Published online before print June 26, 2008, doi: 10.1161/CIRCRESAHA.108.176354
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© 2008 American Heart Association, Inc.
Cellular Biology |
Localized 
4 Integrin Phosphorylation Directs Shear Stress–Induced Endothelial Cell Alignment
From the Divisions of Rheumatology and Hematology-Oncology, Department of Medicine (L.E.G., R.S., K.K., E.T., M.H.G.), the Department of Physics (E.G., A.G.), and the Department of Bioengineering and The Whitaker Institute of Biomedical Engineering (P.N., S.C.), University of California, San Diego; the Department of Cell and Molecular Physiology (E.T.), Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill; and the Core Microscopy Facility (W.B.K.), Scripps Research Institute, La Jolla, Calif.
Correspondence to Lawrence E. Goldfinger, Sol Sherry Thrombosis Research Center and Department of Anatomy & Cell Biology, Temple University School of Medicine, 3400 N. Broad Street, OMS 415, Philadelphia, PA 19140. E-mail goldfinger{at}temple.edu
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Abstract |
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Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to atheroprotection. Here we report that localized
4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress–induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial 4 integrins became phosphorylated on Ser988. In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced 4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a nonphosphorylatable 4(S988A) mutation failed to align in response to shear stress, thus establishing 4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent 4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress.
Key Words: integrin • PKA • endothelial • Rac GTPase • alignment
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exposure of endothelial cells to laminar shear stress elicits directional cellular behaviors. The cells assume a polarized elongated shape parallel to the direction of flow.1 When a wound perpendicular to the direction of flow is introduced into an endothelial cell monolayer, the cells at the upstream margin of the wound migrate approximately 1.5 times faster into the wound than those on the downstream margin.2,3 The aligned morphology associated with laminar flow correlates with responses associated with protection from atherosclerosis.4 Thus, the capacity of endothelial cells to respond to laminar flow, to align along the flow, and to migrate plays a role in vascular physiology.
In vitro studies have analyzed biochemical signaling events associated with flow-induced endothelial cell alignment. In response to the onset of flow a mechanosensory complex comprising VE-Cadherin, VEGFR2, and PECAM-1 leads to PI3-kinase activation, resulting in activation of integrin adhesion receptors. The activated integrins then form new attachments to the subendothelial extracellular matrix.5 These adhesive events result in precise temporal modulation of Rho GTPase activity, which leads to disassembly and reassembly of actin fibers. In particular, localized activation of Rac1 GTPase at the downstream cell edges is required for the alignment of the actin fibers parallel to the direction of flow.6 The mechanisms that control this localized Rac1 activation are obscure.
The new integrin-mediated adhesions formed in response to shear stress contribute to Rac1 GTPase activation.7 In migrating cells, 4 integrins induce highly localized Rac1 activation.8–11 4 integrins bind to paxillin at the trailing edge of migrating cells leading to suppression of adhesion-mediated Rac1 activation. Phosphorylation of the cytoplasmic tail of the 4 integrin subunit by protein kinase A (PKA) at Ser988 is localized to the leading edge of migrating cells where it blocks paxillin binding thus permitting efficient and highly localized Rac1 activation.7,12,13 Because 4 integrins are expressed in endothelial cells,14 we suspected that 4 integrin phosphorylation and its effects on localization of Rac1 activity may contribute to endothelial cell responses to shear stress.15–17 Here we show that PKA-mediated 4 integrin phosphorylation is induced by shear stress at the downstream edge of endothelial cells. This spatially restricted 4 phosphorylation is required for localized activation of Rac1 and for endothelial cell alignment in response to shear. Thus, localized 4 integrin phosphorylation informs the endothelial cell about the direction of blood flow, thereby acting as a "weather vane" of shear stress–induced endothelial cell alignment.
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Materials and Methods |
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Complementary DNAs and Other Reagents
Glutathione-S-transferase fusion of the CS-1 fragment of human fibronectin was generated as described.18 Collagen,
5, and 2 integrin antibodies (BD Biosciences), HP2/1 4 integrin antibodies (Immunotech), and rat -mouse CD31 antibodies (Invitrogen) were purchased. PS4 monoclonal antibodies to Ser988-phospho-4 integrin were as described.19
Generation of 4 Integrin-Null and S988A Knock-In Mice
C57/Bl6 mice harboring an 4 locus flanked by loxP sites were as described.20 Generation of 4(S988A) mice is described in the online supplement (available online at http://circres.ahajournals.org).
