Skip to main content
SpringerLink
Log in

Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation

  • Original Paper
  • Published:
Angiogenesis Aims and scope Submit manuscript

Abstract

The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA expression in two phenotypically distinct cell populations from the same culture, namely in tubule-forming endothelial cells and monolayer endothelial cells not participating in tubule formation. A fibrin-rich 3D matrix derived from human plasma was used to facilitate tubule formation by human foreskin microvascular endothelial cells (hMVEC). After 7 days of stimulation with VEGF, bFGF, and TNF-α, the culture consisted of a monolayer and capillary-like sprouts that had grown into the fibrinous matrix. A method was developed to separate the monolayer and tubule-forming populations of hMVEC, keeping their cellular integrity intact to ensure mRNA extraction and cDNA production. Subsequent array analysis resulted in an inventory of differentially expressed genes that were associated with either tube-forming (angiogenic) or non-angiogenic capacity. Differential gene expression was verified by real-time PCR on the original RNA samples as well as on RNA obtained from laser-capture microdissected cross sections of monolayers and capillary structures in the 3D fibrinous matrix. The expression of CDC42GAP, an inhibitor of active-state small Rho GTPases, was reduced in tubular hMVEC. Overexpression of CDC42GAP in hMVEC attenuated endothelial tubule formation, while its suppression by siRNA slightly enhanced this process. Thus, CDC42GAP was identified as a counter-regulatory mediator for tubule formation

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Carmeliet P (2003) Angiogenesis in health and disease. Nat Med 9:653–660

    Article  PubMed  CAS  Google Scholar 

  2. Ausprunk DH, Folkman J (1977) Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res 14:53–65

    Article  PubMed  CAS  Google Scholar 

  3. Hanahan D, Folkman J (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353–364

    Article  PubMed  CAS  Google Scholar 

  4. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161:1163–1177

    Article  PubMed  CAS  Google Scholar 

  5. van Beijnum JR, Griffioen AW (2005) In silico analysis of angiogenesis associated gene expression identifies angiogenic stage related profiles. Biochim Biophys Acta 1755:121–134

    PubMed  Google Scholar 

  6. Kahn J, Mehraban F, Ingle G, Xin X, Bryant JE, Vehar G, Schoenfeld J, Grimaldi CJ, Peale F, Draksharapu A, Lewin DA, Gerritsen ME (2000) Gene expression profiling in an in vitro model of angiogenesis. Am J Pathol 156:1887–1900

    PubMed  CAS  Google Scholar 

  7. Bell SE, Mavila A, Salazar R, Bayless KJ, Kanagala S, Maxwell SA, Davis GE (2001) Differential gene expression during capillary morphogenesis in 3D collagen matrices: regulated expression of genes involved in basement membrane matrix assembly, cell cycle progression, cellular differentiation and G-protein signaling. J Cell Sci 114:2755–2773

    PubMed  CAS  Google Scholar 

  8. Hahn CN, Su ZJ, Drogemuller CJ, Tsykin A, Waterman SR, Brautigan PJ, Yu S, Kremmidiotis G, Gardner A, Solomon PJ, Goodall GJ, Vadas MA, Gamble JR (2005) Expression profiling reveals functionally important genes and coordinately regulated signaling pathway genes during in vitro angiogenesis. Physiol Genomics 22:57–69

    Article  PubMed  CAS  Google Scholar 

  9. Gerritsen ME, Soriano R, Yang S, Ingle G, Zlot C, Toy K, Winer J, Draksharapu A, Peale F, Wu TD, Williams PM (2002) In silico data filtering to identify new angiogenesis targets from a large in vitro gene profiling data set. Physiol Genomics 10:13–20

    PubMed  CAS  Google Scholar 

  10. Sun XT, Zhang MY, Shu C, Li Q, Yan XG, Cheng N, Qiu YD, Ding YT (2005) Differential gene expression during capillary morphogenesis in a microcarrier-based three-dimensional in vitro model of angiogenesis with focus on chemokines and chemokine receptors. World J Gastroenterol. 11:2283–2290

    Google Scholar 

  11. Grove AD, Prabhu VV, Young BL, Lee FC, Kulpa V, Munson PJ, Kohn EC (2002) Both protein activation and gene expression are involved in early vascular tube formation in vitro. Clin Cancer Res 8:3019–3026

    PubMed  CAS  Google Scholar 

  12. St Croix B, Rago C, Velculescu V, Traverso G, Romans KE, Montgomery E, Lal A, Riggins GJ, Lengauer C, Vogelstein B, Kinzler KW (2000) Genes expressed in human tumor endothelium. Science 289:1197–1202

