Exosomes Research Articles

Biomimetic small exosome with outstanding surgical applications for rapid large-scale wound healing and functional sweat gland restoration

The realization of coordinated infiltration of dermal and epidermal cell types, and the rapid deposition of extracellular matrix are the prerequisites for improved healing quality of large skin defects. However, presently used materials are limited for wound healing due to the inherent poor histocompatibility, low permeability, poor mechanical property and cytotoxicity. It is challenging to develop new therapeutic agents that enable timely and efficient wound healing and meanwhile there are minimal side effects to avoid chronic wounds, wound adhesions, and large scarring. Herein, we fabricated biomimetic exosomes (EMs) that enriched transforming growth factor β1 (TGF-β1) for the rapid healing of large-scale cutaneous wounds. The EM encapsulation allowed well-managed dosing of the TGF-β1 to effectively promote the wound healing process by endowing epidermal keratinocytes with a migratory feature, enhancing their stem cell properties, and resulting in accelerated in vivo wound re-epithelization. Specifically, the EMs could fast restore the function of sweat glands in a thermally injured mice model. Our work provides a promising strategy for improving the healing speed and healing quality for patients with large wounds.

Abstract 274: Deciphering the role of tumor-released microvesicles in glioblastoma mobility and invasion

Abstract To promote cell growth, invasion and therapy resistance, glioblastoma (GBM) makes use of different communication routes with the neighbor environment which include Extracellular Vesicles (EVs). EVs are a heterogeneous group of cell-released membranous structures, which contain a wide mixture of active molecules. Each cell type secretes a unique combination of different EV subpopulations that vary in size, content and function. In GBM, the subfraction of small-EVs derived from multivesicular bodies, also referred as exosomes (EXOs), have received considerable attention for their capacity to create a tumor-supportive microenvironment through their actions on immune cells, vasculature and glial cells. Only recently it has been observed that large-EVs formed by the budding of the cell membrane, classically called microvesicles (MVs), are more abundant than EXOs in the plasma of GBM patients. Large-MVs have been associated with disease progression in the context of prostate cancer, but their functional significance remain largely uncharacterized in GBM. To explore EV migratory potential in the GBM context, an in vitro migration test performed on spheroids of patient-derived Glioma Stem-like Cells (GSC) has been set up. EXOs from GSC culture supernatants exert no migratory effects, whereas MVs triggered remarkable cell migration. Differently from GSC culture supernatants, both EXOs and MVs isolated from surgical washing exerted a remarkable migratory effect suggesting that not only EVs from tumoral cells are actively implicated in GSC mobility but also EVs from the non-tumoral microenvironment act synergistically to sustain GBM invasion. To better understand the contribution of the cell sub-population of the tumor environment, an EV separation based on CD45(leukocyte common antigen) expression was performed. CD45-positive EVs (released by immune cells) and CD45-negative EVs (released by tumor and stroma cells) were employed in the spheroid migration assay. Results showed the absence of any migratory effect of CD45-positive subsets, both EXOs and MVs, indicating that EVs selectively generated by tumor and stroma cells play a key role in tumoral invasion. Furthermore, a multiplex bead-based flow-cytometry analysis performed on EVs from six GBM patients revealed the presence of specific antigen clusters related to migration and could also be investigated as tumoral biomarkers in a liquid biopsy context. Citation Format: Valentino Ribecco, Matteo Tamborini, Elisabetta Stanzani, Marco Pizzocri, Milena Mattioli, Simone Olei, Maria Pia Tropeano, Federico Pessina, Michela Matteoli, Lorena Passoni. Deciphering the role of tumor-released microvesicles in glioblastoma mobility and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 274.

