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1Division of Cardiology, 2Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, and 3Molecular and Systems Pharmacology Program, Emory University, Atlanta, 30322; and the Atlanta Veterans Hospital Medical Center, Decatur, Georgia 30033
Submitted 4 June 2003 ; accepted in final form 14 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.
blood flow; electron spin resonance; hydrogen peroxide; reactive oxygen species
Whereas increased ROS production in response to oscillatory shear stress has been attributed to the endothelial NAD(P)H oxidase, the role of other endothelial sources of ROS has not been examined. This is important, because NADH might be a substrate for enzymes other than the NAD(P)H oxidase such as xanthine oxidoreductase (XOR) (27). Furthermore, the NAD(P)H oxidase could produce ROS that might stimulate other sources of ROS production. For example, ROS produced from the NAD(P)H oxidase have been shown to oxidize the NO synthase (NOS) cofactor tetrahydrobiopterin, resulting in the production of large amounts of by NOS in the endothelium (19). Likewise, XOR can be converted from its dehydrogenase form (XDH) to its oxidase form (XO) by oxidation of critical cysteines (13).
Because of the above considerations, we performed the present study to precisely identify the enzyme system responsible for increased ROS production in response to oscillatory shear stress. Our data indicate that XOR is a major source of ROS produced by endothelial cells in response to oscillatory shear stress and that the NAD(P)H oxidase plays a major role in modulating the cellular balance between XDH and XO.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Cell culture. Bovine aortic endothelial cells (BAECs; Cell Systems, Kirkland, WA) were cultured in medium 199 (M199; Fischer) containing 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT) as previously described (9). Postconfluent BAECs between passages 4 and 8 were used for experiments. Murine aortic endothelial cells (MAECs) from C57Blk/6 and p47phox–/– mice were cultured in high-glucose DMEM (GIBCO) containing endothelial cell growth supplement (Biomedical Technologies) and 10% FCS. The MAECs stained positively for von Willebrand factor, incorporated diacylated LDL (DiAcLDL), and exhibited characteristic endothelial cell morphology. The absence of p47phox expression in cells from p47phox–/– animals has previously been confirmed by Western blot analysis (18). To restore p47phox expression in p47phox–/– endothelial cells, cells were transfected with a full-length p47phox cDNA inserted into the EbopLPP vector. Transfections were performed when the cells were 90% confluent using Lipofectamine. In preliminary experiments, we found that >70% of the cells were transfected, as judged by cotransfection with a vector expressing green fluorescent protein. More importantly, expression of p47phox was restored in these cells as estimated by Western blot analysis (data not shown).
To apply shear, a cone and plate viscometer with a 1° angle was used in all shear stress experiments (7). The cone was driven by a reversible stepping motor and drive (DC Industrial; Atlanta, GA). Oscillations of ±15 dyn/cm2 were applied by reversing the cone at a rate of 1.86 Hz. All shear studies were performed in 5% FCS in an incubator at 37°C with 5% CO2. In experiments where siRNA was used, cells were transfected at 50% confluency and harvested 72 h later.
Electron spin resonance. ESR measurements were performed at room temperature by using an EMX ESR spectrometer (Bruker). The ESR settings were as follows: centerfield = 3,498 G, field sweep = 60 G, microwave frequency = 9.73 GHz, microwave power = 20 mW, modulation amplitude = 1 G, conversion time = 164 ms, detector time constant = 328 ms, and receiver gain = 104. Time scans were performed by monitoring the ESR amplitude of the low-field spectrum component of 2-carboxyl-proxyl nitroxide (CP·). Equal cell numbers (5 x 106) were used for each experiment. Cell counts were taken by an automated cell counter (Beckman Coulter Z1 Cell and Particle Counter) or by a hemacytometer.
Detection of H2O2. Intracellular H2O2 was measured by using DCF-DA fluorescence as previously described (15). Briefly, DCF-DA (30 µM) was added to the media of BAECs. After shear experiments, cells were harvested by being washed twice in 10 ml and then being scraped in 1 ml of cold PBS. Cell counts were taken by hemacytometer, and 5 x 105 cells were loaded into a 96-well plate in triplicate and read with a fluorescence plate reader at excitation (Ex)/emission (Em) = 475/525 nm.
