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. 2003 May;162(5):1431–1439. doi: 10.1016/S0002-9440(10)64276-9

Figure 6.

Figure 6.

DIVAA characterization of angiogenesis inhibition. To demonstrate that DIVAA is useful in a characterization of a variety of angiogenesis inhibitors experiments using endogenous angiogenesis inhibitors (TSP-1) and a genetic model (MMP-2-deficient murine model) are conducted. A: Angioreactors are prepared with FGF-2 alone (FGF-2), FGF-2 plus 0.1 μmol/L final concentration of TSP-1 (FGF+TSP-1), as well as FGF-2 plus 25 μmol/L final concentration of the following TSP-1 peptides, p 416, p545, and p641. DIVAA is performed as described. Results are corrected for negative control (Matrigel without FGF-2) and plotted as relative fluorescence units. DIVAA results demonstrate ∼60% inhibition by 0.1 μmol/L TSP-1, with the following rank order of TSP-1 peptides (25 μmol/L final concentration) for inhibition of angiogenesis: p545> p416. These results are consistent with previous reports describing the relative anti-angiogenic potency of TSP-1 peptides. Data represent the mean ± SD of five replicate determinations. B: DIVAA quantitation of angiogenesis in a murine gene-targeted model. C57/BL6 mice with targeted disruption of the matrix metalloproteinase-2 (MMP-2) gene (KO) and wt C57/BL6 (WT) mice were implanted with angioreactors containing FGF-2 or without (negative control, C) and the angiogenic responses quantitated by DIVAA. No detectable differences in the negative controls (WT versus KO) are observed. The FGF-2-induced angiogenic response in KO mice is reduced by 35% compared with responses observed in WT mice (P < 0.01), consistent with previous reports. Data represent mean ± SD of five replicate determinations. C: Comparison of MMP-2 and MMP-9 expression in angioreactors recovered from WT and KO mice by zymogram analysis. 12 Lane 1, negative control, Matrigel alone; lanes 2 and 3, angioreactors from WT mice; lanes 4 and 5, implants from KO animals; lane 6, MMP-2 and MMP-9 standards. Results show complete loss of MMP-2 (pro- and active forms) in KO mice without significant change in MMP-9 expression. Zymogram gel loading is normalized to total protein concentration.