Substratum topography modulates corneal fibroblast to myofibroblast transformation

Abstract

Purpose: The transition of corneal fibroblasts to the myofibroblast phenotype is known to be important in wound healing. The purpose of this study was to determine the effect of topographic cues on TGFβ-induced myofibroblast transformation of corneal cells.

Methods: Rabbit corneal fibroblasts were cultured on nanopatterned surfaces having topographic features of varying sizes. Cells were cultured in media containing TGFβ at concentrations ranging from 0 to 10 ng/mL. RNA and protein were collected from cells cultured on topographically patterned and planar substrates and analyzed for the myofibroblast marker α-smooth muscle actin (αSMA) and Smad7 expression by quantitative real time PCR. Western blot and immunocytochemistry analysis for αSMA were also performed.

Results: Cells grown on patterned surfaces demonstrated significantly reduced levels of αSMA (P < 0.002) compared with planar surfaces when exposed to TGFβ; the greatest reduction was seen on the 1400 nm surface. Smad7 mRNA expression was significantly greater on all patterned surfaces exposed to TGFβ (P < 0.002), whereas cells grown on planar surfaces showed equal or reduced levels of Smad7. Western blot analysis and αSMA immunocytochemical staining demonstrated reduced transition to the myofibroblast phenotype on the 1400 nm surface when compared with cells on a planar surface.

Conclusions: These data demonstrate that nanoscale topographic features modulate TGFβ-induced myofibroblast differentiation and αSMA expression, possibly through upregulation of Smad7. It is therefore proposed that in the wound environment, native nanotopographic cues assist in stabilizing the keratocyte/fibroblast phenotype while pathologic microenvironmental alterations may be permissive for increased myofibroblast differentiation and the development of fibrosis and corneal haze.

Figure 1.

TGFβ exposure correlates with a measurable increase in αSMA gene expression in fibroblasts. Minimal αSMA expression is seen at the 0 and 0.1 ng/mL concentrations of TGFβ. A threefold increase was observed at 1 ng/mL and a 23-fold increase at 10 ng/mL.

Figure 2.

Exposure to all topographically patterned surfaces decreases αSMA expression in rabbit corneal stromal cells. Expression is presented as the amount of αSMA relative to the planar surface at 10 ng/mL TGFβ. Cells grown on all topographically patterned surfaces demonstrated significantly reduced levels of αSMA compared with the planar surface when exposed to at least 1 ng/mL of TGFβ. Cells on the 1400 nm pitch surface expressed the lowest levels of αSMA, expressing 3.3-fold less than those on planar surfaces at 1 ng/mL TGFβ and 10.7-fold less αSMA at 10 ng/mL TGFβ. Error bars indicate SEM. **P < 0.005; ***P < 0.002.

Figure 3.

αSMA protein is reduced in corneal stromal cells cultured on 1400 nm pitch compared with planar surfaces. (A) Western blot analysis of lysates from cells cultured on surfaces and media as shown (top row); Ponceau S band used for semiquantitative normalization shown below blots. Images are from the same blot reordered for clarity. (B) Graph indicating normalization of αSMA protein signal intensities; signal is significantly reduced on 1400 nm compared with adjacent planar control. Error bars indicate SEM. **P < 0.01.

Figure 4.

Exposure to topographically patterned surfaces decreases transformation of rabbit corneal fibroblasts to myofibroblasts. (A) Representative images of stromal cells cultured on indicated substrates and indicated amounts of exogenous TGFβ added to growth medium. Green signal: αSMA; blue: DAPI. (B) Graph depicting quantification of αSMA-positive cells. Cells grown on the 1400 nm pitch had fewer than 50% the number of transformed cells than those grown on planar surfaces when exposed to 10 ng/mL TGFβ. Error bars indicate SD. *P < 0.05.

Figure 5.

Exposure to all pitch sizes of topographically patterned surfaces increases Smad7 expression in rabbit corneal fibroblasts. Expression is presented as the amount of Smad7 relative to the planar surface at 10 ng/mL TGFβ. Cells grown on all topographically patterned surfaces demonstrated significantly increased levels of Smad7 (**P < 0.002) compared with the planar surface when exposed to at least 1 ng/mL of TGFβ. Cells on 4000 nm pitch expressed the highest levels of Smad7, expressing a 5.8-fold increase in Smad7 expression at 1 ng/mL TGFβ and 10.7-fold increase in Smad7 at 10 ng/mL TGFβ. Representative graph of the experiment completed in triplicate. Error bars indicate SEM.