Giusi Marrone

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.

KLF2 exerts antifibrotic and vasoprotective effects in cirrhotic rat livers: behind the molecular mechanisms of statins

Objective In the liver, the transcription factor, Kruppel-like factor 2 (KLF2), is induced early during progression of cirrhosis to lessen the development of vascular dysfunction; nevertheless, its endogenous expression results insufficient to attenuate establishment of portal hypertension and aggravation of cirrhosis. Herein, we aimed to explore the effects and the underlying mechanisms of hepatic KLF2 overexpression in in vitro and in vivo models of liver cirrhosis. Design Activation phenotype was evaluated in human and rat cirrhotic hepatic stellate cells (HSC) treated with the pharmacological inductor of KLF2 simvastatin, with adenovirus codifying for this transcription factor (Ad-KLF2), or vehicle, in presence/absence of inhibitors of KLF2. Possible paracrine interactions between parenchymal and non-parenchymal cells overexpressing KLF2 were studied. Effects of in vivo hepatic KLF2 overexpression on liver fibrosis and systemic and hepatic haemodynamics were assessed in cirrhotic rats. Results KLF2 upregulation profoundly ameliorated HSC phenotype (reduced α-smooth muscle actin, procollagen I and oxidative stress) partly via the activation of the nuclear factor (NF)-E2-related factor 2 (Nrf2). Coculture experiments showed that improvement in HSC phenotype paracrinally ameliorated liver sinusoidal endothelial cells probably through a vascular endothelial growth factor-mediated mechanism. No paracrine interactions between hepatocytes and HSC were observed. Cirrhotic rats treated with simvastatin or Ad-KLF2 showed hepatic upregulation in the KLF2-Nrf2 pathway, deactivation of HSC and prominent reduction in liver fibrosis. Hepatic KLF2 overexpression was associated with lower portal pressure (–15%) due to both attenuations in the increased portal blood flow and hepatic vascular resistance, together with a significant improvement in hepatic endothelial dysfunction. Conclusions Exogenous hepatic KLF2 upregulation improves liver fibrosis, endothelial dysfunction and portal hypertension in cirrhosis.

Addition of simvastatin to cold storage solution prevents endothelial dysfunction in explanted rat livers.

Pathophysiological alterations in the endothelial phenotype result in endothelial dysfunction. Flow cessation, occurring during organ procurement for transplantation, triggers the endothelial dysfunction characteristic of ischemia/reperfusion injury, partly due to a reduction in the expression of the vasoprotective transcription factor Kruppel‐like Factor 2 (KLF2). We aimed at (1) characterizing the effects of flow cessation and cold storage on hepatic endothelial phenotype, and (2) ascertaining if the consequences of cold stasis on the hepatic endothelium can be pharmacologically modulated, improving liver graft function. Expression of KLF2 and its vasoprotective programs was determined in (i) hepatic endothelial cells (HEC) incubated under cold storage conditions with or without the KLF2‐inducer simvastatin, and (ii) rat livers not cold stored or preserved in cold University of Wisconsin solution (UWS) supplemented with simvastatin or its vehicle. In addition, upon warm reperfusion hepatic vascular resistance, endothelial function, nitric oxide vasodilator pathway, apoptosis, inflammation, and liver injury were evaluated in not cold stored livers or livers preserved in cold UWS supplemented with simvastatin or vehicle. Expression of KLF2 and its vasoprotective programs decrease in HEC incubated under cold storage conditions. Cold‐stored rat livers exhibit a time‐dependent decrease in KLF2 and its target genes, liver injury, increased hepatic vascular resistance, and endothelial dysfunction. The addition of simvastatin to the storage solution, maintained KLF2‐dependent vasoprotective programs, prevented liver damage, inflammation, and oxidative stress and improved endothelial dysfunction. Conclusion: Our results provide a rationale to evaluate the beneficial effects of a vasoprotective preservation solution on human liver procurement for transplantation. (Hepatology 2012)

Sinusoidal communication in liver fibrosis and regeneration

Cellular crosstalk is a process through which a message is transmitted within an individual cell (intracellular crosstalk) or between different cells (intercellular crosstalk). Intercellular crosstalk within the liver microenvironment is critical for the maintenance of normal hepatic functions and for cells survival. Hepatic cells are closely connected to each other, work in synergy, and produce molecules that modulate their differentiation and activity. This review summarises the current knowledge regarding paracrine communication networks in parenchymal and non-parenchymal cells in liver fibrosis due to chronic injury, and regeneration after partial hepatectomy.

