Pericytes modulate endothelial sprouting

William Chang

Jillian Andrejecsk

Martin Kluger

W Saltzman

Jordan Pober

Aim

Angiogenic sprouts arise from microvessels formed by endothelial cells (ECs) invested by pericytes (PCs). The aim of this study was to examine the role of PCs in angiogenic sprouting, an understudied phenomenon.

Methods and results

We adapted a human EC spheroid model to examine PC effects on vascular endothelial growth factor-A-induced EC sprouting in vitro by using Bcl-2-transduced human umbilical vein ECs to reduce apoptosis in collagen gels. Human placental PCs, separated from endothelial spheroids by a transwell, or addition of PC-conditioned media increased EC sprouting primarily through hepatocyte growth factor (HGF). Mixed endothelial–PC spheroids formed similar numbers of endothelial sprouts as endothelial spheroids but the sprouts from mixed spheroids were invested by PCs within 24 h. PCs were recruited to the sprouts by platelet-derived growth factor (PDGF)-BB; inhibition of PDGF signalling reduced PC coverage and increased EC sprouting. Transplanted endothelial spheroids give rise to sprouts in vivo that evolve into perfused microvessels. Mixed endothelial–PC spheroids form similar numbers of microvessels as endothelial-only spheroids, but acquire human PC investment and have reduced average lumen diameter.

Conclusions

PCs promote endothelial sprouting by elaborating HGF, but when recruited to invest endothelial sprouts by PDGF-BB, limit the extent of sprouting in vitro and lumen diameter in vivo.

Supplementary Material

Supplementary Data:

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^{Department of Medicine and Section of Nephrology, Yale University School of Medicine, New Haven, CT 06520, USA}

^{Department of Biomedical Engineering, Yale University, New Haven, CT 06511, USA}

^{Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA}

^{Departments of Immunobiology, Pathology, and Dermatology, Yale University School of Medicine, 10 Amistad Street, Room 401D, New Haven, CT 06520, USA}

^{Corresponding author. Tel: +1 203 737 2292; fax: +1 203 737 2293, Email:}ude.elay@rebop.nadroj

Received 2013 Apr 2; Revised 2013 Aug 23; Accepted 2013 Sep 11.

Abstract

Aim

Methods and results

Conclusions

PCs promote endothelial sprouting by elaborating HGF, but when recruited to invest endothelial sprouts by PDGF-BB, limit the extent of sprouting in vitro and lumen diameter in vivo.

Keywords: Endothelial cells, Pericytes, Bcl-2, Platelet-derived growth factor, Hepatocyte growth factor

Abstract

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Acknowledgements

VEGF-A was obtained from the NCI BRB Preclinical Repository. We thank the Yale Center for Analytical Science for discussion of statistical analysis and Morven Graham (Yale University, New Haven, CT, USA) for assistance with EM.

