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Antifibrotic role of low-dose mitomycin-c-induced cellular senescence in trabeculectomy models.

ABSTRACT: PURPOSE:We assessed whether mitomycin-C (MMC) has different antifibrotic mechanisms in trabeculectomy wound healing. METHODS:We 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 ?-galactosidase (SA-?-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-?-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. RESULTS:In vitro, human Tenon's fibroblast (HTF) senescence was confirmed by observing cell morphologic change, SA-?-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 ?-SMA, COL1A1, and Smad2 signaling in TGF-?1-induced stress fibers were passivated in senescent HTFs. In addition, cellular migration enhanced by TGF-?1was inactivated. In vivo, histological examination indicated increased SA-?-gal accumulation, lower apoptosis ratios, and looser collagen deposition in sites treated with 0.2 ?M MMC. Low-dose MMC-induced cellular senescence prolonged trabeculectomy bleb survival and reduced IOP levels in a rabbit model. CONCLUSION:Low-dose MMC-induced cellular senescence is involved in the antifibrotic mechanism of trabeculectomy wound healing.

SUBMITTER: Lin LT

PROVIDER: S-EPMC7310836 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Antifibrotic role of low-dose mitomycin-c-induced cellular senescence in trabeculectomy models.

Lin Le-Tien LT Chen Jiann-Torng JT Lu Da-Wen DW Tai Ming-Cheng MC Liang Chang-Min CM Chen Ching-Long CL Pao Shu-I SI Hsu Chih-Kang CK Chen Yi-Hao YH

PloS one 20200623 6

<h4>Purpose</h4>We assessed whether mitomycin-C (MMC) has different antifibrotic mechanisms in trabeculectomy wound healing.<h4>Methods</h4>We 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 reac ...[more]

PMID: 32574191

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Project description:Antifibrotic agents are commonly utilized to enhance the success rates of trabeculectomy. Novel approaches to further improve success rates and reduce the risks of complications are needed. The purpose of this study was to compare intraocular pressure (IOP)-lowering efficacy and safety of trabeculectomy or combined phacoemulsification and trabeculectomy with mitomycin-C (MMC) vs. Collagen Matrix (CM).A prospective, multicenter, randomized controlled trial was performed. Ninety-five eyes of 94 patients with uncontrolled glaucoma despite medical therapy, without previous incisional glaucoma surgery underwent trabeculectomy (85 eyes) or combined phacoemulsification and trabeculectomy (10 eyes) and were randomized to MMC or CM. One eye of each subject was analyzed. Patients were followed for 24 months. The criteria for complete success were IOP >5 and ?21 mmHg with at least a 20% reduction below medicated baseline without additional glaucoma surgery or medications. The main outcome measures were complete success rates at 24 months with Kaplan-Meier analysis and incidence of adverse events.The baseline IOPs were 20.4?±?6.0 mmHg and 21.2?±?6.1 (mean?±?standard deviation, p?=?0.49) on 3.2?±?1.1 and 3.1?±?1.0 medications (p?=?0.53) compared to 11.8?±?5.2 and 12.8?±?3.7 (p?=?0.36) on 0.5?±?0.8 and 0.6?±?1.0 medications (p?=?0.63) at 2 years in the MMC and CM groups, respectively. Kaplan-Meier analysis demonstrated complete success rates were similar in both groups at 24 months: 38.4?±?7.6% with MMC and 56.2?±?7.9% with CM (mean?±?standard error, p?=?0.112, log rank test); however, a significantly higher incidence of failure due to persistent hypotony was observed with MMC (p?=?0.002).Use of the CM implant at the time of trabeculectomy or combined phacoemulsification and trabeculectomy is associated with similar complete success rates compared to adjunctive MMC; however, the risk of persistent hypotony is higher with MMC.ClinicalTrials.gov registration number NCT01440751 . Registered 9/14/11.

Project description:Skeletal muscle atrophy is dictated by the balance between protein synthesis and protein breakdown, known as proteostasis. With advanced age, muscle atrophy contributes to development of co-morbidities, loss of strength and independence, and all-cause mortality. Aging also coincides with the accumulation of senescent cells within skeletal muscle that produce inflammatory products, known as the senescence associated secretory phenotype. Previously, we found that a metformin + leucine (MET+LEU) combination treatment had synergistic effects in aged mice to improve skeletal muscle structure and function during disuse atrophy. Therefore, the objective of this study was to determine the mechanisms by which MET+LEU exhibits muscle atrophy protection in vitro and if this occurs through cellular senescence. C2C12 myoblasts differentiated into myotubes were used to determine MET+LEU mechanisms during serum-deprivation (SD) atrophy. Single myofibers were isolated from aged (22-23 mo) C57Bl6/J mice, and human primary myoblasts were extracted from a healthy older adult donor. 25 + 125 µM MET+LEU treatment increased differentiation of myotubes and prevented SD induced atrophy. Low concentration (0.1 + 0.5 µM) MET+LEU had unique effects to prevent myotube atrophy and cause transcriptional reprogramming compared to individual therapies. SD increased inflammatory and cellular senescence pathways that was reversed with MET+LEU treatment. SD resulted in dysregulated proteostasis that was rescued with MET+LEU, and proteasome inhibition with MG-132 also rescued myotube atrophy. Cellular senescence transcriptional pathways and markers increased by SD were reversed with MET+LEU treatment, and dasatinib + quercetin (D+Q) senolytic treatment prevented myotube atrophy similar to MET+LEU. In aged myofibers, MET+LEU prevented SD atrophy, and in human primary myotubes MET+LEU prevented reductions in myonuclei fusion. These data provide evidence that MET+LEU has muscle cell intrinsic properties to prevent atrophy by reversing senescence and improving proteostasis

Dataset Information

Antifibrotic role of low-dose mitomycin-c-induced cellular senescence in trabeculectomy models.

Publications

Antifibrotic role of low-dose mitomycin-c-induced cellular senescence in trabeculectomy models.

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