Cardioprotective and proangiogenic activities of small extracellular vesicles released by amniotic fluid stem cells

Protection against myocardial ischaemia/reperfusion injury and regeneration of the damaged myocardium are long-sought goals. The use of small extracellular vesicles (sEVs) released by mesenchymal stem cells (MSCs) was shown to be of benefit in the myocardial infarction setting. However, MSCs are frequently harvested from aged or diseased patients and suboptimal sEV isolation methods are used. A subtype of young, foetal MSCs, namely spindle-shaped amniotic fluid stem cells (SS-AFSCs), is known to possess better expansion and functional capacity than its adult counterparts. Here, sEVs released by SS-AFSCs were isolated using size-exclusion chromatography (SEC) – an isolation technique that yields vesicles of superior purity – and their cardioprotective and proangiogenic activities were studied. Firstly, using rat blood plasma, it was demonstrated that SEC isolates higher sEV yields with significantly compromised purity, mostly due to the presence of lipoproteins. To overcome this, a serum-free environment was used for sEVs isolation from SS-AFSC-conditioned medium. Comprehensive characterisation experiments showed that the harvested SS-AFSC sEVs are of high purity. Functionally, SS-AFSC sEVs protected the rat myocardium from ischaemia/reperfusion injury in vivo, but not isolated cardiomyocytes in vitro, indicative of indirect cardioprotective effects. Additionally, SS-AFSC sEVs promoted migration of endothelial cells in vitro and recapitulated the promigratory effects of the SS-AFSC-conditioned medium. Using pharmacological inhibition, it was shown that PI3K pathway, a known player in cell migration, mediates the sEV effects, while a series of potential candidates in the sEV cargo were excluded. Finally, cellular sEV uptake was studied by use of lipophilic dye-labelling experiments. Surprisingly, this commonly used approach was found to be unsuitable for sEV tracking due to non-specific dye retention by non-sEV contaminants. Overall, SEC-isolated SS-AFSC sEVs possess cardioprotective potential manifested only in vivo, and promigratory activity which requires PI3K signalling. These data indicate that SS-AFSC sEVs have multifactorial beneficial effects in a myocardial infarction setting

Cross-Linking Mass Spectrometry Uncovers Interactions Between High-Density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein

High-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the life cycle of the hepatitis C virus, their influence on coronavirus (CoV) infections is poorly understood. In this study, we utilize cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. XL-MS of plasma isolated from patients with COVID-19 uncovered HDL protein interaction networks, dominated by acute-phase serum amyloid proteins, whereby serum amyloid A2 was shown to bind to apolipoprotein (Apo) D. XL-MS on isolated HDL confirmed ApoD to interact with SARS-CoV-2 spike but not SARS-CoV-1 spike. Other direct interactions of SARS-CoV-2 spike upon HDL included ApoA1 and ApoC3. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1. Mechanistically, XL-MS coupled with data-driven structural modeling determined that ApoD may interact within the receptor-binding domain of the spike. However, ApoD overexpression in multiple cell-based assays had no effect upon viral replication or infectivity. Thus, SARS-CoV-2 spike can bind to apolipoproteins on HDL, but these interactions do not appear to alter infectivity.</p

SERCA2a protein levels are unaltered in human heart failure

Research lette

PCSK9 Activity Is Potentiated Through HDL Binding

RationaleProprotein convertase subtilisin/kexin type 9 (PCSK9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and high-density lipoprotein (HDL) remains unexplored.ObjectiveThis study sought to interrogate the novel relationship between PCSK9 and HDL in humans.Methods and resultsComparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck (n=656) study revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein-C3, an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 times points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apolipoprotein-C3 from HDL and lower levels of small HDL as measured by nuclear magnetic resonance. Crosslinking mass spectrometry (XLMS) upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin and apolipoprotein-E within the HDL proteome, that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalisation and multimerisation.ConclusionsThis study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipaemia as a driver of PCSK9 and apolipoprotein-C3 release from HDL

Metabolic recovery after weight loss surgery is reflected in serum microRNAs

[Abstract] INTRODUCTION: Bariatric surgery offers the most effective treatment for obesity, ameliorating or even reverting associated metabolic disorders, such as type 2 diabetes. We sought to determine the effects of bariatric surgery on circulating microRNAs (miRNAs) that have been implicated in the metabolic cross talk between the liver and adipose tissue. RESEARCH DESIGN AND METHODS: We measured 30 miRNAs in 155 morbidly obese patients and 47 controls and defined associations between miRNAs and metabolic parameters. Patients were followed up for 12 months after bariatric surgery. Key findings were replicated in a separate cohort of bariatric surgery patients with up to 18 months of follow-up. RESULTS: Higher circulating levels of liver-related miRNAs, such as miR-122, miR-885-5 p or miR-192 were observed in morbidly obese patients. The levels of these miRNAs were positively correlated with body mass index, percentage fat mass, blood glucose levels and liver transaminases. Elevated levels of circulating liver-derived miRNAs were reversed to levels of non-obese controls within 3 months after bariatric surgery. In contrast, putative adipose tissue-derived miRNAs remained unchanged (miR-99b) or increased (miR-221, miR-222) after bariatric surgery, suggesting a minor contribution of white adipose tissue to circulating miRNA levels. Circulating levels of liver-derived miRNAs normalized along with the endocrine and metabolic recovery of bariatric surgery, independent of the fat percentage reduction. CONCLUSIONS: Since liver miRNAs play a crucial role in the regulation of hepatic biochemical processes, future studies are warranted to assess whether they may serve as determinants or mediators of metabolic risk in morbidly obese patients.SS-A was recipient of a travel/visiting scientist fellowship “José Castillejo” from the Ministerio de Educación, Cultura y Deporte-Spain. Project Nº PI16/00884 was awarded to FC and SS-A integrated in the Spanish National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the ISCIII. CG is funded by a British Heart Foundation (BHF) PhD studentship (FS/18/60/34181). MM is a BHF Chair Holder (CH/16/3/32406) with BHF program grant support (RG/16/14/32397). MM was awarded a BHF Special Project grant to participate in the ERA-CVD Transnational Grant “MacroERA: Noncoding RNAs in cardiac macrophages and their role in heart failure” and is part of the Marie Skłodowska-Curie Innovative Training Network TRAIN-HEART (http://train-heart.euhttp://train-heart.eu) as well as a networks on “MicroRNAbased Therapeutic Strategies in Vascular Disease” and on “Defining the Roles of Smooth Muscle Cells and other Extracellular Matrix Producing Cells in Late Stage Atherosclerotic Plaque Formation” funded by the Fondation Leducq. This work was supported by the National Institute of Health Research Biomedical Research Centre based at Guy’s and St Thomas’ NHS (National Health Service) Foundation Trust and King’s College London in partnership with King’s College Hospital and an R&D K-Centre and the excellence initiative VASCage (Centre for Promoting Vascular Health in the Ageing Community, project number 868624) of the Austrian Research Promotion Agency FFG (COMET program—Competence Centers for Excellent Technologies) funded by the Austrian Ministry for Transport, Innovation and Technology, the Austrian Ministry for Digital and Economic Affairs and the federal states Tyrol (via Standortagentur), Salzburg and Vienna (via Vienna Business Agency).info:eu-repo/grantAgreement/MINECO/Programa Estatal de I+D+I Orientada a los Retos de la Sociedad/PI16%2F00884/ES/RELACION ENTRE MARCADORES HORMONALES DE CONTROL DE LA INGESTA Y GASTO METABÓLICO. EFECTO DEL BALANCE ENERGÉTICO NEGATIVO Y LA MODIFICACIÓN DE GHBristish Heart Foundation; FS/18/60/34181Bristish Heart Foundation; RG/16/14/3239

Methods for the identification and characterization of extracellular vesicles in cardiovascular studies - from exosomes to microvesicles

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are released from cells of the cardiovascular system, and are considered important mediators of intercellular and extracellular communications. Two types of EVs of particular interest are exosomes and microvesicles, which have been identified in all tissue and body fluids and carry a variety of molecules including RNAs, proteins, and lipids. EVs have potential for use in the diagnosis and prognosis of cardiovascular diseases and as new therapeutic agents, particularly in the setting of myocardial infarction and heart failure. Despite their promise, technical challenges related to their small size make it challenging to accurately identify and characterize them, and to study EV-mediated processes. Here, we aim to provide the reader with an overview of the techniques and technologies available for the separation and characterization of EVs from different sources. Methods for determining the protein, RNA, and lipid content of EVs are discussed. The aim of this document is to provide guidance on critical methodological issues and highlight key points for consideration for the investigation of EVs in cardiovascular studies

Influence of androgen receptor in vascular cells on reperfusion following hindlimb ischaemia

AIMS:Studies in global androgen receptor knockout (G-ARKO) and orchidectomised mice suggest that androgen accelerates reperfusion of the ischaemic hindlimb by stimulating angiogenesis. This investigation used novel, vascular cell-specific ARKO mice to address the hypothesis that the impaired hindlimb reperfusion in G-ARKO mice was due to loss of AR from cells in the vascular wall. METHODS AND RESULTS:Mice with selective deletion of AR (ARKO) from vascular smooth muscle cells (SM-ARKO), endothelial cells (VE-ARKO), or both (SM/VE-ARKO) were compared with wild type (WT) controls. Hindlimb ischaemia was induced in these mice by ligation and removal of the femoral artery. Post-operative reperfusion was reduced in SM-ARKO and SM/VE-ARKO mice. Immunohistochemistry indicated that this was accompanied by a reduced density of smooth muscle actin-positive vessels but no change in the density of isolectin B4-positive vessels in the gastrocnemius muscle. Deletion of AR from the endothelium (VE-ARKO) did not alter post-operative reperfusion or vessel density. In an ex vivo (aortic ring culture) model of angiogenesis, AR was not detected in vascular outgrowths and angiogenesis was not altered by vascular ARKO or by exposure to dihydrotestosterone (DHT 10(-10)-10(-7)M; 6 days). CONCLUSION:These results suggest that loss of AR from vascular smooth muscle, but not from the endothelium, contributes to impaired reperfusion in the ischaemic hindlimb of G-ARKO. Impaired reperfusion was associated with reduced collateral formation rather than reduced angiogenesis

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly