Tnfɑ‐induced ldl cholesterol accumulation involve elevated ldlr cell surface levels and sr‐b1 downregulation in human arterial endothelial cells

Abstract

Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNFɑ) in modulating the low‐density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNFɑ promoted an up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased125I‐LDL surface binding. Using the non‐hydrolysable label, Dil, TNFɑ could induce a massive increase in Dil‐LDL by over 200 folds. The elevated intracellular Dil‐LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), or apolipoprotein (apoE) depletion. Moreover, the TNFɑ‐induced increase of LDL‐derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil‐LDL accumulation was reduced by over 99%. The effects of TNFɑ in-cluded an LDLR cell surface increase of 138%, and very large increases in ICAM‐1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR‐B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFɑ‐treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFɑ on Dil‐LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose‐dependently, but not by inhibitors against NF‐κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on Transwell inserts, TNFɑ did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNFɑ promotes LDLR functions through combined increase at the cell surface and SR‐B1 downregulation.