The TNF-like weak inducer of the apoptosis/fibroblast growth factor-inducible molecule 14 axis mediates histamine and platelet-activating factor-induced subcutaneous vascular leakage and anaphylactic shock.

BACKGROUND: Anaphylaxis includes mast cell (MC) activation, but less is known about downstream mechanisms (ie, vascular permeability controlled by endothelial cells [ECs]). The TNF-like weak inducer of apoptosis (TWEAK) and its sole receptor, fibroblast growth factor-inducible molecule 14 (Fn14), belong to the TNF superfamily and are involved in proinflammatory responses. OBJECTIVE: We sought to investigate the role of TWEAK/Fn14 axis in anaphylaxis. METHODS: In vivo vascular permeability and mouse models of passive systemic anaphylaxis (PSA) and active systemic anaphylaxis were applied to wild-type (WT), TWEAK- and Fn14-deficient mice (TWEAK-/- and Fn14-/-, respectively). Primary bone marrow-derived mast cells (BMMCs) and ECs from WT and Fn14-/- or TWEAK-/- mice were studied. The TWEAK/Fn14 axis was also investigated in human samples. RESULTS: Mice with PSA and active systemic anaphylaxis had increased Fn14 and TWEAK expression in lung tissues and increased serum soluble TWEAK concentrations. TWEAK and Fn14 deficiencies prevent PSA-related symptoms, resulting in resistance to decreased body temperature, less severe reactions, and maintained physical activity. Numbers of MCs after PSA are similar between genotypes in different tissue regions, such as ear skin and the trachea, tongue, peritoneum, lungs, and bone marrow. Moreover, in vitro studies revealed no differences in degranulation or mediator release between WT and Fn14-/- BMMCs after IgE-FcεRI stimulation. In vivo and in vitro histamine and platelet-activating factor administration increases Fn14 receptor expression in lungs and ECs. Moreover, Fn14 deficiency in ECs maintained in vitro impermeability when stimulated by mediators or activated BMMCs but not by TWEAK-/- BMMCs, indicating that Fn14 is crucial for endothelial barrier function. TWEAK/Fn14 deletion or TWEAK-blocking antibody prevented histamine/platelet-activating factor-induced vascular subcutaneous permeability. Circulating soluble TWEAK levels were increased in patients with anaphylaxis, and plasma from those patients increased Fn14 expression in ECs. CONCLUSION: The TWEAK/Fn14 axis participates in anaphylactic reactions. Inhibition of TWEAK/Fn14 interaction could be efficacious in anaphylaxis therapy.

Cellular Crosstalk between Endothelial and Smooth Muscle Cells in Vascular Wall Remodeling.

Pathological vascular wall remodeling refers to the structural and functional changes of the vessel wall that occur in response to injury that eventually leads to cardiovascular disease (CVD). Vessel wall are composed of two major primary cells types, endothelial cells (EC) and vascular smooth muscle cells (VSMCs). The physiological communications between these two cell types (EC-VSMCs) are crucial in the development of the vasculature and in the homeostasis of mature vessels. Moreover, aberrant EC-VSMCs communication has been associated to the promotor of various disease states including vascular wall remodeling. Paracrine regulations by bioactive molecules, communication via direct contact (junctions) or information transfer via extracellular vesicles or extracellular matrix are main crosstalk mechanisms. Identification of the nature of this EC-VSMCs crosstalk may offer strategies to develop new insights for prevention and treatment of disease that curse with vascular remodeling. Here, we will review the molecular mechanisms underlying the interplay between EC and VSMCs. Additionally, we highlight the potential applicable methodologies of the co-culture systems to identify cellular and molecular mechanisms involved in pathological vascular wall remodeling, opening questions about the future research directions.

Impaired HDL (High-Density Lipoprotein)-Mediated Macrophage Cholesterol Efflux in Patients With Abdominal Aortic Aneurysm-Brief Report.

