Figure 1. Downregulation of preproendothelin-1 mRNA by laminar shear stress in human endothelial cells.

In A, human umbilical vein endothelial cells (HUVEC) were exposed to long-term (24 h) laminar shear stress, and the expression of preproendothelin-1 (ppET-1) mRNA in these cells was determined by Northern analysis. The filter was subsequently stripped and hybridized with a human GAPDH cDNA probe as a control. Results shown are representative of three independent experiments. Laminar shear stress (24 h) caused dose-dependent downregulation of ppET-1 mRNA, compared with control (for 15 and 30 dyn cm⁻², control was supplemented with 5 % dextran medium). B shows the method of quantification of mRNA levels of the human preproendothelin-1 (ppET-1) gene by competitive RT-PCR. The method compares the amplification of a ppET-1 cDNA fragment from reverse transcribed total RNA of HUVEC (461 bp) vs. different concentrations of an internally deleted and reverse transcribed cRNA standard (355 bp) by PCR. The PCR fragments were separated on agarose gels and stained with ethidium bromide. Lane 1: molecular weight marker (100 bp ladder). Amount of standard molecules per reaction: 1 × 10⁸ (lane 2), 3 × 10⁷ (lane 3), 1 × 10⁷ (lane 4), 3 × 10⁶ (lane 5), and 1 × 10⁶ (lane 6). C, downregulation of ppET-1 mRNA by laminar shear stress determined using standard-calibrated, competitive RT-PCR (static condition: open bar, shear stress: dashed bars). Values are given as means ±s.e.m. (aM μg⁻¹ RNA); n= 5, where n is the number of independent experiments; * P < 0.05 vs. static control (con).