Abstract 3932: Central Role Of Toll-like Receptor 4 In Oxidized Low Density Lipoprotein-and Lipopolysaccharide-Induced Bone Morphogenetic Protein-2 Expression In Human Coronary Artery Endothelial Cells
Previous studies demonstrated that osteogenic mediators, in particular bone morphogenetic protein-2 (BMP-2), play an important role in the progression of atherosclerosis and vascular calcification. Although it is known that the atherogenic factor oxidized low density lipoprotein (oxLDL) up-regulates BMP-2 expression in endothelial cells, the signaling mechanisms involved are not well understood. In macrophages, oxLDL up-regulates Toll-like receptor 4 (TLR4) expression, and this innate immunoreceptor appears to be involved in oxLDL-induced macrophage actin polymerization. We earlier found that stimulation of TLR4 with lipopolysac-charide (LPS) increases cellular BMP-2 protein levels in human aortic valve interstitial cells. We hypothesized that the TLR4 pathway plays a central role in mediating BMP-2 expression in human coronary artery endothelial cells (CAECs). The purposes of this study were to examine whether TLR4 mediates oxLDL- and/or LPS-induced BMP-2 expression in human CAECs, and to determine whether the p38 and/or p44/42 MAPKs are involved.
Methods and results: Stimulating human CAECs with LPS (E. coli 0111:B4, 200 ng/ml) for 24 h up-regulated BMP-2 protein expression. Similarly, stimulation with oxLDL (from human plasma, CuSO4-oxidized, LPS-free, 40–160 μg/ml) for 24 h induced BMP-2 expression in a dose-dependent manner. Pretreatment with either TLR4-neutralizing antibody or TLR4 siRNA significantly attenuated oxLDL-induced BMP-2 expression and abrogated the effect of LPS on BMP-2 expression. Over-expression of TLR4 enhanced the cellular BMP-2 response to oxLDL and LPS. Further, immunofluorescent staining co-localized oxLDL with TLR4. Although LPS induced ICAM-1 expression in human CAECs, oxLDL had no effect, indicating that oxLDL and LPS have different pro-inflammatory effects. BMP-2 expression was associated with activation of p38 and p44/42 MAPKs. Inhibiting p44/42, but not p38, reduced BMP-2 expression.
Conclusions: In human CAECs, TLR4 plays a central role in regulating BMP-2 expression induced by either oxLDL or LPS, and the signaling mechanism involves the p44/42 MAPK pathway. These novel findings underscore an important role of TLR4 in atherosclerosis and vascular calcification.