Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: MSC-derived sEV treatment decreases clinical severity scores in murine sepsis (A) Diagram of murine cecal slurry model. Mice were injected with cecal slurry IP, to induce polymicrobial sepsis. At 6 h post-injection, MSC-derived sEVs or sEV-depleted media were administered via tail vein injection. When mice reached a sepsis score of 15 or above or at 24 h post-IP injection, the brain tissue was harvested. (B) MSC-derived sEV treatment ( n = 26) in mice 6 h after the onset of sepsis resulted in improved disease overall severity score as compared with the untreated septic mice ( n = 19) (∗∗∗ p = 0.0005) and lower peak scores (∗∗∗∗ p < 0.0001). (C) MSC-derived sEV treatment improved scores at 24 h in neurological-only parameters (i.e., level of consciousness, activity, response to stimulus) as compared with the untreated septic mice (∗ p = 0.01). Data are represented as mean ± SEM, one-way ANOVA. ANOVA: Analysis of Variance, IP: intraperitoneally, MSC-derived sEV: mesenchymal stem cell-derived small extracellular vesicles, NS: non-significant.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, Injection, Activity Assay

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: Sepsis-induced cerebellar injury is reversed by MSC-derived sEVs (A–D) Representative photomicrographs of H&E and TUNEL staining in the mouse cerebellum show significant histopathological alterations during sepsis. Compared to controls, which exhibited intact cellular architecture with clear, rounded nuclei, the septic mouse cerebellum displayed (A) significant histopathological alterations including shrunken PCs, pyknotic nuclei (black arrows), perineuronal vacuole formation (Materials and Methods: Tissue processing and histological assessment) and (B) increased TUNEL labeled cells (white arrows) indicating DNA fragmentation and cell death. Overall, sepsis resulted in (C) increased neuropathological score and (D) TUNEL+ cells (∗∗∗∗ p < 0.0001 and ∗∗∗∗ p < 0.0001) which both improved with MSC-derived sEV treatment (∗ p = 0.0155 and ∗∗ p = 0.0063). Data are represented as mean ± SEM, one-way ANOVA. Scale bar = 10 μm, Control ( n = 7), Sepsis+media ( n = 8), Sepsis+MSC-derived sEVs ( n = 8). ANOVA: Analysis of Variance, DNA: Deoxyribonucleic Acid, H&E: Hematoxylin and Eosin, NS: non-significant, PC: Purkinje cells, TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling, MSC-derived sEV: mesenchymal stem cell-derived small extracellular vesicles.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, TUNEL Assay, Staining, Labeling, Control, End Labeling

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: RNA-seq reveals MSC-derived sEV-induced changes in cerebellar transcriptome following sepsis (A and B) Predicted activated (green) and inhibited (gray) causal networks and canonical pathways (determined by directional z-scores) in septic mice treated with MSC-derived sEVs compared to untreated septic mice. Ranked based on p value as determined using Fisher’s exact test. (C) Predicted increases (green) and decreases (gray) in cell and molecular functions (determined by directional z-scores) in septic mice treated with MSC-derived sEVs compared to untreated septic mice. Ranked based on p value as determined using Fisher’s exact test. (D) Predicted activated (green) and inhibited (gray) upstream regulators (determined by directional z-scores) in in septic mice treated with MSC-derived sEVs compared to untreated septic mice. Ranked based on p value as determined using Fisher’s exact test. (E) Heatmap of most significantly predicted upstream regulators when septic mice treated with MSC-derived sEVs are compared to untreated septic mice. Boxes are colorized with z-scores (green = activated, gray = inactivated). Source data are provided as a source data file.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: RNA Sequencing Assay, Derivative Assay

