Fig. 3
Angiogenic monocytes present low transmigration in conventional inflammatory conditions. a Prongiogenic (non-classical and intermediate CD16+) versus non-angiogenic (inflammatory CD16-) monocyte transmigration through TNF-activated endothelium under flow assay by live imaging. Non-angiogenic monocytes transmigrate through TNF-activated endothelial monolayer, whereas angiogenic monocytes adhere but remain crawling with very few transmigrating. Scale bar = 10 µm, blue arrows indicate crawling proangiogenic monocytes and brown arrowhead shows a transmigrating non-angiogenic monocyte. b Quantification of the transmigration efficiency of proangiogenic versus non-angiogenic monocytes overtime (top) and the proportion of monocyte subsets within the transmigrated pan-population (bottom) indicating most abluminal monocytes were non-angiogenic under conventional inflammatory conditions. n = 3 independent experiments with monocytes from three different donors, the data presented as mean ± s.e.m., ***p-value = 0.0004 in two-way ANOVA test. c Post-transmigration assay analysis of proangiogenic monocyte localisation by confocal microscopy. Scale bar = 10 µm, orthogonal presentation shows views from x, y and z axes. Basal (bl) and apical (ap) extremities are indicated. HUVEC and monocytes were pre-stained respectively with green and blue cell tracker, and proangiogenic monocytes and cell junctions were stained respectively with anti-CD16 and anti-VE-cadherin after transmigration assays. Orthogonal views show adherent but non-transmigrated proangiogenic monocytes on endothelial luminal surface. d Effect of CD16 labelling on proangiogenic monocytes transmigration analyzed after transmigration assay. n = 4 independent experiments with monocytes from four different donors, 150 individual cells tracked for each condition, the data presented as mean ± s.d., ***p-value <0.0001 in two-way ANOVA. The monocyte positioning was unaffected by labelling with the CD16 antibody used for tracking proangiogenic monocytes
