Adipocyte-derived Extracellular Vesicles Research Articles

Neonatal Adiposity Is Associated with microRNAs in Adipocyte-Derived Extracellular Vesicles in Maternal and Cord Blood

Neonatal Adiposity Is Associated with microRNAs in Adipocyte-Derived Extracellular Vesicles in Maternal and Cord Blood

Adipocyte extracellular vesicles: rescuers of cardiac mitochondrial stress

Nutrient excess induces mitochondrial dysfunction, which participates in obesity-related complications. Obesity also associates with high cardiac oxidative stress, which contributes to myocardial dysfunction. Crewe et al. recently evidenced the pivotal role of adipocyte-derived extracellular vesicles (EVs) in cardiac oxidative stress responses and revealed their unexpected protective effect against ischemia/reperfusion injury.

Adipocyte-derived extracellular vesicles promote breast cancer cell malignancy through HIF-1α activity.

Extracellular vesicles (EVs) are emerging key protagonists in intercellular communication between adipocytes and breast cancer (BC) cells. Here, we described a new mechanism by which EVs released by mature adipocytes promoted breast cancer cell malignancy "in vitro" and "in vivo". We found that adipocyte-derived EVs enhanced growth, motility and invasion, stem cell-like properties, as well as specific traits of epithelial-to-mesenchymal transition in both estrogen receptor positive and triple negative BC cells. Of note, adipocyte-derived EVs aid breast tumor cells in lung metastatic colonization after tail-vein injection in mice. These EV-mediated effects occur via the induction of HIF-1α activity, since they were abrogated by the use of the HIF-1α inhibitor KC7F2 or in cells silenced for HIF-1α expression. Moreover, using an "ex vivo" model of obese adipocytes we found that the depletion of EVs counteracted the ability of obese adipocytes to sustain pro-invasive phenotype in BC cells. Interestingly, EVs released by undifferentiated adipocytes failed to induce aggressiveness and HIF-1α expression. These findings shed new light on the role of adipocyte-derived EVs in breast cancer progression, suggesting the possibility to target HIF-1α activity to block the harmful adipocyte-tumor cell dialogue, especially in obese settings.

The impact of obesity on adipocyte-derived extracellular vesicles.

Recently, the emerging roles of adipocyte-derived extracellular vesicles (EVs) linking obesity and its comorbidities have been recognized. In obese subjects, adipocytes are having hypertrophic growth and are under stressed. The dysfunction adipocytes dysregulate the assembly of the biological components in the EVs including exosomes. This article critically reviews the current findings on the impact of obesity on the exosomal cargo contents that induce the pathophysiological changes. Besides, this review also summarizes the understanding on how obesity affects the biogenesis of adipocyte-derived exosomes and the exosome secretion. Furthermore, the differences of the exosomal contents in different adipose depots, and the impact of obesity on the exosomes that are derived from the stromal vascular fraction such as the adipose tissue macrophages and adipocyte-derived stem cells will also be discussed. The current development and potential application of exosome-based therapy will be summarized. This review provides crucial information for the design of novel exosome-based therapy for the treatment of obesity and its comorbidities.

Fat-Secreted Ceramides Regulate Vascular Redox State and Influence Outcomes in Patients With Cardiovascular Disease

