Wound Healing in Ovariectomized Rats  Effects of Chemically Modified Tetracycline (CMT-8) and Estrogen on Matrix Metalloproteinases -8, -13 and Type I Collagen Expression

Emma      Pirila; Nungavarm      Ramamurthy; Paivi      Maisi; Steve      McClain; Allan      Kucine; Jaana      Wahlgren; Lorne   M   Golub; Tuula      Salo; Timo      Sorsa

Abstract

Cutaneous wound healing is a complex process involving interactions of various cell types. Skin, in addition to certain other organs, is dependent on estrogen and estrogen-deficiency is associated with impaired wound healing. Wound healing involves the action of collagenolytic matrix metalloproteinases (MMPs). We investigated the expression and localization of collagenolytic MMPs -8 and -13 by collagenase activity assay, Western immunoblot analysis, in situ hybridization and immunohistochemical staining as well as type I collagen by hydroxyproline content analysis and immunohistochemical staining in cutaneous wounds from aged Sham and ovarioectomized (OVX) rats. After wounding, OVX rats were treated with either placebo, chemically modified tetracycline-8 (CMT-8) or estrogen. We found that MMP-8 and MMP-13 mRNA were expressed in wound epithelium of all samples examined as evidenced by in situ hybridization. Type I collagen, which was abundant in all groups examined, was decreased in OVX rats, but was increased by both CMT-8 and estrogen treatments to the level of Sham group. Hydroxyproline analysis revealed similar results. Western blot data showed that all forms of MMP-8 and MMP-13 were clearly reduced in the CMT-8 treated group compared to OVX. Analysis of collagenolytic activity confirmed the decreased collagenolysis in skin wound extracts from CMT-treated rats when compared with skin wound extracts from OVX rats. Our results show for the first time that MMP-8 mRNA and protein are expressed in rat wound epithelium. We further show that CMT-8 and estrogen have a beneficial effect on skin wound healing in OVX rats by increasing the collagen content and reducing the MMP-mediated collagenolysis.

Keywords: modified tetracycline, collagen expression, westrn immunobolt analysis, matrix metalloprotenases, misregulated proteinases, non antimicrobial derivative, stegmenn colorimetric assay, bovine serum albumin, immunohistochemistry, endogenous perxidase activity, anti human mmp 8, immunohistochemical staining, suprabasal keratinocytes, murine interstitial collagenase