Publications by authors named "Clément Cochain"

34 Publications

Cytotoxic CD8 T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling.

Nat Commun 2021 03 5;12(1):1483. Epub 2021 Mar 5.

Université de Paris, PARCC, INSERM, F-75015, Paris, France.

Acute myocardial infarction is a common condition responsible for heart failure and sudden death. Here, we show that following acute myocardial infarction in mice, CD8 T lymphocytes are recruited and activated in the ischemic heart tissue and release Granzyme B, leading to cardiomyocyte apoptosis, adverse ventricular remodeling and deterioration of myocardial function. Depletion of CD8 T lymphocytes decreases apoptosis within the ischemic myocardium, hampers inflammatory response, limits myocardial injury and improves heart function. These effects are recapitulated in mice with Granzyme B-deficient CD8 T cells. The protective effect of CD8 depletion on heart function is confirmed by using a model of ischemia/reperfusion in pigs. Finally, we reveal that elevated circulating levels of GRANZYME B in patients with acute myocardial infarction predict increased risk of death at 1-year follow-up. Our work unravels a deleterious role of CD8 T lymphocytes following acute ischemia, and suggests potential therapeutic strategies targeting pathogenic CD8 T lymphocytes in the setting of acute myocardial infarction.
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http://dx.doi.org/10.1038/s41467-021-21737-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935973PMC
March 2021

Immuno-metabolic interfaces in cardiac disease and failure.

Cardiovasc Res 2021 Feb 4. Epub 2021 Feb 4.

Department of Translational Research, Comprehensive Heart Failure Center (CHFC), University Hospital Würzburg, Germany.

The interplay between the cardiovascular system, metabolism, and inflammation plays a central role in the pathophysiology of a wide spectrum of cardiovascular diseases, including heart failure. Here, we provide an overview of the fundamental aspects of the interrelation between inflammation and metabolism, ranging from the role of metabolism in immune cell function to the processes how inflammation modulates systemic and cardiac metabolism. Furthermore, we discuss how disruption of this immuno-metabolic interface is involved in the development and progression of cardiovascular disease, with a special focus on heart failure. Finally, we present new technologies and therapeutic approaches that have recently emerged and hold promise for the future of cardiovascular medicine.
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http://dx.doi.org/10.1093/cvr/cvab036DOI Listing
February 2021

Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction.

Circ Res 2020 Oct 19;127(9):e232-e249. Epub 2020 Aug 19.

Comprehensive Heart Failure Center Wuerzburg (G.R., L.K., A.-P.A.-L., V.A.N., D.J.J.S., C.C.), University Hospital Wuerzburg, Germany.

Rationale: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage.

Objective: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction.

Methods And Results: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (, , ), and putative activity of transcriptional regulators involved in hypoxic response () and emergency granulopoiesis (). At 3 and 5 days, 2 major subsets of (enriched for eg, and ) and () neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of , heart infiltrating neutrophils acquired a unique SiglecF signature. SiglecF neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecF signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecF neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecF neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation.

Conclusions: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecF signature.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317200DOI Listing
October 2020

Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.

Circ Res 2020 Jul 16;127(3):402-426. Epub 2020 Jul 16.

La Jolla Institute for Immunology, CA (C.C.H., Y.G., H.Q.D., K.L.).

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2 foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, , which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.316903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371244PMC
July 2020

The aorta can act as a site of naïve CD4+ T-cell priming.

Cardiovasc Res 2020 02;116(2):306-316

Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow G12 8TA, UK.

Aims: Aortic adaptive immunity plays a role in atherosclerosis; however, the precise mechanisms leading to T-cell activation in the arterial wall remain poorly understood.

Methods And Results: Here, we have identified naïve T cells in the aorta of wild-type and T-cell receptor transgenic mice and we demonstrate that naïve T cells can be primed directly in the vessel wall with both kinetics and frequency of T-cell activation found to be similar to splenic and lymphoid T cells. Aortic homing of naïve T cells is regulated at least in part by the P-selectin glycosylated ligand-1 receptor. In experimental atherosclerosis the aorta supports CD4+ T-cell activation selectively driving Th1 polarization. By contrast, secondary lymphoid organs display Treg expansion.

Conclusion: Our results demonstrate that the aorta can support T-cell priming and that naïve T cells traffic between the circulation and vessel wall. These data underpin the paradigm that local priming of T cells specific for plaque antigens contributes to atherosclerosis progression.
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http://dx.doi.org/10.1093/cvr/cvz102DOI Listing
February 2020

Neutrophils promote atherosclerotic plaque destabilization in a mouse model of endotoxinaemia.

Cardiovasc Res 2018 10;114(12):1573-1574

Institute of Experimental Biomedicine, University Hospital Würzburg, Josef-Schneider-Str. 2, Würzburg, Germany.

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http://dx.doi.org/10.1093/cvr/cvy168DOI Listing
October 2018

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

Circ Res 2018 06 15;122(12):1675-1688. Epub 2018 Mar 15.

Institute of Experimental Biomedicine, University Hospital Würzburg, Germany (C.C., A.Z.)

Rationale: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood.

Objective: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis.

Methods And Results: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed and mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic and mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients.

Conclusions: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.312513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993603PMC
June 2018

Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity of Aortic Macrophages in Murine Atherosclerosis.

Circ Res 2018 06 15;122(12):1661-1674. Epub 2018 Mar 15.

From the Institute of Experimental Biomedicine (C.C., A.Z.)

Rationale: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they have been defined by the expression of a restricted number of markers.

Objective: We have applied single-cell RNA sequencing as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis.

