Supplementary Material for: Intestinal Vascular Endothelial Growth Factor Is Decreased in Necrotizing Enterocolitis

<b><i>Background:</i></b> Decreased intestinal perfusion may contribute to the development of necrotizing enterocolitis (NEC). Vascular endothelial growth factor (VEGF) is an angiogenic protein necessary for the development and maintenance of capillary networks. Whether VEGF is dysregulated in NEC remains unknown. <b><i>Objectives:</i></b> The objective of this study was to determine whether intestinal VEGF expression is altered in a neonatal mouse model of NEC and in human NEC patients. <b><i>Methods:</i></b> We first assessed changes of intestinal VEGF mRNA and protein in a neonatal mouse NEC model before significant injury occurs. We then examined whether exposure to formula feeding, bacterial inoculation, cold stress and/or intermittent hypoxia affected intestinal VEGF expression. Last, we visualized VEGF protein in intestinal tissues of murine and human NEC and control cases by immunohistochemistry. <b><i>Results:</i></b> Intestinal VEGF protein and mRNA were significantly decreased in pups exposed to the NEC protocol compared to controls. Hypoxia, cold stress and commensal bacteria, when administered together, significantly downregulated intestinal VEGF expression, while they had no significant effect when given alone. VEGF was localized to a few single intestinal epithelial cells and some cells of the lamina propria and myenteric plexus. VEGF staining was decreased in murine and human NEC intestines when compared to control tissues. <b><i>Conclusion:</i></b> Intestinal VEGF protein is reduced in human and experimental NEC. Decreased VEGF production might contribute to NEC pathogenesis.