Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice.

Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12(-/-) ApoE(-/-) ) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12(-/-) ApoE(-/-) or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12(+/+) ApoE(-/-) control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12(-/-) ApoE(-/-) . A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12(+/+) ApoE(-/-) mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12(-/-) ApoE(-/-) or anti-TWEAK treatment in TNFSF12(+/+) ApoE(-/-) mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish proinflamatory response associated with atherosclerotic plaque progression and to alter plaque morphology towards a stable phenotype.

HMGB1 expression and secretion are increased via TWEAK-Fn14 interaction in atherosclerotic plaques and cultured monocytes.

OBJECTIVE: High-mobility group box 1 (HMGB1), a DNA-binding cytokine expressed mainly by macrophages, contributes to lesion progression and chronic inflammation within atherosclerotic plaque. It has been suggested that different cytokines could regulate HMGB1 expression in monocytes. We have analyzed the effect of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) on HMGB1 expression both in vivo and in vitro. METHODS AND RESULTS: Expression of TWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) was positively correlated with HMGB1 in human carotid atherosclerotic plaques. TWEAK increased HMGB1 mRNA expression and protein secretion in human acute monocytic leukemia cell line cultured monocytes. TWEAK-mediated HMGB1 increase was only observed in M1 macrophages but not in M2 ones. These effects were reversed using blocking anti-Fn14 antibody or nuclear factor-kappa B and phosphotidylinositol-3 kinase inhibitors. TWEAK also increased monocyte chemoattractant protein-1 secretion in human acute monocytic leukemia cell line cells, an effect blocked with an HMGB1 small interfering RNA. Systemic TWEAK injection in ApoE(-/-) mice increased HMGB1 protein expression in the aortic root and mRNA expression in total aorta of ApoE(-/-) mice. Conversely, TWEAK-blocking antibodies diminished HMGB1 protein and mRNA expression compared with IgG-treated mice. CONCLUSIONS: Our results indicate that TWEAK can regulate expression and secretion of HMGB1 in monocytes/macrophages, participating in the inflammatory response associated with atherosclerotic plaque development.

Indomethacin Prevents the Progression of Thoracic Aortic Aneurysm in Marfan Syndrome Mice.

BACKGROUND: Marfan syndrome (MFS), an inherited disorder of connective tissue characterized by abnormalities in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The high mortality in untreated patients is primarily due to aneurysm and dissection of the ascending aorta. The complex pathogenesis of MFS involves changes in transforming growth factor ß (TGF-ß) signaling, increased matrix metalloproteinase (MMP) expression, and fragmentation of the extracellular matrix. A number of studies have demonstrated increased counts of macrophages and T cells in the ascending aorta of persons or mouse models of MFS, but the efficacy of anti-inflammatory therapy in mouse models of MFS has not yet been assessed. METHODS: FBN1 underexpressing mgR/mgR Marfan mice were treated with oral indomethacin. Treatment was begun at the age of three weeks and continued for 8 weeks, following which the aorta of wild type as well as treated and untreated mgR/mgR mice was compared. RESULTS: Indomethacin treatment led to a statistically significant reduction of aortic elastin degeneration and macrophage infiltration, as well as a lessening of MMP-2, MMP-9, and MMP-12 upregulation. Additionally, indomethacin decreased both cyclooxygenases 2 (COX-2) expression and activity in the aorta of mgR/mgR mice. COX-2-mediated inflammatory infiltrate contributes to the progression of aortic aneurysm in mgR/mgR mice, providing evidence that COX-2 is a relevant therapeutic target in MFS-associated aortic aneurysmal disease. CONCLUSIONS: COX-2 mediated inflammatory infiltration plays an important role in the pathogenesis of aortic aneurysm disease in MFS.

Antagonism of GxxPG fragments ameliorates manifestations of aortic disease in Marfan syndrome mice.

