Faculty Bibliography

Genome-wide association studies (GWAS) have identified a genetic variant at a locus on chromosome 1p13 that is associated with reduced risk of myocardial infarction, reduced plasma levels of LDL cholesterol (LDL-C), and markedly increased expression of the gene sortilin-1 (SORT1) in liver. Sortilin is a lysosomal sorting protein that binds ligands both in the Golgi apparatus and at the plasma membrane and traffics them to the lysosome. We previously reported that increased hepatic sortilin expression in mice reduced plasma LDL-C levels. Here we show that increased hepatic sortilin not only reduced hepatic apolipoprotein B (APOB) secretion, but also increased LDL catabolism, and that both effects were dependent on intact lysosomal targeting. Loss-of-function studies demonstrated that sortilin serves as a bona fide receptor for LDL in vivo in mice. Our data are consistent with a model in which increased hepatic sortilin binds intracellular APOB-containing particles in the Golgi apparatus as well as extracellular LDL at the plasma membrane and traffics them to the lysosome for degradation. We thus provide functional evidence that genetically increased hepatic sortilin expression both reduces hepatic APOB secretion and increases LDL catabolism, providing dual mechanisms for the very strong association between increased hepatic sortilin expression and reduced plasma LDL-C levels in humans.

PURPOSE OF REVIEW: Raising HDL cholesterol (HDL-C) has become an attractive therapeutic target to lower cardiovascular risk in addition to statins. Inhibition of the cholesteryl ester transfer protein (CETP), which mediates the transfer of cholesteryl esters from HDL to apolipoprotein B-containing particles, leads to a substantial increase in HDL-C levels. Various CETP inhibitors are currently being evaluated in phase II and phase III clinical trials. However, the beneficial effect of CETP inhibition on cardiovascular outcome remains to be established. RECENT FINDINGS: Torcetrapib, the first CETP inhibitor tested in a phase III clinical trial (ILLUMINATE), failed in 2006 because of an increase in all-cause mortality and cardiovascular events that subsequently were attributed to nonclass-related off-target effects (particularly increased blood pressure and low serum potassium) related to the stimulation of aldosterone production. Anacetrapib, another potent CETP inhibitor, raises HDL-C levels by approximately 138% and decreases LDL cholesterol (LDL-C) levels by approximately 40%, without the adverse off-targets effects of torcetrapib (DEFINE study). The CETP modulator dalcetrapib raises HDL-C levels by approximately 30% (with only minimal effect on LDL-C levels) and proved safety in the dal-VESSEL and dal-PLAQUE trials involving a total of nearly 600 patients. Evacetrapib, a relatively new CETP inhibitor, exhibited favorable changes in the lipid profile in a phase II study. SUMMARY: The two ongoing outcome trials, dal-OUTCOMES (dalcetrapib) and REVEAL (anacetrapib), will provide more conclusive answers for the concept of reducing cardiovascular risk by raising HDL-C with CETP inhibition.

Introduction Atherosclerosis is characterized by the accumulation of low density lipoprotein (LDL) and recruited macrophages within arterial walls. High macrophage burden is an indicator of greater risk of atherosclerotic tissue rupture and heart attack. As computed tomography (CT) imaging is the best technique for imaging plaque in the coronary arteries, a CT contrast agent able to detect macrophages in the arteries could help identify patients at higher risk. This study investigated whether in vivo macrophage imaging using clinical scanners could be performed with gold core high density lipoprotein (Au-HDL), a macrophage targeted agent (A). Additionally, lowest effective dose and ideal imaging timeframe of AuHDL was probed. Methods & Results Dodecanethiol coated gold cores were prepared following Brust's method and subsequently coated with the phospholipid myristoyl hydroxy phosphocholine (MHPC). The nanoparticles were then purified through centrifugation to remove gold core aggregates and empty MHPC micelles. Negative stain transmission electron microscopy (TEM) images verified removal of empty MHPC micelles from solution after ultracentrifugation (B). Apolipoprotein AI (ApoAI) was added to form the final AuHDL nanoparticle. CT imaging was used to calculate gold concentrations of the samples in mg/ml. To induce atherosclerosis, male New Zealand white rabbits were fed a high fat, high cholesterol diet (4.7% coconut oil and 0.3% cholesterol enriched diet) and underwent a double balloon injury of the aorta. AuHDL was prepared such that five atherosclerotic rabbits were injected with 75 (n=2), 150 (n=2), or 300 (n=1) mg Au/kg. CT images of rabbit aortas were taken at the following three time points: pre-injection, 24 hours post-injection, and 48 hours post-injection. A custom made MATLAB program was created to measure various regions of and around the aorta. CT images of aorta walls after injection exhibited greater radiodensity compared to pre-injection images. For example, CT images taken of one rabbit injected with 150 mg Au/kg showed the radiodensity of the aorta on average to be 38 HU (Hounsfield Units) +/- 1.99 preinjection and 59 HU +/- 1.59 24 hours postinjection (C). The lowest effective dose tested was 75 mg Au/kg and best imaging timeframe tested was 24 hours postinjection. TEM images of rabbit aorta sections confirmed localization of AuHDL nanoparticles in macrophages (D). Conclusion AuHDL increased radiodensity in CT images of aortas 24 and 48 hours post-injection compared to pre-injection images. Electron microscopy showed the nanoparticles to target macrophages. Hence this agent can image macrophages using CT, and has the potential for doing so in patients. Clinical Relevance If translated clinically, AuHDL can be used to image plaques in human aortas with high macrophage burden, thus allowing identification of patients at high risk of a heart attack. In addition, the agent could be of use in studying atherosclerosis and the effect of interventions. (Figure presented)