BackgroundObesity is associated with increased cardiovascular risk; however, the potential role of dysregulations in the adipose tissue (AT) metabolome is unknown. ObjectivesThe aim of this study was to explore the role of dysregulation in the AT metabolome on vascular redox signaling and cardiovascular outcomes. MethodsA screen was conducted for metabolites differentially secreted by thoracic AT (ThAT) and subcutaneous AT in obese patients with atherosclerosis (n = 48), and these metabolites were then linked with dysregulated vascular redox signaling in 633 patients undergoing coronary bypass surgery. The underlying mechanisms were explored in human aortic endothelial cells, and their clinical value was tested against hard clinical endpoints. ResultsBecause ThAT volume was associated significantly with arterial oxidative stress, there were significant differences in sphingolipid secretion between ThAT and subcutaneous AT, with C16:0-ceramide and derivatives being the most abundant species released within adipocyte-derived extracellular vesicles. High ThAT sphingolipid secretion was significantly associated with reduced endothelial nitric oxide bioavailability and increased superoxide generated in human vessels. Circulating C16:0-ceramide correlated positively with ThAT ceramides, dysregulated vascular redox signaling, and increased systemic inflammation in 633 patients with atherosclerosis. Exogenous C16:0-ceramide directly increased superoxide via tetrahydrobiopterin-mediated endothelial nitric oxide synthase uncoupling and dysregulated protein phosphatase 2 in human aortic endothelial cells. High plasma C16:0-ceramide and its glycosylated derivative were independently related with increased risk for cardiac mortality (adjusted hazard ratios: 1.394; 95% confidence interval: 1.030 to 1.886; p = 0.031 for C16:0-ceramide and 1.595; 95% confidence interval: 1.042 to 2.442; p = 0.032 for C16:0-glycosylceramide per 1 SD). In a randomized controlled clinical trial, 1-year treatment of obese patients with the glucagon-like peptide-1 analog liraglutide suppressed plasma C16:0-ceramide and C16:0-glycosylceramide changes compared with control subjects. ConclusionsThese results demonstrate for the first time in humans that AT-derived ceramides are modifiable regulators of vascular redox state in obesity, with a direct impact on cardiac mortality in advanced atherosclerosis. (The Interaction Between Appetite Hormones; NCT02094183)

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 modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,microarrays and can beutilized in functional in vitro/in vivo studies.

Beyond the Extracellular Vesicles: Technical Hurdles, Achieved Goals and Current Challenges When Working on Adipose Cells.

Adipose tissue and its crosstalk with other organs plays an essential role in the metabolic homeostasis of the entire body. Alteration of this communication (i.e., due to obesity) is related to the development of several comorbidities including type 2 diabetes, cardiovascular diseases, or cancer. Within the adipose depot, adipocytes are the main cell type and thus the main source of secreted molecules, which exert modulating effects not only at a local but also at a systemic level. Extracellular vesicles (EVs) have recently emerged as important mediators in cell–cell communication and account for part of the cellular secretome. In recent years, there has been a growing body of research on adipocyte-derived extracellular vesicles (Ad-EVs). However, there is still a lack of standardized methodological approaches, especially regarding primary adipocytes. In this review, we will provide an outline of crucial aspects when working on adipose-derived material, with a special focus on primary adipocytes. In parallel, we will point out current methodological challenges in the EV field and how they impact the transcriptomic, proteomic and functional evaluations of Ad-EVs.

Adipocyte-derived extracellular vesicles regulate survival and function of pancreatic β cells.

Extracellular vesicles (EVs) are implicated in the crosstalk between adipocytes and other metabolic organs, and an altered biological cargo has been observed in EVs from human obese adipose tissue (AT). Yet, the role of adipocyte-derived EVs in pancreatic β cells remains to be determined. Here, we explored the effects of EVs released from adipocytes isolated from both rodents and humans and human AT explants on survival and function of pancreatic β cells and human pancreatic islets. EVs from healthy 3T3-L1 adipocytes increased survival and proliferation and promoted insulin secretion in INS-1E β cells and human pancreatic islets, both those untreated or exposed to cytokines or glucolipotoxicity, whereas EVs from inflamed adipocytes caused β cell death and dysfunction. Human lean adipocyte-derived EVs produced similar beneficial effects, whereas EVs from obese AT explants were harmful for human EndoC-βH3 β cells. We observed differential expression of miRNAs in EVs from healthy and inflamed adipocytes, as well as alteration in signaling pathways and expression of β cell genes, adipokines, and cytokines in recipient β cells. These in vitro results suggest that, depending on the physiopathological state of AT, adipocyte-derived EVs may influence β cell fate and function.

Adipocyte-derived extracellular vesicles in health and diseases: Nano-packages with vast biological properties.

