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Atherosclerosis is a chronic disease of the vascular wall driven by lipid accumulation and inflammation in the intimal layer of arteries [1], [2], and its main complications, myocardial infarction and stroke, are the leading cause of mortality worldwide [3]. Recent studies have identified Triggering receptor expressed on myeloid cells 2 (TREM2), a lipid-sensing receptor regulating several key myeloid cell functions [4], as a highly expressed marker of macrophage foam cells in experimental and human atherosclerosis [5]. However, the function of TREM2 in the development of atherosclerosis is unknown. Here, we show that hematopoietic or global TREM2 deficiency increases necrotic core formation in early experimental atherosclerosis. We further demonstrate that TREM2 is essential for the efferocytosis capacities of macrophages, and to the survival of lipid-laden macrophages, altogether indicating a crucial role of TREM2 in maintaining the balance between foam cell death and their clearance in atherosclerotic lesions, thereby controlling plaque necrosis.

Aims Aging entails profound immunological transformations that can impact myocardial homeostasis and predispose to heart failure. However, preclinical research in the immune-cardiology field is mostly conducted in young healthy animals, which potentially weakens its translational relevance. Herein, we sought to investigate how the aging T-cell compartment associates with changes in myocardial cell biology in aged mice. Methods and results We phenotyped the antigen-experienced effector/memory T cells purified from heart-draining lymph nodes of 2-, 6-, 12-, and 18-month-old C57BL/6J mice using single-cell RNA/T cell receptor sequencing. Simultaneously, we profiled all non-cardiomyocyte cell subsets purified from 2- to 18-month-old hearts and integrated our data with publicly available cardiomyocyte single-cell sequencing datasets. Some of these findings were confirmed at the protein level by flow cytometry. With aging, the heart-draining lymph node and myocardial T cells underwent clonal expansion and exhibited an up-regulated pro-inflammatory transcription signature, marked by an increased interferon-γ (IFN-γ) production. In parallel, all major myocardial cell populations showed increased IFN-γ responsive signature with aging. In the aged cardiomyocytes, a stronger IFN-γ response signature was paralleled by the dampening of expression levels of transcripts related to most metabolic pathways, especially oxidative phosphorylation. Likewise, induced pluripotent stem cells-derived cardiomyocytes exposed to chronic, low grade IFN-γ treatment showed a similar inhibition of metabolic activity. Conclusions By investigating the paired age-related alterations in the T cells found in the heart and its draining lymph nodes, we provide evidence for increased myocardial IFN-γ signaling with age, which is associated with inflammatory and metabolic shifts typically seen in heart failure.

Aims Atherosclerosis is a chronic inflammatory disease of the vessel wall controlled by local and systemic immune responses. The role of interleukin-23 receptor (IL-23R), expressed in adaptive immune cells (mainly T-helper 17 cells) and γδ T cells, in atherosclerosis is only incompletely understood. Here, we investigated the vascular cell types expressing IL-23R and addressed the function of IL-23R and γδ T cells in atherosclerosis. Methods and results IL-23R+ cells were frequently found in the aortic root in contrast to the aorta in low-density lipoprotein receptor deficient IL-23R reporter mice (Ldlr−/−Il23rgfp/+), and mostly identified as γδ T cells that express IL-17 and GM-CSF. scRNA-seq confirmed γδ T cells as the main cell type expressing Il23r and Il17a in the aorta. Ldlr−/−Il23rgfp/gfp mice deficient in IL-23R showed a loss of IL-23R+ cells in the vasculature, and had reduced atherosclerotic lesion formation in the aortic root compared to Ldlr−/− controls after 6 weeks of high-fat diet feeding. In contrast, Ldlr−/−Tcrδ−/− mice lacking all γδ T cells displayed unaltered early atherosclerotic lesion formation compared to Ldlr−/− mice. In both HFD-fed Ldlr−/−Il23rgfp/gfp and Ldlr−/−Tcrδ−/− mice a reduction in the plaque necrotic core area was noted as well as an expansion of splenic regulatory T cells. In vitro, exposure of bone marrow-derived macrophages to both IL-17A and GM-CSF induced cell necrosis, and necroptotic RIP3K and MLKL expression, as well as inflammatory mediators. Conclusions IL-23R+ γδ T cells are predominantly found in the aortic root rather than the aorta and promote early atherosclerotic lesion formation, plaque necrosis, and inflammation at this site. Targeting IL-23R may thus be explored as a therapeutic approach to mitigate atherosclerotic lesion development.

