Functional P2Y2 Nucleotide Receptors Mediate Uridine 5′-Triphosphate–Induced Intimal Hyperplasia in Collared Rabbit Carotid Arteries
Background— Extracellular uridine 5′-triphosphate (UTP) induces mitogenic activation of smooth muscle cells (SMCs) through binding to P2Y2 nucleotide receptors. P2Y2 receptor mRNA is upregulated in intimal lesions of rat aorta, but it is unclear how this G-protein–coupled receptor contributes to development of intimal hyperplasia.
Methods and Results— This study used a silicone collar placed around rabbit carotid arteries to induce vascular injury and intimal thickening. Collar placement caused rapid upregulation of P2Y2 receptor mRNA in medial SMCs before appearance of neointima. Fura-2 digital imaging of single SMCs was used to measure changes in myoplasmic calcium concentration (Cam) in response to P2Y receptor agonists. In contrast to UDP, activation by UTP or adenosine 5′-triphosphate (ATP) greatly increased Cam, which indicates upregulation of functional P2Y2 receptors at which UTP and ATP are equipotent agonists. The number of responsive cells was significantly greater for freshly dispersed SMCs from collared arteries than for controls. Perivascular infusion of UTP (100 μmol/L) within the collar significantly enhanced neointimal development. Intimas that resulted from UTP exposure were infiltrated by macrophages. Moreover, increased expression of osteopontin occurred in response to in situ application of UTP. ATP or UTP also stimulated osteopontin expression in cultured SMCs in a dose-dependent manner. Furthermore, P2Y2 antisense oligonucleotide inhibited osteopontin expression induced by UTP.
Conclusions— These findings indicate for the first time a role for the UTP/ATP receptor, P2Y2, in development of intimal hyperplasia associated with atherosclerosis and restenosis.
Received June 10, 2002; revision received August 13, 2002; accepted August 16, 2002.
Smooth muscle cell (SMC) proliferation and migration play key roles in the development of intimal thickening that accompanies atherosclerosis and restenosis.1,2⇓ Many factors contribute to the development of intimal hyperplasia, although the role of extracellular nucleotides remains largely unknown. Studies indicate that extracellular adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP) mediate SMC activation and proliferation.3–5⇓⇓ Recently, it was shown that UTP induces migration of aortic SMCs via osteopontin expression.6 Effects of extracellular nucleotides are mediated by P2 receptors: ligand-gated ion channels (P2X) and G-protein–coupled receptors (P2Y). P2Y2 receptors (P2Y2R) are activated equipotently by ATP and UTP,7 and P2Y2 mRNA is overexpressed in rat intimal thickenings induced by balloon angioplasty, which suggests a role for P2Y2R in arterial intimal lesion development.8 Organ culture of arteries also elicits functional expression of a UTP receptor.9 In vitro studies showed that extracellular ATP and UTP are released during platelet aggregation10 and from vascular or blood cells under mechanical or chemical stresses.11,12⇓ Alterations in extracellular nucleotide concentrations in response to in vivo injury have been reported.13 Because trauma to vascular endothelium in angioplasty should induce focal release of intracellular nucleotides, it is hypothesized that extracellular nucleotides and P2Y2R promote intimal thickening of arteries, although in vivo studies have not addressed this possibility. The aim of this study was to determine whether activation of P2Y2R promotes intimal hyperplasia under conditions that maintain the vascular endothelium. To directly test this hypothesis, a silicone collar was placed around rabbit carotid arteries to enable local delivery of UTP. Results indicated that collar placement caused rapid upregulation of P2Y2R in SMCs. UTP application to collared carotid arteries induced intimal thickening, expression of osteopontin (OPN), and an inflammatory-proliferative response characteristic of human atherosclerotic lesions. These studies demonstrate a novel role for P2Y2R in development of arterial intimal lesions.
Rabbit SMCs obtained from normal carotid arteries by enzymatic dissociation were maintained in culture as described for rat aortic SMCs8 and used from passages 4 to 6. Phosphorothioate-modified oligonucleotides to rabbit P2Y2 were synthesized and purified by Integrated DNA Technologies (ADT). Sequences including translation initiation site were as follows: sense 5′-GGGCAATGG-CACTCTTCCCTCACCTGACA-3′, antisense 5′-TGTCAGGTGA-GGGAAGAGTGCCATTGCCC-3′. Sequences were checked for uniqueness with the National Center for Biotechnology Information’s Basic Local Alignment Search Tool (BLAST).14 SMCs were incubated with 0.1 or 1 μmol/L P2Y2 sense or antisense S-oligonucleotides for 6 hours in serum-free medium containing 1.4% DOTAP liposomal reagent (Roche Diagnostics). Serum-free medium was added, and cells were cultured for an additional 20 hours. FITC-conjugated P2Y2 antisense and sense oligonucleotides were used to quantify cellular oligonucleotide uptake by SMCs. Cells were rendered quiescent by 24-hour incubation in serum-free medium before stimulation with nucleotides.
Isolation of SMCs and Single-Cell Fura-2 Digital Imaging
SMCs (identified by morphological differences from endothelial cells) expressing α-actin were isolated in enzymatic solution and used for single-cell digital imaging, as described previously.9 Myoplasmic free Ca2+ (Cam) levels were measured with the InCa++ calcium imaging system (Intracellular Imaging Inc). Cells were incubated with 2.5 μmol/L fura-2-acetoxymethylester at 37°C for 25 minutes. Fura-2–loaded cells were placed on a coverslip inside a constant-flow superfusion chamber mounted on an inverted epifluorescent microscope (Nikon; model TMD). Fura-2 was excited by 340 and 380 nm of ultraviolet light, and emitted fluorescence (510 nm) was collected by a monochrome CCD camera (COHU, Inc). Data are expressed as a ratio of emitted light intensity at 340 and 380 nm.
SMCs were continually superfused with physiological salt solution (PSS) containing (mmol/L) NaCl 138, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10, pH 7.4. Depolarizing solution (80K) was composed of (mmol/L) NaCl 65, KCl 80, CaCl2 2, MgCl2 1, HEPES 10, glucose 10, pH 7.4. Caffeine (1,3,7-trimethylxanthine) and nucleotides (Sigma; UTP, UDP, and ATP) were dissolved in PSS and distilled water, respectively, and SMCs were superfused at ≈2 mL/min with PSS that contained the compounds.
Data are expressed as mean±SEM for the number of cells within each group. Analyses of data were done by 1-way ANOVA or Kruskal-Wallis 1-way ANOVA followed by the Bonferroni or Dunn test when more than 2 groups were present. Paired or unpaired Student’s t test was used when 2 groups were compared. SMCs identified as responders had responses to agonist ≥3 SDs above baseline for 5% of agonist exposure time (P≤0.01). Percent responders were analyzed with the χ2 distribution. Statistical analysis was performed with SigmaStat (Jandel Scientific Software). Significance was defined as P<0.05.
Protocols conformed to Animal Care and Use guidelines of the University of Missouri-Columbia. New Zealand White rabbits (42 animals) were anesthetized subcutaneously with ketamine (33 mg/kg body weight) and xylazine (7 mg/kg body weight). Rabbits were maintained under respiration with oxygen and 0.2% isoflurane. A nonocclusive, biologically inert, flexible silicone collar (20 mm long; inlet/outlet diameter 1.8 mm; Silicone MED-4211, Nusil Technology) was placed around a carotid artery, and the contralateral artery was sham operated, ie, isolated from surrounding connective tissue and exposed to similar stretch. Arteries were collected at day 3 (n=12), 7 (n=14), or 14 (n=14). Two collars per animal were implanted in another series of experiments, and each was connected to an osmotic minipump (Alzet 2 ML2; Alza Corporation) implanted subdermally in the thoracic region. The pump continuously delivered (5 μL · h−1) UTP (100 μmol/L) or PBS (mmol/L: NaCl 154, Na2HPO4 8, NaH2PO4 2, EDTA 0.2) for 3 (n=14), 7 (n=14), and 14 (n=14) days. After surgery, analgesic (Buprenex; 0.1 to 0.5 mg/kg body weight) was administered.
