Journal: Journal of Extracellular Vesicles

Article Title: Pharmacological Inhibition of Small Extracellular Vesicle Secretion by ALK5i SD‐208 via Lysosomal Rerouting of CD63+ Compartments

doi: 10.1002/jev2.70362

Figure Lengend Snippet: SD‐208 treatment attenuates myofibroblast activation in primary human cardiac fibroblasts. (A) qRT‐PCR analysis of fibroblast activation markers in primary cardiac fibroblasts treated with SD‐208 (3 µM) or vehicle control for 96 h. Expression levels are normalized to B2M and shown relative to vehicle control. (B) Representative western blot analysis of α‐SMA and COL1A1 protein expression in cardiac fibroblasts treated with SD‐208 or vehicle control. GAPDH served as loading control. (C, D) Densitometric quantification of α‐SMA (C) and COL1A1 (D) protein levels normalized to GAPDH. (E) Flow cytometry analysis showing the percentage of α‐SMA‐positive cells following SD‐208 treatment. (F) Extracellular collagen secretion assay measuring total soluble collagen in conditioned media from SD‐208‐treated and control fibroblasts. (G) Representative immunofluorescence images of α‐SMA and fibronectin expression in cardiac fibroblasts treated with SD‐208 or vehicle control. Cells were counterstained with DAPI. Scale bar; 100 µm. (H, I) Quantitative analysis of α‐SMA (H) and fibronectin (I) fluorescence intensity normalized to cell number. All data represent mean ± SEM, n = 3 independent biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to vehicle control by unpaired t ‐test.

Article Snippet: Immortalised cardiac fibroblast (ICF, ventricular, Innoprot) were cultured in FM‐2 medium (ScienceCell) supplemented with 1% FGS‐2 (v/v), 1% P/S (v/v) and 5% FCS.

Techniques: Activation Assay, Quantitative RT-PCR, Control, Expressing, Western Blot, Flow Cytometry, Immunofluorescence, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Pharmacological Inhibition of Small Extracellular Vesicle Secretion by ALK5i SD‐208 via Lysosomal Rerouting of CD63+ Compartments

doi: 10.1002/jev2.70362

Figure Lengend Snippet: Isolation and characterisation of small extracellular vesicles from primary human cardiac fibroblasts. (A) Schematic of sEV isolation protocol using sequential centrifugation, ultrafiltration, and size exclusion chromatography. (B) Immunophenotyping showing enrichment of EV markers CD9, CD63, CD81, and depletion of calnexin. (C) Western blotting confirmed the presence of luminal sEV markers and depletion of cell abundant proteins in EV preparations. WCL: whole cell lysate. (D) Nanoparticle tracking analysis showing size distribution with modal diameter of 122 nm. (E) Transmission electron microscopy revealing characteristic EV morphology. Scale bar; 100 nm (main).

Article Snippet: Immortalised cardiac fibroblast (ICF, ventricular, Innoprot) were cultured in FM‐2 medium (ScienceCell) supplemented with 1% FGS‐2 (v/v), 1% P/S (v/v) and 5% FCS.

Techniques: Isolation, Centrifugation, Size-exclusion Chromatography, Western Blot, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Pharmacological Inhibition of Small Extracellular Vesicle Secretion by ALK5i SD‐208 via Lysosomal Rerouting of CD63+ Compartments

doi: 10.1002/jev2.70362

Figure Lengend Snippet: SD‐208 reduces EV secretion from primary cardiac fibroblasts without affecting cellular viability. (A) NTA showing reduced particle concentration per cell following SD‐208 treatment (3 µM, 96 h). (B) Protein content per particle in isolated EVs. (C, D) NTA generated histograms for particle size distribution and concentration for sEVs secreted by primary cardiac fibroblasts without (C) and with (D) SD‐208 treatment. (E) Modal EV size remains unchanged between control and SD‐208‐treated groups. (F‐H) Cell viability assessments: (F) metabolic activity (MTS assay), (G, H) viability (trypan blue exclusion), and proliferation (cell number). (I) Phase contrast microscopy showing cell morphology and density at 0, 48, and 96 h. Scale bar; 100 µm. (J) Confocal immunofluorescence of Ki‐67 proliferation marker. Scale bar; 200 µm. Data represent mean ± SEM, n = 3 independent biological replicates. * p < 0.05, ** p < 0.01 by unpaired t ‐test.