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: MSC-derived sEVs affect cytokine concentration in septic mouse cerebellum TNF-α and IL-17α assessed by immunofluorescence. (A) Representative photomicrograph of TNF-α, PV and IL-17α staining in low (x1.4, left) and high (×60, right) magnification of the area included in the hatched box. TNF-α was expressed and co-localized with the PC and their dendrites. (B and C) TNF-α expression significantly increased in SE compared to controls (5.2 ± 1.1 vs. 1.4 ± 0.2, p = 0.0085), however, treatment with MSC-derived sEVs restored its expression by more than 50% (1.9 ± 0.2, p = 0.03). Notably, TNF-α was not expressed in parvalbumin (PV)+ interneurons (a and c) that surround the PCs. (D) The expression of IL-17α was similar in control and septic mice (5.62E+09 ± 2.7E+08 vs. 7.8E+09 ± 3.7E+08, p = 0.1096), however, treatment with MSC-derived sEVs doubled its expression (1.52E+10 ± 1.3E+09, p =<0.0001). Data are represented as mean ± SEM, one-way ANOVA. Scale bar = 10 μm, Control ( n = 6), Sepsis+media [for TNF-α ( n = 7) and IL-17α ( n = 8)], Sepsis+MSC-derived sEVs [(for TNF-α ( n = 5) and IL-17α ( n = 6)]. MSC-derived sEV: mesenchymal stem cell-derived small extracellular vesicles, NS: non-significant, TNFα: tumor necrosis factor alpha, IL-17α: interleukin 17 alpha, ANOVA: Analysis of Variance, PV: parvalbumin.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, Concentration Assay, Immunofluorescence, Staining, Expressing, Control

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: MSC-derived sEVs restore basal and non-mitochondrial respiration in septic mouse cerebellum Cellular respiration measured with Seahorse technology in mice. (A) Basal respiration decreases in sepsis [236.1 ± 19.1 vs. 311.0 ± 38.2 (∗ p = 0.0226)], but significantly improves with MSC-derived sEV treatment (332.4 ± 40.4, ∗ p = 0.0337 ) to levels similar to controls (311.0 ± 38.2, p = 0.3146 ) . (B) Cerebellar tissue shows lower maximum respiration in sepsis that is trending higher with MSC-derived sEV administration but did not reach significance ( p = 0.07). (C) Non-mitochondrial respiration i.e., OCR attributable to ROS production or pentose phosphate pathway increases under septic conditions [268.2 ± 36.4 vs.127.3 ± 19.3 (∗∗∗ p = 0.0051)], but not with treatment (79.3 ± 8.6, ∗∗∗ p = 0.0048), indicating that MSC-derived sEVs favor OXPHOS-linked ATP production. (D) Although the average ATP-linked respiration showed improvement with sEV treatment, there was no statistically significant difference among the groups, likely due to the short period of time in which observations occurred. Data are represented as mean ± SEM, one-way ANOVA. Control+media ( n = 5), Control+MSC-derived sEVs ( n = 5), Sepsis+media ( n = 5), Sepsis+MSC-derived sEVs ( n = 5). OCR: Oxygen consumption rate, ROS: reactive oxygen species, OXPHOS: oxidative phosphorylation, ATP: adenosine triphosphate, MSC-derived sEV: mesenchymal stem cell-derived small extracellular vesicles, NS: non-significant.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, Control

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: MSC-derived sEVs alter the activation of miRNAs in the septic cerebellum (A) Predicted inhibited (gray) miRNAs (determined by directional z-scores) in septic mice compared to controls. Ranked based on p value as determined using Fisher’s exact test. Most miRNAs of interest are inhibited, indicating that they do not have any predicted inhibitory effects on their target mRNA. (B) Predicted activated (green) and inhibited (gray) miRNAs (determined by directional z-scores) in septic MSC-derived sEV-treated mice compared to untreated septic mice. Ranked based on p value as determined using Fisher’s exact test. Several miRNAs inhibited in septic mice that received sEV-depleted media were predicted to be activated in the septic mice that received MSC-derived sEVs. Source data are provided as a source d file.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, Activation Assay

Journal: Extracellular vesicles and circulating nucleic acids

Article Title: Milk-borne small extracellular vesicles: kinetics and mechanisms of transport, distribution, and elimination

doi: 10.20517/evcna.2023.25

Figure Lengend Snippet: Tissue distribution of milk sEVs in mice. (A) Distribution of bovine milk sEVs loaded with fluorophore (ATTO)-labeled miR-375 in Balb/c mice. Modified from Manca et al . with permission from the original publisher, Nature Springer . To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ ; (B) Accumulation of enhanced green fluorescence protein (eGFP)-positive milk sEVs in peripheral tissues and the small intestinal mucosa in wild-type (WT) pups fostered to exosome and cargo tracking (ECT) dams and nursed for 17 days. ECT mice secrete sEVs labeled with an eGFP fusion protein in milk. WT pups fostered to WT dams served as controls. From Zhou et al . with permission from the original publisher, Frontiers Media SA . To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ .

Article Snippet: The pharmaceutical industry has recognized the potential for using bovine milk sEVs to deliver anti-cancer drugs, as evidenced by a $1 billion licensing agreement between Roche, Inc. and PureTech Health, Inc. [ ] .

Techniques: Labeling, Modification, Fluorescence

Journal: bioRxiv

Article Title: Different RNA profiles in plasma derived small and large extracellular vesicles of Neurodegenerative diseases patients

doi: 10.1101/2020.11.23.390591

Figure Lengend Snippet: (a) In AD, of the 33 miRNAs found in SEVs and 13 in LEVs, 6 distribute to both, 4 upregulated-in green and 2 downregulated-in re;. (b) In FTD, of the 88 miRNAs in SEVs and 130 in LEVs, 34 were in common (32 upregulated and 2 downregulated); c) for ALS, of the 109 miRNAs in SEVs and 197 in LEVs, 67 were in common, 45 upregulated and 22 downregulated); d) in PD, of the 104 miRNAs found in SEVs and 109 in LEVs, 34 distribute to both, 30 upregulated and 4 downregulated. Differential miRNA expression analysis by DESeq2 (log2FC > 1, p-value<0.05).

Article Snippet: Figure S1: LEVs and SEVs characterization. a) Nanosight profile of LEVs and SEVs from plasma of a CTR, AD, PD, ALS, FTD patients; b and c) Representative images obtained by transmission electron microscopy (TEM) of LEVs and SEVs from plasma (Scale bar: 100 nm, 50 nm). d) Western Blot of LEVs and SEVs markers in LEVs and SEVs samples from one CTR, an ALS, AD, PD, FTD patients showed the presence of Annexin V only in LEVs pellet and Alix in SEVs fraction.

Techniques: Expressing

Journal: bioRxiv

Article Title: Different RNA profiles in plasma derived small and large extracellular vesicles of Neurodegenerative diseases patients

doi: 10.1101/2020.11.23.390591

Figure Lengend Snippet: (a) For FTD, of the 228 mRNA in SEVs and 114 in LEVs, 39 were in common (36 upregulated, green and 3 downregulated, red). (b) For ALS, of the 522 mRNA in SEVs and 124 in LEVs, 44 were in common (33 upregulated and 11 downregulated). Differential mRNA expression analysis by DESeq2 (log2FC > 1, p-value<0.05).

Article Snippet: Figure S1: LEVs and SEVs characterization. a) Nanosight profile of LEVs and SEVs from plasma of a CTR, AD, PD, ALS, FTD patients; b and c) Representative images obtained by transmission electron microscopy (TEM) of LEVs and SEVs from plasma (Scale bar: 100 nm, 50 nm). d) Western Blot of LEVs and SEVs markers in LEVs and SEVs samples from one CTR, an ALS, AD, PD, FTD patients showed the presence of Annexin V only in LEVs pellet and Alix in SEVs fraction.

Techniques: Expressing

Journal: Heliyon

Article Title: Prostate cancer small extracellular vesicles participate in androgen-independent transformation of prostate cancer by transferring let-7a-5p

doi: 10.1016/j.heliyon.2022.e12114

Figure Lengend Snippet: Characterization of LNCaP and LNCaP AI + F cells and sEVs. (A) CCK-8 assay of the proliferation of LNCaP cells and LNCaP-AI + F cells at 0, 24, 48, and 72 h in an androgen-deprived environment. (B) Flow cytometry analysis of the cell cycle distributions of LNCaP cells and LNCaP-AI + F cells at 72 h in an androgen-deprived environment. (C) Expression levels of AR and PSA proteins in LNCaP cells and LNCaP-AI + F cells determined by western blotting. Original gel data in Supplementary Materials (Figure S3). (D) Transmission electron microscopic images of sEVs isolated from LNCaP and LNCaP-AI + F cells. Scale bar:200 nm (E) Average size distribution of isolated sEVs. (F) Alix protein level was analysed by western blotting. (G) Flow cytometry analysis of the sEVs surface markers CD63 and CD81. Original gel data in Supplementary Materials (Figure S4). sEVs: small extracellular vesicles. Data were analyzed using t -test. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: All sEVs miRNAs primers were obtained from RiboBio (cel-miR-39 was used as a control).

Techniques: CCK-8 Assay, Flow Cytometry, Expressing, Western Blot, Transmission Assay, Isolation

Journal: Heliyon

Article Title: Prostate cancer small extracellular vesicles participate in androgen-independent transformation of prostate cancer by transferring let-7a-5p

doi: 10.1016/j.heliyon.2022.e12114

Figure Lengend Snippet: LNCaP-AI + F sEVs promote AIPC transformation. (A) Western blotting analysis and quantification of AR and PSA protein expressions in LNCaP cells co-cultured with LNCaP-AI + F sEVs for 24 h in an androgen-deprived environment. Original gel data in Supplementary Materials (Figure S5). (B) qRT-PCR analysis of the relative expression of AR and PSA mRNA in LNCaP cells co-cultured with LNCaP-AI + F sEVs for 24 or 48 h in an androgen-deprived environment. (C) CCK-8 assay of the proliferation of LNCaP cells co-cultured with 10 μg/mL, 20 μg/mL, 30 μg/mL LNCaP-AI + F sEVs for 0, 24, 48, and 72 h in an androgen-deprived environment. (D) Cell cycle distributions of LNCaP cells co-cultured with 20 μg/mL LNCaP-AI + F sEVs for 48 h were analysed by flow cytometry in an androgen-deprived environment. sEVs: small extracellular vesicles. Data were analyzed using t -test (A, D) and one-way ANOVA with multiple-comparisons test (B, C). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: All sEVs miRNAs primers were obtained from RiboBio (cel-miR-39 was used as a control).

Techniques: Transformation Assay, Western Blot, Cell Culture, Quantitative RT-PCR, Expressing, CCK-8 Assay, Flow Cytometry

Journal: Heliyon

Article Title: Prostate cancer small extracellular vesicles participate in androgen-independent transformation of prostate cancer by transferring let-7a-5p

doi: 10.1016/j.heliyon.2022.e12114

Figure Lengend Snippet: LNCaP sEVs can reverse AIPC transformation. (A) Western blotting analysis of AR expression in LNCaP-AI + F cells co-cultured with LNCaP sEVs for 6, 12, and 24 h in an androgen-deprived environment. Original gel data in Supplementary Materials (Figure S6). (B) CCK-8 assay of the proliferation of LNCaP-AI + F cells co-cultured with 10 μg/mL, 20 μg/mL, 30 μg/mL LNCaP sEVs for 0, 24, 48, and 72 h in an androgen-deprived environment. (C) The cell cycle distributions of LNCaP-AI + F cells co-cultured with 20 μg/mL LNCaP sEVs for 48 h analysed by flow cytometry in an androgen-deprived environment. sEVs: small extracellular vesicles. Data were analyzed using t- test (C) and one-way ANOVA with multiple-comparisons test (A, B). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: All sEVs miRNAs primers were obtained from RiboBio (cel-miR-39 was used as a control).

Techniques: Transformation Assay, Western Blot, Expressing, Cell Culture, CCK-8 Assay, Flow Cytometry

Journal: Heliyon

Article Title: Prostate cancer small extracellular vesicles participate in androgen-independent transformation of prostate cancer by transferring let-7a-5p

doi: 10.1016/j.heliyon.2022.e12114

Figure Lengend Snippet: Let-7a-5p was transferred by PCa sEVs. (A) Scatter plot of differentially expressed miRNAs between LNCaP sEVs and LNCaP-AI + F sEVs. Red: increased expression; green: decreased expression; grey: equally expression. (B) Heat map of the differentially expressed sEVs miRNAs between LNCaP sEVs and LNCaP-AI + F sEVs. Red: increased expression; green: decreased expression. (C) Relative expression of let-7a-5p in LNCaP sEVs and LNCaP-AI + F sEVs detected by qRT-PCR. (D) Relative expression of let-7a-5p in LNCaP cells and LNCaP-AI + F cells detected by qRT-PCR. (E) Confocal images of LNCaP cells co-cultured with LNCaP-AI + F sEVs for 48–72 h in an androgen-deprived environment. (DIL labelled sEVs; FITC labelled LNCaP cell membranes; DAPI labelled LNCaP cell nuclei) (F) qRT-PCR analysis of the expression of let-7a-5p in LNCaP cells co-cultured with LNCaP-AI + F sEVs for 24, 48 h in an androgen-deprived environment. (G) qRT-PCR analysis of the expression of let-7a-5p in LNCaP-AI + F cells co-cultured with LNCaP sEVs for 6, 12, 24 h in an androgen-deprived environment. sEVs: small extracellular vesicles.Data were analyzed using t - test (C, D) and one-way ANOVA with multiple-comparisons test (F, G). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: All sEVs miRNAs primers were obtained from RiboBio (cel-miR-39 was used as a control).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture

Journal: Heliyon

Article Title: Prostate cancer small extracellular vesicles participate in androgen-independent transformation of prostate cancer by transferring let-7a-5p

doi: 10.1016/j.heliyon.2022.e12114

Figure Lengend Snippet: sEVs encapsuling let-7a-5p regulate the PI3K/Akt signaling pathway. (A) Western blotting analysis of the p -Akt and Akt protein expression in LNCaP cells transfected with 50 nM let-7a-5p mimic for 24 and 48 h. Original gel data in Supplementary Materials (Figure S9). (B) Western blotting analysis of the p -Akt and Akt protein expression in LNCaP-AI + F cells transfected with 100 nM let-7a-5p inhibitor for 24 and 48 h. Original gel data in Supplementary Materials (Figure S10). (C) Western blotting analysis of the p -Akt and Akt protein expression in LNCaP cells co-cultured with LNCaP-AI + F sEVs for 6, 12, 24, 48 h. Original gel data in Supplementary Materials (Figure S11). (D) Western blotting analysis of the p -Akt and Akt protein expression in LNCaP-AI + F cells co-cultured with LNCaP sEVs for 6, 12, 24 h. Original gel data in Supplementary Materials (Figure S12).sEVs: small extracellular vesicles. Data were analyzed using one-way ANOVA with multiple-comparisons test. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: All sEVs miRNAs primers were obtained from RiboBio (cel-miR-39 was used as a control).

Techniques: Western Blot, Expressing, Transfection, Cell Culture

Journal: International Journal of Nanomedicine

Article Title: Advances in Engineered Nanoparticles for the Treatment of Ischemic Stroke by Enhancing Angiogenesis

doi: 10.2147/IJN.S463333

Figure Lengend Snippet: Summary of Nanoparticles for Stroke Treatment by Angiogenesis

Article Snippet: Small extracellular vesicles (sEVs) released from endothelial cells facilitated by ASM inhibitors , Biomimetic nanoparticles , sEVs , Ischemic site , IV , Cerebral microvascular endothelial cells were stimulated to produce and release small extracellular vesicles (sEVs) by the suppression of ASM, which subsequently facilitated angiogenesis , Increased angiogenesis by sEVs released from endothelial cells facilitated by ASM inhibitors as accompanied by brain remodeling response with increased BBB integrity , [ ] .

Techniques: Over Expression, Formulation, In Vitro, Activity Assay, Molecular Weight, Microinjection, Functional Assay, shRNA, Plasmid Preparation, Expressing, Adsorption, Permeability, Comparison, Cell Culture, Membrane, Disruption, Preserving, Liposomes, Derivative Assay, Generated, In Vivo, Control, Knock-Out, Injection

Journal: International Journal of Nanomedicine

Article Title: Advances in Engineered Nanoparticles for the Treatment of Ischemic Stroke by Enhancing Angiogenesis

doi: 10.2147/IJN.S463333

Figure Lengend Snippet: iMSC-sEV promote angiogenesis following stroke and increase HUVEC migration and tube formation after OGD. ( A – D ) Seven days after MCAO, angiogenesis was evaluated by immunofluorescence staining of CD31/EdU and CD34. Scale bar = 100 μm. * P < 0.05. Reproduced from Xia Y, Ling X, Hu G, et al. Small extracellular vesicles secreted by human iPSC-derived MSC enhance angiogenesis through inhibiting STAT3-dependent autophagy in ischemic stroke. Stem Cell Res Ther . 2020;11(1):313. Creative Commons.