Objective- The ability of HDL (high-density lipoprotein) to promote macrophage cholesterol efflux is considered the main HDL cardioprotective function. Abdominal aortic aneurysm (AAA) is usually characterized by cholesterol accumulation and macrophage infiltration in the aortic wall. Here, we aim to evaluate the composition of circulating HDL particles and their potential for promoting macrophage cholesterol efflux in AAA subjects. Approach and Results- First, we randomly selected AAA and control subjects from Spain. The AAA patients in the Spanish cohort showed lower plasma apoA-I levels concomitantly associated with low levels of plasma HDL cholesterol and the amount of preß-HDL particles. We determined macrophage cholesterol efflux to apoB-depleted plasma, which contains mature HDL, preß-HDL particles and HDL regulatory proteins. ApoB-depleted plasma from AAA patients displayed an impaired ability to promote macrophage cholesterol efflux. Next, we replicated the experiments with AAA and control subjects derived from Danish cohort. Danish AAA patients also showed lower apoA-I levels and a defective HDL-mediated macrophage cholesterol efflux. Conclusions- AAA patients show impaired HDL-facilitated cholesterol removal from macrophages, which could be mechanistically linked to AAA.

Increased galectin-3 levels are associated with abdominal aortic aneurysm progression and inhibition of galectin-3 decreases elastase-induced AAA development.

Abdominal aortic aneurysm (AAA) evolution is unpredictable and no specific treatment exists for AAA, except surgery to prevent aortic rupture. Galectin-3 has been previously associated with CVD, but its potential role in AAA has not been addressed. Galectin-3 levels were increased in the plasma of AAA patients (n=225) compared with the control group (n=100). In addition, galectin-3 concentrations were associated with the need for surgical repair, independently of potential confounding factors. Galectin-3 mRNA and protein expression were increased in human AAA samples compared with healthy aortas. Experimental AAA in mice was induced via aortic elastase perfusion. Mice were treated intravenously with the galectin-3 inhibitor modified citrus pectin (MCP, 10 mg/kg, every other day) or saline. Similar to humans, galectin-3 serum and aortic mRNA levels were also increased in elastase-induced AAA mice compared with control mice. Mice treated with MCP showed decreased aortic dilation, as well as elastin degradation, vascular smooth muscle cell (VSMC) loss, and macrophage content at day 14 postelastase perfusion compared with control mice. The underlying mechanism(s) of the protective effect of MCP was associated with a decrease in galectin-3 and cytokine (mainly CCL5) mRNA and protein expression. Interestingly, galectin-3 induced CCL5 expression by a mechanism involving STAT3 activation in VSMC. Accordingly, MCP treatment decreased STAT3 phosphorylation in elastase-induced AAA. In conclusion, increased galectin-3 levels are associated with AAA progression, while galectin-3 inhibition decreased experimental AAA development. Our data suggest the potential role of galectin-3 as a therapeutic target in AAA.

Oxidative Stress in Human Atherothrombosis: Sources, Markers and Therapeutic Targets.

Atherothrombosis remains one of the main causes of morbidity and mortality worldwide. The underlying pathology is a chronic pathological vascular remodeling of the arterial wall involving several pathways, including oxidative stress. Cellular and animal studies have provided compelling evidence of the direct role of oxidative stress in atherothrombosis, but such a relationship is not clearly established in humans and, to date, clinical trials on the possible beneficial effects of antioxidant therapy have provided equivocal results. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the main sources of reactive oxygen species (ROS) in human atherothrombosis. Moreover, leukocyte-derived myeloperoxidase (MPO) and red blood cell-derived iron could be involved in the oxidative modification of lipids/lipoproteins (LDL/HDL) in the arterial wall. Interestingly, oxidized lipoproteins, and antioxidants, have been analyzed as potential markers of oxidative stress in the plasma of patients with atherothrombosis. In this review, we will revise sources of ROS, focusing on NADPH oxidase, but also on MPO and iron. We will also discuss the impact of these oxidative systems on LDL and HDL, as well as the value of these modified lipoproteins as circulating markers of oxidative stress in atherothrombosis. We will finish by reviewing some antioxidant systems and compounds as therapeutic strategies to prevent pathological vascular remodeling.

