http://hdl.handle.net/10171/22962 2013-05-25T13:39:52Z http://hdl.handle.net/10171/27795 Title: Radiomarcaje y estudios de biodistribución de nanopartículas poliméricas como adyuvantes para la vacunación oftálmica frente a la brucelosis Author(s) : Sanchez-Martinez, M. (María); Costa-Martins, R. (Raquel) da; Quincoces, G. (Gemma); Gamazo, C. (Carlos); Caicedo, C. (Carlos); Irache, J.M. (Juan Manuel); Peñuelas, I. (Iván) Abstract: Objetivos: Optimizar el radiomarcaje con 99mTc de nanopartículas de Gantrez® manosiladas y cargadas con el antígeno de Brucella Ovis (Man-NP-HS) y llevar a cabo estudios de biodistribución en ratón tras la administración de las nanopartículas por vía ocular. Metodología: Las Man-NP-HS se obtuvieron por el método de desplazamiento de disolvente. Se purificaron, liofilizaron y caracterizaron. A continuación, se marcaron con 74 MBq de 99mTcO4 - previamente reducido con una disolución ácida de cloruro de estaño, trabajando en ausencia de oxígeno y con un pH final de 4. El rendimiento del marcaje se evaluó mediante TLC. Los estudios de biodistribución se llevaron a cabo en ratones tras la administración oftálmica de la formulación y de un control de 99mTcO4 - libre. Para ello, se sacrificaron los animales a las 2 y a las 24 horas tras la administración ocular y se contaron los órganos en un contador gamma. Resultados: Se obtuvo un rendimiento de marcaje superior al 90%. Los estudios de biodistribución de 99mTc-Man-NP-HS permitieron detectar la actividad concentrada en mucosa nasal y ocular y tracto gastrointestinal tanto a las 2 como a las 24 horas, frente a la biodistribución de 99mTcO4 - libre que permaneció concentrado en la piel alrededor del ojo y en tracto gastrointestinal. Conclusión: Los estudios de biodistribución de 99mTc-Man-NP-HS tras administración oftálmica han permitido demostrar su biodistribución en mucosas y tracto gastrointestinal, característica indispensable como sistema de liberación de antígenos a través de mucosa ocular. Esto, junto con su elevada respuesta inmune, efectiva protección y no virulencia, convierte a estas nanopartículas en una vacuna ideal anti Brucelosis. 2012-12-31T23:00:00Z http://hdl.handle.net/10171/23248 Title: Apoptosis en la cardiopatía hipertensiva Author(s) : Diez, J. (Javier); Fortuño, M.A. (María Antonia); Ravassa, S. (Susana) 1998-12-31T23:00:00Z http://hdl.handle.net/10171/23232 Title: Activation of cAMP signaling transiently inhibits apoptosis in vascular smooth muscle cells in a site upstream of caspase-3 Author(s) : Orlov, S.N. (Sergei N.); Thorin-Trescases, N. (Nathalie); Dulin, N.O. (Nickolai O.); Dam, T.V. (Than-Vinh); Fortuño, M.A. (María Antonia); Tremblay, J. (Johanne); Hamet, P. (Pavel) Abstract: Intracellular signaling pathways that are involved in protection of vascular smooth muscle cells (VSMC) from apoptosis remain poorly understood. This study examines the effect of activators of cAMP/cGMP signaling on apoptosis in non-transfected VSMC and in VSMC transfected with c-myc (VSMC-MYC) or with its functional analogue, E1A-adenoviral protein (VSMC-E1A). Serum-deprived VSMC-E1A exhibited the highest apoptosis measured as the content of chromatin and low molecular weight DNA fragments, phosphatidylserine content in the outer surface of plasma membrane and caspase-3 activity (ten-, five-, four- and tenfold increase after 6 h of serum withdrawal, respectively). In VSMC-E1A, the addition of an activator of adenylate cyclase, forskolin, abolished chromatin cleavage, DNA laddering, caspase-3 activation and the appearance of morphologically-defined apoptotic cells triggered by 6 h of serum deprivation. In non-transfected VSMC and in VSMC-MYC, 6 h serum deprivation led to approximately six- and threefold activation of chromatin cleavage, respectively, that was also blocked by forskolin. In VSMC-E1A, inhibition of apoptosis was observed with other activators of cAMP signaling (cholera toxin, isoproterenol, adenosine, 8-Br-cAMP), whereas 6 h incubation with modulators