Hypoxia regulates the expression of extracellular matrix associated proteins in equine dermal fibroblasts via HIF1

Background: Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. Objective: The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or limb respond differently to hypoxia. Methods: EDFs were isolated and cultured from skin from body or limb under normoxic or hypoxic conditions for up to 7 days. Results: Hypoxia significantly stimulated EDF proliferation, but had no effect on cell survival. The hypoxia-mimetic agent CoCl2 up-regulated COL1A1 expression and down-regulated MMP2 expression, suggesting an increase in ECM synthesis and a decrease in turnover. Both regulatory effects were inhibited by the addition of echinomycin, indicating that they are mediated by the transcriptional regulatory activity of HIF. No differences were observed between EDFs originating from body or limb for any effect of hypoxia or CoCl2, suggesting that EGT development does not depend on intrinsic properties of limb fibroblasts. Conclusions: We conclude that hypoxia regulates ECM remodeling via HIFI in EDFs, and that this may be an important determinant in the pathogenesis of equine EGT. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.