Faculty Bibliography

OBJECTIVE: Emerging evidence suggests that neuronal guidance cues, typically expressed during development, are involved in both physiological and pathological immune responses. We hypothesized that endothelial expression of such guidance cues may regulate leukocyte trafficking into the vascular wall during atherogenesis. Approach and Results- We demonstrate that members of the netrin, semaphorin, and ephrin family of guidance molecules are differentially regulated under conditions that promote or protect from atherosclerosis. Netrin-1 and semaphorin3A are expressed by coronary artery endothelial cells and potently inhibit chemokine-directed migration of human monocytes. Endothelial expression of these negative guidance cues is downregulated by proatherogenic factors, including oscillatory shear stress and proinflammatory cytokines associated with monocyte entry into the vessel wall. Furthermore, we show using intravital microscopy that inhibition of netrin-1 or semaphorin3A using blocking peptides increases leukocyte adhesion to the endothelium. Unlike netrin-1 and semaphorin3A, the guidance cue ephrinB2 is upregulated under proatherosclerotic flow conditions and functions as a chemoattractant, increasing leukocyte migration in the absence of additional chemokines. CONCLUSIONS: The concurrent regulation of negative and positive guidance cues may facilitate leukocyte infiltration of the endothelium through a balance between chemoattraction and chemorepulsion. These data indicate a previously unappreciated role for axonal guidance cues in maintaining the endothelial barrier and regulating leukocyte trafficking during atherogenesis.

OBJECTIVE: The persistence of myeloid-derived cells in the artery wall is a characteristic of advanced atherosclerotic plaques. However, the mechanisms by which these cells are retained are poorly understood. Semaphorins, a class of neuronal guidance molecules, play a critical role in vascular patterning and development, and recent studies suggest that they may also have immunomodulatory functions. The present study evaluates the expression of Semaphorin 3E (Sema3E) in settings relevant to atherosclerosis and its contribution to macrophage accumulation in plaques. Approach and Results- Immunofluorescence staining of Sema3E, and its receptor PlexinD1, demonstrated their expression in macrophages of advanced atherosclerotic lesions of Apoe(-/-) mice. Notably, in 2 different mouse models of atherosclerosis regression, Sema3E mRNA was highly downregulated in plaque macrophages, coincident with a reduction in plaque macrophage content and an enrichment in markers of reparative M2 macrophages. In vitro, Sema3E mRNA was highly expressed in inflammatory M1 macrophages and in macrophages treated with physiological drivers of plaque progression and inflammation, such as oxidized low-density lipoprotein and hypoxia. To explore mechanistically how Sema3E affects macrophage behavior, we treated macrophages with recombinant protein in the presence/absence of chemokines, including CCL19, a chemokine implicated in the egress of macrophages from atherosclerotic plaques. Sema3E blocked actin polymerization and macrophage migration stimulated by the chemokines, suggesting that it may immobilize these cells in the plaque. CONCLUSIONS: Sema3E is upregulated in macrophages of advanced plaques, is dynamically regulated by multiple atherosclerosis-relevant factors, and acts as a negative regulator of macrophage migration, which may promote macrophage retention and chronic inflammation in vivo.

Why patients with chronic kidney disease have elevated cardiovascular risk remains elusive. In this issue of Immunity, Speer et al. (2013) show that natural modification of high density lipoprotein promotes hypertension through a toll-like receptor-dependent mechanism.

Niacin therapy, used either alone or in combination with another agent to reduced the risk of coronary heart disease, is effective in raising and lowering, respectively, HDL-c and apoB/VLDL triglyceride (TG) levels in dyslipidemia. To further understand if part of the mechanism is by reducing the secretion of apoB-containing lipoproteins through autophagy, we did a series of niacin studies in isolated primary hepatocytes or in intact mice. Metabolic labeling and pulse-chase results showed niacin increased intracellular apoB degradation by 30%, and decreased apoB/VLDL TG secretions by 40%. These effects were lost in autophagy-deficient cells. Mice fed with diet containing 3% niacin had 40% increases in plasma HDL-c. Feeding high fat diet (HFD) with niacin lowered LDL-c by 50%. Niacin was able to suppress postprandial plasma triglyceride by 30%. Niacin reduced hepatic triglyceride and cholesterol concentrations by 40% and 50%, respectively, in mice fed with HFD. These data suggest niacin decreases hepatic lipid concentrations, apoB/VLDL secretion, and modifies plasma cholesterol levels. Some of the effects required niacin metabolism and autophagy degradation

Hypoxia has been found in the atherosclerotic plaques of larger mammals, including humans. Whether hypoxia occurs in the plaques of standard mouse models with atherosclerosis has been controversial, given their small size. In this review, we summarize the findings of a recent report demonstrating that direct evidence of hypoxia can indeed be found in the plaques of mice deficient in apolipoprotein E (apoE-/-mice). Furthermore, studies in vitro showed that hypoxia promoted lipid synthesis and reduced cholesterol efflux through the ABCA1 pathway, and that the transcription factor HIF-1alpha mediated many, but not all, of the effects. These results are discussed in the context of the literature and clinical practice.

