Lipoteichoic acid isolated from Lactobacillus plantarum suppresses LPS-mediated atherosclerotic plaque inflammation

Chronic inflammation plays an important role in atherogenesis. Experimental studies have demonstrated the accumulation of monocytes/macrophages in atherosclerotic plaques caused by inflammation. Here, we report the inhibitory effects of lipoteichoic acid (LTA) from Lactobacillus plantarum (pLTA) on atherosclerotic inflammation. pLTA inhibited the production of proinflammatory cytokines and nitric oxide in lipopolysaccharide (LPS)-stimulated cells and alleviated THP-1 cell adhesion to HUVEC by down-regulation of adhesion molecules such as intracellular adhesion molecule-1 (ICAM-I), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. The inhibitory effect of pLTA was mediated by inhibition of NF-κB and activation of MAP kinases. Inhibition of monocyte/macrophage infiltration to the arterial lumen was shown in pLTA-injected ApoE^−/− mice, which was concurrent with inhibition of MMP-9 and preservation of CD31 production. The anti-inflammatory effect mediated by pLTA decreased expression of atherosclerotic markers such as COX-2, Bax, and HSP27 and also cell surface receptors such as TLR4 and CCR7. Together, these results underscore the role of pLTA in suppressing atherosclerotic plaque inflammation and will help in identifying targets with therapeutic potential against pathogen-mediated atherogenesis.     

TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages

Transforming growth factor-beta1 (TGF-beta1), a key cytokine for control of cell growth, extracellular matrix formation, and inflammation control, is secreted by many cells present in the arteriosclerotic plaque. Lipid accumulation in the vessel wall is regarded as an early step in atherogenesis and depends on uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors and their transformation into foam cells. Prominent members of the scavenger receptor family are the class A type I and II receptors (ScR-A), the class B receptor CD36, and the recently detected lectin-like oxidized LDL receptor-1 (LOX-1), which, unlike the native LDL receptor (LDL-R), are not feedback controlled. CD36 is responsible for >50% of modified LDL uptake into human monocyte-derived macrophages. We therefore studied whether TGF-beta1 influences expression and function of ScR-A, CD36, and LOX-1 in monocytes using RT-PCR and flow cytometry. Total uptake of oxidized LDL by monocytoid cells, reflecting the combined function of all scavenger receptors, was significantly reduced by TGF-beta1. At initially low picomolar concentrations, TGF-beta1 decreased CD36 mRNA and protein surface expression and ScR-A mRNA levels in the human monocytic cell line THP-1 and in freshly isolated and cultivated human monocytes, whereas LOX-1 mRNA was increased. Expression of LDL-R and beta-actin was not affected by TGF-beta1. In conclusion, depression of scavenger receptor function in monocytes by TGF-beta1 in low concentrations reduces foam cell formation. Together with matrix control by TGF-beta1, this may be important for atherogenesis and plaque stabilization.     

ACE inhibition and atherogenesis

Recent clinical studies such as HOPE, SECURE, and APRES show that angiotensin-converting enzyme (ACE) inhibitors like ramipril improve the prognosis of patients with a high risk of atherothrombotic cardiovascular events. Atherosclerosis, as a chronic inflammatory condition of the vascular system, can turn into an acute clinical event through the rupture of a vulnerable atherosclerotic plaque followed by thrombosis. ACE inhibition has a beneficial effect on the atherogenic setting and on fibrinolysis. Endothelial dysfunction is the end of a common process in which cardiovascular risk factors contribute to inflammation and atherogenesis. By inhibiting the formation of angiotensin II, ACE inhibitors prevent any damaging effects on endothelial function, vascular smooth muscle cells, and inflammatory vascular processes. An increase in the release of NO under ACE inhibition has a protective effect. Local renin-angiotensin systems in the tissue are involved in the inflammatory processes in the atherosclerotic plaque. Circulating ACE-containing monocytes, which adhere to endothelial cell lesions, differentiate within the vascular wall to ACE-containing macrophages or foam cells with increased local synthesis of ACE and angiotensin II. Within the vascular wall, angiotensin II decisively contributes to the instability of the plaque by stimulating growth factors, adhesion molecules, chemotactic proteins, cytokines, oxidized LDL, and matrix metalloproteinases. Suppression of the increased ACE activity within the plaque can lead to the stabilization and deactivation of the plaque by reducing inflammation in the vascular wall, thus lessening the risk of rupture and thrombosis and the resultant acute clinical cardiovascular events. The remarkable improvement in the long-term prognosis of atherosclerotic patients with increased cardiovascular risk might be the clinical result of the contribution made by ACE inhibition in the vascular wall.     

Platelets: Pleiotropic roles in atherogenesis and atherothrombosis

Platelets are small, anucleate blood elements of critical importance in cardiovascular disease. The ability of platelets to activate and aggregate to form blood clots in response to endothelial injury, such as plaque rupture, is well established. These cells are therefore important contributors to ischaemia in atherothrombosis, and antiplatelet therapy is effective for this reason. However, growing evidence suggests that platelets are also important mediators of inflammation and play a central role in atherogenesis itself. Interactions between activated platelets, leukocytes and endothelial cells trigger autocrine and paracrine activation signals, resulting in leukocyte recruitment at and into the vascular wall. Direct physical interaction may contribute also, through platelet adhesion molecules assisting localization of monocytes to the site of atherogenesis and platelet granule release contributing to the chronic inflammatory milieu which leads to foam cell development and accelerated atherogenesis. Recent studies have shown that antiplatelet therapy in animal models of accelerated atherogenesis can lead to decreased plaque size and improve plaque stability. This review examines the complexity of platelet function and the nature of interactions between activated platelets, leukocytes and endothelial cells. We focus on the growing body of evidence that platelets play a critical role in atherogenesis and contribute to progression of atherosclerosis.     

Enzymology and molecular biology of prokaryotic sulfite oxidation

The intracellular bacterial pathogen Chlamydia pneumoniae causes respiratory tract infection and has been associated with atherosclerosis and coronary artery disease. Since atherosclerosis is a progressive disease and is considered to be a chronic inflammation of the artery vessel wall, the interaction of C. pneumoniae with cells of the vasculature that can result in a local inflammatory response is of paramount importance. In this essay we review the pathophysiology of atherosclerosis in the context of C. pneumoniae infection and present an integrated model that includes the involvement of C. pneumoniae in all stages of atherogenesis including initiation, inflammation, fibrous plaque formation, plaque rupture and thrombosis. We hypothesize that acute and persistent infection of professional immune cells (T-cells, monocytes and macrophages) and non-immune cells (endothelial cells and smooth muscle cells) contributes to a sustained inflammatory response mediated by extensive cellular 'crosstalk' and numerous cytokines/chemokines. This cascade of inflammatory mediators may contribute to cellular dysfunction and tissue remodelling of the arterial intima. An improved understanding of the precise mechanism(s) of C. pneumoniae involvement in atherogenesis may help resolve the question of causality however, at the present time, we interpret the data as favoring a contributory rather than a causal role. Future research directed at the discovery of chlamydial virulence factors necessary for intracellular survival and subsequent alterations in host cell gene expression including signalling pathways may be important for the design of future clinical trials.     

Immunohistochemical evidence for the myeloperoxidase/H2O2/halide system in human atherosclerotic lesions: colocalization of myeloperoxidase and hypochlorite-modified proteins.

The 'oxidation theory' of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although the precise mechanisms of in vivo oxidation are widely unknown, increasing evidence suggests that myeloperoxidase (MPO, EC 1.11.1.7), a protein secreted by activated phagocytes, generates modified/oxidized (lipo)proteins via intermediate formation of hypochlorous acid (HOCl). In vitro generation of HOCl transforms lipoproteins into high uptake forms for macrophages giving rise to cholesterol-engorged foam cells. To identify HOCl-modified-epitopes in human plaque tissues we have raised monoclonal antibodies (directed against human HOCl-modified LDL) that do not cross-react with other LDL modifications, i.e. peroxynitrite-LDL, hemin-LDL, Cu2+-oxidized LDL, 4-hydroxynonenal-LDL, malondialdehyde-LDL, glycated-LDL, and acetylated-LDL. The antibodies recognized a specific epitope present on various proteins after treatment with OCl- added as reagent or generated by the MPO/H2O2/halide system. Immunohistochemical studies revealed pronounced staining for HOCl-modified-epitopes in fibroatheroma (type V) and complicated (type VI) lesions, while no staining was observed in aortae of lesion-prone location (type I). HOCl-oxidation-specific epitopes are detected in cells in the majority of atherosclerotic plaques but not in control segments. Staining was shown to be inside and outside monocytes/macrophages, endothelial cells, as well as in the extracellular matrix. A similar staining pattern using immunohistochemistry could be obtained for MPO. The colocalization of immunoreactive MPO and HOCl-modified-epitopes in serial sections of human atheroma (type IV), fibroatheroma (type V) and complicated (type VI) lesions provides further convincing evidence for MPO/H2O2/halide system-mediated oxidation of (lipo)proteins under in vivo conditions. We propose that MPO could act as an important link between the development of atherosclerotic plaque in the artery wall and chronic inflammatory events.     

Gadolinium-containing phosphatidylserine liposomes for molecular imaging of atherosclerosis

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE^−/− mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.     

Cannabinoid receptor 2 signaling does not modulate atherogenesis in mice

Background

Strong evidence supports a protective role of the cannabinoid receptor 2 (CB₂) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB₂ receptor in Murine atherogenesis.

Methods and Findings

Low density lipoprotein receptor-deficient (LDLR^−/−) mice subjected to intraperitoneal injections of the selective CB₂ receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB₂ activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB₂ ^−/−/LDLR^−/− mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB₂ ^+/+/LDLR^−/− controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-illicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB₂ receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro.

Conclusion

Our study demonstrates that both activation and deletion of the CB₂ receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB₂ in other inflammatory processes. However, in the context of atherosclerosis, CB₂ does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque.     

Differential expression of CD163 on monocyte subsets in healthy and HIV-1 infected individuals

CD163, a haptoglobin-hemoglobin (Hp-Hb) scavenger receptor, expressed by monocytes and macrophages, is important in resolution of inflammation. Age-related non-AIDS co-morbidities in HIV-infected individuals, particularly dementia and cardiovascular disease, result in part from effects of HIV-1 infection on monocyte and macrophage biology. CD163 co-expression on CD14+CD16++ monocytes has been proposed as a useful biomarker for HIV-1 disease progression and the presence of HIV associated dementia. Here we investigated CD163 expression on monocyte subsets ex vivo, on cultured macrophages, and soluble in plasma, in the setting of HIV-1 infection. Whole blood immunophenotyping revealed CD163 expression on CD14++CD16- monocytes but not on CD14+CD16++ monocytes (P = 0.004), supported by CD163 mRNA levels. Incubation with M-CSF induced CD163 protein expression on CD14+CD16++ monocytes to the same extent as CD14++CD16− monocytes. CD163 expression on CD14++CD16+ monocytes from HIV-infected subjects was significantly higher than from uninfected individuals, with a trend towards increased expression on CD14++CD16− monocytes (P = 0.019 and 0.069 respectively), which is accounted for by HIV-1 therapy including protease inhibitors. Shedding of CD163 was shown to predominantly occur from the CD14++CD16− subset after Ficoll isolation and LPS stimulation. Soluble CD163 concentration in plasma from HIV-1 infected donors was similar to HIV-1 uninfected donors. Monocyte CD163 expression in HIV-1 infected patients showed a complicated relationship with classical measures of disease progression. Our findings clarify technical issues regarding CD163 expression on monocyte subsets and further elucidates its role in HIV-associated inflammation by demonstrating that CD163 is readily lost from CD14++CD16− monocytes and induced in pro-inflammatory CD14+CD16++ monocytes by M-CSF. Our data show that all monocyte subsets are potentially capable of differentiating into CD163-expressing anti-inflammatory macrophages given appropriate stimuli. Levels of CD163 expression on monocytes may be a potential biomarker reflecting efforts by the immune system to resolve immune activation and inflammation in HIV-infected individuals.     

