LDL oxidation and human placental trophoblast and macrophage cytotoxicity.

Abstract

We have previously shown that LDL is oxidized by placental cells in primary tissue culture and that this process causes cytotoxicity proportional to LDL oxidation in the presence of sex steroid hormones. Here we describe further experiments linking LDL oxidation to placental cell cytotoxicity. Trophoblast and macrophages were isolated from healthy elective caesarean section placentas by enzymatic digestion and separated by centrifugation on a 40% Percoll gradient and maintained in primary culture for up to 5 days. LDL was oxidized by exposure to 5 microM CuCl2, cells were incubated in the absence of albumen to favor oxidation, and cytotoxicity was measured by 51Cr release from prelabelled cells and cell protein content. Native LDL was oxidized by both cell types with a 10%-50% increase in lipid peroxides and an approximately 4-fold increase in TBARS formation. Increasing concentrations of native LDL and oxidized-LDL increased 51Cr release and diminished protein content in cells incubated in HAM's F-10 medium. Addition of 5 microM Cu2+ augmented cytotoxicity of LDL in macrophage and trophoblast culture, but more in macrophages than trophoblast. Cytotoxicity was diminished by adding 0.001-0.1 mM EDTA to the system, diminishing 51Cr release from 91 +/- 0.5 to 40.8 +/- 1.0% in macrophages and 54.2 +/- 1.2 to 33.1 +/- 1.3% in trophoblast (P < 0.001 in both instances). Similarly, the absence of transition metal ion in culture (MEM medium) blocked an increase in 51Cr release compared to its presence (HAM's F-10 medium). An antioxidant, butylated hydroxytoluene, diminished 51Cr release and LDL electrophoretic mobility in HAM's F-10 medium in placental macrophage culture. LDL oxidation injures placental macrophages and trophoblast, the former more than the latter. The process is LDL- and transition metal ion-dependent and is inhibited by antioxidant. This model of LDL oxidation and placental cell damage in vitro provides a basis for studying mechanisms of placental dysfunction and senescence in human pregnancy.