Fig 2: VEGF165b inhibition induces VEGFR1-STAT3 signaling in in vitro diabetic-PAD model: Immunoblot analysis of (A) pVR1/VR1, (B) pSTAT3/STAT3 in HSS-HUVECs treated with BSA + IgG, PA + IgG, PA + V165b-Ab. n = 3. Unpaired t-test. * p < 0.05 considered significant.

Fig 3: VEGF165b inhibition restores impaired angiogenic capacity and improves endothelial survival that is dependent on VEGFR1 in in vitro diabetic-PAD model: (A) In vitro tube formation assay of HUVECs transfected with non-targeting control siRNA (Con Si) or VEGFR1 siRNA (R1 Si), treated with BSA or BSA-Palmitic acid (500 µM) and IgG or V165b-Ab (10 µg/mL) for 24 h under HSS conditions, n = 4. One-way ANOVA with Bonferroni select pair comparison. * indicates significantly different from Con Si-BSA-IgG, # indicates significantly different from R1 Si-PA-IgG. Unpaired t-test for IgG vs. V165b-Ab comparisons. Scale-100 um. (B) Cell proliferation/survival assay of HUVECs transfected with non-targeting control siRNA (Con Si) or VEGFR1 siRNA (R1 Si), treated with BSA or BSA-Palmitic acid and IgG or V165b-Ab for 24 h under HSS conditions, n = 11. One-way ANOVA with Bonferroni select pair comparison * indicates significantly different from Con Si-BSA-IgG, # indicates significantly different from Con Si-PA-IgG and R1 Si-PA-IgG. Unpaired t-test for IgG vs. V165b-Ab comparisons. * p < 0.05 considered significant. Data Mean ± SEM.

Fig 4: VEGF165b inhibition induces VEGFR1-STAT3 signaling and promotes perfusion recovery in the T2D-PAD model: (A) Immunoblot analysis of VEGF165b (V165b) and total VEGF-A bound to VEGFR1 in the ischemic gastrocnemius muscle of C57BL/6 mice on a HFD treated with IgG or VEGF165b-Ab at day 3 post-HLI. n = 4. Unpaired t-test. (B) Laser Doppler microvascular perfusion imaging of HFD mice treated with IgG or V165b 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. (C) Immunohistochemical analysis of CD31 in HFD-ischemic gastrocnemius muscle treated with IgG or V165b-Ab at day 28 post-HLI. n = 7. Unpaired t-test. (D,E) Immunoblot analysis of pVR1(Y1333)/VR1 in VEGFR1 pull-down fractions and pSTAT3/STAT3 in HFD-ischemic gastrocnemius muscle treated with IgG or V165b-Ab at day 3 post-HLI. n = 5. Unpaired t-test. Significant outliers (p < 0.05) were excluded from the data analysis in Figure 4A by performing Grubbs’ test. Data Mean ± SEM.

Fig 5: 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.