Serum-derived extracellular vesicles (EVs) impact on vascular remodeling and prevent muscle damage in acute hind limb ischemia

Figure 1. In vitro and in vivo angiogenesis in response to sEVs. (A) In vitro quantitative analysis of the % of vessel-like structures formed (tubulogenesis assay) in response to the indicated stimuli. Untreated ECs was the negative control, VEGF (10 ng/ml) was the positive control, while e-sEVs (samples # 12, 14, 15, 17) and i-sEVs (samples # 11, 13, 16, 18) were the stimuli. (Original magnification: ×200; scale bar: 500 μm). (B) In vivo quantitative analysis of vessels counted in 10 sections of matrigel for each experimental condition. Data represent the mean value of untreated (C) (n = 6) or treated mice with: VEGF (n = 6), e-sEVs (samples # 12, 14, 15, 17) (n = 6 each sample) and i-sEVs (samples # 11, 13) (n = 6 each sample). ECs (red), erythrocytes (yellow) and matrigel (light blue) staining in matrigel plugs. Images above the graphs are representative of each condition. (Original magnification: ×20). Arrows indicate vessels. *p < 0.05; **p < 0.01, (One-way ANOVA followed by Tukey’s post hoc test). (Original magnification: ×200; scale bar: 12 μm).

Figure 2. e-sEVs increase blood perfusion and reduced damage in ischemic hind limbs. (A) Representative images of Laser Doppler Blood Flow (LDBF) obtained prior (pre-surg) and immediately after intervention (T0) and at day 7 after surgery (T7) of mice treated with Vehicle, with e-sEVs and i-sEVs. (B) Quantitative analysis of blood perfusion measured by LDBF. Data are expressed as mean ± SD; *p < 0.05 (One-way ANOVA - Newman-Keuls Multiple Comparison Test) (n = 10). (C) Foot damage score was evaluated for the indicated times as reported in Methods. Data are expressed as mean ± SEM, n = 10 (*p < 0.05 ischemic limb of e-sEVs mice vs ischemic limb of vehicle and i-sEVs mice).

Figure 3. e-sEVs increase capillary density and protect muscle from ischemic damage. (A) Representative hematoxylin-eosin images of gastrocnemius muscles of non-ischemic (Normal hind limb) and ischemic hind limb of Vehicle, e-sEVs and i-sEVs treated animals (Original Magnification: ×200; scale bar: 50 µm), (upper panels). Representative immunofluorescence images (confocal microscopy) of capillary density staining. An anti-CD31 mAb was used to analyze gastrocnemius muscles of non-ischemic (Normal hind limb) and ischemic hind limbs as indicated (Original Magnification: ×400; scale bar: 25 µm) (lower panels). (B) Quantitative analysis of muscle damage areas in hematoxilin-eosin stained gastrocnemius muscles of normal and ischemic hind limbs at day 7 after surgery. e-sEVs significantly reduce muscle damage compared to Vehicle. Data are expressed as mean ± SEM; *p < 0.05 e-sEVs vs Vehicle, (One-way ANOVA - Newman-Keuls Multiple Comparison Test), (left panel) (n = 10). Quantitative analysis of capillary density of normal and ischemic hind limbs at day 7 after surgery indicates that e-sEVs significantly increase capillary density compared to Vehicle. Data are expressed as mean ± SEM; *p < 0.05,…

Figure 4. Inflammatory infiltrate in ischemic hind limb. (A) Representative hematoxylin-eosin images of gastrocnemius muscles of non-ischemic (Normal hind limb) and ischemic hind limbs of Vehicle, e-sEVs and i-sEVs treated animals (Original Magnification: ×400; scale bar: 25 µm), showing mononuclear and polymorphonuclear cells. (B) Representative immunofluorescence images of gastrocnemius muscles, stained with an anti CD14 antibody (Original Magnification: ×400; scale bar: 25 µm). (C) Quantitative analysis of muscle damage areas in hematoxylin-eosin stained gastrocnemius muscles of the normal and ischemic hind limbs at day 7 after surgery. e-sEVs significantly reduce the number of inflammatory cells compared to the control (vehicle). Data are expressed as mean ± SEM; *p < 0.05, e-sEVs vs Vehicle, (One-Way ANOVA - Newman-Keuls Multiple Comparison Test) (n = 6).

Figure 5. e-sEV angiogenic mRNA and protein content. (A) Venn diagram to show the first 11 angiogenic mRNAs and proteins expressed by e-sEVs. TGFβ1 was commonly detected as mRNAs and proteins. Data were analyzed by Funrich V3 tool. (B) RT-PCR was used to detect TGFβ1 mRNA in e-sEV cargo. RT-PCR data are expressed as relative quantification (RQ) ± SD (*p < 0.05) (n = 4). (C) e-sEV were analyzed by Western Blot analysis for TGFβ1 content and normalized to CD63 (n = 4) (***p < 0.001). Mesangial cells were used as positive control (+). (D) Network analysis of pathways positively correlated with e-sEV transcripts and proteins. Pathways positively correlated are reported in the boxes. Data were obtained by GeneMANIA analysis performed on Cytoscape.

Figure 6. Gene pattern and representative biological pathways associated with e-sEV and i-sEV administration. (A) Representative Venn diagram of up-regulated and down-regulated mRNA in ECs treated with e-sEVs and i-sEVs. 43 genes up-regulated after e-sEV treatment were down-regulated upon i-sEV treatment; 12 genes down-regulated after e-sEV treatment were up-regulated after i-sEV treatment; 20 genes up-regulated upon e-sEV treatment were up-regulated upon i-sEV treatment and 9 genes down-regulated after e-sEV treatment were down-regulated upon i-sEV treatment. The list of genes is reported in Supplementary Table S4. (B) GO analysis on significantly over-expressed genes induced by e-sEV treatment (genes with a fold increase ≥3 were included). The significant enrichment of TGFβ1-associated pathway was identified. (C) The histogram represents the comparison of GO signaling pathways related to genes reported in the Venn diagram and Supplementary Tables S3 and S4. Columns represent the % of gene clusters involved in each signaling pathway.

Figure 7. e-sEVs, but not i-sEVs, induce SMAD1-5 activation and lead to the formation of a SMAD1/5/ID1 transcriptional complex. (A) Representative SMAD1-5 phosphorylation. ECs treated for the indicated time with TGFβ1, e-sEVs and i-sEVs were lysated and analyzed by WB for p-SMAD 1/5, SMAD 1/5 and normalized to β-actin content. The results are representative of four different experiments (n = 4) **p < 0.01; ***p < 0.001 (One-way ANOVA followed by Tukey’s multicomparison test). (B) ChIP analysis of SMAD 1/5 binding to the ID1 promoter region using ECs treated for four hours with e-sEVs and i-sEVs and 15 minutes with TGFβ1. RQ values of RT-PCR are reported. Selected siRNAs for SMAD1-5 (see Supplementary Fig. S4) were transfected for 48 hours and ChIP analysis was performed as described above. ECs transfected with duplex siRNAs, alone or in combination with e-sEVs, were used as controls. (n = 4) *p < 0.05 C vs TGFβ1 and e-sEVs treatment (One-way ANOVA followed by Tukey’s multicomparison test). (C) In vitro quantitative analysis of the % of vessel-like structures formed (tubulogenesis assay) upon siRNA SMAD duplex transfection alone or in combination with e-sEVs. (n = 4). **P < 0.01 e-sEVs vs…