Targeting activation of specific NF-κB subunits prevents stress-dependent atherothrombotic gene expression.

Psychosocial stress has been shown to be a contributing factor in the development of atherosclerosis. Although the underlying mechanisms have not been elucidated entirely, it has been shown previously that the transcription factor nuclear factor-κB (NF-κB) is an important component of stress-activated signaling pathway. In this study, we aimed to decipher the mechanisms of stress-induced NF-κB-mediated gene expression, using an in vitro and in vivo model of psychosocial stress. Induction of stress led to NF-κB-dependent expression of proinflammatory (tissue factor, intracellular adhesive molecule 1 [ICAM-1]) and protective genes (manganese superoxide dismutase [MnSOD]) via p50, p65 or cRel. Selective inhibition of the different subunits and the respective kinases showed that inhibition of cRel leads to the reduction of atherosclerotic lesions in apolipoprotein(-/-) (ApoE(-/-)) mice via suppression of proinflammatory gene expression. This observation may therefore provide a possible explanation for ineffectiveness of antioxidant therapies and suggests that selective targeting of cRel activation may provide a novel approach for the treatment of stress-related inflammatory vascular disease.

Expression of IL-17A in human atherosclerotic lesions is associated with increased inflammation and plaque vulnerability.

A chronic (auto)immune response is the critical mechanism in atherosclerosis. Interleukin-17A is a pivotal effector cytokine, which modulates immune cell trafficking and initiates inflammation in (auto)immune and infectious diseases. However, expression of IL-17A in the context of human atherosclerosis has hardly been explored. Carotid artery plaques were collected from 79 patients undergoing endarterectomy. Patients were grouped according to their symptomatic status (TIA, stroke), plaque morphology and medication. Quantitative RT-PCR was used to analyze tissue inflammation and immunohistochemistry to assess cellular source of IL-17A expression and lesion morphology. Carotid plaques from patients with ischemic symptoms were characterized by a highly activated inflammatory milieu including accumulation of T cells (p = 0.04) and expression of IL-6 and VCAM1 (p = 0.02, 0.01). Expression of IL-17A and its positive regulators IL-21 and IL-23 was present in atherosclerotic lesions, significantly upregulated in atheromas of symptomatic patients (p = 0.005, 0.004, 0.03), and expression of IL-17A and IL-21 showed a strong correlation (p = 0.002, r = 0.52). The cellular sources of lesional IL-17A expression are T cells, macrophages, B cells and plasma cells. Vulnerable/ruptured (complicated) plaques were significantly associated with IL-17A expression levels (p = 0.003). In addition, IL-17A showed a marked negative correlation with the potent anti-inflammatory/atheroprotective cytokine IL-10 (p = 0.0006, r = -0.46). Furthermore, treatment with a HMG-CoA reductase inhibitor or acetylsalicylic acid showed reduced levels of IL-21, IL-23 and VCAM1 (all p < 0.05), but did not influence IL-17A. The association of IL-17A with ischemic symptoms and vulnerable plaque characteristics suggests that the pro-inflammatory cytokine IL-17A may contribute to atherosclerosis und plaque instability.

Deletion of bone marrow-derived receptor for advanced glycation end products inhibits atherosclerotic plaque progression.