Exosomes deliver lncRNA DARS-AS1 siRNA to inhibit chronic unpredictable mild stress-induced TNBC metastasis

Triple-negative breast cancer (TNBC) is a rapidly recurring and highly metastatic malignancy with high heterogeneity and chemoradiotherapy resistance. Chronic unpredictable mild stress (CUMS) can induce the occurrence of tumors and enhance lymphatic infiltration and distant metastasis through direct interaction with the sympathetic nervous system; however, its relevance in TNBC is yet to be clarified. In this study, DARS-AS1, a newly reported CUMS-responsive lncRNA, was found to be enriched in TNBC clinical tumors and cells and positively correlated with late clinical stage in patients with TNBC. DARS-AS1 overexpression significantly enhanced the migration and invasion of TNBC tumors by inhibiting miR-129-2-3p and upregulated CDK1 to activate the NF-κB/STAT3 signaling pathway both in vitro and in vivo. Treatment with DARS-AS1 siRNA-loaded exosomes (EXOs) substantially slowed CUMS-induced TNBC cell growth and liver metastasis. Therefore, DARS-AS1 represents a potential therapeutic target for metastatic TNBC, and EXOs may serve as siRNA delivery carriers in clinical therapy.

Plasma-Derived Exosomes in Chronic Spontaneous Urticaria Induce the Production of Mediators by Human Mast Cells

Mast cell activation and inflammatory mediators play central roles in the pathogenesis of chronic spontaneous urticaria (CSU). The factors that induce mast cell activation in CSU are still largely unknown. Exosomes (EXs) are extracellular vesicles that activate mast cells. In this study, we enriched the EXs derived from the plasma of healthy volunteers and that of patients with CSU without antihistamine sensitivity (i.e., CSU-derived EXs with antihistamine sensitivity) or resistance (i.e., CSU-derived EXs with antihistamine resistance) using ultracentrifugation. We then incubated these EXs with HMC-1 human mast cells. Notably, CSU-derived EXs with antihistamine sensitivity and CSU-derived EXs with antihistamine resistance increased tryptase-1 expression; histamine production; inflammatory mediator production; and toll-like receptor (TLR) 2, TLR4, and phosphorylated MAPK levels in HMC-1 cells. These effects were more significant in the group with CSU-derived EXs with antihistamine resistance than in the group with CSU-derived EXs with antihistamine sensitivity. TLR2, TLR4, and MAPK inhibitors (CC-401, TAK-715, and SCH772984, respectively) reduced CSU-derived EXs-Stimulated production of inflammatory mediators in HMC-1 cells. Overall, EXs in the plasma of patients with CSU were found to activate mast cells and elicit the production of multiple inflammatory mediators, partly through the TLR2, TLR4, and MAPK pathways. In addition, CSU-derived EXs with antihistamine resistance had more powerful mast cell‒activating and histamine-release abilities. Thus, these EXs may be involved in the pathogenesis of CSU with antihistamine resistance.

Mesenchymal stromal cells in the regeneration of radiation-induced organ sequelae: will they make the difference?

Mesenchymal stromal cells (MSCs) are a stem cell product with good safety that demonstrate significant clinical efficacy in the treatment of different pathologies, including radiation diseases (e.g. radiological burns, pelvic radiation disease). While the first results for some first human applications for the treatment of radiation disease suggest benefit, larger trials with clinically important endpoints are needed before definitive conclusions can be drawn. However, the supply and cost of MSCs remain the two main limitations for this innovative therapeutic product. Exosomes (EXOs), a stem cell product associated with MSC therapy, have shown promising efficacy and safety in humans. MSC-EXO therapeutics represent a promising next-generation approach for treating radiation diseases involving a primary (major) inflammatory component. Provided that conditions for MSC-EXO production and bio-banking are agreed in the near future, the transition to industrial production of MSC-EXOs will be possible, and this is required to initiate well-controlled clinical trials for approval by the European Medicines Agency (EMA) and US Food and Drug Administration (FDA).

Damage-Free and Time-Saving Platform Integrated by a Flow Membrane Separation Device and a Dual-Target Biofuel Cell-Based Biosensor for Continuous Sorting and Detection of Exosomes and Host Cells in Human Serum.

