FN matrix stimulates sEV secretion by VSMCs In Vitro.

A, Immobilised FN but not a soluble FN promotes sEV secretion. Cells were cultured for 24h and sEVs in conditioned media were measured by CD63-beads assay. N=4 with technical triplicates. ANOVA, ****p<0.0001 B, Micrograph showing VSMC plated onto plastic or 3D matrix. C, VSMCs were plated on the 3D matrix for 24h, fixed and labelled for Syndecan-4 (green), F-actin (phalloidin, red) and fibronectin (blue) Size bar, 10µm. D, 3D matrix promote sEV secretion. Cells were cultured for 24h and conditioned media was collected and sEV secretion was measured by CD63-beads assay. N=3, T-test E, FN matrix does not affect sEV mode size. VSMCs were plated on non-coated or FN-coated flasks and incubated for 24h. Isolated sEVs were analysed by Nanoparticle Tracking Analysis. Representative from N=3. F, sEV and EV markers distribution is not altered by FN matrix. Cells were plated on non-coated or FN-coated flasks and AB, EV and Apoptotic bodies (AB, 1.2K pellet), extracellular vesicles (EV, 10K pellet) and small extracellular vesicles (sEVs, 100K pellet) were isolated by differential ultracentrifugation and analysed by western blotting. Equal protein load. Representative image from N=3. G, FN induces secretion of sEVs by activating β1 integrin. VSMCs were plated on non-coated or FN-coated plates in the absence or presence of integrin activating (12G10) or inhibiting (4B4) antibodies for 24h and conditioned media was analysed by CD63-bead assay. N=3, ANOVA, ****p<0.0001 H, Src is required for the sEV secretion. Cells were plated and sEV secretion was measured as in 2A. N=3, ANOVA, **p<0.0001 I, Inhibition of FAK blocks FN-induced sEV secretion. Cells were plated and sEV secretion was measured as in 1A. N=3, ANOVA

sEV secretion depends in branched actin cytoskeleton and occurs via filopodia.

A, Formin inhibitor, SMIFH2 blocking filopodia formation reduces sEV secretion from FN-plated cells only. Cells were plated onto non-coated or FN-coated plates for 24h and conditioned media was analyzed by CD63-bead assay. N=3, ANOVA B, Arp2/3 inhibition with CK666 reduces sEV secretion in VSMC independently on FN plating. Cells were plated onto non-coated or FN-coated plates for 24h and conditioned media was analyzed by CD63-bead assay. N=3, ANOVA C, CD63 MVBs (arrows) are detected in filopodia-like structures. VSMCs were plated on FN-coated plate for 24h and cells were stained for Myo10 (green), CD63 (blue), F-actin (phalloidin, red). Size bar, 10µm. F, Still images of a time-lapse showing that MVBs are transported to the filopodia tip in the live VSMC. Cells were co-transfected by CD63-RFP and Myo10-GFP, cultured for 24h and time-lapse video was captured using confocal spinning disk microscopy. Snapshots were taken at T=3s and T=4s after start of the video. E, Still images of a time-lapse showing that transported MVBs are attached to F-actin tails. VSMCs were co-transfected with CD63-GFP and F-tractin-RFP and time-lapse was captured using confocal spinning-disk microscopy. Arrow head – position of CD63 MVB across time. Time, min. Size bar, 10µm.

Endogenous and exogenous sEVs are trapped by ECM In Vitro.

A, VSMC were plated onto dish not coated (top) or coated with FN (bottom) for 24h and stained for the membrane sEV marker, CD63 and actin. Note an accumulation of CD63 puncta in particular in close proximity to filopodia-like cell projectiles. Size bar, 10µm. B, VSMC were plated on the FN coated dish and Alexa568-labelled sEV were added to the cell media for 3h. Cells were fixed and stained for filopodia marker Myo10 (green) and vinculin (blue). Note perinuclear localisation of internalised sEVs. Size bar, 10µm. Representative image from N = 3. C, VSMC were plated on the FN coated dish pre-coated with Alexa568-labelled sEV and incubated for 24h. Cell staining as in Fig 3B. Note even distribution of sEVs across the matrix and cell area. D, VSMC were plated on the FN coated dish in the absence of Alexa568-labelled sEV and incubated for 24h. Cell staining as in Fig 3B. Note the absence of signal in sEV channel.

Fibronectin deposition in the atherosclerotic plaque is spatially associated with sEV markers.

