Abstract
PurposeWe assessed whether mitomycin-C (MMC) has different antifibrotic mechanisms in trabeculectomy wound healing.MethodsWe identified 2 concentrations of MMC as “low-dose” by using WST-1 assay, Lactic dehydrogenase assay, and fluorescence-activated cell sorting flow cytometry. Senescence-associated β-galactosidase (SA-β-gal) and fibrotic gene expression was examined through immunocytochemistry, flow cytometry, real-time quantitative reverse transcription polymerase chain reaction, Western blotting, zymography, and modified scratch assay in vitro. In vivo, 0.1 mL of MMC or normal saline was injected to Tenon’s capsule before trabeculectomy in a rabbit model. SA-β-gal expression, apoptotic cell death, and collagen deposition in sites treated and not treated with MMC were evaluated using terminal dUTP nick end labeling assay and histochemical staining. Bleb function and intraocular pressure (IOP) levels were examined 3, 7, 14, 21, 28, and 35 days after trabeculectomy.ResultsIn vitro, human Tenon’s fibroblast (HTF) senescence was confirmed by observing cell morphologic change, SA-β-gal accumulation, formation of senescence-associated heterochromatin, increased p16INK4a and p21CIP1/WAF1 expression, lower percentage of Ki-67-positive cells, and decreased COL1A1 release. Increased expression of α-SMA, COL1A1, and Smad2 signaling in TGF-β1-induced stress fibers were passivated in senescent HTFs. In addition, cellular migration enhanced by TGF-β1was inactivated. In vivo, histological examination indicated increased SA-β-gal accumulation, lower apoptosis ratios, and looser collagen deposition in sites treated with 0.2 μM MMC. Low-dose MMC-induced cellular senescence prolonged trabeculectomy bleb survival and reduced IOP levels in a rabbit model.ConclusionLow-dose MMC-induced cellular senescence is involved in the antifibrotic mechanism of trabeculectomy wound healing.
Highlights
Glaucoma is an optic neuropathy that can lead to irreversible blindness [1, 2]
Human Tenon’s fibroblast (HTF) senescence was confirmed by observing cell morphologic change, SA-β-gal accumulation, formation of senescence-associated heterochromatin, increased p16INK4a and p21CIP1/WAF1 expression, lower percentage of Ki-67-positive cells, and decreased COL1A1 release
Increased expression of α-SMA, COL1A1, and Smad2 signaling in TGF-β1-induced stress fibers were passivated in senescent human Tenon’s fibroblast (HTF)
Summary
Glaucoma is an optic neuropathy that can lead to irreversible blindness [1, 2]. Increased intraocular pressure (IOP) is not the only cause of visual impairment, current treatments reduce IOP by using topical medications, lasers, or surgical intervention [3] and can arrest glaucoma [4, 5]. Since its introduction by Cairns in 1968, trabeculectomy, which entails creating a direct channel connecting the anterior chamber and the subconjunctival space, is the benchmark among the available glaucoma surgical techniques [6, 7]. Excessive postoperative scarring at the filtering bleb site can counteract the benefits of trabeculectomy by closing down the artificial route [8,9,10]. Minimizing postoperative scarring has become a vital part of trabeculectomy. No satisfactory method has been developed to resolve this problem
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