Description: Objective— Sphingosine 1-phosphate (S1P) partly accounts for antiatherogenic properties of high-density lipoproteins. We previously demonstrated that FTY720, a synthetic S1P analog targeting all S1P receptors but S1P receptor type 2, inhibits murine atherosclerosis. Here, we addressed the identity of S1P receptor mediating atheroprotective effects of S1P. Approach and Results— Low-density lipoprotein receptor–deficient mice on cholesterol-rich diet were given selective S1P receptor type 1 agonist KRP-203 (3.0 mg/kg per day; 6 and 16 weeks). KRP-203 substantially reduced atherosclerotic lesion formation without affecting plasma lipid concentrations. However, KRP-203 induced lymphopenia, reduced total (CD4 + , CD8 + ) and activated (CD69 + /CD8 + , CD69 + /CD4 + ) T cells in peripheral lymphoid organs, and interfered with lymphocyte function, as evidenced by decreased T-cell proliferation and interleukin-2 and interferon- production in activated splenocytes. Cyto- and chemokine (tumor necrosis factor-α, regulated and normal T cell expressed and secreted) levels in plasma and aortas were reduced by KRP-203 administration. Moreover, macrophages from KRP-203–treated mice showed reduced expression of activation marker MCH-II and poly(I:C)-elicited production of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6. In vitro studies demonstrated that KRP-203 reduced tumor necrosis factor-α, interleukin-6, and interferon-–induced protein-10 production; IB and signal transducer and activator of transcription-1 phosphorylation; and nuclear factor B and signal transducer and activator of transcription-1 activation in poly(I:C)-, lipopolysaccharide-, or interferon-–stimulated bone marrow macrophages, respectively. Conclusions— Present results demonstrate that activation of S1P signaling pathways inhibit atherosclerosis by modulating lymphocyte and macrophage function and suggest that S1P receptor type 1 at least partially mediates antiatherogenic effects of S1P.

Description: Objective— The function of microRNAs is highly context and cell type dependent because of their highly dynamic expression pattern and the regulation of multiple mRNA targets. MicroRNA-155 (miR-155) plays an important role in the innate immune response by regulating macrophage function; however, the effects of miR-155 in macrophages on atherosclerosis are controversial. We hypothesized that the stage-dependent target selection of miR-155 in macrophages determines its effects on atherosclerosis. Approach and Results— The expression of miR-155 increased in lesional macrophages of apolipoprotein E–deficient mice between 12 and 24 weeks of a high-cholesterol diet. Mir155 knockout in apolipoprotein E–deficient mice enhanced lesion formation, increased the lesional macrophage content, and promoted macrophage proliferation after 12 weeks of the high-cholesterol diet. In vitro, miR-155 inhibited macrophage proliferation by suppressing colony-stimulating factor-1 receptor, which was upregulated in lesional macrophages of Mir155 –/– apolipoprotein E–deficient mice. By contrast, Mir155 deficiency reduced necrotic core formation and the deposition of apoptotic cell debris, thereby preventing the progression of atherosclerosis between 12 and 24 weeks of the high-cholesterol diet. miR-155 inhibited efferocytosis in vitro by targeting B-cell leukemia/lymphoma 6 and thus activating RhoA (ras homolog gene family, member A). Accordingly, B-cell leukemia/lymphoma 6 was upregulated in lesional macrophages of Mir155 –/– apolipoprotein E–deficient mice after 24 weeks, but not after 12 weeks of the high-cholesterol diet. Conclusions— Our findings demonstrate a stage-specific role of miR-155 in lesion formation. miR-155 suppressed macrophage proliferation by targeting colony-stimulating factor-1 receptor in early and impaired efferocytosis by downregulating B-cell leukemia/lymphoma 6 in advanced atherosclerosis. Therefore, targeting the interaction between miR-155 and B-cell leukemia/lymphoma 6 may be a promising approach to inhibit the progression of atherosclerosis.

