The ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cellular cholesterol efflux and HDL maturation. ABCA1 mRNA has an unusually long 3’ untranslated region, which makes it highly susceptible to microRNA (miRNA) targeting and repression. As such, multiple miRNAs have been reported to directly target ABCA1, including miR-33a/b, miR-26, miR-106b, and miR-758. Many of these miRNAs participate in feed-forward or feedback networks in controlling cholesterol and lipid homeostasis. Antisense oligonucleotide-based inhibition of miR-33 was found to increase HDL-C levels and regress atherosclerosis in mice and non-human primates. In this edition of Circulation Research, two separate studies identified novel miRNA networks driven by nuclear receptor induced miR-144 targeting of ABCA1 and cholesterol efflux. The first study reports that miR-144 serves to buffer uncontrolled ABCA1 activation in response to high cholesterol states and liver X receptor (LXR) activation in macrophages and liver. The second study highlights the role of miR-144 and ABCA1 in promotion of bile acid secretion in response to farensoid X receptor (FXR) activation in the liver. These studies suggest that anti-miR-144, like anti-miR-33, could be a novel approach to targeting HDL and reverse cholesterol transport.
Editorials; high-density lipoprotein cholesterol; lipoproteins; obesity; adipose tissue; Reverse Cholesterol Transport
The goal of this study was to understand the molecular basis for how the amino acid substitution C112R that distinguishes human apolipoprotein (apo) E4 from apoE3 causes the more pro-atherogenic plasma lipoprotein cholesterol distribution that is known to be associated with expression of apoE4.
Methods and Results
Adeno-associated viruses, serotype 8 (AAV8) were used to express different levels of human apoE3, apoE4 and several C-terminal truncation and internal deletion variants in C57BL/6 apoE-null mice which exhibit marked dysbetalipoproteinemia. Plasma obtained from these mice two weeks after the AAV8 treatment was analyzed for cholesterol and triglyceride levels as well as for the distribution of cholesterol between the lipoprotein fractions. Hepatic expression of apoE3 and apoE4 induced similar dose-dependent decreases in plasma cholesterol and triglyceride to the levels seen in control C57BL/6 mice. Importantly, at the same reduction in plasma total cholesterol, expression of apoE4 gave rise to higher very low density lipoprotein-cholesterol (VLDL-C) and lower high density lipoprotein-cholesterol (HDL-C) levels relative to the apoE3 situation. The C-terminal domain, and residues 261-272 in particular, play a critical role because deleting them markedly affected the performance of both isoforms.
ApoE4 possesses enhanced lipid and VLDL binding ability relative to apoE3 which gives rise to impaired lipolytic processing of VLDL in apoE4-expressing mice. These effects reduce VLDL remnant clearance from the plasma compartment and decrease the amount of VLDL surface components available for incorporation into the HDL pool, accounting for the more pro-atherogenic lipoprotein profile (higher VLDL-C/HDL-C ratio) occurring in apoE4-expressing animals compared to their apoE3 counterparts.
apolipoprotein E; cholesterol; high density lipoprotein; very low density lipoprotein; atherosclerosis
In addition to its effects on cholesterol levels, apoE3 has lipid-independent effects that contribute to cardiovascular protection; one of these effects is the ability to inhibit cell cycling in VSMCs. The goal of this study was to identify and characterize cell cycle-regulatory mechanisms responsible for the anti-mitogenic effect of apoE.
Methods and results
Primary VSMCs were stimulated with serum in the absence or presence of apoE3. apoE3 upregulated expression of the cdk inhibitor, p27kip1, in primary VSMCs, and this effect required Cox2 and activation of PGI2-IP signaling. The microRNA family, miR221/222 has recently been identified as a post-translational regulator of p27, and apoE3 inhibited miR221/222 expression in a Cox2- and PGI2/IP-dependent manner. Moreover, reconstituted miR222 expression was sufficient to override the effects of apoE on p27 expression and S phase entry. The ability to repress expression of miR221/222 is shared by apoE3-containing HDL but is absent from apoA-1, LDL and apoE-depleted HDL. All three apoE isoforms regulate miR221/222, and the effect is independent of the C-terminal lipid-binding domain. miR221/222 levels are increased in the aortae of apoE3-null mice and reduced when apoE3 expression is reconstituted by adeno-associated virus infection. Thus, regulation of miR221/222 by apoE3 occurs in vivo as well as in vitro.
poE inhibits VSMC proliferation by regulating p27 through miR221/222. Control of cell cycle-regulatory microRNAs adds a new dimension to the spectrum of cardiovascular protective effects afforded by apoE and apoE-HDL.
