Monocyte-endothelial cell interactions in the regulation of vascular sprouting and liver regeneration in mouse.

Partial hepatectomy was performed in wild-type mice and those lacking the monocyte adhesion molecule CD11b. Vascular architecture, angiogenesis and macrophage location were analyzed in the whole livers using simultaneous angiography and macrophage staining with fluorescent multiphoton-microscopy. Monocyte adhesion molecule expression and sprouting-related pathways were evaluated.

Materials and Methods

Animal experiments Male

C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA)) and mice lacking CD11b (Strain: B6.129S4-Itgamtm1Myd/J) from the Jackson Laboratory (Bar Harbor, ME). All animals were maintained in a temperature-controlled room (22°C) on a 12-h light-dark cycle under institutional and NIH guidelines. After arrival, mice were continuously fed ad libitum until euthanasia. Partial hepatectomy was performed as described 26 and approved by the Animal Ethics Committee at Massachusetts Institute of Technology, MA, USA. The regenerating right lobe was used for all analyses at different time points. Liver restoration rate was calculated as liver weight/body weight × 100. Whole-mount multiphoton imaging of macrophage presence and angiography in liver Mice (9–12 weeks old) were anesthetized with isoflurane and then injected with 100 µL of 20 mg/mL 70-kDa Texas red-dextran in Dulbecco's PBS into the tail vein in order to load macrophages by phagocytosis. After 2 hours the animals were euthanized by overexposure to CO 2 . Then, mice were perfused via the left ventricle with phosphate buffered saline (PBS) followed by an injection of fluorescein isothiocyanate-labeled dextran (FITC-dextran, MW 2×10 6 Da., Sigma, St. Louis, MO). Finally, vascular and macrophage fluorescence was visualized under a multiphoton microscope (Leica Microsystems, Heerbrugg, Switzerland). Vascular analysis and macrophage presence were determined by capturing 10 µm z-series of whole liver with a 25 x, N.a. 1.05 objective, Olympus FV-1000 MP (Olympus, America Inc, Center Valley, PA, USA) in which the viewing field is 512 × 512 µm. Number of macrophages, vascular diameter and anastomosis quantification were analyzed in all of the z-images with ImageJ manually or using the tool “angiogenesis analyzer” when appropriate. Intrasinusoidal or extrasinusoidal macrophages attached to vessel walls were quantified as positive macrophage-endothelial cell interactions. Dual staining (in yellow) did not interfere with the identification of intravascular staining of attached macrophages (highlighted in red) which were not stained by FITC-dextran at every focal plane. The initial total number of macrophages (sham) was considered as the original number of resident macrophages (KCs). After hepatectomy, the number of KCs was calculated as: total number of macrophages – (number of macrophage-EC interactions – original number of macrophage-EC interactions).

Tissue and cell analysis

Tridimensional reconstruction software analysis, gene expression analysis by Real-time PCR, immunofluorescent staining, Western blotting and “In vitro” studies are all described in the supporting Materials and Methods.

Statistical analysis

Data are expressed as mean ± standard error. Statistical analysis of the results was performed by one-way analysis of variance (ANOVA), the Newman-Keuls test, and the unpaired Student’s t test when appropriate. Differences were considered to be significant at a p value of 0.05 or less. Data sampled from Gaussian populations were used for the calculation of Pearson Correlation Coefficient. A Pearson correlation coefficient (r) value of >0.75 was considered to exhibit strong positive correlation, a value between 0.50 and 0.75 was considered as a moderate correlation whereas value less than 0.5 was considered to demonstrate a weak correlation between two variables.