Published online before print June 21, 2007, doi: 10.1161/ATVBAHA.107.148759
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© 2007 American Heart Association, Inc.
Vascular Biology |
Kininostatin Associates With Membrane Rafts and Inhibits 
vβ3 Integrin Activation in Human Umbilical Vein Endothelial Cells
From The Sol Sherry Thrombosis Research Center (Y.W., Y.L., I.M.S., N.G.S., R.W.C.) and the Cardiovascular Research Center and Department of Anatomy and Cell Biology (V.R.), Temple University School of Medicine, Philadelphia, Pa.
Correspondence to Robert W. Colman, MD, or Yi Wu, MD, PhD, The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 N Broad Street, OMS 418, Philadelphia, PA 19140. E-mail colmanr{at}temple.edu; or yiwu@temple.edu
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Abstract |
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Objective— The cleaved form of high molecular weight kininogen (HKa) is a potent inhibitor of angiogenesis and tumor growth in vivo; the functional domain has been identified as domain 5 (D5, named as kininostatin). We now identify the subcellular targeting site for D5 on endothelial cells (ECs), and investigate D5 inhibition of integrin functions.
Methods and Results— Endothelial membrane rafts were isolated using sucrose density gradient centrifugation. D5, bound to ECs, was predominantly associated with membrane rafts, in which uPAR, a HKa receptor, was also localized. In contrast, other HKa receptors, cytokeratin-1 and gC1q receptor, were not detected in membrane rafts. Colocalization of D5 with caveolin-1 was demonstrated on ECs by confocal microscopy. Disruption of membrane rafts by cholesterol removal decreased D5 binding to ECs. On stimulation with vascular endothelial growth factor, 
vβ3 integrin formed a complex with uPAR and caveolin-1, which was accompanied by an increase in ligand binding affinity of vβ3 integrin. These events were inhibited by D5. Consistently, D5 suppressed specific vβ3 integrin-mediated EC adhesion and spreading as well as small guanosine triphosphatase Rac1 activation.
Conclusions— D5 binds to ECs via membrane rafts and downregulates vβ3 integrin bidirectional signaling and the downstream Rac1 activation pathway.
The cleaved form of high molecular weight kininogen and its functional domain (D5) are potent inhibitors of angiogenesis. This study indicates that D5 binding to endothelial cells depends on membrane rafts, by which it inhibits vβ3 integrin bi-directional signaling and downstream Rac1 activation.
Key Words: kininogen • membrane rafts • integrin • uPAR • angiogenesis
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Introduction |
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Caveolae are specialized components of membrane rafts,1 which are plasmalemmal invaginations formed by the sequestration of cholesterol and glycosphingolipids with self-associating molecules named caveolins. The major structural protein of endothelial caveolae is caveolin-1. Because the definitive identification of caveolae in experimental protocols can be only made by electron microscopy, we use the term membrane rafts in most of this paper and indicate when they contain caveolin. Caveolae are abundant on the surface of endothelial cells (ECs).2 Caveolae and caveolin-1 are widely involved in the regulation of EC function, including EC proliferation, migration, and angiogenesis. Angiogenesis is the process of formation of new blood vessels from the existing microvessels, initiated by stimulation of vascular ECs with growth factors and cytokines. The functional significance of caveolae in angiogenesis has been suggested by defective nitric oxide–dependent angiogenesis in caveolin-1–null mice,3 and by reduced tube formation in the absence of caveolin-1 in vivo and in vitro.4 Integrins, particularly the
v integrin family, are major players in each step of angiogenesis.5 Interaction of integrins with extracellular matrix (ECM) is required for EC function and maturation of newly formed blood vessels. v integrins, highly expressed on activated ECs during angiogenesis, colocalize with caveolin-1 in membrane rafts.6 Downstream effectors of integrins such as Src family kinases, and small guanosine triphosphatases are localized in membrane rafts and are involved in regulation of integrin activation as well as integrin-mediated cytoskeletal rearrangement.7
Human plasma high molecular weight kininogen (HK) is a major component (660 nmol/L) of the plasma kallikrein-kinin contact system,8 and serves as precursor for both bradykinin (BK) and the cleaved form of HK (HKa). The EC plasma membrane is an important site for the generation of BK and HKa, both of which in turn affect EC function.9 HKa exhibits a potent antiangiogenic activity, which is mediated through its Domain 5 (D5), known as kininostatin.10 HKa and D5 inhibition of angiogenesis is associated with their antiadhesive activity, which inhibits EC proliferation and induces EC apoptosis.11 D5 binds to the somatomedin B domain of vitronectin (Vn, aa 1 to 43), thereby masking the RGD region (aa 45 to 47) and preventing integrin ligation.12 D5 also directly binds to domains 2/3 of uPAR, which serves as a binding site for Vn.13 HKa thus competes for Vn interaction with both vβ3 integrin and uPAR. However, considering the potent inhibition of HKa and D5 on in vivo angiogenesis, the selective antiadhesive activity, which is tightly regulated by Vn, is not solely responsible for HKa antiangiogenic effect.