ApoE3; HDL; PGI2; p27; miR221/222; VSMC proliferation
Familial hypercholesterolemia (FH) is a life-threatening genetic disease caused by mutations in the gene encoding low-density lipoprotein receptor (LDLR). As a bridge to clinical trials, we generated a “humanized” mouse model lacking LDLR and apolipoprotein B (ApoB) mRNA editing catalytic polypeptide-1 (APOBEC-1) expression and expressing a human ApoB100 transgene in order to permit more authentic simulation of in vivo interactions between the clinical transgene product, human LDLR (hLDLR), and its endogenous ligand, human ApoB100. On a chow diet, the humanized LDLR-deficient mice have substantial hypercholesterolemia and a lipoprotein phenotype more closely resembling human homozygous FH (hoFH) than in previous mouse models of FH. On injection of an adeno-associated virus serotype 8 (AAV8) vector encoding the human LDLR cDNA, significant correction of hypercholesterolemia was realized at doses as low as 1.5×1011 genome copies (GC)/kg. Given that some patients with heterozygous FH (heFH) cannot be adequately treated with current therapy, we then extended our studies to similarly “humanized” mice that were heterozygous for LDLR deficiency, and that have a lipoprotein phenotype resembling heterozygous FH. Injection of AAV8-hLDLR brought about significant reduction in total and LDL cholesterol at doses as low as 5×1011 GC/kg. Collectively, these data demonstrate the safety and efficacy of the liver-specific AAV8-hLDLR vector in the treatment of humanized mice modeling both hoFH and heFH.
Kassim and colleagues demonstrate that injection of an adeno-associated virus serotype 8 (AAV8) vector encoding the human low-density lipoprotein receptor (LDLR) cDNA results in significant correction of hypercholesterolemia in humanized mouse models of homozygous and heterozygous familial hypercholesterolemia (FH).
Recent studies involving HDL-raising therapeutics have greatly changed our understanding of this field. Despite effectively raising HDL-C levels, niacin remains of uncertain clinical benefit. Synthetic niacin receptor agonists are unlikely to raise HDL-C or have other beneficial effects on plasma lipids. Despite the failure in phase 3 of two CETP inhibitors, two potent CETP inhibitors that raise HDL-C levels by > 100% (and reduce LDL-C substantially) are in late stage clinical development. Infusions of recombinant HDL containing ‘wild-type’ apoA-I or apoA-I Milano, as well as autologous delipidated HDL, all demonstrated promising early results and remain in clinical development. A small molecule that causes upregulation of endogenous apoA-I production is also in clinical development. Finally, upregulation of macrophage cholesterol efflux pathways through agonism of liver X receptors or antagonism of miR-33 remains of substantial interest. The field of HDL therapeutics is poised to transition from the ‘HDL-cholesterol hypothesis’ to the ‘HDL flux hypothesis’ in which the impact on flux from macrophage to feces is deemed to be of greater therapeutic benefit than the increase in steady-state concentrations of HDL cholesterol.
Lipids; HDL; reverse cholesterol transport; niacin; GPR109A; cholesteryl ester transfer protein; CETP; anacetrapib; torcetrapib; dalcetrapib; evacetrapib; apoA-I; recombinant HDL; RVX-208; Liver X receptor; miR-33; cardiovascular disease
Atherosclerosis is a complex and heritable disease involving multiple cell types and the interactions of many different molecular pathways. The genetic and molecular mechanisms of atherosclerosis have in part been elucidated by mouse models; at least 100 different genes have been shown to influence atherosclerosis in mice. Importantly, unbiased genome-wide association studies have recently identified a number of novel loci robustly associated with atherosclerotic coronary artery disease (CAD). Here we review the genetic data elucidated from mouse models of atherosclerosis, as well as significant associations for human CAD. Furthermore, we discuss in greater detail some of these novel human CAD loci. The combination of mouse and human genetics has the potential to identify and validate novel genes that influence atherosclerosis, some of which may be candidates for new therapeutic approaches.
