June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Inhibition of miR-145 as a strategy to prevent corneal scarring
Author Affiliations & Notes
  • Anna Bajor
    Eye Hospital, Hannover Medical School, Hannover, Germany
  • Dominica Ratuszny
    Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
  • Christiane Gras
    Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
  • Amelie Pielen
    Eye Hospital, Hannover Medical School, Hannover, Germany
  • Martin Börgel
    German Society for Tissue Transplantation, Hannover, Germany
  • Carsten Framme
    Eye Hospital, Hannover Medical School, Hannover, Germany
  • Rainer Blasczyk
    Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
  • Constanca Figueiredo
    Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
  • Footnotes
    Commercial Relationships Anna Bajor, None; Dominica Ratuszny, None; Christiane Gras, None; Amelie Pielen, None; Martin Börgel, None; Carsten Framme, None; Rainer Blasczyk, None; Constanca Figueiredo, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4917. doi:
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      Anna Bajor, Dominica Ratuszny, Christiane Gras, Amelie Pielen, Martin Börgel, Carsten Framme, Rainer Blasczyk, Constanca Figueiredo; Inhibition of miR-145 as a strategy to prevent corneal scarring. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4917.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: MicroRNA (miRNA) gene therapies are of particular interest for complex processes such as fibrosis and scarring. The role of miRNAs in corneal wound healing and scar formation upon infection or trauma is unclear. Differentiation of fibroblasts into myofibroblasts may contribute to corneal scar formation causing visual loss. This study focussed on the role of miR-145 in corneal scarring. The aim was to prevent excessive scar formation by inhibiting myofibroblast activity.

Methods: Fibroblasts were isolated from human corneas and differentiated into myofibroblasts in presence of TGF-β1. miR-145 levels in fibroblasts and myofibroblasts were analyzed using miRNA expression assays. Real-time PCR and western blots were used to determine the effect of miR-145 and a miR-145 inhibitor on profibrotic genes such as α-smooth muscle actin (αSMA) and Krüppel-Like Factor 4 (KLF4). miR-145 was previously shown to target KLF4, which is a negative regulator of αSMA. Hence, increased miR-145 levels may cause an upregulation of αSMA expression. To analyze if miR-145 regulates the expression of αSMA, cells were treated with a miR-145 inhibitor prior to TGF-β1 stimulation. Cell contractility was analyzed using collagen contraction assays.

Results: Stimulation of primary corneal fibroblasts with TGF-β1 induced the differentiation into contractile myofibroblasts as shown in contractility assays and de novo expression of αSMA, which is a marker for myofibroblasts. In the miRNA profiling assays, a 6-fold increase of miR-145 in TGF-β1 treated myofibroblasts compared to non-treated fibroblasts was observed (p<0.001). Our results clearly showed that in myofibroblasts the miR-145 inhibitor decreased miR-145 levels by 94% and αSMA expression by 83% on mRNA and by 75% on protein level (p<0.05). In addition, KLF4 expression was restored in presence of the miR-145 inhibitor.

Conclusions: These data suggest that TGF-β1 induces miR-145 expression in fibroblasts, which in turn inhibits KLF4 and hence upregulates αSMA expression. As αSMA plays an important role for the contractility of myofibroblasts, these data strongly suggest that the inhibition of αSMA expression using a miR-145 inhibitor will contribute to decrease myofibroblast contractility. Therefore, miR-145 is a promising therapeutic target to prevent or reduce corneal scarring. Furthermore, our data demonstrate the feasibility of manipulating primary corneal fibroblasts with miRNA inhibitors.

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