Hepatic stellate cell-derived exosomes modulate macrophage inflammatory response.

BACKGROUND: Hepatic stellate cell (HSC) differentiation/activation is central to liver fibrosis and is innately linked to the immune response to liver injury. Exosomes (EXOs) are important means of communication between cell populations. This study sought to characterize EXO release from HSCs and the effect of HSC-EXOs on macrophage cytokine release/function. METHODS: Liver from a rat fibrosis model was analyzed for EXO expression and localization. Quiescent and culture-activated rat and mouse HSCs and activated human HSCs were analyzed for microRNA expression. Mouse, rat, and human HSCs were culture-activated and EXOs purified from culture medium prior to addition to macrophages, and interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) mRNA and protein measured. The effect of activated HSC-EXOs on macrophage migration was assayed. RESULTS: Activation of rat HSCs led to increased EXO production in vivo, an effect mirrored by in vitro rat HSC culture-activation. Culture activation of mouse and rat HSCs led to altered EXO microRNA profiles, with a similar microRNA profile detected in activated human HSCs. Addition of activated HSC-EXOs to macrophages stimulated IL-6 and TNFα mRNA expression and protein secretion in mouse and human macrophages, but not for rat HSC-EXO-macrophages. Addition of human EXOs to macrophages stimulated migration, effects mirrored by the direct addition of rhIL-6 and rhTNFα. CONCLUSIONS: HSC-EXOs associate with macrophages and stimulate cytokine synthesis-release and macrophage migration. Constructing a comprehensive understanding of EXO interactions between liver cell populations in the setting of inflammation/fibrosis increases the potential for developing new diagnostic/therapeutic approaches.

Analysis of Plasma Tenascin-C in Post-HCV Cirrhosis: A Prospective Study.

BACKGROUND AND AIM: Hepatitis C virus (HCV)-related cirrhosis, one of the most common etiologies of liver cirrhosis in the Western world, is a risk factor for hepatocellular carcinoma. To confirm and improve current effectiveness of screening and prognosis of patients with established cirrhosis, a credible, simple plasma biomarker is needed. Hepatic stellate cell activation, a pivotal event in cirrhosis development, results in increased secretion of extracellular matrix proteins, including tenascin-C (TnC). Herein, we tested TnC as a simple biomarker to identify cirrhotic patients with active HCV infection from those with HCV eradication. METHODS: A prospective study of subjects with HCV-related cirrhosis, stratified into two groups, HCV or virologic cure, was conducted. Plasma TnC expression was measured by ELISA and Western blots. TnC values were correlated with markers of liver injury and ROC analyses performed between groups. RESULTS: The HCV cirrhotic cohort, consisting mostly of men (56%), Caucasians (76%), and genotype 1a or 1b (84%), was compared to healthy controls (HCs). Plasma TnC was significantly higher in HCV cirrhotic patients with active infection compared to HCs (P < 0.0001) and virologic cure (P < 0.0001). TnC concentrations in virologic cure subjects were not statistically different from HCs. TnC levels correlated with AST, platelets, MELD, APRI, FIB-4, and Child-Pugh score. TnC and AST together were significantly better indicators of cirrhosis in patients with active HCV infection than other markers tested. CONCLUSIONS: TnC and AST provided the best model for discriminating HCV cirrhotics with active infection from HC and virologic cure cohorts over current liver injury markers, suggesting TnC as a potential indicator of ongoing hepatic injury and inflammation.

Adeno-Associated Virus Serotype 2 Vector-Mediated Reintroduction of microRNA-19b Attenuates Hepatic Fibrosis.

