Svenja Meiler

Protective Role of CXC Receptor 4/CXC Ligand 12 Unveils the Importance of Neutrophils in Atherosclerosis

The CXC ligand (CXCL)12/CXC receptor (CXCR)4 chemokine–receptor axis controls hematopoiesis, organ development, and angiogenesis, but its role in the inflammatory pathogenesis of atherosclerosis is unknown. Here we show that interference with Cxcl12/Cxcr4 by a small-molecule antagonist, genetic Cxcr4 deficiency, or lentiviral transduction with Cxcr4 degrakine in bone marrow chimeras aggravated diet-induced atherosclerosis in apolipoprotein E-deficient (Apoe−/−) or LDL receptor–deficient (Ldlr−/−) mice. Chronic blockade of Cxcr4 caused leukocytosis and an expansion of neutrophils and increased neutrophil content in plaques, associated with apoptosis and a proinflammatory phenotype. Whereas circulating neutrophils were recruited to atherosclerotic lesions, depletion of neutrophils reduced plaque formation and prevented its exacerbation after blocking Cxcr4. Disrupting Cxcl12/Cxcr4 thus promotes lesion formation through deranged neutrophil homeostasis, indicating that Cxcl12/Cxcr4 controls the important contribution of neutrophils to atherogenesis in mice

CCL17-expressing dendritic cells drive atherosclerosis by restraining regulatory T cell homeostasis in mice

Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17+ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.

MicroRNA 302a is a novel modulator of cholesterol homeostasis and atherosclerosis

Objective—Macrophage foam cell formation is a key feature of atherosclerosis. Recent studies have shown that specific microRNAs (miRs) are regulated in modified low-density lipoprotein–treated macrophages, which can affect the cellular cholesterol homeostasis. Undertaking a genome-wide screen of miRs regulated in primary macrophages by modified low-density lipoprotein, miR-302a emerged as a potential candidate that may play a key role in macrophage cholesterol homeostasis. Approach and Results—The objective of this study was to assess the involvement of miR-302a in macrophage lipid homeostasis and if it can influence circulating lipid levels and atherosclerotic development when it is inhibited in a murine atherosclerosis model. We found that transfection of primary macrophages with either miR-302a or anti–miR-302a regulated the expression of ATP-binding cassette (ABC) transporter ABCA1 mRNA and protein. Luciferase reporter assays showed that miR-302a repressed the 3′ untranslated regions (UTR) activity of mouse Abca1 by 48% and human ABCA1 by 45%. In addition, transfection of murine macrophages with miR-302a attenuated cholesterol efflux to apolipoprotein A-1 (apoA-1) by 38%. Long-term in vivo administration of anti–miR-302a to mice with low-density lipoprotein receptor deficiency (Ldlr−/−) fed an atherogenic diet led to an increase in ABCA1 in the liver and aorta as well as an increase in circulating plasma high-density lipoprotein levels by 35% compared with that of control mice. The anti–miR-302a–treated mice also displayed reduced atherosclerotic plaque size by ≈25% and a more stable plaque morphology with reduced signs of inflammation. Conclusions—These studies identify miR-302a as a novel modulator of cholesterol efflux and a potential therapeutic target for suppressing atherosclerosis.

Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice

Background— Atherosclerosis and vascular remodeling after injury are driven by inflammation and mononuclear cell infiltration. Extracellular RNA (eRNA) has recently been implicated to become enriched at sites of tissue damage and to act as a proinflammatory mediator. Here, we addressed the role of eRNA in high-fat diet–induced atherosclerosis and neointima formation after injury in atherosclerosis-prone mice. Methods and Results— The presence of eRNA was revealed in atherosclerotic lesions from high-fat diet–fed low-density lipoprotein receptor–deficient (Ldlr−/−) mice in a time-progressive fashion. RNase activity in plasma increased within the first 2 weeks (44±9 versus 70±7 mU/mg protein; P=0.0012), followed by a decrease to levels below baseline after 4 weeks of high-fat diet (44±9 versus 12±2 mU/mg protein; P<0.0001). Exposure of bone marrow–derived macrophages to eRNA resulted in a concentration-dependent upregulation of the proinflammatory mediators tumor necrosis factor-&agr;, arginase-2, interleukin-1&bgr;, interleukin-6, and interferon-&ggr;. In a model of accelerated atherosclerosis after arterial injury in apolipoprotein E–deficient (ApoE−/−) mice, treatment with RNase1 diminished the increased plasma level of eRNA evidenced after injury. Likewise, RNase1 administration reduced neointima formation in comparison with vehicle-treated ApoE−/− controls (25.0±6.2 versus 46.9±6.9×103 &mgr;m2, P=0.0339) and was associated with a significant decrease in plaque macrophage content. Functionally, RNase1 treatment impaired monocyte arrest on activated smooth muscle cells under flow conditions in vitro and inhibited leukocyte recruitment to injured carotid arteries in vivo. Conclusions— Because eRNA is associated with atherosclerotic lesions and contributes to inflammation-dependent plaque progression in atherosclerosis-prone mice, its targeting with RNase1 may serve as a new treatment option against atherosclerosis.

