CC Chemokine Receptor 5 Targeted Nanoparticles Imaging the Progression and Regression of Atherosclerosis Using Positron Emission Tomography/Computed Tomography.

Chemokines and chemokine receptors play an important role in the initiation and progression of atherosclerosis by mediating the trafficking of inflammatory cells. Chemokine receptor 5 (CCR5) has major implications in promoting the development of plaques to advanced stage and related vulnerability. CCR5 antagonist has demonstrated the effective inhibition of atherosclerotic progression in mice, making it a potential biomarker for atherosclerosis management. To accurately determine CCR5 in vivo, we synthesized CCR5 targeted Comb nanoparticles through a modular design and construction strategy with control over the physiochemical properties and functionalization of CCR5 targeting peptide d-Ala-peptide T-amide (DAPTA-Comb). In vivo pharmacokinetic evaluation through 64Cu radiolabeling showed extended blood circulation of 64Cu-DAPTA-Combs conjugated with 10%, 25%, and 40% DAPTA. The different organ distribution profiles of the three nanoparticles demonstrated the effect of DAPTA on not only physicochemical properties but also targeting efficiency. In vivo positron emission tomography/computed tomography (PET/CT) imaging in an apolipoprotein E knockout mouse atherosclerosis model (ApoE-/-) showed that the three 64Cu-DAPTA-Combs could sensitively and specifically detect CCR5 along the progression of atherosclerotic lesions. In an ApoE-encoding adenoviral vector (AAV) induced plaque regression ApoE-/- mouse model, decreased monocyte recruitment, CD68+ macrophages, CCR5 expression, and plaque size were all associated with reduced PET signals, which not only further confirmed the targeting efficiency of 64Cu-DAPTA-Combs but also highlighted the potential of these targeted nanoparticles for atherosclerosis imaging. Moreover, the up-regulation of CCR5 and colocalization with CD68+ macrophages in the necrotic core of ex vivo human plaque specimens warrant further investigation for atherosclerosis prognosis.

CXCR4-Binding Positron Emission Tomography Tracers Link Monocyte Recruitment and Endothelial Injury in Murine Atherosclerosis.

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 (64Cu-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 64Cu-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 64Cu-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 64Cu-DOTA-FC131, and CXCR4 staining during disease progression and regression, we show endothelial cell expression of CXCR4 is a key target of 64Cu-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.

Limited proliferation capacity of aortic intima resident macrophages requires monocyte recruitment for atherosclerotic plaque progression.

Early atherosclerosis depends upon responses by immune cells resident in the intimal aortic wall. Specifically, the healthy intima is thought to be populated by vascular dendritic cells (DCs) that, during hypercholesterolemia, initiate atherosclerosis by being the first to accumulate cholesterol. Whether these cells remain key players in later stages of disease is unknown. Using murine lineage-tracing models and gene expression profiling, we reveal that myeloid cells present in the intima of the aortic arch are not DCs but instead specialized aortic intima resident macrophages (MacAIR) that depend upon colony-stimulating factor 1 and are sustained by local proliferation. Although MacAIR comprise the earliest foam cells in plaques, their proliferation during plaque progression is limited. After months of hypercholesterolemia, their presence in plaques is overtaken by recruited monocytes, which induce MacAIR-defining genes. These data redefine the lineage of intimal phagocytes and suggest that proliferation is insufficient to sustain generations of macrophages during plaque progression.

Schistosoma mansoni Infection-Induced Transcriptional Changes in Hepatic Macrophage Metabolism Correlate With an Athero-Protective Phenotype.

Hepatic macrophages play an essential role in the granulomatous response to infection with the parasitic helminth Schistosoma mansoni, but the transcriptional changes that underlie this effect are poorly understood. To explore this, we sorted the two previously recognized hepatic macrophage populations (perivascular and Kupffer cells) from naïve and S. mansoni-infected male mice and performed microarray analysis as part of the Immunological Genome Project. The two hepatic macrophage populations exhibited remarkably different genomic profiles. However, this diversity was substantially reduced following infection with S. mansoni, and in fact, both populations demonstrated increases in transcripts of the monocyte lineage, suggesting that both populations may be replenished by monocytes following infection. Pathway analysis showed a profound alteration in global metabolic pathways, including changes to phospholipid and cholesterol metabolism, as well as amino acid biosynthesis and glucagon signaling. These changes suggest a possible mechanism for the previously reported athero-protective effects of S. mansoni infection. Indeed, we find that male ApoE null mice fed a high-fat diet in combination with S. mansoni infection have reduced plaque area and increased glucose tolerance as compared to control mice. Transcript analysis of infected and control high-fat diet fed ApoE-/- mice confirm that ApoC1, Psat1, and Gys1 are all altered by infection, suggesting that altered hepatic macrophage metabolism is associated with S. mansoni- induced protection from hyperlipidemia, atherosclerosis, and glucose intolerance. These results suggest a previously unknown and unreported role of hepatic macrophages in the modulation of whole body lipid and glucose metabolism during infection and provide a template for examining the role of immunomodulation on the long-term metabolism of the host.

