evps  (Cytoskeleton Inc)

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Blockage of Pathological Extracellular Vesicles and Particles From Fibroblast‐Like Synoviocytes for Osteoarthritis Relief: Proteomic Analysis and Cellular Effect

doi: 10.1002/jev2.70162

Figure Lengend Snippet: FLS pathology was accompanied by increased EVP secretion in OA. (A) Gene ontology (GO) cellular component enrichment analysis of differentially expressed genes between normal and OA synovium. (B, C) Violin plots for the expression levels of the exosome secretion marker (RAB27A), senescence markers (CDKN1A, CDKN2A) and inflammation‐related markers (PTGS2, IL6, MMP13), along with Pearson correlation analyses between the expression levels of these markers and RAB27A. Data was normalized as fragments per kilobase of exon model per million mapped fragments (FPKM). (D–G) Multiplex immunohistochemical (mIHC) staining of normal and OA synovium, with magnified views of the boxed areas showing individual colour channels. Scale bar = 100 µm. Line graphs display the relative intensity and co‐localization of each fluorescence along the line in the magnified images. (H, I) Representative RAB27A fluorescence staining images and quantification in the synovial region of sham‐operated mice and mice at 4 and 8 weeks post‐DMM surgery. Scale bar = 25 µm. (J) Schematic diagram of in vitro EVP collection. Briefly, FLSs were pre‐treated with IL‐1β (10 ng/mL) and Bleomycin (25 µg/mL) for 24 h and changed into fresh medium without inducers. EVPs were then isolated from the conditioned medium through ultracentrifugation. (K, L*) Representative MMP13 fluorescence staining and SA‐β‐gal staining images for FLSs after inflammation and senescence induction. (M) Diameter distribution of EVPs from control and pathological FLSs by NTA, with screenshots of the particle flow. (N, O) Representative TEM images for EVPs and Western blot gels for EVP protein markers. Scale bar = 200 µm. (P, Q) Quantification and protein concentration of EVPs isolated from different culture medium, both of which were normalized to original cell counts. ** Indicates p < 0.01, * indicates p < 0.05, ns indicates p > 0.05, versus the indicated groups, one‐way ANOVA.

Article Snippet: For EVP labelling and uptake studies, isolated EVPs (2 × 10 10 particles/mL in PBS) were incubated with 40 nM MemGlow dye (MG02, Cytoskeleton) at room temperature, protected from light.

Techniques: Expressing, Marker, Multiplex Assay, Immunohistochemical staining, Staining, Fluorescence, In Vitro, Isolation, Control, Western Blot, Protein Concentration

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Blockage of Pathological Extracellular Vesicles and Particles From Fibroblast‐Like Synoviocytes for Osteoarthritis Relief: Proteomic Analysis and Cellular Effect

doi: 10.1002/jev2.70162

Figure Lengend Snippet: Proteomic profiling of pathogenic EVPs reflected the pathological changes of the source cells. (A) Schematic diagram for proteomics analysis of EVPs isolated from FLSs after inducing inflammatory and senescent phenotypes, created with Figdraw ( www.figdraw.com ). (B) SDS‐PAGE gel electrophoresis images of proteins lysed from abovementioned EVPs. (C) Number of proteins identified by mass spectrometry in EVPs secreted from control FLSs and FLSs induced with inflammation and senescence. (D, E) Principal component analysis plot and Pearson's Correlation Coefficient heatmap of the protein composition in EVPs from three groups ( n = 3 samples per group). The gradient colours and annotated values represent the Pearson correlation coefficients. (F) Number of differentially expressed proteins in EVPs between the three groups, with screening criteria set at an adjusted p value < 0.01 and a fold change >2 or <0.5. (G) Circos plot visualization of the overlaps among significantly altered proteins that overlap in Inf‐EVP and Sen‐EVP, with lines connecting the commonly altered proteins. (H) Pathway heatmaps of significantly differentially expressed proteins in Inf‐EVP and Sen‐EVP compared to Ctr‐EVP, enriched using Metascape. (I, J) Representative gene set enrichment analysis of proteins in Inf‐EVP or Sen‐EVP relative to those in Ctr‐EVP.

