Korean J Physiol Pharmacol.  2018 Nov;22(6):705-712. 10.4196/kjpp.2018.22.6.705.

Advanced tube formation assay using human endothelial colony forming cells for in vitro evaluation of angiogenesis

• Lee H
• Kang KT

• KMID: 2430093
• DOI: http://doi.org/10.4196/kjpp.2018.22.6.705

Abstract

The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). in vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a 2×2 stitched area from the 4× object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, IC₅₀ values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.

Keyword

Angiogenesis; Endothelial colony forming cells; Real-time cell recorder; Tube formation assay

MeSH Terms

Angiogenesis Inhibitors
Attention
Drug Discovery
Endothelial Cells
Fibroblast Growth Factor 2
Humans*
In Vitro Techniques*
Vascular Endothelial Growth Factor A
Angiogenesis Inhibitors
Fibroblast Growth Factor 2
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Comparison of image areas.(A) A single image taken with a 10× object lens. (B) A single image taken with a 4× object lens. (C) A 2×2 stitched image of 4 side-by-side images taken with a 4× object lens.

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  Fig. 2 Progression of tube formation from human endothelial colony forming cells (ECFCs).(A) Representative images of the tubular structures formed by ECFCs over 48 h (scale bar=500 µm). (B) Graph of the tube number over 48 h (n=3–4). (C) Graph of the length per tube over 48 h (n=3–4).

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  Fig. 3 Dynamic progression of tube formation from human endothelial colony forming cells (ECFCs) with treatment of vascular endothelial growth factor (VEGF).(A) Representative images of the tubular structures formed by ECFCs over 48 h in the presence and absence of VEGF (scale bar=500 µm). (B) Graph of the tube number over 48 h in the presence and absence of VEGF. (C) Graph of the length per tube over 48 h in the presence and absence of VEGF. (D) Statistical analysis of the tube number comparing 6, 24, and 48 h. (E) Statistical analysis of the length per tube comparing 6, 24, and 48 h. The asterisk (*) indicates a significant difference (p≤0.05) between groups (n=3–4).

• 

  Fig. 4 Dynamic progression of tube formation from human endothelial colony forming cells (ECFCs) with treatment of fibroblast growth factor-2 (FGF-2).(A) Representative images of the tubular structures formed by ECFCs over 48 hours in the presence and absence of FGF-2 (scale bar=500 µm). (B) Graph of the tube number over 48 hours in the presence and absence of FGF-2. (C) Graph of the length per tube over 48 hours in the presence and absence of FGF-2. (D) Statistical analysis of the tube number comparing 6, 24, and 48 hours. (E) Statistical analysis of the length per tube comparing 6, 24, and 48 hours. The asterisk (*) indicates a significant difference (p≤0.05) between groups (n=3–4).

• 

  Fig. 5 Synergic effects of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) on tube formation from human endothelial colony forming cells (ECFCs).(A) Representative images of the tubular structures formed by ECFCs over 48 h in the presence and absence of VEGF, FGF-2, and VEGF/FGF-2 (scale bar=500 µm). (B) Graph of the tube number over 48 h in the presence and absence of VEGF, FGF-2, and VEGF/FGF-2. (C) Graph of the length per tube over 48 h in the presence and absence of VEGF, FGF-2, and VEGF/FGF-2. (D) Statistical analysis of the tube number comparing 6, 24, and 48 h. (E) Statistical analysis of the length per tube comparing 6, 24, and 48 h. The asterisk (*) indicates a significant difference (p≤0.05) between groups (n=3–4).

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  Fig. 6 Inhibitory effect of vatalanib on tube formation of ECFCs.(A) Representative images of the tubular structures formed by ECFCs over 24 h in the presence and absence of vatalanib (scale bar=300 µm). (B) Graph of the tube number over 24 h in the presence and absence of vatalanib. (C) Graph of IC50 of vatalanib by examining the inhibition of tube formation. N.A. means “not available.” The asterisk (*) indicates a significant difference (p≤0.05) compared to control. Sharp (#) indicates a notable difference (p≤0.06) compared to control (n=3).

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