La experimentación animal y el progreso de la medicina / Animal experimentation and progress in medicine

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La olvidada investigación clínica / The forgotten clinical investigation

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Effect of angiotensin II and bradykinin inhibition in rat reduced-size liver transplantation.

This study examined whether angiotensin II (Ang II) blockers [Ang II type I receptor antagonist, Ang II type II receptor antagonist, and angiotensin converting enzyme (ACE) inhibitor] could reduce hepatic injury and improve regeneration in reduced-size orthotopic liver transplantation (ROLT) and whether the beneficial effects of ischemic preconditioning (PC) in ROLT could be explained by changes in Ang II. We show that small liver grafts generated Ang II after ROLT and that this was associated with increased angiotensinogen and ACE messenger RNA expression. Furthermore, inhibition of Ang II did not contribute to PC-induced protection in ROLT. All Ang II blockers reduced hepatic injury, but none of them promoted liver regeneration. Bradykinin (BK) receptor antagonist improved liver regeneration but did not reduce hepatic injury in ROLT. Finally, the combination of Ang II blockers and BK receptor antagonists in ROLT reduced hepatic injury and improved liver regeneration. In conclusion, treatments with either Ang II blockers or BK receptor antagonists cannot, on their own, improve the outcome of ROLT. Although Ang II blockers can reduce hepatic ischemia-reperfusion injury and BK receptor antagonists can promote liver regeneration, neither confers both benefits at the same time. Consequently, it may be of clinical interest to apply both treatments simultaneously.

Implantación de la gestión clínica en la organización hospitalaria / Implementation of clinical management in hospital organization

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Carrera profesional del personal médico. Un sistema incentivador útil cuando se mantienen sus principios de aplicación / Medical personnel’s careers. A useful incentive system as long as we adhere to its application principles

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Activation of peroxisome proliferator-activated receptor-alpha inhibits the injurious effects of adiponectin in rat steatotic liver undergoing ischemia-reperfusion.

UNLABELLED: Hepatic steatosis is a major risk factor in ischemia-reperfusion (I/R). Adiponectin acts as an antiobesity and anti-inflammatory hormone. Adiponectin activates peroxisome proliferator-activated receptor-alpha (PPAR-alpha), a transcription factor that regulates inflammation in liver disease. Ischemic preconditioning (PC) based on brief periods of I/R protects steatotic livers against subsequent sustained I/R injury, but just how this is achieved is poorly understood. This study explains the role of PPAR-alpha and adiponectin in the vulnerability shown by steatotic livers to I/R and the benefits of PC in this situation. PPAR-alpha and adiponectin levels in nonsteatotic livers undergoing I/R were similar to those found in the sham group. However, reduced PPAR-alpha and increased adiponectin levels, particularly the high molecular weight isoform, were observed in steatotic livers as a consequence of I/R. Our results suggest that mitogen-activated protein kinases (MAPKs) may be positive regulators of adiponectin accumulation in steatotic livers. The addition of adiponectin small interfering RNA (siRNA) before I/R protected steatotic livers against oxidative stress and hepatic injury. The induction of PC before I/R increased PPAR-alpha and reduced adiponectin levels in steatotic livers. PC, which increased PPAR-alpha, as well as PPAR-alpha agonist pretreatment reduced MAPK expression, adiponectin, oxidative stress, and hepatic injury that follows I/R. In addition, the administration of a PPAR-alpha antagonist in preconditioned steatotic livers eliminated the beneficial effects of PC on MAPKs, adiponectin, oxidative stress, and hepatic injury. CONCLUSION: Steatotic livers are more predisposed to down-regulate PPAR-alpha and overexpress adiponectin when subjected to I/R. PPAR-alpha agonists and adiponectin siRNA are promising candidates to protect steatotic livers. PPAR-alpha agonists as well as PC, through PPAR-alpha, inhibited MAPK expression following I/R. This in turn inhibited adiponectin accumulation in steatotic livers and adiponectin-worsening effects on oxidative stress and hepatic injury.

Serum osteoprotegerin and its ligand in cirrhotic patients referred for orthotopic liver transplantation: relationship with metabolic bone disease.

AIMS: A prospective study was carried out in 22 cirrhotic patients referred for orthotopic liver transplantation, in order to analyze serum osteoprotegerin (OPG) and RANKL levels and their relationship with metabolic bone disease. METHODS: Serum levels of OPG and RANKL were measured in all patients as well as bone markers, serum parathyroid hormone and 25-hydroxyvitamin D levels. OPG and RANKL values were compared with those obtained in 29 healthy controls. Bone mineral density (BMD) of the lumbar spine and proximal femur was measured by dual X-ray absorptiometry and spinal X-rays were obtained to assess vertebral fractures. RESULTS: Serum OPG levels were higher in cirrhotic patients than in controls (6.4+/-2 vs 2.7+/-0.7 pmol/l; P=0.001) and RANKL serum levels were lower in cirrhotic patients (0.215+/-0.6 vs 1.012+/-1.2 pmol/l; P=0.002), with an increased OPG:RANKL ratio when compared with the control group (280.3+/-334.5 vs 113+/-137.6; P=0.04). Ten patients had osteoporosis (45%) and up to 45% skeletal fractures. No differences were found in OPG levels between patients with and without osteoporosis by densitometric criteria or fractures. Negative correlations were found between OPG levels and femoral neck (R-0.46; P=0.03) and total hip BMD (R-0.48; P=0.025). By contrast, OPG values were not related to markers of bone turnover. CONCLUSIONS: OPG values are elevated in cirrhotic patients before liver transplantation, particularly in those with low bone mass at the proximal femur.

