Search

Background: Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound. Methods: Lesion echogenicity was correlated to microarray gene expression profiles from carotid endarterectomies (n=96). The findings were extended into studies of human and mouse atherosclerotic lesions in situ, followed by functional investigations in vitro in human carotid smooth muscle cells (SMCs). Results: Pathway analyses highlighted muscle differentiation, iron homeostasis, calcification, matrix organization, cell survival balance, and BCLAF1 (BCL2 [B-cell lymphoma 2]-associated transcription factor 1) as the most significant signatures. BCLAF1 was downregulated in echolucent plaques, positively correlated to proliferation and negatively to apoptosis. By immunohistochemistry, BCLAF1 was found in normal medial SMCs. It was repressed early during atherogenesis but reappeared in CD68+ cells in advanced plaques and interacted with BCL2 by proximity ligation assay. In cultured SMCs, BCLAF1 was induced by differentiation factors and mitogens and suppressed by macrophage-conditioned medium. BCLAF1 silencing led to downregulation of BCL2 and SMC markers, reduced proliferation, and increased apoptosis. Transdifferentiation of SMCs by oxLDL (oxidized low-denisty lipoprotein) was accompanied by upregulation of BCLAF1, CD36, and CD68, while oxLDL exposure with BCLAF1 silencing preserved MYH (myosin heavy chain) 11 expression and prevented transdifferentiation. BCLAF1 was associated with expression of cell differentiation, contractility, viability, and inflammatory genes, as well as the scavenger receptors CD36 and CD68 . BCLAF1 expression in CD68+/BCL2+ cells of SMC origin was verified in plaques from MYH11 lineage-tracing atherosclerotic mice. Moreover, BCLAF1 downregulation associated with vulnerability parameters and cardiovascular risk in patients with carotid atherosclerosis. Conclusions: Plaque echogenicity correlated with enrichment of distinct molecular pathways and identified BCLAF1 , previously not described in atherosclerosis, as the most significant gene. Functionally, BCLAF1 seems necessary for survival and transdifferentiation of SMCs into a macrophage-like phenotype. The role of BCLAF1 in plaque vulnerability should be further evaluated.

BACKGROUND: The metabolism of tryptophan (Trp) along the kynurenine pathway has been shown to carry strong immunoregulatory properties. Several experimental studies indicate that this pathway is a major regulator of vascular inflammation and influences atherogenesis. Knowledge of the role of this pathway in human atherosclerosis remains incomplete.OBJECTIVES: In this study, we performed a multiplatform analysis of tissue samples, in vitro and in vivo functional assays to elucidate the potential role of the kynurenine pathway in human atherosclerosis.METHODS AND RESULTS: Comparison of transcriptomic data from carotid plaques and control arteries revealed an upregulation of enzymes within the quinolinic branch of the kynurenine pathway in the disease state, whilst the branch leading to the formation of kynurenic acid (KynA) was downregulated. Further analyses indicated that local inflammatory responses are closely tied to the deviation of the kynurenine pathway in the vascular wall. Analysis of cerebrovascular symptomatic and asymptomatic carotid stenosis data showed that the downregulation of KynA branch enzymes and reduced KynA production were associated with an increased probability of patients to undergo surgery due to an unstable disease. In vitro, we showed that KynA-mediated signalling through aryl hydrocarbon receptor (AhR) is a major regulator of human macrophage activation. Using a mouse model of peritoneal inflammation, we showed that KynA inhibits leukocyte recruitment.CONCLUSIONS: We have found that a deviation in the kynurenine pathway is associated with an increased probability of developing symptomatic unstable atherosclerotic disease. Our study suggests that KynA-mediated signalling through AhR is an important mechanism involved in the regulation of vascular inflammation.

BACKGROUND The interplay between innate and adaptive immunity is central in life-threatening clinical complications of atherosclerosis such as myocardial infarction and stroke. The specific mechanisms involved and their protective versus detrimental effects in the disease process remain poorly understood. We have previously shown that higher levels of Toll-like receptor 7 (TLR7) expression in human atherosclerotic lesions are correlated with better patient outcome. OBJECTIVE In this study, we explored whether TLR7 activation can ameliorate disease in experimental atherosclerosis in mice. METHODS Apolipoprotein E deficient mice (Apoe-/- ) with established disease were injected for five weeks intraperitoneally with the TLR7 ligand R848. Local effects were evaluated by characterization of the lesion. Systemic effects of the treatment were investigated by immune composition analysis in the spleen and plasma measurements. RESULTS The in vivo treatment arrested lesion progression in the aorta. We also detected expansion of marginal zone B cells and Treg in the spleen together with increased plasma IgM antibodies against oxidized low-density lipoprotein (oxLDL) and reduced plasma cholesterol levels. These changes were accompanied by increased accumulation of IgM antibodies, decreased necrosis and fewer apoptotic cells in atherosclerotic lesions. CONCLUSIONS Our findings show that TLR7 stimulation could ameliorate atherosclerotic lesion burden and reduce plasma cholesterol in Apoe-/- mice. TLR7 stimulation was associated with an atheroprotective B-cell and Treg response, which may have systemic and local effects within lesions that could prevent arterial lipid accumulation and inflammation.

Concepts of atherogenesis have evolved considerably with time. Early animal experiments showed that a cholesterol-rich diet could induce fatty lesion formation in arteries. The elucidation of lipoprotein metabolism ultimately led to demonstrating the clinical benefits of lipid lowering. The view of atheromata as bland accumulations of smooth muscle cells that elaborated an extracellular matrix that could entrap lipids then expanded to embrace inflammation as providing pathways that could link risk factors to atherogenesis. The characterization of leukocyte adhesion molecules and their control by proinflammatory cytokines, the ability of chemokines to recruit leukocytes, and the identification of inflammatory cell subtypes in lesions spurred the unraveling of innate and adaptive immune pathways that contribute to atherosclerosis and its thrombotic complications. Such pathophysiologic insights have led to the identification of biomarkers that can define categories of risk and direct therapies and to the development of new treatments.

