Local delivery of USC-derived exosomes harboring ANGPTL3 enhances spinal cord functional recovery after injury by promoting angiogenesis

Figures

• 

  Fig. 1. Characterization of isolated USC-Exo. a Representative images of USC-Exo morphology detected by transmission electron microscopy (TEM). Scale bar, 100 nm. b Size distribution assessed by nanoparticle tracking analysis (NTA) using a NanoSight NS3000 system. c Western blot analysis of specific exosomal surface markers

• 

  Fig. 2. Locally delivered labeled USC-Exo home to the injured spinal cord site and promote locomotor and sensory function recovery. a Schematic representation of the procedure used to administer labeled USC-Exo. b In vivo imaging of animals that received a local intrathecal injection with/without DiR-labeled USC-Exo embedded in hydrogels in mice. c Immunofluorescence staining of the T10 spinal segment area in the sham (upper panel) and injured (lower panel) mice. CD31 (green), PKH26-Exo (red), nuclei (blue). Scale bar = 10 μm. d Quantitative PKH26-Exo mean signal intensity in the endothelial cells of the T10 spinal segment in sham and injured mice. e Distribution of the BMS scores per group throughout the 56-day period. f Distribution of sensory recovery per group at 0, 7, 14, 21, 28, and 56 days post-USC-Exo treatment. g Representative transverse HE stained sections 56 days post-SCI at distances 200 μm caudal to the injury epicenter. Scale bar = 1 mm. h Graphical representation showing the quantitative data comparing lesion cavities among different treatment groups. i Representative electrophysiological traces per group at 56 days post-SCI. j Quantification of the amplitude and latent…

• 

  Fig. 3. The administration of USC-Exo promotes angiogenesis after SCI. a Representative 3D image of spinal cord microvasculature at the T10 level per group at 56 days post-SCI visualized by SRμCT. Scale bars = 100 mm. b Quantification of the vascular volume fraction, vascular segment, and bifurcation numbers of the spinal cord among different treatment groups. c Representative immunofluorescence images of CD31 (green) and Ki67 (red) blood vessels in the mouse spinal cord in each group at 7 days post-SCI; scale bars = 50 μm. d Quantification of CD31 Ki67 double-positive cells in the spinal cords of mice among the different treatment groups. The data are presented as the means ± SD; n = 6 per group. *p < 0.05, **p < 0.01 compared among different treatment groups

• 

  Fig. 4. Internalization of USC-Exo by HUVECs promotes their angiogenic activities. a PKH26-labeled USC-Exo uptake by HUVECs was assessed by confocal microscopy. Scale bar, 20 μm. b Representative image of the Ki67 staining results after USC-Exo stimulation. Proliferative cells were stained red with Ki67. Scale bar, 20 μm. c After cultivation with USC-Exo, the number of CD31 Ki67 double-positive cells significantly increased. d Representative images of HUVEC tube formation in vitro after USC-Exo treatment. e Quantitative evaluation of the number of total loops, branch points, and total tube length after treating HUVECs with USC-Exo. f Representative images of HUVEC migration in the PBS and USC-Exo treatment groups in the scratch assay. g The percentage of migration area calculated for the USC-Exo or PBS treatment groups shown in f. The data are presented as the means ± SD; n = 6 per group. *p < 0.05, **p < 0.01 compared among different treatment groups

• 

  Fig. 5. Comparison of 45 different signal transduction pathways following USC-Exo treatment in HUVECs. a Pathway reporters are listed by log2 ratio fold-change values in HUVECs treated with USC-Exo compared to cells treated with PBS. The results revealed that the PI3K/AKT signaling cascade changed the most in HUVECs after treatment with USC-Exo. b The expression of PI3K/AKT signaling pathway components in HUVECs after treatment with USC-Exo was detected by Western blot analysis. c Representative images of HUVEC tube formation in vitro after USC-Exo treatment with added LY294002 and L-NAME. d Quantitative analysis of the number of total loops, branch points, and total tube length shown in c. e Representative images of HUVEC migration in the USC-Exo treatment groups with added LY294002 and L-NAME in the scratch assay. f The percentage of the migration area calculated from e. g Western blot analysis of phosphorylated and total PI3K/AKT levels in HUVECs upon combination LY294002 and L-NAME treatment. The data are presented as the means ± SD; n = 6 per group. *p < 0.05, **p < 0.01 compared among different treatment groups

• 

  Fig. 6. ANGPTL3 mediates the angiogenic effects of USC-Exo on HUVECs. a, b The mRNA and protein levels of ANGPTL3 were assessed by RT-qPCR and Western blot analysis. c Western blot analysis of ANGPTL3 in HUVECs after USC-Exo treatment. d Western blot analysis of ANGPTL3 in exosomes from ANGPTL3-silenced USC and confirmation of the inhibitory efficiency of shRNA targeting ANGPTL3. e The proliferation of HUVECs treated with PBS, USC-Con shRNA-Exo, and USC-shANGPTL3-Exo. f Quantitative analysis of the proliferation of HUVECs in different treatment groups shown in e. g, i Representative images of tube formation and migration in HUVECs treated with PBS, USC-Con shRNA-Exo, and USC-shANGPTL3-Exo. h, j Quantitative analyses of the total tube length, total branching points, and total loops shown in g and migration area shown in i. k Western blot analysis of the protein expression levels of PI3K/AKT components to assess signaling pathway activation in HUVECs among the different treatment groups. The data are presented as the means ± SD; n = 6 per group. *p < 0.05, **p < 0.01 compared among different treatment groups

• 

  Fig. 7. ANGPTL3 present in USC-Exo mediates neurological functional recovery after SCI. a, b Distribution of BMS scores and withdrawal threshold per group throughout the 56 days. c Cross-sectional image of HE staining in the PBS-, USC-Con shRNA-Exo-, and USC-shANGPTL3-Exo-treated mice. Scale bar = 100 μm. d Quantitative data of the lesion cavities shown in c. e, f Representative electrophysiological traces and the quantitative data of amplitudes, latent period in the PBS-, USC-Con shRNA-Exo-, and USC-shANGPTL3-Exo-treated mice. The data are presented as the means ± SD; n = 6 per group. *p < 0.05, **p < 0.01 compared among different treatment groups

• 

  Fig. 8. Exosomal ANGPTL3 in USC-Exo promotes angiogenesis at the spinal cord injury site. a Gross 3D view of spinal cord microvasculature in the injured spinal cord treated with PBS, USC-Con shRNA-Exo, and USC shANGPTL3 post-SCI; scale bar, 200 μm. b Quantitative analysis of the vascular morphological parameters shown in a. c Representative images of ki67/CD31 double staining of injured spinal cord sections treated with PBS, USC-Con shRNA-Exo, and USC shANGPTL3 post-SCI; scale bar, 50 μm. d Quantification of the number of ki67/CD31 double-positive cells shown in c. The data are presented as the means ± SD. *p < 0.05, **p < 0.01 compared among different treatment groups

• 

  Fig. 9. Schematic diagram showing the role of exosomal ANGPTL3 from human USC-derived exosomes in mediating the protective effect on functional recovery after spinal cord injury by promoting angiogenesis. USC-derived exosomes enriched with ANGPTL3 and are imbedded in hydrogel and used to cover the injured spinal cord surface. The exosomes can cross the spinal cord blood-brain barrier (BBB) and be taken up by spinal cord microvasculature endothelial cells, where ANGPTL3 induces PI3K and AKT phosphorylation and subsequently promotes angiogenesis