expression of endothelial cell adhesion molecules on heart valves: up-regulation in degeneration as...
TRANSCRIPT
Original Paper
Expression of endothelial cell adhesion molecules onheart valves: up-regulation in degeneration as well asacute endocarditis
Annette M. MuÈller1,2*, Claus Cronen2, Leon Iri Kupferwasser3, Hellmut Oelert4, Klaus-Michael MuÈller1 and
C. James Kirkpatrick2
1 Institute of Pathology, University Clinic Bergmannsheil, Ruhr-University Bochum, Germany2 Institute of Pathology, Johannes Gutenberg University, Mainz, Germany3 Department of Medicine II, Johannes Gutenberg University, Mainz, Germany4 Department of Cardiovascular and Thoracic Surgery, Johannes Gutenberg University, Mainz, Germany
*Correspondence to:Dr Annette M. MuÈller,BerufsgenossenschaftlicheKlinikenBergmannsheil±UniversitaÈtsklinik,BuÈrkle-de-la-Camp-Platz 1,D±44789 Bochum, Germany.
Received: 1 April 1999
Revised: 12 October 1999
Accepted: 2 November 1999
Abstract
In¯ammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-a),
as well as shear stress, cause endothelial cells (ECs), to undergo not only functional alterations
but also structural reorganizations, which contribute to vascular leakage. Like ECs of the human
aorta, ECs on heart valves are exposed to extreme shear stress. However, while ECs expression of
cell adhesion molecules (CAMs) in large vessels has been widely studied, it seems that there are no
such studies on ECs of heart valves, although this knowledge might be important for our
understanding of the aetiological aspects of local in¯ammatory responses. Using immunohisto-
chemistry, this study characterized the CAM expression of ECs on degenerative, mostly calci®ed
heart valves and on heart valves with ¯orid endocarditis. As expected, the constitutively expressed
molecules (ICAM-1, CD34, CD31) were found both on degenerative and on in¯amed valves.
Furthermore, marked expression of E-selectin and VCAM-1 was found not only on in¯amed
valves, but also on larger portions of the degenerative valves with no morphological evidence of
in¯ammation. This striking ®nding might help to explain why patients with ®brotic heart valves
are susceptible to recurrent endocarditis. Why the endothelial activation markers E-selectin and
VCAM-1 are expressed on degenerative heart valves requires further investigation. Copyright #2000 John Wiley & Sons, Ltd.
Keywords: endothelial cells (EC); heart valve; endocarditis; cytokines; cell adhesion molecule;
E-selectin; VCAM-1; immunohistochemistry; confocal laser scanning microscopy
Introduction
Adhesion molecules promote the participation ofendothelial cells (ECs) in in¯ammatory responsesthrough the active recruitment of immune effectorcells and play a crucial role in the pathomechanisms ofvascular diseases. It is known that they differ in theirantigen expression depending on their location andtheir state of activation [1±3]. Like ECs of large vesselwalls such as of the aorta, ECs of heart valves areexposed to high shear stress. Interestingly, ECs ofheart valves have a speci®c morphological structure;they form surface microvilli [4].
In the pathogenesis of atherosclerosis and ofin¯ammation in general, the interactions of bloodmonocytes and ECs are important [5,6]. Here, celladhesion molecules (CAMs) presented by vascular ECsboth constitutively and after stimulation play a crucialrole; as soon as speci®c adhesion molecules areexpressed, granulocytes and monocytes adhere to theendothelial lining, followed by transmigration acrossthe endothelium [7].
To our knowledge, there are no studies examiningcell adhesion molecules on ECs of heart valves,
although it may be postulated that they play animportant role at least in the pathogenesis of ¯oridendocarditis. Hence the aim of the present study wasto evaluate the expression patterns of the CAMsICAM-1, CD34, PECAM-1 (CD31), E-selectin, andVCAM-1 on ECs of heart valves and to correlate thesewith the morphological status of the valves, especiallythe presence or absence of signs of in¯ammation.
