Figure 8.

Laminar shear stress downregulates S1P lyase expression. A, Quantitative RT-PCR analysis of S1P lyase (Sgpl) transcript in static and shear stress–treated MEECs. Sgpl transcript was normalized to Gapdh mRNA. B, Immunoblot analysis of S1P lyase. MEECs were subjected to laminar shear stress as described in Materials and Methods. Immunoblot analysis on total homogenate was performed with affinity-purified anti-S1P lyase antibody. GAPDH was used as a loading control. Densitometric analysis of immunoblot is shown. C, Downregulation of S1P lyase by double-stranded siRNA in MEECs stimulated intracellular synthesis of [³H]-S1P and its release to extracellular medium (black: scrambled siRNA; gray: S1P lyase si). Down-regulation of S1P lyase by siRNA and corresponding control (scrambled siRNA) is also shown by immunoblot analysis (n=3). D, Transduction of human adeno-Sgpl in HUVECs decreased the formation of [³H]-S1P in intracellular and extracellular compartments (black: AdGFP; gray: AdSgpl). Stimulated expression of S1P lyase is shown by immunoblot analysis (n=3).