AcknowZedgrnents-We acknowledge Dr. Fred Keeley for construc- tive discussion and help with .... 31-47, Academic cress, d w. Urry, D. W. (1984) J. Cell Biol.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry
Vol. 268, No. 2, Issue of January 15, pp. 1405-1413,1993 Printed in U.S.A.
and Molecular Biology, Inc.
The Ductus Arteriosus Migratory Smooth Muscle Cell Phenotype Processes Tropoelastin to a 5%kDa Product Associated with Impaired Assembly of Elastic Laminae* (Received for publication, Aleksander
Hinek
and Marlene
October 28, 1991, and in revised form, July 10, 1992)
RabinovitchS
From the Departments of Pediatrics and Pathology, Research Institute, The Hospital for Sick Children,
University of Toronto, Toronto, Ontario, M5G
We established the identity of a 52-kDa protein secreted by fetal lamb ductus arteriosus (DA) smooth muscle cells (SMC) and suggest how it might be related to structural changes unique to DA development, i.e. reduced assembly of elastic laminae and associated formation of intimal cushions. We produced a monoclonal antibody (HI-20) to the 52-kDa protein and observed, by electron microscopy, immunogold labeling of elastin in both DA and aorta vessel walls. Western immunoblotting showed that HI-20, as well as antibodies to tropoelastin, reacted with the 52-kDa protein secreted by DA SMC, as well as with 68-kDa tropoelastin. The highly specific antibody to the carboxyl-terminal sequence of tropoelastin failed, however, to recognize the 52-kDa protein, although it reacted well with the 6%kDa tropoelastin. Amino acid analysis and sequencing data confirmed the identity of the affinity-purified 52-kDa protein as truncated tropoelastin with an intact amino terminus. Cell-free translation of mRNA extracted from DA and aorta SMC produced a 6%kDa, but not a 52-kDa, immunoprecipitated tropoelastin. When DA and aorta SMC were pulsed with [“Clvaline, we immunoprecipitated, after only a 15-min chase, both 68-kDa and 52-kDa tropoelastin from cell extracts of DA SMC, but only the 6%kDa tropoelastin was present in aorta SMC. There was no evidence of proteolytic degradation of radiolabeled aorta 6%kDa tropoelastin to a 52-kDa species when mixed with DA SMC conditioned medium. This suggests that the 52-kDa tropoelastin is the result of cell-associated processing or degradation of an original 6%kDa product of translation. Furthermore, pulse-chase experiments showed initial secretion of equivalent amounts of 68-kDa and 52-kDa tropoelastins by cultured DA SMC with increasing accumulation of the 52-kDa species, suggesting its impaired insolubilization. The production, in high concentration, of a 52-kDa tropoelastin product that lacks the carboxyl terminus, may prevent its alignment on the * This work was supported by Grant MT 8546 from the Medical Research Council of Canada. This study was presented in part at the Annual Meeting of the American Society of Cell Biolom, San Diego. CA, December, 1990, and was published-in abstract fo& (Hinek, 1.1 and Rabinovitch, M. (1990) J. Cell Biol. 111.445 (abstr.)). The costs of publication of this article were defrayed in part‘by the ‘payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Career Investigator of the Heart and Stroke Foundation of Ontario. To whom correspondence should be addressed: Division of Cardiovascular Research, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, M5G 1X8 Canada. Tel.: 416-813-5918; Fax: 416-813-7480. 1405
and the Division IX8 Canada
of Cardiovascular
Research,
mi&ofibrillar scaffold, resulting in abnormal assembly of elastic laminae in the DA. The accumulation of this soluble tropoelastin may be associated with the previously described property of chemotaxis resulting in the increased SMC migration into the subendothelium associated with DA intimal thickening.
The ductus arteriosus (DA)’ is a fetal vessel which undergoes extensive remodeling associated with the development of intimal cushions in late gestation (Gittenberger-de-Groot et al., 1980, 1985; Yoder et al., 1978). These structures assure that the vessel will close completely when it constricts in the postnatal period. Absence or incomplete development of intimal cushions is associated with persistent patency of the DA (Buchanan, 1978; Patterson, 1979; Gittenberger-de-Groot et al., 1980, 1985). Intimal cushion formation is characterized by migration of smooth muscle cells @MC) into the subendothelium, a process we have shown is related to increased production of specific extracellular matrix components, hyaluronan (De Reeder et al., 1988; Boudreau and Rabinovitch, 1991; Boudreau et al., 1991), chondroitin sulfate, and fibronectin (Boudreau and Rabinovitch, 1991; Boudreau et al., 1991). However, DA SMC migration and intimal cushion formation are also consistently accompanied by striking alterations in elastin fiber assembly (Patterson, 1979; Rabinovitch et al., 1988; De Reeder et al., 1990). Elastic fibers appear fragmented and normal Iaminae are not formed (Fig. 1). We have shown that there is decreased insolubilization of elastin in the DA that is unrelated to reduced lysyl oxidase activity or heightened elastin turnover (Zhu et al., 1993) but that is associated with a reduction in DA SMC surface elastinbinding proteins (Hinek et al., 1991). We reported previously that fetal lamb DA SMC in culture harvested at the time of active remodeling and intimal cushion formation differ from those of the aorta and pulmonary artery by their production of an uncharacterized 52-kDa protein (Rabinovitch et al., 1988). In this study, we produced a monoclonal antibody to the 52-kDa protein isolated from the DA conditioned medium and used it to localize this protein in the vessel wall. We established the identity of the 52-kDa protein as a truncated tropoelastin lacking the carboxyl-terminal region. We went on to show that this protein appears to result from intracellular processing, i.e. post-translational modification or degradation, or it may derive from an isoform susceptible to intracellular cleavage. Alternatively, it may be a tropoelastin which is cleaved at the time of transport ’ The abbreviations used are: DA, ductus arteriosus; SMC, smooth muscle cell(s); PAGE, polyacrylamide gel electrophoresis.
