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Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells

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Abstract

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40 % increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80 % increase in vascular density and pillar number augmentation by 420 %. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturation.

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References

  1. Ribatti D, Crivellato E (2012) “Sprouting angiogenesis”, a reappraisal. Dev Biol 372(2):157–165

    Article  PubMed  CAS  Google Scholar 

  2. Djonov V, Schmid M, Tschanz SA, Burri PH (2000) Intussusceptive angiogenesis: its role in embryonic vascular network formation. Circ Res 86(3):286–292

    Article  PubMed  CAS  Google Scholar 

  3. Djonov V, Baum O, Burri PH (2003) Vascular remodeling by intussusceptive angiogenesis. Cell Tissue Res 314(1):107–117

    Article  PubMed  Google Scholar 

  4. Jakobsson L, Franco CA, Bentley K, Collins RT, Ponsioen B, Aspalter IM, Rosewell I, Busse M, Thurston G, Medvinsky A, Schulte-Merker S, Gerhardt H (2010) Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat Cell Biol 12(10):943–953

    Article  PubMed  CAS  Google Scholar 

  5. Makanya AN, Hlushchuk R, Djonov VG (2009) Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling. Angiogenesis 12(2):113–123

    Article  PubMed  CAS  Google Scholar 

  6. Styp-Rekowska B, Hlushchuk R, Pries AR, Djonov V (2011) Intussusceptive angiogenesis: pillars against the blood flow. Acta Physiol (Oxf) 202(3):213–223

    Article  CAS  Google Scholar 

  7. Dill MT, Rothweiler S, Djonov V, Hlushchuk R, Tornillo L, Terracciano L, Meili-Butz S, Radtke F, Heim MH, Semela D (2012) Disruption of Notch1 induces vascular remodeling, intussusceptive angiogenesis, and angiosarcomas in livers of mice. Gastroenterology 142(4):967–977

    Article  PubMed  CAS  Google Scholar 

  8. Baum O, Suter F, Gerber B, Tschanz SA, Buergy R, Blank F, Hlushchuk R, Djonov V (2010) VEGF-A promotes intussusceptive angiogenesis in the developing chicken chorioallantoic membrane. Microcirculation 17(6):447–457

    PubMed  CAS  Google Scholar 

  9. Konerding MA, Gibney BC, Houdek JP, Chamoto K, Ackermann M, Lee GS, Lin M, Tsuda A, Mentzer SJ (2012) Spatial dependence of alveolar angiogenesis in post-pneumonectomy lung growth. Angiogenesis 15(1):23–32

    Article  PubMed  CAS  Google Scholar 

  10. Schwanbeck R, Martini S, Bernoth K, Just U (2011) The Notch signaling pathway: molecular basis of cell context dependency. Eur J Cell Biol 90(6–7):572–581

    Article  PubMed  CAS  Google Scholar 

  11. High FA, Zhang M, Proweller A, Tu L, Parmacek MS, Pear WS, Epstein JA (2007) An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation. J Clin Invest 117(2):353–363

    Article  PubMed  CAS  Google Scholar 

  12. Napp LC, Augustynik M, Paesler F, Krishnasamy K, Woiterski J, Limbourg A, Bauersachs J, Drexler H, Le NF, Limbourg FP (2012) Extrinsic Notch ligand delta-like 1 regulates tip cell selection and vascular branching morphogenesis. Circ Res 110(4):530–535

    Article  PubMed  CAS  Google Scholar 

  13. 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

    Article  PubMed  CAS  Google Scholar 

  14. Suchting S, Freitas C, Le NF, Benedito R, Breant C, Duarte A, Eichmann A (2007) The Notch ligand delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci USA 104(9):3225–3230

    Article  PubMed  CAS  Google Scholar 

  15. Roca C, Adams RH (2007) Regulation of vascular morphogenesis by Notch signaling. Genes Dev 21(20):2511–2524

    Article  PubMed  CAS  Google Scholar 

  16. Corada M, Nyqvist D, Orsenigo F, Caprini A, Giampietro C, Taketo MM, Iruela-Arispe ML, Adams RH, Dejana E (2010) The Wnt/beta-catenin pathway modulates vascular remodeling and specification by upregulating Dll4/Notch signaling. Dev Cell 18(6):938–949

    Article  PubMed  CAS  Google Scholar 

  17. MacGrogan D, Nus M, de la Pompa JL (2010) Notch signaling in cardiac development and disease. Curr Top Dev Biol 92:333–365

