Microvascular dysfunction, a critical hallmark of sepsis that results in decreased organ perfusion and subsequent development of organ failure, is characterized by disruption of endothelial barriers. An early diagnosis of barrier dysfunction, followed by preventive or therapeutic strategies, may therefore improve sepsis outcome. The current project investigates two interrelated barrier-protecting pathways, the sphingosine 1-phosphate (S1P)/S1P receptor type 1 (S1PR1) axis and the AMP-activated protein kinase (AMPK) pathway, to elucidate their possible diagnostic and therapeutic value in the context of sepsis. We will establish diagnostic tests to determine the status of S1P/S1PR1 signaling and AMPK activity in septic patients in a prospective observational study. In this context, a unique proprietary monoclonal antibody that detects human S1PR1 on the cell surface by flow cytometry (FACS) will be applied. Correlation of AMPK phosphorylation and S1P/S1PR1 signaling with established biomarkers of vascular dysfunction and organ dysfunction will reveal their role in the complex pathophysiology of sepsis. Furthermore, targeting strategies for both pathways will be investigated in experimental models. The proposed project will be the first study exploring the clinical relevance and potential therapeutic use of S1P/S1PR1 signaling and AMPK activation for endothelial barrier maintenance and stabilization in septic patients.