Figure 8: 3PO / SU5416 combo-therapy normalizes pathological angiogenesis.

(a–e) Representative images of retinas (× 20 magnification) stained for isolectinB4 (IB4) in normal conditions (nVEGF, a) and after VEGF injection (hVEGF, b-e). Pups were treated with DMSO (CTR, a,b), 3PO (c), SU5416 (d) or 3PO plus SU5416 (e). Note the widened vessel lumen and fewer tip cells at the vascular forefront upon VEGF injection (b), and the partial (c,d) and complete (e) normalization by single or combined 3PO and SU5416 treatment. Scale bar, 50 μm. (f,g) Quantification of vessel width (f) and tip cell number at the retinal front (g). Data are mean±s.e.m.; n=7 per condition; NS: not significant, *P<0.05, ***P<0.001 versus DMSO-treated vessels in normal conditions. ###, P<0.001 versus DMSO-treated vessels in hVEGF. The statistical significance between the single or combined 3PO/SU5416 treatments is also indicated (NS: not significant, *P<0.05, **P<0.01, ***P<0.001); Student’s t-test. a.u., arbitrary units. (h) VE-cadherin junction classification from ‘active’ (red; irregular/serrated morphology with vesicular/diffuse regions) to ‘inhibited’ (dark blue; straighter morphology and less vesicular staining). (i–p) VE-cadherin morphology of retinal vessels was hand-classified according to the key in h, yielding VE-cadherin heat maps in normal conditions (nVEGF, i) and upon VEGF injection (hVEGF, j–p) of pups, treated with DMSO (j,k), 3PO (l,m), SU5416 (n,o), or 3PO plus SU5416 (p). The colour(s) provide insight into the extent of junctional heterogeneity of the vessels. Compared to the heterogeneous VE-cadherin pattern in nVEGF retinas (i), hVEGF induces clusters of inhibited (j) or activated (k) ECs. These clusters are smaller upon 3PO (inhibited: l; active: m) or SU5416 monotherapy (inhibited: n; active: o) and completely normalized to heterogeneous patterns upon combined 3PO plus SU5416 treatment (p). Scale bar, 20 μm. (q,r) Prevalence of strongly inhibited (q) or activated (r) VE-cadherin junctions in retinal vessels. Data are mean±s.e.m.; n=5–9 per condition; NS, not significant, *P<0.05, ***P<0.001 versus nVEGF (black); #P<0.05, ##P<0.01 versus hVEGF (grey). The statistical significance between the single or combined 3PO/SU5416 treatments is also indicated (*P<0.05); One-tailed Wilcoxon rank-sum test for equal medians.