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

Abstract

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 alfa-Sma proteins and up-regulation of MMP1 and TNF , 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 TNFalfa promoters. A TNFalfa neutralizing antibody or GSH-ethyl ester blocked MMP1 promoter transactivation; whereas TNFalfa or L-buthionine sulfoximine, which depletes GSH, further enhanced it. Pretreatment with SIN-1 or ONOO- reduced the TGF 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 TGF, and reactive oxygen species.

Conclusion: ONOO- could induce a protective mechanism in HSC in early stages of liver injury.