CAD; Lipids; Mice; GWAS; Genome-wide
Inflammation is a critical component of atherosclerosis. IL-1 is a classic proinflammatory cytokine that has been linked to atherosclerosis. A clinical trial has been launched in which an antibody specific for IL-1β is being studied for its effects on cardiovascular events in patients with atherosclerosis. In this issue of the JCI, Alexander et al. report that mice lacking the receptor for IL-1 unexpectedly have features of advanced atherosclerosis that suggest the atherosclerotic plaques may be less stable. These findings illustrate the complexity of inflammatory pathways in atherosclerosis and suggest the need for careful calibration of antiinflammatory approaches to atherosclerosis.
The effect of weight loss on obesity-associated endothelial dysfunction is not clear because of conflicting data, demonstrating both improvement and no change in endothelial function after weight loss in obese subjects.
A two-year prospective study (n=121) was conducted to examine: 1) the effect of obesity and weight loss (either a low-carbohydrate or and low-fat diet) on flow mediated vasodilatation (FMD), a measure of endothelial function.
Participants reduced body weight by 7.1±4.4%, 8.7±6.8% 7.1±7.8% and 4.1±7.7% at 3, 6, 12 and 24 months, respectively with no significant differences between the low-fat and low-carbohydrate groups. Endothelial function was inversely correlated with waist circumference, triglyceride level, and directly correlated with leptin in obese persons prior to weight loss. These weight losses did not confer any improvements in FMD. There were no differences between the low-fat and low-carbohydrate diets in FMD at any time point. At 6 months (r = 0.26, p = 0.04) and one year (r = 0.28, p = 0.03), there were positive correlations between change in FMD and change in leptin but not at two years.
There was no significant improvement in endothelial function after 7.1±7.8% weight loss at one year and 4.1±7.7% at two years, achieved by either a low carbohydrate or a low fat diet.
Endothelium; Obesity; Leptin; Diet
Psoriasis is a Th-1/17 mediated inflammatory disease associated with increased risk of cardiovascular disease (CVD). Inflammation may modulate lipoprotein particle number and directly impair HDL functions, in particular reverse cholesterol transport (RCT). We sought to study how chronic in vivo inflammation modulates lipoprotein particle composition using nuclear magnetic resonance spectroscopy (NMR) and HDL efflux in psoriasis.
Methods and Results
We prospectively enrolled a consecutive sample of patients with psoriasis (n=122) and compared lipoprotein and metabolic risk factors to patients without psoriasis (n=134). Fasting lipids, insulin, glucose were measured by standard assays, and lipoprotein concentration and size were measured by NMR. In a random subset (n=100 each group), HDL efflux capacity was quantified using a validated ex vivo system involving the incubation of macrophages with apolipoprotein B-depleted serum from patients. Traditional lipid concentrations were similar in both groups except for HDL concentration which was lower in psoriasis (43 mg/dL (36–58) vs 50 (42–62), p<0.01). However, NMR showed an atherogenic profile in psoriasis similar to that observed in diabetes, with significant increase in LDL particle concentration [1210.5 (1002–1498) vs 1115 (935–1291), p=0.03] with decrease in LDL size [20.6 (20.3–21.1) vs 21.3 (20.6–21.1), p<0.001] beyond CV risk factors and HOMA-IR (p=0.001). Finally, HDL efflux capacity was lower in psoriasis compared to controls in fully adjusted models (beta −0.14, p=0.001).
These data support a more atherogenic lipoprotein profile by NMR and decreased HDL efflux capacity in psoriasis patients compared to controls beyond CVD risk factors. The abnormal lipoprotein particle composition and HDL efflux capacity in psoriasis may provide a link between psoriasis and CVD.
inflammation; atherosclerosis; HDL efflux; cholesterol; lipoprotein particles
ADAMTS13 cleaves von Willebrand factor (VWF), thereby modulating thrombosis and inflammation. Low plasma ADAMTS13 activity is associated with cardiovascular events including myocardial and cerebral infarction. Here, we investigated the role of ADAMTS13 in the development of early atherosclerosis in a murine model.
