Hypoxia has been previously shown to inhibit the dihydroceramide (DHC) desaturase, leading to the accumulation of DHC. In this study, we used metabolic labeling with [3H]-palmitate, HPLC/MS/MS analysis, and specific inhibitors to show numerous sphingolipid changes after oxygen deprivation in cerebral microendothelial cells. The increased DHC, particularly long-chain forms, was observed in both whole cells and detergent-resistant membranes. This was reversed by reoxygenation and blocked by the de novo sphingolipid synthesis inhibitor myriocin, but not by the neutral sphingomyelinase inhibitor GW-4869. Furthermore, oxygen deprivation of microendothelial cells increased levels of dihydro-sphingosine (DH-Sph), DH-sphingosine1-phosphate (DH-S1P), DH-sphingomyelin (DH-SM), DH-glucosylceramide (DH-GlcCer), and S1P levels. In vitro assays revealed no changes in the activity of sphingomyelinases or sphingomyelin synthase, but resulted in reduced S1P lyase activity and 40% increase in glucosylceramide synthase (GCS) activity, which was reversed by reoxygenation. Inhibition of the de novo sphingolipid pathway (myriocin) or GCS (EtPoD4) induced endothelial barrier dysfunction and increased caspase 3-mediated cell death in response to hypoxia. Our findings suggest that hypoxia induces synthesis of S1P and multiple dihydro-sphingolipids, including DHC, DH-SM, DH-GlcCer, DH-Sph and DH-S1P, which may be involved in ameliorating the effects of stroke .
Progressive hypoxia leads to the accumulation of several dihydrosphingolipids in cerebral microendothelial cells. Hypoxia also increases sphingosine-1-phosphate and the activity of glucosylceramide (Glc-Cer) synthase. These changes reverse by inhibiting the de novo sphingolipid synthesis, which worsens hypoxia-induced endothelial barrier dysfunction and apoptosis, suggesting that the identified sphingolipids may be vasculoprotective.