Alan T. Remaley

MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins

Circulating microRNAs (miRNA) are relatively stable in plasma and are a new class of disease biomarkers. Here we present evidence that high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities. Cellular export of miRNAs to HDL was demonstrated to be regulated by neutral sphingomyelinase. Reconstituted HDL injected into mice retrieved distinct miRNA profiles from normal and atherogenic models. HDL delivery of both exogenous and endogenous miRNAs resulted in the direct targeting of messenger RNA reporters. Furthermore, HDL-mediated delivery of miRNAs to recipient cells was demonstrated to be dependent on scavenger receptor class B type I. The human HDL–miRNA profile of normal subjects is significantly different from that of familial hypercholesterolemia subjects. Notably, HDL–miRNA from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets in cultured hepatocytes. Collectively, these observations indicate that HDL participates in a mechanism of intercellular communication involving the transport and delivery of miRNAs.

PPAR-α and PPAR-γ activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate lipid and glucose metabolism and cellular differentiation. PPAR-α and PPAR-γ are both expressed in human macrophages where they exert anti-inflammatory effects. The activation of PPAR-α may promote foam-cell formation by inducing expression of the macrophage scavenger receptor CD36. This prompted us to investigate the influence of different PPAR- activators on cholesterol metabolism and foam-cell formation of human primary and THP-1 macrophages. Here we show that PPAR-α and PPAR-γ activators do not influence acetylated low density lipoprotein-induced foam-cell formation of human macrophages. In contrast, PPAR-α and PPAR-γ activators induce the expression of the gene encoding ABCA1, a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These effects are likely due to enhanced expression of liver-x-receptor α, an oxysterol-activated nuclear receptor which induces ABCA1- promoter transcription. Moreover, PPAR-α and PPAR-γ activators increase apoAI-induced cholesterol efflux from normal macrophages. In contrast, PPAR-α or PPAR-γ activation does not influence cholesterol efflux from macrophages isolated from patients with Tangier disease, which is due to a genetic defect in ABCA1. Here we identify a regulatory role for PPAR-α and PPAR-γ in the first steps of the reverse-cholesterol-transport pathway through the activation of ABCA1-mediated cholesterol efflux in human macrophages.

Cholesterol Efflux and Atheroprotection Advancing the Concept of Reverse Cholesterol Transport

High-density lipoprotein (HDL) has been proposed to have several antiatherosclerotic properties, including the ability to mediate macrophage cholesterol efflux, antioxidant capacity, antiinflammatory properties, nitric oxide–promoting activity, and ability to transport proteins with their own intrinsic biological activities.1 HDL particles are critical acceptors of cholesterol from lipid-laden macrophages and thereby participate in the maintenance of net cholesterol balance in the arterial wall and in the reduction of proinflammatory responses by arterial cholesterol-loaded macrophages. The pathways that regulate HDL-mediated macrophage cholesterol efflux and disposition of cholesterol involve cell membrane–bound transporters, plasma lipid acceptors, plasma proteins and enzymes, and hepatic cellular receptors (Figure 1). From the earliest proposed concept for HDL-mediated cholesterol efflux,2,3 the concentration of the cholesterol content in HDL particles has been considered a surrogate measurement for the efficiency of the “reverse cholesterol transport” (RCT) process; however, macrophage-derived cholesterol represents a minor component of the cholesterol transported by HDL particles.4–7 One important pathway for cholesterol-mediated efflux from macrophage foam cells involves interaction between the ATP-binding cassette transporter A1 (ABCA1) and cholesterol-deficient and phospholipid-depleted apolipoprotein (apo) A-I complexes (pre-β migrating HDL or very small HDL [HDL-VS]; Figure 2).1,8 Subsequently, the ATP-binding cassette transporter G1 (ABCG1) mediates macrophage cholesterol efflux through interactions (Figure 3) with spherical, cholesterol-containing α-HDL particles (small HDL [HDL-S], medium HDL [HDL-M], large HDL [HDL-L], and very large (HDL-VL).1 In contrast, the scavenger receptor class B type I (SR-BI) is a multifunctional receptor that mediates bidirectional lipid transport in the macrophage, which is dependent on the content of cholesterol in lipid-laden macrophages. A more established role for SR-BI in cholesterol trafficking involves selective uptake of cholesteryl esters from mature HDL by the liver. Recent studies suggest that polymorphisms in SR-BI contribute to the functional capacity of this cholesterol …

