Expression Of Exosomal Markers Research Articles

Activating PKA signaling increases exosome production and attenuates cerebral ischemia-reperfusion injury by regulating Cx43 expression

BackgroundConnexin 43 (Cx43) plays a crucial role in mediating intracellular communication and facitating the interaction between exosomes and recipient cells. This study investigates whether the activation of cAMP/protein kinase A (PKA) can regulate exosomal Cx43 expression and contribute to the functional recovery following ischemia-reperfusion (I/R) injury. MethodsAn intraluminal vascular occlusion was performed on Lewis rats to simulate I/R injury. Concurrently, a PKA activator (8-Bromo-cAMP, 5 mg kg-1) or PKA inhibitor (H 89 2HCl, 20 mg kg-1) was administered intravenously via the tail vein (n = 10). Exosomes were isolated from cerebrospinal fluid, and the expression of exosomal markers (CD63 and CD81) and Cx43 was analyzed using Western blot. The expression of CD63 and CD81 in astrocytes was measured to assess exosome uptake. Spatial learning and memory capability were evaluated using the Morris water maze test. Results8-Bromo-cAMP significantly increased exosome release in cerebrospinal fluid, accompanied by elevated Cx43 expression. Additionally, 8-Bromo-cAMP enhanced exosome uptake by astrocytes, alleviated blood-brain barrier damage and edema, and improved cognitive function. ConclsionsPKA activation enhances exosome production, promotes cognitive function recovery, and attenuates cerebral I/R injury by up-regulating exosomal Cx43 expression.

In vitro maturation using porcine follicular fluid-derived exosomes as an alternative to the conventional method

Extracellular vesicles, also known as exosomes, influence numerous cellular functions by regulating different signaling pathways. However, their role in animal reproduction remains understudied. This study aimed to evaluate the effects of porcine follicular fluid-derived exosomes (pff-Exos) on porcine oocyte in vitro maturation and parthenogenetic embryo development. We obtained pff-Exos through mixed-method ultracentrifugation and size-exclusion chromatography. Transmission electron microscopy revealed an increase in the expression of exosome markers in the first four of thirteen fractions. The number of pff-Exo was 2.2 × 106 particles per microliter. The highest maturation rate of porcine oocytes treated with pff-Exo was observed with 1.1 × 107 particles of pff-Exo in the absence of porcine follicular fluid (pFF) culture conditions. Moreover, increased expression of Gdf9 and Bmp15 was observed. The developmental rate was the highest upon treatment with 1.1 × 107 particles of pff-Exo, which increased the total cell number in blastocysts. Embryonic development to the 2-cell stage was similar between the control and pff-Exo groups; however, development to the 4-cell stage and blastocyst was significantly increased in the pff-Exo group (61.6 ± 6.08 % and 29.72 ± 1.41 %, respectively; P < 0.05) compared with that in the control group (42.0 ± 5.19 % and 18.14 ± 1.78 %, respectively). The expression levels of Oct4, Sox2, Bcl2, Elf4, and Gcn5 significantly increased at the pff-Exo 2-cell stage, whereas those of Bax, Hdac1, Hdac6, and Sirt6 decreased. Specifically, the Oct4, Sox2, Elf4, Gcn5, and Hdac6 levels remained stable in pff-Exo 4-cell embryos, whereas those of p53 and Hat1 were reduced and increased, respectively. Treatment with pffExos significantly increased H3K9 and H3K14 acetylation levels. These results demonstrate that pff-Exo affects the in vitro maturation of porcine oocytes and early embryonic development by regulating gene expression.

Hypoxic bone marrow mesenchymal stem cell exosomes promote angiogenesis and enhance endometrial injury repair through the miR-424-5p-mediated DLL4/Notch signaling pathway.