Although a high marine food intake is considered cardioprotective, randomized trials of ω-3 fatty acids initially generated conflicting results in terms of the role of ω-3 supplementation in cardiovascular prevention. This work demonstrates the results of the 3 most recent clinical trials with ω-3 fatty acids are put into the context of possible mechanisms mediating their beneficial cardiovascular effects. In particular, the randomized Reduction of Cardiovascular Events with EPA Intervention Trial (REDUCE-IT) showed that icosapent ethyl, which is the ethyl ester form of the ω-3 fatty acid eicosapentaenoic acid (EPA), induced a significant reduction of cardiovascular events. Importantly, EPA serves as a substrate for the formation of the specialized proresolving mediator resolvin E1 (RvE1), which stimulates the resolution of inflammation. RvE1 reduces atherosclerosis and intimal hyperplasia by means of its specific receptor ERV1/ChemR23. The decreased levels of proinflammatory and proatherosclerotic leukotrienes by ω-3 fatty acids may further contribute to a beneficial inflammatory balance. Consequently, the Rv/leukotriene ratio is emerging as a marker of nonresolving vascular inflammation. Recent experimental studies have shown that anti-inflammatory and proresolving effects of lipid mediators derived from ω-3 fatty acids inhibit atherosclerosis independently of cholesterol and triglyceride levels. The results of the 3 most recent clinical trials of ω-3 fatty acid supplementation indicate an importance of the type and dose of ω-3 supplementation and highlight the need for risk stratification in the patient selection for ω-3 supplementation for either primary or secondary prevention of cardiovascular disease.-Bäck, M., Hansson, G. K. Omega-3 fatty acids, cardiovascular risk, and the resolution of inflammation.

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

Background The interplay between innate and adaptive immunity is involved in the cause of the life-threatening events in atherosclerosis, such as myocardial infarction and stroke. We have previously shown that higher levels of Toll-like receptor 7 (TLR7) expression in human atherosclerotic lesions are correlated to better patient outcome, emphasizing the role of the innate pattern recognizing receptors in the pathogenesis of atherosclerosis. Purpose Firstly, to investigate if activation of the innate immune receptor TLR7 in vivo in experimental atherosclerosis is a potential way to reduce disease and secondly to identify pathways upon TLR7 ligation treatment. Methods Apolipoprotein E (ApoE−/−) deficient mice with established disease were injected intraperitoneal with a TLR7 ligand. Local effects were evaluated by characterization of atherosclerotic lesions. Systemic effects of the treatment were investigated by immune composition analysis in the spleen and by plasma measurements Results In vivo treatment of ApoE−/−mice with TLR7 ligand arrested atherosclerotic lesion development in the aortic root. Moreover, in the lesions of the treated mice we detected decrease in necrotic area and decrease in accumulation of apoptotic cells. In the spleen we detected an expansion of marginal zone B cells accompanied with an increase in IgM antibodies against oxidized low-density lipoprotein (oxLDL). The treated mice had reduced plasma cholesterol levels. Conclusions In vivo administration of TLR7 ligand ameliorated the atherosclerotic burden in old ApoE−/− mice. Our findings indicate that TLR7 ligation is involved in control of inflammatory responses in atherosclerosis, suggesting that TLR7 is a potential therapeutic target. Further analysis is needed in order to elucidate the underlying mechanisms by which TLR7 exerts its protective role. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Swedish Heart-Lung Foundation

Adaptive as well as innate immune responses contribute to the development of atherosclerosis. Studies performed in experimental animals have revealed that some of these immune responses are protective while others contribute to the progression of disease. These observations suggest that it may be possible to develop novel therapies for cardiovascular disease by selectively modulating such atheroprotective and proatherogenic immunity. Recent advances in cancer treatment using immune check inhibitors and CAR (chimeric antigen receptor) T-cell therapy serve as excellent examples of the possibilities of targeting the immune system to combat disease. LDL (low-density lipoprotein) that has accumulated in the artery wall is a key autoantigen in atherosclerosis, and activation of antigen-specific T helper 1–type T cells is thought to fuel plaque inflammation. Studies aiming to prove this concept by immunizing experimental animals with oxidized LDL particles unexpectedly resulted in activation of atheroprotective immunity involving regulatory T cells. This prompted several research groups to try to develop vaccines against atherosclerosis. In this review, we will discuss the experimental and clinical data supporting the possibility of developing immune-based therapies for lowering cardiovascular risk. We will also summarize ongoing clinical studies and discuss the challenges associated with developing an effective and safe atherosclerosis vaccine.

Rationale: PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens. Objective: Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall. Methods and Results: Genetic analyses revealed association of intronic PCSK6 variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6 −/− versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6 −/− mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration. Conclusions: PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling. Visual Overview: An online visual overview is available for this article.

Supplemental Digital Content is available in the text., Background: In addition to enhanced proinflammatory signaling, impaired resolution of vascular inflammation plays a key role in atherosclerosis. Proresolving lipid mediators formed through the 12/15 lipoxygenase pathways exert protective effects against murine atherosclerosis. n-3 Polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), serve as the substrate for the formation of lipid mediators, which transduce potent anti-inflammatory and proresolving actions through their cognate G-protein–coupled receptors. The aim of this study was to identify signaling pathways associated with EPA supplementation and lipid mediator formation that mediate atherosclerotic disease progression. Methods: Lipidomic plasma analysis were performed after EPA supplementation in Apoe−/− mice. Erv1/Chemr23−/−xApoe−/− mice were generated for the evaluation of atherosclerosis, phagocytosis, and oxidized low-density lipoprotein uptake. Histological and mRNA analyses were done on human atherosclerotic lesions. Results: Here, we show that EPA supplementation significantly attenuated atherosclerotic lesion growth induced by Western diet in Apoe−/− mice and was associated with local cardiovascular n-3 enrichment and altered lipoprotein metabolism. Our systematic plasma lipidomic analysis identified the resolvin E1 precursor 18-monohydroxy EPA as a central molecule formed during EPA supplementation. Targeted deletion of the resolvin E1 receptor Erv1/Chemr23 in 2 independent hyperlipidemic murine models was associated with proatherogenic signaling in macrophages, increased oxidized low-density lipoprotein uptake, reduced phagocytosis, and increased atherosclerotic plaque size and necrotic core formation. We also demonstrate that in macrophages the resolvin E1–mediated effects in oxidized low-density lipoprotein uptake and phagocytosis were dependent on Erv1/Chemr23. When analyzing human atherosclerotic specimens, we identified ERV1/ChemR23 expression in a population of macrophages located in the proximity of the necrotic core and demonstrated augmented ERV1/ChemR23 mRNA levels in plaques derived from statin users. Conclusions: This study identifies 18-monohydroxy EPA as a major plasma marker after EPA supplementation and demonstrates that the ERV1/ChemR23 receptor for its downstream mediator resolvin E1 transduces protective effects in atherosclerosis. ERV1/ChemR23 signaling may represent a previously unrecognized therapeutic pathway to reduce atherosclerotic cardiovascular disease.

