X-box Binding Protein 1 Regulates Unfolded Protein, Acute-Phase, and DNA Damage Responses During Regeneration of Mouse Liver.

BACKGROUND & AIMS: Liver regeneration after partial hepatectomy (PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 (XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. METHODS: We performed studies with C57Bl6-J (control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. RESULTS: Expression of XBP1 by hepatocytes increased immediately after PH (priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response (DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene (Stat3). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X (H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. CONCLUSION: In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.

Fibroblast growth factor 15/19 (FGF15/19) protects from diet-induced hepatic steatosis: development of an FGF19-based chimeric molecule to promote fatty liver regeneration.

OBJECTIVE: Fibroblast growth factor 15/19 (FGF15/19), an enterokine that regulates synthesis of hepatic bile acids (BA), has been proposed to influence fat metabolism. Without FGF15/19, mouse liver regeneration after partial hepatectomy (PH) is severely impaired. We studied the role of FGF15/19 in response to a high fat diet (HFD) and its regulation by saturated fatty acids. We developed a fusion molecule encompassing FGF19 and apolipoprotein A-I, termed Fibapo, and evaluated its pharmacological properties in fatty liver regeneration. DESIGN: Fgf15-/- mice were fed a HFD. Liver fat and the expression of fat metabolism and endoplasmic reticulum (ER) stress-related genes were measured. Influence of palmitic acid (PA) on FGF15/19 expression was determined in mice and in human liver cell lines. In vivo half-life and biological activity of Fibapo and FGF19 were compared. Hepatoprotective and proregenerative activities of Fibapo were evaluated in obese db/db mice undergoing PH. RESULTS: Hepatosteatosis and ER stress were exacerbated in HFD-fed Fgf15-/- mice. Hepatic expression of Pparγ2 was elevated in Fgf15-/- mice, being reversed by FGF19 treatment. PA induced FGF15/19 expression in mouse ileum and human liver cells, and FGF19 protected from PA-mediated ER stress and cytotoxicity. Fibapo reduced liver BA and lipid accumulation, inhibited ER stress and showed enhanced half-life. Fibapo provided increased db/db mice survival and improved regeneration upon PH. CONCLUSIONS: FGF15/19 is essential for hepatic metabolic adaptation to dietary fat being a physiological regulator of Pparγ2 expression. Perioperative administration of Fibapo improves fatty liver regeneration.

Discovery and safety profiling of a potent preclinical candidate, (4-[4-[[(3R)-3-(hydroxycarbamoyl)-8-azaspiro[4.5]decan-3-yl]sulfonyl]phenoxy]-N-methylbenzamide) (CM-352), for the prevention and treatment of hemorrhage.

Discovery of potent and safe therapeutics that improve upon currently available antifibrinolytics, e.g., tranexamic acid (TXA, 1) and aprotinin, has been challenging. Matrix metalloproteinases (MMPs) participate in thrombus dissolution. Then we designed a novel series of optimized MMP inhibitors that went through phenotypic screening consisting of thromboelastometry and mouse tail bleeding. Our optimized lead compound, CM-352 (2), inhibited fibrinolysis in human whole blood functional assays and was more effective than the current standard of care, 1, in the tail-bleeding model using a 30 000 times lower dose. Moreover, 2 reduced blood loss during liver hepatectomy, while 1 and aprotinin had no effect. Molecule 2 displayed optimal pharmacokinetic and safety profiles with no evidence of thrombosis or coagulation impairment. This novel mechanism of action, targeting MMP, defines a new class of antihemorrhagic agents without interfering with normal hemostatic function. Furthermore, 2 represents a preclinical candidate for the acute treatment of bleeding.

Zolmitriptan: a novel portal hypotensive agent which synergizes with propranolol in lowering portal pressure.

OBJECTIVE: Only a limited proportion of patients needing pharmacological control of portal hypertension are hemodynamic responders to propranolol. Here we analyzed the effects of zolmitriptan on portal pressure and its potential interaction with propranolol. METHODS: ZOLMITRIPTAN, PROPRANOLOL OR BOTH WERE TESTED IN TWO RAT MODELS OF PORTAL HYPERTENSION: common bile duct ligation (CBDL) and CCl4-induced cirrhosis. In these animals we measured different hemodynamic parameters including portal venous pressure, arterial renal flow, portal blood flow and cardiac output. We also studied the changes in superior mesenteric artery perfusion pressure and in arterial wall cAMP levels induced by zolmitriptan, propranolol or both. Moreover, we determined the effect of splanchnic sympathectomy on the response of PVP to zolmitriptan. RESULTS: In both models of portal hypertension zolmitriptan induced a dose-dependent transient descent of portal pressure accompanied by reduction of portal flow with only slight decrease in renal flow. In cirrhotic rats, splanchnic sympathectomy intensified and prolonged zolmitriptan-induced portal pressure descent. Also, propranolol caused more intense and durable portal pressure fall when combined with zolmitriptan. Mesenteric artery perfusion pressure peaked for about 1 min upon zolmitriptan administration but showed no change with propranolol. However propranolol enhanced and prolonged the elevation in mesenteric artery perfusion pressure induced by zolmitriptan. In vitro studies showed that propranolol prevented the inhibitory effects of ß2-agonists on zolmitriptan-induced vasoconstriction and the combination of propranolol and zolmitriptan significantly reduced the elevation of cAMP caused by ß2-agonists. CONCLUSION: Zolmitriptan reduces portal hypertension and non-selective beta-blockers can improve this effect. Combination therapy deserves consideration for patients with portal hypertension failing to respond to non-selective beta-blockers.

Identification of fibroblast growth factor 15 as a novel mediator of liver regeneration and its application in the prevention of post-resection liver failure in mice.

OBJECTIVE: Cholestasis is associated with increased liver injury and morbidity after partial hepatectomy (PH), yet bile acids (BAs) are emerging as important mediators of liver regeneration. Fibroblast growth factor 15 (Fgf15, human FGF19) is a BA-induced ileum-derived enterokine that governs BA metabolism. We evaluated the relevance of Fgf15 in the preservation of BA homeostasis after PH and its potential role in the regenerative process. DESIGN: Liver regeneration after PH was studied in Fgf15 (-/-) and Fgf15 (+/+) mice. The effects of the BA sequestrant cholestyramine and adenovirally delivered Fgf15 were examined in this model. The role of Fgf15 in BA-induced liver growth was tested in Fgf15 (-/-) mice upon cholic acid (CA) feeding. The direct mitogenic effect of Fgf15 was evaluated in cultured mouse hepatocytes and cholangiocytes. RESULTS: Fgf15 (-/-) mice showed marked liver injury and mortality after PH accompanied by persistently elevated intrahepatic BA levels. Cholestyramine feeding and adenovirally delivered Fgf15 reduced BA levels and significantly prevented this lethal outcome. Fgf15 also reduced mortality after extensive hepatectomy in Fgf15(+/+) animals. Liver growth elicited by CA feeding was significantly diminished in Fgf15 (-/-) mice. Proliferation of hepatocytes and cholangiocytes was also noticeably reduced in CA-fed Fgf15 (-/-) mice. Fgf15 induced intracellular signalling and proliferation of cultured hepatocytes and cholangiocytes. CONCLUSIONS: Fgf15 is necessary to maintain BA homeostasis and prevent liver injury during liver regeneration. Moreover, Fgf15 is an essential mediator of the liver growth-promoting effects of BA. Preoperative administration of this enterokine to patients undergoing liver resection might be useful to reduce damage and foster regeneration.

Lack of Abcc3 expression impairs bile-acid induced liver growth and delays hepatic regeneration after partial hepatectomy in mice.

BACKGROUND & AIMS: Bile acids (BA) are increasingly recognized as important modulators of liver regeneration. Increased enterohepatic BA flux has been proposed to generate specific signals that activate hepatocyte proliferation after partial hepatectomy (PH). We have investigated the role of the BA membrane transporter Mrp3 (Abcc3), which is expressed in the liver and gut, in the hepatic growth response elicited by BA and in liver regeneration after PH. METHODS: Liver growth and regeneration, and the expression of growth-related genes, were studied in Mrp3(+/+) and Mrp3(-/-) mice fed a cholic acid (CA) supplemented diet and after 2/3 PH. Activation of the BA receptor FXR was measured in mice after in vivo transduction of the liver with a FXR-Luciferase reporter plasmid. BA levels were measured in portal serum and liver tissue by high performance liquid chromatography-tandem mass spectrometry. RESULTS: Liver growth elicited by CA feeding was significantly reduced in Mrp3(-/-) mice. These animals showed reduced FXR activation in the liver after CA administration and decreased portal serum levels of BA. Liver regeneration after PH was significantly delayed in Mrp3-deficient mice. Proliferation-related gene expression and peak DNA synthesis in Mrp3(-/-) mice occurred later than in wild types, coinciding with a retarded elevation in intra-hepatic BA levels. CONCLUSIONS: Lack of Abcc3 expression markedly impairs liver growth in response to BA and after PH. Our data suggest that Mrp3 plays a non-redundant role in the regulation of BA flux during liver regeneration.