Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications.

Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of in vitro and in vivo functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation

Urinary exosome miR-146a is a potential marker of albuminuria in essential hypertension

BACKGROUND: There is increasing interest in using extracellular vesicle-derived microRNAs (miRNAs) as biomarkers in renal dysfunction and injury. Preliminary evidence indicates that miRNAs regulate the progression of glomerular disease. Indeed, exosomes from the renal system have provided novel evidence in the clinical setting of albuminuria. Thus, the aim of this study was to quantify the urinary miRNAs present in exosome and microvesicles (MVs), and to assess their association with the presence of increased urinary albumin excretion in essential hypertension. METHODS: Exosomes were collected from urine specimens from a cohort of hypertensive patients with (n = 24) or without albuminuria (n = 28), and from 20 healthy volunteers as a control group. Urinary exosomes were phenotyped by Western blot, tunable resistive pulse sensing, and electronic microscopy. Expression of miR-146a and miR-335* was analysed by qRT-PCR and any associations between albuminuria and exosomal miRNAs were analysed. RESULTS: Urinary miRNAs are highly enriched in exosome subpopulations compared to MVs, both in patients with or without increased albuminuria (p < 0.001), but not in the control group. High albuminuria was associated with 2.5-fold less miR-146a in exosomes (p = 0.017), whereas miR-146a levels in MV did not change. In addition, exosome miR-146a levels were inversely associated with albuminuria (r = 0.65, p < 0.0001), and discriminated the presence of urinary albumin excretion presence [area under the curve = 0.80, 95% confidence interval: 0.66-0.95; p = 0.0013]. CONCLUSIONS: Our results indicate that miRNAs were enriched in the urinary exosome subpopulation in hypertensive patients and that low miR-146a expression in exosomes was associated with the presence of albuminuria. Thus, urinary exosome miR-146a may be a potentially useful tool for studying early renal injury in hypertension

EVpedia: a community web portal for extracellular vesicles research

Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research.X1110478Ysciescopu

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications

Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption

To prevent dehydration, terrestrial animals and humans have developed a sensitive and versatile system to maintain their water homeostasis. In states of hypernatremia or hypovolemia, the antidiuretic hormone vasopressin (AVP) is released from the pituitary and binds its type-2 receptor in renal principal cells. This triggers an intracellular cAMP signaling cascade, which phosphorylates aquaporin-2 (AQP2) and targets the channel to the apical plasma membrane. Driven by an osmotic gradient, pro-urinary water then passes the membrane through AQP2 and leaves the cell on the basolateral side via AQP3 and AQP4 water channels. When water homeostasis is restored, AVP levels decline, and AQP2 is internalized from the plasma membrane, leaving the plasma membrane watertight again. The action of AVP is counterbalanced by several hormones like prostaglandin E2, bradykinin, dopamine, endothelin-1, acetylcholine, epidermal growth factor, and purines. Moreover, AQP2 is strongly involved in the pathophysiology of disorders characterized by renal concentrating defects, as well as conditions associated with severe water retention. This review focuses on our recent increase in understanding of the molecular mechanisms underlying AVP-regulated renal water transport in both health and disease

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

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

Lysyl oxidase‐like 2 is a regulator of angiogenesis through modulation of endothelial‐to‐mesenchymal transition

Lysyl oxidase‐like 2 (LOXL2) belongs to the family of lysyl oxidases, and as such promotes crosslinking of collagens and elastin by oxidative deamination of lysine residues. In endothelial cells (ECs), LOXL2 is involved in crosslinking and scaffolding of collagen IV. Additionally, several reports have shown a role for LOXL2 in other processes, including regulation of gene expression, tumor metastasis, and epithelial‐to‐mesenchymal transition (EMT). Here, we demonstrate an additional role for LOXL2 in the regulation of angiogenesis by modulation of endothelial‐to‐mesenchymal transition (EndMT). LOXL2 knockdown in ECs results in decreased migration and sprouting, and concordantly, LOXL2 overexpression leads to an increase in migration and sprouting, independent of its catalytic activity. Furthermore, LOXL2 knockdown resulted in a reduced expression of EndMT markers, and inhibition of transforming growth factor‐β (TGF‐β)‐mediated induction of EndMT. Interestingly, unlike in EMT, overexpression of LOXL2 alone is insufficient to induce EndMT. Further investigation revealed that LOXL2 expression regulates protein kinase B (PKB)/Akt and focal adhesion kinase (FAK) signaling, both pathways that have been implicated in the regulation of EMT. Altogether, our studies reveal a role for LOXL2 in angiogenesis through the modulation of EndMT in ECs, independent of its enzymatic crosslinking activity

Lysyl oxidase‐like 2 is a regulator of angiogenesis through modulation of endothelial‐to‐mesenchymal transition

Lysyl oxidase‐like 2 (LOXL2) belongs to the family of lysyl oxidases, and as such promotes crosslinking of collagens and elastin by oxidative deamination of lysine residues. In endothelial cells (ECs), LOXL2 is involved in crosslinking and scaffolding of collagen IV. Additionally, several reports have shown a role for LOXL2 in other processes, including regulation of gene expression, tumor metastasis, and epithelial‐to‐mesenchymal transition (EMT). Here, we demonstrate an additional role for LOXL2 in the regulation of angiogenesis by modulation of endothelial‐to‐mesenchymal transition (EndMT). LOXL2 knockdown in ECs results in decreased migration and sprouting, and concordantly, LOXL2 overexpression leads to an increase in migration and sprouting, independent of its catalytic activity. Furthermore, LOXL2 knockdown resulted in a reduced expression of EndMT markers, and inhibition of transforming growth factor‐β (TGF‐β)‐mediated induction of EndMT. Interestingly, unlike in EMT, overexpression of LOXL2 alone is insufficient to induce EndMT. Further investigation revealed that LOXL2 expression regulates protein kinase B (PKB)/Akt and focal adhesion kinase (FAK) signaling, both pathways that have been implicated in the regulation of EMT. Altogether, our studies reveal a role for LOXL2 in angiogenesis through the modulation of EndMT in ECs, independent of its enzymatic crosslinking activity

Proteomic analysis of machine perfusion solution from brain dead donor kidneys reveals that elevated complement, cytoskeleton and lipid metabolism proteins are associated with 1-year outcome.

Assessment of donor kidney quality is based on clinical scores or requires biopsies for histological assessment. Noninvasive strategies to identify and predict graft outcome at an early stage are, therefore, needed. We evaluated the perfusate of donation after brain death (DBD) kidneys during nonoxygenated hypothermic machine perfusion (HMP). In particular, we compared perfusate protein profiles of good outcome (GO) and suboptimal outcome (SO) 1-year post-transplantation. Samples taken 15&#xA0;min after the start HMP (T1) and before the termination of HMP (T2) were analysed using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hierarchical clustering of the 100 most abundant proteins showed discrimination between grafts with a GO and SO at T1. Elevated levels of proteins involved in classical complement cascades at both T1 and T2 and a reduced abundance of lipid metabolism at T1 and of cytoskeletal proteins at T2 in GO versus SO was observed. ATP-citrate synthase and fatty acid-binding protein 5 (T1) and immunoglobulin heavy variable 2-26 and desmoplakin (T2) showed 91% and 86% predictive values, respectively, for transplant outcome. Taken together, DBD kidney HMP perfusate profiles can distinguish between outcome 1-year post-transplantation. Furthermore, it provides insights into mechanisms that could play a role in post-transplant outcomes