isolation-characterization-human-adipocyte-derived-extracellular

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

University of Oxford

Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs.

Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

Cells naïve to stress can display the effects of stress, such as DNA damage and apoptosis, when they are exposed to signals from stressed cells; this phenomenon is known as the bystander effect. We previously showed that bystander effect induced by ionising radiation are mediated by extracellular vesicles (EVs). Bystander effect can also be induced by other types of stress, including heat shock, but it is unclear whether EVs are involved. Here we show that EVs released from heat shocked cells are also able to induce bystander damage in unstressed populations. Naïve cells treated with media conditioned by heat shocked cells showed higher levels of DNA damage and apoptosis than cells treated with media from control cells. Treating naïve cells with EVs derived from media conditioned by heat shocked cells also induced a bystander effect when compared to control, with DNA damage and apoptosis increasing whilst the level of cell viability was reduced. We demonstrate that treatment of naïve cells with heat shocked cell-derived EVs leads to greater invasiveness in a trans-well Matrigel assay. Finally, we show that naïve cells treated with EVs from heat-shocked cells are more likely to survive a subsequent heat shock, suggesting that these EVs mediate an adaptive response. We propose that EVs released following stress mediate an intercellular response that leads to apparent stress in neighbouring cells but also greater robustness in the face of a subsequent insult.

Extracellular vesicles released following heat stress induce bystander effect in unstressed populations.

Brain injury elicits a systemic acute-phase response (APR), which is responsible for co-ordinating the peripheral immunological response to injury. To date, the mechanisms responsible for signalling the presence of injury or disease to selectively activate responses in distant organs were unclear. Circulating endogenous extracellular vesicles (EVs) are increased after brain injury and have the potential to carry targeted injury signals around the body. Here, we examined the potential of EVs, isolated from rats after focal inflammatory brain lesions using IL-1β, to activate a systemic APR in recipient naïve rats, as well as the behavioural consequences of EV transfer. Focal brain lesions increased EV release, and, following isolation and transfer, the EVs were sequestered by the liver where they initiated an APR. Transfer of blood-borne EVs from brain-injured animals was also enough to suppress exploratory behaviours in recipient naïve animals. EVs derived from brain endothelial cell cultures treated with IL-1β also activated an APR and altered behaviour in recipient animals. These experiments reveal that inflammation-induced circulating EVs derived from endothelial cells are able to initiate the APR to brain injury and are sufficient to generate the associated sickness behaviours, and are the first demonstration that EVs are capable of modifying behavioural responses.

Circulating endothelial cell-derived extracellular vesicles mediate the acute phase response and sickness behaviour associated with CNS inflammation.

Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1-positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion- and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.

Endothelium-derived extracellular vesicles promote splenic monocyte mobilization in myocardial infarction.

Inflammatory lesions in the brain activate a systemic acute-phase response (APR), which is dependent on the release of extracellular vesicles (EVs) into the circulation. The resulting APR is responsible for regulating leukocyte mobilization and subsequent recruitment to the brain. Factors that either exacerbate or inhibit the APR will also exacerbate or inhibit central nervous system (CNS) inflammation as a consequence and have the potential to influence ongoing secondary damage. Here, we were interested to discover how the circulating EV population changes after traumatic brain injury (TBI) and how manipulation of the circulating EV pool impacts on the outcome of TBI. We found the number of circulating EVs increased rapidly post-TBI, and this was accompanied by an increase in CNS and hepatic leukocyte recruitment. In an adoptive transfer study, we then evaluated the outcomes of TBI after administering EVs derived from either in vitro macrophage or endothelial cell lines stimulated with lipopolysaccharide (LPS), or from murine plasma from an LPS challenge using the air-pouch model. By manipulating the circulating EV population, we were able to demonstrate that each population of transferred EVs increased the APR. However, the characteristics of the response were dependent on the nature of the EVs; specifically, it was significantly increased when animals were challenged with macrophage-derived EVs, suggesting that the cellular origins of EVs may determine their function. Selectively targeting EVs from macrophage/monocyte populations is likely to be of value in reducing the impact of the systemic inflammatory response on the outcome of traumatic CNS injury.

Exacerbation of Acute Traumatic Brain Injury by Circulating Extracellular Vesicles.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

Technical challenges of working with extracellular vesicles.

A recently-validated, highly-sensitive T2 mapping magnetic resonance (MRI) technique accurately quantifies carotid plaque lipid. The aims of this study were to determine: (i) the extent of carotid plaque lipid in patients with acute coronary syndromes (ACS); (ii) the effects of initiation of high-intensity statin on plaque lipid content and (iii) whether plaque lipid content is related to standard or 'functional' blood lipid measurements.

T2 mapping MRI technique quantifies carotid plaque lipid, and its depletion after statin initiation, following acute myocardial infarction.

British Cardiovascular Society Young Investigator Award 2019.

