Tumor necrosis factor induces cholesterol accumulation in human arterial endothelial cells through LDL receptor surface redistribution

Tumor necrosis factor (TNFα) is an important mediator of inflammatory processes involved in atherosclerotic development. Here, the effect of TNF? on confluent primary human aortic endothelial cell (pHAEC) cholesterol content and low density lipoprotein (LDL) trafficking were investigated. TNFα, up to 100 ng/ml, did not cause a significant change in cell number, but altered the cell morphology dose-dependently. It induced LDL cholesteryl ester accumulation in a time- and concentration-dependent manner. This increase was accompanied by enhanced 125I-LDL plasma membrane binding. Dil-LDL binding to pHAECs pretreated with TNFα was suppressed by excess unlabeled LDL, but not oxidized LDL. Moreover, the multi-valent metal chelator, 1,10-phenathroline (PhenA), but not reducing agent, tetramethylthiourea (TMTU), inhibited TNFα-induced dil-LDL accumulation. Among receptor inhibitors against scavenger receptor B1 (SR-B1), LDL receptor (LDLR), LDL receptor related proteins (LRPs), using BLT-1, PCSK9, and RAP, respectively, only PCSK9 had a significant effect on dil-LDL uptake. Further, specific antibody against LDLR completely blocked LDL internalization. TNFα did not significantly alter total LDLR protein, but increased surface LDLR. On pHAECs grown on transwell inserts, TNFα did not enhance apical to basolateral LDL cholesterol release. It is concluded that TNFα induces LDLR surface localization and that LDLR does not promote apical to basolateral LDL transport across pHAECs.