Proteomic analysis of small extracellular vesicles purified by three methods
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)
SUBMITTER: Akiko Takahashi
PROVIDER: PXD037822 | JPOST Repository | Sun Oct 29 00:00:00 BST 2023
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Ctrl-EVs_IP.raw | Raw | |||
| Ctrl-EVs_PS.raw | Raw | |||
| Ctrl-EVs_SEC.raw | Raw | |||
| IR-EVs_IP.raw | Raw | |||
| IR-EVs_PS.raw | Raw |
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Project description:Aging significantly affects intercellular communication between vascular endothelial cells (ECs) and hematopoietic cells, leading to vascular inflammation and age-associated diseases. This study determined how senescent ECs communicate with monocytes, whether extracellular vesicles (EVs) released from senescent ECs affect monocyte functions, and investigated the potential for epigallocatechin-3-gallate (EGCG), a flavonoid in green tea, to reverse these effects. Human umbilical vein endothelial cells (HUVECs) were treated with Etoposide (10 µM, 24h) to induce senescence, followed by EGCG (100 µM, 24h) treatment to evaluate its potential as a senotherapeutic agent. The interaction between ECs and monocytes was analyzed using a co-culture system and direct treatment of monocytes with EC-derived EVs. EGCG reduced senescence-associated phenotypes in ECs, as evidenced by decreased senescence-associated (SA)-β-Gal activity and reversal of Etoposide-induced senescence markers. Monocytes co-cultured with EGCG-treated senescent ECs showed decreased pro-inflammatory responses compared to those co-cultured with untreated senescent ECs. Additionally, senescent ECs produced more EVs than non-senescent ECs. EVs from senescent ECs enhanced lipopolysaccharide (LPS)-induced pro-inflammatory activation of monocytes, whereas EVs from EGCG-treated senescent ECs mitigated this activation, maintaining monocyte activation at normal levels. Our findings reveal that EGCG confers anti-senescent effects via modulation of the senescent EC secretome (including EVs) with the capacity to modify monocyte activation. These findings suggest that EGCG could act as a senotherapeutic agent to reduce vascular inflammation related to aging.
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Project description:Senescent cells are beneficial for repairing acute tissue damage but are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) relative to proliferating cells (P-EVs). Interestingly, P-EVs were readily taken up by other proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not; by contrast, macrophages selectively engulfed all EVs (P-EVs, S-EVs). We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We discovered that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to dictate selective uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.
2022-10-09 | MSV000090503 | MassIVE
Project description:Atherosclerosis represents an age-related disease, which remains one of the leading cause of deaths in developed countries. It begins with a local deposition of lipid in the innermost part of the artery – intima. Thereafter a number of immune cells are attracted and infiltrate into the artery wall, where, together with endothelia and smooth muscle cells, form an atherosclerotic plaque (Libby, 2008). Thus, atherosclerosis is considered as an inflammatory disease, characterized by intense immunological activity (Libby P, 2012). One of the main risk factor for atherosclerosis is aging. It was demonstrated that there is an accumulation of senescent cells within atherosclerotic plaque (..). Accordingly, vascular smooth muscle cells (VSMCs) derived from the lesion demonstrate a phenotype characteristic for senescent cells: diminished proliferative potential, shorter telomeres, increased number of DNA damage and upregulation of SA-β-gal activity (Gorenne, 2006; Matthes, 2006, Mahomoudi, 2008). Accumulation of senescent VSMCs within the area of plaque development promotes its instability due to lack of proliferation and impaired production of extracellular matrix, both leading to weakening of fibrous cap (Gorenne, 2006). This has been further confirmed by the observation, that elimination of senescent cells in the atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr-/-) mice slowed down the disease progression (27789842). One of the most important feature of senescent cells, that has the biggest impact on tissue microenvironment, is their ability to secrete a number of cytokines, chemokines, matrix metalloproteinases