Extracellular Vesicles Mediate Anti-Oxidative Response—In Vitro Study in the Ocular Drainage System

The importance of extracellular vesicles (EVs) as signaling mediators has been emphasized for several pathways with only limited data regarding their role as protective messages during oxidative stress (OS). The ocular drainage system is unique by being continuously exposed to OS and having a one-way flow of the aqueous humor carrying EVs taking role in glaucoma disease. Here, we aimed to examine the ability of EVs derived from the non-pigmented ciliary epithelium (NPCE)—the aqueous humor producing cells exposed to OS—to deliver protecting messages to the trabecular meshwork (TM)—the aqueous humor draining cells—a process with significance to the pathophysiology of glaucoma disease. EVs extracted from media of NPCE cells exposed to non-lethal OS and their unstressed control were incubated with TM cells. The effects of EVs derived from oxidative stressed cells on the activation of the nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1 (Nrf2-Keap1), a major OS pathway, and of the Wnt pathway, known for its role in primary open-angle glaucoma, were evaluated. EVs derived from oxidized NPCE cells significantly protected TM cells from direct OS. The TM cells uptake of EVs from oxidized NPCE and their cytosolic Nrf2 levels were significantly higher at 8 h post-exposure. EVs derived from oxidized NPCE cells significantly attenuated Wnt protein expression in TM cells and activated major antioxidant genes as measured by qRT-PCR. TM cells exposed to EVs derived from oxidized NPCE cells exhibited significantly lower OS and higher super oxide dismutase and catalase activity. Finally, we were able to show that carbonylated proteins and products of oxidized protein are presented in significantly higher levels in EVs derived from oxidized NPCE cells, supporting their suggested role in the signaling process. We hypothesize that these findings may have implications beyond understanding the pathophysiology of glaucoma disease and that transmitting signals that activate the antioxidant system in target cells represent a broad response common to many tissues communication.

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Non-Pigmented Ciliary Epithelium-Derived Extracellular Vesicles Loaded with SMAD7 siRNA Attenuate Wnt Signaling in Trabecular Meshwork Cells In Vitro

Pharmaceuticals ◽

10.3390/ph14090858 ◽

2021 ◽

Vol 14 (9) ◽

pp. 858

Author(s):

Saray Tabak ◽

Valeria Feinshtein ◽

Sofia Schreiber-Avissar ◽

Elie Beit-Yannai

Keyword(s):

Extracellular Vesicles ◽

Trabecular Meshwork ◽

Structural Integrity ◽

Open Angle Glaucoma ◽

Drainage System ◽

Ciliary Epithelium ◽

Trabecular Meshwork Cells ◽

Sirna Molecule ◽

Potential Use

Primary open-angle glaucoma is established by the disruption of trabecular meshwork (TM) function. The disruption leads to increased resistance to the aqueous humor (AH), generated by the non-pigmented ciliary epithelium (NPCE). Extracellular vesicles (EVs) participate in the communication between the NPCE and the TM tissue in the ocular drainage system. The potential use of NPCE-derived EVs to deliver siRNA to TM cells has scarcely been explored. NPCE-derived EVs were isolated and loaded with anti-fibrotic (SMAD7) siRNA. EV’s structural integrity and siRNA loading efficiency were estimated via electron microscopy and fluorescence. Engineered EVs were added to pre-cultured TM cells and qRT-PCR was used to verify the transfer of selected siRNA to the cells. Western blot analysis was used to evaluate the qualitative effects on Wnt-TGFβ2 proteins’ expression. EVs loaded with exogenous siRNA achieved a 53% mRNA knockdown of SMAD7 in TM cells, resulting in a significant elevation in the levels of β-Catenin, pGSK3β, N-Cadherin, K-Cadherin, and TGFβ2 proteins in TM cells. NPCE-derived EVs can be used for efficient siRNA molecule delivery into TM cells, which may prove to be beneficial as a therapeutic target to lower intraocular pressure (IOP).

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Properties of a New Food Supplement Containing Actinia equina Extract

Antioxidants ◽

10.3390/antiox9100945 ◽

2020 ◽

Vol 9 (10) ◽

pp. 945

Author(s):

Marika Lanza ◽

Giovanna Casili ◽

Giovanna Loredana La Torre ◽

Daniele Giuffrida ◽

Archimede Rotondo ◽

...

