Polyethylene Glycol Preconditioning: An Effective Strategy to Prevent Liver Ischemia Reperfusion Injury

Hepatic ischemia reperfusion injury (IRI) is an inevitable clinical problem for liver surgery. Polyethylene glycols (PEGs) are water soluble nontoxic polymers that have proven their effectiveness in various in vivo and in vitro models of tissue injury. The present study aims to investigate whether the intravenous administration of a high molecular weight PEG of 35 kDa (PEG 35) could be an effective strategy for rat liver preconditioning against IRI. PEG 35 was intravenously administered at 2 and 10 mg/kg to male Sprague Dawley rats. Then, rats were subjected to one hour of partial ischemia (70%) followed by two hours of reperfusion. The results demonstrated that PEG 35 injected intravenously at 10 mg/kg protected efficiently rat liver against the deleterious effects of IRI. This was evidenced by the significant decrease in transaminases levels and the better preservation of mitochondrial membrane polarization. Also, PEG 35 preserved hepatocyte morphology as reflected by an increased F-actin/G-actin ratio and confocal microscopy findings. In addition, PEG 35 protective mechanisms were correlated with the activation of the prosurvival kinase Akt and the cytoprotective factor AMPK and the inhibition of apoptosis. Thus, PEG may become a suitable agent to attempt pharmacological preconditioning against hepatic IRI.The work was financed by the Fondo de Investigaciones Sanitarias (FIS PI12/00519; FIS PI15/00110), Spain. Eirini Pantazi is fellowship-holder of AGAUR (2012FI B00382), Generalitat de Catalunya, Barcelona, Spain.Peer Reviewe

Machine learning based prediction of soil total nitrogen, organic carbon and moisture content by using VIS-NIR spectroscopy

It is widely known that the visible and near infrared (VIS-NIR) spectroscopy has the potential of estimating soil total nitrogen (TN), organic carbon (OC) and moisture content (MC) due to the direct spectral responses these properties have in the near infrared (NIR) region. However, improving the prediction accuracy requires advanced modelling techniques, particularly when measurement is planned for fresh (wet and un-processed) soil samples. The aim of this work is to compare the predictive performance of two linear multivariate and two machine learning methods for TN, OC and MC. The two multivariate methods investigated included principal component regression (PCR) and partial least squares regression (PLSR), whereas the machine learning methods included least squares support vector machines (LS-SVM), and Cubist. A mobile, fibre type, VIS-NIR spectrophotometer was utilised to collect soil spectra (305–2200 nm) in diffuse reflectance mode from 140 wet soil samples collected from one field in Germany. The results indicate that machine learning methods are capable of tackling non-linear problems in the dataset. LS-SVMs and the Cubist method out-performed the linear multivariate methods for the prediction of all three soil properties studied. LS-SVM provided the best prediction for MC (root mean square error of prediction (RMSEP) = 0.457% and residual prediction deviation (RPD) = 2.24) and OC (RMSEP = 0.062% and RPD = 2.20), whereas the Cubist method provided the best prediction for TN (RMSEP = 0.071 and RPD = 1.96)

Losartan activates sirtuin 1 in rat reduced-size orthotopic liver transplantation

© 2015 Baishideng Publishing Group Inc. All rights reserved. Aim: To investigate a possible association between losartan and sirtuin 1 (SIRT1) in reduced-size orthotopic liver transplantation (ROLT) in rats. Methods: Livers of male Sprague-Dawley rats (200-250 g) were preserved in University of Wisconsin preservation solution for 1 h at 4°C prior to ROLT. In an additional group, an antagonist of angiotensin II type 1 receptor (AT1R), losartan, was orally administered (5 mg/kg) 24 h and 1 h before the surgical procedure to both the donors and the recipients. Transaminase (as an indicator of liver injury), SIRT1 activity, and nicotinamide adenine dinucleotide (NAD+, a co-factor necessary for SIRT1 activity) levels were determined by biochemical methods. Protein expression of SIRT1, acetylated FoxO1 (ac-FoxO1), NAMPT (the precursor of NAD+), heat shock proteins (HSP70, HO-1) expression, endoplasmic reticulum stress (GRP78, IRE1α, p-eIF2) and apoptosis (caspase 12 and caspase 3) parameters were determined by Western blot. Possible alterations in protein expression of mitogen activated protein kinases (MAPK), such as p-p38 and p-ERK, were also evaluated. Furthermore, the SIRT3 protein expression and mRNA levels were examined. Results: The present study demonstrated that losartan administration led to diminished liver injury when compared to ROLT group, as evidenced by the significant decreases in alanine aminotransferase (358.3 133.44 vs 206 33.61, P + (0.87 0.22 vs 1.195 0.144, P < 0.05) the co-factor necessary for SIRT1 activity, as well as with decreases in ac-FoxO1 expression. Losartan treatment also provoked significant attenuation of endoplasmic reticulum stress parameters (GRP78, IRE1α, p-eIF2) which was consistent with reduced levels of both caspase 12 and caspase 3. Furthermore, losartan administration stimulated HSP70 protein expression and attenuated HO-1 expression. However, no changes were observed in protein or mRNA expression of SIRT3. Finally, the protein expression pattern of p-ERK and p-p38 were not altered upon losartan administration. Conclusion: The present study reports that losartan induces SIRT1 expression and activity, and that it reduces hepatic injury in a ROLT model.Supported by Grants from Fondo de Investigaciones Sanitarias, No. FIS PI12/00519; fellowship from Agència de Gestió d’Ajuts Universitaris i de Recerca, No. 2012FI_B00382; Generalitat de Catalunya, Barcelona, Catalonia, Spain (to Pantazi E)Peer Reviewe

Losartan activates sirtuin 1 in rat reduced-size orthotopic liver transplantation

© 2015 Baishideng Publishing Group Inc. All rights reserved. Aim: To investigate a possible association between losartan and sirtuin 1 (SIRT1) in reduced-size orthotopic liver transplantation (ROLT) in rats. Methods: Livers of male Sprague-Dawley rats (200-250 g) were preserved in University of Wisconsin preservation solution for 1 h at 4°C prior to ROLT. In an additional group, an antagonist of angiotensin II type 1 receptor (AT1R), losartan, was orally administered (5 mg/kg) 24 h and 1 h before the surgical procedure to both the donors and the recipients. Transaminase (as an indicator of liver injury), SIRT1 activity, and nicotinamide adenine dinucleotide (NAD+, a co-factor necessary for SIRT1 activity) levels were determined by biochemical methods. Protein expression of SIRT1, acetylated FoxO1 (ac-FoxO1), NAMPT (the precursor of NAD+), heat shock proteins (HSP70, HO-1) expression, endoplasmic reticulum stress (GRP78, IRE1α, p-eIF2) and apoptosis (caspase 12 and caspase 3) parameters were determined by Western blot. Possible alterations in protein expression of mitogen activated protein kinases (MAPK), such as p-p38 and p-ERK, were also evaluated. Furthermore, the SIRT3 protein expression and mRNA levels were examined. Results: The present study demonstrated that losartan administration led to diminished liver injury when compared to ROLT group, as evidenced by the significant decreases in alanine aminotransferase (358.3 133.44 vs 206 33.61, P + (0.87 0.22 vs 1.195 0.144, P < 0.05) the co-factor necessary for SIRT1 activity, as well as with decreases in ac-FoxO1 expression. Losartan treatment also provoked significant attenuation of endoplasmic reticulum stress parameters (GRP78, IRE1α, p-eIF2) which was consistent with reduced levels of both caspase 12 and caspase 3. Furthermore, losartan administration stimulated HSP70 protein expression and attenuated HO-1 expression. However, no changes were observed in protein or mRNA expression of SIRT3. Finally, the protein expression pattern of p-ERK and p-p38 were not altered upon losartan administration. Conclusion: The present study reports that losartan induces SIRT1 expression and activity, and that it reduces hepatic injury in a ROLT model.Supported by Grants from Fondo de Investigaciones Sanitarias, No. FIS PI12/00519; fellowship from Agència de Gestió d’Ajuts Universitaris i de Recerca, No. 2012FI_B00382; Generalitat de Catalunya, Barcelona, Catalonia, Spain (to Pantazi E)Peer Reviewe

Protective Effect of Intravenous High Molecular Weight Polyethylene Glycol on Fatty Liver Preservation