Primary Endothelial Cell Isolation and Cell Culture
Jurkat T leukemia cells and human microvascular endothelial cells were maintained as described.13,21 Primary pulmonary microvascular endothelial cells were isolated from 4(S988A) and 4 (fl/fl) mice as described21 and in the online supplement.
Shear Stress Assays, Immunocytochemistry, and Fluorescence Resonance Energy Transfer
Described in the online supplement.
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Results |
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Shear Stress Induces Phosphorylation of
4 Integrins at the Downstream Edge of Endothelial CellsTo assess a potential role for
4 integrin phosphorylation in endothelial cell alignment in response to shear stress, we first verified the expression of 4 integrins in immortalized human microvascular endothelial cells (HMECs) by fluorescence-activated cell scanning analysis (FACScan). HMECs expressed moderate levels of 4 integrin (
20% as compared to Jurkat T cells; supplemental Figure I). To investigate the role for 4 integrin phosphorylation in endothelial cell responses to shear stress, we plated HMECs onto coverslips coated with the 4-binding CS-1 fragment of fibronectin and subjected the monolayers to a laminar shear stress at 12 dynes/cm2 for time intervals ranging from 5 to 30 minutes. After the shear exposure, cells were fixed immediately and stained with PS4, a monoclonal antibody specific for 4 integrin phosphorylated at Ser988.12 A dramatic increase in phosphorylation of 4 in endothelial cells was observed after all times of exposure to shear, starting from the shortest tested time of 5 minutes (Figure 1). We observed that 4 phosphorylation in response to shear was localized to cell boundaries, in particular at cell edges orthogonal to the direction of flow (Figure 1A.) Labeling with secondary antibody conjugates alone showed undetectable staining (our unpublished results). Antibodies to 4 integrin uniformly labeled the whole cell periphery (Figure 1C), indicating that the increased concentration of phospho-4 at the cell edges was attributable to increased 4 phosphorylation rather than a higher local concentration of 4. Furthermore, we detected a 1.4-fold increase in total cellular 4 phosphorylation after 5 minutes shear stress by Western blotting of 4 immunoprecipitates (Figure 1B), confirming that shear stress upregulates 4 phosphorylation.
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The foregoing experiments showed that shear induced 4 phosphorylation at the cell edges perpendicular to the flow direction, but failed to establish whether it was occurring preferentially on the proximal or distal side. To determine precisely the relationship of shear-induced 4 phosphorylation to flow direction, we generated scratch wounds orthogonal to the flow direction in confluent monolayers of HMECs, immediately subjected the wounded monolayers to shear stress, and stained for phospho-4. Cells at the wound margins on both sides of the scratch wound were scored for phospho-4 staining. In the absence of shear, 4 phosphorylation was observed in 30% of cells at the wound margin. Application of shear induced increased 4 phosphorylation at the downstream cell edges at the wound margin proximal to the flow source but not on the upstream side of cells distal to the flow source. The 4 phosphorylation was observed in 65% (±2.9, P<0.22) of cells at the proximal wound margin beginning 5 minutes after application of shear and persisting for at least 30 minutes (Figure 1D). In contrast, 4 phosphorylation at the upstream edges of cells at the distal margin of the wound showed a trend toward inhibited phosphorylation (13% positive ±0.86, P<0.08, Figure 1D). Similarly, phospho-4 was rarely observed at cell edges lateral with respect to flow direction (16% positive, unpublished results), indicating that flow increases 4 phosphorylation at the downstream edges in endothelial cells.
