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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).

The northern Gulf of Mexico (nGOM) hypoxic zone is a shallow water environment where methane, a potent greenhouse gas, fluxes from sediments to bottom water and remains trapped due to summertime stratification. When the water column is destratified, an active planktonic methanotrophic community could mitigate the efflux of methane, which accumulates to high concentrations, to the atmosphere. To investigate the possibility of such a biofilter in the nGOM hypoxic zone we performed metagenome assembly, and metagenomic and metatranscriptomic read mapping. Methane monooxygenase (pmoA) was an abundant transcript, yet few canonical methanotrophs have been reported in this environment, suggesting a role for non-canonical methanotrophs. To determine the identity of these methanotrophs, we reconstructed six novel metagenome-assembled genomes (MAGs) in the Planctomycetota, Verrucomicrobiota and one putative Latescibacterota, each with at least one pmoA gene copy. Based on ribosomal protein phylogeny, closely related microbes (mostly from Tara Oceans) and isolate genomes were selected and co-analyzed with the nGOM MAGs. Gene annotation and read mapping suggested that there is a large, diverse and unrecognized community of active aerobic methanotrophs in the nGOM hypoxic zone and in the global ocean that could mitigate methane flux to the atmosphere.

Previous studies have demonstrated significant sex differences in vascular surgery outcomes. We assessed stroke or death rates following carotid endarterectomy (CEA) in women versus men.The Vascular Quality Initiative was used to identify all patients who underwent CEA between 2010 and 2019. Demographic, clinical, and procedural characteristics were recorded and differences between women and men were assessed using independent t-test and chi-squared test. The primary outcomes were 30-day and 1-year stroke or death. Associations between sex and outcomes were assessed using univariate/multivariate logistic regression and Cox proportional hazards analysis.Overall, 52,137 women and 79,974 men underwent CEA in Vascular Quality Initiative sites during the study period. Women were younger (70.3 vs. 70.5 years, P 0.001) and more likely to have hypertension (89.2% vs. 88.9%, P 0.05) and diabetes (36.2% vs. 35.8%, P 0.001) but less likely to be diagnosed with coronary artery disease (23.2% vs. 31.0%, P 0.001). A greater proportion of men were receiving cardiovascular risk reduction medications and had symptomatic carotid stenosis (28.5% vs. 26.7%, P 0.001). Women had shorter procedure times (113 vs. 122 min, P 0.001) and were less likely to receive electroencephalography neuromonitoring (27.9% vs. 28.8%, P 0.001), drain (35.9% vs. 37.3%, P 0.001), and protamine (67.4% vs. 68.0%, P 0.01). Stroke or death at 30 days (1.9% vs. 1.8%, P = 0.60) and 1 year (hazard ratio [HR] 0.98, 95% confidence interval [CI] 0.94-1.01, P = 0.20) were similar between groups, which persisted in asymptomatic patients (HR 0.97, 95% CI 0.93-1.01, P = 0.17) and symptomatic patients (HR 0.99, 95% CI 0.93-1.05, P = 0.71). The similarities in 1-year stroke or death rates existed in both the United States (HR 0.96, 95% CI 0.92-1.01, P = 0.09) and Canada (HR 1.21, 95% CI 0.47-3.11, P = 0.70).Despite sex differences in clinical and procedural characteristics, women and men have similar 30-day and 1-year outcomes following CEA.

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.

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.

The choroid is a vascular network that supplies the bulk of the retina's oxygen and nutrient supply. Prior studies have associated changes in the thickness of the choroid with the presence of various cardiovascular diseases. This is the first review that summarizes current knowledge on the relationship between choroidal thickness and cardiovascular diseases while highlighting important findings. Acute hypertension increases choroidal thickness. Chronic hypertension and heart failure may decrease choroidal thickness, but controversy exists. Both coronary artery disease and carotid artery stenosis result in decreased choroidal thickness and blood flow. Carotid endarterectomy may reverse these changes. Choroidal thickening in early stages of carotid stenosis may arise from mechanisms compensating for ischemia. Hyperlipidemia is linked to choroidal thickening, while caffeine intake is linked to choroidal thinning. The effects of smoking and exercise are mixed. Changes in choroidal thickness have been linked to cardiovascular disease. Clarity regarding these changes could lead to the use of choroidal thickness changes as a noninvasive screening or prognostic test for pathological cardiovascular changes. Future studies should also investigate the effect of cardiovascular disease treatments on the choroid.

