ABSTRACT In vitro co-culture of endothelial cells (ECs) with smooth muscle cells (SMCs) induced rapid and sustained increases in EC expression of E-selectin. By using inhibitors, dominant-negative mutants, and siRNA, we found that activations of JNK and p38 are critical for the co-culture-induced E-selectin expression. Gel shifting and chromatin immunoprecipitation assays showed that SMC-co-culture increased the NF-κB-promoter binding activity in ECs. Inhibition of NF-κB activation blocked the co-culture-induced E-selectin promoter activity. Protein arrays and neutralizing antibodies showed that IL-1β and IL-6 produced by EC/SMC co-cultures contribute to the co-culture-induction of EC signaling and E-selectin expression. Pre-shearing of ECs inhibited the co-culture-induced EC signaling and E-selectin expression. These findings serve to elucidate the molecular mechanisms underlying the SMC-induction of EC E-selectin expression and the shear stress-protection against this SMC-induction. INTRODUCTION The aim was to elucidate the mechanisms that regulate the SMC-induced E-selectin expression in ECs and its inhibition by shear stress. This article reviews several publications in our labs [1-5]. MATERIALS AND METHODS Cell culture. ECs were isolated from fresh human umbilical cords. SMCs were obtained from Clonetics (Palo Alto, CA). Preshearing of ECs. ECs were seeded onto the outer side of the membrane (10-μm-thick, 0.4-μm pores, pre-coated with fibronectin) of a transwell. After incubation for 24 h, the membrane with ECs was incorporated into a flow chamber on the underside of the transwell for shear stress applications at a high (HSS, 12 dyn/cm2) or low level (LSS, 0.5 dyn/cm2) for 4 or 24 h. Co-culture of ECs and SMCs. After EC preshearing, the inner side of the membrane was seeded with SMCs under static condition, thus forming an EC/SMC co-culture system. Controls had no cells or ECs instead of SMCs on the inner side. To study the effect of distance of EC/SMC separation, ECs seeded on the membrane were separated by 1 mm from the SMCs plated on an outer chamber (EC/M/SMC). RESULTS AND DISCUSSION Pre-exposure of ECs to HSS, but not LSS, for 24 h inhibits SMC-induced E-selectin expression in ECs. EC/SMC co-culture induced an increase in E-selectin mRNA expression in ECs within 1 h. Separation of ECs from SMCs by 1 mm retarded the E-selectin expression. Pre-shearing of ECs at HSS for 24 h inhibited the co-culture-induced E-selectin expression; this was not seen with LSS. Thus, SMCs induced EC expression of E-selectin via a paracrine effect that can be inhibited by HSS. SMC-induced EC expression of E-selectin and its inhibition by shear stress are mediated by the JNK and p38 pathways. The phosphorylation of ERK, JNK, p38, and Akt in ECs showed transient increases after co-culture with SMCs. The co-culture-induced E-selectin expression was inhibited by inhibitors for only JNK and p38. Pre-shearing at HSS, but not LSS, for 24 h inhibited the co-culture-induced JNK and p38 phosphorylation. JNK- or p38-specific siRNA caused significant inhibition of the co-culture-induced E-selectin expression. The increase in E-selectin-Luc promoter activity in ECs by SMC-co-culture was prevented by pre-shearing at HSS, but not LSS. Thus, the SMC-induction of EC expression of E-selectin is mediated by JNK and p38 and blocked by HSS. SMC-induced EC expression of E-selectin and its inhibition by shear stress are dependent on NF-κB. Inhibition of NF-κB abolished the co-culture-induced E-selectin promoter activity. Co-culture with SMCs increased the NF-κB-DNA binding activity in EC nucleus, which was inhibited by HSS (but not LSS). Thus, the co-culture induced E-selectin expression is mediated by NF-κB, and this effect is inhibited by HSS. IL-1β and IL-6 produced by EC/SMC are the major factors contributing to the SMC-induced signaling and E-selectin expression in ECs. Using a human cytokine array system, we identified IL-1β and IL-6 as the proteins released from EC/SMC at significantly higher levels than EC/EC (>4-fold). Neutralizing antibodies against IL-6 and/or IL-1β inhibited the co-culture-induced increases in E-selectin mRNA, JNK and p38 phosphorylation, and NF-κB-DNA binding activity. IRAK and gp130 are involved in regulatory effects of SMC-co-culture and shear stress on EC E-selectin expression. The SMC-induced E-selectin expression in ECs was suppressed by siRNAs against gp130 (IL-6 receptor) and IRAK (complex with the IL-1β receptor upon its stimulation). The co-culture-induced phosphorylations of gp130 and IRAK were inhibited by pre-shearing at HSS (but not LSS) for 24 h. These results indicate that the SMC-induction of E-selectin in ECs involves the paracrine action of IL6 and IL-1β on their receptors to activate the JNK, p38 and NF-κB, and that this effect can be inhibited by high shear stress.