Methods and Results
ApoE−/− and Adamts13−/−ApoE−/− mice were fed with a high fat Western diet for 12 weeks. Atherosclerotic lesions in the aorta and aortic roots were quantified after staining. Leukocyte rolling and adhesion onto cremaster venules after oxidative injury were determined by intravital microscopy. While plasma cholesterol levels were largely similar in both groups, the extent of atherosclerotic lesions in the aorta en face and in the aortic roots in the Adamts13−/−ApoE−/− mice increased ~5.5 fold (p=0.0017) and ~6.1 fold (p=0.0037), respectively. Also, the ratio of plasma high to low molecular weight VWF multimers increased ~3 fold. The leukocyte rolling velocities were significantly reduced (p<0.001) with an increased number of leukocyte rolling (p=0.0026) and macrophage infiltration into the atherosclerotic lesions in the Adamts13−/−ApoE−/− mice.
Our results suggest that ADAMTS13 plays a critical role in modulating the development of early atherosclerosis, likely through proteolytic cleavage of ultra large VWF multimers, thereby inhibiting platelet deposition and inflammation.
von Willebrand factor cleaving protease; inflammation; and animal model
Peroxisome proliferator-activate receptorα (PPARα) activation has been shown in vitro to increase macrophage cholesterol efflux, the initial step in reverse cholesterol transport (RCT). However, it remains unclear whether PPARα activation promotes macrophage RCT in vivo.
Methods and Results
We demonstrated that a specific potent PPARα agonist GW7647 inhibited atherosclerosis and promoted macrophage RCT in hypercholesterolemic mice expressing the human apoA-I gene. We compared the effect of GW7647 on RCT in human apoA-I transgenic (hA-ITg) mice with wild-type (WT) mice and showed that the PPARα agonist promoted RCT in hA-ITg mice to a much greater extent than in WT mice, indicating that human apoA-I expression is important for PPARα-induced RCT. We further investigated the dependence of the macrophage PPARα-LXR pathway on the promotion of RCT by GW7647. Primary murine macrophages lacking PPARα or LXR abolished the ability of GW7647 to promote RCT in hA-ITg mice. In concert, the PPARα agonist promoted cholesterol efflux and ABCA1/ABCG1 expression in primary macrophages and this was also by the PPARα-LXR pathway.
Our observations demonstrate that a potent PPARα agonist promotes macrophage RCT in vivo in a manner that is enhanced by human apoA-I expression and dependent on both macrophage PPARα and LXR expression.
PPARα; LXR; cholesterol efflux; reverse cholesterol transport; apolipoprotein A-I
The niacin receptor GPR109A is a Gi-protein coupled receptor which mediates the effects of niacin on inhibiting intracellular triglyceride lipolysis in adipocytes. However, the role of GPR109A in mediating the effects of niacin on high density lipoprotein (HDL) metabolism is unclear. We found niacin has no effect on HDL-C in GPR109A knockout mice. Furthermore, niacin lowered intracellular cAMP in primary hepatocytes mediated by GPR109A. We used an adeno-associated viral (AAV) serotype 8 vector encoding GPR109A under the control of the hepatic-specific thyroxine-binding globulin promoter to specifically overexpress GPR109A in mouse liver. Plasma HDL-C, hepatic ABCA1 and the HDL cholesterol production rate were significantly reduced in mice overexpressing GPR109A. Overexpression of GPR109A reduced primary hepatocyte free cholesterol efflux to apoA-I; conversely, GPR109A deficient hepatocytes had increased ABCA1-mediated cholesterol efflux. These data support the concept that the HDL-C lowering effect of niacin in wild-type mice is mediated through stimulation of GPR109A in hepatocytes; such an effect then leads to reduced hepatocyte ABCA1 expression and activity, decreased cholesterol efflux to nascent apoA-I, and reduced HDL-C levels. These results indicate that niacin-mediated activation of GP109A in liver lowers ABCA1 expression leading to reduced hepatic cholesterol efflux to HDL.
niacin; GPR109A; HDL; ABCA1; AAV
Several high profile failures of lipid-related therapeutics in clinical trials have led to intense interest in improved discovery and preclinical prioritization of potential targets. The careful study of patients with rare monogenic disorders has played a key role in establishing the causal role of cholesterol in atherosclerosis and highlighting viable drug targets. Systematic efforts to extend the association of common variants linked with lipid levels to coronary disease allow assessment of the vascular consequences of lifelong differences in lipids due to variation in specific molecules. This application of genetic epidemiology, termed Mendelian randomization, may prove useful in informing ongoing drug development efforts.