BACKGROUND: The multiligand receptor for advanced glycation end products (RAGE) of the immunoglobulin superfamily is expressed on multiple cell types implicated in the inflammatory response in atherosclerosis. We sought to determine the role of bone marrow-derived RAGE in different stages of atherosclerotic development in apolipoprotein E-deficient mice (apoE(-/-)). METHODS: Seven- and 23-week-old apoE(-/-) mice (n = 40) were lethally irradiated and given bone marrow from RAGE null (RAGE(-/-)/apoE(-/-)) or RAGE-bearing (RAGE(+/+)/apoE(-/-)) mice to apoE(-/-) mice to generate double knockout bone marrow chimera (RAGE(-/-)/apoE(-/-bmc) and RAGE(+/+)/apoE(-/-bmc)-, respectively). After 16 weeks on a standard chow diet, mice were sacrificed and atherosclerotic lesion formation was evaluated. RESULTS: Plaques in the aortic root of the young mice showed no significant difference in maximum plaque size (217,470 ± 17,480 µm(2) for the RAGE(-/-) /apoE(-/-bmc) mice compared to 244,764 ± 45,840 µm(2)), whereas lesions in the brachiocephalic arteries of the older RAGE(-/-)/apoE(-/-bmc) mice had significantly smaller lesions (94,049 ± 13,0844 µm(2) vs. 145,570 ± 11,488 µm(2), P < 0.05) as well as reduced average necrotic core area (48,600 ± 9220 µm(2) compared to 89,502 ± 10,032 µm(2), P < 0.05) when compared to RAGE(+/+)/apoE(-/-bmc) mice. Reduced plaque size and more stable plaque morphology was associated with significant reduced expression of VCAM-1, ICAM-1 and MCP-1. Accumulation of the RAGE ligand HMGB-1 was also significantly reduced within the lesions of RAGE(-/-)/apoE(-/-bmc) mice. CONCLUSIONS: This study demonstrates that bone marrow-derived RAGE is an important factor in the progression of atherosclerotic plaques.

Nicotinic acid has anti-atherogenic and anti-inflammatory properties on advanced atherosclerotic lesions independent of its lipid-modifying capabilities.

Inflammation contributes to atherosclerotic plaque initiation and progression. Recent studies suggest that nicotinic acid has anti-inflammatory effects independent of its lipid-modifying capabilities. We assessed the hypothesis that administration of nicotinic acid to older apolipoprotein E (apoE)-deficient mice with established lesions will reduce lesion size and plaque inflammation independent of its lipid-modifying effects. Therefore nicotinic acid was administered to 27-week-old apo E-deficient mice exhibiting advanced atherosclerotic lesions within the innominate artery. After 27 weeks of treatment both animal groups had no significant changes in plasma lipid levels. Mice treated with nicotinic acid (n = 22) demonstrated a 30% reduction in total lesion area compared with controls (n = 20). Furthermore, they revealed a more stable plaque composition with an increase in fibrous cap thickness and a reduction in the size of the necrotic core. Immunohistochemistry demonstrated a reduced accumulation of macrophages and a reduced expression of vascular cell adhesion molecule-1 and tissue factor. Additionally, administration of nicotinic acid significantly reduced tumor necrosis factor alpha expression in the thoracic aorta as demonstrated by real-time PCR. In conclusion, these data suggest that long-term administration of nicotinic acid has anti-atherogenic and anti-inflammatory properties on advanced atherosclerotic lesions, which are independent of its lipid-modifying actions.

Inhibition of IL-17A attenuates atherosclerotic lesion development in apoE-deficient mice.

The importance of an (auto)immune response in atherogenesis is becoming increasingly well understood. IL-17A-expressing T cells modulate immune cell trafficking, initiating inflammation and cytokine production in (auto)immune diseases. In human carotid artery plaques, we previously showed the presence of IL-17A-producing T cells and IL-23; however, IL-17A effects on atherogenesis have not been studied. Aortic root sections from 8-wk-old apolipoprotein E-deficient mice fed a standard chow diet were examined after 12 wk for lesion area, plaque composition, cellular infiltration, cytokine expression, and apoptosis. The treatment group (n = 15) received anti-IL-17A Ab and the controls (n = 10) received irrelevant Abs. Inhibition of IL-17A markedly reduced atherosclerotic lesion area (p < 0.001), maximal stenosis (p < 0.001), and vulnerability of the lesion. IL-17A mAb-treated mice showed reduced cellular infiltration, down-regulation of activation markers on endothelium and immune cells (e.g., VCAM-1), and reduced cytokine/chemokine secretion (e.g., IL6, TNFalpha, CCL5). To investigate possible mechanisms, different atherogenic cell types (e.g., macrophages, dendritic cells, HUVECs, vascular smooth muscle cells) were stimulated with IL-17A in addition to TNF-alpha, IFN-gamma, or LPS to induce cellular activation or apoptosis in vitro. Stimulation with IL-17A induced proinflammatory changes in several atherogenic cell types and apoptotic cell death in murine cells. Functional blockade of IL-17A reduces atherosclerotic lesion development and decreases plaque vulnerability, cellular infiltration, and tissue activation in apolipoprotein E-deficient mice. The present data support a pathogenic role of IL-17A in the development of atherosclerosis by way of its widespread proinflammatory and proapoptotic effects on atherogenic cells.

