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Objective: vMIP-II (viral macrophage inflammatory protein 2)/vCCL2 (viral chemotactic cytokine ligand 2) binds to multiple chemokine receptors, and vMIP-II-based positron emission tomography tracer ( 64 Cu-DOTA-vMIP-II: vMIP-II tracer) accumulates at atherosclerotic lesions in mice. Given that it would be expected to react with multiple chemokine receptors on monocytes and macrophages, we wondered if its accumulation in atherosclerosis lesion-bearing mice might correlate with overall macrophage burden or, alternatively, the pace of monocyte recruitment. Approach and Results: We employed a mouse model of atherosclerosis regression involving adenoassociated virus 8 vector encoding murine Apoe (AAV-mApoE) treatment of Apoe −/− mice where the pace of monocyte recruitment slows before macrophage burden subsequently declines. Accumulation of 64 Cu-DOTA-vMIP-II at Apoe −/− plaque sites was strong but declined with AAV- mApoE -induced decline in monocyte recruitment, before macrophage burden reduced. Monocyte depletion indicated that monocytes and macrophages themselves were not the only target of the 64 Cu-DOTA-vMIP-II tracer. Using fluorescence-tagged vMIP-II tracer, competitive receptor blocking with CXCR4 antagonists, endothelial-specific Cre-mediated deletion of CXCR4, CXCR4-specific tracer 64 Cu-DOTA-FC131, and CXCR4 staining during disease progression and regression, we show endothelial cell expression of CXCR4 is a key target of 64 Cu-DOTA-vMIP-II imaging. Expression of CXCR4 was low in nonplaque areas but strongly detected on endothelium of progressing plaques, especially on proliferating endothelium, where vascular permeability was increased and monocyte recruitment was the strongest. Conclusions: Endothelial injury status of plaques is marked by CXCR4 expression and this injury correlates with the tendency of such plaques to recruit monocytes. Furthermore, our findings suggest positron emission tomography tracers that mark CXCR4 can be used translationally to monitor the state of plaque injury and monocyte recruitment.

HDL is cardioprotective, but plasma HDL levels do not necessarily predict cardiovascular outcomes. The major HDL-associated protein apoA-I picks up its cholesterol from cells within extravascular compartments to return it to plasma and then bile. Yet, tools are lacking to quantify the important step of HDL transit through extravascular spaces. Here, we developed recombinant photoactivatable apoA-I to quantify endogenous HDL recirculation. Using the tool, we studied HDL passage through skin in healthy mice versus those with experimental psoriasis, wherein collagen density increased in the skin in a CD4 + T cell-dependent manner. In control mice, photoactivated HDL mobilized to plasma within 2 h but was retained in collagen-enriched skin of mice with psoriasis. These data suggest that cardiovascular comorbidity in psoriasis might be linked to T cell-mediated structural changes in skin that impedes systemic recirculation of HDL. This new tool is likely to find wide application in HDL research.

Atherosclerosis is an underlying cause of cardiovascular disease and a leading cause of mortality worldwide. Macrophage accumulation in atherosclerotic plaque, their uptake of cholesterol, and subsequent local death drive disease progression. Lipid-laden plaque macrophages are thought to be exclusively derived from blood monocyte progenitors that are recruited following endothelial damage induced by cholesterol exposure. In our current study, we focused on characterization of resident vascular macrophages that reside in the aortic intima in plaque-prone areas, previously identified as ‘vascular dendritic cells’. Using en face whole-mount confocal microscopy of aortas, we confirm a uniform resident CD64 + CD11c + CX3CR1 + MHCII + macrophage population, which is present in C57/BL6 mice resistant to atherosclerosis. Importantly, they do not express dendritic cell restricted genes zBTB46 or L-myc. We find aortic macrophages require M-CSF and Flt3 signaling for survival, but are independent of CCR2, CCR7, and GM-CSF receptor signaling, making them a distinct myeloid population. Lineage-tracing and parabiosis approaches suggest these cells derive from definitive hematopoiesis and are then self-maintained independent of blood-progenitors. Using these characterization data, we developed a labeling strategy to identify resident from recruited macrophages during kinetic studies of lesion progression. We find that resident aortic macrophages are the first cells to take up lipid following high fat diet exposure and expand within the arterial wall to form the initial lesion bed. In the absence of resident macrophages early lipid deposition in the aortic arch is ablated. Finally, utilizing an intravital carotid artery imaging approach, we identify resident aortic macrophages to be potential mediators of monocyte recruitment through direct interactions with rolling monocytes on the endothelial surface under diseased and steady-states. Overall, these results shift our understanding of the cellular mechanisms responsible for plaque construction and maintenance.