Method And Results: We performed single-cell RNA sequencing of total aortic CD45 cells extracted from the nondiseased (chow fed) and atherosclerotic (11 weeks of high-fat diet) aorta of low-density lipoprotein receptor-deficient () mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortas, whereas monocytes, monocyte-derived dendritic cells, and 2 populations of macrophages were almost exclusively detectable in atherosclerotic aortas, comprising inflammatory macrophages showing enrichment in and previously undescribed TREM2 (triggered receptor expressed on myeloid cells 2) macrophages showing enrichment in . Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these 3 macrophage subsets and monocyte-derived dendritic cells and uncovered putative functions of each cell type. Notably, TREM2 macrophages seemed to be endowed with specialized functions in lipid metabolism and catabolism and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in aortas, indicating relevance of our findings in different stages of atherosclerosis and mouse models.

Conclusions: These data unprecedentedly uncovered the transcriptional landscape and phenotypic heterogeneity of aortic macrophages and monocyte-derived dendritic cells in atherosclerotic and identified previously unrecognized macrophage populations and their gene expression signature, suggesting specialized functions. Our findings will open up novel opportunities to explore distinct myeloid cell populations and their functions in atherosclerosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.312509DOI Listing
June 2018

Deletion of Batf3-dependent antigen-presenting cells does not affect atherosclerotic lesion formation in mice.

PLoS One 2017 3;12(8):e0181947. Epub 2017 Aug 3.

Institute of Experimental Biomedicine, University Hospital Würzburg, Würzburg, Germany.

Atherosclerosis is the main underlying cause for cardiovascular events such as myocardial infarction and stroke and its development might be influenced by immune cells. Dendritic cells (DCs) bridge innate and adaptive immune responses by presenting antigens to T cells and releasing a variety of cytokines. Several subsets of DCs can be discriminated that engage specific transcriptional pathways for their development. Basic leucine zipper transcription factor ATF-like 3 (Batf3) is required for the development of classical CD8α+ and CD103+ DCs. By crossing mice deficient in Batf3 with atherosclerosis-prone low density lipoprotein receptor (Ldlr-/-)-deficient mice we here aimed to further address the contribution of Batf3-dependent CD8α+ and CD103+ antigen-presenting cells to atherosclerosis. We demonstrate that deficiency in Batf3 entailed mild effects on the immune response in the spleen but did not alter atherosclerotic lesion formation in the aorta or aortic root, nor affected plaque phenotype in low density lipoprotein receptor-deficient mice fed a high fat diet. We thus provide evidence that Batf3-dependent antigen-presenting cells do not have a prominent role in atherosclerosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181947PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542449PMC
October 2017

Macrophages in vascular inflammation and atherosclerosis.

Pflugers Arch 2017 04 6;469(3-4):485-499. Epub 2017 Feb 6.

Institute of Experimental Biomedicine, University Hospital Würzburg, Josef-Schneider-Str. 2, D16, 97080, Würzburg, Germany.

Atherosclerosis is characterized by lipid accumulation and chronic inflammation of the arterial wall, and its main complications-myocardial infarction and ischemic stroke-together constitute the first cause of death worldwide. Accumulation of lipid-laden macrophage foam cells in the intima of inflamed arteries has long been recognized as a hallmark of atherosclerosis. However, in recent years, an unexpected complexity in the mechanisms of macrophage accumulation in lesions, in the protective and pathogenic functions performed by macrophages and how they are regulated has been uncovered. Here, we provide an overview of the latest developments regarding the various mechanisms of macrophage accumulation in lesion, the major functional features of lesion macrophages, and how the plaque microenvironment may affect macrophage phenotype. Finally, we discuss how best to apprehend the heterogeneous ontogeny and functionality of atherosclerotic plaque macrophages and argue that moving away from a rigid nomenclature of arbitrarily defined macrophage subsets would be beneficial for research in the field.
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http://dx.doi.org/10.1007/s00424-017-1941-yDOI Listing
April 2017

Coagulation factor XII induces pro-inflammatory cytokine responses in macrophages and promotes atherosclerosis in mice.

Thromb Haemost 2017 01 27;117(1):176-187. Epub 2016 Oct 27.

Dr. Alma Zernecke, Universitätsklinikum Würzburg, Institut für Experimentelle Biomedizin, Josef-Schneider-Str. 2, 97080 Würzburg, Germany, Tel: +49 201 48331, Fax: +49 201 648341, E-mail:

Atherosclerosis is considered a chronic inflammatory disease of the vessel wall. Coagulation pathways and immune responses contribute to disease development. The role of coagulation factor XII (FXII) in vascular inflammation, however, remains controversial. We here investigated the function of FXII in atherosclerosis using apolipoprotein E and FXII-deficient (F12Apoe) mice. Compared to F12Apoe controls, atherosclerotic lesion formation was reduced in F12Apoe mice. This was associated with a decrease in serum interleukin (IL)-1β and IL-12 levels and reduced expression of pro-inflammatory cytokines in the aorta in atherosclerotic F12Apoe mice, as well as diminished Th1-cell differentiation in the aorta, blood, and lymphoid organs. No changes in circulating bradykinin, thrombin-antithrombin-complexes or plasminogen were observed. Mechanistically, activated FXII (FXIIa) was revealed to directly induce bone marrow-derived macrophages to secrete pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1β, IL-12, and IL-6. Exposure of bone marrow-derived antigen presenting cells to FXIIa similarly induced pro-inflammatory cytokines, and an enhanced capacity to trigger antigen-specific interferon γ-production in CD4 T cells. Notably, bone-marrow derived macrophages were capable of directly activating FXII. Moreover, the induction of cytokine expression by FXIIa in macrophages occurred independently of FXII protease enzymatic activity and was decreased upon phospholipase C treatment, suggesting urokinase-type plasminogen activator receptor (uPAR) to confer FXIIa-induced cell signalling. These data reveal FXII to play an important role in atherosclerotic lesion formation by functioning as a strong inducer of pro-inflammatory cytokines in antigen-presenting cells. Targeting of FXII may thus be a promising approach for treating cardiovascular disease.
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http://dx.doi.org/10.1160/TH16-06-0466DOI Listing
January 2017

Protective and pathogenic roles of CD8 T cells in atherosclerosis.

