The isolation of endothelial cells (EC) from different organs is a continuing barrier to understanding tumor-EC interactions. We have developed a CD31 based fluorescence-activated cell sorting method which allows isolation of EC from different tissues with a high degree of purity. In an attempt to develop an immortalized cell line, SV40 early genes were introduced into monkey aortic EC. The SV40 transfection resulted in diminished expression of EC adhesion molecules and reduced ability to differentiate and undergo in vitro angiogenesis. Although these were interesting findings, the SV40-induced phenotypic changes rendered the EC ineffective for studies on tumor-EC cell interactions. Tumor cell invasion involves synergistic interactions between a variety of proteinases. The stimulation of ECs by tumor cells within the tumor microenvironment may be an important event during angiogenesis and tumor cell extravasation. ECs are a polarized cell type with distinct apical and basal secretion patterns. Monkey aortic EC (MAEC), grown in a 2- chamber culture system, were used to investigate the polarized secretion of MMP's, PA's and their inhibitors. Proteinase secretion was found to occur with a basal preference. Phorbol ester, interleukin-1 (IL-1), and melanoma conditioned medium (MCM), which has previously been shown to contain IL-1, were used to modulate MAEC proteinase and inhibitor levels. Phorbol ester induced apical MMP-9 expression and increased basal t-PA secretion. IL-1 induced basal MMP-9 secretion and increased basal t-PA activity, MMP-2 and TIMP-2 secretion. These observations correlated with an increased ability of the conditioned medium to degrade basement membrane type IV collagen in frozen tissue sections and within the membrane upon which the EC had been grown, demonstrating that tumor cells have the ability to stimulate EC to degrade basement membrane.
