Faculty Bibliography

Apoprotein B (apoB100) is required for the formation of VLDL, the precursor of LDL, the plasma level of which is a strong risk factor for atherosclerosis and coronary artery disease (CAD). It is an exceptionally large (>4000 aa) and hydrophobic protein. As the nascent polypeptide is translocating across the ER, if lipids are not transferred to stabilize its conformation, we and others have shown that like other abnormally folded proteins in the ER, its quality control is governed by ER-associated ub-proteasome degradation (ERAD). Using a combination of systems from yeast to genetically modified mice, we and our collaborators have shown apoB100 ERAD to involve chaperone and chaperone-like molecules, such as Hsp70, 90, 110, and p58. The full assembly of VLDL does not occur until the Golgi. We have discovered that nascent particles ("pre-VLDL") that do not complete this maturation process (e.g., under the influence of certain dietary fatty acids), are subject to a late stage quality control process mediated by autophagy. In summary, the production of apoB100-lipoproteins, whose plasma levels correlate with the risk of CAD, is determined in part by quality control processes that recognize features of the protein specific to its stage of assembly and that are separated between the ER and Golgi compartments

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.

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)

ER-associated degradation (ERAD) rids the early secretory pathway of misfolded or misprocessed proteins. Some members of the protein disulfide isomerase (PDI) family appear to facilitate ERAD substrate selection and retrotranslocation, but a thorough characterization of PDIs during the degradation of diverse substrates has not been undertaken, in part because there are 20 PDI family members in mammals. PDIs can also exhibit disulfide redox, isomerization, and/or chaperone activity, but which of these activities is required for the ERAD of different substrate classes is unknown. We therefore examined the fates of unique substrates in yeast, which expresses five PDIs. Through the use of a yeast expression system for apolipoprotein B (ApoB), which is disulfide rich, we discovered that Pdi1 interacts with ApoB and facilitates degradation through its chaperone activity. In contrast, Pdi1's redox activity was required for the ERAD of CPY* (a misfolded version of carboxypeptidase Y that has five disulfide bonds). The ERAD of another substrate, the alpha subunit of the epithelial sodium channel, was Pdi1 independent. Distinct effects of mammalian PDI homologues on ApoB degradation were then observed in hepatic cells. These data indicate that PDIs contribute to the ERAD of proteins through different mechanisms and that PDI diversity is critical to recognize the spectrum of potential ERAD substrates.

Movement of free cholesterol between the cellular compartment and acceptor is governed by cholesterol gradients that are determined by several enzymes and reverse cholesterol transport proteins. We have previously demonstrated that adenosine A(2A) receptors inhibit foam cell formation and stimulate production of cholesterol 27-hydroxylase (CYP27A1), an enzyme involved in the conversion of cholesterol to oxysterols. We therefore asked whether the effect of adenosine A(2A) receptors on foam cell formation in vitro is mediated by CYP27A1 or apoE, a carrier for cholesterol in the serum. We found that specific lentiviral siRNA infection markedly reduced apoE or 27-hydroxylase mRNA in THP-1 cells. Despite diminished apoE expression (p < 0.0002, interferon-gamma (IFNgamma) CGS vs. IFNgamma alone, n = 4), CGS-21680, an adenosine A(2A) receptor agonist, inhibits foam cell formation. In contrast, CGS-21680 had no effect on reducing foam cell formation in CYP27A1 KD cells (4 +/- 2%; p < 0.5113, inhibition vs. IFNgamma alone, n = 4). Previously, we reported the A(2A) agonist CGS-21680 increases apoAI-mediated cholesterol efflux nearly twofold in wild-type macrophages. Adenosine receptor activation had no effect on cholesterol efflux in CYP27A1 KD cells but reduced efflux in apoE KD cells. These results demonstrate that adenosine A(2A) receptor occupancy diminishes foam cell formation by increasing expression and function of CYP27A1

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)

The molecular basis for primary hereditary hypertriglyceridemia has been identified in fewer than 5% of cases. Investigation of monogenic dyslipidemias has the potential to expose key metabolic pathways. We describe a hitherto unreported disease in ten individuals manifesting as moderate to severe transient childhood hypertriglyceridemia and fatty liver followed by hepatic fibrosis and the identification of the mutated gene responsible for this condition. We performed SNP array-based homozygosity mapping and found a single large continuous segment of homozygosity on chromosomal region 12q13.12. The candidate region contained 35 genes that are listed in Online Mendelian Inheritance in Man (OMIM) and 27 other genes. We performed candidate gene sequencing and screened both clinically affected individuals (children and adults with hypertriglyceridemia) and also a healthy cohort for mutations in GPD1, which encodes glycerol-3-phosphate dehydrogenase 1. Mutation analysis revealed a homozygous splicing mutation, c.361-1G>C, which resulted in an aberrantly spliced mRNA in the ten affected individuals. This mutation is predicted to result in a truncated protein lacking essential conserved residues, including a functional site responsible for initial substrate recognition. Functional consequences of the mutation were evaluated by measuring intracellular concentrations of cholesterol and triglyceride as well as triglyceride secretion in HepG2 (hepatocellular carcinoma) human cells lines overexpressing normal and mutant GPD1 cDNA. Overexpression of mutant GPD1 in HepG2 cells, in comparison to overexpression of wild-type GPD1, resulted in increased secretion of triglycerides (p = 0.01). This finding supports the pathogenicity of the identified mutation.

Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation

The macrophage is exquisitely sensitive to its microenvironment, as demonstrated primarily through in vitro study. Changes in macrophage phenotype and function within the atherosclerotic plaque have profound consequences for plaque biology, including rupture and arterial thrombosis leading to clinical events such as myocardial infarction. We review the evidence for dynamic changes in macrophage numbers and macrophage differentiation within the atherosclerotic plaque microenvironment and discuss potential approaches to target macrophage differentiation for therapeutic benefit in cardiovascular disease.