As the largest human energy reservoir, adipocytes drive an intense dialog with other cells/organs throughout the body to regulate the size of adipose tissue and to communicate with other metabolic tissues and the brain to regulate energy supply. Adipokines have long been described as mediators of this crosstalk, participating in obesity‐associated complications. Recently, adipocyte‐derived extracellular vesicles (Ad‐EVs) have emerged as new key actors in this communication due to their powerful capacity to convey complex messages between cells. Ad‐EVs convey specific subpopulations of RNA, proteins, and lipids from their parental cells, and can transfer these cargoes into various recipient cells, modulating their metabolism and cell cycle. In healthy individuals, Ad‐EVs actively participate in adipose tissue remodeling to compensate energy supply variations by exchanging information between adipocytes or stroma‐vascular cells, including immune cells. Besides this, recent evidence points out that Ad‐EV secretion and composition from dysfunctional adipocytes are strongly impacted within adipose tissue where they modulate local intercellular communication, contributing to inflammation, fibrosis, abnormal angiogenesis, and at distance with other cells/tissues intrinsically linked to fat (muscle, hepatocytes and even cancer cells). Additionally, some data even suggests that Ad‐EVs might have a systemic action. In this review, we will describe the particular properties of Ad‐EVs and their involvement in health and diseases, with a particular focus on metabolic and cardiovascular diseases as well as cancer.

Adipocyte-Derived Extracellular Vesicles: State of the Art.

White adipose tissue (WAT) is involved in long-term energy storage and represents 10–15% of total body weight in healthy humans. WAT secretes many peptides (adipokines), hormones and steroids involved in its homeostatic role, especially in carbohydrate–lipid metabolism regulation. Recently, adipocyte-derived extracellular vesicles (AdEVs) have been highlighted as important actors of intercellular communication that participate in metabolic responses to control energy flux and immune response. In this review, we focus on the role of AdEVs in the cross-talks between the different cellular types composing WAT with regard to their contribution to WAT homeostasis and metabolic complications development. We also discuss the AdEV cargoes (proteins, lipids, RNAs) which may explain AdEV’s biological effects and demonstrate that, in terms of proteins, AdEV has a very specific signature. Finally, we list and suggest potential therapeutic strategies to modulate AdEV release and composition in order to reduce their deleterious effects during the development of metabolic complications associated with obesity.

MiRNAs in Adipocyte-Derived Extracellular Vesicles: Multiple Roles in Development of Obesity-Associated Disease.

Obesity and overweight are common modern health challenges. Caloric intake greater than that needed for energy production results in excess storage of fat in the abdomen. Visceral fat secretes a wide spectrum of adipokines, and increased adiposity is associated with a higher risk of development of metabolic disorders. In addition, adipose tissue secretes extracellular vesicles (EVs) to communicate with peripheral cells and distant organs, and regulate whole-body metabolism. Furthermore, clinical evidence has shown that adipose tissue-derived EVs are present at low levels in the circulation of healthy individuals. In contrast, individuals with metabolic syndrome have significantly higher levels of circulating adipose-derived EVs. The composition of the contents of EVs is dynamic, and closely mirrors individual daily habits and fasting-fed state metabolic characteristics. In this mini-review, we aimed to elucidate the role of adipocyte-derived EVs in regulation of whole-body metabolism under physiological and pathophysiological conditions. Studies have shown that adipose tissue may be a major source of circulating exosomal miRNAs that regulate metabolic homeostasis and directly promote insulin-resistance in other organs. Furthermore, the composition of adipocyte-derived circulating miRNAs in EVs may change prior to development of metabolic disorder. Adipocyte-derived miRNAs in EVs may also induce obesity-related changes such as M1 polarization and inhibition of M2 polarization of macrophages, which may affect the biological behaviors of surrounding tumor cells.

Biological insight into the extracellular vesicles in women with and without gestational diabetes.

