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(Circulation Research. 1996;79:821-826.)
© 1996 American Heart Association, Inc.

Articles

Vascular Smooth Muscle _vß₃ Integrin Mediates Arteriolar Vasodilation in Response to RGD Peptides

Jon E. Mogford, George E. Davis, Steven H. Platts, Gerald A. Meininger

the Microcirculation Research Institute and Department of Medical Physiology (J.E.M., S.H.P., G.A.M.) and the Department of Pathology and Laboratory Medicine (G.E.D.), Texas A&M University Health Science Center, Texas A&M University, College Station, Tex.

Correspondence to Gerald A. Meininger, PhD, Department of Medical Physiology, Reynold's Medical Building, Texas A&M University Health Science Center, College Station, TX 77843-1114. E-mail gam@tamu.edu.

	Abstract

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Arteriolar vasodilation and the resultant increase in blood flow are characteristic vascular responses to tissue injury. The dilatory mediators signaling these responses are incompletely understood. We show that integrin-binding peptides containing the Arg-Gly-Asp (RGD) tripeptide sequence cause immediate and, in some instances, sustained vasodilation when applied to isolated rat cremaster arterioles. The vasodilation is dependent on interaction of the soluble RGD sequence with the _vß₃ integrin expressed by smooth muscle cells in the arteriolar wall. Possible in vivo sources of soluble RGD sequences are fragments of extracellular matrix proteins that are generated after tissue injury. Indeed, protease-generated fragments of denatured collagen type I (a major source of RGD sequences) also cause cremaster arteriolar vasodilation through the _vß₃ integrin. Thus, extracellular matrix protein fragments containing the RGD sequence may act as vascular wound recognition signals to regulate blood flow to injured tissue.

Key Words: vasodilation • Arg-Gly-Asp • _vß₃ integrin • collagen degradation • vascular wound response

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The integrin family of transmembrane receptors provides a direct adhesive link between cells and the surrounding ECM and also transduces signals involved in a variety of cellular functions.^{1 2 3} A well-recognized integrin-binding sequence is the tripeptide arginine-glycine-aspartic acid (RGD) recognized by a variety of integrins including _vß₃, ₅ß₁, _vß₁, _vß₅, and _IIbß₃.^{4 5 6 7 8} This sequence is found in a variety of ECM components, including the collagens, fibronectin, and vitronectin.^{9 10 11 12}

In contrast to other ECM proteins, collagen RGD sequences are not exposed in the native protein structure.^{13 14} After denaturation of collagens, RGD sequences become exposed, allowing an interaction with integrins.^{13 14 15} It has been proposed that exposure of these latent RGD sites in collagens may be an important signal for cells during tissue injury and may play a role in wound healing, angiogenesis, and tumor invasion, where collagen proteolysis and unfolding are known to occur.¹³ We tested the hypothesis that RGD peptides and fragments of RGD-containing ECM proteins can act on smooth muscle integrins to modulate the contractile function of rat cremaster resistance arterioles.

	Materials and Methods

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Vessel Isolation and Deendothelialization
Male Sprague-Dawley rats (180 to 300 g) were anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg). The right cremaster muscle was excised at the proximal end and pinned flat in a refrigerated silastic-coated Plexiglas chamber containing cold (4°C) PSS (mmol/L): NaCl 145, KCl 4.7, CaCl₂ 2, MgSO₄ 1, NaH₂PO₄ 1.2, dextrose 5, MOPS buffer 3, pyruvate 2, EDTA 0.02, and albumin 0.15, pH 7.4±0.1. A segment of first-order feed arteriole (internal diameter, 150 to 210 µm; passive) was surgically isolated and cannulated as previously described.¹⁶ After 1 hour of rewarming to 34.5°C, vessel pressure was increased to 90 cm H₂O to match in vivo pressure of these vessels. Only vessels without leaks that developed spontaneous tone were studied. Diameter was recorded with a calibrated video caliper coupled to a closed-circuit video microscopy system. At the completion of each experiment, phenylephrine, an ₁-adrenergic receptor agonist (10 µmol/L), and adenosine (1 mmol/L in Ca²⁺-free PBS) were added to confirm arteriolar viability and obtain passive diameter, respectively. All animal handling procedures followed institutional guidelines. Average lumen diameter of all vessels studied (n=43) after development of spontaneous tone was 104±2 µm (64±2% of passive diameter, defined as the steady state diameter in Ca²⁺-free PBS+1 mmol/L adenosine). Average passive diameter was 166±4 µm. Diameter changes to the peptides were quantified as percentage of passive diameter normalized to starting diameter.