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.

Leptin receptor blockade reduces intrahepatic vascular resistance and portal pressure in an experimental model of rat liver cirrhosis

Increased hepatic vascular resistance mainly due to elevated vascular tone and to fibrosis is the primary factor in the development of portal hypertension in cirrhosis. Leptin, a hormone associated with reduction in nitric oxide bioavailability, vascular dysfunction, and liver fibrosis, is increased in patients with cirrhosis. We aimed at evaluating whether leptin influences the increased hepatic resistance in portal hypertension. CCl4-cirrhotic rats received the leptin receptor-blocker ObR antibody, or its vehicle, every other day for 1 wk. Hepatic and systemic hemodynamics were measured in both groups. Hepatic nitric oxide production and bioavailability, together with oxidative stress, nitrotyrosinated proteins, and liver fibrosis, were evaluated. In cirrhotic rats, leptin-receptor blockade significantly reduced portal pressure without modifying portal blood flow, suggesting a reduction in the intrahepatic resistance. Portal pressure reduction was associated with increased nitric oxide bioavailability and with decreased O2(-) levels and nitrotyrosinated proteins. No changes in systemic hemodynamics and liver fibrosis were observed. In conclusion, the present study shows that blockade of the leptin signaling pathway in cirrhosis significantly reduces portal pressure. This effect is probably due to a nitric oxide-mediated reduction in the hepatic vascular tone.

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro.

The adenosine monophosphate–activated protein kinase—vacuolar adenosine triphosphatase–pH axis: A key regulator of the profibrogenic phenotype of human hepatic stellate cells

Liver fibrosis and cirrhosis are characterized by activation of hepatic stellate cells (HSCs), which is associated with higher intracellular pH (pHi). The vacuolar H+ adenosine‐triphosphatase (v‐ATPase) multisubunit complex is a key regulator of pHi homeostasis. The present work investigated the functional role of v‐ATPase in primary human HSC (hHSC) activation and its modulation by specific adenosine monophosphate–activated protein kinase (AMPK) subunits. We demonstrate that the expression of different v‐ATPase subunits was increased in in vivo and in vitro activated hHSCs compared to nonactivated hHSCs. Specific inhibition of v‐ATPase with bafilomycin and KM91104 induced a down‐regulation of the HSC fibrogenic gene profile, which coincided with increased lysosomal pH, decreased pHi, activation of AMPK, reduced proliferation, and lower metabolic activity. Similarly, pharmacological activation of AMPK by treatment with diflunisal, A769662, and ZLN024 reduced the expression of v‐ATPase subunits and profibrogenic markers. v‐ATPase expression was differently regulated by the AMPK α1 subunit (AMPKα1) and AMPKα2, as demonstrated in mouse embryo fibroblasts specifically deficient for AMPK α subunits. In addition, activation of v‐ATPase in hHSCs was shown to be AMPKα1‐dependent. Accordingly, pharmacological activation of AMPK in AMPKα1‐depleted hHSCs prevented v‐ATPase down‐regulation. Finally, we showed that v‐ATPase expression was increased in fibrotic livers from bile duct–ligated mice and in human cirrhotic livers. Conclusion: The down‐regulation of v‐ATPase might represent a promising target for the development of antifibrotic strategies. (Hepatology 2018).Liver fibrosis and cirrhosis are characterized by activation of hepatic stellate cells (HSC) which is associated with higher intracellular pH (pHi). The vacuolar H adenosine-triphosphatase (v-ATPase) multi-subunit complex is a key regulator of intracellular pH homeostasis. The present work was aimed at investigating the functional role of vATPase in primary human HSC (hHSC) activation and its modulation by specific AMPK subunits. Here, we demonstrated that the expression of different v-ATPase subunits was increased in in vivo and in vitro activated hHSC, compared to non-activated hHSC. Specific inhibition of v-ATPase with Bafilomycin and KM91104 induced a downregulation of the HSC fibrogenic gene profile, which coincided with increased lysosomal pH, decreased pHi, activation of AMPK, reduced proliferation, and a lower metabolic activity. Similarly, pharmacological activation of AMPK by treatment with Diflunisal, A769662 and ZLN024, reduced the expression of v-ATPase subunits and pro-fibrogenic markers. V-ATPase expression was differently regulated by AMPKα1 and AMPKα2, as demonstrated in mouse embryo fibroblasts (MEF) specific deficient for AMPKα subunits. Page 6 of 51 Hepatology Hepatology This article is protected by copyright. All rights reserved.

