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.

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)

Background:In chronic hepatitis C (CHC), the transition from mild to moderate fibrosis is a major prognostic step.The aim of this study was to identify serum protein signature to differentiate mild from moderate fibrosis in CHC.Methods: Liver biopsies from 244 untreated CHC patients were studied.Among them, 66% had mild fibrosis (F1, Metavir) and 34% moderate fibrosis (F2).Patients were mainly infected with genotype 1 (55%), 2 (11%), 3 (11%), 4 (20%) and 5-6 (3%) respectively.Real-time quantitative RT-PCR assays were used to analyse the mRNA expression of 51 genes involved in fibrogenesis.The concentration of 6 proteins was assayed in duplicate by ELISA in the serum of 228 patients and reliable data were obtained for 216 (65% with mild fibrosis and 35% with moderate fibrosis).Results: 28 genes were found to be upregulated in F2 patients.These genes were mainly involved in extracellular matrix production and remodelling, in cell-cell and cell-extracellular matrix interactions, in cell cycle, or encode growth factors/cytokines families.We focused our interest on up-regulated genes that code for circulating proteins.The 6 genes with the highest statistical discriminating values were: A2M, CXCL10, SPP1, S100A4, IL8 and ENG.Therefore, we assayed the concentration of these 6 proteins in the serum of patients.The protein signature with the highest discriminating value was composed of A2M, CXCL10, IL8, and SPP1.This 4-protein signature was able to discriminate F2 patients from F1 patients with a sensitivity of 68%, and a specificity of 69% (AUC = 0.684).Addition of clinical parameters to this 4-protein signature allowed reaching 72% sensitivity and 75% specificity (AUC = 0.750). Conclusion:We demonstrated in a large independent cohort that mild and moderate fibrosis have different liver gene expression.The most notable changes occurred mainly in cell-matrix turn-over.Several genes that are up-regulated in the liver encode molecules detected in the serum and provide a logical functional approach for the development of serum markers of fibrosis progression.A 4-protein signature (A2M, CXCL10, IL8, and SPP1) was identified that demonstrates high value for the diagnosis of early fibrosis and particularly the discrimination of F1/F2 stages.

cytokines secretion. In vitro study, DIM inhibited ConA-induced proliferation of splenic CD4+T cells, DIM enhanced the protein expression of CYP1A1 and CYP1B1, and decreased which of TLR4 on Con A-stimulated splenic CD4+T cells. Furthermore, blocking AhR with CH223191 significantly reversed the ability of DIM to promote the differentiation of Tregs, and caused marked inhibition of Th17 differentiation. On the other hand, blockage with single anti-TLR4 neutralizing antibody show the similar immune-regulatory effects of DIM to induc the production of Tregs. Conclusions: DIM may be an efficient theraprutic candidate of liver fibrosis, and high dietary intake of cruciferous vegetables may be an effective nutritional supplement for the patients with chronic liver disease.