Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.

TLDR

VEGF is a key regulator of vasculogenesis and angiogenesis. The study aimed to determine how nitric oxide mediates VEGF‑induced proliferation and angiogenic network formation in human endothelial cells. Human umbilical vein endothelial cells were employed to assess NO production and signaling pathways downstream of VEGF stimulation. VEGF promoted HUVEC proliferation and 3D network formation through NO, with VEGF‑induced NO production dependent on tyrosine kinase, PI‑3K, and calcium signaling, and associated with increased cGMP, eNOS protein, and NO release, indicating NO mediates VEGF’s angiogenic effects.

Abstract

Vascular endothelial growth factor (VEGF) is a regulator of vasculogenesis and angiogenesis. To investigate the role of nitric oxide (NO) in VEGF-induced proliferation and in vitro angiogenesis, human umbilical vein endothelial cells (HUVEC) were used. VEGF stimulated the growth of HUVEC in an NO-dependent manner. In addition, VEGF promoted the NO-dependent formation of network-like structures in HUVEC cultured in three dimensional (3D) collagen gels. Exposure of cells to VEGF led to a concentration-dependent increase in cGMP levels, an indicator of NO production, that was inhibited by nitro-L-arginine methyl ester. VEGF-stimulated NO production required activation of tyrosine kinases and increases in intracellular calcium, since tyrosine kinase inhibitors and calcium chelators attenuated VEGF-induced NO release. Moreover, two chemically distinct phosphoinositide 3 kinase (PI-3K) inhibitors attenuated NO release after VEGF stimulation. In addition, HUVEC incubated with VEGF for 24 h showed an increase in the amount of endothelial NO synthase (eNOS) protein and the release of NO. In summary, both short- and long-term exposure of human EC to VEGF stimulates the release of biologically active NO. While long-term exposure increases eNOS protein levels, short-term stimulation with VEGF promotes NO release through mechanisms involving tyrosine and PI-3K kinases, suggesting that NO mediates aspects of VEGF signaling required for EC proliferation and organization in vitro.