Basic Res Cardiol 2016 11 25;111(6):71. Epub 2016 Oct 25.

Institute of Experimental Biomedicine, University Hospital Würzburg, University of Würzburg, Josef-Schneider-Str. 2, 97080, Würzburg, Germany.

Although infiltration of CD8 T cells in human atherosclerotic lesions has been described 30 years ago, the role of these cells in lesion development has long remained enigmatic. While experimental models hinted at their pro-atherogenic role based on circumstantial evidence, genetic mouse models of cytotoxic CD8 T cell-specific immune deficiency suggested no crucial role of these cells in lesion development. However, in recent years, more refined models of adoptive cell transfer, disruption of specific immune regulatory pathways or monoclonal antibody-mediated cell depletion have proposed both atheroprotective and pro-atherogenic functions for CD8 T cells in atherosclerosis. In particular, MHC class I-restricted CD8 T cell responses may protect from atherosclerosis, and Qa-1 restricted regulatory CD8 T cells have been defined. In addition, regulatory CD8CD25 T cells possess atheroprotective properties. However, CD8 T cells can also promote monopoiesis in hyperlipidemia, and exert prototypical cytotoxic functions to promote vascular inflammation and macrophage accumulation leading to atherosclerotic lesion development. Here, we review these findings, mostly from experimental studies that reveal a previously unrecognized complexity and important role of CD8 T cells in atherosclerosis.
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http://dx.doi.org/10.1007/s00395-016-0589-7DOI Listing
November 2016

Myeloid-Epithelial-Reproductive Receptor Tyrosine Kinase and Milk Fat Globule Epidermal Growth Factor 8 Coordinately Improve Remodeling After Myocardial Infarction via Local Delivery of Vascular Endothelial Growth Factor.

Circulation 2016 Mar 27;133(9):826-39. Epub 2016 Jan 27.

From INSERM UMRS 970, Université Paris Descartes, Sorbonne Paris Cité, France (K.-Y.H., I.Z., C.P., A.N., M.R., J.V., M.L., C.S., Z.M., J.-S.S.); Institute of Clinical Biochemistry and Pathobiochemistry, University Hospital Würzburg, Germany (C.C.); Institute of Physiology I, Life & Brain Center, University of Bonn, Germany (B.K.F.); and Division of Cardiovascular Medicine, University of Cambridge, Addenbrooke's Hospital, UK (Z.M.).

Background: In infarcted heart, improper clearance of dying cells by activated neighboring phagocytes may precipitate the transition to heart failure. We analyzed the coordinated role of 2 major mediators of efferocytosis, the myeloid-epithelial-reproductive protein tyrosine kinase (Mertk) and the milk fat globule epidermal growth factor (Mfge8), in directing cardiac remodeling by skewing the inflammatory response after myocardial infarction.

Methods And Results: We generated double-deficient mice for Mertk and Mfge8 (Mertk(-/-)/Mfge8(-/-)) and challenged them with acute coronary ligature. Compared with wild-type, Mertk-deficient (Mertk(-/-)), or Mfge8-deficient (Mfge8(-/-)) animals, Mertk(-/-)/Mfge8(-/-) mice displayed greater alteration in cardiac function and remodeling. Mertk and Mfge8 were expressed mainly by cardiac Ly6C(High and Low) monocytes and macrophages. In parallel, Mertk(-/-)/Mfge8(-/-) bone marrow chimeras manifested increased accumulation of apoptotic cells, enhanced fibrotic area, and larger infarct size, as well as reduced angiogenesis. We found that the abrogation of efferocytosis affected neither the ability of circulating monocytes to infiltrate cardiac tissue nor the number of resident Ly6C(High) and Ly6C(How) monocytes/macrophages populating the infarcted milieu. In contrast, combined Mertk and Mfge8 deficiency in Ly6C(High)/Ly6C(Low) monocytes/macrophages either obtained from in vitro differentiation of bone marrow cells or isolated from infarcted hearts altered their capacity of efferocytosis and subsequently blunted vascular endothelial growth factor A (VEGFA) release. Using LysMCre(+)/VEGFA(fl/fl) mice, we further identified an important role for myeloid-derived VEGFA in improving cardiac function and angiogenesis.

Conclusions: After myocardial infarction, Mertk- and Mfge8-expressing monocyte/macrophages synergistically engage the clearance of injured cardiomyocytes, favoring the secretion of VEGFA to locally repair the dysfunctional heart.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.115.020857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767109PMC
March 2016

Deficiency of HIF1α in Antigen-Presenting Cells Aggravates Atherosclerosis and Type 1 T-Helper Cell Responses in Mice.

Arterioscler Thromb Vasc Biol 2015 Nov 24;35(11):2316-25. Epub 2015 Sep 24.

From the Institute of Clinical Biochemistry and Pathobiochemistry, University Hospital Würzburg (S.M.C., M.K., C.C., A.Z.), Rudolf Virchow Center (H.D.M., M.B., H.M.H.), Institute of Virology and Immunobiology (M.B.L.), and Division of Hepatology, Medical Clinic II, University Hospital Würzburg (H.M.H.), University of Würzburg, Würzburg, Germany; Department of Pathology, Maastricht University Medical Centre, Cardiovascular Research Institute Maastricht, Maastricht, The Netherlands (J.C.S., T.L.T.); Joslin Diabetes Center, Harvard Medical School, Boston, MA (C.C.-F., S.K.); Experimental and Molecular Pediatric Cardiology, German Heart Center Munich, TU Munich, Munich, Germany (F.V., A.G.); Department of Vascular and Endovascular Surgery, Klinikum Rechts der Isar der Technischen Universität München, Munich, Germany (J.P.); and Department of Pathology, Amsterdam Medical Centre, Amsterdam, The Netherlands (M.J.D.).

