Diana Möckel

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.

Sonoporation enhances liposome accumulation and penetration in tumors with low EPR

The Enhanced Permeability and Retention (EPR) effect is a highly variable phenomenon. To enhance EPR-mediated passive drug targeting to tumors, several different pharmacological and physical strategies have been evaluated over the years, including e.g. TNFα-treatment, vascular normalization, hyperthermia and radiotherapy. Here, we systematically investigated the impact of sonoporation, i.e. the combination of ultrasound (US) and microbubbles (MB), on the tumor accumulation and penetration of liposomes. Two different MB formulations were employed, and their ability to enhance liposome accumulation and penetration was evaluated in two different tumor models, which are both characterized by relatively low levels of EPR (i.e. highly cellular A431 epidermoid xenografts and highly stromal BxPC-3 pancreatic carcinoma xenografts). The liposomes were labeled with two different fluorophores, enabling in vivo computed tomography/fluorescence molecular tomography (CT-FMT) and ex vivo two-photon laser scanning microscopy (TPLSM). In both models, in spite of relatively high inter- and intra-individual variability, a trend towards improved liposome accumulation and penetration was observed. In treated tumors, liposome concentrations were up to twice as high as in untreated tumors, and sonoporation enhanced the ability of liposomes to extravasate out of the blood vessels into the tumor interstitium. These findings indicate that sonoporation may be a useful strategy for improving drug targeting to tumors with low EPR.

Fluorescently labeled microbubbles for facilitating translational molecular ultrasound studies

Microbubbles (MB) are routinely used as contrast agents for functional and molecular ultrasound (US) imaging. For molecular US imaging, MB are functionalized with antibodies or peptides, in order to visualize receptor expression by angiogenic or inflamed endothelium. In general, initial in vitro binding studies with targeted MB are performed using phase contrast microscopy. Difficulties in the identification of MB in standard phase contrast microscopy, however, generally result in high variability, high observer dependency, and low reproducibility. To overcome these shortcomings, we here describe a simple post-loading strategy for labeling polymer-based MB with fluorophores, and we show that the use of rhodamine-loaded MB in combination with fluorescence microscopy substantially reduces the variability and the observer dependency of in vitro binding studies. In addition, we demonstrate that rhodamine-loaded MB can also be used for in vivo and ex vivo experimental setups, e.g., for analyzing MB binding to inflamed carotids using two-photon laser scanning microscopy, and for validating the binding of VEGFR2-targeted MB to tumor endothelium. These findings demonstrate that fluorescently labeled MB substantially facilitate translational molecular US studies, and they suggest that a similar synthetic strategy can be exploited for preparing drug-loaded MB, to enable image-guided, targeted, and triggered drug delivery to tumors and to sites of inflammation.

The CCR2+ macrophage subset promotes pathogenic angiogenesis for tumor vascularization in fibrotic livers

Background & Aims Hepatocellular carcinoma (HCC) typically arises in fibrotic or cirrhotic livers, which are characterized by pathogenic angiogenesis. Myeloid immune cells, specifically tumor-associated macrophages (TAMs), may represent potential novel therapeutic targets in HCC, complementing current ablative or immune therapies. However, the detailed functions of TAM subsets in hepatocarcinogenesis have remained obscure. Methods TAM subsets were analyzed in-depth in human HCC samples and a combined fibrosis–HCC mouse model, established by i.p. injection with diethylnitrosamine after birth and repetitive carbon tetrachloride (CCl4) treatment for 16 weeks. Based on comprehensively phenotyping TAM subsets (fluorescence-activated cell sorter, transcriptomics) in mice, the function of CCR2+ TAM was assessed by a pharmacologic chemokine inhibitor. Angiogenesis was evaluated by contrast-enhanced micro–computed tomography and histology. Results We show that human CCR2+ TAM accumulate at the highly vascularized HCC border and express the inflammatory marker S100A9, whereas CD163+ immune-suppressive TAM accrue in the HCC center. In the fibrosis–cancer mouse model, we identified 3 major hepatic myeloid cell populations with distinct messenger RNA profiles, of which CCR2+ TAM particularly showed activated inflammatory and angiogenic pathways. Inhibiting CCR2+ TAM infiltration using a pharmacologic chemokine CCL2 antagonist in the fibrosis–HCC model significantly reduced pathogenic vascularization and hepatic blood volume, alongside attenuated tumor volume. Conclusions The HCC microenvironment in human patients and mice is characterized by functionally distinct macrophage populations, of which the CCR2+ inflammatory TAM subset has pro-angiogenic properties. Understanding the functional differentiation of myeloid cell subsets in chronically inflamed liver may provide novel opportunities for modulating hepatic macrophages to inhibit tumor-promoting pathogenic angiogenesis.

Targeting and Modulation of Liver Myeloid Immune Cells by Hard‐Shell Microbubbles

Poly n‐butylcyanoacrylate (PBCA)‐based hard‐shell microbubbles (MB) have manifold biomedical applications, including targeted drug delivery or contrast agents for ultrasound (US)‐based liver imaging. MB and their fragments accumulate in phagocytes, especially in the liver, but it is unclear if MB affect the function of these immune cells. Herein, it is shown that human primary monocytes internalize different PBCA‐MB by phagocytosis, which transiently inhibits monocyte migration in vertical chemotaxis assays and renders monocytes susceptible to cytotoxic effects of MB during US‐guided destruction. Conversely, human macrophage viability and function, including cytokine release and polarization, remain unaffected after MB uptake. After intravenous injection in mice, MB predominantly accumulate in liver, especially in hepatic phagocytes (monocytes and Kupffer cells). Despite efficiently targeting myeloid immune cells in liver, MB or MB after US‐elicited burst do not cause overt hepatotoxicity or inflammation. Furthermore, MB application with or without US‐guided burst does not aggravate the course of experimental liver injury in mice or the inflammatory response to liver injury in vivo. In conclusion, PBCA‐MB have immunomodulatory effects on primary human myeloid cells in vitro, but do not provoke hepatotoxicity, inflammation or altered response to liver injury in vivo, suggesting the safety of these MB for diagnostic and therapeutic purposes.