Aims Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h 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 min) 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. Methods and results Here, we show that injury to the myocardium rapidly mobilizes 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 mobilized 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 mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. Conclusions Our findings show a novel EV-dependent mechanism for the rapid mobilization 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.

BACKGROUND:Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS:Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS:In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS:Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Accumulating evidence shows how diverse physiological functions, such as metabolism, immunity, tissue homeostasis, and hematopoiesis, are intricately and profoundly intertwined at multiple levels. This brief review will present evidence from a rapidly expanding field of immunometabolism, highlighting how cells that are relevant to processes at play in determining vascular health and disease can be programmed by changes in their metabolic environment. It will focus on how such changes can be imprinted or trained, particularly through epigenetic modifications, such that adaptations driven by metabolic signals can cause persistent changes in cell function, even after the original stimulus has been corrected or removed. Recognition of these processes and elucidation of the mechanisms underlying them stand to have far-reaching implications for the diagnosis and treatment of diabetes and related metabolic states.

Accumulating evidence shows how diverse physiological functions, such as metabolism, immunity, tissue homeostasis, and hematopoiesis, are intricately and profoundly intertwined at multiple levels. This brief review will present evidence from a rapidly expanding field of immunometabolism, highlighting how cells that are relevant to processes at play in determining vascular health and disease can be programmed by changes in their metabolic environment. It will focus on how such changes can be imprinted or trained, particularly through epigenetic modifications, such that adaptations driven by metabolic signals can cause persistent changes in cell function, even after the original stimulus has been corrected or removed. Recognition of these processes and elucidation of the mechanisms underlying them stand to have far-reaching implications for the diagnosis and treatment of diabetes and related metabolic states.

While obesity and associated metabolic complications are linked to inflammation of white adipose tissue (WAT), the causal factors remain unclear. We hypothesized that the local metabolic environment could be an important determinant. To this end, we compared metabolites released from WAT of 81 obese and non-obese women. This identified glutamine to be downregulated in obesity and inversely associated with a pernicious WAT phenotype. Glutamine administration in vitro and in vivo attenuated both pro-inflammatory gene and protein levels in adipocytes and WAT and macrophage infiltration in WAT. Metabolomic and bioenergetic analyses in human adipocytes suggested that glutamine attenuated glycolysis and reduced uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) levels. UDP-GlcNAc is the substrate for the post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) mediated by the enzyme O-GlcNAc transferase. Functional studies in human adipocytes established a mechanistic link between reduced glutamine, O-GlcNAcylation of nuclear proteins, and a pro-inflammatory transcriptional response. Altogether, glutamine metabolism is linked to WAT inflammation in obesity.

Acute myocardial infarction rapidly increases blood neutrophils (<2 hours). Release of neutrophils 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 are enriched in vascular cell adhesion molecule-1 (VCAM-1) and miRNA-126, 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 their arrival at injured tissue. Ischemic myocardium leads to the generation and release of EC-derived-EVs bearing VCAM-1. EC-EV delivery to the spleen alters inflammatory gene and chemokine protein expression, and mobilises neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing we generated VCAM-1-deficient EV and showed that its deletion removed the ability of EC-EV 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 mechanism for the rapid mobilisation of neutrophils to peripheral blood from a splenic reserve, independent of classical chemokine signalling, and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Background and aims: Imaging studies have relied on the ‘overall’ volumetric quantification of perivascular adipose tissue. We sought to assess the relationship of circumferential distribution between perivascular adipose tissue and adjacent wall thickness of carotid and aortic arteries using dedicated magnetic resonance imaging sequences. Methods: Vessel wall and perivascular adipose tissue were acquired using magnetic resonance imaging (1.5 T). Co-registered images were segmented separately, and measurements of both perivascular adipose tissue and vessel wall were obtained along radii of the vessel spaced at angles of 5° each. Results: In total, 29 patients were recruited. Perivascular adipose tissue thickness of the aorta was 3.34 ± 0.79 mm with specific pattern of ‘double peaks’ distribution, while carotid perivascular adipose tissue had no identifiable pattern with thickness of 0.8 ± 0.91 mm. Although statistically significant, the correlation between perivascular adipose tissue thickness and wall thickness in carotid arteries with normal (r = 0.040, p = 0.001) or with abnormal wall thickness (r = –0.039, p = 0.015) was merely nominal. Similarly, perivascular adipose tissue of the aorta had very weak correlation with normal aortic wall thickness (r = 0.010, p = 0.008) but not with the abnormal ones (r = −0.05, p = 0.29). Conclusion: Dissociation between the spatial distribution of perivascular adipose tissue and arterial wall thickening in the aorta and carotid arteries does not support that perivascular adipose tissue has a causal role in promoting atherosclerotic plaque via a paracrine route. Yet, perivascular adipose tissue functional properties were not examined in this study.