Objective: Although immune responses drive the pathogenesis of atherosclerosis, mechanisms that control antigen-presenting cell (APC)-mediated immune activation in atherosclerosis remain elusive. We here investigated the function of hypoxia-inducible factor (HIF)-1α in APCs in atherosclerosis.

Approach And Results: We found upregulated HIF1α expression in CD11c(+) APCs within atherosclerotic plaques of low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Conditional deletion of Hif1a in CD11c(+) APCs in high-fat diet-fed Ldlr(-/-) mice accelerated atherosclerotic plaque formation and increased lesional T-cell infiltrates, revealing a protective role of this transcription factor. HIF1α directly controls Signal Transducers and Activators of Transcription 3 (Stat3), and a reduced STAT3 expression was found in HIF1α-deficient APCs and aortic tissue, together with an upregulated interleukin-12 expression and expansion of type 1 T-helper (Th1) cells. Overexpression of STAT3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell expansion in chimeric Ldlr(-/-) mice. Notably, deletion of Hif1a in LysM(+) bone marrow cells in Ldlr(-/-) mice did not affect lesion formation or T-cell activation. In human atherosclerotic lesions, HIF1α, STAT3, and interleukin-12 protein were found to colocalize with APCs.

Conclusions: Our findings identify HIF1α to antagonize APC activation and Th1 T cell polarization during atherogenesis in Ldlr(-/-) mice and to attenuate the progression of atherosclerosis. These data substantiate the critical role of APCs in controlling immune mechanisms that drive atherosclerotic lesion development.
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http://dx.doi.org/10.1161/ATVBAHA.115.306171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618712PMC
November 2015

Stimulating arteriogenesis but not atherosclerosis: IFN-α/β receptor subunit 1 as a novel therapeutic target.

Cardiovasc Res 2015 Jul 17;107(2):200-2. Epub 2015 Jun 17.

Institute of Clinical Biochemistry and Pathobiochemistry, University Hospital Würzburg, Josef-Schneider-Str. 2, Würzburg 97080, Germany

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http://dx.doi.org/10.1093/cvr/cvv174DOI Listing
July 2015

Thrombin receptor PAR-1 activation on endothelial progenitor cells enhances chemotaxis-associated genes expression and leukocyte recruitment by a COX-2-dependent mechanism.

Angiogenesis 2015 Jul 31;18(3):347-59. Epub 2015 May 31.

Sorbonne Paris Cité, Université Paris Descartes, Paris, France.

Background: Endothelial colony forming cells (ECFC) represent a subpopulation of endothelial progenitor cells involved in endothelial repair. The activation of procoagulant mechanisms associated with the vascular wall's inflammatory responses to injury plays a crucial role in the induction and progression of atherosclerosis. However, little is known about ECFC proinflammatory potential.

Aims: To explore the role of the thrombin receptor PAR-1 proinflammatory effects on ECFC chemotaxis/recruitment capacity.

Methods And Results: The expression of 30 genes known to be associated with inflammation and chemotaxis was quantified in ECFC by real-time qPCR. PAR-1 activation with the SFLLRN peptide (PAR-1-ap) resulted in a significant increase in nine chemotaxis-associated genes expression, including CCL2 and CCL3 whose receptors are present on ECFC. Furthermore, COX-2 expression was found to be dramatically up-regulated consequently to PAR-1 activation. COX-2 silencing with the specific COX-2-siRNA also triggered down-regulation of the nine target genes. Conditioned media (c.m.) from control-siRNA- and COX-2-siRNA-transfected ECFC, stimulated or not with PAR-1-ap, were produced and tested on ECFC capacity to recruit leukocytes in vitro as well in the muscle of ischemic hindlimb in a preclinical model. The capacity of the c.m. from ECFC stimulated with PAR-1-ap to recruit leukocytes was abrogated when COX-2 gene expression was silenced in vitro (in terms of U937 cells migration and adhesion to endothelial cells) as well as in vivo. Finally, the postnatal vasculogenic stem cell derived from infantile hemangioma tumor (HemSC) incubated with PAR-1-ap increased leukocyte recruitment in Matrigel(®) implant.

Conclusions: PAR-1 activation in ECFC increases chemotactic gene expression and leukocyte recruitment at ischemic sites through a COX-2-dependent mechanism.
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http://dx.doi.org/10.1007/s10456-015-9471-8DOI Listing
July 2015

CD8+ T Cells Regulate Monopoiesis and Circulating Ly6C-high Monocyte Levels in Atherosclerosis in Mice.

Circ Res 2015 Jul 19;117(3):244-53. Epub 2015 May 19.

From the Institute of Clinical Biochemistry and Pathobiochemistry, University Hospital Würzburg (C.C., M.K., S.M.C., A.Z.) and Rudolf Virchow Center (M.B.), University of Würzburg, Würzburg, Germany; Department of Vascular Surgery, Klinikum rechts der Isar, Technical University Munich, Munich, Germany (J.P.); and Bioceros, Utrecht, The Netherlands (L.B.).

Rationale: Proinflammatory adaptive immune responses are recognized as major drivers of atherosclerotic lesion formation. Although CD8(+) T cells have recently been proposed as a proatherogenic cell subset, their full scope of actions remains to be elucidated.