Fibrotic liver injury is a significant healthcare burden in the United States. It represents a major cause of morbidity and mortality for which there are no effective Food and Drug Administration-approved treatment strategies. Fibrosis is considered a disruption of the normal wound healing responses mediated by fibroblastic cells, which are triggered and sustained by pro-fibrotic cytokines such as transforming growth factor beta 1 (TGF-ß1). TGF-ß1-mediated trans-differentiation of hepatic stellate cells (HSCs) from quiescent to activated myofibroblasts is a pivotal event in the development of fibrosis. Activation is accompanied by global changes in microRNA (miR) expression. It has been previously reported that miR19b is decreased in activated HSCs and contributes to increased expression of TGF-ß receptor II and connective tissue growth factor, both confirmed targets of miR19b. An adeno-associated virus serotype 2 vector (AAV2) with a miR19b transgene downstream of enhanced green fluorescent protein under the murine collage alpha 1(I) promoter was developed specifically to target HSCs. Male Sprague Dawley rats (250 g) underwent sham or bile-duct ligation (BDL) surgery. Directly after BDL, rats received AAV2-miR19b, AAV2-control, or vehicle normal saline (NS) by portal-vein injection. After 2 weeks, the animals were euthanized, and blood was collected for alanine and aspartate aminotransferase, total and direct bilirubin, and alkaline phosphatase. Tissue was collected for RNA and protein extraction and histology. Fibrosis and measures of hepatic injury were significantly reduced in AAV2-miR19b-treated rats in combination with significant improvements in total and direct bilirubin. Histological analysis of collagen by PicroSirius Red staining revealed a ∼50% reduction compared to AAV2-control or NS-injected animals. Pro-fibrotic markers, smooth-muscle alpha-actin, TGF-ß receptor II, and collagen alpha 2(I) mRNA and protein were significantly decreased compared to AAV2-control and NS groups. AAV2-mediated reintroduction of miR-19b, specifically expressed in HSCs, improved liver function, inhibited fibrosis, and improved measures of hepatic injury in a BDL model.

Processing of miR17-92 Cluster in Hepatic Stellate Cells Promotes Hepatic Fibrogenesis During Alcohol-Induced Injury.

BACKGROUND: Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through hepatic stellate cell (HSC) activation, leading to global changes in mRNA and microRNA (miR) expression. miRs are expressed in cells or shuttled to exosomes which can be detected in tissue culture media (TCM) and biological fluids. The mechanisms and function underlying the differential expression and processing of miRs and their downstream effects during hepatic injury remain poorly understood. METHODS: Expression of primary (pri)-miR17-92. and individual members of this cluster, miR17a, 18a, 19a, 20a, 19b, and 92, were examined in primary HSCs and human LX2 cells exposed to alcohol-conditioned media (CM), liver tissue from a rodent model of alcoholic injury, and in exosomes from TCM and plasma of rodent models and patients with alcoholic liver disease (ALD). miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter. RESULTS: Profibrotic markers were enhanced in primary HSCs and LX2 cells exposed to alcohol-CM, concomitant with decreased miR19b expression and a significant increase in pri-miR17-92. Increased pri-miR17-92 was confirmed in a rodent model of alcohol-induced liver injury. Individual members of the cluster were inversely proportionate in cells and exosomes. AAV2-mediated miR19b overexpression inhibited miR17-92 and altered expression of individual cluster members in cells and exosomes. Expression of individual miR17-92 cluster members in plasma exosomes isolated from patients with ALD was similar to that seen in a rodent model of alcoholic injury and in vitro. CONCLUSIONS: Reintroduction of miR19b inhibits HSC activation and modulates expression of pri-miR17-92 and the inverse expression of individual cluster members in cells and exosomes. Better understanding of miR17-92 processing may provide mechanistic insights into the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.

Diet-Induced Obesity Enhances Progression of Hepatocellular Carcinoma through Tenascin-C/Toll-Like Receptor 4 Signaling.