Immunohistochemistry and In Situ Hybridization
Rings cut from collar-wrapped or proximal and distal vessel segments outside collars (controls) were formalin fixed, paraffin embedded, or snap-frozen in liquid nitrogen for immunohistochemistry, RNA extraction, or Western analysis. Staining with hematoxylin/eosin was performed. Immunohistochemical detection of SMCs (α-actin; Sigma) and macrophages (CD68; Dako) was performed with monoclonal antibodies visualized by the indirect peroxidase-antibody conjugate technique. To generate P2Y2 riboprobes for in situ hybridization, a T7 promoter adapter was ligated to the 350-bp P2Y2 fragment amplified by reverse transcription–polymerase chain reaction (RT-PCR) with the no-cloning promoter addition kit (Ambion). Antisense and sense riboprobes were obtained by in vitro transcription with digoxigenin-UTP using DIG RNA labeling mix (Roche). Hybridization was performed overnight at 50°C in a humidified chamber, as described previously.8
Intimas were defined as regions between luminal endothelium and the center of internal elastic laminas. Cross-sectional areas of intima and media were measured at 400× magnification with computer-assisted color image analysis (PC-image Color; Foster Findlay Associates). Four sections were analyzed for each artery, and intimal and medial thicknesses at 20 random sites per section were averaged.
Data are expressed as mean±SEM (n=number of arteries). Differences between collared arteries that received UTP or PBS and controls (sham-operated arteries or segments proximal to collars) were evaluated by ANOVA and Student-Newman-Keuls test (significance, P<0.05).
RNA Extraction and RT-PCR
Isolation of RNA, cDNA synthesis, and PCR were performed as described previously.8 PCR was performed with rabbit P2Y2 mRNA oligonucleotide primers (Seye C, Weisman G; Oryctologus Cuniculus P2Y2 receptor partial sequence; GenBank No. AF510345): sense 5′-AACCTGGAACGCCTCCACCA-3′ and antisense 5′-GCACGCTGCTGGTGAACGAAG-3′ or oligonucleotide primers corresponding to positions 901 to 928 and 1302 to 1329 of human P2Y4 cDNA.15 Three different primer pairs used to amplify P2Y6 mRNA corresponded to positions 121 to 146 and 426 to 453; 840 to 865 and 1320 to 1346; and 1120 to 1147 and 1390 to 1415 of rat P2Y6 cDNA.16 Primers for human G3PDH were as follows: sense 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′, antisense 5′CATGTGGGCCATGAGGTCCACCAC-3′. Thirty-five amplification cycles were used, with annealing temperatures of 60°C for P2Y2, 55°C for P2Y4, and 55°C to 62°C for P2Y6. For G3PDH amplification, 25 cycles were used, with 55°C for annealing. PCR products were resolved by 2% agarose gel electrophoresis, and P2Y/G3PDH ratios were determined by densitometry.
Cultured cells were solubilized in 2X Laemmli sample buffer (120 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% sucrose, 1 mmol/L EDTA, 50 mmol/L dithiothreitol, 0.003% bromophenol blue). Rabbit aortas were homogenized with a polytron homogenizer in buffer containing 0.9% NaCl, 20 mmol/L Tris-HCl, pH 7.6, 1 mmol/L PMSF in 0.2% Triton X-100. Equivalent amounts of protein (100 μg) were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting. Detection of OPN (Chemicon International) was performed with rat anti-human OPN (1:1000 dilution) and horse radish-peroxidase–conjugated goat anti-rat IgG (1:1500 dilution) as primary and secondary antibodies, respectively. For signal normalization, membranes were reprobed with anti-mouse α-tubulin antibody (1:1000 dilution; Santa Cruz Biotechnologies).
Upregulation of Functional P2Y2R in Collared Arteries
We examined changes in P2Y2, P2Y4, and P2Y6 mRNA expression in carotid media ex vivo before and after collar implantation. By RT-PCR, P2Y2 transcript was weakly amplified in control arteries and strongly amplified in collared arteries within 3, 7, and 14 days of surgery (Figure 1A). Cultured SMCs expressed high levels of P2Y2 mRNA (Figure 1A). In contrast, P2Y4 mRNA was equivalently expressed in sham-operated and collared arteries or cultured SMCs (Figure 1A), as reported for rat aortic SMC cultures.17 When several oligonucleotide primers were used, the P2Y6 transcript was not detected in carotid arteries or cultured SMCs (Figure 1A). In situ hybridization with sham-operated arteries indicated that P2Y2 mRNA (Figure 1B) was localized to CD31-positive endothelial cells (not shown) and not medial SMCs. High levels of P2Y2 mRNA (Figure 1B) were detected in medial SMCs 3 days after collar placement, before appearance of neointima. At day 14, all intimal and medial cells were P2Y2 positive (Figure 1B).
