Induction of the CXC Chemokine Interferon-γ-Inducible Protein 10 Regulates the Reparative Response Following Myocardial Infarction
Rationale: Interferon-γ-inducible protein (IP)-10/CXCL10, an angiostatic and antifibrotic chemokine with an important role in T-cell trafficking, is markedly induced in myocardial infarcts, and may regulate the reparative response.
Objective: To study the role of IP-10 in cardiac repair and remodeling.
Methods and Results: We studied cardiac repair in IP-10-null and wild-type (WT) mice undergoing reperfused infarction protocols and examined the effects of IP-10 on cardiac fibroblast function. IP-10-deficient and WT animals had comparable acute infarct size. However, the absence of IP-10 resulted in a hypercellular early reparative response and delayed contraction of the scar. Infarcted IP-10−/− hearts exhibited accentuated early dilation, followed by rapid wall thinning during infarct maturation associated with systolic dysfunction. Although IP-10-null and WT mice had comparable cytokine expression, the absence of IP-10 was associated with marked alterations in the cellular content of the infarct. IP-10−/− infarcts had more intense infiltration with CD45+ leukocytes, Mac-2+ macrophages, and α-smooth muscle actin (α-SMA)+ myofibroblasts than WT infarcts but exhibited reduced recruitment of the subpopulations of leukocytes, T lymphocytes and α-SMA+ cells that expressed CXCR3, the IP-10 receptor. IP-10 did not modulate cardiac fibroblast proliferation and apoptosis but significantly inhibited basic fibroblast growth factor-induced fibroblast migration. In addition, IP-10 enhanced growth factor-mediated wound contraction in fibroblast-populated collagen lattices.
Conclusions: Endogenous IP-10 is an essential inhibitory signal that regulates the cellular composition of the healing infarct and promotes wound contraction, attenuating adverse remodeling. IP-10-mediated actions may be due, at least in part, to direct effects on fibroblast migration and function.
Myocardial infarct healing is dependent on activation of an inflammatory cascade, followed by granulation tissue formation and deposition of collagen-based matrix.1,2 Rapid induction of inflammatory chemokines, cytokines and adhesion molecules in the infarcted myocardium results in chemotactic recruitment of leukocytes. Neutrophils and activated macrophages clear the wound from dead cells and debris while secreting mediators that promote fibroblast growth and angiogenesis. The intense, but transient, inflammatory response is followed by repression of cytokine synthesis and activation of fibrogenic and angiogenic pathways. Activated myofibroblasts produce extracellular matrix proteins, whereas neovessels provide oxygen and nutrients necessary for the metabolically active wound. As the infarct matures, fibroblasts and vascular cells undergo apoptosis and a hypocellular collagenous scar is formed. Optimal repair of the infarcted myocardium is dependent on timely induction and suppression of inflammatory pathways and on endogenous mechanisms that ensure containment of the fibrotic response within the area of the infarct. Disturbances in the mechanisms involved in regulation of the reparative process result in formation of a defective scar with altered mechanical properties, leading to increased adverse remodeling.
The chemokines are inflammatory mediators with an essential role in leukocyte trafficking. Several members of the chemokine family are markedly and consistently induced in healing infarcts, modulating the postinfarction inflammatory response through recruitment of leukocyte subpopulations.3,4,5,6,7 The CC chemokine monocyte chemoattractant protein-1/CCL2 plays a crucial role in chemotaxis and activation of mononuclear cells in the infarcted myocardium.5 CXC chemokines containing the ELR motif are potent neutrophil chemoattractants and are critically involved in infiltration of the infarcted heart with granulocytes.6 However, recent evidence suggests that certain chemokines may modulate inflammatory processes through actions beyond their leukocyte chemotactic properties.8 The ELR-negative CXC chemokine CXCL10/interferon-γ-inducible protein (IP)-10 is involved in trafficking of effector T-cells,9 but also modulates fibroblast10 and endothelial cell phenotype,11 exerting angiostatic12 and antifibrotic effects.13 We have previously demonstrated that IP-10 synthesis is markedly induced in reperfused canine14 and murine15 myocardial infarcts and suggested that its upregulation in the infarcted heart may be an essential regulatory mechanism in infarct healing. Fibrogenic and angiogenic growth factors, such as basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β, and vascular endothelial growth factor, are rapidly induced in the infarcted heart; their expression extends in the infarct border zone,16 creating an environment that could promote expansion of the fibrotic response into the noninfarcted myocardium. We hypothesized that through its antifibrotic and angiostatic effects, IP-10 may prevent excessive infiltration of the infarcted heart with fibroblasts and may limit uncontrolled angiogenesis, serving as a “stop signal” that limits expansion of the fibrotic reaction. To test this hypothesis, we studied cardiac injury, repair, and postinfarction remodeling in IP-10-null mice. Our findings suggest that IP-10 plays an essential role in the reparative response following myocardial infarction by modulating the composition of the cellular infiltrate and by enhancing contraction of the healing scar. IP-10 exerts direct actions on growth factor-stimulated fibroblasts, reducing their migratory capacity and enhancing their ability to induce contraction of collagen lattices.
