Fluid shear stress generates oxidative stress in endothelial cells as assayed by aconitase

Fluid shear stress is an important mechanical force which acts upon endothelial cells (EC) present at the interface between the bloodstream and vascular wall. Oxidative stress has been implicated in many of the processes involved in endothelial dysfunction associated with pathological conditions such as atherosclerosis and hypertension. To determine the effect of fluid shear stress on EC redox state, we utilized a novel technique based on aconitase activity that permits evaluation of chronic changes in cellular redox state. Aconitase is an Fe-containing enzyme present in the cytosol and mitochondrial respiratory chain that is inactivated by O₂^- due to reduction of Fe³⁺ in the catalytic site. To measure aconitase activity, bovine aortic EC (BAEC) lysates were prepared, mitochondria pelleted (14,000 rpm x 20 sec), and the conversion of citrate to isocitrate assayed by spectrophotometric absorption at 340 nm. The assay was validated by generation of intracellular superoxide using 1 μM phenylmethyl sulfonate (PMS). In a time-dependent manner, 1 μM PMS caused 100% inhibition of aconitase at 1 hr, which was completely abrogated by addition of 10 mM N-acetyl cysteine. To measure the effect of flow on BAEC redox state, cells were subjected to steady laminar flow (shear stress= 12 dynes/cm²), and aconitase activity determined. Flow caused a time dependent inhibition of aconitase activity: 10 min = 53± 9%, 30 min = 62± 7%, and 60 min = 95± 2% inhibition relative to static control (n=3). In summary, these results demonstrate that steady laminar flow imposes an oxidative stress on EC comparable to 1 μM PMS. Our findings suggest that changes in EC redox state, mediated in part by mitochondrial respiration, are important in the EC response to fluid shear stress.