Currently, bone marrow mesenchymal stem cells (BMSCs) have been reported to promote endometrial regeneration in rat models of mechanically injury-induced uterine adhesions (IUAs), but the therapeutic effects and mechanisms of hypoxic BMSC-derived exosomes on IUAs have not been elucidated. To investigate the potential mechanism by which the BMSCS-derived exosomal miR-424-5p regulates IUA angiogenesis through the DLL4/Notch signaling pathway under hypoxic conditions and promotes endometrial injury repair. The morphology of the exosomes was observed via transmission electron microscopy, and the expression of exosome markers (CD9, CD63, CD81, and HSP70) was detected via flow cytometry and Western blotting. The expression of angiogenesis-related genes (Ang1, Flk1, Vash1, and TSP1) was detected via RT‒qPCR, and the expression of DLL4/Notch signaling pathway-related proteins (DLL4, Notch1, and Notch2) was detected via Western blotting. Cell proliferation was detected by a CCK-8 assay, and angiogenesis was assessed via an angiogenesis assay. The expression of CD3 was detected by immunofluorescence. The endometrial lesions of IUA rats were observed via HE staining, and the expression of CD3 and VEGFA was detected via immunohistochemistry. Compared with those in exosomes from normoxic conditions, miR-424-5p was more highly expressed in the exosomes from hypoxic BMSCs. Compared with those in normoxic BMSC-derived exosomes, the proliferation and angiogenesis of HUVECs were significantly enhanced after treatment with hypoxic BMSC-derived exosomes, and these effects were weakened after inhibition of miR-424-5p. miR-424-5p can target and negatively regulate the expression of DLL4, promote the expression of the proangiogenic genes Ang1 and Flk1, and inhibit the expression of the antiangiogenic genes Vash1 and TSP1. The effect of miR-424-5p can be reversed by overexpression of DLL4. In IUA rats, treatment with hypoxic BMSC exosomes and the miR-424-5p mimic promoted angiogenesis and improved endometrial damage. The hypoxic BMSC-derived exosomal miR-424-5p promoted angiogenesis and improved endometrial injury repair by regulating the DLL4/Notch signaling pathway, which provides a new idea for the treatment of IUAs.

Extracellular vesicles ameliorates sleep deprivation induced anxiety-like behavior and cognitive impairment in mice

The aim of this research was to explore the therapeutic capabilities of extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) that had been subjectedto heat shock pretreatment, in treating psychiatric disorders induced by sleep deprivation in mice. The EVs were isolated and characterized, while western blotting was utilized to assess the expression of exosomal markers and heat shock protein 70 (HSP70). To evaluate the impact of EV treatment on anxiety-like behavior and cognitive impairment in sleep-deprived (SD) mice, the open field test, plus maze test, and Y-maze task were conducted. Heat shock pretreatment significantly increased the expression of HSP70 in EVs. Administration of EVs fromheat shock-pretreated hUC-MSCs improved anxiety-like behavior and cognitive function in SD mice. Furthermore, EV treatment promoted synaptic protein expression, HSP70 expression and inhibited neuroinflammation in the hippocampus of SD mice. Western blotting analysis also revealed that EV treatment reduced the levels of TLR4 and p65 in the hippocampus. EVs from heat shock-pretreated hUC-MSCs have therapeutic potential for sleep deprivation-induced psychiatric disorders by regulating neuroinflammation and synaptic function in mice.

Molecular characterization of small extracellular vesicles in patients with pancreatic cancer treated with neoadjuvant chemotherapy followed by stereotactic body radiation (SBRT).

685 Background: Currently, pancreatic cancer (PanC) is one of the serious gastrointestinal diseases, and more than 90% of the patients die within five years of diagnosis. Therefore, to improve PanC-related mortality, new therapeutic and diagnostic/prognostic measures are urgently needed. In this substudy, we examined the usefulness of plasma small extracellular vesicles (sEV) to discover molecular biomarkers associated with treatment response and overall survival from the plasma samples collected on a prospective clinical trial assessing the efficacy of SBRT following chemotherapy in pancreatic cancers (NCT03600623). Methods: PanCpatients (n=22) with locally advanced and borderline inoperable disease were recruited at the University of Alabama Comprehensive Cancer Center. They were administered either FOLFIRINOX (5-FU, folinic acid, oxaliplatin, and irinotecan) or paclitaxel (gemcitabine-nab-paclitaxel) for 2 months followed by SBRT (33 Gray in 5 fractions). The primary objective of this single-center pilot study was to evaluate the safety and tolerability of this treatment regimen. Further, blood was collected at baseline and, at the end of chemotherapy and radiotherapy. sEV were isolated from archived plasma samples by an established ultracentrifugation method and characterized for size and concentration by nanoparticle tracking analyses (NTA), shape and size by transmission electron microscope (TEM), and surface expression of exosomal tetraspanin markers (CD63, CD9, and CD81) and a PanC marker (CA19-9) by nano-flow cytometry. Lastly, sEV in longitudinal plasma samples were characterized for the expression of specific PanC-related miRNAs (miR196a-5p, miR155-5, miR194-5p, miR301-3p, miR21-5p, miR1246, and miR34a-5p) by real time-PCR (RT-PCR). Results: Neoadjuvant FOLFIRINOX and gemcitabine-nab-paclitaxel followed by SBRT were safe and well tolerated by most patients. NTA data showed that the ultracentrifugation method yielded highly pure sEV (with average diameter of &lt;200 nm) from archived baseline and longitudinal plasma samples. TEM analysis further confirmed the shape and size of the isolated sEV. Nano-flow cytometry showed the expression of exosomal markers, CD63, CD9, and CD81, as well as PanC marker CA19-9 on the surfaces of sEV. The expression of various PanC-related miRNAs in sEV was heterogenous and correlated with corresponding clinical parameters, including treatment response and overall survival. Conclusions: Neoadjuvant chemotherapy in combination with SBRT is safe and tolerable regimen to treat patients with locally advanced and borderline PanC. Further, sEV in the plasma of PanC patients could serve as useful prognostic and predictive markers.

