RESUMO
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.
Assuntos
Cafeína/farmacologia , Cirrose Hepática Experimental/prevenção & controle , Fígado/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ductos Biliares/cirurgia , Biomarcadores/metabolismo , Antígeno CD11b/metabolismo , Catalase/genética , Catalase/metabolismo , Colágeno Tipo I/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Citoproteção , Regulação da Expressão Gênica , Interleucina-1/genética , Interleucina-6/genética , Ligadura , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/etiologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição da Família Snail , Superóxido Dismutase/genética , Tioacetamida , Fator de Crescimento Transformador beta1/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Adenoviridae/genética , Vetores Genéticos/isolamento & purificação , Animais , Linhagem Celular , Centrifugação com Gradiente de Concentração , Césio , Cloretos , Técnicas de Transferência de Genes , Terapia Genética , Masculino , Ratos , EspectrofotometriaRESUMO
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.
Assuntos
Fibrinolisina/fisiologia , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Animais , Linhagem Celular , Colágeno Tipo I/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Proteínas do Tecido Nervoso/metabolismo , Ratos , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Ácido Tranexâmico/farmacologia , Fatores de Transcrição/metabolismo , Transdução Genética , Fator de Crescimento Transformador beta/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
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.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Células Estreladas do Fígado/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cirrose Hepática/prevenção & controle , Metaloproteinase 2 da Matriz/metabolismo , Proteínas Oncogênicas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Repressoras/genética , Adenoviridae/metabolismo , Animais , Peso Corporal , Células Cultivadas , Células Estreladas do Fígado/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Tioacetamida/farmacologia , Transdução Genética , Transgenes , Regulação para CimaRESUMO
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.