Published online before print May 8, 2008, doi: 10.1161/CIRCRESAHA.107.167858
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© 2008 American Heart Association, Inc.
Molecular Medicine |
Hypoxia-Inducible Transcription Factor-1
Triggers an Autocrine Survival Pathway During Embryonic Cardiac Outflow Tract Remodeling
From the Department of Medicine (Cardiology), Case Western Reserve University, Cleveland, Ohio.
Correspondence to Steven A. Fisher, MD, Department of Medicine (Cardiology), Case Western Reserve University, Cleveland, OH, 44106. E-mail steven.fisher{at}case.edu
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Abstract |
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The cardiac outflow tract (OFT) of birds and mammals undergoes complex remodeling in the transition to a dual circulation. We have previously suggested a role of myocardial hypoxia and hypoxia inducible factor (HIF)-1 in the apoptosis-dependent remodeling of the OFT. In the present study, we transduced recombinant adenovirus-mediated HIF-1
in embryonic chick OFT myocardium to test its role in OFT remodeling. HIF-1 reduced the prevalence of apoptosis in OFT cardiomyocytes at stages 25 and 30, as determined by lysosome accumulation and caspase-3 activity. Associated conotruncal defects included malrotation of the aorta and excessive infundibular myocardium. HIF-1 targets induced in these gain-of-function experiments included vascular endothelial growth factor (VEGF), inducible nitric oxide synthase, and stromal cell–derived factor-1. To test the role of VEGF in this context, an adenovirus expressing secreted Flk1 (VEGF receptor 2) that binds and blocks VEGF signaling was targeted to the OFT myocardium. This caused increased cell death in the OFT myocardium at stages 25 and 30. Associated conotruncal heart defects included malrotation of the aorta, defects in the subpulmonic infundibulum associated with a small right ventricle, and increased OFT mesenchyme with failure of semilunar valve formation. We conclude that hypoxia signaling through HIF-1 and VEGF provides an autocrine survival signal in the developing cardiac OFT and that perturbation in this pathway causes OFT defects that model congenital human conotruncal heart defects.
Key Words: HIF-1 • VEGF • apoptosis • OFT remodeling
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Introduction |
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The arterial pole of the heart in birds and mammals, referred to here as the cardiac outflow tract (OFT), undergoes extensive remodeling in the developmental transition to a dual series circulation.1 Spatially and temporally regulated elimination of OFT cardiomyocytes by apoptosis from Hamburger–Hamilton stages 25 to 32 of development is required for the proper shortening and rotation of the OFT.2,3 Inhibition or widespread activation of apoptosis in OFT cardiomyocyte results in failure of the aorta to rotate to its posterior position and thus double-outlet right ventricle, as well as excess or absence of the subpulmonic myocardial infundibulum, respectively. The subpulmonic infundibulum in the mature heart is the remnant of the embryonic OFT myocardium.
A critical question is why, of all the cardiac chambers, does the OFT myocardium undergo apoptosis-dependent remodeling during normal development? Our previous studies suggested that myocardial hypoxia may be involved based on: (1) increased binding of the hypoxia indicator EF5 to the OFT myocardium at Hamburger–Hamilton stages 25 to 324,5; (2) expression of the hypoxia-inducible transcription factor (HIF)-1 specifically in the OFT myocardium at the same stages6; (3) approximately synchronous recruitment of endothelial progenitor cells to the OFT and coronary vasculogenesis6; and (4) incubation of eggs under hyperoxic conditions perturbed OFT remodeling.4 In the present study, we have tested the role of HIF-1 in apoptosis-dependent remodeling of the chick OFT.
HIF-1 is a heterodimer composed of a ubiquitously expressed β subunit (also known as ARNT) and oxygen-regulated subunit.7,8 As oxygen concentrations decrease, HIF-1 accumulates because of reduced prolyl hydroxylase-dependent degradation of the protein. HIF binding to its consensus DNA binding sequence A/GCGTG regulates the transcription of the hypoxia-dependent gene program. Inactivation of the HIF-1 or -β subunits in the mouse results in severe cardiovascular compromise and demise of the embryo by embryonic day 11.9–11 However, the role of hypoxia and HIFs in specific morphogenic processes in the embryonic heart and vascular system remains to be elucidated.