Article Snippet: Immortalised cardiac fibroblast (ICF, ventricular, Innoprot) were cultured in FM‐2 medium (ScienceCell) supplemented with 1% FGS‐2 (v/v), 1% P/S (v/v) and 5% FCS.

Techniques: Concentration Assay, Isolation, Generated, Control, Activity Assay, MTS Assay, Microscopy, Immunofluorescence, Marker

Journal: Journal of Extracellular Vesicles

Article Title: Pharmacological Inhibition of Small Extracellular Vesicle Secretion by ALK5i SD‐208 via Lysosomal Rerouting of CD63+ Compartments

doi: 10.1002/jev2.70362

Figure Lengend Snippet: SD‐208 reduces sEV secretion from immortalised cardiac fibroblasts. (A, B) Quantification of sEV secretion from ICFs treated with SD‐208 (3 µM, 96 h) compared to vehicle control. (A) Particle concentration per cell measured by nanoparticle tracking analysis (NTA) showing significant reduction with SD‐208 treatment. (B) EV protein content per cell demonstrating decreased protein secretion in SD‐208‐treated cultures. (C) Size distribution analysis of secreted sEVs. Representative NTA histograms showing particle size distribution for control (C) and SD‐208‐treated (D) cells. (E) Modal particle size showing no significant difference between groups. (F) Cell viability assessed by trypan blue exclusion. (G, J) Effects of TGF‐β stimulation on sEV secretion. (G) particle concentration per cell showing increased sEV secretion and (H) EV protein per cell following TGF‐β1 treatment (10 ng/mL, 96 h). (I) Representative NTA histogram for TGF‐β‐stimulated cells. (J) Modal particle size remains unchanged with TGF‐β treatment. Data represent mean ± SEM, n = 3 independent biological replicates. * p < 0.05, ** p < 0.01 unpaired t ‐test. ns, not significant.

Article Snippet: Immortalised cardiac fibroblast (ICF, ventricular, Innoprot) were cultured in FM‐2 medium (ScienceCell) supplemented with 1% FGS‐2 (v/v), 1% P/S (v/v) and 5% FCS.

Techniques: Control, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Pharmacological Inhibition of Small Extracellular Vesicle Secretion by ALK5i SD‐208 via Lysosomal Rerouting of CD63+ Compartments

doi: 10.1002/jev2.70362

Figure Lengend Snippet: SD‐208 induces intracellular accumulation of CD63 and upregulates lysosomal, autophagic, and mitochondrial markers. (A–E) Western blot analysis in HEK293 cells treated with SD‐208 (1.5 µM or 3 µM, 96 h). (A) Representative western blot for sEVs secreted by equal cell number showing dose‐dependent decrease in sEV CD63 levels following SD‐208 treatment. (B) Densitometric quantification of sEV‐associated CD63. (C) Representative western blot showing increased intracellular CD63 retention or accumulation in SD‐208‐treated cells. (D) Western blot analysis of LC3A/B, LAMP1, TOM20, and GAPDH in whole cell lysates. (E) Quantification of CD63, LAMP1, TOM20, and LC3A/B protein levels normalized to GAPDH. (F–J) Western blot analysis in immortalised cardiac fibroblasts treated with SD‐208 (3 µM, 96 h). (F) Representative western blot for sEVs secreted by equal cell number showing decrease in sEV‐associated CD63 levels following SD‐208 treatment. (G) Densitometric quantification of sEV‐associated CD63. (H) Representative western blot showing increased intracellular CD63 retention or accumulation in SD‐208‐treated cells. (I) Western blot analysis of LC3A/B, LAMP1, TOM20, and GAPDH with and without SD‐208 treatment. (J) Quantification of CD63, LAMP1, TOM20, and LC3A/B protein levels normalized to GAPDH as the housekeeping gene. Data represent mean ± SEM, n = 3 independent biological replicates. * p < 0.05, by unpaired t‐test or one‐way ANOVA with Tukey's post‐hoc test.

