To the plasma, butylated hydroxytoluene (BHT) in DMSO was added to 45 M (0.01%). TNF-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 TNF included 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 TNF-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNF on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-B, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on Transwell inserts, TNF did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNF promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation. = 5. (F) Cells were pre-treated with 0 or 5 ng/mL TNF in serum medium as described under Section 2. After incubation at 37 C, the amount of 125I-LDL released to serum-free medium at 4 C without (0) or with 20 folds unlabeled LDL competitor (+) was determined as the surface 125I-LDL. (G) The experiment was performed as in (E), and the amount of radioactivity associated with the cell was determined. (C,D) = 3. *, **, ***, 0.05, 0.01, 0.001, relative to the corresponding Ctrl.3.2. ACAT inhibitor does not prevent TNF-induced LDL cholesterol accumulation. It has been reported that TNF induces cholesteryl ester accumulation in monocytes through enhanced ACAT activity in the presence of oxLDL [60]. To evaluate the extent to which enhanced ACAT activity is responsible in raising pHAEC cholesterol in the presence of LDL, the cells were treated with or without ACAT inhibitor, Sandoz 58-035. As reported in Figure 2A,B, the inhibition of ACAT activity slightly raised the unesterified cholesterol, and significantly suppressed cholesteryl ester accumulation. Despite this inhibition, the TNF-induced total cholesterol accumulation, now mainly in the unesterified form, was not prevented (Figure 2A,C). ACAT inhibition also reduced the TH 237A total cholesterol in the cells, with or without TNF stimulation (Figure 2C). This is a known effect of ACAT inhibition. The higher elevated unesterified cholesterol is more likely to be effluxed from the cells than the esterified one [61]. Open in a TH 237A separate window Figure Rabbit polyclonal to GNMT 2 ACAT inhibitor does not prevent TNF-induced LDL cholesterol accumulation. Cells in serum-free medium TH 237A were treated with 0 or 100 ng/mL TNF in the presence of 0.1% DMSO (DMSO) or 10 g/mL ACAT inhibitor (Sandoz 58-035) and 100 g/mL LDL for 24 hrs. After washing with heparin as described under Materials and Methods, the cellular cholesterol content was determined. (A) unesterified cholesterol, (B) esterified cholesterol, and (C) total cholesterol. = 3. *, **, ***, 0.05, 0.01, 0.001, relative to corresponding Ctrl or as indicated with bars. 2.2. ACAT Inhibitor Does Not Prevent TNF-Induced LDL Cholesterol Accumulation It has been reported that TNF induces cholesteryl ester accumulation in monocytes through enhanced ACAT activity in the presence of oxLDL [60]. To evaluate the extent to which enhanced ACAT activity is responsible in raising pHAEC cholesterol in the presence of LDL, the cells were treated with or without ACAT inhibitor, Sandoz 58-035. As is reported in Figure 2A,B, inhibition of ACAT activity slightly raised the unesterified cholesterol, and significantly suppressed cholesteryl ester accumulation. Despite this inhibition, the TNF-induced total cholesterol accumulation, now mainly in the unesterified form, was not prevented (Figure 2A,C). ACAT inhibition also reduced the total cholesterol in the cells, with or without TNF stimulation (Figure 2C). This is a known effect of ACAT inhibition. The higher elevated unesterified cholesterol is more likely to be effluxed from the cells than the esterified one [61]. 2.3. LDL Oxidation Is Not Required for TNF-Induced LDL Accumulation Having demonstrated that TNF promoted LDL binding to pHAECs (Figure 1), the requirement for oxidative modification of LDL was investigated. Particularly, TNF has been reported to promote release of the reactive oxygen species, superoxide and hydrogen peroxide [62,63]. Hence, experiments.
To the plasma, butylated hydroxytoluene (BHT) in DMSO was added to 45 M (0