Liara Gonzalez

The National Research Council has documented a dire national need for veterinary specialists trained in biomedical research. Furthermore, veterinary researchers play a key role in comparative and translational research activities since they naturally bridge basic and clinical research. To address this training need, we request continued NIH funding for 7 fellows per year for 3 years. NC State University will provide funding for up to 2 fellows per year and 2 pre-T32 positions. Trainees will be degree-seeking fellows in the Comparative Medicine and Translational Research training program established by the faculty in the College of Veterinary Medicine (CVM) and the Comparative Medicine Institute (CMI) at North Carolina State University. This training program specifically targets individuals with the DVM degree who have completed specialty training and is designed to prepare trainees to compete for an early career development award and a rapid transition to independence as a principal investigator or in another research-intensive career. Trainees complete requirements leading to the PhD degree in Comparative Biomedical Sciences (CBS) in one of 7 areas of concentration: 1) Immunology 2) Cell Biology, 3) Pharmacology, 4) Neurosciences, 5) Infectious Diseases, 6) Population Medicine and Global Health, and 7) Pathology. Training faculty are well-funded productive scientists that have a strong training track-record and diverse research expertise. Training faculty are all members of the CBS graduate program and the CMI and represent 5 departments from 3 Colleges. Research projects emphasize comparative and translational themes fostered by the CVM and CMI in functional tissue engineering, translational pharmacology and physiology, and emerging and infectious diseases. Program requirements include: (1) a capstone comparative animal models course; (2) professional development courses and workshops; (3) courses in research ethics and research rigor and reproducibility; (4) a grant writing course and (5) annual research symposia. These requirements are in addition to those associated with the graduate program. Twenty four fellows have completed training. Twenty hold faculty positions in academia, one is a research pathologist, one is a clinical pathologist in industry, and one is a postdoctoral fellow. Fellows were awarded 18 NIH or other federal grants as PI and more than 50 extramural research grants and published 125 papers (84 first author) arising from their research while in training.

The Large Animal Core at the College of Veterinary Medicine, North Carolina State University will support the mission of the Center for Gastrointestinal Biology and Disease (CGIBD) by providing CGIBD members with consultation on and development of large animal models that can be used to translate basic science findings to human health. Large animal models are of particular relevance where physiological or anatomical features of large animals more closely resemble humans than small animals, but which are more readily developed at a College of Veterinary Medicine. The Large Animal Models Core provides IACUC support, technical and expert input, and continued scheduling services to conduct large animal research. The Core conducts research in AAALAC-accredited large animal facilities, including state-of-the-art surgical and endoscopy suites.

This proposal addresses the FY18 PRMRP topic area: Immunomonitoring of Intestinal Transplants. Over the last 10 years little improvement has been made in graft and patient survival following intestinal transplantation (IT), which are less frequent than other solid organ transplants, and new strategies are needed to improve outcomes (1, 2). Recent advances in liver transplantation have been achieved through the use of novel organ preservation strategies (3). However, the effects and clinical potential of these organ preservation methods remain unknown in IT. This proposal will address the paucity of data on optimal preservation methods for small bowel (SB) grafts by comparing the effects of normothermic machine perfusion using oxygenated blood versus standard cold storage on the graft immune profile (emphasis on intestine-resident immune cells, including intraepithelial lymphocytes), microbial communities and stem cell viability. Initial measurements will utilize excess intestine from human donors. To understand the effects of preservation on acute immune-remodeling events in the intestine post-transplant, and overall graft survival we will used the well-accepted porcine IT model. The porcine model will provide new insight into intestinal tissue dynamics during the first 7 days post-transplant, a window of time where studies in humans are not possible. This project addresses a FY18 area of encouragement as we seek to improve outcomes in intestinal transplantation by developing preservation strategies that optimize graft immune and microbial factors to improve transplant viability.

1. The Gonzalez lab will isolate ileal crypts from neonatal (1-3 day old) piglets. 2. The Gonzalez lab will perform all monolayer assays. Assays will be conducted on fresh crypt isolations as well as will be frozen and banked for potential future use. Wells will be grouped in triplicate to achieve technical replicates. 3. The Gonzalez lab will perform cell proliferation assays. A dose response curve demonstrating the effect of Protego PDTM on cellular proliferation will be measured. 4. The Gonzalez lab will perform TEER evaluations and permeability assays. 5. The Gonzalez lab will perform all in vitro hypoxia injury assays. The lab is equipped with an inverted microscope fitted with a Tokai hit environmental chamber. 6. The Gonzalez lab will perform all histomorphometric and immunohistochemistry analyses from in vivo studies. 7. The Gonzalez lab will provide consultation and data analysis.

In the United States, colorectal cancer (CRC) is the 3rd leading cause of cancer death among women and men combined, with of 1 in 20 people being diagnosed with CRC each year. Yet, understanding factors controlling disease progression and metastasis has been hampered by the lack of a suitable animal model of CRC. At present, there is considerable information regarding candidate genes that are mutated in CRC and contribute to tumor locations, tumor phenotype and patient response to treatment1. While candidate genes can be studied in mice by use of transgenic lines carrying gain-of-function and loss-of function genetic modifications, mutations in candidate genes such as Apc, result in lesions in mice that differ from those seem in humans. As a result, modifications of the traditional transgenic model have been required to advance our understanding of CRC. In contrast, an analogous APC-/- pig model demonstrates predominantly colon tumors, which are remarkably similar to the human disease. We propose to combine the strengths of an existing gene edited LGR5-H2B-GFP pig line with the power of a Cre-inducible Cas9 system to generate a novel line that will allow a careful dissection both in vivo and in vitro of the molecular pathways associated with the behavior of LGR5 cells in colon cancer. This proposal will allow for the generation and the initial in vitro and in vivo validation of the model. This highly relevant large animal model will greatly improve our understanding of how LGR5 cells behavior is controlled at the molecular level during oncogenic transformation and will be of great use to develop novel strategies to treat human colorectal cancer, as well as other cancers.

Investigating the Impact of Novel Amnion Extracts on Equine Intestinal Epithelial Cell Repair

In the present study, we propose using a porcine model of small intestinal procurement, graft preservation, and IT to evaluate the impact of intestinal cold storage or NMP preservation on immune activation, gut microbiota, ISC proliferative potential and viability, and graft viability. Animals will be used for this study because of the complexity of IT surgery. Development of the improved tissue storage and transportation methodologies that we are interested in cannot be recapitulated using cell culture models, current computer models, or in humans. We have chosen the pig as a model of IT because porcine intestine physiology recapitulates human intestine. For example, we have shown that the pig intestine has similar characteristics to that of human intestine in terms of its response to reduced blood supply and the recovery thereafter.31-33 Our pig model of intestinal injury/repair has been published in both veterinary and human medical journals, both of which have accepted the pig as a model for their respective interests after rigorous peer review.34,35 It is our hope that by using the pig in this way, we can apply our findings directly to human transplantation surgeries. We hypothesize that, when compared to cold storage, intestinal NMP will induce less epithelial injury, better protect ISC proliferative potential and viability, and ultimately improve allograft regenerative potential, resulting in transplantation of healthier bowel and superior recipient survival.

Project Summary/Abstract 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. Tissue reperfusion following ischemia is thought to exacerbate this damage. 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 inducible factor (HIF) pathway regulates cell survival, proliferation and differentiation in environments with 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-reperfusion (I/R) will aid in the translation and ultimate application of IESC research to clinical medicine. This proposal will investigate the hypothesis that distinct IESC populations show differing resistance to I/R injury and that HIF pathway activation in IESCs mediates this resistance and/or IESC-mediated regeneration of severely injured epithelium after I/R. Two specific aims will address this hypothesis by assessing: (1) if IESC cells expressing biomarkers of CBCs or QSCs show differential resistance to I/R 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, reperfusion, and subsequent regeneration. To accomplish specific aim 1, an in vivo porcine mesenteric vascular occlusion model will create increasing degrees of I/R injury. Biomarkers of CBCs and QSCs will be used in quantitative histologic, protein and mRNA analyses to confirm preliminary data for I/R-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 I/R on IESC death. The time course of QSC versus CBC proliferation during I/R and repair with be assessed using co-localization with EdU, PCNA and Phosphohistone 3B. The time course of HIF pathway activation after I/R 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 responses to hypoxia. The successful outcome of this project will define the impact of I/R on IESCs and potentially new therapeutic targets to improve epithelial repair as well as provide additional expertise in cell 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.

Small intestinal strangulation (SIS) is a devastating form of equine colic that causes loss of intestinal blood supply with subsequent intestinal epithelial barrier compromise, sepsis and death. For hope of survival, these patients require surgical intervention to correct the lesion. However, despite resection of grossly abnormal intestine during surgery, severe complications occur postoperatively. Therefore, it is critical to identify an improved method to assess the intestinal epithelial barrier health; this will better inform intraoperative surgical decisions as well as to provide accurate prognosis to owners. The epithelial barrier is comprised of a single layer of epithelial cells that depends on intestinal stem cells (ISCs); they are responsible for continuous regeneration of the intestinal epithelial lining. However, ISCs are sensitive to prolonged ischemia as is found in strangulating injury. This study aims to evaluate ISC expression at both proximal (section closest to the mouth) and distal (section closest to the hind region) resection sites in cases with SIS. We hypothesized the number of stem cell biomarkers at the proximal end to be decreased as the intestine oral to the lesion will be dilated and oedematous due to a physical obstruction. It is hypothesized that the distal end of the anastomosis site will be similar to control tissue as the obstruction lesion will not impede downstream function. Therefore, the number of ISC is decreased within the proximal resection site and is associated with greater numbers of complications. Surgical biopsies will be obtained from horses euthanized for reasons unrelated to colic (control) and from clinical cases diagnosed intraoperatively with SIS. Haemotoxylin and Eosin stain (H&E) will be used for histomorphometric evaluation. ISC populations will be identified using immunofluorescence with biomarkers Sox9, Ki67 and PH3.

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.