Murine Ischemia/Reperfusion Protocols
All animal studies were approved by the animal protocol review committee at Baylor College of Medicine. IP-10−/− mice, and wild-type (WT) C57/BL/6 controls were used for myocardial infarction experiments using an established closed-chest model of coronary occlusion/reperfusion.5
Infarct Size Determination
To assess the size of acute infarcts the Evans blue-triphenyltetrazolium chloride staining method was used.
Immunohistochemistry and Quantitative Histology
For histopathologic analysis murine hearts were fixed in zinc-formalin (Z-fix; Anatech, Battle Creek, Mich), and embedded in paraffin. Immunohistochemical staining was performed using established protocols.17
TUNEL Staining and Immunofluorescence
Identification of apoptotic myofibroblasts in myocardial infarction was performed using the fluorescent In situ Cell Death Detection Kit (Roche) combined with α-smooth muscle actin (SMA) immunofluorescence.
Preparation of Single Cell Suspensions From Myocardial Infarction
Single cell suspensions were obtained from infarcted WT (n=6) and IP-10−/− (n=6) hearts after 72 hours of reperfusion.
Flow cytometric identification of the cells was performed through simultaneous labeling with 2 of the following antibodies: fluorescein isothiocyanate-labeled anti-CD3 (clone BD Biosciences 145 to 2C11); phycoerythrin (PE)-labeled anti-CXCR3 (R&D Systems); PE-Cy5-labeled anti-CD45 (BD Biosciences, clone 30-F11). For intracellular staining, cells were fixed and permeabilized; subsequently, cells prelabeled with surface markers were incubated with anti-α-SMA.
Perfusion Fixation and Assessment of Ventricular Volumes
For assessment of postinfarction remodeling, infarcted hearts after 7 and 28 days of reperfusion were used for perfusion-fixation as previously described.5 Left ventricular end-diastolic volume (LVEDV) and scar size were assessed using established methods. Anterior wall, posterior wall, and midseptal wall thickness were assessed at the midpapillary level.
Short axis M-mode echocardiography was performed before instrumentation and after 7, 14, and 28 days of reperfusion (WT: n=10, IP-10−/−: n=12) using a 25-MHz probe (Vevo 770; Visualsonics. Toronto, Canada). Left ventricular end-diastolic diameter and left ventricular end-systolic dimension were measured as indicators of cardiac remodeling, and left ventricular fractional shortening was calculated as an indicator of systolic function.
RNA Extraction and Ribonuclease Protection Assay
RNA was extracted from sham and infarcted hearts. Ribonuclease protection assays were performed (RiboQuant; Pharmingen) as previously described.17
Cardiac Fibroblast Isolation and Transwell Migration Assay
Mouse cardiac fibroblasts were isolated by enzymatic digestion with a collagenase buffer. Cardiac fibroblast migration was studied using a colorimetric transwell system.
Assessment of Contraction in Collagen Lattices Populated With Cardiac Fibroblasts
Cardiac fibroblasts at passage 3 were used to examine the effects of IP-10 on growth factor-induced collagen lattice contraction.
Cell Proliferation Assay
Proliferation of cardiac fibroblasts was assessed using a colorimetric BrdU Cell Proliferation ELISA kit (Roche Applied Biosciences, Indianapolis, Ind).
Assessment of Cardiac Fibroblast Apoptosis
Apoptosis in isolated cardiac fibroblasts was assessed using the Cell Death Detection ELISAPLUS (Roche Applied Biosciences).
