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. Author manuscript; available in PMC: 2009 Feb 24.
Published in final edited form as: Hypertension. 2008 Dec 22;53(2):189–195. doi: 10.1161/HYPERTENSIONAHA.108.115709

GTP cyclohydrolase I expression and enzymatic activity are present in caveolae of endothelial cells

Timothy E Peterson 1,*, Livius V d’Uscio 1,*, Sheng Cao 2, Xiao-Li Wang 3, Zvonimir S Katusic 1
PMCID: PMC2646898  NIHMSID: NIHMS89730  PMID: 19104007

Abstract

Tetrahydrobiopterin is an essential cofactor required for the synthesis of nitric oxide. GTP cyclohydrolase I (GTPCH I) is the rate limiting enzyme for tetrahydrobiopterin production in endothelial cells, yet little is known about the subcellular localization of this enzyme. In this study, we demonstrate that GTPCH I is localized to caveolar membrane microdomains along with caveolin-1 and endothelial nitric oxide synthase. GTPCH I activity was detected in isolated caveolar membranes from cultured endothelial cells. Confocal and electron microscopy analyses confirmed GTPCH I colocalization with caveolin-1. Consistent with in vitro studies, GTPCH I activity was evident in isolated caveolar microdomains from lung homogenates of wild-type mice. Importantly, a two-fold increase in GTPCH I activity was detected in the aortas of caveolin-1 deficient mice suggesting that caveolin-1 may be involved in the control of GTPCH I enzymatic activity. Indeed, overexpression of caveolin-1 inhibits GTPCH I activity, and tetrahydrobiopterin biosynthesis is activated by disruption of caveolae structure. These studies demonstrate that GTPCH I is targeted to caveolae microdomains in vascular endothelial cells and tetrahydrobiopterin production occurs in close proximity to endothelial nitric oxide synthase. Additionally, our findings provide new insights into the regulation of GTPCH I activity by the caveolar coat protein, caveolin-1.

Keywords: GTP-cyclohydrolase I, tetrahydrobiopterin, endothelium, caveolin-1, nitric oxide

Introduction

The production of the vasodilator nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for the maintenance of normal vasomotor function.1 Tetrahydrobiopterin (BH4) is an essential cofactor required for activity of eNOS.2 We and others have shown that the vascular endothelium is a major source of BH4 in the arterial wall.3–5 BH4 is synthesized from guanosine triphosphate (GTP) in a three step process that is initiated by the enzyme GTP cyclohydrolase I (GTPCH I).6 The molecular mechanisms regulating GTPCH I activity in vascular endothelium are not well understood. GTPCH I activity has been demonstrated to be increased by cytokines, hydrogen peroxide, protein kinase C, and fluid shear stress imposed on endothelium by circulating blood.7–10 Several groups have also demonstrated that protein-protein interactions can influence GTPCH I activity in vitro.11,12 Most notably, the N-terminal sequence of GTPCH I was shown to bind several membrane-associated proteins suggesting that GTPCH I may be involved in membrane trafficking.12

It is now established that eNOS activity is regulated at the post-transcriptional level by the protein caveolin-1, an important structural protein associated with plasma membrane microdomains called caveolae.13–16 In endothelial cells caveolae are flask-like shapes invaginations of the plasma membrane and associated vesicles that provide a platform for many signaling complexes.17,18 The role of caveolin-1 in control of GTPCH I function and BH4 synthesis has not been studied in-vitro or in-vivo. Since proper eNOS function is dependent on both GTPCH I activity and its subcellular localization to caveolae19, we hypothesized that GTPCH I localizes in caveolae and is regulated by caveolin-1.

Materials and Methods

Experimental Animals

Male caveolin-1–deficient mice (Cav1tm1Mls/J; Cav1−/−) and eNOS–deficient mice (B6.129P2-Nos3tm1Unc/J; eNOS−/−) as well as strain matched wild-type mice B6129SF2/J and C57BL/6J, respectively, were obtained from the Jackson Laboratories (Bar Harbor, ME). Lungs of GTPCH I-transgenic mice were provided by Dr. Alex Chen (Michigan State University, East Lansing, MI) with permission of Dr. Keith M. Channon (University of Oxford, Oxford, United Kingdom). Mice were maintained on standard chow with free access to drinking water. Housing facilities and all experimental protocols were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and comply with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Mice were euthanized by overdose of pentobarbital (60 mg/kg, i.p.), and whole aortas and lungs were carefully harvested and dissected free from connective tissue.

