Here we provide a protocol for quantitative three-dimensional ex vivo mouse aortic ring angiogenesis assays, in which developing microvessels undergo many key features of angiogenesis over a timescale similar to that observed in vivo. The aortic ring assay allows analysis of cellular proliferation, migration, tube formation, microvessel branching, perivascular recruitment and remodeling-all without the need for cellular dissociation-thus providing a more complete picture of angiogenic processes compared with traditional cell-based assays. Our protocol can be applied to aortic rings from embryonic stage E18 through to adulthood and can incorporate genetic manipulation, treatment with growth factors, drugs or siRNA. This robust assay allows assessment of the salient steps in angiogenesis and quantification of the developing microvessels, and it can be used to identify new modulators of angiogenesis. The assay takes 6-14 d to complete, depending on the age of the mice, treatments applied and whether immunostaining is performed.

Down Syndrome (DS) is a genetic disorder caused by full or partial trisomy of chromosome 21. It occurs in approximately 1/750 live births and presents with many clinical phenotypes including a reduced incidence of solid tumours1,2. Recent work using the Ts65Dn model of DS, that has orthologs of approximately 50% of the genes on human chromosome 21 (Hsa21), has suggested that three copies of the ETS23 or Down Syndrome candidate region 1 (DSCR1) genes4 (a previously known suppressor of angiogenesis5,6) is sufficient to inhibit tumour growth. We have used the Tc1 transchromosomic mouse model of DS9 to dissect the contribution of extra copies of genes on Hsa21 to tumour angiogenesis. This mouse expresses approximately 81% of Hsa21 genes but not the human DSCR1 region (Supplementary Fig. 1). We transplanted B16F0 and Lewis Lung Carcinoma (LLC) tumour cells into Tc1 mice and showed that growth of these tumours was reduced substantially when compared to wild-type littermate controls. Furthermore, tumour angiogenesis was repressed significantly in Tc1 mice. In particular, in vitro and in vivo angiogenic responses to vascular endothelial growth factor (VEGF) were inhibited. Examination of the genes on the segment of Hsa21 in Tc1 mice identified putative anti-angiogenic genes (ADAMTS17,8 and ERG9) and novel endothelial cell-specific genes10, never shown before to be involved in angiogenesis (JAM-B11 and PTTG1IP) that, when overexpressed, are responsible for the inhibition of angiogenic responses to VEGF. Three copies of these genes within the stromal compartment reduced tumour angiogenesis providing an explanation for the reduced tumour growth in DS. Furthermore, we anticipate that, in addition to the candidate genes that we show to be involved in the repression of angiogenesis, the Tc1 mouse model of DS will likely allow for the identification of other endothelial-specific anti-angiogenic targets relevant to a broad spectrum of cancer patients.

Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that ␤3 integrin can regulate negatively VEGFR2 expression. Here we show that ␤3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of ␣v␤3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when ␤3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of ␤3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that ␣v␤3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that ␤3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2.

Genetic ablation of endothelial Focal Adhesion Kinase (FAK) can inhibit pathological angiogenesis, suggesting that loss of endothelial FAK is sufficient to reduce neovascularisation. Here we show that reduced stromal-FAK expression in FAK-heterozygous mice unexpectedly enhances both B16F0 and CMT19T tumour growth and angiogenesis. We further demonstrate that cell proliferation and microvessel sprouting, but not migration, are increased in serum-stimulated FAK-heterozygous endothelial cells. FAK-heterozygous endothelial cells display an imbalance in FAK phosphorylation at pY397 and pY861 without changes in Pyk2 or Erk1/2 activity. By contrast, serum-stimulated phosphorylation of Akt is enhanced in FAK-heterozygous endothelial cells and these cells are more sensitive to Akt inhibition. Additionally, low doses of a pharmacological FAK inhibitor, although too low to affect FAK autophosphorylation in vitro, can enhance angiogenesis ex vivo and tumor growth in vivo. Our results highlight a potential novel role for FAK as a non-linear, dose-dependent regulator of angiogenesis where heterozygous levels of FAK enhance angiogenesis.

Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. α6β1-and α6β4-integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial α6-integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the α6-integrin-subunit when compared with normal breast tissue. These data suggest that a decrease in α6-integrin-subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial α6-integrin subunit affects tumour growth and angiogenesis, we generated α6fl/fl-Tie1Cre+ mice and showed that endothelial deletion of α6-integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial α6-integrin deficiency elevated significantly VEGF-mediated angiogenesis both in vivo and ex vivo. In particular, α6-integrin-deficient endothelial cells displayed increased levels of VEGF-receptor 2 (VEGFR2) and VEGF-mediated downstream ERK1/2 activation. By developing the first endothelial-specific α6-knockout mice, we show that the expression of the α6-integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo.