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Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling

Abstract

The gut microbiota is a complex ecosystem that has coevolved with host physiology. Colonization of germ-free (GF) mice with a microbiota promotes increased vessel density in the small intestine1, but little is known about the mechanisms involved. Tissue factor (TF) is the membrane receptor that initiates the extrinsic coagulation pathway2, and it promotes developmental and tumour angiogenesis3,4. Here we show that the gut microbiota promotes TF glycosylation associated with localization of TF on the cell surface, the activation of coagulation proteases, and phosphorylation of the TF cytoplasmic domain in the small intestine. Anti-TF treatment of colonized GF mice decreased microbiota-induced vascular remodelling and expression of the proangiogenic factor angiopoietin-1 (Ang-1) in the small intestine. Mice with a genetic deletion of the TF cytoplasmic domain or with hypomorphic TF (F3) alleles had a decreased intestinal vessel density. Coagulation proteases downstream of TF activate protease-activated receptor (PAR) signalling implicated in angiogenesis5. Vessel density and phosphorylation of the cytoplasmic domain of TF were decreased in small intestine from PAR1-deficient (F2r−/−) but not PAR2-deficient (F2rl1−/−) mice, and inhibition of thrombin showed that thrombin–PAR1 signalling was upstream of TF phosphorylation. Thus, the microbiota-induced extravascular TF–PAR1 signalling loop is a novel pathway that may be modulated to influence vascular remodelling in the small intestine.

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Figure 1: TF promotes microbe-induced vascular remodelling in the gut.
Figure 2: The gut microbiota increases TF procoagulant activity and cell-surface localization.
Figure 3: The gut microbiota increases phosphorylation of the cytoplasmic tail of TF, which increases vessel density in the intestine.
Figure 4: PAR1 activation increases vessel density in the small intestine.

References

  1. 1

    Stappenbeck, T. S., Hooper, L. V. & Gordon, J. I. Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc. Natl Acad. Sci. USA 99, 15451–15455 (2002)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Morrissey, J. H., Fakhrai, H. & Edgington, T. S. Molecular cloning of the cDNA for tissue factor, the cellular receptor for the initiation of the coagulation protease cascade. Cell 50, 129–135 (1987)

    CAS  Article  Google Scholar 

  3. 3

    Carmeliet, P. et al. Role of tissue factor in embryonic blood vessel development. Nature 383, 73–75 (1996)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Belting, M. et al. Regulation of angiogenesis by tissue factor cytoplasmic domain signaling. Nature Med. 10, 502–509 (2004)

    CAS  Article  Google Scholar 

  5. 5

    Griffin, T. C., Srinivasan, Y., Zheng, Y.-W., Huang, W. & Coughlin, S. R. A role for thrombin receptor signaling in endothelial cells during embryonic development. Science 293, 1666–1670 (2001)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Hellström, M. et al. Dll4 signalling through Notch 1 regulates formation of tip cells during angiogenesis. Nature 445, 776–780 (2007)

    ADS  Article  Google Scholar 

  7. 7

    Sato, T. N. et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 376, 70–74 (1995)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Suri, C. et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171–1180 (1996)

    CAS  Article  Google Scholar 

  9. 9

    Iwanaga, S. The limulus clotting reaction. Curr. Opin. Immunol. 5, 74–82 (1993)

    CAS  Article  Google Scholar 

  10. 10

    More, L. et al. Immunohistochemical study of tissue factor expression in normal intestine and idiopathic inflammatory bowel disease. J. Clin. Pathol. 46, 703–708 (1993)

    CAS  Article  Google Scholar 

  11. 11

    Luther, T. et al. Tissue factor expression during human and mouse development. Am. J. Pathol. 149, 101–113 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Parry, G. C., Erlich, J. H., Carmeliet, P., Luther, T. & Mackman, N. Low levels of tissue factor are compatible with development and hemostasis in mice. J. Clin. Invest. 101, 560–569 (1998)

    CAS  Article  Google Scholar 

  13. 13

    Snyder, L. A. et al. Expression of human tissue factor under the control of the mouse tissue factor promoter mediates normal hemostasis in knock-in mice. J. Thromb. Haemost. 6, 306–314 (2008)

    CAS  Article  Google Scholar 

  14. 14

    van den Berg, Y. W. et al. Alternatively spliced tissue factor induces angiogenesis through integrin ligation. Proc. Natl Acad. Sci. USA 106, 19497–19502 (2009)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Garabedian, E. M., Roberts, L. J., McNevin, M. S. & Gordon, J. I. Examining the role of Paneth cells in the small intestine by lineage ablation in transgenic mice. J. Biol. Chem. 272, 23729–23740 (1997)

    CAS  Article  Google Scholar 

  16. 16

    Bry, L., Falk, P. G., Midtvedt, T. & Gordon, J. I. A model of host–microbial interactions in an open mammalian ecosystem. Science 273, 1380–1383 (1996)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Krudysz-Amblo, J., Jennings, M. E., II, Mann, K. G. & Butenas, S. Carbohydrates and activity of natural and recombinant tissue factor. J. Biol. Chem. 285, 3371–3382 (2010)

    CAS  Article  Google Scholar 

  18. 18

    Camerer, E. et al. Opposite sorting of tissue factor in human umbilical vein endothelial cells and Madin–Darby canine kidney epithelial cells. Blood 88, 1339–1349 (1996)

    CAS  PubMed  Google Scholar 

  19. 19

    Dorfleutner, A., Hintermann, E., Tarui, T., Takada, Y. & Ruf, W. Cross-talk of integrin α3β1 and tissue factor in cell migration. Mol. Biol. Cell 15, 4416–4425 (2004)