    Article  PubMed  CAS  Google Scholar 

  18. Gridley T (2010) Notch signaling in the vasculature. Curr Top Dev Biol 92:277–309

    Article  PubMed  CAS  Google Scholar 

  19. Lobov IB, Cheung E, Wudali R, Cao J, Halasz G, Wei Y, Economides A, Lin HC, Papadopoulos N, Yancopoulos GD, Wiegand SJ (2011) The Dll4/Notch pathway controls postangiogenic blood vessel remodeling and regression by modulating vasoconstriction and blood flow. Blood 117(24):6728–6737

    Article  PubMed  CAS  Google Scholar 

  20. Armulik A, Abramsson A, Betsholtz C (2005) Endothelial/pericyte interactions. Circ Res 97(6):512–523

    Article  PubMed  CAS  Google Scholar 

  21. Sainson RC, Harris AL (2008) Regulation of angiogenesis by homotypic and heterotypic notch signalling in endothelial cells and pericytes: from basic research to potential therapies. Angiogenesis 11(1):41–51

    Article  PubMed  CAS  Google Scholar 

  22. Scheppke L, Murphy EA, Zarpellon A, Hofmann JJ, Merkulova A, Shields DJ, Weis SM, Byzova TV, Ruggeri ZM, Iruela-Arispe ML, Cheresh DA (2012) Notch promotes vascular maturation by inducing integrin-mediated smooth muscle cell adhesion to the endothelial basement membrane. Blood 119(9):2149–2158

    Article  PubMed  CAS  Google Scholar 

  23. Liu H, Kennard S, Lilly B (2009) NOTCH3 expression is induced in mural cells through an autoregulatory loop that requires endothelial-expressed JAGGED1. Circ Res 104(4):466–475

    Article  PubMed  CAS  Google Scholar 

  24. Egea J, Klein R (2007) Bidirectional Eph-ephrin signaling during axon guidance. Trends Cell Biol 17(5):230–238

    Article  PubMed  CAS  Google Scholar 

  25. Himanen JP, Saha N, Nikolov DB (2007) Cell-cell signaling via Eph receptors and ephrins. Curr Opin Cell Biol 19(5):534–542

    Article  PubMed  CAS  Google Scholar 

  26. Masumura T, Yamamoto K, Shimizu N, Obi S, Ando J (2009) Shear stress increases expression of the arterial endothelial marker ephrinB2 in murine ES cells via the VEGF-Notch signaling pathways. Arterioscler Thromb Vasc Biol 29(12):2125–2131

    Article  PubMed  CAS  Google Scholar 

  27. Sato Y, Watanabe T, Saito D, Takahashi T, Yoshida S, Kohyama J, Ohata E, Okano H, Takahashi Y (2008) Notch mediates the segmental specification of angioblasts in somites and their directed migration toward the dorsal aorta in avian embryos. Dev Cell 14(6):890–901

    Article  PubMed  CAS  Google Scholar 

  28. Krebs LT, Starling C, Chervonsky AV, Gridley T (2010) Notch1 activation in mice causes arteriovenous malformations phenocopied by ephrinB2 and EphB4 mutants. Genesis 48(3):146–150

    PubMed  CAS  Google Scholar 

  29. 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 

  30. Noguera-Troise I, Daly C, Papadopoulos NJ, Coetzee S, Boland P, Gale NW, Lin HC, Yancopoulos GD, Thurston G (2007) Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Novartis Found Symp 283:106–120

    Article  PubMed  CAS  Google Scholar 

  31. Ridgway J, Zhang G, Wu Y, Stawicki S, Liang WC, Chanthery Y, Kowalski J, Watts RJ, Callahan C, Kasman I, Singh M, Chien M, Tan C, Hongo JA, de Sauvage F, Plowman G, Yan M (2006) Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 444(7122):1083–1087

    Article  PubMed  CAS  Google Scholar 

  32. Al Haj ZA, Oikawa A, Bazan-Peregrino M, Meloni M, Emanueli C, Madeddu P (2010) Inhibition of delta-like-4-mediated signaling impairs reparative angiogenesis after ischemia. Circ Res 107(2):283–293

    Article  Google Scholar 

  33. Benedito R, Rocha SF, Woeste M, Zamykal M, Radtke F, Casanovas O, Duarte A, Pytowski B, Adams RH (2012) Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF–VEGFR2 signalling. Nature 484(7392):110–114

    Article  PubMed  CAS  Google Scholar 

  34. Wnuk M, Hlushchuk R, Janot M, Tuffin G, Martiny-Baron G, Holzer P, Imbach-Weese P, Djonov V, Huynh-Do U (2012) Podocyte EphB4 signaling helps recovery from glomerular injury. Kidney Int 81(12):1212–1225