Marfan syndrome (MFS) is an inherited disorder of connective tissue caused by mutations in the gene for fibrillin-1 (FBN1). The complex pathogenesis of MFS involves changes in transforming growth factor beta (TGF-ß) signaling and increased matrix metalloproteinase (MMP) expression. Fibrillin-1 and elastin have repeated Gly-x-x- Pro-Gly (GxxPG) motifs that can induce a number of effects including macrophage chemotaxis and increased MMP activity by induction of signaling through the elastin-binding protein (EBP). In this work, we test the hypothesis that antagonism of GxxPG fragments can suppress disease progression in the Marfan aorta. Fibrillin-1 underexpressing mgR/mgR Marfan mice were treated with weekly intraperitoneal (i.p.) injections of an antibody directed against GxxPG fragments. The treatment was started at 3 weeks of age and continued for 8 weeks. The treatment significantly reduced MMP-2, MMP-9 and pSmad2 activity, as well as fragmentation and macrophage infiltration in the aorta of the mgR/mgR mice. Additionally, airspace enlargement and increased pSmad2 activity in the lungs of mgR/mgR animals were prevented by the treatment. Our findings demonstrate the important role of secondary cellular events caused by GxxPG-containing fragments and matrix-induced inflammatory activity in the pathogenesis of thoracic aortic aneurysm (TAA) in mgR/mgR mice. Moreover, the results of the current study suggest that antagonism of the effects of GxxPG fragments may be a fruitful therapeutic strategy in MFS.

HSP90 inhibition by 17-DMAG attenuates oxidative stress in experimental atherosclerosis.

AIMS: Reactive oxygen species (ROS) participate in atherogenesis through different mechanisms including oxidative stress and inflammation. Proteins implicated in both processes, such as mitogen-activated protein kinase kinase (MEK) and some NADPH oxidase (NOX) subunits, are heat shock protein-90 (HSP90) client proteins. In this work, we investigated the antioxidant properties of the HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) in experimental atherosclerosis. METHODS AND RESULTS: Treatment of ApoE(-/-) mice with 17-DMAG (2 mg/kg every 2 days for 10 weeks) decreased ROS levels and extracellular signal-regulated kinase (ERK) activation in aortic plaques compared with control animals. Accordingly, treatment of rat vascular smooth muscle cells (VSMCs) with 17-DMAG increased HSP27 and HSP70 and inhibited ERK activation. Interestingly, 17-DMAG diminished NADPH oxidase dependent ROS production in VSMCs and monocytes. In addition, a marked reduction in NADPH oxidase dependent ROS production was observed with HSP90siRNA and the opposite pattern with HSP70siRNA. 17-DMAG also diminished the expression of Nox1 and Nox organizer-1 (Noxo1) in VSMCs and monocytes. Interestingly, 17-DMAG was able to modulate ROS-induced monocyte to macrophage differentiation. Finally, higher expression of Nox1 and Noxo1 was found in the inflammatory region of human atherosclerotic plaques, colocalizing with VSMCs, macrophages, and ROS-producing cells. CONCLUSION: Our results suggest that HSP90 inhibitors interfere with oxidative stress and modulate experimental atherosclerosis development through reduction in pro-oxidative factors.

TWEAK-Fn14 interaction enhances plasminogen activator inhibitor 1 and tissue factor expression in atherosclerotic plaques and in cultured vascular smooth muscle cells.

AIMS: atherosclerotic plaque development can conclude with a thrombotic acute event triggered by plaque rupture/erosion. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumour necrosis factor superfamily that, through its receptor, fibroblast growth factor-inducible 14 (Fn14), participates in vascular remodelling, increasing vascular inflammatory responses and atherosclerotic lesion size in ApoE knockout mice. However, the role of the TWEAK-Fn14 axis in thrombosis has not been previously investigated. METHODS AND RESULTS: we have examined whether TWEAK regulates expression of prothrombotic factors such as tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) in atherosclerotic plaques as well as in human aortic vascular smooth muscle cells (hASMCs) in culture. Expression of TF and PAI-1 was colocalized and positively correlated with Fn14 in human carotid atherosclerotic plaques. In vitro, TWEAK increased TF and PAI-1 mRNA, protein expression and activity in hASMCs. All these effects were reversed using blocking anti-TWEAK monoclonal antibody, anti-Fn14 antibody or Fn14 small interfering RNA, indicating that TWEAK increased the prothrombotic state through its receptor, Fn14. Finally, ApoE(-/-) mice were fed a hyperlipidaemic diet for 10 weeks, then randomized and treated with saline (controls), TWEAK (10 microg/kg/day), anti-TWEAK neutralizing monoclonal antibody (1000 µg/kg/day), or non-specific immunoglobulin G (1000 microg/kg/day) daily for 9 days. Systemic TWEAK injection increased TF and PAI-1 protein expression in the aortic root of ApoE(-/-) mice. Conversely, TWEAK blocking antibodies diminished both TF and PAI-1 protein expression compared with non-specific immunoglobulin G-treated mice. CONCLUSIONS: our results indicate that the TWEAK-Fn14 axis can regulate activation of TF and PAI-1 expression in vascular cells. TWEAK-Fn14 may be a therapeutic target in the prothrombotic complications associated with atherosclerosis.

