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In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment

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Abstract

Angiogenesis is regulated by complex interactions between endothelial cells and support cells of the vascular microenvironment, such as tissue myeloid cells and vascular mural cells. Multicellular interactions during angiogenesis are difficult to study in animals and challenging in a reductive setting. We incorporated stromal cells into an established bead-based capillary sprouting assay to develop assays that faithfully reproduce major steps of vessel sprouting and maturation. We observed that macrophages enhance angiogenesis, increasing the number and length of endothelial sprouts, a property we have dubbed “angiotrophism.” We found that polarizing macrophages toward a pro-inflammatory profile further increased their angiotrophic stimulation of vessel sprouting, and this increase was dependent on macrophage Notch signaling. To study endothelial/pericyte interactions, we added vascular pericytes directly to the bead-bound endothelial monolayer. These pericytes formed close associations with the endothelial sprouts, causing increased sprout number and vessel caliber. We found that Jagged1 expression and Notch signaling are essential for the growth of both endothelial cells and pericytes and may function in their interaction. We observed that combining endothelial cells with both macrophages and pericytes in the same sprouting assay has multiplicative effects on sprouting. These results significantly improve bead-capillary sprouting assays and provide an enhanced method for modeling interactions between the endothelium and the vascular microenvironment. Achieving this in a reductive in vitro setting represents a significant step toward a better understanding of the cellular elements that contribute to the formation of mature vasculature.

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References

  1. 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 

  2. Rymo SF, Gerhardt H, Wolfhagen Sand F, Lang R, Uv A, Betsholtz C (2011) A two-way communication between microglial cells and angiogenic sprouts regulates angiogenesis in Aortic Ring cultures. PLoS One 6(1):e15846. doi:10.1371/journal.pone.0015846

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Stefater JA III, Lewkowich I, Rao S, Mariggi G, Carpenter AC, Burr AR, Fan J, Ajima R, Molkentin JD, Williams BO, Wills-Karp M, Pollard JW, Yamaguchi T, Ferrara N, Gerhardt H, Lang RA (2011) Regulation of angiogenesis by a non-canonical Wnt–Flt1 pathway in myeloid cells. Nature 474(7352):511–515. doi:10.1038/nature10085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Armulik A, Genové G, Betsholtz C (2011) Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev Cell 21(2):193–215. doi:10.1016/j.devcel.2011.07.001

    Article  CAS  PubMed  Google Scholar 

  5. Murray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11(11):723–737. doi:10.1038/nri3073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Schmid MC, Varner JA (2012) Myeloid cells in tumor inflammation. Vasc Cell 4(1):14. doi:10.1186/2045-824X-4-14

    Article  PubMed  PubMed Central  Google Scholar 

  7. Wynn TA, Chawla A, Pollard JW (2013) Macrophage biology in development, homeostasis and disease. Nature 496(7446):445–455. doi:10.1038/nature12034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Armulik A, Abramsson A, Betsholtz C (2005) Endothelial/pericyte interactions. Circ Res 97(6):512–523. doi:10.1161/01.RES.0000182903.16652.d7

    Article  CAS  PubMed  Google Scholar 

  9. Radtke F, Schweisguth F, Pear W (2005) The notch ‘gospel’. EMBO rep 6(12):1120–1125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tung JJ, Tattersall IW, Kitajewski J (2012) Tips, stalks, tubes: notch-mediated cell fate determination and mechanisms of tubulogenesis during angiogenesis. Cold Spring Harbor Perspect Med 2(2):a006601. doi:10.1101/cshperspect.a006601

    Article  Google Scholar 

  11. Outtz HH, Wu JK, Wang X, Kitajewski J (2010) Notch1 deficiency results in decreased inflammation during wound healing and regulates vascular endothelial growth factor receptor-1 and inflammatory cytokine expression in macrophages. J Immunol 185(7):4363–4373. doi:10.4049/jimmunol.1000720

    Article  PubMed  Google Scholar 

  12. 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 

  13. Scheppke LL, Murphy EAE, Zarpellon AA, Hofmann JJJ, Merkulova AA, Shields DJD, Weis SMS, Byzova TVT, Ruggeri ZMZ, Iruela-Arispe MLM, Cheresh DAD (2012) Notch promotes vascular maturation by inducing integrin-mediated smooth muscle cell adhesion to the endothelial basement membrane. Blood 119(9):2149–2158. doi:10.1182/blood-2011-04-348706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang Y, Pan L, Moens CB, Appel B (2014) Notch3 establishes brain vascular integrity by regulating pericyte number. Development (Cambridge, England) 141(2):307–317. doi:10.1242/dev.096107