Adenosine monophosphate-activated protein kinase and nitric oxide in rat steatotic liver transplantation.

BACKGROUND/AIMS: Hepatic steatosis is a risk factor for transplantation. We examined the role of AMP-activated protein kinase (AMPK) and nitric oxide (NO) in the benefits of preconditioning in steatotic liver transplantation. METHODS: Steatotic liver transplantation with or without preconditioning was induced in Zucker rats. The activities of AMPK and NO synthase (NOS) were measured and altered pharmacologically. RESULTS: Preconditioning or AMPK activation with aminoimidazole-4-carboxamide ribonucleoside (AICAR) increased AMPK and constitutive NOS activities and protected against lipid peroxidation, nitrotyrosine formation and hepatic injury in both grafts. Inhibition of AMPK activity removed the benefits of preconditioning. NO synthesis inhibition abolished the benefits of preconditioning or AICAR. Therefore, preconditioning or AICAR, through AMPK activation, may induce NO synthesis, thus protecting against hepatic injury in both steatotic and non-steatotic liver transplantation. In non-steatotic grafts, NO donors simulated the benefits of preconditioning. However, in steatotic grafts, NO supplementation was ineffective. CONCLUSIONS: These results indicate (a) a potential relationship between AMPK and NO in the benefits of preconditioning in steatotic liver transplantation, (b) AICAR as a new phamacological strategy in steatotic liver transplantation and (c) a differential effect of NO supplementation in both grafts.

Inhibition of 5-lipoxygenase-activating protein abrogates experimental liver injury: role of Kupffer cells.

Activation of Kupffer cells is a prominent feature of necro-inflammatory liver injury. We have recently demonstrated that 5-lipoxygenase (5-LO) and its accessory protein, 5-LO-activating protein (FLAP), are essential for the survival of Kupffer cells in culture, as their inhibition drives these liver resident macrophages to programmed cell death. In the current study, we explored whether the potent FLAP inhibitor, Bay-X-1005, reduces the number of Kupffer cells in vivo and whether this pharmacological intervention protects the liver from carbon tetrachloride (CCl(4))-induced damage. Rats treated with CCl(4) showed an increased number of Kupffer cells, an effect that was abrogated by the administration of Bay-X-1005 (100 mg/Kg body weight, per oral, daily). Consistent with a role for Kupffer cells in necro-inflammatory liver injury, partial depletion of Kupffer cells following FLAP inhibition was associated with a remarkable hepatoprotective action. Indeed, Bay-X-1005 significantly reduced the intense hepatocyte degeneration and large bridging necrosis induced by CCl(4) treatment. Moreover, Bay-X-1005 induced a reduction in the gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) and a decrease in mRNA expression of tissue inhibitor of MMP-2. The FLAP inhibitor reduced leukotriene (LT)B(4) and cysteinyl LT levels and down-regulated 5-LO and FLAP protein expression in the liver. It is interesting that a significant increase in the hepatic formation of lipoxin A(4), an endogenous, anti-inflammatory lipid mediator involved in the resolution of inflammation, was observed after the administration of Bay-X-1005. These findings support the concept that modulation of the 5-LO pathway by FLAP inhibition may be useful in the prevention of hepatotoxin-induced necro-inflammatory injury.

The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPARgamma activation.

The importance of inflammation in initiating the sequence of events that lead to liver fibrosis is increasingly recognized. In this study, we tested the effects of SC-236, a selective cyclooxygenase (COX)-2 inhibitor, in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Livers from CCl4-treated rats showed increased COX-2 expression and 15-deoxy-prostaglandin (PG)J2 (15d-PGJ2) formation, as well as decreased peroxisome proliferator-activated receptor (PPAR)gamma expression. In these animals, SC-236 reduced liver fibrosis as revealed by histological analysis and by a reduction in hepatic hydroxyproline levels, metalloproteinase-2 activity, and alpha-smooth muscle actin expression. Interestingly, SC-236 normalized 15d-PGJ2 levels and restored PPARgamma expression in the liver of CCl4-treated rats. In isolated hepatic stellate cells (HSCs)--the major player in liver fibrogenesis--and Kupffer cells--the cell type primarily responsible for increased hepatic COX-2-SC-236 exhibited remarkable pro-apoptotic and growth inhibitory properties. Of interest, SC-236 decreased HSC viability to a similar extent than the PPARgamma ligand rosiglitazone. Moreover, SC-236 significantly induced PPARgamma expression in HSCs and acted as a potent PPARgamma agonist in a luciferase-reporter trans-activation assay. These data indicate that, by mechanisms involving non-parenchymal cell apoptosis and PPARgamma activation, the selective COX-2 inhibitor SC-236 might have therapeutic potential for prevention of liver fibrosis.

