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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
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 20, 13905-13912 p.Article in journal (Refereed) Published
Abstract [en]

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 microm 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.

Place, publisher, year, edition, pages
2008. Vol. 283, no 20, 13905-13912 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-89323DOI: 10.1074/jbc.M704917200ISI: 000255728200044PubMedID: 18347025OAI: oai:DiVA.org:uu-89323DiVA: diva2:160091
Available from: 2009-02-11 Created: 2009-02-11 Last updated: 2012-09-05Bibliographically approved
In thesis
1. Directing Angiogenesis: Cellular Responses to Gradients in vitro
Open this publication in new window or tab >>Directing Angiogenesis: Cellular Responses to Gradients in vitro
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood vessels are essential for the delivery of nutrients and oxygen to tissues, as well as for the removal of waste products. Patients with tumors, wounds or diabetes all have active angiogenesis, formation and remodeling of blood vessels, a process that is initiated and manipulated by gradients of secreted signaling proteins.

This thesis describes the development of new microfluidic in vitro assays where directed migration of single endothelial cells and three dimensional vascular structures can be monitored in real time. Combining these assays with live imaging microscopy we have studied the behavior of endothelial cells in gradients of proangiogenic factors as well as directed sprouting in embryonic kidneys and stem cell cultures.

With the 2D assay we have quantified endothelial cell chemotaxis towards FGF2, VEGFA165 and VEGFA121 and we also demonstrate that constant levels of VEGFA165, but not of FGF2, are able to reduce chemokinesis of endothelial cells.

In the 3D migration chamber we have studied directed endothelial cell sprouting in mouse embryonic kidneys and embryoid bodies in response to VEGFA gradients. In both models directed angiogenesis is detected towards increasing levels of growth factor.

Using the microarray technique on differentiating embryonic stem cells we have been able to identify the gene exoc3l2 as potentially involved in angiogenesis and endothelial cell migration and we present evidence that ExoC3l2 is associated with the exocyst complex; an important regulator of cell polarity. We have also shown that siRNA mediated gene silencing of exoc3l2 results in impaired VEGFR2 phosphorylation as well as loss of directionality in response to a VEGFA gradient.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 643
Keyword
Angiogenesis, Endothelial cell, Cell migration, Chemotaxis, Gradients, Microfluidics, VEGFA, VEGFR2, Exocyst, Exocytosis
National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:uu:diva-145525 (URN)978-91-554-8006-6 (ISBN)
Public defence
2011-04-15, B22, BMC, Husargatan 3, 74123, Uppsala, 09:15 (English)
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Note
(Faculty of Medicine)Available from: 2011-03-08 Created: 2011-02-09 Last updated: 2011-05-04

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Barkefors, IrmeliCarlson, GustavKreuger, Johan

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