Hypoxia-induced increase of matrix metalloproteinase-1 synthesis is not restored by reoxygenation in a three-dimensional culture of human dermal fibroblasts.
BACKGROUND
Delayed wound healing is multi-factorial. Although ischemic change is considered to be crucial, little is known about the effects of hypoxia or reoxygenation on the connective tissue metabolism by human dermal fibroblasts.


OBJECTIVE
The aim of this study is to determine whether or not hypoxia (2% O(2)) or reoxygenation (20% O(2)) affects mRNA expression and production of matrix metalloproteinase-1 (MMP-1), type I collagen, tissue inhibitors of metalloproteinase-1 (TIMP-1), and transforming growth factor-beta1 (TGF-beta1) by human dermal fibroblasts in a three-dimensional culture.


METHODS
We introduced the three-dimensional culture of human dermal fibroblasts with experimental wound. After wounding, cells were incubated under hypoxic (2%) or normoxic (20%) condition, and harvested at 24, 36, 48, and 72 h (n=8). In the reoxygenation study (n=4), cells were first exposed to a hypoxic condition for 72 h and further incubated under a normoxic condition for 72 h.


RESULTS
The relative ratio (hypoxia/normoxia) of MMP-1 mRNA expressions were significantly elevated at 36 and 48 h compared with those at 12 h (P<0.05). The relative ratio of proMMP-1 was also significantly increased at 48 and 72 h compared with that at 12 h (P<0.001 and P<0.05, respectively). There were no significant changes in mRNA and protein levels of type I collagen, TGF-beta1, and TIMP-1. In a reoxygenic condition, 72 h reoxygenation after 72 h hypoxia, the hypoxia-induced alterations of MMP-1 and carboxyterminal propeptide of type I procollagen (PIP) were not restored.


CONCLUSIONS
Our results indicate that hypoxia may be responsible for delayed wound healing by inducing an increase of MMP-1 synthesis.
Kan
Chie
C
Department of Dermatology, Gunma University School of Medicine, 3-39-22, Showa-machi, Maebashi, Gunma 371-8511, Japan. ckan@med.gunma-u.ac.jp
Abe
Masatoshi
M
Yamanaka
Masayoshi
M
Ishikawa
Osamu
O
D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics; D017364:Collagenases / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D000860:Hypoxia / Q000201:enzymology* / Q000473:pathology; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology*; D011347:Procollagen / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012867:Skin / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1
D024042:Collagen Type I; D004792:Enzyme Precursors; D011347:Procollagen; D012333:RNA, Messenger; C508960:TGFB1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D010100:Oxygen