T-786C polymorphism of the NOS-3 gene and the endothelial cell response to fluid shear stress-a proteome analysis.

Endothelial dysfunction is a common denominator of cardiovascular disease. Central to endothelial dysfunction is a decrease in the bioavailability of nitric oxide (NO) synthesized by endothelial NO synthase (NOS-3). In vivo, the level of fluid shear stress (FSS) exerted by the flowing blood determines NOS-3 expression. However, in contrast to the -786T variant of the nos-3 gene, the -786C variant is not sensitive to shear stress. Consequently, cells homozygous for this variant have an inadequate capacity to synthesize NO. Therefore, we have compared shear stress-induced protein expression in human primary cultured endothelial cells with TT or CC genotype. Cells with the CC genotype exhibited a greatly reduced FSS-induced NOS-3 expression as well as a diminished NO synthesis capacity when compared to TT genotype cells. Proteome changes in response to FSS (30 dyn/cm(2) for 24 h) were monitored by 2D-gel electrophoresis/densitometry/mass spectrometry. Of a total of 14 FSS-sensitive proteins, 8 were identically expressed in all cells. Four proteins, all of them part of the NO-dependent endoplasmic reticulum-stress response, were up-regulated by FSS only in cells with TT genotype. In contrast, CC genotype cells responded to FSS with a unique increase in manganese-containing superoxide dismutase expression. These differences in protein expression may (i) reflect the low bioavailability of NO in cells homozygous for the -786C variant of the nos-3 gene and (ii) point to a mechanism by which this deficit is counterbalanced by protecting the less abundant NO from rapid degradation.