The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

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A group of flavonoids isolated from medicinal plants and which are selective inhibitors of lipoxygenase activity in vitro: sideritoflavone, cirsiliol, hypolaetin-8-O-beta-D-glucoside, hypolaetin, oroxindin, quercetagetin-7-O-beta-D-glucoside, gossypin, hibifolin and gossypetin, besides leucocyanidol, have been studied for their effects on acute responses induced by carrageenin in mice. The oral administration of flavonoids to mice inhibited dose-dependently the development of paw oedema at 1, 3 and 5 h after carrageenin injection. A similar administration of flavonoids induced a dose-dependent inhibition of leukocyte accumulation in inflammatory exudates following intraperitoneal injection of carrageenin into mice. Some of the flavonoids exhibited a potency against leukocyte infiltration similar to that seen for inhibition of carrageenin oedema at 3 h of induction. In agreement with data reported in rats, indomethacin was much more effective on inhibition of prostaglandin E2 (PGE2) formation than on leukocyte infiltration in mice. The selectivity of flavonoids towards lipoxygenase is not retained in vivo since they behave as dual inhibitors of PGE2 and leukotriene B4 (LTB4) formation in peritoneal exudates. Our data support the inhibition of arachidonic acid metabolism as one of the mechanisms by which flavonoids exert their anti-inflammatory effects.

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Heme oxygenase 1 (HO-1) is induced by oxidative or nitrosative stress, cytokines and other mediators produced during inflammatory processes, likely as part of a defence system in cells exposed to stress to provide a negative feedback for cell activation and the production of mediators, which could modulate the inflammatory response. HO-1 activity results in the inhibition of oxidative damage and apoptosis, with significant reductions in inflammatory events including edema, leukocyte adhesion and migration, and production of inflammatory cytokines. HO-1 is induced by nitric oxide (NO) in different biological systems and can control the increased production of this mediator observed in many inflammatory situations. Regulatory interactions between HO-1 and cyclooxygenase (COX) pathways have also been reported. Modulation of signal transduction pathways by HO-1 or products derived from its activity, such as carbon monoxide (CO), may mediate the anti-inflammatory effects of this protein. Regulation of HO-1 activity may be a therapeutical strategy for a number of inflammatory conditions.

Background/Aims: Chronic inflammation contributes to cartilage degeneration during the progression of osteoarthritis (OA). Adipose tissue-derived mesenchymal stem cells (AD-MSC) show great potential to treat inflammatory and degradative processes in OA and have demonstrated paracrine effects in chondrocytes. In the present work, we have isolated and characterized the extracellular vesicles from human AD-MSC to investigate their role in the chondroprotective actions of these cells. Methods: AD-MSC were isolated by collagenase treatment from adipose tissue from healthy individuals subjected to abdominal lipectomy surgery. Microvesicles and exosomes were obtained from conditioned medium by filtration and differential centrifugation. Chondrocytes from OA patients were used in primary culture and stimulated with 10 ng/ml interleukin(IL)-1β in the presence or absence of AD-MSC microvesicles, exosomes or conditioned medium. Protein expression was investigated by ELISA and immunofluorescence, transcription factor-DNA binding by ELISA, gene expression by real-time PCR, prostaglandin E₂ (PGE₂) by radioimmunoassay, and matrix metalloproteinase (MMP) activity and nitric oxide (NO) production by fluorometry. Results: In OA chondrocytes stimulated with IL-1β, microvesicles and exosomes reduced the production of inflammatory mediators tumor necrosis factor-α, IL-6, PGE₂ and NO. The downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 would lead to the decreased PGE₂ production while the effect on NO could depend on the reduction of inducible nitric oxide synthase expression. Treatment of OA chondrocytes with extracellular vesicles also decreased the release of MMP activity and MMP-13 expression whereas the production of the anti-inflammatory cytokine IL-10 and the expression of collagen II were significantly enhanced. The reduction of inflammatory and catabolic mediators could be the consequence of a lower activation of nuclear factor-κB and activator protein-1. The upregulation of annexin A1 specially in MV may contribute to the anti-inflammatory and chondroprotective effects of AD-MSC. Conclusions: Our data support the interest of AD-MSC extracellular vesicles to develop new therapeutic approaches in joint conditions.

The antiperoxidative effects of 35 phenolic compounds, most of them belonging to the flavonoid class, were investigated using CCl4-induced peroxidation of rat liver microsomes. This system was rather insensitive to gallic acid, methyl gallate and ellagic acid. Nevertheless it was inhibited by flavonoids and structure/activity relationships were established. The most potent compounds were gardenin D, luteolin, apigenin (flavones), datiscetin, morin, galangin (flavonols), eriodictyol (flavanone), amentoflavone (biflavone) and the reference compound, (+)-catechin. The natural polymethoxyflavone gardenin D has shown a potency comparable to that of (+)-catechin and higher than that of silybin. Thus, it may be considered as a new type of natural antioxidant with potential therapeutical applications.

Many in vitro studies have used cell cultures to focus on the relationships between cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) isoforms. We have investigated the time-course of regulation and the role of COX-2 and iNOS in a model of experimental inflammation in mice, the air pouch injected with zymosan. This study demonstrates that there is an early acute phase (4 h) mediated mainly by eicosanoids, with high levels of prostaglandin E2 (PGE2) produced by cyclo-oxygenase-1. In addition, in the later phase (from 12 h) there is a participation of nitric oxide (NO) and PGE2 accompanied by co-induction of both iNOS and COX-2. These enzymes were detected in migrating leukocytes as well as in macrophages lining the air pouch. Administration of NS398 or indomethacin inhibited PGE2 levels and COX activity, but also nitrite levels and iNOS activity, which was accompanied by a reduction in iNOS expression. Aminoguanidine inhibited nitrite levels and iNOS activity in addition to exerting inhibitory effects on the COX pathway. Treatment of animals with dexamethasone reduced nitrite and PGE2 concentrations in air pouch exudates, as well as iNOS and COX-2 expression in migrating cells. Our results indicate that PGE2 and NO may play in vivo mutual modulatory roles in the inflammatory response caused by zymosan injection into the mouse air pouch, a suitable model to study drugs acting on those pathways.