Urokinase plasminogen activator stimulates function of active forms of stromelysin and gelatinases (MMP-2 and MMP-9) in cirrhotic tissue

Background: The authors' previous data support the notion that adenoviral-driven urokinase plasminogen activator (u-PA) expression results in reversion of experimental liver cirrhosis. The specific aim of the present study was to decipher the mechanisms involved in the regulation by endogenous/gene-delivered u-PA of matrix metalloproteinases (MMP) and related proteins engaged in degradation of excessive hepatic connective tissue. Methods: Tissue slices from cirrhotic rat livers were incubated with u-PA-rich supernatants from 24-h-cultured hepatic stellate cells (HSC). Matrix metalloproteinase-2, -9 and tissue inhibitor of metalloproteinases-1 (TIMP-1) were detected by western blot and biologic activity. The HSC that discontinued u-PA production were transfected with the adenovector Adu-PA and serum-free supernatants evaluated for proteolytic activity by MMP-3, MMP-2 and MMP-9. Collagen I, transforming growth factor-?1 (TGF-?1), plasminogen activator inhibitor-1 (PAI-1) and TIMP-1 mRNA levels were also evaluated. Results and Conclusion: Endogenous u-PA from cultured HSC significantly induced the active forms of MMP-2 (68 kDa) and MMP-9 (78 kDa) in cirrhotic tissue slices. The TIMP-1 molecular forms demonstrated that u-PA pushed the presence of 'free' TIMP-1 (not complexed with MMP; 71%) in cirrhotic tissue. When non-producing u-PA-HSC were transfected with adenoviral vector coding for the functional human protein u-PA (Adhu-PA), an overactivation of MMP-3, MMP-2 and MMP-9 (800%, 48% and 100%, respectively) was found as compared with HSC transfected with control adenovirus encoding green fluorescent protein (Ad-GFP). Finally, gene expression of collagen I, TGF-?1, PAI-1 and TIMP-1 were downregulated by Adhu-PA action as well

Plasmin plays a key role in the regulation of profibrogenic molecules in hepatic stellate cells

Background: Plasmin role in transforming growth factor-β (TGF-β)-responsive gene regulation remains to be elucidated. Also, plasmin action on co-repressor Ski-related novel protein N (SnoN) and differential activation of matrix metalloproteinases (MMPs) are unknown. Thus, the role of plasmin on profibrogenic molecule expression, SnoN transcriptional kinetics and gelatinase activation was investigated. Methods:: Hepatic stellate cells (HSC) were transduced with adenovirus-mediated human urokinase plasminogen activator (Ad-huPA) (4 × 109 viral particles/ml). Overexpression of urokinase plasminogen activator and therefore of plasmin, was blocked by tranexamic acid (TA) in transduced HSC. Gene expression was monitored by reverse transcriptase polymerase chain reaction. HSC-free supernatants were used to evaluate MMP-2 and MMP-9 by zymography. SnoN, TGF-β and tissue inhibitor of metalloproteinase (TIMP)-1 were analysed by Western blot. Plasmin and SnoN expression kinetics were evaluated in bile duct-ligated (BDL) rats. Results:: Plasmin overexpression in Ad-huPA-transduced HSC significantly decreased gene expression of profibrogenic molecules [α1(I)collagen 66%, TIMP-1 59%, α-smooth muscle actin 90% and TGF-β 55%]. Interestingly, both SnoN gene and protein expression increased prominently. Plasmin inhibition by TA upregulated the profibrogenic genes, which respond to TGF-β-intracellular signalling. In contrast, SnoN mRNA and protein dropped importantly. Plasmin-activated MMP-9 and MMP-2 in HSC supernatants. Taken together, these findings indicate that MMP-9 activation is totally plasmin dependent. SnoN levels significantly decreased in cholestatic-BDL rats (82%) as compared with control animals. Interestingly, hepatic plasmin levels dropped 46% in BDL rats as compared with control. Conclusion:: Plasmin plays a key role in regulating TGF-β-responding genes. In particular, regulation of TGF-β-co-repressor (SnoN) is greatly affected, which suggests SnoN as a cardinal player in cholestasis-induced fibrogenesis. © 2009 John Wiley & Sons A/S

Effect of short-term carbon tetrachloride administration on blood lactic acid levels

1. 1. A short-term CCl4 administration was used in vivo as a model to produce a rise in lactic acid levels and to explain the probable interaction of CCl4 and lactic acid elevation with hepatic fibrogenesis. 2. 2. A single dose of CCl4 produced an increase in lactic acid levels from 16.6Zapotitlán3.57 to 24.2Zapotitlán4.2mg/dl. Three consecutive doses produced an elevation to 33.28Zapotitlán10.07 mg/dl, thus describing a direct relationship between lactic acid levels and CCl4 administration in a short-term fashion. 3. 3. A morphological evaluation was performed to show hepatic changes caused by CCl4 administration. No clue of fibrogenesis was found. However, we conclude that an elevation in lactic acid exists, prior to cirrhosis. Therefore, chronic presence of lactic acid may lead to cirrhosis. Zapotitlán 1993