Faculty Bibliography

Atherosclerosis can be induced by the injection of a gain-of-function mutant of proprotein convertase subtilisin/kexin type 9 (PCSK9)-encoding adeno-associated viral vector (AAVmPCSK9), avoiding the need for knockout mice models, such as low-density lipoprotein receptor deficient mice. As regression of atherosclerosis is a crucial therapeutic goal, we aimed to establish a regression model based on AAVmPCSK9, which will eliminate the need for germ-line genetic modifications. C57BL6/J mice were injected with AAVmPCSK9 and were fed with Western diet for 16 weeks, followed by reversal of hyperlipidemia by a diet switch to chow and treatment with a microsomal triglyceride transfer protein inhibitor (MTPi). Sixteen weeks following AAVmPCSK9 injection, mice had advanced atherosclerotic lesions in the aortic root. Surprisingly, diet switch to chow alone reversed hyperlipidemia to near normal levels, and the addition of MTPi completely normalized hyperlipidemia. A six week reversal of hyperlipidemia, either by diet switch alone or by diet switch and MTPi treatment, was accompanied by regression of atherosclerosis as defined by a significant decrease of macrophages in the atherosclerotic plaques, compared to baseline. Thus, we have established an atherosclerosis regression model that is independent of the genetic background.

Background: Immunological complexity in atherosclerosis warrants targeted treatment of specific inflammatory cells that aggravate the disease. With the initiation of large phase III trials investigating immunomodulatory drugs for atherosclerosis, cardiovascular disease treatment enters a new era. Accordingly, numerous small molecules have been developed to modulate immune cell function, many of which are promising immunotherapy candidates. However, effective immunotherapies require precise effects only on pathological immune cells without causing side effects on the healthy tissues or other immune cells. Results: We here propose a radically different approach that implement and evaluate in vivo a combinatorial library of nanoparticles with distinct physiochemical properties and differential immune cell specificities. The library's nanoparticles are based on endogenous high-density lipoprotein (HDL), which can preferentially deliver therapeutic compounds to pathological immune cells in atherosclerotic plaques^1,2. Using the Apoe ^-/- mouse model of atherosclerosis, we quantitatively evaluated the library's immune cell specificity by combining nanomaterial characterization, in vitro assays, optical imaging, and immunological techniques (a). These distinct physiochemical properties among the library nanoparticles resulted in an approximate 6-fold difference in promoting cholesterol efflux from macrophages, 10-fold difference among blood half-lives, 3.35-fold difference in relative aorta-to-liver accumulation, and 3.84-fold difference in relative aortic-to-splenic macrophage accumulation. In a proof-of-concept study, we identified an ideal drug-delivery nanoparticle with a long blood half-life, low liver retention, and high specificity to atherosclerotic macrophages. We formulated into the nanoparticle (Rx-HDL) a liver receptor X agonist (GW3965), whose high liver toxicity failed its clinical translation. Compared to an undesirable nanoparticle with poor properties for drug delivery (Rx-PLGA-HDL), Rx-HDL minimally retained in the liver while still efficiently delivered GW3965 to atherosclerotic plaques, revealed by in vivo PET imaging and ex vivo flow cytometry. In a one-week intensive treatment regimen in atherosclerotic mice, Rx-HDL totally abolished GW3965's liver toxicity (b ). Finally, a 6-week long-term treatment with Rx-HDL produced significant therapeutic effects on atherosclerotic plaques (c). Conclusion: In this study, for the first time, we demonstrate a systemic in vivo immune cell screening of a nanoparticle library can produce effective immunotherapy for atherosclerosis. Screening the immune cell specificity of nanoparticles can be employed to develop tailored therapies for atherosclerosis and other inflammatory diseases. [IMAGE PRESENTED]

Immunological complexity in atherosclerosis warrants targeted treatment of specific inflammatory cells that aggravate the disease. With the initiation of large phase III trials investigating immunomodulatory drugs for atherosclerosis, cardiovascular disease treatment enters a new era. We here propose a radically different approach: implementing and evaluating in vivo a combinatorial library of nanoparticles with distinct physiochemical properties and differential immune cell specificities. The library's nanoparticles are based on endogenous high-density lipoprotein, which can preferentially deliver therapeutic compounds to pathological macrophages in atherosclerosis. Using the apolipoprotein E-deficient (Apoe-/-) mouse model of atherosclerosis, we quantitatively evaluated the library's immune cell specificity by combining immunological techniques and in vivo positron emission tomography imaging. Based on this screen, we formulated a liver X receptor agonist (GW3965) and abolished its liver toxicity while still preserving its therapeutic function. Screening the immune cell specificity of nanoparticles can be used to develop tailored therapies for atherosclerosis and other inflammatory diseases.

