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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:762.)
© 2007 American Heart Association, Inc.

Vascular Biology

Role of the Multidrug Resistance Protein-1 in Hypertension and Vascular Dysfunction Caused by Angiotensin II

Julian D. Widder; Tomasz J. Guzik; Cornelius F.H. Mueller; Roza E. Clempus; Harald H.H.W. Schmidt; Sergey I. Dikalov; Kathy K. Griendling; Dean P. Jones; David G. Harrison

From the Emory University Division of Cardiology, Department of Medicine and the Atlanta Veterans Administration Hospital (J.D.W., T.J.G., C.F.H.M., R.E.C., S.I.D., K.K.G., D.P.J., D.G.H.), Ga; and Monash University, Department of Pharmacology & Centre for Vascular Health (H.H.H.W.S.), Melbourne, Australia.

Correspondence to David G. Harrison, Division of Cardiology, Emory University, 101 Woodruff Circle, WMBR 319, Atlanta, GA 30322. E-mail dharr02{at}emory.edu

	Abstract

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Objective— Human endothelial cells use the multidrug resistance protein-1 (MRP1) to export glutathione disulfide (GSSG). This can promotes thiol loss during states of increased glutathione oxidation. We investigated how MRP1 modulates blood pressure and vascular function during angiotensin II-induced hypertension.

Methods and Results— Angiotensin II–induced hypertension altered vascular glutathione flux by increasing GSSG export and decreasing vascular levels of glutathione in wild-type (FVB) but not in MRP1^–/– mice. Aortic endothelium-dependent vasodilatation was reduced in FVB after angiotensin II infusion, but unchanged in MRP1^–/– mice. Aortic superoxide (O₂^·–) production and expression of several NADPH oxidase subunits were increased by angiotensin II in FVB. These effects were markedly blunted in MRP1^–/– vessels. The increase in O₂^·– production in FVB vessels caused by angiotensin II was largely inhibited by L-NAME, suggesting eNOS uncoupling. Accordingly, aortic tetrahydrobiopterin and levels of NO were decreased by angiotensin II in FVB but were unchanged in MRP1^–/–. Finally, the hypertension caused by angiotensin II was markedly blunted in MRP1^–/– mice (137±4 versus 158±6 mm Hg).

Conclusion— MRP1 plays a crucial role in the genesis of multiple vascular abnormalities that accompany hypertension and its presence is essential for the hypertensive response to angiotensin II.

This study demonstrates that glutathione loss via the multidrug resistance protein 1 (MRP1) modulates many aspects of vascular function during chronic angiotensin II infusion, including vascular glutathione levels, NO production, and endothelium-dependent vasodilatation, superoxide production, and tetrahydrobiopterin levels. The hypertension caused by angiotensin II was significantly blunted in MRP1^–/– mice, suggesting that MRP1 could be a target for treatment of vascular oxidative stress and hypertension.

Key Words: endothelial function • glutathione • hypertension • MRP1 • oxidative stress

	Introduction

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Hypertension increases vascular production of reactive oxygen species (ROS).¹ There is accumulating evidence that this contributes to the pathophysiologic consequences of hypertension.² A major intracellular defense against ROS is the tripeptide glutathione (GSH).³ GSH serves as a cosubstrate for the glutathione peroxidases, which scavenge hydrogen peroxide and lipid peroxides. GSH also directly reacts with strong oxidants such as peroxynitrite. Depletion of the glutathione pool or inhibition of glutathione peroxidase alters vascular tone and leads to hypertension.^4,5

The multidrug resistance proteins (MRPs) are members of the ATP-binding cassette (ABC) superfamily which use ATP for active transport of a variety of endogenously produced and exogenously administered molecules.⁶ MRP1 is a transporter of chemotherapeutic agents complexed to glutathione, leukotriene C4, glutathione disulfide (GSSG), and estrogen. Other MRPs have a myriad of cell functions including export of xenobiotics, cyclic nucleotides, lipids, and billirubin. Recently, we found that MRP1 is the major transporter of GSSG in human endothelial cells.⁷ In this prior study, oxidative stress caused by oscillatory shear markedly enhanced the release of GSSG from cultured human aortic endothelial cells in an MRP1 dependent fashion. This led to a depletion of intracellular GSH and ultimately apoptosis. Blockade of MRP1 prevented these untoward effects of oxidative stress. We also showed that endothelial GSSG export was increased in an in vivo model of oxidative stress caused by desoxycorticosterone acetate-salt (DOCA-salt) hypertension in normal but not MRP1^–/– mice. We selected DOCA-salt hypertension as a model of in vivo oxidative stress in this prior study because the increase in ROS is predominantly in the endothelium, mimicking our cell culture model. Moreover, in DOCA-salt hypertension, the degree of hypertension is relatively independent of vasomotor tone,^8,9 and thus was identical between wild-type and MRP1^–/– mice. These studies showed that the difference in GSSG extrusion caused by hypertension was independent of blood pressure levels, but markedly influenced by the presence of MRP1.

