Mice deficient in the putative phospholipid flippase ATP11C exhibit altered erythrocyte shape, anemia, and reduced erythrocyte life span

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11Cshowed a lower rate ofPStranslocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserineexposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.This work was supported in part by National Health and Medical Research Council Grant GNT1061288. Supported by National Health and Medical Research Council Career Development Fellowship GNT1035858 and by the Ramaciotti Foundation

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

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

Background & Aims NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. Methods We fed foz/foz and wild-type mice an atherogenic diet for 16\ua0weeks, gavaged MCC950 or vehicle until 24\ua0weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6\ua0weeks and determined the effects on liver fibrosis. Results In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24\ua0weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. Conclusion MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. Lay summary Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis

Cyclin E facilitates dysplastic hepatocytes to bypass G(1)/S checkpoint in hepatocarcinogenesis

Background and Aim: By array-comparative genomic hybridization, we demonstrated cyclin E as one of seven genes associated with hepatocellular carcinoma (HCC) development in Ku70 DNA repair-deficient mice. We therefore explored the hypothesis that during hepatocarcinogenesis, cyclin E kinase can overcome the inhibitory effects of p53 and establish whether abnormal miRNA(mi-R)-34, a co-regulator of cyclin E and p53, can account for their interactions as " drivers" of HCC. Methods: Dysplastic hepatocytes (DNs) and HCCs were generated from diethylnitrosamine (DEN)-injected C57BL/6 male mice at 3-12 months. Results: Cyclin E/cdk2 was barely expressed in normal liver, but was readily detected in dysplastic hepatocytes, localizing to glutathione-S transferase pi-form positive cells dissected by laser-dissection. Cyclin E kinase activity preceded cyclin D1, proliferating cell nuclear antigen expression in DNs and HCCs despite maximal p53 and p21 expression. We confirmed that cyclin E, rather than cyclin D1, is the proliferative driver in hepatocarcinogenesis by immunoprecipitation experiments demonstrating preferential binding of p21 to cyclin D1, allowing cyclin E-mediated " escape" from G1/S checkpoint. We then showed cyclin E was responsible for regulating wild-type p53 by knockdown experiments in primary HCC cells; cyclin E-knockdown increased p53 and p21, diminished anti-apoptotic Bcl-XL and reduced cell viability. Conversely, blocking p53 augmented cyclin E, Bcl-XL expression and increased proliferation. Physiological interactions between cyclin E/p53/ p21 were confirmed in primary hepatocytes. miR-34a, c were upregulated in dysplastic murine, human liver and HCCs compared with normal liver, and appeared to be linked to cyclin E/p53. Conclusion: Upregulation of functionally active cyclin E via miR34 with loss of p53 function is associated with cell-cycle checkpoint failure increasing proliferative drive that favors hepatocarcinogenesis

Diannexin attenuates release of MPs after hepatic IRI and decreases their pro-inflammatory and deleterious potential.

<p>MPs were obtained from mice pre-treated with 1 mg/kg iv Diannexin 5 min prior to 60 min liver IR and results compared with vehicle-treated/naïve mice subject to IR. <b>A</b>. FACS revealed significantly reduced resting endothelial cell (CD144), resting platelet (CD41) and activated platelet and endothelial cell (CD62P) subsets compared to naïve (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104376#pone-0104376-g001" target="_blank">Fig.1C</a>). <b>Bi</b>. Serum ALT after 60 min ischemia and indicated reperfusion times in naïve and Diannexin-treated mice (n = 10 per cohort). * p<0.05 all experimental groups vs. sham. § p<0.05 Diannexin vs. 24 hr reperfusion. <b>Bii</b>. Platelets were isolated from sham-operated mice and exposed to 60 nM MPs (derived from mice subjected to 60 min ischemia, 2 hr reperfusion) in the absence or presence of 4 µg/ml Diannexin. FACS then performed using CD62P to quantify activated platelet subset as a proportion of total platelets. * p<0.05 experimental vs. control. # p<0.05 Diannexin vs. 60 nM MPs. <b>C, D</b>. Hepatocyte-specific ASPGR, E-selectin and pro-inflammatory VCAM, ICAM-1 protein expression diminished by Diannexin compared to naïve mice subjected to 60 min ischemia and indicated reperfusion times. + p<0.05 Diannexin vs. sham. & p<0.05 Diannexin vs. naïve. <b>E</b>. Pre-treatment of MPs with Diannexin 4 µg/ml inhibited neutrophil transmigration in ThinCert chambers. To ascertain effect of test compounds on transmigration potential, MPs were pre-treated with Diannexin (4 µg/ml) for 1 hr 37°C; thereafter, MPs were resuspended in PBS then recovered by prolonged centrifugation twice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104376#pone.0104376.s004" target="_blank">Materials and Methods S1</a> for detailed MP isolation protocol) prior to the transwell migration assay. * p<0.05 experimental vs. control. # p<0.05 Diannexin vs. 60 nM MPs.</p

Microparticles Mediate Hepatic Ischemia-Reperfusion Injury and Are the Targets of Diannexin (ASP8597)

<div><p>Background & Aims</p><p>Ischemia–reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and other cell types release microparticles during post-ischemic reperfusion, and whether such microparticles have pro-inflammatory, platelet-activating and pro-injurious effects that could contribute to IRI pathogenesis.</p><p>Methods</p><p>C57BL6 mice underwent 60 min of partial hepatic ischemia followed by 15 min–24 hrs of reperfusion. We collected blood and liver samples, isolated circulating microparticles, and determined protein and lipid content. To establish mechanism for microparticle production, we subjected murine primary hepatocytes to hypoxia-reoxygenation. Because microparticles express everted phosphatidylserine residues that are the target of annexin V, we analyzed the effects of an annexin V-homodimer (Diannexin or ASP8597) on post-ischemia microparticle production and function.</p><p>Results</p><p>Microparticles were detected in the circulation 15–30 min after post-ischemic reperfusion, and contained markers of SECs, platelets, natural killer T cells, and CD8⁺ cells; 4 hrs later, they contained markers of macrophages. Microparticles contained F2-isoprostanes, indicating oxidative damage to membrane lipids. Injection of mice with TNF-α increased microparticle formation, whereas Diannexin substantially reduced microparticle release and prevented IRI. Hypoxia-re-oxygenation generated microparticles from primary hepatocytes by processes that involved oxidative stress. Exposing cultured hepatocytes to preparations of microparticles isolated from the circulation during IRI caused injury involving mitochondrial membrane permeability transition. Microparticles also activated platelets and induced neutrophil migration <i>in vitro</i>. The inflammatory properties of microparticles involved activation of NF-κB and JNK, increased expression of E-selectin, P-selectin, ICAM-1 and VCAM-1. All these processes were blocked by coating microparticles with Diannexin.</p><p>Conclusions</p><p>Following hepatic IRI, microparticles circulate and can be taken up by hepatocytes, where they activate signaling pathways that mediate inflammation and hepatocyte injury. Diannexin prevents microparticle formation and subsequent inflammation.</p></div