Description: Objective— Explore aorta B-cell immunity in aged apolipoprotein E-deficient ( ApoE –/– ) mice. Approach and Results— Transcript maps, fluorescence-activated cell sorting, immunofluorescence analyses, cell transfers, and Ig-ELISPOT (enzyme-linked immunospot) assays showed multilayered atherosclerosis B-cell responses in artery tertiary lymphoid organs (ATLOs). Aging-associated aorta B-cell–related transcriptomes were identified, and transcript atlases revealed highly territorialized B-cell responses in ATLOs versus atherosclerotic lesions: ATLOs showed upregulation of bona fide B-cell genes, including Cd19, Ms4a1 (Cd20), Cd79a/b, and Ighm although intima plaques preferentially expressed molecules involved in non–B effector responses toward B-cell–derived mediators, that is, Fcgr3 (Cd16), Fcer1g (Cd23), and the C1q family. ATLOs promoted B-cell recruitment. ATLO B-2 B cells included naive, transitional, follicular, germinal center, switched IgG1 + , IgA + , and IgE + memory cells, plasmablasts, and long-lived plasma cells. ATLOs recruited large numbers of B-1 cells whose subtypes were skewed toward interleukin-10 + B-1b cells versus interleukin-10 – B-1a cells. ATLO B-1 cells and plasma cells constitutively produced IgM and IgG and a fraction of plasma cells expressed interleukin-10. Moreover, ApoE –/– mice showed increased germinal center B cells in renal lymph nodes, IgM-producing plasma cells in the bone marrow, and higher IgM and anti–MDA-LDL (malondialdehyde-modified low-density lipoprotein) IgG serum titers. Conclusions— ATLOs orchestrate dichotomic, territorialized, and multilayered B-cell responses in the diseased aorta; germinal center reactions indicate generation of autoimmune B cells within the diseased arterial wall during aging.

Description: Objective— Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is expressed on CD4 + effector memory T cells and regulatory T cells; however, its role on these functionally opposing cell types in atherosclerosis is not fully understood. Approach and Results— Low-density lipoprotein receptor–deficient mice ( Ldlr –/– ) were lethally irradiated and reconstituted with either bone marrow from B-cell–restricted Gitrl transgenic mice or from wild-type controls and fed a high-cholesterol diet for 11 weeks. Chimeric Ldlr –/– Gitrl tg mice showed a profound increase in both CD4 + effector memory T cells and regulatory T cells in secondary lymphoid organs. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased Gitrl and Il-2 transcript levels. Atherosclerotic lesions of Ldlr –/– Gitrl tg chimeras contained more total CD3 + T cells as well as Foxp3 + regulatory T cells overall, leading to significantly less severe atherosclerosis. Conclusions— These data indicate that continuous GITR stimulation through B cell Gitrl acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4 + T cells.

Description: Objective— Here, we aimed to clarify the role of CXC chemokine receptor (CXCR) 2 in macrophage migration-inhibitory factor (MIF)–mediated effects after myocardial ischemia and reperfusion. As a pleiotropic chemokine-like cytokine, MIF has been identified to activate multiple receptors, including CD74 and CXCR2. In models of myocardial infarction, MIF exerts both proinflammatory effects and protective effects in cardiomyocytes. Similarly, CXCR2 displays opposing effects in resident versus circulating cells. Approach and Results— Using bone marrow transplantation, we generated chimeric mice with Cxcr2 –/– bone marrow–derived inflammatory cells and wild-type (wt) resident cells (wt/Cxcr2 –/– ), Cxcr2 –/– cardiomyocytes and wt bone marrow–derived cells (Cxcr2 –/– /wt), and wt controls reconstituted with wt bone marrow (wt/wt). All groups were treated with anti-MIF or isotype control antibody before they underwent myocardial ischemia and reperfusion. Blocking MIF increased infarction size and impaired cardiac function in wt/wt and wt/CXCR2 –/– mice but ameliorated functional parameters in Cxcr2 –/– /wt mice, as analyzed by echocardiography and Langendorff perfusion. Neutrophil infiltration and angiogenesis were unaltered by MIF blockade or Cxcr2 deficiency. Monocyte infiltration was blunted in wt/Cxcr2 –/– mice and reduced by MIF blockade in wt/wt and Cxcr2 –/– /wt mice. Furthermore, MIF blockade attenuated collagen content in all groups in a CXCR2-independent manner. Conclusions— The compartmentalized and opposing effects of MIF after myocardial ischemia and reperfusion are largely mediated by CXCR2. Although MIF confers protective effects by improving myocardial healing and function through CXCR2 in resident cells, thereby complementing paracrine effects through CD74/AMP-activated protein kinase, it exerts detrimental effects on CXCR2-bearing inflammatory cells by increasing monocyte infiltration and impairing heart function. These dichotomous findings should be considered when developing novel therapeutic strategies to treat myocardial infarction.