Atherosclerosis in ApoE-deficient mice progresses independently of the NLRP3 inflammasome

The interleukin-1 (IL-1) family of cytokines has been implicated in the pathogenesis of atherosclerosis in previous studies. The NLRP3 inflammasome has recently emerged as a pivotal regulator of IL-1β maturation and secretion by macrophages. Little is currently known about a possible role for the NLRP3 inflammasome in atherosclerosis progression in vivo. We generated ApoE−/− Nlrp3−/−, ApoE−/− Asc−/− and ApoE−/− caspase-1−/− double-deficient mice, fed them a high-fat diet for 11 weeks and subsequently assessed atherosclerosis progression and plaque phenotype. No differences in atherosclerosis progression, infiltration of plaques by macrophages, nor plaque stability and phenotype across the genotypes studied were found. Our results demonstrate that the NLRP3 inflammasome is not critically implicated in atherosclerosis progression in the ApoE mouse model.     

In vivo imaging of macrophages during the early-stages of abdominal aortic aneurysm using high resolution MRI in ApoE mice

Background

Angiotensin II (ANG II) promotes vascular inflammation and induces abdominal aortic aneurysm (AAA) in hyperlipidemic apolipoprotein E knock-out (apoE^−/−) mice. The aim of the present study was to detect macrophage activities in an ANG II-induced early-stage AAA model using superparamagnetic iron oxide (SPIO) as a marker.

Methodology/Principal Findings

Twenty-six male apoE^−/− mice received saline or ANG II (1000 or 500 ng/kg/min) infusion for 14 days. All animals underwent MRI scanning following administration of SPIO with the exception of three mice in the 1000 ng ANG II group, which were scanned without SPIO administration. MR imaging was performed using black-blood T2 to proton density -weighted multi-spin multi-echo sequence. In vivo MRI measurement of SPIO uptake and abdominal aortic diameter were obtained. Prussian blue, CD68,α-SMC and MAC3 immunohistological stains were used for the detection of SPIO, macrophages and smooth muscle cells. ANG II infusion with 1000 ng/kg/min induced AAA in all of the apoE^−/− mice. ANG II infusion exhibited significantly higher degrees of SPIO uptake, which was detected using MRI as a distinct loss of signal intensity. The contrast-to-noise ratio value decreased in proportion to an increase in the number of iron-laden macrophages in the aneurysm. The aneurysmal vessel wall in both groups of ANG II treated mice contained more iron-positive macrophages than saline-treated mice. However, the presence of cells capable of phagocytosing haemosiderin in mural thrombi also induced low-signal-intensities via MRI imaging.

Conclusions/Significance

SPIO is taken up by macrophages in the shoulder and the outer layer of AAA. This alters the MRI signaling properties and can be used in imaging inflammation associated with AAA. It is important to compare images of the aorta before and after SPIO injection.     

Bone Marrow Deficiency of MCPIP1 Results in Severe Multi-Organ Inflammation but Diminishes Atherogenesis in Hyperlipidemic Mice

Objective

MCPIP1 is a newly identified protein that profoundly impacts immunity and inflammation. We aim to test if MCPIP1 deficiency in hematopoietic cells results in systemic inflammation and accelerates atherogenesis in mice.

Approach and Results

After lethally irradiated, LDLR^−/− mice were transplanted with bone marrow cells from either wild-type or MCPIP1^−/− mice. These chimeric mice were fed a western-type diet for 7 weeks. We found that bone marrow MCPIP1^−/− mice displayed a phenotype similar to that of whole body MCPIP1^−/− mice, with severe systemic and multi-organ inflammation. However, MCPIP1^−/− bone marrow recipients developed >10-fold less atherosclerotic lesions in the proximal aorta than WT bone marrow recipients, and essentially no lesions in en face aorta. The diminishment in atherosclerosis in bone marrow MCPIP1^−/− mice may be partially attributed to the slight decrease in their plasma lipids. Flow cytometric analysis of splenocytes showed that bone marrow MCPIP1^−/− mice contained reduced numbers of T cells and B cells, but increased numbers of regulatory T cells, Th17 cells, CD11b+/Gr1+ cells and CD11b+/Ly6C^low cells. This overall anti-atherogenic leukocyte profile may also contribute to the reduced atherogenesis. We also examined the cholesterol efflux capability of MCPIP1 deficient macrophages, and found that MCPIP1deficiency increased cholesterol efflux to apoAI and HDL, due to increased protein levels of ABCA1 and ABCG1.