Ischemia reperfusion injury (IRI) leads to significant tissue damage in liver surgery. Polyethylene glycols (PEGs) are water soluble nontoxic polymers that have proved their effectiveness against IRI. The objective of our study was to investigate the potential protective effects of intravenous administration of a high molecular weight PEG of 35 kDa (PEG 35) in steatotic livers subjected to cold ischemia reperfusion. In this study, we used isolated perfused rat liver model to assess the effects of PEG 35 intravenous administration after prolonged cold ischemia (24 h, 4°C) and after reperfusion (2 h, 37°C). Liver injury was measured by transaminases levels and mitochondrial damage was determined by confocal microscopy assessing mitochondrial polarization (after cold storage) and by measuring glutamate dehydrogenase activity (after reperfusion). Also, cell signaling pathways involved in the physiopathology of IRI were assessed by western blot technique. Our results show that intravenous administration of PEG 35 at 10 mg/kg ameliorated liver injury and protected the mitochondria. Moreover, PEG 35 administration induced a significant phosphorylation of prosurvival protein kinase B (Akt) and activation of cytoprotective factors e-NOS and AMPK. In conclusion, intravenous PEG 35 efficiently protects steatotic livers exposed to cold IRI.Eirini Pantazi thanks the fellowship from AGAUR (2012FI_B00382), Generalitat de Catalunya, Barcelona, Catalonia, Spain. The authors would like to thank the Fondo de Investigaciones Sanitarias FIS PI12/00519; FIS PI15/00110 for the economic support.Peer Reviewe

Sirtuin 1 in rat orthotopic liver transplantation: An IGL-1 preservation solution approach

© The Author(s) 2015. AIM: To investigate the possible involvement of Sirtuin 1 (SIRT1) in rat orthotopic liver transplantation (OLT), when Institute Georges Lopez 1 (IGL-1) preservation solution is enriched with trimetazidine (TMZ). METHODS: Male Sprague-Dawley rats were used as donors and recipients. Livers were stored in IGL-1 preservation solution for 8h at 4 °C, and then underwent OLT according to Kamada's cuff technique without arterialization. In another group, livers were stored in IGL-1 preservation solution supplemented with TMZ, at 10-6 mol/L, for 8 h at 4 °C and then underwent OLT. Rats were sacrificed 24 h after reperfusion, and liver and plasma samples were collected. Liver injury (transaminase levels), mitochondrial damage (glutamate dehydrogenase activity) oxidative stress (malondialdehyde levels), and nicotinamide adenine dinucleotide (NAD+), the co-factor necessary for SIRT1 activity, were determined by biochemical methods. SIRT1 and its substrates (ac-FoxO1, ac-p53), the precursor of NAD+, nicotinamide phosphoribosyltransferase (NAMPT), as well as the phosphorylation of adenosine monophosphate activated protein kinase (AMPK), p-mTOR, p-p70S6K (direct substrate of mTOR), autophagy parameters (beclin-1, LC3B) and MAP kinases (p-p38 and p-ERK) were determined by Western blot. RESULTS: Liver grafts preserved in IGL-1 solution enriched with TMZ presented reduced liver injury and mitochondrial damage compared with those preserved in IGL-1 solution alone. In addition, livers preserved in IGL-1 + TMZ presented reduced levels of oxidative stress. This was consistent with enhanced SIRT1 protein expression and elevated SIRT1 activity, as indicated by decreased acetylation of p53 and FoxO1. The elevated SIRT1 activity in presence of TMZ can be attributed to the enhanced NAMPT protein and NAD+/NADH levels. Up-regulation of SIRT1 was consistent with activation of AMPK and inhibition of phosphorylation of mTOR and its direct substrate (p-p70S6K). As a consequence, autophagy mediators (beclin-1 and LC3B) were overexpressed. Furthermore, MAP kinases were regulated in livers preserved with IGL-1 + TMZ, as they were characterized by enhanced p-ERK and decreased p-p38 protein expression. CONCLUSION: Our study shows that IGL-1 preservation solution enriched with TMZ protects liver grafts from the IRI associated with OLT, through SIRT1 up-regulation.Supported by Fondo de Investigaciones Sanitarias, No. FIS PI12/00519; and Eirini Pantazi is the recipient of a fellowship from AGAUR, No. 2012FI_B00382, Generalitat de Catalunya, Barcelona, Catalonia, Spain.Peer Reviewe

