Fig. 1.

Differential effects of laminar and disturbed shear stress on SIA expression. HUVEC were exposed to USS (15 dyn cm^-2), OSS (±5 dyn cm^-2, 1 Hz) or maintained in static conditions for 24 or 48 h, as indicated. (A) Representative electron micrographs of the cross-sectional aspect of the luminal GCX stained with ruthenium red and Alcian blue. Twenty measurements of the luminal GCX depth were averaged from each cell and are expressed as mean ± S.D. (n = 1 donor) from at least 10 different cells per condition. ***P < 0.001 (Student's t-test). L, channel lumen; N, nucleus. Scale bar=200 nm. (B) Representative confocal images of the SIA component of the GCX stained with WGA-CF^TM448A (red) in fixed cells. WGA mean cell intensity (MCI) was quantified in the x-y optical slices and normalized to the respective number of cell nuclei stained with DAPI (blue). Data represent mean ± S.E.M. (n = 5 different donors). *P < 0.05; **P < 0.01 (1-way ANOVA). Scale bar=20 μm. (C) Amount of free SIA in the conditioned culture media was assessed fluorometrically. Data are mean ± S.E.M. (n = 6 different donors). **P < 0.01; ***P < 0.001 (1-way ANOVA). (D) Relative mRNA expression of the genes GNE, CMAS, SLC35A1 and NEU1 encoding enzymes involved in SIA biosynthesis, transport and cleavage was determined by real time-PCR and normalized to 5 reference genes. Data are expressed as fold change from respective mRNA levels in static culture and denote mean ± S.E.M. (n = 4–5 different donors). *P < 0.05; **P < 0.01 (1-way ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)