O2168: Decoding the Mechanism of Stem Cell Therapy in Necrotizing Enterocolitis: The eNOS-Mediated Pathway and Cysteine 440 Residue

Background: Necrotizing enterocolitis (NEC) is a neonatal surgical emergency that results in partial or complete death of affected intestinal tissue. Previous studies have demonstrated that mesenchymal stem cells (MSCs) protect against intestinal injury through the paracrine release of hydrogen sulfide. Previous work from our laboratory suggests that the mechanism by which hydrogen sulfide protects against injury, is by triggering mesenteric vasodilation through endothelial nitric oxide synthase (eNOS), specifically by acting at a particular cysteine residue (Cys440). We hypothesized that MSCs also work through an eNOS-dependent pathway and that a mutation at cysteine 440 would ablate the protective effects of MSCs.

Methods: NEC was induced in C57BL/6 wildtype mice (WT) and mice with a point mutation at Cys440 (eNOSCys440G) using hyperosmolar lipopolysaccharide (LPS) formula and intermittent hypoxia and hypothermia [Indiana University School of Medicine IACUC protocol #22107]. The treatment groups (WT n=12; eNOSCys440G n=8) received 80,000 cells/gram-body weight of inducible pluripotent mesenchymal stem cells (iPMSC) intraperitoneally on post-natal day 7 while control pups received PBS. Changes in weight, clinical sickness, and intestinal injury were scored and analyzed by Mann-Whitney. P value < 0.05 was considered significant.

Results: WT mice treated with iPMSCs were observed to be less ill than their eNOSCys440G iPMSC-treated counterparts. Stem cell therapy also improved macroscopic intestinal injury scores of the WT group [WT PBS 4.7 ± 0.86 vs WT iPMSC 3 ± 0.77, p=0.0034]. This attenuation of injury was not seen in the iPMSC-treated eNOSCys440G group [eNOSCys440G PBS 5.7 ± 0.44 vs eNOSCys440G iPMSC 5.3 ± 0.61, p=0.23]. Blinded evaluation of histology revealed reduced intestinal injury in WT iPMSC treated mice, which was not observed in the eNOSCys440G group.

Conclusion: Herein, we observed that an intact eNOS Cys440 residue in the murine host is required for iPMSCs to exert their therapeutic effects. We saw improved clinical sickness, macroscopic gut injury, and histological injury scores in iPMSC- treated WT animals compared to vehicle, but this effect was lost in eNOS Cys440G mice treated with iPMSCs compared to vehicle. Further studies are required to elucidate the downstream signaling mechanisms that ameliorate the protective effects of iPMSCs in Cys440G mutant mice.