Objectives Apolipoprotein E (apoE) exerts potent anti-inflammatory effects. We here investigated the effect of apoE on the functional phenotype of macrophages. Methods and Results Human apoE receptors VLDL-R or apoER2 were stably expressed in RAW264.7 mouse macrophages. In these cells apoE downregulated markers of the pro-inflammatory M1 phenotype (iNOS, IL-12, MIP-1α), but upregulated markers of the anti-inflammatory M2 phenotype (arginase-I, SOCS3, IL-1RA). In addition, M1 macrophage responses (migration, generation of reactive oxygen species, antibody-dependent cell cytotoxicity, phagocytosis) as well as poly(I:C)- and/or IFN-γ-induced production of pro-inflammatory cytokines, COX-2 expression, and activation of NF-κB, IκB and STAT1 were suppressed in VLDL-R- or apoER2-expressing cells. Conversely, the suppression of 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 LDL receptor-deficient mice. The modulatory effects of apoE on macrophage polarization were inhibited in apoE receptor-expressing RAW264.7 cells exposed to SB220025, a p38MAP kinase inhibitor, and PP1, a tyrosine kinase inhibitor. Accordingly, apoE induced tyrosine kinase-dependent activation of p38MAP 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 pro-inflammatory M1 to the anti-inflammatory M2 phenotype. This effect may represent a novel anti-inflammatory activity of apoE.

Background-Numerous in vitro studies suggest that sphingosine 1-phosphate (S1P), a bioactive lysosphingolipid associated with high-density lipoproteins, accounts at least partly for the potent antiinflammatory properties of high-density lipoprotein and, thereby, contributes to the antiatherogenic potential attributed to high-density lipoproteins. The present study was undertaken to investigate whether modulation of S1P signaling would affect atherosclerosis in a murine model of disease. Methods and Results-Low-density lipoprotein receptor-deficient mice on a cholesterol-rich diet were given FTY720, a synthetic S1P analogue, at low (0.04 mg/kg per day) or high (0.4 mg/kg per day) doses for 16 weeks. FTY720 dose-dependently reduced atherosclerotic lesion formation, both in the aortic root and brachiocephalic artery, and almost completely blunted necrotic core formation. Plasma lipids remained unchanged during the course of FTY720 treatment. However, FTY720 lowered blood lymphocyte count (at a high dose) and significantly interfered with lymphocyte function, as evidenced by reduced splenocyte proliferation and interferon-␥ levels in plasma. Plasma concentrations of proinflammatory cytokines such as tumor necrosis factor-␣, interleukin (IL)-6, IL-12, and regulated on activation normal T cell expressed and secreted were reduced by FTY720 administration. Moreover, lipopolysaccharide-elicited generation of nitrite/nitrate and IL-6 -two markers of classical (M1) macrophage activation-was inhibited, whereas IL-4 -induced production of IL-1-receptor antagonist, a marker of alternative (M2) macrophage activation, was augmented in peritoneal macrophages from FTY720-treated low-density lipoprotein receptor-deficient mice. Conclusions-The present results demonstrate that an S1P analogue inhibits atherosclerosis by modulating lymphocyte and macrophage function, and these results are consistent with the notion that S1P contributes to the antiatherogenic potential of high-density lipoprotein. (Circulation. 2007;115:501-508.)

The TGF-β family member GDF-15 promotes lesion formation and plaque instability in atherosclerosis-prone LDLr-deficient mice.

Atherosclerotic plaque rupture is a leading cause of acute coronary syndromes, such as unstable angina and myocardial infarction ( 1 ). Infl ammatory cells are regarded as key players in the pathogenesis of plaque rupture ( 2, 3 ), and the mast cell, a potent infl ammatory cell, has been shown to accumulate in the rupture-prone shoulder region of human atheromas ( 4 ). Activated mast cells have been identifi ed in the adventitia of vulnerable and ruptured lesions in patients with myocardial infarction, and more importantly, their numbers and degree of activation were found to correlate with the incidence of plaque rupture and erosion ( 5 ). We previously demonstrated that systemic mast cell activation during atherogenesis leads to increased plaque progression in apoE defi cient mice ( 6 ), while others show that the absence of mast cells, and in particular mast cell-derived interleukin (IL)-6 and interferon (IFN)-␥ , attenuated atherosclerotic lesion development in low-density lipoprotein receptor-deficient (LDLr Ϫ / Ϫ ) mice ( 7 ). Moreover, focal activation of mast cells in the adventitia of advanced carotid artery plaques promoted macrophage apoptosis, microvascular leakage, de novo leukocyte infl ux, and the incidence of intraplaque hemorrhage. Mast cell stabilization by cromolyn was seen to prevent these pathophysiological events ( 6 ).Various pathways of mast cell activation have been demonstrated such as cross-linking of the high-affi nity Abstract Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPAmediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell defi cient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability. Press, February 10, 2013 DOI 10.1194 Lysophosphatidic acid triggers mast cell-driven atherosclerotic plaque destabilization by increasing vascular infl ammation Abbreviations: apoE Ϫ / Ϫ , apolipoprotein E-defi...