Rationale: Regulatory T (Treg) cells suppress immune responses and have been shown to attenuate atherosclerosis. The Treg cell lineage-specification factor FOXP3 (forkhead box P3) is essential for Treg cells’ ability to uphold immunologic tolerance. In humans, FOXP3 exists in several different isoforms, however, their specific role is poorly understood. Objective: To define the regulation and functions of the 2 major FOXP3 isoforms, FOXP3fl and FOXP3Δ2, as well as to establish whether their expression is associated with the ischemic atherosclerotic disease. Methods and Results: Human primary T cells were transduced with lentiviruses encoding distinct FOXP3 isoforms. The phenotype and function of these cells were analyzed by flow cytometry, in vitro suppression assays and RNA-sequencing. We also assessed the effect of activation on Treg cells isolated from healthy volunteers. Treg cell activation resulted in increased FOXP3 expression that predominantly was made up of FOXP3Δ2. FOXP3Δ2 induced specific transcription of GARP (glycoprotein A repetitions predominant), which functions by tethering the immunosuppressive cytokine TGF (transforming growth factor)-β to the cell membrane of activated Treg cells. Real-time polymerase chain reaction was used to determine the impact of alternative splicing of FOXP3 in relation with atherosclerotic plaque stability in a cohort of >150 patients that underwent carotid endarterectomy. Plaque instability was associated with a lower FOXP3Δ2 transcript usage, when comparing plaques from patients without symptoms and patients with the occurrence of recent (FOXP3 mRNA between these 2 groups. Conclusions: These results suggest that activated Treg cells suppress the atherosclerotic disease process and that FOXP3Δ2 controls a transcriptional program that acts protectively in human atherosclerotic plaques.

Sortilin-1, a receptor of the VPS10p family, has been associated with cardiovascular disease in genome-wide association studies. It is implicated in lipoprotein metabolism, secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) and secretion of inflammatory cytokines. However, its own regulation remains unclear. Chronic inflammation is a hallmark of atherosclerosis and the absence of regulatory T (Treg) cells is associated with reduced protein expression of sortilin-1 in the liver. Therefore, we postulated that mediator(s) of inflammation known to be downregulated by Treg cells may modulate sortilin-1 expression. In this study, we identify interferon-gamma (IFN-γ) as the key inflammatory mediator controlling sortilin-1 levels. In vitro cultures of murine hepatocytes cell line and in silico experiments showed that the transcription factor Signal transducer and activator of transcription 1 was activated and bound to the Sort-1 gene upon IFN-γ treatment. This reduced the expression of sortilin-1, while disrupting the IFN-γ signaling pathway prevented the effect. These data unravel an intricate mechanism by which inflammation modulates receptors involved in lipoprotein turnover.

In calcific aortic valve disease (CAVD), activated T lymphocytes localize with osteoclast regions; however, the functional consequences of this association remain unknown. We hypothesized that CD8 + T cells modulate calcification in CAVD. CAVD valves ( n = 52) dissected into noncalcified and calcified portions were subjected to mRNA extraction, real-time quantitative PCR, enzyme-linked immunosorbent assay, and immunohistochemical analyses. Compared with noncalcified portions, calcified regions exhibited elevated transcripts for CD8, interferon (IFN)-γ, CXCL9, Perforin 1, Granzyme B, and heat shock protein 60. Osteoclast-associated receptor activator of NK-κB ligand (RANKL), tartrate-resistant acid phosphatase (TRAP), and osteoclast-associated receptor increased significantly. The stimulation of tissue with phorbol-12-myristate-13-acetate and ionomycin, recapitulating CAVD microenvironment, resulted in IFN-γ release. Real-time quantitative PCR detected mRNAs for CD8 + T-cell activation (Perforin 1, Granzyme B). In stimulated versus unstimulated organoid cultures, elevated IFN-γ reduced the mRNAs encoding for RANKL, TRAP, and Cathepsin K. Molecular imaging showed increased calcium signal intensity in stimulated versus unstimulated parts. CD14 + monocytes treated either with recombinant human IFN-γ or with conditioned media-derived IFN-γ exhibited low levels of Cathepsin K, TRAP, RANK, and tumor necrosis factor receptor-associated factor 6 mRNAs, whereas concentrations of the T-cell co-activators CD80 and CD86 increased in parallel with reduced osteoclast resorptive function, effects abrogated by neutralizing anti–IFN-γ antibodies. CD8 + cell–derived IFN-γ suppresses osteoclast function and may thus favor calcification in CAVD.

Rationale: The liver is the central organ that responds to dietary cholesterol intake and facilitates the release and clearance of lipoprotein particles. Persistent hypercholesterolemia leads to immune responses against lipoprotein particles that drive atherosclerosis. However, the effect of hypercholesterolemia on hepatic T-cell differentiation remains unknown. Objective: To investigate hepatic T-cell subsets upon hypercholesterolemia. Methods and Results: We observed that hypercholesterolemia elevated the intrahepatic regulatory T (Treg) cell population and increased the expression of transforming growth factor-β1 in the liver. Adoptive transfer experiments revealed that intrahepatically differentiated Treg cells relocated to the inflamed aorta in atherosclerosis-prone low-density lipoprotein receptor deficient ( Ldlr −/− ) mice. Moreover, hypercholesterolemia induced the differentiation of intrahepatic, but not intrasplenic, Th17 cells in wild-type mice, whereas the disrupted liver homeostasis in hypercholesterolemic Ldlr −/− mice led to intrahepatic Th1 cell differentiation and CD11b + CD11c + leukocyte accumulation. Conclusions: Our results elucidate a new mechanism that controls intrahepatic T-cell differentiation during atherosclerosis development and indicates that intrahepatically differentiated T cells contribute to the CD4 + T-cell pool in the atherosclerotic aorta.

Rationale: In the search for markers and modulators of vascular disease, microRNAs (miRNAs) have emerged as potent therapeutic targets. Objective: To investigate miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. Methods and Results: Using patient material from the BiKE (Biobank of Karolinska Endarterectomies), we profiled miRNA expression in patients with stable versus unstable carotid plaque. A polymerase chain reaction–based miRNA array of plasma, sampled at the carotid lesion site, identified 8 deregulated miRNAs (miR-15b, miR-29c, miR-30c/d, miR-150, miR-191, miR-210, and miR-500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser capture microdissection and in situ hybridization revealed a distinct localization of miR-210 in fibrous caps. We confirmed that miR-210 directly targets the tumor suppressor gene APC (adenomatous polyposis coli), thereby affecting Wnt (Wingless-related integration site) signaling and regulating smooth muscle cell survival, as well as differentiation in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in 2 rodent models of vascular remodeling and plaque rupture. Modulating miR-210 in vitro and in vivo improved fibrous cap stability with implications for vascular disease. Conclusions: An unstable carotid plaque at risk of stroke is characterized by low expression of miR-210. miR-210 contributes to stabilizing carotid plaques through inhibition of APC, ensuring smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic vascular events.

