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Type I Diabetes Delays Perfusion and Engraftment of 3D Constructs by Impinging on Angiogenesis; Which can be Rescued by Hepatocyte Growth Factor Supplementation

  • Wafa Altalhi
  • Rupal Hatkar
  • James B. Hoying
  • Yasaman Aghazadeh
  • Sara S. NunesEmail author
Article

Abstract

Introduction

The biggest bottleneck for cell-based regenerative therapy is the lack of a functional vasculature to support the grafts. This problem is exacerbated in diabetic patients, where vessel growth is inhibited. To address this issue, we aim to identify the causes of poor vascularization in 3D engineered tissues in diabetes and to reverse its negative effects.

Methods

We used 3D vascularized constructs composed of microvessel fragments containing all cells present in the microcirculation, embedded in collagen type I hydrogels. Constructs were either cultured in vitro or implanted subcutaneously in non-diabetic or in a type I diabetic (streptozotocin-injected) mouse model. We used qPCR, ELISA, immunostaining, FACs and co-culture assays to characterize the effect of diabetes in engineered constructs.

Results

We demonstrated in 3D vascularized constructs that perivascular cells secrete hepatocyte growth factor (HGF), driving microvessel sprouting. Blockage of HGF or HGF receptor signaling in 3D constructs prevented vessel sprouting. Moreover, HGF expression in 3D constructs in vivo is downregulated in diabetes; while no differences were found in HGF receptor, VEGF or VEGF receptor expression. Low HGF expression in diabetes delayed the inosculation of graft and host vessels, decreasing blood perfusion and preventing tissue engraftment. Supplementation of HGF in 3D constructs, restored vessel sprouting in a diabetic milieu.

Conclusion

We show for the first time that diabetes affects HGF secretion in microvessels, which in turn prevents the engraftment of engineered tissues. Exogenous supplementation of HGF, restores angiogenic growth in 3D constructs showing promise for application in cell-based regenerative therapies.

Keywords

Endothelial cell Tissue engineering Microvessel Regenerative medicine Revascularization Angiogenesis Hepatocyte growth factor Blood perfusion Diabetes Anastomosis Inosculation 

Notes

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research (CIHR), Institute of Circulatory and Respiratory Health (137352 and PJT153160) and the Heart and Stroke Foundation of Canada (G-14-0006265) to S.S.N; NIH Grant (EB007556) to J.B.H. A Discovery grant from the Natural Sciences and Engineering Research Council (RGPIN 06621-2017) and an Early Researcher Award from the Ministry of Research, Innovation and Science (ER17-13-149) to S.S.N. supported R.H.

Authors contribution

SSN designed the experiments, coordinated the project and contributed to the writing of the manuscript. JBH supported HGF blockage assays. WA designed and performed experiment and contributed to manuscript writing. RH contributed to performing experiments and writing manuscript and YA contributed to performing experiments.

Conflict of interest

J.B.H. is an inventor on a patent regarding the use of adipose-derived microvessels and has equity interest with Advanced Solutions Life sciences, which is commercializing isolated microvessel technology. A version of this technology was used as an experimental model in this manuscript. This equity was obtained after the work for this manuscript was completed. W.A., R.H., Y.A. and S.S.N. declare no conflict of interest.

Ethical standards

All animal studies were carried out in accordance with Institutional guidelines and approved by the Animal Care Committee at the University Health Network (ID 2420 and 2427). No human subjects were used in this study.

Supplementary material

12195_2019_574_MOESM1_ESM.pdf (33 kb)
Supplementary material 1 (PDF 33 kb)

References

  1. 1.
    Altalhi, W., X. Sun, J. M. Sivak, M. Husain, and S. S. Nunes. Diabetes impairs arterio-venous specification in engineered vascular tissues in a perivascular cell recruitment-dependent manner. Biomaterials 119:23–32, 2017.CrossRefGoogle Scholar
  2. 2.
    Blanco, R., and H. Gerhardt. VEGF and notch in tip and stalk cell selection. Cold Spring Harb. Perspect. Med. 3:a006569, 2013.  https://doi.org/10.1101/cshperspect.a006569.CrossRefGoogle Scholar
  3. 3.
    Brem, H., and M. Tomic-Canic. Cellular and molecular basis of wound healing in diabetes. J. Clin. Invest. 117:1219–1222, 2007.  https://doi.org/10.1172/JCI32169.CrossRefGoogle Scholar
  4. 4.
    Cao, L., et al. Modulating notch signaling to enhance neovascularization and reperfusion in diabetic mice. Biomaterials 31:9048–9056, 2010.  https://doi.org/10.1016/j.biomaterials.2010.08.002.CrossRefGoogle Scholar
  5. 5.
    Chang, C. C., et al. Angiogenesis in a microvascular construct for transplantation depends on the method of chamber circulation. Tissue Eng. Part A 16:795–805, 2010.  https://doi.org/10.1089/ten.TEA.2009.0370.CrossRefGoogle Scholar
  6. 6.
    Chang, C. C., et al. Determinants of microvascular network topologies in implanted neovasculatures. Arterioscler. Thromb. Vasc. Biol. 32:5–14, 2012.CrossRefGoogle Scholar
  7. 7.
    Dekker, R. J., et al. Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes. Am. J. Pathol. 167:609–618, 2005.  https://doi.org/10.1016/S0002-9440(10)63002-7.CrossRefGoogle Scholar
  8. 8.
    Eelen, G., P. de Zeeuw, M. Simons, and P. Carmeliet. Endothelial cell metabolism in normal and diseased vasculature. Circ. Res. 116:1231–1244, 2015.  https://doi.org/10.1161/CIRCRESAHA.116.302855.CrossRefGoogle Scholar
  9. 9.
    Gaengel, K., G. Genove, A. Armulik, and C. Betsholtz. Endothelial-mural cell signaling in vascular development and angiogenesis. Arterioscler. Thromb. Vasc. Biol. 29:630–638, 2009.  https://doi.org/10.1161/ATVBAHA.107.161521.CrossRefGoogle Scholar
  10. 10.
    Krishnan, L., et al. Manipulating the microvasculature and its microenvironment. Crit. Rev. Biomed. Eng. 41:91–123, 2013.CrossRefGoogle Scholar
  11. 11.
    le Noble, F., et al. Flow regulates arterial-venous differentiation in the chick embryo yolk sac. Development 131:361–375, 2004.  https://doi.org/10.1242/dev.00929.CrossRefGoogle Scholar
  12. 12.
    Lee, J. S., et al. Klf2 is an essential regulator of vascular hemodynamic forces in vivo. Dev. Cell 11:845–857, 2006.  https://doi.org/10.1016/j.devcel.2006.09.006.CrossRefGoogle Scholar
  13. 13.
    Nakamura, T., and S. Mizuno. The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86:588–610, 2010.CrossRefGoogle Scholar
  14. 14.
    Nicoli, S., et al. MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis. Nature 464:1196–1200, 2010.  https://doi.org/10.1038/nature08889.CrossRefGoogle Scholar
  15. 15.
    Novosel, E. C., C. Kleinhans, and P. J. Kluger. Vascularization is the key challenge in tissue engineering. Adv. Drug Deliv. Rev. 63:300–311, 2011.  https://doi.org/10.1016/j.addr.2011.03.004.CrossRefGoogle Scholar
  16. 16.
    Nunes, S. S., et al. Implanted microvessels progress through distinct neovascularization phenotypes. Microvasc. Res. 79:10–20, 2010.CrossRefGoogle Scholar
  17. 17.
    Nunes, S. S., et al. Angiogenic potential of microvessel fragments is independent of the tissue of origin and can be influenced by the cellular composition of the implants. Microcirculation 17:557–567, 2010.Google Scholar
  18. 18.
    Nunes, S. S., et al. Generation of a functional liver tissue mimic using adipose stromal vascular fraction cell-derived vasculatures. Sci. Rep. 3:2141, 2013.CrossRefGoogle Scholar
  19. 19.
    Nunes, S. S., et al. Generation of a functional liver tissue mimic using adipose stromal vascular fraction cell-derived vasculatures. Sci. Rep. 3:2141, 2013.CrossRefGoogle Scholar
  20. 20.
    Rangasamy, S., R. Srinivasan, J. Maestas, P. G. McGuire, and A. Das. A potential role for angiopoietin 2 in the regulation of the blood-retinal barrier in diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 52:3784–3791, 2011.  https://doi.org/10.1167/iovs.10-6386.CrossRefGoogle Scholar
  21. 21.
    Rask-Madsen, C., and G. L. King. Vascular complications of diabetes: mechanisms of injury and protective factors. Cell Metab. 17:20–33, 2013.  https://doi.org/10.1016/j.cmet.2012.11.012.CrossRefGoogle Scholar
  22. 22.
    Rocha, L. A., D. A. Learmonth, R. A. Sousa, and A. J. Salgado. alphavbeta3 and alpha5beta1 integrin-specific ligands: from tumor angiogenesis inhibitors to vascularization promoters in regenerative medicine? Biotechnol. Adv. 36:208–227, 2018.  https://doi.org/10.1016/j.biotechadv.2017.11.004.CrossRefGoogle Scholar
  23. 23.
    Shin, D., et al. Expression of ephrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization. Dev. Biol. 230:139–150, 2001.CrossRefGoogle Scholar
  24. 24.
    Sun, X., W. Altalhi, and S. S. Nunes. Vascularization strategies of engineered tissues and their application in cardiac regeneration. Adv. Drug Deliv. Rev. 96:183–194, 2016.  https://doi.org/10.1016/j.addr.2015.06.001.CrossRefGoogle Scholar
  25. 25.
    Sun, X., S. Evren, and S. S. Nunes. Blood vessel maturation in health and disease and its implications for vascularization of engineered tissues. Crit. Rev. Biomed. Eng. 43:433–454, 2015.  https://doi.org/10.1615/CritRevBiomedEng.2016016063.CrossRefGoogle Scholar
  26. 26.
    Sun, X., and S. S. Nunes. Overview of hydrogel-based strategies for application in cardiac tissue regeneration. Biomed. Mater. 10:034005, 2015.  https://doi.org/10.1088/1748-6041/10/3/034005.CrossRefGoogle Scholar
  27. 27.
    Taniyama, Y., et al. Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat diabetic hind limb ischemia model: molecular mechanisms of delayed angiogenesis in diabetes. Circulation 104:2344–2350, 2001.CrossRefGoogle Scholar
  28. 28.
    Wythe, J. D., et al. ETS factors regulate Vegf-dependent arterial specification. Dev. Cell 26:45–58, 2013.  https://doi.org/10.1016/j.devcel.2013.06.007.CrossRefGoogle Scholar
  29. 29.
    Zhang, Y. S., et al. 3D bioprinting for tissue and organ fabrication. Ann. Biomed. Eng. 45:148–163, 2017.  https://doi.org/10.1007/s10439-016-1612-8.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2019

Authors and Affiliations

  1. 1.Toronto General Hospital Research InstituteUniversity Health NetworkTorontoCanada
  2. 2.Laboratory of Medicine and PathobiologyUniversity of TorontoTorontoCanada
  3. 3.Advanced Solutions Life SciencesManchesterUSA
  4. 4.Institute of Biomaterials and Biomedical EngineeringUniversity of TorontoTorontoCanada
  5. 5.Heart & Stroke/Richard Lewar Centre of ExcellenceUniversity of TorontoTorontoCanada
  6. 6.Massachusetts General HospitalBostonUSA

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