Paraoxonase-1 overexpression prevents experimental abdominal aortic aneurysm progression.

Abdominal aortic aneurysm (AAA) is a permanent dilation of the aorta due to excessive proteolytic, oxidative and inflammatory injury of the aortic wall. We aimed to identify novel mediators involved in AAA pathophysiology, which could lead to novel therapeutic approaches. For that purpose, plasma from four AAA patients and four controls were analysed by a label-free proteomic approach. Among identified proteins, paraoxonase-1 (PON1) was decreased in plasma of AAA patients compared with controls, which was further validated in a bigger cohort of samples by ELISA. The phenylesterase enzymatic activity of PON1 was also decreased in serum of AAA patients compared with controls. To address the potential role of PON1 as a mediator of AAA, experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and human transgenic PON1 (HuTgPON1) mice. Similar to humans, PON1 activity was also decreased in serum of elastase-induced AAA mice compared with healthy mice. Interestingly, overexpression of PON1 was accompanied by smaller aortic dilation and higher elastin and vascular smooth muscle cell (VSMC) content in the AAA of HuTgPON1 compared with WT mice. Moreover, HuTgPON1 mice display decreased oxidative stress and apoptosis, as well as macrophage infiltration and monocyte chemoattractant protein-1 (MCP1) expression, in elastase-induced AAA. In conclusion, decreased circulating PON1 activity is associated with human and experimental AAA. PON1 overexpression in mice protects against AAA progression by reducing oxidative stress, apoptosis and inflammation, suggesting that strategies aimed at increasing PON1 activity could prevent AAA.

In vitro 3D co-culture model of human endothelial and smooth muscle cells to study pathological vascular remodeling. / Modelo de cocultivo 3D in vitro de células endoteliales y vasculares de músculo liso humanas para el estudio del remodelado vascular patológico.

Pathological vascular remodeling of the vessel wall refers to the structural and functional changes of the vessel wall that occur in response to injury that eventually leads to cardiovascular disease. The vessel wall is composed of two main types of cells, endothelial cells and vascular smooth muscle cells, whose communication is crucial in both the development of the vasculature and the homeostasis of mature vessels. Changes in the dialogue between endothelial cells and vascular smooth muscle cells are associated with various pathological states that triggers remodeling of the vascular wall. For many years, considerable efforts have been made to develop effective diagnoses and treatments for these pathologies by studying their mechanisms in both in vitro and in vivo models. Compared to animal models, in vitro models can provide great opportunities to obtain data in a more homogeneous, economical and massive way, providing an overview of the signaling pathways responsible for these pathologies. The implementation of three-dimensional in vitro co-culture models for the study of other pathologies has been postulated as a potentially applicable methodology, which determines the importance of its application in studies of cardiovascular diseases. In this article we present a method for culturing human endothelial cells and vascular smooth muscle cells, grown under non-adherent conditions, that generate three-dimensional spheroidal structures with greater physiological equivalence to in vivo conditions. This in vitro modeling could be used as a study tool to identify cellular and molecular mechanisms involved in the pathological processes underlying vascular remodeling.

Tweak/Fn14 system is involved in rhabdomyolysis-induced acute kidney injury.

