Association of HDL cholesterol efflux capacity with incident coronary heart disease events: a prospective case-control study.

BACKGROUND: Although HDL cholesterol concentrations are strongly and inversely associated with risk of coronary heart disease, interventions that raise HDL cholesterol do not reduce risk of coronary heart disease. HDL cholesterol efflux capacity-a prototypical measure of HDL function-has been associated with coronary heart disease after adjusting for HDL cholesterol, but its effect on incident coronary heart disease risk is uncertain. METHODS: We measured cholesterol efflux capacity and assessed its relation with vascular risk factors and incident coronary heart disease events in a nested case-control sample from the prospective EPIC-Norfolk study of 25 639 individuals aged 40-79 years, assessed in 1993-97 and followed up to 2009. We quantified cholesterol efflux capacity in 1745 patients with incident coronary heart disease and 1749 control participants free of any cardiovascular disorders by use of a validated ex-vivo radiotracer assay that involved incubation of cholesterol-labelled J774 macrophages with apoB-depleted serum from study participants. FINDINGS: Cholesterol efflux capacity was positively correlated with HDL cholesterol concentration (r=0·40; p<0·0001) and apoA-I concentration (r=0·22; p<0·0001). It was also inversely correlated with type 2 diabetes (r=-0·18; p<0·0001) and positively correlated with alcohol consumption (r=0·12; p<0·0001). In analyses comparing the top and bottom tertiles, cholesterol efflux capacity was significantly and inversely associated with incident coronary heart disease events, independent of age, sex, diabetes, hypertension, smoking and alcohol use, waist:hip ratio, BMI, LDL cholesterol concentration, log-triglycerides, and HDL cholesterol or apoA-I concentrations (odds ratio 0·64, 95% CI 0·51-0·80). After a similar multivariable adjustment the risk of incident coronary heart disease was 0·80 (95% CI 0·70-0·90) for a per-SD change in cholesterol efflux capacity. INTERPRETATION: HDL cholesterol efflux capacity might provide an alternative mechanism for therapeutic modulation of the HDL pathway beyond HDL cholesterol concentration to help reduce risk of coronary heart disease. FUNDING: US National Institutes of Health, UK Medical Research Council, Cancer Research UK.

Macrophage sortilin promotes LDL uptake, foam cell formation, and atherosclerosis.

RATIONALE: Noncoding gene variants at the SORT1 locus are strongly associated with low-density lipoprotein cholesterol (LDL-C) levels, as well as with coronary artery disease. SORT1 encodes a protein called sortilin, and hepatic sortilin modulates LDL metabolism by targeting apolipoprotein B-containing lipoproteins to the lysosome. Sortilin is also expressed in macrophages, but its role in macrophage uptake of LDL and in atherosclerosis independent of plasma LDL-C levels is unknown. OBJECTIVE: To determine the effect of macrophage sortilin expression on LDL uptake, foam cell formation, and atherosclerosis. METHODS AND RESULTS: We crossed Sort1(-/-) mice onto a humanized Apobec1(-/-); hAPOB transgenic background and determined that Sort1 deficiency on this background had no effect on plasma LDL-C levels but dramatically reduced atherosclerosis in the aorta and aortic root. To test whether this effect was a result of macrophage sortilin deficiency, we transplanted Sort1(-/-);LDLR(-/-) or Sort1(+/+);LDLR(-/-) bone marrow into Ldlr(-/-) mice and observed a similar reduction in atherosclerosis in mice lacking hematopoetic sortilin without an effect on plasma LDL-C levels. In an effort to determine the mechanism by which hematopoetic sortilin deficiency reduced atherosclerosis, we found no effect of sortilin deficiency on macrophage recruitment or lipopolysaccharide-induced cytokine release in vivo. In contrast, sortilin-deficient macrophages had significantly reduced uptake of native LDL ex vivo and reduced foam cell formation in vivo, whereas sortilin overexpression in macrophages resulted in increased LDL uptake and foam cell formation. CONCLUSIONS: Macrophage sortilin deficiency protects against atherosclerosis by reducing macrophage uptake of LDL. Sortilin-mediated uptake of native LDL into macrophages may be an important mechanism of foam cell formation and contributor to atherosclerosis development.

Tissue-specific liver X receptor activation promotes macrophage reverse cholesterol transport in vivo.