Urea cycle dysregulation in non-alcoholic fatty liver disease

BACKGROUND & AIMS In non-alcoholic steatohepatitis (NASH), the function of urea cycle enzymes (UCEs) may be affected, resulting in hyperammonemia and the risk of disease progression. We aimed to determine whether the expression and function of UCEs are altered in an animal model of NASH and in patients with non-alcoholic fatty liver disease (NAFLD), and whether this process is reversible. METHODS Rats were first fed a high-fat, high-cholesterol diet for 10 months to induce NASH, before being switched onto a normal chow diet to recover. In humans, we obtained liver biopsies from 20 patients with steatosis and 15 with NASH. Primary rat hepatocytes were isolated and cultured with free fatty acids. We measured the gene and protein expression of ornithine transcarbamylase (OTC) and carbamoylphosphate synthetase (CPS1), as well as OTC activity, and ammonia concentrations. Moreover, we assessed the promoter methylation status of OTC and CPS1 in rats, humans and steatotic hepatocytes. RESULTS In NASH animals, gene and protein expression of OTC and CPS1, and the activity of OTC, were reversibly reduced. Hypermethylation of Otc promoter genes was also observed. Additionally, in patients with NAFLD, OTC enzyme concentration and activity were reduced and ammonia concentrations were increased, which was further exacerbated in those with NASH. Furthermore, OTC and CPS1 promoter regions were hypermethylated. In primary hepatocytes, induction of steatosis was associated with Otc promoter hypermethylation, a reduction in the gene expression of Otc and Cps1, and an increase in ammonia concentration in the supernatant. CONCLUSION NASH is associated with a reduction in the gene and protein expression, and activity, of UCEs. This results in hyperammonemia, possibly through hypermethylation of UCE genes and impairment of urea synthesis. Our investigations are the first to describe a link between NASH, the function of UCEs, and hyperammonemia, providing a novel therapeutic target. LAY SUMMARY In patients with fatty liver disease, the enzymes that convert nitrogen waste into urea may be affected, leading to the accumulation of ammonia, which is toxic. This accumulation of ammonia can lead to scar tissue development, increasing the risk of disease progression. In this study, we show that fat accumulation in the liver produces a reversible reduction in the function of the enzymes that are involved in detoxification of ammonia. These data provide potential new targets for the treatment of fatty liver disease.

O148 KLF2 UP-REGULATION DECREASES HEPATIC FIBROSIS AND AMELIORATES ENDOTHELIAL DYSFUNCTION IN CIRRHOTIC RATS

O148 KLF2 UP-REGULATION DECREASES HEPATIC FIBROSIS AND AMELIORATES ENDOTHELIAL DYSFUNCTION IN CIRRHOTIC RATS G. Marrone, R. Maeso-Diaz, G. Garcia-Cardena, J.C. Garcia-Pagan, J. Bosch, J. Gracia-Sancho. Barcelona Hepatic Hemodynamic Lab, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Hospital Cĺinic de Barcelona, CIBEREHD, University of Barcelona Medical School, Barcelona, Spain; Center for Excellence in Vascular Biology, Harvard University, Boston, MA, United States E-mail: jgracia@clinic.cat