of cGMP signaling (8-Br-cGMP, nitroprusside, atrial natriuretic peptide, L-NAME) did not affect the development of apoptotic machinery. The antiapoptotic effect of forskolin was abolished in 24 h of serum deprivation that was accompanied by normalization of intracellular cAMP content and protein kinase A (PKA) activity. Protection of VSMC-E1A from apoptosis by forskolin was blunted by PKA inhibitors (H-89 and KT5720), whereas transfection of cells with PKA catalytic subunit attenuated apoptosis triggered by serum withdrawal. The protection of VSMC-E1A by forskolin from apoptosis was insensitive to modulators of cytoskeleton assembly (cytochalasin B, colchicine). Neither acute (30 min) nor chronic (24 h) exposure of VSMC to forskolin modified basal and serum-induced phosphorylation of the MAP kinase ERK1/2. Thus, our results show that activation of cAMP signaling delays the development of apoptosis in serum-deprived VSMC at a site upstream of caspase-3 via activation of PKA and independently of cAMP-induced reorganization of the cytoskeleton network and the ERK1/2-terminated MAPK signaling cascade. 1998-12-31T23:00:00Z http://hdl.handle.net/10171/21919 Title: Vascular oxidative stress and endothelial dysfunction Author(s) : Zalba, G. (Guillermo); Gonzalez, A. (Arantxa); Beaumont, J. (Javier); San-Jose, G. (Gorka); Moreno, M.U. (María Ujué); Lopez, B. (Begoña); Ravassa, S. (Susana); Muñiz, P. (Paula); Fortuño, A. (A.); Fortuño, M.A. (María Antonia); Diez, J. (Javier) 2000-12-31T23:00:00Z http://hdl.handle.net/10171/21902 Title: Altered regulation of smooth muscle cell proliferation and apoptosis in small arteries of spontaneously hypertensive rats Author(s) : Diez, J. (Javier); Fortuño, M.A. (María Antonia); Zalba, G. (Guillermo); Etayo, J.C. (Juan Carlos); Fortuño, A. (A.); Ravassa, S. (Susana); Beaumont, J. (Javier) Abstract: AIM: It has been proposed that alterations of the balance between programmed cell death and cell replication might be involved in abnormalities of smooth muscle cell growth in arterial hypertension. This study was designed to analyse some regulators of apoptosis and proliferation in smooth muscle cells of small intra-myocardial arteries from the left ventricle of adult normotensive Wistar-Kyoto rats (WKY) and adult spontaneously hypertensive rats (SHR). Therefore, we assessed the expression of the cytoplasmic proteins Bax and Bcl-2, respectively a promoter and an inhibitor of apoptosis, and the expression of cyclin A, a nuclear protein that induces proliferation of smooth muscle cells. METHODS AND RESULTS: We measured the percentages of smooth muscle cells expressing these proteins using monoclonal antibodies and the avidin-biotin immunoperoxidase method. Compared with WKY, cells from SHR exhibited normal Bax expression, increased (P < 0.001) Bcl-2 expression and increased (P < 0.001) cyclin A expression. The ratio of Bax to Bcl-2, an index of cell susceptibility to apoptosis, was lower (P < 0.001) in SHR than in WKY. Systolic blood pressure was directly correlated (P < 0.01) with Bcl-2 and cyclin A in SHR. CONCLUSION: These results suggest that apoptosis and proliferation of smooth muscle cells might be inhibited and stimulated, respectively, in small arteries of adult SHR. The imbalance between these two processes may account for abnormalities of smooth muscle cell growth in the arterial wall in genetic hypertension. 1997-12-31T23:00:00Z http://hdl.handle.net/10171/21899 Title: Involvement of cardiomyocyte survival-apoptosis balance in hypertensive cardiac remodeling Author(s) : Fortuño, M.A. (María Antonia); Lopez, N. (Natalia); Gonzalez, A. (Arantxa); Diez, J. (Javier) Abstract: The balance between cell death and cell survival is a tightly controlled process, especially in terminally differentiated cells, such as the cardiomyocyte. Accumulating data support a role for cardiomyocyte apoptosis in the development of several cardiac diseases, including the transition from hypertensive compensatory hypertrophy to heart failure. This review briefly summarizes the status of the knowledge regarding the death-survival balance of cardiomyocytes in the context of hypertensive heart disease. Several molecular and cellular aspects as well as the most relevant pathophysiological implications are presented. Moreover, diagnosis tools under development and the possibilities for pharmacological intervention are also examined. 