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J. Biol. Chem., Vol. 283, Issue 20, 13905-13912, May 16, 2008

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Endothelial Cell Migration in Stable Gradients of Vascular Endothelial Growth Factor A and Fibroblast Growth Factor 2

EFFECTS ON CHEMOTAXIS AND CHEMOKINESIS^*

Irmeli Barkefors, Sébastien Le Jan, Lars Jakobsson, Eduar Hejll, Gustav Carlson, Henrik Johansson, Jonas Jarvius, Jeong Won Park^¶, Noo Li Jeon^¶, and Johan Kreuger¹

From the Department of Medical Biochemistry and Microbiology and the Department of Genetics and Pathology, Uppsala University, SE-751 23 Uppsala, Sweden and the ^¶Department of Biomedical Engineering, University of California, Irvine, California 92697

Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 µm was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.

Received for publication, June 14, 2007 , and in revised form, January 22, 2008.

^* This work was supported by Swedish Research Council Project Grants K2006-71X-20225-01-2 and K2006-71P-20224-01-4 (to J. K.), the Swedish Cancer Foundation, the Swedish Foundation for Strategic Research Project A3 05:207g, the Wenner-Gren Foundations, the Magnus Bergvall Foundation, the Jeansson Foundations, the Selander Foundation, and Uppsala University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental S1–S5 and movies 1 and 2.

¹ To whom correspondence should be addressed: Husargatan 3, Box 582, SE-751 23, Uppsala, Sweden. Tel.: 46-18-4714366; Fax: 46-18-4714673; E-mail: Johan.Kreuger{at}imbim.uu.se.