Endocytosis blocks the vesicular secretion of exosome marker proteins.

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.

Syntenin and CD63 Promote Exosome Biogenesis from the Plasma Membrane by Blocking Cargo Endocytosis.

Exosomes are small extracellular vesicles important in health and disease. Syntenin is thought to drive the biogenesis of CD63 exosomes by recruiting Alix and the ESCRT machinery to endosomes, initiating an endosome-mediated pathway of exosome biogenesis. Contrary to this model, we show here that syntenin drives the biogenesis of CD63 exosomes by blocking CD63 endocytosis, thereby allowing CD63 to accumulate at the plasma membrane, the primary site of exosome biogenesis. Consistent with these results, we find that inhibitors of endocytosis induce the exosomal secretion of CD63, that endocytosis inhibits the vesicular secretion of exosome cargo proteins, and that high-level expression of CD63 itself also inhibits endocytosis. These and other results indicate that exosomes bud primarily from the plasma membrane, that endocytosis inhibits their loading into exosomes, that syntenin and CD63 are expression-dependent regulators of exosome biogenesis, and that syntenin drives the biogenesis of CD63 exosomes even in Alix knockout cells.

Extracellular vesicles in Alzheimer's disease: from pathology to therapeutic approaches.

Alzheimer's disease is a progressive and fatal neurodegenerative disorder that starts many years before the onset of cognitive symptoms. Identifying novel biomarkers for Alzheimer's disease has the potential for patient risk stratification, early diagnosis, and disease monitoring in response to therapy. A novel class of biomarkers is extracellular vesicles given their sensitivity and specificity to specific diseases. In addition, extracellular vesicles can be used as novel biological therapeutics given their ability to efficiently and functionally deliver therapeutic cargo. This is critical given the huge unmet need for novel treatment strategies for Alzheimer's disease. This review summarizes and discusses the most recent findings in this field.

Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina.

Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with therapeutic molecules and engineered for retention by specific tissues. However, questions remain on optimal dosing, administration, and pharmacokinetics. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available for larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had longer circulation times in plasma than previously reported in mice and were detectable in cerebrospinal fluid (CSF) after 30-60 minutes. EV association with PBMCs, especially B-cells, was observed as early as one minute post-administration. EVs were detected in liver and spleen within one hour of IV administration. However, IN delivery was minimal, suggesting that pretreatment approaches may be needed in large animals. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling.

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.

Human adipose tissue-derived mesenchymal stem cells and their extracellular vesicles modulate lipopolysaccharide activated human microglia.

Neurodegenerative diseases (NDs), such as Alzheimer's disease (AD), are driven by neuroinflammation triggered by activated microglial cells; hence, the phenotypic regulation of these cells is an appealing target for intervention. Human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) may be a potential therapeutic candidate to treat NDs given their immunomodulatory properties. Evidence suggests that the mechanism of action of hAD-MSCs is through their secretome, which includes secreted factors such as cytokines, chemokines, or growth factors as well as extracellular vesicles (EVs). Recently, EVs have emerged as important mediators in cell communication given, they can transfer proteins, lipids, and RNA species (i.e., miRNA, mRNA, and tRNAs) to modulate recipient cells. However, the therapeutic potential of hAD-MSCs and their secreted EVs has not been fully elucidated with respect to human microglia. In this study, we determined the therapeutic potential of different hAD-MSCs doses (200,000, 100,000, and 50,000 cells) or their secreted EVs (50, 20, or 10 µg/ml), on human microglial cells (HMC3) that were activated by lipopolysaccharides (LPS). Upregulation of inducible nitric oxide synthase (iNOS), an activation marker of HMC3 cells, was prevented when they were cocultured with hAD-MSCs and EVs. Moreover, hAD-MSCs inhibited the secretion of proinflammatory factors, such as IL-6, IL-8, and MCP-1, while their secreted EVs promoted the expression of anti-inflammatory mediators such as IL-10 or TIMP-1 in activated microglia. The present data therefore support a role for hAD-MSCs and their secreted EVs, as potential therapeutic candidates for the treatment of NDs.