2002-12-31T23:00:00Z http://hdl.handle.net/10171/21876 Title: Differential hypertrophic effects of cardiotrophin-1 on adult cardiomyocytes from normotensive and spontaneously hypertensive rats Author(s) : Lopez, N. (Natalia); Diez, J. (Javier); Fortuño, M.A. (María Antonia) Abstract: Cardiotrophin-1 (CT-1) produces longitudinal elongation of neonatal cardiomyocytes, but its effects in adult cardiomyocytes are not known. Recent observations indicate that CT-1 may be involved in pressure overload left ventricular hypertrophy (LVH). We investigated whether the hypertrophic effects of CT-1 are different in cardiomyocytes isolated from adult normotensive and spontaneously hypertensive rats (SHR). Hypertrophy was evaluated by planimetry and confocal microscopy, contractile proteins were quantified by Western blotting and real-time RT-PCR, and intracellular pathways were analyzed with specific chemical inhibitors. CT-1 increased c-fos and ANP expression (p<0.01) and cell area (p<0.01) in cardiomyocytes from both rat strains. In Wistar cells, CT-1 augmented cell length (p<0.01) but did not modify either the transverse diameter or cell depth. In SHR cells, CT-1 increased cell length (p<0.05), cell width (p<0.01) and cell depth, augmented the expression of myosin light chain-2v (MLC-2v) and skeletal alpha-actin (p<0.01) and enhanced MLC-2v phosphorylation (p<0.01). The blockade of gp130 or LIFR abolished CT-1-induced growth in the two cell types. All distinct effects observed in cardiomyocytes from SHR were mediated by STAT3. Baseline angiotensinogen expression was higher in SHR cells, and CT-1 induced a 1.7-fold and 3.2-fold increase of angiotensinogen mRNA in cardiomyocytes from Wistar rats and SHR respectively. In addition, AT1 blockade inhibited the specific effects of CT-1 in SHR cells. Finally, ex vivo determinations revealed that adult SHR exhibited enhanced myocardial CT-1 (mRNA and protein, p<0.01), increased cell width (p<0.01) and concentric LVH compared with pre-hypertensive SHR. These findings reveal a specific cell-broadening effect of CT-1 in cardiomyocytes from adult SHR and suggest that the hypertensive phenotype of these cells may influence the hypertrophic effects of CT-1, probably by means of an exaggerated induction of angiotensinogen expression. We suggest that CT-1 might facilitate LVH in genetic hypertension through a cross-talk with the renin-angiotensin system. 2005-12-31T23:00:00Z http://hdl.handle.net/10171/21860 Title: Loss of myocardial LIF receptor in experimental heart failure reduces cardiotrophin-1 cytoprotection. A role for neurohumoral agonists? Author(s) : Lopez, N. (Natalia); Varo, N. (Nerea); Diez, J. (Javier); Fortuño, M.A. (María Antonia) Abstract: OBJECTIVES: Cardiomyocyte loss is involved in the transition from compensatory left ventricular hypertrophy (LVH) to heart failure (HF). Our aim was to investigate the status of the leukaemia inhibitory factor receptor (LIFR)/gp130 survival pathway and its cytoprotective activity in intact cardiac tissue and in cardiomyocytes obtained from adult spontaneously hypertensive rats (SHR) with LVH (non-failing SHR) and from aged SHR with overt HF (failing SHR). METHODS: Cardiac morphometry was assayed by planimetry in an image analysis system. mRNA and protein expression were quantified by real time RT-PCR and Western blotting. Receptors were localized by immunocytochemistry. Trypan blue staining, TUNEL, and MTT cell viability assays were employed to study the cytoprotective activity of cardiotrophin-1 (CT-1) in isolated caridomyocytes. RESULTS: Compared to non-failing SHR, failing