TWEAK soluble, un nuevo biomarcador de riesgo cardiovascular / Soluble TWEAK, a new biomarker of cardiovascular risk

Introducción y objetivo La valoración del riesgo vascular en pacientes asintomáticos y la respuesta a la intervención terapéutica es uno de los mayores objetivos para la prevención de eventos cardiovasculares. Nuestro objetivo fue evaluar si una nueva proteína descrita de la superfamilia del factor de necrosis tumoral, TWEAK, se secreta diferencialmente por la pared vascular sana en comparación con la lesión aterosclerótica humana, y si las concentraciones plasmáticas de esta proteína pueden servir como biomarcador de aterosclerosis. Material y métodos Mediante técnica de radioinmunoanálisis analizamos el sobrenadante procedente del cultivo de placas carotídeas y de arterias sanas humanas. Además, analizamos la concentración plasmática de TWEAK soluble (sTWEAK) en pacientes con aterosclerosis carotídea (n=30), pacientes que presentaban disfunción endotelial (n=200) y sujetos asintomáticos (n=106), y se compararon con sujetos sanos (n=83). Resultados y conclusión Los niveles de sTWEAK se encontraban disminuidos en el sobrenadante procedente de placas carotídeas comparadas con arterias sanas. Posteriormente, el análisis de la concentración plasmática de sTWEAK mostró que sujetos con estenosis carotídea tenían niveles reducidos en comparación con sujetos sanos. Además, los niveles plasmáticos de sTWEAK se encontraron disminuidos en sujetos asintomáticos en los cuales se midió el espesor íntima-media (un índice de aterosclerosis subclínica) y en sujetos en los cuales se había medido su función endotelial. Estos resultados sugieren que sTWEAK puede ser un nuevo marcador potencial de aterosclerosis subclínica (AU)

Introduction and objective Assessment of vascular risk in asymptomatic patients and the response to medical therapy is a major challenge for prevention of cardiovascular events. Our aim was to investigate if a novel protein of TNF superfamliy, TWEAK, is differentially released by healthy versus atherosclerotic arterial walls. Moreover, we have analysed whether soluble TWEAK (sTWEAK), which could be found in plasma and serve as biomarker of atherosclerosis. Material and methods Supernatants obtained from cultured human carotid plaques and healthy arteries were analysed by ELISA. In addition, sTWEAK plasma levels were measured in subjects with carotid atherosclerosis (N=30), patients with endothelial dysfunction (N=200) and asymptomatic patients (N=106) and compared with healthy subjects (N=28). Results and conclusions TWEAK concentrations were decreased in supernatants from carotid atherosclerotic plaques compared with healthy arteries. Furthermore, measurement of sTWEAK in plasma showed a reduced concentration in subjects with carotid stenosis compared with healthy subjects. Moreover, sTWEAK concentrations were diminished in asymptomatic subjects in whom intima-media thickness was measured and in subjects with endothelial dysfunction.Results and conclusion These results suggest that sTWEAK could be a potential biomarker of atherosclerosis (AU)

Heat shock protein 90 inhibitors attenuate inflammatory responses in atherosclerosis.

