Suberoylanilide hydroxamic acid (vorinostat): its role on equine corneal fibrosis and matrix metalloproteinase activity.
Abstract: To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. Methods: Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFβ1 to induce myofibroblast formation. The cultures were treated with either 5 μm or 10 μm SAHA for 72 h in the presence of TGFβ1. Real-time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying α-smooth muscle actin (αSMA), a marker of myofibroblast formation and fibrosis. Real-time PCR was used to determine the effects of SAHA on MMP2 and MMP9 expression. Cytotoxicity of SAHA was evaluated with phase contrast microscopy and trypan blue exclusion assays. Results: Suberoylanilide hydroxamic acid (SAHA) significantly attenuated TGFβ1-induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3- to 3.5-fold (P < 0.001) decrease in αSMA mRNA and 86-88% (P < 0.001) decrease in αSMA+ immunocytochemical staining. SAHA treatment also resulted in 4.5- to 5.5-fold (P < 0.01) decrease in MMP9 expression. A dose-dependent bimodal effect of SAHA on MMP2 expression was noted (3.5-fold increase with 5 μm dose; 0.5-fold decrease with 10 μm dose). No change in fibroblast viability was observed with a 5 μm SAHA dose, whereas a 10 μm dose resulted in a moderate 17% decrease in cell viability. Conclusions: Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGFβ-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro.
© 2013 American College of Veterinary Ophthalmologists.
Publication Date: 2014-08-16 PubMed ID: 25126665DOI: 10.1111/vop.12129Google Scholar: Lookup
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- Journal Article
Summary
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This study investigates the role of Suberoylanilide hydroxamic acid (SAHA) in affecting equine corneal fibrosis and the activity of matrix metalloproteinases (MMPs). The experiments show that SAHA significantly reduces TGFβ1-induced transformation of corneal fibroblasts into myofibroblasts and influences the modulation of MMPs.
Research Methods
- Corneas from healthy donors were used to create primary cultures of equine corneal fibroblasts.
- Those fibroblasts were exposed to 5 ng/mL TGFβ1, a compound used to stimulate transformation into myofibroblasts, a critical component in the development of fibrosis.
- The cultures were then treated with either 5 μm or 10 μm of SAHA for 72 hours in the presence of TGFβ1.
- The researchers used Real-time PCR and immunocytochemistry to measure the antifibrotic effect of SAHA. This was determined by quantifying α-smooth muscle actin (αSMA), a known marker of myofibroblast formation and fibrosis development.
- The scientists also used Real-time PCR to evaluate the impact of SAHA on the expression of MMP2 and MMP9.
- The cytotoxicity of SAHA was also assessed using phase contrast microscopy and a trypan blue exclusion assay.
Research Findings
- SAHA considerably reduced the TGFβ1-induced conversion of equine fibroblasts to myofibroblasts. This was indicated by a significant decrease in αSMA mRNA and a clear reduction in αSMA+ immunocytochemical staining.
- Furthermore, SAHA treatment resulted in a considerable decrease in MMP9 t expression.
- An interesting dose-dependent bimodal effect on MMP2 expression was observed with SAHA treatment. A lower dose led to an increase, while a higher dose resulted in a decrease.
- The impact of SAHA treatment on fibroblast viability was also assessed. A smaller dose of SAHA had no impact on cell viability, but a larger dose led to a moderate decrease in the number of viable cells.
Conclusions
- The research concludes that Suberoylanilide hydroxamic acid (SAHA) can effectively hamper the TGFβ-induced transformation of equine corneal fibroblasts into myofibroblasts, thereby impeding the progress of fibrosis. SAHA also plays a role in modulating the production of MMPs, proteins that regulate the matrix environment of cells.
Cite This Article
APA
Donnelly KS, Giuliano EA, Sharm A, Mohan RR.
(2014).
Suberoylanilide hydroxamic acid (vorinostat): its role on equine corneal fibrosis and matrix metalloproteinase activity.
Vet Ophthalmol, 17 Suppl 1, 61-68.
https://doi.org/10.1111/vop.12129 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Cell Survival / drug effects
- Cicatrix / drug therapy
- Cicatrix / enzymology
- Cicatrix / veterinary
- Corneal Injuries / drug therapy
- Corneal Injuries / enzymology
- Corneal Injuries / pathology
- Corneal Injuries / veterinary
- Corneal Keratocytes / drug effects
- Corneal Keratocytes / enzymology
- Corneal Keratocytes / pathology
- Histone Deacetylase Inhibitors / therapeutic use
- Horse Diseases / drug therapy
- Horse Diseases / enzymology
- Horse Diseases / pathology
- Horses
- Hydroxamic Acids / therapeutic use
- In Vitro Techniques / veterinary
- Matrix Metalloproteinases / drug effects
- Matrix Metalloproteinases / metabolism
- Vorinostat
Citations
This article has been cited 19 times.- Ghosh A, Singh VK, Singh V, Basu S, Pati F. Recent Advancements in Molecular Therapeutics for Corneal Scar Treatment. Cells 2022 Oct 21;11(20).