Objective: We here addressed the contribution of CD8(+) T cells to monocyte trafficking in atherosclerosis.

Method And Results: We observed that CD8(+) T cells express proinflammatory cytokines (interferon-γ, tumor necrosis factor-α, and interleukin-12) within atherosclerotic lesions and spleens of high-fat diet-fed low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Antibody-mediated CD8(+) T-cell depletion in high-fat diet-fed Ldlr(-/-) mice decreased atherosclerotic plaque formation, associated with decreased macrophage accumulation within lesions. Despite a reduction in vascular chemokine (CC-motif) ligand 2 and chemokine (CXC-motif) ligand 1 expression, CD8(+) T-cell depletion did not directly affect monocyte recruitment to inflamed vessels. However, CD8(+) T-cell depletion decreased chemokine (CC-motif) ligand serum concentrations and circulating Ly6C(high) monocyte counts. We further evidenced that CD8(+) T-cell depletion decreased levels of mature monocytes and myeloid granulocyte-monocyte progenitors in the bone marrow and spleen of hypercholesterolemic mice, effects that were partially reproduced by interferon-γ neutralization, showing a role for interferon-γ.

Conclusions: These data suggest that CD8(+) T cells promote atherosclerosis by controlling monopoiesis and circulating monocyte levels, which ultimately contributes to plaque macrophage burden without affecting direct monocyte recruitment, identifying this cell subset as a critical regulator of proatherogenic innate immune cell responses in atherosclerosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.304611DOI Listing
July 2015

Macrophages and immune cells in atherosclerosis: recent advances and novel concepts.

Basic Res Cardiol 2015 7;110(4):34. Epub 2015 May 7.

Institute of Clinical Biochemistry and Pathobiochemistry, University Hospital Würzburg, Josef-Schneider-Str. 2, 97080, Würzburg, Germany.

Atherosclerotic lesion-related thrombosis is the major cause of myocardial infarction and stroke, which together constitute the leading cause of mortality worldwide. The inflammatory response is considered as a predominant driving force in atherosclerotic plaque formation, growth and progression towards instability and rupture. Notably, accumulation of macrophages in the intima and emergence of a pro-inflammatory milieu are a characteristic feature of plaque progression, and these processes can be modulated by adaptive immune responses. Recently, novel evidences of onsite proliferation of macrophages in lesions and transdifferentiation of smooth muscle cells to macrophages have challenged the prevalent paradigm that macrophage accumulation mostly relies on recruitment of circulating monocytes to plaques. Furthermore, previously unrecognized roles of inflammatory cell subsets such as plasmacytoid dendritic cells, innate response activator B cells or CD8(+) T cells in atherosclerosis have emerged, as well as novel mechanisms by which regulatory T cells or natural killer T cells contribute to lesion formation. Here, we review and discuss these recent advances in our understanding of inflammatory processes in atherosclerosis.
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http://dx.doi.org/10.1007/s00395-015-0491-8DOI Listing
February 2016

Noncoding RNAs in vascular inflammation and atherosclerosis: recent advances toward therapeutic applications.

Curr Opin Lipidol 2014 Oct;25(5):380-6

Institute of Clinical Biochemistry and Pathobiochemistry, University Hospital Würzburg, Würzburg, Germany.

Purpose Of Review: We here highlight recent studies that in vivo demonstrate an involvement of microRNAs in atherosclerotic lesion formation and provide important preclinical evidence of their therapeutic targeting in atherosclerosis, with a particular focus on endothelial cells and macrophages. We also briefly discuss the emerging role of long noncoding RNAs herein.

Recent Findings: Noncoding RNAs have received considerable attention as regulators of different cell types and functions that dictate the inflammatory response in atherosclerosis. In particular, microRNAs have emerged to control endothelial cell functions by acting as mechanosensors that are regulated by flow, determinants of inflammation in the context of cytokine exposure and hypercholesterolemia and guardians of endothelial homeostasis. In addition, microRNAs control macrophage-driven cytokine production and polarization, and regulate cholesterol metabolism and foam cell formation. By these (cell specific) effects, microRNAs contain or drive atherosclerotic lesion formation and progression in animal models of disease and can be harnessed for therapeutic targeting.

Summary: Given their multifaceted and specific contribution to vascular inflammation and atherosclerosis, and proven amenability for successful modulation in preclinical murine models of atheroscleorosis and large animal studies, miRNAs appear as promising therapeutic targets for treating atherosclerosis.
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http://dx.doi.org/10.1097/MOL.0000000000000108DOI Listing
October 2014

Programmed cell death-1 deficiency exacerbates T cell activation and atherogenesis despite expansion of regulatory T cells in atherosclerosis-prone mice.

PLoS One 2014 1;9(4):e93280. Epub 2014 Apr 1.

Institute of Clinical Biochemistry and Pathobiochemistry, University Hospital Würzburg, Würzburg, Germany; Department of Vascular Surgery, Klinikum rechts der Isar, Technical University Munich, Munich, Germany.

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3(+) regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4(+) T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr(-/-)Pd1(-/-)) displayed striking increases in systemic CD4(+) and CD8(+) T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3+ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr(-/-)Pd1(-/-) mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr(-/-) mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093280PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972211PMC
December 2015

On-site education of VEGF-recruited monocytes improves their performance as angiogenic and arteriogenic accessory cells.

J Exp Med 2013 Nov 28;210(12):2611-25. Epub 2013 Oct 28.