This prior study led to several important additional questions. First, it was not clear whether blood pressure could be influenced by MRP1 in a model less dependent on volume and sodium and more dependent on vasoconstriction. Second, because the increase in ROS is predominantly in the endothelium, it was difficult to examine the role of MRP1 in modulation of total vascular GSH. Third, it remained unclear how MRP1 could affect production of ROS and their various sources. In the present study, we examined the role of MRP1 in angiotensin II–induced hypertension. This model of hypertension is associated with an increase in production of ROS in both the endothelium and vascular smooth muscle, and is also dependent on vasomotor tone. This study provides new information about how angiotensin II–dependent hypertension reduces vascular intracellular glutathione and how this in turn affects ROS-producing enzymes. Moreover, our results strongly suggest that MRP1 plays myriad roles in regulation of blood pressure and vascular function and is a potential target for treatment of hypertension and vascular disease.

	Materials and Methods

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Animals
Animal experiments were approved by the Emory University Animal Care and Use Committee. Male MRP1^–/– mice (FVB.129P2-Abcc1a^tm1Bor N12)¹⁰ and congenic wild-type animals (FVB/N) were purchased from Taconic. At 12 weeks of age the mice were anesthetized with Avertin 2.5% (0.3 mL per 25 g body weight, IP) and osmotic minipumps (Alzet Model 2002; Alzet Corp) were implanted to permit subcutaneous infusion of angiotensin II ([Val⁵]angiotensin II, infusion rate 400 ng/kg per min). Sham-operated animals underwent an identical surgical procedure, except that either no pump or an empty osmotic pump was implanted. Blood pressure was measured either noninvasively by a computerized tail-cuff system (Visitech Systems) or invasively by telemetry (Data Sciences International) as previously described.^11,12 For in vitro studies the animals were killed with CO₂ inhalation, and the aortas were removed and dissected free of adherent tissues.

Determination of Glutathione and GSSG Reductase Activity
Aortic GSSG release was determined as previously described.⁷ For measurement of vascular GSH and GSSG, aortas were homogenized in 150 µL ice-cold 5% perchloric acid containing 10 µmol/L -glu-glu as an internal standard and were quantified by high-performance liquid chromatography (HPLC) as described previously.⁷ GSSG reductase activity was determined as described elsewhere, monitoring the disappearance of NADPH spectrophotometrically.¹³

Vascular Reactivity Studies
Two to 3-mm aortic ring segments were studied in organ chambers as previously described.¹¹ Passive tension was adjusted to 1 g, and vessels were preconstricted to equal levels with PGF₂. Relaxations to cumulative concentrations of acetylcholine and nitroglycerin were examined.

Measurements of Vascular O₂^·– and NO Production and Biopterin Content
Vascular O₂^·– was determined by examining the oxidation of dihydroethidium (DHE) to 2-hydroxyethidium as described prevsiously.¹⁴ NO production was measured by electron-spin resonance (ESR) using the specific colloid probe Fe²⁺ diethyldithiocarbamate (Fe²⁺(DETC)₂) in the presence of 10µmol/L A23187 as described.¹⁵ Aortic biopterin content was analyzed using HPLC and a differential oxidation method as performed previously.¹⁶

Western Blot
The MRPr1 antibody was from Kamia Biomedical. The Nox4 antibody was a gift from D. Lambeth, Emory University, Atlanta, Ga. The monoclonal p22^phox and the Nox1 antibodies have been described previously.^12,17 eNOS, Nox2, p47^phox, and p67^phox antibodies were from BD Bioscience. The AT1, AT2, and CDK4 antibodies were from Santa Cruz.

Statistical Analysis
Values are presented as mean±SEM. Comparisons between groups of animals or treatments were made by one-way ANOVA. Comparisons of dose-response curves were performed using one-way ANOVA for repeated measurements. When significance was indicated by ANOVA, the Student-Newman Keuls post hoc-test was used to examine between group differences. In some experiments the Bonferroni post-hoc test was used to compare differences between specific groups.

	Results

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Angiotensin II–Induced Vascular GSSG Export
In preliminary studies, we found that vessels from FVB and MRP1^–/– mice expressed similar amounts of the AT1 and AT2 angiotensin II receptors (supplemental Figure I, available online at http://atvb.ahajournals.org). To determine whether angiotensin II–induced hypertension leads to vascular extrusion of GSSG, aortas were incubated for 6 hours in media and the amount of released GSSG determined using HPLC. Angiotensin II infusion for 2 weeks caused a marked increase in GSSG release from the aorta compared with control conditions (Figure 1A). Endothelial removal increased GSSG export from FVB vessels both at baseline and after angiotensin II infusion, suggesting that the vascular smooth muscle largely contributes to this process in angiotensin II–induced hypertension. Vessels lacking MRP1 had significantly lower export of GSSG than wild-type FVB mice at baseline, and angiotensin II–induced hypertension had no effect on this (Figure 1A). These changes in aortic GSSG extrusion were accompanied by striking alterations in vascular glutathione. In normal mouse aorta, the level of total GSH and GSSG was 30 nmoles/mg protein. Angiotensin II–induced hypertension reduced this by 30% (Figure 1B, P<0.05). This was predominantly attributable to a decrease in GSSG, although GSH was also reduced. In contrast, in MRP1^–/– mice, the intracellular total GSH and GSSG at baseline was 40 nmoles/mg protein and was unaffected by angiotensin II infusion. These results are in keeping with a role of MRP1 in modulating GSSG export from vascular cells.

View larger version (37K): [in this window] [in a new window]   Figure 1. Effect of angiotensin II–induced hypertension on export of GSSG, aortic glutathione levels, glutathione disulfide reductase activity, and MRP1 protein expression. A, Aortas (8 2-mm sections) were incubated in 150 µL media for 6 hours and GSSG levels in the media determined using HPLC. In some aortas endothelium was removed before incubation (endo-). Data are from 5 to 9 experiments. B, Average aortic total glutathione, GSH, and GSSG levels from 5 to 9 separate studies. C, Representative Western blot showing GSSG reductase (GR) expression. D, Aortic GSSG reductase activity. Mean data from 5 separate experiments. E, Representative Western blot showing MRP1 expression in mouse aorta. CDK4 was used as a loading control.

Because GSSG is rapidly converted to GSH by GSSG reductase, we also sought to determine whether the expression or function of this enzyme was altered by angiotensin II–induced hypertension or by the lack of MRP1. Protein levels of GSSG reductase were similar between FVB and MRP1^–/– mice and were not changed by angiotensin II infusion (Figure 1C). The activity of GSSG reductase, however, was significantly increased in mice receiving angiotensin II (Figure 1D). Levels of MRP1 protein were not altered by angiotensin II infusion (Figure 1E). Western analysis also confirmed a complete absence of MRP1 protein in the aorta of MRP1^–/– mice.

Endothelium-Dependent Relaxation
Angiotensin II–induced hypertension is known to diminish endothelium-dependent relaxation. We sought to assess whether reducing thiol export could prevent this effect of angiotensin II. In FVB mice angiotensin II caused a reduction in the acetylcholine evoked endothelium-dependent relaxation as compared with sham-operated animals (Figure 2A). In contrast, in mice lacking MRP1, angiotensin II infusion had absolutely no effect on endothelium-dependent relaxation to acetylcholine (Figure 2A). Endothelium-independent relaxation to nitroglycerin was not different between the groups (Figure 2B).

View larger version (19K): [in this window] [in a new window]   Figure 2. Endothelium-dependent vasodilatation in FVB and MRP1–/– mice with angiotensin II–induced hypertension. Aortic segments (3 mm) were mounted in isolated organ chamber baths and basal tension adjusted to 1 g. Vascular relaxations to acetylcholine (A) and nitroglycerin (B) were measured after preconstriction with PGF-2. Mean data from 4 to 5 separate experiments.

