Figure 6. Endothelial Nogo-B regulates de novo synthesis of sphingolipid during chronic inflammation.

(A) SPT activity assay was performed in heart microsomes from WT and Nogo-A/B–deficient mice at 3 days after transverse aortic constriction (TAC) or sham operation. SPT activity was measured using [³H]-serine as a substrate, followed by TLC separation of sphinganine, a downstream product of SPT. n ≥ 5/group. (B) Assessment of SPT activity in WT and Nogo-A/B–deficient mouse lung endothelial cells (MLEC) treated with TNF-α (50 μg/ml) or vehicle at indicated time points. n ≥ 11 replicates from 5 to 6 independent MLEC isolations. (C) SPT activity assay of human umbilical vein endothelial cells (HUVEC) treated for 24 hours with TNF-α (50 μg/ml) or vehicle. n = 10 replicates/group. (D and E) Total ceramide and dihydrosphingosine (dhSph), sphingosine (Sph), sphingosine-1-phosphate (S1P) levels in WT and Nogo-A/B–deficient (D) MLECs and (E) culture medium. n ≥ 12 replicates from 4 independent isolations of MLECs/group. (F) Levels of dhSph, dhSph-1-phosphate (dhSph-1P), Sph, and S1P in the perfusates of WT and Nogo-A/B–deficient hearts of sham-operated mice or mice 3 days after TAC. n ≥ 9/group. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance was determined by (A and D–F) 1-way ANOVA followed by Tukey’s multiple comparison test, (B) 2-way ANOVA, or (C) unpaired t test.