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. Enzymaticand 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 ␣ 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 dysregulated 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 J

Rationale: Although we and others have recently shown that mast cells play an important role in plaque progressionand destabilization, the nature of the actual trigger for (peri)vascular mast cell activation during atherosclerosis is still unresolved. Objective: 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 Key Words: atherosclerosis Ⅲ mast cells Ⅲ neuropeptides Ⅲ substance P Ⅲ hemorrhage A cute coronary syndromes are generally caused by atherosclerotic plaque rupture. 1,2 Mast cells, major players in allergy and asthma, 3 were found to be abundantly present in the intima and adventitia of unstable human atherosclerotic lesions 4,5 and recently, we and others have conclusively demonstrated that (peri)vascular mast cells contribute to atherosclerotic plaque progression and destabilization in mice. 6,7 However, the nature of potential mast cell triggers in atherosclerosis is still unresolved.Adventitial mast cells in human lesions have been reported to colocalize with nerve fibers, 8 fueling the intriguing option of neuronal regulation of mast cell activation. Neuropeptides such as substance P are predominantly released by peripheral C-type sensory neurons 9 and have been demonstrated to be involved in psychological stress-induced disorders 10 and in stress-related cardiovascular events and inflammation. 11 Mast cell activation by neuropeptides proceeds through dedicated receptors, such as neurokinin-1 receptor (NK1R) or by direct activation of G proteins. 12,13 Because tryptase-positive mast cells were seen to colocalize with substance P-positive nerve fibers in skin 14 and in adventitial tissue, 8 this potent mast cell activator may be a likely candidate to mediate perivascular mast cell recruitment and activation in response to stress. In this study, we addressed the role of substance P in mast cell dependent plaque progression and destabilization. MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.Human coronary artery samples were collected, and 10 m sections were prepared. Mast cell presence was established with a Original

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.

Mature SMCs are characterized by expression of a range of SM-specific contractile proteins, including SM α-actin, SM myosin light chain kinase, SM myosin heavy chain, SM22α, smoothelin, and h1-calponin. The transcription of most SMC marker genes is regulated by serum response factor (SRF) binding to a cis-acting DNA sequence known as a CArG box (CC[A/T] 6 GG). Myocardin is a potent coactivator of SRF found only in smooth and cardiac muscle and regulates gene expression by forming a higher order complex with SRF rather than binding directly to DNA. [3][4][5][6] Mice with homozygous null mutations for myocardin die during early aortic development and embryos fail to express SMC markers, whereas cardiac development seems normal.7 However, myocardin is not absolutely required for SMC development, as homozygous myocardinnull embryonic stem cells can differentiate into SMCs in vitro, and in vivo these myocardin-null cells contribute to vascular smooth muscle in tissues, albeit to a lesser extent than wild-type cells. 8,9 Loss of myocardin might, in part, be compensated for by the myocardin-related transcriptional factors (megakaryoblastic leukemia 1 [MKL1]) and (MKL2).10 Apart from inducing SMC differentiation, myocardin has been reported to inhibit cell proliferation, 4 possibly by its interaction with nuclear factor (NF)-κB and suppression of NF-κB transcriptional activity. Objective-Myocardin, a potent transcriptional coactivator of serum response factor, is involved in vascular development and promotes a contractile smooth muscle phenotype. Myocardin levels are reduced during vascular injury, in association with phenotypic switching of smooth muscle cells (SMCs). However, the direct role of myocardin in vascular disease is unclear. Approach and Results-We show that re-expression of myocardin prevents the vascular injury response in murine carotid arteries, with reduced neointima formation due to decreased SMC migration and proliferation. Myocardin reduced SMC migration by downregulating platelet-derived growth factor receptor-β (PDGFRB) expression. Pdgfrb was regulated by myocardin-induced miR-24 and miR-29a expression, and antagonizing these microRNAs restored SMC migration. Furthermore, using miR-24 and miR-29a mimics, we demonstrated that miR-29a directly regulates Pdgfrb expression at the 3′ untranslated region while miR-24 has an indirect effect on Pdgfrb levels. Myocardin heterozygous-null mice showed an augmented neointima formation with increased SMC migration and proliferation, demonstrating that endogenous levels of myocardin are a critical regulator of vessel injury responses. Conclusions-Our results extend the function of myocardin from a developmental role to a pivotal regulator of SMC phenotype in response to injury, and this transcriptional coactivator may be an attractive target for novel therapeutic strategies in vascular disease.