In many case–control genetic association studies, a set of correlated secondary phenotypes that may share common genetic factors with disease status are collected. Examination of these secondary phenotypes can yield valuable insights about the disease etiology and supplement the main studies. However, due to unequal sampling probabilities between cases and controls, standard regression analysis that assesses the effect of SNPs (single nucleotide polymorphisms) on secondary phenotypes using cases only, controls only, or combined samples of cases and controls can yield inflated type I error rates when the test SNP is associated with the disease. To solve this issue, we propose a Gaussian copula-based approach that efficiently models the dependence between disease status and secondary phenotypes. Through simulations, we show that our method yields correct type I error rates for the analysis of secondary phenotypes under a wide range of situations. To illustrate the effectiveness of our method in the analysis of real data, we applied our method to a genome-wide association study on high-density lipoprotein cholesterol (HDL-C), where “cases” are defined as individuals with extremely high HDL-C level and “controls” are defined as those with low HDL-C level. We treated 4 quantitative traits with varying degrees of correlation with HDL-C as secondary phenotypes and tested for association with SNPs in LIPG, a gene that is well known to be associated with HDL-C. We show that when the correlation between the primary and secondary phenotypes is >0.2, the P values from case–control combined unadjusted analysis are much more significant than methods that aim to correct for ascertainment bias. Our results suggest that to avoid false-positive associations, it is important to appropriately model secondary phenotypes in case–control genetic association studies.
Case–control studies; Statistical genetics; Statistical methods in Epidemiology
Plasma concentrations of high-density lipoprotein cholesterol (HDL-C) are strongly, consistently, and independently inversely associated with risk of atherosclerotic cardiovascular disease (CVD).1 A series of animal studies in the 1990s, primarily involving overexpression of the major HDL protein apolipoprotein A-I (apoA-I) with subsequent increases in HDL-C, showed reduced progression or even regression of atherosclerosis, fitting nicely with the “HDL hypothesis” that raising HDL-C is causally associated with benefit. However, the last decade has seen several observations that do not follow this simple script. Some examples include the following: (1) the demonstration that scavenger receptor class BI knockout mice have increased HDL-C but increased atherosclerosis2; (2) the suggestion that some persons with high HDL-C levels have “dysfunctional” HDL that may not be protective3; and (3) the observation that the cholesteryl ester transfer protein inhibitor torcetrapib raised HDL-C levels considerably but did not decrease, and indeed increased, cardiovascular risk.4,5 These developments have brought into major question the simple hypothesis that higher HDL-C directly and causally results in reduced atherosclerosis and challenge the approach of developing therapies that raise HDL-C levels.
Editorials; atherosclerosis; cholesterol; cholesterol; HDL; lipoproteins
Elevated plasma fibrinogen is a prothrombotic risk factor for cardiovascular disease (CVD). Recent small studies report that fibrinogen oxidative modifications, specifically tyrosine residue nitration, can occur in inflammatory states and may modify fibrinogen function. HDL cholesterol is inversely related to CVD and suggested to reduce the oxidation of LDL cholesterol, but whether these antioxidant functions extend to fibrinogen modifications is unknown. We used a recently validated ELISA to quantify nitrated fibrinogen during experimental human endotoxemia (N=23) and in a cohort of healthy adults (N=361) who were characterized for inflammatory and HDL parameters as well as subclinical atherosclerosis measures, carotid artery intima-medial thickness (IMT) and coronary artery calcification (CAC). Fibrinogen nitration increased following endotoxemia and directly correlated with accelerated ex vivo plasma clotting velocity. In the observational cohort, nitrated fibrinogen was associated with levels of CRP and serum amyloid A. Nitrated fibrinogen levels were not lower with increasing HDL cholesterol and did not associate with IMT and CAC. In humans, fibrinogen nitration was induced during inflammation and was correlated with markers of inflammation and clotting function but not HDL cholesterol or subclinical atherosclerosis in our modest sample. Inflammation-induced fibrinogen nitration may be a risk factor for promoting CVD events.
