Human Apolipoprotein A-II Enrichment Displaces Paraoxonase From HDL and Impairs Its Antioxidant Properties by Vicent Ribas, José Luis Sánchez-Quesada, Rosa Antón, Mercedes Camacho, Josep Julve, Joan Carles Escolà-Gil, Luís Vila, Jordi Ordóñez-Llanos, and Francisco Blanco-Vaca Circulation Research Volume 95(8):789-797 October 15, 2004 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Electrophoretic analyses of the HDL-mediated protection against oxidative modification of murine apoB-containing apolipoproteins. Figure 1. Electrophoretic analyses of the HDL-mediated protection against oxidative modification of murine apoB-containing apolipoproteins. The gels show three series of three lanes in which 1 mmol/L of mouse VLDL cholesterol (V; top) or LDL (L; bottom) were incubated alone (first lane of each series), with copper (5.5 μmol/L; second lane) or with copper and 0.4 mmol/L HDL phospholipids (+H) isolated from the same mouse line (third lane). Vicent Ribas et al. Circ Res. 2004;95:789-797 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Electrophoretic analyses of the HDL-mediated protection against oxidative modification of human LDL. Human LDL (1 mmol/L of cholesterol) was incubated with copper (5.5μmol/L) in presence of HDL isolated from control or transgenic mice standardized for phospholipid, protein, or cholesterol. Figure 2. Electrophoretic analyses of the HDL-mediated protection against oxidative modification of human LDL. Human LDL (1 mmol/L of cholesterol) was incubated with copper (5.5μmol/L) in presence of HDL isolated from control or transgenic mice standardized for phospholipid, protein, or cholesterol. Native and oxidized LDL was loaded in lane 1. Vicent Ribas et al. Circ Res. 2004;95:789-797 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. HDL protection against LDL oxidative modification as analyzed by four different methods. Figure 3. HDL protection against LDL oxidative modification as analyzed by four different methods. A, REM of human LDL after being exposed to oxidation coincubated with HDL from control or transgenic mice (n=7 independent experiments using pools of HDL obtained from 5 to 10 mice). B, 9-HODE and 13-HODE determination of the LDL coincubated with HDL (n=3 experiments); results are expressed as relative amount of LDL HODE incubated without HDL, corrected by phospholipid content. C, Results of the DCF assay (n=4 experiments; see Materials and Methods). D, Lag phase of conjugated diene formation kinetics; results are represented as relative lag phase to the LDL kinetics oxidized without HDL. Bar graphs show mean±SEM. *P<0.05 or **P<0.01 vs control. Vicent Ribas et al. Circ Res. 2004;95:789-797 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Representative apolipoprotein, lipid, and enzymatic HDL fractions isolated by FPLC. A, Apolipoprotein content in milligrams per deciliter for human apoA-II and arbitrary densitometric units for apoA-I determined by Western blot. Figure 4. Representative apolipoprotein, lipid, and enzymatic HDL fractions isolated by FPLC. A, Apolipoprotein content in milligrams per deciliter for human apoA-II and arbitrary densitometric units for apoA-I determined by Western blot. B, Total cholesterol content. C, EDTA-sensitive arylesterase (PON1) activities. Inset, Western blot of PON1 of ultracentrifugally isolated HDL (10 μg of HDL protein loaded per lane). D, PAF-AH activities. Vicent Ribas et al. Circ Res. 2004;95:789-797 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Effect of purified human apoA-II on PON1 activity. Figure 5. Effect of purified human apoA-II on PON1 activity. A, EDTA-sensitive arylesterase (PON1) activities of plasma incubated with increasing concentrations of human apoA-II at 37°C for three hours. B, PON1 activities of HDL and lipoprotein-depleted fractions of plasma isolated by FPLC after the experiment shown in A. C, Western blot using antibodies to PON1 against HDL (1 mmol/L HDL phospholipid) from control mice incubated with purified human apoA-II and separated by native GGE. Vicent Ribas et al. Circ Res. 2004;95:789-797 Copyright © American Heart Association, Inc. All rights reserved.