Abstract Background Exosomes are nanometer-sized vesicles released from cells into the blood, where they can transmit signals throughout the body. Shown to act on the heart, exosomes’ composition and ...the signaling pathways they activate have not been explored. We hypothesized that endogenous plasma exosomes can communicate signals to the heart and provide protection against ischemia and reperfusion injury. Objectives This study sought to isolate and characterize exosomes from rats and healthy volunteers, evaluate their cardioprotective actions, and identify the molecular mechanisms involved. Methods The exosome-rich fraction was isolated from the blood of adult rats and human volunteers and was analyzed by protein marker expression, transmission electron microscopy, and nanoparticle tracking analysis. This was then used in ex vivo, in vivo, and in vitro settings of ischemia-reperfusion, with the protective signaling pathways activated on cardiomyocytes identified using Western blot analyses and chemical inhibitors. Results Exosomes exhibited the expected size and expressed marker proteins CD63, CD81, and heat shock protein (HSP) 70. The exosome-rich fraction was powerfully cardioprotective in all tested models of cardiac ischemia-reperfusion injury. We identified a pro-survival signaling pathway activated in cardiomyocytes involving toll-like receptor (TLR) 4 and various kinases, leading to activation of the cardioprotective HSP27. Cardioprotection was prevented by a neutralizing antibody against a conserved HSP70 epitope expressed on the exosome surface and by blocking TLR4 in cardiomyocytes, identifying the HSP70/TLR4 communication axis as a critical component in exosome-mediated cardioprotection. Conclusions Exosomes deliver endogenous protective signals to the myocardium by a pathway involving TLR4 and classic cardioprotective HSPs.
The past decade has witnessed an exponential increase in the number of publications referring to extracellular vesicles (EVs). For many years considered to be extracellular debris, EVs are now seen ...as novel mediators of endocrine signalling via cell-to-cell communication. With the capability of transferring proteins and nucleic acids from one cell to another, they have become an attractive focus of research for different pathological settings and are now regarded as both mediators and biomarkers of disease including cardio-metabolic disease. They also offer therapeutic potential as signalling agents capable of targeting tissues or cells with specific peptides or miRNAs. In this review, we focus on the role that microvesicles (MVs) and exosomes, the two most studied classes of EV, have in diabetes, cardiovascular disease, endothelial dysfunction, coagulopathies, and polycystic ovary syndrome. We also provide an overview of current developments in MV/exosome isolation techniques from plasma and other fluids, comparing different available commercial and non-commercial methods. We describe different techniques for their optical/biochemical characterization and quantitation. We also review the signalling pathways that exosomes and MVs activate in target cells and provide some insight into their use as biomarkers or potential therapeutic agents. In summary, we give an updated focus on the role that these exciting novel nanoparticles offer for the endocrine community.
Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised ...methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB
+
lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium.
The cytokine release syndrome (CRS) of COVID-19 is associated with the development of critical illness requiring multi-organ support. Further research is required to halt progression of multi-organ ...injury induced by hyper-inflammation.
PubMed/MEDLINE
databases were accessed between May 9
-June 9
, 2020, to review the latest perspectives on the treatment and pathogenesis of CRS.
Over-activity of chemotaxis triggers a macrophage activation syndrome (MAS) resulting in the release of pro-inflammatory cytokines. IL-6 and TNF- α are at the forefront of hyper-inflammation. The inflammatory cascade induces endothelial activation and capillary leak, leading to circulatory collapse and shock. As endothelial dysfunction persists, there is activation of the clotting cascade and microvascular obstruction. Continued endothelial activation results in multi-organ failure, regardless of pulmonary tissue damage. We propose that targeting the endothelium may interrupt this cycle. Immuno-modulating therapies have been suggested, however, further data is necessary to confirm that they do not jeopardize adaptive immunity. Inhibition of IL-6 and the Janus Kinase, signal transducer and activator of transcription proteins pathway (JAK/STAT), are favorable targets. Remote ischemic conditioning (RIC) reduces the inflammation of sepsis in animal models and should be considered as a low risk intervention, in combination with cardiovascular protection.
The coronary circulation is both culprit and victim of acute myocardial infarction. The rupture of an epicardial atherosclerotic plaque with superimposed thrombosis causes coronary occlusion, and ...this occlusion must be removed to induce reperfusion. However, ischaemia and reperfusion cause damage not only in cardiomyocytes but also in the coronary circulation, including microembolization of debris and release of soluble factors from the culprit lesion, impairment of endothelial integrity with subsequently increased permeability and oedema formation, platelet activation and leucocyte adherence, erythrocyte stasis, a shift from vasodilation to vasoconstriction, and ultimately structural damage to the capillaries with eventual no-reflow, microvascular obstruction (MVO), and intramyocardial haemorrhage (IMH). Therefore, the coronary circulation is a valid target for cardioprotection, beyond protection of the cardiomyocyte. Virtually all of the above deleterious endpoints have been demonstrated to be favourably influenced by one or the other mechanical or pharmacological cardioprotective intervention. However, no-reflow is still a serious complication of reperfused myocardial infarction and carries, independently from infarct size, an unfavourable prognosis. MVO and IMH can be diagnosed by modern imaging technologies, but still await an effective therapy. The current review provides an overview of strategies to protect the coronary circulation from acute myocardial ischaemia/reperfusion injury. This article is part of a Cardiovascular Research Spotlight Issue entitled 'Cardioprotection Beyond the Cardiomyocyte', and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225.
Many treatments have been identified that confer robust cardioprotection in experimental animal models of acute ischemia and reperfusion injury. However, translation of these cardioprotective ...therapies into the clinical setting of acute myocardial infarction (AMI) for patient benefit has been disappointing. One important reason might be that AMI is multifactorial, causing cardiomyocyte death via multiple mechanisms, as well as affecting other cell types, including platelets, fibroblasts, endothelial and smooth muscle cells, and immune cells. Many cardioprotective strategies act through common end-effectors and may be suboptimal in patients with comorbidities. In this regard, emerging data suggest that optimal cardioprotection may require the combination of additive or synergistic multitarget therapies. This review will present an overview of the state of cardioprotection today and provide a roadmap for how we might progress towards successful clinical use of cardioprotective therapies following AMI, focusing on the rational combination of judiciously selected, multitarget therapies. This paper emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225.
Exosomes are nanosized lipid vesicles released from cells. They are capable of transferring proteins, mRNA, and miRNA between cells and, therefore, represent a potential means of intercellular ...communication. Exosomes can be proangiogenic and may have cardioprotective properties. In contrast, their larger cousins, microvesicles, seem to have generally detrimental effects that are prothrombotic and proinflammatory. Exosomes are released from multivesicular bodies via an exocytic pathway and have the potential for cell-specific targeting. This normal process is hijacked during various pathological conditions, such as cancer, viral infection, and amyloidopathies. We assess the evidence for a role of exosomes and microvesicles in normal cardiovascular physiology, as well as during cardiovascular disease. In addition to offering a potential source of cardiovascular biomarkers, exosomes may offer a nonimmunogenic means of manipulating the heart.
Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic ...tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.
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