BACKGROUND: Rhabdomyolysis is a severe clinical syndrome associated to acute kidney injury (AKI) and chronic kidney disease (CKD). TWEAK/Fn14 signaling axis regulates renal inflammation and tubular cell death. However, the functional role of TWEAK/Fn14 in rhabdomyolysis remains unknown. METHODS: Rhabdomyolysis was induced in wild-type, TWEAK- and Fn14-deficient mice or mice treated with TWEAK blocking antibody. Renal injury, inflammation, fibrosis and cell death were assessed. Additionally, we performed in vivo and in vitro studies to explore the possible signalling pathways involved in Fn14 regulation. FINDINGS: Fn14 renal expression was increased in mice with rhabdomyolysis, correlating with decline of renal function. Mechanistically, myoglobin (Mb) induced Fn14 expression via ERK and p38 pathway, whereas Nrf2 activation diminished Mb-mediated Fn14 upregulation in cultured renal cells. TWEAK or Fn14 genetic depletion ameliorated rhabdomyolysis-associated loss of renal function, histological damage, tubular cell death, inflammation, and expression of both tubular and endothelial injury markers. Deficiency of TWEAK or Fn14 also decreased long-term renal inflammation and fibrosis in mice with rhabdomyolysis. Finally, pharmacological treatment with a blocking TWEAK antibody diminished the expression of acute renal injury markers and cell death and lessened residual kidney fibrosis and chronic inflammation in rhabdomyolysis. INTERPRETATION: TWEAK/Fn14 axis participates in the pathogenesis of rhabdomyolysis-AKI and subsequent AKI-CKD transition. Blockade of this signaling pathway may represent a promising therapeutic strategy for reducing rhabdomyolysis-mediated renal injury. FUNDING: Spanish Ministry of Science and Innovation, ISCIII and Junta de Andalucía.

Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal aortic aneurysm progression.

OBJECTIVE: Polymorphonuclear neutrophils (PMNs) play a main role in abdominal aortic aneurysm (AAA) progression. We have analyzed circulating PMNs isolated from AAA patients and controls by a proteomic approach to identify proteins potentially involved in AAA pathogenesis. METHODS AND RESULTS: PMNs from 8 AAA patients (4 large AAA >5 cm and 4 small AAA 3-5 cm) and 4 controls were analyzed by 2D differential in-gel electrophoresis. Among differentially expressed spots, several proteins involved in redox balance were identified by mass spectrometry (eg, cyclophilin, thioredoxin reductase, catalase). Diminished catalase expression and activity were observed in PMNs from AAA patients compared with controls. In contrast, PMNs from AAA patients displayed higher H(2)O(2) and myeloperoxidase levels than PMNs from controls. Moreover, a significant decrease in catalase mRNA levels was observed in PMNs after phorbol 12-myristate 13-acetate incubation. Catalase plasma levels were also decreased in large (n=47) and small (n=56) AAA patients compared with controls (n=34). We observed catalase expression in AAA thrombus and thrombus-conditioned medium, associated with PMN infiltration. Furthermore, increased H(2)O(2) levels were observed in AAA thrombus-conditioned medium compared with the media layer. CONCLUSIONS: Diminished catalase levels in circulating PMNs and plasma are observed in AAA patients, supporting an important role of oxidative stress in AAA evolution.

Oxidative stress, macrophage infiltration and CD163 expression are determinants of long-term renal outcome in macrohematuria-induced acute kidney injury of IgA nephropathy.

BACKGROUND: Macroscopic hematuria (MH) may cause acute kidney injury (AKI) in IgA nephropathy. Up to 25% of patients with MH-associated AKI do not recover baseline renal function. Our objective was to identify subjects at high risk for an adverse renal function. METHODS: We examined macrophages, oxidative stress markers (NADPH-p22 and HO-1) and the hemoglobin scavenger receptor (CD163) in renal biopsy specimens from 33 MH-AKI patients with complete recovery (CR, n = 17) or incomplete recovery (IR, n = 16) of renal function after 6.72 (range 0.5-21.5) years of follow-up. RESULTS: CD163-expressing macrophages, HO-1 and NADPH-p22 expression were located in areas surrounding tubules with iron deposits and filled with erythrocyte casts. CD163-positive macrophages score and HO-1- and p22-positive staining correlated positively with percentage of tubules with erythrocyte casts and tubular necrosis. Macrophage infiltration, CD163-positive macrophage score, NADPH-p22- and HO-1-positive staining areas were significantly greater in IR patients when compared with CR patients. The CD163-positive macrophage score and oxidative stress markers (p22 and HO-1) were negatively correlated with renal function outcome, as determined by estimated glomerular filtration rate (eGFR) and proteinuria, at the end of the follow-up period. In multivariate analysis, the CD163-positive macrophage score remained significantly associated with final eGFR and proteinuria after adjustment by age, gender, duration of MH, initial eGFR and proteinuria. CONCLUSIONS: Increased macrophage infiltration, CD163 expression and oxidative stress are significant prognostic factors for an IR of renal function in patients with MH-associated AKI. These molecular pathways may be involved in the renal response to injury and could be useful to improve diagnosis and therapeutics.