Cyclin-dependent kinase 9 as a potential target for anti-TNF resistant inflammatory bowel disease

BACKGROUND AND AIMS: Resistance to single cytokine blockade, namely anti-TNF therapy, is a growing concern for patients with inflammatory bowel disease (IBD). The transcription factor T-bet is a critical regulator of intestinal homeostasis, is genetically linked to mucosal inflammation and controls the expression of multiples genes such as the pro-inflammatory cytokines IFN-γ and TNF. Inhibiting T-bet may therefore offer a more attractive prospect for treating IBD but remains challenging to target therapeutically. In this study, we evaluate the effect of targeting the transactivation function of T-bet using inhibitors of P-TEFb (CDK9-cyclin T), a transcriptional elongation factor downstream of T-bet. METHODS: Using an adaptive immune-mediated colitis model, human colonic lymphocytes from IBD patients and multiple large clinical datasets, we investigate the effect of CDK9 inhibitors on cytokine production and gene expression in colonic CD4+ T cells and link these genetic modules to clinical response in patients with IBD. RESULTS: Systemic CDK9 inhibition led to histological improvement of immune-mediated colitis and was associated with targeted suppression of colonic CD4+ T cell-derived IFN-γ and IL-17A. In colonic lymphocytes from IBD patients, CDK9 inhibition potently repressed genes responsible for pro-inflammatory signalling, and in particular genes regulated by T-bet. Remarkably, CDK9 inhibition targeted genes that were highly expressed in anti-TNF resistant IBD and that predicted non-response to anti-TNF therapy. CONCLUSION: Collectively, our findings reveal CDK9 as a potential target for anti-TNF resistant IBD, which has the potential for rapid translation to the clinic

Interleukin 6 increases production of cytokines by colonic innate lymphoid cells in mice and patients with chronic intestinal inflammation

Background & Aims: Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. Methods: ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. Results: IL17A- and IL22-producing, natural cytotoxicity receptor-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor-positive cells, in a dose-dependent manner. Conclusions: IL6 contributes to activation of colonic natural cytotoxicity receptor-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor-positive cells

Interleukin-22 orchestrates a pathological endoplasmic reticulum stress response transcriptional programme in colonic epithelial cells.

OBJECTIVE: The functional role of interleukin-22 (IL22) in chronic inflammation is controversial, and mechanistic insights into how it regulates target tissue are lacking. In this study, we evaluated the functional role of IL22 in chronic colitis and probed mechanisms of IL22-mediated regulation of colonic epithelial cells. DESIGN: To investigate the functional role of IL22 in chronic colitis and how it regulates colonic epithelial cells, we employed a three-dimentional mini-gut epithelial organoid system, in vivo disease models and transcriptomic datasets in human IBD. RESULTS: As well as inducing transcriptional modules implicated in antimicrobial responses, IL22 also coordinated an endoplasmic reticulum (ER) stress response transcriptional programme in colonic epithelial cells. In the colon of patients with active colonic Crohn's disease (CD), there was enrichment of IL22-responsive transcriptional modules and ER stress response modules. Strikingly, in an IL22-dependent model of chronic colitis, targeting IL22 alleviated colonic epithelial ER stress and attenuated colitis. Pharmacological modulation of the ER stress response similarly impacted the severity of colitis. In patients with colonic CD, antibody blockade of IL12p40, which simultaneously blocks IL12 and IL23, the key upstream regulator of IL22 production, alleviated the colonic epithelial ER stress response. CONCLUSIONS: Our data challenge perceptions of IL22 as a predominantly beneficial cytokine in IBD and provide novel insights into the molecular mechanisms of IL22-mediated pathogenicity in chronic colitis. Targeting IL22-regulated pathways and alleviating colonic epithelial ER stress may represent promising therapeutic strategies in patients with colitis. TRIAL REGISTRATION NUMBER: NCT02749630

New strategies to reduce liver ischemia – reperfusion injury in fatty and non-fatty livers: a focus on sirtuin 1 implication