    Article  PubMed  CAS  Google Scholar 

  13. van Beijnum JR, Dings RP, van der Linden E, Zwaans BM, Ramaekers FC, Mayo KH, Griffioen AW (2006) Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature. Blood 108:2339–2348

    Article  PubMed  CAS  Google Scholar 

  14. Schoenfeld J, Lessan K, Johnson NA, Charnock-Jones DS, Evans A, Vourvouhaki E, Scott L, Stephens R, Freeman TC, Saidi SA, Tom B, Weston GC, Rogers P, Smith SK, Print CG (2004) Bioinformatic analysis of primary endothelial cell gene array data illustrated by the analysis of transcriptome changes in endothelial cells exposed to VEGF-A and PlGF. Angiogenesis 7:143–156

    Article  PubMed  CAS  Google Scholar 

  15. Dvorak HF, Harvey VS, Estrella P, Brown LF, McDonagh J, Dvorak AM (1987) Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Lab Invest 57:673–686

    PubMed  CAS  Google Scholar 

  16. Koolwijk P, van Erck MG, de Vree WJ, Vermeer MA, Weich HA, Hanemaaijer R, van Hinsbergh VWM (1996) Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol 132:1177–1188

    Article  PubMed  CAS  Google Scholar 

  17. Verheul HM, Hoekman K, Lupu F, Broxterman HJ, van der Valk P, Kakkar AK, Pinedo HM (2000) Platelet and coagulation activation with vascular endothelial growth factor generation in soft tissue sarcomas. Clin Cancer Res 6:166–171

    PubMed  CAS  Google Scholar 

  18. van Hinsbergh VWM, Collen A, Koolwijk P (2001) Role of fibrin matrix in angiogenesis. Ann NY Acad Sci 936:426–437

    Article  PubMed  Google Scholar 

  19. Kroon ME, Koolwijk P, van Goor H, Weidle UH, Collen A, van der Pluijm G, van Hinsbergh VWM (1999) Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices. Am J Pathol 154:1731–1742

    PubMed  CAS  Google Scholar 

  20. van Nieuw Amerongen GP, Koolwijk P, Versteilen A, van Hinsbergh VWM (2003) Involvement of RhoA/Rho kinase signaling in VEGF-induced endothelial cell migration and angiogenesis in vitro. Arterioscler Thromb Vasc Biol 23:211–217

    Article  PubMed  Google Scholar 

  21. van Nieuw Amerongen GP, Beckers CM, Achekar ID, Zeeman S, Musters RJ, van Hinsbergh VW (2007) Involvement of Rho kinase in endothelial barrier maintenance. Arterioscler Thromb Vasc Biol 27:2332–2339

    Article  PubMed  CAS  Google Scholar 

  22. Barfod ET, Zheng Y, Kuang WJ, Hart MJ, Evans T, Cerione RA, Ashkenazi A (1993) Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain. J Biol Chem 268:26059–26062

    PubMed  CAS  Google Scholar 

  23. Van Hinsbergh VWM, Sprengers ED, Kooistra T, (1987) Effect of thrombin on the production of plasminogen activators and PA inhibitor-1 by human foreskin microvascular endothelial cells. Thromb Haemostas 57:176-182

    Google Scholar 

  24. Defilippi P, van Hinsbergh V, Bertolotto A, Rossino P, Silengo L, Tarone G (1991) Differential distribution and modulation of expression of alpha 1/beta 1 integrin on human endothelial cells. J Cell Biol 114:855–863

    Article  PubMed  CAS  Google Scholar 

  25. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  PubMed  CAS  Google Scholar 

  26. Low BC, Seow KT, Guy GR (2000) The BNIP-2 and Cdc42GAP homology domain of BNIP-2 mediates its homophilic association and heterophilic interaction with Cdc42GAP. J Biol Chem 275:37742–37751

    Article  PubMed  CAS  Google Scholar 

  27. Gerhardt H, Betsholtz C (2005) How do endothelial cells orientate? EXS 94:3–15

    Google Scholar 

  28. Riento K, Villalonga P, Garg R, Ridley A (2005) Function and regulation of RhoE. Biochem Soc Trans 33:649–651

    Article  PubMed  CAS  Google Scholar 

  29. Fujioka Y, Taira T, Maeda Y, Tanaka S, Nishihara H, Iguchi-Ariga SM, Nagashima K, Ariga H (2001) MM-1, a c-Myc-binding protein, is a candidate for a tumor suppressor in leukemia/lymphoma and tongue cancer. J Biol Chem 276:45137–45144

