Lack of endothelial autophagy does not impair liver regeneration after partial hepatectomy in mice.

Liver sinusoidal endothelial cells (LSEC) are key elements in regulating the liver response to injury and regeneration. While endothelial autophagy is essential to protect endothelial cells from injury-induced oxidative stress and fibrosis, its role in liver regeneration has not been elucidated. This study was intended to investigate the role of endothelial autophagy in liver regeneration in the context of partial hepatectomy (PHx). Analysis of autophagy levels in rat LSEC after PHx indicated a tendency to decrease activity the first 2 days after surgery. PHx performed in mice with impaired endothelial autophagy (Atg7flox/flox ;VE-Cadherin-Cre+ ) and their littermate controls showed no differences neither in liver-to-body weight ratio, histological analysis, hepatocyte proliferation nor vascular integrity during the first 7 days after PH and liver regeneration was completely achieved. Our results indicate that endothelial autophagy does not play an essential role in the coordination of the liver regeneration process after PHx.

Impact of lifestyle interventions targeting physical exercise and caloric intake on cirrhosis regression in rats.

In patients, advanced cirrhosis only regresses partially once the etiological agent is withdrawn. Animal models for advanced cirrhosis regression are missing. Lifestyle interventions (LIs) have been shown to improve steatosis, inflammation, fibrosis, and portal pressure (PP) in liver disease. We aimed at characterizing cirrhosis regression after etiological agent removal in experimental models of advanced cirrhosis and to study the impact of different LI on it. Advanced cirrhosis was induced in rats either by carbon tetrachloride (CCl4) or by thioacetamide (TAA) administration. Systemic and hepatic hemodynamics, liver fibrosis, hepatic stellate cell (HSC) activation, hepatic macrophage infiltration, and metabolic profile were evaluated after 48 h, 4 wk or 8 wk of etiological agent removal. The impact of LI consisting in caloric restriction (CR) or moderate endurance exercise (MEE) during the 8-wk regression process was analyzed. The effect of MEE was also evaluated in early cirrhotic and in healthy rats. A significant reduction in portal pressure (PP), liver fibrosis, and HSC activation was observed during regression. However, these parameters remained above those in healthy animals. During regression, animals markedly worsened their metabolic profile. CR although preventing those metabolic disturbances did not further reduce PP, hepatic fibrosis, or HSC activation. MEE also prevented metabolic disturbances, without enhancing, but even attenuating the reduction of PP, hepatic fibrosis, and HSC activation achieved by regression. MEE also worsened hepatic fibrosis in early-TAA cirrhosis and in healthy rats.NEW & NOTEWORTHY We have developed two advanced cirrhosis regression experimental models with persistent relevant fibrosis and portal hypertension and an associated deteriorated metabolism that mimic what happens in patients. LI, despite improving metabolism, did not enhance the regression process in our cirrhotic models. CR did not further reduce PP, hepatic fibrosis, or HSC activation. MEE exhibited a profibrogenic effect in the liver blunting cirrhosis regression. One of the potential explanations of this worsening could be ammonia accumulation.

Impaired endothelial autophagy promotes liver fibrosis by aggravating the oxidative stress response during acute liver injury.

BACKGROUND & AIMS: Endothelial dysfunction plays an essential role in liver injury, yet the phenotypic regulation of liver sinusoidal endothelial cells (LSECs) remains unknown. Autophagy is an endogenous protective system whose loss could undermine LSEC integrity and phenotype. The aim of our study was to investigate the role of autophagy in the regulation of endothelial dysfunction and the impact of its manipulation during liver injury. METHODS: We analyzed primary isolated LSECs from Atg7control and Atg7endo mice as well as rats after CCl4 induced liver injury. Liver tissue and primary isolated stellate cells were used to analyze liver fibrosis. Autophagy flux, microvascular function, nitric oxide bioavailability, cellular superoxide content and the antioxidant response were evaluated in endothelial cells. RESULTS: Autophagy maintains LSEC homeostasis and is rapidly upregulated during capillarization in vitro and in vivo. Pharmacological and genetic downregulation of endothelial autophagy increases oxidative stress in vitro. During liver injury in vivo, the selective loss of endothelial autophagy leads to cellular dysfunction and reduced intrahepatic nitric oxide. The loss of autophagy also impairs LSECs ability to handle oxidative stress and aggravates fibrosis. CONCLUSIONS: Autophagy contributes to maintaining endothelial phenotype and protecting LSECs from oxidative stress during early phases of liver disease. Selectively potentiating autophagy in LSECs during early stages of liver disease may be an attractive approach to modify the disease course and prevent fibrosis progression. LAY SUMMARY: Liver endothelial cells are the first liver cell type affected after any kind of liver injury. The loss of their unique phenotype during injury amplifies liver damage by orchestrating the response of the liver microenvironment. Autophagy is a mechanism involved in the regulation of this initial response and its manipulation can modify the progression of liver damage.