- Fuchs AA, Balne PK, Giuliano EA, Sinha NR, Mohan RR. Evaluation of a novel combination of TRAM-34 and ascorbic acid for the treatment of corneal fibrosis in vivo. PLoS One 2022;17(1):e0262046.
- Shetty R, Kumar NR, Subramani M, Krishna L, Murugeswari P, Matalia H, Khamar P, Dadachanji ZV, Mohan RR, Ghosh A, Das D. Safety and efficacy of combination of suberoylamilide hydroxyamic acid and mitomycin C in reducing pro-fibrotic changes in human corneal epithelial cells. Sci Rep 2021 Feb 23;11(1):4392.
- Anumanthan G, Sharma A, Waggoner M, Hamm CW, Gupta S, Hesemann NP, Mohan RR. Efficacy and Safety Comparison Between Suberoylanilide Hydroxamic Acid and Mitomycin C in Reducing the Risk of Corneal Haze After PRK Treatment In Vivo. J Refract Surg 2017 Dec 1;33(12):834-839.
- Marlo TL, Giuliano EA, Tripathi R, Sharma A, Mohan RR. Altering equine corneal fibroblast differentiation through Smad gene transfer. Vet Ophthalmol 2018 Mar;21(2):132-139.
- Sarenac T, Trapecar M, Gradisnik L, Rupnik MS, Pahor D. Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-β/Smad pathway of fibrosis in human keratocytes in vitro. Sci Rep 2016 Sep 30;6:34373.
- Zhou H, Zhang W, Bi M, Wu J. The molecular mechanisms of action of PPAR-γ agonists in the treatment of corneal alkali burns (Review). Int J Mol Med 2016 Oct;38(4):1003-11.
- Marlo TL, Giuliano EA, Sharma A, Mohan RR. Development of a novel ex vivo equine corneal model. Vet Ophthalmol 2017 Jul;20(4):288-293.
- Sharma A, Anumanthan G, Reyes M, Chen H, Brubaker JW, Siddiqui S, Gupta S, Rieger FG, Mohan RR. Epigenetic Modification Prevents Excessive Wound Healing and Scar Formation After Glaucoma Filtration Surgery. Invest Ophthalmol Vis Sci 2016 Jun 1;57(7):3381-9.
- Gronkiewicz KM, Giuliano EA, Sharma A, Mohan RR. Molecular mechanisms of suberoylanilide hydroxamic acid in the inhibition of TGF-β1-mediated canine corneal fibrosis. Vet Ophthalmol 2016 Nov;19(6):480-487.
- Gronkiewicz KM, Giuliano EA, Kuroki K, Bunyak F, Sharma A, Teixeira LB, Hamm CW, Mohan RR. Development of a novel in vivo corneal fibrosis model in the dog. Exp Eye Res 2016 Feb;143:75-88.
- Sharma A, Sinha NR, Siddiqui S, Mohan RR. Role of 5'TG3'-interacting factors (TGIFs) in Vorinostat (HDAC inhibitor)-mediated Corneal Fibrosis Inhibition. Mol Vis 2015;21:974-84.
- Koppaka V, Lakshman N, Petroll WM. Effect of HDAC Inhibitors on Corneal Keratocyte Mechanical Phenotypes in 3-D Collagen Matrices. Mol Vis 2015;21:502-14.
- Xie M, Jie Y. Transforming corneal alkali burn treatment: unveiling mechanisms and pioneering therapies from bench to bedside. J Transl Med 2025 Oct 21;23(1):1132.
- Routh BL, Tripathi R, Giuliano E, Lujin P, Sinha PR, Mohan RR. Anti-fibrotic effects of lisinopril (ACE inhibitor) and fasudil (ROCK inhibitor) in combination for canine corneal fibrosis in vitro. Vet Ophthalmol 2026 Jan;29(1):e13304.
- Sood S, Tiwari A, Sangwan J, Vohra M, Sinha NR, Tripathi R, Sangwan VS, Mohan RR. Role of epigenetics in corneal health and disease. Prog Retin Eye Res 2025 Jan;104:101318.
- Chandran C, Santra M, Rubin E, Geary ML, Yam GH. Regenerative Therapy for Corneal Scarring Disorders. Biomedicines 2024 Mar 14;12(3).
- Mohan RR, Kempuraj D, D'Souza S, Ghosh A. Corneal stromal repair and regeneration. Prog Retin Eye Res 2022 Nov;91:101090.
- Tripathi R, Sinha NR, Kempuraj D, Balne PK, Landreneau JR, Juneja A, Webel AD, Mohan RR. Evaluation of CRISPR/Cas9 mediated TGIF gene editing to inhibit corneal fibrosis in vitro. Exp Eye Res 2022 Jul;220:109113.
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