Objective— Plaque macrophages are intricately involved in atherogenesis and plaque destabilization. We sought to identify functional pathways in human plaque macrophages that are differentially regulated in respect of (1) plaque stability and (2) lipid content. We hypothesized that differentially regulated macrophage gene sets would relate to genome-wide association study variants associated with risk of acute complications of atherosclerosis. Approach and Results— Forty patients underwent carotid magnetic resonance imaging for lipid quantification before endarterectomy. Carotid plaque macrophages were procured by laser capture microdissection from (1) lipid core and (2) cap region, in 12 recently symptomatic and 12 asymptomatic carotid plaques. Applying gene set enrichment analysis, a number of gene sets were found to selectively upregulate in symptomatic plaque macrophages, which corresponded to 7 functional pathways: inflammation, lipid metabolism, hypoxic response, cell proliferation, apoptosis, antigen presentation, and cellular energetics. Predicted upstream regulators included IL-1β , TNF-α , and NF-κB . In vivo lipid quantification by magnetic resonance imaging correlated most strongly with the upregulation of genes of the IFN / STAT1 pathways. Cross-interrogation of gene set enrichment analysis and meta-analysis gene set enrichment of variant associations showed lipid metabolism pathways, driven by genes coding for APOE and ABCA1/G1 coincided with known risk-associated SNPs (single nucleotide polymorphisms) from genome-wide association studies. Conclusions— Macrophages from recently symptomatic carotid plaques show differential regulation of functional gene pathways. There were additional quantitative relationships between plaque lipid content and key gene sets. The data show a plausible mechanism by which known genome-wide association study risk variants for atherosclerotic complications could be linked to (1) a relevant cellular process, in (2) the key cell type of atherosclerosis, in (3) a human disease-relevant setting.

Background The mechanisms by which diabetes increases atherosclerosis and cardiovascular disease risk even after glucose normalisation remains unknown. We hypothesised that: hyperglycaemia alters cellular metabolism; these changes drive pro–inflammatory responses, which remain altered after glucose normalisation and these increase atherosclerosis in vivo. Methods and results Hyperglycaemia alters monocyte, macrophage and hematopoietic stem cell (HSC) metabolism, significantly increasing glycolysis (FDR=0.02, human monocyte non-targeted metabolomics screen). In vitro, hyperglycaemia increased pro-inflammatory macrophage gene expression upon LPS +IFNy stimulation (IL-6, p<0.001) and both monocyte adherence to activated endothelium and macrophage uptake of modified lipid (p<0.001); all responses were normalised by the glycolytic inhibitor dichloroacetate (DCA) or 2-deoxy-glucose (2DG). Bone marrow derived macrophages (BMDM) from diabetic mice, grown in physiological glucose retained heightened pro-inflammatory responses, indicating hyperglycaemic memory in the HSC niche as well, as differentiated cells. To understand if diabetic HSC memory has a role in driving disease in vivo, bone marrow from diabetic mice (vs. wild type control) was transplanted into LDLR-/- mice. After 12 weeks, plaque burden in the aortic root (p=0.036) and plaque lipid content (p=0.0076), were greater in the mice receiving cell from the diabetic donor, confirming a memory effect. To investigate the mechanism underlying hyperglycaemic memory, ATAC-seq analysis was performed on diabetic and wild-type (WT) HSCs. Differential peak analyses indicated that cells from diabetic mice had an altered chromatin structure, potentially mediated through the increased histone modifications H3K27ac and H3K4me3 (p<0.01). These histone modifications are normalised by DCA. Motif analysis revealed that binding sites for the transcription factors PU.1, CTCF and RUNX1 are significantly enriched in peaks differentially present in diabetic HSC. In conclusion Diabetic hyperglycaemia alters HSC and macrophage metabolism to induce epigenetic changes which increases their pro-inflammatory responses and drives atherosclerotic disease in vivo. PU.1, CTCF and RUNX1 have been previously associated to chromatin priming elements. This novel demonstration of immunological memory may help to explain why targeting elevated glucose is often ineffective in reducing cardiovascular risk in diabetes.

