Low dose steady state H2O2 induces fibrolytic MMP-3 in vitro and in vivo

Introduction: Inflammation associated oxidative stress such as H2O2 is considered a key event during development of fibrosis/cirrhosis. This contrasts with other fibrogenic diseases such as rheumatoid arthritis where inflammation has been incriminated in opposing effects such as fibrolytic activity and cartilage destruction. We here study the effect of sustained low H2O2 levels on fibroblasts and hepatic stellate cells and in a mouse model.

Methods: Human NIH 3T3 fibroblasts and LX-2 hepatic stellate cells were incubated for 6 or 24h with 0.5–6µM H2O2 using the recently developed GOX/CAT system (enzymatic generation of H2O2 by glucose oxidase and catalase). RNA was isolated and molecules involved in fibrogenesis and fibrolysis were analyzed by qRT-PCR. Additonal Western blotting was performed for MMP3. Experiments were performed under different oxygen tensions using the GOX/CAT system.

Results: In contrast to MMP13, TIMP1 and collagen 1α1, MMP3 was significantly upregulated (x3.5) after 6 hours in both cell types in the presence of non toxic steady state H2O2. The MMP-3 induction could be completely blocked by co-incubation with the antioxidant N-acetylcysteine (2 mM). Notably, MMP-3 was even more drastically upregulated (20x) if the experiment was performed in a slightly lower oxygen environment (10% instead of 21% oxygen) that mimic more closely an inflammatory and hepatic environment. We finally confirm these findings in a recently established mouse model of hepatic oxidative stress by injecting mice with glucose oxidase i.v. (0.2U/g body weight) which homes to the liver and continuously exposes liver cells to non-toxic levels of H2O2. Analysis of MMP-3 in the liver of these mice showed significant upregulation of MMP-3.

Summary: We here demonstrate in vitro and in vivo that non-toxic concentrations of H2O2 comparable to those released by inflammatory cells drastically induced MMP-3, a major fibrolytic player in tissue remodeling.