Fig 2: VEGF165b inhibition improves perfusion recovery in Mg-Tg mice by activating VEGFR1-STAT3 signaling in experimental PAD. (A) Laser Doppler microvascular perfusion imaging of Mg-Tg mice treated with IgG or V165b-Ab over the time course of recovery. n = 7. Repeated Measures ANOVA with Dunnett’s post-test. * p < 0.05 considered significant at specific time points. (B) Immunohistochemical analysis of CD31 in Mg-Tg mice ischemic gastrocnemius muscle treated with IgG or V165b-Ab at day 28 post-HLI. n = 7. Unpaired t-test. (C,D) Immunoblot analysis of pVR1/VR1, pSTAT3/STAT3 in Mg-Tg mice ischemic gastrocnemius muscle treated with IgG or V165b-Ab at day 3 post-HLI. n = 7. Unpaired t-test. * p < 0.05 considered significant. Data Mean ± SEM.

Fig 3: Loss of vascular N-cadherin expression following myeloid-specific ablation of NG2. Double immunostaining for CD31 (red) and N-cadherin (green) was used to assess endothelial cell expression of N-cadherin in control (A–C) and Mac-NG2ko tumor vessels (D–F). (G) Quantification of N-cadherin/CD31 colocalization as a percentage of total CD31. Double immunostaining for PDGFGRß (red) and N-cadherin (green) was used to evaluate pericyte expression of N-cadherin in control (H–J) and Mac-NG2ko tumor vessels (K–M). (N) Quantification of N-cadherin/PDGFRß colocalization as a percentage of total PDGFRß. Scale bars = 20 µm. *p < 0.01 compared to controls.

Fig 4: Histological analysis of the tumor microenvironment. A Confocal microscopy of tumor tissue with injection of dual-sEVs (white). Blue, DAPI; red, vimentin + . B Quantification of the uptake by control and injected (dual-sEVs +) populations: TCs, vimentin + /TR7-; TAMs, F4/80 + ; CAFs, TR7 + ; and ECs, CD31 + . C Mean uptake values in the populations. Data are represented as mean ± SD. **** p < 0.0001

Fig 5: CD93 interacts with MMRN2 in endothelial cells and colocalizes with MMRN2 in tumor vasculature.(A) Western blot against MMRN2 in CD93 coimmunoprecipitated samples (CD93 co-IP) derived from total protein lysates of HDMECs. A nonrelevant IgG antibody was used as negative control (IgG co-IP). CD93 FT and IgG FT represent the flow-through of the unbound fraction. (B) In situ proximity ligation assay (PLA) for CD93 and MMRN2 in cultured HDBECs. Positive signal (green dots) indicates proximity between CD93 and MMRN2. F-actin was stained by phalloidin (red) and nuclei by Hoechst (blue). Scale bar: 20 µm. (C) Quantification of CD93 and MMRN2 interaction based on the number of positive signals per cell (n = 3 independent experiments). Nonspecific signals (PLA negative controls) were also examined. **P < 0.01; 1-way ANOVA with Dunnett’s multiple-comparisons test. (D–F) Immunofluorescent staining of MMRN2, CD93, and CD31 in human grade IV glioma vessels (D), in orthotopic GL261 glioma vasculature (E), and in nontumor brain vasculature adjacent to a GL261 tumor (F). Scale bars in all pictures: 20 µm. (G) MMRN2 quantification in tumor and nontumor vessels of WT (n = 3) and CD93–/– (n = 3) mice. Values represent mean ± SEM expressed as arbitrary units (AU) of MMRN2-positive area normalized by CD31-positive area. **P < 0.01; 2-tailed t test.