Role of Extracellular Vesicles as Potential Diagnostic and/or Therapeutic Biomarkers in Chronic Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the first cause of death worldwide. In recent years, there has been great interest in the analysis of extracellular vesicles (EVs), including exosomes and microparticles, as potential mediators of biological communication between circulating cells/plasma and cells of the vasculature. Besides their activity as biological effectors, EVs have been also investigated as circulating/systemic biomarkers in different acute and chronic CVDs. In this review, the role of EVs as potential diagnostic and prognostic biomarkers in chronic cardiovascular diseases, including atherosclerosis (mainly, peripheral arterial disease, PAD), aortic stenosis (AS) and aortic aneurysms (AAs), will be described. Mechanistically, we will analyze the implication of EVs in pathological processes associated to cardiovascular remodeling, with special emphasis in their role in vascular and valvular calcification. Specifically, we will focus on the participation of EVs in calcium accumulation in the pathological vascular wall and aortic valves, involving the phenotypic change of vascular smooth muscle cells (SMCs) or valvular interstitial cells (IC) to osteoblast-like cells. The knowledge of the implication of EVs in the pathogenic mechanisms of cardiovascular remodeling is still to be completely deciphered but there are promising results supporting their potential translational application to the diagnosis and therapy of different CVDs.

Unbiased plasma proteomics discovery of biomarkers for improved detection of subclinical atherosclerosis.

BACKGROUND: Imaging of subclinical atherosclerosis improves cardiovascular risk prediction on top of traditional risk factors. However, cardiovascular imaging is not universally available. This work aims to identify circulating proteins that could predict subclinical atherosclerosis. METHODS: Hypothesis-free proteomics was used to analyze plasma from 444 subjects from PESA cohort study (222 with extensive atherosclerosis on imaging, and 222 matched controls) at two timepoints (three years apart) for discovery, and from 350 subjects from AWHS cohort study (175 subjects with extensive atherosclerosis on imaging and 175 matched controls) for external validation. A selected three-protein panel was further validated by immunoturbidimetry in the AWHS population and in 2999 subjects from ILERVAS cohort study. FINDINGS: PIGR, IGHA2, APOA, HPT and HEP2 were associated with subclinical atherosclerosis independently from traditional risk factors at both timepoints in the discovery and validation cohorts. Multivariate analysis rendered a potential three-protein biomarker panel, including IGHA2, APOA and HPT. Immunoturbidimetry confirmed the independent associations of these three proteins with subclinical atherosclerosis in AWHS and ILERVAS. A machine-learning model with these three proteins was able to predict subclinical atherosclerosis in ILERVAS (AUC [95%CI]:0.73 [0.70-0.74], p < 1 × 10-99), and also in the subpopulation of individuals with low cardiovascular risk according to FHS 10-year score (0.71 [0.69-0.73], p < 1 × 10-69). INTERPRETATION: Plasma levels of IGHA2, APOA and HPT are associated with subclinical atherosclerosis independently of traditional risk factors and offers potential to predict this disease. The panel could improve primary prevention strategies in areas where imaging is not available. FUNDING: This study was supported by competitive grants from the Spanish Ministry of Science, Innovation and Universities (BIO2015-67580-P, PGC2018-097019-B-I00, PID2019-106814RB-I00 and SAF2016-80843-R), through the Carlos III Institute of Health-Fondo de Investigacion Sanitaria grant PRB3 (IPT17/0019 - ISCIII-SGEFI / ERDF, ProteoRed), CIBERCV and CIBERDEM, the Fundacio MaratoTV3 (grant 122/C/2015) and "la Caixa" Banking Foundation (project HR17-00247). The PESA study is co-funded equally by the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain, and Banco Santander, Madrid, Spain. The ILERVAS study was funded by the Diputacio de Lleida. The study also receives funding from the Instituto de Salud Carlos III (PI15/02019; PI18/00610; RD16/0009) and the FEDER funds. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacion y Universidades (MCNU) and the Pro CNIC Foundation.

