Shear Stress Inhibits H2O2-Induced Apoptosis of Human Endothelial Cells by Modulation of the Glutathione Redox Cycle and Nitric Oxide Synthase
Abstract Physiological levels of shear stress reduce endothelial cell turnover and exert a potent antiatherosclerotic effect. Here we demonstrate that oxidative stress-induced apoptosis of human endothelial cells was inhibited by shear stress exposure (15 dynes/cm2). Incubation with H2O2 (200 μmol/L) for 18 hours induced apoptosis of human umbilical venous endothelial cells as demonstrated by an enzyme-linked immunosorbent assay specific for histone-associated DNA fragments and visual analysis of fluorescence-stained nuclei. Shear stress-mediated inhibition of apoptosis was partially prevented by pharmacological inhibition of glutathione (GSH) biosynthesis with buthionine sulfoximine (BSO) or nitric oxide (NO) synthase with NG-monomethyl-l-arginine (LNMA), whereas inhibition of catalase by aminotriazol did not affect the inhibitory action of shear stress. Combined inhibition of NO synthase and GSH biosynthesis completely reversed the protective effect of shear stress, suggesting that both NO synthase and the GSH redox cycle system are involved in the apoptosis-suppressing effect of shear stress. Similar results were obtained when apoptosis was stimulated by tumor necrosis factor α (TNFα). To gain further insights into the interference of shear stress with apoptosis signal transduction, we measured caspase-3-like activity, a cysteine protease that has been shown to play a predominant role in the cell death effector pathway. Indeed, shear stress prevented the activation of caspase-3-like activity induced by H2O2 or TNFα. The inhibitory effect of shear stress was prevented by LNMA and BSO, suggesting that the reduction of oxidative flux by shear stress prevents the activation of caspase-like proteases and thereby inhibits apoptotic cell death in human endothelial cells.
- Received June 30, 1997.
- Accepted August 20, 1997.
The dysfunction of the vascular endothelium is an important contributor to the development of atherosclerotic lesions and the progression of inflammatory diseases. Regions where atherosclerotic lesions develop are characterized by enhanced endothelial cell turnover,1 which may be due to the activation of the endogenous cell death program, termed apoptosis. Importantly, these lesion-prone regions localize to areas of reduced shear stress or unsteady blood flow, suggesting a link between shear stress and atherogenesis and endothelial cell death. Indeed, we have previously reported that apoptosis of human endothelial cells induced by the inflammatory cytokine TNFα or growth factor withdrawal was potently inhibited by physiological levels of laminar shear stress.2
Vascular endothelial cells are in constant contact with steady-state levels of oxidative metabolites, which are increased in a number of pathophysiological processes that affect the blood vessel such as atherosclerosis and diabetes mellitus. Free radicals can be produced extracellularly via the respiratory burst of neutrophils or macrophages or intracellularly by the activation of the xanthine oxidase system. Clinical studies as well as experimental evidence suggest a causal pathophysiological role of increased oxidative stress in endothelial dysfunction.3 In vitro, oxidative metabolites are involved in the functional inactivation of the endothelial cells with an increase of permeability4 and additionally are potent inducers of endothelial cell death.5 Importantly, the endothelium-derived NO interacts with the oxidative balance of cells and thereby plays a pivotal role as an antiatherosclerotic and anti-inflammatory molecule.6 7 Increased endogenous production or exogenous addition of NO reduces the oxidative flux8 and further prevents cell death induced by the inflammatory cytokine TNFα.9 Increased oxidative stress, on the other hand, reduces the biological available NO, which results in an impaired agonist- and shear stress-mediated vasodilation.10
In this study, we investigated the effect of shear stress on oxidative stress-induced apoptosis of human endothelial cells. We report that the protective shear stress effect is mediated at least in part by enhanced endothelial NO synthesis. However, complete reversion of the shear stress effect was only achieved, when GSH biosynthesis was additionally inhibited, suggesting a combined effect of shear stress on both NO synthesis and the antioxidative capacity of human endothelial cells. In addition, we demonstrate that shear stress also affects TNFα-induced apoptosis by similar mechanisms.
HUVECs, endothelial basal medium, and supplements were purchased from Cell Systems/Clonetics, and fetal calf serum from Gibco. Caspase-3 substrate was supplied by Biomol.
Cell Culture and Shear Stress Exposure
HUVECs were cultured in endothelial basal medium supplemented with hydrocortisone (1 μg/mL), bovine brain extract (3 μg/mL), gentamicin (50 μg/mL), amphotericin B (50 μg/mL), epidermal growth factor (10 μg/mL), and 10% fetal calf serum until the third passage. After detachment with trypsin, cells were grown for at least 18 hours. Confluent monolayers of HUVECs were grown onto 6-cm-wells and exposed to laminar fluid flow in a cone-and-plate apparatus as previously described.11 A constant shear stress of 15 dynes/cm2 was used in all experiments to simulate physiological levels of shear stress.11 12
Determination of Apoptosis and Necrosis
DNA fragmentation was determined with the cell death detection ELISA (Boehringer Mannheim).2 9 Therefore, cells were scraped off the plates and centrifuged at 700g for 10 minutes, washed with phosphate-buffered saline and resuspended in incubation buffer. The histone-associated DNA fragments were linked to the anti-histone antibody from mouse and the DNA part of the nucleosome to the anti-DNA peroxidase. The amount of peroxidase retained in the immunocomplex was determined photometrically.
For morphological staining of nuclei, cells were fixed in 4% formaldehyde and were stained with DAPI (0.2 μg/mL in 10 mmol/L Tris-HCl, pH=7, 10 mmol/L EDTA, and 100 mmol/L NaCl) for 20 minutes. Then nuclei were analyzed by fluorescence microscopy.
For measurement of lactate dehydrogenase levels, a kit was used (Boehringer Mannheim). Cells (1×105) were seeded into 12-well plates. The cell culture supernatant was incubated with pyruvate and NADH, and the lactate dehydrogenase activity was determined photometrically according to the manufacturer’s protocols.
For detection of caspase-3-activity, HUVECs (1×106 cells) were lysed in buffer (1% Triton X-100, 0.32 mol/L sucrose, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 2 mmol/L dithiothreitol, and 10 mmol/L Tris-HCl, pH=8) for 15 minutes at 4°C, followed by centrifugation (20 000g, 10 minutes). Caspase-3 activity was detected in resulting supernatants by measuring the proteolytic cleavage of the fluorogenic substrate 7-amino-4-coumarin-Asp-Glu-Val-Asp (DEVD)13 and 7-amino-4-coumarin as standard in assay buffer (100 mmol/L HEPES, 10% sucrose, 0.1% CHAPS, pH=7.5, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 10 mmol/L dithiothreitol) using an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Protein content was analyzed using the Bio-Rad assay (Bio-Rad).
Statistical analysis was performed with ANOVA followed by least significant differences test (SPSS Software).
Shear Stress Protects From H2O2-Induced Apoptosis of HUVECs
H2O2 (200 μmol/L) induced apoptosis of HUVECs in a time-dependent manner as measured by an ELISA specific for histone-associated DNA fragments (Fig 1⇓) and morphologic analysis of fluorescence staining of nuclei (Fig 2⇓). Maximal apoptosis was achieved after incubation for 18 hours (Fig 1⇓). Quantification of the apoptotic cells by DAPI staining revealed 8.9±1.6% apoptotic cells after H2O2 treatment compared with 2.7±0.6% in controls. At this time point (18 hours), no significant release of lactate dehydrogenase was detectable (115±7% of controls), excluding the induction of necrotic cell death.
To evaluate the effect of shear stress (15 dynes/cm2) on H2O2-induced apoptosis, laminar flow (15 dynes/cm2) was generated in a cone-and-plate apparatus. As shown in Figs 2⇑ and 3⇓, exposure of HUVECs to shear stress completely prevented H2O2-triggered apoptosis. Similar results were obtained by quantification of apoptotic cells by DAPI staining. Shear stress reduced the number of apoptotic cells induced by H2O2 (8.9±1.6% apoptotic cells) to control levels (2.1±1.5% apoptotic cells).
We previously reported that low concentrations of NO potently inhibit endothelial cell apoptosis induced by TNFα.9 Since shear stress correlates with enhanced NO production, we further investigated the role of endothelial-derived and exogenous NO in H2O2-induced apoptosis. Inhibition of NO synthesis by LNMA (1 mmol/L) significantly reduced the inhibitory effect of shear stress on H2O2-triggered apoptosis (Fig 3A⇑ and 3B⇑). The exogenous NO donors sodium nitroprusside or S-nitrosopenicillamine in a concentration of 10 μmol/L ameliorated the H2O2-triggered apoptosis (Fig 4⇓). However, shear stress seems to use additional antiapoptotic pathways besides NO, because both endogenous as well as exogenous NO only partially mediated the effect of shear stress.
Because catalase and the GSH redox cycle are important intracellular antioxidant systems involved in the enzymatic reduction of H2O2, we investigated the contribution of the redox system on shear stress-mediated inhibition of H2O2-induced apoptosis. Specific inhibition of catalase activity with 0.5 mmol/L aminotriazol14 did not reverse the inhibition of H2O2-induced apoptosis by shear stress (Fig 3A⇑), suggesting that catalase does not account for the antiapoptotic effect of shear stress. In contrast, inhibition of GSH biosynthesis with BSO15 partially prevented the protective shear stress effect (Fig 3B⇑).
To test whether NO synthesis and GSH redox status synergistically contribute to the shear stress effect, both pathways were blocked simultaneously. LNMA in combination with BSO completely reversed the antiapoptotic effect of shear stress (Fig 3B⇑). These results were confirmed by visual analysis of apoptotic nuclei (Fig 2⇑). Control experiments showed that the compounds used did not affect basal apoptosis (data not shown) and did not significantly influence the H2O2-induced apoptosis (Fig 3A⇑ and 3B⇑)
Effect of Shear Stress on TNFα-Induced Apoptosis
As previously described, TNFα induces apoptosis of HUVECs,9 which is abrogated by laminar shear stress.2 However, the underlying molecular mechanism is only partially explained by increased NO production (Fig 5⇓).
Because reactive oxygen intermediates generated during mitochondrial respiration have been shown to be involved in TNFα-induced apoptosis,16 we investigated the role of the GSH redox cycle in the antiapoptotic capacity of shear stress. Inhibition of GSH biosynthesis or NO synthase significantly reduced the suppression of TNFα-induced apoptosis by shear stress (Fig 5⇑), and the combination of both substances completely abrogated the apoptosis-suppressive effect of shear stress (Fig 5⇑).
Influence of Shear Stress on Caspase-3
Activation of the protease family of caspases represents the final common pathway of apoptosis signal transduction. Because shear stress has been shown to most likely interfere proximal of caspase-3 and thereby prevent apoptosis,2 we investigated whether shear stress-induced modulation of NO synthesis and the GSH redox cycle contributes to a reduction of caspase-3 activity. H2O2-induced increase of caspase-3 activity was completely prevented by exposure of HUVECs to shear stress (Fig 6⇓). Inhibition of NO synthesis or GSH biosynthesis slightly, but insignificantly, diminished the shear stress-induced caspase-3 suppression (Fig 6⇓), whereas the combination of both inhibitors completely abolished the inhibitory effect of shear stress (Fig 6⇓). Similar results were obtained for TNFα-stimulated apoptosis. Thus, the TNFα-induced increase of caspase-3-like activity (254±50% of controls) was significantly reduced by shear stress (126±38%), but was completely restored in the presence of BSO and LNMA (266±8%).
Fluid shear stress alters diverse transcriptional and post-transcriptional responses of endothelial cells and thereby is an important determinant of endothelial function.12 17 In vivo, physiological levels of shear stress are associated with a diminished risk for the development of atherosclerotic lesions and reduced endothelial cell “turnover.” Thus, shear stress seems to improve endothelial function and prevent endothelial cell death. Here we extend our previous studies by demonstrating that shear stress not only prevents serum depletion or TNFα- induced apoptosis, but also potently interferes with apoptosis due to enhanced exogenous oxidative stress generated by H2O2. Because endothelial cells are constantly exposed to oxidative metabolites, especially in patients with an increased risk for atherosclerotic disease such as diabetes mellitus or hypercholesterolemia,3 these findings may give additional insights to explain the focal nature of atherosclerotic lesion development.
The biological activity of NO is important for the function of the endothelial cells and the modulation of blood pressure. Indeed, NO exerts anti-inflammatory functions by prevention of neutrophil adherence8 18 and downregulation of monocyte chemoattractant protein.7 In addition, NO prevents not only endothelial cell death5 9 but also apoptosis of B-cells19 or hepatocytes20 and interferes with oxidant-induced cell injury.5 It is well established that shear stress upregulates NO production11 even in the presence of TNFα,9 suggesting a link between NO production and suppression of apoptosis. Indeed, inhibition of NO synthesis ameliorated the protective capacity of shear stress on apoptotic cell death. However, the shear stress-induced NO synthesis only partially accounted for the apoptosis-suppressive effects of shear stress. Thus, additional antiapoptotic mechanisms are very likely involved in mediating the apoptosis-suppressive effects of shear stress.
Because exogenous antioxidants are potent inhibitors of apoptosis induced by various kinds of stimuli,21 we determined the role of antioxidative enzymes in the downregulation of apoptosis by shear stress. Inhibition of the GSH redox cycle by BSO partially reversed the antiapoptotic effects of shear stress on H2O2-induced apoptosis, suggesting that shear stress modulates the GSH redox system. In contrast, catalase, the other enzyme, which mediates metabolization of H2O2, seems to play a minor role, since inhibition of catalase activity did not affect the shear stress response. This is in agreement with previous studies demonstrating that the GSH redox cycle represents the most important H2O2-detoxification system in endothelial cells.15 22 In summary, the mechanisms underlying the protective effect of shear stress seem to involve at least two different signals: the upregulation of NO synthase and the interference with the GSH redox status. In the absence of NO, H2O2 might be reduced by an activated GSH redox system. On the other hand, inhibition of the redox system leading to elevated oxidative flux might reduce the biologically active NO and therefore the endogenous NO synthesis might not be sufficient to completely prevent apoptosis.
The cysteine protease family of caspases plays an important role in apoptotic signal transduction especially in TNFα receptor stimulation-mediated DNA fragmentation.23 24 Indeed, we previously demonstrated that the interleukin-1β-converting enzyme recently termed caspase-1 and CPP32/Yama, caspase-3, are involved in TNFα-induced apoptosis of HUVECs.2 9 In addition, caspases also play an obligate role in oxidative stress-induced apoptosis.25 This study shows that shear stress inhibits caspase-3 activation by increasing NO synthesis and modulation of the redox system. The underlying mechanism may include direct inhibition of caspase activity by NO-stimulated S-nitrosylation as previously described,9 whereas the increase of antioxidative enzyme activity might reduce oxidative stress-induced activation of caspases.
A role for the reactive oxygen species to mediate apoptosis of endothelial cells has been recently documented by the demonstration that the radical scavengers vitamin C and N-acetylcysteine inhibit apoptosis of endothelial cells in response to exposure to oxidized LDL, which increases oxidative flux within endothelial cells.26 27 Shear stress is well known to modulate redox-sensitive genes such as VCAM-1,28 which have been implicated in the pathogenesis of atherosclerosis. The results of this study, which demonstrate a potent apoptosis-suppressive effect of shear stress on endothelial cells exposed to oxidative stress, not only considerably extend these previous observations, but, more importantly, for the first time establish the pivotal role of the synergistic upregulation of NO synthase and antioxidative capacity determined by the GSH redox cycle to mediate the effects of shear stress on endothelial cell viability. Given the fundamental importance of functional integrity of endothelial cells to prevent atherosclerotic vascular diseases, the result of this study may give important insights into the mechanisms responsible for the focal nature of atherosclerosis, which preferentially develops in vascular regions with low or unsteady shear stress or turbulent flow.
In summary, shear stress interferes with apoptosis of endothelial cells induced by exogenous addition of H2O2 or endogenously derived oxidative stress. The inhibition of apoptosis by shear stress may make an important contribution to the antiatherosclerotic effects and may explain the enhanced endothelial cell turnover rate in regions with low or unsteady blood flow. The protective effect seems to involve the shear stress-induced increase of NO synthesis and an influence on the redox system, which both act upstream or interfere with activation of the caspases cascade, which is the central effector arm executing the cell death program.
Selected Abbreviations and Acronyms
|ELISA||=||enzyme-linked immunosorbent assay|
|HUVEC||=||human umbilical vein endothelial cell|
|TNFα||=||tumor necrosis factor α|
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Di 600/2-2). S. Dimmeler has a fellowship from the Deutsche Forschungsgemeinschaft. We would like to thank Christine Goebel for expert technical assistance.
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