This manuscript describes the experimental observation of vasculogenesis in chick embryos by means of network analysis. The formation of the vascular network was observed in the area opaca of embryos from 40 to 55 hours of development. On area opaca endothelial cell clusters self-organize as a primitive and approximately regular network of capillaries. The process was observed by bright-field microscopy in control embryos and in embryos treated with Bevacizumab (Avastin), an antibody that inhibits the signalization of the vascular endothelial growth factor VEGF. The sequence of images of the vascular growth were thresholded, and used to quantify the forming network on control and Avastin-treated embryos. This characterization is made by measuring vessels density, number of cell clusters, and the largest cluster density. From the original images the topology of the vascular network was extracted and characterized by means of the usual network metrics such as: the degree distribution, average clustering coefficient, average short path length, and assortativity among others. This analysis allows to monitor how the largest connected cluster of the vascular network evolves in time and provides with quantitative evidences of the disruptive effects that Avastin has on the tree structure of vascular networks.
Table A-Fig2
Area distribution of endothelial cell clusters: the table displays the area distribution of endothelial cell clusters from the binary images, which were identified using the Analyze Particles. The distribution of the Cluster Numbers as a function of the log(A) was obtained using 12 bins. In other to illustrate the emergence of a largest cluster in the system the table displays the area in mm^2 [Area (mm^2)] and the log of the area in mm^2 [log(A)] for a control embryo at:
(a) initial stage 0h,
(b) middle stage 7h,
(c) final Stage 15h.
Table B-Fig4
Statistical analysis of binary patterns: characterize the spatio-temporal self-organization of cell clusters forming the vascular network for three embryos in each group.
(a) Mean vessel density of endothelial cell clusters, p, with the Std. Error as a function of time.
(b) Mean number of cell clusters, N, with the Std. Error as a function of time.
(c) The average largest cluster density, p^LC, with the Std. Error as a function of time.
(d) The largest cluster density, p^LC for a single embryo as a function of time.
Table C-Fig5
The node degree distributions: obtained from three embryos in each group. The symbol (**) indicates the statistical difference of the distributions from the initial stage at t=0h, which have p<0.05 calculated by Smirnov-Kolmogorov test.
(a) The mean node degree distributions for control group with Std. Error.
(b) The mean node degree distributions for Avastin-treated group with Std Error.
Table D-Fig6
Network descriptors: network metrics obtained for three embryos in each group, the tables displays the average in each group with the respective Std. error.
(a) The mean node degree as a function of time measured in a complete vascular network.
(b) The coefficient of assortativity measured on the largest cluster as a function of time.
(c) The average clustering coefficient measured on the largest cluster as a function of time.
(d) The shortest path length measured on the largest cluster as a function of time.
Table E-Fig7
The fraction of nodes in the largest cluster: it is obtained for a single embryo, and the average obtained for three embryos in each group. It is quantified as the number of the nodes in the largest cluster divided for the total number of nodes in the network. The tables displays the values for a single embryo and the average for the two groups with the respective Std. error.
(a) Fraction of nodes in the largest cluster as a function of time for a single embryo.
(b) Fraction of nodes in the largest cluster as a function of time for three embryos in each group with the Std. error.
Table F-Fig8
Structural metrics: it is structural parameters measured on network topologies for three samples in each group. The tables displays the average with the Std. Error.
(a) The mean cord length
(b) The mean fractal dimension measured
(c) Treeness
(d) The gamma index measured on a formed vascular network.