Published online before print April 1, 2004, doi: 10.1161/01.RES.0000126848.54740.4A
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© 2004 American Heart Association, Inc.
Molecular Medicine |
Phosphoinositide-Dependent Kinase 1 and p21-Activated Protein Kinase Mediate Reactive Oxygen Species–Dependent Regulation of Platelet-Derived Growth Factor–Induced Smooth Muscle Cell Migration
From the Department of Medicine (D.S.W., Y.T., P.R., P.N.S., K.K.G.), Division of Cardiology, Emory University, Atlanta, Ga, and Cell and Molecular Biology Program and Department of Pharmacology (M.A.D., W.T.G.), School of Medicine, University of Nevada, Reno, Nev.
Correspondence to Kathy K. Griendling, Emory University, Division of Cardiology, 319 WMB, 1639 Pierce Dr, Atlanta, GA 30322. E-mail kgriend{at}emory.edu
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Abstract |
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Smooth muscle cell migration in response to platelet-derived growth factor (PDGF) is a key event in several vascular pathologies, including atherosclerosis and restenosis. PDGF increases intracellular levels of reactive oxygen species (ROS) in vascular smooth muscle cells (VSMCs), but the ROS sensitivity of migration and of the signaling pathways leading to migration are largely unknown. In VSMCs, PDGF dose-dependently increased migration compared with nonstimulated cells, with a maximum increase at 10 ng/mL. Pretreatment with the antioxidant N-acetyl-cysteine, the flavin-containing enzyme inhibitor diphenylene iodonium, or the glutathione peroxidase mimetic ebselen significantly attenuated migration (PDGF alone, 5.0±1.1-fold; NAC, 1.8±0.2-fold; diphenylene iodonium, 1.4±0.3-fold migration; and ebselen, 2.0±0.5-fold migration), as did overexpression of catalase. Pretreatment of VSMCs with the Src inhibitor PP1 or dominant-negative Rac adenovirus significantly inhibited migration, but only Src activation was attenuated by ROS inhibitors. Phosphorylation of the Src- and Rac-effector p21-activated protein kinase (PAK) 1 on Thr423 (the phosphoinositide-dependent kinase-1 [PDK1] site) was attenuated by ROS inhibition, and infection of VSMCs with dominant-negative PAK1 adenovirus attenuated migration. Moreover, kinase-inactive K111N-PDK1 inhibited PAK1 phosphorylation on Thr423, and both K111N-PDK1 and Y9F-PDK1 significantly inhibited VSMC migration. PDK1 tyrosine phosphorylation was also ROS dependent. These data indicate that PDGF-induced VSMC migration is ROS dependent and identify the Src/PDK1/PAK1 signaling pathway as an important ROS-sensitive mediator of migration. Such information is critical to understanding the role of ROS in vascular diseases in which migration of VSMCs is an important component.
Key Words: growth factors • migration • reactive oxygen species • signaling pathways • vascular smooth muscle cells
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Introduction |
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After artery damage, such as during atherosclerosis and restenosis, the vascular injury is repaired by cells derived from adjacent normal tissue. To repair the injury site, vascular smooth muscle cells (VSMCs) are stimulated to proliferate and migrate by an increase in the generation of several molecules, including basic fibroblast growth factor, platelet-derived growth factor (PDGF), transforming growth factor-ß, and angiotensin II.1 In vivo, PDGF is released by platelets at the injury site and contributes significantly to the formation of neointima after injury by facilitating the movement of VSMCs from the media across the elastic lamina to the intima.1,2 Experimentally, PDGF promotes migration of VSMCs and multiple other cell types as well.3
Several studies have reported an increase in reactive oxygen species (ROS) generation during restenosis after angioplasty4–6 and an impairment of neointimal formation by antioxidants,6 suggesting that ROS may play a central role in several stages of the restenotic process. In particular, it has been shown that PDGF itself stimulates ROS production in VSMCs.7–9 Several years ago, Sundaresan et al9 reported that PDGF-induced migration in VSMCs is inhibited by pretreatment with extracellular catalase, implicating ROS as a mediator of VSMC migration. Since this initial observation, minimal information has been provided as to which signaling mechanisms mediating PDGF-induced VSMC migration are ROS dependent.
Activation of the PDGF receptor by agonist binding results in the autophosphorylation of more than a dozen tyrosine sites on the receptor and the binding of multiple Src-homology (SH)-2 domain–containing signaling molecules to the receptor.3 The role of several of these molecules in mediating PDGF-induced cell chemotaxis has been established, but these mechanisms are not universal, varying significantly depending on cell type (see Rönnstrand and Heldin3 for review). Downstream effectors of PDGF-induced migration include mitogen-activated kinases (MAPKs), p21-activated protein kinases (PAKs), members of the Rho family of small GTPases, and several proteins localized to focal adhesions (ie, focal adhesion kinase and paxillin).10 Another potentially important signaling molecule is 3-phosphoinositide–dependent protein kinase-1 (PDK1). PDK1 is a serine/threonine kinase that was previously reported to mediate actin cytoskeletal reorganization and membrane ruffling in CHO cells after insulin stimulation11 and is activated by the oxidants H2O2 and peroxovanadate.12,13 Although PDK1 activity is regulated mainly by serine phosphorylation,14 our group has recently demonstrated that phosphorylation of PDK1 on Tyr9 is required for angiotensin II–stimulated focal adhesion formation.15 Because cell motility during migration is regulated by a series of dynamic cytoskeletal changes and turnover of focal adhesions, these findings suggest that PDK1 may be a crucial regulator of cell migration.
One potential downstream effector of PDK1 is PAK1.16 PAKs are a family of serine/threonine kinases that are effectors of the small GTPases. PAK activity can also be increased by non–GTPase-dependent mechanisms, including adaptor proteins binding to SH-3 domains, activation by lipids such as sphingosine, PDK1 phosphorylation of PAK1 at Thr423, and activation of heterotrimeric G proteins.16,17 PAK1 mediates cell migration via its effects on the cytoskeleton, which have been reported to be largely independent of the Rho GTPases.18 In response to PDGF, PAK1 redistributes from the cytosol to cortical actin structures,19 and phosphorylated PAK1 has been localized to focal adhesions.20 Although expression of kinase-inactive PAK1 adenovirus has been shown to attenuate PDGF-induced migration of tracheal smooth muscle cells,21 the influence of ROS on PAK activation has not been established.
The goal of the present study was to identify signaling mechanisms involved in PDGF-induced VSMC migration that are ROS dependent. We found that Src, PDK1, and PAK1 are ROS-dependent signaling molecules that are crucial for PDGF-induced migration and established a sequential relationship between their activation. Rac is also an important mediator of VSMC migration, but its activation is ROS independent. Thus, these studies identify novel, ROS-sensitive signaling mechanisms that regulate VSMC migration, a critical component of vascular pathologies, such as those that occur during atherosclerosis or restenosis after balloon injury.
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Materials
Recombinant human PDGF-BB was purchased from R&D Systems, Inc (Minneapolis, Minn). 4,6-Diamidino-2-phenylindole (DAPI) and N-acetylcysteine (NAC) were from Sigma. Diphenylene iodonium (DPI), ebselen, and wortmannin were purchased from Alexis Biochemicals. PP1 was purchased from Bio-Mol. LY29004 was from Calbiochem. Anti-phospho PAK1 (Ser199/204), anti-phospho PAK1 (Thr423), anti-phospho extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204), and anti-PAK1 antibodies were from Cell Signaling Technology (Beverly, Mass). Anti-PDK1 antibody and Rac activation assay kit were from Upstate Biotechnology (Lake Placid, NY). Anti-PKB/PDK1 antibody was from Transduction Laboratories (Lexington, Ky). Anti-Src antibody was from Santa Cruz Biotechnology (Santa Cruz, Calif). PAK and PDK1 adenoviruses were prepared as described previously.15,21
Cell Culture
VSMCs were isolated from rat thoracic aorta by enzymatic digestion as described previously.22 Cells were grown in DMEM supplemented with 10% calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. For VSMCs stably transfected with a vector overexpressing catalase or with an empty vector control,23 geneticin (400 µg/mL) was added to the media. In these cells, agonist-induced H2O2 formation is completely abolished.23
VSMC Migration Assay
Migration was assayed by a modified Boyden chamber method adapted from Brown et al.24 VSMCs were grown to 
85% confluence and then made quiescent in 0.1% calf serum for 24 to 48 hours before migration. VSMCs (106 cells/mL) were added to the upper chamber of the transwell on a collagen-coated polycarbonate membrane containing 8-µm pores (Costar). When pharmacological inhibitors were used, VSMCs were allowed 30 minutes to attach to the membrane before addition of the inhibitors. VSMCs were then exposed to PDGF in the lower chamber for 4 hours, after which nonmigrated cells were removed from the upper chamber using a cotton swab. The cells remaining on the inserts were fluorescently stained with DAPI (1 µg/mL)25 and visualized using a Zeiss Axioskop microscope. Four (x200) images of separate locations were taken of each insert and quantified using Zeiss KS300 software.
Immunoprecipitation and Western Blotting
VSMCs at 80% to 90% confluence were made quiescent by incubation with DMEM containing 0.1% calf serum for 24 to 48 hours. Cells were stimulated with PDGF at 37°C in serum-free DMEM for specified durations. After treatment, cells were washed and lysed with ice-cold lysis buffer as described previously.15,26,27 Western analysis and immunoprecipitation were performed as described previously.15
Statistical Analysis
Results are expressed as mean±SEM. Statistical significance, assessed by 1-way ANOVA with Newman-Keuls multiple-comparison post test, was performed using GraphPad Prism software. A value of P<0.05 was considered to be statistically significant.
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Results |
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PDGF-Induced VSMC Migration Is ROS Dependent
We and others have reported previously that PDGF stimulates ROS production in VSMCs.7–9 Our initial goal was to confirm that VSMC migration in response to PDGF is dependent on ROS generation. PDGF induced a dose-dependent increase in migration with a maximal 5-fold increase at 10 ng/mL (Figure 1A). For all subsequent migration studies, cells were stimulated with 10 ng/mL PDGF. We then tested the effect of a panel of inhibitors known to decrease intracellular ROS levels in these cells,26–28 including NAC, which increases reduced glutathione, DPI, an inhibitor of flavin containing oxidases, and ebselen, a glutathione peroxidase mimetic. PDGF-induced VSMC migration was significantly attenuated after each of these treatments, indicating that ROS are important mediators of PDGF-induced migration (Figure 1B). To additionally test ROS sensitivity, we examined PDGF-induced migration in VSMCs stably transfected to overexpress catalase. Compared with cells transfected with empty vector, migration was decreased by >60% in catalase-overexpressing cells, implicating H2O2 as an important mediator of migration (Figure 1C).
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Significant decrease in migration vs control cells stimulated with PDGF, P<0.01. C, Migration in response to PDGF (10 ng/mL) was measured in VSMCs stably transfected to overexpress catalase and in vector-transfected control cells. Values are mean±SEM of 5 independent observations. *Significant increase in migration versus nonstimulated cells, P<0.001.
Potential Upstream Mediators of PDGF-Induced VSMC Migration
Activation of the PDGF receptor (PDGF-R) results in autophosphorylation of the receptor, facilitating the interaction of the receptor with multiple signal transduction molecules. In the present studies, the goal was to identify targets that satisfy two criteria: (1) they are important in mediating VSMC migration, and (2) their activation is ROS dependent. Initially, we tested whether the activation of PDGF-R itself is ROS dependent. Using immunoprecipitation, we examined both the total tyrosine phosphorylation of the PDGF-R as well as site-specific phosphorylation at Tyr716 (an indicator of intrinsic receptor tyrosine kinase activity) and found that activation of the PDGF-R was not attenuated by inhibitors of ROS formation (data not shown). Both Src and phosphatidylinositol 3 (PI3)-kinase are potential mediators of VSMC migration.29 To test the role of Src in modulating VSMC migration, cells were preincubated with the Src kinase inhibitor PP1. PDGF-induced migration was completely inhibited after PP1 pretreatment (Figure 2A). Moreover, infection with kinase-inactive Src adenovirus significantly inhibited migration (data not shown). In contrast, inhibition of PI3-kinase with either wortmannin (1 µmol/L) or LY294002 (20 µmol/L) did not inhibit PDGF-induced VSMC migration in our system (data not shown), although these inhibitors did block Akt activation.30 To determine if the regulation of Src in response to PDGF is ROS dependent, VSMCs were pretreated with inhibitors of ROS formation before PDGF stimulation. The 3-fold increase in Src tyrosine phosphorylation after stimulation with PDGF was nearly eliminated after preincubation with NAC, DPI, or ebselen, indicating that this phosphorylation is ROS dependent (Figure 2B). As expected, PP1 completely abolished Src activation.
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PDGF has been shown to activate Rac-GEF, which in turn activates Rac and increases ROS.7 Because Rac plays an important role in ROS production and focal adhesion turnover, we also tested the role of Rac in PDGF-induced migration. The infection of VSMCs with dominant-negative Rac adenovirus significantly inhibited PDGF-induced migration (Figure 3A). As expected, Rac activation is apparently upstream of ROS production, because activation of Rac, as measured using a PAK pull-down assay, was unaffected by pretreatment with NAC (Figure 3B).
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Significant decrease in migration vs AdLacZ-infected cells stimulated with PDGF, P<0.05. B, After preincubation with NAC (100 µmol/L for 1 hour), VSMCs were stimulated with PDGF (10 ng/mL) for 5 minutes, and Rac activity was measured by PAK-PBD affinity precipitation followed by immunoblotting with an antibody recognizing Rac. Top, Representative immunoblot; bottom, mean±SEM of 6 to 7 independent observations. *Significant increase in Rac activation compared with nonstimulated cells, P<0.05.
Identification of Downstream Mediators of ROS-Dependent PDGF-Induced VSMC Migration
Several potentially important mediators of VSMC migration have been identified, including ERK1/29 and PAK.21 Although it has previously been reported that ERK1/2 phosphorylation is dependent on H2O2,9,31 in our system the site-specific phosphorylation of ERK1/2 at Thr202/Tyr204 in response to PDGF was ROS independent (data not shown). We next tested the role and ROS sensitivity of PAK1 in mediating VSMC migration using DN-PAK1 (AdPAK1K299R) adenovirus. This adenovirus has been reported previously to inhibit PDGF-induced p38 MAPK phosphorylation.21 In the present study, overexpression of DN-PAK1 significantly attenuated PDGF-induced migration compared with both noninfected cells and cells infected with adenovirus encoding GFP only (Figure 4A).
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To determine whether PAK1 is downstream of Src and if its activation is ROS dependent, VSMCs were pretreated with either PP1 or inhibitors of ROS formation, and Western blots using phosphospecific antibodies were performed to determine which phosphorylation sites on PAK1 (Ser199/204 or Thr423) are ROS dependent. PDGF induced an increase in phosphorylation at Ser199/204, known autophosphorylation sites of PAK1. Activation of these sites was not ROS dependent (Figure 4B). In contrast, PDGF-induced phosphorylation of Thr423 on PAK1 was attenuated after inhibition of ROS formation and Src inhibition (Figure 4C). These findings indicate that the ROS-dependent phosphorylation of PAK1 is site specific. The ROS sensitivity of the Thr423 site of PAK1 is of particular interest because phosphorylation at this site is associated with increased PAK activity, and this site has recently been shown to be phosphorylated by PDK1.16
PDK1 Phosphorylates Thr423 of PAK1
To determine if PDK1 is upstream of PAK1 in our system, VSMCs were infected with AdGFP or an adenovirus encoding a kinase-inactive form of PDK1, AdPDK1K111N,32 and cell lysates were immunoblotted for phospho-PAK1 at site Thr423. Infection with AdPDK1K111N significantly attenuated PDGF-induced phosphorylation of PAK1 at Thr423 (Figure 5), suggesting that this site on PAK1 is regulated by PDK1 and may be an important ROS-dependent downstream regulator of PDGF-induced VMSC migration (Figure 4).
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Role of PDK1 in ROS-Mediated VSMC Migration
In addition to being the kinase responsible for the phosphorylation of Thr423 of PAK1, PDK1 has been reported to be important for cytoskeletal reorganization, a key step in cell chemotaxis.16 Recent work from our group indicates that the tyrosine phosphorylation of PDK1 (Tyr9) is crucial for focal adhesion assembly in response to angiotensin II.15 Thus, to test the role of PDK1 in PDGF-induced VSMC migration, cells were infected with AdPDK1K111N, adenovirus encoding PDK1 mutated at tyrosine 9 (AdPDK1Y9), or a green fluorescent protein lacking an insert (AdGFP). The effectiveness of these PDK1 adenoviruses was confirmed by the decrease in angiotensin II–dependent phosphorylation of Akt in VSMCs after infection (unpublished observations, 2003). In this study, both AdPDK1K111N and AdPDK1Y9 infection significantly attenuated VSMC migration compared with noninfected cells and AdGFP-infected cells (Figure 6A). To determine whether PDK1 phosphorylation is ROS sensitive and to define where in the signaling pathway it is activated, we assessed the effect of antioxidants and PP1 on PDGF-induced tyrosine phosphorylation. PDGF stimulated a 2- to 2.5-fold increase in total tyrosine phosphorylation of PDK1 (Figure 6B). This phosphorylation was completely inhibited by pretreatment with inhibitors of ROS formation or Src activation. These findings indicate that PDK1 tyrosine phosphorylation is both ROS and Src dependent, implicating PDK1 as an important ROS-sensitive target in migratory signaling pathways.
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Discussion |
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In the present study, we identify a novel ROS-dependent signaling pathway that plays a crucial role in PDGF-stimulated VSMC migration (Figure 7). Although PDGF activates many signaling pathways leading to migration, our data indicate for the first time that Src- and ROS-dependent activation of PDK1 leads to site-specific phosphorylation of PAK1, which is critically important for migration. These observations add substantially to our knowledge of how PDGF causes VSMC migration, a process integral to the vascular response to injury that leads to vessel occlusion and plaque formation.
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Using both pharmacological inhibitors as well as stably transfected VSMCs overexpressing catalase, we showed unequivocally that VSMC migration in response to PDGF is mediated by ROS (Figure 1). In response to ligand binding of PDGF to its receptor, the receptors dimerize, resulting in an increase in kinase activity, autophosphorylation, and activation of downstream effectors. Numerous phosphorylation sites on the receptor have been identified, along with their respective downstream signaling molecules. The most proximal potential target of ROS is the receptor itself. One would predict that if ROS alter PDGF-R phosphorylation, all additional signaling events and migration itself should be completely abrogated by antioxidants. This is clearly not the case. Neither total tyrosine phosphorylation of the PDGF-R nor site-specific phosphorylation at Tyr716 (an indicator of intrinsic receptor tyrosine kinase activity) was affected by inhibition of ROS. Furthermore, antioxidants only partially inhibit migration, and certain pathways known to be involved in migration (ERK1/2 and Rac) are not ROS dependent based on the present data, although the ROS dependence of ERK1/2 is controversial.9,33 These observations indicate that migration involves both ROS-sensitive and ROS-insensitive signaling pathways and that ROS sensitivity is conferred downstream of the receptor.
Although there are several effectors that interact with the PDGF-R and ultimately induce cell chemotaxis, these are highly variable depending on cell type.3 In the present studies, we tested the role of both Src and PI3-kinase in mediating VSMC migration. We were able to attenuate VSMC migration using both pharmacological and adenovirus strategies targeting Src (Figure 2). In contrast, inhibition of PI3-kinase with either wortmannin or LY29004, which we have previously shown to inhibit PI3-kinase–dependent signal transduction,30 did not inhibit VSMC migration in response to PDGF. PI3-kinase as a mediator of VSMC migration remains controversial, because several reports have implicated it in VSMC migration,29 whereas this study and others report that PI3-kinase inhibition does not attenuate VSMC migration.34 Src has been less investigated as an effector of migration, but our findings are consistent with those of Yamboliev et al,29 who reported that PP1 inhibits PDGF-induced pulmonary artery smooth muscle cell spreading and migration.
Because Src is a proximal tyrosine kinase, it is likely that Src is responsible for the integration of multiple downstream signaling mechanisms during PDGF-induced VSMC migration. In the present study, we found that Src is proximal to both PDK1 and PAK1, because the activation of both of these kinases in response to PDGF is significantly inhibited by PP1. We also report that Src tyrosine phosphorylation is ROS dependent after PDGF stimulation. This finding is consistent with recent studies in both VSMCs35 and other systems,36,37 in which Src activation was found to be ROS-dependent in response to angiotensin II stimulation. Because the PDGF-R receptor itself is not ROS sensitive in VSMCs, Src seems likely to be the most proximal ROS-dependent signaling event mediating VSMC migration.
Studies with other agonists have shown that Src can mediate activation of PAK1 and PDK1,12,13,38,39 and this also seemed to be the case in the present study, because PP1 completely inhibited phosphorylation of both kinases (Figures 4 and 6
). PAKs are serine/threonine kinases that have been implicated in the regulation of cytoskeletal dynamics and localized to focal adhesions. Recently it has been reported that overexpression of kinase-inactive PAK1 inhibits migration of tracheal smooth muscle cells.21 In the present study, this adenovirus exerted a similar effect, thus implicating PAK1 as a key mediator of VSMC migration in response to PDGF. When ROS formation was inhibited before PDGF stimulation, we observed differential regulation of PAK1 phosphorylation with regard to ROS. Although there was no effect of ROS inhibition on the autophosphorylation site (Ser199), PAK1 phosphorylation at Thr423 was ROS dependent. These sites, although both phosphorylated in response to PDGF, have significantly different roles in PAK activity and suggest that multiple mechanisms may be influencing the role of PAK1 in migration. PAK1 activation is known to be regulated by the binding of GTPases to the amino-terminal regulatory domain and the subsequent autophosphorylation of multiple sites in the autoinhibitory region to maintain kinase activity (ie, Ser199). Thr423 is known to be phosphorylated by PDK1 in vitro and is associated with increased kinase activity of PAK.18 Our finding that Rac mediates VSMC migration, but that its activation (as determined by its association with the PAK binding domain) is unaffected by ROS inhibition, may explain the site specificity of PAK phosphorylation by ROS. It is known that Rac mediates agonist-stimulated NAD(P)H oxidase activity7,27 and that it associates with PAK.17,18 Rac seems to be upstream of ROS production and to interact with PAK1 via ROS-insensitive signaling mechanisms, suggesting that it acts as a point of divergence of ROS-sensitive and ROS-insensitive pathways (Figure 7).
Clearly, a portion of the PAK1 activity in response to PDGF stimulation is mediated by PDK1. It has been previously reported that Thr423-phosphorylated PAK1 can be localized to focal adhesions and that this site is phosphorylated in fibroblasts in response to cell wounding.20 Recently our group localized PDK1 to focal adhesion complexes in VSMCs and demonstrated that angiotensin II–induced formation of focal adhesions is inhibited by mutation of Tyr9 on PDK1.15 Migration is a dynamic process involving the turnover of focal adhesions, and thus the subcellular location of chemotactic mediators is crucial.
In the present studies, we demonstrate for the first time that PDK1 is a critical mediator of PDGF-induced VSMC migration by using adenoviruses overexpressing either kinase-inactive PDK1 or PDK1 mutated at Tyr9. Both of these adenoviruses significantly attenuated VSMC migration (Figure 6). The mechanism of this inhibition seems to be attributable to a compromised kinase activity of PDK1 resulting in decreased phosphorylation of PAK1. This is supported by our finding that the kinase-inactive PDK1 prevented PAK1 phosphorylation at Thr423 and clearly places PDK1 upstream of PAK1 in our model (Figure 7). An alternative explanation of the PDK1 adenovirus inhibition of migration could be that mutated PDK1 was unable to properly translocate to focal adhesion complexes to localize with PAK1. The fact that tyrosine phosphorylation of PDK1 is ROS dependent is consistent with the ROS sensitivity of both Src and PAK Thr423 phosphorylation, because the present experiments have placed these kinases proximal and distal to PDK1, respectively.
In summary, we have demonstrated that PDGF-induced VSMC migration is dependent on the production of ROS and that Src, PDK1, and PAK1 are important mediators of ROS-dependent migration. Thus, a picture emerges of ROS-dependent VSMC migration in which the activated PDGF-R stimulates ROS production, probably via Rac-dependent NAD(P)H oxidase activation, which then stimulates Src kinase activity. Src phosphorylates PDK1, which subsequently stimulates PAK1 kinase activity by phosphorylating Thr423 located in its catalytic domain. PAK1 is a known effector for cytoskeletal regulation and is a crucial mediator of the dynamic events mediating VSMC migration. The present study provides a framework for understanding the integration of migration-related signaling by ROS and provides new targets for therapeutic intervention with antioxidant treatment.
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Acknowledgments |
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This work was supported by NIH grants HL58000 and HL58863 and an American Heart Association Scientist Development Award to D.S.W.
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Footnotes |
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This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received October 25, 2003; revision received March 17, 2004; accepted March 17, 2004.
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