Purchase this article with an account.
Adna Halilovic, Lars Bellner, Kirkland Castellano, Michael W. Dunn, Michal Laniado Schwartzman; Knockdown of HO-2 alters expression of Collagen and MMPs and impairs wound closure in Human Corneal Epithelial (HCE) cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3532.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Heme oxygenase (HO) represents an intrinsic cytoprotective and anti-inflammatory system. We have shown that HO-2 knockdown in vitro attenuates corneal wound healing and also attributes to the remodeling process controlled by a delicate balance between ECM synthesis and degradation by Collagen and MMPs. This study was undertaken to examine the contribution of HO-2 to corneal wound healing in an in vitro epithelial scratch injury model.
HCE cells were stably transfected with HO-2 shRNA or non-target shRNA plasmids. The Human Wound Healing RT² Profiler PCR Array was performed to look at the expression of 84 key genes central to the wound healing process. Genes of the Wound Healing RT Profiler PCR array shown to be affected by altered HO-2 mRNA levels were confirmed by QPCR, and further evaluated in response to scratch injury. Western blot was perfomed to evaluate the expression of proteins involved in migration and proliferation in HO-2 shRNA and non-target shRNA cells.
Real time PCR and Western Blot confirmed a 60-70% HO-2 shRNA-mediated knockdown of HO-2 mRNA and protein levels, respectively. Wound closure in HO-2 shRNA-treated cells was significantly impaired; only 15% of the scratch wound closed at 16h after injury and 44% at 96 h after injury as compared to non-target shRNA transfected cells. Interestingly, expression levels of Collagen Type I and V and MMP-7 were increased at least 3-fold,whereas MMP-1 expression levels were decreased in HO-2 shRNA transfected cells as compared to control. Western blot revealed attenuated levels of protein expression for the activated form of EGFR, p38, ERK1/2 and Akt signaling in HO-2 shRNA compared to non-target shRNA cells.
These results indicate that the knockdown of HO-2 impairs the wound healing process in the cornea; its suppression inhibited MMP-1 but increased Collagen Type I and V and MMP-7 levels. Also, findings suggest that the expression pattern of proteins involved in migration and proliferation is altered in HO-2 shRNA compared to non-target shRNA cells. The mechanisms underlying HO-2 effects may reside in the remodeling controlled by a balance between ECM synthesis and degradation as a physical support to provide for the wound healing process in the cornea.
This PDF is available to Subscribers Only