Gestational diabetes mellitus (GDM) is the most common metabolic disorder in pregnancy, with increasing prevalence worldwide and still unclear pathogenic mechanisms. Extracellular vesicles (EVs) are emerging as potential biomarkers of disease-specific pathways in metabolic disorders, but their potential role in GDM is not fully understood. Therefore, the main aim of this study was to evaluate the link between EVs and hyperglycaemia during pregnancy. We assessed 50 GDM women and 50 controls at the third trimester of pregnancy in whom we collected demographic characteristics and clinical and anthropometric parameters. In addition, the circulating total EVs (tEVs) and their subpopulations were assessed using flow cytometry. The levels of tEVs and EVs subtypes, expressed as median and interquartile range, were not significantly different between two groups; however, adipocyte-derived EVs (aEVs) concentration, expressed as percentage, was higher in controls than in GDM women (p = 0.045). In addition, a significant correlation was observed between aEVs (%) and third trimester total cholesterol (p = 0.022) within the GDM group. Furthermore, a significant correlation between endothelial-derived EVs (eEVs) and platelet-derived EVs (pEVs) within both groups was found, as well as a significant relation between aEVs and pEVs. These data, although preliminary, represent the starting point for further studies to determine the role of circulating EVs in GDM.

Current understanding of the role of Adipose-derived Extracellular Vesicles in Metabolic Homeostasis and Diseases: Communication from the distance between cells/tissues.

Extracellular vesicles (EVs) including exosomes, microvesicles (MVs), and apoptotic bodies, are small membrane vesicular structures that are released during cell activation, senescence, or programmed cell death, including apoptosis, necroptosis, and pyroptosis. EVs serve as novel mediators for long-distance cell-to-cell communications and can transfer various bioactive molecules, such as encapsulated cytokines and genetic information from their parental cells to distant target cells. In the context of obesity, adipocyte-derived EVs are implicated in metabolic homeostasis serving as novel adipokines. In particular, EVs released from brown adipose tissue or adipose-derived stem cells may help control the remolding of white adipose tissue towards browning and maintaining metabolic homeostasis. Interestingly, EVs may even serve as mediators for the transmission of metabolic dysfunction across generations. Also, EVs have been recognized as novel modulators in various metabolic disorders, including insulin resistance, diabetes mellitus, and non-alcoholic fatty liver disease. In this review, we summarize the latest progress from basic and translational studies regarding the novel effects of EVs on metabolic diseases. We also discuss EV-mediated cross-talk between adipose tissue and other organs/tissues that are relevant to obesity and metabolic diseases, as well as the relevant mechanisms, providing insight into the development of new therapeutic strategies in obesity and metabolic diseases.

Adipocyte-derived extracellular vesicles modulate appetite and weight through mTOR signalling in the hypothalamus.

Type 2 diabetes and obesity are diseases related to surplus energy in the body. Abnormal interaction between the hypothalamus and adipose tissues is a key trigger of energy metabolism dysfunction. Extracellular vesicles (EVs) regulate intercellular communication by transporting intracellular cargo to recipient cells thereby altering the function of recipient cells. This study aimed to evaluate whether adipocyte-derived EVs can act on hypothalamic neurons to modulate energy intake and to identify the EV-associated non-coding RNAs. Confocal imaging was used to trace the uptake of labelled adipocyte-derived exosomes by hypothalamic anorexigenic POMC neurons. The effects of adipocyte-derived EVs on the mammalian target of rapamycin (mTOR) signalling pathway in POMC neurons were evaluated based on mRNA and protein expression in vitro using quantitative real-time PCR and western blotting. In addition, adipocyte-derived EVs were injected into recipient mice, and changes in mice body weight and daily food intake were monitored. The biological effects of the EV-associated MALAT1 on POMC neurons were explored. Adipocyte-derived EVs were successfully transferred into POMC neurons in vitro. Results showed that adipocytes of obese mice secreted MALAT1-containing EVs, which increased appetite and weight when administered to lean mice. Conversely, adipocyte-derived EVs from lean mice decreased food intake and weight when administered to obese mice. Adipocyte-derived EVs play important roles in mediating the interaction between adipocytes and hypothalamic neurons. Adipocyte-derived EVs can regulate POMC expression through the hypothalamic mTOR signalling in vivo and in vitro, thereby affecting body energy intake.

