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Cardiac magnetic resonance (CMR) imaging is a valuable modality in the diagnosis and characterization of cardiovascular diseases, since it can identify abnormalities in structure and function of the myocardium non-invasively and without the need for ionizing radiation. However, in clinical practice, it is commonly acquired as a collection of separated and independent 2D image planes, which limits its accuracy in 3D analysis. This paper presents a completely automated pipeline for generating patient-specific 3D biventricular heart models from cine magnetic resonance (MR) slices. Our pipeline automatically selects the relevant cine MR images, segments them using a deep learning-based method to extract the heart contours, and aligns the contours in 3D space correcting possible misalignments due to breathing or subject motion first using the intensity and contours information from the cine data and next with the help of a statistical shape model. Finally, the sparse 3D representation of the contours is used to generate a smooth 3D biventricular mesh. The computational pipeline is applied and evaluated in a CMR dataset of 20 healthy subjects. Our results show an average reduction of misalignment artefacts from 1.82 ± 1.60 mm to 0.72 ± 0.73 mm over 20 subjects, in terms of distance from the final reconstructed mesh. The high-resolution 3D biventricular meshes obtained with our computational pipeline are used for simulations of electrical activation patterns, showing agreement with non-invasive electrocardiographic imaging. The automatic methodologies presented here for patient-specific MR imaging-based 3D biventricular representations contribute to the efficient realization of precision medicine, enabling the enhanced interpretability of clinical data, the digital twin vision through patient-specific image-based modelling and simulation, and augmented reality applications. This article is part of the theme issue ‘Advanced computation in cardiovascular physiology: new challenges and opportunities’.

Background Acute ST-segment elevation myocardial infarction (STEMI) has effects on the myocardium beyond the immediate infarcted territory. However, pathophysiologic changes in the noninfarcted myocardium and their prognostic implications remain unclear. Objectives The purpose of this study was to evaluate the long-term prognostic value of acute changes in both infarcted and noninfarcted myocardium post-STEMI. Methods Patients with acute STEMI undergoing primary percutaneous coronary intervention underwent evaluation with blood biomarkers and cardiac magnetic resonance (CMR) at 2 days and 6 months, with long-term follow-up for major adverse cardiac events (MACE). A comprehensive CMR protocol included cine, T2-weighted, T2∗, T1-mapping, and late gadolinium enhancement (LGE) imaging. Areas without LGE were defined as noninfarcted myocardium. MACE was a composite of cardiac death, sustained ventricular arrhythmia, and new-onset heart failure. Results Twenty-two of 219 patients (10%) experienced an MACE at a median of 4 years (IQR: 2.5-6.0 years); 152 patients returned for the 6-month visit. High T1 (>1250 ms) in the noninfarcted myocardium was associated with lower left ventricular ejection fraction (LVEF) (51% ± 8% vs 55% ± 9%; P = 0.002) and higher NT-pro-BNP levels (290 pg/L [IQR: 103-523 pg/L] vs 170 pg/L [IQR: 61-312 pg/L]; P = 0.008) at 6 months and a 2.5-fold (IQR: 1.03-6.20) increased risk of MACE (2.53 [IQR: 1.03-6.22]), compared with patients with normal T1 in the noninfarcted myocardium (P = 0.042). A lower T1 (