Hypoxia/HIF may function to regulate cell death or cell survival. HIF activates the transcription of prodeath genes such as Bcl family members BNip3 and BMF12 and the death ligand FasL.13 HIF also activates the prosurvival genes including vascular endothelial growth factor (VEGF), insulin-like growth factor, and erythropoietin,14,15 therefore enhancing survival under low oxygen through vasculogenesis and erythropoiesis. Other hypoxia/HIF responsive genes, eg, inducible nitric oxide synthase (iNOS), have prosurvival or prodeath effects depending on the biological context.16
In this study, we used recombinant adenovirus to express a stabilized HIF-1 in the embryonic chick OFT myocardium. Forced expression of HIF-1 reduced the incidence of apoptosis and increased VEGF expression. Interfering with VEGF activity by adenoviral-mediated expression of Flk1-Fc increased apoptosis, suggesting that HIF-1 acting through VEGF provides a survival signal to the cardiomyocytes of the developing OFT. Interference with the hypoxia-dependent gene program in the embryonic OFT through specific gain- and loss-of-function strategies results in OFT defects that model congenital human heart defects.
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Materials and Methods |
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Experimental details are available in the expanded Materials and Methods section in the online data supplement at http://circres. ahajournals.org.
In Ovo Injection of Chicken Heart
Stage 17 to 18 chicken embryos were injected with recombinant adenovirus including AdHIF-1,17 AdFlk1-Fc (AdFlk1)18 and AdGFP as previously described.19
Assays for Apoptosis
Lyso Tracker Red (LTR) accumulation and immunohistochemical detection of active Caspase-3 were performed as previously described.20 Caspase-3/7 activity was measured with OFT protein lysates using caspase-Glo 3/7 Assay kit. Image J and Cell Counter software were used to quantify LTR signal and caspase-3 immunostaining.
Morphological and Histological Analyses
Stage 34 to 35 hearts were photographed with a Leica FLIII stereomicroscope and SpotRT digital camera. Deparaffinized serial frontal sections were stained with hematoxylin/eosin (H&E) as previously described.4
Gene Expression Analysis
Total RNA was isolated using PicoPure RNA isolation kit from OFT tissues of stage 25 hearts injected with AdHIF-1 or AdGFP. Ten nanograms of total RNA were used to perform real-time RT-PCR by standard methods. Data were first normalized to GAPDH and then to the green fluorescent protein (GFP)+ group.
Statistical Analysis
Data are expressed as means±SEM. All statistics were done with Prism software (GraphPad software Inc, San Diego, Calif). One-way ANOVA followed by Bonferroni post test or t test was used for comparison among experimental groups. P<0.05 was considered statistically significant.
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Results |
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Forced Expression of Stabilized HIF-1
Reduces Apoptosis in OFT MyocardiumLTR has been used as among the most sensitive assays for apoptosis in the developing chick heart.3,20,21 In control (uninjected) hearts, a low level of LTR staining was detected in the OFT at stage 25 and increased to a peak at stage 30 (Figure 1A, 1D, 1G), with minimal staining in the remainder of the heart, as we previously reported.6,20 In embryos injected with the control virus AdGFP, LTR staining was modestly increased compared with uninjected embryos at stage 25 but was not different at stage 30 (Figure 1B, 1E, and 1G). Hearts transduced with AdHIF-1
expressed stabilized HIF-1 in the nuclei of the OFT cardiomyocytes (Figure I in the online data supplement). The LTR staining in AdHIF-1–transduced hearts was reduced at stages 25 and 30 as compared with AdGFP transduced hearts, and at stage 30 as compared with uninjected embryos (Figure 1C, 1F, and 1G). The reduction of LTR staining was observed in 16 of 21 AdHIF-1 hearts at stage 25 and 14 of 21 AdHIF-1 hearts at stage 30, respectively, compared with the AdGFP-transduced hearts, as judged by 2 independent observers using a 5-point scoring system for the number of LTR particles (data not shown). These scores correlated well with the quantitative measures of LTR particle numbers (Figure 1G). Caspase-3 activity was used as a second indicator of apoptosis. Activated caspase-3 was detected at low levels in the stage 25 OFT myocardium of AdGFP-injected embryos (Figure 2A and 2B) and increased at stage 30 (Figure 2E and 2F), consistent with our previous observation.22 There was no or minimal staining in the remainder of the heart. At stages 25 and 30, some cells showed colocalization of GFP and caspase-3 signals (Figure 2B and 2F, arrowheads), indicating GFP-labeled cardiomyocytes were executing apoptosis. At both stages 25 (Figure 2C, 2D, and 2I) and 30 (Figure 2G through 2I), there was a marked reduction in the number of active caspase-3–staining cells in the OFT myocardium of AdHIF-1–injected hearts compared with AdGFP-injected stage-matched controls. Cells expressing GFP in AdHIF-1–treated hearts were not active caspase-3–positive.