OBJECTIVES: This study sought to develop magnetic resonance contrast agents based on high-density lipoprotein (HDL) nanoparticles to noninvasively visualize intraplaque macrophages and collagen content in mouse atherosclerotic plaques. BACKGROUND: Macrophages and collagen are important intraplaque components that play central roles in plaque progression and/or regression. In a Reversa mouse model, plaque regression with compositional changes (from high macrophage, low collagen to low macrophage, high collagen) can be induced. METHODS: This study labeled HDL nanoparticles with amphiphilic gadolinium chelates to enable target-specific imaging of intraplaque macrophages. To render HDL nanoparticles specific for the extracellular matrix, labeled HDL nanoparticles were functionalized with collagen-specific EP3533 peptides (EP3533-HDL) via poly(ethylene glycol) spacers embedded in the HDL lipid layers. The association of nanoparticles with collagen was examined in vitro by optical methods. The in vivo magnetic resonance efficacy of these nanoparticles was evaluated in a Reversa mouse model of atherosclerosis regression. Ex vivo confocal microscopy was applied to corroborate the in vivo findings and to evaluate the fate of the different HDL nanoparticles. RESULTS: All nanoparticles had similar sizes (10 +/- 2 nm) and longitudinal relaxivity r (9 +/- 1 s mmol/l). EP3533-HDL showed strong association with collagen in vitro. After 28 days of plaque regression in Reversa mice, EP3533-HDL showed significantly increased (p < 0.05) in vivo magnetic resonance signal in aortic vessel walls (normalized enhancement ratio [NER] = 85 +/- 25%; change of contrast-to-noise ratio [DeltaCNR] = 17 +/- 5) compared with HDL (NER = -7 +/- 23%; DeltaCNR = -2 +/- 4) and nonspecific control EP3612-HDL (NER = 4 +/- 24%; DeltaCNR = 1 +/- 6) at 24 h after injection. Ex vivo confocal images revealed the colocalization of EP3533-HDL with collagen. Immunohistostaining analysis confirmed the changes of collagen and macrophage contents in the aortic vessel walls after regression. CONCLUSIONS: This study shows that the HDL nanoparticle platform can be modified to monitor in vivo plaque compositional changes in a regression environment, which will facilitate understanding plaque regression and the search for therapeutic interventions.

OBJECTIVE: Regression of atherosclerosis is a vital treatment goal of atherosclerotic vascular disease. Inhibitors of the microsomal triglyceride transfer protein (MTP) have been shown to reduce apolipoprotein B (apoB)-containing lipoproteins in animals and humans effectively. Therefore, the major aim of our study is to evaluate the effect of MTP inhibition on atherosclerotic plaque regression. METHODS: LDL-receptor-deficient (LDLr(-/-)) mice were fed a Western diet for 16 weeks and then harvested for baseline (n = 8), switched to chow diet (n = 8) or chow diet containing MTP inhibitor (BMS 212122; n = 8) for 2 weeks before harvesting. RESULTS: Treatment with MTP inhibitor led to rapid reduction in plasma lipid levels, which were accompanied by a significant decrease in lipid content and monocyte-derived (CD68+) cells in atherosclerotic plaques compared to baseline and chow diet control groups. MTP inhibitor-treated mice had increased collagen content, a marker associated with increased stability in human plaques. Furthermore, plaques of these mice showed a significant decrease in tissue factor and pro-inflammatory M1 macrophage marker monocyte chemoattractant protein-1 (MCP-I) and an increase in anti-inflammatory M2 macrophage markers arginase-I and mannose receptor 1 compared to mice in the baseline group. CONCLUSION: Reversal of hyperlipidemia in atherosclerotic mice by inhibition of MTP leads to rapid and beneficial changes in the composition and inflammatory state of the plaque.

Lipoproteins are natural nanoparticles composed of phospholipids and apolipoproteins that transport lipids throughout the body. As key effectors of lipid homeostasis, the functions of lipoproteins have been demonstrated to be crucial during the development of cardiovascular diseases. Therefore various strategies have been used to study their biology and detect them in vivo. A recent approach has been the production of lipoprotein biomimetic particles loaded with diagnostically active nanocrystals in their core. These include, but are not limited to: quantum dots, iron oxide or gold nanocrystals. Inclusion of these nanocrystals enables the utilization of lipoproteins as probes for a variety of imaging modalities (computed tomography, magnetic resonance imaging, fluorescence) while preserving their biological activity. Furthermore as some lipoproteins naturally accumulate in atherosclerotic plaque or specific tumor tissues, nanocrystal core lipoprotein biomimetics have been developed as contrast agents for early diagnosis of these diseases.

OBJECTIVE: Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E-deficient mice. METHODS AND RESULTS: Apolipoprotein-E-deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels. CONCLUSIONS: Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E-deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.

Coronary artery disease, resulting from atherosclerosis, is the leading cause of death in the Western world. Most previous studies have subjected atherosclerotic arteries, a tissue of mixed cellular composition, to homogenization in order to identify the factors in plaque development, thereby obscuring information relevant to specific cell types. Because macrophage foam cells are critical mediators in atherosclerotic plaque advancement, we reasoned that performing gene analysis on those cells would provide specific insight in novel regulatory factors and potential therapeutic targets. We demonstrated for the first time in vascular biology that foam cell-specific RNA can be isolated by laser capture microdissection (LCM) of plaques. As expected, compared to whole tissue, a significant enrichment in foam cell-specific RNA transcripts was observed. Furthermore, because regression of atherosclerosis is a tantalizing clinical goal, we developed and reported a transplantation-based mouse model. This involved allowing plaques to form in apoE-/- mice and then changing the plaque's plasma environment from hyperlipidemia to normolipidemia. Under those conditions, rapid regression ensued in a process involving emigration of plaque foam cells to regional and systemic lymph nodes. Using LCM, we were able to show that under regression conditions, there was decreased expression in foam cells of inflammatory genes, but an up-regulation of cholesterol efflux genes. Interestingly, we also found that increased expression of chemokine receptor CCR7, a known factor in dendritic cell migration, was required for regression. In conclusion, the LCM methods described in this chapter, which have already lead to a number of striking findings, will likely further facilitate the study of cell type-specific gene expression in animal and human plaques during various stages of atherosclerosis, and after genetic, pharmacologic, and environmental perturbations.