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Toll-like receptors (TLR) provide a critical link between innate and adaptive immunity, both important players in atherosclerosis. Since evidence for the role of TLR5 is lacking, we aimed to establish this in the immune axis of atherosclerosis. We assessed the effect of the TLR5-specific ligand Flagellin on macrophage maturation and T-cell polarisation. Next, we generated TLR5−/−LDLr−/− chimeras to study the effect of hematopoietic TLR5 deficiency on atherosclerosis formation. Flagellin stimulation did not influence wildtype or TLR5−/− macrophage maturation. Only in wildtype macrophages, Flagellin exposure increased MCP-1 and IL6 expression. Flagellin alone reduced T-helper 1 proliferation, which was completely overruled in the presence of T-cell receptor activation. In vivo, hematopoietic TLR5 deficiency attenuated atherosclerotic lesion formation by ≈25% (1030*103 ± 63*103 vs. 792*103 ± 61*103 μm2; p = 0.013) and decreased macrophage area (81.3 ± 12.0 vs. 44.2 ± 6.6 μm2; p = 0.011). In TLR5−/− chimeric mice, we observed lower IL6 plasma levels (36.4 ± 5.6 vs. 15.1 ± 2.2 pg/mL; p = 0.003), lower (activated) splenic CD4+ T-cell content (32.3 ± 2.1 vs. 21.0 ± 1.2%; p = 0.0018), accompanied by impaired T-cell proliferative responses. In conclusion, hematopoietic TLR5 deficiency inhibits atherosclerotic lesion formation by attenuated macrophage accumulation and defective T-cell responsiveness.

Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim −/− or wild type bone marrow transplanted ldlr −/− mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim −/− transplanted mice displayed splenomegaly and overt lymphocytosis. CD4+ and CD8+ T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim −/− mice. Bim −/− mice displayed massive intraplaque accumulation of Ig complexes and of lesional T cells, although this did not translate in changes in plaque size or stability features (apoptotic cell and macrophage content). The surprising lack in plaque phenotype despite the profound pro-atherogenic immune effects may be attributable to the sharp reduction of serum cholesterol levels in WTD fed bim −/− mice.

Growth differentiation factor (GDF) 15 is a member of the transforming growth factor β (TGF-β) superfamily, which operates in acute phase responses through a currently unknown receptor. Elevated GDF-15 serum levels were recently identified as a risk factor for acute coronary syndromes. We show that GDF-15 expression is up-regulated as disease progresses in murine atherosclerosis and primarily colocalizes with plaque macrophages. Hematopoietic GDF-15 deficiency in low density lipoprotein receptor-/- mice led to impaired initial lesion formation and increased collagen in later lesions. Although lesion burden in GDF-15-/- chimeras was unaltered, plaques had reduced macrophage infiltrates and decreased necrotic core formation, all features of improved plaque stability. In vitro studies pointed to a TGFβRII-dependent regulatory role of GDF-15 in cell death regulation. Importantly, GDF-15-/- macrophages displayed reduced CCR2 expression, whereas GDF-15 promoted macrophage chemotaxis in a strictly CCR2- and TGFβRII-dependent manner, a phenomenon which was not observed in G protein-coupled receptor kinase 2+/- macrophages. In conclusion, GDF-15 deletion has a beneficial effect both in early and later atherosclerosis by inhibition of CCR2-mediated chemotaxis and by modulating cell death. Our study is the first to identify GDF-15 as an acute phase modifier of CCR2/TGFβRII-dependent inflammatory responses to vascular injury. © 2011 de Jager et al., This work was supported by the Netherlands Heart Foundation (grant D2003T201 to S.C.A. de Jager and E.A.L. Biessen), Marie Curie Grant (PIEF-GA-2008-221836 to B. Bermúdez), and the Spanish Ministry of Science and Innovation (grant AGL2008-028111 to R. Abia).

Aims Mast cells have been shown to accumulate in the adventitia of human atherosclerotic plaques and were recently demonstrated by us to contribute to plaque progression and instability. In this study, we investigated whether selective inhibition of mast cell chymases would affect the lesion development and stability. Methods and results The protease inhibitor RO5066852 appeared to be a potent inhibitor of chymase activity in vitro and ex vivo. With this inhibitor, we provide three lines of evidence that chymase inhibition can prevent many pro-atherogenic activities. First, oral administration of RO5066852 reduced spontaneous atherosclerosis in the thoracic aorta of apoE(-)/(-) mice. Second, chymase inhibition prevented the accelerated plaque progression observed in apoE(-/-) mice that were exposed to repetitive episodes of systemic mast cell activation. Furthermore, RO5066852 enhanced lesional collagen content and reduced necrotic core size. Third, RO5066852 treatment almost completely normalized the increased frequency and size of intraplaque haemorrhages observed in apoE(-/-) mice after acute perivascular mast cell activation in advanced atherosclerosis. Conclusion Our data indicate that chymase inhibition can inhibit pro-atherogenic and plaque destabilizing effects which are associated with perivascular mast cell activation. Our study thus identifies pharmacological chymase inhibition as a potential therapeutic modality for atherosclerotic plaque stabilization.

