ABSTRACT
Tumor necrosis factor alpha (TNFα) and low density lipoprotein (LDL) are important modulators of the atherosclerotic process. Here, the effect of TNFα on confluent primary human aortic endothelial cell (pHAEC) LDL-derived lipids and trafficking were investigated. TNFα promoted up to 2 folds increase in cellular cholesterol and could induce a massive increase in the non-hydrolysable tracer, Dil, by over 200 folds. The lipid increase was associated with increased 125I-LDL surface binding. Further, Dil-LDL cellular association was blocked by excess unlabeled LDL, but not oxidized LDL (oxLDL). Moreover, TNFα-induced Dil and cholesterol increase were enhanced by the endosomal pH-raising agent, chloroquine. Internalization of Dil-LDL was reduced by the scavenger receptor B1 (SR-B1) antagonist, BLT-1, and the LDLR family antagonist, proprotein convertase subtilisin/kexin type 9 (PCSK9), but not receptor associated protein (R AP). Additionally, surface accessible LDLR was higher in TNFα-treated cells by about 30 folds, without a significant change in total LDLR. Correspondingly, specific LDLR antibody blocked Dil-LDL internalization to undetectable levels, and Dil-apoE3-VLDL by 94%, but had no effect on Dil-HDL3 internalization. TNFα did not increase Dil-HDL3 cellular association. Further, ACAT inhibitor reduced cholesteryl esters, but not the total cholesterol increase induced by TNFα. On pHAECs grown on transwell inserts, TNFα did not enhance apical (AP) to basolateral (BL) LDL cholesterol or Dil release. It is concluded that TNFα induces LDLR surface localization and that LDLR does not promote AP to BL LDL transport across pHAECs.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- ACAT
- acyl-CoA cholesterol acyltransferase
- AP
- apical
- apoE
- apolipoprotein E
- BHT
- butylated hydroxytoluene
- BL
- basolateral
- CAB
- cholesterol assay buffer
- DB
- dialysis buffer
- DiL
- 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate
- DMSO
- dimethyl sulfoxide
- GAPDH
- glyceraldehyde 3-phosphate dehydrogenase
- FAF-BSA
- fatty acid-free bovine serum albumin
- FBS
- fetal bovine serum
- LDLR
- low density lipoprotein receptor
- LRP
- ldlr-related protein
- mHBSS
- modified Hanks’balanced salt solution
- PBS
- phosphate buffered saline
- PCSK9
- proprotein convertase subtilisin/kexin type 9
- PhenA
- 1,10-phenanthroline
- pHAECs
- primary human aortic endothelial cells
- RAP
- receptor associated protein
- SDS
- sodium dodecyl sulfate
- TMTU
- tetramethylthiourea
- TNFα
- tumor necrosis factor alpha
- VBM
- vascular basal medium
- VEGF
- vascular endothelial growth factor kit