AIMS: Heat shock protein 90 (HSP90) is a ubiquitous chaperone involved in the folding, activation, and assembly of many proteins. HSP90 inhibitors [17-allylamino-17-demethoxygeldamycin (17-AAG)/17-dimethyl aminothylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG)] bind to and inactivate HSP90, increasing the heat shock response and suppressing different signalling pathways. We aim to investigate the effect of HSP90 inhibitors in the modulation of inflammatory responses during atherogenesis. METHODS AND RESULTS: In human atherosclerotic plaques, HSP90 immunostaining was increased in inflammatory regions and in plaques characterized by lower cap thickness. In cultured human macrophages and vascular smooth muscle cells, treatment with either 17-AAG or 17-DMAG increased HSP70 expression and reduced transcription factor [signal transducers and activators of transcription (STAT) and nuclear factor-kappaB (NF-kappaB)] activation and chemokine expression induced by proinflammatory cytokines. In vivo, hyperlipidaemic ApoE(-/-) mice were randomized to 17-DMAG (2 mg/kg every 2 days, n = 11) or vehicle injected (n = 9) during 10 weeks. Atherosclerotic plaques of mice treated with 17-DMAG displayed increased HSP70 expression and diminished NF-kappaB and STAT activation, along with decreased lesion, lipid, and macrophage content, compared with vehicle-injected mice. In addition, treatment with 17-DMAG significantly reduced monocyte chemoattractant protein-1 levels, both in plaques and in plasma. CONCLUSION: HSP90 expression is associated with features of plaque instability in advanced human lesions. HSP90 inhibitors reduce inflammatory responses in atherosclerosis, suggesting that HSP90 could be a novel therapeutic target in atherosclerosis.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) enhances vascular and renal damage induced by hyperlipidemic diet in ApoE-knockout mice.

OBJECTIVE: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily of cytokines. TWEAK binds and activates the Fn14 receptor, and may regulate apoptosis, inflammation, and angiogenesis, in different pathological conditions. We have evaluated the effect of exogenous TWEAK administration as well as the role of endogenous TWEAK on proinflammatory cytokine expression and vascular and renal injury severity in hyperlipidemic ApoE-knockout mice. METHODS AND RESULTS: ApoE(-/-) mice were fed with hyperlipidemic diet for 4 to 10 weeks, then randomized and treated with saline (controls), TWEAK (10 microg/kg/d), anti-TWEAK neutralizing mAb (1000 microg/kg/d), TWEAK plus anti-TWEAK antibody (10 microg TWEAK +1000 microg anti-TWEAK/kg/d), or nonspecific IgG (1000 microg/kg/d) daily for 9 days. In ApoE(-/-) mice, exogenous TWEAK administration in ApoE(-/-) mice induced activation of NF-kappaB, a key transcription factor implicated in the regulation of the inflammatory response, in vascular and renal lesions. Furthermore, TWEAK treatment increased chemokine expression (RANTES and MCP-1), as well as macrophage infiltration in atherosclerotic plaques and renal lesions. These effects were associated with exacerbation of vascular and renal damage. Conversely, treatment of ApoE(-/-) mice with an anti-TWEAK blocking mAb decreased NF-kappaB activation, proinflammatory cytokine expression, macrophage infiltration, and vascular and renal injury severity, indicating a pathological role for endogenous TWEAK. Finally, in murine vascular smooth muscle cells or tubular cells, either ox-LDL or TWEAK treatment increased expression and secretion of both RANTES and MCP-1. Furthermore, ox-LDL and TWEAK synergized for induction of MCP-1 and RANTES expression and secretion. CONCLUSIONS: Our results suggest that TWEAK exacerbates the inflammatory response associated with a high lipid-rich diet. TWEAK may be a novel therapeutic target to prevent vascular and renal damage associated with hyperlipidemia.

Las proteínas de choque térmico (heat shock proteins) como potenciales dianas terapéuticas en aterosclerosis / Heat shock proteins as potential therapeutic targets in atherosclerosis

