Fig. 7: Blocking IGF-1 signaling decreases intestinal endothelial cell proliferation in vivo; exogenous IGF-1 enhances VEGF protein expression, intestinal endothelial cell proliferation and preserves intestinal microvascular density during experimental NEC.

a, b 24-hour-old wild-type C57BL/6 pups were treated with the IGF-1R inhibitor PPP (n = 8) or vehicle control (n = 7) or submitted to the NEC protocol for 24 h (n = 7). Intestinal LP cells were isolated and the percentage of proliferating endothelial cells (Ki-67⁺CD31⁺CD45⁻/CD31⁺CD45⁻) was determined by flow cytometry. a Typical FACS plots are shown. b Graph represents the results of three experiments combined (mean ± SEM). c–h 1-day-old neonatal mice were injected with IGF-1 (25 μg/kg, i.p., twice) or vehicle control, with a first dose 2 h prior to NEC initiation and a second dose 12 h later (Dam-fed littermates as controls) then submitted to experimental NEC for 24 h (c–f) or 48 h (g, h). c, d Intestinal tissues were assessed for VEGF and VEGFR2 proteins by western blot (c) and band densitometry is shown (d, mean ± SEM), n = 6–9/group (see panel or Source Data file for exact n number). e, f Tissue sections were stained with Ab against Ki-67 and endomucin. e Typical immunofluorescence images. f Graph represents average proliferating endothelial cells (Ki-67⁺ endomucin⁺ cells, arrows in e) counted per ×20 field (mean ± SEM, three fields were taken from each sample), n = 9–12/group (see Source Data file for exact n number). g, h pups were perfused intracardially with WGA-Alexa Fluor 647. Intestinal submucosal vascular networks were imaged, and vascular density was assessed using Photoshop (×10), n = 4–5/group (see dots in panel). P values were calculated using one-way ANOVA followed by Turkey–Kramer multiple-comparison test (b, d, f, and h). i, j Neonatal mice was submitted to the NEC model and treated with IGF-1 or control twice daily (see method section). i 60-hour-survival curve is shown (n = 28 in NEC and 30 in NEC/IGF-1 group), P value were calculated using Gehan–Breslow–Wilcoxon test. j Intestinal injury histological scores (n = 28 in NEC and 29 in NEC-IGF-1 group), P value were calculated using Chi-square test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.