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Rationale: Extracellular vesicles (EVs) contain bioactive cargo including microRNAs (miRNAs) and proteins that are released by cells as a form of cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels and thereby interface with cells in the circulation as well as cells residing in the vascular wall. It is unknown whether ECs have the capacity to release EVs capable of governing recipient cells within two separate compartments, and how this is affected by endothelial activation commonly seen in atheroprone regions.Objective: Given their boundary location, we propose that ECs utilize bidirectional release of distinct EV cargo in quiescent and activated states to communicate with cells within the circulation and blood vessel wall.Methods and Results: EVs were isolated from primary human aortic endothelial cells (ECs) (+/- IL-1β activation), quantified, and analysed by miRNA transcriptomics and proteomics. Compared to quiescent ECs, activated ECs increased EV release, with miRNA and protein cargo that were related to atherosclerosis. RNA sequencing of EV-treated monocytes and smooth muscle cells (SMCs) revealed that EVs from activated ECs altered pathways that were pro-inflammatory and atherogenic. Apical and basolateral EV release was assessed using ECs on transwells. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis.In silicoanalysis determined that compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and SMCs, respectively.Conclusions: The demonstration that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance our ability to design endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.Non-standard Abbreviations and Acronymscryo-EMcryogenic electron microscopyECendothelial cellEVextracellular vesicleGOgene ontologyHAEChuman aortic endothelial cellSMChuman aortic vascular smooth muscle cellIL-1βinterleukin 1 betaKEGGKyoto encyclopedia of genes and genomesLC-MSlabel-free liquid-chromatography mass spectrometryMVBmultivesicular bodymiRNAmicroRNARNAseqRNA sequencingTEMtransmission electron microscopyTIRFtotal interal reflection fluorescence microscopymiRNAmicroRNAGraphical abstract: Polarized endothelial extracellular vesicle communication with luminal and abluminal vascular cellsEndothelial cell small extracellular vesicle (EC-EV) release from apical (luminal) and basolateral (abluminal) surfaces in quiescence and after endothelial activation. Quiescent EC-EVs are depicted in blue (bright blue=apical, light blue=basolateral), while activated EC-EVs are depicted in red (bright red=apical, light red=basolateral). Luminal monocyte is represented in purple with upregulation of pro-inflammatory transcripts (bright purple) after uptake of activated EC-EVs from the apical surface, compared to uptake of quiescent apical EC-EVs (light purple). Basolateral EC-EVs are taken up by an abluminal resident smooth muscle cell depicted in yellow. Smooth muscle cell uptake of activated basolateral EC-EVs with upregulation of pro-inflammatory/pro-atherogenic transcripts (bright yellow), as compared to uptake of quiescent EC-EVs (light yellow).

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is an essential and evolutionarily conserved transcription factor complex primarily involved in innate immunity and inflammation. Transposable elements (TEs) can be co-opted to innovate immune transcriptional regulatory networks; however, the extent to which TEs have contributed to the modulation of NF-κB response in different mammalian lineages is not well established. Here we performed a multi-species analysis of TEs bound by the NF-κB subunit RELA (p65) in response to the pro-inflammatory cytokine TNFα (Tumor Necrosis Factor alpha). Using endothelial cell RELA ChIP-seq data from human, mouse and cow, we found that 55 TE subfamilies were enriched within NF-κB bound regions. These RELA-bound transposons possess multiple active epigenetic features and reside near TNFα-induced genes. A prominent example of lineage-specific contribution of transposons comes from the bovine SINE subfamilies Bov-tA1/2/3 which collectively contributed over 14,000 NF-κB bound regions in cow. By comparing NF-κB binding data across species, we found several examples of NF-κB motif-bearing TEs that appeared to colonize the genome prior to the divergence of the selected mammals, including a DNA transposon MER81, whose ancestral sequence contains two intact RELA motifs. We demonstrate that one NF-κB bound MER81 element can control the TNFα-induced expression ofINFGR2(Interferon Gamma Receptor 2) in human. Lastly, the presence of RELA motifs within MER81 elements appeared to stabilize during human evolution, indicative of purifying selection acting on a subset of these NF-κB bound ancient DNA transposons. Taken together, our results implicate multiple transposons in establishing NF-κB mediated regulatory networks during mammalian evolution.