Single-cell calcium measurements confirmed that newly synthesized P2Y2Rs were functional. SMCs from control and collared (3-day) arteries were exposed to 80 mmol/L KCl to induce Ca2+ influx before treatment with UTP, ATP, or UDP (10 μmol/L) for 4 minutes followed by 3 minutes’ exposure to 5 mmol/L caffeine to release intracellular calcium stores.9,18⇓ Figure 2 shows a typical fura-2 tracing for UTP with control and collared arteries. Percentages of cells responding to agonist in control arteries (Figure 3a) were as follows: UTP, 39% (23/59); ATP, 33% (17/52); and UDP, 2.5% (1/40). In these cells, Cam increased 0.35±0.05, 0.23±0.04, and 0.11±0.02 ratio units above baseline for UTP, ATP, and UDP, respectively (Figure 3b). In contrast, 71% (45/63) and 93% (50/54) of SMCs isolated from collared arteries responded to UTP and ATP, respectively (Figure 3b), with significant increases in Cam (0.62±0.07 and 0.72±0.09 ratio units, respectively) occurring with UTP and ATP, whereas UDP had no significant effect (Figure 3a). A responsiveness index was calculated to include percentages of responding cells and amplitude of responses (Figure 3c) for a more integrative analysis. These results indicate functional upregulation of P2Y2Rs in SMCs of collared arteries. The percentage of SMCs responding to 5 mmol/L caffeine (100%) was consistent in all samples, and amplitudes of caffeine-induced Cam transients were similar between control (0.93±0.06) and collared (0.75±0.04) arteries, which indicates that intracellular Ca2+ stores were relatively equivalent and supports the functional coupling of P2Y2Rs to intracellular Ca2+ stores. Preexposure of SMCs to caffeine abolished the Cam response to 10 μmol/L UTP or ATP (not shown), which confirms that these agonists act at caffeine-sensitive Ca2+ stores, as already reported.9
Effect of Local UTP Application
Collar positioning for 14 days resulted in discrete intimal thickening that encompassed the entire inner surface of vessels (Figure 4), reaching 37.5±8 μm (n=14 arteries), whereas a continuous layer of anti-CD31–stained endothelial cells was maintained (not shown). No neointimas were apparent in sham-operated arteries (n=14). Control vessel segments proximal or distal to collars had normal appearance. Continuous delivery of UTP to vessel segments within collars strongly increased neointimal development by 7-fold (Figure 4), reaching 256±48 μm (n=14 arteries). In contrast, saline infusion (Figure 4) did not significantly increase intimal thickness above collaring alone (39.3±11 μm; n=14 arteries; ANOVA P<0.05).
Collared arteries displayed discrete intimas composed of SMCs, as described previously.19–21⇓⇓ At day 14, macrophages were scattered throughout neointima, representing 2.1±0.1% of total cells (Figure 5A). Enlarged intimas evoked by UTP showed significant macrophage infiltration (12±0.9% of total cells) distributed in the upper neointima beneath the endothelium (Figure 5, A and B). Local saline delivery did not increase intimal macrophage numbers compared with collared arteries without infusion. A few macrophages were occasionally detected in adventitia of collared arteries. However, macrophage infiltration in adventitia was not increased by UTP or saline (Figure 5, A and B). Immunohistochemical staining for CD43 detected no lymphocytes in any collared segments (not shown).
