Novel protocol for the transcriptomic analysis of endothelial extracellular vesicles in atherosclerosis.

INTRODUCTION: Despite the key role of the endothelium in atherosclerosis, there are no direct techniques for its analysis. The study of extracellular vesicles of endothelial origin (EEVs), might lead to the identification of molecular signatures and early biomarkers of atherosclerosis. The aim of this work was to set up the methods for EEVs separation and transcriptomic analysis. METHODS: We adapted an antibody-magnetic-bead based immunocapture protocol for plasma EEVs separation from control (G1), subclinical atherosclerosis (G2) and peripheral artery disease subjects (PAD) (G3), and modified an ultra-low input RNASeq method (n=5/group). By bioinformatics analysis we compared the transcriptome of plasma EEVs with that of human aortic endothelial cells (TeloHAECs), and then, searched for differentially expressed genes (DEG) among EEVs of G1, G2 and G3. From those DEG, UCP2 was selected for further validation in plasma EVs (qPCR), and in vitro, in stimulated TeloHAECs (IL-1ß, TNFα, oxLDL and hypoxia). RESULTS: The RNASeq analysis of plasma EEVs rendered 1667 genes enriched in transcripts expressed by TeloHAECs (NES: 1.93, p adjust=1.4e-73). One hundred seventy DEGs were identified between G2 vs G1, and 180 between G3 vs G1, of which 17 were similarly expressed in G2 and G3 vs control, including UCP2. IL-1ß and TNFα (10ng/mL, p<0.05), hypoxia (1% O2, p=0.05) and oxLDL (100µg/mL, p=0.055) reduced UCP2 expression in TeloHAECs. CONCLUSIONS: We set up a protocol for EEVs separation and sequencing that might be useful for the identification of early markers of endothelial dysfunction in atherosclerosis.

Is tranexamic acid appropriate for all patients undergoing high-risk surgery?

PURPOSE OF REVIEW: Tranexamic acid (TXA), an antifibrinolytic agent, reduces surgical bleeding in a variety of procedures, such as cardiac, orthopedic, abdominal, and urologic surgery, cesarean section, and neurosurgery. However, there are surgical interventions for which its use is not yet widespread, and some caution persists because of concerns regarding thrombotic risk. The purpose of this review is to analyze the most recent evidence in various subgroups of surgical specialties and the association of TXA with thrombotic events and other side effects (e.g. seizures). RECENT FINDINGS: Recent clinical trials and meta-analyses have shown that the efficacy and safety vary according to the clinical context, timing of administration, and dose. Some reports found that TXA reduces major bleeding by 25% without a significant increase in thrombotic events. SUMMARY: Wider use of TXA has the potential to improve surgical safety, avoid unnecessary blood use, and save healthcare funds.

Comparison of assays measuring extracellular vesicle tissue factor in plasma samples: communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19, or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen, but only a few studies have compared some of these assays. OBJECTIVES: The International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity, and reproducibility of these assays. METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without lipopolysaccharide stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared with antigen assays. In addition, there was a large intra-assay and interassay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared with assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

Extracellular vesicles in atherosclerosis: Current and forthcoming impact?

Atherosclerosis is the main pathogenic substrate for cardiovascular diseases (CVDs). Initially categorized as a passive cholesterol storage disease, nowadays, it is considered an active process, identifying inflammation among the key players for its initiation and progression. Despite these advances, patients with CVDs are still at high risk of thrombotic events and death, urging to deepen into the molecular mechanisms underlying atherogenesis, and to identify novel diagnosis and prognosis biomarkers for their stratification. In this context, extracellular vesicles (EVs) have been postulated as an alternative in search of novel biomarkers in atherosclerotic diseases, as well as to investigate the crosstalk between the cells participating in the processes leading to arterial remodelling. EVs are nanosized lipidic particles released by most cell types in physiological and pathological conditions, that enclose lipids, proteins, and nucleic acids from parental cells reflecting their activation status. First considered cellular waste disposal systems, at present, EVs have been recognized as active effectors in a myriad of cellular processes, and as potential diagnosis and prognosis biomarkers also in CVDs. This review summarizes the role of EVs as potential biomarkers of CVDs, and their involvement into the processes leading to atherosclerosis.

SEA 2024 Standards for Global Control of Vascular Risk. / Estándares de la Sociedad Española de Arteriosclerosis 2024 para el control global del riesgo vascular.

One of the objectives of the Spanish Society of Arteriosclerosis is to contribute to the knowledge, prevention and treatment of vascular diseases, which are the leading cause of death in Spain and entail a high degree of disability and health expenditure. Atherosclerosis is a multifactorial disease and its prevention requires a global approach that takes into account the associated risk factors. This document summarises the current evidence and includes recommendations for patients with established vascular disease or at high vascular risk: it reviews the symptoms and signs to evaluate, the laboratory and imaging procedures to request routinely or in special situations, and includes the estimation of vascular risk, diagnostic criteria for entities that are vascular risk factors, and general and specific recommendations for their treatment. Finally, it presents aspects that are not usually referenced in the literature, such as the organisation of a vascular risk consultation.