1406
-
Tropoelastin ti2-kDa Product
:nd Impaired Elastin Assembly
(II
confluence, the SMCfrom aorta and DA were passaged nonenzymatically in 25-ml flasks and maintained in medium 199 supplemented with 20 mM HEPES. 1% antibiotics/antimycotics,and 10”; fetal bovine serum. In the studies related to the isolation of the 52-kDa protein for the preparation of the monoclonal antibody, 25-ml flasks, with confluent SMC cultures a t passage 3, were washed and maintained in serum-free medium 199 and then incuhated with [“Clvaline in valine-free medium for 4 h. The conditioned media were collected, dialyzed against water, concentrated by lyophilization. and the proteins were resolved hy SDS-PACE followed by autoradiography. In all other experiments, 25-1111 flasks with confluent SMC cultures at passage 3 were maintained in medium 199 without serum for 3 days; then conditioned mediawere pooled and further processed. Determinations of secreted protein were related to cell numher obtained by trypsinizing the cells and counting in a Coulter counter (Coulter Electronics, Hialeah, FL). Preparation of Monoclonal Antihdirs to the .52-kl)a IIA PrnteinThe secretion of a 52-kDa protein observed in DA hut not in aorta or pulmonary artery SMC, reported previously following incorporation of a [“C]leucine label (Rabinovitch et a/., 1988), was confirmed with[“Cjvaline labeling. The proteinspresent in the conditioned Flc:. 1. Representative light microscopic photomicrographs media from cultured DA cells were then isolated by fast protein liquid of t h e DA o n t h e left ( A ) a n d the adjacent ascending aorta o n chromatography, resolved hy SDS-PAGE, and then transferred to the right ( B ) , from a 138-day gestation fetal lamb (term = nitrocellulose. Usingtheautoradiograph as atemplate.thehands 145 days). The development of an intimal cushion (hrackrt) and corresponding to the 52-kDa protein were cut from the nitrocellulose the frail appearance of fragmented elasticfibers (arrows) are apparent membrane. Three female RALH/c mice were initiallv immunized by in the DA compared with the aorta. Bar = 30 pm. subcutaneous implantation of these nitrocellulose strips. Initial immunization was followed 4 weeks later by two booster immunizations through the cell membrane, perhaps because it lacks an as- 2 weeks apart, and the mouse with the highest IpG titer was used for sociated“protective”protein(HinekandMecham, 1990). spleen cell harvest. The cells were collected and hyhridized with myeloma cells (PS-x63-AgR-U1) using 5 0 T polyethylene glycol 400. Since it lacks the carboxyl terminus, the 52-kDa truncated The hybridoma cells were selected in medium containing h-ypoxantropoelastin product may have functional significance with thine, aminopterin, and thymidine, and the yield was 20’; of those regard to impaired assembly of elastic fibers in the DA; and originally plated. The hybridomas were then screened for positive IgG-producing clones. Suhcloning of the hvhridomas was performed since it remains insoluble, it maybe chemotactic to SMC by the limiting dilutions method, and five stable hyhridoma cell clones (Senior et al., 1980, 1984; Mecham et al., 1984; Wrenn and (antibody producing after several passages in culture) were subculMecham,1986), a property whichmaybe associatedwith in Iscove’s modified Dulbecco’s medium and then injected (10’ their increased migration related toDA intimal proliferation. tured cells)intotheperitoneum of male RAI,H/c mice pretreated with pristane. The ascitic fluid was collected every other dav starting on EXPERIMENTALPROCEDURES day 21 after hyhridoma injection, and the specificity of the newly Materials-Chemicals and reagents were obtained as follows. Me- produced monoclonal antibodies for the 52-kIhprotein was assessed by Western immunoblot (see details of procedure below). The monodium 199, phosphate-buffered saline, fetal bovine serum, and other tissue culture reagents were obtained from GIRCO. Medium contain- clonal antibody HI-20 was selected for all further studies since it reacted strongly andselectively with a 52-kDa protein secretedin the ing hypoxanthine, aminopterin, and thymidine, and Iscove’s-modified Dulbecco’s medium for maintaininghybridoma cells, chondroitin DA conditioned medium. sulfate, VGVAI’G synthetic peptides, and all reagent grade chemicals Imrnunnelectron Microscopy-We next localized the 52-kDa prowere purchased from Sigma. Polyethylene glycol 400 was supplied by tein in the vessel wall by immunoelectron microscopv using a posMerck, and pristane was ohtained from Wako Pure Chemical Indus- tembedding method described previously (Hinek ef a/., 1988). Briefly, tries, Ltd. (Osaka,.Japan). Affi-Gel 10 for the preparation of affinity 4-mm2 full thickness sections of aorta and DA tissue were fixed with columns, all sodium dodecyl sulfate-polyacrylamide gel electropho- 0.5% glutaraldehydeand 0.5% paraformaldehyde in 0.1 M Trisbuffered saline, pH 7.4. Reactive aldehydes were blocked with 0.5 M resis (SDS-PAGE) reagents, and nitrocellulose transfer membranes were purchasedfrom Hio-Rad. The Immohilon P transfer membranes glycine, the samples were washed with