Beta3-adrenergic receptors modulate vascular endothelial growth factor release in response to hypoxia through the nitric oxide pathway in mouse retinal explants

Beta-adrenergic receptors (beta-ARs) play a role in angiogenic processes that characterize neovascularization-associated retinal diseases, but the role of beta 3-ARs has not been disclosed yet. We used ex vivo retinal explants to investigate the role of beta 3-ARs in regulating vascular endothelial growth factor (VEGF) release associated with hypoxia. Whether nitric oxide (NO) mediates beta 3-AR regulation of VEGF release was also investigated. beta 3-AR activation was obtained using BRL 37344, whereas SR59230A, L-748,337, or specific siRNAs were used to block beta 3-ARs. Pharmacological approaches were used to interfere with the NO pathway. Western blot was used to determine beta-AR levels. Enzyme-linked immunosorbent assay was used to measure VEGF release. NO production was assessed by a colorimetric assay. We found that hypoxia upregulates beta 3-ARs. In addition, we observed that beta 3-AR activation with BRL 37344 increases VEGF release in response to hypoxia. Either beta 3-AR blocker or beta 3-AR silencing downregulates drastically hypoxic levels of VEGF. With experiments using NO synthase (NOS) blockade with L-NAME, NOS activation with fluvastatin or NO supplementation with SNAP, we demonstrated that beta 3-ARs and VEGF are functionally coupled via the NO pathway. In summary, the data presented here support the assumption that beta 3-ARs are involved in the regulation of angiogenic responses to hypoxia through the NO signalling, a key pathway in hypoxic/ischemic diseases. Although extrapolation of these data to the human situation is difficult, these findings may help to explore the possible role of beta 3-ARs in vascularization-associated disorders.