Role of LCN2 in a murine model of hindlimb ischemia and in peripheral artery disease patients, and its potential regulation by miR-138-5P.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is a leading cause of morbimortality worldwide. Lipocalin-2 (LCN2) has been associated with higher risk of amputation or mortality in PAD and might be involved in muscle regeneration. Our aim is to unravel the role of LCN2 in skeletal muscle repair and PAD. METHODS AND RESULTS: WT and Lcn2-/- mice underwent hindlimb ischemia. Blood and crural muscles were analyzed at the inflammatory and regenerative phases. At day 2, Lcn2-/- male mice, but not females, showed increased blood and soleus muscle neutrophils, and elevated circulating pro-inflammatory monocytes (p < 0.05), while locally, total infiltrating macrophages were reduced (p < 0.05). Moreover, Lcn2-/- soleus displayed an elevation of Cxcl1 (p < 0.001), and Cxcr2 (p < 0.01 in males), and a decrease in Ccl5 (p < 0.05). At day 15, Lcn2 deficiency delayed muscle recovery, with higher density of regenerating myocytes (p < 0.04) and arterioles (αSMA+, p < 0.025). Reverse target prediction analysis identified miR-138-5p as a potential regulator of LCN2, showing an inverse correlation with Lcn2 mRNA in skeletal muscles (rho = -0.58, p < 0.01). In vitro, miR-138-5p mimic reduced Lcn2 expression and luciferase activity in murine macrophages (p < 0.05). Finally, in human serum miR-138-5p was inversely correlated with LCN2 (p ≤ 0.001 adjusted, n = 318), and associated with PAD (Odds ratio 0.634, p = 0.02, adjusted, PAD n = 264, control n = 54). CONCLUSIONS: This study suggests a possible dual role of LCN2 in acute and chronic conditions, with a probable role in restraining inflammation early after skeletal muscle ischemia, while being associated with vascular damage in PAD, and identifies miR-138-5p as one potential post-transcriptional regulator of LCN2.

Documento multidisciplinar de consenso sobre el manejo de la hemorragia masiva. Primera actualización 2023 (documento HEMOMAS-II) / Multidisciplinary consensus document on the management of massive haemorrhage. First update 2023 (document HEMOMAS-II)

El presente documento supone una puesta al día del documento multidisciplinar HEMOMAS, publicado en el año 2016 con el aval de las Sociedades Científicas Españolas de Anestesiología y Reanimación (SEDAR), Medicina Intensiva, Crítica y Unidades Coronarias (SEMICYUC) y de Trombosis y Hemostasia (SETH). El objetivo de este documento fue revisar y actualizar las recomendaciones existentes sobre el manejo de la hemorragia masiva (HM). Se siguió una metodología basada en elementos del método ADAPTE (búsqueda y adaptación de guías publicadas en el ámbito específico de la HM desde 2014, más búsqueda bibliográfica en PubMed y EMBASE desde enero-2014 hasta junio-2021). Tras la revisión de nueve guías y 207 artículos seleccionados, se actualizaron las 47 recomendaciones existentes en el artículo original, manteniendo, suprimiendo o modificando cada una de ellas y sus grados de recomendación y evidencia. Consensuadamente, los autores aprobaron la redacción final del artículo y las 41 recomendaciones resultantes.(AU)

This document is an update of the multidisciplinary document HEMOMAS, published in 2016 with the endorsement of the Spanish Scientific Societies of Anaesthesiology (SEDAR), Intensive Care (SEMICYUC) and Thrombosis and Haemostasis (SETH). The aim of this document was to review and update existing recommendations on the management of massive haemorrhage. The methodology of the update was based on several elements of the ADAPTE method by searching and adapting guidelines published in the specific field of massive bleeding since 2014, plus a literature search performed in PubMed and EMBASE from January 2014 to June 2021. Based on the review of 9 guidelines and 207 selected articles, the 47 recommendations in the original article were reviewed, maintaining, deleting, or modifying each of them and the accompanying grades of recommendation and evidence. Following a consensus process, the final wording of the article and the resulting 41 recommendations were approved by all authors.(AU)

Multidisciplinary consensus document on the management of massive haemorrhage. First update 2023 (document HEMOMAS-II).

This document is an update of the multidisciplinary document HEMOMAS, published in 2016 with the endorsement of the Spanish Scientific Societies of Anaesthesiology (SEDAR), Intensive Care (SEMICYUC) and Thrombosis and Haemostasis (SETH). The aim of this document was to review and update existing recommendations on the management of massive haemorrhage. The methodology of the update was based on several elements of the ADAPTE method by searching and adapting guidelines published in the specific field of massive bleeding since 2014, plus a literature search performed in PubMed and EMBASE from January 2014 to June 2021. Based on the review of 9 guidelines and 207 selected articles, the 47 recommendations in the original article were reviewed, maintaining, deleting, or modifying each of them and the accompanying grades of recommendation and evidence. Following a consensus process, the final wording of the article and the resulting 41 recommendations were approved by all authors.