Tris-buffered saline, postfixed were supplied by Millipore (Mississauga, ON, Canada). Species and with 1% osmium tetroxide in 0.1 M cacodylate huffer, dehydrated in type-specific second antibodies, goat ant.i-rabbit, and goat anti-mouse ethanol, and embedded in Epon. Thin sections were placed on nickel conjugated with gold particles for EM-immunolocalization were oh- grids, blocked with 1% bovine serum alhumin, 3% normal goat serum, tained from Janssen Life Science Products (Piscataway,NJ). Horse- and 0.5% Tween 20 in Tris-buffered saline. and reacted with the HIradish peroxidase-conjugated antibodies used in Western immuno20 monoclonal antibody to 52-kDa protein (2 mg/mlJ, diluted 1:200 blotting and proteinase inhibitors were supplied by Roehringer Mann- and, in subsequent studies, with a polyclonal antibodv to tropoelastin heim.Fluorescein isothiocyanat,e-conjugated goat anti-mouseand (5 mg/ml), diluted 1:400 (Prosser et al., 1991). The immune reaction goat anti-rabbit secondary antibodies were obtained from ICN Imwas visualizedwith appropriatesecondaryantibodies(goatantimunobiologicals (Lisle,IL). The radioactive [“CC]leucine and [“C] rabbitorgoatanti-mouse) conjugatedwith 15-nm gold particles. valine were supplied hy Du Pont-New England Nuclear. The in oitro Sections were then stained with uranyl acetate and lead citrate. In translation kit was purchased from Promega (Madison, WI). Insolu- all immunostainingprocedures, controls included suhstitution of nonble elastin was obtained from Elastin Products (Pacific, MO). Mon- immune ascitic fluid or normal rabbit serum for the first antibody. ospecific polyclonal antibody to bovine tropoelastin (Prosser et al., Since the HI-20monoclonal antibody was localized almost exclusively to the elastic fibers and laminae with the same distribution as the 1991) and monoclonal antibody to the VGVAPG sequence on the tropoelastin molecule (Wrenn and Mecham,1986; Wrenn et al., 1986) polyclonal antibody to tropoelastin,we repeated the immunoelectron microscopy following preabsorption of the HI-20 and tropoelastin were gifts from Dr. R. P. Mecham, Washington University, St. Louis, antibodies to an elastin affinity column. MO. The specific polyclonal antibody raised againstasynthetic WesternImrnunoblnts Usin# H I - 2 0 andElastin Antibodirs-To peptide representing the C-propeptide sequence of tropoelastin encoded by exon 36 (Rosenbloom et al., 1986) was kindly supplied by establish whether the 52-kDa protein reacted with the tropoelastin antibody and whether the HI-20 antibodvrecognized purified bovine Dr. J. Rosenbloom, Universit,y of Pennsylvania, Philadelphia, PA. of Western immunohlots Cell Cultures-SMC were harvested from the DA and aorta of 138- ligamentum nuchae tropoelastin, a series day gestation fetal Rambouillet lambs by a protocol described previ- was carried out. Conditioned media were collected in duplicate over ously (Rabinovitch rt al., 1988).Cellsgrownfrom explants of the 3 days from confluent cultures of aorta and DA SMC ( 5 X IO’’ cells/ media were characterizedassmooth muscle usinga monoclonal culture). The media were dialyzed exhaustively against water conantibody specific to smooth muscle actin (Tsukada et al., 1987). At taining proteinase inhibitors and lyophilized. The concentrated sam-
A
52-kDa Tropoelastin Product Impaired andElastin Assembly
1407
ples were suspended in SDS sample buffer with dithiothreitol, and vitro at 25 "C for 60 min using the Promega in uitro translation kit the proteins were resolved by SDS-PAGE using 0.45-mm-thick 12% containing wheat germ extract, ribonuclease inhibitor (40 units/pl), gels (Laemmli, 1970), then transferred to nitrocellulose at 100 V for 1 mM amino acid mixture, 1 mM potassium acetate, and 0.5 mCi/ml 1 h (Towbin et al., 1979) and immunoblotted with the new HI-20 [3H]leucine.Once the reactions were completed, the labeled translamonoclonal antibody (2 mg/ml) or with monospecific polyclonal tion products were immunoprecipitated with anti-tropoelastin antiantibody to bovine tropoelastin (5 mg/ml), both diluted 1:200. The bodies (Wrenn etal., 1987), resolved on SDS-PAGE, and analyzed by reaction was visualized using the peroxidase-conjugated goat anti- autoradiography and by Western immunoblot with HI-20 and antimouse (goat anti-mouse-horseradish peroxidase) or goat anti-rabbit tropoelastin antibodies. Puke-Chose Experiments-Freshly plated aorta and DA SMC (5 (goat anti-rabbit-horseradishperoxidase) antibodies diluted 1:lOO. In each case, the original gels were silver stained to assure that electro- X lo6 cells/dish) were incubated in triplicate for 18 h in medium 199 transfer was even and complete. Similarly, affinity-purified bovine with 10% fetal bovine serum so that they attached and spread; they ligamentum nuchae tropoelastin was resolved by SDS-PAGE, trans- were then pulsed with 15 pCi/ml [I4C]valinein valine-free medium ferred to nitrocellulose, and immunoblotted with HI-20 monoclonal for 1 h followed by a chase in fresh medium 199 for 15 and 30 min. antibody (2 mg/ml) or with a polyclonal antibody to tropoelastin (5 At each time point, the cells were extensively washed and thenscraped