A, Proteomic profiling of pairwise collected carotid atherosclerotic plaques and adjacent intact arterial segments. Venn diagram shows the number of plaque-specific (n = 213) and intact-specific (n = 111) proteins as well as the number of proteins which are common for both vascular regions (1,368). B. Partial least-squares discriminant analysis indicates clear classification pattern between the carotid plaques (indicated by red triangles) and adjacent intact arterial segments (indicated by blue circles). N = 14. C. Volcano plot illustrates that 46 proteins are significantly overexpressed in plaques whilst 13 proteins are significantly upregulated in adjacent intact arterial segments. D, Manders’ split colocalization coefficient for the overlap of FN with CD81 (M1) and CD81 with FN (M2). Neointima region as in Fig. 4E. E, Atherosclerotic plaques were co-stained for fibronectin (FN) and sEV marker, CD81. Cell nuclei were counterstained with DAPI. Main figure: x200 magnification, size bar, 50 µm. Box: x400 magnification, size bar, 15µm. Note an accumulation of FN in the neointima. F, Spatial distribution of FN and CD81 in the neointima. Note high overlap between FN and CD81 in the extracellular matrix. x200 magnification, size bar, 50 µm. G, Quantification of FN content in atherosclerotic plaques. Samples were analysed by western blot and bands intensity was quantified in ImageJ. Fold change was calculated as ratio of band intensity in the atherosclerotic plaque to band intensity in the adjacent intact arterial segments normalised to GAPDH. Note that FN content is elevated in atherosclerotic plaques relative to the adjacent intact arterial segments. Paired t-test. H, FN is presented on the surface of the VSMC-derived sEV along with α5β1 integrin. VSMC sEVs were immobilised on the 4µm beads. sEV-beads and VSMCs were stained with the antibodies (filled graphs) in non-permeabilised conditions and analysed by flow cytometry.

sEV induces directional VSMC invasion.

A, B VSMC migration in 2D assay. VSMC were plated onto FN in the absence or presence of SMPD3 inhibitor (3’-OMS) and/or sEV (25ug). Cells (n>900) were tracked for 4h. ANOVA, N=3. ****,p<0.0001 C. Chemotaxis µ-slide diagram. Yellow, cell chamber, blue chemoattractant-free medium chamber, dark blue – chemoattractant medium chamber. D-H, sEV promote directional VSMC invasion. Cells (n=600) were seeded to the FN-enriched Matrigel matrix in μ-Slide Chemotaxis assay and stained with Draq5. Cell tracking was conducted by OperaPhenix microscope for 12h and cell invasion parameters were quantified using Columbus. Kruskal-Wallis test, N=4, **, p<0.01 I-K, VSMCs were treated with control siRNA (Scramble) or collagen VI-specific siRNA pools for 24h and cell invasion was measure as in panel G-H, respectively. Kolmogorov-Smirnov test, *, p<0.05

sEV induces formation of peripheral FAs.

A, VSMC were plated on FN matrix for 30min and adhered cells were counted by crystal violet staining. N=6, ANOVA, ***p<0.001, ****, p<0.0001 B, C, VSMC spreading onto FN was tracked by using ACEA’s xCELLigence Real-Time Cell Analysis. Note that FN matrix promoted VSMCs adhesion but addition of sEVs inhibited cell spreading. N=3, ANOVA D, VSMCs spread onto FN for 30 min and cells were fixed and stained with CellMask (magenta) and vinculin (green). Size bar, 10µm. E, F, G, I Quantification of FA number, distance from plasma membrane, cell size and mean FA size per cell, respectively. FA were stained as in 5D and quantified. Representative data from N=3, ANOVA, *p<0.05, **p<0.01. J, Focal adhesion turnover is not affected by sEVs. VSMC were transfected with Paxillin-RFP and plated on the FN in the absence or presence of immobilised sEVs. Images were captured for 30min using confocal spinning disk microscopy and FA turnover was quantified using extracted images analysis, N=4, Unpaired T-test. K, sEV induces formation of strong-pulling FAs. VSMC transfected with Paxillin-RFP were plated on the PDMS pillars which were covered with FN and sEVs and pillar displacements were quantified. **p<0.01, Unpaired T-test, Representative data from N=2.

sEVs blocks focal adhesion formation by presenting collagen VI.

A, Proteomic analysis of VSMC-derived sEVs and EV. Venn diagram. N=3. B, Protein enrichment in the EV and sEV proteome. Heat Map. N=3. C, Western blot validation of sEV cargos. EV and sEV were isolated from VSMC’s conditioned media by differential ultracentrifugation and analysed by western blotting. Representative image from N=3. D, VSMC adhesion is regulated by collagen VI loaded to sEV. FN matrices were incubated with sEV and anti-collagen VI antibody (COLVI IgG) or control IgG. Cell adhesion was tracked by using ACEA’s xCELLigence Real-Time Cell Analysis. ANOVA, N=3. E, F, J, sEV promote directional VSMC invasion. VSMCs were treated with control siRNA (Scramble) or collagen VI-specific siRNA pools for 24h and were seeded to the FN-enriched Matrigel matrix in μ-Slide Chemotaxis assay and stained with Draq5. Cell tracking was conducted by OperaPhenix microscope for 12h and cell invasion parameters were quantified using Columbus. Kolmogorov-Smirnov test, *, p<0.05 I, Real-time PCR analysis of expression of CD9, CD63, CD81, COL6A3, EDIL3 and TGFBI in atherosclerotic plaque. *, p<0.05, Paired t-test, N=5.

