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A quantitative neovascularization assay for screening pro- and anti-angiogenic factors
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 Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Radiology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Therapeutic regulation of tissue vascularization has appeared as an attractive approach to treat a number of human diseases. However, promising results obtained in animal models have largely failed when translated into clinical trials. This suggests that current experimental models do not properly reflect the physiological situation and that there is a need for new in vivo assay that allows quantitative and qualitative analysis of non-developmental neovascularization. In this report we present a new assay where the effects of activators and inhibitors of neovascularization can be quantitatively measured. A provisional matrix composed of collagen and fibrin was formed in a plastic cylinder and implanted onto the chick chorioallantoic membrane. A nylon mesh separated the implanted matrix from the underlying tissue to distinguish new from pre-existing vessels. Vascularization of the matrix in response to FGF-2 and PDGF-BB was scored in a double-blinded manner, or vessel density measured using a semi-automated image analysis procedure. Evaluation of thalidomide, fumagillin, wortmannin, cortisol and U0126, which are compounds that has been reported to inhibit angiogenesis, revealed that while some only inhibited ingrowth of neo-vessels into the matrix, others also caused pre-existing vessels to regress. This quantitative, inexpensive and rapid in vivo angiogenesis assay might be a valuable tool in screening and characterizing factors that influence tissue vascularization.

National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
URN: urn:nbn:se:uu:diva-113061OAI: oai:DiVA.org:uu-113061DiVA: diva2:289573
Available from: 2010-01-25 Created: 2010-01-25 Last updated: 2011-06-28
In thesis
1. The Roles of Growth Factor Interactions and Mechanical Tension in Angiogenesis
Open this publication in new window or tab >>The Roles of Growth Factor Interactions and Mechanical Tension in Angiogenesis
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Angiogenesis, the formation of new blood vessels from preexisting ones through creation of new vessel branch points by sprouting or vessel splitting, is an important part of tissue growth in both physiological processes like wound healing and pathological conditions such as cancer. Growth factors like VEGF-A, FGF-2 and PDGF-BB are involved in both types of angiogenesis.

Screening for genes regulated by VEGF-A stimulation in endothelial cells revealed up regulation of the endothelial cell specific glycoprotein endocan. Endocan itself did not stimulate angiogenesis. VEGF was a specific inducer since FGF-2, PDGF-BB, HGF and EGF did not alter expression. The signaling molecule PI3K was a negative regulator of endocan expression. Endocan was expressed in tumor cells and vessels, suggesting that although endocan did not directly regulate angiogenesis it can serve as a marker for angiogenic tumors.

In two models of wound healing angiogenesis, the chick extra-embryonal CAM assay and the mouse cornea assay, we observed that blood vessels grew into avascular areas as functional mural cell covered loops by elongation of preexisting vessels. Loop formation was simultaneous with contraction of the avascular matrix mediated by proto/myofibroblasts. Reducing the contractibility of the stroma reduced vessel ingrowth, showing that contraction was necessary for mediating and directing growth of the vascular loops. These findings suggest a model for biomechanical regulation of vascularization that is complementary to sprouting angiogenesis which is guided by gradients of growth factors.

In defining the role of growth factors, in the CAM assay, we found that FGF-2 and PDGF-BB induced vessel ingrowth, while VEGF-A, EGF and HGF did not. TGF-beta reduced the effect of FGF-2. By use of specific receptor kinase inhibitors we found an absolute requirement VEGF- and PDGF-receptor activity for vascularization while FGF- and TGF-beta-receptor function was dispensable. This suggests that functional VEGF- and PDGF-receptors are needed for vessel elongation.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 67 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 519
Keyword
angiogenesis, myofibroblast, wound healing, VEGF, FGF, PDGF, endocan, endothelial cell, vascularization
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:uu:diva-113238 (URN)978-91-554-7717-2 (ISBN)
Public defence
2010-03-12, C8:301, BMC, Biomedical Center Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2010-02-18 Created: 2010-01-26 Last updated: 2010-02-18Bibliographically approved

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Gerwins, Pär

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