Endothelial cell involvement in arteriolar responses to RGD peptides was determined by removal of the endothelium by physically rubbing the lumen of the vessel with an abrasive pipette.¹⁶ Completeness of endothelial cell removal was confirmed in all vessels by the lack of response to acetylcholine (1 mmol/L), an endothelium-dependent vasodilator.

Peptide Addition
Lyophilized cyclic GPenGRGDSPCA (cyclic RGD, with Pen indicating penicillamine), GRGDSP, GRGDNP, or GRGESP (GIBCO BRL) was solubilized in fresh PSS and carefully added to the vessel bath (abluminal addition) without causing fluctuations in temperature. Cumulative doses (0.21 µmol/L to 0.7 mmol/L) of the peptides were given at 4-minute intervals to determine the concentration-response curve. Maximal diameter changes and minute interval diameter values were recorded.

ß₃ Integrin Function Blockade
Isolated arterioles were pretreated for 15 minutes with 100 µg (50 µg/mL) of either the anti-ß₃ integrin function–blocking monoclonal antibody F11 (Pharmingen) or an anti-rat MHC class I monoclonal antibody (clone R4-8B1, Seikagaku America, Inc) used as a control IgG molecule and a control for the cell surface–binding properties of the anti-ß₃ antibody. Cyclic RGD was then added, and the effects of the antibodies were assessed.

ß₃ Integrin Expression by Arteriolar Vascular Smooth Muscle In Situ and In Culture
Rat cremaster skeletal muscle arterioles were surgically removed, partially digested for 10 minutes with collagenase (250 U/mL, Worthington Biochemical Corp) and elastase (25 U/mL, ICN Pharmaceuticals Inc), and placed flat on polylysine-coated coverslips. The arterioles were fixed with 2% paraformaldehyde/PBS for 15 minutes. After two glycine/PBS washes, the vessels were treated for 2 hours with F11, the anti-rat MHC class I antigen monoclonal antibody, or FITC-conjugated secondary antibody alone. Primary antibodies were diluted (1:10 dilution, 5 µg of antibody) in buffer with 2% goat serum. Vessels were washed with PBS and treated for 1 hour with FITC-conjugated goat anti-mouse antibody (1:50 dilution, 1 µg of antibody). The arterioles were imaged using a Zeiss Axiovert 100 inverted microscope. Fluorescent images were acquired with a Scanalytics CellScan imaging system (Scanalytics, CSP, Inc).

SMCs were isolated from the rat cremaster arterioles by papain digestion and grown in culture using standard techniques. Cells were surface-biotinylated and extracted with 3% octylglucoside in TBS containing 1.5 mmol/L Mg²⁺, 1.5 mmol/L Mn²⁺, and 1 mmol/L phenylmethanesulfonic acid. Cell extracts were incubated in a Sepharose affinity column (1-mL volume) containing GRGDSP peptide (5 mg/mL) for 2 hours with intermittent mixing. The column was washed with 1% octylglucoside/TBS containing 1.5 mmol/L Mg²⁺ and 1.5 mmol/L Mn²⁺ and eluted with 10 mmol/L EDTA in 1% octylglucoside/TBS. Fractions were pooled and precipitated with 5 µg of either anti-ß₃ monoclonal antibody (F11), anti-_v integrin cytoplasmic domain polyclonal antibody (Chemicon), anti-₂ integrin cytoplasmic domain polyclonal antibody (Chemicon), or anti-rat MHC class I antigen monoclonal antibody (Seikagaku). Precipitated proteins were separated by 7% SDS-PAGE under nonreducing conditions. Blots were treated with 1 µg/mL of streptavidin-alkaline phosphatase, washed, and developed with NBT-BCIP (Bio-Rad). For immunoblot analysis, a nonimmunoprecipitated sample from the pooled elutions was separated by electrophoresis as described above. The immunoblot paper was blocked with 5% skim milk, treated for 2 hours with a 1:500 dilution of anti-_v antiserum, washed with 0.1% Tween 20 saline, and treated for 1 hour with a 1:1000 dilution of alkaline phosphatase–conjugated goat anti-rabbit IgG antibody (Sigma Chemical Co). After it was washed, the blot was developed with NBT-BCIP.