The sea surface microlayer (SML) is the upper 1 mm of the ocean, where Earth’s biogeochemical processes occur between the ocean and atmosphere. It is physicochemically distinct from the water below...

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 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.

True hepatic artery aneurysms (HAAs) are rare but have been associated with a significant risk of rupture and associated mortality. The 2020 release of HAA-specific clinical practice guidelines represented an important step toward management standardization. However, it remains essential to build on the body of evidence to further refine these recommendations.The HAA management and outcomes from a single academic center during a 20-year period were retrospectively reviewed. We identified 72 patients from the institutional radiology database (November 24, 1999 to 2019). Pseudoaneurysms were excluded, and 48 patients were found to have had true HAAs. Forty-three HAA patients had sufficient medical records for inclusion in the analysis.Of the 43 patients with HAA included, 65% were male. The mean age was 63 years (range, 22-89 years). Of the HAAs, 72% presented asymptomatically, 16% had ruptured, and 12% were symptomatic at presentation. Most HAAs were of atherosclerotic origin (74%). In addition, 16% of the patients had other visceral aneurysms and 12% had nonvisceral aneurysms on presentation. The mean HAA size overall was 3.3 cm (range, 0.8-10.8 cm), with most being solitary (72%) and involving the common hepatic artery (65%). Rupture was more common in females (40%) and those with vasculitis (67%), with females representing 86% of all patients with rupture. The mean size at intervention was 4.8 cm (21 patients [49%]). Ten patients (23%) had undergone open surgical repair (seven elective and three emergent because of rupture). Eleven patients (26%) had undergone endovascular intervention (64% elective and 36% emergent). Nonoperative management was selected for 22 patients (51%). These patients had a mean HAA diameter of 2.1 cm, and 59% had a life-limiting illness. Of the 18 patients who had been initially monitored for a mean of 3.9 ± 4.1 years, 3 had undergone elective repair and 2 had minimal growth. None of these patients had a subsequently documented rupture.True HAAs are a rare but important clinical phenomenon, with 16% of patients presenting with rupture in this study. Endovascular intervention is a promising alternative to open surgical repair, with no 30-day mortality, and is suitable for ruptured HAAs. Importantly, for the first time, our findings have demonstrated an increased risk of rupture for females, highlighting the need for additional data and ultimately, sex-specific guidelines.

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of “dedifferentiated” vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don’t eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

Traumatic dissection of the innominate artery is a rare clinical entity. Management of a patient with motorsensory compromise and dissection extending to the subclavian and right common carotid arteries is quite rare and can be quite involved. Here we present such a case and discuss the unique peri-operative decision-making in the context of what is reported in the literature. Restoration of motorsensory function is critical and in this case, requiring a multi-disciplinary team.

Introduction: Type A aortic dissection can present with malperfusion syndrome. Lower limb ischaemia may resolve with repair of the dissection; however, the long duration of repair and/or persistent ischaemia may leave tissue at risk of necrosis with potential for substantial morbidity. Report: Here, the case of a 67 year old man who presented with a Type A aortic dissection with malperfusion of the lower extremities is described. The Vascular Surgery and Cardiac Surgery divisions were consulted simultaneously. A decision was made to perform bilateral superficial femoral artery cannulation with perfusion initially via the right axillary artery prior to sternotomy, then via the cardiopulmonary bypass circuit. At completion of the arch replacement, lower extremity inflow was reassessed with formal repair of the femoral arteries. Post-operatively, the patient had complete return of motor-sensory function, despite densely ischaemic symptoms at presentation several hours previously. Discussion: Cannulation of the superficial femoral arteries serves as a means to limit limb ischaemia intra-operatively and potentially improve outcomes in the setting of Type A aortic dissection with lower extremity malperfusion. Keywords: Aortic dissection, Lower extremity ischaemia, Malperfusion, Femoral artery cannulation