Background and Purpose Atherosclerosis is characterized by a chronic non-resolving inflammation in the arterial wall. Aspirin-triggered lipoxin A4 (ATL) is a potent anti-inflammatory mediator, involved in the resolution of inflammation. However, the therapeutic potential of immune targeting by means of ATL in atherosclerosis has not previously been explored. The aim of the present study was to determine the effects of ATL and its receptor Fpr2 on atherosclerosis development and progression in apolipoprotein E deficient (ApoE−/−) mice. Experimental Approach ApoE−/− × Fpr2+/+ and ApoE−/− × Fpr2−/− mice were generated. Four-week-old mice fed a high-fat diet for 4 weeks and 16-week-old mice fed chow diet received osmotic pumps containing either vehicle or ATL for 4 weeks. Atherosclerotic lesion size and cellular composition were measured in the aortic root and thoracic aorta. Lipid levels and leukocyte counts were measured in blood and mRNA was isolated from abdominal aorta and spleen. Key Results ATL blocked atherosclerosis progression in the aortic root and thoracic aorta of ApoE−/− mice. In addition, ATL reduced macrophage infiltration and apoptotic cells in atherosclerotic lesions. The mRNA levels of several cytokines and chemokines in the spleen and aorta were reduced by ATL, whereas circulating leukocyte levels were unchanged. The ATL-induced athero-protection was absent in ApoE−/− mice lacking the Fpr2 receptor. Conclusion and Implications ATL blocked atherosclerosis progression by means of an Fpr2-mediated reduced local and systemic inflammation. These results suggest this anti-inflammatory and pro-resolving agent has therapeutic potential for the treatment of atherosclerosis. Linked Articles This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc

Background: Increased inflammatory activity destabilizes the atherosclerotic lesion and may lead to atherothrombosis and symptomatic cardiovascular disease. Co-stimulatory molecules, such as CD137, are key regulators of inflammation, and CD137 activity regulates inflammation in experimental atherosclerosis. Here, we hypothesized that CD137 activation promotes carotid artery inflammation and atherothrombosis. Methods and Results: In a model of inducible atherothrombosis with surgical ligation of the right carotid artery and a subsequent placement of a polyethene cuff, elevated levels of CD137 and CD137 ligand mRNA in atherothrombotic vs. non-atherothrombotic murine carotid lesions was observed. Mice treated with the CD137 agonistic antibody 2A showed signs of increased inflammation in the aorta and a higher proportion of CD8+ T cells in spleen and blood. In carotid lesions of 2A-treated mice, significantly higher counts of CD8+ and major histocompatibility (MHC)-class II molecule I-Ab+ cells were observed. Treatment with the CD137 agonistic antibody 2A did not significantly affect the atherothrombosis frequency in 16-week-old mice in this model. Conclusions: Levels of CD137 and CD137 ligand mRNA were higher in advanced atherosclerotic disease compared to control vessels, and treatment with the CD137 agonistic antibody 2A, in a murine model for inducible atherothrombosis promoted vascular inflammation, but had no significant effect on atherothrombosis frequency at this early disease stage.

A trait of inflammation pathogenesis in human atherosclerosis: inflammasome driven interleukin-1 signalling in complex atherosclerotic plaques via hyperlipidemia trained innate immunity Objectives We aimed to investigate interleukin (IL)-1 generation and the regulatory role of inflammasome in human advanced atherosclerosis. Background IL-1β is key contributor to the inflammatory process associated with atherosclerosis and its complications. Recent studies suggested that IL-1β blockade reduces the burden of inflammation and recurrence of cardiovascular events. Yet, other cytokines in IL-1 family and the regulation of IL-1 generation in patients with atherosclerosis remains poorly understood. Methods and results A focused transcriptomic analysis in human atherosclerotic specimens discovered that human atherosclerotic plaques host a broad reservoir of inflammasome components, characterised by expression of canonical inflammasome gene NLRP6, NLRP12, NLRC4, NLRP3 and non-canonical inflammasome gene caspase 4 significantly elevated in the symptomatic plaques versus the asymptomatic plaques. Upregulation of NLRP3 inflammasome expression in plaque validated by immunohistochemistry staining suggested it as a distinctive characteristic of plaque vulnerability and complexity. Functional studies on atherosclerotic explants obtained from patients undergoing carotid endarterectomy revealed constitutive generation of IL-1β accompanied by secretion of comparable levels of IL-1α from the majority of the plaques, while IL-18 and IL-33 generation from some of the plaques. Stimulation of the plaques with inflammasome activators showed an inducible generation of both IL-1α and IL-1β, not IL-18 or IL-33, mediated by specific canonical and non-canonical inflammasome pathways. Analysis on the medication records of these patients indicated that plaques from patients with suboptimally controlled hyperlipidemia, imaging signs for plaque instability and inadequate statins therapy possessed higher recruitable production of IL-1β, suggesting the conventional atherogenic factor in regulation of inflammasome immunity and disease activity. Mechanistic studies on tissue and cells isolated from atheromatous plaques demonstrate that generation of mature IL-1β is via a mechanism controlled by NLRP3 and the effector caspase-1. Conclusions The study supports a profound canonical and non-canonical inflammasomes mediated plaque IL-1α/β generation, via a key mechanism by NLRP3 and caspase-1. The results provide biological insights into the clinical merit of high-intensity cholesterol lowering and anti-IL-1 signalling-directed therapies in high-risk patients with atherosclerosis. Acknowledgement/Funding KI-Mayo collaboration project, the Swedish Research Council, the Swedish Heart-Lung Foundation, European Union FP7 projects, the NIH

Atherosclerosis, the formation of fibrofatty lesions in the artery wall, causes much morbidity and mortality worldwide, including most myocardial infarctions and many strokes, as well as disabling peripheral artery disease. Development of atherosclerotic lesions probably requires low-density lipoprotein, a particle that carries cholesterol through the blood. Other risk factors for atherosclerosis and its thrombotic complications include hypertension, cigarette smoking and diabetes mellitus. Increasing evidence also points to a role of the immune system, as emerging risk factors include inflammation and clonal haematopoiesis. Studies of the cell and molecular biology of atherogenesis have provided considerable insight into the mechanisms that link all these risk factors to atheroma development and the clinical manifestations of this disease. An array of diagnostic techniques, both invasive (such as selective coronary arteriography) and noninvasive (such as blood biomarkers, stress testing, CT and nuclear scanning), permit assessment of cardiovascular disease risk and targeting of therapies. An expanding armamentarium of therapies that can modify risk factors and confer clinical benefit is available; however, we face considerable challenge in providing equitable access to these treatments and in maximizing adherence. Yet, the clinical application of the fruits of research has advanced preventive strategies, enhanced clinical outcomes in affected individuals, and improved their quality of life. Rapidly accelerating knowledge and continued research promise to provide further progress in combating this common chronic disease.

