Hypoxia-induced increase of matrix metalloproteinase-1 synthesis is not restored by reoxygenation in a three-dimensional culture of human dermal fibroblasts

Abstract

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-β1 (TGF-β1) 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-β1, 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. Conclusion: Our results indicate that hypoxia may be responsible for delayed wound healing by inducing an increase of MMP-1 synthesis.