Immunoblots of sham-operated arteries indicated lack of OPN (Figure 6A, lane 5), whereas collared arteries at day 14 expressed OPN (Figure 6A, lane 2). Local UTP infusion greatly increased OPN expression (Figure 6A, lane 4) compared with PBS (Figure 6A, lane 3). OPN was not expressed at day 3 in sham-operated controls (Figure 6B, a) or collared arteries with (Figure 6B, b) or without (not shown) saline infusion. In contrast, OPN expression was increased in medial SMCs by local UTP application (Figure 6B, e) at day 3 after collaring before intimal thickening. At days 7 and 14, OPN was detected in intimal SMCs of collared arteries treated with saline (Figure 6B, c and d), whereas further increases in OPN expression were detected in intimal and medial SMCs of UTP-treated collared arteries (Figure 6B, f and g). SMCs and macrophages closest to the lumen showed the strongest OPN reactivity.
UTP-Induced OPN Expression Is Mediated by P2Y2Rs in SMCs
UTP exposure for 8 hours stimulated OPN expression in vascular SMCs (Figure 7A) in a dose-dependent manner (Figure 7B). ATP and 10% FBS (positive control) induced OPN expression, whereas ADP, 2-MeSATP, and UDP did not (Figure 7A), which suggests that P2Y2Rs mediate OPN expression. To conclusively demonstrate the role of P2Y2Rs, SMCs incubated with 0.1 or 1 μg of P2Y2 antisense or sense oligonucleotides were stimulated with 50 μmol/L UTP for 8 hours. Antisense (Figure 7C, lanes 5 and 6) but not sense (Figure 7C, lanes 2 and 3) P2Y2 oligonucleotides inhibited UTP-induced OPN expression in SMCs.
Novel findings in this study demonstrate striking increases in P2Y2 mRNA in collared carotid arteries before intimal thickening. This temporal relationship provides strong evidence for the causal role of P2Y2R in development of intimal hyperplasia, compared with simple correlations obtained between severity of diabetes/atherosclerosis in diabetic, dyslipidemic pigs and functional expression of UTP-sensitive receptors.22 Collaring of rabbit carotid arteries induces discrete medial injury that leads to SMC activation.19–21⇓⇓ Previous studies indicated rapid upregulation of P2Y2 mRNA in activated thymocytes, an immediate-early gene response.23 P2Y2Rs also are upregulated on ligation of salivary gland ducts.24 P2Y2Rs are activated equipotently by ATP and UTP,7 and the present results showed that activation by ATP or UTP strongly increased Cam in SMCs of collared arteries. The pharmacological potency profile of upregulated P2Y receptors in rabbit carotid arteries does not resemble that of other uridine nucleotide receptors. The P2Y4 receptor is activated preferentially by UTP,15 whereas P2Y6 receptors have greater sensitivity to UDP than UTP.25 UDP is also a P2Y4 receptor agonist, although its potency relevant to UTP varies in different studies.15,26⇓ We found that very few SMCs responded to UDP, and SMCs from collared and control arteries were equally unresponsive to increases in Cam caused by UDP, which rules out any significant contribution from functional P2Y4 or P2Y6 receptors. In addition, SMCs of control and collared arteries showed similar levels of P2Y4 mRNA. These data strongly indicate that P2Y2Rs alone are functionally upregulated in collared arteries.
P2Y2R upregulation may relate to the phenotypic status of cells. SMCs of collared carotid arteries display an activated synthetic phenotype by electron microscopy19 and resemble SMCs of human atherosclerotic plaques. Partially dedifferentiated SMCs of angioplasty-induced rat aortic lesions express high levels of P2Y2 mRNA, as do cultured SMCs8,17⇓ that undergo dedifferentiation.27 Moreover, medial SMCs of rat embryo exhibit high levels of P2Y2 expression,8 similar to collared arteries. Finally, organ-cultured coronary SMCs display upregulation of UTP-preferring receptors associated with dedifferentiation and increased DNA synthesis.9 Thus, it is very likely that upregulation of P2Y2Rs in collared arteries is associated with SMC dedifferentiation. Increased P2Y2R expression may be sufficient to enhance local effects of extracellular nucleotides on SMC proliferation, thereby contributing to intimal thickening. Importantly, upregulation of P2Y2Rs is much greater in SMCs than in endothelium, which should promote SMC proliferation and migration to a greater extent than P2Y2R-mediated release of endothelium-derived relaxing factors that could counteract hyperplasia.