Obesity is an independent risk factor for the development of liver fibrosis/cirrhosis and hepatocellular carcinoma (HCC). Tenascin-C (TnC), an extracellular matrix protein, is transiently expressed during tissue injury and plays a role in fibrogenesis and tumorigenesis. However, the mechanistic role of TnC signaling in the development of HCC remains unknown. We developed a diet-induced obesity HCC mouse model and examined TnC expression and liver injury. To determine the cellular mechanism of TnC signaling in promoting inflammation and hepatocyte epithelial-mesenchymal transition and migration, we used primary hepatocytes and hepatoma and macrophage cell lines. Further, to determine whether elevated TnC expression correlated with obesity-associated HCC, we measured plasma TnC in obese patients with various levels of liver injury. Increased tissue inflammation accompanied with elevated hepatic stellate cell-derived TnC and Toll-like receptor 4 expression was observed in the diet-induced obesity HCC animal model. In vitro studies found enhanced Toll-like receptor 4 signaling activated by TnC, promoting an increased inflammatory response, hepatocyte transformation, and migration. Further, obese patients with cirrhosis alone and in combination with HCC showed significant increases in plasma TnC compared with healthy volunteers and patients with less severe liver injury. Overall, these studies suggest TnC/Toll-like receptor 4 signaling as an important regulator in HCC; inhibiting this signaling axis may be a viable therapeutic target for impeding HCC.

Hydrogen sulfide differentially affects the hepatic vasculature in response to phenylephrine and endothelin 1 during endotoxemia.

Despite being protective in many disease states, hydrogen sulfide (H(2)S) contributes to organ injury in sepsis. Like the other gasotransmitters, nitric oxide and carbon monoxide, H(2)S is a modulator of the microcirculation. Because microcirculatory dysfunction is a main cause of organ injury during sepsis, the present study was designed to test the effect of H(2)S on microvascular dysfunction in isolated perfused livers. In most microcirculatory beds, endotoxin activates the endothelium, resulting in hyporesponsiveness to catecholamines and a derangement in blood flow distribution. We demonstrate that H(2)S treatment attenuates the increase in portal pressure during infusion of the α1 adrenergic agonist, phenylephrine (PE) (P < 0.01). Hydrogen sulfide almost completely negated the increase in portal pressure in livers isolated from endotoxemic rats. Treatment with an inhibitor of endogenous H(2)S, DL-propargylglycine (PAG), reversed lipopolysaccharide-induced hyporesponsiveness to PE. Because hepatic microcirculatory dysfunction is associated with excessive sinusoidal vasoconstriction and not dilation, we investigated whether H(2)S affects endothelin 1 (ET-1)-induced vasoconstriction in isolated livers. Contrary to PE treatment, H(2)S did not affect the increase in portal pressure during infusion of ET-1, nor did it attenuate the hypersensitization of the liver to ET-1 during endotoxemia. Hepatic resistance in control rats was increased by PAG treatment during ET-1 infusion, but this increase was not exacerbated during endotoxemia. We monitored hepatic O(2) consumption to assess the effect of vascular changes on oxygen consumption following ET-1 treatment. Low-dose ET-1 infusion caused an increase in hepatic O(2)consumption, whereas low-dose ET-1 infusion decreased O(2) consumption in endotoxemic livers. Interestingly, whereas we observed no effect of PAG on the vascular response to ET-1 infusion during endotoxemia, PAG treatment did maintain O(2), suggesting a more complex effect of H(2)S inhibition. In summary, the discrepancies between the hepatic response to PE and ET-1 suggest that H(2)S differentially contributes to microcirculatory dysfunction in the systemic and hepatic microcirculations. We propose that this is due to H(2)S exerting a differential vasoactive function on presinusoidal and sinusoidal sites within the liver. Moreover, our findings suggest that H(2)S may contribute to the progression of sepsis by contributing to microvascular failure.

The liver as a central regulator of hydrogen sulfide.