Cell Culture and Adenoviral Overexpression Techniques

Human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex (East Rutherford, NJ) and were passaged in EGM-2 growth media (Cambrex). All experiments were performed using HUVECs between passages 3–7. A recombinant adenovirus encoding the human GTPCH I gene (Ad-GTPCH I) driven by a cytomegalovirus promoter20 at a multiplicity of infection (MOI) of 100 was used to overexpress GTPCH I to a level that was detectable by Western blot analysis. In separate studies, adenoviral encoding human caveolin-1 (Ad-Cav1, Vector Biolabs, Philadelphia, PA) at a MOI of 30 was used to overexpress caveolin-1. HUVECs were infected for 12 hours in serum-free media (EBM-2) and were then fed with growth medium for 48 hours prior to analysis. A recombinant adenoviral vector with a deletion of ΔE1 (Ad-ΔE1) was used as a controls.

Western Blot Analysis

Cells were washed twice in cold phosphate buffered saline (PBS) and flash frozen in 200 μL of lysis buffer10 in a ethanol/dry ice bath followed by scraping and a 5 second sonication to achieve a homogeneous solution. 20–30 μg of total protein was separated by 12% SDS-PAGE, transferred to nitrocellulose membrane and stained with Ponceau-S stain (Sigma, St. Louis, MO) to ensure equal protein loading. For Western blot analysis, membranes were probed using primary antibodies against anti-caveolin-1, anti-eNOS (BD Biosciences, San Diego, CA), or anti-GTPCH I.4 Membranes were then incubated for 1 hour with horseradish peroxidase conjugated anti-IgG antibodies and visualized using enhanced chemiluminescence detection (Amersham).

Isolation of Triton X-100 soluble and insoluble fractions

HUVECs were grown in 10cm dishes until 70% confluent. Cells were harvested in lysis buffer containing 1% Triton X-100 and sonicated for 5 seconds. Cell lysates were then centrifuged at 100,000g for 1 hour and the supernatant was collected (soluble fraction). The resulting pellet was washed twice with lysis buffer, resuspended in 100 μL of lysis buffer and sonicated for 30 seconds to achieve a homogeneous solution (insoluble fraction).

Isolation of Caveolae-Enriched Membranes

For each experiment, pooled Ad-GTPCH I transduced HUVECs from 6 dishes or whole mouse lung was homogenized in cold buffer A (0.25 M sucrose, 1 mM EDTA and 20 mM Tricine, pH 7.8) and centrifuged at 1000g for 10 minutes. The supernatant was saved and layered onto 30% Percoll (Sigma) and centrifuged at 84,000g for 30 minutes. The membrane fraction (visualized by an opaque band) was collected and brought to a volume of 2 ml with buffer A and sonicated 3 times for 30 seconds and protein content was determined. Equal amounts of protein from each group were then resuspended in a 23% solution of Optiprep (Accurate Chemical, Westbury, NY) and placed in a centrifuge tube. A 20% to 10% Optiprep gradient was layered on top and centrifuged at 52,000g for 90 minutes. Density gradient fractions were collected in 1.5 mL aliquots from top to bottom with the first four fractions containing the low buoyant density membrane fractions that were enriched in caveolae-associated proteins.

Immunofluorescent imaging

HUVECs were plated onto tissue culture chamber slides and infected with an adenovirus encoding hemagglutinin-tagged GTPCH I (HA-GTPCH I, a gift from Dr. Keith Channon)21 at a MOI of 20 in EBM-2 for 6 hours. Cells were then fed growth medium for 24 hours prior to analysis. Following treatment, cells were fixed with methanol for 15 minutes at 4°C, and blocked with 10% normal goat serum for 30 minutes. Slides were then incubated for 1 hour with a cocktail of mouse anti-caveolin-1 and rabbit anti-HA antibody (Sigma). A secondary antibody cocktail of goat anti-mouse FITC and goat anti-rabbit Texas Red (Invitrogen, Carlsbad, CA) was added and incubated for 45 minutes. Cells were then incubated for 5 minutes with 10 μg/mL Hoechst 33258 (Sigma) to stain for nuclei. Cover slips were mounted using Prolong Gold mounting media (Invitrogen) and the cells were visualized using a Zeiss LSM 510 laser scanning confocal microscope.

