Circulating Extracellular Vesicles Carrying Sphingolipid Cargo for the Diagnosis and Dynamic Risk Profiling of Alcoholic Hepatitis.

BACKGROUND AND AIMS: Alcoholic hepatitis (AH) is diagnosed by clinical criteria, although several objective scores facilitate risk stratification. Extracellular vesicles (EVs) have emerged as biomarkers for many diseases and are also implicated in the pathogenesis of AH. Therefore, we investigated whether plasma EV concentration and sphingolipid cargo could serve as diagnostic biomarkers for AH and inform prognosis to permit dynamic risk profiling of AH subjects. APPROACH AND RESULTS: EVs were isolated and quantified from plasma samples from healthy controls, heavy drinkers, and subjects with end-stage liver disease (ESLD) attributed to cholestatic liver diseases and nonalcoholic steatohepatitis, decompensated alcohol-associated cirrhosis (AC), and AH. Sphingolipids were quantified by tandem mass spectroscopy. The median plasma EV concentration was significantly higher in AH subjects (5.38 × 1011 /mL) compared to healthy controls (4.38 × 1010 /mL; P < 0.0001), heavy drinkers (1.28 × 1011 /mL; P < 0.0001), ESLD (5.35 × 1010 /mL; P < 0.0001), and decompensated AC (9.2 × 1010 /mL; P < 0.0001) disease controls. Among AH subjects, EV concentration correlated with Model for End-Stage Liver Disease score. When EV counts were dichotomized at the median, survival probability for AH subjects at 90 days was 63.0% in the high-EV group and 90.0% in the low-EV group (log-rank P value = 0.015). Interestingly, EV sphingolipid cargo was significantly enriched in AH when compared to healthy controls, heavy drinkers, ESLD, and decompensated AC (P = 0.0001). Multiple sphingolipids demonstrated good diagnostic and prognostic performance as biomarkers for AH. CONCLUSIONS: Circulating EV concentration and sphingolipid cargo signature can be used in the diagnosis and differentiation of AH from heavy drinkers, decompensated AC, and other etiologies of ESLD and predict 90-day survival permitting dynamic risk profiling.

IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis.

BACKGROUND & AIMS: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1αΔhep) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4µ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.

Modulating sphingosine 1-phosphate receptor signaling skews intrahepatic leukocytes and attenuates murine nonalcoholic steatohepatitis.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid associated with nonalcoholic steatohepatitis (NASH). Immune cell-driven inflammation is a key determinant of NASH progression. Macrophages, monocytes, NK cells, T cells, NKT cells, and B cells variably express S1P receptors from a repertoire of 5 receptors termed S1P1 - S1P5. We have previously demonstrated that non-specific S1P receptor antagonism ameliorates NASH and attenuates hepatic macrophage accumulation. However, the effect of S1P receptor antagonism on additional immune cell populations in NASH remains unknown. We hypothesized that S1P receptor specific modulation may ameliorate NASH by altering leukocyte recruitment. A murine NASH model was established by dietary feeding of C57BL/6 male mice with a diet high in fructose, saturated fat, and cholesterol (FFC) for 24 weeks. In the last 4 weeks of dietary feeding, the mice received the S1P1,4,5 modulator Etrasimod or the S1P1 modulator Amiselimod, daily by oral gavage. Liver injury and inflammation were determined by histological and gene expression analyses. Intrahepatic leukocyte populations were analyzed by flow cytometry, immunohistochemistry, and mRNA expression. Alanine aminotransferase, a sensitive circulating marker for liver injury, was reduced in response to Etrasimod and Amiselimod treatment. Liver histology showed a reduction in inflammatory foci in Etrasimod-treated mice. Etrasimod treatment substantially altered the intrahepatic leukocyte populations through a reduction in the frequency of T cells, B cells, and NKT cells and a proportional increase in CD11b+ myeloid cells, polymorphonuclear cells, and double negative T cells in FFC-fed and control standard chow diet (CD)-fed mice. In contrast, FFC-fed Amiselimod-treated mice showed no changes in the frequencies of intrahepatic leukocytes. Consistent with the improvement in liver injury and inflammation, hepatic macrophage accumulation and the gene expression of proinflammatory markers such as Lgals3 and Mcp-1 were decreased in Etrasimod-treated FFC-fed mice. Etrasimod treated mouse livers demonstrated an increase in non-inflammatory (Marco) and lipid associated (Trem2) macrophage markers. Thus, S1P1,4,5 modulation by Etrasimod is more effective than S1P1 antagonism by Amiselimod, at the dose tested, in ameliorating NASH, likely due to the alteration of leukocyte trafficking and recruitment. Etrasimod treatment results in a substantial attenuation of liver injury and inflammation in murine NASH.

