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Oxidized low-density lipoproteins induce apoptosis in macrophages
1Laboratory of Molecular Pharmacology, Hu-nan Medical University, Changsha, China.
Aim:
To examine whether oxidized low density lipoproteins (ox-LDL) might induce apoptosis in mouse peritoneal macrophages (MPM).
Methods:
Low density lipoproteins (LDL) were isolated from healthy human plasma by ultracentrifugation and oxidized by CuSO4 10 mumol.L-1. MPM were incubated in a medium containing ox-LDL, LDL, or phosphate-buffer solution (PBS) as control. DNA fragmentation was visualized by agarose gel electrophoresis and determined quantitatively using Hoechst 33,258 fluorochrome.
Results:
Ox-LDL, not LDL, elicited typical apoptotic morphological changes (shrinkage of cytoplasm and condensation of chromatin) and DNA fragmentation in a time- and dose-dependent manner. Incubation for 24 h was necessary for ox-LDL 200 mg protein.L-1 to induce DNA fragmentation, and the maximal effect reached at 72 h. The DNA fragmentation after 24 h incubation with ox-LDL at concentrations of 100, 200, and 400 mg protein.L-1 amounted to 6.0% (P > 0.05), 9.3% (P < 0.05), and 30.9% (P < 0.01), respectively vs PBS control. Dextran sulfate, a scavenger receptor blocker and cycloheximide, a protein synthesis inhibitor, exhibited no effect on DNA fragmentation. However, antioxidant butylated hydroxytoluene (BHT) 20 mumol.L-1 completely inhibited Cu(2+)-mediated oxidation of LDL as well as the apoptosis-inducing effect of Cu(2+)-exposed LDL. Lysophosphatidylcholine (LPC), an active component in ox-LDL, at concentration up to 60 mumol.L-1, did not elicit DNA fragmentation in MPM.
Conclusion:
Ox-LDL induces apoptosis in MPM without involving LPC.