Reactive Nitrogen Species Switch on Early Extracellular Matrix Remodeling via Induction of MMP1 and TNFα

Background & Aims: Liver injury leads to generation of reactive oxygen and nitrogen species, which can react to produce peroxynitrite (ONOO^-). We investigated whether ONOO^- and its metabolites modulate extracellular matrix remodeling. Methods: Stellate cells (HSC) were incubated with pure ONOO^- or SIN-1 (a ONOO^- donor). Western blot, nuclear in vitro transcription, Northern blot, qPCR, and promoter transactivation analysis for COL1A1 and COL1A2 were carried out. Rats were fed alcohol or injected with CCl₄ to cause alcohol-induced liver injury and an early fibrogenic response. Results: HSC incubated with ONOO^- or SIN-1 showed similar viability, proliferation, COL1A1 and COL1A2 transcription rates, and mRNA levels as controls. There was a time- and dose-dependent down-regulation of collagen I and α-Sma proteins and up-regulation of MMP1 and TNFα, indicating decreased HSC activation. These effects were blocked by ONOO^- scavengers. SIN-1 or ONOO^- increased nitrosylation of MMP1/MMP13 and transactivation of the MMP1, MMP13, and TNFα promoters. A TNFα neutralizing antibody or GSH-ethyl ester blocked MMP1 promoter transactivation; whereas TNFα or l-buthionine sulfoximine, which depletes GSH, further enhanced it. Pretreatment with SIN-1 or ONOO^- reduced the TGFβ pro-fibrogenic response in HSC. In vivo experiments validated the protective role of ONOO^- on the early fibrogenic response. However, highly activated HSC, such as myofibroblasts and HSC from chronic alcohol-fed rats, were resistant to the anti-fibrogenic actions of ONOO^- due to higher levels of GSH, a ONOO^- scavenger, overproduction of pro-fibrogenic TGFβ, and reactive oxygen species. Conclusion: ONOO^- could induce a protective mechanism in HSC in early stages of liver injury.