Association of Loss-of-Function Mutations in the ABCA1 Gene With High-Density Lipoprotein Cholesterol Levels and Risk of Ischemic Heart Disease

CONTEXT Low levels of high-density lipoprotein (HDL) cholesterol are inversely related to cardiovascular risk. Whether this is a causal effect is unclear. OBJECTIVE To determine whether genetically reduced HDL cholesterol due to heterozygosity for 4 loss-of-function mutations in ABCA1 cause increased risk of ischemic heart disease (IHD). DESIGN, SETTING, AND PARTICIPANTS Three studies of white individuals from Copenhagen, Denmark, were used: the Copenhagen City Heart Study (CCHS), a 31-year prospective general population study (n = 9022; 28 heterozygotes); the Copenhagen General Population Study (CGPS), a cross-sectional general population study (n = 31,241; 76 heterozygotes); and the Copenhagen Ischemic Heart Disease Study (CIHDS), a case-control study (n = 16,623; 44 heterozygotes). End points in all 3 studies were recorded during the period of January 1, 1976, through July 9, 2007. MAIN OUTCOME MEASURES Levels of HDL cholesterol in the general population, cellular cholesterol efflux, and the association between IHD and HDL cholesterol and genotype. RESULTS Heterozygotes vs noncarriers for 4 ABCA1 mutations (P1065S, G1216V, N1800H, R2144X) had HDL cholesterol levels of 41 mg/dL (interquartile range, 31-50 mg/dL) vs 58 mg/dL (interquartile range, 46-73 mg/dL), corresponding to a reduction in HDL cholesterol of 17 mg/dL (P < .001). A 17-mg/dL lower HDL cholesterol level in the CCHS was associated with a multifactorially adjusted hazard ratio for IHD of 1.70 (95% confidence interval [CI], 1.57-1.85). However, for IHD in heterozygotes vs noncarriers, the multifactorially adjusted hazard ratio was 0.67 (95% CI, 0.28-1.61; 1741 IHD events) in the CCHS, the multifactorially adjusted odds ratio was 0.82 (95% CI, 0.34-1.96; 2427 IHD events) in the CGPS, and the multifactorially adjusted odds ratio was 0.86 (95% CI, 0.32-2.32; 2498 IHD cases) in the CIHDS. The corresponding odds ratio for IHD in heterozygotes vs noncarriers for the combined studies (n = 41,961; 6666 cases; 109 heterozygotes) was 0.93 (95% CI, 0.53-1.62). CONCLUSION Lower plasma levels of HDL cholesterol due to heterozygosity for loss-of-function mutations in ABCA1 were not associated with an increased risk of IHD.

HDL Measures, Particle Heterogeneity, Proposed Nomenclature, and Relation to Atherosclerotic Cardiovascular Events

BACKGROUND A growing body of evidence from epidemiological data, animal studies, and clinical trials supports HDL as the next target to reduce residual cardiovascular risk in statin-treated, high-risk patients. For more than 3 decades, HDL cholesterol has been employed as the principal clinical measure of HDL and cardiovascular risk associated with low HDL-cholesterol concentrations. The physicochemical and functional heterogeneity of HDL present important challenges to investigators in the cardiovascular field who are seeking to identify more effective laboratory and clinical methods to develop a measurement method to quantify HDL that has predictive value in assessing cardiovascular risk. CONTENT In this report, we critically evaluate the diverse physical and chemical methods that have been employed to characterize plasma HDL. To facilitate future characterization of HDL subfractions, we propose the development of a new nomenclature based on physical properties for the subfractions of HDL that includes very large HDL particles (VL-HDL), large HDL particles (L-HDL), medium HDL particles (M-HDL), small HDL particles (S-HDL), and very-small HDL particles (VS-HDL). This nomenclature also includes an entry for the pre-β-1 HDL subclass that participates in macrophage cholesterol efflux. SUMMARY We anticipate that adoption of a uniform nomenclature system for HDL subfractions that integrates terminology from several methods will enhance our ability not only to compare findings with different approaches for HDL fractionation, but also to assess the clinical effects of different agents that modulate HDL particle structure, metabolism, and function, and in turn, cardiovascular risk prediction within these HDL subfractions.