Conclusions

Hematopoietic deficiency of MCPIP1 resulted in severe systemic and multi-organ inflammation but paradoxically diminished atherogenesis in mice. The reduced atheroegensis may be explained by the decreased plasma cholesterol levels, the anti-atherogenic leukocyte profile, as well as enhanced cholesterol efflux capability. This study suggests that, while atherosclerosis is a chronic inflammatory disease, the mechanisms underlying atherogenesis-associated inflammation in arterial wall versus the inflammation in solid organs may be substantially different.     

Fatty Acid binding protein 4 is associated with carotid atherosclerosis and outcome in patients with acute ischemic stroke

Background and Purpose

Fatty acid binding protein 4 (FABP4) has been shown to play an important role in macrophage cholesterol trafficking and associated inflammation. To further elucidate the role of FABP4 in atherogenesis in humans, we examined the regulation of FABP4 in carotid atherosclerosis and ischemic stroke.

Methods

We examined plasma FABP4 levels in asymptomatic (n = 28) and symptomatic (n = 31) patients with carotid atherosclerosis, as well as in 202 subjects with acute ischemic stroke. In a subgroup of patients we also analysed the expression of FABP4 within the atherosclerotic lesion. In addition, we investigated the ability of different stimuli with relevance to atherosclerosis to regulate FABP4 expression in monocytes/macrophages.

Results

FABP4 levels were higher in patients with carotid atherosclerosis, both systemically and within the atherosclerotic lesion, with particular high mRNA levels in carotid plaques from patients with the most recent symptoms. Immunostaining of carotid plaques localized FABP4 to macrophages, while activated platelets and oxidized LDL were potent stimuli for FABP4 expression in monocytes/macrophages in vitro. When measured at the time of acute ischemic stroke, high plasma levels of FABP4 were significantly associated with total and cardiovascular mortality during follow-up, although we did not find that addition of FABP4 to the fully adjusted multivariate model had an effect on the prognostic discrimination for all-cause mortality as assessed by c-statistics.

Conclusions

FABP4 is linked to atherogenesis, plaque instability and adverse outcome in patients with carotid atherosclerosis and acute ischemic stroke.     

Increased microparticle production and impaired microvascular endothelial function in aldosterone-salt-treated rats: protective effects of polyphenols

We aimed to characterize circulating microparticles in association with arterial stiffness, inflammation and endothelial dysfunction in aldosterone-salt-induced hypertension in rats and to investigate the preventive effects of red wine polyphenols. Uninephrectomized male Sprague-Dawley rats were treated with aldosterone-salt (1 µg.h⁻¹), with or without administration of either red wine polyphenols, Provinols™ (20 mg.kg⁻¹.day⁻¹), or spironolactone (30 mg.kg⁻¹.day⁻¹) for 4 weeks. Microparticles, arterial stiffness, nitric oxide (NO) spin trapping, and mesenteric arterial function were measured. Aldosterone-salt rats showed increased microparticle levels, including those originating from platelets, endothelium and erythrocytes. Hypertension resulted in enhanced aortic stiffness accompanied by increased circulating and aortic NO levels and an upregulation of aortic inducible NO-synthase, NFκB, superoxide anions and nitrotyrosine. Flow-induced dilatation was reduced in mesenteric arteries. These effects were prevented by spironolactone. Provinols™ did not reduce arterial stiffness or systolic hypertension but had effects similar to those of spironolactone on endothelial function assessed by flow-mediated vasodilatation, microparticle generation, aortic NO levels and oxidative stress and apoptosis in the vessel wall. Neither the contractile response nor endothelium-dependent relaxation in mesenteric arteries differed between groups. The in vivo effects of Provinols™ were not mediated by mineralocorticoid receptors or changes in shear stress. In conclusion, vascular remodelling and endothelial dysfunction in aldosterone-salt-mediated hypertension are associated with increased circulating microparticles. Polyphenols prevent the enhanced release of microparticles, macrovascular inflammation and oxidative stress, and microvascular endothelial dysfunction independently of blood pressure, shear stress and mineralocorticoid receptor activation in a model of hyperaldosteronism.     

eNOS protects from atherosclerosis despite relevant superoxide production by the enzyme in apoE mice

Background

All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme.