Aims Accumulation of mononuclear phagocytes [monocytes, macrophages, and dendritic cells (DCs)] in the vessel wall is a hallmark of atherosclerosis. Using integrated single-cell analysis of mouse and human atherosclerosis, we here aimed to refine the nomenclature of mononuclear phagocytes in atherosclerotic vessels and to compare their transcriptomic profiles in mouse and human disease. Methods and results We integrated 12 single-cell RNA-sequencing (scRNA-seq) datasets of immune cells isolated from healthy or atherosclerotic mouse aortas, and data from 11 patients (n = 4 coronary vessels, n = 7 carotid endarterectomy specimens) from two studies. Integration of mouse data identified subpopulations with discrete transcriptomic signatures within previously described populations of aortic resident (Lyve1), inflammatory (Il1b), as well as foamy (Trem2hi) macrophages. We identified unique transcriptomic features distinguishing aortic intimal resident macrophages from atherosclerosis-associated Trem2hi macrophages. Also, populations of Xcr1+ Type 1 classical DCs (cDC1), Cd209a+ cDC2, and mature DCs (Ccr7, Fscn1) with a ‘mreg-DC’ signature were detected. In humans, we uncovered macrophage and DC populations with gene expression patterns similar to those observed in mice. In particular, core transcripts of the foamy/Trem2hi signature (TREM2, SPP1, GPNMB, CD9) mapped to a specific population of macrophages in human lesions. Comparison of mouse and human data and direct cross-species data integration suggested transcriptionally similar macrophage and DC populations in mice and humans. Conclusions We refined the nomenclature of mononuclear phagocytes in mouse atherosclerotic vessels, and show conserved transcriptomic features of macrophages and DCs in atherosclerosis in mice and humans, emphasizing the relevance of mouse models to study mononuclear phagocytes in atherosclerosis.

Immune cells in atherosclerosis include T, B, natural killer (NK) and NKT cells, macrophages, monocytes, dendritic cells (DCs), neutrophils, and mast cells. Advances in single-cell RNA sequencing (sRNA-Seq) have refined our understanding of immune cell subsets. Four recent studies have used scRNA-Seq of immune cells in human atherosclerotic lesions and peripheral blood mononuclear cells (PBMCs), some including cell surface phenotypes revealed by oligonucleotide-tagged antibodies, which confirmed known and identified new immune cell subsets and identified genes significantly up-regulated in PBMCs from HIV+ subjects with atherosclerosis compared to PBMCs from matched HIV+ subjects without atherosclerosis. The ability of scRNA-Seq to identify cell types is greatly augmented by adding cell surface phenotype using antibody sequencing. In this review, we summarize the latest data obtained by scRNA-Seq on plaques and human PBMCs in human subjects with atherosclerosis.

Acute myocardial infarction rapidly increases blood neutrophils (2 hours). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 hours after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (30 minutes) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues.Here, we show that injury to the myocardium rapidly mobilises neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilised splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilisation of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model.Our findings show a novel EV-dependent mechanism for the rapid mobilisation of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.Peripheral blood neutrophils are rapidly elevated following acute myocardial infarction (AMI) and prior to alterations in systemic cytokines. Extracellular vesicles (EVs) are membrane enclosed particles that carry protein and miRNAs and are rapidly liberated from endothelial cells (EC). Here, we show that following AMI EC-derived-EVs (EC-EVs) mediate neutrophil mobilisation from the spleen via EC-EV-VCAM-1 and induce transcriptional activation of neutrophils in the blood to favour miRNA-126-mRNA targets; miRNA-126 antagomir treatment lowers infarct size. EC-EV-VCAM-1 and EC-EV-miRNA-126 are novel mechanisms that mobilise splenic reserve of neutrophils, a previously unidentified source of neutrophils in sterile ischaemic injury.