Fibrinogen; Nitration; Intima-medial thickness; Coronary artery calcification; High-density lipoprotein (HDL)
Traditional risk factors for premature cardiovascular disease such as systemic hypertension and hypercholesterolemia, all described more than half a century ago, are relatively few in number. Efforts to expand the epidemiological canon have met with limited success due to the high hurdle of causality. Fortunately, another solution to current deficiencies in risk assessment – in particular, the underestimation of risk both before and after initiation of pharmacotherapy – may exist. Parallel to the investigation of novel biomarkers, such as high-sensitivity C-reactive protein, ongoing research has yielded improved metrics of known causative conditions. This evolution of traditional risk factors, heralded by measures such as ambulatory blood pressure, central hemodynamics, low density lipoprotein particle concentration, genetic testing, and “vascular age,” may better address the detection gap in cardiovascular disease.
prevention; cholesterol; lipoproteins; blood pressure; cardiovascular risk; coronary artery calcium; carotid intima-media thickness
Autosomal recessive hypercholesterolemia (ARH) is a rare inherited disorder characterized by extremely high total and low-density lipoprotein cholesterol levels that has been previously linked to mutations in LDLRAP1. We identified a family with ARH not explained by mutations in LDLRAP1 or other genes known to cause monogenic hypercholesterolemia. The aim of this study was to identify the molecular etiology of ARH in this family.
Approach and Results
We used exome sequencing to assess all protein coding regions of the genome in three family members and identified a homozygous exon 8 splice junction mutation (c.894G>A, also known as E8SJM) in LIPA that segregated with the diagnosis of hypercholesterolemia. Since homozygosity for mutations in LIPA is known to cause cholesterol ester storage disease (CESD), we performed directed follow-up phenotyping by non-invasively measuring hepatic cholesterol content. We observed abnormal hepatic accumulation of cholesterol in the homozygote individuals, supporting the diagnosis of CESD. Given previous suggestions of cardiovascular disease risk in heterozygous LIPA mutation carriers, we genotyped E8SJM in >27,000 individuals and found no association with plasma lipid levels or risk of myocardial infarction, confirming a true recessive mode of inheritance.
By integrating observations from Mendelian and population genetics along with directed clinical phenotyping, we diagnosed clinically unapparent CESD in the affected individuals from this kindred and addressed an outstanding question regarding risk of cardiovascular disease in LIPA E8SJM heterozygous carriers.
hypercholesterolemia; genetics; myocardial infarction
macrophage cholesterol efflux; polyphenols; phenolic acids; coffee; cardiovascular disease
We previously reported that a systemic liver X receptor (LXR) agonist promoted macrophage reverse cholesterol transport (mRCT) in vivo. Because LXRs 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 where 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 [3H]-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 LXRα/β versus wild-type macrophages. While GW3965 treatment promoted fecal excretion compared with vehicle, its overall ability to promote mRCT was significantly attenuated using LXRα/β KO macrophages.
We demonstrate that intestinal-specific LXR agonism promotes macrophage RCT in vivo, and macrophage LXR itself plays an important, but not predominant, role in promoting RCT in response to an LXR agonist.
To examine the role of lipoprotein-associated phospholipase A2 (Lp-PLA2/PLA2G7) in human inflammation and coronary atherosclerosis.
Lp-PLA2 has emerged as a potential therapeutic target in coronary heart disease (CHD). Data supporting Lp-PLA2 are indirect and confounded by species differences; whether Lp-PLA2 is causal in CHD remains in question.
We examined inflammatory regulation of Lp-PLA2 during experimental endotoxemia in human, probed the source of Lp-PLA2 in human leukocytes under inflammatory conditions, and assessed the relationship of variation in PLA2G7, the gene encoding Lp-PLA2, with coronary artery calcification (CAC).
In contrast to circulating TNFα and CRP, blood and monocyte Lp-PLA2 mRNA decreased transiently, and plasma Lp-PLA2 mass declined modestly during endotoxemia. In vitro, Lp-PLA2 expression increased dramatically during human monocyte to macrophage differentiation and further in inflammatory macrophages and foam like-cells. Despite only a marginal association of SNPs in PLA2G7 with Lp-PLA2 activity or mass, numerous PLA2G7 SNPs were associated with CAC. In contrast, several SNPs in CRP were significantly associated with plasma CRP levels but had no relation with CAC.
