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Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor

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Abstract

Endothelial secretion of von Willebrand factor (VWF) from intracellular organelles known as Weibel-Palade bodies (WPBs) is required for platelet adhesion to the injured vessel wall. Here we demonstrate that WPBs are often found near or within autophagosomes and that endothelial autophagosomes contain abundant VWF protein. Pharmacological inhibitors of autophagy or knockdown of the essential autophagy genes Atg5 or Atg7 inhibits the in vitro secretion of VWF. Furthermore, although mice with endothelial-specific deletion of Atg7 have normal vessel architecture and capillary density, they exhibit impaired epinephrine-stimulated VWF release, reduced levels of high–molecular weight VWF multimers and a corresponding prolongation of bleeding times. Endothelial-specific deletion of Atg5 or pharmacological inhibition of autophagic flux results in a similar in vivo alteration of hemostasis. Thus, autophagy regulates endothelial VWF secretion, and transient pharmacological inhibition of autophagic flux may be a useful strategy to prevent thrombotic events.

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Figure 1: Proximity of autophagosomes and WPBs within endothelial cells.
Figure 2: A role for the essential autophagy proteins Atg5 and Atg7 in endothelial cell secretion of VWF.
Figure 3: Atg7 regulates the processing and maturation of VWF.
Figure 4: Autophagy regulates the pH, morphology and secretion of WPBs.
Figure 5: Endothelial-specific deletion of Atg7 in mice.
Figure 6: Inhibition of autophagy alters secretion of VWF in mice.

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Acknowledgements

We are grateful to S. Gutkind (NIH) for help with primary endothelial cell isolation, T. Carter for the gift of the VWF monomeric GFP plasmid (VWF-mGFP), J. Lippincott-Schwartz (NIH) for the LC3-mCherry plasmid and Y. Fitz (NIH) for help with coagulation measurements. We thank B. Zinselmeyer (NIH) for assistance with the spot cluster analysis and C.A. Brantner (NIH) for help performing cryo-immunogold electron microscopy. This work was supported by NIH Intramural Funds. T.T. is a recipient of a Japan Society for the Promotion of Science Research Fellowship in Biomedical and Behavioral Research at the NIH.

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Contributions

T.T. and K.T. designed, performed and analyzed the experiments and aided in writing the manuscript. I.H.L., J.L., I.I.R., M.M.F. and L.C. contributed to the completion of various experiments. D.M., C.A.C., X.S.W., R.W., P.S.C. and M.P.D. aided in specialized imaging procedures. M.K. provided valuable reagents and advice. T.F. helped conceive the study, supervised the research and contributed to writing the manuscript.

Corresponding author

Correspondence to Toren Finkel.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 (PDF 1155 kb)

Supplementary Video 1

Three dimensional reconstitution of super resolution images of the interaction of autophagosomes and WPBs. Images of LC3 (green) and VWF (red) are shown with a LC3 coated autophagosome apparently encircling a WPB. (MOV 757 kb)

Supplementary Video 2

Dual-color TIRF imaging of LC3-mCherry and VWF-mGFP following histamine stimulation in HUVEC. (MOV 1182 kb)

Supplementary Video 3

TIRF imaging of LC3-GFP following histamine stimulation in HUVEC. (MOV 4025 kb)

Supplementary Video 4

VWF-GFP analysis following histamine stimulation in control knockdown cells. Arrows indicate WPB fusion events. (MOV 3939 kb)

Supplementary Video 5

VWF-GFP analysis following histamine stimulation in Atg7 knockdown cells. (AVI 154375 kb)

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Torisu, T., Torisu, K., Lee, I. et al. Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor. Nat Med 19, 1281–1287 (2013). https://doi.org/10.1038/nm.3288

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