    Article  CAS  Google Scholar 

  15. Tung JJ, Hobert O, Berryman M, Kitajewski J (2009) Chloride intracellular channel 4 is involved in endothelial proliferation and morphogenesis in vitro. Angiogenesis 12(3):209–220. doi:10.1007/s10456-009-9139-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Koh W, Stratman AN, Sacharidou A, Davis GE (2008) In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis. Methods Enzymol 443:83–101. doi:10.1016/S0076-6879(08)02005-3

    Article  CAS  PubMed  Google Scholar 

  17. Nakatsu MN, Hughes CCW (2008) An optimized three-dimensional in vitro model for the analysis of angiogenesis. Methods Enzymol 443:65–82. doi:10.1016/S0076-6879(08)02004-1

    Article  CAS  PubMed  Google Scholar 

  18. Stratman AN, Malotte KM, Mahan RD, Davis MJ, Davis GE (2009) Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation. Blood 114(24):5091–5101. doi:10.1182/blood-2009-05-222364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Investig 52(11):2745–2756. doi:10.1172/JCI107470

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Clausen BE, Burkhardt C, Reith W, Renkawitz R, Förster I (1999) Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res 8(4):265–277

    Article  CAS  PubMed  Google Scholar 

  21. Tu L, Fang TC, Artis D, Shestova O, Pross SE, Maillard I, Pear WS (2005) Notch signaling is an important regulator of type 2 immunity. J Exp Med 202(8):1037–1042. doi:10.1084/jem.20050923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F (2001) Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1:4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nakatsu MN, Davis J, Hughes CCW (2007) Optimized fibrin gel bead assay for the study of angiogenesis. J Visual Exp 3:186. doi:10.3791/186

    Google Scholar 

  24. Nakatsu MN, Sainson RCA, Aoto JN, Taylor KL, Aitkenhead M, Pérez-del-Pulgar S, Carpenter PM, Hughes CCW (2003) Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and Angiopoietin-1. Microvasc Res 66(2):102–112

    Article  CAS  PubMed  Google Scholar 

  25. Wang Y-C, He F, Feng F, Liu X-W, Dong G-Y, Qin H-Y, Hu X-B, Zheng M-H, Liang L, Feng L, Liang Y-M, Han H (2010) Notch signaling determines the M1 versus M2 polarization of macrophages in antitumor immune responses. Cancer Res 70(12):4840–4849. doi:10.1158/0008-5472.CAN-10-0269

    Article  CAS  PubMed  Google Scholar 

  26. Zajac E, Schweighofer B, Kupriyanova TA, Juncker-Jensen A, Minder P, Quigley JP, Deryugina EI (2013) Angiogenic capacity of M1- and M2-polarized macrophages is determined by the levels of TIMP-1 complexed with their secreted proMMP-9. Blood 122(25):4054–4067. doi:10.1182/blood-2013-05-501494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Jetten N, Verbruggen S, Gijbels MJ, Post MJ, De Winther MP, Donners MM (2014) Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo. Angiogenesis 17(1):109–118. doi:10.1007/s10456-013-9381-6

    Article  CAS  PubMed  Google Scholar 

  28. Aplin AC, Ligresti G, Fogel E, Zorzi P, Smith K, Nicosia RF (2014) Regulation of angiogenesis, mural cell recruitment and adventitial macrophage behavior by Toll-like receptors. Angiogenesis 17(1):147–161. doi:10.1007/s10456-013-9384-3

    Article  CAS  PubMed  Google Scholar 

  29. Brancato SK, Albina JE (2011) Wound macrophages as key regulators of repair: origin, phenotype, and function. Am J Pathol 178(1):19–25. doi:10.1016/j.ajpath.2010.08.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Benedito R, Roca C, Sorensen I, Adams S, Gossler A, Fruttiger M, Adams RH (2009) The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137(6):1124–1135. doi:10.1016/j.cell.2009.03.025

    Article  CAS  PubMed  Google Scholar 

  31. Kangsamaksin T, Murtomaki A, Kofler NM, Cuervo H, Chaudhri RA, Tattersall IW, Rosenstiel PE, Shawber CJ, Kitajewski J (2014) Notch decoys that selectively block Dll/Notch or Jagged/Notch disrupt angiogenesis by unique mechanisms to inhibit tumor growth. Cancer discovery. doi:10.1158/2159-8290.CD-14-0650

    PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The authors would like to thank Carrie Shawber for her consultation and advice on experimental methodology and planning. They are additionally grateful to Warren Pear, Boris Reizis, and Andrew Weng for kind gifts of reagents as detailed in the Materials and Methods. In addition, they would like to thank the Columbia University Medical Scientist Training Program (and its MSTP training grant T32GM007367) for its support.