Article Snippet: Immortalised cardiac fibroblast (ICF, ventricular, Innoprot) were cultured in FM‐2 medium (ScienceCell) supplemented with 1% FGS‐2 (v/v), 1% P/S (v/v) and 5% FCS.

Techniques: Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Pharmacological Inhibition of Small Extracellular Vesicle Secretion by ALK5i SD‐208 via Lysosomal Rerouting of CD63+ Compartments

doi: 10.1002/jev2.70362

Figure Lengend Snippet: Quantitative colocalization analysis confirms preferential targeting of CD63+ vesicles to lysosomal compartments. (A, B) CD63‐LAMP1 colocalization in HEK293 cells. (A) Representative high‐resolution confocal images showing CD63 (green), LAMP1 (red), DAPI (blue), merged channels, and colocalization (yellow indicates overlap). Enlarged inset demonstrates extensive CD63‐LAMP1 colocalization in SD‐208‐treated cells. (B) Quantification of Manders' colocalization coefficients in control versus SD‐208‐treated (3 µM, 96 h) cells. Each data point represents one image containing >50 cells. (C‐E) CD63‐LAMP1 colocalization in immortalised cardiac fibroblasts. (C) Representative confocal images showing CD63 (green), LAMP1 (red), DAPI (blue), merged channels, and colocalization panel with enlarged inset. (D) Single‐cell quantification of CD63‐LAMP1 Manders' colocalization coefficients. Each data point represents one individual cell. (E) Image‐based quantification where each data point represents one image containing >15 cells. Scale bars; 50 µm (main images). Data represent mean ± SEM, n = 3 independent biological replicates. **** p < 0.0001 by unpaired t ‐test.

Article Snippet: Immortalised cardiac fibroblast (ICF, ventricular, Innoprot) were cultured in FM‐2 medium (ScienceCell) supplemented with 1% FGS‐2 (v/v), 1% P/S (v/v) and 5% FCS.

Techniques: Control, Single Cell

Journal: Journal of Extracellular Vesicles

Article Title: Pharmacological Inhibition of Small Extracellular Vesicle Secretion by ALK5i SD‐208 via Lysosomal Rerouting of CD63+ Compartments

doi: 10.1002/jev2.70362

Figure Lengend Snippet: Proposed model of SD‐208‐mediated regulation of fibroblast activation and extracellular vesicle trafficking. (1) SD‐208 exerts anti‐fibrotic effects through inhibition of TGF‐β receptor I (ALK5) signalling while also reducing sEV secretion in activated myofibroblasts. TGF‐β1 promotes fibroblast activation and enhances sEV secretion, whereas SD‐208 suppresses both processes. (2) SD‐208 impairs MVB docking at the plasma membrane, reducing exocytosis and sEV release. Retained MVBs are redirected to lysosomes for degradation, with enhanced lysosome biogenesis (LAMP1↑) and dysregulated autophagic flux (LC3A/B↑). ILVs, intraluminal vesicles; MVB, multivesicular body.

Article Snippet: Immortalised cardiac fibroblast (ICF, ventricular, Innoprot) were cultured in FM‐2 medium (ScienceCell) supplemented with 1% FGS‐2 (v/v), 1% P/S (v/v) and 5% FCS.

Techniques: Activation Assay, Inhibition, Clinical Proteomics, Membrane