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P<0.001 compared with both control and AdGFP group. Vent indicates ventricles.
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Caspase-3 activity measured in tissue homogenates increased 2.4-fold in the OFT of AdGFP-injected embryos between stages 30 and 25 (Figure 2J), consistent with our previous reports.2,22 Forced expression of HIF-1 reduced the activity of caspase-3 in the OFT homogenates by 40% to 50% as compared with AdGFP-injected embryos at stages 25 and 30. This is concordant with the reduction in staining for active caspase-3 observed in sections. In total, these observations indicate that forced expression of HIF-1 reduces the incidence of apoptosis in OFT cardiomyocytes.
Forced Expression of HIF-1 Causes OFT Defects
A separate group of embryos injected with AdHIF-1 were examined at stages 34 to 35 when OFT remodeling is nearly complete and effects on morphology can be assessed. Thirty-two AdHIF-1 embryos survived to stages 34 to 35 and 23 had abnormal heart and OFT morphology as observed in whole mount. In 19 of 23 embryos, the aorta is malpositioned anterior and to the right and coursing parallel to the pulmonary artery (Figure 3B and 3D), whereas it is normally posterior to and spirals about the pulmonary artery (Figure 3A and 3C). Also evident is abnormal persistence of a stalk of GFP-labeled OFT myocardium beneath the aorta. In control, the GFP-labeled OFT myocardium has committed to the right heart, where it forms the infundibular connection between the right ventricle and pulmonary artery. In the remaining abnormal hearts (4/23), the aorta is malpositioned almost directly posterior to the pulmonary artery (not shown). In frontal sections from anterior to posterior, the abnormal anterior and rightward position of the aorta and its connection to the right ventricle in a double-outlet right ventricle morphology is evident (Figure 3E through 3G [control] versus Figure 3H through 3J [AdHIF]). The extended nature of the infundibulum is also evident by: (1) the position of the semilunar valves well above the base of the ventricles; (2) staining of sections with the MF20 antibody that specifically recognizes cardiac myosin (Figure 3K and 3L [AdGFP] versus Figure 3M and 3N [AdHIF]). Also noted in sections of the AdHIF-1 hearts is thickening of the semilunar valves with increased mesenchyme in the OFT and a muscular ventricular septal defect (VSD) (Figure 3J). The trabeculation of the ventricles appears normal, although there is variable thinning of the ventricular compact myocardium, likely secondary to the hemodynamic alterations. Thus, forced expression of HIF-1 blocks the normal shortening and rotation of the OFT that is required for the transition to a dual circulation.