Mesenchymal stem cell-derived extracellular vesicles reduce senescence and extend health span in mouse models of aging.

Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow-derived mesenchymal stem cells (BM-MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM-MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM-MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM-MSC CM extended life span of Ercc1-/- mice similarly to injection of young BM-MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC-derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.

Cell type-selective secretome profiling in vivo.

Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Extracellular vesicle-mediated amyloid transfer to neural progenitor cells: implications for RAGE and HIV infection.

Amyloid beta (Aß) deposition was demonstrated to be elevated in the brains of HIV-infected patients and associated with neurocognitive decline; however, the mechanisms of these processes are poorly understood. The goal of the current study was to address the hypothesis that Aß can be transferred via extracellular vesicles (ECVs) from brain endothelial cells to neural progenitor cells (NPCs) and that this process can contribute to abnormal NPC differentiation. Mechanistically, we focused on the role of the receptor for advanced glycation end products (RAGE) and activation of the inflammasome in these events. ECVs loaded with Aß (Aß-ECVs) were readily taken up by NPCs and Aß partly colocalized with the inflammasome markers ASC and NLRP3 in the nuclei of the recipient NPCs. This colocalization was affected by HIV and RAGE inhibition by a high-affinity specific inhibitor FPS-ZM1. Blocking RAGE resulted also in an increase in ECV number produced by brain endothelial cells, decreased Aß content in ECVs, and diminished Aß-ECVs transfer to NPC nuclei. Interestingly, both Aß-ECVs and RAGE inhibition altered NPC differentiation. Overall, these data indicate that RAGE inhibition affects brain endothelial ECV release and Aß-ECVs transfer to NPCs. These events may modulate ECV-mediated amyloid pathology in the HIV-infected brain and contribute to the development of HIV-associated neurocognitive disorders.

Inflammasome Proteins in Serum and Serum-Derived Extracellular Vesicles as Biomarkers of Stroke.

The inflammasome is a key contributor to the inflammatory innate immune response after stroke. We have previously shown that inflammasome proteins are released in extracellular vesicles (EV) after brain and spinal cord injury. In addition, we have shown that inflammasome proteins offer great promise as biomarkers of central nervous system (CNS) injury following brain trauma. In the present study, we used a Simple Plex Assay (Protein Simple), a novel multi-analyte automated microfluidic immunoassay platform, to analyze serum and serum-derived EV samples from stroke patients and control subjects for inflammasome protein levels of caspase-1, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), Interleukins (IL)-1ß, and (IL)-18. Receiver operator characteristic (ROC) curves with associated confidence intervals obtained from the analysis of serum samples revealed that the area under the curve (AUC) for ASC was 0.99 with a confidence interval between 0.9914 and 1.004, whereas the AUC for caspase-1, IL-1ß, and IL-18 were 0.75, 0.61, and 0.67, respectively. Thus, these data indicate that ASC is a potential biomarker of stroke and highlight the role of the inflammasome in the inflammatory response after brain ischemia.