The growth relationship between exosomes (EXOs) and the host cells is highly desired for tumor evaluations, which puts forward high demand on the accurate and convenient acquisition of their individual quantitative information. However, the tedious and destructive separation process and the requirement of dual-channel detection make it become an extremely challenging task. Herein, we integrated an enzymatic biofuel cell (EBFC)-powered biosensor with a flow cell-supported membrane separation device (FMSC) to develop a continuous separation and detection platform for EXOs and host cancer cells in human serum. The FMSC equipped with an aluminum oxide membrane served as a size-dependent sorting unit to nondestructively extract EXOs from human serum within 5 min, representing a 99.3% reduction in isolating time compared to ultracentrifugation. The EBFC-powered biosensors modified with different aptamers on anodes and cathodes were used as a dual-channel sensing unit. By regulating the controlling valves of different fluid passages, the extracted EXOs and residual host cells could be successively inputted into EBFC-powered biosensors, which generated a segmental degradation in output performance due to the EXO-and host cell-caused increase in the steric hindrance of anodes and cathodes, respectively. Based on these degradations, we obtained the quantitative information of EXOs and host cells with a record-breaking sensitivity (EXOs: 5.59 × 103 particles/mL and host cells: 25 cells/mL). Moreover, the growth relationship between EXOs and host cells was also built, which would be beneficial for the disclosure of the growth state or even more detailed biology information of tumor.

Conventional and Nonconventional Sources of Exosomes-Isolation Methods and Influence on Their Downstream Biomedical Application.

Despite extensive study of extracellular vesicles (EVs), specifically exosomes (EXs) as biomarkers, important modulators of physiological or pathological processes, or therapeutic agents, relatively little is known about nonconventional sources of EXs, such as invertebrate or plant EXs, and their uses. Likewise, there is no clear information on the overview of storage conditions and currently used isolation methods, including new ones, such as microfluidics, which fundamentally affect the characterization of EXs and their other biomedical applications. The purpose of this review is to briefly summarize conventional and nonconventional sources of EXs, storage conditions and typical isolation methods, widely used kits and new “smart” technologies with emphasis on the influence of isolation techniques on EX content, protein detection, RNA, mRNA and others. At the same time, attention is paid to a brief overview of the direction of biomedical application of EXs, especially in diagnostics, therapy, senescence and aging and, with regard to the current situation, in issues related to Covid-19.

Astragaloside IV drug-loaded exosomes (AS-IV EXOs) derived from endothelial progenitor cells improve the viability and tube formation in high-glucose impaired human endothelial cells by promoting miR-214 expression.

The high glucose changes caused by diabetes mellitus (DM) can damage the vascular system. Astragaloside IV (AS-IV) can improve diabetes and promote angiogenesis. Exosomes (EXOs) help to carry specific drugs into cells efficiently. However, whether AS-IV loaded EXOs (AS-IV EXOs) can improve damaged endothelial cells through miR-214 remains to be determined. We prepared and identified AS-IV EXOs derived from endothelial progenitor cells (EPCs) and high glucose stimulated endothelial cell models to investigate whether AS-IV EXOs can improve damaged endothelial cells through miR-214. We used a transmission electron microscope (TEM) and DAPI staining to identify the morphology and characteristic expression of EPCs and EXOs, and then prepared AS-IV EXOs. Cell function tests were performed to detect the cloning, proliferation, and migration capabilities of cells. Western blot (WB) and real-time quantitative polymerase chain reaction (qRT-PCR) were used to assess the expression level of Tie-2, Ang-1, and PI3K/Akt-related protein. The DAPI staining results showed that inducing human umbilical vein endothelial cells (HUVECs) could effectively absorb AS-IV EXOs. The results of plate clone formation assay, CCK-8, cell adhesion, and transwell assay of HUVECs stimulated by high glucose showed that AS-IV EXOs had a damage relief effect. By the detection of WB and qRT-PCR, it was found that AS-IV EXOs promoted the expression of miR-214 and proteins related to blood vessel growth. After transfection of miR-214 to pre-treat HUVECs under high glucose stimulation, AS-IV EXOs promoted the tube formation of HUVECs by regulating the level of miR-214. By promoting the expression of miR-214, AS-IV EXOs significantly improved the activity and tubularization of HUVECs under high glucose stimulation.