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HIF-1 Activation of Hypoxia-Responsive Genes
To investigate which genes downstream of hypoxia/HIF-1 regulated cell survival or cell death in this context, transcript abundance was measured by real-time PCR in GFP+ and GFP– cells sorted by flow cytometry. Given the limiting supply of RNA with this approach, the analysis was restricted to transcripts that appeared to be differentially expressed in the developing heart and embryos (data not shown). In control hearts, the expression of VEGF, BMF, RTP801, and iNOS were increased in the GFP+ cells (cardiomyocytes) of the OFT as compared with: (1) the GFP– cells, which are predominately cushion mesenchymal cells; and (2) ventricular myocardium from the same embryos (Figure 4A). Forced expression of HIF-1 in the OFT cardiomyocytes caused a 2- to 4-fold increase in the abundance of the iNOS, RTP801, VEGF, and stromal cell–derived factor (SDF)-1 transcripts as compared with the GFP-only OFT cardiomyocytes, whereas the abundance of BMF was reduced (Figure 4B).
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Blockade of VEGF Induces Apoptosis in OFT Myocardium
We hypothesized that the ability of HIF-1 to promote OFT cardiomyocyte survival may be through VEGF signaling, based on the ability of VEGF to function as an autocrine survival factor in other contexts14,15,23 and the selective expression of the VEGF receptor 2 in the stage 25 to 32 OFT cardiomyocytes, concordant with tissue hypoxia.6 To test this hypothesis, we transduced the embryonic hearts with AdFlk1-Fc (AdFlk1) to trap VEGF and block VEGF signaling.18 Embryonic hearts transduced with AdFlk1 expressed the truncated Flk1 throughout the OFT myocardium (supplemental Figure II). There was increased LTR staining in the OFT myocardium of these hearts as compared with stage-matched controls (Figure 5). Eleven of 11 and 11 of 13 AdFlk1-injected hearts showed increased LTR staining at stages 25 and 30, respectively, in whole mount, as judged by 5-point scoring system (data not shown) and quantitative measurement. In sections, LTR staining was located in the OFT myocardium (Figure 6). No significant increase in LTR staining was observed in other chambers of the heart. The staining for active caspase-3 was also increased in the OFT myocardium of AdFlk1-injected hearts at these stages (Figure 6A through 6I). There was a corresponding increase in caspase-3 activity measured in OFT homogenates of AdFlk1-injected embryos as compared with controls (Figure 6J). Thus, blockade of VEGF signaling increases apoptosis in the OFT myocardium during OFT remodeling.
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Blockade of VEGF Causes OFT Defects
A separate group of embryos injected with AdFlk1 were examined at stages 34 to 35 for morphological assessment. Thirty-eight AdFlk1-injected embryos survived to this stage, and 28 had abnormal heart and OFT morphology. The OFT dysmorphology was more variable than that of the AdHIF-1 hearts, with all of the hearts showing combinations of defects in the OFT and right ventricle. A heart representative of 13 of 28 abnormal hearts is shown in whole mount in Figure 7B, and in section in Figure 7G through 7I. In these hearts, there is a diminutive or absent subpulmonic infundibulum, such that the pulmonic valve is situated at a level below the base of the ventricles. In this heart, as in all of the abnormal AdFlk1 hearts, there is a defect in the positioning of the aorta. It is transposed rightward, so that it originates from the right ventricle and overrides a more posterior located muscular VSD. The pulmonic and aortic valves are not markedly abnormal. A heart representative of 15 of 28 abnormal hearts is shown in whole mount in Figure 7C and in section in Figure 7J through 7L. In these hearts, a bulbous appearing infundibular stalk giving rise to both the aorta and pulmonary artery is evident above the base of the ventricles and is not incorporated into the right ventricle as occurs in normal heart development. In section, the infundibulum can be seen to be composed of myocardial tissue and large amounts of mesenchyme. This large amount of mesenchyme is in the position where the sculpted semilunar valves would normally reside. In these hearts, the right ventricles are markedly diminutive, and there is, again, a more posterior VSD with overriding aorta. All of the abnormal hearts have thinned myocardial walls with reduced trabeculation as evident in these representative embryos.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we used an adenoviral gene delivery system to test the effects of HIF-1
gain-of-function targeted to the OFT myocardium during apoptosis-dependent remodeling (stages 25 to 32). HIF-1 was appropriately expressed in the nuclei of the OFT cardiomyocytes and enhanced the transcription of HIF target genes, indicating that the gain-of-function was achieved. HIF gain-of-function reduced the prevalence of apoptosis as indicated by both LTR staining and caspase-3 activity. Thus, in the context of the developing OFT, hypoxia and HIF signaling promote the survival of the cardiomyocytes. This result is consistent with a study in which transgenic forced expression of HIF-1 in the adult mouse heart reduced myocardial injury in a model of ischemia.24 In contrast, in vitro studies have suggested that HIF-1 triggers cardiomyocyte apoptosis through death ligand or mitochondrial pathways.12,13 One important difference between in vitro and in vivo studies may be the strength of the stimulus. Incubation of stage 25 embryos in 5% to 7.5% oxygen results in the demise of the embryo within 3 to 6 hours. Even with in vitro studies, a second stimulus, eg, acidosis or reoxygenation, appears to be required for the severe hypoxia to trigger a BNip3 and mitochondrial-dependent apoptosis program.12,25 What triggers the cardiomyocyte apoptosis during the normal development of the OFT? One possibility is that tissue hypoxia conditions the myocardium such that a second stimulus later in development triggers apoptosis. What that second stimulus might be is uncertain. One suggestion is that invasion of the OFT by endothelial progenitor cells carrying FasL and their formation of the coronary arteries and coronary orifices is a trigger.26 Neural crest cells that migrate to the heart have a high prevalence of apoptosis27 are also candidates for triggering the apoptosis-dependent remodeling of the OFT.
HIF-1 may trigger a prosurvival pathway through the induction of VEGF, a well-described hypoxia-dependent signaling pathway. The increased expression of VEGF in the OFT cardiomyocytes, and its induction by AdHIF-1, suggested VEGF as a candidate prosurvival factor. Loss-of-function of VEGF signaling using AdFlk1-Fc significantly increased the prevalence of apoptosis in the OFT cardiomyocytes. These results suggest that HIF-1 induction of VEGF functions in autocrine prosurvival signaling in the developing OFT myocardium. Consistent with this interpretation, cotransduction of the embryos with AdHIF-1 and AdFlk1-Fc resulted in an intermediate level of LTR staining and morphological defects similar to that of AdFlk-Fc treatment (supplemental Figures III and IV). A role for receptor tyrosine kinase signaling in the survival of the OFT cardiomyocytes was supported by our previous study, in which gain-and-loss of function of Akt increased and decreased, respectively, the survival of the OFT cardiomyocytes.6 VEGF/Akt may exert prosurvival signals through inactivation of the proapoptotic Bcl-2 family member Bad, or through the suppression of the FOXO and p53-dependent induction of proapoptotic Bcl-2 family members Bim, Puma, and Noxa, and the death ligand FasL.28 Which of these or other gene targets of VEGF or Akt signaling are operative in this developmental context requires further study.
The morphological defects of the OFT observed with the perturbations of hypoxia-dependent signaling are consistent with the effects on apoptosis in the OFT myocardium. Forced expression of HIF-1 reduced apoptosis and resulted in a failure of resorption of the myocardium from under the aorta, in conjunction with a failure of rotation of the aorta to its posterior position. This double-outlet right ventricle morphology with an extended conal myocardium is similar to that observed with direct delivery of caspase inhibitors to the embryonic OFT myocardium.3 In these and other gain-and-loss of function experiments, there is variability in the phenotype. This could reflect interembryo variability in the efficiency of adenoviral gene delivery,19 and/or variability in the tissue response to these perturbations. In a few of the AdHIF-1 embryos, the aorta, rather than being side-by-side the pulmonary artery, was abnormally positioned directly posterior to the pulmonary artery, and again overriding a muscular VSD. We interpret this to represent a spectrum of rotational defects in aortic positioning, although other interpretations may also be plausible. This is not dissimilar to human congenital conotruncal heart defects, that although neatly categorized into double-outlet right ventricle, tetralogy of Fallot and transposition of the great arteries, in actuality, represent a spectrum of defects in the conal myocardium and the positioning of the great vessels with respect to the conus and ventricles.29
Cardiomyocytes are added to the OFT during its normal development from the anterior (or secondary) heart field30–32 before apoptosis-dependent remodeling. Abnormal addition of cardiomyocytes to the OFT may also cause conotruncal heart defects,33 and the addition of myocytes and subsequent remodeling may be linked by, eg, FGF8.34 Whether hypoxia signaling through HIF and VEGF also regulate these multiple aspects of OFT development and remodeling requires further study.
