A Novel Assay of Angiogenesis in the Quail Chorioallantoic Membrane: Stimulation by bFGF and Inhibition by Angiostatin According to Fractal Dimension and Grid Intersection

In a novel assay of angiogenesis in the quail chorioallantoic membrane (CAM), we measured vascular pattern and angiogenic rate after homogeneous exposure of the entire vascular tree to recognized modulators of vessel growth. In comparison to phosphate-buffered saline (PBS)-treated controls, the vasc...

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Published in:	Microvascular research Vol. 55; no. 3; pp. 201 - 214
Main Authors:	Parsons-Wingerter, Patricia, Lwai, Biakhre, Yang, Mong Che, Elliott, Katherine E., Milaninia, Ali, Redlitz, Alexander, Clark, John I., Sage, E.Helene
Format:	Journal Article
Language:	English
Published:	Amsterdam Elsevier Inc 01-05-1998 Elsevier
Subjects:	Allantois - blood supply Allantois - drug effects angiogenesis angiostatin Angiostatins Animals Antineoplastic Agents - pharmacology basic fibroblast growth factor (bFGF) Biological and medical sciences Cell differentiation, maturation, development, hematopoiesis Cell physiology Chickens chorioallantoic membrane Chorion - blood supply Chorion - drug effects Coturnix - embryology Endotoxins - pharmacology Fibroblast Growth Factor 2 - pharmacology fractal dimension Fractals Fundamental and applied biological sciences. Psychology grid intersection Image Processing, Computer-Assisted Molecular and cellular biology Neovascularization, Physiologic - drug effects Ovalbumin - pharmacology Peptide Fragments - pharmacology Plasminogen - pharmacology quail chorioallantoic membrane angiostatin angiogenesis fractal dimension basic fibroblast growth factor (bFGF) grid intersection quail Cell proliferation Cell culture Embryo Basic fibroblast growth factor Quail In vitro Cell density Angiogenesis Vertebrata Fractal dimension Chorioallantoic membrane Neovascularization Aves Morphometry
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Summary:	In a novel assay of angiogenesis in the quail chorioallantoic membrane (CAM), we measured vascular pattern and angiogenic rate after homogeneous exposure of the entire vascular tree to recognized modulators of vessel growth. In comparison to phosphate-buffered saline (PBS)-treated controls, the vascular stimulator, basic fibroblast growth factor (bFGF or FGF-2), increased the rate of angiogenesis by a maximum of 72%, whereas a recently discovered angiogenic inhibitor, angiostatin, decreased the rate of vascular growth by a maximum of 68%. The perturbants were applied in PBS to the CAM of 7-day-old embryos (E7) cultured in petri dishes, and the embryos were cultured further until fixation at E8 or E9. For morphometry of the quasi-two-dimensional CAM vasculature, digital images of arterial endpoints from the middle region of the CAM were acquired in grayscale at a magnification of 10×, binarized to black/white, and skeletonized. The pattern of vessel branching was assessed by measurement of the fractal dimension (Df), and vessel density (ρv), with the method of grid intersection. Correlations between these two statistical techniques were linear (r2ranged from 0.967 to 0.985). For skeletonized images at E9,Dfand ρvof bFGF-treated samples were 1.55 ± 0.01 and 782 ± 26/cm2, respectively (relative to 1.49 ± 0.02 and 583 ± 60/cm2for controls), and of angiostatin-treated samples, 1.43 ± 0.02 and 424 ± 74/cm2(relative to 1.50 ± 0.02 and 616 ± 59/cm2for controls). To establish normalization values for rates of angiogenesis, we analyzed untreated CAMs of E6 to E12. From E7 to E10 in skeletonized images,Dfincreased linearly from 1.37 ± 0.01 to 1.54 ± 0.01 and ρvfrom 311 ± 67 to 746 ± 124/cm2(in both cases,r2= 1.000). Thus, the rates of normal angiogenic growth as measured byDfand ρvwere 0.06/day and 138/cm2-day, respectively. From E10 to E12,Dfand ρvdeclined slightly. Differences between the vasculature of untreated and PBS-treated CAMs were statistically insignificant. In conclusion, vascular branching pattern and density in the quail CAM were stimulated by bFGF and inhibited by angiostatin. We quantified these changes with statistical significance byDfand ρv, which are expressed relative to the rates of normal developmental angiogenesis measured for the two parameters in untreated quail embryos.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0026-2862 1095-9319
DOI:	10.1006/mvre.1998.2073