    Article  PubMed  CAS  Google Scholar 

  35. Djonov VG, Kurz H, Burri PH (2002) Optimality in the developing vascular system: branching remodeling by means of intussusception as an efficient adaptation mechanism. Dev Dyn 224:391–402

    Article  PubMed  Google Scholar 

  36. Jenkins DW, Ross S, Veldman-Jones M, Foltz IN, Clavette BC, Manchulenko K, Eberlein C, Kendrew J, Petteruti P, Cho S, Damschroder M, Peng L, Baker D, Smith NR, Weir HM, Blakey DC, Bedian V, Barry ST (2012) MEDI0639: a novel therapeutic antibody targeting Dll4 modulates endothelial cell function and angiogenesis in vivo. Mol Cancer Ther 11(8):1650–1660

    Article  PubMed  CAS  Google Scholar 

  37. Thurston G, Noguera-Troise I, Yancopoulos GD (2007) The delta paradox: DLL4 blockade leads to more tumour vessels but less tumour growth. Nat Rev Cancer 7(5):327–331

    Article  PubMed  CAS  Google Scholar 

  38. Kalen M, Heikura T, Karvinen H, Nitzsche A, Weber H, Esser N, Yla-Herttuala S, Hellstrom M (2011) Gamma-secretase inhibitor treatment promotes VEGF-A-driven blood vessel growth and vascular leakage but disrupts neovascular perfusion. PLoS ONE 6(4):e18709

    Article  PubMed  CAS  Google Scholar 

  39. Larrivee B, Prahst C, Gordon E, Del TR, Mathivet T, Duarte A, Simons M, Eichmann A (2012) ALK1 signaling inhibits angiogenesis by cooperating with the Notch pathway. Dev Cell 22(3):489–500

    Article  PubMed  CAS  Google Scholar 

  40. Hlushchuk R, Riesterer O, Baum O, Wood J, Gruber G, Pruschy M, Djonov V (2008) Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation. Am J Pathol 173(4):1173–1185

    Article  PubMed  CAS  Google Scholar 

  41. Wnuk M, Hlushchuk R, Tuffin G, Huynh-Do U, Djonov V (2011) The effects of PTK787/ZK222584, an inhibitor of VEGFR and PDGFRbeta pathways, on intussusceptive angiogenesis and glomerular recovery from Thy1.1 nephritis. Am J Pathol 178(4):1899–1912

    Article  PubMed  CAS  Google Scholar 

  42. Kucia M, Reca R, Campbell FR, Zuba-Surma E, Majka M, Ratajczak J, Ratajczak MZ (2006) A population of very small embryonic-like (VSEL) CXCR4(+)SSEA-1(+)Oct-4+ stem cells identified in adult bone marrow. Leukemia 20(5):857–869

    Article  PubMed  CAS  Google Scholar 

  43. Usui F, Yamamoto Y, Nakamura Y, Ono T, Kagami H (2009) Novel system for degeneration of blood vessels by UV irradiation and subsequent regeneration using chick bone marrow cells. Cells Tissues Organs 189(5):348–355

    Article  PubMed  Google Scholar 

  44. Lorusso G, Ruegg C (2008) The tumor microenvironment and its contribution to tumor evolution toward metastasis. Histochem Cell Biol 130(6):1091–1103

    Article  PubMed  CAS  Google Scholar 

  45. Coffelt SB, Lewis CE, Naldini L, Brown JM, Ferrara N, De PM (2010) Elusive identities and overlapping phenotypes of proangiogenic myeloid cells in tumors. Am J Pathol 176(4):1564–1576

    Article  PubMed  Google Scholar 

  46. Camelo S, Raoul W, Lavalette S, Calippe B, Cristofaro B, Levy O, Houssier M, Sulpice E, Jonet L, Klein C, Devevre E, Thuret G, Duarte A, Eichmann A, Leconte L, Guillonneau X, Sennlaub F (2012) Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages. Angiogenesis 15(4):609–622

    Article  PubMed  CAS  Google Scholar 

  47. Lawson ND, Scheer N, Pham VN, Kim CH, Chitnis AB, Campos-Ortega JA, Weinstein BM (2001) Notch signaling is required for arterial–venous differentiation during embryonic vascular development. Development 128(19):3675–3683

    PubMed  CAS  Google Scholar 

  48. Fischer A, Schumacher N, Maier M, Sendtner M, Gessler M (2004) The Notch target genes Hey1 and Hey2 are required for embryonic vascular development. Genes Dev 18(8):901–911

    Article  PubMed  CAS  Google Scholar 

  49. Jin S, Hansson EM, Tikka S, Lanner F, Sahlgren C, Farnebo F, Baumann M, Kalimo H, Lendahl U (2008) Notch signaling regulates platelet-derived growth factor receptor-beta expression in vascular smooth muscle cells. Circ Res 102(12):1483–1491

    Article  PubMed  CAS  Google Scholar 

  50. Cortina C, Palomo-Ponce S, Iglesias M, Fernandez-Masip JL, Vivancos A, Whissell G, Huma M, Peiro N, Gallego L, Jonkheer S, Davy A, Lloreta J, Sancho E, Batlle E (2007) EphB-ephrin-B interactions suppress colorectal cancer progression by compartmentalizing tumor cells. Nat Genet 39(11):1376–1383

    Article  PubMed  CAS  Google Scholar 

  51. Noren NK, Pasquale EB (2007) Paradoxes of the EphB4 receptor in cancer. Cancer Res 67(9):3994–3997

    Article  PubMed  CAS  Google Scholar 

  52. Sato TN, Tozawa Y, Deutsch U, Wolburg-Buchholz K, Fujiwara Y, Gendron-Maguire M, Gridley T, Wolburg H, Risau W, Qin Y (1995) Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 376(6535):70–74

    Article  PubMed  CAS  Google Scholar 

  53. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD (1996) Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87(7):1171–1180

    Article  PubMed  CAS  Google Scholar 

  54. Morrow D, Cullen JP, Cahill PA, Redmond EM (2007) Cyclic strain regulates the Notch/CBF-1 signaling pathway in endothelial cells: role in angiogenic activity. Arterioscler Thromb Vasc Biol 27(6):1289–1296

    Article  PubMed  CAS  Google Scholar 

  55. Salvucci O, Yao L, Villalba S, Sajewicz A, Pittaluga S, Tosato G (2002) Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal-derived factor-1. Blood 99(8):2703–2711

    Article  PubMed  CAS  Google Scholar 

  56. Williams CK, Segarra M, Sierra ML, Sainson RC, Tosato G, Harris AL (2008) Regulation of CXCR4 by the Notch ligand delta-like 4 in endothelial cells. Cancer Res 68(6):1889–1895

    Article  PubMed  CAS  Google Scholar 

  57. Wragg A, Mellad JA, Beltran LE, Konoplyannikov M, San H, Boozer S, Deans RJ, Mathur A, Lederman RJ, Kovacic JC, Boehm M (2008) VEGFR1/CXCR4-positive progenitor cells modulate local inflammation and augment tissue perfusion by a SDF-1-dependent mechanism. J Mol Med (Berl) 86(11):1221–1232

    Article  CAS  Google Scholar 

  58. Arras M, Ito WD, Scholz D, Winkler B, Schaper J, Schaper W (1998) Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb. J Clin Invest 101(1):40–50

    Article  PubMed  CAS  Google Scholar 

  59. De PM, Venneri MA, Galli R, Sergi SL, Politi LS, Sampaolesi M, Naldini L (2005) Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell 8(3):211–226

    Article  Google Scholar 

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Acknowledgments

We are very grateful to Regula Buergy, Werner Graber, Jeannine Wagner, Barbara Krieger, Christoph Lehmann, and Brigitte Scolari for the wonderful laboratory work and technical support! This work is supported by the Swiss National Foundation Grant Nr: 31003A_135740.

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Authors and Affiliations

  1. Institute of Anatomy, University of Fribourg, Fribourg, Switzerland

    Ivanka Dimova, Ruslan Hlushchuk, Andrew Makanya, Beata Styp-Rekowska, Amalia Ceausu, Stefanie Flueckiger, Sonja Lang & Valentin Djonov

  2. Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3000, Bern 9, Switzerland

    Ivanka Dimova, Ruslan Hlushchuk, Andrew Makanya, Beata Styp-Rekowska, Sonja Lang & Valentin Djonov

  3. “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania

    Amalia Ceausu

  4. Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland

    Stefanie Flueckiger

  5. Department of Biomedicine, University Hospital Basel & Kantonsspital, St. Gallen, Switzerland

    David Semela

  6. Max Delbrück Center for Molecular Medicine, Berlin, Germany

    Ferdinand Le Noble

  7. Vascular Biology Lab, AU-KBC Research Centre, Anna University, MIT Campus, Chennai, India

    Suvro Chatterjee

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  1. Ivanka Dimova
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Correspondence to Valentin Djonov.

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10456_2013_9366_MOESM1_ESM.tif

Supplementary material Estimation of vascular permeability by lectin/Hoechst staining 24 h after PBS treatment (control), Notch, ephrinB2, and EphB4 inhibition. (TIFF 4290 kb)

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Dimova, I., Hlushchuk, R., Makanya, A. et al. Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells. Angiogenesis 16, 921–937 (2013). https://doi.org/10.1007/s10456-013-9366-5

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