Collagen Fragment Preparation and Addition to Arterioles
Purified rat-tail collagen type I (Becton-Dickinson) was dialyzed into PSS, heat-denatured, and digested with Sepharose-coupled neutrophil elastase (Calbiochem) for 24 hours at 37°C with constant mixing. After digestion, the beads were removed by centrifugation. Vessel buffer (0.5 mL) was replaced with digested collagen fragment–containing buffer warmed to 34.5°C. Arteriolar diameter was measured until a steady state diameter was maintained for 3 to 4 minutes (total time, <10 minutes). The bath was then washed with fresh buffer to remove fragments. After arterioles returned to baseline diameter, 100 µg (50 µg/mL) of either F11 or anti-MHC class I antigen monoclonal antibody was added to the bath. After a 15-minute incubation with the antibody, fragment addition was repeated.

RGD/Collagen Fragment Integrin-Ligand–Binding Assay
_IIbß₃, purified using previously described techniques,⁸ was bound to polystyrene microwells at 5 µg/mL overnight at 4°C in TBS. The wells were blocked with 0.1% Tween 20 containing 1% BSA in TBS for 30 minutes at 25°C. Denatured collagen and fibrinogen were biotinylated as described previously¹⁷ and added to the microwells at 1 µg/mL with or without unlabeled competitors in 0.1% Tween 20 containing 1% BSA, 1.5 mmol/L Mg²⁺, and 1.5 mmol/L Mn²⁺ in TBS. Competitors added were GRGDSP, cyclic RGD, GRGESP (each at 10 µg/mL), and elastase-generated collagen fragments (dCOL digest, 500 µg/mL). Competitors were added for 30 minutes before the addition of the biotinylated ligands. The competitors and ligands were incubated for 1 hour at 37°C; after which, the wells were washed with 0.1% Tween 20 containing 1.5 mmol/L Mg²⁺ and 1.5 mmol/L Mn²⁺ in TBS. To detect bound biotin, avidin-peroxidase was added at 1 µg/mL in 0.1% Tween 20 containing 1% BSA, 1.5 mmol/L Mg²⁺, and 1.5 mmol/L Mn²⁺ in TBS for 30 minutes at 25°C, and peroxidase was detected using o-phenylenediamine.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolated rat cremaster skeletal muscle arterioles were treated abluminally with increasing concentrations of cyclic RGD, linear GRGDSP, GRGDNP, or the inactive structural analogue GRGESP.¹⁸ The RGD-containing peptides bind integrins and competitively inhibit cell adhesion to RGD-containing substrates such as fibronectin and vitronectin.¹⁸ Addition of the synthetic RGD peptides to the vessel bath caused concentration-dependent dilations (ie, inhibition of myogenic tone) with a potency ranking of cyclic RGD>GRGDSP>GRGDNP (Fig 1A). GRGESP, used as a negative control, had no effect on arteriolar diameter. Dilations to lower concentrations of the RGD peptides were transient (peak response within 20 seconds) with the arterioles returning to, or nearly to, baseline diameter within 1 minute. At higher concentrations of GRGDSP and GRGDNP, dilations continued to be biphasic, with diameters returning only partially toward baseline. By comparison, higher concentrations of cyclic RGD resulted in sustained maximal dilations. Dilations were reversible, with all arterioles returning to baseline diameter within 1 minute after removing the peptides from the vessel bath by washing. The cyclic RGD peptide also caused concentration-dependent vasodilation in rat cremaster skeletal muscle arterioles precontracted with phenylephrine (0.5 µmol/L, n=2) or KCl (35 mmol/L, n=3) (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 1. Synthetic RGD peptides cause endothelium-independent vasodilation of skeletal muscle arterioles. A, Isolated rat cremaster skeletal muscle arterioles that developed spontaneous tone16 were treated with increasing concentrations of cyclic GRGDSP (0.2 µmol/L to 0.2 mmol/L), GRGDSP, GRGDNP, or the control peptide GRGESP (0.7 µmol/L to 0.7 mmol/L). Nonlinear regression analysis shows that significant differences (P<.05) exist between all curves. All diameter changes to the peptides were quantified as a percentage of the dilation achieved with adenosine (1 mmol/L) in Ca2+-free PBS normalized to starting diameter. Data are represented as mean±SEM. B, Possible endothelial cell involvement in the arteriolar dilation to GRGDSP was determined by comparing vessels with the endothelium removed with vessels with an intact endothelium. Vessels denuded of endothelium by physically rubbing the lumen of the vessel with an abrasive pipette showed no difference in response to GRGDSP compared with intact arterioles. Data are represented as mean±SEM.

