Kanishka Hittatiya

CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis

Objectives In chronic liver injury, angiogenesis, the formation of new blood vessels from pre-existing ones, may contribute to progressive hepatic fibrosis and to development of hepatocellular carcinoma. Although hypoxia-induced expression of vascular endothelial growth factor (VEGF) occurs in advanced fibrosis, we hypothesised that inflammation may endorse hepatic angiogenesis already at early stages of fibrosis. Design Angiogenesis in livers of c57BL/6 mice upon carbon tetrachloride- or bile duct ligation-induced chronic hepatic injury was non-invasively monitored using in vivo contrast-enhanced micro computed tomography (µCT) and ex vivo anatomical µCT after hepatic Microfil perfusion. Functional contributions of monocyte-derived macrophage subsets for angiogenesis were explored by pharmacological inhibition of CCL2 using the Spiegelmer mNOX-E36. Results Contrast-enhanced in vivo µCT imaging allowed non-invasive monitoring of the close correlation of angiogenesis, reflected by functional hepatic blood vessel expansion, with experimental fibrosis progression. On a cellular level, inflammatory monocyte-derived macrophages massively accumulated in injured livers, colocalised with newly formed vessels in portal tracts and exhibited pro-angiogenic gene profiles including upregulated VEGF and MMP9. Functional in vivo and anatomical ex vivo µCT analyses demonstrated that inhibition of monocyte infiltration by targeting the chemokine CCL2 prevented fibrosis-associated angiogenesis, but not fibrosis progression. Monocyte-derived macrophages primarily fostered sprouting angiogenesis within the portal vein tract. Portal vein diameter as a measure of portal hypertension depended on fibrosis, but not on angiogenesis. Conclusions Inflammation-associated angiogenesis is promoted by CCL2-dependent monocytes during fibrosis progression. Innovative in vivo µCT methodology can accurately monitor angiogenesis and antiangiogenic therapy effects in experimental liver fibrosis.

Pharmacological inhibition of the chemokine C‐C motif chemokine ligand 2 (monocyte chemoattractant protein 1) accelerates liver fibrosis regression by suppressing Ly‐6C+ macrophage infiltration in mice

Macrophages constitute a major proinflammatory component during chronic liver diseases and are considered a key factor in promoting hepatic fibrosis. However, there is increasing evidence that distinct monocyte and macrophage subsets exert critical functions in regression from organ fibrosis as well. Experimental mouse models of fibrosis regression have identified “restorative” macrophages as Ly‐6C (Ly6C, Gr1) low‐expressing, monocyte‐derived cells. We investigated molecular pathways balancing proinflammatory and restorative macrophages during fibrosis regression as well as pharmacologically augmenting beneficial macrophage functionality in fibrosis resolution. Therefore, we employed a Spiegelmer‐based inhibitor of the chemokine, C‐C motif chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1), termed mNOX‐E36, in the regression phase of two murine models of toxic (CCl4) and metabolic (methionine‐choline–deficient diet) liver fibrosis. Although inflammation rapidly declined after cessation of injury, we observed a transient influx of Ly‐6C+ infiltrating monocytes (iMΦ), which are characterized by typical macrophage morphology, up‐regulated expression of CCR2, and the pro‐inflammatory cytokine, tumor necrosis factor (TNF), in injured liver. By inhibiting the early influx of Ly‐6C+ iMΦ by the CCL2 inhibitor, mNOX‐E36, the intrahepatic macrophage equilibration shifted toward the “restorative” Ly‐6C‐ subset of iMΦ. Consequently, fibrosis resolution was significantly accelerated upon mNOX‐E36 administration in both models. Blocking transient recruitment of infiltrating Ly‐6C+ monocytes, but not direct effects of the inhibitor on the remaining macrophages, resulted in reduced intrahepatic levels of proinflammatory cytokines. Conclusion: Transient CCL2‐dependent recruitment of infiltrating Ly‐6C+ monocytes during fibrosis regression counteracts scar resolution by perpetuating inflammatory reactions through release of proinflammatory cytokines such as TNF. Pharmacological inhibition of Ly‐6C+ monocyte recruitment using the CCL2‐inhibitor, mNOX‐E36, accelerates regression from toxic and metabolic liver fibrosis in two independent experimental models. (Hepatology 2014;59:1060–1072)

Chemokine Receptor CXCR6-Dependent Hepatic NK T Cell Accumulation Promotes Inflammation and Liver Fibrosis

