029 Novel Parameters for Computer-Assisted Image Analysis to Study Angiogenesis

Abstract

Angiogenesis is a dynamic process that requires interaction of proangiogenic and antiangiogenic factors. Several methodologies have been used to evaluate this process. The quail chorioallantoic membrane (CAM) is a suitable model to explore angiogenesis in vivo. Recently, the morphometric parameters of fractal dimension (Df) and grid intersection (ρ) are used to quantify angiogenesis in CAM. However, more information is necessary to understand angiogenic mechanisms. New methods to measure additional parameters such as the average blood vessel thickness, tortuousity, total branch length and complexity (junction node number), are described herein. For the analysis, CAM images were obtained from ex ovo culture of E8-E9 quail embryos. MatLab®(The MathWorks, Natick, MA) software was used for the analyses of branches nodes, thickness and length of the forming branches. Conventional morphometry was done using IPLab (Scanalytics, Fairfax, VA). To validate the new parameters, leptin, VEGF and neutralizing antibodies were evaluated. Leptin and VEGF treated CAM had increased complexity and vascular densities (Df = 1.5 ± 0.005; ρ = 12.3 ± 0.689) and (Df = 1.6 ± 0.006; ρ = 14.3 ± 0.465), respectively, compared to PBS control (Df = 1.3 ± 0.035; ρ = 8.6 ± 0.439). There was a corresponding decrease antileptin and anti-VEGF antibodies. The node-structural map applied to CAM images reveled 53% increase in the number of one-branch nodes and 50% increase in three-branch nodes with leptin compared to controls. Additionally, the total branch length was 30% greater with leptin along with a decrease in vessel thickness, The analyses allowed quantification of branching, tortuousity, thickness, and length of the new blood vessels. Together with the conventional morphometric parameters, these new analyses will help understand the mechanisms exerted by angiogenesis-modulating molecule.