AIMS/OBJECTIVE:Normalization of hypercholesterolaemia, inflammation, hyperglycaemia, and obesity are main desired targets to prevent cardiovascular clinical events. Here we present a novel regulator of cholesterol metabolism, which simultaneously impacts on glucose intolerance and inflammation. METHODS AND RESULTS/RESULTS:Mice deficient for oxygen sensor HIF-prolyl hydroxylase 1 (PHD1) were backcrossed onto an atherogenic low-density lipoprotein receptor (LDLR) knockout background and atherosclerosis was studied upon 8 weeks of western-type diet. PHD1-/-LDLR-/- mice presented a sharp reduction in VLDL and LDL plasma cholesterol levels. In line, atherosclerotic plaque development, as measured by plaque area, necrotic core expansion and plaque stage was hampered in PHD1-/-LDLR-/- mice. Mechanistically, cholesterol-lowering in PHD1 deficient mice was a result of enhanced cholesterol excretion from blood to intestines and ultimately faeces. Additionally, flow cytometry of whole blood of these mice revealed significantly reduced counts of leucocytes and particularly of Ly6Chigh pro-inflammatory monocytes. In addition, when studying PHD1-/- in diet-induced obesity (14 weeks high-fat diet) mice were less glucose intolerant when compared with WT littermate controls. CONCLUSION/CONCLUSIONS:Overall, PHD1 knockout mice display a metabolic phenotype that generally is deemed protective for cardiovascular disease. Future studies should focus on the efficacy, safety, and gender-specific effects of PHD1 inhibition in humans, and unravel the molecular actors responsible for PHD1-driven, likely intestinal, and regulation of cholesterol metabolism.

What is inflammation's big idea? In this brief overview of the role of myeloid cells in inflammation, we will critically discuss what drives the initiation, amplification, and resolution of inflammation in different anatomical sites in response to different pathological stimuli. It can be argued that we have a good understanding of the basic principles that underlie myeloid cell activation and the mobilization of innate immune cells to sites of injury and infection in acute inflammation. The challenge now for inflammation biologists is to understand how resolution of this normal physiological response goes wrong in hyperacute and chronic inflammation. A better understanding of how inflammation is regulated will allow us to develop new anti-inflammatory drugs that will reduce the burden of inflammatory disease without compromising the patient's immune defenses against infectious disease. Ideally such drugs should encourage a return to homeostasis and enhance tissue repair processes.

Apolipoprotein A1 (apoA1) is the major protein component of high-density lipoprotein (HDL) and has well documented anti-inflammatory properties. To better understand the cellular and molecular basis of the anti-inflammatory actions of apoA1, we explored the effect of acute human apoA1 exposure on the migratory capacity of monocyte-derived cells in vitro and in vivo. Acute (20-60 min) apoA1 treatment induced a substantial (50-90%) reduction in macrophage chemotaxis to a range of chemoattractants. This acute treatment was anti-inflammatory in vivo as shown by pre-treatment of monocytes prior to adoptive transfer into an on-going murine peritonitis model. We find that apoA1 rapidly disrupts membrane lipid rafts, and as a consequence, dampens the PI3K/Akt signalling pathway that coordinates reorganization of the actin cytoskeleton and cell migration. Our data strengthen the evidence base for therapeutic apoA1 infusions in situations where reduced monocyte recruitment to sites of inflammation could have beneficial outcomes.

OBJECTIVES: The goal of this study was to develop and validate a noninvasive imaging tool to visualize the in vivo behavior of high-density lipoprotein (HDL) by using positron emission tomography (PET), with an emphasis on its plaque-targeting abilities. BACKGROUND: HDL is a natural nanoparticle that interacts with atherosclerotic plaque macrophages to facilitate reverse cholesterol transport. HDL-cholesterol concentration in blood is inversely associated with risk of coronary heart disease and remains one of the strongest independent predictors of incident cardiovascular events. METHODS: Discoidal HDL nanoparticles were prepared by reconstitution of its components apolipoprotein A-I (apo A-I) and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. For radiolabeling with zirconium-89 (89Zr), the chelator deferoxamine B was introduced by conjugation to apo A-I or as a phospholipid-chelator (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-deferoxamine B). Biodistribution and plaque targeting of radiolabeled HDL were studied in established murine, rabbit, and porcine atherosclerosis models by using PET combined with computed tomography (PET/CT) imaging or PET combined with magnetic resonance imaging. Ex vivo validation was conducted by radioactivity counting, autoradiography, and near-infrared fluorescence imaging. Flow cytometric assessment of cellular specificity in different tissues was performed in the murine model. RESULTS: We observed distinct pharmacokinetic profiles for the two 89Zr-HDL nanoparticles. Both apo A-I- and phospholipid-labeled HDL mainly accumulated in the kidneys, liver, and spleen, with some marked quantitative differences in radioactivity uptake values. Radioactivity concentrations in rabbit atherosclerotic aortas were 3- to 4-fold higher than in control animals at 5 days' post-injection for both 89Zr-HDL nanoparticles. In the porcine model, increased accumulation of radioactivity was observed in lesions by using in vivo PET imaging. Irrespective of the radiolabel's location, HDL nanoparticles were able to preferentially target plaque macrophages and monocytes. CONCLUSIONS: 89Zr labeling of HDL allows study of its in vivo behavior by using noninvasive PET imaging, including visualization of its accumulation in advanced atherosclerotic lesions. The different labeling strategies provide insight on the pharmacokinetics and biodistribution of HDL's main components (i.e., phospholipids, apo A-I).