High-Density Lipoprotein Reduces the Human Monocyte Inflammatory Response

Objective—Whereas the anti–inflammatory effects of high-density lipoprotein (HDL) on endothelial cells are well described, such effects on monocytes are less studied. Methods and Results—Human monocytes were isolated from whole blood followed by assessment of CD11b activation/expression and cell adhesion under shear-flow. HDL caused a dose-dependent reduction in the activation of CD11b induced by PMA or receptor-dependent agonists. The constituent of HDL responsible for the antiinflammatory effects on CD11b activation was found to be apolipoprotein A-I (apoA-I). Cyclodextrin, but not cyclodextrin/cholesterol complex, also inhibited PMA-induced CD11b activation implicating cholesterol efflux as the main mechanism. This was further confirmed with the demonstration that cholesterol content of lipid rafts diminished after treatment with the cholesterol acceptors. Blocking ABCA1 with an anti-ABCA1 antibody abolished the effect of apoA-I. Furthermore, monocytes derived from a Tangier disease patient definitively confirmed the requirement of ABCA1 in apoA-I–mediated CD11b inhibition. The antiinflammatory effects of apoA-I were also observed in functional models including cell adhesion to an endothelial cell monolayer, monocytic spreading under shear flow, and transmigration. Conclusions—HDL and apoA-I exhibit an antiinflammatory effect on human monocytes by inhibiting activation of CD11b. ApoA-I acts through ABCA1, whereas HDL may act through several receptors.

The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice

Identification of mutations in the ABCA1 transporter (ABCA1) as the genetic defect in Tangier disease has generated interest in modulating atherogenic risk by enhancing ABCA1 gene expression. To investigate the role of ABCA1 in atherogenesis, we analyzed diet-induced atherosclerosis in transgenic mice overexpressing human ABCA1 (hABCA1-Tg) and spontaneous lesion formation in hABCA1-Tg × apoE-knockout (KO) mice. Overexpression of hABCA1 in C57BL/6 mice resulted in a unique anti-atherogenic profile characterized by decreased plasma cholesterol (63%), cholesteryl ester (63%), free cholesterol (67%), non-high density lipoprotein (HDL)-cholesterol (53%), and apolipoprotein (apo) B (64%) but markedly increased HDL-cholesterol (2.8-fold), apoA-I (2.2-fold), and apoE (2.8-fold) levels. These beneficial changes in the lipid profile led to significantly lower (65%) aortic atherosclerosis in hABCA1-Tg mice. In marked contrast, ABCA1 overexpression had a minimal effect on the plasma lipid profile of apoE-KO mice and resulted in a 2- to 2.6-fold increase in aortic lesion area. These combined results indicate that overexpression of ABCA1 in C57BL/6 mice on a high cholesterol diet results in an atheroprotective lipoprotein profile and decreased atherosclerosis, and thus provide previously undocumented in vivo evidence of an anti-atherogenic role for the ABCA1 transporter. In contrast, overexpression of ABCA1 in an apoE-KO background led to increased atherosclerosis, further substantiating the important role of apoE in macrophage cholesterol metabolism and atherogenesis. In summary, these results establish that, in the presence of apoE, overexpression of ABCA1 modulates HDL as well as apoB-containing lipoprotein metabolism and reduces atherosclerosis in vivo, and indicate that pharmacological agents that will increase ABCA1 expression may reduce atherogenic risk in humans.

The ClinSeq Project: Piloting large-scale genome sequencing for research in genomic medicine