Article Snippet: For EVP labelling and uptake studies, isolated EVPs (2 × 10 10 particles/mL in PBS) were incubated with 40 nM MemGlow dye (MG02, Cytoskeleton) at room temperature, protected from light.

Techniques: Isolation, SDS Page, Nucleic Acid Electrophoresis, Mass Spectrometry, Control

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Blockage of Pathological Extracellular Vesicles and Particles From Fibroblast‐Like Synoviocytes for Osteoarthritis Relief: Proteomic Analysis and Cellular Effect

doi: 10.1002/jev2.70162

Figure Lengend Snippet: Pathogenic FLS EVPs disrupt chondrocyte and macrophages homeostasis in vitro. (A) Representative fluorescence for the internalization of EVPs by mouse chondrocytes. Scale bar = 20 µm. The red fluorescence represents EVPs labelled with the MemGlow fluorescent dye, and the blue fluorescence represents nuclei stained with DAPI. (B, C) Representative images and quantification of SA‐β‐gal staining for mouse chondrocytes after co‐culturing with different EVPs for 48 h, blue arrow indicting the SA‐β‐gal positive cells. Scale bar = 200 µm. (D) Representative Western blot images showing the senescence‐associated markers P16 and γ‐H2AX in chondrocytes treated with three types of EVPs for 48 h. (E) mRNA expression for OA‐related genes of mouse chondrocytes after co‐culturing with different EVPs for 24 h. (F–K) Representative images and quantification of COL2A1 and TUNEL staining for mouse chondrocytes, as well as EdU staining for ATDC5 cell line after stimulation with different EVPs for 48 h. Scale bar = 200 µm. (L) Internalization of EVPs by RAW264.7 macrophages. Scale bar = 200 µm. (M) mRNA expression for senescence marker ( Cdkn1a ), M1 polarization‐related genes ( Il6 , Tnf , Nos2 and Ptgs2 ), and M2 polarization‐related genes (Arg1 , Cd163 and Cd206 ) of RAW264.7 macrophages after co‐culturing with different EVPs for 24 h. (N–Q) Representative images and quantification of iNOS and P16 fluorescence staining for RAW264.7 macrophages after stimulation with different EVPs for 48 h. Scale bar = 50 µm. (R, S) Concentration of TNF‐α and IL‐6 in the cell culture supernatant of EVP‐stimulated RAW264.7 macrophages. ** Indicates p < 0.01, * indicates p < 0.05, ns indicates p > 0.05, versus the indicated groups, one‐way ANOVA.

Article Snippet: For EVP labelling and uptake studies, isolated EVPs (2 × 10 10 particles/mL in PBS) were incubated with 40 nM MemGlow dye (MG02, Cytoskeleton) at room temperature, protected from light.

Techniques: In Vitro, Fluorescence, Staining, Western Blot, Expressing, TUNEL Assay, Marker, Concentration Assay, Cell Culture

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Blockage of Pathological Extracellular Vesicles and Particles From Fibroblast‐Like Synoviocytes for Osteoarthritis Relief: Proteomic Analysis and Cellular Effect

doi: 10.1002/jev2.70162

Figure Lengend Snippet: Pathogenic FLS EVPs impair chondrogenic differentiation of mesenchymal stem cells. (A) Schematic diagram of EVP stimulation on mouse BMSCs isolated from the femoral bone marrow cavity of mice. (B) Internalization of EVPs by BMSCs. Scale bar = 50 µm. (C) Expression of chondrogenic differentiation‐related genes in chondrogenesis‐induced BMSCs after 7 days of treatment with different EVPs. (D) Representative Western blot images of COL10A1, the marker of chondrocyte hypertrophy. (E, F) Representative images of alcian blue staining and SA‐β‐gal staining in BMSCs after 7 days of stimulation with different EVPs. Scale bar = 1 mm and 200 µm separately. (G) Schematic diagram of section staining observation after inducing BMSCs to form chondrocyte pellets for 21 days while simultaneously stimulating with different EVPs. (H, I) Representative images of SOX9 fluorescence staining, safranin O (SO) staining, alcian blue (AB) staining and toluidine blue (TB) staining of BMSC‐differentiated chondrocyte pellet sections. Scale bar = 50 µm. (J) Schematic diagram of EVP treatment on mouse ADSCs isolated from the iWAT of mice. (K) Internalization of EVPs by ADSCs. Scale bar = 50 µm. (L, M) Representative images of SA‐β‐gal staining and alcian blue staining of ADSCs after 7 days of chondrogenic induction and treatment with different EVPs. (N) Representative images of SOX9 staining in sections of chondrocyte pellets formed by ADSC after 21‐day induction.