X-ray angiography is the most commonly used imaging modality for the detection of coronary stenoses due to its high spatial and temporal resolution of lumen contour and its utility to guide coronary interventions in real time. However, the high inter- and intra-observer variability in interpreting the geometry of 3D vascular structure based on multiple 2D image projections is a limitation in the accurate determination of lesion severity. This could be addressed by the 3D reconstruction of the coronary arterial (CA) tree. The automated reconstruction of 3D CA tree from 2D projections is challenging due to the existence of several imaging artifacts, such as vessel overlap, foreshortening, and most importantly respiratory and cardiac motion. Along with these artifacts, the acquisition geometry introduces the possibility of generating false vessel segments in the reconstruction. Our approach aims to reduce the motion artifacts in angiographic projections by developing a new method for rigid and non-rigid motion correction. A novel point-cloud based approach is subsequently introduced for reconstruction of 3D vessel centerlines by iteratively minimizing the reconstruction error. The performance of the proposed 3D reconstruction is evaluated using angiographic projections from 45 patients, producing average reprojection errors of ${0.092} \pm {0.055}$ mm and ${0.910} \pm {0.352}$ mm for 3D centerlines reconstruction, when co-registered with the parent vessels on projection planes that were/were not used to derive the 3D reconstruction, respectively. A comparison of the reconstructed 3D lumen surface with optical coherence tomography (OCT) measurements has been performed, showing no statistically significant difference in the luminal cross-sections reconstructed with our method, compared to OCT.

Background Myocardial recovery after primary percutaneous coronary intervention in acute myocardial infarction is variable and the extent and severity of injury are difficult to predict. We sought to investigate the role of cardiovascular magnetic resonance T1 mapping in the determination of myocardial injury very early after treatment of ST-segment elevation myocardial infarction (STEMI). Methods STEMI patients underwent 3 T cardiovascular magnetic resonance (CMR), within 3 h of primary percutaneous intervention (PPCI). T1 mapping determined the extent (area-at-risk as %left ventricle, AAR) and severity (average T1 values of AAR) of acute myocardial injury, and related these to late gadolinium enhancement (LGE), and microvascular obstruction (MVO). The characteristics of myocardial injury within 3 h was compared with changes at 24-h to predict final infarct size. Results Forty patients were included in this study. Patients with average T1 values of AAR ≥1400 ms within 3 h of PPCI had larger LGE at 24-h (33% ±14 vs. 18% ±10, P = 0.003) and at 6-months (27% ±9 vs. 12% ±9; P P = 0.016) & [4.0 (0.5–9.5)% vs. 0 (0–3.0)%, P = 0.025]. The average T1 value was an independent predictor of acute LGE (β 0.61, 95%CI 0.13 to 1.09; P = 0.015), extent of MVO (β 0.22, 95%CI 0.03 to 0.41, P = 0.028) and final infarct size (β 0.63, 95%CI 0.21 to 1.05; P = 0.005). Receiver-operating-characteristic analysis showed that T1 value of AAR obtained within 3-h, but not at 24-h, predicted large infarct size (LGE > 9.5%) with 100% positive predictive value at the optimal cut-off of 1400 ms (area-under-the-curve, AUC 0.88, P = 0.006). Conclusion Hyper-acute T1 values of the AAR (within 3 h post PPCI, but not 24 h) predict a larger extent of MVO and infarct size at both 24 h and 6 months follow-up. Delayed CMR scanning for 24 h could not substitute the significant value of hyper-acute average T1 in determining infarct characteristics.

Objectives This study aimed to compare the value of the index of microcirculatory resistance (IMR) and microvascular obstruction (MVO) measured by cardiac magnetic resonance (CMR) in patients treated for and recovering from ST-segment elevation myocardial infarction. Background IMR can identify patients with microvascular dysfunction acutely after primary percutaneous coronary intervention (pPCI), and a threshold of >40 has been shown to be associated with an adverse clinical outcome. Similarly, MVO is recognized as an adverse feature in patients with ST-segment elevation myocardial infarction. Even though both IMR and MVO reflect coronary microvascular status, the interaction between these 2 parameters is uncertain. Methods A total of 110 patients treated with pPCI were included, and IMR was measured immediately at completion of pPCI. Infarct size (IS) as a percentage of left ventricular mass was quantified at 48 h (38.4 ± 12.0 h) and 6 months (194.0 ± 20.0 days) using CMR. MVO was identified and quantified at 48 h by CMR. Results Overall, a discordance between IMR and MVO was observed in 36.7% of cases, with 31 patients having MVO and IMR ≤40. Compared with patients with MVO and IMR ≤40, patients with both MVO and IMR >40 had an 11.9-fold increased risk of final IS >25% at 6 months (p = 0.001). Patients with MVO and IMR ≤40 had a significantly smaller IS at 6 months (p = 0.001), with significant regression in IS over time (34.4% [interquartile range (IQR): 27.3% to 41.0%] vs. 22.3% [IQR: 16.0% to 30.0%]; p = 0.001). Conclusions Discordant prognostic information was obtained from IMR and MVO in nearly one-third of cases; however, IMR can be helpful in grading the degree and severity of MVO.

