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

Abstract

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% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). 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 O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), 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% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.

Fig. 1.

Effects of Po₂ during shear exposure (A) and duration of shear exposure at a specific Po₂ (B) on endothelial cell (EC) respiration. A: bovine aortic ECs (BAECs) were exposed to a wall shear stress of 10 dyn/cm² for 30 min at either 5%, 10%, or 21% O₂, and corresponding controls were left static at either 5% or 21% O₂ for the same period. Po₂ measurements vs. time were performed on EC suspensions (2.4 × 10⁷ cells/ml) at the end of treatment by electron paramagnetic resonance (EPR) oximetry and are representative of 3 experiments with similar results. B: BAECs were sheared (10 dyn/cm²) for either 30 min or 16 h at 21% O₂, and corresponding controls were left static at 21% O₂ for the same periods. Po₂ measurements vs. time were performed on EC suspensions (1.3 × 10⁷ cells/ml) at the end of treatment as before and are representative of 3 experiments with similar results.

Fig. 2.

Analysis of O₂ consumption kinetics. A: BAECs were sheared (10 dyn/cm²) for 30 min at either 5% or 21% O₂, and corresponding controls were left static at either 5% or 21% O₂ for the same period. Po₂ measurements vs. time were performed on EC suspensions (0.8 × 10⁷ cells/ml) as before and are representative of 6 experiments with similar results. B: data in A were analyzed using Eq. 1, and O₂ consumption rate (V̇o₂) vs. Po₂ lines were plotted.

Fig. 3.

Effects of an endothelial nitric oxide (NO) synthase (eNOS) inhibitor, a ONOO⁻ scavenger, and manganese superoxide dismutase (MnSOD) overexpression on the inhibitory effect of shear stress at 21% O₂ on EC respiration. A: BAECs were preincubated with either 100 μM of N^G-nitro-l-arginine methyl ester (l-NAME) for 4 h or 50 μM of uric acid (UA) for 30 min and then sheared at 21% O₂ for 30 min or left static in the incubator with or without the same concentration of the respective drug. Po₂ measurements vs. time were performed on EC suspensions (2.4 × 10⁷ cells/ml) as before and are representative of 3 experiments with similar results. Since there was no difference in respiration between static ECs with or without l-NAME or UA, only data from static ECs without any drug are shown. B: BAECs were transfected with either Ad.MnSOD (multiplicity of infection of 100) or a control virus (Ad.empty) before either shear exposure at 21% O₂ for 30 min or static incubation under the same conditions. Po₂ measurements vs. time were performed on EC suspensions (2.4 × 10⁷ cells/ml) as before and are representative of 2 experiments with identical results. Western blot analysis verified an increase in MnSOD protein levels in static Ad.MnSOD-transfected ECs when compared with static ECs either nontransfected or transfected with the control virus (inset).

Fig. 4.

Intracellular and mitochondrial O₂^·− production by ECs sheared at different Po₂. A: BAECs were sheared at either 5% or 21% O₂ for 30 min, and corresponding controls were left static at each Po₂ for the same period. At the end of shear or static exposure, ECs were incubated with either dihydroethidium (DHE) or MitoSOX red (10 μM) for 10 min, and images (DHE, 20×, 514 nm/585 nm; MitoSOX, 62×, either 514 or 405 nm/585 nm) were obtained by confocal microscopy. Since no difference in DHE and MitoSOX fluorescence was detected between static controls maintained at either 5% or 21% O₂, only static controls at 21% O₂ are shown. B: digital images from 3 fields of view were collected per experiment, and the average fluorescence intensity was calculated. Normalized (to corresponding static controls) fluorescence data from n ≥ 3 independent experiments for ECs labeled with either DHE (514 nm) or MitoSOX (514 and 405 nm) and either left static (21% O₂) or sheared (5% or 21% O₂) are shown as means ± SE. *P < 0.05 vs. static control. †P < 0.05 vs. shear (21% O₂).

Fig. 5.

Regulation of mitochondrial O₂^·− production by shear-induced NO. A: BAECs were sheared at 21% O₂ for 30 min, and corresponding controls were left static under the same conditions. BAECs were preincubated with either l-NAME (100 μM for 4 h), antimycin A (10 μM for 30 min), or both and either left static or sheared in the presence of the respective drug. At the end of shear or static exposure, ECs were incubated with MitoSOX red (10 μM) and DAPI (1 μM) for 10 min, and images (MitoSOX, 62×, 514 nm/585 nm; DAPI, 62×, 359 nm/461 nm) were obtained by confocal microscopy and merged. B: digital images from 3 fields of view were collected per experiment, and the average fluorescence intensity was calculated. Normalized (to corresponding static controls) fluorescence data from n ≥ 3 independent experiments for ECs labeled with MitoSOX (514 nm) and either left static (21% O₂) or sheared (21% O₂) with or without l-NAME, antimycin A, or both are shown as means ± SE. *P < 0.05 vs. static control. †P < 0.05 vs. shear (21% O₂).

Fig. 6.

NO_x production and eNOS phosphorylation (Ser1179) by ECs sheared at different Po₂. A: BAECs were sheared for 30 min at either 5%, 10%, or 21% O₂, and corresponding controls were left static at either 5% or 21% O₂ for the same period. Medium samples were drawn at different time points after either the onset of flow or static incubation, and NO_x accumulation was measured using a chemiluminescence analyzer. Data are means ± SE of 5 independent experiments. *P < 0.05 vs. all other conditions (shear, 5% O₂; shear, 10% O₂; static, 5% O₂; static, 21% O₂) at the same time point. †P < 0.05 vs. all other conditions except shear, 10% O₂ at the same time point. B: BAECs were treated as in A, and lysates were analyzed by Western blot for phospho (p)-eNOS (Ser1179) and total eNOS.