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 CAI regulates VEGF production. CAI regulates VEGF expression and protein production under normoxic and relative hypoxic conditions with a greater effect when stressed by acidosis in melanoma cells. One mechanisms for this may be through recently observed CAI regulation of HIF-1a expression. Reduction in VEGF expression was also observed in small cell lung cancer; CAI was both anti-tumor and anti-proliferative in a small cell lung cancer xenograft model. CAI also regulates response to VEGF by reducing endothelial migration to VEGF as an attractant. These results indicate that as a signal transduction regulator, CAI targets both tumor and stromal elements. In order to further dissect the pathways regulating angiogenesis, two new approaches have been initiated: cDNA microarray and microproteomic pathway profiling. In vitro models of vascular development have been used. cDNA microarray analysis showed little gene expression differences occurred in angiogenesis-related genes during vascular tube formation, including genes in the categories of invasion, proliferation, and signaling. Caldesmon was found to be reduced in gene expression early in tube formation, and reduced at the protein level later in tube formation. It interacts with the RhoA pathway and may be a downstream target of calcium influx regulation. Microproteomics is being applied to profile the signaling pathways driving neovascular events. Wound healing, tubulogenesis, and in vivo vascularization assays are being optimized for application to microdissection and protein array analysis. The effect of signal modulatory agents, such as CAI and 2-methoxyestradiol will be applied. Translational application of these assays will be investigated in upcoming clinical trials of 2-methoxyestradiol, STI-571, and an EGF receptor inhibitor.
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