Figure 2
From: Acid Sphingomyelinase Promotes Endothelial Stress Response in Systemic Inflammation and Sepsis
Inhibition of ceramide generation by (A) desipramine and (B) NB6. Semi-confluent HMECs were metabolically labeled with C6-NBD-sphingomyelin and stimulated with serum (5%) obtained from healthy individuals (n = 12) or patients with severe sepsis for 20 min (n = 12). Monolayers were pretreated with desipramine or NB6 for 30 min (10),20,40 µmolar). Boxes indicate the interquartile range, divided by the median (central horizontal line) as analyzed in twelve independent experiments per group. Significant differences in comparison to monolayers without desipramine or NB6 pretreatment were calculated by Friedmann-Wilcoxon test with adjustment according to Bonferoni-Holm results are indicated by asterisks (*p < 0.05, **p < 0.02). (C) Ceramide generation in proinflammatory stimulated endothelial cells. Semi-confluent HMECs were pretreated with desipramine or NB6 for 60 min (20 µmol each), and stimulated with TNF-α (10 ng/mL) or endotoxin (100 ng/mL) for 240 min in the presence of 5% human serum from healthy individuals. Subsequent to lipid extraction, purification and labeling, ceramides were separated by HPLC. Shown is the total amount of ceramides, since the boxes indicate the interquartile range, divided by the median (central horizontal line) as analyzed in six independent experiments. Significant differences to basal values are indicated by an asterisk (*p < 0.05). (D) Time course of sphingomyelin breakdown in stimulated endothelial cells. Semi-confluent HMECs were metabolically labeled with C6-NBD-sphingomyelin and stimulated with serum (5%) obtained from patients with severe sepsis. Fluorescent sphingolipids such as ceramide as well as GlcCer were separated by TLC quantitated by densitometry. Representative results from three independent experiments are shown. (E) Representative sphingolipid pattern of stimulated endothelial cells used for densitometric analyses. Endothelial monolayers were pretreated with desipramine (upper lines) and NB6 (lower lines) for 30 min (10),20,40 µmolar). Fluorescent sphingolipids such as ceramide (rf 0.8) as well as GlcCer (rf 0.6) were separated by TLC and quantitated by densitometry. Representative results from 24 independent experiments (12 each per group and inhibitor, providing data for A and B) are shown. M, Marker.