The response to soluble RGD peptides did not appear to involve factors released from the endothelium, as mechanical removal of the endothelium did not affect vasodilation (Fig 1B). Removal of the endothelium was pharmacologically verified in each vessel by a lack of dilation in response to the endothelium-dependent vasodilator acetylcholine (1 mmol/L). Thus, vasodilation to RGD peptides resulted from interaction of the peptides with vascular SMCs.

Enhancement of RGD dilatory potency by peptide cyclization implicates the involvement of _v integrins. Cyclic RGD peptide is known to be a more specific antagonist of cell binding to vitronectin compared with ₅ß1-mediated binding to fibronectin.¹⁸ Specific evidence for the involvement of _vß₃ in the RGD-induced dilations was obtained by pretreating arterioles with the anti-rat ß₃ integrin function–blocking monoclonal antibody F11. After a 15-minute incubation with F11, the vasodilatory responses to increasing concentrations of cyclic RGD were significantly inhibited (80% inhibition to the highest concentration, Fig 2). In contrast, the anti-rat MHC class I antibody did not alter the vasodilatory effect of RGD peptides. This antibody served as an immunoglobulin isotype and cell surface–binding control antibody for F11. Neither antibody interfered with the effect of another receptor-mediated vasodilator, adenosine, eliminating the possibility of a nonspecific inhibitory effect of the antibodies on vasodilation. The presence of the ß₃ integrin subunit on the surface of rat cremaster arteriolar SMCs in situ was confirmed by immunofluorescence using F11 (Fig 3A). Stained cells were oriented perpendicular to the long axis of the vessel, consistent with the concentric arrangement of vascular SMCs in the arteriolar wall. Positive SMC immunostaining was also observed with the anti-rat MHC class I antibody (Fig 3B). No positive staining occurred with treatment of FITC-conjugated secondary antibody alone (Fig 3C). The presence of both the _v and ß₃ subunits was also demonstrated by immunoprecipitation of the receptor from cultured rat cremaster arteriolar SMCs (Fig 4).

View larger version (25K): [in this window] [in a new window]   Figure 2. ß3 integrin function–blocking antibody inhibits synthetic RGD–induced vasodilation. Isolated arterioles were pretreated for 15 minutes with 100 µg (50 µg/mL) of either the anti-ß3 integrin function–blocking monoclonal antibody F11 or an anti-rat MHC class I monoclonal antibody, used as a control IgG molecule and a control for the cell surface–binding properties of the anti-ß3 antibody. Cyclic RGD was then added, and the effects of the antibodies were assessed. Nonlinear regression analysis shows that addition of F11 significantly reduced (P<.01) the arteriolar response to cyclic RGD. Neither antibody affected arteriolar dilation to 1 mmol/L adenosine (not shown). Data are represented as mean±SEM.

View larger version (101K): [in this window] [in a new window]   Figure 3. vß3 integrin expression by rat cremaster vascular SMCs in situ. Isolated rat cremaster skeletal muscle arterioles were partially digested, fixed, and treated with anti-rat ß3 integrin subunit monoclonal antibody (F11) (A), anti-rat MHC class I antigen monoclonal antibody (B), or FITC-conjugated secondary antibody alone (C). Single-headed arrows in panels A and B point to the edges of positively stained single cells within the arteriolar wall. Double-headed arrows indicate the long axis of each arteriole. Fluorescence from treatment with secondary antibody alone was no different from tissue autofluorescence (not shown). Bar=20 µm.