SHR exhibited enhanced myocardial cell death (p<0.01) demonstrated by the increase in Bax/Bcl-2 ratio, caspase-3 activation and poly (ADP-ribose) polymerase (PARP) fragmentation. Failing SHR had a 7-fold diminished expression (p<0.01) of LIFR, no changes in gp130, and 1.6-fold increased myocardial expression (p<0.01) of CT-1. In cardiomyocytes isolated from non-failing SHR, recombinant CT-1 inhibited apoptotic and non-apoptotic cell death induced by angiotensin II or hydrogen peroxide. LIFR protein was entirely absent in cardiomyocytes isolated from failing SHR, which were resistant to the cytoprotective effects of CT-1. Finally, stimulation of non-failing SHR cardiomyocytes with angiotensin II, aldosterone, norepinephrine or endothelin-1 significantly decreased (p<0.01) LIFR expression. CONCLUSIONS: These data suggest that loss of CT-1-dependent survival mechanisms may contribute to the increase of cell death associated with HF in SHR. Neurohumoral activation may contribute to this alteration via suppression of LIFR. 2006-12-31T23:00:00Z http://hdl.handle.net/10171/21853 Title: Aldosterone induces cardiotrophin-1 expression in HL-1 adult cardiomyocytes Author(s) : Lopez-Andres, N. (Natalia); Iñigo, C. (Carmen); Gallego, I. (Idoia); Diez, J. (Javier); Fortuño, M.A. (María Antonia) Abstract: Aldosterone (ALDO) may induce cardiac hypertrophy by nonhemodynamic mechanisms that are not completely defined. Cardiotrophin-1 (CT-1) is a cytokine that exerts hypertrophic actions on isolated cardiomyocytes and promotes cardiac hypertrophy in vivo. We investigated whether ALDO induces CT-1 expression in HL-1 cardiomyocytes aiming at the possibility that the cytokine is involved in ALDO-induced cardiomyocyte hypertrophy. mRNA and protein expression were quantified by RT-PCR and Western blot. Cardiomyocyte area, as an index of hypertrophy, was assayed by image analysis in phalloidin-stained HL-1 cells. ALDO addition to adult HL-1 cardiomyocytes increased (P<0.01) CT-1 mRNA and protein expression in a concentration-dependent manner. This effect was abrogated by actinomycin D, the mineralocorticoid and glucocorticoid receptor antagonists spironolactone and RU486, respectively, and the p38 MAPK blocker SB203580. CT-1 signaling pathway blockade with specific antibodies against the cytokine and its two receptor subunits avoided (P<0.01) alpha-sarcomeric actin and c-fos protein overexpression as well as cell size increase induced by ALDO in HL-1 cells. In vivo, a single ALDO injection acutely increased (P<0.01) the myocardial expression of CT-1 in C57BJ6 wild-type mice but not CT-1-null mice. The bolus of the mineralocorticoid increased (P<0.01) ANP and c-fos mRNA expression in the myocardium of wild-type mice, whereas no changes were observed in CT-1-null mice. In summary, ALDO induces CT-1 expression in adult HL-1 cardiomyocytes via genomic and nongenomic mechanisms. CT-1 up-regulation could have relevance in the direct hypertrophic effects of ALDO in cardiomyocytes. 2007-12-31T23:00:00Z http://hdl.handle.net/10171/21846 Title: Cardiotrophin-1 is expressed in adipose tissue and upregulated in the metabolic syndrome Author(s) : Natal, C. (Cristina); Fortuño, M.A. (María Antonia); Restituto, P. (Patricia); Bazan, A. (Antonio); Colina, I. (Inmaculada); Diez, J. (Javier); Varo, N. (Nerea) Abstract: Adipose tissue is a target for cardiotrophin-1 (CT-1), a cytokine member of the IL-6 family of cytokines that is involved in cardiac growth and dysfunction. However, it is unknown whether adipocytes are a source of CT-1 and whether CT-1 is overexpressed in diseases characterized by increased fat depots [i.e., the metabolic syndrome (MS)]. Thus this work aimed 1) to test whether adipose tissue expresses CT-1 and whether CT-1 expression can be modulated and 2) to compare serum CT-1 levels in subjects with and without MS diagnosed by National Cholesterol Education Program Adult Treatment Panel III criteria. Gene and protein expression of CT-1 was determined by real-time