Atherosclerosis is an underlying cause of cardiovascular disease (CVD) and a leading cause of morbidity and mortality worldwide. Atherosclerosis promotes CVD through plaque formation, restricted blood flow, and thrombotic events. Macrophage accumulation in plaques, their uptake of cholesterol, and subsequent local death drive disease progression, however, there is growing appreciation for more diverse roles of myeloid cells during disease progression. Here, we focused on characterization of so-called vascular dendritic cells (DCs), which we refer to as aortic mononuclear phagocytes (MNP), that reside under the endothelium in plaque-prone areas. Using en face whole-mount confocal microscopy of aortas, we confirm a uniform resident CD11c+ CX3CR1+ MHCII+ MNP population, even in C57/BL6 mice resistant to atherosclerosis. We find aortic MNPs require M-CSF and Flt3 signaling for survival, but are independent of CCR2 and GM-CSF receptor signaling, making them a distinct myeloid population. They express macrophage-restricted genes LysM and CD64, but not dendritic cell specific genes zBTB46 or L-myc. Lineage-tracing analysis using CD115creER and Flt3cre reporter mice indicate that aortic MNPs likely differentiate from definitive hematopoiesis and not early yolk sac progenitors. Parabiosis experiments show that aortic MNPs are self-maintained independent of blood-born progenitors, failing to exchange with blood progenitors for up to 5 months. Aortic MNPs are different from either typical DCs or macrophages and instead appear to blend distinguishing features of each, more closely resembling macrophages. Using our characterization data, we hope to develop an inducible cell-tracking and a cell-depletion model to differentiate the function of aortic MNPs during the progression of atherosclerosis. Overall, we anticipate these functional analyses will reveal, for the first time, the role of resident aortic MNPs in atherosclerosis.

Monocytes are central mediators in the advance of atherosclerotic plaque, making them a natural therapeutic target for reducing disease burden. Here, we highlight recent advances in our current understanding of monocyte heterogeneity and its relevance to regulation of monocyte accumulation and function within atherosclerotic plaques. Differences that distinguish monocyte subsets include differential expression of chemokine receptors, especially CCR2 and CX3CR1. Ablation of expression of these 2 receptors (or their ligands) in mice has an additive inhibition on monocyte recruitment to atherosclerotic plaques. Moreover, simultaneously interfering with 3 key pathways—CCR2, CX3CR1, and CCR5—essentially abolishes atherosclerosis in mice. Here, we discuss how these chemokine receptors act at multiple points on at least 1 monocyte subset, regulating their mobilization from bone marrow, survival, or recruitment to plaques. Finally, we discuss how this knowledge may be useful clinically, emphasizing that CX3CR1 may in particular be a viable target for therapeutic manipulation of monocyte-derived cell fate in cardiovascular disease.

Patients with auto-immune disorders have low HDL levels. Mice deficient in genes regulating cholesterol homeostasis, such as liver X receptor (LXR) or apolipoprotein A1 (apoA1), show an autoimmune phenotype. LXR regulates the expression of ATP Binding Cassette A1 and G1 (ABCA1 and ABCG1) that mediate cholesterol efflux to apoA1 and HDL. ABCG1 is highly expressed in dendritic cells (DCs). We hypothesized that ABCA1 and ABCG1 regulate autoimmunity. On a chow diet, 40 weeks old Abca1-/-Abcg1-/- mice showed enlarged lymph nodes (LNs), increased plasma auto-antibodies to dsDNA, and glomerulonephritis, with characteristics typical for lupus nephritis. Using the Cre loxP system, we investigated whether these effects were due to Abca1/g1 deficiency in T-cells, macrophages, or DCs. Only Abca1/g1 deficiency in DCs in CD11cCreAbca1fl/flAbcg1fl/fl mice replicated the auto-immune phenotype found in Abca1-/-Abcg1-/- mice. This suggests a major role for DC cholesterol homeostasis in autoimmunity. DCs present antigens to T-cells, leading to their activation. CD11cCreAbca1fl/flAbcg1fl/fl mice showed increased T-cell activation in blood, spleen, and LNs. After immunization, DCs from CD11cCreAbca1fl/flAbcg1fl/fl mice showed increased antigen presentation to T-cells in vitro and in vivo. CD11cCreAbca1fl/flAbcg1fl/fl mice had increased CD80+ DCs. CD80 is a co-stimulatory molecule required for antigen presentation. Abca1/g1 deficiency in DCs increased endosomal cholesterol accumulation in vitro. Ligands for Toll like receptor (TLR) 3 and 4 increased CD80 mRNA in CD11cCreAbca1fl/flAbcg1fl/fl compared to Abca1fl/flAbcg1fl/fl DCs, where the effect of ligands for TLR3>TLR4. Cholesterol depletion by cyclodextrin decreased CD80 mRNA and antigen presentation in CD11cCreAbca1fl/flAbcg1fl/fl DCs in vitro. The increased CD80 mRNA in CD11cCreAbca1fl/flAbcg1fl/fl DCs was also reversed by a type I interferon (IFN) antibody, suggesting excessive signaling by the known TLR3-IFN-CD80 axis. These studies show for the first time a role for cholesterol efflux pathways in DCs in maintaining immune tolerance.