RationaleAccumulation of mononuclear phagocytes (monocytes, macrophages and dendritic cells) in the vessel wall is a hallmark of atherosclerosis. Although single-cell RNA-sequencing (scRNA-seq) has shed new light on immune cell transcriptional diversity in atherosclerosis, it is still unknown whether the transcriptional states of mononuclear phagocytes are conserved between mouse and human atherosclerosis.ObjectiveTo integrate and compare macrophage and dendritic cell transcriptomes in mouse and human atherosclerosis.Methods and resultsWe integrated 12 scRNA-seq datasets of immune cells isolated from healthy or atherosclerotic mouse aortas, and scRNA-seq data from 11 patients (n=4 coronary vessels, n=7 carotid endarterectomy specimens) from two independent studies. Integration of mouse data recovered previously described macrophage populations and identified novel subpopulations with discrete transcriptomic signatures within populations of aortic resident (Lyve1), inflammatory (Il1b), as well as foamy (Trem2hi) macrophages. We identified unique transcriptomic features distinguishing aortic intimal resident macrophages from atherosclerosis-associated Trem2hi macrophages. Also, populations of Xcr1+ type 1 classical dendritic cells (cDC1), Cd209a+ cDC2 and mature DCs (Ccr7, Fscn1) were detected. In humans, we uncovered macrophage and dendritic cell populations with gene expression patterns similar to those observed in mice in both vascular beds. In particular, core transcripts of the foamy/Trem2hi signature (TREM2, SPP1, GPNMB, CD9) mapped to a specific population of macrophages in human lesions. Cross-species data integration demonstrated transcriptionally proximal macrophage and dendritic cell populations in mice and humans.ConclusionsWe demonstrate conserved transcriptomics features of macrophages and dendritic cells in atherosclerosis in mice and humans, emphasizing the relevance of mouse models to study mononuclear phagocytes in atherosclerosis.

Acute myocardial infarction rapidly increases blood neutrophils (

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, Pf4 , 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.

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 ( Cd177 , Lcn2 , Fpr1 ), and putative activity of transcriptional regulators involved in hypoxic response ( Hif1a ) and emergency granulopoiesis ( Cebpb ). At 3 and 5 days, 2 major subsets of Siglecf hi (enriched for eg, Icam1 and Tnf ) and Siglecf low ( Slpi, Ifitm1 ) 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 Cxcr4 , heart infiltrating neutrophils acquired a unique SiglecF hi signature. SiglecF hi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecF hi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecF hi 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 hi 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 hi signature.

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.

Aims Recent studies have revealed that B cells and antibodies can influence inflammation and remodelling following a myocardial infarction (MI) and culminating in heart failure—but the mechanisms underlying these observations remain elusive. We therefore conducted in mice a deep phenotyping of the post-MI B-cell responses in infarcted hearts and mediastinal lymph nodes, which drain the myocardium. Thereby, we sought to dissect the mechanisms controlling B-cell mobilization and activity in situ. Methods and results Histological, flow cytometry, and single-cell RNA-sequencing (scRNA-seq) analyses revealed a rapid accumulation of diverse B-cell subsets in infarcted murine hearts, paralleled by mild clonal expansion of germinal centre B cells in the mediastinal lymph nodes. The repertoire of cardiac B cells was largely polyclonal and showed no sign of antigen-driven clonal expansion. Instead, it included a distinct subset exclusively found in the heart, herein termed ‘heart-associated B cells’ (hB) that expressed high levels of Cd69 as an activation marker, C-C-chemokine receptor type 7 (Ccr7), CXC-chemokine receptor type 5 (Cxcr5), and transforming growth factor beta 1 (Tgfb1). This distinct signature was not shared with any other cell population in the healing myocardium. Moreover, we detected a myocardial gradient of CXC-motif chemokine ligand 13 (CXCL13, the ligand of CXCR5) on Days 1 and 5 post-MI. When compared with wild-type controls, mice treated with a neutralizing CXCL13-specific antibody as well as CXCR5-deficient mice showed reduced post-MI infiltration of B cells and reduced local Tgfb1 expression but no differences in contractile function nor myocardial morphology were observed between groups. Conclusion Our study reveals that polyclonal B cells showing no sign of antigen-specificity readily infiltrate the heart after MI via the CXCL13-CXCR5 axis and contribute to local TGF-ß1 production. The local B-cell responses are paralleled by mild antigen-driven germinal centre reactions in the mediastinal lymph nodes that might ultimately lead to the production of specific antibodies.

RationaleMonocytes and macrophages have a critical and dual role in post-ischemic cardiac repair, as they can foster both tissue healing and damage. To decipher how monocytes/macrophages acquire heterogeneous functional phenotypes in the ischemic myocardium, we profiled the gene expression dynamics at the single-cell level in circulating and cardiac monocytes/macrophages following experimental myocardial infarction (MI) in mice.Methods and resultsUsing time-series single-cell transcriptome and cell surface epitope analysis of blood and cardiac monocytes/macrophages, as well as the integration of publicly available and independently generated single-cell RNA-seq data, we tracked the transitions in circulating and cardiac monocyte/macrophage states from homeostatic conditions up to 11 days after MI in mice. We show that MI induces marked and rapid transitions in the cardiac mononuclear phagocyte population, with almost complete disappearance of tissue resident macrophages 1 day after ischemia, and rapid infiltration of monocytes that locally acquire discrete and time-dependent transcriptional states within 3 to 7 days. Ischemic injury induced a shift of circulating monocytes towards granulocyte-like transcriptional features (Chil3, Lcn2, Prtn3). Trajectory inference analysis indicated that while conversion to Ly6Clow monocytes appears as the default fate of Ly6Chi monocytes in the blood, infiltrated monocytes acquired diverse gene expression signatures in the injured heart, notably transitioning to two main MI-associated macrophage populations characterized by MHCIIhi and Trem2hiIgf1hi gene expression signatures. Minor ischemia-associated macrophage populations with discrete gene expression signature suggesting specialized functions in e.g. iron handling or lipid metabolism were also observed. We further identified putative transcriptional regulators and new cell surface markers of cardiac monocyte/macrophage states.ConclusionsAltogether, our work provides a comprehensive landscape of circulating and cardiac monocyte/macrophage states and their regulators after MI, and will help to further understand their contribution to post-myocardial infarction heart repair.

ObjectiveAfter myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they can promote both tissue healing and damage. Here, we investigated the dynamics of cardiac neutrophil heterogeneity after infarction.Methods and resultsWe employed single-cell transcriptomics (scRNA-seq) to investigate temporal neutrophil heterogeneity in the heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, neutrophils could be delineated into six distinct clusters with specific time-dependent patterning and proportions. While the majority of neutrophils at day 1 were characterized by high expression of chemokines (e.g.Cxcl3,Ccl6), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb), two major subsets ofSiglecfhi(enriched for e.g.Icam1andTnf) andSiglecflow(Slpi, Ifitm1) neutrophils were found at 3 and 5 days. Flow cytometry analysis confirmed the presence of LY6G+SIGLECFhiand LY6G+SIGLECFlowneutrophils in the heart from 3 days after infarction onwards. LY6G+SIGLECFhineutrophils were absent from the bone marrow, blood and spleen, suggesting local acquisition of surface SIGLECF. Acquisition of the SIGLECFhistate was paralleled by features of neutrophil ageing and activation (ICAM1hiCXCR4hiCD49dhiCD62Llow). scRNA-seq of atherosclerotic aortas revealed two neutrophil subsets with gene expression patterns reminiscent of the majorSiglecfhiandSiglecflowcardiac neutrophil subpopulations, revealing that these populations may be present across distinct contexts of cardiovascular inflammation.ConclusionAltogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse ischemic heart at the single-cell level, and suggests that temporal neutrophil heterogeneity is in part driven by local transition to a SIGLECFhistate.