Department of Developmental Biology and Cancer Research, the Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

Adult neovascularization relies on the recruitment of monocytes to the target organ or tumor and functioning therein as a paracrine accessory. The exact origins of the recruited monocytes and the mechanisms underlying their plasticity remain unclear. Using a VEGF-based transgenic system in which genetically tagged monocytes are conditionally summoned to the liver as part of a VEGF-initiated angiogenic program, we show that these recruited cells are derived from the abundant pool of circulating Ly6C(hi) monocytes. Remarkably, however, upon arrival at the VEGF-induced organ, but not the naive organ, monocytes undergo multiple phenotypic and functional changes, endowing them with enhanced proangiogenic capabilities and, importantly, with a markedly increased capacity to remodel existing small vessels into larger conduits. Notably, monocytes do not differentiate into long-lived macrophages, but rather appear as transient accessory cells. Results from transfers of presorted subpopulations and a novel tandem transfer strategy ruled out selective recruitment of a dedicated preexisting subpopulation or onsite selection, thereby reinforcing active reprogramming as the underlying mechanism for improved performance. Collectively, this study uncovered a novel function of VEGF, namely, on-site education of recruited "standard" monocytes to become angiogenic and arteriogenic professional cells, a finding that may also lend itself for a better design of angiogenic therapies.
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http://dx.doi.org/10.1084/jem.20120690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832929PMC
November 2013

CCR6 selectively promotes monocyte mediated inflammation and atherogenesis in mice.

Thromb Haemost 2013 Dec 10;110(6):1267-77. Epub 2013 Oct 10.

Univ.-Prof. Dr. Alma Zernecke, Klinikum rechts der Isar, Klinik für Gefäßchirurgie, Technische Universität München, Ismaninger Str. 22, 81675 München, Germany, Tel: +49 89 4140 2167, Fax: +49 89 4140 4861, E-mail:

The chemokine receptor CCR6 is expressed by various cell subsets implicated in atherogenesis, such as monocytes, Th17 and regulatory T cells. In order to further define the role of CCR6 in atherosclerosis, CCR6-deficient (Ccr6-/-) mice were crossed with low-density lipoprotein receptor-deficient (Ldlr-/-) mice to generate atherosclerosis-prone mice deficient in CCR6. Compared to Ldlr-/- controls, atherosclerotic burden in the aortic sinus and aorta were reduced in Ccr6-/-Ldlr-/- mice fed a high fat diet, associated with a profound depression in lesional macrophage accumulation. Local and systemic distributions of T cells, including frequencies of Th1, Th17 and regulatory T cells were unaltered. In contrast, circulating counts of both Gr-1(high) and Gr1(low) monocytes were reduced in Ccr6-/-Ldlr-/- mice. Moreover, CCR6 was revealed to promote monocyte adhesion to inflamed endothelium in vitro and leukocyte adhesion to carotid arteries in vivo. Finally, CCR6 selectively recruited monocytes but not T cells in an acute inflammatory air pouch model. We here show that CCR6 functions on multiple levels and regulates the mobilisation, adhesion and recruitment of monocytes/macrophages to the inflamed vessel, thereby promoting atherosclerosis, but is dispensable for hypercholesterolaemia-associated adaptive immune priming. Targeting CCR6 or its ligand CCL20 may therefore be a promising therapeutic strategy to alleviate atherosclerosis.
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http://dx.doi.org/10.1160/TH13-01-0017DOI Listing
December 2013

Angiogenesis in the infarcted myocardium.

Antioxid Redox Signal 2013 Mar 25;18(9):1100-13. Epub 2012 Sep 25.

Paris Cardiovascular Research Center, INSERM UMR-S 970, Paris Descartes University, Paris, France.

Significance: Proangiogenic therapy appeared a promising strategy for the treatment of patients with acute myocardial infarction (MI), as de novo formation of microvessels, has the potential to salvage ischemic myocardium at early stages after MI, and is also essential to prevent the transition to heart failure through the control of cardiomyocyte hypertrophy and contractility.

Recent Advances: Exciting preclinical studies evaluating proangiogenic therapies for MI have prompted the initiation of numerous clinical trials based on protein or gene transfer delivery of growth factors and administration of stem/progenitor cells, mainly from bone marrow origin. Nonetheless, these clinical trials showed mixed results in patients with acute MI.

Critical Issues: Even though methodological caveats, such as way of delivery for angiogenic growth factors (e.g., protein vs. gene transfer) and stem/progenitor cells or isolation/culture procedure for regenerative cells might partially explain the failure of such trials, it appears that delivery of a single growth factor or cell type does not support angiogenesis sufficiently to promote cardiac repair.

Future Directions: Optimization of proangiogenic therapies might include stimulation of both angiogenesis and vessel maturation and/or the use of additional sources of stem/progenitor cells, such as cardiac progenitor cells. Experimental unraveling of the mechanisms of angiogenesis, vessel maturation, and endothelial cell/cardiomyocyte cross talk in the ischemic heart, analysis of emerging pathways, as well as a better understanding of how cardiovascular risk factors impact endogenous and therapeutically stimulated angiogenesis, would undoubtedly pave the way for the development of novel and hopefully efficient angiogenesis targeting therapeutics for the treatment of acute MI.
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http://dx.doi.org/10.1089/ars.2012.4849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567783PMC
March 2013

The chemokine decoy receptor D6 prevents excessive inflammation and adverse ventricular remodeling after myocardial infarction.

Arterioscler Thromb Vasc Biol 2012 Sep 12;32(9):2206-13. Epub 2012 Jul 12.

Université Paris Descartes, INSERM UMR-S970, Paris Cardiovascular Research Center, Hôpital Européen Georges Pompidou. Paris, France.

Objective: Leukocyte infiltration in ischemic areas is a hallmark of myocardial infarction, and overwhelming infiltration of innate immune cells has been shown to promote adverse remodeling and cardiac rupture. Recruitment of inflammatory cells in the ischemic heart depends highly on the family of CC-chemokines and their receptors. Here, we hypothesized that the chemokine decoy receptor D6, which specifically binds and scavenges inflammatory CC-chemokines, might limit inflammation and adverse cardiac remodeling after infarction.

