Vascular-specific quantification in an in vivo Matrigel chamber angiogenesis assay.
The study of angiogenesis as a therapeutic target requires reliable in vivo assays that can provide physiologically relevant data. A murine in vivo Matrigel-based angiogenesis assay is presented here which includes the quantitative assessment of vascular-specific indicators of neovascularization. Matrigel containing 175 ng/ml bFGF is encapsulated in synthetic chambers which are implanted subcutaneously in C57/B16J mice. Ex vivo implants can be imaged to qualitatively view perfused vasculature within the chambers, or histologically processed to confirm the presence of vascular-specific tissue within the Matrigel. Viable cells are recovered from the excised chambers and quantified cytometrically using endothelial cell-specific markers CD34 and CD144, and for a marker of nucleated cells, Hoechst 33342. Thalidomide, 200 mg/kg/day, was tested using the assay and was found to inhibit angiogenesis by 46%. Angiogenesis inhibitors secreted by LL/M27 tumors were also characterized, where tumor-bearing mice showed a 73% inhibition of angiogenesis compared to tumor-free controls. Analysis of the number of nucleated cells in these samples failed to show a strong correlation with the number of endothelial cells, indicating that quantification of nonvascular-specific tissue in in vivo angiogenesis assays may not be sufficient. This new assay provides an objective, comprehensive determination of the vasculature-specific response of both endogenous and exogenous angiogenesis inhibitors in vivo, and also creates new opportunities for obtaining primary murine endothelial cells.
Baker
Jennifer H E
JH
Department of Medical Biophysics, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3.
Huxham
Lynsey A
LA
Kyle
Alastair H
AH
Lam
Kevin K
KK
Minchinton
Andrew I
AI
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D004338:Drug Combinations; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism / Q000494:pharmacology; D007797:Laminin / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D012884:Skin Window Technique; D013792:Thalidomide / Q000494:pharmacology; D014184:Transplantation, Homologous
D020533:Angiogenesis Inhibitors; D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D016222:Fibroblast Growth Factor 2; C059485:matrigel; D013792:Thalidomide; D003094:Collagen