In humans, new-born infants have the ability to regenerate their heart during early life. This is modelled in the mouse, where regenerative capacity is maintained for the first week after birth but lost thereafter. Reactivation of this process holds great therapeutic potential, however, the molecular pathways that might be targeted to extend neonatal regeneration remain elusive. Here, we explore a role for hypoxia and HIF signalling on the regulation of epicardial activity which is essential for heart development and the response to injury. Hypoxic regions were found in the epicardium from mid-gestation, associating with HIF1α and HIF2α, and expression of the epicardial master regulator Wilms’ tumour 1 (WT1). Epicardial deletion of Hif1a reduced WT1 levels, leading to impaired coronary vasculature. Moreover, targeting of the HIF degradation enzyme PHD through pharmacological inhibition with clinically approved drugs or epicardial-specific deletion stabilised HIF and promoted WT1 activity ex vivo. A combination of genetic and pharmacological stabilisation of HIF during neonatal heart injury led to prolonged epicardial activation, increased vascularisation, augmented infarct resolution and preserved function beyond the 7-day regenerative window. Together, these findings suggest pharmacological modulation of HIF signalling may represent a viable therapeutic strategy for treating ischaemic heart disease.

Background The ATI (Age-Thrombus burden-Index of Microvascular Resistance [IMR]) score was developed to predict suboptimal myocardial reperfusion in patients with ST-Elevation Myocardial Infarction (STEMI). When applied in the early phases of revascularization (e.g. before stent insertion), it predicts which patients are most likely to have a larger infarct size. In this study, we assessed the score's utility in determining which STEMI patients are at highest risk of clinical events during follow-up. Methods The ATI-score was calculated prospectively in 254 STEMI patients using age (>50 years = 1 point), pre-stenting IMR (>40 U and < 100 U = 1 point; ≥100 U = 2 points) and angiographic thrombus score (4 = 1 point, 5 = 3 points); the cohort was stratified in high vs. low-intermediate ATI-score strata (≥4 vs. < 4, respectively). Results After 3 years of follow-up, patients with high ATI-score presented a higher rate of Major Adverse Cardiac Events (MACE) defined as the composite of all-cause mortality, resuscitated cardiac arrest and new heart failure diagnosis (Hazard Ratio [HR]: 3.07; 95% Confidence Interval [CI]: 1.19–7.93; p = 0.02). The ATI-score showed a moderate discriminative power (c-stat: 0.69), not significantly different from that of other risk scores used in the STEMI setting. A high ATI-score was an independent predictor of MACE (HR: 3.24; 95% CI: 1.22–8.58; p = 0.018). Conclusions The ATI-score can discriminate patients at higher risk of long-term adverse events. The score allows predication of subsequent events even before coronary stenting, and consequently it may allow the option of individualized therapy in the early stages of the clinical care-pathway.

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.

Extracellular vesicles (EV) are a heterogeneous group of bilipid-enclosed envelopes that carry proteins, metabolites, RNA, DNA and lipids from their parent cell of origin. They mediate cellular communication to other cells in local tissue microenvironments and across organ systems. EV size, number and their biologically active cargo are often altered in response to pathological processes, including infection, cancer, cardiovascular diseases and in response to metabolic perturbations such as obesity and diabetes, which also have a strong inflammatory component. Here, we discuss the broad repertoire of EV produced by neutrophils, monocytes, macrophages, their precursor hematopoietic stem cells and discuss their effects on the innate immune system. We seek to understand the immunomodulatory properties of EV in cellular programming, which impacts innate immune cell differentiation and function. We further explore the possibilities of using EV as immune targeting vectors, for the modulation of the innate immune response, e.g., for tissue preservation during sterile injury such as myocardial infarction or to promote tissue resolution of inflammation and potentially tissue regeneration and repair.

The mechanisms promoting disturbed white adipocyte function in obesity remain largely unclear. Herein, we integrate white adipose tissue (WAT) metabolomic and transcriptomic data from clinical cohorts and find that the WAT phosphocreatine/creatine ratio is increased and creatine kinase-B expression and activity is decreased in the obese state. In human in vitro and murine in vivo models, we demonstrate that decreased phosphocreatine metabolism in white adipocytes alters adenosine monophosphate-activated protein kinase activity via effects on adenosine triphosphate/adenosine diphosphate levels, independently of WAT beigeing. This disturbance promotes a pro-inflammatory profile characterized, in part, by increased chemokine (C-C motif) ligand 2 (CCL2) production. These data suggest that the phosphocreatine/creatine system links cellular energy shuttling with pro-inflammatory responses in human and murine white adipocytes. Our findings provide unexpected perspectives on the mechanisms driving WAT inflammation in obesity and may present avenues to target adipocyte dysfunction.

Atherosclerosis is a disease of chronic inflammation. We investigated the roles of the cytokines IL-4 and IL-13, the classical activators of STAT6, in the resolution of atherosclerosis inflammation. Using Il4-/-Il13-/- mice, resolution was impaired, and in control mice, in both progressing and resolving plaques, levels of IL-4 were stably low and IL-13 was undetectable. This suggested that IL-4 is required for atherosclerosis resolution, but collaborates with other factors. We had observed increased Wnt signaling in macrophages in resolving plaques, and human genetic data from others showed that a loss-of-function Wnt mutation was associated with premature atherosclerosis. We now find an inverse association between activation of Wnt signaling and disease severity in mice and humans. Wnt enhanced the expression of inflammation resolving factors after treatment with plaque-relevant low concentrations of IL-4. Mechanistically, activation of the Wnt pathway following lipid lowering potentiates IL-4 responsiveness in macrophages via a PGE2/STAT3 axis.

Background Acute myocardial infarction (AMI) induces transcriptional activation of monocytes en route to the injured myocardium, in part driven by endothelial cell derived extracellular vesicles (EC-EV), which contain proteins and microRNA (miRNA) cargo. However, neutrophils are the first immune cells to arrive at sites of injury and mediate further damage to the ischemic myocardium. Here, we describe for the first time how neutrophils are released from the spleen in AMI and show that this is driven by EC-EV signalling. Methods and results Experimental AMI in wild-type mice caused a significant increase in peripheral blood neutrophils and a simultaneous reduction in splenic-neutrophil number (P Conclusions (I) Neutrophil deployment from the spleen is a novel finding in acute injury and interactions with (II) EC-EV may mediate their splenic liberation and (III) activation following AMI, en route to the injured myocardium. The splenic neutrophil reserve may be a novel therapeutic target in AMI to modulate the inflammatory response before recruitment of cells to sites of injury.

