Differential gene expression in a coculture model of angiogenesis reveals modulation of select pathways and a role for Notch signaling.

Communication between endothelial and mural cells (smooth muscle cells, pericytes, and fibroblasts) can dictate blood vessel size and shape during angiogenesis, and control the functional aspects of mature blood vessels, by determining things such as contractile properties. The ability of these different cell types to regulate each other's activities led us to ask how their interactions directly modulate gene expression. To address this, we utilized a three-dimensional model of angiogenesis and screened for genes whose expression was altered under coculture conditions. Using a BeadChip array, we identified 323 genes that were uniquely regulated when endothelial cells and mural cells (fibroblasts) were cultured together. Data mining tools revealed that differential expression of genes from the integrin, blood coagulation, and angiogenesis pathways were overrepresented in coculture conditions. Scans of the promoters of these differentially modulated genes identified a multitude of conserved C promoter binding factor (CBF)1/CSL elements, implicating Notch signaling in their regulation. Accordingly, inhibition of the Notch pathway with gamma-secretase inhibitor DAPT or NOTCH3-specific small interfering RNA blocked the coculture-induced regulation of several of these genes in fibroblasts. These data show that coculturing of endothelial cells and fibroblasts causes profound changes in gene expression and suggest that Notch signaling is a critical mediator of the resultant transcription.