We have identified calcium influx as a molecular target in angiogenesis, in part using CAI, an inhibitor of nonvoltage-gated calcium influx. We have advanced that in two directions. Further study of CAI and other molecular targeted agents addressed the differences in signaling events ongoing during tubulogenesis using the in vitro endothelial cell Matrigel assay. Endothelial cells prior to differentiation into tubes and those oriented into tubular structures were subjected to laser capture microdissection and microimmunoblot to measure biochemical activity and quantity of key nodal signaling proteins. Parallel pathways important in the survival, proliferation, and migration of endothelial cells during angiogenesis were examined including those in the MAP kinase, protein kinase C, phospholipase C, nitric oxide synthase, and Akt pathways. Tubulogenesis in the presence of VEGF and straight culture media was examined. Small differences in protein expression and protein activation by phosphorylation were seen between control and VEGF-treated samples. This was consonant with our previous observation that VEGF was inadequate by itself to stimulate tubulogenesis on Matrigel in this model system. While ERK was present and phosphorylation was increased for up to 4 hr into the remodeling process, pharmacologic inhibition of ERK did not abrogate tubulogenesis, consistent with our previous findings. It appears that greater doses of agent are required to alter the phenotype than the signaling events, suggesting a heretofore unstudied signaling complexity. This finding, if confirmed, may provide an explanation for the lack of activity of some anti-angiogenesis directions in patients and underscores the potential complexity of the microenvironmental interactions and signaling events. In order to evaluate this clinically, studies are ongoing to develop tissue lysate array conditions to assess angiogenic activity in microdissected tumor stroma. Surrogate measurements of vascularity, such as stromal CD31 signals, and assessment of activation of receptor tyrosine kinase pathways and intracellular signaling nodal activity will be assessed in both tumor epithelium and stroma from the same biopsy specimens, before and during patient treatment with STI-571, a PDGFR inhibitor, and ZD1839, an EGFR inhibitor. Preclinical in vitro data have shown anti-angiogenic activity for both agents and STI571 was shown to alter intratumoral vascularity by in vivo vascular imaging in GIST patients. This comprehensive approach to angiogenesis profiling in the laboratory and patient samples will provide opportunity for proof of principle of molecularly targeted agents in stromal therapy.
Showing the most recent 10 out of 22 publications