Statistical analysis was performed using ANOVA followed by t test corrected for multiple comparisons (Student-Newman-Keuls). Paired t test was used to compare echocardiographic end points before and after infarction. Data were expressed as means±SEM. Statistical significance was set at P<0.05.
IP-10 Induction in the Infarcted Myocardium Is Associated With Recruitment of CXCR3+ Cells
Ribonuclease protection assay demonstrated marked IP-10 mRNA upregulation in the infarcted myocardium after 6 hours of reperfusion (Figure 1A). Although IP-10 levels were also elevated in the noninfarcted segment, the difference did not reach statistical significance. To examine whether IP-10 upregulation is associated with infiltration of the infarcted myocardium with cells expressing the IP-10 receptor, CXCR3, we performed flow cytometry on single cell suspensions harvested from control and infarcted hearts (1 hour of ischemia/72 hours of reperfusion). Myocardial infarction resulted in a marked increase in the absolute number of nucleated cells isolated from the heart (Table 1). The infarcted myocardium contained a significantly higher number of CD45+ leukocytes, CD3+ T lymphocytes, and α-SMAc+ cells than the control heart (Table 1). Myocardial infarction resulted in intense infiltration with CXCR3+ cells (Figure 1B and Table 2), leading to a marked increase in the number of CXCR3+/CD45+ leukocytes and CXCR3+/CD3+ T lymphocytes (Table 2, Figure 1C and 1D). In addition, infarcts contained abundant CXCR3+ cells that did not express hematopoietic cell markers. Almost 20% of the CXCR3+ cells in the infarcted heart expressed α-SMA (versus less than 5% in control hearts), reflecting the accumulation of myofibroblasts expressing the IP-10 receptor (Table 2 and Figure 1E).
IP-10-Null and WT Mice Exhibit Comparable Acute Infarct Size
In the absence of injury, IP-10-null and WT mice had comparable structural and functional characteristics (Online Data Supplement, available at http://circres.ahajournals.org). Following reperfused infarction, IP-10-null mice and WT animals had comparable mortality rates (WT: 15.6% versus IP-10−/−: 17.4%; P=NS). Infarct size and the ratio of the infarct size to the area at risk (infarct:AAR) were comparable between WT and IP-10-null animals after 24 hours of reperfusion (Figure 1G through 1J), suggesting that the absence of IP-10 did not affect susceptibility of cardiomyocytes to ischemic injury.
IP-10 Absence Is Associated With Enhanced Adverse Remodeling and Early Expansion of the Scar
Both echocardiographic and quantitative morphometric analysis demonstrated that the absence of IP-10 is associated with accentuated early remodeling. After 7 days of reperfusion, LVEDV, as assessed through morphometry (Figure 2B), and echocardiographically derived left ventricular end-diastolic diameter (Online Table I) were significantly higher in infarcted IP-10-null hearts when compared with WT animals. Although there was a trend toward a lower left ventricular fractional shortening in IP-10-null hearts, the difference between IP-10−/− and WT animals did not reach statistical significance at this time point. Seven days after reperfused infarction, IP-10-null mice exhibited significantly larger scars in comparison to WT animals (Figure 2C). Because acute infarct size was comparable between groups (Figure 1), the increased scar area suggested expansion, or impaired contraction, of the fibrotic region. Serial echocardiographic imaging demonstrated that, although both IP-10-null and WT hearts continued to dilate, and cardiac dimensions remained higher in IP-10-null animals after 28 days of reperfusion, the difference was no longer statistically significant (Online Table I). However, after 28 days of reperfusion, IP-10-null hearts had significantly lower left ventricular fractional shortening, indicating accentuated systolic dysfunction (Online Table I). Development of late systolic impairment in IP-10-null hearts was associated with progressive thinning of the left ventricular walls (Figure 2D). Thus, in comparison to WT, IP-10−/− hearts had markedly increased dilative remodeling over the first week following infarction associated with a more extensive fibrotic area. As the scar matured, the differences in chamber dilation between WT and IP-10-null hearts were attenuated; however, IP-10−/− mice developed rapid wall thinning and accentuated systolic dysfunction (Figure 2D and Online Table I).
Effects of IP-10 Loss on the Postinfarction Inflammatory Response
IP-10 deficiency was associated with alterations in the composition of the inflammatory infiltrate in the infarcted heart. IP-10-null mice had increased infiltration with neutrophils after 24 and 72 hours of reperfusion (Figure 3A through 3C) and enhanced macrophage density in the infarcted myocardium after 3 to 7 days (Figure 3D through 3F). The increased recruitment of hematopoietic cells in IP-10-null infarcts was confirmed through flow cytometry of cell suspensions harvested from the infarcted myocardium. After 72 hours of reperfusion, the number of nucleated cells and the number of CD45+ leukocytes were significantly higher in infarcted IP-10−/− hearts when compared with WT infarcts (Table 1); however, the number of CD3+ T lymphocytes was comparable between groups (Figure 3G and 3H). IP-10 deficiency resulted in impaired recruitment of CXCR3+ cells in the healing infarct (Table 2). The number of CXCR3+/CD45+ leukocytes and CXCR3+/CD3+ T cells was markedly reduced in IP-10-null infarcts (Table 2 and Figure 3I through 3L). Despite the significant alterations in cellular composition of the inflammatory infiltrate, the absence of IP-10 had relatively subtle effects on expression of inflammatory cytokines and chemokines (Online Figure I and Online Table II).
IP-10-Null Mice Exhibit an Accentuated Early Fibrotic Response
IP-10 deficiency was associated with an accentuated early fibrotic response. Infarct myofibroblasts were identified as spindle-shaped α-SMA+ cells localized outside the vascular media (Figure 4A and 4B). Myofibroblast density was significantly increased in IP-10-null infarcts after 72 hours of reperfusion (Figure 4C). To examine whether increased myofibroblast accumulation in IP-10−/− infarcts was attributable to enhanced cellular proliferation, we performed dual immunohistochemistry combining α-SMA labeling with staining for ki-67, an indicator of cellular proliferation (Figure 4A and 4B). The number of ki-67+ cells and ki-67+/α-SMA+ myofibroblasts was comparable between groups suggesting that IP-10 deficiency does not affect cellular proliferation (Figure 4D and 4E). Flow cytometry of cell suspensions from the infarcted heart confirmed the immunohistochemical findings demonstrating a significantly higher number of α-SMA+ cells in IP-10-null infarcts (Table 1; Figure 4F and 4G). Despite the increased infiltration of IP-10-null infarcts with myofibroblasts, recruitment of the CXCR3+ subset of α-SMA-expressing cells was markedly reduced in the absence of IP-10 (Table 2; Figure 4H and 4I). Accumulation of myofibroblasts resulted in enhanced deposition of collagen in the infarct and in the neighboring border zone of IP-10-null hearts after 7 days of reperfusion (Figure 4J through 4L). Enhanced fibrotic remodeling of the infarcted heart in IP-10-null animals was not associated with increased expression of TGF-β isoforms (Figure 4M through 4O), FGF-1, and FGF-2 (Online Data Supplement). In addition, assessment of matrix metalloproteinases and tissue inhibitor of metalloproteinases mRNA expression showed only subtle differences between IP-10-null and WT hearts (Online Table III).
To examine whether increased myofibroblast density in the absence of IP-10 is attributable to reduced apoptosis of infarct myofibroblasts, we performed dual fluorescence combining TUNEL staining, to identify apoptotic cells, and α-SMA immunofluorescence (Figure 5A and 5B). After 7 days of reperfusion, the density of apoptotic nuclei was significantly higher in IP-10-null infarcts, when compared with WT scars (Figure 5C). Although only a small number of TUNEL+/α-SMA+ myofibroblasts was detected in the infarcted hearts (perhaps reflecting the rapid clearance of these cells from the infarct or the loss of α-SMA expression as these cells undergo apoptosis), the density of apoptotic myofibroblasts was significantly higher in IP-10-null infarcts (Figure 5D).
IP-10 Deficiency Is Associated With Decreased Microvascular Density in the Infarct and Periinfarct Area
Because IP-10 has potent angiostatic properties,18 we examined whether expansion of fibrosis in IP-10-null infarcts is associated with uncontrolled angiogenesis (Figure 5E through 5G). Surprisingly, IP-10−/− mice showed significantly decreased microvascular density in the infarct and periinfarct area in comparison with WT animals (Figure 5E through 5G).
