Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis

Background & Aims: There is a significant relationship between inheritance of high transforming growth factor (TGF)-beta1 and angiotensinogen-producing genotypes and the development of progressive hepatic fibrosis in patients with chronic hepatitis C. In cardiac and renal fibrosis, TGF-beta1 production may be enhanced by angiotensin II, the principal effector molecule of the renin-angiotensin system. The aim of the present study was to determine the effects of the angiotensin converting enzyme inhibitor, captopril, on the progression of hepatic fibrosis in the rat bile duct ligation model. Methods: Rats were treated with captopril (100 mg kg(-1) day(-1)) commencing 1 or 2 weeks after bile duct ligation. Animals with bile duct ligation only and sham-operated animals sewed as controls. Four weeks after bile duct ligation, indices of fibrosis were assessed. Results: Cap topril treatment significantly reduced hepatic hydroxyproline levels, mean fibrosis score, steady state messenger RNA levels of TGF-beta1 and procollagen alpha1(I), and matrix metalloproteinase 2 and 9 activity. Conclusions: Captopril significantly attenuates the progression of hepatic fibrosis in the vat bile duct ligation model, and its effectiveness should be studied in human chronic liver diseases associated with progressive fibrosis.

Involvement of a transforming-growth-factor-beta-like molecule in tumor-cell-derived inhibition of nitric-oxide synthesis in cerebral endothelial cells.

Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor cells. We have reported that EC219 cells, a rat-brain-microvessel-derived endothelial cell line, produced NO through cytokine-inducible NO synthase (iNOS), the induction of which was significantly decreased by (a) soluble factor(s) secreted by DHD/PROb, an invasive sub-clone of a rat colon-carcinoma cell line. In this study, the DHD/PROb cell-derived NO-inhibitory factor was characterized. Northern-blot analysis demonstrated that the induction of iNOS mRNA in cytokine-activated EC219 cells was decreased by PROb-cell-conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A-Sepharose or heparin-Sepharose, the NO-inhibitory activity was found only in Con A-unbound or heparin-unbound fractions, respectively, indicating that the PROb-derived inhibitory factor was likely to be a non-glycosylated and non-heparin-binding molecule. Pre-incubation of DHD/PROb-cell supernatant with anti-TGF-beta neutralizing antibody completely blocked the DHD/PROb-derived inhibition of NO production by EC219 cells. Addition of exogenous TGF-beta 1 dose-dependently inhibited NO release by EC219 cells. The presence of active TGF-beta in the DHD/PROb cell supernatant was demonstrated using a growth-inhibition assay. Moreover, heat treatment of medium conditioned by the less invasive DHD/REGb cells, which constitutively secreted very low levels of active TGF-beta, increased both TGF-beta activity and the ability to inhibit NO production in EC219 cells. Thus, DHD/PROb colon-carcinoma cells inhibited NO production in EC219 cells by secreting a factor identical or very similar to TGF-beta.

Tratamiento de Artritis reumatoide con antagonistas del factor de necrosis tumoral alfa y su asociación con el desarrollo de melanoma cutáneo: revisión sistemática de la literatura y meta-análisis.

Introducción: El tratamiento con antagonistas del factor de necrosis tumoral alfa (anti TNF) ha impactado el pronóstico y la calidad de vida de los pacientes con artritis reumatoide (AR) positivamente, sin embargo, se interroga un incremento en el riesgo de desarrollar melanoma. Objetivo: Conocer la asociación entre el uso de anti TNF y el desarrollo de melanoma maligno en pacientes con AR. Metodología: Se realizó una búsqueda sistemática en MEDLINE, EMBASE, COCHRANE LIBRARY y LILACS para ensayos clínicos, estudios observacionales, revisiones y meta-análisis en pacientes adultos con diagnóstico de AR y manejo con anti TNF (Certolizumab pegol, Adalimumab, Etanercept, Infliximab y Golimumab). Resultados: 37 estudios clínicos cumplieron los criterios de inclusión para el meta-análisis, con una población de 16567 pacientes. El análisis de heterogeneidad no fue significativo (p=1), no se encontró diferencia en el riesgo entre los grupos comparados DR -0.00 (IC 95% -0.001; -0.001). Un análisis adicional de los estudios en los que se reportó al menos 1 caso de melanoma (4222 pacientes) tampoco mostró diferencia en el riesgo DR -0.00 (IC 95% -0.004 ; -0.003). Conclusión: En la evidencia disponible a la fecha no encontramos asociación significativa entre el tratamiento con anti TNF en pacientes con diagnóstico de AR y el desarrollo de melanoma cutáneo.

Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway

Giachini FR, Zemse SM, Carneiro FS, Lima VV, Carneiro ZN, Callera GE, Ergul A, Webb RC, Tostes RC. Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway. Am J Physiol Heart Circ Physiol 296: H489-H496, 2009. First published December 12, 2008; doi:10.1152/ajpheart.00251.2008.-Interleukin-10 (IL-10) is an anti-inflammatory cytokine with protective actions on the vasculature. On the other hand, endothelin ( ET)-1 has potent vasoconstrictor, mitogenic, and proinflammatory activities, which have been implicated in the pathophysiology of a number of cardiovascular diseases. We hypothesized that, in a condition where ET-1 expression is upregulated, i.e., on infusion of TNF-alpha, IL-10 confers vascular protection from ET-1-induced injury. Aortic rings and first-order mesenteric arteries from male C57BL/6 (WT) and IL-10-knockout (IL-10(-/-)) mice were treated with human recombinant TNF-alpha (220 ng.kg(-1).day(-1)) or vehicle (saline) for 14 days. TNF-alpha infusion significantly increased blood pressure in IL-10(-/-), but not WT, mice. TNF-alpha augmented vascular ET-1 mRNA expression in arteries from WT and IL-10(-/-) mice. ET type A (ETA) receptor expression was increased in arteries from IL-10(-/-) mice, and TNF-alpha infusion did not change vascular ETA receptor expression in control or IL-10(-/-) mice. Aorta and mesenteric arteries from TNF-alpha-infused IL-10(-/-) mice displayed increased contractile responses to ET-1, but not the ET type B receptor agonist IRL-1620. The ETA receptor antagonist atrasentan completely abolished responses to ET-1 in aorta and mesenteric vessels, whereas the ERK1/2 inhibitor PD-98059 abrogated increased contractions to ET-1 in arteries from TNF-alpha-infused IL-10(-/-) mice. Infusion of TNF-alpha, as well as knockdown of IL-10 (IL-10(-/-)), induced an increase in total and phosphorylated ERK1/2. These data demonstrate that IL-10 counteracts ET(A)-mediated vascular responses to ET-1, as well as activation of the ERK1/2 pathway.

Effect of nωnla, an inhibitor of No-synthase, on the release of tissue-plasminogem activator from rat aorta

Alterations in the synthesis or enhanced inactivation of nitric oxide (NO) and increase in fibrin deposition in the vascular bed lead to an imbalance that can induced intravascular coagulation. NO is produced through L-arginine pathway by constitutive and inducible nitric oxide synthase (NOS). The inducible isoform can be activated by cytokines such as tumor necrosis factor alfa. We evaluated NO-induced tissue-plasminogen activator (t-PA) release from isolated aortic segments of Wistar rats measuring the fibrinolytic activity in the fibrin plate. Inhibition of NO biossynthesis with Nω-nitro-L-arginine (NωNLA) significantly attenuated the fibrinolytic activity (FA) evoked by aortic segments of this group (GII) compared to the saline group (GI). The administration of L-arginine produced restoration of FA in this group (GIII) treated with NωNLA suggesting that t-PA arising from segments of rat aorta is influenced by NO.

Natural history of markers of collagen turnover in patients with early diastolic dysfunction and impact of eplerenone

OBJECTIVES: This study was designed to evaluate the impact of eplerenone on collagen turnover in preserved systolic function heart failure (HFPSF).

BACKGROUND: Despite growing interest in abnormal collagen metabolism as a feature of HFPSF with diastolic dysfunction, the natural history of markers of collagen turnover and the impact of selective aldosterone antagonism on this natural history remains unknown.

METHODS: We evaluated 44 patients with HFPSF, randomly assigned to control (n = 20) or eplerenone 25 mg daily (n = 24) for 6 months, increased to 50 mg daily from 6 to 12 months. Serum markers of collagen turnover and inflammation were analyzed at baseline and at 6 and 12 months and included pro-collagen type-I and -III aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha. Doppler-echocardiographic assessment of diastolic filling indexes and tissue Doppler analyses were also obtained.

RESULTS: The mean age of the patients was 80 +/- 7.8 years; 46% were male; 64% were receiving an angiotensin-converting enzyme inhibitor, 34% an angiotensin-II receptor blocker, and 68% were receiving beta-blocker therapy. Pro-collagen type-III and -I aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha increased with time in the control group. Eplerenone treatment had no significant impact on any biomarker at 6 months but attenuated the increase in pro-collagen type-III aminoterminal peptide at 12 months (p = 0.006). Eplerenone therapy was associated with modest effects on diastolic function without any impact on clinical variables or brain natriuretic peptide.

CONCLUSIONS: This study demonstrates progressive increases in markers of collagen turnover and inflammation in HFPSF with diastolic dysfunction. Despite high background utilization of renin-angiotensin-aldosterone modulators, eplerenone therapy prevents a progressive increase in pro-collagen type-III aminoterminal peptide and may have a role in management of this disease. (The Effect of Eplerenone and Atorvastatin on Markers of Collagen Turnover in Diastolic Heart Failure; NCT00505336).

Effects of glutamine on the nuclear factor-kappaB signaling pathway of murine peritoneal macrophages

The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappaB (NF-kappa B) signaling pathway of murine peritoneal macrophages. Since glutamine is essential for the normal functioning of macrophages, it was hypothesized that in vitro glutamine supplementation would increase NF-kappa B activation. Peritoneal macrophages were pretreated with glutamine (0, 0.6, 2 and 10 mM) before incubation with lipopolysaccharide (LPS), and the effects of glutamine on the production of tumor necrosis factor-alpha and on the expression and activity of proteins involved in the NF-kappa B signaling pathway were studied by an enzyme linked immuno-sorbent assay, Western blotting, and an electrophoretic mobility shift assay. Glutamine treatment (2 and 10 mM) increased the activation of NF-kappa B in LPS-stimulated peritoneal macrophages (P < 0.05). In non-stimulated cells, glutamine treatment (2 and 10 mM) significantly reduced I kappa B-alpha protein expression (P < 0.05). Glutamine modulates NF-kappa B signaling pathway by reducing the level of I kappa B-alpha, leading to an increase in NF-kappa B within the nucleus in peritoneal macrophages.

Increased activities of cardiac matrix metalloproteinases matrix metalloproteinase (MMP)-2 and MMP-9 are associated with mortality during the acute phase of experimental Trypanosoma cruzi infection

The strong inflammatory reaction that occurs in the heart during the acute phase of Trypanosoma cruzi infection is modulated by cytokines and chemokines produced by leukocytes and cardiomyocytes. Matrix metalloproteinases (MMPs) have recently emerged as modulators of cardiovascular inflammation. In the present study we investigated the role of MMP-2 and MMP-9 in T. cruzi-induced myocarditis, by use of immunohistochemical analysis, gelatin zymography, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction to analyze the cardiac tissues of T. cruzi-infected C57BL/6 mice. Increased transcripts levels, immunoreactivity, and enzymatic activity for MMP-2 and MMP-9 were observed by day 14 after infection. Mice treated with an MMP inhibitor showed significantly decreased heart inflammation, delayed peak in parasitemia, and improved survival rates, compared with the control group. Reduced levels of cardiac tumor necrosis factor-alpha, interferon-gamma, serum nitrite, and serum nitrate were also observed in the treated group. These results suggest an important role for MMPs in the induction of T. cruzi-induced acute myocarditis.

Role of cytokines (TNF-alpha, IL-1 beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide

Introduction Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. Purpose Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11. Materials and methods Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-alpha and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and KC ELISA. Results CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-alpha, IL-1 beta and KC level and TNF-alpha immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-alpha, IL-1 beta and KC and TNF-alpha immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-alpha tissue level and TNF-alpha immuno-staining, but did not reduce the severity of diarrhea. Conclusion These results suggest an important role of TNF-alpha, IL-1 beta and KC in the pathogenesis of intestinal mucositis induced by CPT-11.

Pharmacokinetics and posterior segment biodistribution of ESBA105, an anti-TNF-alpha single-chain antibody, upon topical administration to the rabbit eye.

PURPOSE: The purpose of this study was to characterize local distribution and systemic absorption of the tumor necrosis factor (TNF)-alpha inhibitory single-chain antibody fragment (scFv) ESBA105 following topical administration to the eye in vivo. METHODS: Rabbits received ESBA105 as topical eye drops in two dosing regimens. First, pharmacokinetics after the topical route of administration was compared to the intravenous (i.v.) route by means of applying the identical cumulative daily dose of ESBA105. In a second study rabbits received five eye drops daily for six consecutive days in a lower frequency topical dosing regimen. Kinetics and biodistribution of ESBA105 in ocular tissues and fluids as well as in sera were determined in all animals. RESULTS: After topical administration to the eye, ESBA105 quickly reaches therapeutic concentrations in all ocular compartments. Systemic exposure after topical administration is 25,000-fold lower than exposure after i.v. injection of the identical cumulative daily dose. ESBA105 levels in vitreous humor and neuroretina are significantly higher on topical administration than after i.v. injection. Absolute and relative intraocular biodistribution of ESBA105 is different with topical and systemic delivery routes. Compared to its terminal half-life in circulation (7 hours), the vitreal half-life of ESBA105 is significantly enhanced (16-24 hours). CONCLUSIONS: On topical administration, ESBA105 is efficiently absorbed and distributed to all compartments of the eye, whereby systemic drug exposure is very low. Based on its unique intraocular biodistribution and pharmacokinetics and the absolute intraocular levels reached, topical ESBA105 appears highly attractive for treatment of various ophthalmological disorders.

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha.

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis.

Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand- and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a Glu --&gt; Ala mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases.

Oxidants positively or negatively regulate nuclear factor kappaB in a context-dependent manner.

Redox-based mechanisms play critical roles in the regulation of multiple cellular functions. NF-kappaB, a master regulator of inflammation, is an inducible transcription factor generally considered to be redox-sensitive, but the modes of interactions between oxidant stress and NF-kappaB are incompletely defined. Here, we show that oxidants can either amplify or suppress NF-kappaB activation in vitro by interfering both with positive and negative signals in the NF-kappaB pathway. NF-kappaB activation was evaluated in lung A549 epithelial cells stimulated with tumor necrosis factor alpha (TNFalpha), either alone or in combination with various oxidant species, including hydrogen peroxide or peroxynitrite. Exposure to oxidants after TNFalpha stimulation produced a robust and long lasting hyperactivation of NF-kappaB by preventing resynthesis of the NF-kappaB inhibitor IkappaB, thereby abrogating the major negative feedback loop of NF-kappaB. This effect was related to continuous activation of inhibitor of kappaB kinase (IKK), due to persistent IKK phosphorylation consecutive to oxidant-mediated inactivation of protein phosphatase 2A. In contrast, exposure to oxidants before TNFalpha stimulation impaired IKK phosphorylation and activation, leading to complete prevention of NF-kappaB activation. Comparable effects were obtained when interleukin-1beta was used instead of TNFalpha as the NF-kappaB activator. This study demonstrates that the influence of oxidants on NF-kappaB is entirely context-dependent, and that the final outcome (activation versus inhibition) depends on a balanced inhibition of protein phosphatase 2A and IKK by oxidant species. Our findings provide a new conceptual framework to understand the role of oxidant stress during inflammatory processes.

TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

A secreted high-affinity inhibitor of human TNF from Tanapox virus.

A class of secreted poxvirus tumor necrosis factor (TNF)-binding proteins has been isolated from Tanapox-infected cell supernatants. The inhibitor bound to a TNF-affinity column and was identified as the product of the 2L gene. Sequence analysis of 2L family members from other yatapoxviruses and swinepox virus yielded no sequence homology to any known cellular gene. The expressed Tanapox virus 2L protein bound to human TNF with high affinity (K(d) = 43 pM) and exhibits an unusually slow off-rate. However, 2L is unable to bind to a wide range of human TNF family members. The 2L protein can inhibit human TNF from binding to TNF receptors I and II as well as block TNF-induced cytolysis. Thus, Tanapox virus 2L represents an inhibitor of human TNF and offers a unique strategy with which to modulate TNF activity.