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Rasip1 is essential to blood vessel stability and angiogenic blood vessel growth

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Cardiovascular function depends on patent, continuous and stable blood vessel formation by endothelial cells (ECs). Blood vessel development initiates by vasculogenesis, as ECs coalesce into linear aggregates and organize to form central lumens that allow blood flow. Molecular mechanisms underlying in vivo vascular ‘tubulogenesis’ are only beginning to be unraveled. We previously showed that the GTPase-interacting protein called Rasip1 is required for the formation of continuous vascular lumens in the early embryo. Rasip1−/− ECs exhibit loss of proper cell polarity and cell shape, disrupted localization of EC–EC junctions and defects in adhesion of ECs to extracellular matrix. In vitro studies showed that Rasip1 depletion in cultured ECs blocked tubulogenesis. Whether Rasip1 is required in blood vessels after their initial formation remained unclear. Here, we show that Rasip1 is essential for vessel formation and maintenance in the embryo, but not in quiescent adult vessels. Rasip1 is also required for angiogenesis in three models of blood vessel growth: in vitro matrix invasion, retinal blood vessel growth and directed in vivo angiogenesis assays. Rasip1 is thus necessary in growing embryonic blood vessels, postnatal angiogenic sprouting and remodeling, but is dispensable for maintenance of established blood vessels, making it a potential anti-angiogenic therapeutic target.

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  1. Sacharidou A, Stratman AN, Davis GE (2012) Molecular mechanisms controlling vascular lumen formation in three-dimensional extracellular matrices. Cells Tissues Organs 195(1–2):122–143. doi:10.1159/000331410

    Article  CAS  PubMed  Google Scholar 

  2. 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(Pt 6):1123–1136

    CAS  PubMed  Google Scholar 

  3. Sacharidou A, Koh W, Stratman AN, Mayo AM, Fisher KE, Davis GE (2010) Endothelial lumen signaling complexes control 3D matrix-specific tubulogenesis through interdependent Cdc42- and MT1-MMP-mediated events. Blood 115(25):5259–5269. doi:10.1182/blood-2009-11-252692

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Datta A, Bryant DM, Mostov KE (2011) Molecular regulation of lumen morphogenesis. Curr Biol 21(3):R126–R136. doi:10.1016/j.cub.2010.12.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kesavan G, Sand FW, Greiner TU, Johansson JK, Kobberup S, Wu X, Brakebusch C, Semb H (2009) Cdc42-mediated tubulogenesis controls cell specification. Cell 139(4):791–801. doi:10.1016/j.cell.2009.08.049

    Article  CAS  PubMed  Google Scholar 

  6. Zovein AC, Luque A, Turlo KA, Hofmann JJ, Yee KM, Becker MS, Fassler R, Mellman I, Lane TF, Iruela-Arispe ML (2010) Beta1 integrin establishes endothelial cell polarity and arteriolar lumen formation via a Par3-dependent mechanism. Dev Cell 18(1):39–51. doi:10.1016/j.devcel.2009.12.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Xu K, Sacharidou A, Fu S, Chong DC, Skaug B, Chen ZF, Davis GE, Cleaver O (2011) Blood vessel tubulogenesis requires Rasip1 regulation of GTPase signaling. Dev Cell 20(4):526–539

  8. Koh W, Mahan RD, Davis GE (2008) Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling. J Cell Sci 121(Pt 7):989–1001. doi:10.1242/jcs.020693

    Article  CAS  PubMed  Google Scholar 

  9. Strilic B, Kucera T, Eglinger J, Hughes MR, McNagny KM, Tsukita S, Dejana E, Ferrara N, Lammert E (2009) The molecular basis of vascular lumen formation in the developing mouse aorta. Dev Cell 17(4):505–515. doi:10.1016/j.devcel.2009.08.011

