Increased lysophosphatidylcholine and non-esterified fatty acid content in LDL induces chemokine release in endothelial cells: Relationship with electronegative LDL

Electronegative low-density lipoprotein (LDL(−)) is a plasma-circulating LDL subfraction with proinflammatory properties that induces the production of chemokines in cultured endothelial cells. However, the specific mechanism of LDL(−)-mediated chemokine release is presently unknown. A characteristic feature of LDL(−) is an increased content of lysophosphatidylcholine (LPC) and non-esterified fatty acids (NEFA). The effect of increasing amounts of LPC and NEFA associated with LDL on the release of chemokines by endothelial cells was studied. Total LDL was subfractionated by anion-exchange chromatography in electropositive (LDL(+)) and LDL(−). LDL(−) contained two-fold more LPC and NEFA than LDL(+) and induced two- to four-fold more (p< 0.05) interleukin-8 (IL-8, 11.5 ± 8.2 ng/105 cells) and monocyte chemotactic protein-1 (MCP-1, 10.8 ± 3.8 ng/105 cells) release by human umbilical vein endothelial cells (HUVEC) than LDL(+) (IL-8: 3.4 ± 1.5 ng/105 cells, MCP-1: 5.8 ± 2.9 ng/105 cells). The content of LPC and NEFA in LDL(+) was increased by enzymatic treatment with secretory phospholipase A2 (sPLA2) at 5 ng/mL or 20 ng/mL or by incubation with NEFA at 2 mmol/L. Modification of LDL(+) by both methods did not result in oxidative modification as demonstrated by the lack of change in antioxidants, conjugated dienes and malondialdehyde content. sPLA2 treatment resulted in an increase in LPC and NEFA in LDL(+) which enhanced its ability to release IL-8 and MCP-1 by HUVEC in a concentration-dependent manner (sPLA2(5)-LDL; IL-8: 7.1 ± 3.8 ng/105 cells, MCP-1: 8.0 ± 5.1 ng/105 cells; sPLA2(20)-LDL; IL-8: 20.8 ± 11.2 ng/105 cells, MCP-1: 15.0 ± 7.5 ng/105 cells). NEFA loading of LDL(+) also favored the release of IL-8 and MCP-1 (IL-8: 7.8 ± 6.1 ng/105 cells, MCP-1: 8.4 ± 2.7 ng/105 cells, p< 0.05 versus LDL(+)). These effects were observed when modified LDL(+) reached a content of LPC and/or NEFA similar that of LDL(−). These data indicate that non-oxidized polar lipids associated with LDL promote an inflammatory response in endothelial cells and suggest that increased NEFA and LPC could be involved in the inflammatory activity of LDL(−).