Transmission electron microscopy assessment of a novel method for isolating pure exosomes from serum

ABSTRACT Serum exosomes frequently are used for liquid biopsy. Serum exosomes normally are isolated using ultracentrifugation; however, ultracentrifugation is time-consuming, labor intensive and requires a high-speed centrifuge. Many commercial kits use a precipitation-based method; however, this process can result in substantial contamination. We developed a new method to isolate pure serum exosomes. We isolated serum exosomes using precipitation, extracted them using acetone, then isolated them again by precipitation. We used transmission electron microscopy (TEM) to examine the morphology of serum exosomes. TEM indicated that our isolated exosomes were pure with typical morphology and with a size ranging from 40 to 150 nm. Flow cytometry revealed expression of exosome markers, CD63, CA81 and CD9. Our double precipitation method enables ready extraction of pure exosomes from serum. Our double precipitation method simplifies detection of serum exosomal biomarkers for diagnosis and prognosis of disease.

Associations of maternal and placental extracellular vesicle miRNA with preeclampsia.

Introduction: Gestational vascular complications (GVCs), including gestational hypertension and preeclampsia, are leading causes of maternal morbidity and mortality. Elevated levels of extracellular vesicles (EVs), in GVC have been linked to vascular injury. This study aims to characterize placental and circulating EV miRNA in GVCs, and explores the involvement of EV-miRNA in GVC, and whether they may be used to distinguish between placental and maternal pathologies. Methods: Blood samples were obtained from 15 non-pregnant (NP), 18 healthy-pregnant (HP), and 23 women with GVC during the third trimester. Placental sections were obtained after caesarian section. Platelet-poor-plasma (PPP) and EV pellets were characterized: EV size/concentration, protein content and miRNA expression were measured by nanoparticle tracking analysis, western blot, nano-string technology and RT-PCR. The effects of EVs on trophoblasts and EC miRNA expression were evaluated. Results: Higher EVs concentrations were observed in HP-PPP and GVC-PPP (p < 0.0001) compared to the NP-PPP. The concentration of large EVs (>100nm) was higher in PPP and EV pellets of HP and GVC compared to the NP group. EV pellets of pregnant women demonstrated lower expression of exosomal markers CD63/CD81 compared to NP-EVs. GVC-EVs expressed more human placental lactogen (hPL) hormone than HP-EVs, reflecting their placental origin. Screening of miRNAs in EV pellets and in PPP identified certain miRNAs that were highly expressed only in EVs pellets of the HP (13%) and GVC groups (15%), but not in the NP group. Differences were detected in the expression of hsa-miR-16-5p, hsa-miR-210, and hsa-miR-29b-3p. The expression of hsa-miR-16-5p and hsa-miR-210 was low in EV pellets obtained from NP, higher in HP-EVs, and significantly lower in GVC-EVs. Except for hsa-miR-29b-3p, which was upregulated in GVC, no significant differences were found in the levels of other miRNAs in placental sections. Exposure to GVC-EVs resulted in higher expression of hsa-miR-29b-3p compared to cells exposed to HP-EVs in villous trophoblasts, but not in EC. Conclusion: Expression of hsa-miR-16-5p and hsa-miR-210 reflects maternal pathophysiological status, while hsa-miR-29b-3p reflects placental status. These findings suggest that EV-miRNA are involved in GVC, and that they may be used to distinguish between pathologies of placental and maternal origins in preeclampsia.

Single-cell transcriptomics reveal extracellular vesicles secretion with a cardiomyocyte proteostasis signature during pathological remodeling

Aberrant Wnt activation has been reported in failing cardiomyocytes. Here we present single cell transcriptome profiling of hearts with inducible cardiomyocyte-specific Wnt activation (β-catΔex3) as well as with compensatory and failing hypertrophic remodeling. We show that functional enrichment analysis points to an involvement of extracellular vesicles (EVs) related processes in hearts of β-catΔex3 mice. A proteomic analysis of in vivo cardiac derived EVs from β-catΔex3 hearts has identified differentially enriched proteins involving 20 S proteasome constitutes, protein quality control (PQC), chaperones and associated cardiac proteins including α-Crystallin B (CRYAB) and sarcomeric components. The hypertrophic model confirms that cardiomyocytes reacted with an acute early transcriptional upregulation of exosome biogenesis processes and chaperones transcripts including CRYAB, which is ameliorated in advanced remodeling. Finally, human induced pluripotent stem cells (iPSC)-derived cardiomyocytes subjected to pharmacological Wnt activation recapitulated the increased expression of exosomal markers, CRYAB accumulation and increased PQC signaling. These findings reveal that secretion of EVs with a proteostasis signature contributes to early patho-physiological adaptation of cardiomyocytes, which may serve as a read-out of disease progression and can be used for monitoring cellular remodeling in vivo with a possible diagnostic and prognostic role in the future.

Malaria-derived exosomes exacerbate liver injury during blood stage of Plasmodium berghei infection

Liver injury is a common clinical feature of Plasmodium spp. infection and contributes to multi-organ failure of severe malaria. Malaria-derived exosomes (MD-Exos) have recently engaged as key mediators in parasite-host interactions, modulating the subsequent pathogenic process. However, the role of MD-Exos in malaria-related liver injury and the underlying mechanisms remain unclear. Herein, exosomes from C57BL/6 mice infected with or without P. berghei ANKA serum (namely inf-Exos or un-Exos) were isolated and characterized by transmission electron microscopy, western blotting, and nanoparticle tracking analysis. The miRNAs profiling between inf-Exos and un-Exos were generated using RNA-seq and qPCR. The functions of inf-Exos on liver injury were investigated after two types of exosomes injected into mice intravenously (i.v.), by examining histopathological and apoptotic changes, macrophage polarization, and pro-inflammatory response. The infected red blood cells-stimulated mouse Raw264.7 macrophage cells targeted by inf-Exos or un-Exos were cultured for further study and verification the potential mechanisms. We found that both inf-Exos and un-Exos displayed a typical cup-shaped structure with a diameter of 60–200 nm, and had a positive expression of exosomal markers (e.g., CD9, CD63, and CD81). Compared with infected control mice, the treatment of inf-Exos but not un-Exos dramatically enhanced peripheral blood parasitemia and ECM incidence, exacerbated liver histopathological damage, elevated numbers of liver apoptotic cells, CD68+and CD86+ macrophages. The CD68+-TREM-1+ macrophages in liver tissues and the mRNA levels of pro-inflammatory cytokines (e.g., iNOS, TNF-α, IL-1β, and IL-6) were increased by inf-Exos treatment in vivo. Meanwhile, the treatment of inf-Exos resulted in a substantial increase of the mRNA levels of CD86, iNOS, TNF-α, IL-1β, and IL-6, but led to a remarkable decrease of Bcl-6 and SOCS-1 in Raw264.7 cells stimulated with iRBC in vitro. Notably, compared to un-Exos, five types of miRNAs (including miR-10a-5p, miR-10b-5p, miR-155–5p, miR-205–5p, and miR-21a-5p), that were previously reported to target Bcl-6 or SOCS-1, present higher abundance on inf-Exos, as demonstrated by RNA-seq and qPCR. Collectively, our data suggest that inf-Exos exacerbate malaria-induced liver pathology via triggering excessive pro-inflammatory response and promoting macrophage M1 polarization. Our findings will provide new insights into the roles of inf-Exos in malaria parasite-host interaction and pathogenesis of liver injury.