Evaluation of plaque stability of advanced atherosclerotic lesions in apo E-deficient mice after treatment with the oral factor Xa inhibitor rivaroxaban.

AIM: Thrombin not only plays a central role in thrombus formation and platelet activation, but also in induction of inflammatory processes. Activated factor X (FXa) is traditionally known as an important player in the coagulation cascade responsible for thrombin generation. We assessed the hypothesis that rivaroxaban, a direct FXa inhibitor, attenuates plaque progression and promotes stability of advanced atherosclerotic lesions in an in vivo model. METHODS AND RESULTS: Rivaroxaban (1 or 5 mg/kg body weight/day) or standard chow diet was administered for 26 weeks to apolipoprotein E-deficient mice (n = 20 per group) with already established atherosclerotic lesions. There was a nonsignificant reduction of lesion progression in the high-concentration group, compared to control mice. FXa inhibition with 5 mg Rivaroxaban/kg/day resulted in increased thickness of the protective fibrous caps (12.3 ± 3.8 µm versus 10.1 ± 2.7 µm; P < .05), as well as in fewer medial erosions and fewer lateral xanthomas, indicating plaque stabilizing properties. Real time-PCR from thoracic aortas revealed that rivaroxaban (5 mg/kg/day) treatment reduced mRNA expression of inflammatory mediators, such of IL-6, TNF-α, MCP-1, and Egr-1 (P < .05). CONCLUSIONS: Chronic administration of rivaroxaban does not affect lesion progression but downregulates expression of inflammatory mediators and promotes lesion stability in apolipoprotein E-deficient mice.

Hypercoagulability inhibits monocyte transendothelial migration through protease-activated receptor-1-, phospholipase-Cbeta-, phosphoinositide 3-kinase-, and nitric oxide-dependent signaling in monocytes and promotes plaque stability.

BACKGROUND: Clinical studies failed to provide clear evidence for a proatherogenic role of hypercoagulability. This is in contrast to the well-established detrimental role of hypercoagulability and thrombin during acute atherosclerotic complications. These seemingly opposing data suggest that hypercoagulability might exert both proatherogenic and antiatherogenic effects. We therefore investigated whether hypercoagulability mediates a beneficial effect during de novo atherogenesis. METHODS AND RESULTS: De novo atherogenesis was evaluated in 2 mouse models with hyperlipidemia and genetically imposed hypercoagulability (TM(Pro/Pro)ApoE(-/-) and FVL(Q/Q)ApoE(-/-) mice). In both mouse models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Importantly, plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. Long-term anticoagulation reversed these changes. The reduced frequency of intraplaque macrophages in hypercoagulable mice is explained by an inhibitory role of thrombin and protease-activated receptor-1 on monocyte transendothelial migration in vitro. This is dependent on phospholipase-Cbeta, phosphoinositide 3-kinase, and nitric oxide signaling in monocytes but not in endothelial cells. CONCLUSIONS: Here, we show a new function of the coagulation system, averting stenosis and plaque destabilization during de novo atherogenesis. The in vivo and in vitro data establish that thrombin-induced signaling via protease-activated receptor-1, phospholipase-Cbeta, phosphoinositide 3-kinase, and nitric oxide in monocytes impairs monocyte transendothelial migration. This likely accounts for the reduced macrophage accumulation in plaques of hypercoagulable mice. Thus, in contrast to their role in unstable plaques or after vascular injury, hypercoagulability and thrombin convey a protective effect during de novo atherogenesis.

FGF-inducible 14-kDa protein (Fn14) is regulated via the RhoA/ROCK kinase pathway in cardiomyocytes and mediates nuclear factor-kappaB activation by TWEAK.