Reverse cholesterol transport (RCT) refers to mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used two models of disrupted lymphatic drainage from skin -one surgical and the other genetic- to quantitatively track RCT following injection of [3H]cholesterol-loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analogue (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aorta of donor atherosclerotic apoE-/- mice with [2H]6-labeled cholesterol and surgically transplanting these aortas into recipient apoE-/- mice that were treated with anti-VEGFR3 mAb to block lymphatic regrowth or with control mAb to allow such regrowth. [2H]-Cholesterol was retained in aortas of anti-VEGFR3 treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. Therefore, supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis.

Resolution of acute inflammation is a key step in the return to homeostasis after initiation of tissue injury, and its failure ultimately leads to a state of chronic inflammation known to favor the initiation and/or the progression of numerous diseases such as atherosclerosis. However, the cellular mechanisms governing inflammatory macrophages (iM f ) clearance during the process of resolution are not completely understood. To better investigate iM f clearance in acute inflammation, we for the first time compared the importance of emigration through lymphatics to that of local death using a classical model of acute resolving peritonitis induced by thioglycollate. Of note, Ly-6C hi monocyte-derived iM f that accumulated in the peritoneum became CD11c+ cells, but gene expression profiling revealed these cells as macrophages, not dendritic cells. Although these iM f migrate to draining lymph nodes, this appeared in a kinetic and a quantity that doesn’t fit with the onset and extend of macrophage contraction during resolution. We found that migration out of the peritoneal cavity surprisingly did not rely on CCR7 but was partially dependent on CCR2. Moreover, blocking migration using a Gαi signaling inhibitors (that block all chemokine-dependent migrations and others) minimally impact on iM f clearance. By contrast, competition assays between WT and CD68-Bcl2 transgenic or Bim -/- iM f revealed that macrophage protected from apoptosis were much less susceptible to be cleared from the cavity during resolution. Thus, we conclude that in a model previously claimed to remove macrophages via emigration, local death of macrophages is quantitatively the main mode of clearance. As we recently reported that iM f removal from regressing atherosclerotic plaques is an emigration-independent process, we are now completing experiments to study the role of apoptosis in this phenomenon. Overall, removal by local death and control of new recruitment may be universally dominant mechanisms for macrophage clearance.

Introduction: HDL accepts free cholesterol from cells within extravascular tissues and transports it to the liver for excretion. Little is known about how HDL-C leaves such tissues to reach the blood. HDL-C is found in lymph, but it is unclear if lymphatics are critical for mobilization of HDL-C from tissues. Methods & Results: We employed models of disrupted lymphatic drainage--one a surgical resection of lymphatics in the mouse tail and the other a genetic lack of skin lymphatics due to a mutation in VEGFR3--to quantify the role of lymphatic vessels in movement of HDL-C out of skin to plasma. We implanted bone marrow macrophages loaded with [ 3 H]-cholesterol in skin sites and then quantified [ 3 H]-cholesterol in plasma, liver, and feces. [ 3 H]-cholesterol movement into plasma was reduced by 80% in both models of disrupted lymphatics, and this result was unaffected by conditions of a leaky blood vessels. To ensure that this reduction reflected events between efflux and appearance of cholesterol in plasma rather than altered macrophage cholesterol efflux, we substituted [ 3 H]-cholesterol-loaded macrophages with congenic macrophages loaded with a fluorescently modified cholesterol compound that allowed us to monitor ABCA1/ABCG1-dependent efflux from individual macrophages in the same assays and models. Flow cytometric analysis of bodipy-cholesterol-loaded macrophages retrieved from injection sites revealed similar efflux in mice lacking functional lymphatics as in controls. Conclusions: Mobilization of HDL-C from extravascular tissues to plasma occurs through the lymphatic vasculature. Recent studies show that transport through lymphatic vessels is compromised by hypercholesterolemia. Hypercholesterolemia-induced impairments in lymphatic function may retard clearance of cholesterol from extravascular tissues, possibly including atherosclerotic plaques.

Neural guidance cues are attracting attention in atherosclerosis. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Wanschel et al1 add yet another neural guidance molecule, Semaphorin 3E, to the growing list of neural guidance cues that may contribute to atherosclerosis. Indeed, this team of scientists, led by Kathryn Moore at New York University, has in the last year implicated 3 other neural guidance cues in atherosclerosis, including netrin-1,2,3 semaphorin 3A,3 and ephrin B3 in the regulation of inflammatory atherosclerosis. See accompanying article on page 886 In the first of a series of papers on these molecules, this group showed that netrin-1–deficient bone marrow transplanted into LDLR–/− mice led to markedly reduced atherosclerotic plaques.2 Using a method to track monocyte entry and persistence in a quantitative manner,4,5 the authors argued that netrin-1 was essential for retaining monocyte-derived foamy macrophages in plaques. In the absence of netrin-1, monocytes could …