Liver failure causes blood-brain-barrier (BBB) breakdown leading to central nervous system damage, however the mechanisms whereby the liver influences BBB-integrity remain elusive. One possibility is that the liver secretes an as-yet to be identified molecule(s) that circulate in the serum to directly promote BBB integrity. We developed light-sheet imaging for three-dimensional study of BBB function. We show that liver- or muscle-specific knockout of Hfe2 induces BBB breakdown, leading to accumulation of toxic-blood-derived fibrinogen in the brain, lower cortical neuron numbers, and behavioral deficits. In healthy animals, soluble Hfe2 competes with its homologue RGMa for binding to Neogenin, thereby blocking RGMa-induced downregulation of PDGF-B and Claudin-5 in endothelial cells and the ensuing BBB disruption. Hfe2 administration in an animal model of multiple sclerosis prevented paralysis and immune cell infiltration by inhibiting RGMa-mediated BBB alteration. This study has implications for the pathogenesis and potential treatment of diseases associated with BBB dysfunction such as multiple sclerosis.

The regulatory elements controlling gene expression during acute inflammation are not fully elucidated. Here we report the identification of a set of NF-κB-bound elements and common chromatin landscapes underlying the acute inflammatory response across cell-types and mammalian species. Using primary vascular endothelial cells (human/mouse/bovine) treated with the pro−inflammatory cytokine, Tumor Necrosis Factor-α, we identify extensive (~30%) conserved orthologous binding of NF-κB to accessible, as well as nucleosome-occluded chromatin. Regions with the highest NF-κB occupancy pre-stimulation show dramatic increases in NF-κB binding and chromatin accessibility post-stimulation. These ‘pre-bound’ regions are typically conserved (~56%), contain multiple NF-κB motifs, are utilized by diverse cell types, and overlap rare non-coding mutations and common genetic variation associated with both inflammatory and cardiovascular phenotypes. Genetic ablation of conserved, ‘pre-bound’ NF-κB regions within the super-enhancer associated with the chemokine-encoding CCL2 gene and elsewhere supports the functional relevance of these elements., Genetic elements that control inflammatory gene expression are not fully elucidated. Here the authors conduct a multi-species analysis of chromatin landscape and NF-κB binding in response to the proinflammatory cytokine TNFα, finding that conserved NF-κB bound regions are linked to enhancer activity and disease.

Rationale: We previously identified somatic activating mutations in the KRAS ( Kirsten rat sarcoma viral oncogene homologue ) gene in the endothelium of the majority of human sporadic brain arteriovenous malformations; a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to arteriovenous malformations, remains unknown. Objective: To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for arteriovenous malformations. Methods and Results: Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce brain arteriovenous malformations. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity but instead seem to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent arteriovenous malformations in zebrafish are refractory to inhibition of the downstream effector PI3K but instead require active MEK (mitogen-activated protein kinase kinase 1) signaling. Conclusions: We demonstrate that active KRAS expression in the endothelium is sufficient for brain arteriovenous malformations, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for arteriovenous malformation patients. Graphical Abstract: A graphical abstract is available for this article.

Objective: Coronavirus disease 2019 (COVID-19) is a global pandemic involving >5 500 000 cases worldwide as of May 26, 2020. The culprit is the severe acute respiratory syndrome coronavirus-2, which invades cells by binding to ACE2 (angiotensin-converting enzyme 2). While the majority of patients mount an appropriate antiviral response and recover at home, others progress to respiratory distress requiring hospital admission for supplemental oxygen. In severe cases, deterioration to acute respiratory distress syndrome necessitating mechanical ventilation, development of severe thrombotic events, or cardiac injury and dysfunction occurs. In this review, we highlight what is known to date about COVID-19 and cardiovascular risk, focusing in on the putative role of the endothelium in disease susceptibility and pathogenesis. Approach and Results: Cytokine-driven vascular leak in the lung alveolar-endothelial interface facilitates acute lung injury in the setting of viral infection. Given that the virus affects multiple organs, including the heart, it likely gains access into systemic circulation by infecting or passing from the respiratory epithelium to the endothelium for viral dissemination. Indeed, cardiovascular complications of COVID-19 are highly prevalent and include acute cardiac injury, myocarditis, and a hypercoagulable state, all of which may be influenced by altered endothelial function. Notably, the disease course is worse in individuals with preexisting comorbidities that involve endothelial dysfunction and may be linked to elevated ACE2 expression, such as diabetes mellitus, hypertension, and cardiovascular disease. Conclusions: Rapidly emerging data on COVID-19, together with results from studies on severe acute respiratory syndrome coronavirus-1, are providing insight into how endothelial dysfunction may contribute to the pandemic that is paralyzing the globe. This may, in turn, inform the design of biomarkers predictive of disease course, as well as therapeutics targeting pathogenic endothelial responses.

