Search

Extracellular vesicles (EVs) are promising tools to transfer macromolecular therapeutic molecules to recipient cells, however, efficient functional intracellular protein delivery by EVs remains challenging. Here, we have developed novel and versatile systems that leverage selected molecular tools to engineer EVs for robust cytosolic protein delivery bothin vitroandin vivo. These systems, termed VSV-G plus EV-sorting Domain-Intein-Cargo (VEDIC) and VSV-G-Foldon-Intein-Cargo (VFIC), exploit an engineered mini-intein (intein) protein with self-cleavage activity to link cargo to an EV-sorting domain and release it from the EV membrane inside the EV lumen. In addition, we utilize the fusogenic protein VSV-G to facilitate endosomal escape and cargo release from the endosomal system to the cytosol of recipient cells. Importantly, we demonstrate that the combination of the self-cleavage intein, fusogenic protein and EV-sorting domain are indispensable for efficient functional intracellular delivery of cargo proteins by engineered EVs. As such, nearly 100% recombination and close to 80% genome editing efficiency in reporter cells were observed by EV-transferred Cre recombinase and Cas9/sgRNA RNPs, respectively. Moreover, EV-mediated Cre delivery by VEDIC or VFIC engineered EVs resulted in significantin vivorecombination in Cre-LoxP R26-LSL-tdTomato reporter mice following both local and systemic injections. Finally, we applied these systems for improved treatment of LPS-induced systemic inflammation by delivering a super-repressor of NF-ĸB activity. Altogether, this study describes a platform by which EVs can be utilized as a vehicle for the efficient intracellular delivery of macromolecular therapeutics for treatments of disease.Abstract FigureGraphic summary: Development of VEDIC and VFIC systems for high-efficiency intracellular protein delivery in vitro and in vivo.Intein in tripartite fusion protein (EV-sorting Domain-Intein-Cargo) performs C-terminal cleavage during the process of EV-biogenesis, resulting in enriched free cargo proteins inside of vesicles. Together with fusogenic protein, VSV-G, these engineered EVs achieve high-efficiency intracellular delivery of cargo protein (Cre and super repressor of NF-ĸB) or protein complex (Cas9/sgRNA RNPs) both in reporter cells and in mice models.

The therapeutic use of RNA interference is limited by the inability of siRNA molecules to reach their site of action, the cytosol of target cells. Lipid nanoparticles, including liposomes, are commonly employed as siRNA carrier systems to overcome this hurdle, although their widespread use remains limited due to a lack of delivery efficiency. More recently, nature's own carriers of RNA, extracellular vesicles (EVs), are increasingly being considered as alternative siRNA delivery vehicles due to their intrinsic properties. However, they are difficult to load with exogenous cargo. Here, EV-liposome hybrid nanoparticles (hybrids) are prepared and evaluated as an alternative delivery system combining properties of both liposomes and EVs. It is shown that hybrids are spherical particles encapsulating siRNA, contain EV-surface makers, and functionally deliver siRNA to different cell types. The functional behavior of hybrids, in terms of cellular uptake, toxicity, and gene-silencing efficacy, is altered as compared to liposomes and varies among recipient cell types. Moreover, hybrids produced with cardiac progenitor cell (CPC) derived-EVs retain functional properties attributed to CPC-EVs such as activation of endothelial signaling and migration. To conclude, hybrids combine benefits of both synthetic and biological drug delivery systems and might serve as future therapeutic carriers of siRNA.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss of survival motor neuron (SMN) protein. While SMN restoration therapies are beneficial, they are not a cure. We aimed to identify potentially novel treatments to alleviate muscle pathology combining transcriptomics, proteomics, and perturbational data sets. This revealed potential drug candidates for repurposing in SMA. One of the candidates, harmine, was further investigated in cell and animal models, improving multiple disease phenotypes, including lifespan, weight, and key molecular networks in skeletal muscle. Our work highlights the potential of multiple and parallel data-driven approaches for the development of potentially novel treatments for use in combination with SMN restoration therapies.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss of survival motor neuron (SMN) protein. While SMN restoration therapies are beneficial, they are not a cure. We aimed to identify novel treatments to alleviate muscle pathology combining transcriptomics, proteomics and perturbational datasets. This revealed potential drug candidates for repurposing in SMA. One of the lead candidates, harmine, was further investigated in cell and animal models, improving multiple disease phenotypes, including SMN expression and lifespan. Our work highlights the potential of multiple, parallel data driven approaches for development of novel treatments for use in combination with SMN restoration therapies.

Supplemental Digital Content is available in the text., Background. Delayed graft function (DGF) after kidney transplantation is negatively associated with long-term graft function and survival. Kidney function after transplantation depends on multiple factors, both donor- and recipient-associated. Prediction of posttransplantation graft function would allow timely intervention to optimize patient care and survival. Currently, graft-based predictions can be made based on histological and molecular analyses of 0-hour biopsy samples. However, such analyses are currently not implemented, as biopsy samples represent only a very small portion of the entire graft and are not routinely analyzed in all transplantation centers. Alternatives are thus required. Methods. We analyzed whether donor organ preservation fluid contain small extracellular vesicles (sEV) and whether the RNA content of these vesicles could be used as a source for potential biomarkers for posttransplantation kidney function. Results. We provide proof of principle that sEVs are present in preservation fluid, which contain RNAs associated with donor origin. Furthermore, sEV micro RNA profiles could be associated with graft function during the first 7 days posttransplantation, but no significant correlation with DGF could be established based on the current dataset. Conclusions. Overall, the predictive potential of sEV RNA biomarkers together with relatively easy and noninvasive sample collection and analysis methods could pave the way towards universal screening of donor kidney-associated risk for DGF, optimized patient treatment, and subsequently improved short- and long-term graft function and survival.

[Image: see text] Extracellular vesicles are nanoparticles produced by cells. They are composed of cellular membrane with associated membrane proteins that surrounds an aqueous core containing soluble molecules such as proteins and nucleic acids, like miRNA and mRNA. They are important in many physiological and pathological processes as they can transfer biological molecules from producer cells to acceptor cells. Preparation of the niche for cancer metastasis, stimulation of tissue regeneration and orchestration of the immune response are examples of the diverse processes in which extracellular vesicles have been implicated. As a result, these vesicles have formed a source of inspiration for many scientific fields. They could be used, for example, as liquid biopsies in diagnostics, as therapeutics in regenerative medicine, or as drug delivery vehicles for transport of medicines. In this Account, we focus on drug delivery applications. As we learn more and more about these vesicles, the complexity increases. What originally appeared to be a relatively uniform population of cellular vesicles is increasingly subdivided into different subsets. Cells make various distinct vesicle types whose physicochemical aspects and composition is influenced by parental cell type, cellular activation state, local microenvironment, biogenesis pathway, and intracellular cargo sorting routes. It has proven difficult to assess the effects of changes in production protocol on the characteristics of the cell-derived vesicle population. On top of that, each isolation method for vesicles necessarily enriches certain vesicle classes and subpopulations while depleting others. Also, each method is associated with a varying degree of vesicle purity and concomitant coisolation of nonvesicular material. What emerges is a staggering heterogeneity. This constitutes one of the main challenges of the field as small changes in production and isolation protocols may have large impact on the vesicle characteristics and on subsequent vesicle activity. We try to meet this challenge by careful experimental design and development of tools that enable robust readouts. By engineering the surface and cargo of extracellular vesicles through chemical and biological techniques, favorable characteristics can be enforced while unfavorable qualities can be overruled or masked. This is coupled to the precise evaluation of the interaction of extracellular vesicles with cells to determine the extracellular vesicle uptake routes and intracellular routing. Sensitive reporter assays enable reproducible analysis of functional delivery. This systematic evaluation and optimization of extracellular vesicles improves our insight into the critical determinants of extracellular vesicle activity and should improve translation into clinical application of engineered extracellular vesicles as a new class of drug delivery systems.

