Choroid sprouting assay: an ex vivo model of microvascular angiogenesis.
Angiogenesis of the microvasculature is central to the etiology of many diseases including proliferative retinopathy, age-related macular degeneration and cancer. A mouse model of microvascular angiogenesis would be very valuable and enable access to a wide range of genetically manipulated tissues that closely approximate small blood vessel growth in vivo. Vascular endothelial cells cultured in vitro are widely used, however, isolating pure vascular murine endothelial cells is technically challenging. A microvascular mouse explant model that is robust, quantitative and can be reproduced without difficulty would overcome these limitations. Here we characterized and optimized for reproducibility an organotypic microvascular angiogenesis mouse and rat model from the choroid, a microvascular bed in the posterior of eye. The choroidal tissues from C57BL/6J and 129S6/SvEvTac mice and Sprague Dawley rats were isolated and incubated in Matrigel. Vascular sprouting was comparable between choroid samples obtained from different animals of the same genetic background. The sprouting area, normalized to controls, was highly reproducible between independent experiments. We developed a semi-automated macro in ImageJ software to allow for more efficient quantification of sprouting area. Isolated choroid explants responded to manipulation of the external environment while maintaining the local interactions of endothelial cells with neighboring cells, including pericytes and macrophages as evidenced by immunohistochemistry and fluorescence-activated cell sorting (FACS) analysis. This reproducible ex vivo angiogenesis assay can be used to evaluate angiogenic potential of pharmacologic compounds on microvessels and can take advantage of genetically manipulated mouse tissue for microvascular disease research.
Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
Zhuo
Z
Shao
Mollie
M
Friedlander
Christian G
CG
Hurst
Zhenghao
Z
Cui
Dorothy T
DT
Pei
Lucy P
LP
Evans
Aimee M
AM
Juan
Houda
H
Tahiri
Houda
H
Tahir
François
F
Duhamel
Jing
J
Chen
Przemyslaw
P
Sapieha
Sylvain
S
Chemtob
Jean-Sébastien
JS
Joyal
Lois E H
LE
Smith
D000375:Aging / Q000502:physiology; D043925:Angiogenesis Inducing Agents / Q000494:pharmacology; D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D002829:Choroid / Q000098:blood supply* / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D055806:Microvessels / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D020286:Pericytes / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D015203:Reproducibility of Results; D055213:Retinal Pigment Epithelium / Q000502:physiology
D043925:Angiogenesis Inducing Agents; D003470:Culture Media