Nrf2 and Snail-1 in the prevention of experimental liver fibrosis by caffeine.

AIM: To determine the molecular mechanisms involved in experimental hepatic fibrosis prevention by caffeine (CFA). METHODS: Liver fibrosis was induced in Wistar rats by intraperitoneal thioacetamide or bile duct ligation and they were concomitantly treated with CFA (15 mg/kg per day). Fibrosis and inflammatory cell infiltrate were evaluated and classified by Knodell index. Inflammatory infiltrate was quantified by immunohistochemistry (anti-CD11b). Gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction for collagen I (Col-1), connective tissue growth factor (CTGF), transforming growth factor ß1 (TGF-ß1), tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, superoxide dismutase (SOD) and catalase (CAT). Activation of Nrf2 and Snail-1 was analyzed by Western-blot. TNF-α expression was proved by enzyme-linked immunosorbant assay, CAT activity was performed by zymography. RESULTS: CFA treatment diminished fibrosis index in treated animals. The Knodell index showed both lower fibrosis and necroinflammation. Expression of profibrogenic genes CTGF, Col-1 and TGF-ß1 and proinflammatory genes TNF-α, IL-6 and IL-1 was substantially diminished with CFA treatment with less CD11b positive areas. Significantly lower values of transcriptional factor Snail-1 were detected in CFA treated rats compared with cirrhotic rats without treatment; in contrast Nrf2 was increased in the presence of CFA. Expression of SOD and CAT was greater in animals treated with CFA showing a strong correlation between mRNA expression and enzyme activity. CONCLUSION: Our results suggest that CFA inhibits the transcriptional factor Snail-1, down-regulating profibrogenic genes, and activates Nrf2 inducing antioxidant enzymes system, preventing inflammation and fibrosis.

Genotoxic evaluation of pirfenidone using erythrocyte rodent micronucleus assay.

Pirfenidone is a non-steroidal antifibrotic compound that has been proposed in clinical protocols and experimental studies as a pharmacological treatment for fibroproliferative diseases. The objective of this study was to determine the genotoxicity or cytotoxicity of three doses of pirfenidone using the micronuclei test in peripheral blood erythrocytes of rodent models. Pirfenidone was administered orally to Balb-C mice for 3 days, and also was administered topically to hairless Sprague Dawley rats during the final stage of gestation. Mice were sampled every 24 h over the course of 6 days; pregnant rats were sampled every 24 h during the last 6 days of gestation, and pups were sampled at birth. Blood smears were analyzed and the frequencies of micronucleated erythrocytes (MNEs), micronucleated polychromatic erythrocytes (MNPCEs), and the proportion of polychromatic erythrocytes (PCEs), were recorded in samples from mice, pregnant rats and rat neonates. Increases in MN frequencies (p<0.03) were noted only in the positive control groups. No genotoxic effects or decreased PCE values were observed neither in newborn rats transplacentally exposed to pirfenidone, or in two adult rodent models when pirfenidone was administered orally or topically.

Pirfenidone restricts Th2 differentiation in vitro and limits Th2 response in experimental liver fibrosis.

Polarized T helper type 2 (Th2) response is linked with fibrosis. Here, we evaluated the effect of the anti-fibrotic agent pirfenidone on Th type 1 (Th1) and Th2 responses. For in vivo testing; Wistar rats were made cirrhotic by intraperitoneal administration of thioacetamide. Once hepatic damage was established, pirfenidone was administered intragastrically on a daily basis during three weeks. Gene expression of Th marks was evaluated by RT-PCR and Western blot assays from liver homogenates. Pirfenidone therapy induced down-regulation of Th2 transcripts and proteins (GATA3 and IL-4), without affecting significantly Th1 genes expression (T-bet and IFN-γ). We found that the activated form of p38 MAPK (identified by Western blot) was reduced by pirfenidone treatment, which is consistent with the anti-Th2 activity observed. Pirfenidone reduced GATA3 nuclear localization without modifying its DNA binding activity (evaluated by electrophoretic mobility shift assay). For in vitro testing; human naive CD4+ T cells were cultured in either Th1 or Th2 polarizing conditions in the presence of pirfenidone and flow cytometric analysis of intracellular synthesis of IFN-γ and IL-4 was conducted. Pirfenidone impaired development of Th2 subpopulation. In conclusion, pirfenidone is capable of impairing Th2 differentiation and limits Th2 profibrogenic response. The mechanism involves p38 inhibition and regulation of GATA3 expression and translocation.

Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.

Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdßGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹4 VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl4-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdßGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture.

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

BACKGROUND: Plasmin role in transforming growth factor-beta (TGF-beta)-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 x 10(9) 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-beta 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 [alpha1(I)collagen 66%, TIMP-1 59%, alpha-smooth muscle actin 90% and TGF-beta 55%]. Interestingly, both SnoN gene and protein expression increased prominently. Plasmin inhibition by TA upregulated the profibrogenic genes, which respond to TGF-beta-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-beta-responding genes. In particular, regulation of TGF-beta-co-repressor (SnoN) is greatly affected, which suggests SnoN as a cardinal player in cholestasis-induced fibrogenesis.

Adenoviral delivery of dominant-negative transforming growth factor beta type II receptor up-regulates transcriptional repressor SKI-like oncogene, decreases matrix metalloproteinase 2 in hepatic stellate cell and prevents liver fibrosis in rats.

BACKGROUND: Dominant-negative transforming growth factor beta type II receptor (TbetaRIIDeltacyt) is a protein that blocks transforming growth factor (TGF-beta) signaling. Because the consequences of blocking TGF-beta have not been completely elucidated in liver fibrosis, we analysed the effects of adenoviral delivery of TbetaRIIDeltacyt on profibrogenic genes and matrix metalloproteinase (MMP) proteins, as well as on TGF-beta signal repressor SKI-like oncogene (SnoN), in cultured hepatic stellate cells (HSCs) and in a rat model of liver fibrosis. METHODS: To induce liver fibrosis, rats were treated with thioacetamide for 7 weeks and administrated once with Ad-TbetaRIIDeltacyt or Ad-betagal through the iliac vein. Fibrosis was measured by morphometric analysis. We evaluated SnoN by western blot, immunocytochemistry and immunohistochemistry; MMP activity was determined by zymography and profibrogenic gene expression by the real-time reverse transcriptase-polymerase chain reaction in cultured HSCs and liver tissue. RESULTS: Profibrogenic gene expression of collagen alpha1 (I), TGF-beta1, platelet-derived growth factor-B, plasminogen activator inhibitor (PAI)-1, tissue inhibitor of matrix metalloproteinase-1 and MMP-2 was down-regulated; whereas MMP-3 was over-expressed in response to Ad-TbetaRIIDeltacyt in HSCs. Moreover, zymography assays corroborated MMP-2 and MMP-3 changes in activity. Surprisingly, anti-TGF-beta molecular intervention increased nuclear SnoN in HSCs. In vivo, Ad-TbetaRIIDeltacyt reduced liver fibrosis, increased nuclear SnoN in sinusoidal cells, and also produced significant suppression in collagen alpha1 (I), TGF-beta1, PAI-1, MMP-2 and over-expression in MMP-3 in thioacetamide-intoxicated animals. CONCLUSIONS: The results obtained in the present study suggest that the molecular mechanism for the blocking effects of Ad-TbetaRIIDeltacyt in TGF-beta signaling acts via up-regulation of the transcriptional repressor SnoN, which antagonizes TGF-beta signaling (TGF-beta/Smad-pathway inhibitor). Consequently, profibrogenic genes are down-regulated.

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-beta1 (TGF-beta1), 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-beta1, PAI-1 and TIMP-1 were downregulated by Adhu-PA action as well.