Codelivery of CPT and siPHB1 with GSH/ROS Dual-Responsive Hybrid Nanoparticles Based on a [12]aneN3-Derived Lipid for Synergistic Lung Cancer Therapy.

The combination of small-interfering RNA (siRNA)-mediated gene silencing and chemotherapeutic agents for lung cancer treatment has attracted widespread attention in terms of a greater therapeutic effect, minimization of systemic toxicity, and inhibition of multiple drug resistance (MDR). In this work, three amphiphiles, CBN1-CBN3, were first designed and synthesized as a camptothecin (CPT) conjugate and gene condensation agents by the combination of CPT prodrugs and di(triazole-[12]aneN3) through the ROS-responsive phenylborate ester and different lengths of alkyl chains (with 6, 9, 12 carbon chains for CBN1-CBN3, respectively). CBN1-CBN3 were able to be self-assembled into liposomes with an average diameter in the range of 320-240 nm, showing the ability to effectively condense siRNA. Among them, CBN2, with a nine-carbon alkyl chain, displayed the best anticancer efficiency in A549 cells. In order to give nanomedicines a stealth property and PEGylation/dePEGylation transition, a GSH-responsive PEGylated TPE derivative containing a disulfide linkage (TSP) was further designed and prepared. A combination of CBN2/siRNA complexes and DOPE with TSP resulted in GSH/ROS dual-responsive lipid-polymer hybrid nanoparticles (CBN2-DP/siRNA NPs). In present GSH and H2O2, CBN2-DP/siRNA NPs were decomposed, resulting in the controlled release of CPT drug and siRNA. In vitro, CBN2-DP/siPHB1 NPs showed the best anticancer activity for suppression of about 75% of A549 cell proliferation in a serum medium. The stability of CBN2-DP/siRNA NPs was significantly prolonged in blood circulation, and they showed effective accumulation in the A549 tumor site through an enhanced permeability and retention (EPR) effect. In vivo, CBN2-DP/siPHB1 NPs demonstrated enhanced synergistic cancer therapy efficacy and tumor inhibition as high as 71.2%. This work provided a strategy for preparing lipid-polymer hybrid NPs with GSH/ROS dual-responsive properties and an intriguing method for lung cancer therapy.

[12]aneN3-modified camptothecin and PEGylated AIEgens co-assembly into core-shell nanoparticles with ROS/NTR dual-response for enhanced cancer therapy.

A novel dual-responsive nanoparticle (NP) system was aimed to be developed for the co-delivery of camptothecin (CPT) and plasmid encoding TNF-related apoptosis-inducing ligand (pTRAIL) DNA in cancer therapy. The combination of the prodrug CPT and the nucleic acid condensing di-(triazole-[12]aneN3) unit with 4-nitrobenzyl ester through alkyl chains resulted in three nitroreductase (NTR) responsive amphiphiles, CNN1-CNN3 (with 5, 8, and 11 carbon chains, respectively). Among them, CNN2 was the most effective in inhibiting the proliferation of HeLa cells in the presence of fusogenic lipid DOPE. The NPs composed of CNN2, pDNA, and DOPE were further co-assembled with ROS-responsive thioketal-linked amphiphilic polymer (TTP) to afford the core-shell NPs (CNN2-DT/pDNA) with an average size of 118 nm, which exhibited high drug-loading capacity, excellent serum tolerance, and good biocompatibility. In the presence of ROS, NTR, and NADH, the core-shell NPs were decomposed, leading to the efficient release of 80% CPT and abundant pDNA. The self-assembly and delivery process of CNN2-DT NPs and DNA were clearly observed through the AIE fluorescent imaging. In vitro and in vivo results demonstrated that the CNN2-DT/pTRAIL NPs synergistically promoted 68% apoptosis of tumor cells and inhibited tumor growth with negligible toxic side effects. This study showed that the combination of prodrug and nucleic acid through dual-responsive core-shell NPs provide a spatially and temporally-controlled strategy for cancer therapy.

Extracellular Vesicle-Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart.

BACKGROUND: Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. METHODS: We developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. We compared the gene delivery and therapeutic efficacy of EV-AAVs with an equal titer of free AAVs in the presence of NAbs, both in vitro and in vivo. In addition, we investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging. RESULTS: Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAbs, both to human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9-sarcoplasmic reticulum calcium ATPase 2a to infarcted hearts in preimmunized mice significantly improved ejection fraction and fractional shortening compared with AAV9-sarcoplasmic reticulum calcium ATPase 2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Trafficking studies using human induced pluripotent stem cell-derived cells in vitro and mouse hearts in vivo showed significantly higher expression of EV-AAV6/9-delivered genes in cardiomyocytes compared with noncardiomyocytes, even with comparable cellular uptake. Using cellular subfraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. CONCLUSIONS: Together, using 5 different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared with free AAVs in the presence of NAbs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure.

Competition between FLS and DFR regulates the distribution of flavonols and proanthocyanidins in Rubus chingii Hu.

Rubus chingii Hu is a berry plant of the genus Rubus of the Rosaceae family, which has high nutritional and medicinal value and is rich in flavonoids. Flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) compete for the common substrate dihydroflavonols to regulate the metabolic flux of flavonoids. However, the competition between FLS and DFR based on enzyme is rarely reported. Here, we isolated and identified two FLS genes (RcFLS1 and RcFLS2) and one DFR gene (RcDFR) from Rubus chingii Hu. RcFLSs and RcDFR were highly expressed in stems, leaves, and flowers, although the flavonol accumulation in these organs was significantly higher than that of proanthocyanidins (PAs). The recombinant RcFLSs demonstrated bifunctional activities via hydroxylation and desaturation at the C-3α position having a lower Michaelis constant (Km) for dihydroflavonols than RcDFR. We also found that a low concentration of flavonols could significantly inhibit RcDFR activity. To investigate the competitive relationship between RcFLSs and RcDFR, we used a prokaryotic expression system (E. coli) to co-express these proteins. The transgenic cells expressing recombinant proteins were incubated with substrates, and the reaction products were analyzed. Furthermore, two transient expression systems (tobacco leaves and strawberry fruits) and a stable genetic system (Arabidopsis thaliana) were used to co-express these proteins in vivo. The results showed that RcFLS1 was dominant in the competition with RcDFR. Our results demonstrated that the competition between FLS and DFR regulated the metabolic flux distribution of flavonols and PAs, which will be of great significance for the molecular breeding of Rubus plants.

Esterase-Responsive Polymeric Micelles Containing Tetraphenylethene and Poly(ethylene glycol) Moieties for Efficient Doxorubicin Delivery and Tumor Therapy.

Enzyme-responsive drug delivery systems have drawn much attention in the field of cancer theranostics due to their high sensitivity and substrate specificity under mild conditions. In this study, an amphiphilic polymer T1 is reported, which contains a tetraphenylethene unit and a poly(ethylene glycol) chain linked by an esterase-responsive phenolic ester bond. In aqueous solution, T1 formed stable micelles via self-assembly, which showed an aggregation-induced emission enhancement of 32-fold at 532 nm and a critical micelle concentration of 0.53 µM as well as esterase-responsive activity. The hydrophobic drug doxorubicin (DOX) was efficiently encapsulated into the micelles with a drug loading of 21%. In the presence of the esterase, the selective decomposition of drug-loaded T1 micelles was observed, and DOX was subsequently released with a half-life of 5 h. In vitro antitumor studies showed that T1@DOX micelles exhibited good therapeutic effects on HeLa cells, while normal cells remained mostly intact. In vivo anticancer experiments revealed that T1@DOX micelles indeed suppressed tumor growth and had reduced side effects compared to DOX·HCl. The present work showed the potential clinical application of esterase-responsive drug delivery in cancer therapy.

New Therapeutics for Extracellular Vesicles: Delivering CRISPR for Cancer Treatment.

Cancers are defined by genetic defects, which underlines the prospect of using gene therapy in patient care. During the past decade, CRISPR technology has rapidly evolved into a powerful gene editing tool with high fidelity and precision. However, one of the impediments slowing down the clinical translation of CRISPR-based gene therapy concerns the lack of ideal delivery vectors. Extracellular vesicles (EVs) are nano-sized membrane sacs naturally released from nearly all types of cells. Although EVs are secreted for bio-information conveyance among cells or tissues, they have been recognized as superior vectors for drug or gene delivery. Recently, emerging evidence has spotlighted EVs in CRISPR delivery towards cancer treatment. In this review, we briefly introduce the biology and function of the CRISPR system and follow this with a summary of current delivery methods for CRISPR applications. We emphasize the recent progress in EV-mediated CRISPR editing for various cancer types and target genes. The reported strategies for constructing EV-CRISPR vectors, as well as their limitations, are discussed in detail. The review aims to throw light on the clinical potential of engineered EVs and encourage the expansion of our available toolkit to defeat cancer.

A NIR Aggregation-Induced Emission Fluoroamphiphile as Visually Trackable and Serum-Tolerant Nonviral Gene Carrier.

Functional fluorescence (FL) nonviral gene vectors with high serum tolerance bear broad application prospects in gene delivery. Fluorination has been widely utilized as an effective strategy to enhance serum tolerance. Herein, we show the combination of fluorination and aggregation-induced emission (AIE) for the construction of a nonviral gene vector with low cytotoxicity, visual tracking ability, and high serum tolerance. Large π-conjugation triphenylamine (TPA) derivative with a characteristic D-π-A structure was modified with two polar [12]aneN3 heads and a long fluorocarbon tail, giving the vector molecule FluoTPA. FluoTPA features near-infrared (NIR) emission, large Stokes shift, and strong binding affinity toward nucleic acids. Liposomes consisting of FluoTPA and dioleoylphosphatidylethanolamine (DOPE) (FluoTPA/DOPE) can effectively deliver both plasmid DNAs (pDNAs) and siRNAs into cells. Impressively, FluoTPA/DOPE showed comparable transfection efficiency (TE) in the presence of serum content up to 30% with that in the serum-free condition and achieved 7.4 times higher TE than the commercial transfection agent lipofectamine 2000 at the same condition. Finally, spatiotemporal tracking of the delivery process in cells was demonstrated. The results in this work suggest that FluoTPA could be a reliable theranostic platform for the nonviral delivery of nucleic acid therapeutics in serum condition.

H2O2-Responsive amphiphilic polymer with aggregation-induced emission (AIE) for DOX delivery and tumor therapy.

Stimuli-responsive drug delivery systems (DDSs) based on amphiphilic polymers have attracted much attention. In this study, we reported an innovative H2O2-responsive amphiphilic polymer (TBP), bearing a H2O2-sensitive phenylboronic ester, AIE fluorophore tetraphenylethene (TPE) hydrophobic, and polyethylene glycol hydrophilic (PEG) moieties. TBP could self-assemble into micelles with an encapsulation efficiency as high as 74.9% for doxorubicin (DOX) in aqueous solution. In the presence of H2O2, TBP micelles was decomposed by oxidation, hydrolysis and rearrangement, leading to almost 80% DOX release from TBP@DOX micelles. TBP and the corresponding degradation products were biocompatible, while TBP@DOX micelles only displayed obvious toxicity toward cancer cells. Drug delivery process was clearly monitored by confocal laser scanning microscopic (CLSM) and flow cytometry (FCM) analysis. Moreover, in vivo anticancer study showed that TBP@DOX micelles were accumulated in tumor region of nude mice and effectively inhibited tumor growth. The results suggested that the reported H2O2-responsive amphiphilic polymer displayed great potential in drug delivery and tumor therapy.

AAV-Containing Exosomes as a Novel Vector for Improved Gene Delivery to Lung Cancer Cells.

Lung carcinoma is the most common type of cancer and the leading cause of cancer-related death worldwide. Among the numerous therapeutic strategies for the treatment of lung cancer, adeno-associated virus (AAV)-mediated gene transfer has been demonstrated to have the potential to effectively suppress tumor growth or reverse the progression of the disease in a number of preclinical studies. AAV vector has a safety profile; however, the relatively low delivery efficacy to particular subtypes of lung carcinoma has limited its prospective clinical translation. Exosomes are nanosized extracellular vesicles secreted from nearly all known cell types. Exosomes have a membrane-enclosed structure carrying a range of cargo molecules for efficient intercellular transfer of functional entities, thus are considered as a superior vector for drug delivery. In the present study, we developed a novel strategy to produce and purify AAV-containing exosomes (AAVExo) from AAV-packaging HEK 293T cells. The cellular uptake capacity of exosomes assisted and enhanced AAV entry into cells and protected AAV from antibody neutralization, which was a serious challenge for AAV in vivo application. We tested a list of lung cancer cell lines representing non-small-cell lung cancer and small-cell lung cancer and found that AAVExo apparently improved the gene transfer efficiency compared to conventional AAV vector. Our in vitro results were supported in vivo in a lung cancer xenograft rodent model. Additionally, we evaluated the gene delivery efficiency in the presence of neutralizing antibody on lung cancer cells. The results demonstrated that AAVExo-mediated gene transfer was not impacted, while the AAV vectors were significantly blocked by the neutralizing antibody. Taken together, we established an efficient methodology for AAVExo purification, and the purified AAVExo largely enhanced gene delivery to lung cancer cells with remarkable resistance to antibody neutralization.

Emerging methods in biomarker identification for extracellular vesicle-based liquid biopsy.

Extracellular vesicles (EVs) are released by many cell types and distributed within various biofluids. EVs have a lipid membrane-confined structure that allows for carrying unique molecular information originating from their parent cells. The species and quantity of EV cargo molecules, including nucleic acids, proteins, lipids, and metabolites, may vary largely owing to their parent cell types and the pathophysiologic status. Such heterogeneity in EV populations provides immense challenges to researchers, yet allows for the possibility to prognosticate the pathogenesis of a particular tissue from unique molecular signatures of dispersing EVs within biofluids. However, the inherent nature of EV's small size requires advanced methods for EV purification and evaluation from the complex biofluid. Recently, the interdisciplinary significance of EV research has attracted growing interests, and the EV analytical platforms for their diagnostic prospect have markedly progressed. This review summarizes the recent advances in these EV detection techniques and methods with the intention of translating an EV-based liquid biopsy into clinical practice. This article aims to present an overview of current EV assessment techniques, with a focus on their progress and limitations, as well as an outlook on the clinical translation of an EV-based liquid biopsy that may augment current paradigms for the diagnosis, prognosis, and monitoring the response to therapy in a variety of disease settings.

Two-Photon Near-Infrared AIE Luminogens as Multifunctional Gene Carriers for Cancer Theranostics.

Construction of multifunctional nonviral gene vectors to execute defined tasks holds great potential for the precise and effective treatment of gene-associated diseases. Herein, we have developed four large π-conjugation triphenylamine derivatives bearing two polar [12]aneN3 heads and a lipophilic tail for applications in gene delivery, one/two-photon-triggered near-infrared (NIR) fluorescence bioimaging, and combined photodynamic therapy (PDT) and gene therapy of cancer. These compounds possess typical NIR aggregation-induced emission characteristics, mega Stokes shifts, strong two-photon excitation fluorescence, and excellent DNA condensation abilities. Among them, vector 4 with a tail of n-hexadecane realized a transfection efficiency as high as 6.7 times that of the commercial transfection agent Lipofectamine 2000 in HEK293T cell lines. Using vector 4 as an example, transfection process tracking and ex vivo/in vivo tumoral imaging and retention with high resolution, high brightness, deep tissue penetration, and good biosafety were demonstrated. In addition, efficient singlet oxygen (1O2) generation by the DNA complex formed by vector 4 (4/DNA) resulted in effective PDT. Combined with anticancer gene therapy, collaborative cancer treatment with a dramatically enhanced cancer cell-killing effect was achieved. The development of this "three birds, one stone" approach suggests a new and promising strategy for better cancer treatment and real-time tracking of gene delivery.

Functional equivalence of stem cell and stem cell-derived extracellular vesicle transplantation to repair the irradiated brain.

Cranial radiotherapy, although beneficial for the treatment of brain tumors, inevitably leads to normal tissue damage that can induce unintended neurocognitive complications that are progressive and debilitating. Ionizing radiation exposure has also been shown to compromise the structural integrity of mature neurons throughout the brain, an effect believed to be at least in part responsible for the deterioration of cognitive health. Past work has shown that cranially transplanted human neural stem cells (hNSCs) or their extracellular vesicles (EVs) afforded long-term beneficial effects on many of these cognitive decrements. To provide additional insight into the potential neuroprotective mechanisms of cell-based regenerative strategies, we have analyzed hippocampal neurons for changes in structural integrity and synaptic remodeling after unilateral and bilateral transplantation of hNSCs or EVs derived from those same cells. Interestingly, hNSCs and EVs similarly afforded protection to host neurons, ameliorating the impact of irradiation on dendritic complexity and spine density for neurons present in both the ipsilateral and contralateral hippocampi 1 month following irradiation and transplantation. These morphometric improvements were accompanied by increased levels of glial cell-derived growth factor and significant attenuation of radiation-induced increases in postsynaptic density protein 95 and activated microglia were found ipsi- and contra-lateral to the transplantation sites of the irradiated hippocampus treated with hNSCs or hNSC-derived EVs. These findings document potent far-reaching neuroprotective effects mediated by grafted stem cells or EVs adjacent and distal to the site of transplantation and support their potential as therapeutic agents to counteract the adverse effects of cranial irradiation.

FTO-Dependent N6-Methyladenosine Regulates Cardiac Function During Remodeling and Repair.

BACKGROUND: Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. METHODS: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. RESULTS: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. CONCLUSIONS: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Fetal Bovine Serum-Derived Extracellular Vesicles Persist within Vesicle-Depleted Culture Media.

It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 108 ± 1.39 × 108 EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

miR-146a Suppresses SUMO1 Expression and Induces Cardiac Dysfunction in Maladaptive Hypertrophy.