Ly9 (SLAMF3) receptor differentially regulates iNKT cell development and activation in mice.

Invariant natural killer T (iNKT) cells develop into three subsets (NKT1, NKT2, and NKT17) expressing a distinct transcription factor profile, which regulates cytokine secretion upon activation. iNKT cell development in the thymus is modulated by signaling lymphocytic activation molecule family (SLAMF) receptors. In contrast to other SLAMF members, Ly9 (SLAMF3) is a non-redundant negative regulator of iNKT cell development. Here, we show that Ly9 influences iNKT cell lineage differentiation. Ly9-deficient mice on a BALB/c background contained a significantly expanded population of thymic NKT2 cells, while NKT1 cells were nearly absent in BALB/c.Ly9-/- thymus. Conversely, the number of peripheral NKT1 cells in BALB/c.Ly9-/- mice was comparable to that in wild-type mice, indicating that the homeostasis of the different iNKT cell subsets may have distinct requirements depending on their tissue localization. Importantly, Ly9 absence also promoted NKT2 cell differentiation in the NKT1-skewed C57BL/6 background. Furthermore, treatment of wild-type mice with an agonistic monoclonal antibody directed against Ly9 impaired IL-4 and IFN-γ production and reduced by half the number of spleen iNKT cells, with a significant decrease in the proportion of NKT2 cells. Thus, anti-Ly9 targeting could represent a novel therapeutic approach to modulate iNKT cell numbers and activation.

Mitochondria-targeted antioxidant mitoquinone deactivates human and rat hepatic stellate cells and reduces portal hypertension in cirrhotic rats.

BACKGROUND & AIMS: In cirrhosis, activated hepatic stellate cells (HSC) play a major role in increasing intrahepatic vascular resistance and developing portal hypertension. We have shown that cirrhotic livers have increased reactive oxygen species (ROS), and that antioxidant therapy decreases portal pressure. Considering that mitochondria produce many of these ROS, our aim was to assess the effects of the oral mitochondria-targeted antioxidant mitoquinone on hepatic oxidative stress, HSC phenotype, liver fibrosis and portal hypertension. METHODS: Ex vivo: Hepatic stellate cells phenotype was analysed in human precision-cut liver slices in response to mitoquinone or vehicle. In vitro: Mitochondrial oxidative stress was analysed in different cell type of livers from control and cirrhotic rats. HSC phenotype, proliferation and viability were assessed in LX2, and in primary human and rat HSC treated with mitoquinone or vehicle. In vivo: CCl4 - and thioacetamide-cirrhotic rats were treated with mitoquinone (5 mg/kg/day) or the vehicle compound, DecylTPP, for 2 weeks, followed by measurement of oxidative stress, systemic and hepatic haemodynamic, liver fibrosis, HSC phenotype and liver inflammation. RESULTS: Mitoquinone deactivated human and rat HSC, decreased their proliferation but with no effects on viability. In CCl4 -cirrhotic rats, mitoquinone decreased hepatic oxidative stress, improved HSC phenotype, reduced intrahepatic vascular resistance and diminished liver fibrosis. These effects were associated with a significant reduction in portal pressure without changes in arterial pressure. These results were further confirmed in the thioacetamide-cirrhotic model. CONCLUSION: We propose mitochondria-targeted antioxidants as a novel treatment approach against portal hypertension and cirrhosis.

Elafibranor upregulates the EMT-inducer S100A4 via PPARß/δ.

