Abstract 5538: Glutathione Peroxidase-1 Modulates Human Microvascular Endothelial Cell Response to Lipopolysaccharide Through Altering CD14 Expression
To determine the pathways by which glutathione peroxidase-1 (GPx-1) modulates cellular responses to reactive oxygen species (ROS), we performed microarray analysis in human microvascular endothelial cells (HMVEC) treated with 20 ng/ml TNF-α for 2 hr following GPx-1 knockdown (siGPx-1) or control (siControl) transfection. Gene set enrichment analysis identified over 430 genes up- or down-regulated by more than 2-fold. Among these changes, CD14 mRNA was 6.8-fold higher in siGPx-1 compared to siControl (P<0.0001). Additional analysis by qRT-PCR indicated that siGPx-1 in the absence of cytokine stimulation increased CD14 expression 1.2-fold after 24 hr and 4.2-fold after 48 hr of transfection (P<0.0001). A role for endogenously expressed CD14 in endothelial cells is unclear, as CD14 is not highly expressed in these cells. CD14 may contribute to LPS signaling through formation of Toll-like receptor 4 (TLR-4)/CD14 receptor complexes. In HMVEC, 1 μg/ml LPS treatment increased ICAM-1 (4.3-fold, P<0.0001) and VCAM-1 protein (11.8-fold, P<0.0001) after 12 hr. siGPx-1 cells decreased GPx-1 mRNA by 98.4% (P<0.0001) with corresponding reductions in GPx-1 protein (74.8%) and activity (75.2%), and was accompanied by an increase in ICAM-1 and VCAM-1 mRNA (5.7-fold and 4.1-fold, respectively, P<0.0001) and protein (2.7-fold and 2.5-fold, respectively) in the absence of LPS stimulation. GPx-1 deficiency had no effect on expression of TLR-4 or MyD88. After 24 hr LPS treatment, GPx-1 deficiency further augmented ICAM-1 mRNA 2.3-fold (P<0.0001) and protein 1.9-fold compared to LPS-treated control cells. VCAM-1 expression was similarly enhanced in LPS treated siGPx-1 and siControl cells. GPx-1 deficiency increased LPS-induced intracellular ROS accumulation as measured by dichlorodi-hydrofluorescein (DCF) fluorescence, whereas adenoviral overexpression of GPx-1 attenuated LPS-mediated responses. siCD14 decreased CD14 mRNA by 99.5% (P<0.0001) and was accompanied by a decrease in ICAM-1 mRNA (2.4-fold) and protein (4.2-fold) following LPS stimulation. Double knockdown of GPx-1 and CD14 decreased ICAM-1 mRNA and protein to a lesser extent in the presence of LPS. These data suggest that GPx-1 modulates the cellular response to LPS by CD14-mediated effects.