ClinSeq is a pilot project to investigate the use of whole-genome sequencing as a tool for clinical research. By piloting the acquisition of large amounts of DNA sequence data from individual human subjects, we are fostering the development of hypothesis-generating approaches for performing research in genomic medicine, including the exploration of issues related to the genetic architecture of disease, implementation of genomic technology, informed consent, disclosure of genetic information, and archiving, analyzing, and displaying sequence data. In the initial phase of ClinSeq, we are enrolling roughly 1000 participants; the evaluation of each includes obtaining a detailed family and medical history, as well as a clinical evaluation. The participants are being consented broadly for research on many traits and for whole-genome sequencing. Initially, Sanger-based sequencing of 300-400 genes thought to be relevant to atherosclerosis is being performed, with the resulting data analyzed for rare, high-penetrance variants associated with specific clinical traits. The participants are also being consented to allow the contact of family members for additional studies of sequence variants to explore their potential association with specific phenotypes. Here, we present the general considerations in designing ClinSeq, preliminary results based on the generation of an initial 826 Mb of sequence data, the findings for several genes that serve as positive controls for the project, and our views about the potential implications of ClinSeq. The early experiences with ClinSeq illustrate how large-scale medical sequencing can be a practical, productive, and critical component of research in genomic medicine.

Inflammation and iron deficiency in the hypoferremia of obesity

Context:Obesity is associated with hypoferremia, but it is unclear if this condition is caused by insufficient iron stores or diminished iron availability related to inflammation-induced iron sequestration.Objective:To examine the relationships between obesity, serum iron, measures of iron intake, iron stores and inflammation. We hypothesized that both inflammation-induced sequestration of iron and true iron deficiency were involved in the hypoferremia of obesity.Design:Cross-sectional analysis of factors anticipated to affect serum iron.Setting:Outpatient clinic visits.Patients:Convenience sample of 234 obese and 172 non-obese adults.Main outcome measures:Relationships between serum iron, adiposity, and serum transferrin receptor, C-reactive protein, ferritin, and iron intake analyzed by analysis of covariance and multiple linear regression.Results:Serum iron was lower (75.8±35.2 vs 86.5±34.2 g/dl, P=0.002), whereas transferrin receptor (22.6±7.1 vs 21.0±7.2 nmol/l, P=0.026), C-reactive protein (0.75±0.67 vs 0.34±0.67 mg/dl, P<0.0001) and ferritin (81.1±88.8 vs 57.6±88.7 μg/l, P=0.009) were higher in obese than non-obese subjects. Obese subjects had a higher prevalence of iron deficiency defined by serum iron (24.3%, confidence intervals (CI) 19.3–30.2 vs 15.7%, CI 11.0–21.9%, P=0.03) and transferrin receptor (26.9%, CI 21.6–33.0 vs 15.7%, CI 11.0–21.9%, P=0.0078) but not by ferritin (9.8%, CI 6.6–14.4 vs 9.3%, CI 5.7–14.7%, P=0.99). Transferrin receptor, ferritin and C-reactive protein contributed independently as predictors of serum iron.Conclusions:The hypoferremia of obesity appears to be explained both by true iron deficiency and by inflammatory-mediated functional iron deficiency.

Detection and Quantitation of Serum Mesothelin, a Tumor Marker for Patients with Mesothelioma and Ovarian Cancer

Purpose: To determine whether mesothelin, a cell surface protein highly expressed in mesothelioma and ovarian cancer, is shed into serum and if so to accurately measure it. Experimental Design: We developed a sandwich ELISA using antibodies reacting with two different epitopes on human mesothelin. To quantitate serum mesothelin levels, a standard curve was generated using a mesothelin-Fc fusion protein. Sera from 24 healthy volunteers, 95 random hospital patients, 56 patients with mesothelioma, and 21 patients with ovarian cancer were analyzed. Serum mesothelin levels were also measured before and after surgical cytoreduction in six patients with peritoneal mesothelioma. Results: Elevated serum mesothelin levels were noted in 40 of 56 (71%) patients with mesothelioma and in 14 of 21 (67%) patients with ovarian cancer. Serum mesothelin levels were increased in 80% and 75% of the cases of mesothelioma and ovarian cancer, respectively, in which the tumors expressed mesothelin by immunohistochemistry. Out of the six patients with peritoneal mesothelioma who underwent surgery, four had elevated serum mesothelin levels before surgery. Out of these four patients, three had cytoreductive surgery and the serum mesothelin level decreased by 71% on postoperative day 1 and was undetectable by postoperative day 7. Conclusions: We developed a serum mesothelin assay that shows that mesothelin is elevated in patients with mesothelioma and ovarian cancer. The rapid decrease in mesothelin levels after surgery in patients with peritoneal mesothelioma suggests that serum mesothelin may be a useful test to monitor treatment response in mesothelin-expressing cancers.