GLOBAL PROTEOMICS OF EXPERIMENTAL NEC REVEAL DISPRUPTION IN NOVEL PATHWAYS INCLUDING MITOCHONDRIAL FUNCTION AND IDENTIFY POTENTIAL THERAPEUTIC TARGETS

Background
Necrotizing enterocolitis is devastating intestinal disease that affects up to 10% of premature infants. To date, effective treatment options remain elusive and new clinically translatable approaches are urgently needed. Our group has previously demonstrated that a placental stem cell-based therapeutic approach can repair experimental NEC damage. Further, we have shown extracellular vesicles (EV) from human placental stem cells (hPSC) can effectively serve as a cell-free approach. We have now hypothesized that identification of novel molecular pathways in NEC pathogenesis and the specific biological processes ameliorated with the hPSC and EV therapies will allow further optimization of a more targeted NEC therapy.
Methods
Newborn rats were divided into 4 groups: breastfed control (BF), NEC, NEC-hPSC, and NEC-EV. NEC was induced in all NEC groups via hypertonic formula feeding, hypoxic stress, and oral LPS. At 32 and 56 hours of NEC induction, NEC-hPSC and NEC-EV pups received IP injections of hPSC or EV from hPSC, respectively. At 4 days, intestinal damage of the ileum was assessed. Global protein identification and abundance within each group was also assessed via LC-MS/MS.
Results
NEC tissue had significant increases in expected signaling pathways including immune response (regulation of complement cascade-lectin pathway, IL7 signaling, NFκB transcription factor activity), responses to hypoxia and ischemia, and decreased epithelial cell motility (integrin cell-matrix interactions). Novel disrupted processes were also identified in NEC disease. First, proteins involved in mucin maturation were disrupted in NEC disease including reduced TFF3 and Neu5AC but increased TFF1. AB-PAS staining of ileal tissue further confirmed disruption of goblet cell localization within the crypt-villus axis. In NEC, goblet cells were concentrated closer to the crypts rather than along the villi. In addition, decreases in mitochondrial function, specifically fatty acid beta-oxidation, were observed in NEC disease. Upon hPSC or EV therapy, a subset of proteins involved in metabolic function were increased, though not to the extent found in NEC disease. Other novel biological targets of the therapies were involved in angiogenesis and VEGF signaling.
Conclusions
Our data has highlighted mitochondrial function as a potential NEC pathogenesis pathway. The therapeutic approaches did not fully ameliorate this pathway. Thus, future therapeutic approaches may include additional targeting of this pathway. Additionally, our data again illustrated the importance of mucin maturation in NEC. Altered goblet cell localization may further suggest changed in mucin function in NEC. Mucin maturation may be a potential target for a prophylactic approach as well as a combined therapeutic approach. Overall, our study, has elucidated key pathways both in NEC pathogenesis and in repair.