mg/ml), both diluted 1:200. The reaction was visualized using the and extracted overnight at 4 "C with 0.5 M acetic acid in the presence peroxidase-conjugated goat anti-mouse (goat anti-mouse-horseradish of proteinase inhibitors. After removal of insoluble material by centrifugation the supernatantwas dialyzed exhaustively (12,000-14,000 peroxidase) antibody diluted 1:lOO. Affinity purification was subsequently carried out to obtain large molecular weight cutoff membrane) a t 4 "C against water containing amounts of the 52-kDa protein. We also determined whether the 52- proteinase inhibitors and then lyophilized and immunoprecipitated kDa protein could be detected in conditioned media from a much with anti-tropoelastin antibody as described by Wrenn et al. (1987). larger number of aorta SMC. Conditioned media collected from five The identity of radiolabeled immunoprecipitated tropoelastins was 75-ml flasks of confluent DA or aorta SMC (50 X lo6 cells) were established by SDS-PAGE followed by autoradiography. Further pulse-chase experiments enabled comparison of the procmixed with the HI-20 orwith anti tropoelastin antibody affinity resin and rotary shaken for 4 h at 4 "C. The antibody affinity resins were ess of tropoelastin secretion and insolubilization by cultured aorta prepared using Affi-Gel 10 and coupled by mixing 10 mgof each and DA SMC. Densely plated (5 X 106cells/dish)confluent dishes of antibody/ml of resin according to the manufacturer's directions. At aorta and DA SMC a t passage 3 were incubated in quadruplicate in the end of the 4-h incubation with antibody affinity resins, the serum-free medium 199 for 18 h and thenpulsed with 15 pCi/ml ["C] unbound material was removed by washing with 0.1 M sodium bicar- valine in valine-free medium for 1 h. The cultures were then rinsed bonate buffer, pH 8, followed by 1 M sodium chloride washing until well and chased in fresh medium 199 for 0.5, 1,3, 6, and 24 h. At the the absorption AZmof the eluent returned to background level. The end of each chase period, culture media and cell layers including proteins that bound to theHI-20 or to theanti-tropoelastin antibody matrices were removedseparately and processed separately. Proteinaffinity resins were eluted with 4 M urea and 2 M acetic acid, pH 3.3, ase inhibitors were added to each sample prior to the processing. dialyzed exhaustively against water, concentrated by lyophilization Media were dialyzed against water with proteinase inhibitors and and then separated by SDS-PAGE, electrotransferred to nitrocellu- then lyophilized and immunoprecipitated with anti-tropoelastin anlose, and immunoblotted with HI-20 and anti-tropoelastin antibodies tibody as described above. The identity of radiolabeled tropoelastins was similarly confirmed by SDS-PAGE followed by Western immuas described above. To address whether the 52-kDa protein may be a truncated form of the 68-kDa tropoelastin lacking the carboxyl- noblot with anti-tropoelastin antibodies and also by autoradiography. terminal region, we also carried out Western immunoblots using the The amounts of 68- and 52-kDa tropoelastins were assessed quantispecific antibody against a synthetic peptide representing 17 amino tatively after the appropriate bands were dissected from the gels, acids at the carboxyl-terminal sequence of tropoelastin (exon 36) (2 solubilized in scintillation mixture, and counted in a Beckman LS 8000 scintillation counter. mg/ml diluted 1:200) (Rosenbloom et at., 1986). Extraction of Soluble Elastin from Aorta and DA Tissue-Since To extract insoluble elastin, the cells were quickly washed in preparation of the tissue for immunoelectron microscopy effectively phosphate-buffered saline, scraped with their associated matrices removed soluble elastin, it was necessary to confirm that the52-kDa from the dishes, and digested with (CNBr, 50 mg/ml in 70% formic protein was also present in the intact DA and not the result of an in acid) at 20 "C for 18 h to dissolve all non-elastin cellular and matrix vitro phenotypic change. Tissue samples similar in size to those used components (Todorovich-Hunter et al., 1988). The CNBr residues for cell harvest (approximately 50mg)were extracted with 0.5 M were collected by centrifugation (10,000 x g, 20 min, 4 "C), washed acetic acid in the presence of proteinase inhibitors in the following in water, and then solubilized in 5.7 N HCl at 110 "C for 30 min and final concentrations: 2 mM benzamidine, 2 mM c-amino-n-caproic counted as described above. The identity of CNBr residue as insoluble elastin was confirmed by amino acid analysis. The radioactivity acid, 2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and 1 mg/ ml Trasylol. Extraction was carried out overnight at 4 "C with con- present in each sample was expressed as cpm/dish, and the mean f stant stirring, and the insoluble material was pelleted by centrifuga- S.D. was calculated. Search for Tropoelastin Degrading Enzyme in DA Conditioned tion. The supernatantwas dialyzed exhaustively (12,000-14,000 molecular weight cutoff membrane) at 4 "C against water containing Medium-To confirm further the absence in DA SMC conditioned proteinase inhibitors andthen lyophilized. Concentrated samples medium of a proteolytic enzyme which was responsible for cleavage were suspended in SDS sample buffer with dithiothreitol, and pro- of 68-kDa tropoelastin to a 52-kDa product, the followingexperiments teins were resolved by SDS-PAGE, transferred to nitrocellulose, and were carried out. Concentrated samples of aorta SMC conditioned immunoblotted with HI-20 and anti-tropoelastin antibodies. medium containing ['4C]valine-labeled tropoelastin were incubated Amino Acid Analysis of the 52-kDa Protein, Sequencing of Frag- for 18 h at 37 "C with aliquots of unlabeled DA conditioned medium ments-To analyze the amino acid composition of the 52-kDa protein in dilutionsof 1:1, 1:2, 1:5, and 1:lO. The tropoelastin present in each and to obtain some sequence data, the following experiments were sample was then analyzed by SDS-PAGE followed by autoradiogracarried out. Proteins were eluted from both HI-20 and anti-tropoe- phy and Western immunoblotting with the anti-tropoelastin antilastin antibody affinity columns and electrotransferred onto Immo- body. bilon P membranes using a constant current of 250 mA for 1.5 h. The Is Accumulation of 52-kDa Tropoelastin Directly Linked t o Reduced proteins were then stained with Coomassie Blue, and the band of Cell Surface Elastin-binding Proteins?-Since we previously docuinterest (52-kDa) was cut from the membrane and used for amino mented a reduction in the concentration of the 67-kDa elastin-binding acid analysis and sequencing. The 52-kDa protein band was addi- protein on DA SMC surfaces associated with increased accumulation tionally treated for 10 min with 5 pg/100 pl of trypsin (from bovine of tropoelastin (Hinek et al., 1991), we hypothesized that this feature pancreas type 111, Sigma) at 37 "C prior to sequencing in an attempt might be directly linked to production of a 52-kDa tropoelastin. We to overcome possible blocking of the amino terminuswhich frequently therefore repeated anexperiment in which we used chondroitin occurs in proteinsseparated by SDS-PAGE (Stone et al., 1989). sulfate, an N-acetylgalactosamine-containingglycosaminoglycan to Sequencing was performed on aPorton Instruments model2090 release the 67-kDa elastin-binding proteinsfrom aorta SMC surfaces (Tarzana, CA) gas phase sequenator. in culture, causing impaired elastin fiber assembly and increased In Vitro Translation-In vitro translation was carried out to deter- accumulation of tropoelastin (Hinek et al., 1991), and then determine whether the 52-kDa protein was a unique low molecular weight mined whether the tropoelastin produced was a 52-kDa protein. We translation product of DA tropoelastin mRNA. From fetal lamb aorta cultured aorta SMC with and without chondroitin sulfate (200 pg/ and DA tissue (approximately 50 mg each), RNA was extracted with ml) for 5 days, then purified and concentrated the conditioned meguanidine as described by MacDonald et al. (1987) and translated in dium using HI-20 antibody affinity columns, and assessed the molec-
1408
52-kDa Tropoelastin Product Impaired andElastin Assembly
ular weight of tropoelastin in the conditioned medium by Western blot using an anti-tropoelastin antibody.
A
B
C
D
RESULTS
Immurwelectron Microscopy-Using the HI-20 antibody to the 52-kDa protein in DA and aorta tissue, we observed that in both vessels immunoreactivity was limited to elastic fibers (Fig. 2, A and B ) . The distribution and the density of the immunogold particles were similar to thatobserved in sections stained with the anti-elastin antibody (Fig. 2C). To determine whether this represented co-localization of two different proteins or similar immunoreactivity, we preabsorbed both antibodies onto an elastin affinity column prior to immunogold labeling of the tissue. This procedure completely prevented labeling of the tissue with either HI-20 or the anti-tropoelastin antibodies (data notshown). We therefore concluded that our newly produced monoclonal antibody HI-20 reacted with elastin rather than with other proteinsassociated with the elastic fiber, e.g. microfibrillar proteins. WesternImmurwblotting-When the conditioned media from DA and aortaSMC cultures (5 x IO6cells) were analyzed by Western immunoblots, we confirmed that our new HI-20 monoclonal antibody and a polyclonal antibody to tropoelastin recognized a 52-kDa protein present in large amounts in DA conditioned media as well as smaller quantities of a 68kDa protein (Fig. 3). Both antibodies recognized only a 68kDa protein in aorta SMC conditioned media. Furthermore, the HI-20 monoclonal antibody raised against the 52-kDa
e 6 8 kD
*52
kD
FIG. 3. Proteins from conditioned media of aortic ( A and C) and ductus arteriosus ( B and D ) SMC (5 X 10' cells) resolved by SDS-PAGE and immunoblotted using a polyclonal antibody to tropoelastin ( A and B ) and HI-20 monoclonal antibody to 52-kDa protein (Cand D ) . Both antibodies react with the 68kDa band present in all conditioned media. Both antibodiesalso react with a prominent 52-kDa protein band which is present in the DA but notobserved in the aortaconditioned medium.
A
B
C
-.
D
-.
