Thymosin β4 induces adult epicardial progenitor mobilization and neovascularization

Abstract

Cardiac failure has a principal underlying aetiology of ischaemic damage arising from vascular insufficiency. Molecules that regulate collateral growth in the ischaemic heart also regulate coronary vasculature formation during embryogenesis. Here we identify thymosin β4 (Tβ4) as essential for all aspects of coronary vessel development in mice, and demonstrate that Tβ4 stimulates significant outgrowth from quiescent adult epicardial explants, restoring pluripotency and triggering differentiation of fibroblasts, smooth muscle cells and endothelial cells. Tβ4 knockdown in the heart is accompanied by significant reduction in the pro-angiogenic cleavage product N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). Although injection of AcSDKP was unable to rescue Tβ4 mutant hearts, it significantly enhanced endothelial cell differentiation from adult epicardially derived precursor cells. This study identifies Tβ4 and AcSDKP as potent stimulators of coronary vasculogenesis and angiogenesis, and reveals Tβ4-induced adult epicardial cells as a viable source of vascular progenitors for continued renewal of regressed vessels at low basal level or sustained neovascularization following cardiac injury.

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Figure 1: Tβ4 knockdown results in numerous cardiac defects.
Figure 2: Tβ4 is required for coronary vasculature and great vessel formation.
Figure 3: Tβ4 promotes migration of adult EPDCs and enables their differentiation into vasculogenic cells.
Figure 4: AcSDKP is not sufficient to compensate for loss of Tβ4 in coronary vessel development.
Figure 5: Model for the role of Tβ4 in coronary vessel development.

References

  1. 1

    Smart, N., Hill, A. A., Cross, J. C. & Riley, P. R. A differential screen for putative targets of the bHLH transcription factor Hand1 in cardiac morphogenesis. Mech. Dev. 119, S65–S71 (2002)

    Article  Google Scholar 

  2. 2

    Bock-Marquette, I., Saxena, A., White, M. D., Dimaio, J. M. & Srivastava, D. Thymosin β4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432, 466–472 (2004)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Moses, K., DeMayo, F., Braun, R., Reecy, J. & Schwartz, R. Embryonic expression of an Nkx2–5/Cre gene using ROSA26 reporter mice. Genesis 31, 176–180 (2001)

    CAS  Article  Google Scholar 

  4. 4

    Zeisberg, E. M. et al. Morphogenesis of the right ventricle requires myocardial expression of Gata4. J. Clin. Invest. 115, 1522–1531 (2005)

    CAS  Article  Google Scholar 

  5. 5

    Chen, J. et al. Selective requirement of myosin light chain 2v in embryonic heart function. J. Biol. Chem. 273, 1252–1256 (1998)

    CAS  Article  Google Scholar 

  6. 6

    von Kodolitsch, Y. et al. Coronary artery anomalies Part I: Recent insights from molecular embryology. Z. Kardiol. 93, 929–937 (2004)

    CAS  Article  Google Scholar 

  7. 7

    Ward, N. L. & Dumont, D. J. The angiopoietins and Tie2/Tek: adding to the complexity of cardiovascular development. Semin. Cell Dev. Biol. 13, 19–27 (2002)

    CAS  Article  Google Scholar 

  8. 8

    Merki, E. et al. Epicardial retinoid X receptor α is required for myocardial growth and coronary artery formation. Proc. Natl Acad. Sci. USA 102, 18455–18460 (2005)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Yamashita, J. et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92–96 (2000)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Carmeliet, P. Angiogenesis in health and disease. Nature Med. 9, 653–660 (2003)

    CAS  Article  Google Scholar 

  11. 11

    Tevosian, S. G. et al. FOG-2, a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary vessels from epicardium. Cell 101, 729–739 (2000)

    CAS  Article  Google Scholar 

  12. 12

    Giordano, F. J. et al. A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function. Proc. Natl Acad. Sci. USA 98, 5780–5785 (2001)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Chen, T. H. et al. Epicardial induction of fetal cardiomyocyte proliferation via a retinoic acid-inducible trophic factor. Dev. Biol. 250, 198–207 (2002)

    CAS  Article  Google Scholar 

  14. 14

    Morabito, C. J., Dettman, R. W., Kattan, J., Collier, J. M. & Bristow, J. Positive and negative regulation of epicardial-mesenchymal transformation during avian heart development. Dev. Biol. 234, 204–215 (2001)

    CAS  Article  Google Scholar 

  15. 15

    Poelmann, R. E., Lie-Venema, H. & Gittenberger-de Groot, A. C. The role of the epicardium and neural crest as extracardiac contributors to coronary vascular development. Tex. Heart Inst. J. 29, 255–261 (2002)

    PubMed  PubMed Central  Google Scholar 

  16. 16

    Luttun, A. & Carmeliet, P. De novo vasculogenesis in the heart. Cardiovasc. Res. 58, 378–389 (2003)

    CAS  Article  Google Scholar 

  17. 17

    Kamihata, H. et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 104, 1046–1052 (2001)

    CAS  Article  Google Scholar 

  18. 18

    Saiura, A., Sata, M., Hirata, Y., Nagai, R. & Makuuchi, M. Circulating smooth muscle progenitor cells contribute to atherosclerosis. Nature Med. 7, 382–383 (2001)

    CAS  Article  Google Scholar 

  19. 19

    Grillon, C. et al. Involvement of thymosin β4 and endoproteinase Asp-N in the biosynthesis of the tetrapeptide AcSerAspLysPro a regulator of the hematopoietic system. FEBS Lett. 274, 30–34 (1990)

    CAS  Article  Google Scholar 

  20. 20

    Rieger, K. J. et al. Involvement of human plasma angiotensin I-converting enzyme in the degradation of the haemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline. Biochem. J. 296, 373–378 (1993)

    CAS  Article  Google Scholar 

  21. 21

    Goldstein, A. L., Hannappel, E. & Kleinman, H. K. Thymosin β4: actin-sequestering protein moonlights to repair injured tissues. Trends Mol. Med. 11, 421–429 (2005)

    CAS  Article  Google Scholar 

  22. 22

    Wang, D. et al. N-acetyl-seryl-aspartyl-lysyl-proline stimulates angiogenesis in vitro and in vivo. Am. J. Physiol. Heart Circ. Physiol. 287, H2099–H2105 (2004)

    CAS  Article  Google Scholar 

  23. 23

    Rasoul, S. et al. Antifibrotic effect of Ac-SDKP and angiotensin-converting enzyme inhibition in hypertension. J. Hypertens. 22, 593–603 (2004)

    CAS  Article  Google Scholar 

  24. 24

    Pokharel, S. et al. Increased myocardial collagen content in transgenic rats overexpressing cardiac angiotensin-converting enzyme is related to enhanced breakdown of N-acetyl-ser-asp-lys-pro and increased phosphorylation of Smad2/3. Circulation 110, 3129–3135 (2004)

    CAS  Article  Google Scholar 

  25. 25

    Peng, H. et al. Angiotensin-converting enzyme inhibitors: A new mechanism of action. Circulation 112, 2436–2445 (2005)

    CAS  Article  Google Scholar 

  26. 26

    Tomanek, R., Zheng, W. & Yue, X. Growth factor activation in myocardial vascularization: Therapeutic implications. Mol. Cell. Biochem. 264, 3–11 (2004)

    CAS  Article  Google Scholar 

  27. 27

    Kunath, T. et al. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nature Biotechnol. 21, 559–561 (2003)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This research was supported by the British Heart Foundation and the Medical Research Council. We acknowledge the collaboration of K. Thiam and S. Millet of genOway (France) in generating the Tβ4shRNAflox line and G. Gish for providing RasGAPshRNA plasmid. We thank J. Clark for providing adult myocardial infarction heart samples and S. Bhattacharya for comments. Author Contributions N.S. carried out the majority of experimental work and data analyses with contributions from C.A.R., A.A.D.M. and P.R.R. K.M. and R.J.S. provided the Nkx2.5Cre mouse strain and K.R.C. provided the MLC2vCre mouse strain. P.R.R. devised and planned the project and the manuscript was written by P.R.R. and N.S.

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Correspondence to Paul R. Riley.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

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Supplementary Notes

This file contains Supplementary Methods detailing all the protocols used in the paper and Supplementary Figure Legends. (PDF 220 kb)

Supplementary Figures

Supplementary Figures 1–9. (PDF 1070 kb)

Supplementary Table 1

A table summarising the penetrance and severity of the Tβ4 knockdown embryo phenotype. (PPT 29 kb)

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Smart, N., Risebro, C., Melville, A. et al. Thymosin β4 induces adult epicardial progenitor mobilization and neovascularization. Nature 445, 177–182 (2007). https://doi.org/10.1038/nature05383

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