View larger version (74K): [in this window] [in a new window]   Figure 4. SDS-PAGE analysis of cell extracts from surface-biotinylated cultured rat cremaster arteriolar SMCs after incubation in a Sepharose affinity column containing GRGDSP peptide. EDTA-eluted fractions were precipitated with either anti-ß3 monoclonal antibody (lane 2), anti-rat MHC class I cell surface antigens (lane 3), anti-v integrin cytoplasmic domain polyclonal antibody (lane 4), or anti-2 integrin cytoplasmic domain polyclonal antibody (lane 5). Lane 1 shows total eluted protein used as starting material for immunoprecipitation. Lane 6 shows immunoblot analysis of the starting material with an anti-v integrin cytoplasmic domain polyclonal antibody. Control antibodies did not react with the band (not shown). Molecular masses (in kilodaltons) and positions of the v and ß3 chains are indicated.

We speculated that collagens are a possible in vivo ECM source of soluble RGD-containing peptides. Collagens are the most abundant proteins in the ECM and are a major source of RGD sequences.¹³ To potentially mimic in vivo generation of RGD-containing fragments, heat-denatured rat collagen type I was digested for 24 hours with Sepharose-coupled neutrophil elastase. Neutrophil elastase is known to degrade a variety of ECM proteins and participates in neutrophil-induced tissue injury.¹⁹ SDS-PAGE analysis of the elastase-treated collagen confirmed its degradation into numerous smaller fragments that were <30 kD (not shown). Addition of these fragments to the isolated arteriole bath caused a biphasic sustained dilation similar to that seen with synthetic RGD peptides (Fig 5A). Dilations to denatured collagen fragments were inhibited by 90% after pretreatment with F11 but were not blocked by the control anti-MHC class I antibody, implicating the involvement of _vß₃ in the arteriolar response to the collagen fragments. No change in arteriolar diameter was observed after application of control collagen-free buffer similarly incubated with the Sepharose-coupled elastase. The collagen fragments were also found to competitively block the binding of labeled denatured collagen and fibrinogen to another RGD-binding integrin, _IIbß₃, in a purified integrin-ligand–binding assay (Fig 5B). Ligand binding was similarly blocked by linear GRGDSP and cyclic RGD peptides but not by the control RGE peptide. The ability of the collagen fragments to induce arteriolar dilation and to competitively interfere with the binding function of an RGD-binding integrin strongly supports the concept that biologically active RGD-containing fragments exist in proteolytic digests of collagen type I.

View larger version (18K): [in this window] [in a new window]   Figure 5. Neutrophil elastase–generated fragments of denatured collagen cause vß3-mediated arteriolar dilation and competitively block ligand binding to the RGD-binding integrin IIbß3. A, Vessel buffer (0.5 mL) was replaced with digested collagen fragment–containing buffer warmed to 34.5°C. Arteriolar diameter was measured until a steady state diameter was maintained for 3 to 4 minutes (total time, <10 minutes). The bath was then washed with fresh buffer to remove fragments, and 100 µg (50 µg/mL) of either the anti-ß3 integrin subunit function–blocking antibody F11 or an MHC class I antigen monoclonal antibody was added to the bath. After a 15-minute incubation with the antibody, fragment addition was repeated. Pretreatment of arterioles with F11 significantly inhibited (P<.01) fragment-induced vasodilation determined by two-sample t test, assuming unequal variances. B, Collagen fragments, cyclic RGD, GRGDSP, and GRGDNP competitively inhibit the binding of biotinylated denatured collagen (dCOL) and fibrinogen to immobilized IIbß3 using an integrin-ligand–binding assay. Bars indicate triplicate values (±SD) with respect to control wells with no competitors.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
An initial and often sustained response to tissue injury is local vasodilation leading to an increase in blood flow to the injured area. Increased local blood flow supports the vascular injury response by delivering inflammatory cells and plasma components to the injured site. Although several mediators of injury-induced arteriolar dilation and increased blood flow are well described,^{20 21} we show data supporting another potential mechanism for this response. Our data show that RGD-containing peptides induce vasodilation by interacting with arteriolar smooth muscle _vß₃ integrin. The effect of RGD peptides appears to be mediated by the vascular SMCs, since removal of the endothelium did not alter the vasodilatory response. RGD peptide also caused vasodilation of KCl- and phenylephrine-constricted vessels, indicating that the vasodilatory response of RGD is not a selective antagonism of myogenic tone.