Circulating Lp-PLA2 did not increase during acute phase response in human, while inflammatory macrophages and foam cells, but not circulating monocytes, are major leukocyte sources of Lp-PLA2. Common genetic variation in PLA2G7 is associated with sub-clinical coronary atherosclerosis. These data link Lp-PLA2 to atherosclerosis in human while highlighting the challenge in using circulating Lp-PLA2 as a biomarker of Lp-PLA2 actions in the vasculature.
Lp-PLA2; PLA2G7; CAC
Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple is unknown. Here, we show that apolipoprotein E (apoE) and apoE-containing HDL maintain arterial elasticity by suppressing the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop which increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain and transduced by Cox2 and miR-145. Arterial stiffness is increased in apoE-null mice, this stiffening can be reduced by administration of the lysyl oxidase inhibitor, BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. We conclude that apoE and apoE-containing HDL promote healthy arterial biomechanics, and this confers protection from cardiovascular disease independent of the established apoE-HDL effect on cholesterol.
Lecithin:cholesterol acyltransferase (LCAT) catalyzes the formation of plasma cholesteryl ester, plays a key role in high density lipoprotein (HDL) metabolism, and has been believed to be critical in the process of reverse cholesterol transport (RCT).
Method and Results
The role of LCAT in RCT from macrophages was quantitated using a validated assay involving intraperitoneal injection in mice of 3H-cholesterol-labeled J774 macrophages and monitoring appearance of tracer in plasma, liver, bile and feces. Human LCAT overexpression in human apoA-I transgenic mice substantially increased plasma HDL-C levels, but, surprisingly, did not increase macrophage RCT. Even in the setting of co-expression of SR-BI or CETP, both of which promoted the transfer of LCAT-derived HDL-CE to the liver, LCAT overexpression still had no effect on RCT. Serum from LCAT overexpressing mice had reduced ability to promote cholesterol efflux from macrophages ex vivo via ABCA1. To determine the effect of LCAT deficiency on macrophage RCT, LCAT-/- and LCAT+/- mice were compared to wild-type mice. Despite extremely low plasma levels of HDL-C, LCAT deficient mice had only a 50% reduction in RCT. LCAT+/- mice had normal RCT despite a significant reduction in HDL-C. Serum from LCAT deficient mice had increased ability to promote ABCA1-mediated cholesterol efflux from macrophages ex vivo.
These results demonstrate that LCAT overexpression does not promote an increased rate of macrophage RCT. While LCAT activity does become rate-limiting in the context of complete LCAT deficiency, RCT is reduced by only 50% even in the absence of LCAT. These data suggest that macrophage RCT may not be as dependent on LCAT activity as has been previously believed.
apolipoproteins; atherosclerosis; lipids; metabolism
Reverse cholesterol transport (RCT) is the process by which excess cholesterol is removed from peripheral tissue by high density lipoprotein (HDL) and delivered to the liver for excretion. Presently, methods of measuring in vivo RCT do so by monitoring the appearance in the feces of labeled cholesterol that originated from peripheral macrophage foam cells. These methods do not account for changes in macrophage cholesterol mass. We have developed an in vivo assay to measure cholesterol mass changes in atherosclerotic foam cells.
Methods and Results
Macrophages are entrapped in semi-permeable (pore size 0.2μm) hollow fibers and surgically implanted into the peritoneum of recipient mice. The fibers are removed from the peritoneum 24h after implantation. This method allows the complete recovery of the macrophages for quantification of changes in cholesterol mass and cellular protein. In wild type mice we measured a significant reduction in total cell cholesterol (TC) when hollow fibers containing cholesterol-enriched macrophage cells were implanted (TC before implantation = 105±18μg/mg cell protein, TC 24h after implantation = 60±16μg/mg protein). Additionally, there was an increase in cholesterol content when hollow fibers containing cholesterol-normal macrophages were implanted in an atherogenic mouse model (LDLr/apobec dko) compared to a wild type mouse (initial TC content = 57±24μg/mg protein, TC 24h after implantation: wild type mice = 52±10μg/mg protein; LDLr/apobec dko mice = 118±27μg/mg protein).
This assay can quantify in vivo both cholesterol mass accumulation, and reduction, in macrophages. This method permits quantitative analysis of the progression and regression of foam cells.
Atherosclerosis; lipoprotein; lipids; reverse cholesterol transport