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Large-scale time series microscopy of neovessel growth during angiogenesis

Abstract

During angiogenesis, growing neovessels must effectively navigate through the tissue space as they elongate and subsequently integrate into a microvascular network. While time series microscopy has provided insight into the cell activities within single growing neovessel sprouts, less is known concerning neovascular dynamics within a large angiogenic tissue bed. Here, we developed a time-lapse imaging technique that allowed visualization and quantification of sprouting neovessels as they form and grow away from adult parent microvessels in three dimensions over cubic millimeters of matrix volume during the course of up to 5 days on the microscope. Using a new image acquisition procedure and novel morphometric analysis tools, we quantified the elongation dynamics of growing neovessels and found an episodic growth pattern accompanied by fluctuations in neovessel diameter. Average elongation rate was 5 μm/h for individual vessels, but we also observed considerable dynamic variability in growth character including retraction and complete regression of entire neovessels. We observed neovessel-to-neovessel directed growth over tens to hundreds of microns preceding tip-to-tip inosculation. As we have previously described via static 3D imaging at discrete time points, we identified different collagen fibril structures associated with the growing neovessel tip and stalk, and observed the coordinated alignment of growing neovessels in a deforming matrix. Overall analysis of the entire image volumes demonstrated that although individual neovessels exhibited episodic growth and regression, there was a monotonic increase in parameters associated with the entire vascular bed such as total network length and number of branch points. This new time-lapse imaging approach corroborated morphometric changes in individual neovessels described by us and others, as well as captured dynamic neovessel behaviors unique to days-long angiogenesis within the forming neovascular network.

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Acknowledgments

This research was supported by NIH Grants R01HL077683 and S10RR023737. We thank Richard A. Cordova (Ironwood Ridge High School, Tucson Arizona) for registering datasets and selecting regions of interests for visualization. He also contributed to the vessel length measurements. Nathan Galli (SCI Institute, University of Utah) provided the artwork in Fig. 1. The developers of the FluoRender software were supported by National Institutes of Health (NIH) Grant R01GM098151. Carlos Lois (California Institute of Technology) developed the ubiquitous EGFP expressing transgenic rat that was provided by the RRRC (University of Missouri).

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Correspondence to Urs Utzinger.

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Utzinger, U., Baggett, B., Weiss, J.A. et al. Large-scale time series microscopy of neovessel growth during angiogenesis. Angiogenesis 18, 219–232 (2015). https://doi.org/10.1007/s10456-015-9461-x

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Keywords

  • 5D imaging
  • Angiogenesis
  • Extracellular matrix
  • Inosculation
  • Matrix remodeling
  • Sprouting
  • Regression
  • Neovessel