TNFalpha and IL‐1beta Synergically Decrease VEGFR2 and Increase VEGFR1 Protein Expression in Endothelial Cells

Abstract

Necrotizing enterocolitis (NEC) is the most serious gastrointestinal disease affecting premature infants. Among other inflammatory cytokines, TNFalpha and IL1beta are significantly increased in intestinal tissues during NEC induction in a neonatal mouse model. We previously found that intestinal villous endothelial cell (EC) proliferation and VEGFR2 expression are decreased, and mucosal vascular network development impaired during experimental NEC induction. Furthermore, inhibiting VEGFR2 kinase activity increased NEC severity. However, whether cytokines directly affect VEGFR2 expression in neonatal intestinal EC remains unknown. Here, we hypothesize that increased inflammatory cytokines may affect microvascular development by decreasing intestinal EC VEGFR2 signaling. To test our hypothesis, EC isolated from mouse neonatal intestine tissue and HUVEC were cultured in vitro and incubated with TNFalpha and/or IL1beta and VEGFR1 and 2 assessed by western blot. We found that: 1) TNFalpha treatment of both mouse EC and HUVEC for 12 hours dramatically decreased their VEGFR2 expression while inducing an increase in VEGFR1 expression. 2) IL‐1beta treatment for 12 hours decreased VEGFR2 expression without affecting VEGFR1 in both mouse EC and HUVEC. 3) When EC (both neonatal mouse intestinal EC and HUVEC) were incubated with IL1beta and TNFalpha together, there was a synergistic decrease in VEGFR2 expression and a synergistic increase in VEGFR1 protein expression. Other inflammatory cytokines found to be increased in NEC, such as IL6, IL10, CCL2 and CCL20 had no effect on HUVEC VEGFR1 and 2 expression. This suggests that TNFalpha and IL1beta produced during NEC induction may promote disease progression by impairing the development of the mucosal intestinal microvasculature, thus further affecting intestinal tissue perfusion.Support or Funding InformationNIH grant RO1HD060876 and Ann & Robert H. Lurie Children's Hospital of Chicago Foundation