Inflammatory adipocyte-derived extracellular vesicles promote leukocyte attachment to vascular endothelial cells

Background and aimsObesity is associated with an increased risk of cardiovascular disease, but the mechanisms involved are not completely understood. In obesity, the adipocyte microenvironment is characterised by both hypoxia and inflammation. Therefore, we sought to determine whether extracellular vesicles (EVs) derived from adipocytes in this setting might be involved in mediating cardiovascular disease, specifically by promoting leukocyte attachment to vascular endothelial cells. MethodsMature 3T3-L1 adipocytes were incubated for 24 h under control, TNF-α (30 ng/mL), hypoxia (1% O2), or TNF-α+hypoxia (30 ng/mL, 1% O2) conditions. EVs were isolated by differential ultracentrifugation and analysed by nanoparticle tracking analysis. Primary human umbilical vein endothelial cells (HUVECs) were treated with EVs for 6 h before being lysed for Western blotting to investigate changes in adhesion molecule production, or for use in leukocyte attachment assays. ResultsEVs from adipocytes treated with TNF-α and TNF-α+hypoxia increased vascular cell adhesion molecule (VCAM-1) production in HUVECs compared to basal level (4.2 ± 0.6 and 3.8 ± 0.3-fold increase, respectively (p < 0.05)), an effect that was inhibited by an anti-TNF-α neutralising antibody. Production of other adhesion molecules (E-selectin, P-selectin, platelet endothelial cell adhesion molecule and VE-Cadherin) was unchanged. Pre-incubating HUVECs with TNF-α+hypoxia EVs significantly increased leukocyte attachment compared to basal level (3.0 ± 0.4-fold increase (p < 0.05)). ConclusionsInflammatory adipocyte EVs induce VCAM-1 production in vascular endothelial cells, accompanied by enhanced leukocyte attachment. Preventing adipocyte derived EV-induced VCAM-1 upregulation may offer a novel therapeutic target in the prevention of obesity-driven cardiovascular disease.

Anaphylatoxin Receptors C3aR and C5aR1 Are Important Factors That Influence the Impact of Ethanol on the Adipose Secretome

Background and aims: Chronic ethanol exposure results in inflammation in adipose tissue; this response is associated with activation of complement as well as the development of alcohol-related liver disease (ALD). Adipose communicates with other organs, including liver, via the release of soluble mediators, such as adipokines and cytokines, characterized as the “adipose secretome.” Here we investigated the role of the anaphaylatoxin receptors C3aR and C5aR1 in the development of adipose tissue inflammation and regulation of the adipose secretome in murine ALD (mALD).Methods: Wild-type C57BL/6 (WT), C3aR−/−, and C5aR1−/− mice were fed Lieber-DeCarli ethanol diet for 25 days (6% v/v, 32% kcal) or isocaloric control diets; indicators of inflammation and injury were assessed in gonadal adipose tissue. The adipose secretome was characterized in isolated adipocytes and stromal vascular cells.Results: Ethanol feeding increased the expression of adipokines, chemokines and leukocyte markers in gonadal adipose tissue from WT mice; C3aR−/− were partially protected while C5aR1−/− mice were completely protected. In contrast, induction of CYP2E1 and accumulation of TUNEL-positive cells in adipose in response to ethanol feeding was independent of genotype. Bone marrow chimeras, generated with WT and C5aR1−/− mice, revealed C5aR1 expression on non-myeloid cells, likely to be adipocytes, contributed to ethanol-induced adipose inflammation. Chronic ethanol feeding regulated both the quantity and distribution of adipokines secreted from adipocytes in a C5aR1-dependent mechanism. In WT mice, chronic ethanol feeding induced a predominant release of pro-inflammatory adipokines from adipocytes, while the adipose secretome from C5aR1−/− mice was characterized by an anti-inflammatory/protective profile. Further, the cargo of adipocyte-derived extracellular vesicles (EVs) was distinct from the soluble secretome; in WT EVs, ethanol increased the abundance of pro-inflammatory mediators while EV cargo from C5aR1−/− adipocytes contained a greater diversity and more robust expression of adipokines.Conclusions: C3aR and C5aR1 are potent regulators of ethanol-induced adipose inflammation in mALD. C5aR1 modulated the impact of chronic ethanol on the content of the adipose secretome, as well as influencing the cargo of an extensive array of adipokines from adipocyte-derived EVs. Taken together, our data demonstrate that C5aR1 contributes to ethanol-mediated changes in the adipose secretome, likely contributing to intra-organ injury in ALD.

