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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.

Older age is a major risk factor for damage to many tissues, including liver. Aging undermines resiliency and impairs liver regeneration. The mechanisms whereby aging reduces resiliency are poorly understood. Hedgehog is a signaling pathway with critical mitogenic and morphogenic functions during development. Recent studies indicate that Hedgehog regulates metabolic homeostasis in adult liver. The present study evaluates the hypothesis that Hedgehog signaling becomes dysregulated in hepatocytes during aging, resulting in decreased resiliency and therefore, impaired regeneration and enhanced vulnerability to damage. Partial hepatectomy (PH) was performed on young and old wild‐type mice and Smoothened (Smo)‐floxed mice treated with viral vectors to conditionally delete Smo and disrupt Hedgehog signaling specifically in hepatocytes. Changes in signaling were correlated with changes in regenerative responses and compared among groups. Old livers had fewer hepatocytes proliferating after PH. RNA sequencing identified Hedgehog as a top downregulated pathway in old hepatocytes before and after the regenerative challenge. Deleting Smo in young hepatocytes before PH prevented Hedgehog pathway activation after PH and inhibited regeneration. Gene Ontogeny analysis demonstrated that both old and Smo‐deleted young hepatocytes had activation of pathways involved in innate immune responses and suppression of several signaling pathways that control liver growth and metabolism. Hedgehog inhibition promoted telomere shortening and mitochondrial dysfunction in hepatocytes, consequences of aging that promote inflammation and impair tissue growth and metabolic homeostasis. Hedgehog signaling is dysregulated in old hepatocytes. This accelerates aging, resulting in decreased resiliency and therefore, impaired liver regeneration and enhanced vulnerability to damage. It is not well understood why older age is a major risk factor for damage to many tissues, including liver. Aging‐related impairments in regeneration may be involved as this limits recovery from injury. Our studies identify a mechanism that explains why aging inhibits liver regeneration by demonstrating that the activity of Hedgehog, a signaling pathway that is necessary for liver growth, is reduced in old hepatocytes.

In cirrhosis, liver microvascular dysfunction is a key factor increasing hepatic vascular resistance to portal blood flow, which leads to portal hypertension. De‐regulated inflammatory and pro‐apoptotic processes due to chronic injury play important roles in the dysfunction of liver sinusoidal cells. The present study aimed at characterizing the effects of the pan‐caspase inhibitor emricasan on systemic and hepatic hemodynamics, hepatic cells phenotype, and underlying mechanisms in preclinical models of advanced chronic liver disease. We investigated the effects of 7‐day emricasan on hepatic and systemic hemodynamics, liver function, hepatic microcirculatory function, inflammation, fibrosis, hepatic cells phenotype, and paracrine interactions in rats with advanced cirrhosis due to chronic CCl4 administration. The hepato‐protective effects of emricasan were additionally investigated in cells isolated from human cirrhotic livers. Cirrhotic rats receiving emricasan showed significantly lower portal pressure than vehicle‐treated animals with no changes in portal blood flow, indicating improved vascular resistance. Hemodynamic improvement was associated with significantly better liver function, reduced hepatic inflammation, improved phenotype of hepatocytes, liver sinusoidal endothelial cells, hepatic stellate cells and macrophages, and reduced fibrosis. In vitro experiments demonstrated that emricasan exerted its benefits directly improving hepatocytes’ expression of specific markers and synthetic capacity, and ameliorated nonparenchymal cells through a paracrine mechanism mediated by small extracellular vesicles released by hepatocytes. Conclusion: This study demonstrates that emricasan improves liver sinusoidal microvascular dysfunction in cirrhosis, which leads to marked amelioration in fibrosis, portal hypertension and liver function, and therefore encourages its clinical evaluation in the treatment of advanced chronic liver disease. One‐week emricasan promoted a significant amelioration in portal hypertension and hepatic microcirculation in experimental cirrhosis. Underlying mechanisms included direct improvement in hepatocytes phenotype, which paracrinally leads to fibrosis improvement, better endothelial function, and less inflammation.

Non-alcoholic steatohepatitis (NASH) is a major cause of chronic liver disease. However, most available animal models fail to reflect the whole spectrum of the disease. Liver fibrosis and portal hypertension are the strongest prognostic markers in advanced NASH. We herein aimed at developing a new model of NASH in male rats, obtained using a multi-hit protocol that combines the administration of a high fat and high-cholesterol diet with CCl4 and phenobarbital. Following this protocol, rats showed the full characteristics of advanced human NASH after 10 weeks and NASH with cirrhosis by 24 weeks. Specifically, our NASH rats exhibited: steatosis and metabolic syndrome, lipotoxicity, hepatocellular ballooning necrosis, inflammation and importantly, marked hepatic fibrosis and significant portal hypertension. Furthermore, a whole transcriptomic analysis of liver tissue from our rat model using next generation sequencing was compared with human NASH and illustrated the similarity of this pre-clinical model with the human disease. Pathway enrichment analysis showed that NASH animals shared a relevant number of central pathways involved in NASH pathophysiology, such as those related with cell death, as well as inflammatory or matrix remodeling. The present study defines a pre-clinical model of moderate and advanced NASH that mimics the human disease, including pathophysiologic characteristics and transcriptomic signature.

