Molecular Pathways Governing the Termination of Liver Regeneration.

The liver has the unique capacity to regenerate, and up to 70% of the liver can be removed without detrimental consequences to the organism. Liver regeneration is a complex process involving multiple signaling networks and organs. Liver regeneration proceeds through three phases: the initiation phase, the growth phase, and the termination phase. Termination of liver regeneration occurs when the liver reaches a liver-to-body weight that is required for homeostasis, the so-called "hepatostat." The initiation and growth phases have been the subject of many studies. The molecular pathways that govern the termination phase, however, remain to be fully elucidated. This review summarizes the pathways and molecules that signal the cessation of liver regrowth after partial hepatectomy and answers the question, "What factors drive the hepatostat?" SIGNIFICANCE STATEMENT: Unraveling the pathways underlying the cessation of liver regeneration enables the identification of druggable targets that will allow us to gain pharmacological control over liver regeneration. For these purposes, it would be useful to understand why the regenerative capacity of the liver is hampered under certain pathological circumstances so as to artificially modulate the regenerative processes (e.g., by blocking the cessation pathways) to improve clinical outcomes and safeguard the patient's life.

Experimental Conditions That Influence the Utility of 2'7'-Dichlorodihydrofluorescein Diacetate (DCFH2-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status.

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2',7'-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2',7'-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2',7'-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol.

Acute Liver Injury and Acute Liver Failure following Bariatric Surgery.

Bariatric surgery is the most effective treatment for obesity and improves several manifestations of the metabolic syndrome, including nonalcoholic fatty liver disease. Strict nutritional counseling after bariatric surgery is a key in realizing these outcomes. When postoperative nutrient intake or nutrient uptake is compromised, bariatric surgery can also lead to severe hepatic complications. Here, we describe 3 cases of acute liver injury and acute liver failure caused by bariatric surgery, all with different management strategies and outcomes.

Optimal Use of 2',7'-Dichlorofluorescein Diacetate in Cultured Hepatocytes.

Oxidative stress is a state that arises when the production of reactive transients overwhelms the cell's capacity to neutralize the oxidants and radicals. This state often coincides with the pathogenesis and perpetuation of numerous chronic diseases. On the other hand, medical interventions such as radiation therapy and photodynamic therapy generate radicals to selectively damage and kill diseased tissue. As a result, the qualification and quantification of oxidative stress are of great interest to those studying disease mechanisms as well as therapeutic interventions. 2',7'-Dichlorodihydrofluorescein-diacetate (DCFH2-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH2-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2',7'-dichlorodihydrofluorescein (DCFH2). The nonfluorescent DCFH2 is subsequently oxidized by reactive transients to form the fluorescent 2',7'-dichlorofluorescein (DCF). The use of DCFH2-DA in hepatocyte-derived cell lines is more challenging because of membrane transport proteins that interfere with probe uptake and retention, among several other reasons. Cancer cells share some of the physiological and biochemical features with hepatocytes, so probe-related technical issues are applicable to cultured malignant cells as well. This study therefore analyzed the in vitro properties of DCFH2-DA in cultured human hepatocytes (HepG2 cells and differentiated and undifferentiated HepaRG cells) to identify methodological and technical features that could impair proper data analysis and interpretation. The main issues that were found and should therefore be accounted for in experimental design include the following: (1) both DCFH2-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH2 oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH2-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH2-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH2-DA was applied to visualize and quantify oxidative stress in real time in HepG2 cells subjected to anoxia/reoxygenation as a source of reactive transients.

Liver Injury and Acute Liver Failure After Bariatric Surgery: An Overview of Potential Injury Mechanisms.

The obesity epidemic has caused a surge in the use of bariatric surgery. Although surgery-induced weight loss is an effective treatment of nonalcoholic fatty liver disease, it may precipitate severe hepatic complications under certain circumstances. Acute liver injury (ALI) and acute liver failure (ALF) following bariatric surgery have been reported in several case series. Although rare, ALI and ALF tend to emerge several months after bariatric surgery. If so, it can result in prolonged hospitalization, may necessitate liver transplantation, and in some cases prove fatal. However, little is known about the risk factors for developing ALI or ALF after bariatric surgery and the mechanisms of liver damage in this context are poorly defined. This review provides an account of the available data on ALI and ALF caused by bariatric surgery, with emphasis on potential injury mechanisms and the outcomes of liver transplantation for ALF after bariatric surgery.

Analysis and Optimization of Conditions for the Use of 2',7'-Dichlorofluorescein Diacetate in Cultured Hepatocytes.

Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2',7'-dichlorodihydrofluorescein-diacetate (DCFH2-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other reasons. The properties of DCFH2-DA were analyzed in hepatocytes (HepG2, HepaRG) to characterize methodological issues that could hamper data interpretation and falsely skew conclusions. Experiments were focused on probe stability in relevant media, cellular probe uptake/retention/excretion, and basal oxidant formation and metabolism. DCFH2-DA was used under optimized experimental conditions to intravitally visualize and quantify oxidative stress in real-time in HepG2 cells subjected to anoxia/reoxygenation. The most important findings were that: (1) the non-fluorescent DCFH2-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH2 oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH2-DA in cell culture medium relied on medium composition. The use of DCFH2-DA to measure oxidative stress in cultured hepatocytes comes with methodological and technical challenges, which were characterized and solved. Optimized in vitro and intravital imaging protocols were formulated to help researchers conduct proper experiments and draw robust conclusions.

Unaltered Liver Regeneration in Post-Cholestatic Rats Treated with the FXR Agonist Obeticholic Acid.

: In a previous study, obeticholic acid (OCA) increased liver growth before partial hepatectomy (PHx) in rats through the bile acid receptor farnesoid X-receptor (FXR). In that model, OCA was administered during obstructive cholestasis. However, patients normally undergo PHx several days after biliary drainage. The effects of OCA on liver regeneration were therefore studied in post-cholestatic Wistar rats. Rats underwent sham surgery or reversible bile duct ligation (rBDL), which was relieved after 7 days. PHx was performed one day after restoration of bile flow. Rats received 10 mg/kg OCA per day or were fed vehicle from restoration of bile flow until sacrifice 5 days after PHx. Liver regeneration was comparable between cholestatic and non-cholestatic livers in PHx-subjected rats, which paralleled liver regeneration a human validation cohort. OCA treatment induced ileal Fgf15 mRNA expression but did not enhance post-PHx hepatocyte proliferation through FXR/SHP signaling. OCA treatment neither increased mitosis rates nor recovery of liver weight after PHx but accelerated liver regrowth in rats that had not been subjected to rBDL. OCA did not increase biliary injury. Conclusively, OCA does not induce liver regeneration in post-cholestatic rats and does not exacerbate biliary damage that results from cholestasis. This study challenges the previously reported beneficial effects of OCA in liver regeneration in cholestatic rats.

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion.

OBJECTIVE AND BACKGROUND: Activation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice. METHODS: Plasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury. RESULTS: In patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects. CONCLUSION: HMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.

FXR agonist obeticholic acid induces liver growth but exacerbates biliary injury in rats with obstructive cholestasis.

Cholestasis impairs liver regeneration following partial liver resection (PHx). Bile acid receptor farnesoid X-receptor (FXR) is a key mediator of liver regeneration. The effects of FXR agonist obeticholic acid (OCA) on liver (re)growth were therefore studied in cholestatic rats. Animals underwent sham surgery or reversible bile duct ligation (rBDL). PHx with concurrent internal biliary drainage was performed 7 days after rBDL. Animals were untreated or received OCA (10 mg/kg/day) per oral gavage from rBDL until sacrifice. After 7 days of OCA treatment, dry liver weight increased in the rBDL + OCA group, indicating OCA-mediated liver growth. Enhanced proliferation in the rBDL + OCA group prior to PHx concurred with a rise in Ki67-positive hepatocytes, elevated hepatic Ccnd1 and Cdc25b expression, and an induction of intestinal fibroblast growth factor 15 expression. Liver regrowth after PHx was initially stagnant in the rBDL + OCA group, possibly due to hepatomegaly prior to PHx. OCA increased hepatobiliary injury markers during BDL, which was accompanied by upregulation of the bile salt export pump. There were no differences in histological liver injury. In conclusion, OCA induces liver growth in cholestatic rats prior to PHx but exacerbates biliary injury during cholestasis, likely by forced pumping of bile acids into an obstructed biliary tree.