Intestinal mucosal epithelial injury compromises barrier function and can cause sepsis and death. Ischemia and the resulting hypoxia contribute significantly to epithelial damage in diseases such as neonatal necrotizing enterocolitis, volvulus, cardiopulmonary disease and hemorrhagic shock. Intestinal epithelial stem cells (IESCs) offer a promising therapeutic target due to their capacity to regenerate the mucosal epithelial barrier. Current evidence supports the existence of two IESC populations: a) Crypt Base Columnar (CBC) cells (Lgr5 enriched); and b) reserve or more quiescent stem cells (QSC; concentrated at the +4 position; Hopx enriched; slower cycling). Understanding their contribution to epithelial repair is critical to optimal therapeutic targeting. The hypoxia inducibe factor (HIF) pathway regulates cell survival, proliferation and differentiation in environments wit decreased oxygen. The HIF pathway activates the Wnt-/-catenin pathway which is known to be critical to IESC survival, proliferation and intestinal epithelial hemostasis. We propose that a porcine model of ischemia will aid in the translation and ultimate application of IESC research to clinical medicine. This proposal will investigate the hypothesis that distinct IESC populations wil show differing resistance to ischemic injury and that HIF pathway activation in IESCs mediates this resistance and/or IESC-mediated regeneration of severely injured epithelium after ischemia.
Two specific aims will address this hypothesis by assessing: (1) if IESC cells expressing biomarkers of CBCs or QSCs show differential resistance to ischemic injury and different contributions to the subsequent repair and regenerative response; and (2) the role of the HIF pathway within the IESC populations during ischemia and subsequent regeneration. To accomplish specific aim 1, an in vivo porcine mesenteric vascular occlusion model will create increasing degrees of ischemic injury. Biomarkers of CBCs and QSCs will be used in quantitative histologic, protein and mRNA analyses to confirm preliminary data for ischemia-induced loss of CBCs but preservation of QSCs. Co-localization of the specific biomarkers of the two IESCs with expression of apoptosis markers will assess the impact of ischemia on IESC death. The time course of QSC versus CBC proliferation during ischemia and repair with be assessed using co- localization with EdU, PCNA and Phosphohistone 3B. The time course of HIF pathway activation after ischemia and during repair will be defined by assays of mediators of protein and mRNA levels. Crypt and cell culture systems will be utilized in specific aim 2 to manipulate and define the functional role of the HIF pathway in response to hypoxia. The successful outcome of this project will define the impact of ischemia on IESCs and potentially new therapeutic targets to improve epithelial repair as well as provide additional expertise in cel and molecular biology focused specifically on IESC and hypoxia-related pathways. These training and career development activities will promote a successful transition to independence as an academic research scientist.
Intestinal ischemic injury is associated with high mortality rates, and severe complications such as short bowel syndrome resulting from extensive surgical resection. Recent ground breaking advances in the field of intestinal epithelial stem cell (IESC) biology have highlighted the possibility of stem cell-targeted epithelial regeneration as a means of repairing severely injured intestine. This project aims to determine the impact of ischemic injury on IESCs, and identify the mechanisms and mediators that improve IESC-mediated epithelial repair following injury in a novel, non-rodent porcine model relevant to humans.
