Joel D. Morrisett

A molecular theory of lipid—protein interactions in the plasma lipoproteins

Lipid-protein interactions are of fundamental importance in the structure of biological membranes and of plasma lipoproteins. From previous studies [1-3] it seems reasonable to assume that the interactions between phospholipids and lipoprotein-protein (apoprotein) constituents are fundamental to the binding of neutral lipid by the plasma lipoproteins. For example, there is an insignificant binding of cholesteryl ester by the apoproteins of human HDL * in the absence of phospholipids. We have recently presented studies describing the probable location of phospholipid-binding site(s) in the MN-glycoprotein of the human red cell membrane [4-6] and of plasma lipoproteins [7-12]. Phospholipid-binding regions do not appear to be uniformly distributed along the length of the polypeptide chain [7-11,13] ; certain fragments of apoLP-Ala, apoLP-Gln-I and apoLP-Gln-II preferentially bind phospholipid as corn-

Quantitation and localization of apolipoproteins [a] and B in coronary artery bypass vein grafts resected at re-operation.

Lp[a] is a lipoprotein whose plasma concentration is highly correlated with cardiovascular disease. Its protein moiety, apoLp[a], consists of two large polypeptides, apo[a] and apo B. The possible contribution of Lp[a] to atherosclerosis in saphenous vein aortocoronary bypass grafts was studied in a population of patients undergoing coronary re-bypass surgery. The vein graft tissue levels of apoLp[a] were compared with graft duration, gross and light microscopic pathology, as well as plasma levels of apoLp[a]. The localization pattern of apo[a] and apo B in vein graft tissue was determined. In addition, the plasma levels of cholesterol, triglycerides, apoproteins (apo) A-I, A-II, and E were measured. In a representative subpopulation of 17 patients with a mean age of 63 years from whom grafts with a mean duration of 112 months were resected, the mean total plasma cholesterol level was 221 mg/dl, the mean high density lipoprotein cholesterol level was 31 mg/dl, and the mean plasma triglyceride level was 228 mg/dl. In normal saphenous veins, the level of apoLp[a] was below measurable limits (less than 2 ng/mg), and the level of apo B was very low (3.3 ng/mg). In resected grafts, the mean tissue level of apoLp[a] was 32 ng/mg, and that of apo B was 70 ng/mg, demonstrating the net accumulation of these apoproteins in veins from the time of their grafting into the arterial bed. The apoLp[a]/apo B ratio was determined in 77 tissue segments from 59 grafts (28 patients) and was found to be 0.313. This tissue ratio was significantly higher (p = 0.02) than the plasma apoLp[a]/apo B ratio from these patients, which was 0.132. Immunostaining showed co-localization of apo[a] and apo B in the neointima of grafts. The most abundant pathologic features observed in resected grafts were proliferated intima (43/52), thrombus (28/52), and atherosclerotic core regions (21/52). The level of tissue apo B correlated well with the abundance of core regions (r = 0.501), whereas the level of tissue apoLp[a] did not correlate as well with this feature (r = 0.233). Although apo[a] and apo B are almost absent from normal saphenous vein, these apoproteins (and presumably the lipoproteins Lp[a] and low density lipoprotein) accumulate in bypass vein grafts. The data support the view that these lipoproteins play a significant role in vein graft atherosclerosis.

Hypertriglyceridemic Very Low Density Lipoproteins Induce Triglyceride Synthesis and Accumulation in Mouse Peritoneal Macrophages

Triglyceride-rich lipoproteins may be responsible for the lipid accumulation in macrophages that can occur in hypertriglyceridemia. Chylomicrons and very low density lipoproteins (VLDL, total and with flotation constant [S(f)] 100-400) from fasting hypertriglyceridemic subjects induced a massive accumulation of oil red O-positive inclusions in unstimulated peritoneal macrophages. Cell viability was not affected. The predominant lipid that accumulated in cells exposed to hypertriglyceridemic VLDL was triglyceride. Hypertriglyceridemic VLDL stimulated the incorporation of [(14)C]oleate into cellular triglyceride up to ninefold in 16 h, but not into cholesteryl esters. Mass increase in cellular triglyceride was 38-fold. The stimulation of cellular triglyceride formation was dependent on time, temperature, and concentration of hypertriglyceridemic VLDL. By contrast, VLDL, low density, and high density lipoproteins from fasting normolipemic subjects had no significant effect on oleate incorporation into neutral lipids or on visible lipid accumulation.(125)I-Hypertriglyceridemic VLDL (S(f) 100-400) were degraded by macrophages in a dose-dependent manner, with 50 and 100% saturation observed at 3 and 24 mug protein/ml (2.5 and 20 nM), respectively. Hypertriglyceridemic VLDL inhibited the internalization and degradation of (125)I-hypertriglyceridemic VLDL (4 nM) by 50% at 3 nM. Cholesteryl ester-rich VLDL from cholesterol-fed rabbits gave 50% inhibition at 5 nM. Low density lipoproteins (LDL) inhibited by 10% at 5 nM and 40% at 47 nM. Acetyl LDL at 130 nM had no effect. We conclude that the massive triglyceride accumulation produced in macrophages by hypertriglyceridemic VLDL is a direct consequence of uptake via specific receptors that also recognize cholesteryl ester-rich VLDL and LDL but are distinct from the acetyl LDL receptor. Uptake of these triglyceride-rich lipoproteins by monocyte-macrophages in vivo may play a significant role in the pathophysiology of atherosclerosis.