A Comparative Proteomic Analysis of Extracellular Vesicles Associated With Lipotoxicity.

Extracellular vesicles (EVs) are emerging mediators of intercellular communication in nonalcoholic steatohepatitis (NASH). Palmitate, a lipotoxic saturated fatty acid, activates hepatocellular endoplasmic reticulum stress, which has been demonstrated to be important in NASH pathogenesis, including in the release of EVs. We have previously demonstrated that the release of palmitate-stimulated EVs is dependent on the de novo synthesis of ceramide, which is trafficked by the ceramide transport protein, STARD11. The trafficking of ceramide is a critical step in the release of lipotoxic EVs, as cells deficient in STARD11 do not release palmitate-stimulated EVs. Here, we examined the hypothesis that protein cargoes are trafficked to lipotoxic EVs in a ceramide-dependent manner. We performed quantitative proteomic analysis of palmitate-stimulated EVs in control and STARD11 knockout hepatocyte cell lines. Proteomics was performed on EVs isolated by size exclusion chromatography, ultracentrifugation, and density gradient separation, and EV proteins were measured by mass spectrometry. We also performed human EV proteomics from a control and a NASH plasma sample, for comparative analyses with hepatocyte-derived lipotoxic EVs. Size exclusion chromatography yielded most unique EV proteins. Ceramide-dependent lipotoxic EVs contain damage-associated molecular patterns and adhesion molecules. Haptoglobin, vascular non-inflammatory molecule-1, and insulin-like growth factor-binding protein complex acid labile subunit were commonly detected in NASH and hepatocyte-derived ceramide-dependent EVs. Lipotoxic EV proteomics provides novel candidate proteins to investigate in NASH pathogenesis and as diagnostic biomarkers for hepatocyte-derived EVs in NASH patients.

Improving medication safety by cloud technology: Progression and value-added applications in Taiwan.

OBJECTIVES: To develop and implement an integrated cloud technology with the aim of ensuring medication reconciliation during transitions of care and improve medication safety in aged societies. METHODS: PharmaCloud is a new technical platform adopted by the National Health Insurance Administration of Taiwan to collect patients' medication information via cloud technology. Using this platform, healthcare providers can access patients' medication-related information with patient consent. Our hospital applied this technology and developed several approaches to collect and detect medication-related information and alert physicians for the purpose of enhancing patients' medication safety. In addition, pharmacists were involved in the admission process to access medication data and provide optimal suggestions to physicians. Several indicators, including a reduction in the number of drug items in each prescription and medication expenditure, were employed to evaluate the overall effects of the cloud inquiry. RESULTS: After the application of PharmaCloud, the average number of prescribed drug items significantly decreased (change of 0.04 to -0.35 per prescription, p < 0.05), and the median medication expenditure significantly decreased by an average of 3.55 USD, (p < 0.05) per prescription. Intra-hospital medication duplication rates also showed a downward trend. CONCLUSIONS: The use of the cloud technology and value-added applications significantly improved patient medication safety. Further long-term beneficial effects in terms of medication safety and medical cost savings are expected.

