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The transcription factor KLF2 mediates hepatic endothelial protection and paracrine endothelial–stellate cell deactivation induced by statins

BACKGROUND & AIMS Statins improve hepatic endothelial function and liver fibrosis in experimental models of cirrhosis, thus they have been proposed as therapeutic options to ameliorate portal hypertension syndrome. The transcription factor Kruppel-like factor 2 (KLF2) may be induced by statins in liver sinusoidal endothelial cells (SEC), orchestrating an efficient vasoprotective response. The present study aimed at characterizing whether KLF2 mediates statins-derived hepatic protection. METHODS Expression of KLF2 and its vasoprotective target genes was determined in SEC freshly isolated from control or CCl(4)-cirrhotic rats treated with four different statins (atorvastatin, mevastatin, simvastatin, and lovastatin), in the presence of mevalonate (or vehicle), under static or controlled shear stress conditions. KLF2-derived vasoprotective transcriptional programs were analyzed in SEC transfected with siRNA for KLF2 or siRNA-control, and incubated with simvastatin. Paracrine effects of SEC highly-expressing KLF2 on the activation status of rat and human hepatic stellate cells (HSC) were evaluated. RESULTS Statins administration to SEC induced significant upregulation of KLF2 expression. KLF2 upregulation was observed after 6h of treatment and was accompanied by induction of its vasoprotective programs. Simvastatin vasoprotection was inhibited in the presence of mevalonate, and was magnified in cells cultured under physiological shear stress conditions. Statin-dependent induction of vasoprotective genes was not observed when KLF2 expression was muted with siRNA. SEC overexpressing KLF2 induced quiescence of HSC through a KLF2-nitric oxide-guanylate cyclase-mediated paracrine mechanism. CONCLUSIONS Upregulation of hepatic endothelial KLF2-derived transcriptional programs by statins confers vasoprotection and stellate cells deactivation, reinforcing the therapeutic potential of these drugs for liver diseases that course with endothelial dysfunction.

Effects of simvastatin administration on rodents with lipopolysaccharide-induced liver microvascular dysfunction

Endothelial dysfunction drives vascular derangement and organ failure associated with sepsis. However, the consequences of sepsis on liver sinusoidal endothelial function are largely unknown. Statins might improve microvascular dysfunction in sepsis. The present study explores liver vascular abnormalities and the effects of statins in a rat model of endotoxemia. For this purpose, lipopolysaccharide (LPS) or saline was given to: (1) rats treated with placebo; (2) rats treated with simvastatin (25 mg/kg, orally), given at 3 and 23 hours after LPS/saline challenge; (3) rats treated with simvastatin (25 mg/kg/24 h, orally) from 3 days before LPS/saline injection. Livers were isolated and perfused and sinusoidal endothelial function was explored by testing the vasodilation of the liver circulation to increasing concentrations of acetylcholine. The phosphorylated endothelial nitric oxide synthase (PeNOS) / endothelial nitric oxide synthase (eNOS) ratio was measured as a marker of eNOS activation. LPS administration induced an increase in baseline portal perfusion pressure and a decrease in vasodilation to acetylcholine (sinusoidal endothelial dysfunction). This was associated with reduced eNOS phosphorylation and liver inflammation. Simvastatin after LPS challenge did not prevent the increase in baseline portal perfusion pressure, but attenuated the development of sinusoidal endothelial dysfunction. Treatment with simvastatin from 3 days before LPS prevented the increase in baseline perfusion pressure and totally normalized the vasodilating response of the liver vasculature to acetylcholine and reduced liver inflammation. Both protocols of treatment restored a physiologic PeNOS/eNOS ratio. Conclusion: LPS administration induces intrahepatic endothelial dysfunction that might be prevented by simvastatin, suggesting that statins might have potential for liver protection during endotoxemia. (HEPATOLOGY 2013)

Simvastatin maintains function and viability of steatotic rat livers procured for transplantation