Methods And Results: D6 was expressed in human and murine infarcted myocardium. In a murine model of myocardial infarction, D6 deficiency led to increased chemokine (C-C motif) ligand 2 and chemokine (C-C motif) ligand 3 levels in the ischemic heart. D6-deficient (D6(-/-)) infarcts displayed increased infiltration of pathogenic neutrophils and Ly6Chi monocytes, associated with strong matrix metalloproteinase-9 and matrix metalloproteinase-2 activities in the ischemic heart. D6(-/-) mice were cardiac rupture prone after myocardial infarction, and functional analysis revealed that D6(-/-) hearts had features of adverse remodeling with left ventricle dilation and reduced ejection fraction. Bone marrow chimera experiments showed that leukocyte-borne D6 had no role in this setting, and that leukocyte-specific chemokine (C-C motif) receptor 2 deficiency rescued the adverse phenotype observed in D6(-/-) mice.

Conclusions: We show for the first time that the chemokine decoy receptor D6 limits CC-chemokine-dependent pathogenic inflammation and is required for adequate cardiac remodeling after myocardial infarction.
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http://dx.doi.org/10.1161/ATVBAHA.112.254409DOI Listing
September 2012

Sympathetic nervous system regulates bone marrow-derived cell egress through endothelial nitric oxide synthase activation: role in postischemic tissue remodeling.

Arterioscler Thromb Vasc Biol 2012 Mar 19;32(3):643-53. Epub 2012 Jan 19.

Paris Cardiovascular Research Center, INSERM U970, Université Paris Descartes, 56 rue Leblanc, 75015 Paris, France.

Objective: Catecholamines have been shown to control bone marrow (BM)-derived cell egress, yet the cellular and molecular mechanisms involved in this effect and their subsequent participation to postischemic vessel growth are poorly understood.

Methods And Results: Tyrosine hydroxylase mRNA levels, as well as dopamine (DA) and norepinephrine (NE) contents, were increased in the ischemic BM of mice with right femoral artery ligation. Angiographic score, capillary density, and arteriole number were markedly increased by treatments with DA (IP, 50 mg/kg, 5 days) or NE (IP, 2.5 mg/kg, 5 days). Using chimeric mice lethally irradiated and transplanted with BM-derived cells from green fluorescent protein mice, we showed that DA and NE enhanced by 70% (P<0.01) and 62% (P<0.001), respectively, the number of green fluorescent protein-positive BM-derived cells in ischemic tissue and promoted their ability to differentiate into cells with endothelial and inflammatory phenotypes. Similarly, both DA and NE increased the in vitro differentiation of cultured BM-derived cells into cells with endothelial phenotype. This increase was blunted by the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester. DA and NE also upregulated the number of CD45-positive cells in blood 3 days after ischemia and that of macrophages in ischemic tissue 21 days after ischemia. Of interest, DA and NE increased BM endothelial nitric oxide synthase (eNOS) mRNA levels and were unable to promote BM-derived cell mobilization in chimeric eNOS-deficient mice lethally irradiated and transplanted with BM-derived cells from wild-type animals. Furthermore, administration of a β2 adrenergic agonist (clenbuterol, IP, 2 mg/kg, 5 days) and that of a dopaminergic D1/D5 receptor agonist (SKF-38393, IP, 2.5 mg/kg, 5 days) also enhanced BM-derived cell mobilization and subsequently postischemic vessel growth. CONCLUSION These results unravel, for the first time, a major role for the sympathetic nervous system in BM-derived cell egress through stromal eNOS activation.
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http://dx.doi.org/10.1161/ATVBAHA.111.244392DOI Listing
March 2012

C/EBP homologous protein-10 (CHOP-10) limits postnatal neovascularization through control of endothelial nitric oxide synthase gene expression.

Circulation 2012 Feb 20;125(8):1014-26. Epub 2012 Jan 20.

Department of Medicine, Division of Cardiovascular Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK.

Background: C/EBP homologous protein-10 (CHOP-10) is a novel developmentally regulated nuclear protein that emerges as a critical transcriptional integrator among pathways regulating differentiation, proliferation, and survival. In the present study, we analyzed the role of CHOP-10 in postnatal neovascularization.

Methods And Results: Ischemia was induced by right femoral artery ligation in wild-type and CHOP-10(-/-) mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes mellitus. Angiographic score, capillary density, and foot perfusion were increased in CHOP-10(-/-) mice compared with wild-type mice. This effect was associated with a reduction in apoptosis and an upregulation of endothelial nitric oxide synthase (eNOS) levels in ischemic legs of CHOP-10(-/-) mice compared with wild-type mice. In agreement with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 short interfering RNA-transfected human endothelial cells, whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using a chromatin immunoprecipitation assay, we also showed that CHOP-10 was bound to the eNOS promoter. Interestingly, enhanced postischemic neovascularization in CHOP-10(-/-) mice was fully blunted in CHOP-10/eNOS double-knockout animals. Finally, we showed that induction of diabetes mellitus is associated with a marked upregulation of CHOP-10 that substantially inhibited postischemic neovascularization.

Conclusions: This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.111.041830DOI Listing
February 2012

Regulation of monocyte subset systemic levels by distinct chemokine receptors controls post-ischaemic neovascularization.

Cardiovasc Res 2010 Oct 25;88(1):186-95. Epub 2010 May 25.

Paris Cardiovascular Research Center, Université Paris Descartes, UMR-S970 Paris, France.