Mesenchymal Stem Cell Secreted-Extracellular Vesicles are Involved in Chondrocyte Production and Reduce Adipogenesis during Stem Cell Differentiation

Background:Extracellular vesicles (EVs) are derived from internal cellular compartments, and have potential as a diagnostic and therapeutic tool in degenerative disease associated with aging. Mesenchymal stem cells (MSCs) have become a promising tool for functional EVs production. This study investigated the efficacy of EVs and its effect on differentiation capacity.Methods:The characteristics of MSCs were evaluated by flow cytometry and stem cell differentiation analysis, and a production mode of functional EVs was scaled from MSCs. The concentration and size of EVs were quantitated by Nanoparticle Tracking Analysis (NTA). Western blot analysis was used to assess the protein expression of exosome-specific markers. The effects of MSC-derived EVs were assessed by chondrogenic and adipogenic differentiation analyses and histological observation.Results:The range of the particle size of adipose-derived stem cells (ADSCs)- and Wharton’s jelly -MSCs-derived EVs were from 130 to 150 nm as measured by NTA, which showed positive expression of exosomal markers. The chondrogenic induction ability was weakened in the absence of EVs in vitro. Interestingly, after EV administration, type II collagen, a major component in the cartilage extracellular matrix, was upregulated compared to the EV-free condition. Moreover, EVs decreased the lipid accumulation rate during adipogenic induction.Conclusion:The results indicated that the production model could facilitate production of effective EVs and further demonstrated the role of MSC-derived EVs in cell differentiation. MSC-derived EVs could be successfully used in cell-free therapy to guide chondrogenic differentiation of ADSC for future clinical applications in cartilage regeneration.

Oxidative stress state inhibits exosome secretion of hPDLCs through a specific mechanism mediated by PRMT1.

Periodontitis, the most common chronic inflammation characterized by persistent alveolar bone resorption in the periodontitis, affects almost half of the adult population worldwide. Oxidative stress is one of the pathophysiological mechanisms underlying periodontitis, which affects the occurrence and development of periodontitis. Exosomes are increasingly recognized as vehicles of intercellular communication and are closely related to periodontitis. However, the effects of oxidative stress on exosome secretion and the specific mechanisms remain elusive in human periodontal ligament cells (hPDLCs). The relationship between exosome secretion and the osteogenic differentiation of hPDLCs also needs to be investigated. Isolated PDLSCs were identified using flow cytometry. Osteogenesis was measured using alizarin red staining and ALP staining. Expression of exosomal markers and PRMT1 was analyzed using western blot. Immunofluorescence was used to measure exosome uptake and the expression of EEA1. The secretion capacity of exosomes was markedly suppressed under oxidative stress. Protein arginine methyltransferase 1 (PRMT1) has been strongly associated with both oxidative stress and inflammation, and PRMT1 was significantly upregulated under oxidative stress conditions. Lentivirus-mediated overexpression of PRMT1 caused a significant reduction in the secretion of exosomes, but multivesicular bodies (MVBs) containing a large number of intraluminal vesicles (ILVs) were increased. Rab11a and Rab27a expression, which mediate MVBs fusion with cell membranes, decreased, although this phenomenon was restored after knocking down PRMT1 expression under oxidative stress. These results indicated that PRMT1 mediated a decrease in exosome secretion of hPDLCs. The decrease in Rab11a and Rab27a leads to a large accumulation of MVBs in cells and is one of the main reasons for impaired exosome secretion. The decrease in osteogenic differentiation of hPDLCs caused by H2 O2 may originate in part from the inhibition of exosome secretion.

Mouse mesenchymal stem cell-derived exosomal miR-466f-3p reverses EMT process through inhibiting AKT/GSK3\u03b2 pathway via c-MET in radiation-induced lung injury

BackgroundRadiation-induced lung fibrosis (RILF) is a common complication of thoracic radiotherapy. Alveolar epithelial cells play a crucial role in lung fibrosis via epithelial-mesenchymal transition (EMT). Exosomes derived from mesenchymal stem cells own the beneficial properties to repair and regeneration of damaged tissues, however the underlying mechanisms remain poorly understood.MethodsMouse mesenchymal stem cells-derived exosomes (mMSCs-Exo) were isolated by differential centrifugation, and their protective effects were assessed in vivo and in vitro, respectively. EMT-associated proteins were measured via western blot assay and/or immunofluorescence staining. The miRNA expression was measured by microarray assay and qPCR. Furthermore, bioinformatics prediction with KEGG analysis, luciferase assay, and rescue experiments were performed to explore the molecular mechanism underlying miR-466f-3p.ResultsmMSCs-Exos were efficiently isolated ranging from 90-150 nm with high expression of exosomal markers (CD63, TSG101, and CD9). mMSCs-Exos administration efficiently relieved radiation-induced lung injury with less collagen deposition and lower levels of IL-1β and IL-6. Meanwhile, in vitro results showed mMSCs-Exos treatment obviously reversed EMT process induced by radiation. Among enriched miRNA cargo in exosomes, miR-466f-3p was primarily responsible for the protective effects via inhibition of AKT/GSK3β pathway. Our mechanistic study further demonstrated that c-MET was the direct target of miR-466f-3p, whose restoration partially abrogated mMSCs-Exo-mediated inhibition in both EMT process and AKT/GSK3β signaling activity induced by radiation.ConclusionsOur findings indicated that exosomal miR-466f-3p derived from mMSCs may possess anti-fibrotic properties and prevent radiation-induced EMT through inhibition of AKT/GSK3β via c-MET, providing a promising therapeutic modality for radiation-induced lung fibrosis.