Aims The SDF-1α/CXCR4 dyad was previously shown by us and others to be instrumental in intimal hyperplasia as well as early stage atherosclerosis. We here sought to investigate its impact on clinically relevant stages of atherosclerosis in mouse and man. Methods and results Immunohistochemical analysis of CXCR4 expression in human atherosclerotic lesions revealed a progressive accumulation of CXCR4+ cells during plaque progression. To address causal involvement of CXCR4 in advanced stages of atherosclerosis we reconstituted LDLr−/− mice with autologous bone marrow infected with lentivirus encoding SDF-1α antagonist or CXCR4 degrakine, which effects proteasomal degradation of CXCR4. Functional CXCR4 blockade led to progressive plaque expansion with disease progression, while also promoting intraplaque haemorrhage. Moreover, CXCR4 knockdown was seen to augment endothelial adhesion of neutrophils. Concordant with this finding, inhibition of CXCR4 function increased adhesive capacity and reduced apoptosis of neutrophils and resulted in hyperactivation of circulating neutrophils. Compatible with a role of the neutrophil CXCR4 in end-stage atherosclerosis, CXCR4 expression by circulating neutrophils was lowered in patients with acute cardiovascular syndromes. Conclusion In conclusion, CXCR4 contributes to later stages of plaque progression by perturbing neutrophil function.

Abstract Aims Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1−/−) deficiency on leukocyte subsets relevant to atherosclerosis. Methods and Results LDL receptor deficient mice that were transplanted with Sgpl1−/− bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1−/− chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response. Conclusions Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.

Objective— Despite common disbelief that neutrophils are involved in atherosclerosis, evidence is accumulating for a causal role of neutrophils in atherosclerosis. CC chemokine ligand (CCL)3 is an inflammatory chemokine and its expression is significantly increased during atherosclerotic lesion formation in mice. It has recently been shown that under conditions of inflammation neutrophils can migrate along a CCL3 gradient. In this study, we aimed to elucidate the role of leukocyte-derived CCL3 in atherogenesis. Methods and Results— Irradiated low density lipoprotein receptor –/– mice, reconstituted with CCL3 –/– or littermate bone marrow showed markedly reduced CCL3 response to lipopolysaccharide treatment, establishing the critical relevance of leukocytes as source of CCL3. Hematopoietic deficiency of CCL3 significantly reduced aortic sinus lesion formation by 31% after 12 weeks of western-type diet. Interestingly, whereas plaque macrophage, collagen, and vascular smooth muscle cell content were unchanged, neutrophil adhesion to and presence in plaques was significantly attenuated in CCL3 –/– chimeras. These mice had reduced circulating neutrophil numbers, which could be ascribed to an increased neutrophil turnover and CCL3 –/– neutrophils were shown to be less responsive toward the neutrophil chemoattractant CXC chemokine ligand 1. Conclusion— Our data indicate that under conditions of acute inflammation leukocyte-derived CCL3 can induce neutrophil chemotaxis toward the atherosclerotic plaque, thereby accelerating lesion formation.

Objective The ABCA2 transporter shares high structural homology to ABCA1, which is crucial for the removal of excess cholesterol from macrophages and, by extension, in atherosclerosis. It has been suggested that ABCA2 sequesters cholesterol inside the lysosomes, however, little is known of the macrophage-specific role of ABCA2 in regulating lipid homeostasis in vivo and in modulating susceptibility to atherosclerosis. Methods Chimeras with dysfunctional macrophage ABCA2 were generated by transplantation of bone marrow from ABCA2 knockout (KO) mice into irradiated LDL receptor (LDLr) KO mice. Results Interestingly, lack of ABCA2 in macrophages resulted in a diminished lesion size in the aortic root (−24.5%) and descending thoracic aorta (−36.6%) associated with a 3-fold increase in apoptotic cells, as measured by both caspase 3 and TUNEL. Upon oxidized LDL exposure, macrophages from wildtype (WT) transplanted animals developed filipin-positive droplets in lysosomal-like compartments, corresponding to free cholesterol (FC) accumulation. In contrast, ABCA2-deficient macrophages displayed an abnormal diffuse distribution of FC over peripheral regions. The accumulation of neutral sterols in lipid droplets was increased in ABCA2-deficient macrophages, but primarily in cytoplasmic clusters and not in lysosomes. Importantly, apoptosis of oxLDL loaded macrophages lacking ABCA2 was increased 2.7-fold, probably as a consequence of the broad cellular distribution of FC. Conclusions Lack of functional ABCA2 generates abnormalities in intracellular lipid distribution/trafficking in macrophages consistent with its lysosomal sequestering role, leading to an increased susceptibility to apoptosis in response to oxidized lipids and reduced atherosclerotic lesion development.