Dual mechanism of type VII collagen transfer by bone marrow mesenchymal stem cell extracellular vesicles to recessive dystrophic epidermolysis bullosa fibroblasts.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe blistering disease resulting from a lack of type VII collagen production. Recent clinical trials have shown efficacy of bone marrow-derived mesenchymal stem cells (BM-MSCs) in the treatment of epidermolysis bullosa, including improved basement membrane restructuring and cutaneous wound healing. The mechanism as to how type VII collagen is transferred from donor stem cell to recipient RDEB cells has not been defined. Here, we submit the model that BM-MSC-derived extracellular vesicles serve at least two roles: 1) to help transport type VII collagen within the extracellular space; and 2) to feed RDEB fibroblasts with messenger RNA that codes for type VII collagen, resulting in COL7A1 translation and synthesis of type VII collagen alpha chain proteins by RDEB fibroblasts. Utilizing a chemoselective ligation detection method, we found RDEB cells that were treated simultaneously with BM-MSC EVs and an l-methionine analog, l-homopropargylglycine (HPG), synthesized collagen VII alpha chain protein that contained the alkyne group of HPG to react (i.e. undergo the Click-iT® reaction) with azide-modified Alexa 594, suggesting de novo synthesis of type VII collagen by RDEB fibroblasts. Thus, our results support a model in which BM-MSC EVs help increase type VII collagen levels available to recipient cells by 1) donating BM-MSC type VII collagen protein and 2) inducing RDEB fibroblasts to make their own type VII collagen protein. These findings allow us to hypothesize that the secretome of BM-MSCs could have therapeutic value in the treatment of RDEB-related skin disorders.

Comprehensive Metabolomics Study To Assess Longitudinal Biochemical Changes and Potential Early Biomarkers in Nonobese Diabetic Mice That Progress to Diabetes.

A global nontargeted longitudinal metabolomics study was carried out in male and female NOD mice to characterize the time-profile of the changes in the metabolic signature caused by onset of type 1 diabetes (T1D) and identify possible early biomarkers in T1D progressors. Metabolomics profiling of samples collected at five different time-points identified 676 and 706 biochemicals in blood and feces, respectively. Several metabolites were expressed at significantly different levels in progressors at all time-points, and their proportion increased strongly following onset of hyperglycemia. At the last time-point, when all progressors were diabetic, a large percentage of metabolites had significantly different levels: 57.8% in blood and 27.8% in feces. Metabolic pathways most strongly affected included the carbohydrate, lipid, branched-chain amino acid, and oxidative ones. Several biochemicals showed considerable (>4×) change. Maltose, 3-hydroxybutyric acid, and kojibiose increased, while 1,5-anhydroglucitol decreased more than 10-fold. At the earliest time-point (6-week), differences between the metabolic signatures of progressors and nonprogressors were relatively modest. Nevertheless, several compounds had significantly different levels and show promise as possible early T1D biomarkers. They include fatty acid phosphocholine derivatives from the phosphatidylcholine subpathway (elevated in both blood and feces) as well as serotonin, ribose, and arabinose (increased) in blood plus 13-HODE, tocopherol (increased), diaminopimelate, valerate, hydroxymethylpyrimidine, and dulcitol (decreased) in feces. A combined metabolic signature based on these compounds might serve as an early predictor of T1D-progressors.

Plasma-derived exosome characterization reveals a distinct microRNA signature in long duration Type 1 diabetes.

Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing ß cells which leads to chronic hyperglycemia. Exosomes are lipid vesicles derived from cellular multivesicular bodies that are enriched in specific miRNAs, potentially providing a disease-specific diagnostic signature. To assess the value of exosome miRNAs as biomarkers for T1DM, miRNA expression in plasma-derived exosomes was measured. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of plasma-derived exosomes (EXOs) isolated by differential centrifugation. Total RNA extracted from plasma-derived EXOs of 12 T1DM and 12 control subjects was hybridized onto Nanostring human v2 miRNA microarray array and expression data were analyzed on nSolver analysis software. We found 7 different miRNAs (1 up-regulated and 6 down-regulated), that were differentially expressed in T1DM. The selected candidate miRNAs were validated by qRT-PCR analysis of cohorts of 24 T1DM and 24 control subjects. Most of the deregulated miRNAs are involved in progression of T1DM. These findings highlight the potential of EXOs miRNA profiling in the diagnosis as well as new insights into the molecular mechanisms involved in T1DM.

Bone Marrow Mesenchymal Stem Cell-Derived CD63+ Exosomes Transport Wnt3a Exteriorly and Enhance Dermal Fibroblast Proliferation, Migration, and Angiogenesis In Vitro.

Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space. To help answer this question, we utilized flow cytometry of magnetic beads coated with anti-CD63 antibodies and found, for the first time, that Wnt3a protein is detectable exteriorly on CD63+ exosomes derived from BM-MSCs over-secreting Wnt3a into serum-free conditioned media (Wnt3a CM). Our data suggest that CD63+ exosomes significantly help transport exterior Wnt3a signal to recipient cells to promote fibroblast and endothelial functions. During purification of exosomes, we unexpectedly found that use of ultracentrifugation alone significantly decreased the ability to detect exteriorly bound Wnt3a on CD63+ exosomes, however, polyethylene glycol (PEG)-mediated exosome-enrichment before exosome-purification (with ultracentrifugation into a sucrose cushion) resulted in exosomes more likely to retain exterior Wnt3a detectability and downstream Wnt/beta-catenin activity. Our findings indicate the important role that purification methods may have on stem cell-derived Wnt-exosome activity in downstream assays. The ability for BM-MSC Wnt3a CM and exosomes to stimulate dermal fibroblast proliferation and migration, and endothelial angiogenesis in vitro, was significantly decreased after CD63+-exosome depletion or knockdown of Wnt coreceptor LRP6 in recipient cells, suggesting both are required for optimal Wnt-exosome activity in our system. Thus, BM-MSC-derived CD63+ exosomes are a significant carrier of exterior Wnt3a within high Wnt environments, resulting in downstream fibroblast proliferation, migration, and angiogenesis in vitro.

Metabolomics Study of the Effects of Inflammation, Hypoxia, and High Glucose on Isolated Human Pancreatic Islets.

The transplantation of human pancreatic islets is a therapeutic possibility for a subset of type 1 diabetic patients who experience severe hypoglycemia. Pre- and post-transplantation loss in islet viability and function, however, is a major efficacy-limiting impediment. To investigate the effects of inflammation and hypoxia, the main obstacles hampering the survival and function of isolated, cultured, and transplanted islets, we conducted a comprehensive metabolomics evaluation of human islets in parallel with dynamic glucose-stimulated insulin release (GSIR) perifusion studies for functional evaluation. Metabolomics profiling of media and cell samples identified a total of 241 and 361 biochemicals, respectively. Metabolites that were altered in highly significant manner in both included, for example, kynurenine, kynurenate, citrulline, and mannitol/sorbitol under inflammation (all elevated) plus lactate (elevated) and N-formylmethionine (depressed) for hypoxia. Dynamic GSIR experiments, which capture both first- and second-phase insulin release, found severely depressed insulin-secretion under hypoxia, whereas elevated baseline and stimulated insulin-secretion was measured for islet exposed to the inflammatory cytokine cocktail (IL-1ß, IFN-γ, and TNF-α). Because of the uniquely large changes observed in kynurenine and kynurenate, they might serve as potential biomarkers of islet inflammation, and indoleamine-2,3-dioxygenase on the corresponding pathway could be a worthwhile therapeutic target to dampen inflammatory effects.

Diet and Inflammation: Possible Effects on Immunity, Chronic Diseases, and Life Span.

Chronic inflammation negatively impacts all physiological functions, causing an array of degenerative conditions including diabetes; cancer; cardiovascular, osteo-articular, and neurodegenerative diseases; autoimmunity disorders; and aging. In particular, there is a growing knowledge of the role that gene transcription factors play in the inflammatory process. Obesity, metabolic syndrome, and diabetes represent multifactorial conditions resulting from improper balances of hormones and gene expression. In addition, these conditions have a strong inflammatory component that can potentially be impacted by the diet. It can reduce pro-inflammatory eicosanoids that can alter hormonal signaling cascades to the modulation of the innate immune system and gene transcription factors. Working knowledge of the impact of how nutrients, especially dietary fatty acids and polyphenols, can impact these various molecular targets makes it possible to develop a general outline of an anti-inflammatory diet that offers a unique, nonpharmacological approach in treating obesity, metabolic syndrome, and diabetes. Several important bioactive dietary components can exert their effect through selected inflammatory pathways that can affect metabolic and genetic changes. In fact, dietary components that can modulate glucose and insulin levels, as well as any other mediator that can activate nuclear factor-kB, can also trigger inflammation through common pathway master switches.

