Faculty Bibliography

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.

Atherosclerosis, caused in part by monocytes in plaques, continues to be a disease that afflicts the modern world. Whilst significant steps have been made in treating this chronic inflammatory disease, questions remain on how to prevent monocyte and macrophage accumulation in atherosclerotic plaques. Junctional Adhesion Molecule C (JAM-C) expressed by vascular endothelium directs monocyte transendothelial migration in a unidirectional manner leading to increased inflammation. Here we show that interfering with JAM-C allows reverse-transendothelial migration of monocyte-derived cells, opening the way back out of the inflamed environment. To study the role of JAM-C in plaque regression we used a mouse model of atherosclerosis, and tested the impact of vascular JAM-C expression levels on monocyte reverse transendothelial migration using human cells. Studies in-vitro under inflammatory conditions revealed that overexpression or gene silencing of JAM-C in human endothelium exposed to flow resulted in higher rates of monocyte reverse-transendothelial migration, similar to antibody blockade. We then transplanted atherosclerotic, plaque-containing aortic arches from hyperlipidemic ApoE-/- mice into wild-type normolipidemic recipient mice. JAM-C blockade in the recipients induced greater emigration of monocyte-derived cells and further diminished the size of atherosclerotic plaques. Our findings have shown that JAM-C forms a one-way vascular barrier for leukocyte transendothelial migration only when present at homeostatic copy numbers. We have also shown that blocking JAM-C can reduce the number of atherogenic monocytes/macrophages in plaques by emigration, providing a novel therapeutic strategy for chronic inflammatory pathologies.

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)

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).

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.

BACKGROUND: High density lipoproteins (HDLs) are thought to be atheroprotective and to reduce the risk of cardiovascular disease (CVD). Besides their antioxidant, antithrombotic, anti-inflammatory, anti-apoptotic properties in the vasculature, HDLs also improve glucose metabolism in skeletal muscle. SCOPE OF THE REVIEW: Herein, we review the functional role of HDLs to improve metabolic disorders, especially those involving insulin resistance and to induce regression of CVD with a particular focus on current pharmacological treatment options as well as lifestyle interventions, particularly exercise. MAJOR CONCLUSIONS: Functional properties of HDLs continue to be considered important mediators to reverse metabolic dysfunction and to regress atherosclerotic cardiovascular disease. Lifestyle changes are often recommended to reduce the risk of CVD, with exercise being one of the most important of these. Understanding how exercise improves HDL function will likely lead to new approaches to battle the expanding burden of obesity and the metabolic syndrome.

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.

Cardinal events in atherogenesis are the retention of apolipoprotein B-containing lipoproteins in the arterial wall and the reaction of macrophages to these particles. My laboratory has been interested in both the cell biological events producing apolipoprotein B-containing lipoproteins, as well as in the reversal of the damage they cause in the plaques formed in the arterial wall. In the 2013 George Lyman Duff Memorial Lecture, as summarized in this review, I covered 3 areas of my past, present, and future interests, namely, the regulation of hepatic very low density lipoprotein production by the degradation of apolipoprotein B100, the dynamic changes in macrophages in the regression of atherosclerosis, and the application of nanoparticles to both image and treat atherosclerotic plaques.