.
m
.'%
. .
r :'
* 6 8 kD e 5 2 kD
Ao
D
DA
Ao
DA
FIG.4. Western immunoblots of proteins from the conditioned media from 5 0 X 10' aorta and DA SMC purified by HI-20 antibody affinity column (lanes A and B ) or purified by anti-elastin antibody affinity column (lanes C and D, respectively) and stained with anti-tropoelastin antibody. Similar amounts of 68-kDa tropoelastin are seen in DA and aorta (Ao) conditioned media purified by both affinity columns. The 52-kDa a small protein observed in DA conditioned medium is prominent, but amount of 52-kDaproteinis now apparent in aorta conditioned medium from a large number of cells after affinity purification.
protein also reacted with tropoelastin isolated from bovine ligamentum nuchae (data not shown). To determine whether the 52-kDa protein was unique to the DA or was also produced in small quantities by aorta SMC, we used both HI-20 and anti-elastin antibody affinity columns to purify proteins from the conditioned media of 10fold greater number of cells (50 X IO6). Similar amounts of the 68-kDa tropoelastin were purified by both HI-20 and FIG.2. Representative immunoelectron micrographs of tropoelastin affinity columns using DA and aorta SMC convessel tissue from the ductus arteriosus ( A )and aorta ( B ) .HI- ditioned media; the 52-kDa protein was, however, abundant 20 monoclonal antibody to the 52-kDa protein localizes exclusively to elasticlaminaeinboth vessels. Similar immunolocalization is in DA conditioned medium with only trace amounts evident evident with the anti-tropoelastin antibody in a DA section (C). A in aorta (Fig. 4). We were also able to verify that the52-kDa control DA sectionincubatedwithnonimmuneascitic fluid and motein reacting with HI-20 antibody was extractable in large secondary antibody (D)shows no immunogold label. Bar = 0.5 pm. amounts from the intact DA tissuebut was virtually absent
52-kDa Tropoelastin Product and Impaired Elastin Assembly in aorta tissue samples (data not shown). To assess whether the 52-kDa protein might represent a truncated form of tropoelastin lacking the amino- or carboxylterminal sequences, we carried out further Western immunoblots using the monospecific polyclonal antibody to 17 amino acids at the carboxyl terminus of tropoelastin (exon 36). This antibody failed to recognize the 52-kDa protein isolated from DA conditioned medium, while it reacted3well with the 68-kDa tropoelastin (Fig. 5). These data suggested that the 52-kDa protein might be an elastin lacking amino acids at the carboxyl-terminal region present in the 68-kDa tropoelastin. 172 Amino Acid Analysis and Sequence Data-We next con-114 firmed by amino acid analysis and sequencing that the 52kDa protein was in fact truncated tropoelastin. The amino 16 acid composition of the 52-kDa protein purified by HI-20 33 antibody affinity resin resembled that of bovine tropoelastin 6 reported by Prosser et al. (1991) and that predicted from 15 bovine cDNA (Yeh et al., 1989) i.e. with similar high residue content of glycine, alanine, valine, and proline. The 3high content of lysine 27/mol and the lack of desmosines suggest that this protein is not derived from insoluble (cross-linked) elastin (Starcher and Galione, 1976). In Table I we compare the amino acid composition of 52-kDa protein with the composition of tropoelastin as predicted from the full-length cDNA (Yoon et al., 1985; Raju and Anwar, 1987). Although 68-kDa tropoelastin contains 720 amino acids, our analysis showed that thetruncated 52-kDa protein contained no more then 563 residues, and, with an intact amino terminus, this suggests a cleavage betweenexons 26 and 27. Our calculations were adjusted accordingly. Lack of cysteine residues further supported loss of amino acids at thecarboxyl terminus. The 16-amino acid long sequence obtained from the affinity-purified 52-kDa protein (Table 11) matches sequences at the amino terminus of tropoelastin as reported by others (Indik et al., 1987; Davidson et al., 1982a; Sandberg et al., 1985; Raju and Anwar, 1987). The loss of the first 4 amino
A
B .
UA
-
DA
FIG. 5. Proteins extracted from conditioned' media of DA cells (SO X 10' cells) and purifiedby HI-20antibody affinity column were resolvedbySDS-PAGEandimmunoblotted using a polyclonal antibody to tropoelastin ( A ) and an antibody specific to tropoelastin carboxyl terminus ( B ) .The 68and 52-kDa bandsreactedwith the anti-tropoelastin antibody, whereasonly the 68-kDa bandreactedwith the antibody to the carboxyl terminus.
1409
TABLEI Amino acid composition of 52-kDa proteinsecreted by DA SMC compared with the composition predicted from full-length cDNAfor 68-kDa tropoelnstin 68-kDa tropoelastin
52-kDa tropoelastin
residu.?s/mol
residues/mol
CYS Asx Thr Ser Glx Pro + Hyp GlY Ala Val Met Ile Leu TYr Phe LYS A% Isodes" Desb
2 3 8 6 8 89 229 151 93 0 18 45 6 21 37 4 0 0 720 ".
0
.
7 5 10 74 78 0
21 0 0 -
563
Isodes, isodesmosine.
* Des, desmosine.