Exosomes Derived from Dental Pulp Stem Cells Show Different Angiogenic and Osteogenic Properties in Relation to the Age of the Donor

Craniofacial tissue reconstruction still represents a challenge in regenerative medicine. Mesenchymal stem cell (MSC)-based tissue engineering strategies have been introduced to enhance bone tissue repair. However, the risk of related complications is limiting their usage. To overcome these drawbacks, exosomes (EXOs) derived from MSCs have been recently proposed as a cell-free alternative to MSCs to direct tissue regeneration. It was hypothesized that there is a correlation between the biological properties of exosomes derived from the dental pulp and the age of the donor. The aim of the study was to investigate the effect of EXOs derived from dental pulp stem cells of permanent teeth (old donor group) or exfoliated deciduous teeth (young donor group) on MSCs cultured in vitro. Proliferation potential was evaluated by doubling time, and commitment ability by gene expression and biochemical quantification for tissue-specific factors. Results showed a well-defined proliferative influence for the younger donor aged group. Similarly, a higher commitment ability was detected in the young group. In conclusion, EXOs could be employed to promote bone regeneration, likely playing an important role in neo-angiogenesis in early healing phases.

The Impact of Exosomes/Microvesicles Derived from Myeloid Dendritic Cells Cultured in the Presence of Calcitriol and Tacalcitol on Acute B-Cell Precursor Cell Lines with MLL Fusion Gene.

Vitamin D analogs (VDAs) may directly inhibit the growth of normal and malignant (derived from acute lymphoblastic leukemia (ALL)) B cells, as both types of cells express vitamin D receptor (VDR). We performed anti-proliferative, morphology tests and phenotyping to evaluate the sensitivity of monocytes and iDCs (immature myeloid-derived dendritic cells) on calcitriol and tacalcitol treatment, phenotyping, morphology, and size distribution measurement to determine the characteristics of microvesicles (MVs) and exosomes (EXs) derived from them and, finally, phenotyping and Elisa test to determine the effects of VDAs on modulation of the phenotype of B cells through extracellular vesicles (EVs) released by iDCs. Our results confirmed that both SC cells and iDCs were sensitive to the VDAs and showed altered surface expression of markers associated with monocyte differentiation, which was resulting in the phenotypic changes in EVs derived from them. We also showed that obtained EVs could change the morphology and phenotype of ALL-B-derived precursor cells in a different way, depending on their origin. The differential effect of VDAs on ALL-B cells, which was associated with increased or decreased expression of CD27, CD24, CD38, and CD23 expression, was observed. Hence, further studies to explain the modulation in the composition of EVs by VDAs are required.

Exosomes Derived from Mesenchymal Stem Cells Ameliorate the Progression of Atherosclerosis in ApoE-/- Mice via FENDRR.

Exosomes (EXO) are extracellular vesicles with lipid bilayer membrane structure containing noncoding RNA, DNA, and other molecules which mediate biological functions. The importance of EXO derived from mesenchymal stem cells (MSCs) has been underlined in cardiovascular diseases. However, the functional role of long non-coding RNA (lncRNA) released by MSCs-EXO on atherosclerosis (AS) was unknown. We aimed to investigate the effects of lncRNA fetal-lethal non-coding developmental regulatory RNA (FENDRR) released from MSC-derived EXO on AS. The accumulation of oxidized low-density lipoprotein (oxLDL) caused AS in mice and damage to human vascular endothelial cells (HUV-EC-C). MSC-EXO restored HUV-EC-C activity and alleviated arterial injury. LncRNA microarrays revealed that FENDRR was delivered to cells and tissues by MSC-EXO. FENDRR bound to microRNA (miR)-28 to regulate TEA domain transcription factor 1 (TEAD1) expression. Moreover, FENDRR knockdown exacerbated cell injury and arterial injury in mice. miR-28 inhibitor reversed the effects of FENDRR silencing and reduced atherosclerotic plaque formation. While loss of TEAD1 mitigated the effect of miR-28 inhibitor and accentuated HUV-EC-C injury in vitro and AS symptoms in vivo. Our results demonstrated that MSC-EXO secreted FENDRR to treat AS. FENDRR competed with TEAD1 to bind tomiR-28, thereby reducing HUV-EC-C injury and atherosclerotic plaque formation.

Human Amnion Epithelial Cells and Their Derived Exosomes Alleviate Sepsis-Associated Acute Kidney Injury via Mitigating Endothelial Dysfunction.

