Vascular endothelial cells (ECs), which exist in close proximity to vascular smooth muscle cells (SMCs), are constantly subjected to blood flow induced shear stress. We examined the role of SMCs in regulating the shear stress-induced gene expression in ECs, using a parallel-plate co-culture flow system in which these 2 types of cells were separated by a porous membrane with only the EC side subjected to the flow condition. In this co-culture system, SMCs tended to orient perpendicularly to the flow direction, whereas the ECs were elongated and aligned with the flow direction. Under static condition, DNA microarrays identified 23 inflammation-relevant genes in ECs whose expression was significantly affected by co-culture with SMCs, with 18 upregulated and 5 down-regulated. These results were confirmed by reverse-transcription polymerase chain reaction. SMCs were less responsive than ECs in inflammation-relevant gene expression in co-cultures. Application of shear stress (12 dynes/cm2) to the EC side of the co-culture for 6 h inhibited most of the pro-inflammatory gene expressions in ECs induced by co-culture with SMCs. The EC responses under static and shear conditions were not observed in the absence of close communication between ECs and SMCs, and they were also not observed when ECs were co-cultured with fibroblasts instead of SMCs. Inhibition of nuclear factor-kappaB (NF-kappaB) activation by the p65-antisense, lactacystin, and N-acetyl-cysteine blocked the co-culture-induced EC expression of pro-inflammatory genes, indicating that the NF-kappaB binding sites in the promoters of these genes play a significant role in their expression as a result of co-culture with SMCs. Chromatin immunoprecipitation assays demonstrated the in vivo regulation of NF-kappaB recruitment to selected target promoters. Shear stress inhibited the SMC-co-culture-induced NF-kappaB activation in ECs and monocytic THP-1 cell adhesion to ECs. Our findings suggest that shear stress may serve as a down-regulator for the pro-inflammatory gene expression in ECs located in close proximity to SMCs.
