For the in vitro angiogenesis assay
, HUVECs (3 x [10.sup.4] cells/well) were seeded into 96-well culture plates, which were coated with Matrigel (BD Biosciences), and cultured with the following media: (a) supernatant from the LentiLacZ group, (b) supernatant from the Lenti-PDGF group supplemented with PD98059 (10 [micro]M), (c) supernatant from the Lenti-PDGF group supplemented with LY294002 (10 [micro]M), or (d) supernatant from the Lenti-PDGF group.
In the independent studies, culture media of RPE cells were placed in contact with ECs during angiogenesis assays, intending to evaluate the effects of GF released by RPE.
Some studies used different matrices from several angiogenesis assays since nowadays there are several laboratories purchasing angiogenesis kits, all based on the matrix method.
Yuan, "Quantitative angiogenesis assays
: progress and problems," Nature Medicine, vol.
The corneal angiogenesis assay involves creating a pocket in the cornea of a rabbit or mouse wherein test tumors or tissues are placed to induce the development of vasculature (Gimbrone et al., 1974; Muthukkaruppan and Auerbach, 1979; Muthukkaruppan et al., 1982).
(1995, 1996) demonstrated, using the tumor cell-induced angiogenesis assay, that 1,25-dihydroxyvitamin D3 (1,25[[OH].sub.2]D3), a derivative of vitamin D3, acted synergistically with retinoids, derivatives of vitamin A, to inhibit angiogenesis in mice.
The most frequently used measure, the thymidine incorporation assay, will serve to introduce several of the key problems of validating in vitro angiogenesis assays
Interestingly, in the in vitro angiogenesis assays
, the inhibitory effects of [beta]APN treatment were more pronounced in tumor-associated pericytes than in their normal counterparts, given that not only a lower amount of total tube-like structures was observed but also a longer time to achieve the peak of such structures.
In further experiments, SV-EC were pretreated with either Akt inhibitor LY294002 (1 [micro]M; Merck-Millipore, Watford, UK), eNOS inhibitor (L-NG-nitroarginine methyl ester, L-NAME; 500 [micro]M; Sigma-Aldrich), high glucose (25 mM; Sigma-Aldrich), TNF-[alpha] (1 ng/mL; Life Technologies), palmitate (100 [micro]M; Sigma-Aldrich), or appropriate control vehicle for 24 h prior to angiogenesis assays
. Cells were incorporated into assays without replenishment of the stimulus/inhibitor and tube formation was quantified after 8h.
Various in vitro (endothelial cells proliferation assay, endothelial cells migration assay, tube formation assay and aortic ring assay) and in vivo (matrigel plug assay, corneal angiogenesis assay
and CAM assay) methods are available to evaluate pro-angiogenic and anti-angiogenic potentials of molecules (Auerbach et al., 2003).