Involvement of the integrin _vß₃ was implicated by the finding that cyclic RGD and GRGDSP were more potent vasodilators than GRGDNP. Furthermore, pretreatment of the arterioles with a ß₃ integrin subunit function–blocking antibody markedly reduced the vasodilatory activity of cyclic RGD. Therefore, the vasodilatory properties of the RGD peptides appear to result from interaction with vascular smooth muscle _vß₃. However, the possible involvement of other RGD-binding integrins such as ₅ß₁ cannot be excluded.

The mechanism by which RGD peptides cause vasodilation is unknown at present. One possibility is that the RGD peptides disrupt vascular SMC-ECM interactions by competing with existing binding sites. This could lead to a decrease in force transmission between the cells and the vascular wall.²² Similarly, the peptides may antagonize mechanosensor sites involved in signaling the development or maintenance of myogenic tone. Alternatively, RGD peptides may possess "agonist" properties by inducing _vß₃ integrin–dependent signaling. Experimental support for this concept comes from the finding that RGD peptides block the production of platelet-derived growth factor by vascular SMCs adherent to fibrillar collagen.²³ One possible mechanism relates to the finding that RGD-binding integrins can be recruited to focal contacts after addition of soluble RGD peptides.²⁴ Furthermore, these peptides have been shown to facilitate the assembly of focal adhesion complexes in certain contexts.²⁵ Since focal adhesion complexes regulate cell-signaling pathways,³ RGD peptides may alter the makeup of such complexes in vascular smooth muscle, leading to signals that change the contractile behavior of the cells.

As the most abundant proteins in the ECM, collagens are a major potential source of RGD-containing peptide fragments in vivo. Members of the collagen family contain numerous RGD sequences.¹³ Although not exposed under normal conditions, the RGD site(s) is exposed by denaturation and consequently supports _vß₃-mediated cell binding.^{13 14} Collagen is degraded and denatured as a result of, and in response to, tissue injury, including myocardial infarction and burn injury.^{26 27} Various proteolytic enzymes of tissue and inflammatory cell origin can extensively degrade denatured collagens, which may release cryptic RGD sequences as soluble peptide fragments. These fragments could then interact with the local microvasculature causing vasodilation in a manner similar to the synthetic RGD peptides.

Fragments of ECM are known to provide signals to cells during tissue injury and repair,^{28 29 30 31} inviting the possibility that these peptides may be more widely involved in injury-related microvascular events. Examples include fragments of SPARC, fibrin, collagen, and elastin that regulate angiogenesis, vascular permeability, neutrophil chemotaxis, and monocyte/fibroblast chemotaxis, respectively.^{28 29 30 31} Using the isolated arteriole system, we found that neutrophil-elastase–generated fragments of denatured rat collagen type I have vasodilatory properties similar to synthetic RGD peptides. Also, the arteriolar response to the collagen fragments was significantly inhibited by pretreatment of the vessel with a ß₃ integrin function–blocking antibody. These results support the hypothesis that vasodilation associated with tissue injury could in part result from the interaction of RGD-containing fragments of ECM proteins with vascular smooth muscle _vß₃.

	Selected Abbreviations and Acronyms

 

ECM = extracellular matrix MHC = major histocompatibility complex PSS = physiological saline solution SMC = smooth muscle cell TBS = Tris-buffered saline

	Acknowledgments

 
Dr Gerald A. Meininger is supported by National Institutes of Health grant HL-33324 and Texas A & M University FMG 96-41. Dr George E. Davis is supported by the American Heart Association, Texas Affiliate, Inc, grant 94R025. The authors gratefully acknowledge H.J. Granger and J.L. Leibowitz for critical review of this manuscript and J.P. Trzeciakowski for statistical advice.

Received February 20, 1996; accepted July 3, 1996.

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