Figure 4

In vivo-like endothelial ECM-integrin cascade induced by BM-COL1 and applications of the synthetic endothelial ECM system.

(a) Expression of integrin α6 (green) in COL1 and synthetic endothelial ECM systems. ECs in the synthetic endothelial ECM system strongly expressed integrin α6 on their cell membranes. Scale bar, 100 μm. (right) Average fluorescent intensities of the integrin alpha6 expressions in the synthetic endothelial ECM (red bar) and the COL1 (blue bar) systems at culture day5. Error bars represent standard deviation (***P = 0.001, n = 4 ~ 6). (b) Absolute vascular permeability values for the blocking antibody (the GoH3 rat monoclonal antibody against the integrin alpha 6 subunit) treated synthetic endothelial ECM system were significantly increased to the levels of the COL1 compared with those of negative control (IgG2a). (*P = 0.030, **P = 0.001 on culture day4; *P = 0.042 on culture day5). Error bars, ± SEM (n = 8 ~ 20). (c) Tension force was assessed by monitoring the expression levels of diphosphorylated regulatory myosin light chain 2 (RLC-pp; green). White arrows in the upper image indicate overexpressed RLC-pp in ECs cultured on COL1. (right) Average fluorescent intensities of the RLC-pp expressions in the synthetic endothelial ECM (red bar) and the COL1 (blue bar) systems at culture day5. Error bars represent standard deviation (***P = 0.001, n = 4 ~ 6). (d) Schematic of the mechanism underlying the stabilization and tightening of ECs by strengthening EC-ECM adhesion. (e) Neutrophil-like cells (differentiated human HL-60 promyelocytic leukemia cells [dHL-60]), could pass through the EC monolayer and even the BM-COL1 layer. The phase-contrast image was taken 1 hour after seeding dHL-60 cells. Scale bar, 100 μm. (f) The phase-contrast image shows sprouting angiogenesis in the presence of BM-COL1. The image was taken 3 days after stimulation with VEGF. (g). Total sprouting lengths of the sprouting angiogenesis in the synthetic endothelial ECM and the COL1 systems under the angiogenic condition (50-20 ng/ml of VEGF gradient) and the control condition (20-20 ng/ml, no VEGF gradient). Error bar represents standard error (**P = 0.006, n = 2). (h). A confocal microscopic image of the dotted square area in Fig. 4f. Scale bars, 100 μm (a,c) and 150 μm (e,f,h).