Chronic liver injury characteristically results in hepatic inflammation, which represents a prerequisite for organ fibrosis. Although NKT cells are abundantly present in liver and involved in hepatic inflammation, molecular mechanisms of their recruitment in liver fibrosis remained elusive. We hypothesized that chemokine receptor CXCR6 and its ligand CXCL16 control NKT cell migration and functionality in liver fibrosis. In patients with chronic liver diseases (n = 58), CXCR6 and CXCL16 expression was intrahepatically upregulated compared with controls. In murine liver, Cxcl16 was strongly expressed by endothelium and macrophages, whereas lymphocyte populations (NKT, NK, CD4 T, CD8 T cells) expressed CXCR6. Intravital two-photon microscopy imaging of Cxcr6+/gfp and Cxcr6gfp/gfp mice and chemotaxis studies in vitro revealed that CXCR6 specifically controls hepatic NKT cell accumulation during the early response upon experimental liver damage. Hepatic invariant NKT cells expressed distinct proinflammatory cytokines including IFN-γ and IL-4 upon injury. CXCR6-deficient mice were protected from liver fibrosis progression in two independent experimental models. Macrophage infiltration and protein levels of inflammatory cytokines IFN-γ, TNF-α, and IL-4 were also reduced in fibrotic livers of Cxcr6−/− mice, corroborating that hepatic NKT cells provide essential cytokine signals perpetuating hepatic inflammation and fibrogenesis. Adoptive transfer of NKT cells, but not CD4 T cells, isolated from wild type livers restored hepatic fibrosis in Cxcr6−/− mice upon experimental steatohepatitis. Our results demonstrate that hepatic NKT cells accumulate CXCR6-dependent early upon injury, thereby accentuating the inflammatory response in the liver and promoting hepatic fibrogenesis. Interfering with CXCR6/CXCL16 might therefore bear therapeutic potential in liver fibrosis.

Fluorescent cell-traceable dexamethasone-loaded liposomes for the treatment of inflammatory liver diseases

Liposomes are routinely used carrier materials for delivering drug molecules to pathological sites. Besides in tumors and inflammatory sites, liposomes also strongly accumulate in liver and spleen. The potential of using liposomes to treat acute and chronic liver disorders, however, has not yet been evaluated. We here explored the therapeutic potential of dexamethasone (Dex)-loaded liposomes for inflammatory liver diseases, using experimental models of acute and chronic liver injury in mice. Fluorescently labeled liposomes predominantly accumulated in hepatic phagocytes, but also in T cells. Importantly, Dex-loaded liposomes reduced T cells in blood and liver, more effectively than free Dex, and endorsed the anti-inflammatory polarization of hepatic macrophages. In experimental chronic liver damage, Dex-loaded liposomes significantly reduced liver injury and liver fibrosis. In immune-mediated acute hepatitis Dex-loaded liposomes, but not free Dex, significantly reduced disease severity. T cells, not macrophages, were significantly depleted by Dex liposomes in liver disease models in vivo, as further supported by mechanistic cell death in vitro studies. Our data indicate that Dex liposomes may be an interesting treatment option for liver diseases, in particular for immune-mediated hepatitis. The depletion of T cells might represent the major mechanism of action of Dex liposomes, rather than their macrophage-polarizing activities.

Seven weeks of Western diet in apolipoprotein-E-deficient mice induce metabolic syndrome and non-alcoholic steatohepatitis with liver fibrosis.

Non-alcoholic steatohepatitis (NASH) is characterised by hepatic steatosis, inflammation and fibrosis, which might progress to cirrhosis. Human NASH is associated with metabolic syndrome (MS). Currently, rodent NASH models either lack significant fibrosis or MS. ApoE−/− mice are a MS model used in cardiovascular research. The aim of this work was to establish and characterise a novel mouse NASH model with significant fibrosis and MS. ApoE−/− and wild-type mice (wt) were fed either a western-diet (WD), methionine-choline-deficient-diet (MCD) or normal chow. Liver histology, RT-PCR, hepatic hydroxyproline content, triglycerides and cholesterol levels, and fasting glucose levels assessed hepatic steatosis, inflammation and fibrosis. Further, portal pressure was measured invasively, and kidney pathology was assessed by histology. ApoE−/− mice receiving WD showed abnormal glucose tolerance, hepatomegaly, weight gain and full spectrum of NASH including hepatic steatosis, fibrosis and inflammation, with no sign of renal damage. MCD-animals showed less severe liver fibrosis, but detectable renal pathological changes, besides weight loss and unchanged glucose tolerance. This study describes a murine NASH model with distinct hepatic steatosis, inflammation and fibrosis, without renal pathology. ApoE−/− mice receiving WD represent a novel and fast model with all characteristic features of NASH and MS well suitable for NASH research.

Pharmacological inhibition of the chemokine CXCL16 diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury.

Non-alcoholic fatty liver disease (NAFLD) is a major cause of morbidity and mortality in developed countries, resulting in steatohepatitis (NASH), fibrosis and eventually cirrhosis. Modulating inflammatory mediators such as chemokines may represent a novel therapeutic strategy for NAFLD. We recently demonstrated that the chemokine receptor CXCR6 promotes hepatic NKT cell accumulation, thereby controlling inflammation in experimental NAFLD. In this study, we first investigated human biopsies (n = 20), confirming that accumulation of inflammatory cells such as macrophages is a hallmark of progressive NAFLD. Moreover, CXCR6 gene expression correlated with the inflammatory activity (ALT levels) in human NAFLD. We then tested the hypothesis that pharmacological inhibition of CXCL16 might hold therapeutic potential in NAFLD, using mouse models of acute carbon tetrachloride (CCl4)- and chronic methionine-choline-deficient (MCD) diet-induced hepatic injury. Neutralizing CXCL16 by i.p. injection of anti-CXCL16 antibody inhibited the early intrahepatic NKT cell accumulation upon acute toxic injury in vivo. Weekly therapeutic anti-CXCL16 administrations during the last 3 weeks of 6 weeks MCD diet significantly decreased the infiltration of inflammatory macrophages into the liver and intrahepatic levels of inflammatory cytokines like TNF or MCP-1. Importantly, anti-CXCL16 treatment significantly reduced fatty liver degeneration upon MCD diet, as assessed by hepatic triglyceride levels, histological steatosis scoring and quantification of lipid droplets. Moreover, injured hepatocytes up-regulated CXCL16 expression, indicating that scavenging functions of CXCL16 might be additionally involved in the pathogenesis of NAFLD. Targeting CXCL16 might therefore represent a promising novel therapeutic approach for liver inflammation and steatohepatitis.

Janus-kinase-2 relates directly to portal hypertension and to complications in rodent and human cirrhosis.

Objective Angiotensin II (AngII) activates via angiotensin-II-type-I receptor (AT1R) Janus-kinase-2 (JAK2)/Arhgef1 pathway and subsequently RHOA/Rho-kinase (ROCK), which induces experimental and probably human liver fibrosis. This study investigated the relationship of JAK2 to experimental and human portal hypertension. Design The mRNA and protein levels of JAK2/ARHGEF1 signalling components were analysed in 49 human liver samples and correlated with clinical parameters of portal hypertension in these patients. Correspondingly, liver fibrosis (bile duct ligation (BDL), carbon tetrachloride (CCl4)) was induced in floxed-Jak2 knock-out mice with SM22-promotor (SM22Cre+-Jak2f/f). Transcription and contraction of primary myofibroblasts from healthy and fibrotic mice and rats were analysed. In two different cirrhosis models (BDL, CCl4) in rats, the acute haemodynamic effect of the JAK2 inhibitor AG490 was assessed using microsphere technique and isolated liver perfusion experiments. Results Hepatic transcription of JAK2/ARHGEF1 pathway components was upregulated in liver cirrhosis dependent on aetiology, severity and complications of human liver cirrhosis (Model for End-stage Liver disease (MELD) score, Child score as well as ascites, high-risk varices, spontaneous bacterial peritonitis). SM22Cre+- Jak2f/f mice lacking Jak2 developed less fibrosis and lower portal pressure (PP) than SM22Cre−-Jak2f/f upon fibrosis induction. Myofibroblasts from SM22Cre+-Jak2f/f mice expressed less collagen and profibrotic markers upon activation. AG490 relaxed activated hepatic stellate cells in vitro. In cirrhotic rats, AG490 decreased hepatic vascular resistance and consequently the PP in vivo and in situ. Conclusions Hepatic JAK2/ARHGEF1/ROCK expression is associated with portal hypertension and decompensation in human cirrhosis. The deletion of Jak2 in myofibroblasts attenuated experimental fibrosis and acute inhibition of JAK2 decreased PP. Thus, JAK2 inhibitors, already in clinical use for other indications, might be a new approach to treat cirrhosis with portal hypertension.