Acknowledgements

References

1. Simons MAngiogenesis: where do we stand now? Circulation. 2005;111:1556–1566.[PubMed][Google Scholar]
2. Adams RH, Alitalo KMolecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol. 2007;8:464–478.[PubMed][Google Scholar]
3. Lieu C, Heymach J, Overman M, Tran H, Kopetz SBeyond VEGF: inhibition of the fibroblast growth factor pathway and antiangiogenesis. Clin Cancer Res. 2011;17:6130–6139.[Google Scholar]
4. Potente M, Gerhardt H, Carmeliet PBasic and therapeutic aspects of angiogenesis. Cell. 2011;146:873–887.[PubMed][Google Scholar]
5. Ribatti D, Nico B, Crivellato EThe role of pericytes in angiogenesis. Int J Dev Biol. 2011;55:261–268.[PubMed][Google Scholar]
6. Hellström M, Gerhardt H, Kalén M, Li X, Eriksson U, Wolburg H, et al Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol. 2001;153:543–553.[Google Scholar]
7. Hammes HP, Lin J, Wagner P, Feng Y, Vom Hagen F, Krzizok T, et al Angiopoietin-2 causes pericyte dropout in the normal retina: evidence for involvement in diabetic retinopathy. Diabetes. 2004;53:1104–1110.[PubMed][Google Scholar]
8. Morikawa S, Baluk P, Kaidoh T, Haskell A, Jain RK, McDonald DMAbnormalities in pericytes on blood vessels and endothelial sprouts in tumors. Am J Pathol. 2002;160:985–1000.[Google Scholar]
9. Goodwin AMIn vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents. Microvasc Res. 2007;74:172–183.[Google Scholar]
10. Korff T, Augustin HIntegration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J Cell Biol. 1998;143:1341–1352.[Google Scholar]
11. Korff T, Kimmina S, Martiny-Baron G, Augustin HGBlood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J. 2001;15:447–457.[PubMed][Google Scholar]
12. Ilan N, Mahooti S, Madri JADistinct signal transduction pathways are utilized during the tube formation and survival phases of in vitro angiogenesis. J Cell Sci. 1998;111(Pt 24):3621–3631.[PubMed][Google Scholar]
13. Schechner J, Nath A, Zheng L, Kluger M, Hughes C, Sierra-Honigmann M, et al In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse. Proc Natl Acad Sci USA. 2000;97:9191–9196.[Google Scholar]
14. Enis D, Shepherd B, Wang Y, Qasim A, Shanahan C, Weissberg P, et al Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells. Proc Natl Acad Sci USA. 2005;102:425–430.[Google Scholar]
15. Zheng L, Ben LH, Pober JS, Bothwell ALPorcine endothelial cells, unlike human endothelial cells, can be killed by human CTL via Fas ligand and cannot be protected by Bcl-2. J Immunol. 2002;169:6850–6855.[PubMed][Google Scholar]
16. Maier C, Shepherd B, Yi T, Pober JExplant outgrowth, propagation and characterization of human pericytes. Microcirculation. 2010;17:367–380.[Google Scholar]
17. Gimbrone MACulture of vascular endothelium. Prog Hemost Thromb. 1976;3:1–28.[PubMed][Google Scholar]
18. Shepherd BR, Jay SM, Saltzman WM, Tellides G, Pober JSHuman aortic smooth muscle cells promote arteriole formation by coengrafted endothelial cells. Tissue Eng Part A. 2009;15:165–173.[Google Scholar]
19. Grant DS, Kleinman HK, Goldberg ID, Bhargava MM, Nickoloff BJ, Kinsella JL, et al Scatter factor induces blood vessel formation in vivo. Proc Natl Acad Sci USA. 1993;90:1937–1941.[Google Scholar]
20. Bussolino F, Di Renzo MF, Ziche M, Bocchietto E, Olivero M, Naldini L, et al Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol. 1992;119:629–641.[Google Scholar]
21. Levéen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz CMice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev. 1994;8:1875–1887.[PubMed][Google Scholar]
22. Soriano PAbnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev. 1994;8:1888–1896.[PubMed][Google Scholar]
23. Lindahl P, Johansson BR, Levéen P, Betsholtz CPericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science. 1997;277:242–245.[PubMed][Google Scholar]
24. Alajati A, Laib AM, Weber H, Boos AM, Bartol A, Ikenberg K, et al Spheroid-based engineering of a human vasculature in mice. Nat Methods. 2008;5:439–445.[PubMed][Google Scholar]
25. Stratman AN, Schwindt AE, Malotte KM, Davis GEEndothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization. Blood. 2010;116:4720–4730.[Google Scholar]
26. Trusolino L, Bertotti A, Comoglio PMMET Signalling: principles and functions in development, organ regeneration and cancer. Nat Rev Mol Cell Biol. 2010;11:834–848.[PubMed][Google Scholar]
27. Newman AC, Chou W, Welch-Reardon KM, Fong AH, Popson SA, Phan DT, et al Analysis of stromal cell secretomes reveals a critical role for stromal cell-derived hepatocyte growth factor and fibronectin in angiogenesis. Arterioscler Thromb Vasc Biol. 2013;33:513–522.[Google Scholar]
28. Waters JP, Kluger MS, Graham M, Chang WG, Pober JSIn vitro self-assembly of human pericyte-supported endothelial microvessels in three-dimensional co-culture: a simple model for interrogating endothelial-pericyte interactions. J Vasc Res. 2013;50:324–331.[Google Scholar]