Evidence for Adipocyte-Derived Extracellular Vesicles in the Human Circulation.

Adipocyte-derived extracellular vesicles (EVs) may serve as novel endocrine mediators of adipose tissue and impact upon vascular health. However, it is unclear whether adipocyte-derived EVs are present in the human circulation. Therefore, the purpose of this study was to seek evidence for the presence of adipocyte-derived EVs in circulating plasma. Size-exclusion chromatography of platelet-free plasma identified fractions 5 to 10 as containing EVs by a peak in particle concentration, which corresponded with the presence of EV and adipocyte proteins. Pooling fractions 5 to 10 and subjecting to ultracentrifugation yielded a plasma EV sample, as verified by transmission electron microscopy (TEM) showing EV structures and Western blotting for EV (e.g., CD9 and Alix) and adipocyte markers. Magnetic beads and a solid-phase assay were used to deplete the EV sample of the four major families of circulating EVs: platelet-derived, leukocyte-derived, endothelial-derived, and erythrocyte-derived EVs. Postdepletion samples from both techniques contained EV structures as visualized by TEM, as well as CD9, Alix, and classic adipocyte proteins. Postdepletion samples also contained a range of other adipocyte proteins from an adipokine array. Adipocyte proteins and adipokines are expressed in optimally processed plasma EV samples, suggesting that adipocyte-derived EVs are secreted into the human circulation.

Circulating adipocyte-derived extracellular vesicles are novel markers of metabolic stress.

• Extensive characterization of 3T3L1 EVs identified perilipin A in their composition. • Circulating EVs are elevated in obese mice and associated with glucose intolerance. • Circulating EVs are elevated in obese human and correlated with metabolic factors. • Perilipin A and EV levels are increased in the circulation of obese mice and human. • Circulating EV and perilipin A levels decrease with low calorie intervention.

SAT-298 - Perilipin a is a Novel Biomarker of Circulating Adipocyte-Derived Extracellular Vesicles in Both Murine Models of Obesity and Obese Humans

SAT-298 - Perilipin a is a Novel Biomarker of Circulating Adipocyte-Derived Extracellular Vesicles in Both Murine Models of Obesity and Obese Humans

Characterisation of adipocyte-derived extracellular vesicles released pre- and post-adipogenesis

Extracellular vesicles (EVs) are submicron vesicles released from many cell types, including adipocytes. EVs are implicated in the pathogenesis of obesity-driven cardiovascular disease, although the characteristics of adipocyte-derived EVs are not well described. We sought to define the characteristics of adipocyte-derived EVs before and after adipogenesis, hypothesising that adipogenesis would affect EV structure, molecular composition and function. Using 3T3-L1 cells, EVs were harvested at day 0 and day 15 of differentiation. EV and cell preparations were visualised by electron microscopy and EVs quantified by nanoparticle tracking analysis (NTA). EVs were then assessed for annexin V positivity using flow cytometry; lipid and phospholipid composition using 2D thin layer chromatography and gas chromatography; and vesicular protein content by an immuno-phenotyping assay. Pre-adipogenic cells are connected via a network of protrusions and EVs at both time points display classic EV morphology. EV concentration is elevated prior to adipogenesis, particularly in exosomes and small microvesicles. Parent cells contain higher proportions of phosphatidylserine (PS) and show higher annexin V binding. Both cells and EVs contain an increased proportion of arachidonic acid at day 0. PREF-1 was increased at day 0 whilst adiponectin was higher at day 15 indicating EV protein content reflects the stage of adipogenesis of the cell. Our data suggest that EV production is higher in cells before adipogenesis, particularly in vesicles <300 nm. Cells at this time point possess a greater proportion of PS (required for EV generation) whilst corresponding EVs are enriched in signalling fatty acids, such as arachidonic acid, and markers of adipogenesis, such as PREF-1 and PPARγ.