Effect of sirolimus on the metabolism of apoB100- containing lipoproteins in renal transplant patients.

BACKGROUND Sirolimus (Rapamune, rapamycin, RAPA) is a potent immunosuppressive drug that has reduced the rate of acute rejection episodes by more than 40% in phase III trials when added to an immunosuppression regimen of cyclosporine (CsA) and prednisone. However, RAPA treatment tends to increase lipid levels, particularly among patients with pre-existing hyperlipidemia. METHODS To identify the metabolic pathway(s) leading to RAPA-mediated hyperlipidemia, five patients with renal transplants maintained on CsA+/-prednisone+/- azathioprine (AZA) were studied before and after 6 weeks of treatment with RAPA (off RAPA and on RAPA, respectively). Each study patient was infused with a single bolus of [2H4]-lysine to derive metabolic parameters for apoB100-containing lipoproteins by using kinetic analysis based upon quantitation of isotopic enrichment by gas chromatography-mass spectrometry. RESULTS Serial lipid measurements revealed that four patients displayed increased plasma triglyceride levels after RAPA treatment, which coincided with significantly higher plasma VLDL-apoB100 concentrations (21.7+/-12.1 mg/dl off RAPA vs. 38.7+/-14.8 mg/dl on RAPA, mean+/-SD, P<0.05). Kinetic analysis showed that the RAPA-induced increase in VLDL-apoB100 concentrations was due to a significant reduction in the fractional catabolic rate (FCR) of very low-density lipoprotein (VLDL) apoB100 (0.83+/-0.65 off RAPA vs. 0.24+/-0.10 on RAPA, mean+/-SD, P<0.05), rather than an enhanced VLDL-apoB100 synthesis. In one patient, RAPA treatment induced hypercholesterolemia but not hypertriglyceridemia. This hypercholesterolemia was due to elevated low-density lipoprotein (LDL) cholesterol levels, which coincided with a decreased FCR of LDL-apoB100. Heparin-induced lipoprotein lipase activity was significantly lower in the immunosuppressed hyperlipidemic patients than in normolipidemic controls. However, RAPA treatment did not significantly alter basal lipoprotein lipase activity in renal transplant patients in this study. CONCLUSIONS This study indicates that for renal transplant patients in whom RAPA treatment induces hyperlipidemia, this effect is the result of reduced catabolism of apoB100-containing lipoproteins.

Sample Size Calculation for Clinical Trials Using Magnetic Resonance Imaging for the Quantitative Assessment of Carotid Atherosclerosis

PURPOSE To provide sample size calculation for the quantitative assessment of carotid atherosclerotic plaque using non-invasive magnetic resonance imaging in multi-center clinical trials. METHODS. As part of a broader double-blind randomized trial of an experimental pharmaceutical agent, 20 asymptomatic placebo-control subjects were recruited from 5 clinical sites for a multi-center study. Subjects had 4 scans in 13 weeks on GE 1.5 T scanners, using TOF, T1-/PD-/T2- and contrast-enhanced Tl-weighted images. Measurement variability was assessed by comparing quantitative data from the index carotid artery over the four time points. The wall/outer wall (W/OW) ratio was calculated as wall volume divided by outer wall volume. The percent lipid-rich/necrotic core (%LR/NC) and calcification (%Ca) were measured as a proportion of the vessel wall. For %LR/NC and %Ca, only those subjects that exhibited LR/NC or Ca components were used in the analysis. RESULTS Measurement error was 5.8% for wall volume, 3.2% for W/OW ratio, 11.1% for %LR/NC volume and 18.6% for %Ca volume. Power analysis based on these values shows that a study with 14 participants in each group could detect a 5% change in W/OW ratio, 10% change in wall volume, and 20% change in %LR/NC volume (power = 80%, p < .05). The calculated measurement errors presume any true biological changes were negligible over the 3 months that subjects received placebo. CONCLUSION In vivo MRI is capable of quantifying plaque volume and plaque composition, such as %lipid-rich/necrotic core and %calcification, in the clinical setting of a multi-center trial with low inter-scan variability. This study provides the basis for sample size calculation of future MRI trials.

