Exosomes Extracellular Vesicles Research Articles

MicroRNA Expression in Exosome Extracellular Vesicles as Targets for Personalized Medicine in Diffuse Large B Cell Lymphoma Patients with HIV Infection.

Extracellular Vesicles (EVs), more specifically exosomes (xEVs), have been associated with Diffuse Large B-cell Lymphoma (DLBCL). These xEVs contain a variety of biomolecules, such as proteins and nucleic acids (e.g., microRNA, LncRNA, and DNA). The expressions of these vesicles in the setting of Human Immunodeficiency Virus (HIV) have been linked to disease progression. Studies have explored the use of EVs in more practical clinical settings. Several studies have found that biomolecules within xEVs can serve to detect disease progression. The biomolecule content within xEVs is useful in prognostication and has even been associated with mechanisms of resistance for some DLBCL treatment modalities. This review article explores the role of xEV biomolecule content in DLBCL progression in the context of HIV infection and its applied use in practical disease management.

Systematic compositional analysis of exosomal extracellular vesicles produced by cells undergoing apoptosis, necroptosis and ferroptosis.

Formation of extracellular vesicles (EVs) has emerged as a novel paradigm in cell-to-cell communication in health and disease. EVs are notably produced during cell death but it had remained unclear whether different modalities of regulated cell death (RCD) influence the biogenesis and composition of EVs. To this end, we performed a comparative analysis of steady-state (ssEVs) and cell death-associated EVs (cdEVs) following TNF-induced necroptosis (necEVs), anti-Fas-induced apoptosis (apoEVs), and ML162-induced ferroptosis (ferEVs) using the same cell line. For each RCD condition, we determined the biophysical and biochemical characteristics of the cell death-associated EVs (cdEVs), the protein cargo, and the presence of methylated ribosomal RNA. We found that the global protein content of all cdEVs was increased compared to steady-state EVs. Qualitatively, the isolated exosomal ssEVs and cdEVs, contained a largely overlapping protein cargo including some quantitative differences in particular proteins. All cdEVs were enriched for proteins involved in RNA splicing and nuclear export, and showed distinctive rRNA methylation patterns compared to ssEVs. Interestingly, necEVs and apoEVs, but strikingly not ferEVs, showed enrichment of proteins involved in ribosome biogenesis. Altogether, our work documents quantitative and qualitative differences between ssEVs and cdEVs.

Composition and Possibility of Application in Practical Medicine of Exosom/Extracellular Vesicles from Multipotent Stromal Cells

The therapeutic effect of multipotent stem cells (MSCs) is largely mediated by the secretion of exosomes/extracellular vesicles (EMSCs), which reflect the biophysical characteristics of MSC-producers and are considered more effective. The use of EMSCs can help overcome practical and ethical issues that limit cell therapy. The importance of EMSCs is also recognized because of their ability to transfer various proteins, DNA and RNA to target cells and change the behavior of them and neighboring cells. EMSCs contribute to cellular processes such as transcription, proliferation, adhesion, migration and differentiation. EMSCs are involved in the induction of angiogenesis, inhibition of fibrosis, stimulation of extracellular matrix remodeling, abolition of the local inflammatory response, and also in the regulation of immune cell activity. A deeper understanding of the content of EMSC and its dynamics may affect the study and treatment of various diseases. However, EMSCs, even obtained from MSCs of the same origin and cultivated under the same conditions, can differ significantly in their components and, accordingly, in efficiency. Modification, including genetic modification, of the initial cellular elements is of certain importance for purposeful and, therefore, predictable changes in the content of EMSCs, but this approach also does not solve the problem of sufficient standardization of their components. Perhaps more promising is the artificial creation of exosome-like structures with a predetermined composition and, as a result, an accurate and predictable effect.

What do we actually know about exosomal microRNAs in kidney diseases?

There are several types of kidney diseases with complex causes. If left untreated, these diseases irreversibly progress to end-stage renal disease. Thus, their early diagnosis and targeted treatment are important. Exosomes—extracellular vesicles released by a variety of cells—are ideal carriers for DNA, RNA, proteins, and other metabolites owing to their bilayer membranes. Studies have shown that almost all renal cells can secrete exosomes. While research on exosomal microRNAs in the context of renal diseases begun only recently, rapid progress has been achieved. This review summarizes the changes in exosomal microRNA expression in different kidney diseases. Thus, it highlights the diagnostic and prognostic value of these exosomal microRNAs. Further, this review analyzes their roles in the development of different kidney diseases, guiding research on molecular mechanisms and therapeutic strategies.

