The modification of low density lipoprotein by the flavonoids myricetin and gossypetin.

Myricetin and gossypetin, two hexahydroxylated flavonoids, are capable of modifying low density lipoprotein (LDL) to increase greatly its uptake by macrophages. When human 125I-labelled LDL was incubated with 100-1000 microM myricetin or gossypetin, it was subsequently endocytosed much faster by mouse peritoneal macrophages. This modification did not occur at a concentration of 10 microM. Nine other flavonoids containing up to five hydroxyl substituents did not modify LDL to any great extent at 100 microM. The modification of LDL by 100 microM myricetin was time-dependent and complete by 6 hr. Flavonoids can sometimes act as pro-oxidants but myricetin did not act by oxidizing the LDL, as the LDL lipid hydroperoxide content was not increased by myricetin, nor did it promote the depletion of the endogenous antioxidant alpha-tocopherol in the LDL. High concentrations of myricetin caused the aggregation of LDL particles, as judged by light microscopy, agarose gel electrophoresis, retention by a membrane filter and sedimentability by centrifugation. SDS-PAGE indicated that the apolipoprotein B-100 molecules of LDL particles were covalently crosslinked. The uptake and degradation by macrophages of myricetin-modified 125I-labelled LDL reached saturation at about 10 micrograms protein/mL, suggesting the existence of a high affinity uptake process for the modified LDL. The uptake of myricetin-modified 125I-labelled LDL was not competed for by a large excess of non-labelled native LDL or acetylated LDL. We conclude that myricetin and gossypetin at high concentrations are capable of modifying LDL by a novel non-oxidative mechanism to a form taken up by macrophages by a high affinity process.

Influence of the cytocidal macrophage phenotype on the degradation of acetylated low density lipoproteins: dual regulation of scavenger receptor activity and of intracellular degradation of endocytosed ligand.

The appearance of lipid-laden macrophages is a characteristic feature in the development of the atherosclerotic plaque. The functional status of macrophages located within the intima of atherosclerotic lesions is as yet unknown; nevertheless, macrophages are known to be exceedingly responsive to their environment and can differentiate to different functional states. The objective of this study was to determine the influence of two definable macrophage functional states, namely the IFN-primed state and the cytocidal state, on the capacity of macrophages to bind and degrade lipoproteins. We report that priming of macrophages with IFN-beta or IFN-gamma failed to influence the ability of macrophages to degrade native low density lipoprotein or acetylated low density lipoprotein (AcLDL). However, challenge with stimuli that induce expression of the cytocidal state (poly[I:C] and LPS) resulted in a marked inhibition of the capacity of the cells to degrade both lipoproteins. The poly[I:C]-induced inhibition of 125I-AcLDL degradation was accompanied by a proportional decrease in the binding of the ligand to its receptor which Scatchard analysis revealed was due to a decrease in receptor number rather than a change in receptor affinity for 125I-AcLDL. However, in addition to the down-regulation of receptor activity, the degradation of endocytosed 125I-AcLDL was also suppressed in macrophages that had been exposed to poly[I:C]. This latter observation suggests that the degradation of endocytosed lipid is also regulated at a second, previously unidentified level, independent of the availability of cell surface ligand receptors. We speculate that this down-regulation in the intracellular hydrolysis of endocytosed lipid may account for the observed accumulation of 125I-AcLDL in these cells.

Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages.

Low density lipoproteins (LDL) can be oxidatively modified in vitro by macrophages and certain other cell types so that macrophages will take them up much faster. This process may be important in the formation of cholesterol-laden foam cells derived from macrophages in atherosclerotic lesions. In this study, we have shown that certain flavonoids, plant constituents found in the diet, are potent inhibitors of the modification of 125I-labelled LDL by macrophages, with IC50 values in the micromolar range (e.g. morin and fisetin 1 microM; quercetin and gossypetin 2 microM). The potencies of individual flavonoids in inhibiting LDL modification did not correlate with their previously determined potencies as inhibitors of 5-lipoxygenase and cyclo-oxygenase. The modification of LDL by macrophages exhibits a lag period of about 4-6 hr before enhanced uptake is detected. During this time, there is a rapid depletion in its content of alpha-tocopherol (an endogenous antioxidant found in lipoproteins) followed by a large increase in the level of hydroperoxides. The flavonoids conserved the alpha-tocopherol content of LDL and delayed the onset of detectable lipid peroxidation. Flavonoids also inhibited the cell-free oxidation of LDL mediated by CuSO4. These findings raise the possibility that flavonoids may protect LDL against oxidation in atherosclerotic lesions and may therefore be natural anti-atherosclerotic components of the diet, although this will depend to a large extent on their pharmacokinetics.

Alpha-tocopherol consumption during low-density-lipoprotein oxidation.

1. The kinetics of the depletion of alpha-tocopherol in human low-density lipoprotein (LDL) were measured during macrophage-mediated and cell-free oxidation. The formation of oxidatively modified, high-uptake species of LDL in these systems was not detectable until all of the endogenous alpha-tocopherol had been consumed. 2. Supplementation of the alpha-tocopherol content of LDL by loading in vivo extended the duration of the lag period during which no detectable oxidative modification occurred. 3. The addition of a flavonoid (morin) prevented both alpha-tocopherol consumption and oxidative modification of LDL. 4. The alpha-tocopherol contents of LDLs from a range of individual donors could not be used to predict their relative resistance to oxidation, indicating that other endogenous antioxidants may also be present, and quantitatively significant, in human LDL.