Reports of effects of extracellular nucleotides in vivo are limited. Because cellular ATP and UTP are presumably released during angioplasty, we examined whether activation of P2Y2Rs contributed to development of intimal hyperplasia. Intimal thickening of collared carotid arteries was greatly enhanced by in situ UTP application and was closely associated with OPN expression in medial SMCs by day 3, when neointimas were not apparent. OPN is chemotactic for SMCs and is associated with arterial SMC proliferation.28 The present data suggest a role for OPN in proliferation and migration of SMCs, processes involved in intimal thickening. Furthermore, UTP and ATP increased OPN expression in cultured SMCs, whereas ADP, UDP, and 2-MeSATP were ineffective, which suggests a role for P2Y2R in which ATP and UTP are equipotent. Direct evidence for involvement of P2Y2R is provided by inhibition by P2Y2 antisense oligonucleotides of UTP-induced OPN expression in cultured SMCs. Furthermore, UTP induced OPN expression in P2Y2-transfected human 1321N1 astrocytoma cells (not shown).
P2Y2Rs interact with αvβ3-integrin,29 and anti-αvβ3-antibodies inhibited UTP-induced migration of vascular SMCs associated with OPN expression.6 Recent reports indicate that UTP-induced SMC migration in vitro is mediated by P2Y2Rs.30 A hypothesis is that arterial injury caused by silicone collar placement induces P2Y2R expression, whereupon receptor activation increases OPN expression and activates αvβ3-integrin to promote SMC migration in arteries.
Local UTP delivery induced intimal accumulation of macrophages similar to oxidized LDL.20 Macrophage distribution indicated that inflammatory responses induced by UTP were localized to neointima. Although mechanisms of UTP-induced monocyte/macrophage infiltration into intima remain unclear, leukocyte migration depends on activities of adhesion proteins (eg, selectins and integrins) on leukocytes and vascular endothelial cells. Preliminary data (C.I.S., unpublished results, 2001) indicate that UTP stimulates expression of vascular cell adhesion molecule-1 in human endothelial cells. Nucleotides also regulate leukocyte adhesion to endothelium,31 which suggests a role for UTP in early stages of leukocyte infiltration.
It is difficult to estimate locally effective concentrations of agonists in vivo. A tissue-free space corresponding to 100 μL is available inside the carotid artery collar, and with delivery of 100 μmol/L UTP at 5 μL/h, UTP concentrations of ≈5 μmol/L should be maintained around carotid arteries. However, disappearance of UTP through hydrolysis and lymphatic drainage makes estimations suspect. Continuous periarterial delivery of UTP within collars should enable UTP to reach SMCs in outer and inner medial layers. UTP (1 to 100 μmol/L) can stimulate various cellular events, eg, proliferation and migration of vascular cells.3–6⇓⇓⇓ Although these concentrations appear high compared with reported circulating levels, such concentrations might be locally achieved in vivo on cell lysis that occurs during transluminal angioplasty or atherosclerosis. Thus, UTP concentrations used in the present study may be relevant to in vivo arterial injury.
In conclusion, these studies demonstrate for the first time that extracellular UTP and P2Y2R contribute to development of arterial lesions associated with atherosclerosis and restenosis.
This study was supported by the American Heart Association, American Diabetes Association, National Institutes of Health, and F21C and Molecular Biology Programs of University of Missouri–Columbia. The authors thank Jean Camden and Marieme Ndiaye for excellent technical assistance.
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- ↵Kockx MM, De Meyer GRY, Jacob WA, et al. Triphasic sequence of neointimal formation in the cuffed carotid artery of the rabbit. Arterioscler Thromb. 1992; 12: 1447–1457.
- ↵Matthys KE, Van Hove CE, Kockx MM, et al. Local application of LDL promotes intimal thickening in the collared carotid artery of the rabbit. Arterioscler Thromb Vasc Biol. 1997; 17: 2423–2429.
- ↵De Meyer GRY, Kockx MK, Cromheeke KM, et al. Periadventitial inducible nitric oxide synthase expression and intimal thickening. Atheroscler Thromb Vasc Biol. 2000; 20: 1896–1902.
- ↵Hill BFJ, Dixon JL, Weisman GA, et al. Diabetes upregulates a UTP-preferring P2 nucleotide receptor in coronary smooth muscle cells. Diabetes. 1998; 47 (suppl 1): A443. Abstract.
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