Hierarchical paracrine interaction of breast cancer associated fibroblasts with cancer cells via hMAPK-microRNAs to drive ER-negative breast cancer phenotype.

Multiple juxtacrine and paracrine interactions occur between cancer cells and non-cancer cells of the tumor microenvironment (TME) that direct tumor progression. Cancer Associated Fibroblasts (CAFs) are an integral component of the TME, and the majority of breast tumor stroma is comprised of CAFs. Heterotypic interactions between cancer cells and non-cancer cells of the TME occur via soluble agents, including cytokines, hormones, growth factors, and secreted microRNAs. We previously identified a microRNA signature indicative of hyperactive MAPK signaling (hMAPK-miRNA signature) that significantly associated with reduced recurrence-free and overall survival. Here we report that the hMAPK-miRNA signature associates with a high metric of stromal cell infiltrate, and we investigate the role of microRNAs, particularly hMAPK-microRNAs, secreted by CAFs on estrogen receptor (ER) expression in breast cancer cells. ER-positive MCF-7/ltE2- cells were treated with conditioned media (CM) from CAFs derived from breast cancers of different PAM50 subtypes (CAFBAS, CAFHER2, and CAFLA). CAF CM isolated specifically from ER-negative primary breast tumors led to ER repression in vitro. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of CAF-secreted exosomes in CM and the uptake of these exosomes by the ER+ MCF-7/ltE2- cells. Differentially expressed microRNAs in CAF CM as well as in MCF-7/ltE2- cells treated with this CM were identified. Knockdown of miR-221/222 in CAFBAS resulted in knockdown of miR221/222 levels in the conditioned media and the CM from CAFBAS; miR221/222 knockdown rescued ER repression in ER-positive cell lines treated with CAFBAS-CM. Collectively, our results demonstrate that CAF-secreted microRNAs are directly involved in ER-repression, and may contribute to the MAPK-induced ER repression in breast cancer cells.

Therapeutic potential of human adipose-derived stem cells (ADSCs) from cancer patients: a pilot study.

Mesenchymal stem cells from adipose tissue (ADSCs) are an important source of cells for regenerative medicine. The therapeutic effect of culture-expanded adipose derived stem cells has been shown; however, optimal xeno-free culture conditions remain to be determined. Cancer patients, specifically those undergoing invasive surgery, constitute a subgroup of patients who could benefit from autologous stem cell transplantation. Although regenerative potential of their ADSCs could be affected by the disease and/or treatment, we are not aware of any study that has evaluated the therapeutic potential of ADSCs isolated from cancer patients in reference to that of ADSCs derived from healthy subjects. Here we report that ADSCs isolated from subabdominal adipose tissue of patients with urological neoplasms yielded similar growth kinetics, presented equivalent mesenchymal surface markers and showed similar differentiation potential into distinct mesodermal cell lineages: adipocytes, chondroblasts and osteoblasts than ADSCs isolated from adipose tissue of age-matched non-oncogenic participants, all under xeno-free growth culture conditions. Molecular karyotyping of patient expanded ADSCs genomes showed no disease-related alterations indicating their safety. In addition, vesicles <100 nm identified as exosomes (EXOs) which may be at least partly responsible for the attributed therapeutic paracrine effects of the ADSCs were effectively isolated from ADSCs and showed equivalent miRNA content regardless they were derived from cancer patients or non-oncogenic participants indicating that the repair capabilities of xeno-free expanded ADSCs are not compromised by patient condition and therefore their xeno-free culture expanded ADSCs should be suitable for autologous stem cell transplantation in a clinical setting.