Annexins: Involvement in cholesterol homeostasis, inflammatory response and atherosclerosis. / Anexinas: implicación en la homeostasis del colesterol, la respuesta inflamatoria y la aterosclerosis.

The annexin superfamily consists of 12 proteins with a highly structural homology that binds to phospholipids depending on the availability of Ca2+-dependent. Different studies of overexpression, inhibition, or using recombinant proteins have linked the main function of these proteins to their dynamic and reversible binding to membranes. Annexins are found in multiple cellular compartments, regulating different functions, such as membrane trafficking, anchoring to the cell cytoskeleton, ion channel regulation, as well as pro- or anti-inflammatory and anticoagulant activities. The use of animals deficient in any of these annexins has established their possible functions in vivo, demonstrating that annexins can participate in relevant functions independent of Ca2+ signalling. This review will focus mainly on the role of different annexins in the pathological vascular remodelling that underlies the formation of the atherosclerotic lesion, as well as in the control of cholesterol homeostasis.

Fcγ receptor activation mediates vascular inflammation and abdominal aortic aneurysm development.

BACKGROUND: Abdominal aortic aneurysm (AAA), a degenerative vascular pathology characterized by permanent dilation of the aorta, is considered a chronic inflammatory disease involving innate/adaptive immunity. However, the functional role of antibody-dependent immune response against antigens present in the damaged vessel remains unresolved. We hypothesized that engagement of immunoglobulin G (IgG) Fc receptors (FcγR) by immune complexes (IC) in the aortic wall contributes to AAA development. We therefore evaluated FcγR expression in AAA lesions and analysed whether inhibition of FcγR signaling molecules (γ-chain and Syk kinase) influences AAA formation in mice. METHODS: FcγR gene/protein expression was assessed in human and mouse AAA tissues. Experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and γ-chain knockout (γKO) mice (devoid of activating FcγR) in combination with macrophage adoptive transfer or Syk inhibitor treatment. To verify the mechanisms of FcγR in vitro, vascular smooth muscle cells (VSMC) and macrophages were stimulated with IgG IC. RESULTS: FcγR overexpression was detected in adventitia and media layers of human and mouse AAA. Elastase-perfused γKO mice exhibited a decrease in AAA incidence, aortic dilation, elastin degradation, and VSMC loss. This was associated with (1) reduced infiltrating leukocytes and immune deposits in AAA lesions, (2) inflammatory genes and metalloproteinases downregulation, (3) redox balance restoration, and (4) converse phenotype of anti-inflammatory macrophage M2 and contractile VSMC. Adoptive transfer of FcγR-expressing macrophages aggravated aneurysm in γKO mice. In vitro, FcγR deficiency attenuated inflammatory gene expression, oxidative stress, and phenotypic switch triggered by IC. Additionally, Syk inhibition prevented IC-mediated cell responses, reduced inflammation, and mitigated AAA formation. CONCLUSION: Our findings provide insight into the role and mechanisms mediating IgG-FcγR-associated inflammation and aortic wall injury in AAA, which might represent therapeutic targets against AAA disease.

Protective effect of suppressor of cytokine signalling 1-based therapy in experimental abdominal aortic aneurysm.