Article Snippet: Small extracellular vesicles (sEVs) released from endothelial cells facilitated by ASM inhibitors , Biomimetic nanoparticles , sEVs , Ischemic site , IV , Cerebral microvascular endothelial cells were stimulated to produce and release small extracellular vesicles (sEVs) by the suppression of ASM, which subsequently facilitated angiogenesis , Increased angiogenesis by sEVs released from endothelial cells facilitated by ASM inhibitors as accompanied by brain remodeling response with increased BBB integrity , [ ] .

Techniques: Migration, Immunofluorescence, Staining, Derivative Assay

Journal: International Journal of Nanomedicine

Article Title: Advances in Engineered Nanoparticles for the Treatment of Ischemic Stroke by Enhancing Angiogenesis

doi: 10.2147/IJN.S463333

Figure Lengend Snippet: When PMNs are absent in vivo, sEVs produced from hypoxic MSCs promote microvascular remodeling after ischemic stroke. ( A – C ) The microvascular network properties in the ischemic cerebral cortex were examined using 3D light sheet microscopy in mice that were subjected to 40 minutes of MCAO and then survived for 14 days. A, microvascular length density; B, branch point density; and C, mean branch length. ( D ) Magnified axial views of the striatum and cortex in an ischemic mouse brain showing the regions of interest for determining the properties of the microvascular network. Images of the ischemic cortex taken using maximum intensity projection ( E ) from each of the five groups. * p <0.05 compared with isotype/vehicle; # p <0.05 compared with isotype/sEV hypoxic . Scale bars=500 µm (in ( D )/100 µm (in ( E ). Reproduced from Gregorius J, Wang C, Stambouli O, et al. Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice. Basic Res Cardiol . 2021;116(1):40. Creative Commons.

Article Snippet: Small extracellular vesicles (sEVs) released from endothelial cells facilitated by ASM inhibitors , Biomimetic nanoparticles , sEVs , Ischemic site , IV , Cerebral microvascular endothelial cells were stimulated to produce and release small extracellular vesicles (sEVs) by the suppression of ASM, which subsequently facilitated angiogenesis , Increased angiogenesis by sEVs released from endothelial cells facilitated by ASM inhibitors as accompanied by brain remodeling response with increased BBB integrity , [ ] .

Techniques: In Vivo, Produced, Microscopy

Journal: Journal of Extracellular Biology

Article Title: Chemo‐small extracellular vesicles released in cisplatin‐resistance ovarian cancer cells are regulated by the lysosomal function

doi: 10.1002/jex2.157

Figure Lengend Snippet: CDDP promotes the secretion of small EVs in A2780cis CDDP‐resistant OvCa cells, with chemoresistance transference capacity. (a) Analysis of detergent‐soluble protein extracts by Western blot from cell lysates and small EVs (sEVs) obtained by differential ultracentrifugation (dUC) from 72 h supernatant medium (SM) of A2780 (sEVs‐A2780), A2780cis (sEVs‐A2780cis) and A2780cis cells treated with 1 μM CDDP during 72 h (sEVs‐A2780cis+CDDP). To Western blot, we used anti‐CD63, anti‐TSG101, anti‐Syntenin, anti‐GM130 and anti‐GRP94. * Indicate unspecified band. (b) Nano‐tracking analysis (NTA) of sEVs‐A2780, sEVs‐A2780cis and sEVs‐A2780cis+CDDP obtained by dUC. The quantifications represent the distribution size of the sEVs calculated by the mean concentration of the sEVs of each size ( n = 5. Five independent experiments). (c) Representative images of TEM micrograph of sEVs‐A2780, sEVs‐A2780cis and sEVs‐A2780cis+CDDP, obtained by dUC. ScaleBar 0.5 μm. (d) Analysis of the number of sEVs‐A2780, sEVs‐A2780cis and sEVs‐A2780cis+CDDP per 10 6 cells obtained by dUC. Bars indicated the mean with SEM; ** p < 0.01, *** p < 0.001, NS: not significant; one‐tiled, unpaired, non‐parametric Man‐Whitney test ( n = 5. Five independent experiments). (e,f) Live/Dead cell stain analysis by (e) flow cytometry and (f) trypan blue analysis of A2780 cells treated with 3 μM CDDP for 48 h after 16 h stimulation with PBS (Non‐sEVs) or with sEVs‐A2780, sEVs‐A2780cis and sEVs‐A2780cis+CDDP obtained by dUC. The analysis represents the percentage of A2780 cells dead for each condition. Bars indicated the mean with SEM; * p < 0.05, ** p < 0.01, NS: not significant; one‐tiled, paired, non‐parametric Mann‐Whitney test ( n = 3).