Stroke is the leading cause of serious disability in the world and a large number of ischemic strokes are due to thromboembolism from unstable carotid artery atherosclerotic plaque. As it is difficult to predict plaque rupture and surgical treatment of asymptomatic disease carries a risk of stroke, carotid disease continues to present major challenges with regard to clinical decision-making and revascularization. There is therefore an imminent need to better understand the molecular mechanisms governing plaque instability and rupture, as this would allow for the development of biomarkers to identify at-risk asymptomatic carotid plaque prior to disease progression and stroke. Further, it would aid in creation of therapeutics to stabilize carotid plaque. MicroRNAs (miRNAs) have been implicated as key protagonists in various stages of atherosclerotic plaque initiation, development and rupture. Notably, they appear to play a crucial role in carotid artery thromboembolism. As the molecular pathways governing the role of miRNAs are being uncovered, we are learning that their involvement is complex, tissue- and stage-specific, and highly selective. Notably, miRNAs can be packaged and secreted in extracellular vesicles (EVs), where they participate in cell–cell communication. The measurement of EV-encapsulated miRNAs in the circulation may inform disease mechanisms occurring in the plaque itself, and therefore may serve as sentinels of unstable plaque as well as therapeutic targets.

The damping of short gravity-capillary waves (Bragg waves) due to surfactant accumulation under low wind speed conditions results in the formation of natural sea slicks. These slicks are detectable visually and in synthetic aperture radar satellite imagery. Surfactants are produced by natural life processes of many marine organisms, including bacteria, phytoplankton, seaweed, and zooplankton. In this work, samples were collected in the Gulf of Mexico during a research cruise on the R/V F.G. Walton Smith to evaluate the relative abundance of Bacillus spp., surfactant-associated bacteria, in the sea surface microlayer compared to the subsurface water at 0.2 m depth. A method to reduce potential contamination of microlayer samples during their collection on polycarbonate filters was implemented and advanced, including increasing the number of successive samples per location and changing sample storage procedures. By using DNA analysis (real-time polymerase chain reaction) to target Bacillus spp., we found that in the slick areas, these surfactant-associated bacteria tended to reside mostly in subsurface waters, lending support to the concept that the surfactants they may produce move to the surface where they accumulate under calm conditions and enrich the sea surface microlayer.

Cells contributing to atherosclerotic disease are highly plastic and can shift their phenotype in a changing microenvironment. A study in Nature Metabolism now reveals that transforming growth factor-β (TGF-β) can transform endothelial cells into pro-inflammatory cells and that inhibition of TGF-β-receptor signalling in the endothelium can reverse atherosclerosis in mice.

Intestinal epithelia regulate barrier integrity when challenged by inflammation, oxidative stress, and microbes. Transforming growth factor-β1 (TGF-β1) is a cytokine with known beneficial effects on intestinal epithelia, including barrier enhancement, after exposure to proinflammatory cytokines and infectious agents. The aim of this study was to determine whether TGF-β1 directly protects intestinal epithelia during hypoxia-reoxygenation (HR). Intestinal epithelial monolayers (T84, Caco-2) were exposed to either hypoxia (1% O2, 1 h) or oxidative stress (hydrogen peroxide, 1 mM), followed by normoxic atmosphere for different time points in the absence and presence of varying concentrations of TGF-β1. Transepithelial electrical resistance (TER) assessed barrier function, with RNA extracted for reverse transcription polymerase chain reaction analysis of GPx-1, HIF-1, heme-oxygenase-1 (HO-1), and NOX-1. In some experiments, intestinal epithelia were exposed to enterohemorrhagic Escherichia coli (EHEC) O157:H7 during the reoxygenation period and TER recorded 7 h after the infectious challenge. Hypoxia-reoxygenation significantly decreased TER in intestinal epithelia compared with normoxic controls. Transforming growth factor-β1 pretreatment ameliorated HR-induced epithelial barrier dysfunction in T84 (at 1 - 3 h) and Caco-2 (1 h) monolayers. Transforming growth factor-β1 preserved barrier integrity for up to 16 h after challenge with hydrogen peroxide. In TGF-β1-treated epithelial monolayers, only HO-1 mRNA significantly increased after HR (P < 0.05 vs. normoxic controls). The EHEC-induced epithelial barrier dysfunction was significantly worsened by intestinal HR (P < 0.05 vs. normoxia-EHEC-infected cells), but this was not protected by TGF-β1 pretreatment. Transforming growth factor-β1 preserves loss of epithelial barrier integrity caused by the stress of HR via a mechanism that may involve the upregulation of HO-1 transcription. Targeted treatment with TGF-β could lead to novel therapies in enteric diseases characterized by HR injury.