IP-10 Does Not Modulate Cardiac Fibroblast Proliferation
To dissect the mechanisms responsible for the antifibrotic actions of IP-10, we examined whether recombinant mouse IP-10 modulates proliferative activity of isolated mouse cardiac fibroblasts. Incubation with 1% to 5% FCS markedly enhanced cardiac fibroblast proliferation (Figure 5H). IP-10 (10 to 250 ng/mL) had no effect on the proliferative activity of serum-stimulated cardiac fibroblasts (Figure 5H). Stimulation of serum-starved cells with bFGF, induced dose-dependent fibroblast proliferation (Figure 5I). Coincubation with IP-10 did not affect bFGF-mediated proliferative activity (Figure 5I).
IP-10 Does Not Induce Cardiac Fibroblast Apoptosis
Because fibroblast apoptosis plays an important role in regulation of myofibroblast density in the infarct, we examined the effects of IP-10 on cardiac fibroblast apoptosis. Using a cell death detection ELISA, we found that incubation with IP-10 (10 to 100 ng/mL) did not induce cardiac fibroblast apoptosis (Figure 5J). Cycloheximide was used as a positive control and markedly increased cardiac fibroblast apoptosis.
IP-10 Attenuates bFGF-Mediated Fibroblast Migration
Because migration of fibroblasts in the infarcted area plays an important role in the development of periinfarct fibrosis we examined the effects of IP-10 in modulating cardiac fibroblast migratory activity. Using a transwell migration assay, we demonstrated that bFGF induces cardiac fibroblast migration. Preincubation with IP-10 one hour before bFGF stimulation prevented growth factor-mediated cardiac fibroblast migration (Figure 6A through 6D).
IP-10 Enhances Growth Factor-Mediated Wound Contraction in Fibroblast-Populated Collagen Lattices
Infarct healing is associated with contraction of the wound. Because IP-10-null animals have increased scar size in comparison with WT infarcts, we examined whether IP-10 modulates contraction of fibroblast-populated collagen lattices. Fibroblast stimulation with 1% serum, TGF-β1 (50 ng/mL), or bFGF (50 ng/mL) induced collagen lattice contraction. IP-10 had no effects in the absence of serum or growth factors. However, incubation with IP-10 (100 ng/mL) significantly enhanced the effects of serum, TGF-β1 and bFGF on collagen lattice contraction (Figure 6E through 6M).
Our study suggests that the CXC chemokine IP-10 is an essential molecular signal in regulation of the reparative response following myocardial infarction. In the absence of IP-10, formation of a hypercellular wound with impaired contractile properties is associated with accelerated early dilative remodeling. At a later stage, during the maturation phase of healing, clearance of the abundant macrophages and myofibroblasts recruited in IP-10-null infarcts, results in rapid wall thinning and leads to the development of systolic dysfunction (Figure 2). The deleterious effects of IP-10 deficiency are not attributable to increased susceptibility of cardiomyocytes to ischemic injury because acute infarct size was comparable between WT and IP-10-null mice (Figure 1). The mechanisms responsible for the effects of IP-10 gene disruption on the remodeling infarcted heart appear to involve alterations in recruitment of inflammatory cells and changes in functional activation of myofibroblasts that ultimately may result in perturbation of the mechanical properties of the wound.
A growing body of evidence suggests that IP-10 is involved in the pathogenesis of several chronic inflammatory conditions, including multiple sclerosis,19 sarcoidosis, atherosclerosis,20,21 heart, lung, and small bowel transplant rejection.22 The proinflammatory effects of IP-10 appear to be mediated predominantly through recruitment of effector T cells9 in sites of inflammation. IP-10 gene disruption in hypercholesterolemic ApoE−/− mice results in a 2-fold reduction in atherosclerotic lesion formation, associated with increased density of regulatory T cells and enhanced expression of the inhibitory cytokines interleukin (IL)-10 and TGF-β.21 We found that the absence of IP-10 resulted in significant alterations in the composition of the inflammatory infiltrate in the infarcted heart (Figure 3). Histology and flow cytometry demonstrated that IP-10-null infarcts contain higher numbers of CD45+ leukocytes and Mac-2+ macrophages than WT infarcts. In addition, the absence of IP-10 markedly reduced recruitment of leukocyte subsets expressing its main receptor, CXCR3. However, in contrast to the important role of IP-10-induced alterations in modulating inflammatory-mediator synthesis in models of chronic inflammation, the absence of IP-10 did not affect the balance between proinflammatory and antiinflammatory cytokines in acute myocardial infarction. Infarcted IP-10-null and WT hearts showed comparable expression of the inflammatory cytokines tumor necrosis factor-α, IL-1β, and IL-6 and of the inhibitory cytokines TGF-β and IL-10 at all time points examined (Online Table II). Thus, the role of the altered inflammatory cell content in mediating the protective actions of endogenous IP-10 in postinfarction cardiac remodeling is unclear.
