Figure 1.
Non-denaturing gradient gel of fluorescently-labeled apoA-I added to human plasma.
ApoA-I labeled with Alexa350 was incubated at 37°C at increasing concentrations with plasma from a healthy human donor and examined by polyacrylamide gel electrophoresis on a 4–20% Tris-glycine gel. Lanes 1 and 2 contain reference samples of purified LDL and HDL, respectively. Fluorescence imaging was performed prior to staining with Coomassie (excitation light, 365 nm; emission blue filter 440–480 nm). The figure is a composite of fluorescence and protein stain images of the same gel.
Figure 2.
Representative EPR spectra of spin-labeled apoA-I probe added to human plasma.
The spectrum of spin-labeled apoA-I in apoB depleted plasma from a healthy human donor at 6°C (blue line) is compared to the spectra after a 15 minute incubation at 37°C (red line). The maximal nitroxide spectral response is obtained from spin-labeled apoA-I in an extended lipid-bound conformation (orange line). Sample response was normalized between instruments using a proprietary internal standard. Sample response was calculated by subtracting the peak amplitude at 6°C from the amplitude at 37°C and dividing by the amplitude of the 100% response standard.
Figure 3.
Oxidation of HDL by myeloperoxidase inhibits HDL-ApoA-I exchange.
Human HDL from healthy, fasted volunteers (n = 3) was incubated at 37°C with increasing concentrations of H2O2 in the presence (open squares) or absence (closed circles) of 50 nM MPO. Reactions were performed at a constant apoA-I concentration of 10 mg/dL. Following oxidation, HAE was analyzed by EPR as described in materials and methods. Statistically significant differences are indicated (*P<0.05 and **P<0.01). Two-tailed Student's t-tests were performed on each pair of samples at their respective concentration.
Figure 4.
Progression of atherosclerosis is associated with reduced HAE in rabbits.
[A] HDL-ApoA-I exchange, normalized to HDL-C in rabbits fed normal rabbit chow, then switched to a high fat, high cholesterol diet (0.3% cholesterol, 4.7% coconut oil). Blood was collected at 2 months and 3 months following onset of the diet. HDL was isolated by ultracentrifugation from plasma and HAE was normalized to HDL-C concentration. P<0.0001, one-way ANOVA. [B] Atherosclerotic plaque area, as measured by histology analysis in the aortic arch. [C] Relationship between the change in HAE over months 2 and 3 on the high fat diet and the size of atherosclerotic plaque in the aortic arch. Correlation was determined by linear regression analysis using Pearson's correlation coefficient. [D] Total serum efflux capacity from J774 macrophage cells in rabbits on chow diet and after 3 months on the high fat, high cholesterol diet (P = 0.003). Statistical significance was determined by two-tailed Student's t-test.
Figure 5.
HDL-C, ApoA-I and HDL-ApoA-I exchange (HAE) measured in control and ACS subjects.
[A] HDL-C and [B] apoA-I levels in ACS and control subjects. Significant differences (P = 0.002) were observed between the two groups. [C] HAE measured in apoB-depleted plasma in control and ACS subjects. Closed circles are males and open circles are females. Statistical significance for [A–C] was determined by performing a two-tailed Student's t-tests.
Table 1.
Clinical characteristics of study subjects.
Figure 6.
Analysis of HAE in ACS and control subjects with respect to apoA-I and HDL-C levels.
[A] Analysis of HAE with respect to apoA-I for ACS (open circles) and control subjects (closed circles). HAE is highly correlated to apoA-I (r = 0.89; P<0.0001) and increases by 0.23±0.07% for every 1 mg/dL increase in apoA-I in both ACS and control subjects (P = 0.002). HAE of ACS subjects is on average 20±4% lower than control patients with the same apoA-I concentration. [B] Analysis of HAE with respect to HDL-C shows that HAE is also highly correlated to HDL-C (r = 0.90; P<0.0001). For ACS patients, HAE increases by 0.69±0.17% for every 1 mg/dL increase in HDL-C and 0.23±0.19% for control patients (P = 0.0006), but the difference between the slopes is not significant (P = 0.09). Statistical significance was determined by two-way ANOVA, and post hoc comparisons by custom tests of parameters. Interaction p-values greater than 0.20 were dropped in favor of pooled estimates.
Figure 7.
Comparison of HDL-ApoA-I exchange to ABCA1-mediated cholesterol efflux capacity in subjects with metabolic syndrome.
[A] In-plasma HAE is compared to ABCA1-mediated cholesterol efflux capacity in BHK cell expressing mifepristone-inducible human ABCA1 for subjects with metabolic syndrome (squares) and healthy controls (circles). Black circles or squares are males and open circles or squares are females. [B] Correlation of ABCA1-mediated cholesterol efflux and HAE, [C] HDL-C and HAE, and [D] HDL-C and ABCA1-mediated cholesterol efflux. Correlation was determined by linear regression analysis using Pearson's correlation coefficient.