Macrophages are components of the innate immune system with key roles in tissue inflammation and repair. It is now evident that macrophages also support organogenesis, but few studies have characterized their identity, ontogeny and function during heart development. Here, we show that the distribution and prevalence of resident macrophages in the subepicardial compartment of the developing heart coincides with the emergence of new lymphatics, and that macrophages interact closely with the nascent lymphatic capillaries. Consequently, global macrophage deficiency led to extensive vessel disruption, with mutant hearts exhibiting shortened and mis-patterned lymphatics. The origin of cardiac macrophages was linked to the yolk sac and foetal liver. Moreover, the Cx3cr1 + myeloid lineage was found to play essential functions in the remodelling of the lymphatic endothelium. Mechanistically, macrophage hyaluronan was required for lymphatic sprouting by mediating direct macrophage-lymphatic endothelial cell interactions. Together, these findings reveal insight into the role of macrophages as indispensable mediators of lymphatic growth during the development of the mammalian cardiac vasculature. This work was funded by the British Heart Foundation (chair award CH/11/1/28798 and programme grant RG/08/003/25264 to PRR) and supported by the BHF Oxbridge Centre of Regenerative Medicine (RM/13/3/30159); a Wellcome Trust Doctoral Training Fellowship 106334/Z/14/Z to TJC; a Wellcome Trust Four year PhD Studentship 215103/Z/18/Z to KK; a BHF Intermediate Basic Science Research Fellowship FS/19/31/34158 to JMV; a British Israel Research and Academic Exchange Partnership (BIRAX) Grant 13BX14PRET; a Leducq Foundation Transatlantic Network of Excellence Program 14CVD04 and MRC Unit funding to DGJ. Sí

Background Obesity is associated with increased cardiovascular risk; however, the potential role of dysregulations in the adipose tissue (AT) metabolome is unknown. Objectives The aim of this study was to explore the role of dysregulation in the AT metabolome on vascular redox signaling and cardiovascular outcomes. Methods A screen was conducted for metabolites differentially secreted by thoracic AT (ThAT) and subcutaneous AT in obese patients with atherosclerosis (n = 48), and these metabolites were then linked with dysregulated vascular redox signaling in 633 patients undergoing coronary bypass surgery. The underlying mechanisms were explored in human aortic endothelial cells, and their clinical value was tested against hard clinical endpoints. Results Because ThAT volume was associated significantly with arterial oxidative stress, there were significant differences in sphingolipid secretion between ThAT and subcutaneous AT, with C16:0-ceramide and derivatives being the most abundant species released within adipocyte-derived extracellular vesicles. High ThAT sphingolipid secretion was significantly associated with reduced endothelial nitric oxide bioavailability and increased superoxide generated in human vessels. Circulating C16:0-ceramide correlated positively with ThAT ceramides, dysregulated vascular redox signaling, and increased systemic inflammation in 633 patients with atherosclerosis. Exogenous C16:0-ceramide directly increased superoxide via tetrahydrobiopterin-mediated endothelial nitric oxide synthase uncoupling and dysregulated protein phosphatase 2 in human aortic endothelial cells. High plasma C16:0-ceramide and its glycosylated derivative were independently related with increased risk for cardiac mortality (adjusted hazard ratios: 1.394; 95% confidence interval: 1.030 to 1.886; p = 0.031 for C16:0-ceramide and 1.595; 95% confidence interval: 1.042 to 2.442; p = 0.032 for C16:0-glycosylceramide per 1 SD). In a randomized controlled clinical trial, 1-year treatment of obese patients with the glucagon-like peptide-1 analog liraglutide suppressed plasma C16:0-ceramide and C16:0-glycosylceramide changes compared with control subjects. Conclusions These results demonstrate for the first time in humans that AT-derived ceramides are modifiable regulators of vascular redox state in obesity, with a direct impact on cardiac mortality in advanced atherosclerosis. (The Interaction Between Appetite Hormones; NCT02094183 )