    CAS  Article  Google Scholar 

  20. 20

    Zioncheck, T. F., Soumitra, R. & Vehar, G. A. The cytoplasmic domain of tissue factor is phosphorylated by a protein kinase C-dependent mechanism. J. Biol. Chem. 267, 3561–3564 (1992)

    CAS  PubMed  Google Scholar 

  21. 21

    Dorfleutner, A. & Ruf, W. Regulation of tissue factor cytoplasmic domain phosphorylation by palmitoylation. Blood 102, 3998–4005 (2003)

    CAS  Article  Google Scholar 

  22. 22

    Melis, E. et al. Targeted deletion of the cytoplasmic domain of tissue factor in mice does not affect development. Biochem. Biophys. Res. Commun. 286, 580–586 (2001)

    CAS  Article  Google Scholar 

  23. 23

    Blackburn, J. S. & Brinckerhoff, C. E. Matrix metalloproteinase-1 and thrombin differentially activate gene expression in endothelial cells via PAR-1 and promote angiogenesis. Am. J. Pathol. 173, 1736–1746 (2008)

    CAS  Article  Google Scholar 

  24. 24

    Schaffner, F. et al. Cooperation of tissue factor cytoplasmic domain and PAR2 signaling in breast cancer development. Blood 116, 6106–6113 (2010)

    CAS  Article  Google Scholar 

  25. 25

    Bäckhed, F. et al. The gut microbiota as an environmental factor that regulates fat storage. Proc. Natl Acad. Sci. USA 101, 15718–15723 (2004)

    ADS  Article  Google Scholar 

  26. 26

    Connolly, A. J., Ishihara, H., Kahn, M. L., Farese, R. V., Jr & Coughlin, S. R. Role of the thrombin receptor in development and evidence for a second receptor. Nature 381, 516–519 (1996)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Damiano, B. P. et al. Cardiovascular responses mediated by protease-activated receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1. J. Pharmacol. Exp. Ther. 288, 671–678 (1999)

    CAS  PubMed  Google Scholar 

  28. 28

    Perreault, N. & Beaulieu, J.-F. Primary cultures of fully differentiated and pure human intestinal epithelial cells. Exp. Cell Res. 245, 34–42 (1998)

    CAS  Article  Google Scholar 

  29. 29

    Furlan-Freguia, C., Marchese, P., Gruber, A., Ruggeri, Z. M. & Ruf, W. P2X7 receptor signaling contributes to tissue factor-dependent thrombosis in mice. J. Clin. Invest. 121, 2932–2944 (2011)

    CAS  Article  Google Scholar 

  30. 30

    Falcón, B. L. et al. Contrasting actions of selective inhibitors of angiopoietin-1 and angiopoietin-2 on the normalization of tumor blood vessels. Am. J. Pathol. 175, 2159–2170 (2009)

    Article  Google Scholar 

  31. 31

    Stappenbeck, T. S. et al. Laser capture microdissection of mouse intestine: characterizing mRNA and protein expression, and profiling intermediary metabolism in secified cell populations. Methods Enzymol. 356, 167–196 (2002)

    CAS  Article  Google Scholar 

  32. 32

    Petersen, L. C. et al. Characterization of recombinant murine factor VIIa and recombinant murine tissue factor: a human–murine species compatibility study. Thromb. Res. 116, 75–85 (2005)

    CAS  Article  Google Scholar 

  33. 33

    Söderberg, O. et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nature Methods 3, 995–1000 (2006)

    Article  Google Scholar 

  34. 34

    Kirchhofer, D., Moran, P., Bullens, S. & Peale, F. A monoclonal antibody that inhibits mouse tissue factor function. J. Thromb. Haemost. 3, 1098–1099 (2005)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank R. Perkins for editing the manuscript; C. Arvidsson, A. Hallén, S. Wagoner, M. Karlsson, D. O’Donell, S. Islam, N. Hörmann and A. Mohammadzadeh for technical assistance; A. Hallén for providing Supplementary Fig. 16; and P. Lindahl, J. Gordon, C. Betsholtz, M. Bergö, A. Wichmann, V. Tremaroli, M. Levin and S. Massberg for comments and suggestions. We are grateful to D. Kirchhofer for the gift of 1H1 monoclonal anti-mouse TF antibody, J. Nichols at Amgen for mL4-3, N. Mackman for the low-TF mice, M. Anderson for the human TF knock-in mice, and J. Gordon for providing CR2-tox176 mice. This study was supported by the Swedish Foundation for Strategic Research, the Swedish Research Council, Torsten and Ragnar Söderberg’s foundation, Petrus and Augusta Hedlund’s foundation, and the Swedish federal government under the LUA/ALF agreement to F.B., National Institutes of Health grants HL-60742 and HL-77753 to W.R., and a Marie Curie Fellowship, a Marie Curie Reintegration Grant from the European Union and the German Federal Ministry of Education and Research (BMBF 01EO1003) to C.R.

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C.R. was responsible for conception and study design, biochemical analysis of TF, analysis of vessel densities, data assembly and analysis, and writing the manuscript. M.B., T.U.G., F.S. and G.Ö.L. performed data collection, analysis and interpretation and commented on the manuscript. L.C.P. provided material. W.R. and F.B. were responsible for conception and study design, data analysis and interpretation, and writing the manuscript.

Corresponding author

Correspondence to Fredrik Bäckhed.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-17 and Supplementary Table 1. (PDF 2185 kb)

Supplementary Movie 1

This movie shows a three dimensional rendering of a small intestinal epithelial cell stained for tissue factor (red), cytokeratin (green), and nuclei (blue). (MOV 5 kb)

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Reinhardt, C., Bergentall, M., Greiner, T. et al. Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling. Nature 483, 627–631 (2012). https://doi.org/10.1038/nature10893

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