We have adapted a zebrafish (Danio rerio) tumor xenograft model for use in the study of oncolytic virotherapy. Following implantation of mammalian cancer cells into the perivitelline space of developing zebrafish embryos, both local and intravenous oncolytic virus treatments produce a tumor-specific infection with measurable antitumor effects. Tumor cells are injected at 48 h post fertilization, with oncolytic virus treatment then being administered 24 h later to allow for an initial period of tumor development and angiogenesis. Confocal fluorescent imaging is used to quantify dynamics within the tumor environment. The natural translucency of zebrafish at the embryo stage, coupled with the availability of strains with fluorescent immune and endothelial cell reporter lines, gives the model broad potential to allow for real time, in vivo investigation of important events within tumors throughout the course of virotherapy. Zebrafish xenografts offer a system with biologic fidelity to processes in human cancer development that influence oncolytic virus efficacy, and to our knowledge this is the first demonstration of the model’s use in the context of virotherapy. Compared with other models, our protocol offers a powerful, inexpensive approach to evaluating novel oncolytic viruses and oncolytic virus-based combination therapies, with potential application to investigating the impacts of virotherapy on immune response, tumor vasculature, and metastatic disease.

Coronary artery disease is one of the major causes of morbidity and mortality worldwide. Coronary stents, tube-shaped medical implants that are placed in narrowed coronary arteries, have been used successfully in the management of this condition. However, re-narrowing (i.e. restenosis) of the artery can occur which is instigated by an immune response towards the implanted ‘foreign’ material. A new approach to prevent restenosis and reduce the stent-induced immune response has been proposed previously, which involves re-endothelialization of the implanted stent. In the present study a proof-of-concept experiment involving surface-modified medical grade steel was employed in order to examine the best surface chemistry for in vitro cell capture. Steel coupons were coated with a silanized adlayer, followed by attachment of whole antibodies, followed by culturing of human umbilical vein endothelial cells (HUVECs) or human aortic endothelial cells (HAECs or HAoECs). With regard to the adlayer-antibody configuration, HUVECs adhered and grew with normal morphology, and a significantly increased number of HUVECs proliferated on the coupons compared to the ‘bare’ surface. Similar effects were observed with HAECS grown on adlayer-antibody modified substrates, with a significantly higher number of cells proliferating. These results demonstrate a successful strategy for re-endothelialization of the steel surface that may prevent immune response with respect to the behaviour of steel-based stents in vivo.

Background: Stroke is a devastating consequence of plaque rupture from the carotid arteries. Current management of carotid plaques involves waiting for symptoms (e.g., stroke or mini-stroke), as intervention itself has risk of stroke and not all plaques are vulnerable to rupture. There is a need to better risk-stratify plaque that causes stroke. Carotid ultrasound (US) is a non-invasive and inexpensive visualization of plaques but is limited by human interpretation. We hypothesize that convolutional neural networks (CNNs) will identify unique features of carotid plaques for automated risk stratification. Methods: Our workflow is illustrated in Fig. a. A total of 141 B-mode US images of carotid arteries were included; 64 high-risk with symptomatic carotid plaques and 75 low-risk with no significant plaque. Data was cropped and divided into training (70%) and holdout test (30%) subsets. During model training, an ensemble of ResNet-18 CNNs learned classification of low-risk and high-risk cases using five-fold stratified cross validation and was used to predict on the holdout test set. The model was evaluated using ROC-AUC and sensitivity. Saliency maps were used for model interpretability to highlight relevant pixels for model decisions. Results: The cross-validation AUC was 0.995 ± 0.010. The testing AUC was 0.909 and class-wise sensitivities were 0.88 (low-risk) and 0.79 (high-risk). The density plot (Fig. b) shows that the classifier correctly identifies both classes with confidence. Model interpretability using saliency maps (Fig. c) shows pixels corresponding to carotid artery vessel edges and in high-risk cases, carotid plaques. Conclusions: Using this proof-of-concept model, carotid US long axis images are sufficient to identify high-risk plaques in symptomatic patients - we now need to determine whether we can identify high-risk plaques before symptoms to prevent devastating stroke caused by carotid disease.