Article Snippet: For EVP labelling and uptake studies, isolated EVPs (2 × 10 10 particles/mL in PBS) were incubated with 40 nM MemGlow dye (MG02, Cytoskeleton) at room temperature, protected from light.

Techniques: Isolation, Expressing, Western Blot, Marker, Staining, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Blockage of Pathological Extracellular Vesicles and Particles From Fibroblast‐Like Synoviocytes for Osteoarthritis Relief: Proteomic Analysis and Cellular Effect

doi: 10.1002/jev2.70162

Figure Lengend Snippet: Intra‐articular injection of FLS‐targeting AAV for delivering Rab27a ‐shRNA to specifically reduce EVP secretion. (A) Rab27a expression levels in FLSs after transfection with control shRNA and Rab27a knockdown shRNA. (B, C) EVP size distribution and quantification after transfection with control shRNA and Rab27a knockdown shRNA. ** Indicates p < 0.01, versus the indicated groups, student’s t ‐test. (D) Representative Western blot images for positive and negative surface markers of EVPs at equal concentration after shRNA transfection. (E) Schematic diagram of constructing a virus targeting FLS to inhibit Rab27a for intra‐articular injection. The synovium‐affinity peptide HAP‐1 is fused with the AAV9 viral capsid viral protein 2 (VP2), and the AAV9 vector is designed to simultaneously carry the gene encoding the mScarlet fluorescent protein and an expression cassette for shRNA. (F) Representative images of mScarlet fluorescence in the synovium and cartilage regions of mouse knee joints. Scale bar = 200 µm and 100 µm separately. (G) Representative images of mScarlet fluorescence in sections of multiple mouse organs. Scale bar = 200 µm. (H) Representative images and quantitative results of RAB27A expression in the synovial region after intra‐articular AAV injection. ** Indicates p < 0.01, versus the indicated groups, two‐way ANOVA.

Article Snippet: For EVP labelling and uptake studies, isolated EVPs (2 × 10 10 particles/mL in PBS) were incubated with 40 nM MemGlow dye (MG02, Cytoskeleton) at room temperature, protected from light.

Techniques: Injection, shRNA, Expressing, Transfection, Control, Knockdown, Western Blot, Concentration Assay, Virus, Plasmid Preparation, Fluorescence

Journal: bioRxiv

Article Title: Astrocytes mediate the pro-cognitive value of α7nAChRs and of α7nAChR-targeting therapeutics

doi: 10.64898/2026.04.16.719027

Figure Lengend Snippet: A , Pharmacokinetic studies of EVP-6124 in the brain of wild-type male and female mice after i.p. administration (0.3 mg/kg in 0.36% DMSO in saline, i.p. at time 0). N.Q.: non quantifiable. B , Schematic of the NOR paradigm and EVP-6124 injection timeline. C , Dose-response curve of EVP-6124 (0.1, 0.3, 0.6 and 1.0 mg/kg) on the performance in the NOR relative to vehicle-treated cage-mates. Naïve: untreated animals. Vehicle: all vehicle-treated animals (n=97). The performance of EVP-6124-treated animals was normalized to that of their respective vehicle-treated cage mates (n = 16 to 34 per group). D, Left : Time spent interacting with the novel (n) and familiar (f) object during the probe phase of the NOR by mice treated with vehicle or 0.3mg/kg EVP-6124. Right : Computed object discrimination index and representative trajectories. E, F , same as D in control (E) and A-α7 KO (F) mice. G , Bar graphs summarizing the enhancing effect of EVP-6124, relative to vehicle treatment, in control and A-α7 KO mice. Unpaired Student’s t -test (D right, E right, F right, G), paired Student’s t -test (D left, E left, F left), Wilcoxon test (D left, F left), Kruskal-Wallis test with Dunn’s test (C) were used.