Chemical modification of the surface of viruses, both the interior and the exterior, imparts new functionalities, that have potential applications in nanomedicine. In this study, we developed novel virus-based nanomaterials as a contrast agent for computed tomography (CT) imaging in vitro. The gold-coated cowpea mosaic virus (Au-CPMV) particles were generated by the electrostatic adsorption of positively charged electrolyte on the virus capsid with the subsequent incubation and reduction of anionic gold complexes. Au-CPMV particles as a CT contrast agent offer a fast scan time (less than 2 min), low cost, and biocompatibility and allow for high-resolution imaging with ca. 150 Hounsfield units (HU). The Au-CPMV surface was further modified allowing for the incorporation of targeting molecules of specific cell types.

Gold-coated plant virus as computed tomography imaging contrast agent.

Extracellular vesicles (EVs) are submicron-sized lipid envelopes that are produced and released from a parent cell and can be taken up by a recipient cell. EVs are capable of mediating cellular signalling by carrying nucleic acids, proteins, lipids and cellular metabolites between cells and organs. Metabolic dysfunction is associated with changes in plasma concentrations of EVs as well as alterations in their EV cargo. Since EVs can act as messengers between parent and recipient cells, they could be involved in cell-to-cell and organ-to-organ communication in metabolic diseases. Recent literature has shown that EVs are produced by cells within metabolic tissues, such as adipose tissue, pancreas, muscle and liver. These vesicles have therefore been proposed as a novel intercellular communication mode in systemic metabolic regulation. In this review, we will describe and discuss the current literature that investigates the role of adipose-derived EVs in the regulation of obesity-associated metabolic disease. We will particularly focus on the EV-dependent communication between adipocytes, the vasculature and immune cells in type 2 diabetes.

Extracellular vesicles in metabolic disease.

Obesity and insulin resistance are risk factors for nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide. Because no approved medication nor an accurate and noninvasive diagnosis is currently available for NAFLD, there is a clear need to better understand the link between obesity and NAFLD. Lipid accumulation during obesity is known to be associated with oxidative stress and inflammatory activation of liver macrophages (LMs). However, we show that although LMs do not become proinflammatory during obesity, they display signs of oxidative stress. In livers of both humans and mice, antioxidant nuclear factor erythroid 2-related factor 2 (NRF2) was down-regulated with obesity and insulin resistance, yielding an impaired response to lipid accumulation. At the molecular level, a microRNA-targeting NRF2 protein, , was elevated in the livers of obese insulin-resistant humans and mice, and specific silencing of  in murine and human LMs was sufficient to restore NRF2 protein expression and the antioxidant response. These results highlight the pathological role of LMs and their therapeutic potential to restore the impaired endogenous antioxidant response in obesity-associated NAFLD.

Liver macrophages inhibit the endogenous antioxidant response in obesity-associated insulin resistance.

Atherosclerosis is a disease of chronic inflammation. We investigated the roles of the cytokines IL-4 and IL-13, the classical activators of STAT6, in the resolution of atherosclerosis inflammation. Using  mice, resolution was impaired, and in control mice, in both progressing and resolving plaques, levels of IL-4 were stably low and IL-13 was undetectable. This suggested that IL-4 is required for atherosclerosis resolution, but collaborates with other factors. We had observed increased Wnt signaling in macrophages in resolving plaques, and human genetic data from others showed that a loss-of-function Wnt mutation was associated with premature atherosclerosis. We now find an inverse association between activation of Wnt signaling and disease severity in mice and humans. Wnt enhanced the expression of inflammation resolving factors after treatment with plaque-relevant low concentrations of IL-4. Mechanistically, activation of the Wnt pathway following lipid lowering potentiates IL-4 responsiveness in macrophages via a PGE/STAT3 axis.

Article	Year
Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation. Stroke\| PubMed ID: 28536169	2017
Extracellular vesicles released following heat stress induce bystander effect in unstressed populations. Journal of extracellular vesicles\| PubMed ID: 28717426	2017
Circulating endothelial cell-derived extracellular vesicles mediate the acute phase response and sickness behaviour associated with CNS inflammation. Scientific reports\| PubMed ID: 28851955	2017
Endothelium-derived extracellular vesicles promote splenic monocyte mobilization in myocardial infarction. JCI insight\| PubMed ID: 28878126	2017
Exacerbation of Acute Traumatic Brain Injury by Circulating Extracellular Vesicles. Journal of neurotrauma\| PubMed ID: 29149810	2018
Technical challenges of working with extracellular vesicles. Nanoscale\| PubMed ID: 29265147	2018
T2 mapping MRI technique quantifies carotid plaque lipid, and its depletion after statin initiation, following acute myocardial infarction. Atherosclerosis\| PubMed ID: 30227984	2018
British Cardiovascular Society Young Investigator Award 2019. Heart (British Cardiac Society)\| PubMed ID: 31542722	2019
Gold-coated plant virus as computed tomography imaging contrast agent. Beilstein journal of nanotechnology\| PubMed ID: 31667046	2019
Extracellular vesicles in metabolic disease. Diabetologia\| PubMed ID: 31690986	2019
Liver macrophages inhibit the endogenous antioxidant response in obesity-associated insulin resistance. Science translational medicine\| PubMed ID: 32102936	2020
Wnt signaling enhances macrophage responses to IL-4 and promotes resolution of atherosclerosis. eLife\| PubMed ID: 33720008	2021

Naveed Akbar

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