The variability in morphological defects in AdFlk1 embryos was greater than that of HIF-1. In some embryos, the myocardial infundibulum was diminutive, associated with defects in the posterior rotation of the aorta similar to that observed when apoptosis is directly triggered through the delivery of the death ligand.2 However, in other embryos, OFT myocardium appeared to be "hung-up," a protuberant stalk above the base of the ventricles, associated with a mass of mesenchyme, again with a defect in the position of the aorta. We speculate that temporally coordinated remodeling of the OFT myocardium and cushion mesenchyme may be required for the transition to a dual in-series circulation. Interference with either of these processes may contribute to the conotruncal heart defects with malposition of the aorta. Interestingly, these embryos also had a markedly diminutive right ventricle. Cell-marking and fate analyses have indicated that cardiomyocytes from anterior heart field contribute to the formation of both the OFT and right ventricle.35,36 We suggest that the OFT cardiomyocytes are hung up by the dense mesenchyme and cannot contribute to the right ventricle in these AdFlk1 embryos, leading to its small size. Alternatively, it is possible that VEGF, like FGF8,34 provides a direct growth stimulus to the right ventricular myocardium, which is abrogated by AdFlk1. In this and other models of loss-of-VEGF function thinned ventricular myocardium is noted, but it remains to be established if this is a direct or secondary effect.
A dramatic increase in OFT cushion mesenchyme was observed in the AdFlk1 embryos and, to a lesser extent, in AdHIF-1 embryos. Mesenchymal cell proliferation was modestly but significantly increased as assessed by 5-bromodeoxyuridine incorporation in the AdFlk1 embryos (data not shown). In vitro studies have shown that VEGF, depending on the dose, positively or negatively regulates endothelial-to-mesenchymal transformation.37,38 The increased mesenchyme could reflect a defect in later remodeling of the cushions to form the semilunar valves. It is well established that myocardial signaling to the endocardium through the release of TGF-β and other molecules affects these processes.38,39 Further study is indicated to define the cell and molecular basis of the cushion and valve defects with VEGF loss-of-function.
Experiments in mouse models also support a role of VEGF in OFT development. Exclusive expression of the VEGF120 (soluble) isoform resulted in a spectrum of conotruncal defects, some of which resembled Tetralogy of Fallot.40 Interestingly, increased apoptosis in the ED12.5 subpulmonic myocardium and hyperplasia of the OFT cushions were noted, quite similar to the effects of trapping VEGF in chick in the present study. The VEGF gene has been proposed as a disease modifying locus for Tbx1 in DiGeorge syndrome and OFT defects in human and animal models.41 In contrast, conditional inactivation of HIF-1 and VEGF in the embryonic mouse myocardium have not induced significant morphological defects42,43 (and data not shown). Loss-of-function studies of HIF family members in the diverse cell types of the OFT are required to further define its role in the complex morphogenesis of this structure.
In conclusion, the present targeted HIF-1 gain-of-function study suggests that hypoxia signaling though HIF-1 and VEGF exerts a prosurvival effect on the OFT myocardium. Interference with the hypoxia-dependent signaling alters the timing and prevalence of apoptosis in the OFT. Perturbations in the hypoxia-dependent signaling pathway may contribute to conotruncal heart defects in warm-blooded animals.
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Acknowledgments |
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The authors thank Dr. Gregg Semenza for generously providing AdHIF-1
and Drs. Kenneth Walsh and Calvin Kuo for providing AdFlk1-Fc. We thank Michael Payne for technical assistance and Dr. Michiko Watanabe for helpful discussions. Sources of Funding
This research was supported by NIH grant R01 HL65314.
Disclosures
None.
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Footnotes |
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Original received November 14, 2007; revision received April 14, 2008; accepted April 28, 2008.
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