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Although arachidonic acid (AA) has diverse vascular effects, the mechanisms that mediate these effects are incompletely defined. The goal of the present study was to use genetic approaches to examine the role of hydrogen peroxide (H2O2), glutathione peroxidase (Gpx1, which degrades H2O2), and CuZn-superoxide dismutase (SOD1, which produces H2O2 from superoxide) in mediating and determining vascular responses to AA. In basilar arteries in vitro, AA produced dilation in non-transgenic mice and this response was markedly reduced in transgenic mice overexpressing Gpx1 (Gpx1 Tg) or in mice genetically deficient in SOD1. For example, AA (1 nM to 1 µM) dilated the basilar artery and this response was reduced by about 90% in Gpx1 Tg mice (P<0.01), while responses to acetylcholine were not altered. Dilation of cerebral arterioles in vivo in response to AA was inhibited by ~50% by treatment with catalase (300 U/ml)(P<0.05) and reduced by as much as 90% in Gpx1 Tg mice compared to controls (P<0.05). These results provide the first evidence that Gpx1 has functional effects in the cerebral circulation and that AA-induced vascular effects are mediated by H2O2 produced by SOD1. In contrast, cerebral vascular responses to the endothelium-dependent agonist acetylcholine are not mediated by H2O2.
Arachidonic acid plays a major role in vascular biology and elicits a variety of effects, including changes in vascular tone and permeability. Such effects may be mediated by multiple mechanisms. Arachidonic acid can potentially alter cell function without being metabolized by acting on specific targets such as ion channels (Meves, 2008). Arachidonic acid can also be metabolized through several major pathways including the cyclooxygenase, cytochrome P450 monooxygenase, and lipoxygenase pathways (Meves, 2008). In addition to producing eicosanoids, the metabolism of arachidonic acid also results in the generation of reactive oxygen species (ROS)(Kontos, 1990), which have both acute and chronic vascular effects (Faraci, 2006a; Chrissobolis and Faraci, 2008).
In blood vessels from several organs, including heart and brain, arachidonic acid increases levels of ROS (Didion et al, 2001a; Oltman et al, 2003; Zafari et al, 1999). Previous studies using pharmacological approaches have provided evidence both for (Oltman et al, 2003; Sobey et al, 1998) and against (Bryan et al, 2006; Pomposiello et al, 1999) a role for ROS as mediators of changes in vascular tone in response to arachidonic acid. In the present study we examined the role of ROS, and specifically hydrogen peroxide (H2O2), as a mediator of vascular effects of arachidonic acid using pharmacological and new genetic approaches. Steady state levels of H2O2 are determined in part by the activity of glutathione peroxidases (Gpx), which metabolizes H2O2 to water (Arthur, 2000; Briggelius-Flohe et al, 2003; Faraci, 2006a). The functional importance of Gpx in most vascular beds including the cerebral circulation is unknown.
The goal of these experiments was to examine the role of H2O2 in mediating effects of arachidonic acid on vascular tone. We sought to define the functional importance of the cytosolic form of Gpx (Gpx1) as a determinant of this vascular response. We tested the hypothesis that vasodilation to arachidonic acid is mediated by H2O2 and that genetic overexpression of Gpx1 would inhibit this response. As part of these experiments, we also tested whether overexpression of Gpx1 affected dilation of cerebral arteries to the classic endothelium-dependent agonist acetylcholine.
Superoxide dismutases (SOD) are a major cellular source of H2O2 as they convert superoxide to H2O2 (Faraci and Didion, 2004). To further define mechanisms involved, we tested the hypothesis that vascular responses to arachidonic acid are dependent on expression of the cytosolic form of SOD (CuZn-SOD or SOD1). SOD1 accounts for the majority of total SOD activity in vascular tissue (Faraci and Didion, 2004; Didion et al, 2001b). Overall, our findings provide the first evidence that Gpx1 has functional effects in the cerebral circulation and that vascular effects of arachidonic acid, but not acetylcholine, are mediated by H2O2 produced by SOD1.
Gpx1 transgenic mice (Gpx1 Tg)(Chrissobolis et al, 2008) were derived from breeding C57Bl/6 and Gpx1 Tg mice. Gpx1 Tg and their non-transgenic littermates (non-Tg) were studied. SOD1-deficient mice were derived from breeding heterozygous SOD1 deficient mice (Didion et al, 2001b). We studied homozygous SOD1 deficient (SOD1−/−) mice and their wild-type (SOD1+/+) littermates. Transgenic mice that overexpress renin and angiotensinogen (R+A+) were derived as described previously (Faraci et al, 2006a). Additional C57BL6 mice were used in some studies. All breeding was performed in a virus- and pathogen-free barrier-facility at the University of Iowa. Genotyping was performed as described previously for these strains (Chrissobolis et al, 2008; Didion et al, 2001b; Didion et al, 2002). Mice were fed regular chow and water was available ad libitum. All experimental protocols and procedures conform to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Iowa.
After an overdose of anesthesia (pentobarbital, 150 mg/kg ip), the brain was rapidly removed and placed in ice-cold Krebs buffer. As described previously (Faraci et al, 2006a, Yamada et al, 2001), the basilar artery was isolated, cannulated onto glass micropipettes filled with Krebs buffer in an organ chamber, and secured with nylon monofilament. Arteries were pressurized to 60 mmHg. Using a microscope and a video camera, vessel images were projected on a video monitor. An electronic dimension analyzer was used to measure lumen diameter.
Once prepared, basilar arteries were allowed to equilibrate for at least 30 min at a distending pressure of 60 mmHg before protocols were initiated. We examined changes in diameter of the basilar artery in response to KCl (50 mM). For studies testing vasodilator responses, basilar arteries were constricted by approximately 30% (~60% of the response to 50 mM KCl) with U-46619, a thromboxane A2 mimetic. Acetylcholine was used as an endothelium-dependent agonist and is known to produce NO-mediated dilation of the basilar artery (Faraci and Heistad, 1998; Faraci et al, 2006b). After development of a stable baseline diameter, cumulative dose-response curves were obtained. At the end of the protocols, papaverine (an endothelium-independent vasodilator, 100 µM) was used to produce maximal vasodilation.
Mice were anesthetized with pentobarbital sodium (75–90 mg/kg ip), supplemented regularly at approximately 20 mg/kg/h. Animals were ventilated mechanically with supplemental oxygen, and arterial blood pressure and blood gases were monitored as described previously (Sobey and Faraci, 1997). A cranial window was made over the left parietal cortex, the window constantly suffused with artificial CSF, and a pial arteriole (branches of the middle cerebral artery) was exposed. Arteriolar diameter was measured using a microscope equipped with a camera coupled to a video monitor and an image-shearing device. The diameter of one arteriole per animal was measured under control conditions and during topical application of drugs. Mean arterial pressure was similar in all groups of mice and averaged 69±1 mmHg (mean ± SE). Arterial blood gases were monitored and were also similar (PCO2 = 41±1 mmHg, PO2 = 128±10 mmHg, and pH = 7.28±0.01). The gases and pH of the artificial CSF were PCO2 = 40±1 mmHg, PO2 = 73±2 mmHg, and pH = 7.32±0.02. In these studies, responses of arterioles to topical application of arachidonic acid (1 and 10 µM), acetylcholine (1 and 10 µM), and papaverine (10 and 100 µM) were examined in the absence or presence of catalase (300 U/ml, a scavenger of H2O2) or diethyldithiocarbamate (10 mM, an inhibitor of SOD1 and SOD3)(Faraci and Didion, 2004). These latter experiments were performed to examine whether responses to arachidonic acid and the mechanisms involved in the mouse were similar to previous studies in other species (Didion et al, 2001a; Sobey et al, 1998).
For the basilar artery, constriction in response to KCl was determined by calculating the percent reduction in vessel diameter from the baseline control level. For vasodilator responses, results are expressed as percent dilation (% of induced tone), with 100% representing the difference between the resting value under basal conditions and the constricted value with U-46619. For cerebral arterioles in vivo, results are expressed as percent dilation (% change). As appropriate, comparisons were made using paired or unpaired t-tests or ANOVA with repeated measures followed by Student-Newman-Keuls test to detect individual differences. A P<0.05 was defined as significant.
Baseline diameter of cerebral arterioles in all C57Bl6/J mice in vivo was 32±1 µm. Catalase had no significant effect on baseline diameter (33±1 versus 34±1 µm). Dilation of cerebral arterioles in response to arachidonic acid (n=9), but not papaverine, was inhibited by catalase (Figure 1). These data provide pharmacological evidence that arachidonic acid-induced vasodilation is mediated by H2O2. In time control experiments in a separate group of mice (n=5), responses of cerebral arterioles to arachidonic acid were reproducible in the absence of any inhibitor (data not shown).
Baseline diameter of the basilar artery in C57Bl6/J mice was 128±10 µm. Dilation of the basilar artery in response to arachidonic acid was completely inhibited by indomethacin (10 µM, n=4)(Figure 2). We have shown previously that indomethacin does not alter dilation of the mouse basilar to 14,15-epoxyeicosatrienoic acid (Fang et al, 2006).
We have shown previously that vascular expression of Gpx1 is increased by ~ 3-fold in Gpx1 Tg mice compared to non-Tg controls (Chrissobolis et al, 2008). Baseline diameters of the basilar artery in non-Tg and Gpx1 Tg mice were 132±1 and 130±2 µm (n=10 for each genotype), respectively. Dilator responses of the basilar artery to arachidonic acid were reduced by almost 90% in Gpx1 Tg mice (Figure 3). For example, 1 µM arachidonic acid dilated the basilar artery by 53±2 and 7±1% in control and Gpx1 Tg mice, respectively. H2O2 is known to dilate cerebral arterioles (Faraci, 2006b; Sobey et al, 1997). Exogenous H2O2 produced dilation of the basilar artery and this response was inhibited by about 50% in Gpx1 Tg mice (Figure 3). These inhibitory effects were selective, as the basilar artery constricted similarly to KCl (Figure 3) and U46619 (data not shown) and dilated similarly to acetylcholine and papaverine in both groups of mice (Figure 3).
In vivo, baseline diameter was similar in cerebral arterioles from control and Gpx1 Tg mice [28±1 and 27±1 µm in non-Tg (n=9) and Gpx1 Tg mice (n=9), respectively]. Dilation of cerebral arterioles in response to arachidonic acid, but not papaverine, was markedly reduced in Gpx1 Tg mice compared to controls (Figure 4).
Baseline diameter of the basilar artery in wild-type and SOD1 deficient mice were 135±2 and 133±2 µm (n=8–9), respectively. Maximal dilator responses of the basilar artery to arachidonic acid were reduced by approximately 75% in SOD1 deficient mice (Figure 5). For example, 1 µM arachidonate dilated the basilar artery by 70±3 and 18±1% in wild-type and SOD1 deficient mice, respectively (Figure 5).
We and others have shown that genetic deficiency in SOD1 reduces formation of H2O2, increases superoxide, and impairs NO-mediated vascular responses (Didion et al, 2001b; Baumbach et al, 2006; Yada et al, 2008). Consistent with previous studies in cerebral arterioles (Didion et al, 2001a), diethyldithiocarbamate inhibited dilation of cerebral arterioles in response to acetylcholine (Figure 5). Dilation of the basilar artery in response to acetylcholine was reduced in SOD1 deficient mice (Figure 5). This change was selective as constriction of the basilar artery to KCl (Figure 5) and U46619 (data not shown) and dilation to papaverine was similar in both groups of mice (Figure 5).
Because deficiency in SOD1 produces oxidative stress, we considered the possibility that reductions in the response to arachidonic acid in SOD1 deficient mice was simply a consequence of oxidative stress. To examine this possibility, we studied basilar arteries from a genetic model of angiotensin II-dependent hypertension (R+A+ mice)(Didion et al, 2002). R+A+ mice have oxidative stress, increased vascular superoxide, and superoxide-mediated vascular dysfunction (Didion et al, 2002; Faraci et al, 2006a). Vasodilation in response to arachidonic acid was not altered in R+A+ mice compared to littermate controls (Figure 5) suggesting that the presence of oxidative stress per se does not impair responses to arachidonic acid.
There are several novel findings in this study. Arachidonic acid dilated cerebral blood vessels both in vivo and in vitro. The in vitro data demonstrate that effects of arachidonic acid reflect direct actions on the vessel wall and are dependent on activity of cyclooxygenase. Overexpression of Gpx1 markedly reduced responses to arachidonic acid, providing genetic evidence that H2O2 mediates the majority of the effect on vascular tone. Our findings with exogenous application of catalase support this concept. Using Gpx1 Tg mice, similar findings were obtained in a cerebral artery studied in vitro as well as in small microvessels with myogenic tone studied in vivo. To our knowledge, this is first evidence of any kind that Gpx1 is functionally important in the cerebral circulation. We also found that genetic deficiency in SOD1 markedly reduced responses to arachidonic acid suggesting the majority of the vascular response is mediated by H2O2 produced by this form of SOD. Our study provides new genetic evidence regarding the role of H2O2 and mechanisms that mediate vascular effects of arachidonic acid. Lastly, our data suggest strongly that responses of cerebral blood vessels to the endothelium-dependent agonist acetylcholine are not mediated by H2O2.
Arachidonic acid is generally a vasodilator including in the cerebral circulation. Although arachidonic acid has the potential to produce vascular effects through direct actions on ion channels and following metabolism through multiple pathways, most previous studies in multiple species including primates indicate that arachidonic acid produces changes in tone of cerebral arteries and arterioles through a cyclooxygenase-dependent mechanism (Busija and Heistad, 1983; Ellis et al, 1990; Hayashi et al, 1985; Niwa et al, 2001; Sobey et al, 1998). In this study, we found that indomethacin completely inhibited vascular effects of arachidonic acid indicating that the response was also dependent on activity of cyclooxygenase in the mouse basilar artery. Because vascular effects of arachidonic acid are reduced by cyclooxygenase inhibitors, some investigators assume that prostacyclin or other eicosanoids mediates these responses (Hayashi et al, 1985; Ospina et al, 2003).
The metabolism of arachidonic acid is known to be a major source of ROS (Kontos, 1990). Superoxide is produced during cyclooxygenase activity (ie, the conversion of arachidonic acid to prostaglandin H2)(Kontos, 1990). For example, arachidonic acid produces cyclooxygenase-dependent superoxide formation in cerebral and coronary blood vessels (Didion et al, 2001a; Oltman et al, 2003). Once produced, superoxide may have direct effects (Faraci, 2006b; Faraci and Didion, 2004) or may be converted to H2O2 primarily by SOD’s (Faraci and Didion, 2004; Faraci 2006a). While some previous studies suggest that vasodilation in response to arachidonic acid is inhibited by scavengers of ROS, there is controversy regarding the role of ROS and which ROS mediate the response (Bryan et al, 2006; Kontos et al, 1984; Niwa et al, 2001; Pomposiello et al, 1999; Sobey et al, 1998). The present finding that dilation of cerebral arterioles in response to arachidonic acid is inhibited by catalase supports the concept that ROS are a key component and that H2O2 is a mediator of this response (Kontos et al, 1984; Sobey et al, 1998).
In the cerebral circulation and other vascular beds, H2O2 is generally a dilator in both large arteries and microvessels (see Faraci, 2006a and 2006b for reviews). A similar effect was seen here as H2O2 was a potent dilator of the basilar artery. Although there is little evidence to suggest that ROS affect cerebral vascular tone under resting conditions, some pharmacological studies suggested that dilation of cerebral arterioles to both arachidonic acid and bradykinin are mediated by endogenous formation of ROS. We provided the first evidence that H2O2 dilates cerebral arterioles by activation of potassium channels (Sobey et al, 1997; Sobey et al, 1998) and thus may function as an endothelium-derived hyperpolarizing factor. In addition to these stimuli, H2O2 may contribute to flow-mediated vasodilation (Paravicini et al, 2006) and increases in cerebral blood flow in diseases with an inflammatory component, including meningitis (Hoffmann et al, 2007).
As noted above, H2O2 is formed primarily from superoxide as a result of activity of the various SODs (Faraci and Didion, 2004). In blood vessels, there are three isoforms of SOD: SOD1, manganese SOD localized in mitochondria (SOD2), and an extracellular form of CuZn-SOD (SOD3)(Faraci and Didion, 2004). Vasodilation of cerebral arterioles in response to arachidonic acid was markedly reduced by diethyldithiocarbamate (Didion et al, 2001a), an inhibitor of SOD1 and SOD3 (Faraci and Didion, 2004) suggesting the source of H2O2 that mediates the response is a copper-containing SOD. However, this approach is limited and provides no insight into the relative importance of specific isoforms of SOD. In addition, diethyldithiocarbamate is a chelator of copper and thus may have non-selective effects (Faraci and Didion, 2004). In the present experiments, we found that vasodilation to arachidonic acid was markedly reduced in mice deficient in SOD1, suggesting this response is mediated very predominately by H2O2 produced by SOD1.
We and others have shown that pharmacological inhibition of SOD1 and SOD3 or genetic deficiency in SOD1 increase vascular superoxide (Baumbach et al, 2006; Didion et al, 2001a; Didion et al, 2001b), reduce vascular H2O2, (Yada et al, 2008) and impair NO-mediated vascular responses (Baumbach et al, 2006; Didion et al, 2001a; Didion et al, 2001b). Consistent with this concept, we found that dilation of cerebral arterioles to acetylcholine was inhibited by diethyldithiocarbamate and that responses of the basilar artery to acetylcholine was impaired in SOD1 deficient mice. In both these vascular segments, multiple lines of evidence suggest that vasodilation to acetylcholine is normally mediated by endothelium-derived nitric oxide (Faraci and Heistad, 1998; Sobey and Faraci, 1997).
Gpx’s are part of a family of selenium-dependent enzymes that use glutathione to metabolize H2O2 and lipid peroxides to water and respective alcohols (Arthur, 2000; Briggelius-Flohe et al, 2003). Similar to the SODs, there are multiple isoforms of Gpx, including the cytosolic Gpx1 (Arthur, 2000; Briggelius-Flohe et al, 2003). Gpx is expressed in vascular cells and blood vessels including cerebral arteries (Chrissobolis et al, 2008; Kobayashi et al, 2002; Lapenna et al, 1998; Napoli et al, 1999). Expression of Gpx is relatively high in endothelium (Kobayashi et al, 2002) and may change within the vessel wall depending on genetic factors and in disease (Lapenna et al, 1998; Ulker et al, 2003). Although catalase also degrades H2O2, human vascular cells reportedly express little or no catalase (Shingu et al, 1985).
Although exogenous catalase had inhibitory effects on vascular responses to arachidonic acid, that approach may underestimate the role of intracellularly produced H2O2 and provides no insight into the subcellular action of H2O2 or the functional importance of GPx1. The existence of multiple isoforms of SOD and GPx implies specific functions exist in different subcellular locations. Increasing evidence suggests that the effects of ROS are compartmentalized with different signaling consequences in different locations. Our findings with Gpx1 Tg mice provide genetic evidence that H2O2 is the mediator of the majority of the vascular response to arachidonic acid, particularly with lower concentrations of arachidonate. We previously provided evidence that Gpx1 has functional effects in the carotid artery and was important in protecting against angiotensin II-induced vascular dysfunction (Chrissobolis et al, 2008). The present study provides the first evidence that Gpx1 has functional effects in the cerebral circulation (in intracranial blood vessels). It is perhaps not surprising that the response to arachidonic acid is dependent on activity of both SOD1 and Gpx1 as both these enzymes are cytosolic as opposed to other forms of SOD and Gpx.
Many studies suggest that NO produced by the endothelial isoform of NO synthase (eNOS) affects basal tone and mediates responses to varied endothelium-dependent stimuli (Faraci and Heistad, 1998; Cipolla and Bullinger, 2008; Cipolla et al, 2004), In contrast, a recent study suggests that under normal conditions, eNOS is uncoupled in cerebral arteries and thus produces superoxide due to deficiency in substrate (Drouin et al, 2007). Under this scenario, superoxide is produced by eNOS and then converted to H2O2 by SOD(s). With this postulated mechanism, H2O2 is the actual mediator of endothelium-dependent relaxation (Drouin et al, 2007). Our finding that overexpression of Gpx1 greatly reduces vascular responses to arachidonic acid and exogenous H2O2, but not acetylcholine, do not support the concept that H2O2 is the mediator of responses to this neurotransmitter and muscarinic agonist. The present findings are consistent with previous pharmacologically-based studies that concluded that responses to acetylcholine in the cerebral circulation are not mediated by ROS (see Katusic et al 1989; Kontos et al, 1988; for examples). Uncoupled eNOS may well contribute to oxidative stress in vascular disease including in brain but there has been little evidence to suggesting that this form of dysfunction occurs under normal conditions in vivo (Chrissobolis and Faraci, 2008).
In summary, this study provides the first genetic evidence that vascular effects of arachidonic acid are mediated by H2O2 produced by SOD1. Our study suggests that Gpx1 is a major determinant of vascular effects of arachidonic acid both in vitro and in vivo. Effects of lower concentrations of arachidonic acid (1 µM and less) were almost completely inhibited following overexpression of Gpx1 in both the basilar artery and cerebral arterioles. These findings support the concept that blood vessels can use ROS as key signaling molecules under normal conditions. The concentrations of arachidonic acid used in these experiments are well within the physiological range (Meves, 2008). Higher local concentrations of arachidonic acid may occur with disease or cellular injury and may affect blood vessels via additional mechanisms including activation of two-pore-domain potassium channels (Bryan et al, 2006).
This work was supported by National Institutes of Health grants HL-38901, NS-24621, HL-62984 and HL-63943 as well as support from the American Heart Association in the form of a Bugher Award (0575092N). R+A+ mice were kindly provided by Dr. Curt Sigmund, University of Iowa.