Oxidative stress plays a key role in liver fibrosis. Both inflammatory cells and activated Kupffer cells produce H₂O₂, an oxidant involved in the activation of hepatic stellate cells (HSC). Increased production of reactive oxygen intermediates (ROIs) in fibrotic livers is associated in part with the up–regulation of transforming growth factor β (TGF–β), and this cytokine enhances collagen production by cultured HSC. However, the possible link between oxidative stress and the molecular mechanisms by which TGF–β induces collagen gene expression in HSC remains to be elucidated. To address this question, we investigated whether H₂O₂ is a mediator of TGF–β–elicited α1(I) collagen gene (col1a1) up–regulation. We demonstrated that TGF–β induces the accumulation of H₂O₂, and that this oxidant is, in turn, directly involved in up–regulating the expression of the col1a1 gene. While the addition of H₂O₂ to HSC induced the expression of α1(I) procollagen mRNA, catalase, an H₂O₂ enzyme scavenger, abrogated TGF–β–mediated col1a1 gene up–regulation. We transfected HSC with chimeric plasmids driven by different segments of the mouse col1a1 promoter and mapped a cis–acting element (-370 to -344) essential for TGF–β responsiveness. We further showed that TGF–β induced the activation and binding of a C/EBPβ–containing transcriptional complex to this sequence, an effect that was also mimicked by the addition of H₂O₂. Taken together, these data demonstrate a direct connection between TGF–β–mediated accumulation of H₂O₂ and the up–regulation of col1a1 gene in HSC.

S-adenosylmethionine (AdoMet) is an essential compound in cellular transmethylation reactions and a precursor of polyamine and glutathione synthesis in the liver. In liver injury, the synthesis of AdoMet is impaired and its availability limited. AdoMet administration attenuates experimental liver damage, improves survival of alcoholic patients with cirrhosis, and prevents experimental hepatocarcinogenesis. Apoptosis contributes to different liver injuries, many of which are protected by AdoMet. The mechanism of AdoMet's hepatoprotective and chemopreventive effects are largely unknown. The effect of AdoMet on okadaic acid (OA)-induced apoptosis was evaluated using primary cultures of rat hepatocytes and human hepatoma cell lines. AdoMet protected rat hepatocytes from OA-induced apoptosis dose dependently. It attenuated mitochondrial cytochrome c release, caspase 3 activation, and poly(ADP-ribose) polymerase cleavage. These effects were independent from AdoMet-dependent glutathione synthesis, and mimicked by 5'-methylthioadenosine (MTA), which is derived from AdoMet. Interestingly, AdoMet and MTA did not protect HuH7 cells from OA-induced apoptosis; conversely both compounds behaved as proapoptotic agents. AdoMet's proapoptotic effect was dose dependent and observed also in HepG2 cells. In conclusion, AdoMet exerts opposing effects on apoptosis in normal versus transformed hepatocytes that could be mediated through its conversion to MTA. These effects may participate in the hepatoprotective and chemopreventive properties of this safe and well-tolerated drug.

—It has been suggested that left ventricular fibrosis in spontaneously hypertensive rats (SHR) is the result of both exaggerated collagen synthesis and insufficient collagen degradation. We have shown previously that chronic treatment with the angiotensin II type 1 receptor antagonist losartan results in diminished synthesis of collagen type I molecules and reversal of myocardial fibrosis in SHR. This study was designed to investigate whether losartan also affects the extracellular degradation of collagen type I fibers in the left ventricle of SHR. The study was performed in 30-week-old normotensive Wistar-Kyoto rats (WKY), untreated SHR, and SHR treated with orally administered losartan (20 mg/kg per day) for 14 weeks before they were killed. Ventricular collagenase activity was determined by degradation of [ ¹⁴ C]collagen with tissue extracts. Ventricular expression of tissue inhibitor of metalloproteinases 1 (TIMP-1) mRNA was analyzed by Northern blot. A histomorphometric study of the left ventricle was performed in all rats. Compared with WKY, SHR exhibited left ventricular hypertrophy, increased ( P <0.05) blood pressure, left ventricular collagen volume fraction and TIMP-1 mRNA, and diminished ( P <0.05) collagenase activity. After the treatment period, blood pressure was higher ( P <0.05) in losartan-treated SHR than in WKY, and no significant differences were noted in the remaining parameters between the 2 strains of rats. Compared with untreated SHR, treated SHR showed no left ventricular hypertrophy, diminished ( P <0.05) blood pressure, left ventricular collagen volume fraction and TIMP-1 mRNA, and increased ( P <0.05) collagenase activity. These results suggest that the transcription of the TIMP-1 gene is upregulated in the hypertrophied and fibrotic left ventricle of adult SHR. Upregulation of TIMP-1 may account for diminished collagenase activity in the myocardium of those rats. Chronic angiotensin II type 1 receptor blockade with losartan resulted in inhibition of TIMP-1 expression and stimulation of collagenase activity in the left ventricle of SHR. It is proposed that angiotensin II may facilitate myocardial fibrosis in SHR by depressing the collagenase-mediated extracellular degradation of collagen fibers.

Previous studies have shown that as well as left ventricular hypertrophy, myocardial fibrosis develops early in rats with spontaneous hypertension (SHR). The present study was designed to investigate whether chronic treatment with the angiotensin II type 1 (AT1) receptor antagonist losartan modifies collagen type I metabolism and reverses left ventricular fibrosis in young SHR with left ventricular hypertrophy. The study was performed in 30-week-old normotensive Wistar–Kyoto (WKY) rats, untreated SHR and SHR treated with losartan (20 mg/kg per day, orally) for 14 weeks before they were killed. Ventricular pro-α1(I) collagen messenger RNA was analyzed by Northern blot. Serum levels of the carboxy-terminal propeptide of procollagen type I (PIP) and the pyridoline cross-linked telopeptide domain of collagen type I (CITP) were determined by specific radioimmunoassays as markers of collagen type I synthesis and degradation, respectively. Collagen volume fraction was determined in the left ventricle by quantitative morphometry. Compared with WKY rats, SHR exhibited increased (P < 0.05) mean arterial pressure, pro-α1(I) collagen messenger RNA, PIP and left ventricular collagen volume fraction, and similar CITP values. After the treatment period, mean arterial pressure was higher (P < 0.05) in losartan-treated SHR than in WKY rats. Compared with untreated SHR, treated SHR showed no left ventricular hypertrophy and diminished (P < 0.05) values of mean arterial pressure, PIP and left ventricular collagen volume fraction. No changes in pro-α1(I) collagen messenger RNA and CITP values were observed with treatment in SHR. No significant differences in the left ventricular collagen volume fraction were observed between treated SHR with normal blood pressure and treated SHR with abnormally high blood pressure at the end of the treatment period. These results suggest that chronic AT1 blockade with losartan decreases the post-transcriptional synthesis of fibril-forming collagen type I molecules in young SHR. This effect may be involved in the ability of this drug to reverse left ventricular fibrosis in young rats with genetic hypertension. Apart from its antihypertensive action, other mechanisms may mediate the antifibrotic effect of losartan in this animal model.