Abstract 3430: Breast tumor cells inhibit vascular gap junction communication by downregulation of mural cell Connexin 43

A functional blood vessel is composed of the endothelium, the cell layer that forms the channel through which blood flows, and mural cells, the vascular smooth muscle cells (vSMC) and pericytes that serve to support and stabilize the endothelium. Proper physical and functional association between mural cells and endothelial cells (EC) causes EC to become quiescent. In contrast, the vasculature formed by a tumor is highly disorganized and exhibits decreased and abnormal association with mural cells, which allows the vessel to be unstable and proliferative. However, the mechanism(s) by which tumors prevent proper association between mural cells and the endothelium have not been well elucidated. Gap junctions (GJ) between EC and mural cells are required for EC-induced mural cell differentiation during vessel development and are involved in maintaining vasomotor tone and electrical conductance. Since GJ play an important role in cell-cell contacts and contact between mural cells and EC is disrupted in tumors, we investigated whether inhibitory mural cell interactions with EC require functional gap junction communication. Using a parachute dye transfer assay, we have demonstrated that exposure to media conditioned by MDA-MB-231 breast cancer cells substantially decreases gap junction communication between vSMC and EC. Although levels of the GJ proteins connexin 40, 43, and 45 are not altered in tumor-exposed EC, we observed that connexin 43 (Cx43) protein is downregulated in vSMC by tumor conditioned media in a time- and dose-dependent manner. A similar decrease in Cx43 is observed in response to 4T1 cells, but not MCF7 or SKBR3 cells. Quantitative real-time PCR suggests that Cx43 loss results at least partially from a decrease in Cx43 mRNA levels. Pharmacological inhibitors of MAPK, Src, and PKC indicate that these signaling pathways are not involved in tumor-induced Cx43 downregulation. We further demonstrate that expression of Cx43 by mural cells is required for the mural cell-induced inhibition of EC proliferation, as mural cells in which Cx43 levels have been reduced by Cx43 siRNA transfection have decreased ability to attenuate EC proliferation compared to control siRNA-transfected mural cells. This effect is observed with both undifferentiated mural precursor C3H10T1/2 cells as well as fully differentiated vSMC mural cells. These data suggest that tumor-induced loss of mural cell Cx43 may both disrupt mural cell-EC contacts in a differentiated, mature ve