Modulation by pathophysiological stimuli of the shear stress-induced up-regulation of endothelial nitric oxide synthase expression in endothelial cells

Abstract

Objective: Fluid shear stress (the frictional force resulting from blood flow) is a principal regulator of endothelial nitric oxide synthase (eNOS) expression. We examined the responses of eNOS messenger ribonucleic acid (mRNA) levels to dynamic shear stimuli in the presence of pathological risk modifiers.

Methods: Confluent bovine aortic endothelial cells were subjected in vitro to shear stress (using a cone-plate viscometer) and to hydrostatic pressure (using a custom-built pressure chamber device). eNOS mRNA levels were quantitated by densitometric analysis of Northern blots.

Results: In contrast to steady laminar shear stress, which elevated eNOS mRNA levels in a time- and dose-dependent manner (2.9- and 3.6-fold after 6 h at 4 and 20 dyn/cm2, respectively), steady hydrostatic pressure of 150 mm Hg decreased eNOS mRNA levels by 46%. eNOS mRNA up-regulation by shear stress was reversible after cessation of flow, although it was not influenced by previous shear exposure, and it was not mediated by a stable transferable factor. eNOS mRNA up-regulation by sinusoidal shear stress was frequency-dependent, with a moderate response at 1-Hz oscillating shear and no change at 0.3 Hz. Hypoxia (3% O2) suppressed eNOS mRNA expression by 78% under static conditions and by 72% under shear conditions but did not alter the fold induction by shear. Elevated glucose concentrations reduced eNOS mRNA levels in both resting and shear stress-exposed cells but did not reduce the fold induction by shear; the protein kinase C inhibitor calphostin C was without effect. Shear-induced up-regulation of eNOS mRNA was unaffected by changes in the medium partial pressure of CO2/pH, by the Na+/H+-exchanger inhibitor HOE694, or by aspirin. In contrast, the shear response was potentiated by homocysteine.

Conclusion: Both physical and chemical stimuli regulate eNOS mRNA levels in endothelial cells. Although eNOS mRNA expression is increased by shear stress, it is decreased by hydrostatic pressure, hypoxia, and elevated glucose levels. The effect of shear on eNOS mRNA expression involves a reversible, frequency-dependent process. These in vitro findings suggest possible contributions of the eNOS flow response to atherosclerosis, in the presence of systemic risk factors.