Immunogold labeling of mouse aorta by electron microscopy

Isolated aortic ring segments from wild-type mice were fixed in 4% formaldehyde plus 1% glutaraldehyde overnight, dehydrated in a series of ethanol from 35% to absolute while progressively lowering the temperature to −20°C, embedded in LR White and polymerized at 55°C. Thin sections were mounted on nickel grids and labeled for GTPCH I and double-labeled for caveolin-1. GTPCH I was labeled by blocking free aldehydes with 1% glycine, and blocking with phosphate buffered saline containing Tween-20 and 2% normal goat serum (PBST+NGS). A rabbit polyclonal antibody specific for mouse GTPCH I4 was diluted 1:2 in PBST+NGS and grids were incubated at room temperature for 2 hours. Grids were rinsed extensively in PBST and incubated in a goat anti-rabbit secondary antibody conjugated to 10 nm gold beads (Amersham) for 1 hour at room temperature. Caveolin-1 was labeled by blocking in glycine and PBST+NGS similarly to GTPCH I labeling. Mouse anti-caveolin-1 was diluted 1:5 in PBST+NGS and incubated 2 hours at room temperature. After rinsing in PBST, the sections were incubated in goat anti-mouse secondary antibody conjugated to 5 nm gold beads (Amersham). When double-labeling was completed, the sections were stained in lead and uranyl for transmission electron microscopy.

Measurements of Biopterin Levels and GTP-Cyclohydrolase I Activity

BH4 and 7,8-dihydrobiopterin (7,8-BH2) levels, and GTPCH I enzymatic activity were determined in fresh aortas using reverse-phase HPLC method as described previously.4,22

Statistical Analysis

Data are expressed as means ± SEM and “n” indicates the number of animals from which tissues were harvested. Single values were compared by one-way ANOVA with Bonferroni’s correction for multiple comparisons. For simple comparisons between two groups, an unpaired Student’s t-test was used where appropriate. A value of P<0.05 was considered significant.

Results

In-vitro analysis of GTPCH I subcellular localization

In order to track GTPCH I protein localization in vitro, an adenoviral construct encoding human GTPCH I was used in HUVECs at a MOI of 100 (Figure 1A). Initial experiments demonstrated that GTPCH I protein expression was equally partitioned to both Triton X-100 soluble and Triton X-100 insoluble fractions suggesting that a significant fraction of GTPCH I protein is associated with cellular membranes along with caveolin-1 and eNOS (Figure 1A). Subsequent studies using sucrose gradient ultracentrifugation demonstrated that both GTPCH I protein expression and activity was localized to caveolar microdomains as well as non-caveolar-associated membrane fractions (Figure 1B). Confocal microscopic analysis of HUVECs infected with an adenovirus encoding an HA-tagged GTPCH I demonstrated that GTPCH I staining was localized to perinuclear regions of the cell (Figure 1C) as previously reported21, however, there was also a considerable amount of co-localization of GTPCH I with caveolin-1 at the cell membrane (Figure 1C).

Figure 1.

Figure 1

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A) Western blot analysis of eNOS, GTPCH I and caveolin-1 in Triton X-100 soluble (S) and Triton X-100 insoluble (I) fractions following adenoviral GTPCH I overexpression of HUVECs (MOI=100). B) Western blot analysis of eNOS, caveolin-1 and GTPCH I in sucrose gradient membrane fractions of HUVECs following adenoviral GTPCH I overexpression of HUVECs (MOI=100). Enrichment of caveolin-1 in fraction 2 indicated the successful separation of caveolae fraction (lower panel). The results showed that GTPCH I, eNOS, and caveolin-1 were co-fractionated to the low buoyant density fraction. C) Confocal microscopic analysis of HUVECs following adenoviral overexpression of HA-tagged GTPCH I (MOI=20). Immunofluorescent staining of GTPCH I (green) and caveolin-1 (red) demonstrates partial colocalization of the two proteins (yellow). Data are representative of at least three independent experiments.