Introduction: Endothelial cells (EC) must maintain an effective physiologic barrier in a highly dynamic environment. Capable of regulating other cells within the vasculature (e.g. smooth muscle cells), ECs govern response to injury and inflammation. microRNAs (miR) are emerging as critical regulators of vascular disease, implicating EC miRs in homeostasis maintenance. Increased EC permeability has been documented in atherosusceptible regions of the aorta suggesting it plays a role in disease development, but whether this is a cause or consequence is unknown. Hypothesis: We hypothesized EC barrier dysfunction is a critical event that perpetuates chronic vascular disease via altered miR profiles. Methods: Human aortic endothelial cells (HAEC) cultured in transwells were exposed to thrombin (0.5, 1, 2U/ml) and permeability measured by fluorescence flux across monolayers at 90 minutes. Total HAEC RNA was isolated at various timepoints with individual miR transcripts counted using nanoString nCounter® (n=6 samples). miR that was altered >2-fold compared to controls were analyzed using nSolver ™ software and Ingenuity® Pathway Analysis (IPA). Results: Thrombin exposure increased EC permeability to 130±8.4% of untreated controls (2U/ml thrombin; p Conclusions: In response to a permeability insult, HAEC miR levels are significantly altered. Although our preliminary data need to be further substantiated, they highlight a possible mechanism whereby EC barrier dysfunction is a critical event that perpetuates chronic vascular disease. In this way, we might envision a scenario not unlike cancer (which has been compared to atherosclerosis), where an initial appropriate endothelial repair response becomes dysregulated in the context of ongoing injury (e.g. permeability) in atherosusceptible regions.

Contexte: L'activite chirurgicale est probablement la composante la plus importante de la formation en chirurgie. La premiere annee de residence en chirurgie constitue une occasion de developper des habiletes chirurgicales tot au cours de la formation, avant que les disparites entre les habiletes des residents n'augmentent avec les annees. Il est vraisemblable que les habiletes chirurgicales sont liees au volume chirurgical. Il n'existe pas de lignes directrices publiees qui quantifient le nombre de cas chirurgicaux requis pour parvenir a la competence en chirurgie. Le but de cette etude etait de decrire les tendances actuelles concernant l'activite chirurgicale dans une cohorte recente de residents canadiens en neurochirurgie au cours de leur premiere annee de formation. Methodologie: Nous avons procede a une revue retrospective d'une base de donnees et a une enquete pour decrire la situation actuelle de la formation en chirurgie chez les residents de premiere annee en neurochirurgie. Un comite compose de cinq residents a concu cette enquete afin d'identifier les facteurs qui pourraient influencer l'activite chirurgicale des residents. Resultats: Neuf de 20 residents canadiens en neurochirurgie qui ont commence leur formation en juillet 2008 ont participe a cette etude. Le nombre median de cas completes par un resident pendant les 3 premiers mois de la rotation en neurochirurgie etait de 66, dont 12 cas au cours desquels le resident etait identifie comme etant le chirurgien principal. Les types les plus frequents d'interventions au cours desquelles le resident etait identifie comme le chirurgien principal etaient des interventions pour hemorragie intracrânienne et des derivations du liquide cephalorachidien. Conclusion: Selon cette etude preliminaire, il semble que l'activite chirurgicale des residents canadiens au cours de leur premiere annee de formation est equivalente a celle de residents participant a d'autres programmes de formation quant au volume et a la diversite de l'activite chirurgicale.

Transforming growth factor beta (TGFbeta) is a multifunctional cytokine with effects on many cell types. We recently showed that in addition to epithelial barrier enhancing properties, TGFbeta causes diminished cAMP-driven chloride secretion in colonic epithelia, in a manner that is p38 MAPK-dependent. In this study, we sought to further delineate the mechanism behind TGFbeta diminution of chloride secretion. Using colonic and kidney epithelial cell lines, we found that exposure to TGFbeta causes dramatic changes in the expression and localization of the apical membrane chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR). In TGFbeta-treated colonic epithelia (T84 and HT-29), CFTR mRNA was significantly reduced 2-24 h post-cytokine exposure. At a time consistent with decreased colonic epithelial secretory responses (16 h), TGFbeta treatment caused diminished intracellular CFTR protein expression (confocal microscopy) and reduced channel expression in the apical membrane during stimulated chloride secretion (biotinylation assay). In comparison, polarized kidney epithelia (MDCK) treated with TGFbeta displayed similarly reduced secretory responses to cAMP stimulating agents; however, a perinuclear accumulation of CFTR was observed, contrasting the diffuse cytoplasmic CFTR expression of control cells. Our data indicate that TGFbeta has profound effects on the expression and subcellular localization of an important channel involved in cAMP-driven chloride secretion, and thus suggest TGFbeta represents a key regulator of fluid movement.

