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Aims: To study the role of lipocalin-2 (Lcn2) and the effect of Lcn2 blockade via anti-Lcn2 antibody in the development of abdominal aortic aneurysm (AAA). Methods and results: Expression mRNA and protein levels of Lcn2 and its human orthologue neutrophil gelatinase-associated lipocalin (NGAL) in aortic wall samples from experimental mouse and human AAA samples, respectively, were analysed by real-time PCR and immunohistochemistry. Experimental AAA was induced by aortic elastase perfusion in wild-type mice (WT) and Lcn2-deficient mice (Lcn2-/-). NGAL/Lcn2 mRNA and protein levels in human and murine AAA samples were increased compared with healthy aortas. Decreased AAA incidence and reduced aortic expansion were observed in Lcn2-/- mice or mice preoperative treated with a polyclonal anti-Lcn2 antibody compared with WT mice or mice treated with control IgG, respectively, at Day 14 after elastase perfusion. Moreover, immunohistochemical analysis of AAA tissues from Lcn2-/- or anti-Lcn2-treated mice showed diminished elastin damage, reduced microvessels and polymorphonuclear neutrophil (PMN) infiltration, and enhanced preservation of vascular smooth muscle cells compared with WT aortas. Fluorescent molecular tomography revealed decreased MMP activity in AAA of Lcn2-/- mice compared with WT controls. Therapeutic administration of anti-Lcn2 antibody to WT mice 3 days after elastase perfusion decreased aortic dilatation and PMN infiltration compared with WT mice treated with control IgG. Conclusion: Either Lcn2 deficiency or anti-Lcn2 antibody blockade limits AAA expansion in mice by decreasing PMN infiltration in the aorta. Lcn2 modulation may therefore be a viable new therapeutic option for the treatment of AAA. [ABSTRACT FROM AUTHOR]

Aim: The interaction between TNF-like weak inducer of apoptosis (TWEAK, Tnfsf12) and the receptor, fibroblast growth factor-inducible 14 (Fn14), regulates vascular damage through different mechanisms, including inflammation. Oxidative stress plays a major role in inflammation and the development of atherosclerosis, but the relationship between TWEAK and oxidative stress is, however, poorly understood. Methods and results: In this study, we found that TWEAK and Fn14 are co-localized with the NADPH subunits, p22phox and Nox2, in human advanced atherosclerotic plaques. Using primary human macrophages and a murine macrophage cell line, we demonstrate that TWEAK promotes ROS production and enhances NADPH oxidase activity. Hence, we show a direct involvement of the TWEAK-Fn14 axis in oxidative stress, as genetic silencing of Fn14 or Nox2 abrogates the TWEAKinduced ROS production. Furthermore, our results point at Rac1 as an upstream mediator of TWEAK during oxidative stress. Finally, using an in vivo murine model we confirmed the major role of TWEAK in oxidative stress, as genetic silencing of Tnfsf12 in an ApoE-/- background reduces the number of DHE and 8-hydroxydeoxyguanosine-positive macrophages by 50%. Conclusions Our results suggest that TWEAK regulates vascular damage by stimulating ROS production in an Nox2-dependent manner. These new insights into the TWEAK/Fn14 axis underline their potential use as therapeutic targets in atherosclerosis. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Complemento II-CM La Caixa Aim Previous human and experimental studies have suggested a role of complement activation in abdominal aortic aneurysm (AAA).  We recently described an increase in complement C5 levels in plasma of subjects with subclinical atherosclerosis (1). Our aim was to investigate the potential association of C5 with AAA presence and progression. Material and methods Tissue and tissue-conditioned media from AAA (thrombus and wall) or healthy samples were obtained from surgical repair or brain-deceased organ donors, respectively. Blood samples from 490 AAA patients (maximal aortic diameter ≥30 mm) were obtained within a population-based ultrasound-screening trial in Danish men and from 176 age-matched screened negative controls. C5 protein levels were assessed by immunohistochemistry and ELISA. Results High C5 positive immunostaining was observed in AAA thrombus and media, while the staining in healthy arteries was faint. C5 levels were higher in tissue-conditioned media from AAA thrombus and AAA media as compared to healthy arteries (7.1±1 vs 2.9 ±0.5 vs 1.2±0.2ug/ml, p<0.0001 for both). C5 plasma levels in AAA patients were higher than in controls (116±8 vs 61±7 ug/ml, P<0.001). AAA patients at the upper tertile of C5 at baseline had a 25% higher risk of needing surgical repair during the follow-up (Hazard Ratio=1.25, 95% confidence interval, 1.045;1.511, P<0.05). Conclusions C5 is associated to AAA presence and progression suggesting its potential use as a prognostic marker. Future studies are needed to clarify the pathogenic role of C5 in AAA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 µg/kg/day), anti-TWEAK neutralizing monoclonal antibody (1000 µg/kg/day), or non-specific immunoglobulin G (1000 µg/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. [ABSTRACT FROM AUTHOR]

