Intestinal transplantation is the only cure for patients suffering from intestinal failure that can no longer be maintained on total parenteral nutrition and is the only treatment that re-establishes a patient's capacity to receive oral nutrition. Despite a highly concerning level of graft failure, limited research has been conducted on mechanisms to prevent intestinal injury and failure associated with transplantation. A key contributor to allograft failure is intestinal ischemia-reperfusion (IR). In serial surgical biopsies from clinical cases with graft failure, epithelial loss extends into the crypt base, similar to observations in animal models of IR. Within the crypt exist two intestinal stem cell (ISC) populations critical to epithelial repair: a) Active ISC (aISC; highly proliferative; Lgr5+; sensitive to injury) and b) reserve ISC (rISC; less proliferative; marked by Hopx; resistant to injury). Preliminary, K01 derived data, demonstrates that following prolonged ischemic injury: 1. aISC undergo apoptosis, 2. rISC (HOPX+) are preserved, 3. high levels of Hopx expression correlate with inhibition of spheroid proliferation, and 4. decreased Hopx expression correlates with a `release' of spheroids to proliferate. Taken together, these data indicate that Hopx+ cells are resistant to injury, and that Hopx may operate as a `molecular switch' to control cellular proliferation. Interestingly, although Hopx expression has been exclusively used in the ISC field as a biomarker to identify rISC, strong evidence exists in cancer biology that Hopx plays a functional role as a regulator of cellular proliferation. What has not been investigated is if changes in Hopx expression in rISC modulate cellular quiescence or activation to preserve ISC during IR injury and release ISC to proliferate during regeneration. Therefore, this proposal will investigate the hypothesis that high levels of Hopx gene expression in rISC confer resistance to IR injury by prohibiting cellular proliferation and that a decrease in Hopx expression in rISC during repair allows those cells to become more proliferative and contribute to repair. The hypothesis will be tested by two specific aims: (1) Determine the role Hopx+ cells play in epithelial regeneration following severe IR injury; and (2) Determine the role Hopx expression plays in modulating rISC proliferation during and following I/R injury. To accomplish specific aim 1, transgenic mouse models will be used to assess cellular mechanisms that regulate rISC resistance to ischemic injury and determine Hopx+ rISC fate.
In Aim 2, the in vivo porcine mesenteric vascular occlusion model, utilized in the K01, will be used to evaluate the impact of Hopx expression changes on ISC growth patterns and proliferation. The successful outcome of this project will determine if the Hopx cellular pathway confers rISC resistance to ischemic injury and plays a role in rISC mediated regeneration following IR injury which could greatly advance our knowledge of the mechanisms of intestinal graft failure. Therapeutically targeting the potential Hopx `switch' to both preserve rISC and enhance their proliferation may provide a highly innovative approach to enhance graft survival in the face of IR injury. This proposal will contribute data and training for an R01 submission to study therapies that modulate ISC resistance to injury and contribution to repair in vivo.
A key contributor to intestinal allograft failure is ischemia-reperfusion, however limited research has been conducted on mechanisms to prevent intestinal injury and failure associated with transplantation. Intestinal stem cells (ISC) are the source of epithelial renewal and although a reserve population (rISC), identified by the biomarker Hopx, has been shown to be resistant to injury, the mechanism that confers this resistance is not known. This project aims to determine the role Hopx+ cells play in epithelial regeneration following severe ischemic injury and the role Hopx expression plays in modulating rISC proliferation during and following ischemia-reperfusion injury by utilizing a complimentary transgenic mouse and a porcine model relevant to humans.