Proinflammatory cytokines, including TNF family members, have been shown to play a critical role in cardiac remodeling. FGF-inducible 14-kDa protein (Fn14, TNFrsf12a or TWEAKR) is the smallest member of the TNF-receptor family. Currently, little is known about the functional role of Fn14 and its only known ligand TNF-like weak inducer of apoptosis (TWEAK) in the heart. We therefore evaluated the expression and regulation of Fn14 in cardiomyocytes and in experimental myocardial infarction. In order to study the regulation of Fn14, myocardial infarction was induced in CD-1 mice and neonatal rat cardiomyocytes were used for in vitro studies. TWEAK and Fn14 were markedly upregulated in the remodeling myocardium after experimental myocardial infarction in vivo. Likewise, fibroblast growth factor 1, norepinephrine and angiotensin II as well as mechanical stretch were able to strongly induce Fn14 expression in cardiomyocytes. This induction is mediated via the Rho/ROCK pathway, since the known inhibitors C3 exoenzyme for RhoA and Y27632 for ROCK prevented the upregulation of Fn14 in cardiomyocytes. Consistently, pretreatment of cardiomyocytes with siRNA against Rho A and ROCK also abolished Fn14 induction. Moreover, stimulation of cardiomyocytes with TWEAK promoted nuclear translocation of NF-kappaB and subsequent induction of NF-kappaB dependent genes such as RANTES and MCP-1. Conversely, when cells were pretreated with siRNA against Fn14, NF-kappaB activation by TWEAK was inhibited. We here provide the first evidence of a stress-induced regulation of the TWEAK/Fn14 axis in cardiomyocytes implying a role of the TWEAK/Fn14 pathway in cardiac remodeling.

Resuscitation Guidelines 2005: does experienced nursing staff need training and how effective is it?

INTRODUCTION: Even among health care professionals, resuscitation performance has been shown to be poor. So far, it remains unclear whether cardiac arrest staff with frequent practice in resuscitation requires training to adapt to the new International Liaison Committee on Resuscitation (ILCOR) guidelines of 2005. This study evaluated the need for basic life support training in nurses with emergency experience. METHODS AND RESULTS: Nurses (N = 24) recruited from an intensive care unit self-assessed their resuscitation skills and performed a cardiac arrest scenario using a manikin. After a theoretical instruction and hands-on training followed by feedback, participants once again performed a resuscitation scenario in addition to completing posttraining self-assessments. Participating nurses considered resuscitation skills training--in particular in adapting to the new ILCOR guidelines of 2005--to be important. Pretraining data revealed performance deficits even in this sample of emergency-experienced nursing staff. Training resulted in significant improvement in ventilation volume (P < .001), rate of compressions with correct depth (P < .031) and full release (P < .001), and a reduction in total hands-off time (P < .050). Objective data were mirrored in participants' self-assessed competencies. CONCLUSION: Results suggest that basic life support training based on the ILCOR guidelines of 2005 is necessary even in nurses with emergency experience. Training followed by the application of a feedback algorithm seems to improve short-term resuscitation performance and is well accepted by experienced nurses who work on an intensive care unit and who also comprise the inner-hospital cardiac arrest team.

Rhizoma Coptidis inhibits LPS-induced MCP-1/CCL2 production in murine macrophages via an AP-1 and NFkappaB-dependent pathway.

INTRODUCTION: The Chinese extract Rhizoma coptidis is well known for its anti-inflammatory, antioxidative, antiviral, and antimicrobial activity. The exact mechanisms of action are not fully understood. METHODS: We examined the effect of the extract and its main compound, berberine, on LPS-induced inflammatory activity in a murine macrophage cell line. RAW 264.7 cells were stimulated with LPS and incubated with either Rhizoma coptidis extract or berberine. Activation of AP-1 and NFkappaB was analyzed in nuclear extracts, secretion of MCP-1/CCL2 was measured in supernatants. RESULTS: Incubation with Rhizoma coptidis and berberine strongly inhibited LPS-induced monocyte chemoattractant protein (MCP)-1 production in RAW cells. Activation of the transcription factors AP-1 and NFkappaB was inhibited by Rhizoma coptidis in a dose- and time-dependent fashion. CONCLUSIONS: Rhizoma coptidis extract inhibits LPS-induced MCP-1/CCL2 production in vitro via an AP-1 and NFkappaB-dependent pathway. Anti-inflammatory action of the extract is mediated mainly by its alkaloid compound berberine.

High-mobility group box-1 in ischemia-reperfusion injury of the heart.