Aim: Abdominal aortic aneurysm (AAA) contributes to significant post-rupture mortality in aging populations. AAA management with watchful waiting and surgical repair is based on our limited understanding of disease processes, and current research foci including extracellular vesicles (EVs; nano-sized packages of proteins, RNAs, and lipids that facilitate intercellular communication) may aid in developing novel AAA therapies. To characterize this regulatory cargo, we isolated EVs from human AAA tissue or control aortic punch biopsies and profiled EV content with microRNA (miRNA) sequencing to identify dysregulated pathways. Methods: The study was approved by the University Health Network Research Ethics Board. EVs were isolated from human AAA or aortic punch tissue and enriched using size exclusion chromatography (SEC; qEVoriginal columns 70 nm, Izon Science Ltd.) (n=3). EV size and concentration were determined using nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Panalytical Ltd.). EV-miRNA sequencing was performed with Illumina NextSeq (HTG Molecular Diagnostics Inc.) and analyzed using Partek Genomics Suite (v.10) and MIENTURNET (19-11-25). Results: Patients were selected for infrarenal AAA requiring surgical repair (AAA) or coronary artery disease requiring bypass graft surgery (control). EV size and concentration were confirmed with NTA. Principal components and gene set analyses revealed distinct clustering of tissue types with 901 and 687 miRNAs enriched in AAA and control samples, respectively. Pathway prediction using established AAA miRNAs (e.g., miR-122, miR-146a, and miR-503) identified significant interactions with proaneurysmal signaling pathways (e.g., PI3K-AKT, JAK-STAT, and HIF-1) as well as cell senescence and adhesion processes (FDR < 0.05). Conclusion: EV-derived miRNAs from patients with AAA prominently associate with cell signaling, senescence, and adhesion pathways in aneurysm pathogenesis. To our knowledge, this is the first study to profile the EV-miRNA landscape in human AAA tissue. Further investigation will explore EV-miRNAs as mediators of communication between distinct vascular cell populations that contribute to AAA development.

Coronavirus disease 2019 (COVID-19) was primarily identified as a novel disease causing acute respiratory syndrome. However, as the pandemic progressed various cases of secondary organ infection and damage by severe respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, including a breakdown of the vascular barrier. As SARS-CoV-2 gains access to blood circulation through the lungs, the virus is first encountered by the layer of endothelial cells and immune cells that participate in host defense. Here, we developed an approach to study SARS-CoV-2 infection using vasculature-on-a-chip. We first modeled the interaction of virus alone with the endothelialized vasculature-on-a-chip, followed by the studies of the interaction of the virus exposed-endothelial cells with peripheral blood mononuclear cells (PBMCs). In an endothelial model grown on a permeable microfluidic bioscaffold under flow conditions, both human coronavirus (HCoV)-NL63 and SARS-CoV-2 presence diminished endothelial barrier function by disrupting VE-cadherin junctions and elevating the level of pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, and angiopoietin-2. Inflammatory cytokine markers were markedly more elevated upon SARS-CoV-2 infection compared to HCoV-NL63 infection. Introduction of PBMCs with monocytes into the vasculature-on-a-chip upon SARS-CoV-2 infection further exacerbated cytokine-induced endothelial dysfunction, demonstrating the compounding effects of inter-cellular crosstalk between endothelial cells and monocytes in facilitating the hyperinflammatory state. Considering the harmful effects of SARS-CoV-2 on endothelial cells, even without active virus proliferation inside the cells, a potential therapeutic approach is critical. We identified angiopoietin-1 derived peptide, QHREDGS, as a potential therapeutic capable of profoundly attenuating the inflammatory state of the cells consistent with the levels in non-infected controls, thereby improving the barrier function and endothelial cell survival against SARS-CoV-2 infection in the presence of PBMC.