and growth factors collectively known as the Senescence Associated Secretory Phenotype (SASP) (20078217, 28165648). Apart from soluble factors, senescent cells secret also an increased number of extracellular vesicles (EVs), research of which has only recently begun (32461143). EVs are small membranous vesicles that can be categorized based on their size and origin, into: exosomes, microvesicles and apoptotic bodies. EVs play crucial role as carriers of proteins, different types of RNA and DNA, lipids and metabolites that, take part in intercellular communication (23420871). They were shown to participate in many physiological but also pathological conditions. Studies performed over the last decade proved that EVs affect atherosclerosis progression at different stages by several distinct mechanisms (as reviewed by Knokhot, 2020, 33261815). The increased concentrations of EVs have been found in plasma of patients suffering from cardiovascular diseases as well as in atherosclerotic plaque itself (Yin, 2015, 26142082; Rautou, 2011, 21852557). However, the role of EVs derived from senescent cells in atherosclerosis, particularly in the context of the plaque development, remains unknown. The functioning of immune cells within atherosclerotic plaque is modulated by local milieu. Thus, senescent cells present in the lesion can influence plaque development by non-cell autonomous mechanism. Indeed, recently it was demonstrated that SASP factors secreted by senescent VSMCs promote recruitment of monocytes, induce expression of adhesion receptors on endothelial cells and activate adjacent normal VSMCs, promoting inflammation in the plaque (Gardner, 2015). However, the role of EVs in this context has not been studied. Thus, we have undertaken research aimed at investigating the role of senescent cell derived EVs on immune cells activity. To this end, we isolated and characterized the EVs secreted by human VSMCs undergoing senescence. We performed unbiased quantitative proteomic analysis of both soluble and insoluble SASP components. Moreover, we analyzed the influence of EVs derived from senescent and non-senescent cells on T lymphocytes and monocytes. Altogether our studies revealed that EVs produced by senescent VSMCs strengthen inflammatory response of the immune cells, what would have a tremendous significance in atherosclerotic plaque development.
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Project description:Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. However, the altered secretome of senescent cells can promote the growth of the surrounding cancer cells. Although extracellular vesicles (EVs) have emerged as new players in intercellular communication, their role in the function of senescent cell secretome has been largely unexplored. Here, we show that exosome-like small EVs (sEVs) are important mediators of the pro-tumorigenic function of senescent cells. sEV-associated EphA2 secreted from senescent cells binds to ephrin-A1 that is highly expressed in several types of cancer cells and promotes cell proliferation through EphA2/ephrin-A1 reverse signalling. sEV sorting of EphA2 is increased in senescent cells due to its enhanced phosphorylation resulting from oxidative inactivation of PTP1B phosphatase. Our results demonstrate a novel mechanism of reactive oxygen species (ROS)-regulated cargo sorting into sEVs, which is critical for the potentially deleterious growth-promoting effect of the senescent cell secretome.
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Project description:In arthritis, synovial fibroblast (SF) senescence is linked to the activation of a pro-inflammatory phenotype contributing to chronic arthritis pathogenesis. Additionally, senescent cells accumulate in ageing tissues further promoting ageing and inflammation. These cells have dysregulated mitochondrial function and metabolism, limited tissue regeneration, produce reactive oxygen species (ROS) and secrete bioactive molecules, including pro-inflammatory cytokines, chemokines and matrix-remodelling enzymes known as SASP (senescence-associated secretory phenotype). In vitro, senescent cells can induce a senescent phenotype in surrounding bystander cells. Interestingly, extracellular vesicles (EVs) have critical roles in cellular senescence and ageing and are mediators in intercellular communication. We hypothesize that senescent cells in osteoarthritic SFs induce senescence and/or a proinflammatory phenotype in non-senescent osteoarthritic SFs, mediated through EV cargo.