Protein Kinase A Activity Is Required for Shear-Induced 4 Phosphorylation
PKA phosphorylates 4 integrins at Ser988 and inhibition of PKA activity blocks 4 phosphorylation in fibroblasts and T cells.12,19 To assess the contribution of PKA to shear-induced 4 phosphorylation in endothelial cells, we pretreated cells plated on CS-1 with the PKA inhibitor, H-89, for 15 minutes, scratch-wounded and applied flow in medium containing the inhibitor. Cultures were fixed and stained for phospho-4. Five-minute exposure to shear induced 4 integrin phosphorylation at the downstream cell edges, and addition of H-89 to the flow medium abrogated the 4 phosphorylation response (Figure 2). At the upstream and lateral cell edges facing the interior of a wound, where 4 phosphorylation was low, treatment with H-89 had no apparent effect on the levels of phospho-4 staining. As additional confirmation of this effect of PKA inhibition, we repeated these experiments using a second pharmacological PKA inhibitor, KT-5720 at 1 µmol/L; it also blocked shear-induced 4 phosphorylation (result not shown). Thus, shear-induced 4 phosphorylation requires PKA activity.
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Localized Rac1 Activation After Shear Stress Requires PKA Activity
PKA-dependent 4 integrin phosphorylation helps to localize Rac1 activation to the leading edge of migrating cells.7,12 Furthermore, Rac1 becomes activated at the downstream edge in a subconfluent endothelial monolayer within five minutes of exposure to laminar shear stress.6 This transient localized Rac1 activation is required for subsequent stress fiber alignment. Using a FRET-based assay, we found that shear stress induced a polarized increase of activated GTP-bound Rac1 at the downstream edge of HMECs as previously reported.6 This polarized Rac activation occurred concurrently with shear-mediated 4 integrin phosphorylation (Figures 3 and 1
D). Treatment with the PKA inhibitor (H-89) that blocked shear-induced 4 phosphorylation inhibited the localized Rac1 activation, indicating that PKA activity is required for the shear induction of polarized Rac1 activation at the downstream edge of endothelial cells (Figure 3).
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Shear-Induced Cell Alignment of the Actin Cytoskeleton Requires PKA Activity
Endothelial cells respond to prolonged exposure to shear stress by remodeling their actin cytoskeleton along a dominant longitudinal cell axis, a phenomenon referred to as endothelial cell alignment.22 This response requires dynamic spatio-temporal regulation of Rac1 activation, as expression of either constitutively active or dominant negative Rac1 blocks the alignment.6 Because PKA activity was required for polarized Rac1 activation, we assessed whether PKA activity is necessary for alignment. HMECs were subjected to shear, and actin filaments were labeled with rhodamine-phalloidin. Cells subjected to prolonged (20 hour) laminar shear stress at 12 dynes/cm2 developed an elongated bipolar shape with actin stress fibers aligned in the direction of flow. However, alignment and elongation did not occur if H-89 was added to the flow medium (Figure 4A). To quantify this morphological observation, we assessed stress fiber alignment by measuring the angles of actin filament bundles relative to the direction of flow.
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The average angles from the flow direction of actin filaments in control and H-89–treated cells under static conditions were 42±1.6 (17.15°=S.D.) and 41±1.1° (11.58°=S.D.), respectively, both very close to the value of 45° expected for randomly oriented fibers. Thus, actin fibers in static cells were not aligned. Actin fibers in control cells subjected to shear aligned to an average angle of 14.3±0.69° (9.23°=S.D.) from the flow direction, indicating a major bias in the alignment of actin filaments toward the flow direction. Furthermore, the reduction in standard deviation (17.15
9.23) indicates a marked reduction in the variability of the orientation of the actin fibers, providing an independent measure of the alignment response. In contrast, actin filaments in sheared H-89–treated cells aligned to a much lesser extent with an average radial displacement of 38±1° (10.61°=S.D.) (Figure 4B and supplemental Table I). Similar to H-89, addition of KT-5720 also dramatically reduced the alignment (supplemental Table I). Thus, PKA activity is required for actin stress fiber reorientation and morphological alignment of endothelial cells in response to shear stress.