IMPORTANCE: Although myocardial injury can occur with acute COVID-19, there is limited understanding of changes with myocardial metabolism in recovered patients. OBJECTIVE: To examine myocardial metabolic changes early after recovery from COVID-19 using fluorodeoxyglucose–positron emission tomography (PET) and associate these changes to abnormalities in cardiac magnetic resonance imaging (MRI)–based function and tissue characterization measures and inflammatory blood markers. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study took place at a single-center tertiary referral hospital system. A volunteer sample of adult patients within 3 months of a diagnosis of COVID-19 who responded to a mail invitation were recruited for cardiac PET/MRI and blood biomarker evaluation between November 2020 and June 2021. EXPOSURES: Myocardial inflammation as determined by focal fluorodeoxyglucose (FDG) uptake on PET. MAIN OUTCOMES AND MEASURES: Demographic characteristics, cardiac and inflammatory blood markers, and fasting combined cardiac (18)F-FDG PET/MRI imaging were obtained. All patients with focal FDG uptake at baseline returned for repeated PET/MRI and blood marker assessment 2 months later. RESULTS: Of 47 included patients, 24 (51%) were female, and the mean (SD) age was 43 (13) years. The mean (SD) interval between COVID-19 diagnosis and PET/MRI was 67 (16) days. Most patients recovered at home during the acute infection (40 [85%]). Eight patients (17%) had focal FDG uptake on PET consistent with myocardial inflammation. Compared with those without FDG uptake, patients with focal FDG uptake had higher regional T2, T1, and extracellular volume (colocalizing with focal FDG uptake), higher prevalence of late gadolinium enhancement (6 of 8 [75%] vs 9 of 39 [23%], P = .009), lower left ventricular ejection fraction (mean [SD], 55% [4%] vs 62% [5%], P

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)–dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)–dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB–dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB–dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe −/− mice fed a cholesterol-rich (Western) diet for 8 weeks. Results: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P P RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet–induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B , E-selectin , and monocyte attachment. Conclusions: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains poorly understood. Here, we performed single-cell RNA sequencing of 599,215 freshly isolated endothelial, perivascular and other tissue-derived cells from 47 fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We uncover extensive molecular heterogeneity of healthy fetal and adult human brains and across eight vascular-dependent CNS pathologies including brain tumors and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict numerous endothelial-to-perivascular cell ligand-receptor crosstalk including immune-related and angiogenic pathways, thereby unraveling a central role for the endothelium within brain neurovascular unit signaling networks. Our single-cell brain atlas provides insight into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Background Type 2 diabetes (T2D) is associated with coronary microvascular dysfunction, which is thought to contribute to compromised diastolic function, ultimately culminating in heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms remain incompletely understood, and no early diagnostics are available. We sought to gain insight into biomarkers and potential mechanisms of microvascular dysfunction in obese mouse (db/db) and lean rat (Goto-Kakizaki) pre-clinical models of T2D-associated diastolic dysfunction. Methods The microRNA (miRNA) content of circulating extracellular vesicles (EVs) was assessed in T2D models to identify biomarkers of coronary microvascular dysfunction/rarefaction. The potential source of circulating EV-encapsulated miRNAs was determined, and the mechanisms of induction and the function of candidate miRNAs were assessed in endothelial cells (ECs). Results We found an increase in miR-30d-5p and miR-30e-5p in circulating EVs that coincided with indices of coronary microvascular EC dysfunction (i.e., markers of oxidative stress, DNA damage/senescence) and rarefaction, and preceded echocardiographic evidence of diastolic dysfunction. These miRNAs may serve as biomarkers of coronary microvascular dysfunction as they are upregulated in ECs of the left ventricle of the heart, but not other organs, in db/db mice. Furthermore, the miR-30 family is secreted in EVs from senescent ECs in culture, and ECs with senescent-like characteristics are present in the db/db heart. Assessment of miR-30 target pathways revealed a network of genes involved in fatty acid biosynthesis and metabolism. Over-expression of miR-30e in cultured ECs increased fatty acid β-oxidation and the production of reactive oxygen species and lipid peroxidation, while inhibiting the miR-30 family decreased fatty acid β-oxidation. Additionally, miR-30e over-expression synergized with fatty acid exposure to down-regulate the expression of eNOS, a key regulator of microvascular and cardiomyocyte function. Finally, knock-down of the miR-30 family in db/db mice decreased markers of oxidative stress and DNA damage/senescence in the microvascular endothelium. Conclusions MiR-30d/e represent early biomarkers and potential therapeutic targets that are indicative of the development of diastolic dysfunction and may reflect altered EC fatty acid metabolism and microvascular dysfunction in the diabetic heart.