    Article  CAS  PubMed  Google Scholar 

  10. Strilic B, Eglinger J, Krieg M, Zeeb M, Axnick J, Babal P, Muller DJ, Lammert E (2010) Electrostatic cell-surface repulsion initiates lumen formation in developing blood vessels. Curr Biol 20(22):2003–2009. doi:10.1016/j.cub.2010.09.061

    Article  CAS  PubMed  Google Scholar 

  11. Mitin NY, Ramocki MB, Zullo AJ, Der CJ, Konieczny SF, Taparowsky EJ (2004) Identification and characterization of rain, a novel Ras-interacting protein with a unique subcellular localization. J Biol Chem 279(21):22353–22361

    Article  CAS  PubMed  Google Scholar 

  12. Xu K, Chong DC, Rankin SA, Zorn AM, Cleaver O (2009) Rasip1 is required for endothelial cell motility, angiogenesis and vessel formation. Dev Biol 329(2):269–279. doi:10.1016/j.ydbio.2009.02.033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Xu K, Cleaver O (2011) Tubulogenesis during blood vessel formation. Semin Cell Dev Biol 22(9):993–1004. doi:10.1016/j.semcdb.2011.05.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wilson CW, Parker LH, Hall CJ, Smyczek T, Mak J, Crow A, Posthuma G, De Maziere A, Sagolla M, Chalouni C, Vitorino P, Roose-Girma M, Warming S, Klumperman J, Crosier PS, Ye W (2013) Rasip1 regulates vertebrate vascular endothelial junction stability through Epac1–Rap1 signaling. Blood 122(22):3678–3690. doi:10.1182/blood-2013-02-483156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Post A, Pannekoek WJ, Ross SH, Verlaan I, Brouwer PM, Bos JL (2013) Rasip1 mediates Rap1 regulation of Rho in endothelial barrier function through ArhGAP29. Proc Natl Acad Sci USA 110(28):11427–11432. doi:10.1073/pnas.1306595110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Post A, Pannekoek WJ, Ponsioen B, Vliem MJ, Bos JL (2015) Rap1 spatially controls ArhGAP29 to inhibit rho signaling during endothelial barrier regulation. Mol Cell Biol 35(14):2495–2502. doi:10.1128/MCB.01453-14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC (1995) Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376(6535):62–66. doi:10.1038/376062a0

    Article  CAS  PubMed  Google Scholar 

  18. Hayashi S, Lewis P, Pevny L, McMahon AP (2002) Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain. Mech Dev 119(Suppl 1):S97–S101

    Article  PubMed  Google Scholar 

  19. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M (2001) Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol 230(2):230–242. doi:10.1006/dbio.2000.0106

    Article  CAS  PubMed  Google Scholar 

  20. Monvoisin A, Alva JA, Hofmann JJ, Zovein AC, Lane TF, Iruela-Arispe ML (2006) VE-cadherin-CreERT2 transgenic mouse: a model for inducible recombination in the endothelium. Dev Dyn 235(12):3413–3422. doi:10.1002/dvdy.20982

    Article  CAS  PubMed  Google Scholar 

  21. Hayashi S, McMahon AP (2002) Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 244(2):305–318. doi:10.1006/dbio.2002.0597

    Article  CAS  PubMed  Google Scholar 

  22. Barry DM, Xu K, Meadows SM, Zheng Y, Norden PR, Davis GE, Cleaver O (2015) Cdc42 is required for cytoskeletal support of endothelial cell adhesion during blood vessel formation in mice. Development 142(17):3058–3070. doi:10.1242/dev.125260

    Article  CAS  PubMed  Google Scholar 

  23. Pitulescu ME, Schmidt I, Benedito R, Adams RH (2010) Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat Protoc 5(9):1518–1534. doi:10.1038/nprot.2010.113

    Article  CAS  PubMed  Google Scholar 

  24. Stratman AN, Davis MJ, Davis GE (2011) VEGF and FGF prime vascular tube morphogenesis and sprouting directed by hematopoietic stem cell cytokines. Blood 117(14):3709–3719. doi:10.1182/blood-2010-11-316752