Article Snippet: The cell‐cell communication supported by sEVs is associated with the transference of messenger RNAs (mRNAs), miRNAs, DNAs, lipids and proteins (Steinbichler et al., ).

Techniques: Western Blot, Concentration Assay, Staining, Flow Cytometry, MANN-WHITNEY

Journal: Journal of Extracellular Biology

Article Title: Chemo‐small extracellular vesicles released in cisplatin‐resistance ovarian cancer cells are regulated by the lysosomal function

doi: 10.1002/jex2.157

Figure Lengend Snippet: Induction of lysosomal biogenesis by rapamycin prevents CDDP effect on A2780cis CDDP‐resistant OvCa cells in the secretion of sEVs and chemoresistance transference capacity. (a) Analysis of detergent‐soluble protein extracts obtained from A2780cis control, treated with 1 μM CDDP, 100 nM rapamycin (RAPA) or 1 μM CDDP (Cisplatin) plus 100 nM RAPA (rapamycin) during 72 h, by Western blot with anti‐S6K, anti‐S6K (T389) and β ‐actin. (b) Densitometric quantification of the signal of p‐S6K (T389) with respect to total S6K. Bars indicated the mean with SEM; Control samples were normalized to 1 since the biological replicates were run in different Western blot; * p < 0.05, NS: not significant; one‐tiled, paired, non‐parametric Mann‐Whitney test ( n = 3. Three independent experiments). (c) Confocal microscopy images of A2780cis control, treated with 1 μM CDDP, 100 nM RAPA or 1 μM CDDP plus 100 nM RAPA during 72 h. PFA‐fixed cells immunofluorescent stained with anti‐LAMP1 and anti‐Cathepsin D. Scale Bar 10 μm. (d,e) Analysis of average intensity of structures of LAMP1 (d) and CathD per cell (e) from images as those shown in (c). Bars indicated the mean with SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, NS: not significant; one‐tiled, unpaired, parametric t ‐test ( n > 50 cells, from three independent experiments). (f) NTA of sEVs obtained by dUC from 72 h CM of A2780cis (sEVs‐A2780cis), A2780cis cells treated with 1 μM CDDP during 72 h (sEVs‐A2780cis+CDDP), treated with 100 nM RAPA during 72 h (sEVs‐A2780cis+RAPA), and treated with 1 μM CDDP plus 100 nM RAPA during 72 h (sEVs‐A2780cis+CDDP+RAPA). The quantifications represent the distribution size of the sEVs calculated by the mean concentration of the sEVs of each size ( n = 5). (g) Analysis of the number of sEVs‐A2780cis, sEVs‐A2780cis+CDDP, sEVs‐A2780cis+RAPA and sEVs‐A2780cis+CDD+RAPA per 10 6 cells of obtained by dUC. Bars indicated the mean with SEM; * p < 0.05, ** p < 0.01, NS: not significant; one‐tiled, unpaired, non‐parametric Mann‐Whitney test ( n = 5. Five independent experiments). (h,i) Live/Dead cell stain analysis by flow cytometry (h) and trypan blue analysis (i) of A2780 cells treated with 3 μM CDDP for 48 h after 16 h of stimulation with PBS (Non‐sEVs) or with sEVs‐A2780cis, sEVs‐A2780cis+CDDP, sEVs‐A2780cis+RAPA or sEVs‐A2780cis+CDD+RAPA obtained by dUC. The analysis represents the percentage of A2780 cells dead for each condition. Bars indicated the mean with SEM; * p < 0.05, NS: not significant; one‐tiled, paired, non‐parametric Mann‐Whitney test ( n = 3. Three independent experiments).

Article Snippet: The cell‐cell communication supported by sEVs is associated with the transference of messenger RNAs (mRNAs), miRNAs, DNAs, lipids and proteins (Steinbichler et al., ).

Techniques: Control, Western Blot, MANN-WHITNEY, Confocal Microscopy, Staining, Concentration Assay, Flow Cytometry