BACKGROUND: High-mobility group box-1 (HMGB1) is a nuclear factor released by necrotic cells and by activated immune cells. HMGB1 signals via members of the toll-like receptor family and the receptor for advanced glycation end products (RAGE). Although HMGB1 has been implicated in ischemia/reperfusion (I/R) injury of the liver and lung, its role in I/R injury of the heart remains unclear. METHODS AND RESULTS: Here, we demonstrate that HMGB1 acts as an early mediator of inflammation and organ damage in I/R injury of the heart. HMGB1 levels were already elevated 30 minutes after hypoxia in vitro and in ischemic injury of the heart in vivo. Treatment of mice with recombinant HMGB1 worsened I/R injury, whereas treatment with HMGB1 box A significantly reduced infarct size and markers of tissue damage. In addition, HMGB1 inhibition with recombinant HMGB1 box A suggested an involvement of the mitogen-activated protein kinases jun N-terminal kinase and extracellular signal-regulated kinase 1/2, as well as the nuclear transcription factor nuclear factor-kappaB in I/R injury. Interestingly, infarct size and markers of tissue damage were not affected by administration of recombinant HMGB1 or HMGB1 antagonists in RAGE(-/-) mice, which demonstrated significantly reduced damage in reperfused hearts compared with wild-type mice. Coincubation studies using recombinant HMGB1 in vitro induced an inflammatory response in isolated macrophages from wild-type mice but not in macrophages from RAGE(-/-) mice. CONCLUSIONS: HMGB1 plays a major role in the early event of I/R injury by binding to RAGE, resulting in the activation of proinflammatory pathways and enhanced myocardial injury. Therefore, blockage of HMGB1 might represent a novel therapeutic strategy in I/R injury.

Critical role of macrophages in glucocorticoid driven vascular calcification in a mouse-model of atherosclerosis.

OBJECTIVE: Macrophage-derived products are known to play a crucial role during atherogenesis and vascular calcification. Glucocorticoids (GC) are important modulators of immune cell functions, but their specific effects on macrophages behavior during plaque formation are not defined. The present study was therefore designed to investigate the effects of macrophage-specific deletion of the glucocorticoid receptor (GR(LysMCre)) on atherogenesis and vascular calcification in a hyperlipidemic mouse-model. METHODS AND RESULTS: Bone marrow was isolated from GR(LysMCre) mice and wild-type controls (GR(flox)) and subsequently transplanted into lethally irradiated LDL-receptor-deficient mice. Animals were fed a Western-type diet for 15 or 24 weeks, and atherosclerotic lesions within the aortic sinus were evaluated. At both time points, no significant difference in serum lipid and corticosterone concentrations, atherosclerotic lesion size and macrophage-content within the lesions could be observed. However, GR(LysMCre) mice showed less calcification as well as a significant reduction of RANKL, BMP2, and Msx2 expression within the vasculature. In vitro studies using conditioned media from macrophages which had been stimulated with dexamethasone demonstrated a dose-dependent increase in calcium deposition by vascular smooth muscle cells. CONCLUSIONS: This study demonstrates that macrophage-specific glucocorticoid receptor inactivation reduces vascular calcification without affecting atherosclerotic lesion size in LDL receptor-deficient mice.

Sphingosine-1-phosphate analogue FTY720 causes lymphocyte redistribution and hypercholesterolemia in ApoE-deficient mice.

OBJECTIVE: Resident immune cells are a hallmark of atherosclerotic lesions. The sphingolipid analogue drug FTY720 mediates retrafficking of immune cells and inhibits their homing to inflammatory sites. We have evaluated the effect of FTY720 on atherogenesis and lipid metabolism. METHODS AND RESULTS: ApoE-/- mice on a normal laboratory diet received oral FTY720 for 12 weeks, which led to a 2.4-fold increase in serum cholesterol (largely VLDL fraction) and a 1.8-fold increase in hepatic HMGCoA reductase mRNA. FTY720 increased plasma sphingosine-1-phosphate and induced marked peripheral blood lymphopenia. A discoordinate modulation of B, T and monocyte cell numbers was found in peripheral lymphoid organs. Overall depletion of T cells was accompanied by a relative (2-fold) increase in regulatory T cell content paralleled by a similar increase in effector memory T cells (CD4+ CD44hi CD62lo) as absolute numbers of both subpopulations remained essentially unchanged. Lymphocyte function was unaltered as indicated by anti-OxLDL antibodies and T cell proliferation. There were no changes in atherosclerotic lesions in early and established atherosclerosis. CONCLUSIONS: FTY720 mediated peripheral lymphocyte depletion and retrafficking without altering function and overall balance of pro- and antiatherogenic lymphocyte populations. A net decrease in lymphocyte numbers occurred concomitantly with a more proatherogenic hypercholesterolemia resulting in unaltered atherogenesis.