In-vivo analysis of GTPCH I subcellular localization

In order to determine if similar results could be obtained in vivo, initial studies using electron microscopy were performed to determine if GTPCH I and caveolin-1 might be localized in similar subcellular compartments to each other in endothelial cells from wild-type mouse aorta. Indeed, substantial amount of GTPCH I labeling was found both in the cytoplasm as well as at the plasma membrane of mouse aortic endothelial cells. Dual labeling for both caveolin-1 and GTPCH I showed that the two proteins were often associated in close proximity to each other (Figure 2A). Analysis of membrane-associated proteins using detergent-free sucrose density gradient ultra-centrifugation demonstrated that GTPCH I activity is concentrated in the caveolae-rich fraction of wild-type mouse lung (Figure 2B).

Figure 2.

Figure 2

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A) Electron microscopic analysis of wild-type (B6129SF2/J) mouse aorta. Immuno-gold labeling of caveolin-1 (5nm gold particles) and GTPCH I (10nm gold particles) demonstrate that these proteins reside in close proximity to each other (inset). L: Lumen, E: endothelium, SM: smooth muscle. B) Enzymatic activity of GTPCH I in density gradient fractions of lung lysates from wild-type mice. Data are representative of at least three independent experiments. Results are mean ± SEM. * P<0.05 vs. other fractions.

To further investigate the functional significance of GTPCH I localization to caveolar microdomains, we measured GTPCH I protein expression and activity in the aortas of Cav1−/− mice which lack any morphological caveolar structures.23 Western blot analysis demonstrated similar levels of GTPCH I protein expression in the aorta from wild-type and Cav1−/− mice (Figure 3A), while HPLC analysis revealed a significant increase in GTPCH I activity in the aorta and lung from Cav1−/− mice when compared to wild-type controls (Figure 3B). In contrast, GTPCH I activity was unaltered in other organs such as brain and liver of Cav1−/− mice (Table 1). Along with the increases in GTPCH I activity, we also observed an increase in BH4 levels from the aorta of Cav1−/− mice compared to wild-type controls while oxidative products of BH4, 7,8-BH2 levels, were unchanged (data not shown), indicating that the selective increase in BH4 levels was due to the increased de novo biosynthesis of BH4 via GTPCH I (Figure 3C). Furthermore, we could not detect a difference in BH4 levels in the aortas of eNOS−/− mice (Figure 3D). Together, this data demonstrates that caveolin-1 may act as a functional inhibitor of GTPCH I activity in vivo.

Figure 3.

Figure 3

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Determination of GTPCH I activity in the aorta of wild-type (WT) and Cav1−/− mice. A) Western blot analysis of caveolin-1 and GTPCH I protein levels in the aorta (n=4). B) Enzymatic activity of GTPCH I in wild-type (B6129SF2/J) and Cav1−/− mouse aorta (n=4). C) Tetrahydrobiopterin (BH4) levels in wild-type (B6129SF2/J) and Cav1−/− mice aortas (n=4). D) BH4 levels in the aorta of strain matched wild-type (C57BL/6J) and eNOS-deficient mice (n=5). Results are mean ± SEM. * P<0.05 vs. wild-type.

Table 1.

Enzymatic activity of GTP-cyclohydrolase I in wild-type and Cav1-deficient mice.

Organ WT Cav1 −/−
Lung 15.5±0.9 25.2±1.6 *
Brain 0.6±0.3 0.3±0.1
Liver 250.2±30.1 202.3±18.8
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WT indicates wild-type (B6129SF2/J). Data are means ± SEM (n=4) and are expressed in pmol neopterin/mg protein.

*

P<0.05 vs. WT.

Interaction between caveolin-1 and GTPCH I activity

Ad-Cav1-transduced cells showed increased Cav1 protein expression as detected by Western blot analysis (Figure 4A). Interestingly, enzymatic activity of GTPCH I was reduced by about 50% in HUVECs transduced with Ad-Cav1 as compared to control Ad-ΔE1 vector (Figure 4B).

Figure 4.

Figure 4

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Effect of caveolin-1 overexpression on GTPCH I activity. A) Protein expression of caveolin-1 in HUVECs transfected by Ad-Cav1 and Ad-ΔE1 for 48 hours. B) Bar graphs showing enzymatic activity of GTPCH I in HUVECs transfected by 30 MOI of Ad-Cav1 or Ad-ΔE1 for 48 hours. Results are mean ± SEM (n=8). * P<0.05 vs. Ad-ΔE1.