    Article  PubMed  CAS  Google Scholar 

  30. Folkman J (1996–1997) Endogenous inhibitors of angiogenesis. Harvey Lect 92:65–82

    PubMed  Google Scholar 

  31. Marneros AG, Olsen BR (2005) Physiological role of collagen XVIII and endostatin. FASEB J 19:716–728

    Article  PubMed  CAS  Google Scholar 

  32. Autiero M, De Smet F, Claes F, Carmeliet P (2005) Role of neural guidance signals in blood vessel navigation. Cardiovasc Res 65:629–638

    Article  PubMed  CAS  Google Scholar 

  33. Eichmann A, Le Noble F, Autiero M, Carmeliet P (2005) Guidance of vascular and neural network formation. Curr Opin Neurobiol 15:108–115

    Article  PubMed  CAS  Google Scholar 

  34. Hashizume A, Ueno T, Furuse M, Tsukita S, Nakanishi Y, Hieda Y (2004) Expression patterns of claudin family of tight junction membrane proteins in developing mouse submandibular gland. Dev Dyn 231:425–431

    Article  PubMed  CAS  Google Scholar 

  35. Shi Y, Reitmaier B, Regenbogen J, Slowey RM, Opalenik SR, Wolf E, Goppelt A, Davidson JM (2005) CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue. Am J Pathol 166:303–312

    PubMed  CAS  Google Scholar 

  36. Schwartz M (2004) Rho signalling at a glance. J Cell Sci 117:5457–5458

    Article  PubMed  CAS  Google Scholar 

  37. Bayless KJ, Davis GE (2002) The Cdc42 and Rac1 GTPases are required for capillary lumen formation in three-dimensional extracellular matrices. J Cell Sci 115:1123–1136

    PubMed  CAS  Google Scholar 

  38. Bishop AL, Hall A (2000) Rho GTPases and their effector proteins. Biochem J 348:241–255

    Article  PubMed  CAS  Google Scholar 

  39. Zhang B, Zheng Y (1998) Regulation of RhoA GTP hydrolysis by the GTPase-activating proteins p190, p50RhoGAP, Bcr, and 3BP-1. Biochemistry 37:5249–5257

    Article  PubMed  CAS  Google Scholar 

  40. Zhang B, Chernoff J, Zheng Y (1998) Interaction of Rac1 with GTPase-activating proteins and putative effectors. A comparison with Cdc42 and RhoA. J Biol Chem 273:8776–8782

    Article  PubMed  CAS  Google Scholar 

  41. Su ZJ, Hahn CN, Goodall GJ, Reck NM, Leske AF, Davy A, Kremmidiotis G, Vadas MA, Gamble JR (2004) A vascular cell-restricted RhoGAP, p73RhoGAP, is a key regulator of angiogenesis. Proc Natl Acad Sci USA 101:12212–12217

    Article  PubMed  CAS  Google Scholar 

  42. Aitsebaomo J, Wennerberg K Der CJ, Zhang C, Kedar V, Moser M, Kingsley-Kallesen ML, Zeng GQ, Patterson C (2004) p68RacGAP is a novel GTPase-activating protein that interacts with vascular endothelial zinc finger-1 and modulates endothelial cell capillary formation. J Biol Chem 279:17963–17972

    Article  PubMed  CAS  Google Scholar 

  43. Fryer BH, Field J (2005) Rho, Rac, Pak and angiogenesis: old roles and newly identified responsibilities in endothelial cells. Cancer Lett 229:13–23

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants of ZON-MW (902-90-017) and the European Vascular Genomics Network (LSHM-CT-2003-503254).

Author information

Authors and Affiliations

  1. Department for Physiology, Institute for Cardiovascular Research, VU University medical center, Amsterdam, The Netherlands

    Marten A. Engelse, Nancy Laurens, Robert E. Verloop, Pieter Koolwijk & Victor W. M. van Hinsbergh

  2. Gaubius laboratory, TNO Quality of Life, Leiden, The Netherlands

    Pieter Koolwijk

  3. Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, 1081 BT, Amsterdam, The Netherlands

    Victor W. M. van Hinsbergh

Authors
  1. Marten A. Engelse
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nancy Laurens
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Robert E. Verloop
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pieter Koolwijk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Victor W. M. van Hinsbergh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Victor W. M. van Hinsbergh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Engelse, M.A., Laurens, N., Verloop, R.E. et al. Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation. Angiogenesis 11, 153–167 (2008). https://doi.org/10.1007/s10456-007-9086-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10456-007-9086-9

Keywords

Search

Navigation