Extracellular H2O2 was measured using the horseradish peroxidase-linked Amplex Red fluorescence assay as previously described (20). Briefly, Amplex Red (50 µM) and horseradish peroxidase type II (0.1 U/ml) were added to the media of BAECs before and during shear. Fluorescence readings were made in triplicate in a 96-well plate at Ex/Em = 530/580 nm using 200-µl samples of media. H2O2 concentration was calculated by using a standard curve and was normalized to cellular protein as measured by the Bradford assay.
Western blot analysis of XOR. Western blot analysis was performed as previously described (23). Protein (100 µg) was electrophoresed at 110 V for 1.5 h on a 7.5% SDS polyacrylamide gel. The blots were incubated with a polyclonal, biotin-labeled 1° antibody anti-XOR (1:15,000 in 0.5% milk; Rockland), and 2° probe streptavidin-horseradish peroxidase (1: 50,000 in 1.0% milk; GIBCO-BRL). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham) and were quantified using densitometry.
Assessment of XO/XDH activity and xanthine-dependent ROS production. Xanthine-driven ROS production was detected using ESR. Cells were lysed by sonication (5 s), and protein concentration was quantified by Bradford assay. CPH (final concentration of 5 mM) was added to 30 µg of cellular protein homogenate. The oxypurinol (100 µM)-inhibitable portion of the xanthine (100 µM)-dependent signal was used to determine the cellular activity of XO.
To separately determine XO and XDH activity, a pterine-based assay was used as previously described (16). Briefly, XO activity was determined by the addition of pterine (10 µM) to 1 mg protein lysate. The electron donor methylene blue (10 µM) was added to determine the total XO + XDH activity. Conversion of pterine to the fluorescent product isoxanthopterin was monitored over 2.5 h (Ex/Em = 345/390 nm at 37°C).
Statistical analysis. Data are presented as means ± SE. Differences between groups and control were analyzed by using ANOVA. A post hoc Dunnett's test was employed when significance was indicated, except in the studies using siRNA and scrambled RNA, where a Tukey's post hoc analysis was used for multiple comparisons. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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at a rate approximately threefold greater than cells exposed to either laminar shear or no shear (Fig. 1C).
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The spin label CPH can be oxidized to CP· by , peroxynitrite (OONO–), and other strong oxidants (8). In addition, it is cell permeable and thus can detect intracellular ROS. To define the precise oxidant that was increased by oscillatory shear stress and its site of production, cells were exposed to a variety of specific ROS scavengers before and during ESR measurements (Fig. 1D). The CP· signal was not decreased by cell-impermeable MnSOD (2,000 U/ml) but was abolished by membrane-permeable scavengers PEG-SOD (100 U/ml) and Tiron (10 mM), suggesting that CPH was oxidized by intracellular . Ebselen (50 µM), a glutathione peroxidase mimetic that scavenges both H2O2 and OONO–, did not alter the signal.
Determination of the enzymatic source of ROS production. The above data indicate that oscillatory shear stress increases endothelial cell production of . We next sought to define the sources of that are activated by oscillatory shear stress by exposing cells to various pharmacological inhibitors immediately after shear and during ESR analysis. Neither miconazole (100 µM), NG-nitro-L-arginine methyl ester (100 µM), rotenone (2 µM), nor apocynin (600 µM) diminished the ESR signal caused by oscillatory shear stress (Fig. 2A). These experiments excluded cytochrome P-450, uncoupled endothelial NOS, mitochondrial electron transport, and the NAD(P)H oxidase as sources of activated by oscillatory shear stress. In contrast, oxypurinol (100 µM), an inhibitor of XO, significantly decreased the ESR signal caused by oscillatory shear (Fig. 2A).
