Abstract
The pathophysiology of sepsis involves activation of acid sphingomyelinase (SMPD1) with subsequent generation of the bioactive mediator ceramide. We herein evaluated the hypothesis that the enzyme exerts biological effects in endothelial stress response. Plasma-secreted sphingomyelinase activity, ceramide generation and lipid raft formation were measured in human microcirculatory endothelial cells (HMEC-1) stimulated with serum obtained from sepsis patients. Clustering of receptors relevant for signal transduction was studied by immuno staining. The role of SMPD1 for macrodomain formation was tested by pharmacological inhibition. To confirm the involvement of the stress enzyme, direct inhibitors (amino bisphosphonates) and specific downregulation of the gene was tested with respect to ADAMTS13 expression and cytotoxicity. Plasma activity and amount of SMPD1 were increased in septic patients dependent on clinical severity. Increased breakdown of sphingomyelin to ceramide in HMECs was observed following stimulation with serum from sepsis patients in vitro. Hydrolysis of sphingomyelin, clustering of receptor complexes, such as the CD95L/Fas-receptor, as well as formation of ceramide enriched macrodomains was abrogated using functional inhibitors (desipramine and NB6). Strikingly, the stimulation of HMECs with serum obtained from sepsis patients or mixture of proinflammatory cytokines resulted in cytotoxicity and ADAMTS13 downregulation which was abrogated using desipramine, amino bisphosphonates and genetic inhibitors. SMPD1 is involved in the dysregulation of ceramide metabolism in endothelial cells leading to macrodomain formation, cytotoxicity and downregulation of ADAMTS13 expression. Functional inhibitors, such as desipramine, are capable to improve endothelial stress response during sepsis and might be considered as a pharmacological treatment strategy to favor the outcome.