Abstract
Sphingolipid metabolites, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes. In particular, sphingosine-1-phosphate (S1P) the product of sphingosine phosphorylation, through transactivation of five cognate G protein-coupled receptors namely endothelial cell differentiation gene receptors (EDGs/S1P1-5), promotes proliferation, differentiation, enhances cell survival and inhibits apoptosis in different cellular systems. Modulation of sphingolipids and their receptors has been shown in some stroke models to be neuroprotective and anti-inflammatory. We undertook this study to elucidate the role of S1P receptor subtypes and sphinosine kinase (SPHK) in models of brain derived endothelial cells and microglia. We studied mono and co-cultures where endotoxin (LPS) activated microglia stimulate endothelial cell death. After documenting SPHK1/ 2 expression in microglia by immunofluorescence, we found that an agonist of SPHK ((2-(p-Hydrozyanilino)-4(p-chlorophenyl) tiazole, SKI) preserved microvascular endothelial cells, and markedly prevented microglia-induced endothelial cell injury. Furthermore, SKI inhibited iNOS and NO generation in microglia by both endotoxin as well as a second TLR4 agonist (Kdo2-Lipid (ADi[3-deoxy-D-manno-octulosonyl]-lipid A) (*P<0.01). S1P (1) agonists SEW-1 and FTY720 and endogenous ligand S1P had no significant effect in on microglia-mediated cell injury or NO induction by LPS. However, preincubation with the S1P (3) antagonist ( CAY10444 ) dose dependently prevented NO accumulation, while JTE-013 (S1P2 antagonist) & VPC23019 (S1P1 antagonist) were effective in preventing NO release. Our findings indicate that sphingosine phosphorylation promotes immune responses in microglia and its blockade, especially SPHK1 and to lesser extent S1P (3) attenuated these responses. This inhibition also led to decreased cell death due to immune cell activation. Our data suggests that targeting aspects of the sphingolipid pathway may be a new strategy to inhibit microglia activation and preserving endothelial cell integrity in relevant cerebrovascular pathologies such as stroke, trauma, or sepsis
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