Western blot analysis of membrane fractions of wild-type mouse lung demonstrated that GTPCH I was present in the caveolae-rich fraction (fraction 2) (Figure 5A, upper panel), again confirming that GTPCH I is targeted to the cholesterol-rich, low buoyant density caveolae-rich fraction of the membrane. In contrast, GTPCH I was significantly reduced in the fraction 2 of Cav1−/− mice (Figure 5A, lower panel). To further demonstrate that GTPCH I activity is localized in cholesterol-enriched membrane microdomains, isolated mouse aortas were treated in-vitro with methyl-β-cyclodextrin (β-CD) that binds cholesterol and cause reversible disassembly of caveolae. Indeed, treatment with β-CD significantly increased BH4 levels in wild-type but not in Cav1−/− mice (Figure 5B).

Figure 5.

Figure 5

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A) Density gradient fractions of lung lysates from strain-matched wild-type (B6129SF2/J) and Cav1−/− mice was performed that separates caveolar microdomains from other cellular constituents. Western blot analysis with anti-GTPCH I antibody was performed to determine the localization of GTPCH I in gradient fractions. Please note that GTPCH I was fractionated to the low buoyant density fraction in wild-type mice (upper panel) and this was almost eliminated in fraction 2 of Cav1−/− mice (lower panel). B) Effect of cholesterol depletion on BH4 levels. Isolated mouse aortas of wild-type (WT, B6129SF2/J) and Cav1−/− mice were exposed in-vitro with methyl-β-cyclodextrin (β-CD, 10 mM) for 1 hour at 37°C and BH4 levels were then determined. Bar graph showing BH4 levels after treatment without or with β-CD. Results are mean ± SEM (n=6). * P<0.05 vs. untreated wild-type mice.

Caveolar activity of GTPCH I in GTPCH I transgenic mice

Since overexpression of GTPCH I in endothelium has well established vascular protective effects, we performed experiments on transgenic mice with endothelial-targeted overexpression of GTPCH I. Consistent with findings on cultured endothelial cells, we found that GTPCH I activity was significantly increased in caveolae-rich fractions (and non-caveolae membrane fractions) of GTPCH I transgenic mice as compared to wild-type mice (Figure 6).

Figure 6.

Figure 6

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Enzymatic activity of GTPCH I in caveolar (A) and in non-caveolar (B) membrane after density gradient fractions of lung lysates from wild-type (C57BL/6J) and GTPCH I transgenic (GTPCH-tg) mice. Results are mean ± SEM (n=3). * P<0.05 vs. wild-type mice.

Discussion

The results of this study reveal several novel findings regarding the regulation of BH4 synthesis in endothelial cells. First, we demonstrate that GTPCH I protein expression and enzymatic activity is localized in both caveolar and non-caveolar membrane compartments. Second, enzymatic activity of GTPCH I and BH4 levels were increased in the aorta of Cav1−/− mice. Third, caveolin-1 overexpression inhibits GTPCH I activity, and BH4 biosynthesis is activated by disruption of caveolae structure. Fourth, overexpression of GTPCH I increased GTPCH I activity in caveolar microdomains of endothelial cells. Given that optimal NO synthesis occurs in caveolar microdomains, we propose that the close spatial localization of eNOS and GTPCH I may help to ensure proper NO synthesis in endothelial cells. In addition, it is likely that caveolar localization contributes to well established vascular protective effect of GTPCH I in experimental models of diabetes, hypercholesterolemia, as well as in pulmonary and systemic hypertension.24–27

Although GTPCH I is known to be critical for the synthesis of BH4 in multiple cell types, there is little information about the subcellular localization of this enzyme in endothelial cells. Using an adenoviral construct to overexpress human GTPCH I in endothelial cells, we were able to demonstrate that GTPCH I can be equally partitioned into both Triton X-100 soluble and Triton X-100 insoluble fractions. It is unclear at this time what are the differences between these two distinct subpopulations of GTPCH I, however, it is possible that there are post-translational modifications to GTPCH I which may affect its solubility in Triton X-100. Further investigation of the membrane-associated pool of GTPCH I using sucrose gradient ultracentrifugation revealed that GTPCH I can be targeted to low buoyant density membrane fractions called caveolae and other membrane domains such as Golgi, ER, and clatherin coated pits along with eNOS and caveolin-1. Confocal microscopic analysis confirmed that GTPCH I was partially co-localized with caveolin-1 under basal conditions in vitro. However, confocal microscopy also revealed GTPCH I in membrane patches and in cytosol that did not contain caveolin-1. Based on these studies, GTPCH I localization in caveolae represents only a portion of the GTPCH I pool within the cell. Furthermore, we observed a loss of GTPCH I from caveolar and non-caveolar membranes in Cav1−/− mice whereas Western blot analysis did not show any change in GTPCH I expression between aortas obtained from wild-type and Cav1−/− mice. These observations are consistent with our conclusion regarding caveolar localization of GTPCH I. Whether trafficking28 of GTPCH I, or BH4 biosynthesis, occur in caveolae remains to be determined in the future studies.

Since caveolin-1 has been reported to be an important regulator of eNOS activity29, and eNOS activity is enhanced in Cav1−/− mice23,30, we determined basal GTPCH I activity in the aortas of Cav1−/− mice. Surprisingly, we found that there was a two-fold increase in GTPCH I activity in aortas of Cav1−/− mice when compared to wild-type mice. We also observed a selective increase in BH4 levels from the aorta of Cav1−/− mice suggesting that this increase was caused by the increased de novo biosynthesis of BH4 via GTPCH I. In contrast, GTPCH I activity was unchanged in the liver or brain of Cav1−/− mice when compared to wild-type controls, suggesting that caveolin-1 may exert an inhibitory effect on basal GTPCH I activity in-vivo, in an organ specific manner. Alternatively, this observation could also be explained by differential regulation of BH4 biosynthesis between large conduit and small resistance arteries. Indeed, it is generally accepted that NO (and possibly BH4) plays more prominent functional role in control of large conduit arteries.31 The inhibitory effect of caveolin-1 on GTPCH I activity was further demonstrated by the fact that transduction of HUVECs with Ad-Cav1 resulted in significant suppression of GTPCH I activity. On the other hand, treatment with cholesterol-binding drug β-CD, which prevents formation of functional caveolae by depletion of cholesterol32, increased BH4 biosynthesis in the aorta of wild-type mice. Of note, β-CD treatment did not further increase BH4 levels in the aorta of Cav1−/− mice reinforcing our conclusion that caveolin-1 has negative regulatory effect on enzymatic activity of GTPCH I.

Over the past decade, studies from several groups have reported the beneficial effects of increasing endothelial BH4 levels in various models of vascular disease. Indeed, a number of investigations have demonstrated that supplementation with BH4 can prevent endothelial dysfunction. Accordingly, acute and chronic supplementation of BH4 in experimental models of oxidative stress and in patients with cardiovascular disease improved endothelium-dependent relaxations and increased eNOS activity.5,33–36 In the present study, GTPCH I enzymatic activity was increased in low buoyant density membrane fraction of transgenic mice with endothelial-targeted overexpression of GTPCH I. This observation is important because several recent studies demonstrated that endothelial overexpression of GTPCH I reduces superoxide anion production and preserves NO release suggesting that endothelial dysfunction can be restored by increasing local concentration of BH4.24,25,27 Relevant to interpretation of our results, previous studies have demonstrated that caveolar microdomains are sensitive to oxidative and nitrosative stress.37,38 Whether GTPCH I localization in the caveolar membrane is critical for protection of caveolae against oxidative stress in vivo remains to be determined.

Perspectives

Results of the present study have several important implications for understanding of vascular endothelial function. In addition to well established role of BH4 in activity of eNOS, our findings underscore the importance of cellular localization of GTPCH I, a critical enzyme responsible for biosynthesis of BH4. Co-localization of GTPCH I and eNOS in caveolae is most likely designed to provide optimal local concentration of BH4 required for biosynthesis of endothelial NO. Since elevated concentration of superoxide anion and subsequent formation of peroxynitrite is one of the most important mechanisms underlying endothelial dysfunction, the relevance of BH4 in preservation of caveolar architecture and function should be investigated in the future studies.

Acknowledgments

Source of Funding

This work was supported by National Institutes of Health grant HL-53524, by Roche Foundation for Anemia Research, and by the Mayo Foundation. Dr. d’Uscio is the recipient of Scientist Development Grant from the American Heart Association (07-30133N).

Footnotes

Conflict on Interest Disclosures

None

References

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