Our current study demonstrates that D5 binding to ECs depends on membrane rafts, by which it exerts inhibitory effect on vβ3 integrin functions, which represents a novel mechanism of D5 antiangiogenic activity.
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Materials and Methods |
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HUVECs (Clonetics, Wakersville, MD) were cultured in endothelial growth culture medium (EGM, Clonetics) with additives (Bullet Kit, Clonetics) provided by the manufacturer and 10% fetal bovine serum (FBS). HUVECs from 3 to 6 passages were used in individual experiments. To establish a stable cell line expressing
vβ3 integrin, CS-1 cells, provided by Dr. D.A. Cheresh (The Scripps Research Institute, La Jolla, CA), were transfected with pcDNA3 expressing human integrin β3 subunit (a gift from Dr. S. Santoso, Justus-Liebig University, Germany) using lipofectamine 2000. The sequence of human uPAR cDNA (5'-GGTGAAGAAGGGCGTCCAA-3') was selected as the targeting region, and a nonsilencing siRNA, 5'-AACCTGCGGGAAGAAGTGG-3', was used as a control. Control siRNA or uPAR siRNA (Qiagen) was transfected into HUVECs with HiPerfect (Qiagen) following the manufacturers’ instructions. Immunoprecipitation and Western blotting were performed as described previously.11 GST-free D5 (aa 420-513) was prepared10 and binding to endothelial cells performed as described.13 Caveolin-1-enriched membrane rafts were isolated and purified as described previously.16 Evaluation of vβ3 integrin binding affinity (WOW-1 binding) using HUVECs cultured on gelatin-coated 96-well plates after incubation with or without 600 nmol/L D5 for 30 minutes followed by stimulated with 20 ng/mL of VEGF for 5 minutes. WOW-1 Fab (30 µg/mL) was then added, and followed by addition of 10 µg/mL of Alexa Fluor 488-F(ab’)2 fragment of anti-mouse IgG (Molecular Probes, Eugene, Oregon). After 30 minutes, the cells were washed and the fluorescence intensity was immediately measured by 1420 Multilabel Counter (Wallac-Victor 2). The data were calculated as average±SEM from experiments done at least 3 times, and statistically analyzed by Students’ t test (two groups only) or One Way Analysis of variance (ANOVA) and Student-Newman-Keuls test (multiple groups). The materials and methods used in this study are fully described in the supplemental material (available online at http://atvb.ahajournals.org). |
Results |
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D5 Binds to Caveolin-1–Enriched Membrane Rafts via Association With uPAR
In this study, we focus on the function of D5, which is the functional region of HKa in antiangiogenesis and contains an EC binding site. Because uPAR, which is a HKa receptor, is localized in membrane rafts in tumor cells, and colocalized with caveolin-1,14 we tested whether the targeting site of D5 on ECs is associated with membrane rafts. To monitor D5 binding, we labeled recombinant human D5 with biotin. Biotin-D5, bound to human umbilical vein endothelial cells (HUVECs), was cross-linked to the receptor by 3, 3'-dithiobis [sulfosuccinimidylpropionate] (DTSSP), a cleavable disulfide cross-linking reagent. The concentration of D5 chosen (200 nmol/L), 30% of the plasma HK concentration (660 nmol/L), has been observed in human sepsis syndrome at the molecular weight of HKa (100 kDa) compared with HK (120 kDa).15 Membrane raft fractions were separated by sucrose density gradient centrifugation. Figure 