Limited Macrophage Positional Dynamics in Progressing or Regressing Murine Atherosclerotic Plaques-Brief Report.

Objective- Macrophages play important roles in the pathogenesis of atherosclerosis, but their dynamics within plaques remain obscure. We aimed to quantify macrophage positional dynamics within progressing and regressing atherosclerotic plaques. Approach and Results- In a stable intravital preparation, large asymmetrical foamy macrophages in the intima of carotid artery plaques were sessile, but smaller rounded cells nearer plaque margins, possibly newly recruited monocytes, mobilized laterally along plaque borders. Thus, to test macrophage dynamics in plaques over a longer period of time in progressing and regressing disease, we quantified displacement of nondegradable phagocytic particles within macrophages for up to 6 weeks. In progressing plaques, macrophage-associated particles appeared to mobilize to deeper layers in plaque, whereas in regressing plaques, the label was persistently located near the lumen. By measuring the distance of the particles from the floor of the plaque, we discovered that particles remained at the same distance from the floor regardless of plaque progression or regression. The apparent deeper penetration of labeled cells in progressing conditions could be attributed to monocyte recruitment that generated new superficial layers of macrophages over the labeled phagocytes. Conclusions- Although there may be individual exceptions, as a population, newly differentiated macrophages fail to penetrate significantly deeper than the limited depth they reside on initial entry, regardless of plaque progression, or regression. These limited dynamics may prevent macrophages from escaping areas with unfavorable conditions (such as hypoxia) and pose a challenge for newly recruited macrophages to clear debris through efferocytosis deep within plaque.

PET/MRI of Hypoxic Atherosclerosis Using 64Cu-ATSM in a Rabbit Model.

The macrophage-rich core of advanced human atheroma has been demonstrated to be hypoxic, which may have implications in plaque stability. The goal of this study was to determine the feasibility of the hypoxia PET imaging agent 64Cu-ATSM to detect hypoxia in a rabbit model of atherosclerosis imaged on a simultaneous PET/MR scanner, using MR for both attenuation correction and depiction of lesion location. METHODS: New Zealand White rabbits fed a Western diet for 4-6 wk underwent endothelial denudation of the right femoral artery by air desiccation to induce an atherosclerotic-like lesion and underwent a sham operation on the left femoral artery. Four and 8 wk after injury, a 0- to 60-min dynamic whole-body PET/MR examination was performed after injection of approximately 111 MBq of 64Cu-ATSM. After 24 h, a 0- to 75-min dynamic PET/MR examination after injection of approximately 111 MBq of 18F-FDG was performed. The rabbits were euthanized, and the injured femoral artery (IF) and sham-operated femoral artery (SF) were collected for immunohistochemistry assessment of hypoxic macrophages (hypoxia marker pimonidazole, macrophage marker RAM-11, and hypoxia-inducible factor-1 α subunit [HIF-1α]). Regions of interest of IF, SF, and background muscle (BM) were drawn on fused PET/MR images, and IF-to-BM and SF-to-BM SUV ratios were compared using the Student t test. RESULTS: Elevated uptake of 64Cu-ATSM was found in the rabbits' IF compared with the SF. 64Cu-ATSM imaging demonstrated IF-to-SF SUVmean ratios (±SD) of 1.75 ± 0.21 and 2.30 ± 0.26 at 4 and 8 wk after injury, respectively. 18F-FDG imaging demonstrated IF-to-SF SUVmean ratios of 1.84 ± 0.12 at 8 wk after injury. IF-to-BM SUVmean ratios were significantly higher (P < 0.001) than SF-to-BM SUVmean ratios both 4 and 8 wk after injury for 64Cu-ATSM and 8 wk after injury for 18F-FDG (P < 0.05). Pimonidazole immunohistochemistry at 8 wk colocalized to RAM-11 and HIF-1α. CONCLUSION: The results show that hypoxia is present in this rabbit model of atherosclerosis and suggest that 64Cu-ATSM PET/MR is a potentially promising method for the detection of hypoxic and potentially vulnerable atherosclerotic plaque in human subjects.