Flow Pulsatility Is a Critical Determinant of Oxidative Stress in Endothelial Cells
Atherosclerotic plaques are found in regions exposed to disturbed flow, suggesting the active participation of the hemodynamic environment in atherogenesis. Indeed, unidirectional and oscillatory flow patterns (ie, bidirectional) have been shown to induce contrasting effects on endothelial function. The purpose of the present study was to evaluate the effect of these 2 flow patterns characterizing plaque-free and plaque-prone regions, respectively, on the oxidative stress of endothelial cells. NADH-dependent oxidase activity was shown to be equally induced (2- to 3-fold) in endothelial cells exposed to pulsatile unidirectional or oscillatory flow patterns. Under these flow conditions, an increase in endothelial cell oxidative state compared with static cultures was observed. Pulsatility of flow, but not cyclic stretch, was a critical determinant of flow-induced superoxide anion production. P22phox mRNA level increased in cells exposed to both unidirectional and oscillatory shear stress, suggesting that p22phox gene expression upregulation contributes to flow-induced increase in superoxide anion production in endothelial cells. In conclusion, we demonstrate a flow-induced increase in oxidative stress in endothelial cells. This chronic increase is dependent on the pulsatile nature of flow and is mediated in part by upregulation of an NADH-dependent oxidase expression.
A whole body of evidence shows that the endothelium is both quantitatively and qualitatively responsive to mechanical forces. Indeed, contrasting responses were observed when endothelial cells (ECs) were exposed to an oscillatory shear stress, characterized by a low mean shear stress value and a cyclic flow reversal, compared with ECs exposed to unidirectional shear stress. Lack of cell alignment in the flow direction,1 increased expression of adhesion molecules,2 lack of endothelial NO synthase (NOS III) gene expression upregulation,3–5 and, finally, chronic increase in superoxide anion production6 were observed under oscillatory flow conditions. All of these changes are of particular interest because of the focal nature of atherosclerosis along the vascular tree with a particular tropism for regions exposed to oscillatory shear stress. Several studies have demonstrated that the early stages of atherosclerosis are characterized by a decreased NO bioavailability as a possible consequence of inactivation by superoxide anion.7,8
In the present study, we further characterize the effects of mechanical forces on the oxidative state of ECs and on NADH-dependent oxidase activity using a unique flow device developed in our laboratory.1,3 The major advantage of this device consists of exposure of ECs to different combinations of shear stress, cyclic stretch, and pressure. The effects of these hemodynamic forces on superoxide anion production were analyzed. In particular, the contribution of flow pulsatility, an important mechanical property of arterial circulation, has not been carefully considered so far. We further evaluated the effect of flow on the expression of a subunit of vascular NADH-dependent oxidase; p22phox. Finally, we analyzed the effect of flow patterns on the protein carbonylation rate and the heme-oxygenase-1 (HO-1) protein expression, which can be considered a hallmark of total cellular oxidative state.
Cell Culture, Transfection, and Perfusion System
Bovine aortic ECs (BAECs) were isolated as previously described and were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 100 U/mL penicillin/streptomycin (Life Technologies). All experiments were performed with cells between passages 3 and 6 from the same aorta. The perfusion system has been described in more detail elsewhere.1,3
NADH-Dependent Oxidase Activity Measurement
A lucigenin assay was used to assess NADH-dependent superoxide anion production in ECs subjected to mechanical forces for 24 hours as previously described,6 with minor modifications. The assay was performed in 1 mL of reaction buffer containing 5 μmol/L lucigenin (Sigma Chemical Co) and 100 μmol/L NADH (Calbiochem). Levels of photon emission in the extracts in absence of NADH were similar to the levels measured in reaction buffer alone. NADH-dependent photon emission was then transformed in superoxide anion nanomoles per minute per milligram of protein by comparison with a standard curve generated with xanthine/xanthine oxidase and normalized to the protein content of the extracts.
Oxidative State of the Cells
The protein carbonylation rate in BAECs exposed to flow was determined using the OxyBlot kit system (Oncor Appligene) based on the detection of carbonyl groups on proteins. Protein extracts (15 μg) was processed according to the instruction provided by the manufacturer.
To analyze HO-1 gene expression, 15 μg proteins was separated on 8% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond-ECL; Amersham) using a semidry transfer system (Bio-Rad). Membranes were hybridized with a mouse anti-human HO-1 (Calbiochem) for 15 to 18 hours at 4°C in 10 mmol/L Tris, pH 7.5, 100 mmol/L NaCl, and 0.1% Tween 20. After 3 washes in the same buffer, membranes were hybridized with a secondary antibody, rabbit anti-mouse IgG coupled to horseradish peroxidase (Amersham) for 60 minutes at room temperature in the same buffer. After final washes, peroxidase activity was revealed using ECL (Amersham). After exposure, HO-1 protein levels were quantified by scanning the x-ray film (Apple Onescanner) followed by densitometric analysis (NIH Image, NIH).
RNA Extraction and Semiquantitative RT-PCR
RNA was extracted from trypsinized cells using the RNAeasy kit (Qiagen) according to the manufacturer’s instructions. To analyze p22phox mRNA expression, we performed reverse transcription-polymerase chain reaction (RT-PCR). Total RNA (1 μg) was denatured in the presence of 1 μg random hexamers (Promega) at 95°C for 5 minutes. After cooling on ice, RNA was reverse transcribed in presence of 50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L DTT, 0.5 mmol/L concentration of each dNTP, 20U RNase inhibitor (Promega), and 200 U Moloney murine leukemia virus reverse transcriptase (Promega). The reaction was incubated for 60 minutes at 37°C. One fifth of the RT-PCR was used to amplify p22phox and GAPDH mRNAs. This amplification was performed using HotStartTaq DNA polymerase (Qiagen), in the presence of 1.5 mmol/L MgCl2, 0.2 mmol/L concentration of each dNTP, 1 μCi [α-32P]dCTP (Amersham), and 0.5 μmol/L concentration of the respective forward and backward primers. The sequence of the primers used were p22phox forward, 5′-CCTACTCCATAGCAGCAGGC-3′; p22phox backward, 5′-ATGGCTGCCAGCAGATAGAT-3′; GAPDH forward, 5′-GGGTCATCATCTCTGCACCT-3′; and GAPDH backward,5′-ATCCACAGTCTTCTGGGTGG-3′. After an initial incubation of 15 minutes at 95°C, mRNAs were amplified for 24 (for p22phox) or 18 (for GAPDH) cycles including a first step of 1 minute at 95°C, followed by an annealing step of 1 minute at 57°C and a final amplification step of 1 minute at 72°C. The lengths of the amplified fragments were 259 bp for p22phox and 215 bp for GAPDH. Identities of the amplified fragments were verified by partial sequencing. To analyze NOS III gene expression, 10 μg total RNA was denatured for 10 minutes at 65°C in 50% formamide, 2.1 mol/L formaldehyde, and 1× MOPS buffer (40 mmol/L 3-N-morpholinopropanesulfonic acid, pH 7.0, 10 mmol/L sodium acetate, 1 mmol/L EDTA) and loaded onto a 1.2% agarose, 1.1 mol/L formaldehyde gel. After migration, RNA was transferred onto a Biodyne A membrane (Pall) in 20× SSC (1× SSC=150 mmol/L NaCl, 15 mmol/L trisodium citrate dihydrate) for 12 hours. The prehybridization and hybridization steps were performed as previously described,3 using specific probes for bovine NOS III (gift of Dr Harrison) and human GAPDH (Clontech). All cDNAs were labeled by random priming in the presence of [32P]dATP (Amersham) before their use. NOS III gene expression was normalized to GAPDH gene expression.
Data are represented as mean±SEM. ANOVAs were performed using a Kruskal-Wallis test for independent samples. When positive (P<0.05), the groups were compared with the reference using a nonparametric Mann-Whitney test.
Unidirectional and Oscillatory Shear Stress Increases Superoxide Anion Production in ECs
Using lucigenin chemiluminescence, we were able to detect a superoxide anion production (688±74 nmol · min−1 · mg of protein extract−1) in BAECs maintained in static cultures. This measurement was performed using 5 μmol/L lucigenin, a concentration at which spontaneous generation of superoxide anion by lucigenin has been shown to remain undetectable.9 This activity, completely dependent on the concomitant presence of NADH (100 μmol/L), was inhibited in the presence of 100 μmol/L Tiron (Sigma Chemical Co), a superoxide scavenger, and in the presence of diphenylene iodonium (DPI; Alexis), an inhibitor of flavin-containing enzymes, in a dose-dependent way (Figure 1). No inhibition was observed in the presence of rotenone (100 μmol/L; (Sigma Chemical Co), allopurinol (100 μmol/L; (Sigma Chemical Co), and L-NAME (1 mmol/L; (Sigma Chemical Co) (data not shown).
Superoxide anion production in BAECs was increased by exposure to pulsed unidirectional shear stress (0.3±0.1 and 6±3 dynes/cm2) and to a pulsed oscillatory shear stress (0.3±3 dynes/cm2) for 24 hours (Figure 2A, open columns), whereas at 4 hours, we observed an increase in superoxide anion production only in cells exposed to the higher level of unidirectional shear stress (6±3 dynes/cm2) (Figure 2A, filled columns). The combination of these mechanical forces with a 6% cyclic stretch value did not reveal significant differences (Figure 2B).
Our results obtained after 24 hours of exposure to pulsed unidirectional shear stress were in partial contradiction to published data.6 To investigate the reasons of this paradox, we exposed ECs to a nonpulsed unidirectional flow (0.3 and 6 dynes/cm2) for 24 hours. This particular flow pattern is identical to that generated in the parallel-plate device used in the cited studies.6 Under these flow conditions, no difference in NADH-dependent superoxide anion production was observed in flow-exposed cells compared with cells maintained in static cultures (Figure 2C).
Increased superoxide anion production was shown to correlate with the upregulation of p22phox gene expression in cells exposed to different flow patterns (Figure 3A and 3B).
Both Unidirectional and Oscillatory Shear Stress Induce an Increase in the Oxidative State of ECs
The rate of protein carbonylation, an hallmark of global cellular oxidation,10 was observed to be increased by ≈2.5- to 3-fold in BAECs exposed to different flow patterns for 24 hours compared with the static cultures (Figure 4A). However, protein carbonylation has been questioned as a valid unambiguous marker of oxidative stress.11 We therefore verified and confirmed the global increase in cellular oxidation by analyzing the expression of a redox-sensitive protein: HO-1.12,13 Indeed, a strong induction of HO-1 protein level was observed after exposure to flow independent from its pattern (Figure 4B). Induction was particularly marked in cells exposed to 6±3 dynes/cm2 for 24 hours.
Antioxidant Treatment Inhibits Unidirectional Flow-Induced NOS III Gene Expression Upregulation
We next evaluated the relationship between the observed increase in oxidative state and the regulation of NOS III gene expression by shear stress. The effect of a well-known antioxidant, N-acetyl-l-cysteine (L-NAC), on oxidative stress of ECs submitted to flow was determined by establishing a dose-response curve; 20 mmol/L L-NAC was necessary to inhibit the flow-mediated induction of oxidative stress in ECs (data not shown).
The relation between NOS III gene expression in ECs exposed to shear stress and the increase in oxidative state was then investigated using 20 mmol/L L-NAC. At these concentrations, L-NAC did not affect basal expression of NOS III (data not shown). Presence of the antioxidant in the flow experiment did not affect the weak induction of NOS III mRNA in cells exposed to oscillatory flow compared with the level in static cultures (Figure 5). On the contrary, the sustained induction of NOS III gene expression by pulsed unidirectional flow was partially inhibited in the presence of 20 mmol/L L-NAC (Figure 5).
Increased oxidative state is encountered in many pathological situations that affect the vasculature, such as angiotensin II-induced hypertension,14–16 diet-induced atherosclerosis,17 and mechanical injury of the vessel wall.18 In situ hybridization on vascular tissue sections localized superoxide anion production in smooth muscle cells in the media and in fibroblasts in the adventitia.16 In vitro experiments have revealed that ECs and infiltrated monocytes were also able to produce superoxide anions.6,19–21
The aim of the present study was to examine the effects of different hemodynamic forces characteristic of plaque-free and plaque-prone regions on the oxidative stress in ECs by using a perfusion system that was developed in our laboratory. The device allows exposure of cells to different combinations of shear stress, cyclic stretch, and pressure. We first detected superoxide anion production in BAECs maintained is static cultures, as a result of an NADH-dependent enzymatic activity sensitive to DPI, a flavin-containing enzyme inhibitor. Transient transfection analysis suggested p22phox to be a component of the NADH-oxidase (data not shown). BAECs were then exposed to different laminar pulsatile flow conditions: unidirectional and oscillatory flows, characteristic of plaque-free and plaque-prone zones, in combination with a mean pressure of 100±30 mm Hg. After 24 hours’ exposure under these conditions, an induction of superoxide anion production, increased p22phox gene expression, and protein carbonylation as well as HO-1 protein levels were observed. Superoxide anion production was unaltered by the combination of shear stress with cyclic stretch, suggesting that cyclic stretch is not playing a major role on ECs oxidative stress. Interestingly, the increase in superoxide anion production was dependent on the magnitude of the applied shear stress only after 4 hours of flow exposure but not at 24 hours. This observation suggests the existence of different kinetics of mechanotransduction pathways triggered by low and moderate pulsatile unidirectional shear stress. Moreover, these data further support the critical role of pulsatility over the shear rate and/or pattern on the control of the redox state of ECs.
Lack of pulsatility of unidirectional flow abolished the increase in superoxide anion production after 24 hours’ flow exposure. This observation stresses the importance of flow pulsatility when assessing the redox state of the ECs exposed to different hemodynamic conditions. De Keulenaer et al6 reported an NADH-oxidase activity induction only in ECs exposed to oscillatory shear stress, whereas unidirectional shear stress was unable to maintain a sustained enzyme activity. However, their data were obtained using a parallel-plate perfusion system generating a nonpulsed unidirectional shear stress and a pulsed oscillatory shear stress. The pulsatile nature of the flow in vivo should not be overlooked when addressing the role of hemodynamic forces in the pathogenesis of atherosclerosis.
The enhanced superoxide anion production observed in BAECs exposed to the different flow patterns correlates positively with an increase in p22phox mRNA level, suggesting that flow-induced superoxide anion production is achieved, at least in part, via upregulation of the expression of different subunits of NADH-oxidase. These data do not exclude posttranslational regulation of such subunits, which have been shown to play an important role in regulation of enzymatic activity.22
The increase in superoxide anion production also resulted in a global enhanced oxidative state of ECs, as determined by analysis of the protein carbonylation level and expression of the redox-sensitive gene HO-1.
In conclusion, these data suggest that pulsatile flow induces a general increase in the oxidative state of ECs, which is mediated by upregulation of the expression of vascular NADH-dependent oxidase subunits with a consequent increase in superoxide anion production. Taken together with our previous results regarding NOS III gene expression regulation by different hemodynamic environments, we can speculate that in cells exposed to a plaque-free flow regimen, the balance between NO and superoxide anion productions is well preserved, whereas under oscillatory flow, it is in favor of superoxide anion.
We and others were able to show that pulsed unidirectional flow upregulated NOS III gene expression via a mechanism that partially involved activation of nuclear factor (NF)-κB activation.3–5 On the contrary, oscillatory flow failed to induce NOS III gene expression in ECs. To analyze the possible relationship between increased oxidative state in cells exposed to flow patterns and regulation of NOS III gene expression, we exposed BAECs to flow conditions in the presence of L-NAC, a well-known antioxidant. First, we determined the concentration that effectively affected flow-induced upregulation of the oxidative state of ECs. Second, we analyzed by Northern blot the expressions of NOS III gene in ECs exposed to flow in combination with antioxidant treatment. These analyses revealed a partial involvement of increased superoxide production in NOS III gene expression upregulation by unidirectional shear stress. This requirement can be explained by the contribution of NF-κB on the induction of NOS III promoter activity via its binding to the shear stress responsive element, as demonstrated in our previous work.5 Finally, the use of an antioxidant did not affect the lack of induction of NOS III gene expression observed in cells exposed to oscillatory shear stress.
Interestingly, Gosgnach et al23 recently demonstrated the participation of oxidative stress in the upregulation of NOS II in smooth muscle cells exposed to shear stress.23 In this case, too, oxidative stress exerted its inductive action through the activation of NF-κB.
When antioxidants were used, we observed, for the higher concentrations (>20 mmol/L for LNAC), detachments of the cells from the tubes during flow exposure. This effect was even enhanced by the use of pyrrolidine dithiocarbamate (>25 μmol/L), an antioxidant that inhibits NF-κB activation.24 Interestingly, NF-κB has been involved in antiapoptotic response in ECs.25 It is therefore tempting to speculate about a possible protective role played by a certain level of oxidative stress in ECs against apoptosis.
This work was supported by grants to Drs Silacci and Hayoz from Swiss National Foundation (32-54612.98), by the Leenaards Foundation, and by the Fondation pour la Recherche Cardiovasculaire.
- Received April 28, 2001.
- Revision received June 18, 2001.
- Accepted July 5, 2001.
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