    Article  PubMed  PubMed Central  Google Scholar 

  25. Chong DC, Koo Y, Xu K, Fu S, Cleaver O (2011) Stepwise arteriovenous fate acquisition during mammalian vasculogenesis. Dev Dyn 240(9):2153–2165. doi:10.1002/dvdy.22706

    Article  PubMed  PubMed Central  Google Scholar 

  26. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M (2001) Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Developmental biology 230(2):230–242. doi:10.1006/dbio.2000.0106

    Article  CAS  PubMed  Google Scholar 

  27. Wang Y, Nakayama M, Pitulescu ME, Schmidt TS, Bochenek ML, Sakakibara A, Adams S, Davy A, Deutsch U, Luthi U, Barberis A, Benjamin LE, Makinen T, Nobes CD, Adams RH (2010) Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465(7297):483–486. doi:10.1038/nature09002

    Article  CAS  PubMed  Google Scholar 

  28. Stahl A, Connor KM, Sapieha P, Chen J, Dennison RJ, Krah NM, Seaward MR, Willett KL, Aderman CM, Guerin KI, Hua J, Lofqvist C, Hellstrom A, Smith LE (2010) The mouse retina as an angiogenesis model. Investig Ophthalmol Vis Sci 51(6):2813–2826. doi:10.1167/iovs.10-5176

    Article  Google Scholar 

  29. 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(6):1163–1177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tammela T, Zarkada G, Nurmi H, Jakobsson L, Heinolainen K, Tvorogov D, Zheng W, Franco CA, Murtomaki A, Aranda E, Miura N, Yla-Herttuala S, Fruttiger M, Makinen T, Eichmann A, Pollard JW, Gerhardt H, Alitalo K (2011) VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling. Nat Cell Biol 13(10):1202–1213. doi:10.1038/ncb2331

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Tammela T, Zarkada G, Wallgard E, Murtomaki A, Suchting S, Wirzenius M, Waltari M, Hellstrom M, Schomber T, Peltonen R, Freitas C, Duarte A, Isoniemi H, Laakkonen P, Christofori G, Yla-Herttuala S, Shibuya M, Pytowski B, Eichmann A, Betsholtz C, Alitalo K (2008) Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature 454(7204):656–660. doi:10.1038/nature07083

    Article  CAS  PubMed  Google Scholar 

  32. Fantin A, Vieira JM, Gestri G, Denti L, Schwarz Q, Prykhozhij S, Peri F, Wilson SW, Ruhrberg C (2010) Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction. Blood 116(5):829–840. doi:10.1182/blood-2009-12-257832

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Radu M, Chernoff J (2013) An in vivo assay to test blood vessel permeability. J Vis Exp 73:e50062. doi:10.3791/50062

    PubMed  Google Scholar 

  34. Bates DO (2010) Vascular endothelial growth factors and vascular permeability. Cardiovasc Res 87(2):262–271. doi:10.1093/cvr/cvq105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Guedez L, Rivera AM, Salloum R, Miller ML, Diegmueller JJ, Bungay PM, Stetler-Stevenson WG (2003) Quantitative assessment of angiogenic responses by the directed in vivo angiogenesis assay. Am J Pathol 162(5):1431–1439. doi:10.1016/S0002-9440(10)64276-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Seo DW, Li H, Guedez L, Wingfield PT, Diaz T, Salloum R, Wei BY, Stetler-Stevenson WG (2003) TIMP-2 mediated inhibition of angiogenesis: an MMP-independent mechanism. Cell 114(2):171–180

    Article  CAS  PubMed  Google Scholar 

  37. Napoli C, Giordano A, Casamassimi A, Pentimalli F, Ignarro LJ, De Nigris F (2011) Directed in vivo angiogenesis assay and the study of systemic neoangiogenesis in cancer. Int J Cancer 128(7):1505–1508. doi:10.1002/ijc.25743