TABLE I1 Amino-terminal sequence from large tryptic fragment of sheep 52kDa protein(1) demonstrates homology with the amino-terminal sequence of bovine (2), porcine (3), sheep (4), and human (5) tropoelnstins Sequence 1 was from a large tryptic fragment of sheep (52-kDa tropoelastin). Sequence 2 was deduced from bovine tropoelastin cDNA (Fhjuand Anwar, 1987). Sequence 3 wasfrom a large tryptic fragment of porcine tropoelastin (Sandberget d,1985). Sequence 4 is the cell-free translation product of sheep elastin RNA (Davidsonet d,1982a, 1982b). Sequence 5 was deduced from human tropoelastin cDNA (Indik et al.. 1987). 1 GAVPGGVPGGVFFPGA 2 GGVF'GAVPGGVPGGVFFPGAGL 3 GGVPGAVPGGVPGGVFFPGAGLGGLG 4 GGVPGAVPGGVPGGVFFXXXXL 5 GGVPGAIPGGVPGGVFYPGAGL
acids is very likely because of their cleavage by proteinases present in the trypsin preparation. I n Vitro Translation-To determine whether the 52-kDa tropoelastin was a short isoform not described previously, in vitro translation was carried out. We observed that both DA and aortaRNA extracts produced similar translation products of 68-72 kDa which immunoprecipitated with anti-tropoelastin antibody (Fig. 6). Post-translational Modification of Tropoelastin-We next determined whether the 52-kDa tropoelastin was more likely a product of intracellular post-translational processing of the 68-kDa species or was producedby extracellular cleavage. We determined that the anti-tropoelastin antibody precipitates both 68- and 52-kDa proteins from cell extracts of 5 X 10' DA SMC pulsed with [14C]valineafter only a 15- or 30-min chase, whereas in cell extracts of aorta SMC the same antibody precipitated only the 68-kDa tropoelastin (Fig. 7). Pulse-chase experiments provided further information concerning the timing of appearance of 52- and 68-kDa tropoelastin in the conditioned media and the relation to insoluble elastin deposition in cultures of aorta and DA SMC. In aorta SMC cultures (Fig. 8A), significant amounts of ["Clvaline-
52-kDa TropoelustinProduct and Impaired Elastin Assembly
1410
A
& - 3 4 8 kD
112
1
3
6
24
Chaselhours
B
= 1 n
A o . DA FIG.6. Products ofcell-free translation usingRNA isolated from 10 X 10' DA and aorta (Ao) cells and immunoprecipiThat both aorta and DA tated with anti-tropoelastin antibody. produce 68-72-kDa tropoelastins and no 52-kDa product is evident.
6000
112
1
3
6
24
Chaselhours
A
B
C
D
4 68 kD 4 52 kD
FIG.8. Valuesofincorporated['%]valine(cpm)are expressed as mean f S.D. of quadruplicate dishes (each = 5 X 10' cells). In A, aorta SMC cultures ["Clvaline is incorporated into a secreted soluble 68-kDa tropoelastin (0)detected in the media as early as the first 30 min of chase. It reaches a peak level after 1 h, then values steadily decrease over the subsequent 24-h period. Coincident with the latter is an increase in the incorporation of ["C] valine into insoluble elastin (A) measured in the cell-associated matrices. Only trace amounts of radiolabeled 52-kDa tropoelastin (0) are detectable in the culture media during the 24-h chase. In B, DA SMC cultures ["Clvaline is incorporated into similar amounts of 52kDa (0)and 68-kDa (0)tropoelastin detected in the media within the first 30 min of chase. Levels of 52-kDa tropoelastin increased over the first 3 h and persisted over the 24-h period of chase. The level of 68-kDa tropoelastin was only half that observed in the aorta SMC but showed the same time course of appearance and disappearance, and the latterwas associated with increasing insoluble elastin in the matrix (A). The amount of insoluble elastin produced by DA cells after the first 3 h of chase as detected in cell associated matrices was consistently approximately one-third that observed in aorta SMC cultures.
