Conjunctival fibrosis remains the major impediment to the success of glaucoma filtration surgery. Anti-metabolites remain the gold standard for mitigating post-surgical fibrosis, but they are associated with high complication rates and surgical failure rates. Establishing a more targeted approach to attenuate conjunctival fibrosis may revolutionize the surgical approach to glaucoma. A new strategy is needed to prevent progressive tissue remodeling and formation of a fibrotic scar, subsequently increasing surgical success and reducing the prevalence of glaucoma-related vision loss. Advancements in our understanding of molecular signaling and biomechanical cues in the conjunctival tissue architecture are broadening the horizon for new therapies and biomaterials for the mitigation of fibrosis. This review aims to highlight the strategies and current state of promising future approaches for targeting fibrosis in glaucoma filtration surgery.

Sulfur mustard gas (SM) is a vesicating and alkylating agent used as a chemical weapon in many mass-casualty incidents since World War I. Ocular injuries were reported in >90% of exposed victims. The mechanisms underlying SM-induced blindness remain elusive. This study tested the hypothesis that SM-induced corneal fibrosis occurs due to the generation of myofibroblasts from resident fibroblasts via the SMAD2/3 signaling pathway in rabbit eyes in vivo and primary human corneal fibroblasts (hCSFs) isolated from donor corneas in vitro. Fifty-four New Zealand White Rabbits were divided into three groups (Naïve, Vehicle, SM-Vapor treated). The SM-Vapor group was exposed to SM at 200 mg-min/m3 for 8 min at the MRI Global facility. Rabbit corneas were collected on day 3, day 7, and day 14 for immunohistochemistry, RNA, and protein lysates. SM caused a significant increase in SMAD2/3, pSMAD, and ɑSMA expression on day 3, day 7, and day 14 in rabbit corneas. For mechanistic studies, hCSFs were treated with nitrogen mustard (NM) or NM + SIS3 (SMAD3-specific inhibitor) and collected at 30 m, 8 h, 24 h, 48 h, and 72 h. NM significantly increased TGFβ, pSMAD3, and SMAD2/3 levels. On the contrary, inhibition of SMAD2/3 signaling by SIS3 treatment significantly reduced SMAD2/3, pSMAD3, and ɑSMA expression in hCSFs. We conclude that SMAD2/3 signaling appears to play a vital role in myofibroblast formation in the cornea following mustard gas exposure.

Mitochondria regulate cellular activity in a tissue-selective manner. The role of mitochondria in corneal fibrosis is elusive. This study investigated changes in mitochondrial DNA (mtDNA) and mitochondrial transcription factor A (TFAM) in human corneal stromal fibroblasts (CSFs) and corneal myofibroblast (CMFs) and effects on corneal fibrosis in vitro and ex vivo. Healthy donor human corneas were used to generate CSFs and ex vivo culture. CMF formation was induced by transforming growth factor beta-1 (TGFβ1) in vitro and human cornea by nitrogen mustard (NM) ex vivo. mtTFA/TFAM CRISPR/Cas9 KO plasmid, Lipofectamine CRISPRMAX, and TrueCut Cas9 Protein v2 were used for gene editing. Long-range PCR and quantitative reverse-transcription PCR (qRT-PCR) measured mtDNA transcription, mtDNA quantity, and ratios of mtDNA to nuclear DNA (nDNA). Immunofluorescence and immunoblotting quantified protein expression. The MitoSOX assay was used to analyze mitochondrial reactive oxygen species (mtROS). Human CMFs showed significantly reduced mtDNA copies (P < 0.01) and mtDNA-to-nDNA ratios (P < 0.05) compared to CSFs. Significant time-dependent increases in mRNA levels of α-smooth muscle actin (αSMA) and nDNA-transcribed genes and decreases in TFAM and mtDNA-transcribed genes were noted during CSF transdifferentiation to CMFs (P < 0.05, P < 0.001, or P < 0.0001). Correspondingly, time-dependent decreases in TFAM and increases in Rieske iron-sulfur (Fe-S) and αSMA protein (P < 0.0001) and mtROS and ROS levels (P < 0.0001) were observed. TFAM silencing arrested fibrotic events and exhibited reduced αSMA and enhanced mtDNA (P < 0.001). The NM-induced fibrotic human cornea showed decreased TFAM and increased αSMA compared to naïve corneas (P < 0.01). We observed that mtDNA plays an important role in corneal fibroblast transdifferentiation to myofibroblast and that TFAM has the potential to modulate this process in an injured cornea. Additional studies are warranted.