BackgroundSepsis is characterized by organ dysfunction resulting from a patient’s dysregulated response to infection. Sepsis-associated acute kidney injury (S-AKI) is the most frequent complication contributing to the morbidity and mortality of sepsis. The prevention and treatment of S-AKI remains a significant challenge worldwide. In the recent years, human amnion epithelial cells (hAECs) have drawn much attention in regenerative medicine, yet the therapeutic efficiency of hAECs in S-AKI has not been evaluated.MethodsSeptic mice were induced by cecal ligation and puncture (CLP) operation. hAECs and their derived exosomes (EXOs) were injected into the mice via tail vein right after CLP surgery. The 7-day survival rate was observed. Serum creatinine level was measured and H&E staining of tissue sections were performed 16 h after CLP. Transmission electron microscopy was used to examine the renal endothelial integrity in CLP mice. Human umbilical vein endothelial cells (HUVECs) were treated with lipopolysaccharide (LPS) and EXOs. Zonula occludens-1 (ZO-1) localization was observed by immunofluorescence staining. Expression of phosphor-p65 (p-p65), p65, vascular cell adhesion molecule-1 (VCAM-1), and ZO-1 in the kidney were determined by Western blot.ResultshAECs decreased the mortality of CLP mice, ameliorated septic injury in the kidney, and improved kidney function. More precisely, hAECs suppressed systemic inflammation and maintained the renal endothelial integrity in septic animals. EXOs from hAECs exhibited similar renal protective effects as their parental cells. EXOs maintained endothelial cell adhesion junction in vitro and inhibited endothelial cell hyperactivation in vivo. Mechanistically, EXOs suppressed proinflammatory nuclear factor kappa B (NF-κB) pathway activation in LPS-treated HUVECs and in CLP mice kidneys.ConclusionOur results indicate that hAECs and their derived EXOs may ameliorate S-AKI via the prevention of endothelial dysfunction in the early stage of sepsis in mice. Stem cell or exosome-based therapy targeting endothelial disorders may be a promising alternative for treatment of S-AKI.

Milk-Derived Exosomes as Nanocarriers to Deliver Curcumin and Resveratrol in Breast Tissue and Enhance Their Anticancer Activity.

Dietary (poly)phenols are extensively metabolized, limiting their anticancer activity. Exosomes (EXOs) are extracellular vesicles that could protect polyphenols from metabolism. Our objective was to compare the delivery to breast tissue and anticancer activity in breast cancer cell lines of free curcumin (CUR) and resveratrol (RSV) vs. their encapsulation in milk-derived EXOs (EXO-CUR and EXO-RSV). A kinetic breast tissue disposition was performed in rats. CUR and RSV were analyzed using UPLC-QTOF-MS and GC-MS, respectively. Antiproliferative activity was tested in MCF-7 and MDA-MB-231 breast cancer and MCF-10A non-tumorigenic cells. Cell cycle distribution, apoptosis, caspases activation, and endocytosis pathways were determined. CUR and RSV peaked in the mammary tissue (41 ± 15 and 300 ± 80 nM, respectively) 6 min after intravenous administration of EXO-CUR and EXO-RSV, but not with equivalent free polyphenol concentrations. Nanomolar EXO-CUR or EXO-RSV concentrations, but not free CUR or RSV, exerted a potent antiproliferative effect on cancer cells with no effect on normal cells. Significant (p < 0.05) cell cycle alteration and pro-apoptotic activity (via the mitochondrial pathway) were observed. EXO-CUR and EXO-RSV entered the cells primarily via clathrin-mediated endocytosis, avoiding ATP-binding cassette transporters (ABC). Milk EXOs protected CUR and RSV from metabolism and delivered both polyphenols to the mammary tissue at concentrations compatible with the fast and potent anticancer effects exerted in model cells. Milk EXOs enhanced the bioavailability and anticancer activity of CUR and RSV by acting as Trojan horses that escape from cancer cells’ ABC-mediated chemoresistance.

