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Home / Equine Research Bank / Vet Ophthalmol / Altering equine corneal fibroblast differentiation through S...
Veterinary ophthalmology2017; 21(2); 132-139; doi: 10.1111/vop.12485

Altering equine corneal fibroblast differentiation through Smad gene transfer.

Abstract: To explore the impact of equine corneal fibroblast (ECF) to myofibroblast (ECM) differentiation by altering the expression of the Smad genes either individually or in combination. Specifically, we sought to examine the ECF differentiation after (a) silencing of Smad2, 3, and 4 profibrotic genes individually and (b) overexpression of antifibrotic Smad7 gene and in a combination with pro- and antifibrotic Smad genes. Methods: Equine corneal fibroblast primary cultures were generated as previously described. ECFs were transfected with individual plasmids which silenced gene expression of either Smad2, 3, or 4 or in combination with a plasmid overexpressing Smad7 using Lipofectamine 2000™ or Lipofectamine BLOCK-iT™. Smad-transfected clones were then exposed to TGF-β1 to induce differentiation to myofibroblasts. Immunofluorescence and qRT-PCR techniques quantified levels of ECF differentiation to ECM by measuring alpha smooth muscle actin, a known marker of ECM transdifferentiation. Results: Silencing of individual Smad2, 3, or 4 genes or overexpression of Smad7 showed significant inhibition of ECF transdifferentiation (73-83% reduction). Silencing of Smad2 showed the greatest inhibition of ECF transdifferentiation in (a) and was therefore utilized for the combination gene transfer testing. The combination gene transfer consisting of Smad7 overexpression and Smad2 silencing attenuated ECF differentiation significantly; however, the level was not significant compared to the overexpression of Smad7 individually. Conclusions: Using gene transfer technology involving profibrotic Smad silencing, antifibrotic Smad overexpression or its combination is a novel strategy to control TGF-β1-mediated fibrosis in equine fibroblasts. Combination gene therapy was not better than single gene therapy in this study.
© 2017 American College of Veterinary Ophthalmologists.
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Publication Date: 2017-07-06 PubMed ID: 28685927PubMed Central: PMC5756533DOI: 10.1111/vop.12485Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research explores the impact of manipulating Smad genes on the differentiation of equine corneal fibroblasts (ECF) to myofibroblasts (ECM). The findings suggest that gene therapy techniques involving the silencing of profibrotic Smad genes and overexpression of antifibrotic Smad genes could potentially control fibrosis in equine fibroblasts.

Methodology

  • Primary cultures of ECF were created in a laboratory setting.
  • The fibroblasts were genetically modified either by plasmids that silenced the expression of profibrotic Smad2, 3, or 4 genes individually or by a combination of these with the overexpression of the antifibrotic Smad7 gene. This was done using Lipofectamine 2000™ or Lipofectamine BLOCK-iT™.
  • The transfections used the Smad genes which are responsible for cell differentiation and growth.
  • After the genetic modification, the cultures were exposed to a protein known as TGF-β1, which is known to induce the ECFs to differentiate into ECMs.
  • ECF differentiation to ECM was measured using immunofluorescence and qRT-PCR techniques, focusing on the levels of alpha smooth muscle actin, which is a known marker of ECM transdifferentiation.

Findings

  • The silencing of individual Smad2, 3, or 4 genes, as well as the overexpression of the Smad7 gene, resulted in a significant decrease (73-83%) in the transdifferentiation of ECF to ECM.
  • Of the individual genes, Smad2 silencing showed the greatest reduction in transdifferentiation and was consequently used for combination gene transfer testing.
  • When the antifibrotic Smad7 gene was overexpressed in combination with the silencing of the profibrotic Smad2 gene, there was a significant decrease in ECF differentiation. However, the level was not statistically significant compared to overexpression of Smad7 alone.

Conclusions

  • The research concluded that manipulating Smad gene expression can control fibrosis in equine fibroblasts, this is in regards to TGF-β1-mediated fibrosis.
  • However, combination gene therapy of both Profibrotic Smad silencing and Antifibrotic Smad overexpression was not found to be more effective than individual gene therapies.

Cite This Article

APA
Marlo TL, Giuliano EA, Tripathi R, Sharma A, Mohan RR. (2017). Altering equine corneal fibroblast differentiation through Smad gene transfer. Vet Ophthalmol, 21(2), 132-139. https://doi.org/10.1111/vop.12485

Publication

Veterinary ophthalmology
ISSN: 1463-5224
NlmUniqueID: 100887377
Country: England
Language: English
Volume: 21
Issue: 2
Pages: 132-139

Researcher Affiliations

Marlo, Todd L
  • Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Giuliano, Elizabeth A
  • Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Tripathi, Ratnakar
  • Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
  • Harry S. Truman Veterans Memorial Hospital, Columbia, MO, USA.
Sharma, Ajay
  • Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
  • Harry S. Truman Veterans Memorial Hospital, Columbia, MO, USA.
Mohan, Rajiv R
  • Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
  • Harry S. Truman Veterans Memorial Hospital, Columbia, MO, USA.
  • Mason Eye Institute, University of Missouri, 1 Hospital Drive, Columbia, MO, USA.

MeSH Terms

  • Animals
  • Cell Differentiation / genetics
  • Cells, Cultured
  • Cornea / cytology
  • Fibroblasts / cytology
  • Fibrosis / genetics
  • Fibrosis / therapy
  • Fibrosis / veterinary
  • Gene Silencing
  • Gene Transfer Techniques
  • Genetic Therapy / veterinary
  • Horses
  • Myofibroblasts / cytology
  • RNA, Messenger / antagonists & inhibitors
  • Smad Proteins / economics
  • Smad Proteins / genetics

Grant Funding

  • I01 BX000357 / BLRD VA
  • R01 EY017294 / NEI NIH HHS

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Citations

This article has been cited 2 times.
  1. Mohan RR, Martin LM, Sinha NR. Novel insights into gene therapy in the cornea.. Exp Eye Res 2021 Jan;202:108361.
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  2. Kamil S, Mohan RR. Corneal stromal wound healing: Major regulators and therapeutic targets.. Ocul Surf 2021 Jan;19:290-306.
    doi: 10.1016/j.jtos.2020.10.006pubmed: 33127599google scholar: lookup
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