Background: Atherosclerosis progression is modulated by interactions with the adaptive immune system. Humoral immunity can help protect against atherosclerosis formation; however, the existence, origin, and function of putative atherogenic antibodies are controversial. How such atherosclerosis-promoting antibodies could affect the specific composition and stability of plaques, as well as the vasculature generally, remains unknown. Methods: We addressed the overall contribution of antibodies to atherosclerosis plaque formation, composition, and stability in vivo (1) with mice that displayed a general loss of antibodies, (2) with mice that had selectively ablated germinal center-derived IgG production, or (3) through interruption of T–B-cell interactions and further studied the effects of antibody deficiency on the aorta by transcriptomics. Results: Here, we demonstrate that atherosclerosis-prone mice with attenuated plasma cell function manifest reduced plaque burden, indicating that antibodies promote atherosclerotic lesion size. However, the composition of the plaque was altered in antibody-deficient mice, with an increase in lipid content and decreases in smooth muscle cells and macrophages, resulting in an experimentally validated vulnerable plaque phenotype. Furthermore, IgG antibodies enhanced smooth muscle cell proliferation in vitro in an Fc receptor–dependent manner, and antibody-deficient mice had decreased neointimal hyperplasia formation in vivo. These IgG antibodies were shown to be derived from germinal centers, and mice genetically deficient for germinal center formation had strongly reduced atherosclerosis plaque formation. mRNA sequencing of aortas revealed that antibodies are required for the sufficient expression of multiple signal-induced and growth-promoting transcription factors and that aortas undergo large-scale metabolic reprograming in their absence. Using an elastase model, we demonstrated that absence of IgG results in an increased severity of aneurysm formation. Conclusions: We propose that germinal center–derived IgG antibodies promote the size and stability of atherosclerosis plaques, through promoting arterial smooth muscle cell proliferation and maintaining the molecular identity of the aorta. These results could have implications for therapies that target B cells or B–T-cell interactions because the loss of humoral immunity leads to a smaller but less stable plaque phenotype.

The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis.The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb−/−Apoe−/− mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb−/−Apoe−/− macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb−/−Apoe−/− CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb−/−Apoe−/− bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells. Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.

Background and aims Endothelin-1 (ET-1) and arginase are both suggested to be involved in the inflammatory processes and development of endothelial dysfunction in atherosclerosis. However, information regarding the roles of ET-1 and arginase, as well as the interactions between the two in human atherosclerosis, is scarce. We investigated the expression of ET-1 and its receptors, ETA and ETB, as well as arginase in human carotid atherosclerotic plaques and determined the functional interactions between ET-1 and arginase in endothelial cells and THP-1-derived macrophages. Methods Carotid plaques and blood samples were retreived from patients undergoing surgery for symptomatic or asymptomatic carotid stenosis. Plaque gene and protein expression was determined and related to clinical characteristics. Functional interactions between ET-1 and arginase were investigated in endothelial cells and THP-1 cells. Results Expression of ET-1 and ETB receptors was increased in plaques from patients with symptomatic carotid artery disease. ET-1 was co-localized with arginase 1 and arginase 2 in the necrotic core, together with macrophage markers CD163 and CD68. Arginase 2, ET-1 and ETB receptors were expressed in endothelial cells as well as in smooth muscle cells in the fibrous cap. ET-1 increased arginase 2 mRNA expression and arginase activity in endothelial cells and arginase activity in macrophages. Moreover, ET-1 stimulated formation of reactive oxygen species (ROS) in THP-1-derived macrophages via an arginase-dependent mechanism. Conclusions This is the first study that demonstrates co-localization of ET-1 and arginase 2 in human atherosclerotic plaques. ET-1 stimulated arginase 2 expression and activity in endothelial cells, as well as arginase activity and ROS formation in macrophages via an arginase-dependent mechanism. These results indicate an important interaction between the ET pathway and arginase in human atherosclerotic plaques.

Aims: Processes in the development of atherosclerotic lesions can lead to plaque rupture or erosion, which can in turn elicit myocardial infarction or ischaemic stroke. The aims of this study were to determine whether Toll-like receptor 7 (TLR7) gene expression levels influence patient outcome and to explore the mechanisms linked to TLR7 expression in atherosclerosis. Methods and Results: Atherosclerotic plaques were removed by carotid endarterectomy (CEA) and subjected to gene array expression analysis (n=123). Increased levels of TLR7 transcript in the plaques were associated with better outcome in a follow-up study over a maximum of 8 years. Patients with higher TLR7 transcript levels had a lower risk of experiencing major cardiovascular and cerebrovascular events (MACCE) during the follow-up period after CEA (hazard ratio: 2.38, p=0.012, 95% CI 1.21-4.67). TLR7 was expressed in all plaques by T cells, macrophages and endothelial cells in capillaries, as shown by immunohistochemistry. In short-term tissue cultures, ex vivo treatment of plaques with the TLR7 ligand imiquimod elicited dose-dependent secretion of IL-10, TNF-α, GM-CSF, and IL-12/IL-23p40. This secretion was blocked with a TLR7 inhibitor. Immunofluorescent tissue analysis after TLR7 stimulation showed IL-10 expression in T cells, macrophages and vascular smooth muscle cells. TLR7 mRNA levels in the plaques were correlated with IL-10 receptor (r=0.4031, p

Objective— Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. Approach and Results— Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7 , and PLN expression positively correlated to typical SMC markers in plaques (Pearson r >0.6, P r >0.8, P PDLIM7, PLN , and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. Conclusions— We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation.