Hematopoietic Interferon Regulatory Factor 8-Deficiency Accelerates Atherosclerosis in Mice

Objective—Inflammatory leukocyte accumulation drives atherosclerosis. Although monocytes/macrophages and polymorphonuclear neutrophilic leukocytes (PMN) contribute to lesion formation, sequelae of myeloproliferative disease remain to be elucidated. Methods and Results—We used mice deficient in interferon regulatory factor 8 (IRF8−/−) in hematopoietic cells that develop a chronic myelogenous leukemia-like phenotype. Apolipoprotein E-deficient mice reconstituted with IRF8−/− or IRF8−/− apolipoprotein E-deficient bone marrow displayed an exacerbated atherosclerotic lesion formation compared with controls. The chronic myelogenous leukemia-like phenotype in mice with IRF8−/− bone marrow, reflected by an expansion of PMN in the circulation, was associated with an increased lesional accumulation and apoptosis of PMN, and enlarged necrotic cores. IRF8−/− compared with IRF8+/+ PMN displayed unaffected reactive oxygen species formation and discharge of PMN granule components. In contrast, accumulating in equal numbers at sites of inflammation, IRF8−/− macrophages were defective in efferocytosis, lipid uptake, and interleukin-10 cytokine production. Importantly, depletion of PMN in low-density lipoprotein receptor or apolipoprotein E-deficient mice with IRF8−/− or IRF8−/− apolipoprotein E-deficient bone marrow abrogated increased lesion formation. Conclusion—These findings indicate that a chronic myelogenous leukemia-like phenotype contributes to accelerated atherosclerosis in mice. Among proatherosclerotic effects of other cell types, this, in part, is linked to an expansion of functionally intact PMN.

Selenoprotein K is required for palmitoylation of CD36 in macrophages: implications in foam cell formation and atherogenesis

Selk is an ER transmembrane protein important for calcium flux and macrophage activation, but its role in foam cell formation and atherosclerosis has not been evaluated. BMDMs from Selk−/− mice exhibited decreased uptake of modLDL and foam cell formation compared with WT controls, and the differences were eliminated with anti‐CD36 blocking antibody. CD36 expression was decreased in TNF‐α‐stimulated Selk−/− BMDMs compared with WT controls. Fluorescence microscopy revealed TNF‐α‐induced clustering of CD36 in WT BMDMs indicative of lipid raft localization, which was absent in Selk−/− BMDMs. Fractionation revealed lower levels of CD36 reaching lipid rafts in TNF‐α‐stimulated Selk−/− BMDMs. Immunoprecipitation showed that Selk−/− BMDMs have decreased CD36 palmitoylation, which occurs at the ER membrane and is crucial for stabilizing CD36 expression and directing its localization to lipid rafts. To assess if this phenomenon had a role in atherogenesis, a HFD was fed to irradiated Ldlr−/− mice reconstituted with BM from Selk−/− or WT mice. Selk was detected in aortic plaques of controls, particularly in macrophages. Selk−/− in immune cells led to reduction in atherosclerotic lesion formation without affecting leukocyte migration into the arterial wall. These findings suggest that Selk is important for stable, localized expression of CD36 in macrophages during inflammation, thereby contributing to foam cell formation and atherogenesis.

Lymphocytic tumor necrosis factor receptor superfamily co-stimulatory molecules in the pathogenesis of atherosclerosis.