RT-PCR, ELISA, and Western blotting. CT-1 expression progressively increased, along with differentiation time from preadipocyte to mature adipocyte in 3T3-L1 cells. CT-1 expression was enhanced by glucose in a dose-dependent manner in these cells. mRNA and protein CT-1 expression was also demonstrated in human adipose biopsies. Immunostaining showed positive staining in adipocytes. Finally, increased CT-1 serum levels were observed in patients with MS compared with control subjects (127 +/- 9 vs. 106 +/- 4 ng/ml, P < 0.05). Circulating levels of CT-1 were associated with glucose levels (r = 0.2, P < 0.05). Taken together, our data suggest that adipose tissue can be recognized as a source of CT-1, which could account for the high circulating levels of CT-1 in patients with MS. 2008-12-31T23:00:00Z http://hdl.handle.net/10171/21665 Title: The epidermal growth factor receptor ligand amphiregulin is a negative regulator of hepatic acute-phase gene expression Author(s) : Pardo-Saganta, A. (Ana); Latasa, M.U. (María Ujué); Castillo, J. (Josefa); Alvarez-Asiain, L. (Laura); Perugorria, M.J. (María J.); Sarobe, P. (Pablo); Rodriguez-Ortigosa, C.M. (Carlos M.); Prieto, J. (Jesús); Berasain, C. (Carmen); Santamaria, M. (Mónica); Avila, M.A. (Matías Antonio) Abstract: BACKGROUND/AIMS: The modulation of the hepatic acute-phase reaction (APR) that occurs during inflammation and liver regeneration is important for allowing normal hepatocellular proliferation and the restoration of homeostasis. Activation of acute-phase protein (APP) gene expression by interleukin-6 (IL-6)-type cytokines is thought to be counteracted by growth factors released during hepatic inflammation and regeneration. The epidermal growth factor receptor (EGFR) ligand amphiregulin (AR) is readily induced by inflammatory signals and plays a nonredundant protective role during liver injury. In this paper, we investigated the role of AR as a modulator of liver APP gene expression. METHODS: Expression of APP genes was measured in the livers of AR(+/+) and AR(-/-)mice during inflammation and regeneration and in cultured liver cells treated with AR and oncostatin M (OSM). Crosstalk between AR and OSM signalling was studied. RESULTS: APP genes were overexpressed in the livers of AR(-/-) mice during inflammation and hepatocellular regeneration. In cultured AR-null hepatocytes and human hepatocellular carcinoma (HCC) cells after AR knockdown, APP gene expression is enhanced. AR counteracts OSM-triggered signal transducer and activator of transcription 3 signalling in hepatocytes and attenuates APP gene transcription. CONCLUSIONS: Our data support the relevance of EGFR-mediated signalling in the modulation of cytokine-activated pathways. We have identified AR as a key regulator of hepatic APP gene expression during inflammation and liver regeneration. 2008-12-31T23:00:00Z http://hdl.handle.net/10171/20329 Title: Effects of antihypertensive agents on the left ventricle: clinical implications Author(s) : Diez, J. (Javier); Gonzalez, A. (Arantxa); Lopez, B. (Begoña); Ravassa, S. (Susana); Fortuño, M.A. (María Antonia) Abstract: Hypertensive heart disease (HHD) is characterized by left ventricular hypertrophy (LVH), alterations of cardiac function, and coronary flow abnormalities. LVH is an independent cardiovascular risk factor related to cardiovascular complications in patients with hypertension. Therefore, a decrease in left ventricular mass is a therapeutic goal in these patients. The effect of the different antihypertensive agents on LVH regression has been studied in nearly 500 clinical trials. Most studies conclude that there is regression of LVH after significant decrease in blood pressure with most commonly prescribed antihypertensive agents. However, the ability to regress LVH is different between antihypertensive drug classes. ACE inhibitors and calcium channel antagonists are more potent in reducing left ventricular mass than beta-blockers, with diuretics falling in the intermediate