Intravenous delivery of autologous mesenchymal stem cells limits infarct size and improves left ventricular function in the infarcted porcine heart.

Systemic delivery of bone marrow-derived mesenchymal stem cells (MSCs) is a noninvasive approach for myocardial repair. We aimed to test this strategy in a pig model of myocardial infarction. Pigs (n = 8) received autologous MSCs (1 x 10(6)/kg body weight) labeled with fluorescent dye 48 h post proximal left anterior descending artery (LAD) occlusion. Hemodyamics, infarct size, and myocardial function were assessed at baseline and after 1 month. Morphologic analysis revealed that labeled MSCs migrated in the peri-infarct region, resulting in smaller infarct size (32 +/- 7 vs. 19 +/- 7%, p = 0.01), higher fractional area shortening (23 +/- 3 vs. 34.0 +/- 7%, p = 0.001), lower left ventricular end diastolic pressure (18.7 +/- 5 vs. 10.2 +/- 4 mmHg, p = 0.02) and higher +dp/dt (4,570 +/- 540 vs. 6,742 +/- 700 mmHg/s, p = 0.03) during inotropic stimulation. Systemic intravenous delivery of MSCs to pigs limits myocardial infarct size and is an attractive approach for tissue repair.

Interleukin-10 suppresses tissue factor expression in lipopolysaccharide-stimulated macrophages via inhibition of Egr-1 and a serum response element/MEK-ERK1/2 pathway.

Atherosclerosis is considered to be an inflammatory disease. Tissue factor (TF), a prothrombotic molecule expressed by various cell types within atherosclerotic plaques, is thought to play an essential role in thrombus formation after atherosclerotic plaque rupture. Recent studies suggest that the antiinflammatory cytokine interleukin-10 (IL-10) has many antiatherosclerotic properties. Therefore, the effects of IL-10 on TF expression in response to inflammation were investigated. Mouse macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of IL-10. Pretreatment with IL-10 resulted in a 50% decrease in TF mRNA expression and TF promoter activity. Binding of early growth response gene-1 (Egr-1) to the consensus DNA sequence, a key transcriptional activator of TF expression in response to inflammation, and the expression of Egr-1 mRNA were also inhibited by IL-10. This inhibition was independent of the induction of suppressor of cytokine signaling protein-3 by IL-10. Macrophages that had been transfected with luciferase reporter constructs containing the murine Egr-1 5'-flanking sequence exhibited reduced reporter gene activity in response to LPS stimulation with IL-10 pretreatment. Studies with deletion constructs of the Egr-1 promoter identified the proximal serum response element SRE3 as a potential regulatory site for the IL-10 mediated suppression of Egr-1 expression. Furthermore, activation of the upstream signal-transduction elements, such as mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase 1/2, and Elk-1 were also inhibited by IL-10 pretreatment. Taken together, these results demonstrate a pathway for the IL-10 mediated inhibition of TF expression during inflammation and may explain the antiatherosclerotic effects of IL-10.

Melagatran reduces advanced atherosclerotic lesion size and may promote plaque stability in apolipoprotein E-deficient mice.