FN matrix stimulates sEV secretion by VSMCs.

A, FN and collagen I but not laminin stimulate secretion of CD63-enriched exosomes. VSMCs were plated on the various matrices for 24h and sEV secretion was measured by CD63-beads assay. N=3, ANOVA **p<0.01 B, Inhibition of SMPD3 blocks sEV secretion by VSMC plated onto FN matrix. VSMCs were plated on non-coated or FN-coated plates for 24h and conditioned media was analysed by CD63-bead assay. N=2 with n=4 for each, ANOVA, ***, p<0.001, ****p<0.0001

A, sEV secretion detected with CD63-pHluorin. VSMCs were co-transfected with CD63-pHluorin and incubated for 24h. Time-lapse was captured using confocal spinning-disk microscopy. Arrows, typical “burst”-like appearance of sEV secretion at the cell-ECM interface. Arrows, an intense CD63-pHluorin staining along filopodia-like structures indicating that sEV release can occur in filopodia. B, Still images of a time-lapse showing that Arp2/3 and F-actin form tails in VSMC cytosol. VSMCs were co-transfected by ARPC2–GFP and F-tractin-RFP and cultured for 24h. Time-lapse video was captured using confocal spinning disk microscopy. Note, that Arp2/3 and F-actin are observed in lamellipodia but also detected in the cytosol with the unknown activity (arrow). Size bar, 10µm

SMPD3-dependent sEVs are trapped in ECM.

A. VSMCs were plated onto gelatin-covered plates and treated with control or SMPD3 siRNA for 72 hrs. 3D matrices were generated as described in “Materials and methods” and stained for CD63 and fibronectin. Images were acquired using Nikon AX inverted confocal microscope. Oil 60x Objective. Note the decrease in CD63-positive sEVs associated with the FN fibrils. (B) VSMC were plated on the FN-coated dish and Alexa568-labelled sEV were added to the cell media for 3 h. Cells were fixed and stained for filopodia marker Myo10 (green) and vinculin (blue). Note perinuclear localisation of internalised sEVs. Size bar, 10µm. 3D projection. Myo10 staining channel is removed. Representative image from N=3. C, VSMC were plated on the FN-coated dish pre-coated with Alexa568-labelled sEV and incubated for 24h. Cell staining as in Fig 3B. Note even distribution of sEVs across the extracellular matrix and cell area.

Fibronectin deposition in the atherosclerotic plaque is spatially associated with sEV marker CD81.

A. Expression of FN in atherosclerotic plaques. Atherosclerotic plaques (A) and adjacent intact arterial segments (I) were homogenised and analysed by Western blotting. FN is abundantly presented in both regions and 50 kDa FN fragment (Homandberg et al., 1992) intensity was used to quantify FN amount. Representative image for N=4 B. Quantification of FN content in atherosclerotic plaques. Heatmap for figure S4A. D, E, Exogenously added FN-Alexa555 can be detected in the early endosomes and MVBs. VSMC were incubated with FN-Alexa555 for 30min (D) or 3h (E) and stained for EEA-1 (D, green) or CD63 (E, blue). Size bar, 10um, F, FN is presented in sEV along with β1 integrin. Western blot analysis of isolated VSMC-derived sEVs.

sEVs regulate VSMC motility and invasion.

A, Centripetal FAs are linked to actin stress fibers. VSMCs were plated on FN-coated plate for 24h and cells were stained for Myo10 (green), CD63 (blue) and F-actin (phalloidin, red). Note the dot-like focal complexes in lamellipodium which are not associated with the contractile actin bundles (arrow) and an appearance of elongated FAs associated with the mature actin bundles (arrowhead). Dotted line, approximate position of the lamellipodium boundaries. Size bar, 10µm. B, Mature focal adhesion turnover is not affected by sEVs. VSMC were transfected with Paxillin-RFP and plated on the FN in the absence or presence of immobilised sEVs. Images were captured for 30min using confocal spinning disk microscopy. Note the appearance of the mature FAs in the lamellipodium (Arrowhead). Arrow, direction of the VSMC movement. N=4. C, sEV induces formation of FAs with the enhanced pulling force. VSMC transfected with Paxillin-RFP were plated on the PDMS pillars which were covered with FN and sEVs and pillar displacements were quantified. Representative image from N=2.

Proteomic analysis of VSMC-derived sEVs and EV. VSMC-derived EVs and sEVs were isolated from cells and analyzed by protein mass-spectrometry. N=3. A. Clustered proteomic heatmap for EV and sEV. B, GO functional enrichment analysis