Dissociation of atherogenesis from aortic accumulation of lipid hydro(pero)xides in Watanabe heritable hyperlipidemic rabbits

Abstract

Antioxidants can inhibit atherosclerosis, but it is unclear how inhibition of intimal lipid oxidation relates to atherogenesis. Here we tested the effect of probucol and its metabolite bisphenol on aortic lipid (per)oxidation and atherogenesis in Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL and aortas from rabbits fed probucol contained bisphenol at concentrations comparable to those in bisphenol-treated animals. Bisphenol treatment increased plasma cholesterol slightly, and plasma and aortic alpha-tocopherol more substantially; these parameters were unaffected by probucol. Bisphenol and probucol treatment both enhanced the resistance of circulating LDL to peroxyl radical-induced lipid peroxidation; this was due to bisphenol, not probucol. Only probucol enhanced LDL's resistance to Cu(2+)-induced oxidation. Both bisphenol and probucol treatment strongly inhibited aortic accumulation of hydroperoxides and hydroxides of cholesteryl esters and triglycerides [LO(O)H]. Despite this, however, probucol had a modestly significant effect on the extent of lesion formation; bisphenol had no inhibitory effect. In addition, the extent of atherosclerosis did not correlate with amounts of aortic LO(O)H present, but, as expected, it did correlate with aortic alpha-tocopherol and cholesterol. Together, these results suggest that aortic accumulation of LO(O)H is not required for, nor is alpha-tocopherol depleted during, the initiation and progression of atherogenesis in WHHL rabbits.

Figure 1

LDL from WHHL rabbits treated with probucol or bisphenol is resistant to ex vivo lipid peroxidation induced by AAPH. Pooled LDL (0.5-0.6 mg protein/mL) from rabbits fed probucol (filled circles), bisphenol (filled squares), control diets (filled triangles), or control LDL + BHT (10 μM) (filled diamonds) was treated at 37°C with 2 mM AAPH. Aliquots were removed and analyzed for α-TOH (a) and LO(O)H (b). LDL from bisphenol-treated (c) and probucol-treated (d) rabbits was also analyzed for bisphenol (open squares), diphenoquinone (open circles), and probucol (cross-hatched squares). Data represent mean ± SD of 4 independent studies. Initial concentrations (i.e., 100%) of α-TOH were 13 ± 2, 13 ± 3, 15 ± 2, and 15 ± 6 for probucol-treated, bisphenol-treated, control, and BHT-treated LDL, respectively. Initial levels of bisphenol and diphenoquinone were 10 ± 2 and 8 ± 1 μM and 12 ± 2 and 10 ± 3 μM for LDL from bisphenol- and probucol-treated rabbits, respectively. Initial levels of diphenoquinone were 1.6 ± 0.7 and 1.0 ± 0.1 μM for bisphenol and probucol samples, respectively. Probucol was expressed as percent peak area relative to that before oxidation. BHT-free control LDL oxidized to LO(O)H in a chain reaction, with chain length υ∼15 (see ref. for determination of υ).

Figure 2

Probucol and bisphenol inhibit aortic lipid oxidation, while aortic levels of LO(O)H correlate poorly with atherosclerotic lesion size. (a) Aortas from control (A), probucol-treated (B), and bisphenol-treated (C) rabbits were homogenized, and the organic extracts were analyzed by HPLC with chemiluminescence detection. Representative results are shown with hydroperoxides of triglycerides and CE eluting at 5–7 minutes and ∼8 minutes, respectively. (b) Fractional intima-to-media volume derived from control (open circles; n = 11), bisphenol (open squares; n = 11), and probucol (open diamonds; n = 12) groups vs. aortic LO(O)H. The coefficient of variation determined by linear regression is r = 0.11.