Purpose of review The role of lymphocytes in the chronic inflammatory disease atherosclerosis has emerged over the past decade. Co-stimulatory molecules of the heterogeneous tumor necrosis factor receptor superfamily play a pivotal role in lymphocyte activation, proliferation and differentiation. Here we describe the immune modulatory properties and mechanisms of four tumor necrosis factor receptor superfamily members in atherosclerosis. Recent findings CD40/CD40L, OX40L/OX40, CD70/CD27 and CD137/CD137L are present in human atherosclerotic plaques and have shown strong immune modulatory functions in atherosclerosis, resulting in either atherogenic or atheroprotective effects in mouse models of atherosclerosis. Summary Insight into the immune modulatory mechanisms of co-stimulatory interactions in atherosclerosis can contribute to clinical exploitation of these interactions in the treatment of cardiovascular disease.

Liposomal prednisolone promotes macrophage lipotoxicity in experimental atherosclerosis

Atherosclerosis is a lipid-driven inflammatory disease, for which nanomedicinal interventions are under evaluation. Previously, we showed that liposomal nanoparticles loaded with prednisolone (LN-PLP) accumulated in plaque macrophages, however, induced proatherogenic effects in patients. Here, we confirmed in low-density lipoprotein receptor knockout (LDLr(-/-)) mice that LN-PLP accumulates in plaque macrophages. Next, we found that LN-PLP infusions at 10mg/kg for 2weeks enhanced monocyte recruitment to plaques. In follow up, after 6weeks of LN-PLP exposure we observed (i) increased macrophage content, (ii) more advanced plaque stages, and (iii) larger necrotic core sizes. Finally, in vitro studies showed that macrophages become lipotoxic after LN-PLP exposure, exemplified by enhanced lipid loading, ER stress and apoptosis. These findings indicate that liposomal prednisolone may paradoxically accelerate atherosclerosis by promoting macrophage lipotoxicity. Hence, future (nanomedicinal) drug development studies are challenged by the multifactorial nature of atherosclerotic inflammation.

CD70 limits atherosclerosis and promotes macrophage function

The co-stimulatory molecule CD70 is expressed on activated immune cells and is known to modulate responses of T, B, and NK cells via its receptor CD27. Until now, there is only limited data describing the role of CD70 in atherosclerosis. We observed that ruptured human carotid atherosclerotic plaques displayed higher CD70 expression than stable carotid atherosclerotic plaques, and that CD70 expression in murine atheroma localized to macrophages. Lack of CD70 impaired the inflammatory capacity (e. g. reactive oxygen species and nitric oxide production) of bone marrow-derived macrophages, increased both M1-like and M2-like macrophage markers, and rendered macrophages metabolically inactive and prone to apoptosis. Moreover, CD70-deficient macrophages expressed diminished levels of scavenger receptors and ABC-transporters, impairing uptake of oxidised low-density lipoprotein (oxLDL) and cholesterol efflux, respectively. Hyperlipidaemic Apoe-/- mice reconstituted with CD70-deficient bone marrow displayed a profound increase in necrotic core size, plaque area, and number of lesional macrophages as compared to mice receiving control bone marrow. Accordingly, 18 week-old, chow diet-fed CD70-deficient Apoe-/- mice displayed larger atheroma characterised by lower cellularity and more advanced plaque phenotype than Apoe-/- mice. In conclusion, CD70 promotes macrophage function and viability and is crucial for effective phagocytosis and efflux of oxLDL. Deficiency in CD70 results in more advanced atheroma. Our data suggest that CD70 mitigates atherosclerosis at least in part by modulating macrophage function.

Response to Letter Regarding Article “Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice”

We acknowledge the interest and comments of Dr Chen et al regarding our findings that self-extracellular RNA (eRNA) significantly contributes to atherogenesis (as demonstrated in 2 established animal models) by inducing a prominent inflammatory response in situ and in bone marrow–derived macrophages (BMDM), as well.1 In particular, Chen et al question whether eRNA-dependent effects may have been mediated by Toll-like receptor (TLR)–related signaling, because they recently reported that BMDM responses toward the RNA analogue poly(IC) were significantly dampened in TLR3-deficient cells.2 During the past decade, our laboratory has characterized a number of new functions of eRNA in inflammation and cardiovascular diseases. In the indicated study, we aimed to characterize a causal role of natural eRNA, which may also serve as a cell-injury marker, in the onset and progression of atherosclerosis. It …