Co-Culture of Bone Marrow Mesenchymal Stem Cell (BMSC) and Osteoclasts Regulates Cell Differentiation and Alleviates Osteoporosis by Up-Regulating miR-211

Bone marrow mesenchymal stem cells (BMSCs) can release a large amount of exosomes (EXO) during bone remodeling by osteoclasts. EXO contains miRNA-211, which has a variety of biological effects. However, little is known about whether miR-211 from BMSC-EXO affects the surrounding cells. Therefore, we aim to study the role of miRNA-211 derived from BMSC-EXO in regulating osteoclasts differentiation. Macrophage colony stimulating factor (M-CSF) and nuclear factor kappa B receptor activator (RANKL) were used to stimulate bone marrow macrophages (BMM) to obtain osteoclasts, which were treated with BMSC-EXO or LPS followed by analysis of osteoclast-related genes expression by PCR, ROS release by flow cytometry, actin ring formation by immunofluorescence, and osteoclast differentiation by anti-tartrate acid phosphatase (TRAP) staining. Finally, an in vivo experiment was conducted to verify BMSC-EXO’s effect on osteoporosis. BMSC-EXO significantly inhibited RNAKL-induced osteoclast differentiation of BMMs. During osteoclasts formation, BMSC-EXO inhibited ROS production induced by RANKL and the subsequent activation of NF-κB signaling pathway induced by ROS. In addition, BMSC-EXO significantly down-regulated the osteoclast genes including nuclear factor, cytoplasmic 1 (NFATc1), C-fos, tartrate-resistant acid phosphatase (TRAP) and osteoclast-associated immunoglobulin-like receptor (OSCAR) in activated T cells. BMSC-EXO inhibited ROS release by promoting miR-211 expression, thereby inhibiting the NF-κB signaling and ultimately participating in osteoclasts differentiation. In LPS-induced mouse osteoporosis models, BMSC-EXO inhibited LPS-induced bone loss and exerted a protective effect. In conclusion, microRNA-211 derived from BMSC-EXO can regulate osteoclasts differentiation, suggesting that it might be used as a potential approach for treating osteoporosis.

Distinguishing functional exosomes and other extracellular vesicles as a nucleic acid cargo by the anion-exchange method.

The development of a new large‐scale purification protocol is required for research on the reliable bioactivity and drug discovery of extracellular vesicles (EVs). To address this issue, herein, we propose an effective method for preparing high‐performance exosomes (EXOs) by using an anion‐exchange method. Cytotoxic T‐lymphocyte (CTL) EVs from 4 L of culture supernatant through a 220 nm cut‐off filter are divided into two populations at a deproteinization rate of over 99.97%, which are eluted at low (0.15 M–0.3 M) and high (0.3 M–0.5 M) NaCl concentrations (approximately 2 × 1012 and 1.5 × 1012 particles, respectively) through the anion‐exchange column chromatography. The former are abundant in EXO proteins, including late endosome‐associated proteins and rab‐family and integrin‐family proteins, and functional micro (mi) RNAs, and have bioactivity for preventing tumour metastasis by depleting mesenchymal cell populations in the primary tumour lesions. By contrast, the latter is microvesicle (MV)‐like particles including DNA, core histone and ribosomal proteins, and GC‐rich miRNAs with unknown function, and are easily phagocytosed by mannose receptor+ Kupffer cells. Thus, the anion‐exchange method is suitable for the large‐scale separation of bioactive EXOs and MV‐like EVs as a cargo for dangerous nucleic acids at high‐purity.

Tumor-Derived Extracellular Vesicles Activate Normal Human Fibroblasts to a Cancer-Associated Fibroblast-Like Phenotype, Sustaining a Pro-Tumorigenic Microenvironment.

Fibroblasts in the tumor microenvironment have been proven to actively participate in tumor progression; they can be “educated” by cancer cells acquiring an activated state and, as such, are identified as cancer-associated fibroblasts (CAFs); CAFs, in turn, remodel tumor stroma to be more advantageous for cancer progression by modulating several processes, including angiogenesis, immunosuppression, and drug access, presumably driving the chemoresistance. That is why they are believed to hamper the response to clinical therapeutic options. The communication between cancer cells and fibroblasts can be mediated by extracellular vesicles (EVs), composed of both exosomes (EXOs) and microvesicles (MVs). To verify the role of different subpopulations of EVs in this cross-talk, a nearly pure subpopulation of EXO-like EVs and the second one of mixed EXO- and MV-like EVs were isolated from ovarian cancer cells and administered to fibroblasts. It turned out that EVs can activate fibroblasts to a CAF-like state, supporting their proliferation, motility, invasiveness, and enzyme expression; EXO-like EV subpopulation seems to be more efficient in some of those processes, suggesting different roles for different EV subpopulations. Moreover, the secretome of these “activated” fibroblasts, composed of both soluble and EV-associated molecules, was, in turn, able to modulate the response of bystander cells (fibroblasts, tumor, and endothelial cells), supporting the idea that EVs sustain the mutual cross-talk between tumor cells and CAFs.

