Augmentation of vascular permeability of VEGF is enhanced by KDR-binding proteins

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Abstract

VEGF165 is a key regulator of angiogenesis and a potent vascular permeability factor. Using snake venom proteins as tools, we demonstrate the enhanced vascular leakage of VEGF by KDR-binding proteins. The snake venom-derived KDR-specific VEGF, vammin, potently enhanced vascular leakage compared with other known permeability-enhancing factors including VEGF165, while KDR-bp from snake venom, a KDR antagonist of endothelial cell growth was a very weak permeability enhancer. Unexpectedly when co-administrated, KDR-bp synergistically enhanced either vammin or VEGF165-stimulated vascular leakage, despite its antagonistic effect on cell growth. This augmenting effect was specifically observed in the combined administration of KDR-bp with either VEGF165 or vammin, but not other combination of known permeability-enhancing factors. We further demonstrated a similar increased vascular leakage by the combined administration of VEGF165 and TIMP-3, the only known endogenous antagonist of KDR. Our findings implicate TIMP-3 as a critical player in the vascular leakage-enhancing effect of VEGF165 in vivo.

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Materials and methods

Materials. Snake venom proteins, vammin and KDR-bp, were purified from crude venom of the snakes as described previously [19], [20]. Recombinant human VEGF165 and TIMP-3 were purchased from PeproTech (Rocky Hill, NJ, USA) and R&D Systems (Minneapolis, MN, USA), respectively. Nω-nitro-l-arginine (l-NNA) was obtained from Sigma (St. Louis, MO, USA).

Miles assay. Vascular permeability was measured by using Miles assay [22]. After anesthesia by ketamine, guinea pigs (400 g) were shaved on the back

VEGF-F is a highly potent enhancer of vascular permeability

To clarify the correlation between vascular permeability-enhancing effects and receptor-binding of VEGFs, we first examined the effect of vammin, a snake venom-derived KDR-specific VEGF-F [19], on vascular leakage. Permeability-enhancing activity was measured by the Miles dye leakage assay [22]. While both vammin and VEGF165 similarly provoked dye leakage when injected intradermally at low concentrations (<10−8 M) vammin was a more potent enhancer of vascular leakage than VEGF165 at higher

Acknowledgments

We thank Dr. Craig M. Jackson for helping to revise the language of the manuscript. This work was supported in part by Scientific Research Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan (T.M.), Mitsubishi Pharma Research Foundation (Y.Y.), and President’s Special Research Grant from Meiji Pharmaceutical University (Y.Y.).

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