Keyword(s):

In Vitro Study ◽

Neutrophil Infiltration ◽

Food Supplement ◽

Biologically Active ◽

In Vivo Model ◽

Related Factor ◽

Nuclear Factor ◽

Anti Inflammatory

Marine species represent a great source of biologically active substances; Actinia equina (AE), an Anthozoa Cnidaria belonging to the Actinidiae family, have been proposed as original food and have already been included in several cooking recipes in local Mediterranean shores, and endowed with excellent nutraceutical potential. The aim of this study was to investigate some unexplored features of AE, through analytical screening and an in-vitro and in-vivo model. An in-vitro study, made on RAW 264.7 stimulated with H2O2, showed that the pre-treatment with AE exerted an antioxidant action, reducing lipid peroxidation and up-regulating antioxidant enzymes. On the other hand, the in-vivo study over murine model demonstrated that the administration of AE extracts is able to reduce the carrageenan (CAR)-induced paw edema. Furthermore, the histological damage due to the neutrophil infiltration is prevented, and this highlights precious anti-inflammatory features of the interesting food-stuff. Moreover, it was assessed that AE extract modulated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and The nuclear factor erythroid 2–related factor 2 (Nrf-2) pathways. In conclusion, our data demonstrated that thanks to the antioxidant and anti-inflammatory properties, AE extract could be used as a new food supplement for inflammatory pathology prevention.

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Very Long-Chain C24:1 Ceramide Is Increased in Serum Extracellular Vesicles with Aging and Can Induce Senescence in Bone-Derived Mesenchymal Stem Cells

Cells ◽

10.3390/cells8010037 ◽

2019 ◽

Vol 8 (1) ◽

pp. 37 ◽

Cited By ~ 21

Author(s):

Andrew Khayrullin ◽

Priyanka Krishnan ◽

Luis Martinez-Nater ◽

Bharati Mendhe ◽

Sadanand Fulzele ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Extracellular Vesicles ◽

Cardiovascular Health ◽

Rhesus Macaques ◽

Cell Communication ◽

Size Exclusion ◽

Target Cells ◽

Serum Samples

Extracellular vesicles (EVs), including exosomes and microvesicles, function in cell-to-cell communication through delivery of proteins, lipids and microRNAs to target cells via endocytosis and membrane fusion. These vesicles are enriched in ceramide, a sphingolipid associated with the promotion of cell senescence and apoptosis. We investigated the ceramide profile of serum exosomes from young (24–40 yrs.) and older (75–90 yrs.) women and young (6–10 yrs.) and older (25–30 yrs.) rhesus macaques to define the role of circulating ceramides in the aging process. EVs were isolated using size-exclusion chromatography. Proteomic analysis was used to validate known exosome markers from Exocarta and nanoparticle tracking analysis used to characterize particle size and concentration. Specific ceramide species were identified with lipidomic analysis. Results show a significant increase in the average amount of C24:1 ceramide in EVs from older women (15.4 pmol/sample) compared to those from younger women (3.8 pmol/sample). Results were similar in non-human primate serum samples with increased amounts of C24:1 ceramide (9.3 pmol/sample) in older monkeys compared to the younger monkeys (1.8 pmol/sample). In vitro studies showed that primary bone-derived mesenchymal stem cells (BMSCs) readily endocytose serum EVs, and serum EVs loaded with C24:1 ceramide can induce BMSC senescence. Elevated ceramide levels have been associated with poor cardiovascular health and memory impairment in older adults. Our data suggest that circulating EVs carrying C24:1 ceramide may contribute directly to cell non-autonomous aging.

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MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

International Journal of Molecular Sciences ◽

10.3390/ijms21238888 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8888

Author(s):

Bárbara Melo-Baez ◽

Yat S. Wong ◽

Constanza J. Aguilera ◽

Joel Cabezas ◽

Ana C. F. Mançanares ◽

...

Keyword(s):

Extracellular Vesicles ◽

Fatty Acid Biosynthesis ◽

Culture Media ◽

Blastocyst Stage ◽

Developmental Competence ◽

Target Cells ◽

Bovine Embryos ◽

Developmental Potential ◽

Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

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Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing

Stem Cells International ◽

10.1155/2020/8889379 ◽

2020 ◽

Vol 2020 ◽

pp. 1-18

Author(s):

Cinzia Maria Chinnici ◽

Giandomenico Amico ◽

Alessia Gallo ◽

Gioacchin Iannolo ◽

Nicola Cuscino ◽

...

