The Korean Journal of Physiology and Pharmacology

The Korean Society of Pharmacology (대한약리학회)
권호 표지
DOI QR코드

DOI QR Code

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

  • Lee, Hyunsook (College of Pharmacy, Duksung Innovative Drug Center, Duksung Women's University) ;
  • Kang, Kyu-Tae (College of Pharmacy, Duksung Innovative Drug Center, Duksung Women's University)
  • Received : 2018.06.16
  • Accepted : 2018.08.06
  • Published : 2018.11.01
Download PDF
⟨ Previous Next ⟩

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 $2{\times}2$ stitched area from the $4{\times}$ 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, $IC_{50}$ 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.

Keywords

References

  1. Gumkowski F, Kaminska G, Kaminski M, Morrissey LW, Auerbach R. Heterogeneity of mouse vascular endothelium. In vitro studies of lymphatic, large blood vessel and microvascular endothelial cells. Blood Vessels. 1987;24:11-23.
  2. Arnaoutova I, Kleinman HK. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc. 2010;5:628-635. https://doi.org/10.1038/nprot.2010.6
  3. Melero-Martin JM, Khan ZA, Picard A, Wu X, Paruchuri S, Bischoff J. In vivo vasculogenic potential of human blood-derived endothelial progenitor cells. Blood. 2007;109:4761-4768. https://doi.org/10.1182/blood-2006-12-062471
  4. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964-967. https://doi.org/10.1126/science.275.5302.964
  5. Fadini GP, Losordo D, Dimmeler S. Critical reevaluation of endothelial progenitor cell phenotypes for therapeutic and diagnostic use. Circ Res. 2012;110:624-637. https://doi.org/10.1161/CIRCRESAHA.111.243386
  6. Ingram DA, Mead LE, Tanaka H, Meade V, Fenoglio A, Mortell K, Pollok K, Ferkowicz MJ, Gilley D, Yoder MC. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood. 2004;104:2752-2760. https://doi.org/10.1182/blood-2004-04-1396
  7. Murasawa S, Asahara T. Endothelial progenitor cells for vasculogenesis. Physiology (Bethesda). 2005;20:36-42.
  8. Rafii S, Lyden D, Benezra R, Hattori K, Heissig B. Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy? Nat Rev Cancer. 2002;2:826-835. https://doi.org/10.1038/nrc925
  9. Iruela-Arispe ML, Dvorak HF. Angiogenesis: a dynamic balance of stimulators and inhibitors. Thromb Haemost. 1997;78:672-677. https://doi.org/10.1055/s-0038-1657610
  10. Melero-Martin JM, Bischoff J. Chapter 13. An in vivo experimental model for postnatal vasculogenesis. Methods Enzymol. 2008;445:303-329.
  11. Kim MK, Park HJ, Kim SR, Choi YK, Bae SK, Bae MK. Involvement of heme oxygenase-1 in orexin-A-induced angiogenesis in vascular endothelial cells. Korean J Physiol Pharmacol. 2015;19:327-334. https://doi.org/10.4196/kjpp.2015.19.4.327
  12. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669-676. https://doi.org/10.1038/nm0603-669
  13. Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci. 2001;22:201-207. https://doi.org/10.1016/S0165-6147(00)01676-X
  14. Wood JM, Bold G, Buchdunger E, Cozens R, Ferrari S, Frei J, Hofmann F, Mestan J, Mett H, O'Reilly T, Persohn E, Rosel J, Schnell C, Stover D, Theuer A, Towbin H, Wenger F, Woods-Cook K, Menrad A, Siemeister G, et al. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res. 2000;60:2178-2189.
  15. Bikfalvi A, Bicknell R. Recent advances in angiogenesis, anti-angiogenesis and vascular targeting. Trends Pharmacol Sci. 2002;23:576-582. https://doi.org/10.1016/S0165-6147(02)02109-0
  16. Kim DY, Jung SY, Kim YJ, Kang S, Park JH, Ji ST, Jang WB, Lamichane S, Lamichane BD, Chae YC, Lee D, Chung JS, Kwon SM. Hypoxia-dependent mitochondrial fission regulates endothelial progenitor cell migration, invasion, and tube formation. Korean J Physiol Pharmacol. 2018;22:203-213. https://doi.org/10.4196/kjpp.2018.22.2.203

Cited by

  1. Tumour-associated macrophages mediate the invasion and metastasis of bladder cancer cells through CXCL8 vol.8, pp.None, 2018, https://doi.org/10.7717/peerj.8721
  2. Oroxylin a Attenuates Limb Ischemia by Promoting Angiogenesis via Modulation of Endothelial Cell Migration vol.12, pp.None, 2018, https://doi.org/10.3389/fphar.2021.705617
  3. CCL8 mediates crosstalk between endothelial colony forming cells and triple-negative breast cancer cells through IL-8, aggravating invasion and tumorigenicity vol.40, pp.18, 2018, https://doi.org/10.1038/s41388-021-01758-w
  4. Tropoelastin Promotes the Formation of Dense, Interconnected Endothelial Networks vol.11, pp.9, 2018, https://doi.org/10.3390/biom11091318