Extracellular vesicles (EVs) are increasingly being recognized as mediators of intercellular signaling via the delivery of effector molecules. Interestingly, certain types of EVs are also capable of inducing therapeutic responses. For these reasons, the therapeutic potential of EVs is a topic of intense research, both in the context of drug delivery and regenerative medicine. However, to fully utilize EVs for therapeutic purposes, an improved understanding of the mechanisms by which they function would be highly advantageous. Here, the current state of knowledge regarding the cellular uptake and trafficking of EVs is reviewed, along with a consideration of how these pathways potentially influence the functions of therapeutic EVs. Furthermore, the natural cell-targeting abilities, biodistribution profiles, and pharmacokinetics of exogenously administered EVs, along with the components responsible for these features are discussed. An overview of the potential clinical applications and preclinical examples of their successful use is also provided. Finally, examples of EV modifications that have successfully been employed to improve their therapeutic characteristics receive a particular focus. We suggest that, in addition to investigation of EV cell targeting and routes of uptake, future research into the routes of intracellular trafficking in recipient cells is required to optimally utilize EVs for therapeutic purposes., Therapeutics: Helping vesicles to deliver drugs inside cells An increased understanding of how extracellular vesicles (EVs) enter cells and deliver molecules will enable promising new therapies, according to researchers in the Netherlands, UK and France. EVs are liquid-filled sacs secreted by cells that transport proteins, lipids and RNA between cells, and therefore have potential for delivering drugs. Pieter Vader at UMC Utrecht and co-workers review recent research into EVs, focusing on how EVs are distributed around the body, and how they target and enter cells. However, there is little known about EV biology once they are inside cells, and it is likely that many EVs simply degrade without delivering their cargo. Further research in this area could help identify features that improve cargo escape from EVs, thus ensuring that future therapies can be effective.

Tissue-engineered grafts for cardiovascular structures experience biochemical stimuli and mechanical forces that influence tissue development after implantation such as the immunological response, oxidative stress, hemodynamic shear stress, and mechanical strain. Endothelial cells are a cell source of major interest in vascular tissue engineering because of their ability to form a luminal antithrombotic monolayer. In addition, through their ability to undergo endothelial to mesenchymal transition (EndMT), endothelial cells may yield a cell type capable of increased production and remodeling of the extracellular matrix (ECM). ECM is of major importance to the mechanical function of all cardiovascular structures. Tissue engineering approaches may employ EndMT to recapitulate, in part, the embryonic development of cardiovascular structures. Improved understanding of how the environment of an implanted graft could influence EndMT in endothelial cells may lead to novel tissue engineering strategies. This review presents an overview of biochemical and mechanical stimuli capable of influencing EndMT, discusses the influence of these stimuli as found in the direct environment of cardiovascular grafts, and discusses approaches to employ EndMT in tissue-engineered constructs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules, including proteins and RNA, between cells. As a result of their suggested natural capability to functionally deliver RNA, EVs may be harnessed as therapeutic RNA carriers. One major limitation for their translation to therapeutic use is the lack of an efficient, robust, and scalable method to load EVs with RNA molecules of interest. Here, we evaluated and optimized methods to load EVs with cholesterol-conjugated small interfering RNAs (cc-siRNAs) by systematic evaluation of the influence of key parameters, including incubation time, volume, temperature, and EV:cc-siRNA ratio. EV loading under conditions that resulted in the highest siRNA retention percentage, incubating 15 molecules of cc-siRNA per EV at 37°C for 1 hr in 100 μL, facilitated concentration-dependent silencing of human antigen R (HuR), a therapeutic target in cancer, in EV-treated cells. These results may accelerate the development of EV-based therapeutics.

Exosomes are cell-derived extracellular vesicles of 30-150 nm in size and are involved in intercellular communication. Because of their bioactive cargo, consisting of proteins, RNA and lipids, and their natural ability to deliver these biomolecules to recipient cells, exosomes are increasingly being studied as novel drug delivery vehicles or as cell-free approaches to regenerative medicine. However, one of the major hurdles for clinical translation of therapeutic strategies based on exosomes is their low yield when produced under standard culture conditions. Exosomes are vesicles of endocytic origin and are released when multivesicular endosomes fuse with the plasma membrane. Here, we demonstrate that interfering with endolysosomal trafficking significantly increases exosome release. Furthermore, these exosomes retain their regenerative bioactivity as demonstrated by pro-survival and angiogenesis assays using both cardiomyocytes and endothelial cells. These results may be employed to increase exosome production for studying biological functions or to improve clinical translation of exosome-based therapeutics.

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.

Background Endothelial colony forming cells (ECFCs) have shown a promise in tissue engineering of vascular constructs, where they act as endothelial progenitor cells. After implantation, ECFCs are likely to be subjected to elevated reactive oxygen species (ROS). The transcription factor Nrf2 regulates the expression of antioxidant enzymes in response to ROS. Methods Stable knockdown of Nrf2 and Keap1 was achieved by transduction with lentiviral shRNAs; activation of Nrf2 was induced by incubation with sulforaphane (SFN). Expression of Nrf2 target genes was assessed by qPCR, oxidative stress was assessed using CM-DCFDA, and angiogenesis was quantified by scratch-wound and tubule-formation assays Results. Nrf2 knockdown led to a reduction of antioxidant gene expression and increased ROS. Angiogenesis was disturbed after Nrf2 knockdown even in the absence of ROS. Conversely, angiogenesis was preserved in high ROS conditions after knockdown of Keap1. Preincubation of ECFCs with SFN reduced intracellular ROS in the presence of H2O2 and preserved scratch-wound closure and tubule-formation. Results Nrf2 knockdown led to a reduction of antioxidant gene expression and increased ROS. Angiogenesis was disturbed after Nrf2 knockdown even in the absence of ROS. Conversely, angiogenesis was preserved in high ROS conditions after knockdown of Keap1. Preincubation of ECFCs with SFN reduced intracellular ROS in the presence of H2O2 and preserved scratch-wound closure and tubule-formation. Conclusion The results of this study indicate that Nrf2 plays an important role in the angiogenic capacity of ECFCs, particularly under conditions of increased oxidative stress. Pretreatment of ECFCs with SFN prior to implantation may be a protective strategy for tissue-engineered constructs or cell therapies.

Background: The healthy vascular endothelium, which forms the barrier between blood and the surrounding tissues, is known to efficiently respond to stress signals like hypoxia and inflammation by adaptation of cellular physiology and the secretion of (soluble) growth factors and cytokines. Exosomes are potent mediators of intercellular communication. Their content consists of RNA and proteins from the cell of origin, and thus depends on the condition of these cells at the time of exosome biogenesis. It has been suggested that 20 exosomes protect their target cells from cellular stress through the transfer of RNA and proteins. We hypothesized that endothelium-derived exosomes are involved in the endothelial response to cellular stress, and that exosome RNA and protein content reflect the effects of cellular stress induced by hypoxia, inflammation or hyperglycemia. Methods: We exposed cultured endothelial cells to different types of cellular stress (hypoxia, TNF-a-induced 25 activation, high glucose and mannose concentrations) and compared mRNA and protein content of exosomes produced by these cells by microarray analysis and a quantitative proteomics approach. Results: We identified 1,354 proteins and 1,992 mRNAs in endothelial cell-derived exosomes. Several proteins and mRNAs showed altered abundances after exposure of their producing cells to cellular stress, which were confirmed by immunoblot or qPCR analysis. 30 Conclusion: Our data show that hypoxia and endothelial activation are reflected in RNA and protein exosome composition, and that exposure to high sugar concentrations alters exosome protein composition only to a minor extend, and does not affect exosome RNA composition. Keywords: extracellular vesicles; exosomes; RNA; proteomics; hypoxia; tumor necrosis factor alpha. (Published: 16 April 2012) Citation: Journal of Extracellular Vesicles 2012, 1 : 18396 - DOI: 10.3402/jev.v1i0.18396 To access the supplementary material to this article: Tables SI-SIV and Figures S1-2, please see Supplementary files in the column to the right (under Article Tools)

Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome‐ECM interactions is limited. Here, we investigate whether the exosome‐associated lysyl oxidase family member lysyl oxidase‐like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)‐derived exosomes, placing it in direct vicinity of the ECM. It is up‐regulated twofold in EC‐derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome‐producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC‐derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia‐regulated focal ECM remodelling, a key process in both fibrosis and wound healing.

PD-1 plays an important role in T cell exhaustion during HIV infection. PD-1 has two ligands: PD-L1, expressed on hematopoietic and nonhematopoietic cells, and PD-L2, limited to DCs and macrophages. Little is known about PD-L1 expression and regulation in human macrophages. Previous reports have found few immediate effects of macrophage exposure to HIV, suggesting that macrophages lack PRRs for this virus. Using quantitative confocal microscopy and a multiplexed cytokine bead array, we measured induction of PD-L1, PD-L2, and innate response cytokines in human MDMs in response to chemically inactivated HIV virions. Consistent with previous reports, no cytokines were induced by HIV virion exposure. Whereas PD-L1 and PD-L2 had low baseline expression, TLR ligands (LPS and CL097) up-regulated PD-L1 but not PD-L2. Unlike what we found for cytokine expression, PD-L1 and PD-L2 were up-regulated in response to exposure with inactivated HIV virions or with replication-competent HIV. Expression of PD-L1 was differentially modulated by IL-10, which induced up-regulation of PD-L1 but not of PD-L2, and IL-10 blockade enhanced only PD-L2 expression. We discuss implications for innate recognition of HIV by macrophages and potential, different roles for PD-L1 and PD-L2 in immunity and pathogenesis.

Background: Patients with Critical Limb Ischemia (CLI) have a high risk of amputations and mortality. We hypothesize that inflammation is involved in atherosclerotic disease progression. In this study we investigate whether levels of plasma cytokines are associated with disease progression in CLI. Methods: Data were collected from a randomized controlled trial cohort investigating cell therapy for CLI (the JUVENTAS study) from 2006 to 2012. The primary outcome measures were major amputation and mortality at 6 months; secondary outcomes included Ankle/Brachial Index (ABI) and Transcutaneous O2 Pressure. Plasma was collected at inclusion in the study and a panel of cytokines consisting of GROa, HGF, LIF, SCF, SCGFb, SDF1a, TRAIL, IL-6, IL-8, FGFb, GCSF, GMCSF, IP10, MCP1, PDGFbb, RANTES, TNFa and VEGF was measured and evaluated for predictive power. Results: Data on 108 patients was collected with a follow-up of 6 months. Patients who underwent a major outcome had significantly higher levels of IL-6, IL-8 and GROa (p=0.0004, 0.006 and 0.009 resp.). Univariate Kaplan-Meier analysis showed that amputation-free survival was 94.4% in the lowest tertile, 74% in the middle tertile and 64% in the highest tertile of plasma IL-6 levels (p=0.009, Fig A). Adjustment for potential confounders in multivariate Cox proportional hazards models showed that IL-6 remained independent predictor of major outcome. The ROC of a model using Il-6 as single predictor for major outcomes is 0.73 (Fig. B) Conclusion: Plasma levels of inflammatory cytokines, in particular IL-6 are associated with disease progression in CLI and can serve as an independent predictor of amputation-free survival.

Extracellular vesicles (EV) consist of exosomes, which are released upon fusion of the multivesicular body with the cell membrane, and microvesicles, which are released directly from the cell membrane. EV can mediate cell-cell communication and are involved in many processes, including immune signaling, angiogenesis, stress response, senescence, proliferation, and cell differentiation. The vast amount of processes that EV are involved in and the versatility of manner in which they can influence the behavior of recipient cells make EV an interesting source for both therapeutic and diagnostic applications. Successes in the fields of tumor biology and immunology sparked the exploration of the potential of EV in the field of regenerative medicine. Indeed, EV are involved in restoring tissue and organ damage, and may partially explain the paracrine effects observed in stem cell-based therapeutic approaches. The function and content of EV may also harbor information that can be used in tissue engineering, in which paracrine signaling is employed to modulate cell recruitment, differentiation, and proliferation. In this review, we discuss the function and role of EV in regenerative medicine and elaborate on potential applications in tissue engineering.

Signaling between endothelial cells, endothelial progenitor cells, and stromal cells is crucial for the establishment and maintenance of vascular integrity and involves exosomes, among other signaling pathways. Exosomes are important mediators of intercellular communication in immune signaling, tumor survival, stress responses, and angiogenesis. The ability of exosomes to incorporate and transfer messenger RNAs (mRNAs) encoding for “acquired” proteins or micro RNAs (miRNAs) repressing “resident” mRNA translation suggests that they can influence the physiological behavior of recipient cells. We demonstrate that miR-214, an miRNA that controls endothelial cell function and angiogenesis, plays a dominant role in exosome-mediated signaling between endothelial cells. Endothelial cell–derived exosomes stimulated migration and angiogenesis in recipient cells, whereas exosomes from miR-214–depleted endothelial cells failed to stimulate these processes. Exosomes containing miR-214 repressed the expression of ataxia telangiectasia mutated in recipient cells, thereby preventing senescence and allowing blood vessel formation. Concordantly, specific reduction of miR-214 content in exosome-producing endothelial cells abolishes the angiogenesis stimulatory function of the resulting exosomes. Collectively, our data indicate that endothelial cells release miR-214–containing exosomes to stimulate angiogenesis through the silencing of ataxia telangiectasia mutated in neighboring target cells.