Keyword(s):

Wound Healing ◽

Signaling Pathways ◽

Extracellular Vesicles ◽

Target Genes ◽

Fold Increase ◽

Target Cells ◽

Published Data ◽

Dermal Cell ◽

Thrombospondin 1

The use of cell secreted factors in clinical settings could be an alternative to conventional cell therapy, with the advantage of limiting concerns generally associated with traditional cell transplantation, such as tumorigenicity, immunoreactivity, and carrying of infections. Based on our published data, we predict a potential role for extracellular vesicles (EVs) in contributing to the proangiogenic activity of human fetal dermal cell secretome. Depletion of nanosized EVs from secretome significantly impaired its ability to induce formation of mesh-like structures in vitro. The isolated EVs were characterized for size and concentration by nanoparticle tracking analysis, and for protein markers (Rab5+, Alix+, CD63+, and calnexin-). The microRNA profile of EVs revealed 87 microRNAs significantly upregulated (≥15-fold increase) in fetal compared to adult dermal cell-derived EVs. Interestingly, these upregulated microRNAs included microRNAs with a validated role in angiogenesis according to literature. Moreover, the DIANA-TarBase v7.0 analysis confirmed enrichment in the KEGG signaling pathways associated with angiogenesis and wound healing, with the identification of putative target genes including thrombospondin 1. To validate the in silico data, EVs were also characterized for total protein contents. When tested in in vitro angiogenesis, fetal dermal cell-derived EVs were more effective than their adult counterpart in inducing formation of complete mesh-like structures. Furthermore, treatment of fibroblasts with fetal dermal-derived EVs determined a 4-fold increase of thrombospondin 1 protein amounts compared with the untreated fibroblasts. Finally, visualization of CSFE-labeled EVs in the cytosol of target cells suggested a successful uptake of these particles at 4-8 hours of incubation. We conclude that EVs are important contributors of the proangiogenic effect of fetal dermal cell secretome. Hence, EVs could also serve as vehicle for a successful delivery of microRNAs or other molecules of therapeutic interest to target cells.

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VP5: Plasma Derived Extracellular Vesicles as a Treatment Option in Nerve Regeneration- An In-Vitro Study

Plastic & Reconstructive Surgery Global Open ◽

10.1097/01.gox.0000817700.12755.26 ◽

2022 ◽

Vol 10 (1S) ◽

pp. 8-8

Author(s):

Anton Borger ◽

Paul Supper ◽

Maximilian Härtinger ◽

Flavia Millesi ◽

Lorenz Supper ◽

...

Keyword(s):

Nerve Regeneration ◽

Extracellular Vesicles ◽

Treatment Option ◽

In Vitro Study ◽

Vitro Study

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Mesenchymal Stem Cell-Derived Extracellular Vesicles and Their Therapeutic Potential

Stem Cells International ◽

10.1155/2020/8825771 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Ashley G. Zhao ◽

Kiran Shah ◽

Brett Cromer ◽

Huseyin Sumer

Keyword(s):

Tissue Engineering ◽

Regenerative Medicine ◽

Extracellular Vesicles ◽

Therapeutic Potential ◽

Target Cells ◽

Therapeutic Effects ◽

Multipotent Cells ◽

Membrane Bound

Extracellular vesicles (EVs) are cell-derived membrane-bound nanoparticles, which act as shuttles, delivering a range of biomolecules to diverse target cells. They play an important role in maintenance of biophysiological homeostasis and cellular, physiological, and pathological processes. EVs have significant diagnostic and therapeutic potentials and have been studied both in vitro and in vivo in many fields. Mesenchymal stem cells (MSCs) are multipotent cells with many therapeutic applications and have also gained much attention as prolific producers of EVs. MSC-derived EVs are being explored as a therapeutic alternative to MSCs since they may have similar therapeutic effects but are cell-free. They have applications in regenerative medicine and tissue engineering and, most importantly, confer several advantages over cells such as lower immunogenicity, capacity to cross biological barriers, and less safety concerns. In this review, we introduce the biogenesis of EVs, including exosomes and microvesicles. We then turn more specifically to investigations of MSC-derived EVs. We highlight the great therapeutic potential of MSC-derived EVs and applications in regenerative medicine and tissue engineering.

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Hydrangenol Isolated from the Leaves of Hydrangea serrata Attenuates Wrinkle Formation and Repairs Skin Moisture in UVB-Irradiated Hairless Mice

Nutrients ◽

10.3390/nu11102354 ◽

2019 ◽

Vol 11 (10) ◽

pp. 2354 ◽

Cited By ~ 3

Author(s):

Da-Bin Myung ◽

Hee-Soo Han ◽

Ji-Sun Shin ◽

Ji Yeon Park ◽

Han Jun Hwang ◽

...