1A shows that fractions 3 and 4 represented the caveolin-1–enriched membrane rafts, as evidenced by the presence of caveolin-1. The cytoskeletal protein, β-tubulin, was not detected in this fraction and served as a control for nonraft proteins (Figure 1A). The majority of Biotin-D5 bound to HUVECs was recovered in the membrane rafts, which also contained uPAR (Figure 1A). Moreover, D5 was detected in uPAR immunoprecipitates from membrane rafts (Figure 1B), suggesting D5 association with uPAR in the membrane rafts. In contrast, neither gC1q receptor (gC1qR) nor cytokeratin 1 (CK1), 2 other receptors for HKa, were identified in the membrane rafts (Figure 1A). Compared with the control, the association of D5 with uPAR in membrane rafts was enhanced by DTSSP treatment (supplemental Figure IA). This treatment seemed to prevent dissociation of D5 from the receptor during subsequent ultracentrifugation and solubilization, whereas it did not affect total D5 binding to ECs, as well as D5 association with membrane raft fraction and nonmembrane raft fraction (supplemental Figure IB and IC). We cannot exclude additional binding sites for D5 in the membrane rafts to explain the larger amount of D5 in fraction 3 than in fraction 4 (Figure 1A).
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D5 Association With Caveolin-1
The ultracentrifugation method yields a heterogenous population of membrane rafts. Membrane rafts (fractions 3 and 4 shown in Figure 1A) were pooled (designated as starting materials, SM), and incubated with Dynal magnetic beads preabsorbed with anti–caveolin-1 mAb. This method allowed successful separation of caveolae from other membrane rafts (Figure 1C). The majority of D5 bound to the membrane rafts was retained in caveolar rafts (Figure 1C).
To confirm the physical association of D5 with caveolin-1–enriched membrane rafts, HUVECs were incubated with 200 nmol/L GST or GST-D5. In the cell lysates, caveolin-1 was precipitated with GST-D5, but not GST (Figure 2A). The colocalization of D5 with caveolin-1 was demonstrated in a single ECs by confocal microscopy. Figure 2B shows that when HUVECs were labeled with fluorescein isothiocyanate (FITC)-D5 and stained with a fluorescent antibody to caveolin-1, an intense immunofluorescence signal was obtained in certain stretches of the plasma membrane, overlay of the immunofluorescent images for each label showed a high degree of colocalization. The binding of D5 to HUVECs and its colocalization with caveolin-1 were inhibited in the absence of Zn2+ (Figure 2C, i and data not shown), consistent with previous observations that D5 binding to HUVECs is specifically Zn2+-dependent.8 The integrity of membrane rafts depends on plasma membrane cholesterol. Cholesterol depletion by methyl-β-cyclodextrin (MβCD) results in their disappearance. D5 binding to ECs was inhibited by MβCD in a concentration-dependent manner (Figure 2C, ii), although MβCD did not affect cell viability, as determined by trypan blue dye exclusion (dead cells/live cells ratio, data not shown). MβCD altered membrane localization of uPAR and caveolin-1 (supplemental Figure II; see also Yang and Rizzo16), suggesting that the depletion of cholesterol reduces D5 binding to ECs because of the disturbed uPAR and caveolin-1 localization on membrane. Because uPAR is a GPI-anchored protein, MβCD depletion of membrane cholesterol prevents uPAR localization on the membrane surface.
