We have identified calcium influx as a molecular target in angiogenesis, in part using CAI, an inhibitor of nonvoltage-gated calcium influx. CAI inhibits endothelial cell proliferation, motility, collagenase expression, and regulates adhesion and spreading on matrix. We previously implicated the FAK, RhoA, and RhoGAP pathways in this function. We have now demonstrated that oral CAI treatment reduces circulating concentrations of human IL-8 and human VEGF in a murine melanoma xenograft model and that oral administration of CAI abrogated vascular in growth in a VEGF-driven Matrigel implant murine model. Our laboratory findings confirm that this is related to CAI treatment, wherein CAI treatment reduces VEGF secretion, protein production, message production and the message and nuclear localization of HIF-1alpha. Further, endothelial cells have reduced spontaneous and VEGF-stimulated migration when CAI is provide environmentally (in attractant solution) or when the cells are pretreated with CAI. These findings support the in vivo observations. We are now applying proteomic dissection, micro-immunoblot, and protein lysate array technology to further profile the activation state of the signaling pathways downstream of CAI and other angiogenesis modulators. Wound healing, tubulogenesis, and in vivo vascularization assays are being optimized for laser capture microdissection and array analysis. The effect of signal modulatory agents, such as CAI and 2-methoxyestradiol will be examined. Parallel pathways important in the survival, proliferation, and migration of endothelial cells during angiogenesis are being examined. Proteins under study include those in the MAP kinase, protein kinase C, phospholipase C, nitric oxide synthase, and Akt/p70S6kinase pathways. These were chosen because there are available inhibitors for these events to allow proof of concept and against which to compare activity of other putative molecular therapeutics for which collateral regulation may be found. These findings can then also be used for screening other putative molecular therapeutics. The comparison of the in vitro and in vivo Matrigel model will also provide a mechanism to vet the in vitro model which provides a more rapid turnaround for screening. Further, this approach will allow modeling for simultaneous regulation of multiple angiogenesis signaling pathways.
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