Atherosclerosis is a chronic inflammatory disease that is initiated by the retention and accumulation of cholesterol-containing lipoproteins, particularly low-density lipoprotein, in the artery wall. In the arterial intima, lipoprotein components that are generated through oxidative, lipolytic, and proteolytic activities lead to the formation of several danger-associated molecular patterns, which can activate innate immune cells as well as vascular cells. Moreover, self- and non-self-antigens, such as apolipoprotein B-100 and heat shock proteins, can contribute to vascular inflammation by triggering the response of T and B cells locally. This process can influence the initiation, progression, and stability of plaques. Substantial clinical and experimental data support that the modulation of adaptive immune system may be used for treating and preventing atherosclerosis. This may lead to the development of more selective and less harmful interventions, while keeping host defense mechanisms against infections and tumors intact. Approaches such as vaccination might become a realistic option for cardiovascular disease, especially if they can elicit regulatory T and B cells and the secretion of atheroprotective antibodies. Nevertheless, difficulties in translating certain experimental data into new clinical therapies remain a challenge. In this review, we discuss important studies on the function of T- and B-cell immunity in atherosclerosis and their manipulation to develop novel therapeutic strategies against cardiovascular disease.

Supplemental Digital Content is available in the text., Objective— The V1 (VHS107.1.42) immunoglobulin heavy chain gene is thought to be critical in producing IgM natural antibodies of the T15-idiotype that protect against both atherosclerosis and infection from Streptococcus pneumoniae. Our aim was to determine whether genetic loss of the V1 gene increased atherosclerotic plaque burden in vivo because of a reduction in the T15-idiotype or other atheroprotective antibodies. Approach and Results— We crossed VHS107.1.42-deficient mice with the atherosclerosis-prone Apoe−/− and Ldlr−/− strains. Although these double knockout strains manifested no defects in B-cell development, we did observe a substantial reduction in early immune responses against phosphocholine after immunization. However, the titers of plasma antibodies reacting against defined atherosclerotic antigens such as oxidized low-density lipoprotein, as well as the T15-idiotype, were unaffected by loss of the VHS107.1.42 gene in hypercholesterolemic mice. Furthermore, we observed no increase in atherosclerotic lesion formation, either within the aortic arch or aortic root. Robust deposition of IgM within atherosclerotic plaques could also be readily observed in both control and experimental mice. Conclusions— Our data indicate that IgM-dependent protection against atherosclerosis is unlikely to be dependent on antibodies that use the VHS107.1.42 gene, in contrast to the acute immune response conferred by this heavy chain in the response to phosphocholine and in providing resistance against lethal S pneumoniae infection.

Atherosclerosis is a chronic inflammatory disease that is mediated by innate and adaptive immune responses. The disease is characterized by sub-endothelial accumulation and modification of lipids in the artery wall triggering an inflammatory reaction which promotes lesion progression and eventual plaque rupture, thrombus formation, and the respective clinical sequelae such as myocardial infarction or stroke. During the past decade, T-cell-mediated immune responses, especially control of pro-inflammatory signals by regulatory T cells (Tregs), have increasingly attracted the interest of experimental and clinical researchers. By suppression of T cell proliferation and secretion of anti-inflammatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor-β, Tregs exert their atheroprotective properties. Atherosclerosis-prone, hyperlipidemic mice harbor systemically less Tregs compared to wild-type mice, suggesting an imbalance of immune cells which affects local and systemic inflammatory and potentially metabolic processes leading to atherogenesis. Restoring or increasing Treg frequency and enhancing their suppressive capacity by various modulations may pose a promising approach for treating inflammatory conditions such as cardiovascular diseases. In this review, we briefly summarize the immunological basics of atherosclerosis and introduce the role and contribution of different subsets of T cells. We then discuss experimental data and current knowledge pertaining to Tregs in atherosclerosis and perspectives on manipulating the adaptive immune system to alleviate atherosclerosis and cardiovascular disease.

Does inflammation matter in coronary artery disease? Is it a driving force in atherosclerosis or merely an epiphenomenon? Is there space for novel therapies besides those targeting cholesterol? The CANTOS trial (Canakinumab Anti-inflammatory Thrombosis Outcomes Study), which was presented at the European Society of Cardiology meeting in Barcelona a few weeks ago, provides answers to these important questions. Indeed, the results of this trial open up a new avenue for cardiovascular prevention. Atherosclerosis, the underlying pathology in most cases of myocardial infarction, ischemic stroke, and ischemic gangrene, is a disease of complex genesis that has remained controversial for decades. Clinical and experimental studies have provided unequivocal evidence for an etiologic role of cholesterol and also offered strong support for a pathogenic role of inflammation.1 By targeting cholesterol, blood pressure, and cigarette smoking, cardiovascular prevention has become one of the great success stories in medicine. We have witnessed a reduction in the incidence of myocardial infarction by ≈40% in large parts of the world during the past 2 decades, and a substantial part of this decrease can be ascribed to preventive medicine.1 Cholesterol-lowering statin therapy reduces the incidence of major adverse cardiovascular events by 25% to 50% in individuals with elevated low-density lipoprotein cholesterol and has become standard therapy for …

AimsAtherosclerosis is a chronic inflammatory disease involving immunological and metabolic processes. Metabolism of tryptophan (Trp) via the kynurenine pathway has shown immunomodulatory properties and the ability to modulate atherosclerosis. We identified 3-hydroxyanthranilic acid (3-HAA) as a key metabolite of Trp modulating vascular inflammation and lipid metabolism. The molecular mechanisms driven by 3-HAA in atherosclerosis have not been completely elucidated. In this study, we investigated whether two major signalling pathways, activation of SREBPs and inflammasome, are associated with the 3-HAA-dependent regulation of lipoprotein synthesis and inflammation in the atherogenesis process. Moreover, we examined whether inhibition of endogenous 3-HAA degradation affects hyperlipidaemia and plaque formation.Methods and resultsIn vitro, we showed that 3-HAA reduces SREBP-2 expression and nuclear translocation and apolipoprotein B secretion in HepG2 cell cultures, and inhibits inflammasome activation and IL-1β production by macrophages. Using Ldlr−/− mice, we showed that inhibition of 3-HAA 3,4-dioxygenase (HAAO), which increases the endogenous levels of 3-HAA, decreases plasma lipids and atherosclerosis. Notably, HAAO inhibition led to decreased hepatic SREBP-2 mRNA levels and lipid accumulation, and improved liver pathology scores.ConclusionsWe show that the activity of SREBP-2 and the inflammasome can be regulated by 3-HAA metabolism. Moreover, our study highlights that targeting HAAO is a promising strategy to prevent and treat hypercholesterolaemia and atherosclerosis.

