, Volume 11, Issue 2, pp 141–151

RGS5 expression is a quantitative measure of pericyte coverage of blood vessels

  • Tracy S. Mitchell
  • John Bradley
  • Gregory S. Robinson
  • David T. Shima
  • Yin-Shan Ng
Original Paper


Pericytes play a key role in the process of vascular maturation and stabilization however, the current methods for quantifying pericyte coverage of the neovasculature are laborious and subjective in nature. In this study, we have developed an objective, sensitive, and high-throughput method for quantifying pericyte coverage of angiogenic vessels by analyzing the expression of the pericyte-specific gene, the regulator of G-protein signaling 5 (RGS5). We determined that RGS5 expression was up-regulated during a defined developmental time period in which nascent vessel sprouts acquired a pericyte covering. Furthermore, RGS5 expression was dramatically reduced in vessels with poor pericyte coverage compared to normal angiogenic vasculature. Finally, we determined that the susceptibility of nascent vessels to regression by vascular endothelial growth factor (VEGF) inhibition was significantly reduced following RGS5 up-regulation, further implicating RGS5 in pericyte-endothelial cell interactions and the vascular maturation process. These studies establish the use of RGS5 gene expression as a quantitative and robust measure of pericyte coverage of neovasculature. This method provides a tool for vascular biologists studying pericyte-endothelial cell interactions and vascular maturation in both normal and pathological conditions, such as diabetic retinopathy and cancer.


Angiogenesis Endothelial cell Mural cell Pericyte RGS5 Vascular maturation Vascular smooth muscle Vascular stabilization 