Supplemental Digital Content is available in the text., 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 Ly6CHigh 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 Ly6CHigh and Ly6CHow monocytes/macrophages populating the infarcted milieu. In contrast, combined Mertk and Mfge8 deficiency in Ly6CHigh/Ly6CLow 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+/VEGFAfl/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.

Atherosclerosis, an inflammatory disease of large arteries, is - through its clinical manifestations stroke and myocardial infarction - globally the leading cause of morbidity and mortality. The interplay of pro- and anti-inflammatory leukocytes in the aorta modulates and drives atherosclerosis. Although cells of the innate and adaptive immune system are found in atherosclerotic plaques, their phenotypic and functional diversity is poorly understood. Here, we applied single cell RNA-sequencing (scRNAseq) and mass cytometry (CyTOF) to assess leukocyte diversity in depth, thus defining an immune cell atlas in atherosclerosis. Single cell transcriptional profiling of aortic leukocytes from 20-week old chow (CD) and western diet (WD) fed Apoe -/- and Ldlr -/- mice revealed 11 phenotypically different leukocyte clusters. Atherosclerotic aortas exhibited enhanced leukocyte diversity, whilst WD further changed the abundance of leukocyte subpopulations. Gene set enrichment analysis of single cells established that multiple pathways, e.g. for lipid metabolism, proliferation, and cytokine secretion, pertained to particular leukocyte clusters. Applying a novel 35-marker CyTOF panel with metal-labelled antibodies confirmed the phenotypic diversity of aortic leukocytes. Among lymphocytes, we detected three principal B-cell subsets defined by scRNAseq, CyTOF, and flow cytometry. These B cell subsets harbor distinct surface marker expression, functional gene pathways, and ex vivo cytokine production. Finally, we used leukocyte cluster gene signatures to assess leukocyte frequencies in 121 human plaques by a transcriptomic deconvolution strategy. This approach revealed a similar immune cell complexity in human carotid plaques with a higher percentage of monocytes and macrophages. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunological mechanisms and cell-type specific pathways, and may result in novel diagnostic risk stratification tools.

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 Apoe −/− and Ldlr −/− 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 Apoe −/− and Ldlr −/− 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.

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.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.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.

SummaryAtherosclerosis 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 (F12−/−Apoe −/−) mice. Compared to F12 +/+ Apoe −/− controls, atherosclerotic lesion formation was reduced in F12−/−Apoe −/− 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 F12−/−Apoe −/− 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.Supplementary Material to this article is available online at www.thrombosis-online.com.

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.

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 CD8+CD25+ 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.

SummaryThe 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-1high and Gr1low 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.Note: The review process for this manuscript was fully handled by G. Y. H. Lip, Editor in Chief.

This editorial refers to ‘MAb therapy against the IFN-α/β receptor subunit 1 stimulates arteriogenesis in a murine hindlimb ischaemia model without enhancing atherosclerotic burden’ by P.F.A. Teunissen et al ., pp. 255–266. In ischaemic diseases such as myocardial infarction (MI), restoration of sufficient blood supply to ischaemic areas through arteriogenesis is critical for preservation of tissue integrity and function.1 Although ischaemia activates natural mechanisms promoting arteriogenesis, these are often insufficient to alleviate the consequences of ischaemia in patients, prompting the need for novel therapeutic strategies.1 One significant hurdle, however, is that several pro-arteriogenic mechanisms and therapeutic approaches also promote atherosclerotic plaque development.1,2 Teunissen et al. 3 now show that targeting the IFN-α/β receptor subunit 1 (IFNAR1) stimulates functional perfusion of ischaemic tissue while only minimally affecting atherosclerosis. Arteriogenic and atherosclerotic processes often engage similar mechanisms. For example, limb ischaemia induces the mobilization of Ly6Chigh monocytes to the bloodstream, which then infiltrate ischaemic tissue to promote blood flow recovery in mice,4 but also constitutes the main …

VEGF-driven neovascularization transiently recruits Ly6Chigh monocytes, which subsequently alter their phenotype and exert angiogenic function to enlarge small vessels., 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 Ly6Chi 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.