Macroscopic haematuria of glomerular origin has been associated with acute kidney injury. We report a patient with IgA nephropathy, macroscopic haematuria and acute kidney injury. Systemic anticoagulation may have aggravated haematuria. There was extensive interstitial and intratubular red blood cell extravasation, and interstitial haemosiderin deposits. The abundant presence of macrophages expressing the haemoglobin scavenger receptor CD163 and of cells stained for oxidative stress markers (NADPH-p22 phox and heme-oxigenase-1) in areas of interstitial haemorrhage and red blood cell cast-containing tubules provided evidence for a role for free haemoglobin in tubulointerstitial renal injury in human glomerular disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

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-κB (NF-κB) 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-α 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-κB-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. [ABSTRACT FROM AUTHOR]

Abstract: Atherosclerotic plaque rupture triggers the onset of cardiovascular complications such as myocardial infarction and stroke, which represent the main cause of death in western countries. Atherogenesis is a complex process characterized by lipid retention, proteolytic injury and a chronic inflammatory response. The resulting pathological vascular remodeling involves inflammatory cell recruitment, fibrosis, smooth muscle cell proliferation, neovascularization and intraplaque hemorrhage. However, the cellular and molecular mechanisms underlying cardiovascular dysfunction remain widely unknown. The development of differential proteomics allows the identification of novel proteins whose association with the genesis of atherosclerotic plaques is at present unforeseen in the light of available data. Moreover, different strategies have been used to discover new potential biomarkers which could be related to cardiovascular risk. The multi-factorial nature of cardiovascular diseases necessitates the use of biomarkers for early detection, for monitoring the response to therapy and to predict clinical outcome. In this review, we summarize the different proteomic approaches and recent findings that will help us to understand the mechanisms implicated in the pathogenesis of atherothrombosis. [Copyright &y& Elsevier]

Objective: To study whether simvastatin reduces inflammation in atherosclerosis beyond its hypolipidemic effects. Methods: Twenty-four rabbits with induced femoral injury and on an atherogenic diet were randomized to normolipidemic diet (n=9), or to continue the atherogenic diet while receiving simvastatin 5 mg/kg/day (n=9) or no treatment (n=6) for 4 weeks. Results: As compared with no treatment, the normolipidemic diet significantly reduced lipid levels, while simvastatin produced nonsignificant reductions. In spite of this, NF-κB binding activity in peripheral mononuclear cells was reduced in the simvastatin group [2,958±5,123 arbitrary units (a.u.)] as compared with no treatment (49,267±20,084 a.u.; P<0.05) and normolipidemic groups (41,492±15,876 a.u.; P<0.05) (electrophoretic mobility shift assay). NF-κB activity in the atherosclerotic lesions was also reduced by simvastatin as compared to nontreated animals (4,108±3,264 vs. 8,696±2,305 nuclei/mm2; P<0.05), while the normolipidemic diet induced only a nonsignificant diminution (P>0.05) (Southwestern histochemistry). Similarly, simvastatin decreased macrophage infiltration (4.6±12 vs. 19±12% of area staining positive; P<0.05) and the expression of interleukin-8 (24±12 vs. 63±21%; P<0.05) and metalloproteinase-3 (16±3 vs. 42±28%; P<0.05) (immunohistochemistry), while the reduction achieved by normolipidemic diet in all these parameters was again nonsignificant (P>0.05). Conclusions: These findings suggest that simvastatin reduces inflammation in atherosclerotic plaques and in blood mononuclear cells more than expected for the lipid reduction achieved. [Copyright &y& Elsevier]

: 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-κB (NF-κB) 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-κB in both VSMC and macrophages. In cultured VSMC, CD74 expression was induced by interferon γ (IFNγ). Incubation with an agonistic anti-CD74 antibody or with IFNγ elicited MCP-1 expression, which was prevented by AKT and γ-secretase inhibitors. Moreover, CD74 small-interfering RNA decreased NF-κB activation and MCP-1 production induced by IFNγ 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. [ABSTRACT FROM AUTHOR]