[spa] La lesión por isquemia-reperfusion (LIR) hepática es causa de una significativa mortalidad en caso de resecciones hepáticas y en el trasplante hepático. La LIR está relacionada con el desarrollo de fallo primario y con la disfunción primaria del injerto. Ella se está desarrollando por la privación del oxígeno durante la fase de isquemia y la nueva entrada de oxígeno, durante la reperfusión, la cual daña más el órgano sometido a isquemia. La LIR es un proceso muy complejo y varias investigaciones intensivas se han llevado a cabo para explorar los mecanismos de su patogénesis. Entre los más estudiados son el estrés oxidativo, la inflamación y la apoptosis. Además, debido a la gran demanda por órganos, muchos centros de trasplante se han visto obligados a utilizar injertos que anteriormente eran considerados inapropiados para trasplantar. Los hígados esteatósicos son un ejemplo; se caracterizan de una acumulación exagerada de lípidos y son más vulnerables frente la LIR. Además, están correlacionados con una incidencia elevada de disfunción después del trasplante. Consecuentemente, es necesario la identificación de nuevas estrategias que confieren una protección eficaz frente la LIR y especialmente en caso de hígados esteatósicos. La sirtuina 1 (SIRT1) es una enzima que elimina el grupo acetilo en varias proteínas y así regula varios procesos celulares, como la respuesta frente varios tipos de estrés, el ciclo celular y el metabolismo. Estudios recientes en el corazón y el cerebro han asociado la activación de la SIRT1 con una aumentada resistencia frente la LIR. No obstante, no se ha reportado todavía una posible implicación de la SIRT1 en la LIR hepática o en el trasplante. La presenta tesis tiene como objetivo investigar la implicación de la SIRT1 en la isquemia-reperfusión caliente en hígados esteatósicos de rata y su correlación con el precondicionamiento isquémico, una estrategia previamente reportada para prevenir la LIR. Además, se ha investigado la implicación de la SIRT1 en un modelo de trasplante ortotópico y en un modelo de trasplante con tamaño reducido en la rata. Así, el presente estudio demuestra que SIRT1 está involucrada en la LIR hepática y su activación podría ser una estrategia prometedora para disminuir los efectos adversos de la LIR.[eng] Ischemia-reperfusion injury (IRI) is an inevitable situation in clinical settings such as liver transplantation and hepatic resection. It develops when blood flow is interrupted for a long period of time (ischemia) and then it restarts (reperfusion). IRI pathophysiology is highly complex and includes a number of mechanisms, such as oxidative stress, inflammation, apoptosis, microcirculatory disturbances, that contribute to organ damage and limit the clinical outcome. In addition, in order to reduce the gap between the need and availability of donors, many transplantation teams now use liver grafts, that previously have been considered unsuitable for transplantation, such as the steatotic livers. Steatotic livers are characterized by an excessive lipid accumulation and present higher vulnerability against IRI in comparison to normal ones. Subsequently, the use of steatotic liver grafts has been associated with poorest transplantation outcome and higher incidence of dysfunction. In spite of the extensive investigations, IRI remains an unsolved problem and it is necessary the identification of new targets that could mitigate its adverse effects. Recently, sirtuin 1, (SIRT1) a nicotinamide adenosine dinucleotide (NAD+)-dependent deacetylase has gained increasing attention from researchers, due to its involvement in various cellular functions, including cell response to stress, cell cycle and metabolism. Although SIRT1 has been associated with beneficial effects against IRI in heart and brain, no data have been reported concerning its potential implication in hepatic IRI and liver transplantation. Consequently, the present thesis aims to investigate the potential role of SIRT1 in rat liver IRI in a model of warm ischemia-reperfusion in steatotic livers (first study), in a model of rat orthotopic liver transplantation, OLT, (second study) and in reduced-size orthotopic liver transplantation, ROLT, (third study). In the first study, SIRT1 has been associated with the beneficial effects of ischemic preconditioning (a well-known surgical strategy to prevent IRI). In rat OLT SIRT1 has been correlated with the activation of autophagic pathway. In ROLT SIRT1 has been shown to contribute to the beneficial effects exerted by angiotensin II inhibition. According these results, the present thesis concludes that SIRT1 is implicated in the hepatic IRI and that strategies that enhance its activity can be a promising approach to reduce liver IRI