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. Conclusion— 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.

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 P N ω -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.

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.

We hypothesized that activation of angiogenesis by chronic hypoxia may affect vascular resistance and, subsequently, blood pressure levels in spontaneously hypertensive rats (SHRs). Five-week-old prehypertensive SHRs and age-matched normotensive Wistar–Kyoto (WKY) rats (n=8 per group) were maintained under normobaric normoxic or hypoxic (10% O 2 ) conditions for 8 weeks. Three weeks later, the systolic blood pressure was lower by 26% in hypoxic SHRs compared to normoxic SHRs ( P P P P P P

Cardiovascular risk factors are associated with reduction in both the number and function of vascular progenitor cells. We hypothesized that 1) hypertension abrogates postnatal vasculogenesis, and 2) antihypertensive treatment based on the combination of perindopril (angiotensin-converting enzyme inhibitor) and indapamide (diuretic) may counteract hypertension-induced alteration in progenitor cell-related effects. Postischemic neovascularization was significantly lower in untreated spontaneously hypertensive rats (SHRs) compared with Wistar Kyoto (WKY) rats (p < 0.05). Treatment of SHRs with perindopril and the combination of perindopril/indapamide reduced the blood pressure levels and normalized vessel growth in ischemic area. Cotreatment with perindopril and indapamide increased vascular endothelial growth factor and endothelial nitric-oxide synthase protein contents, two key proangiogenic factors. It is interesting to note that 14 days after bone marrow mononuclear cell (BM-MNC) transplantation, revascularization was significantly lower in ischemic SHRs receiving BM-MNCs isolated from SHRs compared with those receiving BM-MNCs isolated from WKY rats (p < 0.05). Alteration in proangiogenic potential of SHR BM-MNCs was probably related to the reduction in their ability to differentiate into endothelial progenitor cells in vitro. Furthermore, the number of circulating endothelial progenitor cells (EPCs) was reduced by 3.1-fold in SHRs compared with WKY rats (p < 0.001). Treatments with perindopril or perindopril/indapamide restored the ability of BM-MNCs to differentiate in vitro into EPCs, increased the number of circulating EPCs, and re-established BM-MNC proangiogenic effects. Therefore, hypertension is associated with a decrease in the number of circulating progenitor cells and in the BM-MNC proangiogenic potential, probably leading to vascular complications in this setting. The combination of perindopril and indapamide counteracts hypertension-induced alterations in progenitor cell-related effects and restores blood vessel growth.

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.

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.

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. To explore the role of the thrombin receptor PAR-1 proinflammatory effects on ECFC chemotaxis/recruitment capacity. 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. PAR-1 activation in ECFC increases chemotactic gene expression and leukocyte recruitment at ischemic sites through a COX-2-dependent mechanism.

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.

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.

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.

Auteur(s) : Clement Cochain PARCC Inserm U790. Il est aujourd’hui bien demontre que le developpement de la plaque d’atherosclerose, menant finalement a sa rupture et aux complications atherothrombotiques associees, est intimement lie a la reaction immunitaire et inflammatoire. Le role de nombreuses cellules immunitaires, ainsi que les mecanismes par lesquels elles sont recrutees dans la plaque, puis participent a sa progression et a sa destabilisation font toujours l’objet d’intenses [...]

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. Antioxid. Redox Signal. 18, 1100–1113.

