June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Transcriptomic and Immunocytochemical comparison of 2D and 3D in vitro angiogenesis assays
Author Affiliations & Notes
  • Jan Niklas Ness
    Eye Center, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
    Institute for Pharmaceutical Sciences, Albert-Ludwigs-Universitat Freiburg Fakultat fur Chemie und Pharmazie, Freiburg, Baden-Württemberg, Germany
  • Julian Rapp
    Eye Center, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
    Albert-Ludwigs-Universitat Freiburg Medizinische Fakultat, Freiburg, Baden-Württemberg, Germany
  • Julian Wolf
    Eye Center, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
    Albert-Ludwigs-Universitat Freiburg Medizinische Fakultat, Freiburg, Baden-Württemberg, Germany
  • Paula Liang
    Eye Center, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
    Albert-Ludwigs-Universitat Freiburg Medizinische Fakultat, Freiburg, Baden-Württemberg, Germany
  • Hansjüergen T Agostini
    Eye Center, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
    Albert-Ludwigs-Universitat Freiburg Medizinische Fakultat, Freiburg, Baden-Württemberg, Germany
  • Gunther R Schlunck
    Eye Center, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
    Albert-Ludwigs-Universitat Freiburg Medizinische Fakultat, Freiburg, Baden-Württemberg, Germany
  • Martin J. Hug
    Hospital Pharmacy, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
    Institute for Pharmaceutical Sciences, Albert-Ludwigs-Universitat Freiburg Fakultat fur Chemie und Pharmazie, Freiburg, Baden-Württemberg, Germany
  • Clemens Lange
    Eye Center, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
    Albert-Ludwigs-Universitat Freiburg Medizinische Fakultat, Freiburg, Baden-Württemberg, Germany
  • Felicitas Bucher
    Eye Center, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
    Albert-Ludwigs-Universitat Freiburg Medizinische Fakultat, Freiburg, Baden-Württemberg, Germany
  • Footnotes
    Commercial Relationships   Jan Ness None; Julian Rapp None; Julian Wolf None; Paula Liang None; Hansjüergen Agostini None; Gunther Schlunck None; Martin J. Hug None; Clemens Lange None; Felicitas Bucher Hoffmann-La Roche, Bayer AG, Novartis, Code F (Financial Support)
  • Footnotes
    Support  DFG (German Research Foundation), Dr. Werner Jackstädt Nachwuchspreis (German Retina Society), Berta-Ottenstein-Fellowship, Volker Homann Stiftung Freiburg (Private Foundation)
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1178 – A0032. doi:
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    • Get Citation

      Jan Niklas Ness, Julian Rapp, Julian Wolf, Paula Liang, Hansjüergen T Agostini, Gunther R Schlunck, Martin J. Hug, Clemens Lange, Felicitas Bucher; Transcriptomic and Immunocytochemical comparison of 2D and 3D in vitro angiogenesis assays. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1178 – A0032.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Retinal vascular diseases represent one of the most common causes for vision loss in the western world. To study angiogenic processes and identify novel therapeutic targets, researchers utilize a variety of in vitro angiogenesis assays in either 2D or 3D settings. Interestingly, research results from 2D and 3D in vitro assays do not necessarily align. In this study, we compare transcriptomic and immunocytochemical changes in vascular endothelial cells from common 2D and 3D in vitro angiogenesis assays to unravel advantages and disadvantages and, based on this, the preferred use of the respective assay.

Methods : The 2D Migration Assay and 3D Spheroid Sprouting Assay were both performed using human umbilical vein endothelial cells (HUVEC). Human VEGF165 was used to stimulate the endothelial cell migration and sprouting. We applied RNA Seq analysis to assess the transcriptomic modulations in HUVECs following stimulation with hVEGF165 in the respective 2D and 3D setting. Immunocytochemistry was used to characterize differences at the protein level in the assays performed.

Results : In both 2D and 3D assays, genes related to angiogenesis, migration and vascular development were enriched after stimulation with hVEGF165. Interestingly, in the 3D setting vascular endothelial cells showed a clearer transcriptomic shift in response to hVEGF165 treatment (foldchange: 3.96, differentially expressed genes (DEG): 763) than in the 2D setting (foldchange: 1.34, DEG: 167). Upregulation of common proangiogenic genes including KDR and VCAM1 was comparable in both assays. In contrast, transcriptomic changes related to cell-matrix interaction and glycolysis were both significantly increased only in the 3D setting. Immunocytochemical stainings revealed that exclusively in the 3D setting, vascular endothelial cells were able to generate an in vivo like tip cell behavior visualized by localized CD34 expression in the spheroid sprouts.

Conclusions : Our data underline that the transcriptomic profile of vascular endothelial cells and their responsiveness to VEGF and most likely other test substances is influenced by the assay microenvironment. For scientific questions regarding matrix interactions or glycolytic switches researchers should consider the 3D setting. For high throughput screening experiments and investigations of main angiogenic pathways the 2D setting may be sufficient.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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