis in rat Tenon's fibroblasts. Gel-Exo-siEN1 treatment significantly increased functional bleb area, reduced IOP, and decreased collagen deposition and inflammatory cell infiltration in the conjunctiva after GFS.

Conclusions:

Gel-Exo-siEN1 is a promising strategy for preventing postsurgical scarring and improving the outcomes of glaucoma surgery.

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