BACKGROUND & AIMS Liver grafts obtained from healthy rat donors develop acute microcirculatory dysfunction due to cold-storage and warm-reperfusion injuries. These detrimental effects are avoided adding simvastatin to the cold-storage solution. Considering the importance of increasing organ donor pool for transplantation, we characterized whether simvastatin pretreatment can protect steatotic grafts from cold-storage and warm-reperfusion injuries. METHODS Rats fed with high-fat diet received a single dose of simvastatin, or its vehicle, 30 min before liver procurement. Grafts were then cold stored for 0 h (control group) or 16 h and warm reperfused. At the end of the reperfusion period, hepatic vascular resistance, endothelial function, nitric oxide pathway, cell death, oxidative stress, autophagy, and liver injury were evaluated. Hepatic vascular resistance and endothelial function were determined in a group of simvastatin-treated livers in the presence of the nitric oxide synthase inhibitor L-NNA. RESULTS Cold-stored rat steatotic livers exhibit increased hepatic vascular resistance and marked endothelial dysfunction, together with liver damage, oxidative stress, and low nitric oxide. Simvastatin markedly improved liver injury and prevented hepatic endothelial dysfunction. The beneficial effects of simvastatin were associated with cell death diminution, autophagy induction, and nitric oxide release. Statin-derived liver microcirculation protection was not observed when nitric oxide production was blunted. CONCLUSIONS Pretreatment of steatotic liver donors with simvastatin shortly before procurement of the liver graft strongly protects both parenchymal and endothelial components of the liver after warm reperfusion. Our data reinforce the use of statins to protect liver grafts undergoing transplantation.

Modulation of autophagy for the treatment of liver diseases.

Introduction: Autophagy is a cellular process essential for survival and homeostasis that confers cellular protection toward a wide range of deleterious stimuli. It has a highly complex regulation with several autophagic proteins also belonging to other main signaling pathways as cell proliferation or apoptosis. In addition, autophagy has an important role in cell metabolism. Interest in the study of this process is rapidly rising and, in the past few years, autophagy has been implicated in a variety of hepatic diseases. Areas covered: The review covers the research and investigational use of pharmacological strategies that modify autophagy in the treatment of liver diseases. Autophagy modulation in steatosis, steatohepatitis, viral hepatitis, fibrogenesis, cirrhosis, hepatocellular carcinoma and ischemia/reperfusion injury will be described, critically analyzed and discussed. Papers included in the present manuscript were selected from the PubMed search: liver + (macro)autophagy + each of the pathologies described above. Expert opinion: The complexity of autophagy creates significant controversy on the potential of its pharmacological modulation. A major requirement for drugs regulating autophagy in the treatment of liver diseases is that these should be liver-specific; moreover, they should primarily target one specific hepatic cell type.

A novel form of the human manganese superoxide dismutase protects rat and human livers undergoing ischaemia and reperfusion injury

Hepatic microcirculatory dysfunction due to cold storage and warm reperfusion (CS+WR) injury during liver transplantation is partly mediated by oxidative stress and may lead to graft dysfunction. This is especially relevant when steatotic donors are considered. Using primary cultured liver sinusoidal endothelial cells (LSECs), liver grafts from healthy and steatotic rats, and human liver samples, we aimed to characterize the effects of a new recombinant form of human manganese superoxide dismutase (rMnSOD) on hepatic CS+WR injury. After CS+WR, the liver endothelium exhibited accumulation of superoxide anion (O2-) and diminished levels of nitric oxide (NO); these detrimental effects were prevented by rMnSOD. CS+WR control and steatotic rat livers exhibited markedly deteriorated microcirculation and acute endothelial dysfunction, together with liver damage, inflammation, oxidative stress, and low NO. rMnSOD markedly blunted oxidative stress, which was associated with a global improvement in liver damage and microcirculatory derangements. The addition of rMnSOD to CS solution maintained its antioxidant capability, protecting rat and human liver tissues. In conclusion, rMnSOD represents a new and highly effective therapy to significantly upgrade liver procurement for transplantation.

Effects of warm ischemia and reperfusion on the liver microcirculatory phenotype of rats: underlying mechanisms and pharmacological therapy

Warm ischemia and reperfusion (WIR) causes hepatic damage and may lead to liver failure, however the mechanisms involved are largely unknown. Here we have characterized the microcirculatory status and endothelial phenotype of livers undergoing WIR, and evaluated the use of simvastatin in WIR injury prevention. Male Wistar rats received simvastatin, or vehicle, 30 min before undergoing 60 min of partial warm ischemia (70%) followed by 2 h or 24 h of reperfusion. Hepatic and systemic hemodynamics, liver injury (AST, ALT, LDH), endothelial function (vasodilatation in response to acetylcholine), KLF2 and nitric oxide pathways, oxidative stress, inflammation (neutrophil and macrophage infiltration) and cell death were evaluated. Profound microcirculatory dysfunction occurred rapidly following WIR. This was evidenced by down-regulation of the KLF2 vasoprotective pathway, impaired vasodilatory capability and endothelial activation, altogether leading to increased hepatic vascular resistance and liver inflammation, with significant leukocyte infiltration, oxidative stress and cell death. Simvastatin preserved the hepatic endothelial phenotype, and blunted the detrimental effects of WIR on liver hemodynamics and organ integrity. In conclusion, WIR-induced injury to liver sinusoidal endothelial cells is mitigated by pre-treatment with Simvastatin probably through a KLF2-dependent mechanism.