OBJECTIVE: Inflammatory mechanisms are involved in atherosclerotic plaque rupture and subsequent thrombin formation. Thrombin not only plays a central role in thrombus formation and platelet activation, but also in the induction of inflammatory processes. We assessed the hypothesis that melagatran, a direct thrombin inhibitor, attenuates plaque progression and promotes stability of advanced atherosclerotic lesions. METHODS AND RESULTS: Melagatran (500 micromol/kg/d) or control diet was administered to apolipoprotein E-deficient mice (n=54) with advanced atherosclerotic lesions. Treatment reduced lesion progression in brachiocephalic arteries (P<0.005). Morphometric analysis confirmed that thrombin inhibition promoted plaque stability and resulted in thicker fibrous caps (28.4+/-14.2 microm versus 20.8+/-12.0 microm; P<0.05), increased media thickness (29.3+/-9.6 microm versus 24.4+/-6.7 microm; P<0.05), and smaller necrotic cores (73,537+/-41,301 microm2 versus 126,819+/-51,730 microm2; P<0.0005). Electro mobility shift assays revealed reduced binding activity of nuclear factor kappaB (P<0.05) and activator protein-1 (P<0.05) in aortas of treated mice. Furthermore, immunohistochemistry demonstrated reduced staining for matrix metalloproteinase (MMP)-9 (P<0.05). Melagatran had no significant effect on early lesion formation in C57BL/6J mice. CONCLUSIONS: The direct thrombin inhibitor melagatran reduces lesion size and may promote plaque stability in apolipoprotein E-deficient mice, possibly through reduced activation of proinflammatory transcription factors and reduced synthesis of MMP-9.

Induction of glutathione synthesis in macrophages by oxidized low-density lipoproteins is mediated by consensus antioxidant response elements.

The uptake of oxidized low-density lipoproteins (oxLDL) by macrophages leading to conversion into foam cells is a seminal event in atherogenesis. Excessive accumulation of oxLDL can cause oxidative stress in foam cells leading to cell death and the progression and destabilization of atherosclerotic lesions. Oxidative stress induces a protective compensatory increase in the synthesis of the endogenous antioxidant glutathione (GSH). Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in GSH synthesis and is composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM), which are products of separate genes. Treatment of RAW 264.7 mouse macrophages and mouse peritoneal macrophages with oxLDL (30 microg/mL) induces increased expression of both Gclc and Gclm in vitro. The increase in mRNA occurs in part via increased transcription as demonstrated with luciferase reporter constructs. The promoters for both GCLC and GCLM contain consensus antioxidant response elements (AREs). Electrophoretic mobility shift assays revealed induction of nuclear factor binding to these AREs after treatment of RAW 264.7 cells and mouse peritoneal macrophages with oxLDL. Nuclear factor binding to the AREs is diminished by a single base pair substitution in the core sequence. Site-directed mutagenesis of the AREs within the Gclc and Gclm promoters resulted in a decrease of oxLDL-induced luciferase activity. Supershift analyses revealed that oxLDL stimulates binding of the transcription factors Nrf1, Nrf2, and c-jun to the AREs. These data suggest that AREs play a direct role in mediating the induction of GSH synthesis by oxLDL and in protecting macrophages against oxidized lipid-induced oxidative stress.

Chlamydia pneumoniae induces tissue factor expression in mouse macrophages via activation of Egr-1 and the MEK-ERK1/2 pathway.

Recent studies have suggested that infection with Chlamydia pneumoniae (C pneumoniae) may contribute to the instability of atherosclerotic plaques and thrombosis and is associated with acute coronary events. Tissue factor (TF), a potent prothrombotic molecule, is expressed by macrophages and other cell types within atherosclerotic lesions and plays an essential role in thrombus formation after plaque rupture. Therefore the effects of C pneumoniae on induction of TF expression in macrophages were investigated. Infection of RAW mouse macrophages with C pneumoniae induced a time-dependent increase in procoagulant activity, expression of TF protein, and TF mRNA. C pneumoniae infection stimulated increased binding of nuclear proteins to the consensus DNA sequence for Egr-1, a key response element within the TF promoter, and increased the expression of Egr-1 protein. Transient transfections of RAW cells with mutated TF promoter constructs showed that the Egr-1 binding region is an important transcriptional regulator of C pneumoniae-induced TF expression. Furthermore, C pneumoniae-stimulated phosphorylation of ERK1/2 and Elk-1 and pharmacological inhibition of mitogen-activated protein kinase activity reduced the expression of TF and Egr-1. Antibody and polymyxin B blocking of the Toll-like receptor 4 (TLR4) partially reduced the C pneumoniae-induced expression of TF and Egr-1. In conclusion, the C pneumoniae-induced increase in TF expression in macrophages is mediated in part by Egr-1, signaling through TLR4, and activation of the MEK-ERK1/2 pathway.