Traditionally, the British Cardiovascular Society (BCS) Annual Conference hosted two research competitions for young cardiologists or cardiovascular scientists: the Young Investigators Prize competition administered by the British Society of Cardiovascular Research (BSCR) and the British Atherosclerosis Society (BAS), and the Young Research Workers Prize competition administered by the BCS. Although the former competition was clearly aimed at basic scientists and the latter more broadly at clinical scientists, in the era of translational medicine the boundaries have become increasingly blurred. In line with the aims of BCS, BSCR, BAS and the British Heart Foundation (BHF) to encourage greater dialogue and collaboration between basic and clinical scientists, these competitions were amalgamated into one in 2016. For the 2018 BCS Young Investigator Award (YIA) competition, there were 48 submissions from some of the UK’s leading clinical and basic science cardiovascular researchers. The BCS Academic and Research Committee, Chaired by Professor Rod Stables, selected four finalists, who each gave a 10 min oral presentation, followed by questions from a panel of independent judges. All finalists were guests of the BCS at the Society’s Annual Dinner, and first prize was awarded to Laurienne Edgar and presented by Dr Sarah Clarke, BCS President. The details of the four YIA finalists and their academic training and interests can be seen below. Laurienne is currently in her final, fourth year of a BHF-funded DPhil in Cardiovascular …

Background Following acute myocardial infarction (AMI), monocytes are rapidly mobilised from the spleen to peripheral blood, from where they undergo transcriptional activation and infiltrate injured tissue, with potential to contribute to both injury and repair. The mechanism by which the injured myocardium signals splenic-monocyte mobilisation remains poorly understood. Recent work shows extracellular vesicles (EV, which carry proteins, microRNA/mRNA) are a means of rapid cell-to-cell communication, which, combined with knowledge of their composition and propensity to be taken up by other cells, suggests a possible role in signalling. Here we show that AMI results in a net increase in circulating endothelial cell (EC)-EV that induce splenic monocyte motility in vivo and cellular transcription. Methods Platelet-poor plasma was collected from patients with ST-segment elevation-AMI (STEMI) and mice subjected to AMI. EV were isolated by ultra-centrifugation and analysed for size/number by Nanoparticle Tracking Analysis, western blot (EV-markers: ALIX, TSG101, CD69, CD9 and Hsp70), ELISA for EC markers (CD31, ICAM-1, P-selectin, E-selectin and VCAM-1), electron microscopy and for EV-miRNAs. Human and mouse EC were used in vitro to evaluate EV release, injected into wild-type or CD68GFP+ naïve mice to assess bio-distribution, splenic-monocyte mobilisation, uptake by monocytes, cellular mRNA transcription and cell motility. Results Acutely (24 hours) after AMI there is a significant increase in circulating EV in humans (p<0.01) and mice (p<0.001) that later subsides. Plasma EV number correlates with myocardial injury in humans (R2=0.52, p<0.01). Plasma EV display EC-surface markers and show enrichment for vascular cell adhesion moleculae-1 (VCAM-1) in AMI (p<0.05). In vitro pro-inflammatory cytokines significantly increase EV production by EC, whereas ‘anti-inflammatory’ IL-4 and IL-6 had no effect. Inflammatory-EC-EV displayed significant enrichment of VCAM-1 (p<0.05). In-vitro labelled EC-EV accumulate in monocytes. Inflammatory-EC-EV significantly enhanced macrophage chemokineses (p<0.05) and chemotaxis to MCP-1 (p<0.05), a response that was abolished by pre-incubating EC-EV with an anti-VCAM-1 antibody (p<0.05). Injected labelled EC-EV accumulate in the spleen, interact with splenic monocytes and induce splenic-monocyte mobilisation and peripheral monocytosis in-vivo (p<0.01). Human plasma-EV show enrichment for 12 miRNAs in AMI, including EC-associated miR-126–3p/5p. miRNA-mRNA target gene prediction and functional enrichment analysis show roles for these miRNAs in the positive regulation of chemotaxis, cellular growth and proliferation. EC-EV significantly induced alterations in mRNA of motility genes by reducing PLEXIN-B2 (p<0.001), a negative regulator of motility and increasing ITGB2 (p<0.001) expression in monocytes. In conclusion (1) AMI surges plasma EV; (2) Plasma-EV protein composition is consistent with EC origin. (3) Injected EV localise to the spleen and (4) mobilise splenic monocytes. (5) In culture, EC increase EV release, enhance monocyte motility and (6) regulate genes that are important in cellular movement. These demonstrate a novel role for EC-derived EV in monocyte activation after AMI.

Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1-positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion- and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.