Keyword(s):

Heme Oxygenase 1 ◽

Skin Thickness ◽

Related Factor ◽

Activator Protein 1 ◽

Hairless Mice ◽

Glutamate Cysteine Ligase ◽

Antioxidant Genes ◽

Activation Of Transcription ◽

Mitogen Activated Protein

Our previous study showed that hydrangenol isolated from Hydrangea serrata leaves exerts antiphotoaging activity in vitro. In this study, we determined its antiphotoaging effect in UVB-irradiated HR-1 hairless mice. We evaluated wrinkle formation, skin thickness, histological characteristics, and mRNA and protein expression using qRT-PCR and Western blot analysis in dorsal skins. Hydrangenol mitigated wrinkle formation, dorsal thickness, dehydration, and collagen degradation. Hydrangenol increased the expression of involucrin, filaggrin, and aquaporin-3 (AQP3) as well as hyaluronic acid (HA) production via hyaluronidase (HYAL)-1/-2 downregulation. Consistent with the recovery of collagen composition, the expression of Pro-COL1A1 was increased by hydrangenol. Matrix metalloproteinase (MMP)-1/-3, cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) expression was reduced by hydrangenol. Hydrangenol attenuated the phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK and p38, activator protein 1 (AP-1) subunit, and signal transduction and activation of transcription 1 (STAT1). Hydrangenol upregulated the expression of nuclear factor-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO-1), glutamate cysteine ligase modifier subunit (GCLM), and glutamate cysteine ligase catalysis subunit (GCLC). Taken together, our data suggest that hydrangenol can prevent wrinkle formation by reducing MMP and inflammatory cytokine levels and increasing the expression of moisturizing factors and antioxidant genes.

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The Triad Hsp60-miRNAs-Extracellular Vesicles in Brain Tumors: Assessing Its Components for Understanding Tumorigenesis and Monitoring Patients

Applied Sciences ◽

10.3390/app11062867 ◽

2021 ◽

Vol 11 (6) ◽

pp. 2867

Author(s):

Francesca Graziano ◽

Domenico Gerardo Iacopino ◽

Giacomo Cammarata ◽

Gianluca Scalia ◽

Claudia Campanella ◽

...

Keyword(s):

Brain Tumors ◽

Extracellular Vesicles ◽

Current Knowledge ◽

Disease Status ◽

Experimental Models ◽

Host Cells ◽

Target Cells ◽

Original Cell

Brain tumors have a poor prognosis and progress must be made for developing efficacious treatments, but for this to occur their biology and interaction with the host must be elucidated beyond current knowledge. What has been learned from other tumors may be applied to study brain tumors, for example, the role of Hsp60, miRNAs, and extracellular vesicles (EVs) in the mechanisms of cell proliferation and dissemination, and resistance to immune attack and anticancer drugs. It has been established that Hsp60 increases in cancer cells, in which it occurs not only in the mitochondria but also in the cytosol and plasma-cell membrane and it is released in EVs into the extracellular space and in circulation. There is evidence suggesting that these EVs interact with cells near and far from their original cell and that this interaction has an impact on the functions of the target cell. It is assumed that this crosstalk between cancer and host cells favors carcinogenesis in various ways. We, therefore, propose to study the triad Hsp60-related miRNAs-EVs in brain tumors and have standardized methods for the purpose. These revealed that EVs with Hsp60 and related miRNAs increase in patients’ blood in a manner that reflects disease status. The means are now available to monitor brain tumor patients by measuring the triad and to dissect its effects on target cells in vitro, and in experimental models in vivo.

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Human hepatocyte-derived extracellular vesicles attenuate the carbon tetrachloride-induced acute liver injury in mice

Cell Death and Disease ◽

10.1038/s41419-021-04204-7 ◽

2021 ◽

Vol 12 (11) ◽

Author(s):

Masatoshi Kakizaki ◽

Yuichiro Yamamoto ◽

Shunya Nakayama ◽

Kazuaki Kameda ◽

Etsuko Nagashima ◽

...

Keyword(s):

Liver Transplantation ◽

Liver Injury ◽

Immune Responses ◽

Extracellular Vesicles ◽

Acute Liver Injury ◽

Human Hepatocytes ◽

Target Cells ◽

Surgical Costs ◽

Chemokine Ligand

AbstractAcute liver injury (ALI) induced by chemicals or viruses can progress rapidly to acute liver failure (ALF), often resulting in death of patients without liver transplantation. Since liver transplantation is limited due to a paucity of donors, expensive surgical costs, and severe immune rejection, novel therapies are required to treat liver injury. Extracellular vesicles (EVs) are used for cellular communication, carrying RNAs, proteins, and lipids and delivering them intercellularly after being endocytosed by target cells. Recently, it was reported that EVs secreted from human hepatocytes have an ability to modulate the immune responses; however, these roles of EVs secreted from human hepatocytes were studied only with in vitro experiments. In the present study, we evidenced that EVs secreted from human hepatocytes attenuated the CCL4-induced ALI by inhibiting the recruitment of monocytes through downregulation of chemokine receptor in the bone marrow and recruitment of neutrophils through the reduction of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2 expression levels in the liver.

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