Background: Immune cells, particularly myeloid-derived ones, play a pivotal role in the microenvironment of breast cancer. Because of the high diagnostic and therapeutic values of these immune cells, they have been extensively investigated, mostly invasively. Therefore, non-invasive breast cancer immune cell imaging methods can have great impact on diagnosis, disease management, and evaluation of therapy. Here, we describe the development of a high-density lipoprotein (HDL) -based positron emission tomography (PET) nano-tracer to noninvasively image immune cells in a breast cancer model. Methods: Radiolabeled HDL-based nano-tracers were developed by using two different approaches that incorporated the long-lived positron-emitting nuclide ⁸⁹Zr into HDL. The nano-tracers are composed of the phospholipid DMPC and apolipoprotein A-I (apoA-I) in a 2.5 : 1 weight ratio. DFO chelators, conjugated to either phospholipids or apoA-I proteins, were used to complex with ⁸⁹Zr to generate ⁸⁹Zr-PL-HDL (phospholipid-labeled) or ⁸⁹Zr-AI-HDL (apoA-1- labeled). In vivo evaluation was carried out in an orthotropic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo radioautography and histology analyses of tumor tissues were performed to assess regional distribution of the nano-tracers. Fluorescent analogs of the nanotracers were used to determine cell-targeting specificity by using flow cytometry. Results: ⁸⁹Zr-PL-HDL (phospholipid-labeled) was produced in 79 +/- 13% (n = 6) radiochemical yield; ⁸⁹Zr-AI-HDL (apoA-I-labeled), 94 +/- 6% (n = 6). Both nano-tracers had at least 99% radiochemical purity. Intravenous administration of both nano-tracers resulted in high tumor radioactivity accumulation (16.5 +/- 2.8 %ID/g for ⁸⁹Zr-PL-HDL and 8.6 +/- 1.3 %ID/g for ⁸⁹Zr-AI-HDL) at 24 hours post injection. Radioautography and histology analyses showed high colocalization of radioactivity with macrophage-rich areas in tumors. Flow cytometry revealed high accumulation of the nano-tracers in myeloid-derived immune cells (preferentially in tumor-associated macrophages and monocytes, followed by dendritic cells and neutrophils), whereas low uptake was observed in endothelial cells and tumor cells (n = 4). Conclusions: Based on natural HDL particles, we have developed immune cell-targeting PET nano-tracers. In an orthotropic mouse model of breast cancer, we have demonstrated their specificity for myeloid-derived immune cells. Quantitative immune cell PET imaging with our ⁸⁹Zr-PET nano-tracers could be valuable for non-invasive diagnosis of breast cancer and evaluation of immunotherapy response. (Figure Presented)

TRPV4 ion channel mediates vascular mechanosensitivity and vasodilation. Here, we sought to explore whether non-mechanical activation of TRPV4 could limit vascular inflammation and atherosclerosis. We found that GSK1016790A, a potent and specific small-molecule agonist of TRPV4, induces the phosphorylation and activation of eNOS partially through the AMPK pathway. Moreover, GSK1016790A inhibited TNF-alpha-induced monocyte adhesion to human endothelial cells. Mice given GSK1016790A showed increased phosphorylation of eNOS and AMPK in the aorta and decreased leukocyte adhesion to TNF-alpha-inflamed endothelium. Importantly, oral administration of GSK1016790A reduced atherosclerotic plaque formation in ApoE deficient mice fed a Western-type diet. Together, the present study suggests that pharmacological activation of TRPV4 may serve as a potential therapeutic approach to treat atherosclerosis.

Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport (RCT) through upregulation of ATP-binding Cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRalpha complexes, and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRalpha and LXRbeta. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow derived macrophages, but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor, 3-aminobenzamide, enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI (apoA-I). These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis.