Substratum topography modulates corneal fibroblast to myofibroblast transformation.
OBJECTIVE
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.
Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Kathern E
KE
Myrna
Rima
R
Mendonsa
Paul
P
Russell
Simon A
SA
Pot
Sara J
SJ
Liliensiek
James V
JV
Jester
Paul F
PF
Nealey
Donald
D
Brown
Christopher J
CJ
Murphy
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D003318:Corneal Opacity / Q000235:genetics / Q000473:pathology*; D003319:Corneal Stroma / Q000473:pathology*; D019781:Corneal Topography; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000473:pathology*; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D058628:Myofibroblasts / Q000473:pathology*; D012313:RNA / Q000235:genetics; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051906:Smad7 Protein / Q000096:biosynthesis / Q000235:genetics
C541116:ACTA2 protein, human; D000199:Actins; D051906:Smad7 Protein; D012313:RNA