Aims: The transcription factor ERG has emerged as an important regulator of vascular function through its ability to repress inflammation in endothelial cells (ECs) and ERG dysregulation is associated with chronic inflammation in atherosclerosis and aortic aneurysms. To characterize the cellular mechanisms that underlie this regulation, we deleted ERG in human aortic ECs and assessed genome-wide chromatin accessibility and gene expression to identify dysregulated pathways. Furthermore, we have begun to investigate ERG expression in mouse atherosclerotic plaque. Methods: CRISPR/Cas9 was used to delete ERG exon 6 in immortalized human aortic ECs. Wildtype or Δ ERG ECs were profiled with ATAC-seq and RNA-seq (n=2-4). GREAT and clusterProfiler were used to identify enriched pathways (log 2 FC>|1|; FDRLdlr −/− mice were fed a 12-week high-cholesterol diet (1.25%) before sacrifice. Ascending aortic sections were stained with anti-CD68 and anti-ERG (n=2). Images were obtained using a Leica SP8 confocal microscope. Results: Pathway analysis of >21,000 differentially accessible chromatin regions identified 67 enriched processes in Δ ERG ECs. Of these, the top 20% included disrupted wound healing, abnormal aorta morphology, and aneurysm, which were driven by association with proatherogenic and proaneurysmal genes including PDGFRB and TGFBR1 . Gene expression analysis revealed that loss of ERG contributes to endothelial to mesenchymal transition (increased KLF4 and SNAI2 ), inflammation (increased IL18 ), and dysregulated extracellular matrix and cell adhesion (increased FBLN2 and VCAN ). In the mouse aorta, preliminary data suggest that ERG expression may be elevated in EC nuclei overlying plaque compared to non-atherosclerotic regions. Conclusions: The loss of ERG has a broad impact on chromatin accessibility and gene expression in aortic ECs, with enrichment of vascular dysfunction pathways. Further investigation will establish how altered ERG expression contributes to in vivo models of atherosclerosis and aortic aneurysms.

Oral tyrosine kinase inhibitors have revolutionized the treatment of chronic myelogenous leukemia, with many patients achieving major clinical and molecular responses without complications. While typically well-tolerated, clinical experience with tyrosine kinase inhibitors (particularly those of the second and third generations) has highlighted unanticipated associations with serious non-cancer adverse effects on various organs, particularly the cardiovascular system. Herein, we review the current literature surrounding the major cardiovascular toxicities of BCR-ABL1 tyrosine kinase inhibitors in chronic myelogenous leukemia, discuss potential mechanisms underpinning their development, and suggest future research directions to uncover novel ways to reduce cardiovascular events in patients treated with tyrosine kinase inhibitors. As a whole, while cardiovascular toxicities are well-documented, the mechanistic basis of these clinical observations remains poorly defined. In turn, to provide safe and effective treatment to all patients, it is necessary to close the knowledge gap regarding mechanisms that drive toxicity and elucidate the complex interactions that predispose specific individuals to these toxicities.

Significant improvements in cancer survival have brought to light unintended long-term adverse cardiovascular effects associated with cancer treatment. Although capable of manifesting a broad range of cardiovascular complications, cancer therapy-related cardiac dysfunction (CTRCD) remains particularly common among the mainstay anthracycline-based and human epidermal growth factor receptor-targeted therapies. Unfortunately, the early asymptomatic stages of CTRCD are difficult to detect by cardiac imaging alone, and the initiating mechanisms remain incompletely understood. More recently, circulating inflammatory markers, cardiac biomarkers, microRNAs, and extracellular vesicles (EVs) have been considered as early markers of cardiovascular injury. Concomitantly, the role of the endothelium in regulating cardiac function in the context of CTRCD is starting to be understood. In this review, we highlight the impact of breast cancer therapies on the cardiovascular system with a focus on the endothelium, and examine the status of circulating biomarkers, including inflammatory markers, cardiac biomarkers, microRNAs, and endothelial cell-derived EVs. Investigation of these emerging biomarkers may uncover mechanisms of injury, detect early stages of cardiovascular damage, and elucidate novel therapeutic approaches.

We have adapted a zebrafish (Danio rerio) tumor xenograft model for use in the study of oncolytic virotherapy. Following implantation of mammalian cancer cells into the perivitelline space of developing zebrafish embryos, both local and intravenous oncolytic virus treatments produce a tumor-specific infection with measurable antitumor effects. Tumor cells are injected at 48 h post fertilization, with oncolytic virus treatment then being administered 24 h later to allow for an initial period of tumor development and angiogenesis. Confocal fluorescent imaging is used to quantify dynamics within the tumor environment. The natural translucency of zebrafish at the embryo stage, coupled with the availability of strains with fluorescent immune and endothelial cell reporter lines, gives the model broad potential to allow for real time, in vivo investigation of important events within tumors throughout the course of virotherapy. Zebrafish xenografts offer a system with biologic fidelity to processes in human cancer development that influence oncolytic virus efficacy, and to our knowledge this is the first demonstration of the model's use in the context of virotherapy. Compared with other models, our protocol offers a powerful, inexpensive approach to evaluating novel oncolytic viruses and oncolytic virus-based combination therapies, with potential application to investigating the impacts of virotherapy on immune response, tumor vasculature, and metastatic disease.

Background Sporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown. Methods We analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase-chain-reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations,...

Objective— During inflammation, macrophages secrete vesicles carrying RNA, protein, and lipids as a form of extracellular communication. In the vessel wall, extracellular vesicles (EVs) have been shown to be transferred between vascular cells during atherosclerosis; however, the role of macrophage-derived EVs in atherogenesis is not known. Here, we hypothesize that atherogenic macrophages secrete microRNAs (miRNAs) in EVs to mediate cell–cell communication and promote proinflammatory and proatherogenic phenotypes in recipient cells. Approach and Results— We isolated EVs from mouse and human macrophages treated with an atherogenic stimulus (oxidized low-density lipoprotein) and characterized the EV miRNA expression profile. We confirmed the enrichment of miR-146a, miR-128, miR-185, miR-365, and miR-503 in atherogenic EVs compared with controls and demonstrate that these EVs are taken up and transfer exogenous miRNA to naive recipient macrophages. Bioinformatic pathway analysis suggests that atherogenic EV miRNAs are predicted to target genes involved in cell migration and adhesion pathways, and indeed delivery of EVs to naive macrophages reduced macrophage migration both in vitro and in vivo. Inhibition of miR-146a, the most enriched miRNA in atherogenic EVs, reduced the inhibitory effect of EVs on macrophage migratory capacity. EV-mediated delivery of miR-146a repressed the expression of target genes IGF2BP1 (insulin-like growth factor 2 mRNA-binding protein 1) and HuR (human antigen R or ELAV-like RNA-binding protein 1) in recipient cells, and knockdown of IGF2BP1 and HuR using short interfering RNA greatly reduced macrophage migration, highlighting the importance of these EV-miRNA targets in regulating macrophage motility. Conclusions— EV-derived miRNAs from atherogenic macrophages, in particular miR-146a, may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall.

Rationale: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor κ light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)–derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. Objective: To define the role of endogenous miR-146a during atherogenesis. Methods and Results: Paradoxically, Ldlr −/− (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr −/− ;miR-146a −/− mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr −/− mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1( Sort1 ), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. Conclusions: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells.

Emerging evidence suggests that long non-coding RNAs (lncRNAs) represent a cellular hub coordinating various cellular processes that are critical in health and disease. Mechanical stress triggers changes in vascular smooth muscle cells (VSMCs) that in turn contribute to pathophysiological changes within the vasculature. We sought to evaluate the role that lncRNAs play in mechanical stretch-induced alterations of human aortic smooth muscle cells (HASMCs). RNA (lncRNA and mRNA) samples isolated from HASMCs that had been subjected to 10 or 20% elongation (1 Hz) for 24 h were profiled with the Arraystar Human LncRNA Microarray V3.0. LncRNA expression was quantified in parallel via qRT-PCR. Of the 30,586 human lncRNAs screened, 580 were differentially expressed (DE, P