Objectives: This study sought to evaluate the long-term prognostic implications of coronary microvascular dysfunction (CMD) when assessed with both cardiovascular magnetic resonance (CMR) and index of microcirculatory resistance (IMR) in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). Background: Post-ischemic CMD can be assessed using the pressure-wire based IMR and/or by the presence of microvascular obstruction (MVO) on CMR. Methods: A total of 198 patients with STEMI underwent IMR and MVO assessment. Patients were classified as follows: Group 1, no significant CMD (low IMR [≤40 U] and no MVO); Group 2, CMD with either high IMR (>40 U) or MVO; Group 3, CMD with both IMR >40 U and MVO. The primary endpoint was the composite of all-cause mortality, diagnosis of new heart failure, cardiac arrest, sustained ventricular tachycardia/fibrillation, and cardioverter defibrillator implantation. Results: CMD with both high IMR and MVO was present in 23.7% of the cases (Group 3) and CMD with either high IMR or MVO was observed in 40.9% of cases (Group 2). At a median follow-up of 40.1 months, the primary endpoint occurred in 34 (17%) cases. At 1 year of follow-up, Group 3 (hazard ratio [HR]: 12.6; 95% confidence interval [CI]: 1.6 to 100.6; p = 0.017) but not Group 2 (HR: 7.2; 95% CI: 0.9 to 57.9; p = 0.062) had worse clinical outcomes compared with those with no significant CMD in Group 1. However, in the long-term, patients in Group 2 (HR: 4.2; 95% CI: 1.4 to 12.5; p = 0.009) and those in Group 3 (HR: 5.2; 95% CI: 1.7 to 16.2; p = 0.004) showed similar adverse outcomes, mainly driven by the occurrence of heart failure. Conclusions: Post-ischemic CMD predicts a more than 4-fold increase in long-term risk of adverse outcomes, mainly driven by the occurrence of heart failure. Defining CMD by either invasive IMR >40 U or by CMR-assessed MVO showed similar risk of adverse outcomes.

Supplemental Digital Content is available in the text., 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.

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte- derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances including, obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis including, transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing, microarray and utilized in functional in vitro/in vivo studies.SUMMARYWe describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Acute myocardial infarction rapidly increases blood neutrophils (

Background Myocardial infarction (MI) induces activation of immune cells and alters their gene expression en route to the injured myocardium but the underlying mechanisms coordinating immune cell programming following MI remain unknown. Plasma extracellular vesicle (EV) numbers are elevated in MI, correlate with the extent of myocardial injury and mobilises immune cells from the splenic reserve to peripheral blood. Here, we describe the role of plasma EV-microRNAs (miRs) in the modulation of peripheral blood mononuclear cell (PBMC) transcriptomes post-MI. Methods PBMCs were exposed to plasma EVs followed by whole transcriptome RNA-sequencing. Plasma EVs were isolated by size-exclusion chromatography and ultra-centrifugation (2 hours at 120,000 x g) from patients presenting with ST-segment elevation MI (STEMI) (N=9) and non-STEMI (NSTEMI) (N=11) control patients. Plasma EVs were characterised by western blot and Nanoview for EV markers CD9 and CD63, transmission electron microscopy (TEM) for morphology and Nanoparticle Tracking Analysis for size and concentration. High sensitive C-reactive protein (hs-CRP) and PCSK9 were determined in plasma by ELISA and compared to plasma EV number using Pearson's correlation. Plasma EV-miRs were measured by Agilent microarray and miR-mRNA putative targets assessed by TargetScanHuman. Results Plasma EVs were positive for EV markers CD9 and CD63, displayed typical EV morphology by TEM and had a heterogeneous size and concentration distribution profile as determined by Nanoparticle Tracking Analysis. Plasma EV number correlated significantly with hs-CRP at presentation (R2= 0.20 and P Conclusions Plasma EVs mediating immune cell transcriptional programming following MI by promoting inflammatory pathways in PBMCs is a novel finding. Targeting PBMCs with EVs may allow modulation of the immune response following myocardial injury, to perturb inflammatory immune mediated damage following ischaemic injury. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation Centre of Research Excellence Awards, British Heart Foundation Project Grant, Novo Nordisk Fonden the Tripartite Immunometabolism Consortium and Wellcome Institutional Strategic Support Fund (ISSF)