Inhibition of phosphodiesterase (PDE) activity is beneficial in models of arthritis and airway inflammation. Here we assessed the ability of PDE inhibitors to modulate colitis by exposing mice to 4% (w/v) dextran sulfate sodium (DSS) drinking water for 5 days with or without rolipram, an inhibitor of PDE type 4, or the nonselective PDE inhibitor, pentoxifylline (both at 5 mg/kg, i.p., twice daily). Controls received saline, vehicle, or drug only. Colonic histology, myeloperoxidase (MPO) and tumor necrosis factor-alpha (TNF-alpha) levels, and epithelial ion transport (baseline and stimulated by electrical nerve stimulation, carbachol, and forskolin) were examined. DSS-treated mice displayed a variable diarrhea, significant histopathology in the mid-distal colon, elevated MPO activity, and reduced (50%) responses to all three pro-secretory stimuli. Treatment with rolipram, and to a lesser extent pentoxifylline, significantly reduced the severity of the colonic histopathology and MPO levels. Neither PDE inhibitor had any affect on the diminished ion transport events caused by DSS-induced colitis. However, although stimulated ion transport events were still reduced 3 days after DSS treatment, colonic segments from DSS + rolipram-treated mice displayed enhanced recovery in their secretory responsiveness, particularly to carbachol. These findings indicate that specific PDE4 inhibition can significantly reduce the tissue damage that accompanies colitis and enhance recovery of normal colonic function.

Awake craniotomy for brain tumor resection has the benefit of avoiding a general anesthetic and decreasing associated costs (e.g., intensive care unit beds and intravenous line insertion). In low- and middle-income countries, significant resource limitations for the system and individual make awake craniotomy an ideal tool, yet it is infrequently used. We sought to determine if awake craniotomy could be effectively taught and implemented safely and sustainably in low- and middle-income countries.A neurosurgeon experienced in the procedure taught awake craniotomy to colleagues in China, Indonesia, Ghana, and Nigeria during the period 2007-2012. Patients were selected on the basis of suspected intraaxial tumor, absence of major dysphasia or confusion, and ability to tolerate the positioning. Data were recorded by the local surgeons and included preoperative imaging, length of hospital admission, final pathology, postoperative morbidity, and mortality.Awake craniotomy was performed for 38 cases of suspected brain tumor; most procedures were completed independently. All patients underwent preoperative computed tomography or magnetic resonance imaging. In 64% of cases, patients remained in the hospital10 days. The most common pathology was high-grade glioma, followed by meningioma, low-grade glioma, and metastasis. No deaths occurred, and no case required urgent intubation. The most common perioperative and postoperative issue was seizure, with 1 case of permanent postoperative deficit.Awake craniotomy was successfully taught and implemented in 6 neurosurgical centers in China, Indonesia, Ghana, and Nigeria. Awake craniotomy is safe, resource-sparing, and sustainable. The data suggest awake craniotomy has the potential to significantly improve access to neurosurgical care in resource-challenged settings.

Objective Contributing to medical practice in developing countries has become increasingly prevalent. Primary care and preventative health initiatives have been most visible, although attention has recently shifted to surgical disease, which represents a large burden in resource-poor settings. Typically dominated by individual efforts, there is now a more concerted approach, with surgical care being included in the comprehensive primary health care plan set by the World Health Organization. Although ethical dilemmas in international surgery have been discussed sporadically in the context of specific missions from the visiting surgeon/team perspective, we are missing a comprehensive evaluation of these issues in the literature. Here we have chosen to systematically categorize ethical issues confronted while teaching and operating in a developing country into 2 broad categories: venue (i.e., host) and visitor related. Methods For each category, topics within follow an ordinal sequence that one might use when designing a surgical education mission. Illustrative examples are provided, as well as a depiction of the ethical principles or theories involved. Results and Conclusions This article provides a discussion written from visiting and host surgeon perspectives on diverse ethical challenges for which there is limited literature, including location selection, unmet needs at home, role of sponsors, and personal gain. In addition to candid discussion and a solutions-focused approach, the reader is provided with an “ethical checklist” for international surgical education, akin to the World Health Organization surgical safety checklist, to serve as a framework for the design of surgical missions that avoid ethical pitfalls. • Peer-Review Article