Las heat shock proteins (HSP) son una familia de proteínas que se producen en grandes cantidades por la mayoría de las células como respuesta al estrés. Las HSP se localizan tanto en arterias sanas, como en placas ateroscleróticas, aunque su papel en la aterosclerosis está aún por definir. En el presente estudio, nuestro objetivo fue analizar el efecto de la modulación de las HSP en procesos involucrados en la formación de la placa de ateroma, como el estrés oxidativo, la inflamación y la apoptosis. Para ello, hemos usado un inhibidor de la HSP90 (17-AAG), el cual es capaz de inducir un aumento en los valores de expresión de la HSP70/HSP27. Inicialmente, hemos observado que el tratamiento con 17-AAG es capaz de reducir la actividad de la NAD(P)H oxidasa inducida por factor de necrosis tumoral alfa. Asimismo, el tratamiento moduló distintas vías de señalización proliferativas (p. ej., MAPK), así como la activación del factor nuclear kappa B (NF-κB) inducidas por un cócktail de citocinas (interferón gamma/interleucina 6 [IL-6]). En estas condiciones, se observó una disminución en los niveles de citocinas proinflamatorias (p. ej., IL-6). Por último, mientras el tratamiento con 17-AAG provocó una disminución en la apoptosis después de la incubación con diversos estímulos proapoptóticos, la inhibición de la HSP27 mediante transfección con un siARN específico aumentó la apoptosis celular inducida por elastasa. Estos resultados indican que la modulación de los valores de ciertas HSP puede representar una nueva aproximación terapéutica en el tratamiento de enfermedades inflamatorio-proliferativas como la aterosclerosis (AU)

Heat shock proteins (HSP) are a family of proteins present in the majority of cells and are produced as a cell protection mechanism against adverse conditions. HSP are present in both atherosclerotic plaques and healthy arteries, although their role in atherosclerosis remain to be defined. Our objective was to investigate the effect of HSP90 inhibitors in the modulation of the different processes involved in atherogenesis. We have used an HSP90 inhibitor (17-AAG), which is able to increase the heat shock response. Treatment of monocytes and vascular smooth muscle cells with 17-AAG increased HSP70 expression. Under these conditions, 17-AAG was able to decrease NADPH oxidase activity induced by TNF-α. Stimulation of cells with cytokines induced the activation of different signalling pathways (e.g. MAPKs and NF-κB) and increased the levels of cytokines such as IL-6, which was diminished by HSP90 inhibitors. Finally, while treatment with 17-AAG prevented the apoptosis of cells induced proapoptotic stimuli, HSP27 silencing increased apoptosis induced by elastase. HSP90 inhibitors decrease pro-atherogenic processes, suggesting that these drugs may have beneficial effects in the treatment of atherosclerosis (AU)

Biomarkers in cardiovascular medicine.

Cardiovascular disease is the principal cause of death in developed countries. The underlying pathological process is arterial wall thickening due to the formation of atherosclerotic plaque, which is frequently complicated by thrombus, thereby giving rise to the possibility of acute coronary syndrome or stroke. One of the major challenges in cardiovascular medicine is to find a way of predicting the risk that an individual will suffer an acute thrombotic event. During the last few decades, there has been considerable interest in finding diagnostic and prognostic biomarkers that can be detected in blood. Of these, C-reactive protein is the best known. Others, such as the soluble CD40 ligand, can be used to predict cardiovascular events. However, to date, no biomarker has been generally accepted for use in clinical practice. At present, there are a number of high-performance techniques, such as proteomics, that have the ability to detect multiple potential biomarkers. In the near future, these approaches may lead to the discovery of new biomarkers that, when used with imaging techniques, could help improve our ability to predict the occurrence of acute vascular events.

Considering TWEAK as a target for therapy in renal and vascular injury.

TWEAK is a cytokine of the TNF superfamily that activates the Fn14 receptor. TWEAK may regulate cell proliferation, cell death, cell differentiation, angiogenesis and inflammation. The expression of TWEAK and Fn14 is increased during vascular and renal injury. Inflammatory cytokines increase Fn14 receptor expression in tubular and vascular smooth muscle cells. Moreover, TWEAK induces tubular cell apoptosis under proinflammatory conditions. TWEAK itself contributes to renal and vascular inflammation by promoting chemokine and inflammatory cytokine secretion. Confirmation of its role in acute kidney injury and atherosclerotic lesions formation came from functional studies in experimental animal models. The available evidence suggests that TWEAK might be a target for therapeutic intervention in renal and vascular injury and its role in different forms of tissue damage should be further explored.

