Faculty Bibliography

OBJECTIVE: Hypoxia is intimately linked to atherosclerosis and has become recognized as a primary impetus of inflammation. We recently demonstrated that the neuroimmune guidance cue netrin-1 (Ntn1) inhibits macrophage emigration from atherosclerotic plaques, thereby fostering chronic inflammation. However, the mechanisms governing netrin-1 expression in atherosclerosis are not well understood. In this study, we investigate the role of hypoxia in regulating expression of netrin-1 and its receptor uncoordinated-5-B receptor (Unc5b) in plaque macrophages and its functional consequences on these immune cells. APPROACH AND RESULTS: We show by immunostaining that netrin-1 and Unc5b are expressed in macrophages in hypoxia-rich regions of human and mouse plaques. In vitro, Ntn1 and Unc5b mRNA are upregulated in macrophages treated with oxidized low-density lipoprotein or inducers of oxidative stress (CoCl2, dimethyloxalylglycine, 1% O2). These responses are abrogated by inhibiting hypoxia-inducible transcription factor (HIF)-1alpha, indicating a causal role for this transcription factor in regulating Ntn1 and Unc5b expression in macrophages. Indeed, using promoter-luciferase reporter genes, we show that Ntn1- and Unc5b-promoter activities are induced by oxidized low-density lipoprotein and require HIF-1alpha. Correspondingly, J774 macrophages overexpressing active HIF-1alpha show increased netrin-1 and Unc5b expression and reduced migratory capacity compared with control cells, which was restored by blocking the effects of netrin-1. Finally, we show that netrin-1 protects macrophages from apoptosis under hypoxic conditions in a HIF-1alpha-dependent manner. CONCLUSIONS: These findings provide a molecular mechanism by which netrin-1 and its receptor Unc5b are expressed in atherosclerotic plaques and implicate hypoxia and HIF-1alpha-induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages.

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.

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.

Both in humans and animal models, an acute increase in plasma insulin levels, typically following meals, leads to transient depression of hepatic secretion of very low density lipoproteins (VLDL). One contributing mechanism for the decrease in VLDL secretion is enhanced degradation of apolipoprotein B100 (apoB100), which is required for VLDL formation. Unlike the degradation of nascent apoB100, which occurs in the endoplasmic reticulum (ER), insulin-stimulated apoB100 degradation occurs post-ER and is inhibited by pan-phosphatidylinositol (PI)3-kinase inhibitors. It is unclear, however, which of the three classes of PI3-kinases is required for insulin-stimulated apoB100 degradation, as well as the proteolytic machinery underlying this response. Class III PI3-kinase is not activated by insulin, but the other two classes are. By using a class I-specific inhibitor and siRNA to the major class II isoform in liver, we now show that it is class II PI3-kinase that is required for insulin-stimulated apoB100 degradation in primary mouse hepatocytes. Because the insulin-stimulated process resembles other examples of apoB100 post-ER proteolysis mediated by autophagy, we hypothesized that the effects of insulin in autophagy-deficient mouse primary hepatocytes would be attenuated. Indeed, apoB100 degradation in response to insulin was significantly impaired in two types of autophagy-deficient hepatocytes. Together, our data demonstrate that insulin-stimulated apoB100 degradation in the liver requires both class II PI3-kinase activity and autophagy.

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.

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.

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.

Rationale: Statins have been shown to have anti-inflammatory pleiotropic effects in experimental settings. However, the bioavailability of orally administered statins is poor, which limits the direct therapeutic effects on atherosclerotic plaques. We have developed a simvastatin loaded high-density lipoprotein nanoparticle platform ([S]-rHDL), with the similar properties to natural HDL (A). The formulation has potent anti-inflammatory effects in vitro (S-A), produces significantly higher therapeutic effects in apolipoprotein E knockout (ApoE KO) mice than oral simvastatin or rHDL treatment, and induces rapid regression when administered at a high dose (S-B). The purpose of the current study is to understand the mechanism of action by which this formulation exerts its potent antiinflammatory effects. Methods and Results: To investigate the protecting effects of [S]-rHDL on encapsulated simvastatin in blood, the nanoparticles were incubated in mouse serum and intact simvastatin was measured over time with HPLC. [S]-rHDL drastically protected the encapsulated simvastatin (B). To evaluate the in vivo targeting of atherosclerotic plaques, [S]-rHDL was additionally labeled with Gd-DPTA-lipids and Cy5.5-DMPE, and intravenously administered in ApoE KO mice. In vivo T1-weighted magnetic resonance imaging (MRI) revealed [S]-rHDL accumulation in atherosclerotic plaques in the abdominal aortas (C), while ex vivo nearinfrared fluorescence imaging (NIRF) of excised aortas demonstrated pronounced accumulation in plaque rich lesions such as the aortic roots and arches (D). To investigate the cellular targeting specificity of [S]-rHDL, flow cytometry and fluorescence microscopy were used to analyze the blood, spleen, and atherosclerotic plaques. [S]-rHDL was preferentially taken up by monocytes and macrophages in the blood and spleen (E), but not by other leukocytes (S-D). It also extensively accumulated in atherosclerotic plaques, and was taken up by plaque macrophages (F and S-C). Altogether these data suppor!

Rationale: Although statins have been shown to have anti-inflammatory pleiotropic effects in experimental studies, the poor plaque targeting of orally administered statins limits their direct anti-inflammatory therapeutic effect. To that end, we have developed a simvastatin loaded high-density lipoprotein nanoparticle ([S]-rHDL), which has an improved plaque bioavailability and therefore exerts a higher therapeutic effect in apolipoprotein E knockout (ApoE KO) mice than orally administered simvastatin. The purpose of the current study is to understand the mechanism of this potent anti-inflammatory effect. Methods and Results: [S]-rHDL was found to specifically target plaque macrophages by fluorescence microscopy (a). To investigate the targeting efficiency of [S]-rHDL to monocytes/macrophages, we intravenously injected [S]-rHDL in ApoE KO mice (n=3/time point) and analyzed the cells from aortas and blood by flow cytometry. [S]-rHDL was found to target macrophages and monocytes in aortas (b), and to target inflammatory Gr-1hi monocytes more efficiently than anti-inflammatory Gr-1lo monocytes in blood (c). Last, laser capture microdissection was used to isolate plaque macrophages (n=7), and quantitative RT-PCR was used to measure their mRNA level of TNF-alpha, the hallmark of macrophage inflammation, which was significantly reduced (d). All the above data support our hypothesis that [S]-rHDL acts on inflammatory monocytes and plaque macrophages and thereby exerts a strong anti-inflammatory effect on atherosclerotic plaque. Conclusion: In ApoE KO mice, [S]-rHDL specifically targets plaque macrophages. [S]-rHDL also locally acts on macrophages in plaque and preferentially targets pro-inflammatory monocytes in blood, which results in a strong anti-inflammatory effect. This nanotherapy may represent a potent addition to the current standard of care for atherosclerosis patients