On ECs, ICAM-1 (CD57) elicits moderate basalexpression and is stimulated by interleukin-1 (IL-1),tumour necrosis factor-alpha (TNF-a), and interferon-gamma (IFN-c) [8±10]. Lukacs et al. [11] demonstratedthat ICAM-1-mediated monocyte interactions withactivated HUVECs (human umbilical vein endothelialcells) increase monocyte-derived chemoattractant pro-tein-1 production. CD34 is a transmembrane proteinpresented on immature haematopoietic precursor cells.It is also presented by vascular ECs of normal andneoplastic tissues [12±15]. PECAM-1 (CD31), anadhesion molecule of the immunoglobulin superfamily,is constitutively expressed by all vascular ECs inadults. It is concentrated near the intercellular bordersof adjacent cells, mediating leukocyte±endothelialinteractions and playing a role during the emigration
Journal of PathologyJ Pathol 2000; 191: 54±60.
Copyright # 2000 John Wiley & Sons, Ltd.
of leukocytes into a focus of in¯ammation [16±18]. Inin vitro models and in animal models, E-selectin andVCAM-1 were recognized as surface receptors, induci-ble, for example, by IL-1, TNF-a or lipopolysacchar-ides (LPS) [7,9,19±23]. E-selectin mediates neutrophiladhesion to cytokine-activated ECs and plays a role inleukocyte traf®cking in acute and chronic in¯amma-tory responses [20]. Cultured ECs express E-selectinwithin 1±2 h after stimulation with interleukins [9,24].Maximal expression is found 4±6 h later [25].VCAM-1, induced more slowly than E-selectin, bindsmonocytes and lymphocytes, but not neutrophils. Theexpression of both E-selectin and VCAM-1 is asso-ciated with a variety of in¯ammatory processes andwith atherosclerosis [26,27].
In the present study, we characterized the endo-thelial linings of heart valves by their expression ofthese adhesion molecules. As FVIII-related antigen isconstitutively expressed in ECs, this marker was usedas an identi®cation marker for the control stainingreaction for ECs. The co-expression of FVIII-relatedantigen and of ICAM-1 and E-selectin was veri®ed byconfocal laser scanning microscopy.
Materials and methods
Forty aortic valves excised during a prosthetic valveoperation and two aortic valves from post-mortem,both taken not later than 6 h after death, were in-vestigated. The two valves from post-mortem and sixof the surgically resected valves showed macroscopicsigns of acute ulcerative endocarditis (Figure 1A).Thirty-four of the surgically resected valves (all weretricuspid, none was bicuspid) showed no macroscopicsigns of in¯ammation, but ®brotic degeneration, withor without calci®ed nodules in the stroma (Figure 1B).The mean age of the patients was 67 years (range57±81 years); 26 of the patients were men, 16 women(Table 1).
The specimens were obtained by cutting the valvesfrom the free edge towards the insertion rim; they werethen snap-frozen in isopentane, pre-cooled in liquidnitrogen (x196uC), and stored at x70uC. Then cryo-sections of 5 mm were cut and mounted on gelatin-coated glass slides, dried at room temperature over-night, and ®xed in 100% acetone for 10 min at 0uC.The sections were either stained immediately or stored,wrapped in aluminium foil, at x20uC until staining. Inorder to obtain large areas of intact ECs in bothgroups, the samples were taken from regions without
macroscopic signs of ulceration or calci®cation. Themacroscopic examination was performed with thenaked eye, without the use of a hand lens or dissectingmicroscope. Because of the danger of destroying theendothelium in areas with marked calci®cation, thespecimens were taken from adjacent areas. Ulcerationwas treated in a similar fashion. This preselection wascontrolled by subsequent microscopy of the specimens.Specimens without an intact endothelial layer, or withextensive ulceration, were excluded. To evaluate themorphological status, each specimen was stainedadditionally with haematoxylin and eosin (H&E stain-ing). As all patients had received pre-operative anti-biotic treatment over several days, no staining forbacteria was performed.
For the immunohistochemical study, only sampleswith intact or at least long stretches of ECs werechosen. Thus, only specimens from the neighbourhoodof an ulcerative lesion were taken, never from theulcerated region itself.