Exosomes from dental pulp cells attenuate bone loss in mouse experimental periodontitis.

Exosomes are small vesicles secreted from many cell types. Their biological effects largely depend on their cellular origin and the physiological state of the originating cells. Exosomes secreted by mesenchymal stem cells exert therapeutic effects against multiple diseases and may serve as potential alternatives to stem cell therapies. We previously established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this study, we aimed to investigate the effect of local administration of HHH-DPC exosomes in a mouse model of periodontitis. Exosomes purified from HHH-DPCs were subjected to particle size analysis, and expression of exosome markers was confirmed by western blotting. We also confirmed the effect of exosomes on the migration of both HHH-DPCs and mouse osteoblastic MC3T3-E1 cells. A mouse experimental periodontitis model was used to evaluate the effect of exosomes in vivo. The morphology of alveolar bone was assessed by micro-computed tomography (μCT) and histological analysis. The effect of exosomes on osteoclastogenesis was evaluated using a co-culture system. The exosomes purified from HHH-DPCs were homogeneous and had a spherical membrane structure. HHH-DPC exosomes promoted the migration of both human DPCs and mouse osteoblastic cells. The MTT assay showed a positive effect on the proliferation of human DPCs, but not on mouse osteoblastic cells. Treatment with HHH-DPC exosomes did not alter the differentiation of osteoblastic cells. Imaging with µCT revealed that the exosomes suppressed alveolar bone resorption in the mouse model of periodontitis. Although no change was apparent in the dominance of TRAP-positive osteoclast-like cells in decalcified tissue sections upon exosome treatment, HHH-DPC exosomes significantly suppressed osteoclast formation in vitro. HHH-DPC exosomes stimulated the migration of human DPCs and mouse osteoblastic cells and effectively attenuated bone loss due to periodontitis.

Dihydroceramide desaturase promotes the formation of intraluminal vesicles and inhibits autophagy to increase exosome production

SummaryExosomes are important for cell–cell communication. Deficiencies in the human dihydroceramide desaturase gene, DEGS1, increase the dihydroceramide-to-ceramide ratio and cause hypomyelinating leukodystrophy. However, the disease mechanism remains unknown. Here, we developed an in vivo assay with spatially controlled expression of exosome markers in Drosophila eye imaginal discs and showed that the level and activity of the DEGS1 ortholog, Ifc, correlated with exosome production. Knocking out ifc decreased the density of the exosome precursor intraluminal vesicles (ILVs) in the multivesicular endosomes (MVEs) and reduced the number of exosomes released. While ifc overexpression and autophagy inhibition both enhanced exosome production, combining the two had no additive effect. Moreover, DEGS1 activity was sufficient to drive ILV formation in vitro. Together, DEGS1/Ifc controls the dihydroceramide-to-ceramide ratio and enhances exosome secretion by promoting ILV formation and preventing the autophagic degradation of MVEs. These findings provide a potential cause for the neuropathy associated with DEGS1-deficient mutations.

Dihydroceramide Desaturase Promotes the Formation of Intraluminal Vesicles and Inhibits Autophagy to Increase Exosome Production

Exosomes are important for cell-cell communication. Deficiencies in the human dihydroceramide desaturase gene, DEGS1 , increase the dihydroceramide-to-ceramide ratio and causes hypomyelinating leukodystrophy. However, the disease mechanism remains unknown. Here, we developed an in vivo assay with spatially controlled expression of exosome markers in Drosophila eye imaginal discs and showed that the level and activity of the DEGS1 ortholog, ifc , correlated with exosome production. Knocking out ifc decreased the density of the exosome precursor intraluminal vesicles (ILVs) in the multivesicular endosomes and reduced the number of exosomes released. While ifc overexpression and autophagy inhibition both enhanced exosome production, combining the two had no additive effect. Moreover, DEGS1 activity was sufficient to drive ILV formation in vitro . Together, DEGS1/Ifc controls the dihydroceramide-to-ceramide ratio and enhances exosome secretion by promoting ILV formation and preventing the autophagic degradation of MVEs. These findings provide a potential cause for the neuropathy associated with DEGS1-deficient mutations.