BACKGROUND AND PURPOSE: Abdominal aortic aneurysm (AAA) is a multifactorial disease characterized by chronic inflammation, oxidative stress and proteolytic activity in the aortic wall. Targeting JAK/signal transducer and activator of transcription (JAK/STAT) pathway is a promising strategy for chronic inflammatory diseases. We investigated the vasculo-protective role of suppressor of cytokine signalling-1 (SOCS1), the negative JAK/STAT regulator, in experimental AAA. EXPERIMENTAL APPROACH: A synthetic, cell permeable peptide (S1) mimic of SOCS1 kinase inhibitory domain to suppress STAT activation was evaluated in the well-established mouse model of elastase-induced AAA by monitoring changes in aortic diameter, cellular composition and gene expression in abdominal aorta. S1 function was further evaluated in cultured vascular smooth muscle cells (VSMC) and macrophages exposed to elastase or elastin-derived peptides. KEY RESULTS: S1 peptide prevented AAA development, evidenced by reduced incidence of AAA, aortic dilation and elastin degradation, partial restoration of medial VSMC and decreased inflammatory cells and oxidative stress in AAA tissue. Mechanistically, S1 suppressed STAT1/3 activation in aorta, down-regulated cytokines, metalloproteinases and altered the expression of cell differentiation markers by favouring anti-inflammatory M2 macrophage and contractile VSMC phenotypes. In vitro, S1 suppressed the expression of inflammatory and oxidative genes, reduced cell migration and reversed the phenotypic switch of macrophages and VSMC. By contrast, SOCS1 silencing promoted inflammatory response. CONCLUSION AND IMPLICATIONS: This preclinical study demonstrates the therapeutic potential of SOCS1-derived peptide to halt AAA progression by suppressing JAK/STAT-mediated inflammation and aortic dilation. S1 peptide may therefore be a valuable option for the treatment of AAA.

Malondialdehyde-modified HDL particles elicit a specific IgG response in abdominal aortic aneurysm.

High Density Lipoprotein (HDL) plays a protective role in abdominal aortic aneurysm (AAA); however, recent findings suggest that oxidative modifications could lead to dysfunctional HDL in AAA. This study aimed at testing the effect of oxidized HDL on aortic lesions and humoral immune responses in a mouse model of AAA induced by elastase, and evaluating whether antibodies against modified HDL can be found in AAA patients. HDL particles were oxidized with malondialdehyde (HDL-MDA) and the changes were studied by biochemical and proteomics approaches. Experimental AAA was induced in mice by elastase perfusion and then mice were treated with HDL-MDA, HDL or vehicle for 14 days. Aortic lesions were studied by histomorphometric analysis. Levels of anti-HDL-MDA IgG antibodies were measured by an in-house immunoassay in the mouse model, in human tissue-supernatants and in plasma samples from the VIVA cohort. HDL oxidation with MDA was confirmed by enhanced susceptibility to diene formation. Proteomics demonstrated the presence of MDA adducts on Lysine residues of HDL proteins, mainly ApoA-I. MDA-modification of HDL abrogated the protective effect of HDL on cultured endothelial cells as well as on AAA dilation in mice. Exposure to HDL-MDA elicited an anti-HDL-MDA IgG response in mice. Anti-HDL-MDA were also detected in tissue-conditioned media from AAA patients, mainly in intraluminal thrombus. Higher plasma levels of anti-HDL-MDA IgG antibodies were found in AAA patients compared to controls. Anti-HDL-MDA levels were associated with smoking and were independent predictors of overall mortality in AAA patients. Overall, MDA-oxidized HDL trigger a specific humoral immune response in mice. Besides, antibodies against HDL-MDA can be detected in tissue and plasma of AAA patients, suggesting its potential use as surrogate stable biomarkers of oxidative stress in AAA.

Proteomic and metabolomic profiles in atherothrombotic vascular disease.