Description: Objective— Unstable atherosclerotic lesions are prone to rupture, which leads to atherothrombosis. Chemokine (C-X-C motif) ligand 12 (CXCL12) promotes the mobilization and neointimal recruitment of smooth muscle progenitor cells (SPCs), and thereby mediates vascular repair. Moreover, treatment with SPCs stabilizes atherosclerotic lesions in mice. We investigated the role of CXCL12 in the treatment of unstable atherosclerotic lesions. Approach and Results— Intravenous injection of CXCL12 selectively increased the level of Sca1 + Lin platelet derived growth factor receptor-β + SPCs in the circulation as determined by flow cytometry. Macrophage-rich lesions were induced by partial ligation of the carotid artery in Apoe –/– mice. Repeated injection of CXCL12 reduced the macrophage content, increased the number of smooth muscle cells, increased the fibrous cap thickness, and increased the collagen content in these lesions. However, CXCL12 did not alter the lesion size or the luminal diameter of the carotid artery as determined by planimetry and micro-computed tomography, respectively. Recruitment of bone marrow–derived SPCs to the lesions was increased after treatment with CXCL12 in chimeric mice that expressed SM22-LacZ in bone marrow cells as determined by quantification of the number of lesional β-galactosidase–expressing cells. CXCL12 expression was upregulated in atherosclerotic arteries after CXCL12 treatment. Silencing of arterial CXCL12 expression during atherosclerosis promoted lesion formation and reduced the lesional smooth muscle cell content in CXCL12-treated mice. Conclusions— Systemic treatment with CXCL12 promotes a more stable atherosclerotic lesion phenotype and enhances the accumulation of SPCs in these lesions without promoting atherosclerosis. Thus, CXCL12-induced SPC mobilization appears a promising approach to treat unstable atherosclerosis.

Description: Objective— The Cxcl12/Cxcr4 chemokine ligand/receptor axis mediates the mobilization of smooth muscle cell progenitors, driving injury-induced neointimal hyperplasia. This study aimed to investigate the role of endothelial Cxcr4 in neointima formation. Approach and Results— β-Galactosidase staining using bone marrow x kinase ( Bmx ) -CreER T2 reporter mice and double immunofluorescence revealed an efficient and endothelial-specific deletion of Cxcr4 in Bmx-CreER T2+ compared with Bmx-CreER T2– Cxcr4-floxed apolipoprotein E–deficient ( Apoe –/– ) mice (referred to as Cxcr4 EC-KO ApoE –/– and Cxcr4 EC-WT ApoE –/– , respectively). Endothelial Cxcr4 deficiency significantly increased wire injury–induced neointima formation in carotid arteries from Cxcr4 EC-KO ApoE –/– mice. The lesions displayed a higher number of macrophages, whereas the smooth muscle cell and collagen content were reduced. This was associated with a significant reduction in reendothelialization and endothelial cell proliferation in injured Cxcr4 EC-KO ApoE –/– carotids compared with Cxcr4 EC-WT ApoE –/– controls. Furthermore, stimulation of human aortic endothelial cells with chemokine (C-X-C motif) ligand 12 (CXCL12) significantly enhanced their wound-healing capacity in an in vitro scratch assay, an effect that could be reversed with the CXCR4 antagonist AMD3100. Also, flow cytometric analysis showed a reduced mobilization of Sca1 + Flk1 + Cd31 + and of Lin – Sca1 + progenitors in Cxcr4 EC-KO ApoE –/– mice after vascular injury, although Cxcr4 surface expression was unaltered. No differences could be detected in plasma concentrations of Cxcl12, vascular endothelial growth factor, sphingosine 1-phosphate, or Flt3 (fms-related tyrosine kinase 3) ligand, all cytokines with an established role in progenitor cell mobilization. Nonetheless, double immunofluorescence revealed a significant reduction in local endothelial Cxcl12 staining in injured carotids from Cxcr4 EC-KO ApoE –/– mice. Conclusions— Endothelial Cxcr4 is crucial for efficient reendothelialization after vascular injury through endothelial wound healing and proliferation, and through the mobilization of Sca1 + Flk1 + Cd31 + cells, often referred to as circulating endothelial progenitor cells.

Description: Chemokines play important roles in atherosclerotic vascular disease. Expressed by not only cells of the vessel wall but also emigrated leukocytes, chemokines were initially discovered to direct leukocytes to sites of inflammation. However, chemokines can also exert multiple functions beyond cell recruitment. Here, we discuss novel and recently emerging aspects of chemokines and their involvement in atherosclerosis. While reviewing newly identified roles of chemokines and their receptors in monocyte and neutrophil recruitment during atherogenesis and atheroregression, we also revisit homeostatic functions of chemokines, including their roles in cell homeostasis and foam cell formation. The functional diversity of chemokines in atherosclerosis warrants a clear-cut mechanistic dissection and stage-specific assessment to better appreciate the full scope of their actions in vascular inflammation and to identify pathways that harbor the potential for a therapeutic targeting of chemokines in atherosclerosis.