Dataset Information

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

ABSTRACT:

Purpose

The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFβ1 inhibitor SB-431542 (SB) to MMC.

Methods

To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC or 20 µM SB followed by incubation with 2 ng/mL TGFβ1. Total protein extracted from experimentally treated HTCFs underwent immunoblotting for α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP-9), and EDA splice-variant fibronectin (EDA-FN) expression. Cytotoxicity and cell metabolism were assessed using LIVE/DEAD staining, lactate dehydrogenase (LDH) assay, and methylthiazole tetrazolium (MTT) assay.

Results

Collagen lattice contraction in TGFβ1-induced HTCFs was significantly lowered by SB and MMC. Pretreatment with SB and MMC significantly lowered protein expression of α-SMA, MMP-9, and EDA-FN in HTCFs relative to TGFβ1 alone. HTCF viability in collagen lattices was significantly reduced with MMC pretreatment but not SB pretreatment. MMC-pretreated HTCFs had a significant increase in LDH release after 3 hours and a decrease in MTT activity after 20 minutes, while SB-pretreated HTCFs showed no significant changes via MTT or LDH assay during the same treatment period.

Conclusions

SB shows comparable efficacy to MMC in reducing expression of fibrosis-promoting proteins in HTCFs and in vitro scarring activity. SB distinguishes itself from MMC by exhibiting less cytotoxicity in both two-dimensional and three-dimensional in vitro assays.

Translational relevance

This study demonstrates in vitro the potential of SB as a safer alternative ocular antifibrotic agent.

SUBMITTER: Teplitsky JE

PROVIDER: S-EPMC9973532 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Publications

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

Teplitsky Jack E JE Vinokurtseva Anastasiya A Armstrong James J JJ Denstedt James J Liu Hong H Hutnik Cindy M L CML

Translational vision science & technology 20230201 2

<h4>Purpose</h4>The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFβ1 inhibitor SB-431542 (SB) to MMC.<h4>Methods</h4>To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC o ...[more]

PMID: 36826843

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Project description:PurposeTo synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting IkappaB kinase subunit beta (IKKbeta) on the proliferation of human Tenon's capsule fibroblasts (HTFs) in vitro.MethodsFirst, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKbeta (IKKBeta-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT-PCR) subsequently assessed the mRNA level of IKKbeta, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to evaluate the proliferation of HTFs.ResultsThe diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKbeta was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-kappaB (NF-kappaB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-kappaB pathway.ConclusionsAll the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.

Project description:Glaucoma filtration surgery is one of the most effective methods for lowering intraocular pressure in glaucoma. The surgery efficiently reduces intra-ocular pressure but the most common cause of failure is scarring at the incision site. This occurs in the conjunctiva/Tenon's capsule layer overlying the scleral coat of the eye. Currently used antimetabolite treatments to prevent post-surgical scarring are non-selective and are associated with potentially blinding side effects. Developing new treatments to target scarring requires both a better understanding of wound healing and scarring in the conjunctiva, and new means of delivering anti-scarring drugs locally and sustainably. By combining plastic compression of collagen gels with a soft collagen-based layer, we have developed a physiologically relevant model of the sub-epithelial bulbar conjunctiva/Tenon's capsule interface, which allows a more holistic approach to the understanding of subconjunctival tissue behaviour and local drug delivery. The biomimetic tissue hosts both primary human conjunctival fibroblasts and an immune component in the form of macrophages, morphologically and structurally mimicking the mechanical proprieties and contraction kinetics of ex vivo porcine conjunctiva. We show that our model is suitable for the screening of drugs targeting scarring and/or inflammation, and amenable to the study of local drug delivery devices that can be inserted in between the two layers of the biomimetic. We propose that this multicellular-bilayer engineered tissue will be useful to study complex biological aspects of scarring and fibrosis, including the role of inflammation, with potentially significant implications for the management of scarring following glaucoma filtration surgery and other anterior ocular segment scarring conditions. Crucially, it uniquely allows the evaluation of new means of local drug delivery within a physiologically relevant tissue mimetic, mimicking intraoperative drug delivery in vivo.

Dataset Information

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

Purpose

Methods

Results

Conclusions

Translational relevance

Publications

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

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