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Endothelial cell expression of haemoglobin α regulates nitric oxide signalling

Abstract

Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems1,2,3. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone4, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle3,5. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe3+ state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe2+ state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1)6. Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells7,8,9,10,11,12,13.

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Figure 1: Monomeric Hb α is expressed in ECs and enriched at the MEJ.
Figure 2: Hb α regulates vessel tone, NO diffusion and associates with eNOS.
Figure 3: The oxidation state of Hb α resides in a mixture of Fe2+ and Fe3+.
Figure 4: CYB5R3 expression and activity are crucial for vasomotor tone and NO diffusion.

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Acknowledgements

We thank the Advanced Microscopy and Histology core at the University of Virginia and the Yale Proteomic Facility. We acknowledge V. Balasubramaniam, S. Lewis and D. Singel for discussions of the data and B. Duling for critical evaluation of experiments and the manuscript. We also thank M. Weiss for the Hb α-stabilizing protein antibody and for discussions. This work was supported by an American Heart Association Scientist Development Grant (B.E.I.), National Institutes of Health grants HL088554 (B.E.I.), HL107963 (B.E.I.) HL059337 (B.G.), HL101871 (B.G.), HL112904 (A.C.S.) and HL007284 (A.W.L. and A.C.S.). M.B. and S.R.J. were supported by American Heart Association postdoctoral fellowships, and A.W.L. and M.Y.L. were supported by American Heart Association predoctoral fellowships.

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A.C.S. performed most of the experiments and data analysis. A.W.L. performed vessel transfections. Vascular reactivity was executed by A.W.L. and M.B. S.R.J. carried out immunofluorescence studies and S.T.D. assisted in NO diffusion and consumption assays. M.Y.L. and P.S.B. performed real-time PCR experiments, and A.K.B. helped with all cell culture experiments. L.C. performed the modelling experiments. B.G. helped with experimental design, provided use of the NO analyser and NO, and assisted with final manuscript preparation. B.E.I. initiated, directed and supported the work through all levels of development. All the authors discussed the results and commented on the manuscript.

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Correspondence to Brant E. Isakson.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-18, Supplementary Tables 1-2 and Supplementary Methods, which include Supplementary References and Tables 1-3. (PDF 4557 kb)

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Straub, A., Lohman, A., Billaud, M. et al. Endothelial cell expression of haemoglobin α regulates nitric oxide signalling. Nature 491, 473–477 (2012). https://doi.org/10.1038/nature11626

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