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D5 Dissociates uPAR-vβ3 Integrin Complex in the Membrane Rafts and Downregulates vβ3 Integrin Ligand Binding Affinity
The association of D5 with membrane rafts stimulated us to hypothesize that D5 exerts its inhibitory effect via raft localization. In HUVECs, vβ3 integrin formed a complex with uPAR and caveolin-1, which was enhanced by VEGF stimulation (Figure 3A). However, VEGF did not change the expression of each molecule during the period of stimulation (data not shown). MβCD treatment markedly dissociated vβ3 integrin from caveolin-1, but not uPAR (Figure 3B), suggesting that vβ3 integrin association with uPAR is direct, and its binding to caveolin-1 is dependent on intact structure of the rafts/caveolae. D5, like HKa,11 inhibited the formation of vβ3 integrin–uPAR–caveolin-1 complex (Figure 3C), implying that D5 is the functional moiety. The concentration (600 nmol/L) used is lower than the dose necessary for the inhibition of EC migration.17 As indicated in Figure 3D, vβ3 integrin formed a complex with uPAR and caveolin-1 only in the membrane rafts, but not in the nonraft fractions. D5 dissociated uPAR and caveolin-1 from vβ3 integrin in the membrane rafts (Figure 3D), suggesting D5 disrupts this complex formation via binding to membrane rafts.
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uPAR and caveolin-1 are required for integrin activation.18 We further found that D5 disruption of this complex was accompanied by its downregulation of vβ3 integrin binding affinity. WOW-1 is a Fab fragment which specifically recognizes activated vβ3 and vβ5 integrins.19 VEGF stimulated WOW-1 binding to ECs (Figure 3E), supporting that VEGF increases active integrin recycling. D5 significantly inhibited the increase in WOW-1 binding to ECs stimulated by VEGF, but did not affect the overall expression of vβ3 or vβ5 integrin on membrane surface (Figure 3E). To confirm that the complex formation of vβ3 integrin–uPAR–caveolin-1 is required for v integrin function, uPAR was depleted by silencing RNA (siRNA) approach, in which case, the 3-molecule complex no longer existed. Nonsilencing siRNA served as control for nonspecific effects of double-strand RNA. Transfection of HUVECs with uPAR-siRNA reduced uPAR expression by 91.3% versus 8.74±4.58% (control, n=3) and did not affect FAK and β3 integrin expression (Figure 4A). In the absence of uPAR, VEGF-mediated HUVEC migration on Vn was dramatically suppressed (Figure 4B, compare b and d). The reduction of migration on Vn was about 70%, whereas there was less than 30% decrease in migration on collagen (Figure 4C), suggesting that uPAR is necessary for v integrin-mediated EC migration, and the physical association of uPAR with vβ3 integrin is functional. The decreased EC migration on collagen in the absence of uPAR supports that uPAR is also required for collagen-mediated migration,20 although Vn-mediated EC migration seems more dependent on uPAR.
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D5 Inhibits vβ3 Integrin Ligation-Dependent Cytoskeletal Reorganization and Rac1 Activation
D5 downregulation of v integrin ligand binding affinity raises a possibility that D5 inhibits vβ3 integrin outside-in signaling. We measured EC spreading on fibrinogen (Fbg)-coated surface, as Fbg does not bind to either D5 or uPAR, but is a ligand for vβ3 integrin. D5 blocked Fbg-mediated cell spreading, particularly lamellipodial formation (Figure 5A). In contrast, D5 did not inhibit EC spreading on collagen surface (data not shown). The inhibitory pattern was similar to that with cRGDfK, an inhibitory peptide specific for vβ3 integrin (Figure 5A), and both D5 and cRGDfK lowered EC spreading surface area by 60% to 70% (Figure 5B), suggesting that D5 suppresses actin cytoskeletal reorganization of ECs, possibly by inhibiting vβ3 integrin activation. Consistently, D5 abrogated Fbg-mediated Rac1 activation (Figure 5C). Supplemental Figure III further indicates that HUVECs rapidly underwent spreading on Fbg surface, which was accompanied by Rac1 activation. D5 significantly inhibited these 2 activation processes, especially at early time points (0.5, 1, and 2 hours). The inhibitory effect decreased at 4 hours. The inhibitory effect of D5 on spreading is more striking than on EC adhesion, and the inhibitory pattern is different. Because human ECs express 2 receptors for Fbg, vβ3 and 5β1 integrins,21 the delay induced by D5 in Fbg-mediated cell spreading and Rac1 activation seems attributable to its inhibition of vβ3 but not 5β1 integrin. The interaction of 5β1 integrin with Fbg may reverse D5 inhibition of vβ3 integrin-mediated outside-in signaling.
