Molecular mechanisms mediating colonic anastomotic leakage.

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This study aims to identify the role of prostaglandin signalling in anastomotic leakage and to test interventional strategies to support healing pathways. During abdominal surgery, the surgeon's intraoperative handling of visceral organs unavoidably exerts a surgical trauma, and the resulting tissue damage can affect regular organ function. Our group has extensive experience studying the pathological mechanisms of several surgical trauma-induced disorders, including postoperative ileus, intra-abdominal adhesion formation and anastomotic leakage. In this project, we will explore the molecular mechanisms of anastomotic leakage that significantly disturb colonic surgery patients' recovery. Colorectal anastomotic leakage (CAL) is a dreaded surgical complication and corresponds to one-third of all deaths after colorectal surgery. CAL has been recognised as a complex process involving diverse cellular responses. Prostaglandins (PG) are produced from arachidonic acid via cyclooxygenases (COX) and are crucial for regulating wound healing processes. Consistently, inhibition of COX activity in a clinical setting is known to augment CAL considerably and is therefore often circumvented in the clinical workup. To assess the molecular pathways driving CAL, we analysed CAL in mice through transcriptional profiling and identified PGE2 receptors EP2 and EP4 as potential mediators of CAL. We hypothezised that EP2 or EP4 signalling regulates anastomotic healing and disturbances of these pathways are likely to mediate CAL. Since EP2 and EP4 are expressed by various cell types and influence numerous cellular processes, a detailed investigation is necessary to identify the molecular mechanisms of CAL to allow interrogation of its pathology. In this study, we have three main objectives: i) Identification of the cellular source and location of PGE2 and cytokine production in CAL by single-cell RNAseq, desorption electrospray mass spectrometry imaging (DESI) and spatial transcriptomics. ii) To study the inhibition of intrinsic PGE2 degrading pathways, EP2 and EP4 receptor agonists and inhibition of cell-specific EP2 and EP4 signalling in two different CAL models to identify potentially novel pathways in CAL prevention. iii) Translational analyses of human anastomotic healers and leakers blood samples by UPLC-MS-based oxylipin panel assay to determine COX-derived mediator patterns and potential association with different healing outcomes in humans undergoing colorectal anastomotic surgery. Overall, the datasets yielded by this project will provide novel insight into the signalling pathways of murine anastomosis during healing and CAL with a focus on PGE2 signalling. The data generated herein include comprehensive transcriptional characterisation at the single cell level, lipidomic profiling, spatial transcriptomics and interventional strategies in CAL. Together, these data will help to develop targeted drugs to prevent CAL prevention in the future.