Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC-iCaspase-9), but not control cells (HDMEC-LXSN). Here, we generated blood vessels containing several HDMEC-iCaspase-9:HDMEC-LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF165) did not prevent apoptosis of HDMEC-iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.
Bibliographical noteFunding Information:
We thank ARIAD Pharmaceuticals ( www.ariad.com/regulationkits ) for the dimerizer agent AP20187; the Biological Resources Branch of the NIH/NCI for the rhVEGF; and Dr. H. Weber (Spherogenex, Freiburg, Germany) for help with the spheroid-based endothelial sprouting assay. This work was supported by grants R01-DE14601, R01-DE15948, R01-DE16586 from the NIH/NIDCR (JEN), grant from the American Dental Association Health Foundation (JEN), and grants R01-CA70057 (GN) and R01-CA77266 (DMS) from the NIH/NCI.
- Mathematical modeling