ObjectiveIn 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.DesignActivation 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.ResultsKLF2 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.ConclusionsExogenous hepatic KLF2 upregulation improves liver fibrosis, endothelial dysfunction and portal hypertension in cirrhosis.

Susceptibility to the biological consequences of aging varies among organs and individuals. We analyzed hepatocyte transcriptomes of healthy young and aged male mice to generate an aging hepatocyte gene signature, used it to deconvolute transcriptomic data from humans and mice with metabolic dysfunction-associated liver disease, validated findings with functional studies in mice and applied the signature to transcriptomic data from other organs to determine whether aging-sensitive degenerative mechanisms are conserved. We discovered that the signature enriches in diseased livers in parallel with degeneration. It is also enriched in failing human hearts, diseased kidneys and pancreatic islets from individuals with diabetes. The signature includes genes that control ferroptosis. Aged mice develop more hepatocyte ferroptosis and liver degeneration than young mice when fed diets that induce metabolic stress. Inhibiting ferroptosis shifts the liver transcriptome of old mice toward that of young mice and reverses aging-exacerbated liver damage, identifying ferroptosis as a tractable, conserved mechanism for aging-related tissue degeneration.

Portal hypertension (PH) is a common complication of chronic liver disease, and it determines most complications leading to death or liver transplantation in patients with liver cirrhosis. PH results from increased resistance to portal blood flow through the cirrhotic liver. This is caused by two mechanisms: (a) distortion of the liver vascular architecture and (b) hepatic microvascular dysfunction. Increment in hepatic resistance is latterly accompanied by splanchnic vasodilation, which further aggravates PH. Hepatic microvascular dysfunction occurs early in the course of chronic liver disease as a consequence of inflammation and oxidative stress and determines loss of the normal phenotype of liver sinusoidal endothelial cells (LSEC). The cross-talk between LSEC and hepatic stellate cells induces activation of the latter, which in turn proliferate, migrate and increase collagen deposition around the sinusoids, contributing to fibrogenesis, architectural disruption and angiogenesis. Therapy for PH aims at correcting these pathophysiological abnormalities: liver injury, fibrogenesis, increased hepatic vascular tone and splanchnic vasodilatation. Continuing liver injury may be counteracted specifically by etiological treatments, while architectural disruption and fibrosis can be ameliorated by a variety of anti-fibrogenic drugs and anti-angiogenic strategies. Sinusoidal endothelial dysfunction is ameliorated by statins and other drugs increasing NO availability. Splanchnic hyperemia can be counteracted by non-selective beta-blockers (NSBBs), vasopressin analogs and somatostatin analogs. Future treatment of portal hypertension will evolve to use etiological treatments together with anti-fibrotic agents and/or drugs improving microvascular function in initial stages of cirrhosis (pre-primary prophylaxis), while NSBBs will be added in advanced stages of the disease.

The burden of senescent hepatocytes correlates with the severity of metabolic dysfunction-associated steatotic liver disease (MASLD), but the mechanisms driving senescence and how it exacerbates MASLD are poorly understood. Hepatocytes experience lipotoxicity and become senescent when Smoothened (Smo) is deleted to disrupt Hedgehog signaling. We aimed to determine whether the secretomes of Smo-deficient hepatocytes perpetuate senescence to drive MASLD progression. RNA-Seq analysis of liver samples from human and murine cohorts with MASLD confirmed that hepatocyte populations in MASLD livers were depleted of Smo+ cells and enriched with senescent cells. When fed a choline-deficient, amino acid-restricted high-fat diet (CDA-HFD) to induce MASLD, Smo- mice had lower antioxidant markers and developed worse DNA damage, senescence, steatohepatitis, and fibrosis than did Smo+ mice. Sera and hepatocyte-conditioned medium from Smo- mice were depleted of thymidine phosphorylase (TP), a protein that maintains mitochondrial fitness. Treating Smo- hepatocytes with TP reduced senescence and lipotoxicity, whereas inhibiting TP in Smo+ hepatocytes had the opposite effect and exacerbated hepatocyte senescence, steatohepatitis, and fibrosis in CDA-HFD-fed mice. We conclude that inhibition of Hedgehog signaling in hepatocytes promoted MASLD by suppressing hepatocyte production of proteins that prevent lipotoxicity and senescence.

Cellular senescence is a stress-induced, stable cell cycle arrest phenotype which generates a pro-inflammatory microenvironment, leading to chronic inflammation and age-associated diseases. Determining the fundamental molecular pathways driving senescence instead of apoptosis could enable the identification of senolytic agents to restore tissue homeostasis. Here, we identify thrombomodulin (THBD) signaling as a key molecular determinant of the senescent cell fate. Although normally restricted to endothelial cells, THBD is rapidly upregulated and maintained throughout all phases of the senescence program in aged mammalian tissues and in senescent cell models. Mechanistically, THBD activates a proteolytic feed-forward signaling pathway by stabilizing a multi-protein complex in early endosomes, thus forming a molecular basis for the irreversibility of the senescence program and ensuring senescent cell viability. Therapeutically, THBD signaling depletion or inhibition using vorapaxar, an FDA-approved drug, effectively ablates senescent cells and restores tissue homeostasis in liver fibrosis models. Collectively, these results uncover proteolytic THBD signaling as a conserved pro-survival pathway essential for senescent cell viability, thus providing a pharmacologically exploitable senolytic target for senescence-associated diseases.

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.