Calcification of advanced atherosclerotic lesions in the innominate arteries of ApoE-deficient mice: potential role of chondrocyte-like cells.

OBJECTIVE: Advanced atherosclerotic lesions in the innominate arteries of chow-fed apolipoprotein E-deficient mice become highly calcified with 100% frequency by 75 weeks of age. The time course, cell types, and mechanism(s) associated with calcification were investigated. METHODS AND RESULTS: The deposition of hydroxyapatite is preceded by the formation of fibro-fatty nodules that are populated by cells that morphologically resemble chondrocytes. These cells are spatially associated with small deposits of hydroxyapatite in animals between 45 and 60 weeks of age. Immunocytochemical analyses with antibodies recognizing known chondrocyte proteins show that these cells express the same proteins as chondrocytes within developing bone. Histological and electron microscopic analyses of lesions from animals between 45 and 60 weeks of age show that the chondrocyte-like cells are surrounded by dense connective tissue that stains positive for type II collagen. Nanocrystals of hydroxyapatite can be seen within matrix vesicles derived from the chondrocyte-like cells. In mice between 75 and 104 weeks of age, the lesions have significantly reduced cellularity and contain large calcium deposits. The few remaining chondrocyte-like cells are located adjacent to or within the large areas of calcification. CONCLUSIONS: Calcification of advanced lesions in chow-fed apolipoprotein E-deficient mice occurs reproducibly in mice between 45 and 75 weeks of age. The deposition of hydroxyapatite is mediated by chondrocytes, which suggests that the mechanism of calcification may in part recapitulate the process of endochondral bone formation.

Ticagrelor promotes atherosclerotic plaque stability in a mouse model of advanced atherosclerosis.

OBJECTIVE: There is increasing evidence supporting the role of platelets in atherosclerotic vascular disease. The G-protein-coupled receptor P2Y12 is a central mediator of platelet activation and aggregation but has also been linked to platelet-independent vascular disease. Ticagrelor is an oral P2Y12 antagonist that is used as a standard treatment in patients after acute myocardial infarction. However, the effects of ticagrelor on advanced atherosclerosis have not been investigated. MATERIALS AND METHODS: Twenty-week-old apolipoprotein-E-deficient mice received standard chow or standard chow supplemented with 0.15% ticagrelor (approximately 270 mg/kg/day) for 25 weeks. The lesion area was evaluated in the aortic sinus by Movat's pentachrome staining and lesion composition, thickness of the fibrous cap, and size of the necrotic core evaluated by morphometry. RAW 264.7 macrophages were serum starved and treated with ticagrelor in vitro for the detection and quantification of apoptosis. In addition, oxLDL uptake in RAW 264.7 macrophages was evaluated. RESULTS: A trend toward the reduction of total lesion size was detected. However, data did not reach the levels of significance (control, n=11, 565,881 µm(2) [interquartile range {IQR} 454,778-603,925 µm(2)] versus ticagrelor, n=13, 462,595 µm(2) [IQR 379,740-546,037 µm(2)]; P=0.1). A significant reduction in the relative area of the necrotic core (control, n=11, 0.46 [IQR 0.4-0.51] versus ticagrelor, n=13, 0.34 [IQR 0.31-0.39]; P=0.008), and a significant increase in fibrous caps thickness (control, n=11, 3.7 µm [IQR 3.4-4.2 µm] versus ticagrelor, n=13, 4.7 [IQR 4.3-5.5 µm], P=0.04) were seen in ticagrelor-treated mice. In vitro studies demonstrated a reduction in apoptotic RAW 264.7 macrophages (control 0.07±0.03 versus ticagrelor 0.03±0.03; P=0.0002) when incubated with ticagrelor. Uptake of oxLDL in RAW 264.7 was significantly reduced when treated with ticagrelor (control 9.2 [IQR 5.3-12.9] versus ticagrelor 6.4 [IQR 2.5-9.5], P=0.02). CONCLUSION: The present study demonstrates for the first time a plaque-stabilizing effect of ticagrelor in a model of advanced vascular disease, potentially induced by a reduction of oxLDL uptake or an inhibition of apoptosis as seen in vitro.