Rationale: Inflammation drives progression and destabilization of atherosclerotic plaques. Statins constitute the backbone for strategies to lower cardiovascular risk because of their potent cholesterol lowering capability. Whereas preclinical studies have shown that statins also have anti-inflammatory effects, the clinical relevance is hampered by the limited bioavailability of orally administered statins. To enhance the anti-inflammatory effects we developed statin-loaded reconstituted high-density lipoprotein nanoparticle ([s]-rHDL). The advantages of [s]-rHDL comprise its long half-life in plasma and the targeting to macrophages in atherosclerotic plaques. Methods & Results: To focus on anti-inflammatory effects, we used ApoE KO mice, whose cholesterol level is unaffected by statins. First, to evaluate macrophage targeting by [s]-rHDL, Gd-DTPA labeled [s]-rHDL was administered intravenously to ApoE KO mice (n=3). In vivo T1-weighted MR imaging (9.4 T Bruker MRI scanner) revealed strong signal enhancement in the abdominal aortic wall (Suppl. Fig a-d). Moreover, accumulation of [s]-rHDL was observed in the aortic valve and branching areas in the mice administered with Cy5.5 labeled [s]-rHDL by NIRF imaging (Suppl. Fig e, f) and specific uptake of [s]-rHDL by macrophages was revealed by fluorescence microscopy (Suppl. Fig g-l). Furthermore, flow cytometry confirmed that macrophages robustly took up rHDL in plaques, and the more differentiated macrophages took up more rHDL than less differentiated macrophages (Suppl. Fig m-r). Second, to assess the anti-inflammatory effects of [s]-rHDL, mice (n=62) were put on a high fat diet from 4 weeks of age onwards. At 14 weeks after diet initiation, mice were randomized to receive either placebo (n=15), oral simvastatin (10 mg/kg per day; n=15), intravenous rHDL (10 mg/kg ApoAI twice per week; n=16), or intravenous [s]-rHDL (15 mg/kg simvastatin with 10 mg/kg ApoAI twice a week; n=16) for 12 weeks. In vivo MR imaging of abdominal aorta was performed in 8 mice of each group at baseline, 6, and 12 weeks after randomization. Progression of vessel wall thickness was significantly inhibited in [s]-rHDL-treated animals compared to oral simvastatin, rHDL, and placebo groups (panel a). To objectively and quantitatively analyze histological sections (n4000), we built an automated Matlab procedure. Histology results at termination showed that plaque size (hematoxylin phloxine saffron staining) in the [s]-rHDL treated group was significantly reduced compared to rHDL and placebo groups (panel b). Importantly, the macrophage positive area (anti-CD68 immunostaining) in the [s]- rHDL treated group was profoundly reduced compared to all other groups (panel c). Conclusion: [s]-rHDL successfully delivers simvastatin to macrophages in atherosclerotic plaques as revealed by in vivo MRI imaging, ex vivo imaging, and histology. As a consequence, the [s]-rHDL formulation improves the anti-inflammatory effects of statins, which can be expected to improve its atheroprotective effects compared to oral statin therapy. These data warrant further studies in patients at increased cardiovascular risk. (Figure presented)

We have developed a mouse model of atherosclerotic plaque regression in which an atherosclerotic aortic arch from a hyperlipidemic donor is transplanted into a normolipidemic recipient, resulting in rapid elimination of cholesterol and monocyte-derived macrophage cells (CD68+) from transplanted vessel walls. To gain a comprehensive view of the differences in gene expression patterns in macrophages associated with regressing compared with progressing atherosclerotic plaque, we compared mRNA expression patterns in CD68+ macrophages extracted from plaque in aortic aches transplanted into normolipidemic or into hyperlipidemic recipients. In CD68+ cells from regressing plaque we observed that genes associated with the contractile apparatus responsible for cellular movement (e.g. actin and myosin) were up-regulated whereas genes related to cell adhesion (e.g. cadherins, vinculin) were down-regulated. In addition, CD68+ cells from regressing plaque were characterized by enhanced expression of genes associated with an anti-inflammatory M2 macrophage phenotype, including arginase I, CD163 and the C-lectin receptor. Our analysis suggests that in regressing plaque CD68+ cells preferentially express genes that reduce cellular adhesion, enhance cellular motility, and overall act to suppress inflammation.