Abstract P-43: Effect of Glucocerebrosidase Dysfunction on the Pool of Plasma Exosomes of Patients with Gaucher Disease

Background: Extracellular vesicles (EVs) are small membrane vesicles released from different types of cells. EVs are found in many human biological fluids. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies with the plasma membrane. This type of vesicles is characterized by specific exosomal markers. Exosomes extracted from peripheral body liquids could have specific properties associated with different physiological conditions as well as human disorders, including neurodegenerative diseases. Gaucher disease (GD) – is the most common form of lysosomal storage disorders caused by mutations in the glucocerebrosidase (GBA) gene. Lysosome functionality is critical for the regulation of extracellular vesicle secretion and content. In model animals, the inhibition of glucocerebrosidase has been shown to increase the secretion of extracellular vesicles in brain tissues. Amount evaluation of EVs and their size in the biological fluids of patients with GD has not been early performed; therefore, it is unknown whether lysosomal dysfunction found in GD patients influences the plasma pool of EVs. The aim of this study was to evaluate the amount of blood plasma EVs in patients with GD and their characterization for morphology and size. Methods: EVs were isolated from the blood plasma of 8 GD patients and 8 controls by ultracentrifugation, and were characterized using cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). Also, the presence of exosomal markers CD9, CD63, CD81, and HSP70 was analyzed by flow cytometry and western blot. Results: Here, it was first shown an increased proportion of exosome fraction in EVs from plasma of GD patients compared to controls by DLS and cryo-EM (p&lt;0.001) that was confirmed by mode size detected by NTA (p&lt;0.02). Moreover, an increased number of double and multilayer vesicles in plasma EVs from GD patients was demonstrated by cryo-EM. We also detected an increase in the expression of exosomal markers on the surface of vesicles from the blood plasma of patients with GD compared to controls. Conclusion: Here, we firstly report that the exosomes obtained from the blood plasma of GD patients have a larger size and altered morphology. Thus, we have shown that lysosomal dysfunction in GD patients leads to a striking alteration of blood plasma extracellular vesicle pool.

RAB5A effect on metastasis of hepatocellular carcinoma cell line via altering the pro-invasive content of exosomes

Tumor microenvironment exerts a critical role in cancer progression and metastasis. Exosomes, cell-cell communicators and major players of the tumor microenvironment are considered as a serious mediator of cancer metastasis. Here, we determined the effect of RAB5A gene on the hepatocellular carcinoma (HCC) cells particularly whether RAB5A could affect HCC metastasis via regulating the pro-invasive content of exosomes. In response to RAB5A knockdown, we analyzed the proliferation rate and migration capability of HCC cells. Then, we estimated changes in the total protein composition of exosomes via analyzing the expression of exosomal markers, CD63 and Alix. Thereafter, alterations of the pro-invasive content of exosomes were functionally evaluated using matrigel invasion assay. Our results revealed that knockdown of RAB5A could decrease HCC cell proliferation rate and migration capability significantly. Moreover, no significant changes in the expression of exosomal CD63 and Alix reflected that no differences might be occurred in protein composition of RAB5A knockdown cell-derived exosomes. Matrigel invasion assay functionally showed that exosomes-derived from RAB5A knockdown cells still had pro-invasive properties and their pro-invasive content was not affected in response to RAB5A knockdown. In conclusion, we believe that our results propose a new explanation about RAB5A and metastatic potentials of exosomes-derived from HCC cells.