Hepatocyte-Derived Lipotoxic Extracellular Vesicle Sphingosine 1-Phosphate Induces Macrophage Chemotaxis.

Background: The pathophysiology of non-alcoholic steatohepatitis involves hepatocyte lipotoxicity due to excess saturated free fatty acids and concomitant proinflammatory macrophage effector responses. These include the infiltration of macrophages into hepatic cords in response to incompletely understood stimuli. Stressed hepatocytes release an increased number of extracellular vesicles (EVs), which are known to participate in intercellular signaling and coordination of the behavior of immune cell populations via their cargo. We hypothesized that hepatocyte-derived lipotoxic EVs that are enriched in sphingosine 1-phosphate (S1P) are effectors of macrophage infiltration in the hepatic microenvironment. Methods: Lipotoxic EVs were isolated from palmitate treated immortalized mouse hepatocytes and characterized by nanoparticle tracking analysis. Lipotoxic EV sphingolipids were quantified using tandem mass spectrometry. Wildtype and S1P1 receptor knockout bone marrow-derived macrophages were exposed to lipotoxic EV gradients in a microfluidic gradient generator. Macrophage migration toward EV gradients was captured by time-lapse microscopy and analyzed to determine directional migration. Fluorescence-activated cell sorting along with quantitative PCR and immunohistochemistry were utilized to characterize the cell surface expression of S1P1 receptor on intrahepatic leukocytes and hepatic expression of S1P1 receptor, respectively. Results: Palmitate treatment induced the release of EVs. These EVs were enriched in S1P. Palmitate-induced S1P enriched EVs were chemoattractive to macrophages. EV S1P enrichment depended on the activity of sphingosine kinases 1 and 2, such that, pharmacological inhibition of sphingosine kinases 1 and 2 resulted in a significant reduction in EV S1P cargo without affecting the number of EVs released. When exposed to EVs derived from cells treated with palmitate in the presence of a pharmacologic inhibitor of sphingosine kinases 1 and 2, macrophages displayed diminished chemotactic behavior. To determine receptor-ligand specificity, we tested the migration responses of macrophages genetically deleted in the S1P1 receptor toward lipotoxic EVs. S1P1 receptor knockout macrophages displayed a marked reduction in their chemotactic responses toward lipotoxic palmitate-induced EVs. Conclusions:Palmitate-induced lipotoxic EVs are enriched in S1P through sphingosine kinases 1 and 2. S1P-enriched EVs activate persistent and directional macrophage chemotaxis mediated by the S1P1 receptor, a potential signaling axis for macrophage infiltration during hepatic lipotoxicity, and a potential therapeutic target for non-alcoholic steatohepatitis.

CLL2-1, a chemical derivative of orchid 1,4-phenanthrenequinones, inhibits human platelet aggregation through thiol modification of calcium-diacylglycerol guanine nucleotide exchange factor-I (CalDAG-GEFI).

CalDAG-GEFI is a guanine nucleotide exchange factor, which actives small GTPase Rap1 and plays an important role in platelet aggregation. Our previous study has shown that CalDAG-GEFI contains redox-sensitive thiols, and its function can be inhibited by thiol modification. In the present study, the effect of CLL2-1, a 1,4-phenanthrenequinone, on CalDAG-GEFI and platelet functions was investigated. In human platelets, CLL2-1 prevented platelet aggregation caused by various stimulators. Flow cytometric analysis revealed that CLL2-1 inhibited GPIIb/IIIa activation and P-selectin secretion. Moreover, CLL2-1 prevented Rap1 activation caused by thrombin, the Ca(2+) ionophore A23187, and the diacylglycerol mimetic phorbol 12-myristate 13-acetate, while only slightly inhibited thrombin-induced increases in [Ca(2+)]i and did not inhibit protein kinase C activation. Western blots after reducing SDS-PAGE showed that treatment of either platelets or platelet lysates with CLL2-1 led to a decrease of monomeric CalDAG-GEFI and appearance of cross-linked oligomers of CalDAG-GEFI, and these effects were inhibited by pretreatment of platelets or lysates with thiol reducing agents prior to the addition of CLL2-1, indicating thiol modification of CalDAG-GEFI by CLL2-1. Furthermore, the thiol reducing agents also prevented the inhibitory effect of CLL2-1 on Rap1 activation, GPIIb/IIIa activation, and platelet aggregation. In CalDAG-GEFI-overexpressing human embryonic kidney 293T cells, CLL2-1 also inhibited CalDAG-GEFI-mediated Rap1 activation. Taken together, our results suggest that the antiplatelet effect of CLL2-1 is due to, at least in part, inhibition of CalDAG-GEFI-mediated Rap1 activation, and provide the basis for development of novel antiplatelet drugs.

