Oxidative-modified and acetylated low-density lipoproteins differ in their effects on cholesterol synthesis and stimulate synthesis of apolipoprotein E in rat peritoneal macrophages by different mechanisms

Abstract

Apolipoprotein (apo) E plays an important role in the recognition of lipoproteins by cellular lipoprotein receptors. Unlike other apolipoproteins, apo E is expressed by many extrahepatic tissue including macrophages (MΦ). Resident MΦ express low levels of apo E. However, their synthesis of apo E is substantially increased after MΦ have been incubated with acetylated low-density lipoprotein (LDL). But acetylation of LDL is not known to occur in vivo. On the other hand, modification of LDL by oxidation and by enzymatic action is believed to happen physiologically. In this report, we compared the effects of various modified LDLs on the synthesis of apo E by MΦ. Freshly isolated human LDL was modified by (1) repeated addition of acetic anhydride (Ac-LDL); (2) incubation with 20 μmol/L CuSO 4 at 37°C for 24 hours (Ox-LDL); and (3) incubation with phospholipase C at 37°C for 1 hour (PI-LDL). Resident peritoneal MΦ were collected by lavage from rats and allowed to attach to plastic culture dishes. Although native LDL had no effect, treatment with Ac-, Ox-, and PI-LDL (50 μg/mL each) was found to increase medium apo E by (-fold) 4.19 ± 0.26, 4.20 ± 0.34, and 2.02 ± 0.20 (mean ± SEM, n = 5), respectively, as compared with untreated cells. Northern blot analysis revealed that cellular apo E mRNA was increased in parallel to apo E protein by Ac-LDL and PI-LDL. However, increases of apo E protein and mRNA by Ox-LDL were not equal. Ac-, Ox-, and PI-LDL-treated cells each contained 2.66-, 1.95- and 2.10-fold more total cholesterol than untreated cells (20.5 ± 2.1 μg/mg cell protein, n = 11). Interestingly, newly synthesized cholesterol esters in MΦ were increased (-fold) 19.20 ± 2.50 (mean ± SEM, n = 4) by Ac-LDL and 5.16 ± 0.19 by PI-LDL, but only 1.81 ± 0.19 by Ox-LDL. It is concluded that (1) similar to Ac-LDL, Ox- and PI-LDL also stimulate MΦ apo E synthesis; (2) all three modified LDLs are effective in loading cholesterol into macrophages, but vary greatly in their abilities to stimulate cholesterol synthesis; (3) stimulation of apo E synthesis in rat peritoneal MΦ by modified LDLs does not parallel cellular cholesterol contents nor correspond to de novo cholesterol synthesis; and (4) Ox-LDL and Ac-LDL stimulate MΦ apo E synthesis by different mechanisms.