COVID-19 therapy with mesenchymal stromal cells (MSC) and convalescent plasma must consider exosome involvement: Do the exosomes in convalescent plasma antagonize the weak immune antibodies?

Exosome extracellular vesicles as biologic therapy for COVID‐19 are discussed for two areas. The first involves the growing use of mesenchymal stromal cells (MSCs) for the profound clinical cytokine storm and severe pneumonia in COVID‐19 patients. Instead, it is recommended to treat alternatively with their MSC‐released exosomes. This is because many reports in the literature and our data have shown that the release of exosomes from the in vivo administered MSC is actually responsible for their beneficial effects. Further, the exosomes are superior, simpler and clinically more convenient compared to their parental MSC. Additionally, in the context of COVID‐19, the known tendency of MSC to intravascularly aggregate causing lung dysfunction might synergize with the pneumonia aspects, and the tendency of MSC peripheral vascular micro aggregates might synergize with the vascular clots of the COVID‐19 disease process, causing significant central or peripheral vascular insufficiency. The second exosome therapeutic area for severe COVID‐19 involves use of convalescent plasma for its content of acquired immune antibodies that must consider the role in this therapy of contained nearly trillions of exosomes. Many of these derive from activated immune modulating cells and likely can function to transfer miRNAs that acting epigenetically to also influence the convalescent plasma recipient response to the virus. There is sufficient evidence, like recovery of patients with antibody deficiencies, to postulate that the antibodies actually have little effect and that immune resistance is principally due to T cell mechanisms. Further, COVID‐19 convalescent plasma has remarkably weak beneficial effects if compared to what was expected from many prior studies. This may be due to the dysfunctional immune response to the infection and resulting weak Ab that may be impaired further by antagonistic exosomes in the convalescent plasma. At the least, pre selection of plasma for the best antibodies and relevant exosomes would produce the most optimum therapy for very severely affected COVID‐19 patients.

N-Terminal Fatty Acids of NEFMUT Are Required for the CD8+ T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

We recently described a cytotoxic CD8+ T lymphocyte (CTL) vaccine platform based on the intramuscular (i.m.) injection of DNA eukaryotic vectors expressing antigens of interest fused at the C-terminus of HIV-1 Nefmut, i.e., a functionally defective mutant that is incorporated at quite high levels into exosomes/extracellular vesicles (EVs). This system has been proven to elicit strong CTL immunity against a plethora of both viral and tumor antigens, as well as inhibit both transplantable and orthotopic tumors in mice. However, a number of open issues remain regarding the underlying mechanism. Here we provide evidence that hindering the uploading into EVs of Nefmut-derived products by removing the Nefmut N-terminal fatty acids leads to a dramatic reduction of the downstream antigen-specific CD8+ T-cell activation after i.m. injection of DNA vectors in mice. This result formally demonstrates that the generation of engineered EVs is part of the mechanism underlying the in vivo induced CD8+ T-cell immunogenicity. Gaining new insights on the EV-based vaccine platform can be relevant in view of its possible translation into the clinic to counteract both chronic and acute infections as well as tumors.

Exosome Extracellular Vesicles: A Vehicle for Simultaneous Immune and Genetic Therapy

Exosome Extracellular Vesicles: A Vehicle for Simultaneous Immune and Genetic Therapy

Exosome Extracellular Vesicles: A Vehicle for Simultaneous Immune and Genetic Therapy

Exosome Extracellular Vesicles: A Vehicle for Simultaneous Immune and Genetic Therapy

Magnetic Nanoparticle and Exosomal Therapeutic (M-NEXT) Effects on HIV-Associated Neurotoxicity.

The human immunodeficiency virus (HIV) envelope glycoprotein protein 120 (gp120) induces neurotoxicity associated with HIV-associated neurocognitive disorders (HAND). Mechanism of Gp120-mediated neurotoxicity is primarily apoptosis. Currently, there are no therapeutics that address gp120 neurotoxicity. A biocompatible, efficacious therapeutic that easily crosses the blood-brain barrier (BBB) is needed to treat neuronal toxicity observed in HIV-infected individuals. Magnetic nanoparticles (MNPs) have successfully delivered anti-HIV agents across in vitro BBB transwell model. However, MNPs at high doses may damage cells. Exosomal extracellular vesicles (xEVs) are endogenous nanocarriers capable of crossing the BBB. Unlike MNPs, xEVs interact with cells in a paracrine or juxtracrine manner, lacking long-range site specificity. Here we investigated the efficacy of an MNP and xEV-coupled therapeutic (M-NEXT) as a nanocarrier for targeted delivery of anti-HIV fusion agent across the BBB to inhibit HIV-gp120 associated neuropathology. M-NEXT consisting of MNPs encapsulated within xEV carrying T20 peptide on the surface was synthesized and characterized via zeta potential, dynamic light scattering, and TEM imaging. Preliminary efficacy studies using SH-SY5Y cocultured with the in vitro BBB model showed that the M-NEXT-T20-fusion peptide protected neurons from HIV gp120-mediated neurotoxicity. Additionally, BBB integrity and permeability assessed via trans-endothelial resistance (TEER) and a Dextran-FITC transport assay was unaffected. SH-SY5Y viability measured by XTT assay was not significantly modulated by M-NEXT. In summary, preliminary findings support M-NEXT as effective nanocarriers for delivery of anti-HIV gp120 associated neurotoxicity agents.