Human Amniotic Epithelial Cells and Their Derived Exosomes Protect Against Cisplatin-Induced Acute Kidney Injury Without Compromising Its Antitumor Activity in Mice.

Background: Cisplatin is a widely used chemotherapeutic drug, whereas the clinical application is greatly limited by its nephrotoxic side effect. Currently, there has been no effective treatment to prevent cisplatin-induced acute kidney injury (cisplatin-AKI). Human amniotic epithelial cells (hAECs) and their derived exosomes (EXOs) have been proven to effectively protect against ischemia reperfusion-induced AKI, yet their roles in cisplatin-AKI are still unknown. Methods: C57BL/6J mice were given two doses of cisplatin at 20 or 15 mg/kg of body weight to induce AKI with or without mortality. hAECs or EXOs were injected via tail vein 1 day after cisplatin administration. Serum and kidney tissues were collected on the fourth day after 15 mg/kg cisplatin treatment to explore the nephro-protective effects of hAECs and EXOs on cisplatin-AKI. Lung cancer xenograft model was built by subcutaneous injection of A549 cells into BALB/c nude mice to evaluate the effect of hAECs or EXOs on cisplatin chemotherapy. Results: Cisplatin nephrotoxicity was significantly attenuated by hAECs and EXOs as evidenced by reduced mortality rate and decreased serum creatinine (sCr) and reduced tubular injury score. hAECs or EXOs exerted the nephro-protective effects via suppression of TNF-α/MAPK and caspase signaling pathways. In the A549 lung cancer xenograft mouse model, administration of hAECs or EXOs did not promote tumor growth or compromise the therapeutic effects of cisplatin on tumors. Conclusion: This study is the first to demonstrate that hAECs and their derived exosomes have nephro-protective effects in cisplatin-AKI in vivo. Importantly, neither hAECs nor EXOs compromise the antitumor activity of cisplatin. These results potentially support the use of hAECs and their derived EXOs as nephro-protectors against cisplatin-induced nephrotoxicity clinically.

Perivascular adipose-derived exosomes reduce macrophage foam cell formation through miR-382-5p and the BMP4-PPARγ-ABCA1/ABCG1 pathways

BackgroundPerivascular adipose tissue (PVAT) releases exosomes (EXOs) to regulate vascular homeostasis. PVAT-derived EXOs reduce macrophage foam cell formation, but the underlying molecular mechanism has yet to be fully elucidated. We hypothesize that PVAT release miRNA through EXOs and regulate the expression of cholesterol transporter of macrophages, thereby reducing foam cell formation.Methods and resultsThrough RT-qPCR, we identified that miR-382-5p, which was expressed at lower levels in PVAT-EXOs from coronary atherosclerotic heart disease patients than healthy individuals, was expressed at higher levels in wild-type C57BL/6 J mouse aortic PVAT-EXOs than in subcutaneous adipose tissue-derived EXOs. We explored macrophage lipid accumulation through oil red O staining, assessed cholesterol uptake and efflux, and verified cholesterol transporter expression. We found that transfection with a miR-382-5p inhibitor offset PVAT-EXO-related reductions in macrophage foam cell formation and increases in cholesterol efflux mediated by ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1). In addition, bone morphogenetic protein 4 (BMP4) pretreatment and si-peroxisome proliferator-activated receptor γ (PPARγ) transfection showed that BMP4-PPARγ participated in PVAT-EXO-mediated upregulation of the cholesterol efflux transporters ABCA1 and ABCG1.ConclusionsPVAT-EXOs reduce macrophage foam cell formation through miR-382-5p- and BMP4-PPARγ-mediated upregulation of the cholesterol efflux transporters ABCA1 and ABCG1. This finding suggests a promising strategy for the prevention and treatment of atherosclerosis.