Vascular O₂^·– Production
One explanation for the preservation of endothelial function in the MRP1^–/– mice could be a modulation of O₂^·– levels. As previously described, chronic angiotensin II infusion caused an increase in aortic O₂^·– production in wild-type FVB mice. In contrast, in mice lacking MRP1, angiotensin II had no effect on O₂^·– production (Figure 3A). To investigate the contribution of uncoupled nitric oxide synthase on aortic superoxide production, vessels were treated with L-N-arginine methyl ester (L-NAME, 100 µmol/L) for 30 minutes before exposure to DHE. L-NAME significantly reduced vascular O₂^·– in FVB mice previously treated with angiotensin II, while not significantly altering O₂^·– production in MRP1^–/– mice treated with angiotensin II (Figure 3A). In vessels from angiotensin II–treated mice, O₂^·– production remained 2-fold higher in FVB as compared with MRP1^–/– mice vessels after L-NAME treatment.

View larger version (42K): [in this window] [in a new window]   Figure 3. Aortic O2·– production and NADPH oxidase subunit protein expression in sham animals and in animals with angiotensin II–induced hypertension. A, Superoxide production was measured by monitoring the conversion of dihydroethidium to 2-hydroxyethidium with an HPLC based method with and without L-NAME (100µmol/L). Data are from 5 to 10 separate experiments. B, Representative Western blot showing NADPH oxidase subunits protein expression in the aorta. CDK4 was used as a loading control. C, Mean data from 4 separate experiments for densitometry of NADPH oxidase subunits/CDK4.

NADPH-Oxidase Subunit Protein Expression
It has previously been shown that angiotensin II–mediated O₂^·– production is in large part attributable to activation and increased expression of the NADPH-oxidase.^18,19 Further, oxidative stress is known to increase expression of NADPH oxidase subunits.^12,20 We therefore hypothesized that prevention of thiol export would blunt the increase in NADPH oxidase subunit expression caused by angiotensin II. At baseline, protein levels of Nox1, Nox2, Nox4, p22^phox, p47^phox, and p67^phox were similar between FVB and MRP1^–/– mice, as determined by Western blotting (Figure 3B and 3C). Nox4 expression was not altered by angiotensin II infusion in either group of mice. In contrast, chronic angiotensin II infusion increased Nox1, Nox2, p22^phox, p47^phox, and p67^phox expression in FVB mice. These effects of angiotensin II was markedly blunted in the MRP1^–/– mice (Figure 3B and 3C).

Effect of Angiotensin II on Vascular Tetrahydrobiopterin and NO Levels
As noted above, L-NAME blunted O₂^·– production in wild-type mice. NADPH oxidase-derived ROS can lead to H₄B oxidation and uncoupling of nitric oxide synthase.¹¹ We therefore sought to determine whether the reduced levels of vascular O₂^·– in MRP1^–/–mice might be associated with preservation of aortic H₄B. Even in the absence of angiotensin II, the amount of oxidized pterin was higher in FVB as compared with MRP1^–/– vessels. In FVB mice chronic angiotensin II infusion caused a reduction in aortic H₄B and a slight increase in the ratio of oxidized to reduced H₄B, whereas in MRP1^–/– mice angiotensin II had no effect on pterin levels or the ratio of reduced to oxidized H₄B (Figure 4A). These changes in H₄B levels were mirrored by the capacity of the vessels to produce NO (Figure 4B and 4C). Angiotensin II–induced hypertension caused a decline in vascular NO production in FVB mice, but had no effect in MRP1^–/– vessels. The levels of eNOS protein were not significantly affected by angiotensin II or the presence of MRP1 (Figure 4D).

View larger version (30K): [in this window] [in a new window]   Figure 4. Aortic biopterin and nitric oxide levels in MRP1–/– and FVB mice after sham-surgery or angiotensin II–induced hypertension. A, Aortic biopterin levels were measured using differential oxidation condition and HPLC. Data are from 4 to 5 separate experiments. B, Representative Fe2+(DETC)2-ESR aortic spectra. C, Mean data from aortic nitric oxide production as measured with ESR using Fe2+(DETC)2. Data are from 5 to 7 experiments. D, Top: Western blot showing eNOS expression at baseline and after angiotensin II infusion in wild-type FVB and MRP1–/– mice. Bottom: Mean data for densitometry of eNOS/CDK4. Data are from 5 experiments.

Role of MRP1 in Modulating Blood Pressure
To determine whether the preservation of endothelium-dependent relaxation or reduce vascular O₂^·– production blunted the blood pressure response to angiotensin II, we monitored arterial blood pressure using telemetry and noninvasively by the tail cuff method. While the baseline pressure was similar between FVB and MRP1^–/– mice the increase in blood pressure caused by angiotensin II infusion was significantly greater in wild-type FVB mice as compared with MRP1^–/– mice (Figure 5 and supplemental Figure II).

View larger version (13K): [in this window] [in a new window]   Figure 5. Blood pressure in FVB and MRP1–/– animals with or without chronic angiotensin II infusion. A,: Blood pressure was measured continuously every hour for 10 minutes in conscious mice using a telemetry system. Mean arterial blood pressure in mice on the 7th day of angiotensin II infusion. Data are from 5 to 6 separate experiments. B, Tail cuff blood pressure measured on the 13th day of angiotensin II infusion. Data are from 8 to 10 separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study we demonstrated that the multidrug resistance protein-1 is a major exporter of oxidized glutathione and contributes to angiotensin II–induced hypertension. Vascular levels of GSH were higher in MRP1^–/– mice than in FVB mice and during angiotensin II–induced hypertension, the lack of MRP1 prevented the decline in vascular glutathione levels, preserved endothelium-dependent relaxation and prevented the increase in O₂^·– caused by chronic angiotensin II infusion. This was associated with a blunted increase in Nox1, Nox2, p22^phox, p47^phox, and p67^phox and a preservation of H₄B in MRP1^–/–-vessels. The absence of MRP1 also prevented the decrease in NO production caused by angiotensin II treatment. Finally, these favorable effects on vascular function were accompanied by a reduction in the hypertension caused by angiotensin II in MRP1^–/– mice. These data clearly demonstrate that MRP1 plays an important role in regulation of blood pressure in response to angiotensin II, likely via modulation of intracellular thiol levels.

When GSH is oxidized, mammalian cells have two options for removing the newly formed GSSG. One is recycling by the enzyme GSSG reductase. In the present study, we showed that the protein level of this enzyme wasn’t altered, but that its activity was modestly enhanced by angiotensin II–induced hypertension. This finding is in keeping with previous studies showing that GSSG reductase activity is modulated by substrate availability.²¹ The second mechanism for GSSG removal is export. Our current results show that vascular cells predominantly use MRP1 for this process. Of note, MRP1 protein expression wasn’t changed in vessels of wild-type mice during angiotensin II infusion. It is likely that the Km of MRP1 for GSSG is sufficiently high that the expression of this transporter does not need to be increased to handle higher levels of GSSG formed during persistent oxidative stress. Before the present study, it wasn’t clear which of these two pathways would be superior in terms of maintaining normal vascular function. Our current results indicate that recycling of GSSG to GSH by the reductase is clearly beneficial and that excessive thiol extrusion is deleterious. In addition to a reduction of total intracellular thiol and the attendant oxidant stress, export of GSSG by MRP1 requires that vascular cells synthesize new GSH de novo, which is metabolically demanding. The induction of GSSG reductase likely enhances GSSG recycling and thus preservation of higher levels of GSH. Of note, in MRP1^–/– mice, the vascular levels of glutathione were substantially higher at baseline than in wild-type animals suggesting that MRP1 not only modulates intracellular thiol levels during pathological states but also under normal conditions. This is in keeping with data by Lorico et al in other tissues of MRP1^–/– mice.²²

Our current findings also indicate that in angiotensin II–induced hypertension, MRP1 has a significant impact on vascular NO production. This is manifested as preserved endothelium-dependent vasodilatation and NO production as measured by ESR when MRP1 is absent. Unlike the case in wild-type vessels, angiotensin II failed to stimulate an increase in O₂^·– in MRP1^–/– vessels. Because of the rapid reaction between NO and O₂^·–, this would likely preserve NO. We also found that the ratio of reduced to oxidized H₄B was higher at baseline and during angiotensin II infusion in MRP1^–/– mice as compared with wild-type animals and that the depletion of H₄B observed in wild-type mice did not occur in MRP1^–/– mice. This was not attributable to a change in eNOS expression; however, angiotensin II failed to stimulate an increase in O₂^·– in MRP1^–/– vessels. A major source of H₄B oxidation is peroxynitrite (ONOO^–), which in turn is effectively scavenged by GSH.^23,24 Thus, preservation of H₄B could maintain eNOS function and NO production. Finally, as discussed below, the reduced induction of several NADPH oxidase components in MRP1^–/– mice could reduce O₂^·– production and preserve NO.

Our results also indicate that the increase in expression of NADPH oxidase subunits caused by angiotensin II is modulated by MRP1. Previously, it has been shown that angiotensin II–induced hypertension is associated with an 6- to 7-fold increase in Nox1 mRNA expression, but only a modest change in Nox4 message.^{17, 18} Desouki et al have shown that reactive oxygen species produced by the mitochondria can stimulate Nox1 expression.²⁵ Further, we have shown that treatment of rats with membrane targeted superoxide dismutase prevents upregulation of aortic p22 ^phox mRNA during angiotensin II infusion.²⁶ Recently, multiple NADPH oxidase subunits have been found to be increased in vessels of humans with coronary artery disease—a setting in which ROS production is increased.²⁷ These studies suggest that expression of various components of the NADPH oxidase is redox sensitive and raise the possibility that the initial increase in ROS production caused by angiotensin II can lead to a further increase in NADPH oxidase subunit expression in a feed-forward fashion. It is interesting to speculate that in MRP1^–/– mice, prevention of thiol export in response to angiotensin II favorably affected the endothelial intracellular redox status and suppressed the redox stimulus for Nox1 expression.

Several prior studies have supported the concept that various pathophysiological stimuli can activate multiple sources of ROS production and that there is an interaction between these systems.²⁸ Our current findings are in keeping with this observation. In addition to the increase in NADPH oxidase subunits and activity caused by angiotensin II, we and others¹⁸ have found clear-cut evidence of eNOS uncoupling, as evidenced by partial inhibition of vascular O₂^·– production by L-NAME. Oxidation of H₄B can lead to an accumulation of dihydrobiopterin or, when extensive, destruction of the pterin structure and loss of total cellular biopterin.²³ It has been reported that GSH can serve to protect against H₄B oxidation and that various oxidized species of glutathione can react with H₄B.^24,29 In keeping with these prior studies, we found that angiotensin II–induced hypertension is associated with a decrease in H₄B levels. In contrast, H₄B levels were not diminished in MRP1^–/– mice, also likely contributing to the normal aortic O₂^·– production in these animals.

These favorable effects on vascular function in MRP1^–/– mice were associated with a blunted increase in blood pressure caused by angiotensin II. Interestingly, this effect on blood pressure is similar to that we have previously observed in p47^phox–/– mice.¹⁹ Overexpression of either Nox1 or p22^phox augments hypertension caused by angiotensin II, and knockout of Nox1 has been shown in two independent studies to blunt hypertension.^30–32 Moreover, treatment with H₄B has proven effective in lowering blood pressure in animals.^11,33 The blunting of Nox1 expression and preservation of H₄B therefore likely contributed to the lower blood pressure in response to angiotensin II in MRP1^–/– mice. Other undefined mechanisms may also contribute.

In summary, our data indicate that the MRP1 plays a major role in modulating intracellular glutathione levels, NO production, O₂^·– flux, gene expression, vascular function, and blood pressure. The loss of thiols in response to angiotensin II is associated with myriad untoward vascular effects that are prevented when MRP1 is absent. These data strongly suggest that specific MRP1 blockade might prove effective in the treatment of hypertension and perhaps other vascular diseases.

	Acknowledgments

 
We gratefully acknowledge excellent technical support by Louise McCann and Graciela Gamez.

Sources of Funding

This work was supported by NIH grants HL390006 and HL59248, NIH Program Project Grant HL58000 and a Department of Veterans Affairs Merit Grant, and the NH&MRC Australia. Dr Widder was supported by the Deutsche Akademie der Naturforscher Leopoldina (BMBF-LPD 9901/8-97). Dr Mueller was supported by the Deutsche Forschungsgemeinschaft (MU 1731/1-1).

Disclosures

None.

	Footnotes

 
Original received October 23, 2006; final version accepted January 5, 2007.

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