Histidine-rich glycoprotein promotes macrophage activation and inflammation in chronic liver disease

Pathogen‐ and injury‐related danger signals as well as cytokines released by immune cells influence the functional differentiation of macrophages in chronic inflammation. Recently, the liver‐derived plasma protein, histidine‐rich glycoprotein (HRG), was demonstrated, in mouse tumor models, to mediate the transition of alternatively activated (M2) to proinflammatory (M1) macrophages, which limit tumor growth and metastasis. We hypothesized that liver‐derived HRG is a critical endogenous modulator of hepatic macrophage functionality and investigated its implications for liver inflammation and fibrosis by comparing C57BL/6N wild‐type (WT) and Hrg−/− mice. In homeostatic conditions, hepatic macrophages were overall reduced and preferentially polarized toward the anti‐inflammatory M2 subtype in Hrg−/− mice. Upon chronic liver damage induced by CCl4 or methionine‐choline‐deficient (MCD) diet, liver injury and fibrosis were attenuated in Hrg−/−, compared to WT, mice. Macrophage populations were reduced and skewed toward M2 polarization in injured livers of Hrg−/− mice. Moreover, HRG‐deficient mice showed significantly enhanced hepatic vascularization by micro‐computed tomography and histology, corroborating proangiogenic activities of M2‐polarized liver macrophages. Purified HRG protein induced, but HRG‐deficient serum prevented, M1 macrophage differentiation in vitro. Accordingly, Hrg−/− mice transplanted with Hrg+/+ bone marrow, but not Hrg−/−‐transplanted Hrg+/+ mice, remained protected from experimental steatohepatitis. Consistent with these findings, patients with chronic hepatitis C and nonalcoholic steatohepatitis significantly up‐regulated hepatocytic HRG expression, which was associated with M1 polarization of adjacent macrophages. Conclusions: Liver‐derived HRG, similar to alarmins, appears to be an endogenous molecular factor promoting polarization of hepatic macrophages toward the M1 phenotype, thereby promoting chronic liver injury and fibrosis progression, but limiting angiogenesis. Therefore, controlling tissue levels of HRG or PGF might be a promising strategy in chronic inflammatory liver diseases. (Hepatology 2016;63:1310‐1324)

Statins improve NASH via inhibition of RhoA and Ras

Nonalcoholic steatohepatitis (NASH), especially as part of the metabolic syndrome (MS), is an increasing burden in Western countries. Statins are already used in MS and seem to be beneficial in liver diseases. The aim of this study was to investigate the molecular mechanisms underlying pleiotropic effects on small GTPases of statins in NASH. NASH within MS was induced in 12-wk-old apoE-/- mice after 7 wk of Western diet (NASH mice). Small GTPases were inhibited by activated simvastatin (SMV), NSC23766 (NSC), or Clostridium sordellii lethal toxin (LT) by using subcutaneous osmotic minipumps. Hepatic steatosis, inflammation, and fibrosis were assessed by histology, Western blot, and RT-PCR measurements of cholesterol and hydroxyproline content. SMV treatment significantly decreased hepatic inflammation and fibrosis, but had no significant effect on steatosis and hepatic cholesterol content in NASH. SMV blunted fibrosis due to inhibition of both RhoA/Rho kinase and Ras/ERK pathways. Interestingly, inhibition of RAC1 and Ras (by LT) failed to decrease fibrosis to the same extent. Inhibition of RAC1 (by NSC) showed no significant effect at all. Inhibition of RhoA and Ras downstream signaling by statins is responsible for the beneficial hepatic effects in NASH.

Novel Rat Model of Repetitive Portal Venous Embolization Mimicking Human Non-Cirrhotic Idiopathic Portal Hypertension

Background Non-cirrhotic idiopathic portal hypertension (NCIPH) is characterized by splenomegaly, anemia and portal hypertension, while liver function is preserved. However, no animal models have been established yet. This study assessed a rat model of NCIPH and characterized the hemodynamics, and compared it to human NCIPH. Methods Portal pressure (PP) was measured invasively and coloured microspheres were injected in the ileocecal vein in rats. This procedure was performed weekly for 3 weeks (weekly embolization). Rats without and with single embolization served as controls. After four weeks (one week after last embolization), hemodynamics were investigated, hepatic fibrosis and accumulation of myofibroblasts were analysed. General characteristics, laboratory analyses and liver histology were collected in patients with NCIPH. Results Weekly embolization induced a hyperdynamic circulation, with increased PP. The mesenteric flow and hepatic hydroxyproline content was significantly higher in weekly embolized compared to single embolized rats (mesenteric flow +54.1%, hydroxyproline +41.7%). Mesenteric blood flow and shunt volumes increased, whereas splanchnic vascular resistance was decreased in the weekly embolization group. Fibrotic markers αSMA and Desmin were upregulated in weekly embolized rats. Discussion This study establishes a model using repetitive embolization via portal veins, comparable with human NCIPH and may serve to test new therapies.