OBJECTIVE: We previously reported that a systemic liver X receptor (LXR) agonist promoted macrophage reverse-cholesterol transport (mRCT) in vivo. Because LXR are expressed in multiple tissues involved in RCT (macrophages, liver, intestine), we analyzed the effect of tissue-specific LXR agonism on mRCT. METHODS AND RESULTS: In initial studies, the systemic LXR agonist GW3965 failed to promote mRCT in a setting in which LXR was expressed in macrophages but not in liver or intestine. To evaluate the effect of LXR activation specifically in small intestine on mRCT, wild-type mice were treated with either intestinal-specific LXR agonist (GW6340) or systemic LXR agonist (GW3965). Both GW3965 and GW6340 significantly promoted excretion of [(3)H]-sterol in feces by 162% and 52%, respectively. To evaluate the requirement for macrophage LXR activation, we assessed the ability of GW3965 to promote mRCT in wild-type mice using primary macrophages deficient in LXR alpha/beta vs wild-type macrophages. Whereas GW3965 treatment promoted fecal excretion compared with vehicle, its overall ability to promote mRCT was significantly attenuated using LXR alpha/beta knockout macrophages. CONCLUSIONS: We demonstrate that intestinal-specific LXR agonism promotes macrophage RCT in vivo and that macrophage LXR itself plays an important, but not predominant, role in promoting RCT in response to an LXR agonist.

Fish oil promotes macrophage reverse cholesterol transport in mice.

OBJECTIVE: Fish oil (FO), and specifically omega 3 fatty acids, has favorable effects on cardiovascular outcomes. The aim of this study was to investigate the effects of FO on the process of macrophage reverse cholesterol transport (RCT) in an in vivo mouse model. METHODS AND RESULTS: C57BL/6J mice were fed a FO diet, whereas control mice were fed diets containing alternative sources of fats, soybean oil (SO), and coconut oil (CO) for 4 weeks. Macrophage RCT was assessed by injecting [(3)H]cholesterol-labeled J774 macrophages intraperitoneally into mice. After 48 hours, tissues were harvested and feces were collected. An increase in the excretion of macrophage-derived [(3)H]-tracer recovered in fecal neutral sterols for FO-fed mice was observed (273% versus SO and 182% versus CO). FO also decreased [(3)H]-tracer in hepatic cholesteryl ester compared to SO and CO by 76% and 56%, respectively. To specifically determine the effect of FO on the fate of HDL-derived cholesterol, mice fed FO or SO diets were injected with HDL labeled with [(3)H]cholesteryl oleate, and the disappearance of [(3)H]-tracer from blood and its excretion in feces was measured. There was no significant difference in the fractional catabolic rate of [(3)H]cholesteryl oleate-HDL between the 2 groups. However, there was a 242% increase in the excretion of HDL-derived [(3)H]-tracer recovered in fecal neutral sterols in FO-fed mice, concordant with significantly increased expression of hepatic Abcg5 and Abcg8 mRNA. CONCLUSIONS: As measured by this tracer-based assay, FO promoted reverse cholesterol transport, primarily by enhancement of the hepatic excretion of macrophage-derived and HDL-derived cholesterol.

Flaxseed reduces plasma cholesterol levels in hypercholesterolemic mouse models.

OBJECTIVE: We examined the effects of whole ground flaxseed added to a Western diet on plasma and hepatic lipids and hepatic gene expression in male and female human apolipoprotein B-100 transgenic (hApoBtg) mice which have a plasma lipid profile more closely resembling man than wild type mice and in mice lacking the low density lipoprotein receptor (LDLr) and apolipoprotein B mRNA editing enzyme complex 1 (LDLr(-/-)/apobec(-/-)). METHODS: The Westernized control diet containing 0.1% cholesterol and 30% kcal as fat was fed for 10 days to hApoBtg mice and for 14 days to LDLr(-/-)/apobec(-/-) mice. Animals from each genetic background were then divided into 2 groups based on gender and mean plasma total cholesterol (TC). The hApoBtg and LDLr(-/-)/apobec(-/-) mice either continued on the control diet for a total of 31 and 35 days, respectively or were fed 20% w/w whole ground flaxseed (flax) with comparable caloric, macronutrient and fiber content for 21 days. Blood was obtained after a 4 hour fast from all mice prior to feeding both control and flax diets, after 10 days on the flax diet, and after 21 days on the flax at which time all mice were exsanguinated. RESULTS: The control diet increased TC by >100 mg/dl in the hApoBtg with a greater increase observed in males and by 800 mg/dl in mice lacking the LDLr. After 3 weeks, the flax diet significantly reduced plasma TC by 19% and 22% in hApoBtg and LDLr(-/-)/apobec(-/-), respectively and non-high density lipoprotein cholesterol (non-HDL-C) by 24% in both models (p for all <0.05). Flax significantly reduced hepatic cholesterol in hApoBtg by 32% and 47% in males and females, respectively and LDLr(-/-)/apobec(-/-) mice by 66%. Flax had no effect on the expression of the following hepatic genes: LDLr, 3-hydroxy-3-methylglutaryl (HMG) CoA reductase, phospholipid transfer protein, cholesterol 7alpha hydroxylase, fatty acid synthase, and acyl CoA oxidase in either mouse model. CONCLUSIONS: Flaxseed reduces plasma and hepatic cholesterol in hApoBtg mice, but had no effect on hepatic lipogenic genes and was equally effective in mice lacking LDLr. The combined data suggest that the lipid lowering effect of flax is not hepatic mediated and may be at the level of cholesterol absorption and/or bile acid reabsorption.