Anthony Blikslager - US grants

The long range goal of this research program is to study mechanisms responsible for maintenance and restoration of the intestinal barrier. Intestinal ischemia/reperfusion injury is characterized by acute breakdown of the intestinal barrier. However, preliminary studies indicate that prostaglandins signal a remarkably rapid recovery of epithelial barrier function via the second messengers Ca2+ and cyclic AMP. Our central hypothesis is that prostaglandins I2 and E2 (PGI2 and PGE2) are elaborated by cyclooxygenase-2 (COX-2) during intestinal injury from sub-epithelial fibroblasts and restore barrier function by signaling closure of inter-epithelial tight junctions. Tight junctions close in response, to a synergistic signal between intracellular and cyclic AMP, which are increased by PGI2-stimulated cholinergic nerves and PGE2 receptor-linked adenylate cyclase respectively. The aims of this research are: to determine if PGI2 and PGE2 increase transepithelial resistance in ischemic-injured epithelium by a specific action on tight junctions; to determine the neural and epithelial receptor pathways by which PGI2 and PGE2 signal recovery of transepithelial resistance; and to determine the cellular and enzymatic source of prostaglandins responsible for rescuing epithelial barrier function. Two distinct injury models will be used: ischemic-injured intestinal mucosa and oxidant-damaged epithelial monolayers. Tissues will be mounted in Ussing chambers, and transepithelial electrical resistance will be monitored as an indicator of epithelial integrity. Radio-labeled fluxes will be used to determine the contribution of tight junction closure and restitution. Mechanisms by which prostaglandins signal repair will be tested by measuring second messengers and by blocking epithelial and neural signaling pathways. The cellular source of reparative prostaglandins will be determined by immunohistochemistry and fibroblast monolayer studies. Inhibition of COX-2 will be used to determine the contribution of distinct cyclooxygenases in the repair response. These experiments will provide insight into the signaling pathways and mechanisms by which prostaglandins trigger repair of acutely-injured intestinal epithelium.

DESCRIPTION (provided by applicant): The human inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, affect over one million Americans and significant unmet medical needs still exist. Activation of NF??B transcription factors are central events in the initiation and perpetuation of chronic inflammation in IBD. TheraLogics, Inc., have been at the forefront of NF??B research and hold intellectual property pertaining to novel NF??B inhibitor peptides including TLX1423. TLX1423 is a peptide comprised of a 8 lysine (8K) protein transduction domain (PTD) with an I?B kinase (IKK) inhibitory sequence, NF??B essential modulator (NEMO) binding domain (NBD). Compared to other NF??B inhibitors, TLX1423 has the advantages of inhibiting activated NF??B, a hallmark of chronic inflammation, but not inhibiting basal NF??B activity, involved in fundamental cellular processes thus correlating with toxicity. During phase 1, we achieved important milestones in the development of TLX1423 as a therapy for IBD and published these findings in the Journal of Immunology. We demonstrated transduction of TLX1423 into cells and tissues. In-vivo, TLX1423 inhibited LPS-activated NF??B in the ileum, but did not inhibit basal NF??B in Peyer's patches. IL-10-/- mice treated systemically with TLX1423 demonstrated amelioration of established colitis and decreased NF??B activation in the lamina propria. In phase 1, we also demonstrated that intrarectal administration of TLX1423 results in amelioration of intestinal inflammation in two experimental IBD models. The ideal therapeutic agent to treat IBD would be administered by mouth. However, drug delivery to the inflamed intestine remains a challenge for two main reasons: 1) lack of highly effective immunomodulatory agents that can be delivered locally and inhibit their targets in intestinal immune cells and, 2) lack of vehicles to carry these agents to the site of inflammation with minimal degradation in the GI tract. The multidisciplinary team assembled for this phase 2 proposal has developed innovative solutions to these hurdles. This would be an important advancement to minimize toxicity, increase patient compliance, and improve quality of life. To address these challenges, we have developed microemulsion (ME)- based delivery systems suitable for local administration of TLX1423, and via enteric release strategies, target the peptide to inflamed regions of the GI tract. We show preliminary data that PTD peptides in water-in-oil (w/o) MEs are efficiently delivered to the large intestine in mice as compared to free PTD peptides. TheraLogics has enlisted CMC, regulatory and clinical consultants to translate results of this phase 2 program into the next phases, including GMP manufacturing, GLP safety/toxicity studies, and an IND submission.

PROJECT SUMMARY/ ABSTRACT Breaches in the intestinal mucosal barrier lead to sepsis and death if epithelial coverage is not rapidly restored, particularly in neonates. The reason for higher mortality in infants as compared to more mature individuals with intestinal injury has not been explained. High infant mortality results from diseases associated with ischemia/ reperfusion (I/R) injury, including necrotizing enterocolitis and volvulus. In juvenile pigs (6-8-weeks of age), we have extensively studied the remarkably rapid intestinal repair marked by epithelial cell migration (restitution). However, in a recently published study featured in the revised version of this proposal, we have shown an intriguing age-dependent deficiency in the recovery of mucosal repair following ischemic injury with a near-total lack of restitution in neonatal piglets. Interestingly, this defect in repair can be rescued by a homogenate of ischemic injured juvenile homogenate, suggesting more mature cell populations stimulate restitution. One cell population of particular interest is the enteric glial cell (EGC) network, which we have shown matures postnatally in pigs. In the revised proposal, we have now shown that porcine EGC-conditioned media stimulates cell migration (restitution) in wounded neonatal porcine IPEC-J2 cells. However, a gap remains in our knowledge regarding the signaling mechanisms between epithelium and underlying EGC that results in restitution. To address this gap, we have performed RNAseq analyses of ischemic-injured neonatal and juvenile mucosa, and created an unbiased discovery pipeline approach to focus on significantly deficient signaling pathways in the neonate. One such pathway includes EGF, which is secreted by the EGC as proEGF, and epithelial annexin A2, which initiates cell migration by signaling internalization of ?1-integrin. Our central hypothesis is that repair of ischemic-injured mucosa is reliant on postnatal migration of EGC into the lamina propria in order to signal wounded epithelium to efficiently restitute. We will test this hypothesis with two specific aims: 1) Determine age-dependent defects in migration signaling mechanisms in wound-adjacent intestinal epithelial cells. Our working hypothesis is that there is an age-dependent development of epithelial restitution in response to injury at least in part via annexin A2 signaling. 2) Determine perinatal changes in mucosal EGC network structure, density and secretome. Our working hypothesis is that there are insufficient EGCs secreting barrier-promoting factors, including proEGF, in proximity to the epithelium to stimulate restitution in neonates. To examine these specific aims, we will use our unique age-dependent porcine model of mucosal repair, a wounded porcine neonatal IPEC-J2 cell model, primary culture of porcine EGC, EGC secretome analyses via mass spectrometry, and state of the art imaging techniques for glial cells, including immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). Using an unbiased discovery pipeline approach, we expect to be able to define signaling mechanisms that are deficient in neonatal injured intestine that can ultimately be targeted to enhance survival of patients with intestinal failure

PROJECT SUMMARY - LARGE ANIMAL MODELS CORE The Large Animal Models Core was developed at the time of the last competing renewal to address the concern that in vitro cell culture systems and small animal models have not always translated well to solving human health digestive disease problems. The core was designed to provide models more physiologically similar to people. Over the past 5-years, the CGIBD has developed and strengthened a Large Animal Models Core at the NC State University College of Veterinary Medicine focused on medical and surgical technologies in pigs. The overall goal of the core is to provide a cost-effective central resource for Center investigators wishing to understand mechanisms of digestive disease using large animals. This has been particularly successful for CGIBD members interested in esophageal disease, in large part because the pig has proven to be an excellent translational model of diseases such as eosinophilic esophagitis and gastroesophageal reflux disease. The pig esophagus contains esophageal submucosal glands as does the human esophagus, while mice lack these glands. Recent work through the core has helped to establish these glands as a progenitor cell source in the esophagus. Disease models of intestinal ischemia have also continued to be a critical component of the Core, again because pigs are thought to more closely resemble humans in terms of their response to ischemia. Ex vivo (Ussing chambers) and in vitro studies, particularly those involving large animal complex cell culture (2D and 3D) are now being offered to bolster the basic science output from large animals. Furthermore, because of novel ideas related to the interaction of the gastrointestinal tract and the microbiome, we now plan to introduce gnotobiotic pigs as an additional resource. The specific aims of the core are: 1) to provide consultation with Core veterinarians to assess feasibility, logistics and study design; 2) to assist with procurement of animals, and 3) to coordinate animal procedures, including anesthesia, endoscopy, and surgery. The Core directors (Blikslager and Gonzalez) have a demonstrated track record in digestive disease and are board-certified veterinary specialists in surgery. Veterinary board-certified specialists in laboratory animal medicine, internal medicine, anesthesia, and advanced imaging (MRI, CT, nuclear medicine) are readily available from the NC State Veterinary Hospital. Newly renovated facilities funded in large part by an NIH G20 grant are crucial to the Core?s ability to offer medical, surgical and gnotobiotic capabilities adjacent to large animal research housing. Based on funded projects developed with the Large Animal Models Core and CGIBD investigators during the prior funded period, the Core will aim to have 4-6 projects running at any one time. Although the Core currently serves a small number of members, it provides a remarkable scientific impact on their research which would not be possible without the core.