Intestinal diseases which involve ischemia such as volvulus or necrotizing enterocolitis cause mucosal barrier damage and are associated with poor survival in neonatal patients. The cause of higher mortality in neonates as compared to more mature patients has not been fully explained and represents a critical gap in our knowledge. Using a translational pig model of the human infant, I have found that while juvenile animals (6-weeks-old) repair rapidly by villus contraction and epithelial restitution, barrier repair is markedly impaired in neonates (2-weeks- old), characterized by a complete lack of epithelial restitution. Importantly, I found that the restitution defect in neonates can be rescued by the direct application of homogenized mucosal tissue from ischemia-injured small intestine from juvenile pigs. The mechanisms responsible for the age-dependent defect in restitution in neonates and the components of the juvenile mucosal tissues responsible for rescue of restitution are incompletely understood and form the basis of this proposal. Identifying how specific repair signaling mechanisms mature during the postnatal period has the potential to promote innovative discovery efforts leading to effective clinical interventions in neonatal patients with intestinal disease characterized by defective epithelial barrier function. A mixed population of subepithelial cells modulate epithelial cell functions in health and disease by secreting paracrine factors into the mucosal microenvironment. Of these cells, the subepithelial glial network is known to mature postnatally, and has been shown to directly regulate epithelial repair. I examined the glia within porcine jejunum and found an underdeveloped glial network in mucosa of neonates as compared to juveniles. Additionally, I found that epidermal growth factor and intracellular mediators of epithelial migration annexin A2 and focal adhesion kinase are reduced in neonatal mucosa. In order to study glial-epithelial interactions more closely, I will shift my studies to include advanced in vitro techniques. In a preliminary in vitro experiment, I found that media conditioned by glia cultured from juvenile pigs enhanced wound restitution in IPEC-J2 cells, a neonatal-derived intestinal epithelial cell line. These findings suggest glia may play an important role in postnatal development of barrier repair mechanisms. Therefore, I hypothesize that developmental glial factors within the subepithelial microenvironment signal restitution. To examine age-dependent differences in the effects of glia on epithelial repair, I will optimize and study primary enteric glial (aim 1) culture and primary intestinal epithelial culture on cutting-edge biomimetic collagen hydrogel scaffolds (aim 2) in the pig. As a young investigator completing these studies under new mentorship at a new top-ranking medical institution, I will expand my repertoire of skills in experimental design, execution and analysis to include basic science approaches to epithelial restitution mechanisms using advanced in vitro models. These studies will produce data to support new research grants (R03, R01) as I transition to research independence and provide the foundation for a productive career in comparative biomedical research to advance both human and veterinary medicine.
Intestinal ischemic injury to the epithelial barrier is associated with poorer survival outcomes in younger patients and, in our highly comparative pig model of intestinal ischemia, neonate epithelium fails to repair while juvenile epithelium repairs rapidly. Remarkably, direct application of juvenile intestinal tissue can induce repair in neonates, but the mechanisms responsible for defective neonatal restitution and the components of the juvenile mucosa responsible for its rescue are unknown and form the basis of this research. Understanding how repair mechanisms develop after birth will foster development of novel therapeutic strategies for treatment of neonatal patients affected by ischemic intestinal disease.