The sections were stained by the avidin±biotincomplex (ABC), immunoperoxidase technique or bythe indirect immunoperoxidase technique. Slides werewashed in phosphate-buffered saline (PBS, pH 7.4) andincubated with 0.3% H2O2 for 30 min at roomtemperature. After washing in PBS, the slides wereincubated for 20 min with 1% horse serum in PBS. Thesections were incubated with primary antibodiesdiluted in 1% bovine serum albumin (BSA)/PBS for60 min. The dilutions of the monoclonal antibodiesanti-FVIII, anti-ICAM-1, anti-CD34, anti-PECAM-1,anti-E-selectin, and anti-VCAM-1 are listed in Table 2.The slides were washed in PBS and, for indirectimmunoperoxidase staining, overlaid with a 1 : 50dilution of peroxidase-conjugated rabbit anti-mouseIg for all antibodies except anti-FVIII-Ig. For FVIIIstaining, swine anti-rabbit IgG in a dilution of 1 : 50was used. In the ABC staining, this step was omittedand instead a 1 : 200 dilution of biotinylated rabbitanti-mouse IgG was incubated for 30 min. Afterwashing in PBS, the slides were incubated for 30 minwith peroxidase-conjugated avidin±biotin complex andwashed again in PBS. The colour reaction used 3,3-diaminobenzidine hydrochloride and H2O2 for 10 min.Slides were counterstained with haematoxylin for 30 s,washed in water, and mounted in a water-solublemedium, prior to routine light microscopy. Two non-speci®c binding control slides were prepared each timea tissue was examined.
Table 1. Age and sex of the patients with degenerative®brotic aortic valves and acute endocarditis
Fibrotic, degenerative
valves with calci®cation
Valves with acute
endocarditis
Female 14 2
Male 20 6Mean age (years) 71 63
Table 2. Monoclonal antibodies used in this study
First antibody Dilution Purchased from
FVIII 1 : 3000 DAKO, DenmarkICAM-1 1 : 400 Immunotech S.A., France
E-selectin 1 : 100 Immunotech S.A., France
VCAM-1 1 : 100 Immunotech S.A., FrancePECAM-1 1 : 100 Immunotech S.A., France
CD34 1 : 400 Immunotech S.A., France
Endothelial cell adhesion molecules on heart valves 55
Copyright # 2000 John Wiley & Sons, Ltd. J Pathol 2000; 191: 54±60.
The co-expression of E-selectin and FVIII, as well asthat of FVIII and VCAM-1, was demonstrated byCLSM (confocal laser scanning microscopy). By thismethod, the co-expression of cell adhesion mole-cules and FVIII could be demonstrated in threedimensions. This was important because especially inulcerated valves, the precise morphology of the ECson the valve surface might be dif®cult to interpret.Double immuno-staining was performed by combiningthe detection of antibodies labelled with ¯uoresceinisothiocyanate (FITC; DAKO, Denmark) and with
Texas red (Dianova, Germany) respectively. Brie¯y,the ®rst unlabelled antibody, anti-E-selectin immuno-globulin, diluted 1 : 50 in a blocking kit (Boehringer,Germany) or anti-ICAM-1 antibodies, diluted 1 : 100in a blocking kit, was mixed with anti-FVIII anti-body in a concentration of 1 : 500. After incubationfor 12 h at 4uC, the specimens were washed in PBS.Then the FITC-labelled second antibody in a concen-tration of 1 : 50, or the Texas red-labelled secondantibody in a dilution of 1 : 100 was applied. After-wards the slides were mounted in a water-soluble
A B
C D
E F
Figure 1. (A, B) Macroscopic appearance of an aortic valve with acute endocarditis (A) and a valve with ®brotic degeneration withcalci®ed nodules in the stroma (B). (C) Strong immunohistochemical reaction of endothelial cells with antibodies against ICAM-1 onan ulcerated valve (r99). The tissue is derived from the neighbourhood of an ulcerated lesion. (D) Strong staining of endothelialcells of a degenerative valve for E-selectin (r189). There is no leukocyte in®ltrate in the subendothelial stroma. (E) Moderatestaining of endothelial cells with antibodies against VCAM-1 on degenerative heart valves (r99). (F) Endothelial cells stained by¯uorescein isothiocyanate (FITC) (green) and Texas red (red) for confocal laser scanning microscopy: the endothelial lining on adegenerative valve demonstrates the co-expression of FVIII (green) and E-selectin (red). The co-expression of both molecules resultsin a yellow colour (r189).
56 A. M. MuÈ ller et al.
Copyright # 2000 John Wiley & Sons, Ltd. J Pathol 2000; 191: 54±60.
medium and examined using a Leica confocal laserscanning microscope.