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D5 Inhibits Specific vβ3 Integrin Adhesive Function
To confirm that D5 indeed regulates specific vβ3 integrin function, we generated a cell line expressing vβ3 integrin using CS-1 cells (murine melanoma cell line). CS-1 cells maintain an intracellular pool of v subunit, they do not express β3 or β5 integrin, and thus cannot adhere on culture dishes (Figure 6A, i). Once CS-1 cells were transfected with human β3 subunit cDNA, vβ3 heterodimer was expressed on the cell membrane (Figure 6A, ii), which allowed the CS-1 cells to acquire adhesion capacity (Figure 6A, i). LM609, a mAb directed to vβ3 integrin, inhibited adhesion of vβ3CS-1 cells to culture dishes as well as Fbg and Vn (data not shown). Thus, the acquired adhesion property indicates specific vβ3 integrin function. In a concentration-dependent manner, D5 inhibited adhesive property of vβ3CS-1 cells on culture plate (Figure 6B). The inhibition was also effective on Fbg or Fn coated surface, both of them are ligands for vβ3 integrin (Figure 6C). The detachment is not the result of anoikis because tumor cells (CS-1) are resistant to that form of apoptosis.
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Discussion |
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In this study we demonstrate that the subcellular binding site of D5 on ECs is localized in membrane rafts. D5 is associated with caveolin-1–enriched membrane rafts. In addition, colocalization of D5 with caveolin-1 was observed in a live ECs using confocal microscopy. Disruption of membrane rafts decreases D5 binding to HUVECs. Caveolin-1 is coprecipitated with D5 in HUVECs. Although there are several surface proteins on ECs known to bind D5 such as gC1qR, cytokeratin-1, and uPAR, the relative contribution of each binding moiety has been uncertain. We found that gC1qR is not localized in membrane rafts, which is consistent with a previous finding that gC1qR is prominently localized in vesicular fractions associated with mitochondria, microsomal fractions, and cytoplasm in ECs.22 The failure to detect CK1 in membrane rafts might be attributable to any of the following reasons: CK1 is part of a large family of cytoskeletal proteins, most of which are not released from cytoskeletal constraints during ultracentrifugation.23 Only a small fraction of total CK1 forms a complex with uPAR. This complex is too low to be detected by the membrane raft isolation procedures. uPAR is more highly expressed on HUVEC membrane than CK1 (about 250 000 molecules per cell for uPAR versus 72 000 molecules per cell for CK1).24 However, further studies are required to determine how uPAR and other receptors are assembled in the D5-receptor complex in membrane rafts.
Caveolae and their principal protein caveolin-1 are closely involved in the various processes of EC activation. Caveolin-1 is not only a structural protein, but also serves as a scaffold to assemble many signaling molecules and generate preassembled signaling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin-1 binding may functionally regulate the activation state of caveolae-associated signaling molecules.2 The identification of membrane rafts/caveolae as a major membrane subdomain for D5 binding is intriguing. These membrane microdomains are responsible for subcellular localization and regulation of integrins in angiogenesis.18 Integrins interact closely with caveolae/caveolin-1 in the context of EC proliferation, migration, and tube formation.25 We now show that D5, whose binding to ECs depends on membrane rafts, is able to inhibit vβ3 integrin activation in both outside-in and inside-out signaling. This inhibitory effect is independent of D5 antiadhesive activity, which is regulated by D5 interaction with ECM components. D5 suppresses Fbg-induced Rac1 activation and EC spreading, similarly to cRGDfK, an inhibitory peptide of vβ3 integrin. D5 inhibition of specific vβ3 integrin function is confirmed by the finding in vβ3CS-1 transfection model (Figure 6). As demonstrated by WOW-1 binding assay, D5 inhibition of vβ3 is attributable to downregulation of its ligand binding affinity (Figure 3E). WOW-1 Fab is monomeric, and thus insensitive to changes in integrin clustering and indicates only changes in affinity. WOW-1 binds only to unoccupied, high-affinity integrins. Like other integrins, vβ3 can exist in different functional states with respect to ligand binding, which is modulated by growth factors such as VEGF, through inside-out signaling.26 Although D5 did not change the total expression of vβ3 and vβ5 integrins on the membrane, D5 inhibition of WOW-1 binding to ECs presumably results from blocking activated integrin recycling.27
In the context of the crucial role of integrins in angiogenesis, understanding the connection between D5 targeting site and its inhibition of integrin function becomes important. The interaction between integrins and membrane rafts is necessary for integrin-mediated EC adhesion, spreading, and migration to the ECM on growth factor stimulation. The association of v integrins with caveolin-1 in membrane rafts is disrupted by MβCD, thereby suppressing vβ3 integrin signaling.28 Given that vβ3 integrin association with caveolin-1 is sensitive to cholesterol depletion,27 and that caveolin-1 does not extend outside the cell, this association must be indirect, and mediated by receptors in membrane rafts. VEGF enhances vβ3 integrin association with uPAR and caveolin-1. Recruitment of vβ3 integrin into membrane rafts allows its proximal localization to downstream effectors including Src and Rac1 GTPases, as well as its partners such as VEGF receptor. The dynamic redistribution of vβ3 integrin is required for its functional crosstalk with growth factor receptors and EC function. We found that D5 blockade of VEGF-stimulated recruitment of vβ3 integrin to membrane rafts is accompanied by a decrease in vβ3 ligand binding affinity, which may account for D5 inhibition downstream Rac1 activation and EC spreading. D5 disrupts the formation of vβ3 integrin–caveolin-1–uPAR complex in membrane rafts, suggesting that D5 exerts its inhibitory effect on vβ3 integrin through membrane rafts.
Functional interaction between uPAR and vβ3 integrins has become an important issue in tumor-induced angiogenesis. Both proteins are localized in membrane rafts of several cell types and form a complex.28–31 uPAR has no cytoplasmic domain and is bound to the plasma membrane by a glycosylphosphatidylinositol anchor. Therefore, its signaling is mediated by integrins, in particular vβ3.32 On the other hand, uPAR behaves as an important modulator of integrin function. This concept is supported by our finding that uPAR is required for v integrin-mediated EC migration (Figure 4B and 4C). Because Vn-mediated migratory signal does not depend on uPAR interaction with Vn,29 the decrease of EC migration represents inhibition of v integrin. VEGF activates vβ3 integrin forming a complex with uPAR and caveolin-1 in ECs. Because the association of uPAR with vβ3 integrin is resistant to removal of cholesterol, the interaction between uPAR and vβ3 integrin seems direct, in agreement with the finding of Degryse et al that direct binding of uPAR to vβ3 integrin occurs a region of uPAR domain 2.29 Because D5 binds to the D2 domain of uPAR, and an antibody against uPAR restores HKa inhibition of EC migration on Vn,17 uPAR is a primary binding site for HKa inhibition of vβ3 integrin function. D5 occupancy of uPAR may not only block internalization of both receptors and the turnover of focal adhesion plaques during EC migration, but also prevent the assembly of reexpressed uPAR and integrin on the membrane surface during receptor cycling. Thus, D5 blockage of vβ3 integrin-uPAR interaction inhibits the signaling of each receptor as well as their functional crosstalk.
During the dynamic process of angiogenesis, vβ3 integrin acts as a biosensor and modulates cell survival and death as a function of ECM composition.5,33 Ligation of the upregulated vβ3 integrin during neovascularization represents a potential mechanism to promote angiogenesis. Our current studies support a model in which D5 downregulates vβ3 integrin function by targeting membrane rafts, which represents a new mechanism underlying D5 inhibition of EC function.
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Acknowledgments |
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Sources of Funding
This work was supported by the following grants: R01CA083121, R01AR051713, and T32HL07777 from National Institute of Health (to R.W.C.).
Disclosures
None.
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Footnotes |
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Original received January 4, 2007; final version accepted June 8, 2007.
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