Abstract 434: LDL-like Particles in HDL Density Have Small Diameters and Stimulate Macrophages Similar to LDL

Abstract

We have been detected small portion of LDL-like lipoprotein in HDL density (1.063 <d< 1.21 g/mL) from human plasma. This β-migrating lipoprotein has affinity to Dextrin Sulfate (DS) column of which use for LDL apheresis (LA-15, Kanenka Medix Corp, Japan). Here, we isolated and examined character of this human dense LDL (d-LDL) isolated by ultracentrifugation and DS column then evaluated its potential on mouse macrophages foam cell development. Protein composition of d-LDL fraction detected by SDS-PAGE was similar to LDL fraction rather than HDL DS (remaining after DS column). This d-LDL particle size was measured by Transmission Electron Microscopic-negative staining method (JEM-1011J, x200,000) and this digital image data were further analyzed by Developer Toolbox TM (GE Healthcare) for particle area and diameter. The calibration speed of average 420.4 lipoprotein particles per 2440 x 3571 pixels image was less than 10 sec by this program. The d-LDL contained smaller particles compared to the LDL fraction, significantly. Small dense LDL (sdLDL) detected by DENKA assay system revealed increased sdLDL/LDL ratio in HDL DS fraction than d-LDL or LDL fractions. Perhaps, the sdLDL has low affinity to the DS column. In the other hand, Lp(a) was highly bound to DS column. The acetylated LDLs (ac-LDL, ac-d-LDL) were load on mouse peritoneal resident macrophage for 48 hrs to produce the foam cells. Both acetylated LDLs successfully induced foam cells while cellular choline labeled phosphatidylcholine was twice as high as in ac-d-LDL treated foam cells. The cholesterol content in the macrophage was also increased by ac-d-LDL. d-LDL fraction was re-isolated by “masked” DS column from non-specifically bound plasma proteins (d-LDL ri ). Difference of phospholipid synthesis during macrophages foam cell development was abolished between by ac-LDL and by ac-d-LDL ri . Thus, original DS-Cellulose column bounds LDL but also inducer/inducers of macrophage lipid synthesis from human plasma HDL fraction. These results indicate LDL-like lipoprotein in HDL fraction, d-LDL, has smaller diameter than LDL. The acetylated d-LDL ri activated phospholipid synthesis in mouse peritoneal macrophage as the same extent to Ac-LDL.