Associations Between Lipoprotein(a) Levels and Cardiovascular Outcomes in Black and White Subjects The Atherosclerosis Risk in Communities (ARIC) Study

Background— On the basis of studies with limited statistical power, lipoprotein(a) [Lp(a)] is not considered a risk factor for cardiovascular disease (CVD) in blacks. We evaluated associations between Lp(a) and incident CVD events in blacks and whites in the Atherosclerosis Risk in Communities (ARIC) study. Methods and Results— Plasma Lp(a) was measured in blacks (n=3467) and whites (n=9851). Hazards ratios (HRs) for incident CVD events (coronary heart disease and ischemic strokes) were calculated. Lp(a) levels were higher with wider interindividual variation in blacks (median [interquartile range], 12.8 [7.1–21.7] mg/dL) than whites (4.3 [1.7–9.5] mg/dL; P<0.0001). At 20 years of follow-up, 676 CVD events occurred in blacks, and 1821 events occurred in whites. Adjusted HRs (95% confidence interval) per race-specific 1-SD–greater log-transformed Lp(a) were 1.13 (1.04–1.23) for incident CVD, 1.11 (1.00–1.22) for incident coronary heart disease, and 1.21 (1.06–1.39) for ischemic strokes in blacks. For whites, the respective HRs (95% confidence intervals) were 1.09 (1.04–1.15), 1.10 (1.05–1.16), and 1.07 (0.97–1.19). Quintile analyses showed that risk for incident CVD was graded but statistically significant only for the highest compared with the lowest quintile (HR [95% confidence interval], 1.35 [1.06–1.74] for blacks and 1.27 [1.10–1.47] for whites). Similar results were obtained with the use of Lp(a) cutoffs of ⩽10 mg/dL, >10 to ⩽20 mg/dL, >20 to ⩽30 mg/dL, and >30 mg/dL. Conclusions— Lp(a) levels were positively associated with CVD events. Associations were at least as strong, with a larger range of Lp(a) concentrations, in blacks compared with whites.

Race and Gender Differences in the Association of Lp(a) With Carotid Artery Wall Thickness The Atherosclerosis Risk in Communities (ARIC) Study

The association of lipoprotein(a) [Lp(a)] with preclinical atherosclerotic disease is not well established in any race group, particularly African Americans. This report examined the association of Lp(a) with preclinical extracranial carotid atherosclerosis in middle-aged black and white participants in the Atherosclerosis Risk in Communities (ARIC) Study. Study participants (15 124: 2417 black women, 1522 black men, 5907 white women, and 5278 white men) who were 45 to 64 years old at baseline were examined during the period 1987 to 1989. Carotid intimal-medial far-wall thickness was determined by B-mode ultrasonography and expressed as the overall wall thickness mean at six sites to approximate atherosclerosis in the carotid system. Lp(a) was measured as its total protein component, Lp(a) protein, by a double-antibody ELISA for apolipoprotein(a) detection. Mean Lp(a) protein levels were higher in blacks than whites (169.1 and 147.0 microgram/mL in black women and black men, respectively, compared with 86.6 and 75.1 micrograms/mL in white women and white men). Mean carotid wall thickness (in millimeters) varied by race and gender: 0.798 in white men, 0.779 in black men, 0.718 in black women and 0.695 in white women. Multivariable-adjusted Lp(a) protein was independently associated with wall thickness (in millimeters) in white men and black men; among women, however, this association appeared to be stronger when smoking and diabetes were present. A 100-microgram/mL difference in Lp(a) protein was associated with 0.049- and 0.043-mm higher wall thickness values in black men and white men, respectively. Among white women who smoked, the difference in wall thickness was 0.051 mm compared with 0.032 mm for former/never smokers and 0.21 mm in black female diabetics compared with 0.031 mm in black female nondiabetics. These results suggest that Lp(a) is associated with preclinical carotid atherosclerosis in both blacks and whites, but that this association may be affected by the presence of other cardiovascular risk factors, particularly in women.

