Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO−) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the “hyperoxic state” of 21% O2, compared with more physiological O2 tensions (Po2), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O2·−) generation leading to ONOO− formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O2 consumption rates of bovine aortic ECs sheared (10 dyn/cm2, 30 min) at 5%, 10%, or 21% O2 or left static at 5% or 21% O2. Respiration was inhibited to a greater extent when ECs were sheared at 21% O2 than at lower Po2 or left static at different Po2. Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO− scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O2·− production was higher in ECs sheared at 21% than at 5% O2, as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O2 was modestly increased compared with ECs sheared at lower Po2, suggesting that eNOS activity may be higher at 21% O2. Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O2·− production, leads to enhanced ONOO− formation intramitochondrially and suppression of respiration.

Shear-induced reactive nitrogen species inhibit mitochondrial respiratory complex activities in cultured vascular endothelial cells

There is evidence that nitric oxide (NO), superoxide (O2•−), and their associated reactive nitrogen species (RNS) produced by vascular endothelial cells (ECs) in response to hemodynamic forces play a role in cell signaling. NO is known to impair mitochondrial respiration. We sought to determine whether exposure of human umbilical vein ECs (HUVECs) to steady laminar shear stress and the resultant NO production modulate electron transport chain (ETC) enzymatic activities. The activities of respiratory complexes I, II/III, and IV were dependent on the presence of serum and growth factor supplement in the medium. EC exposure to steady laminar shear stress (10 dyn/cm2) resulted in a gradual inhibition of each of the complexes starting as early as 5 min from the flow onset and lasting up to 16 h. Ramp flow resulted in inhibition of the complexes similar to that of step flow. When ECs were sheared in the presence of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 100 μM), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; 100 μM), or the peroxynitrite (ONOO−) scavenger uric acid (UA; 50 μM), the flow-inhibitory effect on mitochondrial complexes was attenuated. In particular, l-NAME and UA abolished the flow effect on complex IV. Increased tyrosine nitration was observed in the mitochondria of sheared ECs, and UA blocked the shear-induced nitrotyrosine staining. In summary, shear stress induces mitochondrial RNS formation that inhibits the electron flux of the ETC at multiple sites. This may be a critical mechanism by which shear stress modulates EC signaling and function.

A New In Vitro Model to Evaluate Differential Responses of Endothelial Cells to Simulated Arterial Shear Stress Waveforms

In the circulation, flow-responsive endothelial cells (ECs) lining the lumen of blood vessels are continuously exposed to complex hemodynamic forces. To increase our understanding of EC response to these dynamic shearing forces, a novel in vitro flow model was developed to simulate pulsatile shear stress waveforms encountered by the endothelium in the arterial circulation. A modified waveform modeled after flow patterns in the human abdominal aorta was used to evaluate the biological responsiveness of human umbilical vein ECs to this new type of stimulus. Arterial pulsatile flow for 24 hours was compared to an equivalent time-average steady laminar shear stress, using no flow (static) culture conditions as a baseline. While both flow stimuli induced comparable changes in cell shape and alignment, distinct patterns of responses were observed in the distribution of actin stress fibers and vinculin-associated adhesion complexes, intrinsic migratory characteristics, and the expression of eNOS mRNA and protein. These results thus reveal a unique responsiveness of ECs to an arterial waveform and begin to elucidate the complex sensing capabilities of the endothelium to the dynamic characteristics of flows throughout the human vascular tree.

Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K+ and Cl− channels

Steady laminar shear stress has been shown previously to markedly increase Na-K-Cl cotransporter mRNA and protein in human umbilical vein endothelial cells and also to rapidly increase endothelial K+ and Cl− channel conductances. The present study was done to evaluate the effects of shear stress on Na-K-Cl cotransporter activity and protein expression in bovine aortic endothelial cells (BAEC) and to determine whether changes in cotransporter expression may be dependent on early changes in K+ and Cl− channel conductances. Confluent BAEC monolayers were exposed in a parallel-plate flow chamber to either steady shear stress (19 dyn/cm2) or purely oscillatory shear stress (0 ± 19 dyn/cm2) for 6–48 h. After shearing, BAEC monolayers were assessed for Na-K-Cl cotransporter activity or were subjected to Western blot analysis of cotransporter protein. Steady shear stress led to a 2- to 4-fold increase in BAEC cotransporter protein levels and a 1.5- to 1.8-fold increase in cotransporter activity, increases that were sustained over the longest time periods studied. Oscillatory flow, in contrast, had no effect on cotransporter protein levels. In the presence of flow-sensitive K+ and Cl− channel pharmacological blockers, the steady shear stress-induced increase in cotransporter protein was virtually abolished. These results suggest that shear stress modulates the expression of the BAEC Na-K-Cl cotransporter by mechanisms that are dependent on flow-activated ion channels.

Endothelial albumin permeability is shear dependent, time dependent, and reversible

Altered permeability of vascular endothelium to macromolecules may play a role in vascular disease as well as vascular homeostasis. Because the shear stress of flowing blood on the vascular wall is known to influence many endothelial cell properties, an in vitro system to measure transendothelial permeability (Pe) to fluorescein isothiocyanate conjugated bovine serum albumin under defined physiological levels of steady laminar shear stress was developed. Bovine aortic endothelial cells grown on polycarbonate filters pretreated with gelatin and fibronectin constituted the model system. Onset of 1 dyn/cm2 shear stress resulted in a Pe rise from 5.1 +/- 1.3 x 10(-6) cm/s to 21.9 +/- 4.6 X 10(-6) cm/s at 60 min (n = 6); while 10 dyn/cm2 shear stress increased Pe from 4.8 +/- 1.5 X 10(-6) cm/s to 50.2 +/- 6.8 X 10(-6) cm/s at 30 min and 49.6 +/- 8.9 X 10(-6) cm/s at 60 (n = 9). Pe returned to preshear values within 120 and 60 min after removal of 1 and 10 dyn/cm2 shear stress, respectively. The data show that endothelial cell Pe in vitro is acutely sensitive to shear stress.