Liraglutide improves liver microvascular dysfunction in cirrhosis: Evidence from translational studies

Hepatic stellate cells (HSC) play a key role in the development of chronic liver disease (CLD). Liraglutide, well-established in type 2 diabetes, showed anti-inflammatory and anti-oxidant properties. We evaluated the effects of liraglutide on HSC phenotype and hepatic microvascular function using diverse pre-clinical models of CLD. Human and rat HSC were in vitro treated with liraglutide, or vehicle, and their phenotype, viability and proliferation were evaluated. In addition, liraglutide or vehicle was administered to rats with CLD. Liver microvascular function, fibrosis, HSC phenotype and sinusoidal endothelial phenotype were determined. Additionally, the effects of liraglutide on HSC phenotype were analysed in human precision-cut liver slices. Liraglutide markedly improved HSC phenotype and diminished cell proliferation. Cirrhotic rats receiving liraglutide exhibited significantly improved liver microvascular function, as evidenced by lower portal pressure, improved intrahepatic vascular resistance, and marked ameliorations in fibrosis, HSC phenotype and endothelial function. The anti-fibrotic effects of liraglutide were confirmed in human liver tissue and, although requiring further investigation, its underlying molecular mechanisms suggested a GLP1-R-independent and NF-κB-Sox9-dependent one. This study demonstrates for the first time that liraglutide improves the liver sinusoidal milieu in pre-clinical models of cirrhosis, encouraging its clinical evaluation in the treatment of chronic liver disease.

Effects of aging on liver microcirculatory function and sinusoidal phenotype

The socioeconomic and medical improvements of the last decades have led to a relevant increase in the median age of worldwide population. Although numerous studies described the impact of aging in different organs and the systemic vasculature, relatively little is known about liver function and hepatic microcirculatory status in the elderly. In this study, we aimed at characterizing the phenotype of the aged liver in a rat model of healthy aging, particularly focusing on the microcirculatory function and the molecular status of each hepatic cell type in the sinusoid. Moreover, major findings of the study were validated in young and aged human livers. Our results demonstrate that healthy aging is associated with hepatic and sinusoidal dysfunction, with elevated hepatic vascular resistance and increased portal pressure. Underlying mechanisms of such hemodynamic disturbances included typical molecular changes in the cells of the hepatic sinusoid and deterioration in hepatocyte function. In a specific manner, liver sinusoidal endothelial cells presented a dysfunctional phenotype with diminished vasodilators synthesis, hepatic macrophages exhibited a proinflammatory state, while hepatic stellate cells spontaneously displayed an activated profile. In an important way, major changes in sinusoidal markers were confirmed in livers from aged humans. In conclusion, our study demonstrates for the first time that aging is accompanied by significant liver sinusoidal deregulation suggesting enhanced sinusoidal vulnerability to chronic or acute injuries.

Oxidative Stress in Liver Diseases

Abstract This chapter describes the implication of oxidative stress in the progression of the liver diseases with higher prevalence worldwide: nonalcoholic fatty liver disease, cholestasis, hepatitis C and B infections, cirrhosis, and finally hepatocellular carcinoma. The molecular mechanisms of some of these chronic diseases are still unclear; however, oxidative and nitrosative damages are implicated in the progression of all these liver diseases. Interestingly, all chronic liver diseases, independently from its etiology, are accompanied by a highly oxidative hepatic environment that favors the progression to fibrosis, cirrhosis, and ultimately hepatocellular carcinoma. In these advanced disease stages, cirrhosis and hepatocellular carcinoma, oxidative stress contributes to aggravate the cellular modifications present in these pathologies. The cellular impairment induced by oxidative stress in the liver is related with mitochondrial dysfunction as well as with the depletion of antioxidant proteins such as glutathione or superoxide dismutase. Moreover, the cellular redox imbalance produced in liver diseases induces the activation of apoptotic and inflammation signaling pathways enhancing cell death. Altogether, this chapter reviews the contribution of oxidative stress in the pathophysiology of chronic liver disease.