Introduction The study of nanoparticle-nanoparticle and nanoparticle-cell interactions is of paramount importance to better understand biological processes and to improve the design of nanomaterials to be applied for molecular imaging. Forster resonance energy transfer (FRET) between a nanoparticle core and its coating allows studying the aforementioned phenomena with a variety of optical techniques. In the current study we applied to lipoproteins, natural nanoparticles comprised of lipids and apolipoproteins that transport fats throughout the body. We developed a high-density lipoprotein (HDL) based nanoparticle that consists of a quantum dot (QD) core and Cy5.5 labeled lipidic coating. The methodology allows judicious tuning of the QD/Cy5.5 ratio, which enabled us to optimize FRET between the QD core and the Cy5.5 labeled coating (Fig.1A). FRET allowed us to study lipoprotein-lipoprotein interactions, lipid exchange dynamics and the influence of apolipoproteins on these processes. Moreover, we were able to study HDL-cell interactions and exploit FRET to visualize HDL uptake by and dissociation in live macrophage cells. Methods and Results Exceptionally stable CdSe-CdS-ZnS core-shell-shell (CSS) QDs were synthesized, capped with oleic acid and coated with an appropriate mixture of Cy5.5 labeled and unlabeled phospholipids. Subsequently, ApoA-I was incorporated in the lipid corona to render stable dual labeled hybrid nanoparticles that consist of one QD core per particle, schematically depicted in Fig. 1A. To investigate the occurrence of FRET we acquired emission spectra and life time measurements of QD-HDL nanoparticles that contained varying amounts of Cy5.5 in their lipid coating (Fig. 1B). Next, we studied the particle-particle interactions through FRET facilitated optical measurements. Particle-cell interactions with cell membranes (Fig. 1C-D) were studied using the same methodology and ultimately, we performed a proof-ofprinciple study where we aimed to visualize FRET of QD-HDL-Cy5.5 nanoparticles in living adhered J774A.1 macrophages via fluorescence microscopy. This allowed us to study and visualize the temporal fate of QD-HDL once associated with macrophages (Supplemental Data). We observed the lipid coating to very differently interact with the cells from the QD core The fluorescence from the lipids primarily integrated in the cell membrane, while the QD core was predominantly found in the cytoplasm, suggesting a dissociation of the HDL nanoparticle upon association with the macrophage cells. Lastly, FRET fluorescence microscopy convincingly corroborated the aforementioned and proved to be a valuable tool to study the disassembly of the HDL nanoparticle. Conclusion Both the increasing interest in lipid-coated nanocrystals and the need to better understand HDL biology in detail inspired us to develop a hybrid nanoarchitecture that resembles HDL. We show that these lipid-coated and dye labeled QDs represent a versatile probe to study FRET and also allow the study of fundamental and biological processes via FRET, including lipid-exchange between nanoparticles and nanoparticle uptake by cells. (Figure presented)

Very low density lipoproteins (VLDL) are a major secretory product of the liver. They serve to transport endogenously synthesized lipids, mainly triglycerides (but also some cholesterol and cholesteryl esters) to peripheral tissues. VLDL is also the precursor of LDL. ApoB100 is absolutely required for VLDL assembly and secretion. The amount of VLDL triglycerides secreted by the liver depends on the amount loaded onto each lipoprotein particle, as well as the number of particles. Each VLDL has one apoB100 molecule, making apoB100 availability a key determinant of the number of VLDL particles, and hence, triglycerides, that can be secreted by hepatic cells. Surprisingly, the pool of apoB100 in the liver is typically regulated not by its level of synthesis, which is relatively constant, but by its level of degradation. It is now recognized that there are multiple opportunities for the hepatic cell to intercept apoB100 molecules and to direct them to distinct degradative processes. This mini-review will summarize progress in understanding these processes, with an emphasis on autophagy, the most recently described pathway of apoB100 degradation, and the one with possibly the most physiologic relevance to common metabolic perturbations affecting VLDL production. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

Introduction: Atherosclerosis is the leading cause of death in the Western world. Despite huge advances in understanding its pathophysiological mechanisms, current treatment is mostly based on 'traditional' risk factors. The introduction of statins more than 20 years ago reduced morbidity and mortality of atherosclerosis by 30%, leaving a residual cardiovascular risk. Therefore, efforts continue toward the development of novel therapies that can be added to established treatments. Besides targeting dyslipidemia, recent focus has been put on preventing or resolving inflammatory processes involved in atherosclerosis. Areas covered: The article discusses therapeutic and diagnostic targets in atherosclerosis and how they can be discovered and studied in preclinical animal models. The roles of immune cells, specifically macrophages and monocytes, in plaque inflammation are discussed. The article also describes current preclinical models of atherosclerosis, specifically the mouse, study designs (for progression and regression studies), basic and advanced methods of analysis of atherosclerotic lesions, and discusses the challenges of translating the findings to humans. Expert opinion: Advances in genomics, proteomics, lipidomics and the development of high-throughput screening techniques help to improve our understanding of atherosclerosis disease mechanisms immensely and facilitate the discovery of new diagnostic and therapeutic targets. Preclinical studies in animals are still indispensable to uncover pathways involved in atherosclerotic disease and to evaluate novel drug targets. The translation of these targets, however, from animal studies to humans remains challenging. There is a strong need for novel biomarkers that can be used to prove the concept of a new target in humans.