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Investigating endothelial invasion and sprouting behavior in three-dimensional collagen matrices

Nature Protocols volume 4, pages 1888–1898 (2009)Cite this article

Abstract

Seeding a monolayer of primary human endothelial cells on the surface of a polymerized three-dimensional collagen matrix in the presence of pro-angiogenic stimuli allows manipulation and analysis of rapid sprouting responses. This protocol is useful for elucidating incompletely defined intracellular mechanisms downstream of pro-angiogenic factors that regulate sprout formation and initiation, and can also be used to test the efficacy of pro-and anti-angiogenic compounds. We present protocols to culture endothelial cells, prepare three-dimensional collagen matrices and quantify and image rapid endothelial sprouting responses (24 h). This protocol can be carried out using either type I or type II collagen matrices with primary endothelial cells isolated from macrovascular and microvascular sources of varying species.

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Figure 1: Schematic illustrating steps to carry out invasion assays.
Figure 2: Endothelial cell invasion in type II collagen matrices is driven by sphingosine 1-phosphate (S1P) and growth factors and is integrin dependent.
Figure 3: Schematic illustration of imaging chamber preparation.
Figure 4: Photographs illustrating expected responses following 24 h of invasion.
Figure 5: Comparable responses are observed with human umbilical vein (HUVEC), dermal and retinal endothelial cells.
Figure 6: Seeding proper endothelial cell density is critical for successful invasion responses.
Figure 7: Still images from time-lapse analyses.

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Acknowledgements

This work was supported by American Heart Association Scientist Development Grant 053020N (K.J.B.) and start up funds from Texas A&M HSC. We thank Dr. George Davis (University of Missouri-Columbia) for reading the manuscript and Pei-Hsing Chen and Chih-Po Su for assistance with illustrations in Figure 1.

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Authors and Affiliations

  1. Department of Molecular & Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA

    Kayla J Bayless, Hyeong-Il Kwak & Shih-Chi Su

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Contributions

K.J.B., S.-C.S. and H.-I.K. designed the study; K.J.B. and H.-I.K. carried out the experiments; K.J.B., S.-C.S and H.-I.K collected and analyzed data; K.J.B., S.-C.S. and H.-I.K wrote the manuscript.

Corresponding author

Correspondence to Kayla J Bayless.

Supplementary information

Supplementary Movie 1

Differential interference contrast (DIC) analyses of invading endothelial cells stably expressing green fluorescent protein with time. Experiments were established as described in Box 2. The movie is compiled from 87 frames captured at 5 minute intervals. Movies were initiated at 12 hours of invasion and were allowed to proceed until 19.25 hours. Retracting processes, extending processes, individual endothelial cells, and large vacuoles are indicated in Figure 7. (WMV 2162 kb)

Supplementary Movie 2

Time-lapse analyses of invading endothelial cells stably expressing green fluorescent protein (GFP) using epifluorescence with time. Movie is compiled from 87 frames captured at 5 minute intervals. Movies were initiated at 12 hours of invasion and were allowed to proceed until 19.25 hours. Retracting processes, extending processes, individual endothelial cells, and vacuoles are indicated in Figure 7. (WMV 701 kb)

Supplementary Movie 3

Combination of Differential interference contrast (DIC; movie 1) and epifluorescence (movie 2) analyses of invading endothelial cells stably expressing green fluorescent protein with time. (WMV 2154 kb)

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Bayless, K., Kwak, HI. & Su, SC. Investigating endothelial invasion and sprouting behavior in three-dimensional collagen matrices. Nat Protoc 4, 1888–1898 (2009). https://doi.org/10.1038/nprot.2009.221

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