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.
Highlights
Sepsis is characterized by an overwhelming host response and development of remote organ failure [1]
A more pronounced rise was observed in patients fulfilling systemic inflammatory response syndrome (SIRS) criteria complicated by organ dysfunction on day 1 (291.7 pmol/(mL*h); 75.2 pmol/(mL*h), n = 14)
The increase in SMPD1 activity in inflammatory host response of different origin was found increased in patients with systemic inflammation (SIRS without organ dysfunction (OD)), with further increasing values in those with OD or severe sepsis/septic shock compared with intensive care unit (ICU) controls (Kruskal-Wallis-test, p ≤ 0.001)
Summary
Sepsis is characterized by an overwhelming host response and development of remote organ failure [1]. Once an inflammatory response has been instigated by endogenous mediators, such as cytokines, or components of the bacterial cell wall [4, 5], a large number of host-derived mediators are capable to further activate endothelial cells. The proinflammatory phenotype results in promotion of endothelial dysfunction, leukocyte trafficking and a prothrombotic state which potentially may lead to thrombotic microangiopathy (TMA), remote organ dysfunction and death [6,7,8]. SMPD1 activation and rapid ceramide generation have been demonstrated in response to various stressors [9, 10] including ligation of death receptors, viral, bacterial and parasitic pathogens and proinflammatory cytokines [11,12,13,14]. The lipid mediator ceramide consisting of a sphingosine base linked to a fatty acid of different length, exhibits the tendency for spontaneous self-association, the formation
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