Biomarcadores en la medicina cardiovascular / Biomarkers in Cardiovascular Medicine

Las enfermedades cardiovasculares son la primera causa de muerte en el mundo occidental. El proceso patológico que subyace a ellas es un engrosamiento de la pared arterial debido a la formación de placas ateroscleróticas, las cuales se complican frecuentemente con un trombo y pueden dar lugar a síndrome coronario agudo o accidente cerebrovascular. Uno de los mayores retos de la medicina cardiovascular es encontrar la manera de predecir el riesgo de un sujeto de sufrir un evento trombótico agudo. En las últimas décadas, hay un gran interés en la búsqueda de biomarcadores diagnósticos y pronósticos que puedan ser identificados en sangre. Entre ellos, la proteína C reactiva es la más conocida. Otros, como el ligando de CD40 soluble, pueden predecir eventos cardiovasculares. En cambio, hasta el momento no hay un biomarcador aceptado en la práctica clínica. Actualmente, existen diversas técnicas de alto rendimiento como la proteómica, que permite la detección de múltiples biomarcadores potenciales. Estas aproximaciones pueden identificar en un futuro próximo nuevos biomarcadores que, junto con las técnicas de imagen, pueden ayudar a mejorar la predicción de eventos vasculares agudos (AU)

Cardiovascular disease is the principal cause of death in developed countries. The underlying pathological process is arterial wall thickening due to the formation of atherosclerotic plaque, which is frequently complicated by thrombus, thereby giving rise to the possibility of acute coronary syndrome or stroke. One of the major challenges in cardiovascular medicine is to find a way of predicting the risk that an individual will suffer an acute thrombotic event. During the last few decades, there has been considerable interest in finding diagnostic and prognostic biomarkers that can be detected in blood. Of these, C-reactive (..) (AU)

The CD163-expressing macrophages recognize and internalize TWEAK: potential consequences in atherosclerosis.

BACKGROUND: CD163 is a new potential scavenger receptor of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) which elicits diverse biologic actions involved in atherosclerosis. We have analyzed the importance of TWEAK-CD163 interaction in atherosclerosis. METHODS: TWEAK and CD163 interaction was studied in cultured human macrophages. Moreover, TWEAK and CD163 expression was analyzed in carotid atherosclerotic plaques (immunohistochemistry) and plasma (ELISA). We have also assessed their potential association with intima/media thickness (IMT) in asymptomatic subjects. RESULTS: In vitro studies revealed that CD163-expressing macrophages can bind and internalize TWEAK protein exogenously added from supernatants. Accordingly, we observed an inverse correlation between the expression of CD163 and TWEAK (r=-0.51; p=0.008) in the shoulder region of atherosclerotic plaques obtained from 25 patients undergoing carotid endarterectomy. The same trend was observed when we analyzed the plasma concentration of both proteins in 90 subjects free from clinical cardiovascular disease (r=-0.25; p=0.016) in which carotid ultrasonography was performed to determine IMT. In these subjects, we found a positive correlation between sCD163 and IMT (r=0.36; p<0.001) and between sCD163-sTWEAK ratio and IMT (r=0.51; p<0.001). This association remained significant after adjusting for traditional cardiovascular risk factors and inflammatory markers explaining 39% (sCD163) or 48% (sCD163-sTWEAK ratio) of IMT variance. CONCLUSIONS: Our results suggest that TWEAK-CD163 interaction takes place in vivo, probably decreasing TWEAK plasma concentration. Furthermore, we have observed that CD163-TWEAK plasma ratio is a potential biomarker of clinical and subclinical atherosclerosis.

Increased CD74 expression in human atherosclerotic plaques: contribution to inflammatory responses in vascular cells.

