Proteomic signatures of extracellular vesicles secreted by non-mineralizing and mineralizing human osteoblasts and stimulation of tumor cell growth
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Jeroen van de Peppel
PROVIDER: E-GEOD-56172 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Beyond forming bone, osteoblasts play pivotal roles in various biological processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have recently been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. Here, we focused on the proteomic characterization of non-mineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent five-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to two-fold increase in PC3 cell growth compared to cell-free culture medium-derived vesicle controls. Our findings elucidate the mineralization stage-specific protein content of osteoblast-secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention.
2014-10-28 | GSE56172 | GEO
Project description:Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Here, we characterize the miRNA profile of EVs secreted by human osteoblasts and study their biological effect of on human umbilical cord blood-derived CD34+ HSPCs by sequencing, gene expression and biochemical analyses. Using next-generation sequencing we show that osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and show successful engraftment in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.
2016-10-05 | E-MTAB-4560 | biostudies-arrayexpress
Project description:Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EV play decisive roles in establishing a connection with recipient cells, and they are putative targets in diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers. The surface proteins of intact seminal fluid ssmall EV (SF-sEVs and PC3) sEVs were labeled using sulfo-NHS-SS-biotin, a membrane-impermeable reagent reacting with primary amines. The fractions obtained from the purification process were: (i) protein in total sEV lysate (Total); (ii) proteins isolated from sEV surfaces (Surface), and (iii) supernatant proteins left after isolation of surface proteins (Total minus Surface). Proteins from each fraction were digested by trypsin and analyzed by HRMS. A total PC3 cell lysate was prepared andanalyzed as a control.
2023-03-11 | PXD037791 | Pride
Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.
2023-04-03 | E-MTAB-11641 | biostudies-arrayexpress
Project description:Patient-derived prostate fibroblast primary cultures PCF-54 and PCF-55 were established from two specimens of PC tissues. Urinary EVs were isolated from urine samples of 3 patients with PC and 2 healthy males and used for the treatment of prostate fibroblast primary cultures and normal foreskin fibroblasts. Normoxic and hypoxic EVs were isolated from cell culture medium of PC3 and LNCaP prostate cancer cell lines, cultivated in normoxic and hypoxic conditions respectively. The EV-treated fibroblasts were subjected to RNA sequencing analysis.
2023-04-01 | E-MTAB-11317 | biostudies-arrayexpress
Project description:Introduction: Cell-derived extracellular vesicles ([EVs], i.e., exosomes and microparticles) have been reported to mediate the cardioprotective effects of stem cells. However, a head-to-head comparison of their effects with those of the cells from which they derive has not been reported. Hypothesis: The effects of the EV content of human embryonic stem cell (hESC)-derived cardiac progenitors may be equivalent to those of the parent cells. Methods: Two series of immunodeficient (nude) mice underwent permanent coronary artery ligation. Three weeks later, those with an echocardiographically-determined LV ejection fraction ⤠50% were randomly allocated to receive transcutaneous echo-guided peri-infarct injections of alpha-MEM media (controls), hESC-derived SSEA-1+ cardiac progenitors (500000 cells in 30 μL), or total EV secreted by those 500000 cells in 48 hours in an equivalent volume. The second series also included sham-operated animals for which the needle was inserted into the myocardium in three places without injection. EVs were collected from the cell-secreted medium by ultracentrifugation and characterized by flow cytometry, and nanosight NTS. Outcomes were assessed after 6 weeks by echocardiography, taking LV end-systolic volume (ESV) as the primary end point. Hearts were processed for the assessment of fibrosis and angiogenesis. Total RNA was extracted from carefully selected animals for gene expression analysis by Affymetrix chip. Only animals with similar VTS at baseline were used for this analysis. All data were collected and analyzed blindly. Results: Cardiac progenitors were successfully generated by exposure of the pluripotent ESC to bone morphogenetic protein-2, purified by anti-CD15 immunomagnetic sorting and then cultured on vitronectin for 48 hours, after which cells or EVs derived from the same cell batch were injected. After 6 weeks in the first series, LVESV [m±SEM] in controls did not significantly differ from the pre-injection value (difference: -2.64 ± 1.54 µL, p=0.12, n= 12). Conversely, in the cell-treated group, LVESV significantly decreased by -4.20 ± 0.96 µL (p=0.0007, n=16). A similar decrease was seen in the EV-treated group: -5.73 ± 1.21 µL (p=0.0003, n=15). Similar patterns were seen for LV end-diastolic volumes: controls (-2.46 ± 1.19 µL, p=NS), cells (-4.48 ± 1.47 µL, p=0.009), EV (-4.29 ± 1.31 µL, p=0.005). For the second series, the VTD of cell- and EV-treated animals remained stable (cell: -1.85 ± 3.27 µL, p=NS, n= 4; EV: +0.05 ± 1.53 µL, p=NS, n = 4), whereas sham- and control-treated animals deteriorated significantly (sham: +2.20 ± 0.35 µL, p = 0.003, n = 5; control: +3.27 ± 0.87 µL, p = 0.03, n = 4). The trend held for VTS, though differences were not significant. Analysis of gene expression data revealed interesting pathways that were more highly expressed in cell- and EV-treated animals than in controls and sham-operated animals. Conclusions: These data support the hypothesis that EVs may be critical mediators of the paracrine effects of cell therapy, and for the sake of streamlining translational processes, deserve to be considered as potential alternatives to stem cell transplantation. Gene expression levels were compared between mouse hearts.
2016-05-29 | E-GEOD-69401 | biostudies-arrayexpress
Project description:Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impacts EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and non-coding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and glial communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation.
2023-08-12 | GSE240294 | GEO
Project description:Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impacts EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and non-coding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and glial communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation.
2023-08-12 | GSE240293 | GEO
Project description:Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impacts EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and non-coding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and glial communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation.
2023-08-12 | GSE240290 | GEO
Project description:This study assessed exosomal and cellular miRNA profiles in GFP-PC3 cells and ectopically expressed cavin-1 PC3 cell lines. To understand the consequences of cavin-1 on extracellular vesicle (EV)-contained miRNAs, a small RNA-seq experiment was performed on cells and secreted EVs for GFP-PC3 and cavin-1-PC3 cells. Small RNA were selected for sequencing using the NEBNext Small RNA library Prep Kit. Libraries were size selected using a Perkin Elmer Labchip XT. Whole cell and EV libraries were multiplexed from three biological replicates and sequenced on an Illumina NextSeq500 mid-output run at a read depth of 1million reads per sample. While the raw data was mapped to the human miRNAome using RNA-STAR and miRBase (v21) as reference, 317 mature miRNAs were identified in EVs at varying abundances depending on cell of origin. The 317 miRNAs identified in both cell and EVs were further processed. After RUVseq normalization, DESeq2 analysis was used to identify differences between cellular miRNA profiles and EV miRNA profiles from the two different cell lines. This analysis ultimately defined the repertoire of selectively and passively exported exosomal miRNAs mediated by non-caveolar caveolin-1 and cavin-1 in a prostate cancer cell line. Aim: To assess exosomal miRNA compositional changes evoked by ectopic expression of cavin-1 in the PC3 advanced prostate cancer cell line. Objectives: 1. Determine differential expression of miRNAs in cells and 2. determine differential abundance of miRNAs in extracellular vesicles secreted from cavin-1 expressing or GFP expressing cells.
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