Fig. 3.

CPS49 targets angiogenic blood vessels. (A–L) Mouse aortic ring cultures at day 2 (A–F) and day 5 (G–L) stained with IsolectinB4-AlexaFluor488 conjugate to label endothelial cells (A–C, E–I, K, and L) or anti-alpha-Smooth muscle actin (D and J). Vessels in 2 D.I.V. cultures lack smooth muscle (D), whereas at 5 D.I.V. smooth muscle is present (J; indicated by arrowheads, compare with G). Cultures were treated with DMSO (B, H, E, and K) or CPS49 (C, F, I, and L) and fixed after 6 h (B, C, H, and I) or 24 h (E, F, K, and L). (M and N) Mean ± SEM microvessel count of day 2 and day 5 cultures. CPS49 induces a significant decrease in microvessel number from 2 D.I.V. (compare C and F with B and E; arrows highlight vessels) but not 5 D.I.V. cultures (compare H and I; arrowheads indicate vessels). (O) Percentage change in surviving vessel after addition of CPS49. In both 2 D.I.V. and 5 D.I.V., surviving vessels retract initially after CPS49 treatment, resulting in a decrease in vessel length 6 h posttreatment. Outgrowth resumes subsequently, but by 24 h posttreatment vessels are still stunted compared with controls. (P–T) Cultures of HUVEC cells treated with DMSO (P and S) or 10 μg/mL CPS49 (Q and T). Addition of CPS49 at the time of plating prevented vascular tube formation (P and Q). (Q) Boxed area i is enlarged (ii), indicating no connections or tube formations between HUVEC cells, whereas addition after 8 h in culture, when a vascular network has formed already (R), had no impact on preexisting vessel tubes but prevented further elaboration of the network (S and T). *, P < 0.01 compared with control (Student's unpaired t test). (Scale bars: A–C, 200 μm; D–L, 400 μm; R–T, 300 μm.)