Lipoprotein(a) and Incident Ischemic Stroke: The Atherosclerosis Risk in Communities (ARIC) Study

Background and Purpose— Numerous case-control and cross-sectional studies have reported higher median lipoprotein(a) [Lp(a)] levels among stroke patients than controls, but existing prospective studies have not consistently shown an association. We sought to examine the relationship between plasma Lp(a) levels and the incidence of ischemic stroke among blacks and whites. Methods— Between 1987 and 1989, 14 221 men and women (3647 blacks and 10 574 whites) aged 45 to 64 years and free of clinical cardiovascular disease, took part in the first examination of the Atherosclerosis Risk in Communities (ARIC) study cohort. Lp(a) and other risk factors for cardiovascular disease were measured at baseline. Results— During the 13.5-year follow-up, 496 ischemic strokes occurred. Participants with Lp(a) ≥300 &mgr;g/mL had a 79% higher age, sex, and race-adjusted rate ratio (RR) of ischemic stroke than did those with Lp(a) levels <100 &mgr;g/mL. Compared with Lp(a) <100 &mgr;g/mL, the multivariate adjusted RRs for Lp(a) ≥300 &mgr;g/mL were 1.84 (95% CI, 1.05 to 3.07) in black women, 1.72 (95% CI, 0.86 to 3.48) in black men, 2.42 (95% CI, 1.30 to 4.53) in white women, and 1.18 (95% CI, 0.47 to 2.90) in white men. There was no significant increment in the RRs for 100 to 199 &mgr;g/mL and 200 to 299 &mgr;g/mL groups. Conclusions— A high Lp(a) concentration is associated with a higher incidence of ischemic stroke in blacks and white women, but not in white men.

Quantitation and Localization of Matrix Metalloproteinases and Their Inhibitors in Human Carotid Endarterectomy Tissues

Background—Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) play a central role in arterial wall remodeling, affecting stability of fibrous caps covering atherosclerotic plaques. The objective of this study was to determine the spatial distribution of TIMP mass and MMP mass and activity of carotid endarterectomy (CEA) tissues and relate it to the distribution of atherosclerotic lesions. Methods and Results—Fresh CEA tissues were imaged by multicontrast MRI to generate 3D reconstructions. Tissue segments were cut transversely from the common, bifurcation, internal, and external regions. Segments were subjected to total protein extractions and analyzed by ELISA for MMP-2 and -9 and TIMP-1 and -2 mass and by zymography for gelatinase activity. Segments at or near the bifurcation with highly calcified lesions contained higher MMP levels and activity than segments distant from the bifurcation; highly fibrotic or necrotic plaque contained lower MMP levels and activity and higher TIMP levels. Fatty streak, fibroatheroma with hemorrhage and calcification, and fully occluded lesions were enriched in MMP-2, MMP-9, and TIMP-1 and TIMP-2, respectively. Conclusion—The spatial distribution of MMPs and TIMPs in carotid atherosclerotic lesions is highly heterogeneous, reflecting lesion location, size, and composition. This study provides the first semi-quantitative maps of differential distribution of MMPs and TIMPs over atherosclerotic plaques.

The relation of lipoprotein[a] concentrations and apolipoprotein[a] phenotypes with asymptomatic atherosclerosis in subjects of the Atherosclerosis Risk in Communities (ARIC) Study.

Plasma levels of lipoprotein[a] (Lp[a]) are associated with increased risk of coronary artery disease and show an inverse correlation with apolipoprotein[a] (apo[a]) molecular weight. We determined Lp[a] levels and apo[a] phenotypes in 171 cases with preclinical extracranial carotid atherosclerosis as ascertained by B-mode ultrasound and in 274 control subjects free of carotid atherosclerosis. Lp[a] protein levels measured by enzyme-linked immunosorbent assay ranged from 4 to 361 micrograms/mL in cases and from 2 to 392 micrograms/mL in controls, but median levels of Lp[a] were higher in cases than in controls (51 micrograms/mL versus 33 micrograms/mL, P < .003). In both groups, all 11 apo[a] polymorphs that are resolved by the procedure used were present, resulting in 43 and 39 different apo[a] phenotypes in cases and controls, respectively. An inverse relation between apo[a] polymorph size and Lp[a] level was observed in both cases (r = -0.49, P < .001) and controls (r = -0.34, P < .001). Apo[a] phenotype distributions were similar in cases and controls. However, in 17 phenotypes with three or more subjects per group, the difference of mean Lp[a] concentrations between cases and controls was 32 +/- 36 micrograms/mL (mean +/- SD). Thus, the higher Lp[a] levels in cases were not associated with a greater prevalence of small apo[a] polymorphs. Stepwise logistic regression analyses of known risk factors for coronary heart disease showed that plasma Lp[a] concentration was an independent predictor of case-control status, while Lp[a] phenotype was not, irrespective of the presence or absence of Lp[a] concentration in the model.(ABSTRACT TRUNCATED AT 250 WORDS)