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N. Guo, M.L. Funderburgh, M.M. Mann, Y. Du, J.L. Funderburgh; Hyaluronan Is a Potent Enhancer of the Keratocyte Fibrotic Response to TGF–ß1 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2738.
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Transforming growth factor beta (TGFß) is a key inducer of the fibrotic response typifying wound healing in many tissues including cornea. Keratocytes in vitro respond to TGFß by secretion of a number of matrix components characteristic of corneal scars, including the EDA–splice form of fibronectin, collagen III, and biglycan, in addition to displaying the myofibroblastic phenotype marked by alpha–smooth muscle actin. Hyaluronan (HA) is observed in healing corneas in vivo and is also induced in keratocytes by TGFß in vitro. We found recently that the mRNA for HA synthase was strongly and rapidly upregulated in keratocytes by TGFß1. HA is also known to be active in induction of motility and mitosis in a number of cells. The purpose of this study was to test the hypothesis that HA acts in concert with TGFß1 to induce the wound–healing and fibrotic responses of keratocytes.
Primary bovine keratocytes were treated with TGFß1 in the presence of inhibitors of HA synthesis, and/or siRNA to HA synthase. HA synthesis was measured by Fluorophore–assisted Carbohydrate Electrophresis (FACE) analysis. Smooth muscle actin, EDA–fibronectin, and collagen III mRNA and protein levels were monitored by qRT–PCR, western blot, and immunohistology. Cell division was monitored by Ki–67 antibody.
Inhibition of HA synthesis with 4–methylumbelliferone blocked upregulation by TGFß of both mRNA and proteins of fibrotic components in a dose–dependent fashion. Suppression of HA synthase mRNA expression with siRNA had the similar effects on fibrotic response. Increases in smooth muscle actin synthesis and cell division were also blocked by inhibition of HA secretion.
As a matrix component inducing activation of multiple keratocyte functions, corneal HA fulfils the role of a potent enhancer for fibrotic response. The very early appearance of HA in response to TGFß and the dependence of the fibrogenic response on HA biosynthesis suggests that HA may be important in mediating response to TGFß during wound healing in vivo.
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