Nerve growth factor (NGF) supports the survival and differentiation of sympathetic and sensory neurons and is also mitogenic for a variety of tumors. K252a, an antagonist of NGF receptor TrkA, was previously used as a pharmacological tool to study NGF actions and as a lead compound for developing anti-tumor drugs. Since recently, NGF was characterized as an angiogenic factor, we sought to investigate the angiostatic properties of K252a on endothelial cells (ECs). For this purpose, we used a murine brain microcapillary ECs model in which we found autocrine release of NGF in the culture medium and activation of TrkA receptor-induced downstream signaling molecules Erk1/2, Akt, and PLCγ. In this model, we demonstrated the angiostatic property of K252a based on its ability to affect several important angiogenic steps. K252a, but not its cell membrane impermeable analogue K252b at 100 nM: (i) inhibited the proliferation of the ECs by 45 ± 9%; (ii) reduced by 70 ± 4% the migration of the ECs measured in a wound-closure model; (iii) reduced by 29 ± 9% the formation of tube-like structures of the ECs cultured on Matrigel; (iv) stimulated by 100 ± 25% the collagen deposition by the ECs, a process responsible for the increased endothelial barrier functions expressed by 22 ± 5% reduction of paracellular permeability and by 17 ± 3% elevation of transendothelial electrical resistance. These data suggest that NGF/TrkA may represent a target for the development of novel, K252a-derived multikinase inhibitors drugs with anti-tumor and angiostatic dual activities.
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Acknowledgments This work was supported in part by a grant-in-aid form the Stein Family Foundations (PL and PIL); PL is affiliated and partially supported by the David R. Bloom Center for Pharmacy; and the Dr. Adolf and Klara Brettler Center for Research in Molecular Pharmacology and Therapeutics at The Hebrew University of Jerusalem, Israel; SL is supported by ‘‘Eshkol fellowship’’ from the Israeli Ministry of Science and Technology. The authors would like to acknowledge the help of Mrs. Zehava Cohen for graphics preparation and the constructive remarks of the referees.
- Nerve growth factor
- TrkA receptor