Article Snippet: EVP-6124 (EVP, MedChem Express, HY-15430A, 5 mg) was dissolved in DMSO into 10mg/mL or 50mg/mL stock solutions as needed.

Techniques: Saline, Injection, Control

Journal: Nature Communications

Article Title: Serotonin 2A receptor attenuates psoriatic inflammation by suppressing IL-23 secretion in monocyte-derived Langerhans cells

doi: 10.1038/s41467-025-63971-5

Figure Lengend Snippet: A Heatmap showing 140 differentially expressed genes (DEGs) between wild-type moLCs ( GSE222197 ) treated with Imiquimod and KO moLCs treated with Imiquimod ( GSE274941 ) with genes affected by NFκB shown on the right. The experiments generating the datasets of GSE222197 and GSE274941 were done in parallel. MoLCs were pooled from the ears of five mice treated with IMQ for two days, constituting a single biological sample. B Bar chart showing transcription factors enriched among the DEGs from ( A ) as analyzed using Generally Applicable Gene-set/pathway Analysis (GAGE) on the TRRUST Transcription Factor database. C Western blot of cultured WT and KO moLCs lysate treated with or without DOI and stained with anti-p100/p52, anti-RelB, and anti-GAPDH. D IL23p19 ELISA of 2 × 10 5 WT or KO moLCs (5 biological replicates) stimulated for 24 h with Imiquimod (10 μg/mL) and treated with either DOI (10 μg/mL) or EVP 4593 (0.1 mM). E Differentiation of Vγ4 T cells into IL-17 + IL-22+ cells after coculture with WT or KO moLCs (5 biological replicates) for 3 days. F Intracellular cytokine staining of IL23A of WT moLCs treated with Imiquimod (10 μg/mL) or CD40 stimulating antibody (CD40L) (1 μg/mL) with or without DOI (10 μg/mL) treatment. G Intracellular cytokine staining of IL23A of WT or KO moLCs, stimulated with Imiquimod (10 mg/mL) and treated with DOI (10 μg/mL) or B022 (5 μM). H Intracellular cytokine staining of IL23A of human moLCs (from 3 different individuals) treated with Imiquimod (10 μg/mL), serotonin (10 μM) or DOI (10 μg/mL). Data are a summary of two independent experiments ( C – G ). p values were determined by one-way ANOVA followed by Dunnett’s post-hoc test ( D , E , and H ) and one-way ANOVA followed by Tukey’s post-hoc test ( F , G ). Mean ± or SEM ( D – H ). Source data are provided as a Source Data file.

Article Snippet: LCs were stimulated with 10 μg/mL Imiquimod (Enzo, ALX-420-039-M100) and 10 μg/ml of DOI (Merck, D101) or 0.1 μM of EVP 4593 (MCE, HY-13812) were added for 24 h to investigate IL-23 expression and its ability to induce differentiation of Vγ4 T cells in a coculture assay.

Techniques: Western Blot, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: The Role of Extracellular Vesicles in the Control of Vascular Checkpoints for Cancer Metastasis

doi: 10.3390/cancers17121966

Figure Lengend Snippet: Convergence of EVP-mediated vascular regulatory pathways and the canonical angiogenesis signalling programs on tumour-associated endothelial cells. While endothelial cells are pre-programmed to respond to canonical cues triggered by VEGF, VEGF receptors (especially VEGFR2) and other physiological regulators (NRP, NOTCH, TIE2, integrins, VE-cadherin), cancer EVPs and EVPs from cancer-associated stromal cells insert additional elements into the endothelial cell signalling apparatus. For example, ectopic transfer of oncogenic EGFR may abort standard angiogenic responses and trigger alternative processes, such as vasectasia . It is not clear whether such ectopic EGFR signalling involves standard molecular intermediates and whether/how it intersects with the VEGF-driven angiogenesis pathway . Endothelial responses are also influenced by other cargo of EVPs (RNA, DNA, proteins; see text). Collectively, these changes impact the involvement of the vasculature at different steps of cancer progression and metastasis.

Article Snippet: The role of EVPs in tumour–vascular interaction also includes angiocrine effects [ , ].

Techniques: