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. 2014 Dec;63(12):1960-1971.
doi: 10.1136/gutjnl-2013-306294. Epub 2014 Feb 21.

CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis

Josef Ehling #  1   2 , Matthias Bartneck #  3 , Xiao Wei  3 , Felix Gremse  1 , Viktor Fech  3 , Diana Möckel  1 , Christer Baeck  3 , Kanishka Hittatiya  4 , Dirk Eulberg  5 , Tom Luedde  3 , Fabian Kiessling  1 , Christian Trautwein  3 , Twan Lammers  1   6   7 , Frank Tacke  3
Affiliations

CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis

Josef Ehling et al. Gut. 2014 Dec.

Abstract

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.

Keywords: ANGIOGENESIS; CHEMOKINES; COMPUTER TOMOGRAPHY; FIBROSIS; MACROPHAGES.

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Figures

Figure 1
Figure 1. Association among chronic liver injury, inflammation, hepatic fibrosis and angiogenesis
(A) Chronic toxic liver injury was induced by repetitive i.p. injections of CCl4 in c57BL/6 mice, and mice were sacrificed 48 hours after the last injection of CCl4. Control mice received corn oil for 6 weeks. Representative H&E staining, Sirius red (fibrotic fibers in red), CD45 immunohistochemistry (leukocytes), and CD31 immunofluorescence (blood vessels) of controls and after 4 or 8 weeks CCl4. (B) Serum ALT activity (liver injury), hepatic hydroxyproline content (collagen deposition), CD45+ cells in liver sections (hepatic inflammation), and area fraction of CD31+ endothelial cells (quantification of neovascularisation). Data are shown as mean±SD (n=15 mice). ***P<0.001, **P<0.01 and *P<0.05 (Student’s t test).
Figure 2
Figure 2. Functional and anatomical μCT imaging of angiogenesis in CCl4-induced liver fibrosis
(A) Visualization of hepatic blood vessels by in vivo μCT using an iodine-based blood pool contrast agent (eXIA 160XL), resulting in a spatial resolution of 35μm voxel side length (2D cross-sectional images in transversal (I), sagittal (II) and coronal (III) planes, as well as representative pictures of 3D volume renderings). (B) Non-invasive μCT-based quantification of the relative blood volume (rBV) in fibrotic and healthy livers. (C) A highly significant correlation was found between hepatic rBV determined using in vivo μCT and area fraction of CD31 determined using ex vivo immunofluorescence (IF) staining. Data are shown as mean±SD; n=15 mice; ***P<0.001 (Student’s t test). Correlation analyses were performed by calculating R2 (square of Pearson correlation coefficient). (D) High-resolution ex vivo μCT imaging (after perfusion with Microfil, a lead-containing radiopaque contrast agent) enables a detailed 3D examination of vascular microarchitecture of healthy liver (left) and after 6 weeks CCl4 (right). Spatial resolution: 12μm voxel side length.
Figure 3
Figure 3. Association between fibrosis and angiogenesis in bile duct ligation-induced cholestatic liver injury
Chronic cholestatic liver injury was induced by surgical bile duct ligation (BDL) in c57BL/6 mice. Control animals received sham operations. Mice were imaged and sacrificed 21 days after surgery. (A) H&E staining, Sirius red staining (fibrosis), CD45 immunohistochemistry (inflammation) and CD31 immunofluorescence (blood vessel formation). (B-C) Functional in vivo (B) and morphological high-resolution ex vivo μCT imaging (C) of liver blood vessels from control and from BDL-treated mice (I: transversal, II: sagittal, III: coronal 2D cross-sectional images). Segmented gall bladders (green) illustrate gall bladder hydrops and cholestasis after BDL. (D) Quantification of liver injury via ALT activity in serum, of liver fibrosis by hepatic hydroxyproline levels, of hepatic inflammation by quantifying CD45+ cells, of hepatic blood vessels by determining the CD31 area fraction, and of the hepatic rBV, determined non-invasively using contrast-enhanced in vivo μCT. Results are shown as mean±SD (n=8 mice). ***P<0.001, **P<0.01 and *P<0.05 (Student’s t test).
Figure 4
Figure 4. Role of hepatic macrophage subsets in fibrosis-associated angiogenesis
Chronic toxic liver injury was induced by repetitive i.p. administrations of CCl4 in c57BL/6 mice, control mice received corn oil. Mice were sacrificed 48 hours after the last injection of CCl4 (n=6 mice per condition and time-point). (A) Representative microscopy images of F4/80 immunohistochemistry (macrophages). (B) Quantification of total F4/80+ macrophages in sections from control and chronically injured livers. (C) Representative F4/80 (red) and CD31 (green) co-stainings, demonstrating periportal localization of inflammatory macrophages and co-localization of macrophages with newly formed small blood vessels in progressive CCl4 or BDL injury. (D) The relative amount of intrahepatic CD11b+ F4/80+ inflammatory macrophages (iMΦ) isolated by FACS sorting is early and persistently increased in chronic liver injury. (E+F) Expression of VEGF-A (E) and MMP9 (F) by primary murine inflammatory macrophages, Kupffer cells, hepatocytes, endothelial cells and hepatic stellate cells. Cells were isolated from injured (n=12) and control (n=12) livers using FACS sorting, and expression levels were normalized to iMΦ isolated from corn oil-treated control livers. Data are shown as mean±SD. ***P<0.001 and **P<0.01 (Student’s t test).
Figure 5
Figure 5. Effect of pharmacological inhibition of CCL2-dependent inflammatory monocytes on fibrosis-associated angiogenesis
Chronic toxic liver injury was induced by repetitive i.p. administrations of CCl4 in c57BL/6 mice, and half of these animals received thrice weekly s.c. injections of the specific CCL2 inhibitor mNOX-E36, to block the CCL2-dependent infiltration of inflammatory monocytes. Analyses were performed 48 hours after the last CCl4 injection. Control mice received corn oil for 6 weeks. (A) Representative H&E staining, Sirius red, F4/80 immunohistochemistry, and F4/80-CD31 immunofluorescence co-stainings. (B) Quantification of F4/80+ macrophages and CD31+ blood vessels in livers of chronically injured and mNOX-E36 treated mice. (C) Representative FACS plots and statistical analysis showing the increase of intrahepatic inflammatory macrophages (iMΦ) in chronically injured livers and their significant reduction in mNOX-E36 treated livers. iMΦ were separated from Kupffer cells on the basis of differential expression of F4/80 and CD11b. (D) Liver vascularization visualized by contrast-enhanced in vivo μCT. (E) Quantification of the rBV in livers and spleens using functional in vivo μCT imaging. Data are shown as mean±SD. ***P<0.001, **P<0.01 and *P<0.05 for comparing CCl4 vs. CCl4+mNOX-E36; ###P<0.001, ##P<0.01 and #P<0.05 for comparing CCl4 or CCl4+mNOX-E36 vs. corresponding control groups (i.e. 6 weeks oil or 6 weeks oil+mNOX-E36) (n=30 mice; Student’s t test).
Figure 6
Figure 6. Vascular branching analysis of sprouting angiogenesis in the central and portal vein system in progressive liver fibrosis and upon pharmacological inhibition of CCL2
(A) Representative high-resolution ex vivo μCT images of chronically injured and mNOX-E36-treated livers after systematic Microfil perfusion. (B) Overview and magnification of segmented blood vessels of the liver after semi-automated discrimination between vessels related to the central (blue) or portal vein (red) system. Arrows schematically depict the order of rising branching points along the course of blood vessels, from center to periphery. (C) μCT-based quantification of the mean total number of branching points in livers from corn oil-, CCl4- and CCl4+mNOX-E36-treated mice (all for 6 weeks). Branching points from five representative blood vessels were quantified for both the central and the portal vein system. (D) μCT-based quantification of the percentage of branching points per increasing order (1st to 11th branching order) for livers from corn oil-, CCl4- and CCl4+mNOX-E36-treated mice. Data are shown as mean±SD. ***P<0.001, **P<0.005 and *P<0.05 (Student’s t test) for comparing CCl4 vs. CCl4+mNOX-E36; ###P<0.001, and #P<0.05 for comparing CCl4 or CCl4+mNOX-E36 vs. control (Student’s t test) (D).
Figure 7
Figure 7. In vivo quantification of the portal vein diameter using contrast-enhanced μCT imaging
(A) Cross-sectional images in transversal planes exemplifying the determination of the portal vein diameter 3-4 slices above the junction of the superior mesenteric and splenic vein in control, chronically injured and mNOX-E36 treated livers. (B) Quantification of the portal vein diameter using functional μCT imaging in combination with an iodine-based large molecular weight blood pool contrast agent. (C) A highly significant correlation was found between in vivo quantified portal vein diameters and ex vivo determined hydroxyproline content. (D) No significant correlation was found for the comparison between portal vein diameters and relative blood volumes in CCl4 treated, CCl4 + mNOX-E36 treated or control mice. Data are shown as mean±SD. ***P<0.001, **P<0.01 for comparing CCl4 or mNOX-E36 vs. corresponding control groups (i.e. 6 weeks oil or 6 weeks oil + mNOX-E36) (n=30 mice; Student’s t test).
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