Bioengineered Exosomal Extracellular Vesicles in Cancer Therapeutics.

Liquid or blood-based biopsy is a less invasive and more efficient method in which to clinicians can identify diagnostic, prognostic, and therapeutic responsive biomarkers in cancer patients. Circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), RNAs, proteins, metabolites, and extracellular vesicles (EVs) are all potential biomarkers found in liquid biopsies. All nucleated cells including healthy, virally infected, and cancer cells release EVs. Since the early 1980s, evidence has mounted to support the pathophysiological role of EVs in cancer. Here we focus on the smallest of the EV, the exosome, and their clinical relevance as nanotherapeutics for cancers. Exosomes obtained from tumors have been reported to promote and/or facilitate malignancy of cancers especially in terms of metastatic potential. Exosomal EVs have also contributed to the development of therapeutic resistance. Recent studies demonstrate that intrinsic and bioengineered exosomes can serve as effective therapeutic agents that disrupt cancer progression. Here we review the current literature regarding the utilization of bioengineered exosomes for therapeutics to treat prevalent cancers such as melanoma, glioma, breast, pancreatic, hepatic, cervical, prostate, and colon cancers. Overall, studies reviewed show that bioengineered exosomes are effective and promising for targeted cancer therapy.

Premenopausal Purified Exosome Products from Women Protect against Male Dominant Cardiomyopathies Myocarditis and DCM

Introduction An estimated 3.1 cases of myocarditis were diagnosed in 2017 worldwide. Patients with myocarditis are at risk of sudden death from acute heart failure and may progress to dilated cardiomyopathy (DCM) and chronic heart failure, often requiring a heart transplant. Currently, no disease-specific therapies exist to reduce myocarditis or prevent progression to DCM. Hypothesis Exosomes extracellular vesicles released from stem cells or other cells like platelets are thought to be able to confer cardioprotection. Because more men develop myocarditis (3.5:1 men to women) and DCM we hypothesized that purified exosome product (PEP) from premenopausal women (pmPEP) could improve and/or prevent myocarditis and prevent progression to dilated cardiomyopathy (DCM) using a preclinical mouse model of myocarditis/DCM. Methods We administered PEP (from men and women of all ages), pmPEP (premenopausal women) or control ip to male BALB/c mice at day -1, 0 and +1 and injected virus on day 0 and harvested mice at day 10 post infection (pi) during peak myocarditis or day 35pi during DCM. Next we treated male BALB/c mice with PEP, pmPEP, or control ip on day 7, 8 and 9 pi (a clinically relevant timepoint) and harvested on day 10pi during acute myocarditis or day 35pi during DCM. Results pmPEP, but not PEP, significantly decreased acute myocarditis based on histology (ANOVA p=0.0009) and globally decreased immune cells (CD45 p=0.008, F4/80 p=0.009, CD11b p=0.04, etc.) but had no effect on viral replication in the heart. We found that pmPEP reduced myocardial inflammation by increasing complement receptor (CR)1 (p=0.004)- a master regulator of inflammation. Both PEP and pmPEP treatment given during myocarditis- a clinically relevant timepoint- significantly reduced myocardial inflammation compared to control (ANOVA PEP p=0.005, pmPEP p=0.005). Both PEP and pmPEP globally downregulated inflammation during myocarditis (PEP CD45 p=0.03, pmPEP CD45 p=0.04, and other immune markers); however, pmPEP was more effective than PEP at reducing inflammation. Likewise, PEP and pmPEP given during myocarditis also decreased cardiac inflammation and fibrosis at day 35pi during DCM (PEP p=0.02, pmPEP p=0.04). Interestingly, PEP administration during myocarditis decreased dilatation assessed using echocardiography, while pmPEP decreased cardiac remodeling genes during DCM. Conclusion These findings suggest that PEP regenerative medicine therapies could successfully reduce myocardial inflammation associated with acute onset myocarditis and reduce DCM. Premenopausal PEP therapies from women may be more effective at reducing cardiomyopathy in men.