4 Integrin Is Required for Shear-Induced Alignment
The experiments reported above showed that PKA activation was required for localized 4 integrin phosphorylation, for localized GTP loading of Rac1, and for cytoskeletal alignment in response to shear stress. These results suggested that 4 integrin phosphorylation may provide cues for endothelial cell reorientation and stress fiber alignment. Integrin ligation by the subendothelial matrix is necessary for shear-induced Rac1 activation,6 and thus presumably for alignment. Therefore we hypothesized that blocking 4 integrin binding to ligands in the extracellular matrix would inhibit stress fiber alignment induced by shear. To test this hypothesis, we subjected confluent HMEC monolayers cultured on slides coated with fibronectin (a ligand for 4 and 5 integrins23) to shear stress for 20 hours in medium containing either control IgG or function-blocking antibodies to 4, 5, or 2 integrin subunits. In all cases the cells remained attached to the substratum in monolayers. After shear exposure, cellular alignment was observed in the presence of control IgG or antibodies to 2 or 5 integrin subunits, with the mean stress fiber angles relative to the flow direction of 20.8±0.42° (2.95°=S.D.), 23±1.6° (11.46°=S.D.), and 26±1.6° (13.84°=S.D.), respectively (Figure 5). However, blocking antibodies to 4 integrins inhibited endothelial elongation and stress fiber reorientation in the flow direction, with the mean angle±SEM of 48±2.4° (16.68°=S.D.; Figure 5). In separate control experiments, these antibodies blocked the ability of endothelial cells to adhere to the relevant substrates CS-1 (anti-4), the cell-binding domain of fibronectin (anti-5), or collagen (anti-2; supplemental Figure II). These data indicate that 4 integrin interaction with the subendothelial matrix is required for shear-induced cytoskeletal alignment in microvascular endothelial cells.
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To verify separately that 4 integrins are required for shear-induced alignment, we generated endothelial cells deficient in 4 integrin expression and compared the alignment responses of these cells with those of 4-expressing endothelial cells. To do this, we isolated pulmonary endothelial cells (MLECs) from the lungs of wild-type mice and mice with a conditional 4-null allele in which exon 28 (which includes the 4 polyadenylation signal) is flanked by loxP recombination sites.20 CD31-positive cell cultures were then infected with adenovirus encoding Cre recombinase, leading to loss of surface 4 in the mutant cells as determined by FACS (supplemental Figure III); however, these cells adhered and spread on fibronectin-coated surfaces (Figure 5B), presumably via other fibronectin-binding integrins. Wild-type and 4-null MLECs were plated onto fibronectin-coated slides and subjected to laminar shear stress for 20 hours. Wild-type MLECs either infected with Cre or with empty adenovirus aligned in response to shear stress (Figure 5B). In contrast, the 4-null (4 fl/fl CRE) endothelial cells maintained a polygonal morphology and did not align in the flow direction. These cells also displayed few discernable stress fibers, suggesting a disruption in the ability of actin to reorganize in 4-null cells in response to shear. Thus, 4 integrin contributes to shear stress-induced morphological alignment in primary pulmonary endothelial cells.
4 Integrin Phosphorylation Is Necessary for Alignment
As shown above, PKA activity is required for 4 phosphorylation in response to shear in wounded monolayers and for cell alignment in prolonged shear. Therefore, we considered that 4 phosphorylation may be an early event in establishing the directionality of shear stress-induced cytoskeletal alignment. To test this possibility, we isolated endothelial cells from the lungs of mice expressing 4 integrin that harbors a Ser988Ala mutation (S988A), which disrupts the PKA phosphorylation site in 4.
The antiphospho-4 PS4 antibody did not react in Western blots of lysates from endothelial cells derived from these mice, nor did these cells display any edge PS4 staining in sheared, wounded cultures (our unpublished results and supplemental Figure IV). Whereas wild-type endothelial cells exposed to laminar shear for 20 hours aligned, 4(S988A) endothelial cells did not (Figure 6A). As shown in Figure 6B, the average angle from the flow direction of actin filaments in wild-type and mutant cells under static conditions was 37.6±0.85° (10.7°=S.D.) and 44±1.° (12.2°=S.D.), respectively (Figure 6B). Filaments in wild-type cells subjected to shear stress were orientated at an average angle of 16.9±0.25° (3.2°=S.D.) from the flow direction, which was close to the value obtained earlier (14.3±0.69 (9.23°=S.D.), indicating that most actin filaments aligned parallel to the flow direction. In contrast, filaments in 4(S988A) endothelial cells subjected to shear stress maintained an average angle of 47±0.92° (14.3°=S.D.; Figure 6B), showing that they did not align after shear. Thus, 4 integrin phosphorylation is required for stress fiber alignment induced by shear stress.
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As a further test of the role of 4 integrin phosphorylation in the endothelial cell responses to shear stress, we assessed the effect of the 4(S988A) mutation on shear-induced Rac1 activation in endothelial cells. HMECs were cotransfected with a Rac FRET reporter plasmid and plasmids encoding wild-type 4 or 4(S988A). These cells were plated on 4 ligands, scratch wounded, and immediately subjected to 12 dynes/cm2 shear stress for 5 minutes, fixed, and analyzed for Rac1 activation. In cells expressing recombinant wild-type 4 integrins, Rac1 activation was observed at the downstream edge of 78±8.2% of cells at the wound margins, consistent with earlier observations (Figures 3 and 7
A). In contrast, Rac1 activation was observed at the downstream edge in only 5±5% of the cells expressing recombinant 4(S988A) (Figure 7B). Thus, ectopic expression of 4(S988A) in endothelial cells exerts a dominant inhibitory effect on the localization of shear-induced Rac1 activation. These results demonstrate that 4 integrin phosphorylation is required for shear-induced polarized Rac1 activation and consequently for endothelial cell alignment.
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Discussion |
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Endothelial cell alignment in the direction of blood flow has been known for many years; recent work indicated that localized activation of Rac1 GTPase at the downstream side of the endothelial cell is a critical event in flow-induced alignment.6 Here we report that localized
4 integrin phosphorylation leads to this localized Rac1 activation and subsequent stress fiber alignment and endothelial cell elongation parallel to the flow direction in response to shear stress. 4 integrins were phosphorylated within 5 minutes of shear stress exposure and phosphorylation occurred predominantly at the downstream edges of the cells. Inhibition of PKA blocked 4 phosphorylation and prevented both localized Rac1 activation and stress fiber alignment in the flow direction. Furthermore, 4 integrins are required for endothelial cell alignment because deletion of 4 or addition of antibodies against 4 inhibited stress fiber alignment. Most importantly, PKA phosphorylation of 4 is involved in alignment because endothelial cells bearing 4(S988A), a mutation which disrupts the PKA phosphorylation site, fail to align in the flow direction. Together these results show that shear-induced PKA-dependent 4 phosphorylation is localized to the downstream edge of endothelial cells. The localized 4 phosphorylation leads to localized Rac1 activation at the downstream edge, enabling endothelial cell reorientation in the flow direction. Previous studies showed how a mechanosensory complex led to integrin activation that resulted in Rac1 activation.5,6 The present studies elucidate the pathway whereby 4 integrin phosphorylation informs the endothelial cell about the flow direction by localizing this Rac1 activation to the downstream edge, thereby acting as a "weather vane" of shear-induced endothelial cell reorientation.
The tangential drag forces imposed by laminar shear stress induce 4 integrin phosphorylation at Ser988 in endothelial cells. Using a phospho-specific anti-4 antibody, we observed phosphorylation as early as 5 minutes after application of 12 dynes/cm2 shear stress, which is within the ranges of shear stresses in medium-sized arteries.24 Previous studies have suggested that other key signaling events occur within similar time frames, including c-Src activation (1 minute, peak at 10 minutes),25 VEGFR2 phosphorylation (1 minute, peak at 5 minutes),26,27 and Ras28 and Rac1 activation (5 minutes).6 Furthermore, 4 is phosphorylated at Ser988, a known PKA phosphorylation site,19 and blocking PKA abolishes phosphorylation. The dependence of 4 phosphorylation on the application of shear and on PKA activity suggests that shear stress may activate PKA; however, we cannot exclude the possibility that there is tonic PKA activity and that shear stress acts to suppress phosphatase activity. Nevertheless, we favor the former possibility because PKA is known to be activated in endothelial cells by shear stress, which also induces phosphorylation of several PKA substrates such as VASP29 and endothelial nitric oxide synthase.30 Shear stress exerts force on endothelial cell attachments to the substrate, attachments mediated by integrins. Mechanical strain on integrins can result in enhancement of intracellular cAMP concentration leading to PKA activation.31 The role of integrin attachments in initiating 4 phosphorylation warrants future study. In sum, we conclude that fluid shear stress results in PKA-dependent 4 integrin phosphorylation in microvascular endothelial cells.
Localized phosphorylation of 4 integrins induced by shear stress is an important cue for alignment directionality. After 5 minutes in shear, phosphorylation was observed only at the downstream cell edge. Furthermore, stress fiber alignment required both the presence of 4 integrin and its phosphorylation by PKA. 4 integrins strongly promote cell migration. The 4 cytoplasmic tail is sufficient for a promigratory response,8 and we have shown that phosphorylation at Ser988 at the cell’s leading edge is the key determinant of this function; Ser988 is also the only identified 4 phosphorylation site in vivo.12,13 Shear stress accelerates endothelial wound closure.32 When a wound is orthogonal to the flow, cells on the wound margin proximal to the flow source (in which 4 is phosphorylated) migrate into the wound space faster than cells on the distal margin (in which 4 phosphorylation does not occur).2,3 Those latter cells have to move against the flow. Indeed, reendothelialization occurs fastest parallel to the flow direction following endothelial wounds in vivo, indicating a flow-induced enhancement of cell migration.33,34 Shear stress can promote migration of endothelial cells from the upstream edge of wounds; our studies now suggest that localized 4 phosphorylation can contribute to this enhanced directional migration.
The restriction of 4 phosphorylation to the downstream side of endothelial cells under shear serves to localize other signaling responses required for proper cytoskeletal alignment. Blocking 4 phosphorylation disrupts localized Rac1 activation to the downstream edge, which is essential for endothelial cytoskeletal alignment.6 One clue to the mechanism for this effect on Rac1 comes from our previous studies in migrating cells. In particular, 4 cytoplasmic tail phosphorylation at Ser988 by PKA enables that integrin to activate Rac1 because it prevents the binding of a protein complex that blocks Rac1 activation.7,12 Other signaling events are induced or enhanced by shear and play a role in cell migration. For example, PI3-kinase activity is increased by shear.35 PI3-K is also typically localized to the leading edge of migrating cells36 and can promote Rac1 activation, but it is not required for the alignment response.37 Both cell migration and morphological alignment under shear are driven by cytoskeletal rearrangements22,38 in response to Rho GTPases. 4 phosphorylation by shear stress coordinates localized Rho GTPase signaling to favor cytoskeletal alignment along the flow and migration in the flow direction.
4 integrins and PKA play important roles in endothelial functions such as neovascularization. Endothelial cells express 4 integrins in vitro and in vivo, and fibronectin, a ligand for 4 integrins, is highly expressed in the vasculature.15,39,40 4 integrins mediate endothelial cell adhesion, spreading, proliferation, and migration in vitro.39,41 In vivo, 4 expression is required for hematopoietic and endothelial progenitor cell homing and for efficient angiogenesis in developing embryos and in tumors. 4 antagonists also block angiogenesis in a chick chorioallantoic membrane model confirming this function of 4 integrins, although the requirement for 4 in angiogenesis may result from combined contributions in vascular endothelial and smooth muscle cells, as well as from paracrine effects in macrophages.41–43 Similarly, PKA regulates vascular endothelial cell adhesion, migration and survival,44,45 and angiogenesis.46,47 New blood vessel formation is stimulated by shear stress and requires flow conditions which promote cytoskeletal parallel alignment, indicating the importance of endothelial alignment in angiogenesis.24,48 Therefore shear-induced PKA-dependent 4 phosphorylation may be an important regulatory step in endothelial functions during vascular development and remodeling.
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Acknowledgments |
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Sources of Funding
This work was supported by American Heart Association SDG 0435295 (to L.E.G.) and 0635228N (to E.T.), National Science Foundation NIRT 0608863 (to A.G.), and NIH grants HL088632 (to E.T.), GM 68524 (to E.G.), HL085159 (to S.C.), and AR27214, HL078784, and HL31950 (to M.H.G.). E.T. is an Ellison Medical Foundation New Scholar in Aging.
Disclosures
None.
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Footnotes |
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*These authors contributed equally to this study.
Original received December 11, 2007; resubmission received March 26, 2008; revised resubmission received May 8, 2008; accepted June 16, 2008.
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