A novel aptamer targeting TGF-β receptor II inhibits transdifferentiation of human tenon's fibroblasts into myofibroblast

Invest Ophthalmol Vis Sci. 2012 Oct 5;53(11):6897-903. doi: 10.1167/iovs.12-10198.

Abstract

Purpose: To isolate aptamers that were bound to the extracellular segment of TGF-β receptor II (TβRII) and evaluate their effect on the TGF-β-induced transdifferentiation of fibroblasts.

Methods: TβRII-binding aptamers were screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) from a single stranded DNA (ssDNA) library. Human Tenon's fibroblasts (HTFs) were cultured and treated with TGF-β2, TGF-β2 and aptamer S58/68, or aptamer S58/68 alone. Western blot analysis was performed to determine levels of α-smooth muscle actin (α-SMA) and the signaling protein phosphorylated Smad2 (p-Smad2). α-SMA and p-Smad2 subcellular distribution and fibrous actin (F-actin) with rhodamine-phalloidin staining were evaluated by confocal immunofluorescence microscopy. Cell contractility was assessed in collagen gel contraction assays.

Results: Twenty-one sequences were obtained after eight rounds of selection. Two preferential sequences, aptamer S58 and S68, were isolated and used in the following experiments. Aptamer S58 significantly inhibited α-SMA expression and incorporation into actin stress fibers, as induced by TGF-β2. Aptamer S58 also suppressed TGF-β2-induced cell contraction. Furthermore, aptamer S58 inhibited the TGF-β2-induced phosphorylation and nuclear translocation of Smad2. However, we did not find any effect of aptamer S68 on TGF-β2 activity in vitro.

Conclusions: Our study revealed that a novel aptamer binding TβRII inhibited TGF-β2-induced myofibroblast transdifferentiation in HTFs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Aptamers, Nucleotide / pharmacology*
  • Base Sequence
  • Blotting, Western
  • Cell Transdifferentiation / drug effects*
  • Cells, Cultured
  • DNA, Single-Stranded / genetics
  • Fibroblasts / cytology*
  • Fluorescent Antibody Technique, Indirect
  • Humans
  • Microscopy, Confocal
  • Molecular Sequence Data
  • Myofibroblasts / cytology*
  • Phosphorylation
  • Polymerase Chain Reaction
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein-Serine-Threonine Kinases / metabolism
  • Radioimmunoassay
  • Receptor, Transforming Growth Factor-beta Type II
  • Receptors, Transforming Growth Factor beta / antagonists & inhibitors*
  • Receptors, Transforming Growth Factor beta / metabolism
  • SELEX Aptamer Technique
  • Sequence Analysis, DNA
  • Smad2 Protein / metabolism
  • Tenon Capsule / cytology*
  • Transforming Growth Factor beta / antagonists & inhibitors*
  • Transforming Growth Factor beta / metabolism

Substances

  • ACTA2 protein, human
  • Actins
  • Aptamers, Nucleotide
  • DNA, Single-Stranded
  • Receptors, Transforming Growth Factor beta
  • SMAD2 protein, human
  • Smad2 Protein
  • Transforming Growth Factor beta
  • Protein-Serine-Threonine Kinases
  • Receptor, Transforming Growth Factor-beta Type II