group. Recent data suggest that angiotensin AT(1) receptor antagonists reduce left ventricular mass to a similar extent as ACE inibitors or calcium channel antagonists. Although a large number of studies have established that reversal of LVH decreases the occurrence of adverse cardiovascular events in patients with hypertension, the hypothesis that LVH regression is beneficial has not yet been conclusively proven. On the other hand, the time has come to revisit the current management of HHD simply focused on controlling blood pressure and reducing left ventricular mass. In fact, it is necessary to develop new approaches aimed to repair myocardial structure and protect myocardial perfusion and function and, in doing so, to reduce in a more effective manner, adverse risk associated with HHD. The identification of genes involved in both the process of HHD and the response to therapy may be critical for the development of these new approaches. This article will review briefly the available data on the effects of antihypertensive agents on HHD. In addition, the emerging new concepts on the pharmacology of hypertensive myocardial remodeling and the pharmacogenetic basis of the treatment of HHD will be also considered. 2000-12-31T23:00:00Z http://hdl.handle.net/10171/20250 Title: Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger Author(s) : Banales, J.M. (Jesús M.); Arenas, F. (Fabián); Rodriguez-Ortigosa, C.M. (Carlos M.); Saez, E. (Elena); Uriarte, I. (Iker); Doctor, R.B. (R. Brian); Prieto, J. (Jesús); Medina, J.F. (Juan Francisco) Abstract: Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl- efflux and subsequent biliary HCO3- secretion, possibly via Cl-/HCO3- anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl-/HCO3- exchange in secretin-stimulated HCO3- secretion all remain unclear. Here, secretin was administered to normal rats with maintained bile acid pool via continuous taurocholate infusion. Bile flow and biliary HCO3- and Cl- excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl- channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) or the Cl-/HCO3- exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion of HCO3- and Cl-. Interestingly, secretin effects were not observed in the absence of taurocholate. Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin-induced increases in bile flow and HCO3- excretion but not the increased Cl- excretion, revealing a role of biliary Cl-/HCO3- exchange in secretin-induced, bicarbonate-rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na+-independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused a marked inhibition of unstimulated and secretin-stimulated Cl-/HCO3- exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate-rich choleresis in the normal rat and that this occurs via a chloride-bicarbonate exchange process consistent with AE2 function. 2005-12-31T23:00:00Z http://hdl.handle.net/10171/20239 Title: Biliary secretion of S-nitrosoglutathione is involved in the hypercholeresis induced by ursodeoxycholic acid in the normal rat Author(s) : Rodriguez-Ortigosa, C.M. (Carlos M.); Banales, J.M. (Jesús M.); Olivas, I. (Israel); Uriarte, I. (Iker); Marin, J.J.G (Jose J.G.); Corrales, F.J. (Fernando José); Medina, J.F. (Juan Francisco); Prieto, J. (Jesús) Abstract: Ursodeoxycholic acid (UDCA) induces bicarbonate-rich hypercholeresis by incompletely defined mechanisms that involve the stimulation of adenosine triphosphate (ATP) release from cholangiocytes. As nitric oxide (NO) at a low concentration can stimulate a variety of secretory processes, we investigated whether this mediator could be implicated in the choleretic response to UDCA. Our in vivo experiments with the in situ perfused rat liver model in anesthetized rats, showed that UDCA infusion increased the biliary secretion of NO derivatives, hepatic inducible NO synthase expression, and NO synthase activity in liver tissue. UDCA also stimulated NO release by isolated rat hepatocytes. In contrast to