2192-P: Extracellular Vesicles as Novel Mediators of Proinflammatory Cytokine-Induced ß-Cell Dysfunction

The presence of a proinflammatory islet microenviroment and consequent dysregulation of glucose-stimulated insulin secretion (GSIS) is a hallmark feature of both type 1 and type 2 diabetes. Recent evidence has implicated islet cell-derived extracellular vesicles (EVs) as novel mediators of cytokine-derived inflammatory stress in diabetes, however the mechanisms governing this process remain largely unknown. Therefore, we set out to test the hypothesis that cytokine-mediated β-cell dysfunction is mediated in part through β-cell autocrine release of proinflammatory EVs which promote inflammation and inhibit GSIS. Particle size analysis of EVs from conditioned media of cytokine treated (IL-1β, TNF-α, and IFNγ) Min6 β-cells (CytoEV) showed an overall increase in β-cell EV secretion (∼2 fold increase, P<0.05) and a decrease in the average size of secreted EVs (P<0.05) implicating a shift towards a more exosomal EV profile. Subsequently, functional assessment of isolated mouse islets treated (48h) with CytoEVs resulted in a significant suppression of GSIS (∼80%, P<0.05 vs. control). Moreover, acute exposure to CytoEVs also recapitulated the transcriptional signature of β-cell failure in diabetes characterized by increased expression of key proinflammatory genes (e.g., Cxcl10, Tlr1, Tlr4), and a decrease in Insulin and Pdx-1 expression (∼40%, P<0.05 for CytoEVs vs. control). Finally, proteomics analysis of CytoEVs from Min6 and INS-1 832/13 lines (compared to control EVs from each line) revealed a distinct proinflammatory profile consisting of proteins implicated in type 1 diabetes and dendritic cell maturation pathways (e.g., HLA-A, STAT1, INS, CPE etc., P<0.05). Taken together, this work provides a novel mechanism of β-cell dysfunction in diabetes mediated by the autocrine exchange of proinflammatory EVs which has implications for the detection and treatment of β-cell failure in diabetes. Disclosure N. Javeed: None. T.K. Her: None. P.M. Vanderboom: None. I.R. Lanza: None. A. Matveyenko: None.

Abstract 34: Human Cardiac Mesenchymal Cell Derived Extracellular Vesicles Promote Angiogenesis Through Angiopoietin-TIE2 Signaling

Cell-based therapy is considered a promising approach to treat the damaged heart due to myocardial infarction. Although the mechanisms for their beneficial action are not yet clear, exosome/extracellular vesicles (EVs) secreted by these cells may be involved in their reparative paracrine signaling. Previous studies have suggested that EVs isolated from several cell types (e.g. cardiosphere-derived cells, embryonic stem cell, CD34+ stem cells) induce angiogenic activity both in vitro and in vivo . Here, we investigated whether EVs secreted by adult human cardiac mesenchymal cells (hCMCs) exhibit pro-angiogenic activity, and if so, what signaling molecules are involved in this process. hCMCs were isolated from right atrial appendage of patients undergoing cardiac procedures and were characterized by the expression of classical mesenchymal markers- CD29 (99.1%), CD73 (99.0%), CD90 (20.4%), CD105 (99.3%), CD 31 (16.8%), CD34 (0.9%) and CD45 (0.1%). EVs isolated from serum-free 24-hour hCMC conditioned media using PEG4000-based precipitation technique exhibited two distinct population of particles with size range of 10-60nm and 100-500nm in diameter; expressed characteristic exosomal markers- CD63, HSP70, Flotillin-1 and were negative for cellular organelle markers- calreticulin (ER and apoptotic bodies), prohibitin (mitochondria), GM130 (Golgi), Lamin B (nuclear protein), β-actin (cytoskeleton) and PMP70 (peroxisomes) as determined by immunoblotting. In vitro assays revealed that hCMC EVs promote human umbilical cord endothelial cells (HUVECs) proliferation, transwell migration in Boyden chamber and tube formation on Matrigel, indicative of enhanced angiogenesis. Angiogenic proteomic array identified that angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) proteins are highly enriched in EVs secreted by hCMCs. Furthermore, hCMC EV mediated HUVEC migration and tube formation was inhibited by TIE2 kinase inhibitor. Overall, these findings suggest that ANG-1 and ANG-2 are the key component of hCMC secreted EVs and they promote angiogenesis by activating TIE2 receptor in endothelial cells.