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Angiogenesis

, Volume 19, Issue 2, pp 201–215 | Cite as

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

  • 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
Original Paper

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.

Keywords

Notch In vitro Endothelial cell Pericyte Macrophage 

Notes

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.).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

10456_2016_9501_MOESM1_ESM.pdf (243 kb)
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)
10456_2016_9501_MOESM2_ESM.pdf (2.2 mb)
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)
10456_2016_9501_MOESM3_ESM.pdf (54 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)
10456_2016_9501_MOESM4_ESM.pdf (6.6 mb)
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)
10456_2016_9501_MOESM5_ESM.pdf (33 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)
10456_2016_9501_MOESM6_ESM.pdf (3.1 mb)
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)
10456_2016_9501_MOESM7_ESM.pdf (55 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)
10456_2016_9501_MOESM8_ESM.pdf (38 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)
10456_2016_9501_MOESM9_ESM.pdf (11.9 mb)
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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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Ian W. Tattersall
    • 1
  • Jing Du
    • 1
    • 2
  • Zhuangzhuang Cong
    • 1
  • Bennet S. Cho
    • 1
  • Alyssa M. Klein
    • 1
  • Chelsea L. Dieck
    • 1
  • Reyhaan A. Chaudhri
    • 1
  • Henar Cuervo
    • 1
  • James H. Herts
    • 1
  • Jan Kitajewski
    • 1
    • 2
    • 3
    • 4
  1. 1.Obstetrics/GynecologyColumbia UniversityNew YorkUSA
  2. 2.Pathology and Cell BiologyColumbia UniversityNew YorkUSA
  3. 3.Physiology and BiophysicsUniversity of Illinois Chicago, College of MedicineChicagoUSA
  4. 4.Columbia University Medical CenterNew YorkUSA

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