The selective cyclooxygenase-2 inhibitor celecoxib modulates the formation of vasoconstrictor eicosanoids and activates PPARgamma. Influence of albumin.

BACKGROUND/AIMS: Selective cyclooxygenase (COX)-2 inhibitors do not adversely affect renal function in experimental cirrhosis. In the current study, we investigated the molecular mechanisms underlying the effects of the selective COX-2 inhibitor, celecoxib, and assessed the influence of albumin on its actions. METHODS: Rat mesangial cells (RMC) were incubated with celecoxib in the absence or presence of albumin, and levels of selected vasoconstrictor eicosanoids, renin release and alpha-smooth muscle actin (alpha-SMA) expression were determined. The effects of celecoxib on PPARgamma were assessed in RMC co-transfected with PPARgamma and luciferase reporter constructs. RESULTS: Under resting conditions, RMC expressed COX-1, COX-2 and 12/15-lipoxygenase and mainly generated prostaglandin (PG)E2, thromboxane (TX)B2, 12-hydroxyeicosatetraenoic acid (12-HETE) and 8-epi-PGF2alpha. Celecoxib, in addition to reducing PGE2, significantly decreased 8-epi-PGF2alpha formation. In the presence of albumin, celecoxib also reduced TXB2 and 12-HETE. Albumin per se inhibited PGE2 as well as renin release. In trans-activation assays, celecoxib acted as a PPARgamma agonist whereas albumin inhibited PPARgamma as well as 15d-PGJ2-induced PPARgamma activation. Finally, celecoxib and albumin potentiated the inhibitory effect of 15d-PGJ2 on alpha-SMA expression. CONCLUSIONS: These data provide novel molecular mechanisms of celecoxib and their modulation by albumin, that may be relevant to prevent renal dysfunction in conditions of unbalanced effective blood volume.

Ischemic preconditioning affects interleukin release in fatty livers of rats undergoing ischemia/reperfusion.

The present study evaluates the effect of ischemic preconditioning on interleukin-1 (IL-1) and interleukin-10 (IL-10) generation following hepatic ischemia/reperfusion (I/R) in normal and steatotic livers as well as the role of nitric oxide (NO) in this process. Increased IL-1beta and IL-10 levels were observed in normal livers after I/R. Steatotic livers showed higher IL-1beta levels than normal livers, and IL-10 at control levels. The injurious role of IL-1beta and the benefits of IL-10 on hepatic I/R injury was shown with the use of IL-1 receptor antagonist (IL-1ra), anti-IL-10 polyclonal antibody against IL-10 (anti-IL-10) and exogenous IL-10. The effective dose of these treatments was different in both types of livers. Preconditioning prevented IL-1beta release and increased IL-10 generation after I/R in normal and steatotic livers. IL-1beta or anti-IL-10 pretreatments reversed the benefits of preconditioning. IL-1beta action inhibition in a preconditioned group that was pretreated with anti-IL-10 did not modify the benefits of preconditioning. In addition, anti-IL-10 pretreatment in the preconditioned group resulted in IL-1beta levels comparable to those observed after I/R. NO inhibition eliminated the benefits of preconditioning on IL-10 release, IL-1beta levels, and hepatic injury. In conclusion, preconditioning, through IL-10 overproduction, inhibits IL-1beta release and the ensuing hepatic I/R injury in normal and steatotic livers. IL-10 generation induced by preconditioning could be mediated by NO.

Inhibition of 5-lipoxygenase induces cell growth arrest and apoptosis in rat Kupffer cells: implications for liver fibrosis.

The existence of an increased number of Kupffer cells is recognized as critical in the initiation of the inflammatory cascade leading to liver fibrosis. Because 5-lipoxygenase (5-LO) is a key regulator of cell growth and survival, in the current investigation we assessed whether inhibition of the 5-LO pathway would reduce the excessive number of Kupffer cells and attenuate inflammation and fibrosis in experimental liver disease. Kupffer cells were the only liver cell type endowed with a metabolically active 5-LO pathway (i.e., expressed mRNAs for 5-LO, 5-LO-activating protein [FLAP], and leukotriene [LT] C4 synthase and generated LTB4 and cysteinyl-LTs). Both the selective 5-LO inhibitor AA861 and the FLAP inhibitor BAY-X-1005 markedly reduced the number of Kupffer cells in culture. The antiproliferative properties of AA861 and BAY-X-1005 were associated with the occurrence of condensed nuclei, fragmented DNA, and changes in DNA content and cell cycle frequency distribution consistent with an apoptotic process. In vivo, in carbon tetrachloride-treated rats, BAY-X-1005 had a significant antifibrotic effect and reduced liver damage and the hepatic content of hydroxyproline. Together, these findings indicate a novel mechanism by which inactivation of the 5-LO pathway could disrupt the sequence of events leading to liver inflammation and fibrosis.