Atherothrombosis remains a major cause of morbidity and mortality in the western world. The underlying processes associated with clinical expression of atherothrombosis include oxidative stress and proteolysis in relation to neovascularisation and intraplaque hemorrhages, leading to immuno-inflammatory response, cell death, and extracellular matrix breakdown. The complex biological multifactorial nature of atherothrombosis requires the development of novel technologies that allow the analysis of cellular and molecular processes responsible for the transition to disease phenotypes and the discovery of new diagnostic and prognostic biomarkers. In the present article, we have reviewed recent advances in the application of proteomic and metabolomic techniques to the study of atherothrombosis. We have focused on recent studies analyzing cells involved in hemo-thrombus formation (platelets, red blood cells, and polymorphonuclear cells), as well as tissues, tissue-conditioned media, and plasma of atherothrombotic patients. In the future, the application of these high-throughput technologies, along with imaging techniques, in systems biology approaches will help to individualize medicine.

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.

Macrophage Cholesterol Efflux Downregulation Is Not Associated with Abdominal Aortic Aneurysm (AAA) Progression.

Recent studies have raised the possibility of a role for lipoproteins, including high-density lipoprotein cholesterol (HDLc), in abdominal aortic aneurysm (AAA). The study was conducted in plasmas from 39 large size AAA patients (aortic diameter > 50 mm), 81 small/medium size AAA patients (aortic diameter between 30 and 50 mm) and 38 control subjects (aortic diameter < 30 mm). We evaluated the potential of HDL-mediated macrophage cholesterol efflux (MCE) to predict AAA growth and/or the need for surgery. MCE was impaired in the large aortic diameter AAA group as compared with that in the small/medium size AAA group and the control group. However, no significant difference in HDL-mediated MCE capacity was observed in 3 different progression subgroups (classified according to growth rate < 1 mm per year, between 1 and 5 mm per year or >5 mm per year) in patients with small/medium size AAA. Moreover, no correlation was found between MCE capacity and the aneurysm growth rate. A multivariate Cox regression analysis revealed a significant association between lower MCE capacity with the need for surgery in all AAA patients. Nevertheless, the significance was lost when only small/medium size AAA patients were included. Our results suggest that MCE, a major HDL functional activity, is not involved in AAA progression.

Complement C5 Protein as a Marker of Subclinical Atherosclerosis.

BACKGROUND: The mechanisms underlying early atherosclerotic plaque formation are not completely understood. Moreover, plasma biomarkers of subclinical atherosclerosis are lacking. OBJECTIVES: The purpose of this study was to analyze the temporal and topologically resolved protein changes taking place in human aortas with early atherosclerosis to find new potential diagnostic and/or therapeutic targets. METHODS: The protein composition of healthy aortas (media layer) or with early atheroma (fatty streak and fibrolipidic, media and intima layers) was analyzed by deep quantitative multiplexed proteomics. Further analysis was performed by Western blot, immunohistochemistry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. Plasma levels of complement C5 were analyzed in relation to the presence of generalized (>2 plaques) or incipient (0 to 2 plaques) subclinical atherosclerosis in 2 independent clinical cohorts (PESA [Progression of Early Subclinical Atherosclerosis] [n = 360] and NEFRONA [National Observatory of Atherosclerosis in Nephrology] [n = 394]). RESULTS: Proteins involved in lipid transport, complement system, immunoglobulin superfamily, and hemostasis are increased in early plaques. Components from the complement activation pathway were predominantly increased in the intima of fibrolipidic plaques. Among them, increased C5 protein levels were further confirmed by Western blot, enzyme-linked immunosorbent assay and immunohistochemistry, and associated with in situ complement activation. Plasma C5 was significantly increased in individuals with generalized subclinical atherosclerosis in both PESA and NEFRONA cohorts, independently of risk factors. Moreover, in the PESA study, C5 plasma levels positively correlated with global plaque volume and coronary calcification. CONCLUSIONS: Activation of the complement system is a major alteration in early atherosclerotic plaques and is reflected by increased C5 plasma levels, which have promising value as a novel circulating biomarker of subclinical atherosclerosis.