RATIONALE: Abnormal SUMOylation has emerged as a characteristic of heart failure (HF) pathology. Previously, we found reduced SUMO1 (small ubiquitin-like modifier 1) expression and SERCA2a (sarcoplasmic reticulum Ca2+-ATPase) SUMOylation in human and animal HF models. SUMO1 gene delivery or small molecule activation of SUMOylation restored SERCA2a SUMOylation and cardiac function in HF models. Despite the critical role of SUMO1 in HF, the regulatory mechanisms underlying SUMO1 expression are largely unknown. OBJECTIVE: To examine miR-146a-mediated SUMO1 regulation and its consequent effects on cardiac morphology and function. METHODS AND RESULTS: In this study, miR-146a was identified as a SUMO1-targeting microRNA in the heart. A strong correlation was observed between miR-146a and SUMO1 expression in failing mouse and human hearts. miR-146a was manipulated in cardiomyocytes through AAV9 (adeno-associated virus serotype 9)-mediated gene delivery, and cardiac morphology and function were analyzed by echocardiography and hemodynamics. Overexpression of miR-146a reduced SUMO1 expression, SERCA2a SUMOylation, and cardiac contractility in vitro and in vivo. The effects of miR-146a inhibition on HF pathophysiology were examined by transducing a tough decoy of miR-146a into mice subjected to transverse aortic constriction. miR-146a inhibition improved cardiac contractile function and normalized SUMO1 expression. The regulatory mechanisms of miR-146a upregulation were elucidated by examining the major miR-146a-producing cell types and transfer mechanisms. Notably, transdifferentiation of fibroblasts triggered miR-146a overexpression and secretion through extracellular vesicles, and the extracellular vesicle-associated miR-146a transfer was identified as the causative mechanism of miR-146a upregulation in failing cardiomyocytes. Finally, extracellular vesicles isolated from failing hearts were shown to contain high levels of miR-146a and exerted negative effects on the SUMO1/SERCA2a signaling axis and hence cardiomyocyte contractility. CONCLUSIONS: Taken together, our results show that miR-146a is a novel regulator of the SUMOylation machinery in the heart, which can be targeted for therapeutic intervention.

A novel community driven software for functional enrichment analysis of extracellular vesicles data.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Angiogenic Mechanisms of Human CD34+ Stem Cell Exosomes in the Repair of Ischemic Hindlimb.

RATIONALE: Paracrine secretions seem to mediate therapeutic effects of human CD34+ stem cells locally transplanted in patients with myocardial and critical limb ischemia and in animal models. Earlier, we had discovered that paracrine secretion from human CD34+ cells contains proangiogenic, membrane-bound nanovesicles called exosomes (CD34Exo). OBJECTIVE: Here, we investigated the mechanisms of CD34Exo-mediated ischemic tissue repair and therapeutic angiogenesis by studying their miRNA content and uptake. METHODS AND RESULTS: When injected into mouse ischemic hindlimb tissue, CD34Exo, but not the CD34Exo-depleted conditioned media, mimicked the beneficial activity of their parent cells by improving ischemic limb perfusion, capillary density, motor function, and their amputation. CD34Exo were found to be enriched with proangiogenic miRNAs such as miR-126-3p. Knocking down miR-126-3p from CD34Exo abolished their angiogenic activity and beneficial function both in vitro and in vivo. Interestingly, injection of CD34Exo increased miR-126-3p levels in mouse ischemic limb but did not affect the endogenous synthesis of miR-126-3p, suggesting a direct transfer of stable and functional exosomal miR-126-3p. miR-126-3p enhanced angiogenesis by suppressing the expression of its known target, SPRED1, simultaneously modulating the expression of genes involved in angiogenic pathways such as VEGF (vascular endothelial growth factor), ANG1 (angiopoietin 1), ANG2 (angiopoietin 2), MMP9 (matrix metallopeptidase 9), TSP1 (thrombospondin 1), etc. Interestingly, CD34Exo, when treated to ischemic hindlimbs, were most efficiently internalized by endothelial cells relative to smooth muscle cells and fibroblasts, demonstrating a direct role of stem cell-derived exosomes on mouse endothelium at the cellular level. CONCLUSIONS: Collectively, our results have demonstrated a novel mechanism by which cell-free CD34Exo mediates ischemic tissue repair via beneficial angiogenesis. Exosome-shuttled proangiogenic miRNAs may signify amplification of stem cell function and may explain the angiogenic and therapeutic benefits associated with CD34+ stem cell therapy.