Investigations in models of fibrosis demonstrated that IP-10/CXCR3 interactions play an important regulatory role in the pathogenesis of fibroproliferative processes. Both IP-10-null and CXCR3−/− mice exhibit increased fibrosis in a model of bleomycin-induced lung injury,13,23 whereas CXCR3 deficiency was associated with formation of hypercellular and weakened wounds in a model of full-thickness cutaneous wounding.24 On the other hand, transgenic mice overexpressing IP-10 in keratinocytes had delayed and disorganized repair of cutaneous wounds associated with impaired blood vessel formation.25 Both quantitative histology and flow cytometry demonstrated increased accumulation of myofibroblasts in the infarcted heart (Figure 4). Enhanced fibrosis in IP-10-null infarcts was not attributed to altered synthesis of TGF-β isoforms.
What is the mechanism responsible for enhanced myofibroblast accumulation in IP-10-null infarcts? IP-10 may regulate myofibroblast infiltration by inhibiting proliferation and migration of cardiac fibroblasts or by enhancing fibroblast apoptosis. Our in vivo studies showed comparable density of proliferating myofibroblasts in IP-10-null and WT infarcts. In vitro experiments demonstrated that IP-10 does not inhibit growth factor-mediated cardiac fibroblast proliferation (Figure 5) and does not exert proapoptotic actions but markedly attenuates fibroblast migration (Figure 6). Similar IP-10-mediated inhibitory effects on fibroblast migration were observed in mouse pulmonary fibroblasts.13 In addition, Shiraha et al demonstrated that IP-10-induced inhibition of motility in epidermal growth factor-stimulated human dermal fibroblasts was not attributable to a disruption of epidermal growth factor receptor signaling at the ligand or receptor level but was associated with inhibition of calpain activation.10
Although enhanced inflammatory cell and myofibroblast infiltration may alter the reparative response following myocardial infarction,26 these alterations do not explain the increased adverse remodeling observed in infarcted IP-10-null hearts. Our experiments identified a novel mechanism that may be responsible for the effects of IP-10 in cardiac remodeling. Using a fibroblast-populated collagen lattice model, we found that IP-10 enhanced the ability of growth factor-stimulated murine cardiac fibroblasts to induce collagen contraction (Figure 6E through 6M). Thus, in addition to inhibition of the migratory potential of cardiac fibroblasts, IP-10 appears to increase their wound-contracting properties. IP-10-mediated reduction of the volume of the healing wound may alter the mechanical properties of the scar playing a role in the pathogenesis of dilative remodeling. The cellular basis for the effects of IP-10 on contraction of the collagen matrix is unknown. Wound contraction is a complex process that involves cell contraction, cell tractional forces, and cell elongation ultimately resulting in elimination of water from between the collagen fibers.27 Previous studies demonstrated that IP-10 stimulation did not affect basal cell morphometry but prevented epidermal growth factor-mediated dermal fibroblast compaction resulting in an elongated cell morphology10; this mechanism may be responsible, in part, for enhanced collagen lattice contraction. Modulation of integrin-mediated interactions between transdifferentiated myofibroblasts and the matrix may also be implicated.
We have identified a chemokine-mediated pathway that plays an essential role in infarct healing, through mechanisms that may involve alterations in recruitment of inflammatory cells and modulation of fibroblast phenotype and function. Our study highlights the importance of endogenous IP-10 as a key orchestrator of the fibrotic reparative response following myocardial infarction. Defects in the pathways involved in regulation of infarct healing may be associated with impaired wound contraction and perturbation of the mechanical properties of the infarcted heart leading to accentuated remodeling in patients with myocardial infarction.
Sources of Funding
This work was supported by NIH grants R01 HL-76246 and R01 HL-85440, the Alkek Foundation, and the Medallion Foundation (to N.G.F.); and NIH grant R01 CA-69212 (to A.D.L.).
↵*Both authors contributed equally to this work.
Original received April 20, 2009; revision received September 15, 2009; accepted September 15, 2009.
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