Effect of melatonin on exosomal dynamics in bovine cumulus cells

In this study, the effects of melatonin were investigated on the exosome release from bovine cumulus cells after 7 days. Cells were randomly allocated into three groups as follows; Control, 0.1 nM, and 0.1 mM Met groups. The physicochemical properties of exosomes such as size and zeta potential were measured by the dynamic light scattering. Both scanning electron microscope (SEM) and transmission electron microscope (TEM) were used to characterize exosome morphology. The effect of melatonin on cell viability was studied using an MTT assay. The expression of exosome markers like Alix, CD63, Rab27a, and Rab27b was monitored using real-time PCR analysis. We also investigated fatty acid composition by the gas chromatography method. TEM and SEM confirmed typical exosome's morphology. By increasing the concentration of melatonin, the viability of cumulus cells was significantly increased compared to the control group (p < 0.05). These changes coincided with the significant reduction of exosome diameter (p < 0.05). Zeta potentials of exosomes were not changed after treatment with 0.1 nM, and 0.1 mM melatonin. Compared to the control cells, 0.1 mM melatonin increased the expression of mRNA for Rab27b whereas suppression of CD63 was found simultaneously (p < 0.05). The expression of Alix and Rab27a was not changed in groups that received 0.1 nM, and 0.1 mM melatonin. Melatonin increased the percent of unsaturated fatty acids such as Arachidonic acid, Oleic acid, and Linoleic acid. These data showed that melatonin could change the exosomal dynamics in bovine cumulus cells.

The effects of neoadjuvant chemoradiation on exosomal markers (CD63 and CD9) expression in patients with locally advanced rectal adenocarcinoma.

101 Background: Exosomes mediate intercellular communications and have pivotal roles in cancer development. CD63 and CD9 are widely accepted exosomal markers. The effect of concurrent chemoradiation on the expression of exosomal markers is unknown. Here we explored the effect of neoadjuvant concurrent chemoradiation (NCCR) on exosomal markers (CD63 and CD9) expression in patients with locally advanced rectal cancer (LARC). Methods: Between 2015 and 2018, 33 patients had LARC treated with NCCR and had pre NCCR biopsy and post NCCR resected rectum examined for exosomal markers expression using immunohistochemistry. Two pathologists independently scored CD63 and CD9 staining in the tumor. Staining intensity was graded from 1-3. Staining percentage was estimated in 10% increments. Mean quick-score (Q-score) was calculated (intensity x percentage). Un-Paired t test was used for statistical analysis. Results: In our cohort, median age was 59 years. Males represented 79% of the patients. Caucasians, African American and other ethnic groups represented 70%, 27% and 3% respectively.The mean tumor CD63 score in pre NCCR biopsy vs post NCCR resected rectum was 106 vs 165 (p = 0.0022). The mean tumor CD9 score in pre NCCR biopsy vs post NCCR resected rectum was 136 vs 215 (p &lt; 0.0001). The exosomal markers expression in the adjacent normal mucosa (ANM) from pre NCCR biopsy and post NCCR resected rectum was only performed in16 out of 33 patients (due to ANM tissue availability). The mean ANM CD63 score in pre NCCR biopsy vs post NCCR resected rectum was 166 vs 183 (p = 0.37). The mean ANM CD9 score in pre NCCR biopsy vs post NCCR resected rectum was 104 vs 145 (p = 0.0897). Conclusions: In patients with LARC, the expression of exosomal markers (CD63 and CD9) increased after treatment with NCCR. Our results show that the expression of CD63 and CD9 is relatively higher in rectal cancer specimens treated with NCCR and thus suggest a possible role of these exosomes in adaptive response to NCCR. Further follow-up and laboratory studies are required to precisely understand the underlying mechanism(s).

Utility of Claudin-3 in extracellular vesicles from human bile as biomarkers of cholangiocarcinoma

Extracellular vesicles (EVs) are released from all cells. Bile directly contacts bile duct tumor; bile-derived EVs may contain high concentrations of cancer biomarkers. We performed a proteomic analysis of human bile-derived EVs and identified a novel biomarker of cholangiocarcinoma (CCA). EVs were isolated using ultracentrifugation, and chelating agents, ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid (EDEG) and phosphate buffered saline (PBS) were used as dissolution solutions. Bile was collected from 10 CCA and 10 choledocholithiasis (stones) cases. Proteomic analysis was performed; subsequently, ELISA was performed using the candidate biomarkers in a verification cohort. The vesicles isolated from bile had a typical size and morphology. The expression of exosome markers was observed. RNA was more abundant in the EDEG group. The proportion of microRNA was higher in the EDEG group. EDEG use resulted in the removal of more contaminants. Proteomic analysis identified 166 proteins as CCA-specific. ELISA for Claudin-3 revealed statistically significant difference. The diagnostic accuracy was AUC 0.945 and sensitivity and specificity were 87.5%. We report the first use of EDEG in the isolation of EVs from human bile and the proteomic analysis of human bile-derived EV-proteins in CCA. Claudin-3 in bile-derived EVs is a useful biomarker for CCA.