Objective— Dyslipidemia is a component of the metabolic syndrome, an established risk factor for atherosclerotic cardiovascular disease, and is also observed in various autoimmune and chronic inflammatory conditions. However, there are limited opportunities to study the impact of acquired dyslipidemia on cardiovascular and immune pathology. Approach and Results— We designed a model system that allows for the conversion to a state of acute hyperlipidemia in adult life, so that the consequences of such a transition could be observed, through conditionally deleting APOE (apolipoprotein E) in the adult mouse. The transition to hypercholesterolemia was accompanied by adaptive immune responses, including the expansion of T lymphocyte helper cell 1, T follicular helper cell, and T regulatory subsets and the formation of germinal centers. Unlike steady-state Apoe −/− mice, abrupt loss of APOE induced rapid production of antibodies recognizing rheumatoid disease autoantigens. Genetic ablation of the germinal center reduced both autoimmunity and atherosclerosis, indicating that the immune response that follows loss of APOE is independent of atherosclerosis but nevertheless promotes plaque development. Conclusions— Our findings suggest that immune activation in response to hyperlipidemia could contribute to a wide range of inflammatory autoimmune diseases, including atherosclerosis.

T-cell activation is characteristic during the development of atherosclerosis. While overall T-cell responses have been implicated in disease acceleration, regulatory T cells (Tregs) exhibit atheroprotective effects. The expression of the enzyme indoleamine 2,3-dioxygenase-1 (IDO1), which catalyzes the degradation of tryptophan (Trp) along the kynurenine pathway, has been implicated in the induction and expansion of Treg populations. Hence, Tregs can reciprocally promote IDO1 expression in dendritic cells (DCs) via reverse signaling mechanisms during antigen presentation. In this study, we hypothesize that triggering the "Treg/IDO axis" in the artery wall is atheroprotective. We show that apolipoprotein B100-pulsed tumor growth factor beta 2-treated tolerogenic DCs promote de novo FoxP3+ Treg expansion in vivo. This local increase in Treg numbers is associated with increased vascular IDO1 expression and a robust reduction in the atherosclerotic burden. Using human primary cell cultures, we show for the first time that IDO1 expression and activity can be regulated by cytotoxic T-lymphocyte associated protein-4, which is a constitutive molecule expressed and secreted by Tregs, in smooth muscle cells, endothelial cells, and macrophages. Altogether, our data suggest that Tregs and IDO1-mediated Trp metabolism can mutually regulate one another in the vessel wall to promote vascular tolerance mechanisms that limit inflammation and atherosclerosis.

Objective: Understanding molecular processes behind carotid plaque instability is necessary to develop methods that can identify patients and lesions at risk of stroke. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound (US). Results: Plaque echogenicity measured by US was correlated to microarray profiles from lesions retrieved at surgery (n=96). Pathway analyses highlighted enrichment of cell apoptosis and proliferation, and BCLAF1 (BCL2 associated factor 1) as the most significantly dysregulated gene (adjusted pIn vitro , stimulation of SMCs with pro-survival factors EGF, bFGF, PDGFB resulted in induction of BCLAF1, while it was suppressed by macrophage-conditioned medium. Moreover, BCLAF1 silencing in SMCs led to downregulation of BCL2 and SMC markers, and a decrease in proliferation and adhesion (p Conclusions: Carotid plaque echogenicity correlated with enrichment of molecular pathways associated with cell survival and apoptosis and identified BCLAF1, previously not described in atherosclerosis, as the most dysregulated gene. Functionally, BCLAF1 appeared to promote SMC survival by transdifferentiation into macrophage-like phenotype, by interacting with BCL2 and THRAP3.

Radiotherapy is an established therapeutic method in many different cancer types. Its success raises a new problem, namely radiotherapy-induced vascular disease (vRTx), which typically manifests as coronary disease, heart failure or carotid stenosis. A chronic inflammatory response likely underlies vRTx, involving complex processes, such as vascular smooth muscle cell de-differentiation, oxidative stress, and ECM remodeling. These processes are tightly regulated and thus far difficult to influence therapeutically. In other diseases, a set of microRNAs (miRs) is known to orchestrate these processes. We hypothesized that they similarly could play a role in vRTx and be targets for treatment or prevention of the disease. We performed qRT-PCR screening of 11 pre-selected miRs, which identified miR-29b as significantly decreased in irradiated arteries collected from patients undergoing free tissue transfer reconstruction, compared with non-irradiated arteries from the same patient (n=15). In a vascular biology context, miR-29b is known as inhibitor of collagen- and ECM associated mRNA targets, and has been shown to play a detrimental role in aneurysm disease and advanced atherosclerotic lesions. Consistent with human tissue data, vascular smooth muscle and endothelial cells in vitro receiving two radiation doses of 2 Gy, showed decreased miR-29b upon 24 hours after exposure. In these irradiated SMCs, miR-29b induction reduced soluble collagen levels, while inhibition further increased them. Array-based tissue gene expression analysis showed that Pentraxin-3 (PTX) and dipeptidyl-peptidase 4 (DPP4), both targets of miR-29b and pivotal in inflammation and adverse wound healing, were downregulated in the same patient cohort. Carotid arteries of Apoe -/- mice treated with miR-29b mimic, 24h before and 24h after irradiation (14 Gy to the upper chest and neck), displayed a downward trend (non-significant) in Ptx3 and Dpp4 gene expression. Our results suggest that miR-29b overexpression therapy could have a place in the prevention of vRTx. To further strengthen this conclusion, additional Apoe -/- mice irradiation experiments are currently ongoing, to further establish PTX3 and DPP4 as mediators of anti-inflammatory and anti-fibrotic strategies.