Therapeutic neovascularization represents an alternative treatment modality for patients with advanced ischemic coronary disease or critical limb ischemia (CLI). Upregulation of HIF-1α, through specific PHDs inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. The aim of this study was to unravel the efficiency of specific prolyl-hydroxylase domain protein (PHDs) silencing in both cell and non cell-based therapeutic strategies in CLI. We first showed that PHD2 and PHD3 mRNA levels were up-regulated whereas that of HIF-1α were down-regulated in mononuclear circulating cells and in ischemic legs of diabetic and non diabetic patients with CLI. We then used short hairpin RNAs (shRNAs) to downregulate murine PHD1, PHD2 or PHD3 levels and analyzed their efficiency as a therapeutic strategy in diabetic mice with hindlimb ischemia. PHDs silencing increased post-ischemic neovascularization. This increase was associated with an up-regulation of two key pro-angiogenic factors, VEGF-A and endothelial NOS and with that of pro-arteriogenic factors including fractalkine, MCP-1, and SDF-1 leading to activation of postischemic immunoinflammatory response. In addition, concomitant silencing of PHD2 and HIF-1α abrogated the shPHD2-related effects, demonstrating that activation of HIF-1α signaling mediated the pro-angiogenic and pro-arteriogenic effects of shPHD2. Finally, we assessed the putative beneficial effects of PHDs silencing on human endothelial progenitor cells (hEPC) or bone-marrow derived mesenchymal stem cells (hMSC)-based therapies. Cells were transfected with siRNA directed against PHDs and injected in Nude mice with hind-limb ischemia. We showed that PHDs silencing enhanced human cells therapeutic effect in the ischemic leg of Nude mice compared to animals receiving untreated cells. In conclusion, our study reveals, for the first time, that silencing of PHDs by the transient and local upregulation of endogenous HIF-1α improves therapeutic angiogenesis. This study also paves the way for future strategy based on administration of siRNAs directed against PHDs to enhance the efficiency of cell-based strategy.

Aims Monocyte systemic levels are known to be a major determinant of ischaemic tissue revascularization, but the mechanisms mediating mobilization of different monocyte subsets—Ly6Chi and Ly6Clo—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 Ly6Chi and Ly6Clo monocyte circulating levels were increased after femoral artery ligation. CCL2/CCR2 activation enhanced blood Ly6Chi and Ly6Clo monocyte counts, although the opposite effect was seen in mice with CCL2 or CCR2 deficiency. CX3CL1/CX3CR1 strongly impacted Ly6Clo 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 Ly6Chi—but not Ly6Clo—monocytes enhanced vessel growth and blood flow recovery. Conclusion Altogether, our data demonstrate that regulation of proangiogenic Ly6Chi monocytes systemic levels by CCL2/CCR2 controls post-ischaemic vessel growth, whereas Ly6Clo monocytes have no major role in this setting.

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-1α (shHIF-1α), 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-1α. 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-1α-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 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.

Background— We hypothesized that microparticles (MPs) released after ischemia are endogenous signals leading to postischemic vasculogenesis. Methods and Results— MPs from mice ischemic hind-limb muscle were detected by electron microscopy 48 hours after unilateral femoral artery ligation as vesicles of 0.1- to 1-μm diameter. After isolation by sequential centrifugation, flow cytometry analyses showed that the annexin V + MP concentration was 3.5-fold higher in ischemic calves than control muscles (1392±406 versus 394±180 annexin V + MPs per 1 mg; P 144+ ). MPs isolated from ischemic muscles induced more potent in vitro bone marrow–mononuclear cell (BM-MNC) differentiation into cells with endothelial phenotype than those isolated from control muscles. MPs isolated from atherosclerotic plaques were ineffective, whereas those isolated from apoptotic or interleukin-1β–activated endothelial cells also promoted BM-MNC differentiation. Interestingly, MPs from ischemic muscles produced more reactive oxygen species and expressed significantly higher levels of NADPH oxidase p47 (6-fold; P P P Conclusion— MPs produced during tissue ischemia stimulate progenitor cell differentiation and subsequently promote postnatal neovascularization.