UDCA, cholic acid was a poor inducer of NO secretion, and tauroursodeoxycholic acid showed no effect on NO secretion. Upon UDCA administration, NO was found in bile as low-molecular-weight nitrosothiols, of which S-nitrosoglutathione (GSNO) was the predominant species. UDCA-stimulated biliary NO secretion was abolished by the inhibition of inducible NO synthase with N(omega)-nitro-L-arginine methyl ester in isolated perfused livers and also in rats whose livers were depleted of glutathione with buthionine sulfoximine. Moreover, the biliary secretion of NO species was significantly diminished in UDCA-infused transport mutant [ATP-binding cassette C2 (ABCC2)/multidrug resistance-associated protein 2 (Mrp2)-deficient] rats, and this finding was consistent with the involvement of the glutathione carrier ABCC2/Mrp2 in the canalicular transport of GSNO. It was particularly noteworthy that in cultured normal rat cholangiocytes, GSNO activated protein kinase B, protected against apoptosis, and enhanced UDCA-induced ATP release to the medium; this effect was blocked by phosphoinositide 3-kinase inhibition. Finally, retrograde GSNO infusion into the common bile duct increased bile flow and biliary bicarbonate secretion. Conclusion: UDCA induces biliary secretion of GSNO, which contributes to stimulating ductal secretion. 2009-12-31T23:00:00Z http://hdl.handle.net/10171/20237 Title: Oral methylthioadenosine administration attenuates fibrosis and chronic liver disease progression in Mdr2-/- mice Author(s) : Latasa, M.U. (María Ujué); Gil-Puig, C. (Carmen); Fernandez-Barrena, M. G. (Maite G.); Rodriguez-Ortigosa, C.M. (Carlos M.); Banales, J.M. (Jesús M.); Urtasun, R. (Raquel); Goñi, S. (Saioa); Mendez, M. (Miriam); Arcelus, S. (Sara); Juanarena, N. (Nerea); Recio, J.A. (Juan A.); Lotersztajn, S. (Sophie); Prieto, J. (Jesús); Berasain, C. (Carmen); Corrales, F.J. (Fernando José); Lecanda, J. (Jon); Avila, M.A. (Matías Antonio) Abstract: BACKGROUND: Inflammation and fibrogenesis are directly related to chronic liver disease progression, including hepatocellular carcinoma (HCC) development. Currently there are few therapeutic options available to inhibit liver fibrosis. We have evaluated the hepatoprotective and anti-fibrotic potential of orally-administered 5'-methylthioadenosine (MTA) in Mdr2(-/-) mice, a clinically relevant model of sclerosing cholangitis and spontaneous biliary fibrosis, followed at later stages by HCC development. METHODOLOGY: MTA was administered daily by gavage to wild type and Mdr2(-/-) mice for three weeks. MTA anti-inflammatory and anti-fibrotic effects and potential mechanisms of action were examined in the liver of Mdr2(-/-) mice with ongoing fibrogenesis and in cultured liver fibrogenic cells (myofibroblasts). PRINCIPAL FINDINGS: MTA treatment reduced hepatomegaly and liver injury. α-Smooth muscle actin immunoreactivity and collagen deposition were also significantly decreased. Inflammatory infiltrate, the expression of the cytokines IL6 and Mcp-1, pro-fibrogenic factors like TGFβ2 and tenascin-C, as well as pro-fibrogenic intracellular signalling pathways were reduced by MTA in vivo. MTA inhibited the activation and proliferation of isolated myofibroblasts and down-regulated cyclin D1 gene expression at the transcriptional level. The expression of JunD, a key transcription factor in liver fibrogenesis, was also reduced by MTA in activated myofibroblasts. CONCLUSIONS/SIGNIFICANCE: Oral MTA administration was well tolerated and proved its efficacy in reducing liver inflammation and fibrosis. MTA may have multiple molecular and cellular targets. These include the inhibition of inflammatory and pro-fibrogenic cytokines, as well as the attenuation of myofibroblast activation and proliferation. Downregulation of JunD and cyclin D1 expression in myofibroblasts may be important regarding the mechanism of action of MTA. This compound could be a good candidate to be tested for the treatment of (biliary) liver fibrosis. 2009-12-31T23:00:00Z