Supplemental Digital Content is available in the text., Objective— Androgen deprivation therapy has been associated with increased cardiovascular risk in men. Experimental studies support that testosterone protects against atherosclerosis, but the target cell remains unclear. T cells are important modulators of atherosclerosis, and deficiency of testosterone or its receptor, the AR (androgen receptor), induces a prominent increase in thymus size. Here, we tested the hypothesis that atherosclerosis induced by testosterone deficiency in male mice is T-cell dependent. Further, given the important role of the thymic epithelium for T-cell homeostasis and development, we hypothesized that depletion of the AR in thymic epithelial cells will result in increased atherosclerosis. Approach and Results— Prepubertal castration of male atherosclerosis-prone apoE−/− mice increased atherosclerotic lesion area. Depletion of T cells using an anti-CD3 antibody abolished castration-induced atherogenesis, demonstrating a role of T cells. Male mice with depletion of the AR specifically in epithelial cells (E-ARKO [epithelial cell-specific AR knockout] mice) showed increased thymus weight, comparable with that of castrated mice. E-ARKO mice on an apoE−/− background displayed significantly increased atherosclerosis and increased infiltration of T cells in the vascular adventitia, supporting a T-cell–driven mechanism. Consistent with a role of the thymus, E-ARKO apoE−/− males subjected to prepubertal thymectomy showed no atherosclerosis phenotype. Conclusions— We show that atherogenesis induced by testosterone/AR deficiency is thymus- and T-cell dependent in male mice and that the thymic epithelial cell is a likely target cell for the antiatherogenic actions of testosterone. These insights may pave the way for new therapeutic strategies for safer endocrine treatment of prostate cancer.

Aims Radiotherapy-induced cardiovascular disease is an emerging problem in a growing population of cancer survivors where traditional treatments, such as anti-platelet and lipid-lowering drugs, have limited benefits. The aim of the study was to investigate vascular inflammatory patterns in human cancer survivors, replicate the findings in an animal model, and evaluate whether interleukin-1 (IL-1) inhibition could be a potential treatment. Methods and results Irradiated human arterial biopsies were collected during microvascular autologous free tissue transfer for cancer reconstruction and compared with non-irradiated arteries from the same patient. A mouse model was used to study the effects of the IL-1 receptor antagonist, anakinra, on localized radiation-induced vascular inflammation. We observed significant induction of genes associated with inflammasome biology in whole transcriptome analysis of irradiated arteries, a finding supported by elevated protein levels in irradiated arteries of both, pro-caspase and caspase-1. mRNA levels of inflammasome associated chemokines CCL2, CCL5 together with the adhesion molecule VCAM1, were elevated in human irradiated arteries as was the number of infiltrating macrophages. A similar pattern was reproduced in Apoe−/− mouse 10 weeks after localized chest irradiation with 14 Gy. Treatment with anakinra in irradiated mice significantly reduced Ccl2 and Ccl5 mRNA levels and expression of I-Ab. Conclusion Anakinra, administered directly after radiation exposure for 2 weeks, ameliorated radiation induced sustained expression of inflammatory mediators in mice. Further studies are needed to evaluate IL-1 blockade as a treatment of radiotherapy-induced vascular disease in a clinical setting.

Visual Abstract, Highlights • Multiomics profiling of gene and protein expression in carotid plaques, together with protein expression in peripheral and “local” plasma sampled around the lesion, was performed using a large cohort of patients from BiKE in discovery analyses. • BLVRB was identified as a biomarker of intraplaque hemorrhage and plaque instability in carotid atherosclerosis that was further validated in population samples. • BLVRB was characterized as a hitherto unappreciated key enzyme, functionally linked with HMOX1, in the hemoglobin catabolism under pathological conditions. • The novel translational approach applied for biomarker discovery in this study permits causal interpretation of candidates directly in relation to the underlying disease and paves the way for similar investigations in other vascular territories., Summary Clinical tools to identify individuals with unstable atherosclerotic lesions are required to improve prevention of myocardial infarction and ischemic stroke. Here, a systems-based analysis of atherosclerotic plaques and plasma from patients undergoing carotid endarterectomy for stroke prevention was used to identify molecular signatures with a causal relationship to disease. Local plasma collected in the lesion proximity following clamping prior to arteriotomy was profiled together with matched peripheral plasma. This translational workflow identified biliverdin reductase B as a novel marker of intraplaque hemorrhage and unstable carotid atherosclerosis, which should be investigated as a potential predictive biomarker for cardiovascular events in larger cohorts.

Background Embolism from unstable atheromas in the carotid bifurcation is a major cause of stroke. Here, we analysed gene expression in endarterectomies from patients with symptomatic (S) and asymptomatic (AS) carotid stenosis to identify pathways linked to plaque instability. Methods Microarrays were prepared from plaques (n = 127) and peripheral blood samples (n = 96) of S and AS patients. Gene set enrichment, pathway mapping and network analyses of differentially expressed genes were performed. Results These studies revealed upregulation of haemoglobin metabolism (P = 2.20E-05) and bone resorption (P = 9.63E-04) in S patients. Analysis of subgroups of patients indicated enrichment of calcification and osteoblast differentiation in S patients on statins, as well as inflammation and apoptosis in plaques removed >1 month compared to

Atherosclerosis is a maladaptive, nonresolving chronic inflammatory disease that occurs at sites of blood flow disturbance. The disease usually remains silent until a breakdown of integrity at the arterial surface triggers the formation of a thrombus. By occluding the lumen, the thrombus or emboli detaching from it elicits ischaemic symptoms that may be life-threatening. Two types of surface damage can cause atherothrombosis: plaque rupture and endothelial erosion. Plaque rupture is thought to be caused by loss of mechanical stability, often due to reduced tensile strength of the collagen cap surrounding the plaque. Therefore, plaques with reduced collagen content are thought to be more vulnerable than those with a thick collagen cap. Endothelial erosion, on the other hand, may occur after injurious insults to the endothelium instigated by metabolic disturbance or immune insults. This review discusses the molecular mechanisms involved in plaque vulnerability and the development of atherothrombosis.

Toll-like receptors (TLRs) have been implicated in the pathogenesis of type 2 diabetes. We examined the function of TLR3 in glucose metabolism and type 2 diabetes–related phenotypes in animals and humans. TLR3 is highly expressed in the pancreas, suggesting that it can influence metabolism. Using a diet-induced obesity model, we show that TLR3-deficient mice had enhanced glycemic control, facilitated by elevated insulin secretion. Despite having high insulin levels, Tlr3−/− mice did not experience disturbances in whole-body insulin sensitivity, suggesting that they have a robust metabolic system that manages increased insulin secretion. Increase in insulin secretion was associated with upregulation of islet glucose phosphorylation as well as exocytotic protein VAMP-2 in Tlr3−/− islets. TLR3 deficiency also modified the plasma lipid profile, decreasing VLDL levels due to decreased triglyceride biosynthesis. Moreover, a meta-analysis of two healthy human populations showed that a missense single nucleotide polymorphism in TLR3 (encoding L412F) was linked to elevated insulin levels, consistent with our experimental findings. In conclusion, our results increase the understanding of the function of innate receptors in metabolic disorders and implicate TLR3 as a key control system in metabolic regulation.