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Project description:Tissue-resident stem cell senescence leads to stem cell exhaustion, which is a major cause of physiological and pathological aging. Stem cells-derived extracellular vesicles(SCs-EVs) have been reported to possess therapeutic potential in preclinical studies for diverse diseases. However, whether SCs-EVs could rejuvenate senescent tissue stem cell to prevent age-related disorders still remains unknown. Here, we showed that chronic application of human embryonic stem cells-derived extracellular vesicles (hESCs-sEVs) rescues senescent bone marrow MSCs (BM-MSCs) function and prevents age-related bone loss in aging mice. Transcriptome analysis revealed that hESCs-sEVs treatment up-regulates the expression of genes involved in anti-aging, stem cell proliferation and osteogenic differentiation in BM-MSCs. Furthermore, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified 4122 proteins encapsulated in hESCs-sEVs. Bioinformatics analysis predicated that protein components in the hESCs-sEVs function in a synergistic way to induce the activation of several canonical signaling pathways, including Wnt, Sirtuin, AMPK, PTEN signaling, which results in the up-regulation of anti-aging genes in BM-MSCs and then the recovery of senescent BM-MSCs function. Collectively, our findings reveal the effect of hESCs-sEVs in reversing BM-MSCs senescence and age-related osteogenic dysfunction, and thereby preventing age-related bone loss. Given that hESCs-sEVs could alleviate senescence of tissue-resident stem cells, it might be a promising therapeutic candidate for age-related diseases.
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Project description:<p><strong>BACKGROUND:</strong> Periodontal ligament mesenchymal stem cells (PDLSCs) are a promising cell resource for cell-based regenerative medicine in dentistry. PDLSCs inevitably acquire a senescent phenotype after prolonged in vitro expansion, and the key regulators of cells during replicative senescence remain unclear.</p><p><strong>METHODS:</strong> We cultured periodontal ligament stem cells to passages 4, 10 and 20 (P4, P10, P20). The senescent phenotypes, proliferation and migration ability of PDLSCs (P4, P10, P20) were detected, and non-targeted metabonomic sequencing was performed. We treated PDLSCs with AICAR and detected the expression of FOXO1, FOXO3a, FOXO6 and AMPK phosphorylation (p-AMPK) levels of replicating senescent PDLSCs to explore the correlation between the metabolic changes and the AMPK pathway.</p><p><strong>RESULTS:</strong> Immunofluorescence staining of γ-H2AX, β-galactosidase staining, cell scratch test and qPCR were performed to confirm the occurrence of replicative senescence in PDLSCs during passaging. Three groups of cells at passage 4 (P4), passage 10 (P10) and passage 20 (P20) were collected for non-targeted metabolomics analysis. Metabonomic sequencing showed that the metabolism of replicative senescence in PDLSCs varied significantly. In particular, the content of fatty acid metabolites decreased with senescence, including capric acid, stearic acid, myristic acid and dodecanoic acid. KEGG pathway analysis showed that the AMPK signaling pathway was closely related to AMP levels. The AMP:ATP ratio increased in senescent PDLSCs; however, the levels of p-AMPK and the profile of FOXO1 and FOXO3a, which are downstream of the AMPK signaling pathway, decreased with senescence. We treated PDLSCs with AICAR, an activator of the AMPK pathway, and the phosphorylated AMPK level at P20 PDLSCs was partially restored. </p><p><strong>CONCLUSION:</strong> In summary, our study suggests that the metabolic process of PDLSCs is active in the early stage of senescence, prefers to consume fatty acids, and is attenuated in the later stages of senescence. AMP accumulates in replicative senescent PDLSCs; however, the sensitivity of AMPK phosphorylation sites is impaired, causing senescent PDLSCs to fail to respond to changes in energy metabolism. Our findings provide a new basis for the clinical application of periodontal ligament stem cells.</p>
2022-11-15 | MTBLS4568 | MetaboLights
Project description:We studied extracellular vesicles (EVs) from synovial fibroblast (SF). EVs were isolated from the secretome of non-senescent and irradiation-induced senescent SFs using size exclusion chromatography. EV RNA was extracted and subject to small RNA sequencing on an Illumina NovaSeq SP using 100bp, single end reads. After mapping against GRCh38.p12 and miRBase v22.1 differential expression analysis was undertaken with edgeRv3.28 using a quasi-likelihood negative binomial generalized log-linear model. We identified a panel of differentially expressed small non-coding RNAs.
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2011-08-09 | E-GEOD-27404 | biostudies-arrayexpress