Direct-contact co-culture between smooth muscle and endothelial cells inhibits TNF-α-mediated endothelial cell activation

We used a direct-contact endothelial cell-smooth muscle cell (EC-SMC) co-culture to examine whether quiescent SMCs regulate the EC inflammatory response to tumor necrosis factor (TNF)-α. ECs were cultured under static and physiological flow conditions. Compared with TNF-α-treated ECs in monoculture, TNF-α-treated ECs in co-culture had less NF-κB nuclear translocation; less intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin surface protein expression; no change in TNF receptor expression, but greater Kruppel-like factor 2 (KLF2) gene expression. After flow preconditioning for 24 h at 15 dyne/cm2, and exposure of ECs to flow and TNF-α for 4.5 h, ECs in co-culture had less ICAM-1, VCAM-1, and E-selectin surface protein expression. Exposure to flow greatly increased KLF2 gene expression levels in both EC cultures; as a result, ECs in co-culture and monoculture had similar levels of post-flow KLF2 gene expression. The reduced levels of TNF-α-induced adhesion molecule expression in co-culture required the presence of quiescent SMCs; adhesion to decellularized extracellular matrix (ECM) or co-culture with fibroblasts produced only a modest reduction in EC adhesion molecule expression. Furthermore, co-culture of quiescent SMCs and ECs on the opposite side of a 10-μm-thick porous membrane did not alter the TNF-α-mediated ICAM-1 surface protein expression. Although the ECM produced by SMCs plays some role in reducing TNF-α-mediated inflammation, these results suggest that the direct contact between ECs and quiescent SMCs is required to inhibit TNF-α-mediated activation.