PECAM1-Normalized Regulator of G-protein signaling 5


Platelet-derived growth factor-B


Platelet-derived growth factor receptor β


Regulator of G-protein signaling 5


Smooth muscle actin


Vascular endothelial growth factor, vascular permeability factor


  1. 1.
    Gerhardt H, Betsholtz C (2003) Endothelial–pericyte interactions in angiogenesis. Cell Tissue Res 314(1):15–23PubMedCrossRefGoogle Scholar
  2. 2.
    Alon T, Hemo I et al (1995) Vascular endothelial growth-factor acts as a survival factor for newly formed retinal-vessels and has implications for retinopathy of prematurity. Nat Med 1(10):1024–1028PubMedCrossRefGoogle Scholar
  3. 3.
    Benjamin LE, Golijanin D et al (1999) Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest 103(2):159–165PubMedCrossRefGoogle Scholar
  4. 4.
    Benjamin LE, Hemo I et al (1998) A plasticity window for blood vessel remodeling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 125(9):1591–1598PubMedGoogle Scholar
  5. 5.
    Darland DC, D’Amore PA (1999) Blood vessel maturation: vascular development comes of age. J Clin Invest 103(2):157–158PubMedCrossRefGoogle Scholar
  6. 6.
    Baluk P, Morikawa S et al (2003) Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors. Am J Pathol 163(5):1801–1815PubMedGoogle Scholar
  7. 7.
    Morikawa S, Baluk P et al (2002) Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors. Am J Pathol 160(3):985–1000PubMedGoogle Scholar
  8. 8.
    Nehls V, Drenckhahn D (1991) Heterogeneity of microvascular pericytes for smooth-muscle type alpha-actin. J Cell Biol 113(1):147–154PubMedCrossRefGoogle Scholar
  9. 9.
    Ozerdem U, Grako KA et al (2001) NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis. Dev Dyn 222(2):218–227PubMedCrossRefGoogle Scholar
  10. 10.
    Nehls V, Denzer K et al (1992) Pericyte involvement in capillary sprouting during angiogenesis in situ. Cell Tissue Res 270(3):469–474PubMedCrossRefGoogle Scholar
  11. 11.
    Berger M, Bergers G et al (2005) Regulator of G-protein signaling-5 induction in pericytes coincides with active vessel remodeling during neovascularization. Blood 105(3):1094–1101PubMedCrossRefGoogle Scholar
  12. 12.
    Cho H, Kozasa T et al (2003) Pericyte-specific expression of Rgs5: implications for PDGF and EDG receptor signaling during vascular maturation. FASEB J 17(1):440–442PubMedGoogle Scholar
  13. 13.
    Lindahl P, Johansson BR et al (1997) Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277(5323):242–245PubMedCrossRefGoogle Scholar
  14. 14.
    Goodwin A. M. a. P. A. D. A. (2008) Vessel maturation and perivascular cells. Tumor Angiogenesis: Mechanisms and Cancer Therapy. N. a. D. M. Fusenig, Springer (in press)Google Scholar
  15. 15.
    Bondjers C, Kalen M et al (2003) Transcription profiling of platelet-derived growth factor-B-deficient mouse embryos identifies RGS5 as a novel marker for pericytes and vascular smooth muscle cells. Am J Pathol 162(3):721–729PubMedGoogle Scholar
  16. 16.
    Chen X, Higgins J et al (2004) Novel endothelial cell markers in hepatocellular carcinoma. Mod Pathol 17(10):1198–1210PubMedCrossRefGoogle Scholar
  17. 17.
    Furuya M, Nishiyama M et al (2004) Expression of regulator of G protein signaling protein 5 (RGS5) in the tumour vasculature of human renal cell carcinoma. J Pathol 203(1):551–558PubMedCrossRefGoogle Scholar
  18. 18.
    Paik JH, Skoura A et al (2004) Sphingosine 1-phosphate receptor regulation of N-cadherin mediates vascular stabilization. Genes Dev 18(19):2392–2403PubMedCrossRefGoogle Scholar
  19. 19.
    Hobson JP, Rosenfeldt HM et al (2001) Role of the sphingosine-1-phosphate receptor EDG-1 in PDGF-induced cell motility. Science 291(5509):1800–1803PubMedCrossRefGoogle Scholar
  20. 20.
    Rosenfeldt HM, Hobson JP et al (2001) EDG-1 links the PDGF receptor to Src and focal adhesion kinase activation leading to lamellipodia formation and cell migration. FASEB J 15(14):2649–2659PubMedCrossRefGoogle Scholar
  21. 21.
    Rosenfeldt HM, Hobson JP et al (2001) The sphingosine-I-phosphate receptor EDG-I is essential for platelet-derived growth factor-induced cell motility. Biochem Soc Trans 29:836–839PubMedCrossRefGoogle Scholar
  22. 22.
    Abramsson A, Lindblom P et al (2003) Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors. J Clin Invest 112(8):1142–1151PubMedGoogle Scholar
  23. 23.
    Armulik A, Abramsson A et al (2005) Endothelial/pericyte interactions. Circ Res 97(6):512–523PubMedCrossRefGoogle Scholar
  24. 24.
    Heldin CH, Eriksson U et al (2002) New members of the platelet-derived growth factor family of mitogens. Arch Biochem Biophys 398(2):284–290PubMedCrossRefGoogle Scholar
  25. 25.
    Hellstrom M, Kalen M et al (1999) Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 126(14):3047–3055PubMedGoogle Scholar
  26. 26.
    Hirschi KK, Rohovsky SA et al (1998) PDGF, TGF-beta, and heterotypic cell–cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol 141(3):805–814PubMedCrossRefGoogle Scholar
  27. 27.
    Bjarnegard M, Enge M et al (2004) Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities. Development 131(8):1847–1857PubMedCrossRefGoogle Scholar
  28. 28.
    Jo N, Mailhos C et al (2006) Inhibition of platelet-derived growth factor B signaling enhances the efficacy of anti-vascular endothelial growth factor therapy in multiple models of ocular neovascularization. Am J Pathol 168(6):2036–2053PubMedCrossRefGoogle Scholar
  29. 29.
    Bergers G, Song S et al (2003) Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 111(9):1287–1295PubMedGoogle Scholar
  30. 30.
    Green LS, Jellinek D et al (1996) Inhibitory DNA ligands to platelet-derived growth factor B-chain. Biochemistry 35(45):14413–14424PubMedCrossRefGoogle Scholar
  31. 31.
    Ruckman J, Green LS, Beeson J et al (1998) 2’-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF-165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem 273:20556–20567PubMedCrossRefGoogle Scholar
  32. 32.
    Fruttiger M (2007) Development of the retinal vasculature. Angiogenesis 10:77–88PubMedCrossRefGoogle Scholar
  33. 33.
    Uemura AK, Kusuhara S et al (2006) Angiogenesis in the mouse retina: a model system for experimental manipulation. Exp Cell Res 312(5):676–683PubMedCrossRefGoogle Scholar
  34. 34.
    Ishida S, Usui T et al (2003) VEGF(164)-mediated inflammation is required for pathological, but not physiological, ischemia-induced retinal neovascularization. J Exp Med 198(3):483–489PubMedCrossRefGoogle Scholar
  35. 35.
    Usui T, Isbida S et al (2004) VEGF(164(165)) as the pathological isoform: differential leukocyte and endothelial responses through VEGFR1 and VEGFR2. Invest Ophthalmol Vis Sci 45(2):368–374PubMedCrossRefGoogle Scholar
  36. 36.
    von Tell D, Armulik A et al (2006) Pericytes and vascular stability. Exp Cell Res 312(5):623–629CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Tracy S. Mitchell
    • 1
    • 2
  • John Bradley
    • 1
    • 3
  • Gregory S. Robinson
    • 1
    • 4
  • David T. Shima
    • 1
    • 5
  • Yin-Shan Ng
    • 1
    • 6
  1. 1.OSI EyetechLexingtonUSA
  2. 2.Adnexus TherapeuticsWalthamUSA
  3. 3.Institute for Nutrisciences and HealthCharlottetownCanada
  4. 4.Shire HGTCambridgeUSA
  5. 5.The Institute of OphthalmologyUniversity College LondonLondonUK
  6. 6.Pervasis TherapeuticsCambridgeUSA

Personalised recommendations