The intensity and the extent of endothelial cellstaining for ICAM-1, E-selectin, VCAM-1, PECAM-1and CD34 were scored semiquantitatively based on ascale from x to +++ as follows: grade 0 (x): nostaining; grade 1 (+): mild but de®nite staining, grade2 (++): moderate staining, grade 3 (+++): stronglypositive.
The immunoperoxidase-stained tissue sections werereviewed in blinded fashion by two observers. Anydifferences of grades assigned by the two observerswere resolved by joint examination.
Results
On H&E staining, valves with acute endocarditisshowed thrombotic ®brinous layers over ulcers. Thestroma underneath showed signs of acute in¯amma-tion, with in®ltrates of granulocytes, plasma cells, andlymphocytes. The endothelial layers in the neighbour-hood of the ulcers were intact. The ®brotic valvesshowed a hyaline stroma with calci®ed areas andsometimes with in®ltrates of lymphocytes and plasmacells next to these foci.
On immunohistochemistry, anti-FVIII elicited mod-erate to strong staining for the ECs of all 42 valves.
ICAM-1
There was mild to moderate staining for ICAM-1 ofECs on 15 degenerative ®brotic valves. Seven valvesshowed moderate and 12 valves mild but de®nitelypositive staining of the endothelial layer. Of the valveswith acute endocarditis, the ECs of two gave a stronglypositive (Figure 1C), ®ve a moderate, and one a mildbut de®nite immunohistochemical reaction.
CD34
CD34 was mildly or moderately expressed by the ECsof 17 out of 19 ®brotic valves: the endothelial layers ofthree of the 17 valves were moderately stained, whilethe other 14 showed mild but unequivocal reactions.The ECs of two valves gave no staining. The samplesfrom seven of the eight valves with acute endocarditiswere positive: the ECs of three valves showed moderatestaining and the ECs of four valves were mildly butde®nitely positive. One valve gave a negative stainingreaction.
PECAM-1
PECAM-1 was expressed mildly to strongly on theECs of degenerative valves with ®brosis: the ECs of®ve valves showed strong, 15 moderate, and four mildbut de®nite immunohistochemical reactions. Moderateto strong staining of the ECs was found in samplestaken from valves with acute endocarditis: the ECs oftwo valves were strongly, and of six moderatelystained.
E-selectin
The ECs of 21 of the degenerative ®brotic valves werepositive for E-selectin: three valves showed a strongreaction (Figure 1D), 11 showed moderate, and sevenmild but de®nite staining of the endothelium. The ECsof 14 valves were completely negative.
In samples from valves with ulcerative endocarditis,E-selectin was strongly positive on the endothelium ofone valve, four were moderately stained and three weremildly but de®nitely positive.
VCAM-1
For VCAM-1 we found moderate reactions of the ECson 11 of the degenerative ®brotic valves (Figure 1E);the endothelium of 15 valves was mildly but de®nitelystained. Eight valves with ®brosis were negative forVCAM-1. Of the valves with acute endocarditis, theendothelial layer of two valves showed a strong, ofthree valves a moderate, and of three valves a mild butde®nite staining reaction.
CLSM
The strong co-expression of ICAM-1 and FVIII and ofE-selectin and FVIII was demonstrated by CLSM(Figure 1F).
Discussion
According to their location, function, and state ofactivation, ECs differ in their antigen expression[1±3,7]. To our knowledge, up to now there have beenno studies concerning the expression patterns of CAMsof ECs on heart valves with pathological lesions. Thismay be due to the dif®culties of obtaining andpreparing the specimens.