Principal Findings

Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE^−/−/eNOS^−/−), while P/E-interactions did not differ, compared to apoE^−/−. eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE^−/− vessels.

Conclusion

Overt plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE^−/−/eNOS^−/− vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE^−/− atherosclerosis but does not negate the enzyme''s strong protective effects.     

NOS1-mediated macrophage and endothelial cell interaction in the progression of atherosclerosis

Atherosclerosis is a chronic inflammatory disease arising due to an imbalance in lipid metabolism and maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Interactions between monocytes/macrophages and endothelial cells play an essential role in the pathogenesis of atherosclerosis. In our current study, nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) has been identified as a regulator of macrophage and endothelial cell interaction. Oxidized LDL (OxLDL) activates NOS1, which results in the expression of CD40 ligand in macrophages. OxLDL-stimulated macrophages produce some soluble factors which increase the CD40 receptor expression in endothelial cells. This increases the interaction between the macrophages and endothelial cells, which leads to an increase in the inflammatory response. Inhibition of NOS1-derived NO might serve as an effective strategy to reduce foam cell formation and limit the extent of atherosclerotic plaque expansion.     

Elevated expression of phospholipid transfer protein in bone marrow derived cells causes atherosclerosis

Background

Phospholipid transfer protein (PLTP) is expressed by various cell types. In plasma, it is associated with high density lipoproteins (HDL). Elevated levels of PLTP in transgenic mice result in decreased HDL and increased atherosclerosis. PLTP is present in human atherosclerotic lesions, where it seems to be macrophage derived. The aim of the present study is to evaluate the atherogenic potential of macrophage derived PLTP.

Methods and Findings

Here we show that macrophages from human PLTP transgenic mice secrete active PLTP. Subsequently, we performed bone marrow transplantations using either wild type mice (PLTPwt/wt), hemizygous PLTP transgenic mice (huPLTPtg/wt) or homozygous PLTP transgenic mice (huPLTPtg/tg) as donors and low density lipoprotein receptor deficient mice (LDLR−/−) as acceptors, in order to establish the role of PLTP expressed by bone marrow derived cells in diet-induced atherogenesis. Atherosclerosis was increased in the huPLTPtg/wt→LDLR−/− mice (2.3-fold) and even further in the huPLTPtg/tg→LDLR−/− mice (4.5-fold) compared with the control PLTPwt/wt→LDLR−/− mice (both P<0.001). Plasma PLTP activity levels and non-HDL cholesterol were increased and HDL cholesterol decreased compared with controls (all P<0.01). PLTP was present in atherosclerotic plaques in the mice as demonstrated by immunohistochemistry and appears to co-localize with macrophages. Isolated macrophages from PLTP transgenic mice do not show differences in cholesterol efflux or in cytokine production. Lipopolysaccharide activation of macrophages results in increased production of PLTP. This effect was strongly amplified in PLTP transgenic macrophages.

Conclusions

We conclude that PLTP expression by bone marrow derived cells results in atherogenic effects on plasma lipids, increased PLTP activity, high local PLTP protein levels in the atherosclerotic lesions and increased atherosclerotic lesion size.     

Overexpression of TGF-ß1 in macrophages reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE^−/− mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.     

Type I interferon signaling regulates Ly6C(hi) monocytes and neutrophils during acute viral pneumonia in mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1 ^−/−) mice develop significant defects in the infiltration of Ly6C^hi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6C^hi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6C^int monocytes of Ifnar1 ^−/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1 ^−/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6C^hi monocytes. By using BM chimeric mice (WT BM into Ifnar1 ^−/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6C^hi monocytes. Of note, WT BM reconstituted Ifnar1 ^−/− chimeric mice with increased numbers of Ly6C^hi monocytes survived longer than influenza-infected Ifnar1 ^−/− mice. In contrast, WT mice that received Ifnar1 ^−/− BM cells with alternative Ly6C^int monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.