AIMS: The purpose of this study was to analyse the expression of CD74 in human atherosclerotic plaques and peripheral blood mononuclear cells (PBMC) as well as its potential participation in proinflammatory responses in cultured human vascular smooth muscle cells (VSMC). METHODS AND RESULTS: CD74 expression was analysed in human atherosclerotic plaques (immunohistochemistry), PBMC (real-time PCR), and human aortic VSMC (real-time PCR and western blotting). Nuclear factor-kappaB (NF-kappaB) activation was assessed by southwestern histochemistry and electrophoretic mobility shift assay. Monocyte chemoattractant protein-1 (MCP-1) levels were studied by both real-time PCR and enzyme-linked immunosorbent assay. CD74 immunostaining was increased in the inflammatory vs. the fibrous region of atherosclerotic plaques (n = 70, 18.2 +/- 1.3 vs. 7.8 +/- 0.6% positive staining/mm2, P < 0.001). CD74 colocalized with the transcription factor NF-kappaB in both VSMC and macrophages. In cultured VSMC, CD74 expression was induced by interferon gamma (IFNgamma). Incubation with an agonistic anti-CD74 antibody or with IFNgamma elicited MCP-1 expression, which was prevented by AKT and gamma-secretase inhibitors. Moreover, CD74 small-interfering RNA decreased NF-kappaB activation and MCP-1 production induced by IFNgamma in VSMC. Finally, CD74 mRNA levels in PBMC from patients with carotid stenosis were higher than in healthy subjects (n = 20, 3 +/- 0.5 vs. 2 +/- 0.5 AU, P < 0.001). Additionally, a linear trend between CD74 mRNA expression tertiles and intima-media thickness (IMT) was observed in PBMC from asymptomatic subjects (n = 185, P < 0.001). CONCLUSION: CD74 levels are increased in plaques and PBMC from patients with carotid stenosis and are associated with IMT in subjects free from clinical cardiovascular diseases. CD74 could be a novel therapeutic target to decrease the inflammatory response in atherosclerosis.

Suppressors of cytokine signaling modulate JAK/STAT-mediated cell responses during atherosclerosis.

OBJECTIVE: Suppressors of cytokine signaling (SOCS) proteins are intracellular regulators of receptor signal transduction, mainly Janus kinase/signal transducers and activators of transcription (JAK/STAT). We investigated the effects of SOCS modulation on the JAK/STAT-dependent responses in vascular cells, and their implication in atherosclerotic plaque development. METHODS AND RESULTS: Immunohistochemistry in human plaques revealed a high expression of SOCS1 and SOCS3 by vascular smooth muscle cells (VSMCs) and macrophages in the inflammatory region of the shoulders, when compared to the fibrous area. SOCS were also increased in aortic lesions from apoE(-/-) mice. In cultured VSMCs, endothelial cells, and monocytes, SOCS1 and SOCS3 were transiently induced by proinflammatory cytokines, proatherogenic lipoproteins, and immune molecules. Furthermore, overexpression of SOCS suppressed STAT activation and reduced inflammatory gene expression and cell growth, whereas SOCS knockdown increased these cell responses. In vivo, antisense oligodeoxynucleotides targeting SOCS3 exacerbated the atherosclerotic process in apoE(-/-) mice by increasing the size, leukocyte content, and chemokine expression in the lesions. CONCLUSIONS: SOCS expressed in atherosclerotic lesions are key regulators of vascular cell responses. Activation of this endogenous antiinflammatory pathway might be of interest in the treatment of atherosclerosis.

Leukotriene B4 enhances the activity of nuclear factor-kappaB pathway through BLT1 and BLT2 receptors in atherosclerosis.

AIMS: Leukotriene B4 (LTB4) is a powerful chemoattractant and pro-inflammatory mediator in several inflammatory diseases, including atherosclerosis. It acts through its two membrane receptors, BLT1 and BLT2. The aim of this study was to determine the molecular mechanism involved in the proatherogenic effect of LTB4, BLT1 and BLT2 in atherosclerosis. Moreover, we characterized the expression of 5-lipoxygenase (5-LO) pathway and LTB4 receptors in blood and plaques from patients with carotid atherosclerosis. METHODS AND RESULTS: In cultured monocytic cells, LTB4 induced a rapid phosphorylation of mitogen-activated protein kinases (MAPKs ERK1/2 and JNK1/2) and PI3K/Akt via BLT1 and BLT2 in a pertussis toxin (PTX)-dependent mechanism (assessed via western blotting) and also increased nuclear factor-kappaB (NF-kappaB) DNA binding activity (assessed via EMSA) in a MAPK- and reactive oxygen species-dependent mechanism. Furthermore, LTB4 elicited interleukin-6, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha mRNA overexpression also via BLT1 and BLT2 by a PTX- and NF-kB-dependent mechanism (assessed by real-time PCR), promoting an inflammatory environment. When compared with healthy subjects, patients with carotid atherosclerosis showed a significant increase in the expression of all the components of the 5-LO pathway and BLT1 and BLT2 mRNA (real-time PCR) in peripheral blood mononuclear cells and LTB4 plasma levels (ELISA). In these patients, an overexpression of 5-LO, leukotriene A-4 hydroxylase (LTA4-H) and BLT1 was noted in the inflammatory region of carotid plaques when compared with the fibrous cap (assessed by immunohistochemistry). CONCLUSION: The 5-LO pathway is enhanced in patients with carotid atherosclerosis. Furthermore, its product LTB4 phosphorylates MAPKs and stimulates NF-kappaB-dependent inflammation via BLT1 and BLT2 receptors in cultured monocytic cells. The blockade of this pathway could be a novel and potential therapeutic target in atherothrombosis.