Spinal cord injury target-immunotherapy with TNF-α autoregulated and feedback-controlled human umbilical cord mesenchymal stem cell derived exosomes remodelled by CRISPR/Cas9 plasmid

Human umbilical cord mesenchymal stem cell (hucMSC) derived exosomes (EXOs) have been investigated as a new treatment for spinal cord injury (SCI) because of their anti-inflammatory, anti-apoptotic, angiogenesis-promoting, and axonal regeneration properties. The CAQK peptide found in the brains of mice and humans after trauma has recently been found to specifically bind to the injured site after SCI. Thus, we developed a nanocarrier system called EXO-C@P based on hucMSC exosomes remodelled by the CRISPR/Cas9 plasmid to control inflammation and modified by the CAQK peptide. EXO-C@P was shown to effectively accumulate at the injury site and saturate the macrophages to significantly reduce the expression of inflammatory cytokines in a mouse model of SCI. Moreover, EXO-C@P treatment improved the performance of mice in behavioural assessments and upregulated soluble tumour necrosis factor receptor-1 (sTNFR1) in serum and at the trauma site after SCI surgery, but lowered the proportion of iNOS+ cells and the concentration of proinflammatory factors. In conclusion, EXO-C@P provides an effective alternative to multiple topical administration and drug delivery approaches for the treatment of SCI. Statement of significanceSCI is a serious disease characterised by a high incidence, high disability rate, and high medical costs, and has become a global medical problem. Several studies have shown that the inflammatory response is the critical inducer of secondary injury after SCI. The inflammatory cytokine TNF-α is considered to be one of the most significant therapeutic targets for autoimmune diseases. Antibodies targeting TNF-α and sTNFR1 are capable of neutralising free TNF-α. In this study, exosomes in the CRISPR/Cas9 system were used to establish stem cells with an autoregulated and feedback-controlled TNF-α response, with these cells secreting sTNFR1, which neutralised TNF-α and antagonised the inflammation stimulated by TNF-α. Moreover, the plasmid was combined with CAQK, which targeted the injury site and promoted the recovery of SCI function.

Myeloma cells regulate miRNA transfer from fibroblast-derived exosomes by expression of lncRNAs.

Multiple myeloma (MM) progression and drug resistance depend on the crosstalk between MM cells and bone marrow (BM) fibroblasts (FBs). During monoclonal gammopathy of undetermined significance (MGUS) to MM transition, MM cell-derived exosomes (EXOs) reprogram the miRNA (miR) profile of FBs, inducing the overexpression miR-23b-3p, miR-27b-3p, miR-125b-5p, miR-214-3p, and miR-5100. Here, we demonstrate that the miR content of MM FB-derived EXOs (FB-EXOs) overlaps the miR profile of parental FBs by overexpressing comparable levels of miR-23b-3p, miR-27b-3p, miR-125b-5p, miR-214-3p, and miR-5100. Recipient MM cells co-cultured with MM FB-EXOs selectively overexpress only miR-214-3p and miR-5100 but not miR-23b-3p, miR-27b-3p, and miR-125b-5p, suggesting a putative selective transfer. MM cells express HOTAIR, TOB1-AS1, and MALAT1 lncRNAs. Transient transfection of MM cells with lnc·siRNAs demonstrates that HOTAIR, TOB1-AS1, and MALAT1 lncRNAs are sponges for miR-23b-3p, miR-27b-3p, and miR-125b-5p. Indeed, lncRNA knockdown significantly increased miR levels in U266 MM cells co-cultured with MM FB-EXOs. Selective miR-214-3p and miR-5100 overexpression modulates MAPK, PI3K/AKT/mTOR, and p53 pathways in MM cells. Interrogation using the DIANA tools algorithm and transient overexpression using miR mimic probes confirmed the involvement of miR-214-3p and miR-5100 and their target genes, PTEN and DUSP16, respectively, in the modulation of these intracellular pathways. Finally, the uptake of EXOs as well as miR-214-3p and miR-5100 overexpression increase MM cell proliferation and resistance to bortezomib-induced apoptosis by switching the balance between pro-/anti-apoptotic proteins. Overall, these data show that MM cells are not simply a container into which EXOs empty their cargo. On the contrary, tumour cells finely neutralize exosomal miRs via lncRNA expression to ensure their survival. © 2021 The Pathological Society of Great Britain and Ireland.