Multidisciplinary consensus document on the management of massive haemorrhage. First update 2023 (document HEMOMAS-II).

This document is an update of the multidisciplinary document HEMOMAS, published in 2016 with the endorsement of the Spanish Scientific Societies of Anaesthesiology (SEDAR), Intensive Care (SEMICYUC) and Thrombosis and Haemostasis (SETH). The aim of this document was to review and update existing recommendations on the management of massive haemorrhage. The methodology of the update was based on several elements of the ADAPTE method by searching and adapting guidelines published in the specific field of massive bleeding since 2014, plus a literature search performed in PubMed and EMBASE from January 2014 to June 2021. Based on the review of 9 guidelines and 207 selected articles, the 47 recommendations in the original article were reviewed, maintaining, deleting, or modifying each of them and the accompanying grades of recommendation and evidence. Following a consensus process, the final wording of the article and the resulting 41 recommendations were approved by all authors.

Documento multidisciplinar de consenso sobre el manejo de la hemorragia masiva. Primera actualización 2023 (documento HEMOMAS-II) / Multidisciplinary consensus document on the management of massive haemorrhage. First update 2023 (document HEMOMAS-II)

El presente documento supone una puesta al día del documento multidisciplinar HEMOMAS, publicado en el año 2016 con el aval de las Sociedades Científicas Españolas de Anestesiología y Reanimación (SEDAR), Medicina Intensiva, Crítica y Unidades Coronarias (SEMICYUC) y de Trombosis y Hemostasia (SETH). El objetivo de este documento fue revisar y actualizar las recomendaciones existentes sobre el manejo de la hemorragia masiva (HM). Se siguió una metodología basada en elementos del método ADAPTE (búsqueda y adaptación de guías publicadas en el ámbito específico de la HM desde 2014, más búsqueda bibliográfica en PubMed y EMBASE desde enero-2014 hasta junio-2021). Tras la revisión de nueve guías y 207 artículos seleccionados, se actualizaron las 47 recomendaciones existentes en el artículo original, manteniendo, suprimiendo o modificando cada una de ellas y sus grados de recomendación y evidencia. Consensuadamente, los autores aprobaron la redacción final del artículo y las 41 recomendaciones resultantes (AU)

This document is an update of the multidisciplinary document HEMOMAS, published in 2016 with the endorsement of the Spanish Scientific Societies of Anaesthesiology (SEDAR), Intensive Care (SEMICYUC) and Thrombosis and Haemostasis (SETH). The aim of this document was to review and update existing recommendations on the management of massive haemorrhage. The methodology of the update was based on several elements of the ADAPTE method by searching and adapting guidelines published in the specific field of massive bleeding since 2014, plus a literature search performed in PubMed and EMBASE from January 2014 to June 2021. Based on the review of 9 guidelines and 207 selected articles, the 47 recommendations in the original article were reviewed, maintaining, deleting, or modifying each of them and the accompanying grades of recommendation and evidence. Following a consensus process, the final wording of the article and the resulting 41 recommendations were approved by all authors (AU)

Immunothrombosis: Molecular Aspects and New Therapeutic Perspectives.

Thromboinflammation or immunothrombosis is a concept that explains the existing link between coagulation and inflammatory response present in many situations, such as sepsis, venous thromboembolism, or COVID-19 associated coagulopathy. The purpose of this review is to provide an overview of the current data regarding the mechanisms involved in immunothrombosis in order to understand the new therapeutic strategies focused in reducing thrombotic risk by controlling the inflammation.

Hemostatic Biomarkers and Volumetry Help to Identify High-Risk Abdominal Aortic Aneurysms.

Predicting the progression of small aneurysms is a main challenge in abdominal aortic aneurysm (AAA) management. The combination of circulating biomarkers and image techniques might provide an alternative for risk stratification. We evaluated the association of plasma TAT complexes (TAT) and D-dimer with AAA severity in 3 groups of patients: group 1, without AAA (n = 52), group 2, AAA 40−50 mm (n = 51) and group 3, AAA > 50 mm (n = 50). TAT (p < 0.001) and D-dimer (p < 0.001) were increased in patients with AAA (groups 2 and 3) vs. group 1. To assess the association between baseline TAT and D-dimer concentrations, and AAA growth, aortic diameter and volume (volumetry) were measured by computed tomography angiography (CTA) in group 2 at recruitment (baseline) and 1-year after inclusion. Baseline D-dimer and TAT levels were associated with AAA diameter and volume variations at 1-year independently of confounding factors (p ≤ 0.044). Additionally, surgery incidence, recorded during a 4-year follow-up in group 2, was associated with larger aneurysms, assessed by aortic diameter and volumetry (p ≤ 0.036), and with elevated TAT levels (sub-hazard ratio 1.3, p ≤ 0.029), while no association was found for D-dimer. The combination of hemostatic parameters and image techniques might provide valuable tools to evaluate AAA growth and worse evolution.