Figure 3
Characterization of mCRP-induced vascular activation:
(A) BAEC spheroids were generated to examine the effect of mCRP on sprout structure and formation in a 3-dimensional system. In normal culture conditions, sprouting was slower, sprouts had a thicker appearance and cell-cell junctions were maintained (left panel). In the presence of mCRP, sprouts formed more quickly, were notably thinner in appearance and the inter-cellular gaps between cells was notably larger (right panel). (B) Dorsal matrigel implants containing mCRP (10 μg/ml; 72 h) produced strong and visible haemorrhagic angiogenesis (iv; arrows) compared with a typical, normal looking vascular response seen in the presence of VEGF (ii; 25 ng/ml), whilst nCRP (10 μg/ml) produced very little angiogenic response (p < 0.05 increase in the presence of mCRP and VEGF compared with control implants) (iii). In (C) the graph shows a significant increase in monolayer permeability in the presence of mCRP (10 μg/ml; 8 h) using a Millipore-based filter assay, similar to that produced by 10% DMSO (p < 0.01 increase in FITC dextran penetrating the monolayer in the presence of either mCRP or the positive control DMSO) and lighter regions in the images shown indicate areas of increased permeability. (D) Expression of adhesion molecules was examined in BAEC treated with mCRP (10 μg/ml; 24 h). NCAM expression was increased by approximately 2.8 fold whilst VCAM, ICAM and integrins were not affected (data not shown). β-tubulin was used as the house keeping control (gel and bar chart shown). These experiments were repeated at least twice and a representative example is shown.