Treatment with amlodipine and atorvastatin has additive effect on blood and plaque inflammation in hypertensive patients with carotid atherosclerosis.

Since previous studies have reported a beneficial effect of amlodipine and atorvastatin treatment in experimental atherosclerosis, we aimed to investigate the effect of the combination of both drugs on blood and plaque inflammation in patients with carotid stenosis. For that purpose, twenty six hypertensive patients undergoing carotid endarterectomy were randomized to receive either atorvastatin 20 mg/day alone (ATV, n=12) or in combination with amlodipine 20 mg/day (ATV+AML, n=14) before scheduled carotid endarterectomy. At the end of follow-up (4-6 weeks), there was a significant decrease in total and LDL-cholesterol levels, but not in blood pressure levels. In contrast, decreased MCP-1 plasma levels, NF-kappaB activation (EMSA) and MCP-1 mRNA expression (quantitative PCR) was only observed in blood from ATV+AML treated-patients. Moreover, carotid atherosclerotic plaques from ATV+AML group demonstrated a significant reduction in macrophage infiltration in relation to ATV group (immunohistochemistry). Our results suggest that combined treatment with atorvastatin and amlodipine decreases inflammatory status of atherosclerotic patients more than atorvastatin treatment alone, suggesting that co-administration of both drugs could have beneficial additive effects.

TWEAK and Fn14. New players in the pathogenesis of atherosclerosis.

Atherosclerosis is currently described as an inflammatory disease given that the main components of chronic inflammation are present in this process: cell recruitment, proliferation, neovascularization, and sclerosis. Vascular lesions are caused by inflammatory and fibroproliferative responses to injury of the endothelium and vascular smooth muscle cells. Interaction between members of the tumor necrosis factor (TNF) superfamily and their receptors elicits diverse biologic actions that participate in atherosclerosis development. These responses include the expression of adhesion molecules, proinflammatory cytokines, matrix metalloproteinases, and tissue factor, which are known to increase plaque instability. TNF-like weak inducer of apoptosis (TWEAK) is a recently described member of the TNF superfamiliy, which is involved in induction of inflammation, activation of cell growth, and stimulation of apoptosis. In this review, we summarize the potential proatherogenic consequences of the interaction of TWEAK with its receptor Fn14 in the vascular wall.

Trail and vascular injury.

Cardiovascular diseases are the leading cause of mortality in the Western world. The underlying pathological process is a thickening of the arterial wall due to the formation of atheromatous plaques which contain a lipid core covered by a fibrous cap. The main mechanisms involved in atherogenesis are: lipoprotein retention, endothelial cell activation, vascular smooth muscle cell proliferation, macrophage infiltration, proteolytic injury, neovascularization and apoptosis. Different members of the tumor necrosis factor family (TNF) of proteins have been detected in human atherosclerotic plaques, among these are TNF-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-Rs and osteoprotegerin, OPG). In this review, the involvement of TRAIL and its receptors in the mechanisms underlying atherothrombosis is reviewed. In this respect, there are still some controversial data on the effects of TRAIL on inflammation and apoptosis of vascular cells. However, recent in vivo studies have suggested a potential proinflammatory and proapoptotic role of TRAIL in vascular injury. In addition, soluble forms of the TNF-superfamily can be released extracellularly and have been detected in human plasma. For this reason, we different studies evaluating the potential use of TRAIL and OPG plasma levels as markers of vascular injury are discussed.