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

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Abstract

Beta-adrenergic receptors (β-ARs) play a role in angiogenic processes that characterize neovascularization-associated retinal diseases, but the role of β3-ARs has not been disclosed yet. We used ex vivo retinal explants to investigate the role of β3-ARs in regulating vascular endothelial growth factor (VEGF) release associated with hypoxia. Whether nitric oxide (NO) mediates β3-AR regulation of VEGF release was also investigated. β3-AR activation was obtained using BRL 37344, whereas SR59230A, L-748,337, or specific siRNAs were used to block β3-ARs. Pharmacological approaches were used to interfere with the NO pathway. Western blot was used to determine β-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 β3-ARs. In addition, we observed that β3-AR activation with BRL 37344 increases VEGF release in response to hypoxia. Either β3-AR blocker or β3-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 β3-ARs and VEGF are functionally coupled via the NO pathway. In summary, the data presented here support the assumption that β3-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 β3-ARs in vascularization-associated disorders.

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Acknowledgments

This work was supported by the Meyer Foundation, “A. Meyer” University Children’s Hospital (PB) and the Ente Cassa di Risparmio of Florence (LF). The authors thank Prof. Giovanni Casini for critical reading of the manuscript and Dr. Angelo Gazzano and Gino Bertolini for assistance with the mouse colonies.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Biology, University of Pisa, Via San Zeno, 31, 56127, Pisa, Italy

    Massimo Dal Monte & Paola Bagnoli

  2. Neonatal Intensive Care Unit, Department of Critical Care Medicine, “A. Meyer” University Children’s Hospital, Viale Gaetano Pieraccini, 24, Florence, Italy

    Luca Filippi

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  1. Massimo Dal Monte
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  2. Luca Filippi
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  3. Paola Bagnoli
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Correspondence to Paola Bagnoli.

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β3-AR mRNA and protein after siRNA transfection in hypoxic retinal explants (PDF 764 kb)

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Dal Monte, M., Filippi, L. & Bagnoli, P. Beta3-adrenergic receptors modulate vascular endothelial growth factor release in response to hypoxia through the nitric oxide pathway in mouse retinal explants. Naunyn-Schmiedeberg's Arch Pharmacol 386, 269–278 (2013). https://doi.org/10.1007/s00210-012-0828-x

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