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Capturing membrane trafficking events during 3D angiogenic development in vitro.

Caitlin R Francis1, Erich J Kushner1

  • 1Department of Biological Sciences, University of Denver, Denver, Colorado, USA.

Microcirculation (New York, N.Y. : 1994)
|August 20, 2021
PubMed
Summary

A novel 3D in vitro sprouting model allows high-resolution imaging of endothelial membrane trafficking. This model reveals distinct polarity and secretion events crucial for angiogenic growth.

Keywords:
Rab27aWeibel-Palade bodyangiogenesisapical membraneblood vesseldevelopmentendothelialendotheliumexocytosisimaginglumensproutingtraffickingvascularvesiclevon Willebrand factor

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Area of Science:

  • Cell Biology
  • Biophysics
  • Developmental Biology

Background:

  • Vesicular trafficking is crucial for protein localization and function during tissue development.
  • Endothelial-specific trafficking signatures are poorly understood due to visualization challenges.
  • Existing 2D models limit the study of complex endothelial cell behaviors.

Purpose of the Study:

  • To explore a 3D in vitro sprouting model for imaging endothelial membrane trafficking.
  • To investigate endothelial-specific trafficking signatures in a more physiologically relevant context.
  • To overcome limitations in visualizing endothelial trafficking events.

Main Methods:

  • Utilized a fibrin bead assay to induce endothelial cell sprouting in 3D.
  • Transfected sprouts with fluorescent proteins and stained for cell markers.
  • Employed live and fixed-cell microscopy to image trafficking events.

Main Results:

  • Demonstrated strong apicobasal polarity in 3D sprouts, with apical localization of moesin and podocalyxin.
  • Successfully imaged vesicular carriers with high resolution, showing proper membrane polarity exclusively in 3D sprouts.
  • Observed distinct imaging advantages for monitoring exocytic events, such as von Willebrand Factor secretion, in 3D sprouts compared to 2D cultures.

Conclusions:

  • The fibrin bead sprouting assay is highly suitable for imaging trafficking events during angiogenesis.
  • This 3D model provides critical insights into endothelial cell polarity and secretion.
  • The findings advance our understanding of endothelial-specific trafficking in developmental processes.