Role of Flt-1 in necrotizing enterocolitis development - ABSTRACT: Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality among premature infants that commonly leads to overwhelming sepsis and extensive intestinal tissue necrosis requiring surgery. NEC pathogenesis is not completely understood, and no specific therapies currently exist to prevent or treat NEC. We found novel evidence that defective intestinal microvascular development plays a crucial role in NEC and that inhibiting monocyte recruitment preserves gut microvascular network and decreases the incidence of NEC. Our preliminary data suggest that: (1) in the neonatal intestine, monocytes and monocyte-derived macrophages (mMf) are the major cells expressing Fms-like tyrosine kinase-1 (Flt-1) which is known to inhibit vascular endothelial growth factor receptor-2 (VEGFR2) signaling and angiogenesis by trapping its ligand VEGF; (2) TNF-activated myeloid cells produce soluble Flt-1 (sFlt-1); (3) pups subjected to experimental NEC have increased circulating sFlt-1 and increased intestinal Flt-1+ monocytes; (4) the number of Flt-1+ Mϕ is increased in human NEC gut and (5) blocking Flt-1 decreases NEC in mouse pups. Together, this suggests that, in the gut, recruited monocytes and mMf may downregulate endothelial cell (EnC) VEGFR2 signaling via an Flt-1-dependent mechanism. Furthermore, our data suggest that EnC PGD (6-phosphogluconate dehydrogenase), an enzyme required for DNA synthesis, is dependent on VEGFR2/FoxM1 signaling. PGD is significantly higher in neonatal EnC and is decreased during experimental NEC. These data support our overarching premise that, in the intestine of infants susceptible to NEC, monocytes recruited upon stress and mMϕ express or release Flt-1, thus impairing EnC VEGFR2-dependent proliferation and angiogenesis. Thus, the gut microvasculature remains underdeveloped and inadequate to cope with postnatal metabolic demands, such as enteral feeding and immune challenge, resulting in ischemia, necrosis and NEC. In this proposal, using novel approaches, we will test the hypothesis that, during NEC development in the intestine, recruited monocytes and mMϕ produce Flt-1 which traps VEGF, decreases EnC VEGFR2 signaling and subsequently decreases FoxM1- mediated PGD expression. The resulting decrease in EnC proliferation causes abnormal gut microvascular development and predisposes premature neonates to NEC. We will test our hypothesis with the following 3 aims: (1) Define the mechanism by which recruited monocytes and mMf inhibit microvascular development to promote intestinal injury during NEC induction; (2) Determine the clinical relevance of monocyte/mMϕ-derived Flt-1-mediated inhibition of EnC proliferation in human NEC; (3) Define the mechanism mediating Flt-1-induced microvascular impairment in the neonatal intestine during NEC development. The goal of this proposal is to characterize the myeloid cell-EnC crosstalk and key anti-angiogenic signaling mechanism(s) that impair intestinal microvascular development, thus promoting NEC. These studies will provide a solid foundation for testing novel therapeutic strategies that can preserve local VEGFR2 signaling to prevent NEC development.