Identification of CalDAG-GEFI as an intracellular target for the vicinal dithiol binding agent phenylarsine oxide in human platelets.

CalDAG-GEFI, a guanine nucleotide exchange factor activating Rap1, is known to play a key role in Ca2+-dependent glycoprotein (GP)IIb/IIIa activation and platelet aggregation. Although inhibition of CalDAG-GEFI could be a potential strategy for antiplatelet therapy, no inhibitor of this protein has been identified. In the present study, phenylarsine oxide (PAO), a vicinal dithiol blocker, potently prevented Rap1 activation in thrombin-stimulated human platelets without significantly inhibiting intracellular Ca2+ mobilisation and protein kinase C activation. PAO also prevented the Ca2+ ionophore-induced Rap1 activation and platelet aggregation, which are dependent on CalDAG-GEFI. In the biotin-streptavidin pull-down assay, CalDAG-GEFI was efficiently pull-downed by streptavidin beads from the lysates of biotin-conjugated PAO-treated platelets, suggesting that PAO binds to intracellular CalDAG-GEFI with high affinity. The above effects of PAO were reversed by a vicinal dithiol compound 2,3-dimercaptopropanol. In addition, CalDAG-GEFI formed disulfide-linked oligomers in platelets treated with the thiol-oxidant diamide, indicating that CalDAG-GEFI contains redox-sensitive thiols. In a purified recombinant protein system, PAO directly inhibited CalDAG-GEFI-stimulated GTP binding to Rap1. Using CalDAG-GEFI and Rap1-overexpressed human embryonic kidney 293T cells, we further confirmed that PAO abolished Ca2+-mediated Rap1 activation. Taken together, these results have demonstrated that CalDAG-GEFI is one of the targets of action of PAO, and propose an important role of vicinal cysteines for the functions of CalDAG-GEFI.

The roles and mechanisms of PAR4 and P2Y12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation.

BACKGROUND AND PURPOSE: Activation of human platelets by thrombin is mediated predominately through two proteinase-activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3-kinase (PI3K) inhibition leads to reversible PAR1-mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated. EXPERIMENTAL APPROACH: The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC-1. Western blotting and fluo-3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca(2+) mobilization respectively. KEY RESULTS: When PAR4 function was inhibited either by the PAR4 antagonist YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin-elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD-3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin-stimulated platelets. The aggregation-reversing activity mainly resulted from inhibition of both PI3K-dependent PKC activation and PAR4-mediated sustained intracellular Ca(2+) rises. Blockade of ADP P2Y(12) receptor with 2-methylthioadenosine 5'-monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K-dependent PKC activation and its ability to reverse PAR1-activating peptide-induced platelet aggregation. Co-administration of 2-methylthioadenosine 5'-monophosphate triethylammonium salt with YD-3 also decreased the stability of thrombin-induced platelet aggregation. CONCLUSIONS AND IMPLICATIONS: These results suggest that PAR4 acts in parallel with the P2Y(12)/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.