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Directing Angiogenesis: Cellular Responses to Gradients in vitro
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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 [en]
Angiogenesis, Endothelial cell, Cell migration, Chemotaxis, Gradients, Microfluidics, VEGFA, VEGFR2, Exocyst, Exocytosis
National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-145525ISBN: 978-91-554-8006-6OAI: oai:DiVA.org:uu-145525DiVA: diva2:396365
Public defence
2011-04-15, B22, BMC, Husargatan 3, 74123, Uppsala, 09:15 (English)
Opponent
Supervisors
Note
(Faculty of Medicine)Available from: 2011-03-08 Created: 2011-02-09 Last updated: 2011-05-04
List of papers
1. Endothelial cell migration in stable gradients of vascular endothelial growth factor A and fibroblast growth factor 2: effects on chemotaxis and chemokinesis
Open this publication in new window or tab >>Endothelial cell migration in stable gradients of vascular endothelial growth factor A and fibroblast growth factor 2: effects on chemotaxis and chemokinesis
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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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89323 (URN)10.1074/jbc.M704917200 (DOI)000255728200044 ()18347025 (PubMedID)
Available from: 2009-02-11 Created: 2009-02-11 Last updated: 2012-09-05Bibliographically approved
2. A fluidic device to study directional angiogenesis in complex tissue and organ culture models
Open this publication in new window or tab >>A fluidic device to study directional angiogenesis in complex tissue and organ culture models
2009 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 9, no 4, 529-535 p.Article in journal (Refereed) Published
Abstract [en]

Many signals that induce angiogenesis have been identified; however, it is still not clear how these signals interact to shape the vascular system. We have developed a fluidic device for generation of molecular gradients in 3-dimensional cultures of complex tissues and organs in order to create an assay for precise induction and guidance of growing blood vessels. The device features a centrally placed culture chamber, flanked by channels attached to a perfusion system used to generate gradients. A separate network of vacuum channels permits reversible attachment of the device to a flat surface. We show that the fluidic device can be used to create growth factor gradients that induce directional angiogenesis in embryonic mouse kidneys and in clusters of differentiating stem cells. These results demonstrate that the device can be used to accurately manipulate complex morphogenetic processes with a high degree of experimental control.

National Category
Medical and Health Sciences Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-89325 (URN)10.1039/b814691h (DOI)000263465900005 ()19190788 (PubMedID)
Available from: 2009-02-11 Created: 2009-02-11 Last updated: 2016-04-14Bibliographically approved
3. Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells
Open this publication in new window or tab >>Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 27, 24189-24199 p.Article in journal (Refereed) Published
Abstract [en]

The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections. We detect an up-regulation of exoc3l2 mRNA synthesis in primary human endothelial cells in response to VEGFA, and this response is enhanced when the cells are grown on a three-dimensional collagen I matrix. Myc-tagged EXOC3L2 co-precipitates with the exocyst protein EXOC4, and immunofluorescence detection of EXOC3L2 shows partial subcellular colocalization with EXOC4 and EXOC7. Finally, we show that exoc3l2 silencing inhibits VEGF receptor 2 phosphorylation and VEGFA-directed migration of cultured endothelial cells.

Keyword
Angiogenes, VEGF, Cell migration, Chemotaxis, Exocyst, Microarray
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
urn:nbn:se:uu:diva-145508 (URN)10.1074/jbc.M110.212209 (DOI)000292294900061 ()
Available from: 2011-02-09 Created: 2011-02-09 Last updated: 2015-07-24Bibliographically approved

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