Macrophages are components of the innate immune system with key roles in tissue inflammation and repair. It is now evident that macrophages also support organogenesis, but few studies have characterized their identity, ontogeny and function during heart development. Here, we show that resident macrophages in the subepicardial compartment of the developing heart coincide with the emergence of new lymphatics and interact closely with the nascent lymphatic capillaries. Consequently, global macrophage-deficiency led to extensive vessel disruption with mutant hearts exhibiting shortened and mis-patterned lymphatics. The origin of cardiac macrophages was linked to the yolk sac and fetal liver. Moreover, Csf1r+ and Cx3cr1+ myeloid sub-lineages were found to play essential functions in the remodeling of the lymphatic endothelium. Mechanistically, macrophage hyaluronan was found to be required for lymphatic sprouting by mediating direct macrophage-lymphatic endothelial cell interactions. Together, these findings reveal insight into the role of macrophages as indispensable mediators of lymphatic growth during the development of the mammalian cardiac vasculature.Summary statementTissue-resident macrophages are indispensable mediators of lymphatic vessel formation during heart development and function to remodel the vascular plexus.

Objective: The visual appearance of coronary thrombi may be clinically informative in ST elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (pPCI). However, subjective assessment is poorly reproducible and cannot provide an objective basis for treatment decisions or patient stratification. We have assessed the feasibility of a novel reflectance spectroscopy technique to systematically characterize coronary artery thrombi retrieved by aspiration during pPCI in patients with STEMI, and the clinical utility for predicting distal microvascular obstruction. Approach: Patients with STEMI treated with pPCI and thrombus aspiration (n = 288) were recruited from the Oxford Acute Myocardial Infarction (OxAMI) Study. Of these, 158 patients underwent cardiac magnetic resonance imaging within 48 h for assessment of microvascular obstruction (MVO). Coronary thrombi were imaged by reflectance spectroscopy across wavelengths 500–800 nm. Main results: Spectral data were analysed using function fitting and multivariate models. The coefficient 'c red' determined from the fitting procedure correlated with the visually-assessed colour of thrombi ('red' or 'white') and with MVO. When applied to a reduced data set, consisting of spectra from 20 patients with the largest MVO and from 20 propensity-score-matched patients with no MVO, three multivariate analysis methods were able to discriminate spectra of thrombi from patients without MVO and with the largest MVO. Significance: Reflectance spectral analysis of coronary thrombus provides new insights into the pathology of STEMI, with potential clinical implications for emergency patient care. Further studies are warranted for validation as a point-of-care stratification tool in predicting the degree of microvascular injury and clinical outcomes in STEMI.

Atherosclerotic plaques are not randomly distributed, but tend to form at the inner curvatures and branch points of arteries, where laminar flow is either disturbed or insufficient to support the normal, quiescent state of the endothelium (the lining of endothelial cells that separates the circulating blood from the arterial wall). This is an inflammatory process that involves the contribution of endothelial cells, lymphocytes, monocytes, and smooth muscle cells in conjunction with the deposition of atherogenic lipoproteins in the intimal layer of the vascular wall. The initial stage involves activation of the endothelium at regions of non-laminar flow in vessels resulting in increased permeability to Apo B-containing lipoproteins. Inflammatory cells, in particular monocytes, are recruited into the intimal layer of the vessel wall via the action of chemokines and adhesion molecules mobilized by activated endothelium.

Atherosclerosis is a complex disease process. It is increasingly recognised that both lipoprotein retention and inflammatory cellular components are intricately related in the initiation and development of atherosclerotic plaque. LDL-c (cholesterol) has been long established as a cause for atherosclerosis; additionally, inflammatory cells such as monocytes and subsequently foam cells have also been directly linked to the progression of atherosclerotic disease. Emerging data suggest that structures outside vascular intima and media are also closely related to atherosclerosis. Perivascular adipose tissue (PVAT) may be a determinant of the inflammatory status of the atherosclerotic plaque. All these features are becoming extremely relevant as therapies against atherosclerosis are targeting both lipid retention and inflammation. Recently, there has been some success in these novel therapies, such as the proprotein convertase subtilisin-kexin type 9 (PCSK-9) inhibitor evolocumab and the interleukin-1ß neutralising antibody, canakinumab, in reducing cardiovascular events when added to standard therapy such as statin. This review will discuss the pathogenesis of atherosclerosis, including some novel features, and its management using new anti-atherosclerotic drugs.