    Article  CAS  PubMed  Google Scholar 

  38. Ando K, Fukuhara S, Moriya T, Obara Y, Nakahata N, Mochizuki N (2013) Rap1 potentiates endothelial cell junctions by spatially controlling myosin II activity and actin organization. J Cell Biol 202(6):901–916. doi:10.1083/jcb.201301115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Outtz HH, Tattersall IW, Kofler NM, Steinbach N, Kitajewski J (2011) Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice. Blood 118(12):3436–3439. doi:10.1182/blood-2010-12-327015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hellstrom M, Phng LK, Hofmann JJ, Wallgard E, Coultas L, Lindblom P, Alva J, Nilsson AK, Karlsson L, Gaiano N, Yoon K, Rossant J, Iruela-Arispe ML, Kalen M, Gerhardt H, Betsholtz C (2007) Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445(7129):776–780

    Article  PubMed  Google Scholar 

  41. Murakami M (2012) Signaling required for blood vessel maintenance: molecular basis and pathological manifestations. Int J Vasc Med 2012:293641. doi:10.1155/2012/293641

    PubMed  PubMed Central  Google Scholar 

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We thank the following for mouse lines: Ralf Adams for Cdh5(PAC)-CreERT2, Tom Sato for Tie2-Cre, Janet Rossant for Flk1-eGFP and Thomas Carroll for Sox2-Cre and CAG-CreERT2. We thank Dr. Hiromi Yanagisawa for use of cell culture equipment. The authors would like to thank Stephen Fu and Katherine Speichinger for excellent technical assistance. We thank the TIG group, and the Carroll, Olson, MacDonald and Cleaver labs for invaluable discussions and assistance. Finally, we are very grateful to Dr. Bob Hammer and the UTSW Transgenic Core for help with generating the Rasip1 conditional mice.

Author contributions

Most experiments were performed by Y.K. K.X. initiated experiments by crossing mouse lines and making initial observations. D.M.B. and S.F. carried out supportive experiments and D.M.B. finished key studies. K.T. and C.M. assisted with DIVAA implants. G.E.D. contributed to underlying ideas and analysis, contributed 3D in vitro data and read manuscript critically. O.C. supervised the overall project and contributed to the analysis. Y.K., D.M.B and O.C. wrote the manuscript.


This work was supported by NIH R01HL113498 to DMB, R01HL109604 to CM, R01HL105606 and R01HL108670 to GED, and CPRIT RP110405, R01HL113498 and R01DK079862 to OC.

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Correspondence to Ondine Cleaver.

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The authors declare no competing or financial interests.

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Yeon Koo and David M. Barry have contributed equally to this work.

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Figure S1

Generation of conditional Rasip1 knockout mouse. Schematic showing the wild-type allele of Rasip1 and the targeting vector (KOMP) used to generate the floxed allele. A LacZNeo cassette was used to interrupt the gene. Flippase recombinase (FLP) was used to remove the LacZ and Neo cassette which is flanked by flippase recognition target (FRT) sequences. The Rasip1 coding region is interrupted by removal of the third exon, which is flanked by loxP sites (floxed exon), in the presence of Cre recombinase (JPEG 435 kb)

Figure S2

Disrupted Rasip1 allele prevents lumen formation and disrupts junctional polarity. (A-F) Immunofluorescence staining for Flk1-eGFP and ZO1 in aortic ECs (at E8.25) showing that the LacZNeo cassette can disrupt Rasip1 function, thereby affecting lumen formation and adhesion polarity. Scale bars: A–F 15 μm (JPEG 954 kb)

Figure S3

Rasip1 is necessary for arterial fate. (A-B) Deletion of Rasip1 using Sox2-Cre disrupts maintenance of arterial fate by E9.0, as indicated by expression of the flow-responsive gene connexin 40 (CX40), as detected by RNA in situ hybridization. Scale bars: A–B 1 mm (JPEG 332 kb)

Figure S4

R26-YFP reporter shows that Cre recombinase is expressed in newly born angioblasts using Tie2-Cre. (A-B) Aortic ECs from 0-4 somite embryos were stained using a GFP antibody (red) and DAPI (blue) to mark Rosa26-YFP reporter expressing angioblasts in Tie2-Cre transgenic mice before and after lumen formation. M = mesoderm, EC = endothelial cell, End = endoderm. Scale bars: A–B 15 μm (JPEG 660 kb)

Figure S5

Rasip1 is necessary to polarize adhesion complexes away from the apical membrane but is not necessary for apical polarity. (A-F) Staining of ZO-1 and Rasip1 in aortae of E8.25 Rasip1f/f;Tie2-Cre and Rasip1f/+;Tie2-Cre embryos showing that junction polarity (junctions are abnormally localized apically, rather than their normal peripheral localization) is lost after deletion of Rasip1. Asterisks indicate the aortic lumen. (G-L) Staining of claudin5 and VEcad show that tight junction and adherens junction polarity, localization away from the apical membrane, are lost after deletion of Rasip1 using Tie2-Cre. (M-R) Staining of PECAM, endomucin and Podxl shows that apical polarity is normal after deletion of Rasip1 using Tie2-Cre. B, blood autofluorescence. Scale bars: A–R 15 μm (JPEG 1574 kb)

Figure S6

Rasip1 f/f ;Tie2-Cre embryos become hypoplastic at E9.0 and die from failed lumen formation. (A-B) Embryos become hypoplastic at E9.0 after Rasip1 deletion using Tie2-Cre and exhibit hemorrhages. (C-D) The dorsal aorta marked by PECAM and endomucin staining collapses after deletion of Rasip1 using Tie2-Cre at E9.0. Asterisks denote open lumens. Scale bars: A–B 1 mm, C-D 15 μm (JPEG 1950 kb)

Figure S7

CAG-Cre ERT2 (CAG) conditionally deletes Rasip1 in the retina. (A) In situ hybridization with anti-Rasip1 RNA Dig-labeled probe showing Rasip1 expression is vascular specific (throughout retinal vessels). (B) A diagram for tamoxifen treatment of neonatal mice for Rasip1 ablation. (C-E) Tamoxifen efficiency of recombination in blood vessels was verified by co-expression of isolectin B4 and the Rosa26-YFP reporter. Scale bars: A 500 μm, C–E 125 μm (JPEG 1496 kb)

Figure S8

Rasip1 is dispensable in adult vessels. (A) Rasip1 deletion confirmation in adult mice by detection of the deleted floxed allele using PCR. (B) Reverse transcriptase PCR of adult lung tissue showing that Rasip1 transcripts are ablated after treatment of Rasip1f/f;Cad5-CreERT2 adult mice with tamoxifen. (C) Western blot analysis showing Rasip1 protein levels are significantly reduced in adult Rasip1f/f;Cad5-CreERT2 mice treated with tamoxifen (lung). (D) The Rosa26-Tomato allele was used to visualize Rasip1-deficient ECs in the adult mouse ear vasculature. PECAM immunofluorescence shows that vessels are grossly normal. Scale bars: D 25 μm (JPEG 971 kb)

Figure S9

Rasip1 regulates blood vessel lumen formation in vitro. (A-D) 3D collagen vasculogenesis assay after reduction in Rasip1, Arhgap29 or both together with siRNA. Arrowheads denote open lumens and arrows denote failed lumen formation. (E) Quantification showing that lumen formation fails after reduction in Rasip1, Arhgap29 or both together. * = p < 0.01, n = 15. Scale bars: A–D 100 μm (JPEG 1442 kb)

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Koo, Y., Barry, D.M., Xu, K. et al. Rasip1 is essential to blood vessel stability and angiogenic blood vessel growth. Angiogenesis 19, 173–190 (2016).

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