Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity.

BACKGROUND: Obesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity's impact on EV secretion from human AT remains limited. METHODS: We investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography-mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry. RESULTS: EVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation. CONCLUSIONS: We are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

MCF10CA Breast Cancer Cells Utilize Hyaluronan-Coated EV-Rich Trails for Coordinated Migration.

Invasion of tumor cells through the stroma is coordinated in response to migratory cues provided by the extracellular environment. One of the most abundant molecules in the tumor microenvironment is hyaluronan, a glycosaminoglycan known to promote many hallmarks of tumor progression, including the migratory potential of tumor cells. Strikingly, hyaluronan is also often found to coat extracellular vesicles (EVs) that originate from plasma membrane tentacles of tumor cells crucial for migration, such as filopodia, and are abundant in tumor niches. Thus, it is possible that hyaluronan and hyaluronan-coated EVs have a cooperative role in promoting migration. In this work, we compared the hyaluronan synthesis, EV secretion and migratory behavior of normal and aggressive breast cell lines from MCF10 series. Single live cell confocal imaging, electron microscopy and correlative light and electron microscopy experiments revealed that migrating tumor cells form EV-rich and hyaluronan -coated trails. These trails promote the pathfinding behavior of follower cells, which is dependent on hyaluronan. Specifically, we demonstrated that plasma membrane protrusions and EVs left behind by tumor cells during migration are strongly positive for CD9. Single cell tracking demonstrated a leader-follower behavior, which was significantly decreased upon removal of pericellular hyaluronan, indicating that hyaluronan promotes the pathfinding behavior of follower cells. Chick chorioallantoic membrane assays in ovo suggest that tumor cells behave similarly in 3D conditions. This study strengthens the important role of extracellular matrix production and architecture in coordinated tumor cell movements and validates the role of EVs as important components and regulators of tumor matrix. The results suggest that tumor cells can modify the extracellular niche by forming trails, which they subsequently follow coordinatively. Future studies will clarify in more detail the orchestrated role of hyaluronan, EVs and other extracellular cues in coordinated migration and pathfinding behavior of follower cells.

Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes.

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1-3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.

Characterization of hyaluronan-coated extracellular vesicles in synovial fluid of patients with osteoarthritis and rheumatoid arthritis.

BACKGROUND: Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA-EV), but their concentration and function in joint pathologies remain unknown. METHODS: The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA-EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA-EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal-Wallis analysis of variance. RESULTS: The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. CONCLUSIONS: CLSM analysis offers a useful tool to assess HA-EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.

Orotic acid-treated hepatocellular carcinoma cells resist steatosis by modification of fatty acid metabolism.

BACKGROUND: Orotic acid (OA) has been intensively utilized to induce fatty liver in rats. Although the capacity of OA to cause steatosis is species-specific, previous in vitro studies indicate that humans could also be susceptible to OA-induced fatty liver. The aim of the present study was to re-elucidate the potential of OA exposure to modulate the cellular mechanisms involved in both non-alcoholic fatty liver disease pathogenesis and cellular protection from lipid accumulation. In addition, alterations in detailed fatty acid (FA) profiles of cells and culture media were analyzed to assess the significance of lipid metabolism in these phenomena. METHODS: In our experiments, human hepatocellular carcinoma HepG2 cells were exposed to OA. Bacterial endotoxin, lipopolysaccharide (LPS), was used to mimic hepatic inflammation. The lipogenic and inflammatory effects of OA and/or LPS on cells were assessed by labeling cellular lipids with Nile red stain and by performing image quantifications. The expression levels of key enzymes involved in de novo lipogenesis (DNL) and of inflammatory markers related to the disease development were studied by qRT-PCR. FA profiles of cells and culture media were determined from total lipids with gas chromatography-mass spectrometry. RESULTS: Our data indicate that although OA possibly promotes the first stage of DNL, it does not cause a definite lipogenic transformation in HepG2 cells. Reduced proportions of 16:0, increased stearoyl-Coenzyme A desaturase 1 mRNA expression and relatively high proportions of 16:1n-7 suggest that active delta9-desaturation may limit lipogenesis and the accumulation of toxic 16:0. Inflammatory signaling could be reduced by the increased production of long-chain n-3 polyunsaturated FA (PUFA) and the active incorporation of certain FA, including 18:1n-9, into cells. In addition, increased proportions of 20:4n-6 and 22:6n-3, total PUFA and dimethyl acetal 18:0 suggest that OA exposure may cause increased secretion of lipoproteins and extracellular vesicles. CONCLUSIONS: The present data suggest that, apart from the transcription-level events reported by previous studies, modifications of FA metabolism may also be involved in the prevention of OA-mediated steatosis. Increased delta9-desaturation and secretion of lipoproteins and extracellular vesicles could offer potential mechanisms for further studies to unravel how OA-treated cells alleviate lipidosis.

Hyaluronan histochemistry-a potential new tool to assess the progress of liver disease from simple steatosis to hepatocellular carcinoma.

Nonalcoholic fatty liver disease is among the most common liver diseases worldwide and one cause of cirrhosis that can result in the development of hepatocellular carcinoma (HCC). Hyaluronan (HA) is a high-molecular-mass glycosaminoglycan with diverse functions in tissue injury and repair, for instance, in inflammation and fibrogenesis. The aim of the present study was to investigate the relationships between the HA synthesizing and degrading enzymes in a spectrum of liver pathologies. This was realized by histological staining of liver sections from controls and patients with simple steatosis, steatohepatitis, cirrhosis and HCC (n = 90). HA-positive staining intensified in connective tissue in all liver pathologies, and staining of CD44, the major HA receptor, similarly increased in steatohepatitis and cirrhosis. HA synthase 1 (HAS1)-positive staining was reduced in steatosis, steatohepatitis and HCC. Staining of HAS3, which produces HA of a lower molecular mass, promotes inflammation and is pathogenic in animal models, increased in all diagnoses. The responses in staining intensity of HAS2 and hyaluronidases 1-2 were specific for different cell types. These findings suggest that HAS1-2 are responsible for HA synthesis in healthy livers, while HAS3 increases in importance in liver diseases. It is noteworthy that the pathological changes in HA metabolism are already visible in simple steatosis and, thus, precede the histological changes of inflammation and fibrosis. It could be possible to intervene in disease progression at an early stage by influencing HA metabolism. The results could have potential clinical applications with HAS3 immunostaining supplementing the existing HCC diagnostics.

Extracellular vesicles are integral and functional components of the extracellular matrix.

Extracellular vesicles (EV) are small plasma membrane-derived particles released into the extracellular space by virtually all cell types. Recently, EV have received increased interest because of their capability to carry nucleic acids, proteins, lipids and signaling molecules and to transfer their cargo into the target cells. Less attention has been paid to their role in modifying the composition of the extracellular matrix (ECM), either directly or indirectly via regulating the ability of target cells to synthesize or degrade matrix molecules. Based on recent results, EV can be considered one of the structural and functional components of the ECM that participate in matrix organization, regulation of cells within it, and in determining the physical properties of soft connective tissues, bone, cartilage and dentin. This review addresses the relevance of EV as specific modulators of the ECM, such as during the assembly and disassembly of the molecular network, signaling through the ECM and formation of niches suitable for tissue regeneration, inflammation and tumor progression. Finally, we assess the potential of these aspects of EV biology to translational medicine.