Background: Bacterial vaginosis (BV) has been associated with increased HIV cervicovaginal shedding. We hypothesized that this might relate to BV-associated increases in mucosal activated CD4 + T cells, which could enhance local HIV replication. Methods: Vaginal flora, cytokine/chemokine levels, and mucosal immune cell populations collected by cervical cytobrush were analyzed in 15 HIV-infected Kenyan female sex workers, before and after BV therapy with oral metronidazole. Results: Therapy reduced the Nugent score in all but 1 participant, and BV elimination was associated with reduced genital levels of interleukin 1β(IL1β), interleukin 8 (IL-8), and Regulated Upon Activation Normal T-cell Expressed and Secreted (RANTES). In addition, BV elimination reduced the total number of cervical CD4 + T cells, including those expressing the HIV coreceptor CCR5 and the activation marker CD69. Conclusions: BV induces significant and reversible alterations in cervical immune cell populations and local inflammatory cytokines that would be expected to enhance local HIV replication.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an enteric pathogen that causes potentially fatal symptoms after intimate adhesion, modulation of intestinal epithelial signal transduction, and alteration of epithelial function (eg, barrier disruption). Although the epithelial barrier is critical to gut homeostasis, only a few agents, such as transforming growth factor (TGF)-beta, can enhance or protect epithelial barrier function. Our aims were to delineate the mechanism(s) behind TGF-beta-induced barrier enhancement and to determine whether TGF-beta could prevent EHEC-induced barrier disruption. Using monolayers of the human T84 colonic epithelial cell line, we found that TGF-beta induced a significant increase in transepithelial electrical resistance (a measure of paracellular permeability) through activation of ERK MAPK and SMAD signaling pathways and up-regulation of the tight junction protein claudin-1. Additionally, TGF-beta pretreatment of epithelia blocked the decrease in transepithelial electrical resistance and the increase in transepithelial passage of [(3)H]-mannitol caused by EHEC infection. EHEC infection was associated with reduced expression of zonula occludens-1, occludin, and claudin-2 (but not claudin-1 or claudin-4); TGF-beta pretreatment prevented these changes. These studies provide insight into EHEC pathogenesis by illustrating the mechanisms underlying TGF-beta-induced epithelial barrier enhancement and identifying TGF-beta as an agent capable of blocking EHEC-induced increases in epithelial permeability via maintenance of claudin-2, occludin, and zonula occludens-1 levels.

The normal gut flora has been implicated in the pathophysiology of inflammatory bowel disease and there is increased interest in the role that stress can play in gut disease. The chemical stressor dinitrophenol (DNP, uncouples oxidative phosphorylation) was injected into the ileum of laparotomized rats and mitochondria structure, epithelial permeability, and inflammatory cell infiltrate were examined 6 and 24 hours later. Monolayers of human colonic epithelial cells (T84, HT-29) were treated with DNP +/- commensal Escherichia coli, followed by assessment of epithelial permeability, bacterial translocation, and chemokine (ie, interleukin-8) synthesis. Delivery of DNP into rat distal ileum resulted in disruption of epithelial mitochondria; similar changes were noted in mildly inflamed ileal resections from patients with Crohn's disease. Also, DNP-treated ileum displayed increased gut permeability and immune cell recruitment. Subsequent studies revealed deceased barrier function, increased bacterial translocation, increased production of interleukin-8, and enhanced mobilization of the transcription factor AP-1 in the model epithelial cell lines exposed to commensal bacteria (E. coli strains HB101 or C25), but only when the monolayers were pretreated with DNP (0.1 mmol/L). These data suggest that enteric epithelia under metabolic stress perceive a normally innocuous bacterium as threatening, resulting in loss of barrier function, increased penetration of bacteria into the mucosa, and increased chemokine synthesis. Such responses could precipitate an inflammatory episode and contribute to existing enteric inflammatory disorders.