Aims: Monocyte systemic levels are known to be a major determinant of ischaemic tissue revascularization, but the mechanisms mediating mobilization of different monocyte subsets-Ly6C(hi) and Ly6C(lo)-to the blood and their respective role in post-ischaemic neovascularization are not clearly understood. Here, we hypothesized that distinct chemokine/chemokine receptor pathways, namely CCL2/CCR2, CX3CL1/CX3CR1, and CCL5/CCR5, differentially control monocyte subset systemic levels, and might thus impact post-ischaemic vessel growth.

Methods And Results: In a model of murine hindlimb ischaemia, both Ly6C(hi) and Ly6C(lo) monocyte circulating levels were increased after femoral artery ligation. CCL2/CCR2 activation enhanced blood Ly6C(hi) and Ly6C(lo) monocyte counts, although the opposite effect was seen in mice with CCL2 or CCR2 deficiency. CX3CL1/CX3CR1 strongly impacted Ly6C(lo) monocyte levels, whereas CCL5/CCR5 had no role. Only CCL2/CCR2 signalling influenced neovascularization, which was increased in mice overexpressing CCL2, whereas it markedly decreased in CCL2-/- mice. Moreover, adoptive transfer of Ly6C(hi)-but not Ly6C(lo)-monocytes enhanced vessel growth and blood flow recovery.

Conclusion: Altogether, our data demonstrate that regulation of proangiogenic Ly6C(hi) monocytes systemic levels by CCL2/CCR2 controls post-ischaemic vessel growth, whereas Ly6C(lo) monocytes have no major role in this setting.
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http://dx.doi.org/10.1093/cvr/cvq153DOI Listing
October 2010

Regulatory T cells modulate postischemic neovascularization.

Circulation 2009 Oct 21;120(14):1415-25. Epub 2009 Sep 21.

Paris-Cardiovascular Research Center, INSERM U970, Hôpital Européen Georges Pompidou, Université Paris 5, 75015 Paris, France.

Background: CD4+ and CD8+ T lymphocytes are key regulators of postischemic neovascularization. T-cell activation is promoted by 2 major costimulatory signalings, the B7/CD28 and CD40-CD40 ligand pathways. Interestingly, CD28 interactions with the structurally related ligands B7-1 and B7-2 are also required for the generation and homeostasis of CD4+CD25+ regulatory T cells (Treg cells), which play a critical role in the suppression of immune responses and the control of T-cell homeostasis. We hypothesized that Treg cell activation may modulate the immunoinflammatory response to ischemic injury, leading to alteration of postischemic vessel growth.

Methods And Results: Ischemia was induced by right femoral artery ligation in CD28-, B7-1/2-, or CD40-deficient mice (n=10 per group). CD40 deficiency led to a significant reduction in the postischemic inflammatory response and vessel growth. In contrast, at day 21 after ischemia, angiographic score, foot perfusion, and capillary density were increased by 2.0-, 1.2-, and 1.8-fold, respectively, in CD28-deficient mice, which showed a profound reduction in the number of Treg cells compared with controls. Similarly, disruption of B7-1/2 signaling or anti-CD25 treatment and subsequent Treg deletion significantly enhanced postischemic neovascularization. These effects were associated with enhanced accumulation of CD3-positive T cells and Mac-3-positive macrophages in the ischemic leg. Conversely, treatment of CD28(-/-) mice with the nonmitogenic anti-CD3 monoclonal antibody enhanced the number of endogenous Treg cells and led to a significant reduction of the postischemic inflammatory response and neovascularization. Finally, coadministration of Treg cells and CD28(-/-) splenocytes in Rag1(-/-) mice with hindlimb ischemia abrogated the CD28(-/-) splenocyte-induced activation of the inflammatory response and neovascularization.

Conclusions: Treg cell response modulates postischemic neovascularization.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.109.875583DOI Listing
October 2009

Inhibition of prolyl hydroxylase domain proteins promotes therapeutic revascularization.

Circulation 2009 Jul 22;120(1):50-9. Epub 2009 Jun 22.

Paris Cardiovascular Research Center, INSERM U970, Hôpital Européen Georges Pompidou, Université Paris 5, Paris, France.

Background: The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O(2)-dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization.

Methods And Results: Mice with right femoral artery ligation were treated, by in vivo electrotransfer, with plasmids encoding for an irrelevant short hairpin RNA (shRNA) (shCON [control]) or specific shRNAs directed against HIF-1alpha (shHIF-1alpha), PHD1 (shPHD1), PHD2 (shPHD2), and PHD3 (shPHD3). The silencing of PHDs induced a specific and transient downregulation of their respective mRNA and protein levels at day 2 after ischemia and, as expected, upregulated HIF-1alpha. As a consequence, 2 key hypoxia-inducible proangiogenic actors, vascular endothelial growth factor-A and endothelial nitric oxide synthase, were upregulated at the mRNA and protein levels. In addition, monocyte chemotactic protein-1 mRNA levels and infiltration of Mac-3-positive macrophages were enhanced in ischemic leg of mice treated with shPHD2 and shPHD3. Furthermore, activation of HIF-1alpha-related pathways was associated with changes in postischemic neovascularization. At day 14, silencing of PHD2 and PHD3 increased vessel density by 2.2- and 2.6-fold, capillary density by 1.8- and 2.1-fold, and foot perfusion by 1.2- and 1.4-fold, respectively, compared with shCON (P<0.001). shPHD1 displayed a lower proangiogenic effect. Of interest, coadministration of shHIF-1alpha with shPHD3 abrogated shPHD3-related effects, suggesting that activation of endogenous HIF-1-dependent pathways mediated the proangiogenic effects of PHD silencing.

Conclusions: We demonstrated that a direct inhibition of PHDs, and more particularly PHD3, promoted therapeutic revascularization. Furthermore, we showed that activation of the HIF-1 signaling pathway is required to promote this revascularization.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.108.813303DOI Listing
July 2009