SummarySphingosine 1-phosphate (S1P), a lysosphingolipid associated with high-density lipoprotein (HDL), contributes to the anti-atherogenic potential attributed to this lipoprotein. This study examined whether a reduction of S1P plasma levels affects atherosclerosis in a murine model of disease. LDL-R−/−mice on Western diet were given ABC294640, an inhibitor of sphingosine kinase (SphK) for 16 weeks. ABC294640 decreased plasma S1P by approximately 30%. However, ABC294640 failed to affect atherosclerotic lesion formation. Plasma triglycerides were reduced whereas total and HDL-cholesterol remained unchanged in course of ABC294640 treatment. ABC294640 increased plasma interleukin (IL)-12p70 and RANTES concentration as well as IL-12p70, RANTES and interferon (IFN)-γ production by peritoneal cells and this was paralleled by enhanced activity of peritoneal and spleen dendritic cells as evidenced by up-regulation of CD86 and MHC-II on CD11c+ cells. As a consequence, increased T-cell activation was noted in ABC294640-treated mice as indicated by enhanced CD4+ splenocyte proliferation, IFN-γ and IL-2 production, and CD69 expression. Con-comitantly, however, ABC294640 treatment redistributed CD4+ and CD8+ cells from blood to lymphatic organs and reduced T-cell number within atherosclerotic lesions. In addition, plasma sVCAM-1, sICAM-1, and MCP-1 levels as well as in vivo leukocyte adhesion and CCL19-induced T-cell penetration into peritoneum were lower in ABC294640-treated animals. In vitro experiments demonstrated reduced VCAM-1 and ICAM-1 expression and lymphocyte adhesion to endothelial cells exposed to ABC294640. In conclusion, treatment with SphK inhibitor leads to both pro- and anti-atherogenic effects in LDL-R−/− mice. As a consequence, SphK inhibition fails to affect atherosclerosis despite significant S1P reduction in plasma.

Rationale: Nuclear factor of activated T-cells (NFAT) is importantly implicated in pathological cardiac remodeling and vascular lesion formation. NFAT functionality is mainly regulated by calcineurin, a Ca 2+ -dependent multi-effector phosphatase. Calcineurin inhibitors such as cyclosporine A (CsA) were shown to be effective in the treatment of restenosis and vascular inflammation but with adverse side effects. Objective: This prompted the design of more selective inhibitors such as VIVIT and inhibitors of NFAT-calcineurin association, which unfortunately have a poor potency precluding clinical use. Methods and Results: Here, we describe the rational design of a potent bipartite inhibitor of NFAT–calcineurin interaction, MCV1, which targets two separate calcineurin docking motifs. Modeling, site-directed mutagenesis, and functional studies demonstrated that MCV1 acts by allosteric modulation of calcineurin. Comparable to CsA, MCV1 prevents NFAT activation at nanomolar potency without impairing calcineurin phosphatase activity, nuclear factor-κB nuclear import, and general cell signaling. In contrast, CsA but not MCV1-activated basal level extracellular signal-regulated kinases activity and prevented nuclear import of calcineurin, independent of NFAT activation. In vivo MCV1 abrogated NFAT-mediated T-cell activation in a model of PMA-elicited peritonitis, whereas topical application of MCV1 markedly reduced neointima formation in a mouse model of restenosis. Conclusions: We designed a bipartite NFAT inhibitor that is more potent than VIVIT and more selective than CsA. MCV1 constitutes not only a powerful tool to unravel NFAT function but also a potential drug candidate for the treatment of diseases implicating NFAT activation.

Objective— Apolipoprotein E (apoE) exerts potent antiinflammatory effects. Here, we investigated the effect of apoE on the functional phenotype of macrophages. Methods and Results— Human apoE receptors very-low-density lipoprotein receptor (VLDL-R) and apoE receptor-2 (apoER2) were stably expressed in RAW264.7 mouse macrophages. In these cells, apoE downregulated markers of the proinflammatory M1 phenotype (inducible nitric oxide synthase, interleukin [IL]-12, macrophage inflammatory protein-1α) but upregulated markers of the antiinflammatory M2 phenotype (arginase I, SOCS3, IL-1 receptor antagonist [IL-1RA]). In addition, M1 macrophage responses (migration, generation of reactive oxygen species, antibody-dependent cell cytotoxicity, phagocytosis), as well as poly(I:C)- or interferon-γ-induced production of proinflammatory cytokines; cyclooxygenase-2 expression; and activation of nuclear factor-κB, IκB, and STAT1, were suppressed in VLDL-R- or apoER2-expressing cells. Conversely, the suppression of the M2 phenotype and the enhanced response to poly(I:C) were observed in apoE-producing bone marrow macrophages derived from VLDL-R-deficient mice but not wild-type or low-density lipoprotein receptor–deficient mice. The modulatory effects of apoE on macrophage polarization were inhibited in apoE receptor-expressing RAW264.7 cells exposed to SB220025, a p38 mitogen-activated protein kinase inhibitor, and PP1, a tyrosine kinase inhibitor. Accordingly, apoE induced tyrosine kinase-dependent activation of p38 mitogen-activated protein kinase in VLDL-R- or apoER2-expressing macrophages. Under in vivo conditions, apoE −/− mice transplanted with apoE-producing wild-type bone marrow showed increased plasma IL-1RA levels, and peritoneal macrophages of transplanted animals were shifted to the M2 phenotype (increased IL-1RA production and CD206 expression). Conclusion— ApoE signaling via VLDL-R or apoER2 promotes macrophage conversion from the proinflammatory M1 to the antiinflammatory M2 phenotype. This effect may represent a novel antiinflammatory activity of apoE.

Lysophosphatidic acid (LPA) accumulates in the central atheroma of human atherosclerotic plaques and is the primary platelet-activating lipid constituent of plaques. Here, we investigated the enzymatic regulation of LPA homeostasis in atherosclerotic lesions at various stages of disease progression. Atherosclerotic lesions were induced in carotid arteries of low-density lipoprotein receptor deficient mice by semiconstrictive collar placement. At 2-week intervals after collar placement, lipids and RNA were extracted from the vessel segments carrying the plaque. Enzymatic-and liquid chromatography-mass spectrometry based lipid profiling revealed progressive accumulation of LPA species in atherosclerotic tissue preceded by an increase in lysophosphatidylcholine, a precursor in LPA synthesis. Plaque expression of LPA-generating enzymes cytoplasmic phospholipase A(2)IVA (cPLA(2)IVA) and calcium-independent PLA(2)VIA (iPLA(2)VIA) was gradually increased, whereas that of the LPA-hydrolyzing enzyme LPA acyltransferase alpha was quenched. Increased expression of cPLA(2)IVA and iPLA(2)VIA in advanced lesions was confirmed by immunohistochemistry. Moreover, LPA receptors 1 and 2 were 50% decreased and sevenfold upregulated, respectively. Therefore, key proteins in LPA homeostasis are increasingly dysregulined in the plaque during atherogenesis, favoring intracellular LPA production. This might at least partly explain the observed progressive accumulation of this thrombogenic proinflammatory lipid in human and mouse plaques. Thus, intervention in the enzymatic LPA production may be an attractive measure to lower intraplaque LPA content, thereby reducing plaque progression and thrombogenicity. (Am Pathol 2010, 176:3073-3084; DOI: 10.2353/ajpath.2010.090009)

Although we and others have recently shown that mast cells play an important role in plaque progression and destabilization, the nature of the actual trigger for (peri)vascular mast cell activation during atherosclerosis is still unresolved.In this study, we confirm that perivascular mast cell content correlates with the number of nerve fibers in the adventitia of human coronary atherosclerotic plaque specimen. Because peripheral C-type nerve fibers secrete, among others, substance P, a potent mast cell activator, we set out to study effects of adventitial administration of this neuropeptide on mast cell dependent destabilization of carotid artery plaques in apolipoprotein E-deficient (apoE(-/-)) mice.Substance P treatment significantly enhanced the number and activation status of adventitial mast cells compared to controls and promoted intraplaque hemorrhages. These phenomena could be prevented by coadministration of the neurokinin-1 receptor antagonist spantide I and did not occur in mast cell deficient apoE(-/-) mice, establishing the critical involvement of mast cells in substance P-elicited plaque destabilization.Our data suggest that neurotransmitters such as substance P are capable of promoting mast cell dependent plaque destabilization and provide a new, direct link between neural factors and vascular inflammation.