Elafibranor is a dual peroxisome proliferator-activated receptor (PPAR)α and ß/δ agonist that has reached a phase III clinical trial for the treatment of metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we examined the effects of elafibranor in mice fed a choline-deficient high-fat diet (CD-HFD), a model of metabolic dysfunction-associated steatohepatitis (MASH) that presents obesity and insulin resistance. Our findings revealed that elafibranor treatment ameliorated steatosis, inflammation, and fibrogenesis in the livers of CD-HFD-fed mice. Unexpectedly, elafibranor also increased the levels of the epithelial-mesenchymal transition (EMT)-promoting protein S100A4 via PPARß/δ activation. The increase in S100A4 protein levels caused by elafibranor was accompanied by changes in the levels of markers associated with the EMT program. The S100A4 induction caused by elafibranor was confirmed in the BRL-3A rat liver cells and a mouse primary hepatocyte culture. Furthermore, elafibranor reduced the levels of ASB2, a protein that promotes S100A4 degradation, while ASB2 overexpression prevented the stimulating effect of elafibranor on S100A4. Collectively, these findings reveal an unexpected hepatic effect of elafibranor on increasing S100A4 and promoting the EMT program.

GDF15 mediates the metabolic effects of PPARß/δ by activating AMPK.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) activates AMP-activated protein kinase (AMPK) and plays a crucial role in glucose and lipid metabolism. Here, we examine whether PPARß/δ activation effects depend on growth differentiation factor 15 (GDF15), a stress response cytokine that regulates energy metabolism. Pharmacological PPARß/δ activation increases GDF15 levels and ameliorates glucose intolerance, fatty acid oxidation, endoplasmic reticulum stress, and inflammation, and activates AMPK in HFD-fed mice, whereas these effects are abrogated by the injection of a GDF15 neutralizing antibody and in Gdf15-/- mice. The AMPK-p53 pathway is involved in the PPARß/δ-mediated increase in GDF15, which in turn activates again AMPK. Consistently, Gdf15-/- mice show reduced AMPK activation in skeletal muscle, whereas GDF15 administration results in AMPK activation in this organ. Collectively, these data reveal a mechanism by which PPARß/δ activation increases GDF15 levels via AMPK and p53, which in turn mediates the metabolic effects of PPARß/δ by sustaining AMPK activation.

Spermidine Supplementation Protects the Liver Endothelium from Liver Damage in Mice.

Chronic liver diseases are multifactorial and the need to develop effective therapies is high. Recent studies have shown the potential of ameliorating liver disease progression through protection of the liver endothelium. Polyamine spermidine (SPD) is a caloric restriction mimetic with autophagy-enhancing properties capable of prolonging lifespan and with a proven beneficial effect in cardiovascular disease in mice and humans. We evaluated the use of dietary supplementation with SPD in two models of liver disease (CCl4 and CDAAH diet). We analyzed the effect of SPD on endothelial dysfunction in vitro and in vivo. C57BL/6J mice were supplemented with SPD in the drinking water prior and concomitantly with CCl4 and CDAAH treatments. Endothelial autophagy deficient (Atg7endo) mice were also evaluated. Liver tissue was used to evaluate the impact of SPD prophylaxis on liver damage, endothelial dysfunction, oxidative stress, mitochondrial status, inflammation and liver fibrosis. SPD improved the endothelial response to oxidative injury in vitro and improved the liver endothelial phenotype and protected against liver injury in vivo. SPD reduced the overall liver oxidative stress and improved mitochondrial fitness. The absence of benefits in the Atg7endo mice suggests an autophagy-dependent effect of SPD. This study suggests SPD diet supplementation in early phases of disease protects the liver endothelium from oxidative stress and may be an attractive approach to modify the chronic liver disease course and halt fibrosis progression.

The Endothelium as a Driver of Liver Fibrosis and Regeneration.

Liver fibrosis is a common feature of sustained liver injury and represents a major public health problem worldwide. Fibrosis is an active research field and discoveries in the last years have contributed to the development of new antifibrotic drugs, although none of them have been approved yet. Liver sinusoidal endothelial cells (LSEC) are highly specialized endothelial cells localized at the interface between the blood and other liver cell types. They lack a basement membrane and display open channels (fenestrae), making them exceptionally permeable. LSEC are the first cells affected by any kind of liver injury orchestrating the liver response to damage. LSEC govern the regenerative process initiation, but aberrant LSEC activation in chronic liver injury induces fibrosis. LSEC are also main players in fibrosis resolution. They maintain liver homeostasis and keep hepatic stellate cell and Kupffer cell quiescence. After sustained hepatic injury, they lose their phenotype and protective properties, promoting angiogenesis and vasoconstriction and contributing to inflammation and fibrosis. Therefore, improving LSEC phenotype is a promising strategy to prevent liver injury progression and complications. This review focuses on changes occurring in LSEC after liver injury and their consequences on fibrosis progression, liver regeneration, and resolution. Finally, a synopsis of the available strategies for LSEC-specific targeting is provided.