ICAM-1
We found a positive antibody reaction for ICAM-1 on®brotic as well as on in¯amed valves, while culturedECs and ECs in an organ culture show a low basicexpression of ICAM-1 that can be increased bytreatment with cytokines, such as IL-1 and TNF-a, aswell as UV irradiation or ionizing radiation [28].Lukacs et al. [11] demonstrated that ICAM-1-mediatedinteractions on activated HUVECs increase monocyte-derived monocyte chemoattractant protein-1 produc-tion. Combe et al. [29] provided evidence that mono-cytes stimulate and amplify their adhesion to ECsby induction of ICAM-1, thus constituting a self-perpetuating positive feedback system. The binding ofleukocytes by this adhesion molecule and the trans-migration of leukocytes are regarded as part of theinitiation and progression of atherosclerosis: Ridkeret al. [30] found support for the hypothesis that allcellular mediators of in¯ammation have a role inatherogenesis. As alterations in the valvular stroma ofdegenerative valves show some morphological similar-
Endothelial cell adhesion molecules on heart valves 57
Copyright # 2000 John Wiley & Sons, Ltd. J Pathol 2000; 191: 54±60.
ity to vascular atherogenesis, the question is stillunresolved as to whether ICAM-1 plays a role indegenerative processes in heart valves.
Another explanation for the mostly strong constitu-tive expression of ICAM-1, regardless of the presence ofunderlying in¯ammation, is given by the study of Chiuet al. [31]. They proved that shear ¯ow to ECs can induceintracellular reactive oxygen species (acting as intracel-lular second messengers), which may result in anincrease of ICAM-1 mRNA levels via transcriptionalevents. Hwang et al. [32] found that recruitment ofcirculating leukocytes at sites of atherosclerosis ismediated through adhesion molecules and that plasmalevels of ICAM-1 and E-selectin may serve as molecularmarkers for atherosclerosis and the development ofcoronary heart disease. ICAM-1 expression onHUVECs reaches a maximum at 24 h after stimulationor later [9]. Although we found the same intensity forICAM-1 on degenerative and ®brotic valves, and onvalves with acute endocarditis, an uneven expression hasbeen described in the literature. Lee et al. [33] correlatedthis heterogeneous expression on unstimulated andstimulated HUVECs with the DNA synthetic activityof ECs; thus, little DNA activity was found in stronglyICAM-1-positive cells, but active DNA synthesis wasobserved in faintly ICAM-1-positive ECs.
CD34
CD34 is a haematopoietic stem cell antigen, as well asan endothelial transmembrane protein which appearsto be involved in leukocyte adhesion and endothelialcell migration during angiogenesis [34]. Immuno-electron microscopic studies suggest that CD34 isconcentrated on membranes, most of which interdigi-tate between adjacent ECs, although CD34 is locatedat a distance from the tight junctions themselves [12].As CD34 staining elicited the same intensity as FVIIIin all valve specimens examined by us, this antigenseems to be constantly expressed by ECs of cardiacvalves. According to Suster and Wong [35], theexpression of CD34 proves that ECs of heart valvesare mature cells. In this study we could not ®ndsupport for the theories of Delia et al. [14], that CD34might have a negative modulating role on adhesionfunctions of ECs by a reciprocal regulation ofE-selectin and CD34. On the contrary, we foundstrong expression of both CD34 and E-selectin in anin vivo study [36].
PECAM-1
PECAM-1 is concentrated at the intercellular bordersof adjacent cells and seems to play a distinct role in theemigration of leukocytes into a focus of in¯ammationand vascular permeability [16,37,38]. According toDeLisser et al. [39], the activity of PECAM-1 isregulated by variations in its cytoplasmic domain.Like Romer et al. [16] and Scholz et al. [40], we foundthe same staining intensity for PECAM-1 on valveswith and without signs of acute in¯ammation. Here
our observations correlate with those of Gibbs et al. [41],who found no change in the pattern of PECAM-1expression in renal biopsies following kidney transplan-tation, although in the same biopsies there was strongVCAM-1 and E-selectin expression, suggesting that theECs were in an activated state. Likewise, Heckmann et al.[28] saw no change in vascular immunoreactivity forPECAM-1 in skin organ culture after stimulation byionizing radiation. Wong and Dorovini-Zis [42] foundthat in primary cultures of human brain microvessels,endothelial cell surface expression of PECAM-1 was notaltered by lipopolysaccharide treatment. Henninger et al.[43] also describe unaltered expression of PECAM-1 inthe microvasculature of different organs in the mouseafter cytokine stimulation, although Stewart et al. [38]found that treatment of ECs with TNF-a, and/or IFN-cled to dramatic decreases in the steady-state levels ofPECAM-1 mRNA transcripts. According to Romer et al.[16], TNF-a and INF-c do not alter PECAM-1 transcrip-tion or total surface PECAM-1, but elicit a change inPECAM-1 cytoskeletal association, with a change inlocalization at the cell surface. Moreover, engagement ofPECAM-1 may alter a number of ECs functions,including the secretion of vasoactive mediators [37].
VCAM-1 and E-selectin
Cytokines such as IL-1 and TNF-a, as well as LPSinduce E-selectin and VCAM-1 expression onHUVECs, as well as in vivo [44±48]. The transferabilityof these results to animal models was shown by Redlet al. [22] in baboons with septic shock. Up to now, E-selectin has been regarded as being expressed by ECs inearly and active states of in¯ammation. VCAM-1expression, on the other hand, is thought to character-ize later phases of in¯ammation. The expression of E-selectin and VCAM-1 on in¯amed, focally ulceratedvalves was therefore expected, although we couldnot con®rm a time-dependent correlation. However,according to Fries et al. [47], the induced expression ofE-selectin and VCAM-1 in animals can persist forlonger than 3 days. On the other hand, the studies ofHuang et al. [49] indicate that endothelial activationand the expression of E-selectin correlates with acontinuous presence of cytokines.
An unexpected ®nding is the expression of E-selectinand VCAM-1 by ECs on degenerative heart valves, asthese valves neither clinically, macroscopically normicroscopically gave any morphological sign of acuteor chronic in¯ammation.
Local cytokine release, such as IL-1 or TNF-a, cantherefore be ruled out. None of the patients in our studyhad clinical signs of a systemic in¯ammatory response orsepsis. One explanation might be that degenerativealterations of the valvular stroma initiate pathogeneticmechanisms similar to those in atherosclerosis.
Davies et al. [50] emphasized that the same riskfactors that enhance atherosclerosis may enhance valvecalci®cation. Moreover, Otto et al. [51] found that inearly excision of degenerative aortic stenosis, an active
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in¯ammatory process can be seen with similarities toatherosclerosis, such as leucocyte in®ltration.
The expression of VCAM-1 and E-selectin has beenproven in atherosclerotic lesions [52±55]. Animal model®ndings have provided evidence that the expression ofVCAM-1 in arterial endothelium is up-regulated beforethe accumulation of intimal macrophages [55]. In ourstudy, only a proportion of the degenerative valvesshowed macrophages. It is known that cardiopulmonarysurgery induces a systemic in¯ammatory response inwhich E-selectin, VCAM-1, and ICAM-1 appear to beof considerable importance [56]. However, if thisexplanation were valid, all of our degenerative ®broticheart valves should have had positive staining for E-selectin and VCAM-1. This was not the case.
Another explanation for the expression of activatedadhesion molecules might be the constant exposure ofthe ECs to shear stress. Gonzales and Wick [6] demon-strated that shear stress induces endothelial VCAM-1expression and increases monocytic cell adherence via aVCAM-1/alpha 4 beta 1 mechanism. When we reviewedthe medical history of the patients whose valves stainedfor E-selectin and VCAM-1, we found a broad spectrumof co-existing diseases such as bronchial carcinoma,diabetes mellitus or ankylosing spondylitis. In theliterature, low E-selectin expression has been describedfor chronic diseases, including chronic in¯ammatoryprocesses and autoimmune responses: Mrowka andSieberth [57] suggested that levels of circulating adhesionmolecules might re¯ect different pathophysiologicalprocesses in systemic vasculitis and endothelial/immuneactivation in non-in¯ammatory renal diseases. Wenischet al. [58] presented evidence that the targeting andrecruitment of in¯ammatory cells to vascular endothe-lium in thyrotoxicosis is mediated by ICAM-1, E-selectin and VCAM-1. In addition, al-Saffar et al. [59]demonstrated that there is a constant up-regulation of E-selectin on the vascular ECs at bone±implant interfaces.
Regardless of the underlying signal transductionmechanisms, the expression of E-selectin and VCAM-1may be a major component of the pathogenesis ofheart valve in¯ammation and ulceration. It is alsopossible that the expression of E-selectin and VCAM-1might be a prognostic factor for those valves at risk ofbecoming in¯amed. Finally, screening for the serumlevels of E-selectin and VCAM-1 could prove to be amarker for patients at risk of developing acuteendocarditis. To clarify this, a prospective clinicalstudy is required.
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