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XOR exists in two interconvertable forms, both of which catalyze the oxidation of hypoxanthine to xanthine and subsequently xanthine to uric acid (14). In addition, XDH reduces NAD+ to NADH, whereas XO reduces oxygen to and H2O2. The above results suggest that increased flux of xanthine through XO might be responsible for enhanced production by endothelial cells following oscillatory shear stress. To confirm this finding, we employed an alternative strategy for inhibiting XO. The active molybdenum center of XOR can be replaced by prolonged exposure to tungstic acid, resulting in inhibition of XO activity (21). Culture of endothelial cells with tungstic acid (50 µM) for two passages before shear completely prevented the increase in production caused by oscillatory shear stress (Fig. 2B).
In addition, XO can generate H2O2 through the two-electron reduction of oxygen (11). It would therefore be expected that endothelial cells exposed to oscillatory shear stress would also produce increased amounts of H2O2. To test this hypothesis, we used DCF-DA to detect intracellular H2O2 generation. Oscillatory shear caused a twofold increase in DCF-DA fluorescence, a measure of intracellular H2O2 (Fig. 3A). Likewise, the release of H2O2 into the media was increased by oscillatory shear stress, as monitored by the peroxidase-linked Amplex Red assay (Fig. 3B). These effects of oscillatory shear stress were prevented by oxypurinol and pretreatment with PEG-catalase. These data further support XO as the major source of endothelial ROS generation in response to oscillatory shear stress.
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Determination of XOR expression and activity in response to oscillatory shear stress. The above experiments indicate that XO is most likely responsible for increased ROS generation by oscillatory shear stress. We next sought to determine the mechanism by which XOR increases ROS production. XOR might increase ROS via changes in expression or activity of the enzyme. As reported previously (26), Western blot analysis revealed two bands corresponding to XDH (150 kDa) and XO (130 kDa). Oscillatory shear decreased XDH protein levels by approximately threefold, whereas XO remained unchanged (Fig. 4A). In keeping with these findings, the pterine activity assay revealed decreased XDH activity, whereas XO activity remained constant in cells exposed to oscillatory shear stress (Fig. 4B).
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Decreased XDH expression in the setting of unchanged XO could lead to increased formation by promoting the flux of xanthine and hypoxanthine through the latter enzyme. To test this possibility, we compared xanthine-driven production in homogenates of cells exposed to oscillatory or no shear stress. ESR analysis revealed an increased xanthine-dependent, oxypurinol-inhibitable signal after 4 and 12 h of oscillatory shear stress (Fig. 4C). These data suggest that oscillatory shear stress most likely increases ROS generation by decreasing XDH expression and increasing purine flux through XO.
To more specifically inhibit XOR, we designed a siRNA (AS-XOR) targeted to the bovine XOR mRNA sequence. Our objective was to reduce expression of both XDH and XO. No alteration of either XDH or XO was observed when cells were transfected with a scrambled siRNA sequence. Surprisingly, transfection of 25 nM AS-XOR downregulated XDH expression and activity, whereas XO levels were only slightly affected (Fig. 5, A and B). Fortuitously, this effect of antisense siRNA mimicked that observed when cells were exposed to oscillatory shear. Analogous to oscillatory shear, AS-XOR transfection increased ROS generation by threefold compared with scrambled transfection (Fig. 6). In addition, in cells transfected with AS-XOR the fold increase in ROS after oscillatory shear stress was substantially less than in cells transfected with scrambled siRNA (1.28-fold vs. 4.09-fold, Fig. 6). These data support the hypothesis that lower levels of XDH can increase ROS generation through XO. In the setting of siRNA-reduced XDH expression, purine metabolites are most likely shunted through the XO pathway. In this situation, the effect of further XDH suppression by oscillatory shear is minimal.
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Determination of the role of p47phox in superoxide production and XOR expression. Previous studies have suggested that oscillatory shear stress stimulates ROS production from an NADH-dependent oxidase (5, 24). To directly examine the role of the NAD(P)H oxidase in response to oscillatory shear stress, we studied MAECs cultured from wild-type and p47phox–/– mice. In cells from wild-type C57Blk/6 mice, oscillatory shear stress increased production by an amount similar to that observed in BAECs, and this response was completely prevented by oxypurinol (Fig. 7A).
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The above finding is in keeping with our studies in BAECs suggesting that XO is responsible for the increase in endothelial cell production in response to oscillatory shear stress. In contrast to this conclusion, we found that p47phox–/– MAEC produced dramatically lower levels than wild-type cells under both static and oscillatory shear stress conditions (Fig. 7B).
At first glance, the markedly reduced production in response to oscillatory shear stress in endothelial cells from p47phox–/– mice would seem to contradict our findings that XO serves as a major source of production in response to this stimulus. Previous studies have indicated that both enzymes can be activated by ROS, indicating that these two oxidases could act in series. Furthermore, XDH can be converted to its oxidase form by oxidation of critical cysteine residues and subsequent proteolytic cleavage (2, 6, 26). To determine whether the NAD(P)H oxidase might modulate levels of either XDH or XO, we examined the expression of these in MAECs. Western blot analysis revealed dramatically lower XO expression in p47phox–/– MAECs compared with wild-type MAECs (Fig. 7C). In addition, transfection of p47phox–/– MAECs with a cDNA vector encoding p47phox increased XO protein expression to levels similar to those observed in wild-type MAECs.
These experiments suggest that XO levels are dependent on a functional NAD(P)H oxidase. To confirm this, we treated BAECs for varying periods of time with the NAD(P)H oxidase inhibitor apocynin. In contrast to its absence of short-term effect (Fig. 2A), long-term treatment with apocynin (600 µM, 4 or more days) lowered XO expression and prevented an increase in generation in response to oscillatory shear (Fig. 7D). These data suggest that XO levels are maintained by ROS produced by the NAD(P)H oxidase.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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. Our data suggest that the XOR is important in this process, because inhibiting this enzyme with either oxypurinol or culture with tungstic acid completely abolished this response. Interestingly, we found that oscillatory shear stress leads to a reduction of the XDH form of XOR, whereas it does not change the XO form. We speculate that the downregulation of XDH and the subsequent increase in the XO/XDH ratio increases ROS production by promoting the metabolism of xanthine and hypoxanthine through XO. In addition, our findings indicate that the presence of endothelial XO is dependent on a functional NAD(P)H oxidase. In cells lacking the p47phox subunit of the NAD(P)H oxidase, there is marked reduction of XO, and these cells have dramatically reduced production both at baseline and in response to oscillatory shear stress. Likewise, prolonged treatment of BAECs with the NAD(P)H oxidase inhibitor apocynin reduced XO protein levels and production in response to oscillatory shear. In this study, we used a cone and plate viscometer driven by a reversible stepping motor to study the effects of oscillatory flow on cultured endothelial cells. In preliminary studies, we demonstrated that this produced reversing laminar flow by visualizing the distribution of small boluses (<50 µl) of India ink after injection between the cone and plate. These studies demonstrated that the India ink followed an arc radiating in both directions from the site of injection for several cycles before dissolving in the media. Turbulence is highly dispersive, and the visualization clearly showed that no ink dispersion existed, verifying that flow was laminar. Computational fluid dynamics have previously demonstrated that the distribution of shear forces is quite uniform from the center to near the periphery of the cone in such a device (7). The oscillatory shear stress we employed (±15 dyn/cm2) is similar to that observed in models of the carotid bulb and is thought also to occur at other sites prone to atherosclerosis, including the proximal coronary arteries and the distal aorta (17). Thus we believe the model system we employed has relevance to sites in the circulation where oscillatory shear stress occurs.
To our knowledge, these are the first studies using ESR to detect changes in endothelial cell ROS production in response to oscillatory shear stress. The spin label we employed (CPH) is partially cell permeable and also can be oxidized by several ROS (8). The fact that the signal generated by cells exposed to oscillatory shear stress was inhibited by preincubation of cells with PEG-SOD and with the SOD mimetic Tiron strongly suggests that the oxidant responsible for this signal was . The produced was likely intracellular because MnSOD, which remains extracellular, failed to inhibit this signal. These findings are in keeping with previous studies by De Keulenaer et al. (5), who demonstrated that oscillatory shear stress increases endothelial cell dihydroethidium-mediated fluorescence, which reflects intracellular production. We also found that oscillatory shear stress increased endothelial cell H2O2 production, as reflected by DCF-DA fluorescence. This finding is consistent with either the formation of H2O2 by dismutation of the increased levels of or increased production of H2O2. XO is capable of performing both one- and two-electron reductions of oxygen, generating both and H2O2 (11).