Funding

This study was funded by NIH grants: 1R01HL112626 (J.K.) and 1R01HL119043 (J.K.).

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Authors and Affiliations

  1. Obstetrics/Gynecology, Columbia University, New York, NY, USA

    Ian W. Tattersall, Jing Du, Zhuangzhuang Cong, Bennet S. Cho, Alyssa M. Klein, Chelsea L. Dieck, Reyhaan A. Chaudhri, Henar Cuervo, James H. Herts & Jan Kitajewski

  2. Pathology and Cell Biology, Columbia University, New York, NY, USA

    Jing Du & Jan Kitajewski

  3. Physiology and Biophysics, University of Illinois Chicago, College of Medicine, Chicago, IL, USA

    Jan Kitajewski

  4. Columbia University Medical Center, 1130 St. Nicholas Avenue, ICRC 926, New York, NY, 10032, USA

    Jan Kitajewski

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  1. Ian W. Tattersall
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Correspondence to Jan Kitajewski.

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Electronic supplementary material

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Online Resource 1

Schematic of the incorporation of macrophages and pericytes in the capillary sprouting assay. a Schematic of an endothelial cell-only capillary sprouting assay, involving co-culture of HUVEC with an overlying D551 fibroblast feeder layer. b Schematics of capillary sprouting assays incorporating bone marrow macrophages (directly into the fibrin matrix) and pericytes (bound to the bead on top of the endothelial monolayer) (PDF 242 kb)

Online Resource 2

a EOC2 inclusion increases number and length of endothelial sprouts at early timepoint day 3. b Sprout number and length remains increased through late timepoint day 5. c Quantification of sprout number. d Quantification of frequency of longer (> 200um) sprouts. Scale bars represent 200um. Error bars represent standard error. * = p < 0.05 (PDF 2231 kb)

Online Resource 3

Macrophage polarization was confirmed via qPCR. Myeloid cells treated with pro-inflammatory factors LPS and IFNγ show increased mRNA expression of pro-inflammatory marker iNOS/NOS2, while cells treated with alternative activator cytokine IL-4 show increased mRNA expression of alternative activation marker Arginase. Error bars represent standard error. * = p < 0.05 (PDF 54 kb)

Online Resource 4

LPS treatment does not alter angiogenesis in endothelial cell-only capillary sprouting assays. a Treatment with 0.1, 1.0, or 10 ng/mL LPS did not significantly alter sprout number or tip cell number. b Quantification of sprout number. No relationships are statistically significant for p < 0.05. This experiment was performed once (PDF 6775 kb)

Online Resource 5

Macrophage expression of DNMAM-GFP was confirmed via FACS. Bone marrow macrophages derived from LysMcre/+ ; DNMAML-GFPfl/+ mice show widespread expression of GFP, indicating synthesis of the DNMAML-GFP gene product. LysMcre/+ control mice do not express GFP (PDF 33 kb)

Online Resource 6

Pericyte dispersal in fibrin gel does not recapitulate effects of direct bead binding. HBVP resuspension in fibrin gel, rather than direct binding to the endothelial-coated beads, does not produce the profound phenotypic changes observed in the case of direct pericyte association with endothelial cells. Scale bar represents 200 μm (PDF 3194 kb)

Online Resource 7

Pericyte/endothelial knockdown of Jagged1 qPCR was validated via Western blot. Protein expression of Jagged1 in cells coinfected with RFP and the Jagged1KD shRNA construct are approximately half that of RFP/Scramble controls (PDF 55 kb)

Online Resource 8

Knockdown of Jagged1 in pericytes reduces cell growth in monoculture. Growth assessed 4 days after plating. Error bar represents standard deviation. * = p<0.05 (PDF 38 kb)

Online Resource 9

Dll4 inhibition increases sprouting in endothelial cell-only capillary sprouting assays and in endothelial–pericyte co-cultures. a Protein expression of Dll4 in cells coinfected with RFP and the Dll4KD shRNA construct are greatly reduced compared to RFP/Scramble control. b Dll4KD HUVEC show increased sprouting in endothelial cell-only capillary sprouting assay. c Dll4KD HUVEC show increased sprouting in endothelial and pericyte co-culture capillary sprouting assay. d Quantification of sprout number in both experiments. Error bars represent standard error. * = p < 0.05. Experiments were performed once (PDF 12178 kb)

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Tattersall, I.W., Du, J., Cong, Z. et al. In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment. Angiogenesis 19, 201–215 (2016). https://doi.org/10.1007/s10456-016-9501-1

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