The liver is likely exposed to high levels of hydrogen sulfide (H2S) from endogenous hepatic synthesis and exogenous sources from the gastrointestinal tract. Little is known about the consequence of H2S exposure on the liver or hepatic regulation of H2S levels. We hypothesized that the liver has a high capacity to metabolize H2S and that H2S oxidation is decreased during sepsis, a condition in which hepatic O2 is limited and H2S synthesis is increased. Using a nonrecirculating isolated and perfused liver system, we demonstrated rapid hepatic H2S metabolism up to an infusion concentration of 200' µM H2S. Hydrogen sulfide metabolism was associated with an increase in O2 consumption from a baseline 96.7 ± 7.6 µmol O2/min/kg to 109 ± 7.4 µmol O2/min/kg at an infusion concentration of 150 µM H2S (P < 0.001). Removal of O2 from the perfusate decreased H2S clearance from a maximal 97% to only 23%. Livers isolated from rats subjected to cecal ligation and puncture (CLP) did not differ significantly from control livers in their capacity to metabolize H2S, suggesting that H2S oxidation remains a priority during sepsis. To test whether H2S induces O2 consumption in vivo, intravital microscopy was utilized to monitor the oxygen content in the hepatic microenvironment. Infusion of H2S increased the NADH/NAD+ ratio (645 gray-scale-unit increase, P = 0.035) and decreased hepatic O2 availability visualized with Ru(Phen)3(2+) (439 gray-scale-unit increase, P = 0.040). We conclude that the liver has a high hepatic capacity for H2S metabolism. Moreover, H2S oxidation consumes available oxygen and may exacerbate the tissue hypoxia associated with sepsis.

Improved preservation of warm ischemic livers by hypothermic machine perfusion with supplemented University of Wisconsin solution.

Hypothermic machine perfusion (HMP) has the potential to improve recovery and preservation of Donation after Cardiac Death (DCD) livers, including uncontrolled DCD livers. However, current perfusion solutions lack the needed substrates to improve energy recovery and minimize hepatic injury, if warm ischemic time (WIT) is extended. This proof-of-concept study tested the hypothesis that the University of Wisconsin (UW) solution supplemented with anaplerotic substrates, calcium chloride, thromboxane A2 inhibitor, and antioxidants could improve HMP preservation and minimize reperfusion injury of warm ischemic livers. Preflushed rat livers subjected to 60 min WIT were preserved for 5 h with standard UW or supplemented UW (SUW) solution. Post preservation hepatic functions and viability were assessed during isolated perfusion with Krebs-Henseleit solution. Livers preserved with SUW showed significantly (p < .001) improved recovery of tissue ATP levels (micromol/g liver), 2.06 +/- 0.10 (mean +/- SE), as compared to the UW group, 0.70 +/- 0.10, and the level was 80% of that of fresh control livers (2.60 +/- 0.13). At the end of 1 h of rewarming, lactate dehydrogenase (U/L) in the perfusate was significantly (p < .05) lower in the SUW group (429 +/- 58) as compared to ischemia-reperfusion (IR) (781 +/- 12) and the UW group (1151 +/- 83). Bile production (microg/min/g liver) was significantly (p < .05) higher in the SUW group (280 +/- 13) as compared to the IR (224 +/- 24) and the UW group (114 +/- 14). The tissue edema formation assessed by tissue wet-dry ratio was significantly (p < .05) higher in UW group. Histology showed well-preserved hepatic structure in the SUW group. In conclusion, this study suggests that HMP with SUW solution has the potential to restore and preserve livers with extended WIT.

Contribution of non-parenchymal cells to the performance of micropatterned hepatocytes.

Hepatocytes within current bioartificial liver designs are unable to maintain the wide range of differentiated functions observed for the liver in vivo. As recent studies suggest, the absence of controlled interactions between hepatocytes and non-parenchymal cell populations of the liver may contribute to this hepatocyte dedifferentiation. The current study investigates the effect of Kupffer cells, fibroblasts, and human umbilical vein endothelial cells on hepatocyte function. To effectively study the effect of these heterotypic cell-to-cell interactions, it is necessary to establish a culture environment that controls the interactions between multiple cell types. Micropatterning is such a technique and was used in the current study. In addition, to elucidate the influence of soluble factors on hepatocyte function, the micropatterned results were compared with those of the trans-well culture system. Specifically, we compared the morphological and functional changes of hepatocytes cultured with these cells at varying ratios. Our results suggest that direct heterotypic cell-to-cell contact between hepatocytes and fibroblasts, and soluble factor exchanges between hepatocytes and human umbilical vein endothelial cells, significantly enhanced hepatocyte performance.