The precise mechanism whereby oscillatory shear stress stimulates increased ROS production by XO is not entirely clear. Interestingly, oscillatory shear stress caused a marked decrease in both XDH protein expression and activity, whereas XO expression and activity remained unchanged. An alteration in the XO/XDH ratio has been implicated as a cause of increased production following liver radiation (25), hepatic ischemia (10), or exposure of intestinal tissues to platelet-activating factor (22). In most of these cases, this alteration in the XO/XDH ratio has been attributed to proteolytic cleavage of XDH, leading to formation of the smaller molecular weight XO. Unlike these previous examples, the change in XO/XDH ratio caused by oscillatory shear was not associated with an increase in XO but rather a marked decrease in XDH. This provides a situation in which hypoxanthine and xanthine can be preferentially metabolized by XO, favoring transfer of electrons to oxygen rather than NAD+. In support of this, the addition of xanthine to cytoplasmic extracts of cells previously exposed to oscillatory shear stress led to a greater production of than in extracts of cells not exposed to oscillatory shear stress. This xanthine-dependent production was completely blocked with oxypurinol, confirming that the source was XO.
The role of XO in the production of in cells exposed to oscillatory shear stress was further studied by using siRNA targeted against bovine xanthine oxidoreducatase (AS-XOR). Whereas our original goal was to inhibit both XDH and XO, siRNA transfection reduced expression and activity of XDH while having minimal effect on the levels and activity of XO. This effect was observed with several different siRNA sequences, durations of transfection, and methods of transfection. It is possible that the relative insensitivity of XO to siRNA transfection was due to a longer XO protein half-life. AS-XOR transfection led to an increase in endothelial cell production that was almost identical to that of oscillatory shear stress. Interestingly, after AS-XOR transfection, oscillatory shear stress failed to significantly increase endothelial cell production. This is consistent with the hypothesis that oscillatory shear stress leads to a loss of XDH. After siRNA transfection, purine flux through XO is likely near maximum, minimizing the effect of oscillatory shear stress.
An important concept arising from our present studies is that endothelial cell levels of XO are modulated by ROS derived from the NAD(P)H oxidase. This conclusion is based on three lines of evidence. First, protein levels and activity of XO were markedly reduced in endothelial cells from p47phox–/– mice compared with endothelial cells from wild-type C57Blk/6 mice, whereas XDH protein expression and activity were identical in these cells. Second, transfection of p47phox–/– MAECs with a vector encoding p47phox increased XO protein levels. Finally, inhibition of the NAD(P)H oxidase for 4 days with apocynin reduced XO levels and prevented production in response to oscillatory shear stress. It has been shown that XO can be formed from XDH by reversible sulfhydryl oxidation (13). It is interesting to speculate that ROS production by the NAD(P)H oxidase can facilitate proteolyic cleavage of XDH and therefore represents a major regulator of the relative levels of XDH and XO in endothelial cells.
Interestingly, we have recently shown that ROS produced by the NAD(P)H oxidase play a role in oxidation of tetrahydrobiopterin and uncoupling of endothelial NOS in the setting of hypertension (19). The present studies point to an additional role of the NAD(P)H oxidase in modulating ROS production by another enzyme, the XOR. Taken together, it seems that the NAD(P)H oxidase serves a role as a "master oxidase" that modulates the production of ROS by other potential enzymatic sources. Furthermore, our data suggest that inhibition of either XO or the NAD(P)H oxidase could prove beneficial in preventing the development of atherosclerosis at sites of the circulation where disturbed flow profiles exist. It is likely that newer methodologies, such as magnetic resonance imaging, to detect flow profiles in the human circulation might permit identification of sites of disturbed flow and that inhibition of the NAD(P)H oxidase or XO might prevent development of focal atherosclerosis at these sites.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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