Only trace amounts of 52-kDa tropoelastin were detected in these cultures during the 24-h chase. In DA SMC cultures (Fig. 8B), similar amounts of both ["Clvaline-labeled 52- and 68-kDa tropoelastins were detected after a30-minchase. Increasing amounts of the 52-kDa species, however,persisted over the 24-h chase, whereas there was a drop in the amount FIG.7. Aorta and DA SMC (5 X 10' cells/dish) were pulsed of the 68-kDa species detected concurrent with increasing with ["C]valine for 1 h and chased in fresh medium199 for insolubilization of elastin. The amount of insolubilized elastin 15 and 30 min and thenextracted with0.5 M acetic acidand produced by DA SMC after the first 3 h was approximately resolved on SDS-PAGE.The autoradiograph shows that the anti- one-third that observed in the aorta SMC cultures during tropoelastin antibody precipitates only a 68-kDa tropoelastin from the cell extracts of aorta SMC (lanes A and B are 15- and 30-min further chase. ,These results suggest that the truncated 52time points, respectively), whereas both 68- and 52-kDa proteins are kDa tropoelastin product appears early and remains soluble precipitated from the cell extracts of cultured DA SMC (lanes C and and accumulates in the conditioned medium perhaps because D are the 15- and 30 min time points, respectively). it cannot be insolubilized. To further exclude the possibility that accumulation of the labeled soluble 68-kDa tropoelastin were detected in theme- 52-kDa tropoelastin product did not result from extracellular dium even after a 30-min chase. Peak values were reached cleavage, we investigated whether in DA SMC-conditioned after 1 h, and thenlevels steadily decreased over the following medium there was proteolytic activity resulting in the break24 h concurrent with increasing insolubilization of elastin. down of the 68-kDa tropoelastin. We did not, however, detect
52-kDa Tropoelastin Product and Impaired Elastin Assembly
1411
lastin described previously (Davidson et al., 1982a, 198213, 1984; Yoon et al., 1984) in that it lacks amino acids at the carboxyl-terminal region. The 52-kDa tropoelastin product could only be found in very small amounts in conditioned media harvested from much higher concentrations of aorta SMC. The identity of the 52-kDa protein as tropoelastin is basedupon amino acidcomposition, amino acidsequence data, andimmunoreactivitywith anti-tropoelastin antibodies (except the one which is specificto thetropoelastin carboxyl terminus, exon36). In vitro translation showed that the initially synthesizedtropoelastin in DA is 68 kDa in molecular mass and suggested that the52-kDa protein might represent an intra- or extracellular cleavageproduct. In pulse-chase DISCUSSION experiments, we showed that a 52-kDa tropoelastin could be Our findings suggest that the DA, a fetal vesselwhich immunoprecipitated from DA cell extracts, suggesting that it assembles elastin poorly in association with intimal prolifer- was a product of cell-associated post-translational processing ation, produces abundant quantities of a 52-kDa tropoelastin involving cleavage of the carboxyl-terminal region. Further which appears to result from intracellular processing or deg- studies showed that in contrast to the 68-kDa tropoelastin, radation of one or more of the 68-72-kDa isoforms of tropoe- the secreted 52-kDa product accumulates and is not insolubilized. The mixing experiments further supported lack of involvement of extracellular proteinases in the production of A B C D E the 52-kDa tropoelastin. Thus, the52-kDa tropoelastin could arise if the intracellular environment of the DA, compared with the aorta,was conduciveto post-translational processing or degradation of the 68-kDa species,or if DA SMC produce, in high concentration, a 68-kDa isoform that is particularly susceptible to enzymatic or nonenzymaticdegradation. It could also occur if for some reason extracellular transport of C 68 kD the 68-kDa tropoelastin requires association with a binding or “companion” protein that is deficient in the DA. The functional significance of the 52-kDa tropoelastin is that it lacks the carboxyl-terminal and associatedcysteines that appear to be necessary for proper alignment and cross-linking of insoluble elastin, and thismay at least in part explain the defective assembly of elastic fibers in the DA. In addition, tropoelastin and related peptides are potent chemotactic factors, and this may be important in the mechanism of SMC migration associated withintimal cushion formation. FIG. 9. A representative autoradiograph showing that in Current evidence favors the presence of a single elastin aorta SMC cultures [“Clvalineis incorporated into a secretedgene in bovine (Ciciliaet aL, 19851, ovine (Olliveret aL, 1987), 68-kDa protein immunoprecipitated with the anti-tropoelastin antibody (lane A ) . Concentrated samples of aorta conditioned and in the human species (Emanuel et al., 1985; Fazio et aL, media containing the [“C]valine-labeled 68-kDa tropoelastin incu- 1988). Indik et al. (1987), however, suggested that through bated for 18 h with aliquots of unlabeled DA conditioned medium extensive alternative splicing of tropoelastin mRNA, there is diluted 1:1, 1:2, 1:5, and 1:lO (lanesB, C, D,and E, respectively) do the potential for manydifferent isoforms of the final protein. not show degradation of radiolabeled tropoelastin. The existence of multiple isoforms of tropoelastin of different molecular mass (68-72 kDa) has been detected following cellA B free translation of mRNAisolatedfrom different elastinproducing cells, including fetal tissues (Foster et al., 1980, 1981, 1986; Burnett et al., 1980; Parks et al., 1988; Davidson et al., 1982a; Chipman et aL, 1985; Wrenn et aZ., 1987; Raju and Anwar,1987;Yeh et al., 1987,1989).Although these isoforms are developmentally regulated (Parks et. al., 1988), their functional properties which might berelated to possible post-translational modifications, e.g. susceptibility to enzy4-68 kD matic or nonenzymatic degradation, have not been explored. The amino acid composition of the 52-kDa protein (being high in lysine and containing no desmosines) suggestedthat it is a product or tropoelastin rather than of insoluble crosslinked elastin (Table I). Since 52-kDa tropoelastin is not an initial product of in uitro translation, it appears to result from FIG. 10. Western immunoblot with an anti-tropoelastin an- post-translational processing of a higher molecular weight tibody showing that both chondroitin sulfate-treated( A )and tropoelastin isoform. The existence of sequence-specific trountreated ( B )aortic SMC produce only 68-kDa tropoelastin. poelastin proteinases might suggest a mechanism of postThe proteins from conditioned media were affinity purified by HI-20 antibody column, separated by SDS-PAGE, electrotransferred onto translational processing of tropoelastin isoforms in vascular nitrocellulose, and immunostained with an anti-tropoelastin anti- tissues (Mecham and Foster, 1977). The expression of such body. A larger amount of tropoelastin is suggested in lane A . enzymes might be tissue-specific or developmentally regu-
any signs of degradation of radiolabeled 68-kDa tropoelastin in concentrated samples of aorta conditioned medium even when incubated for 18 h with concentrated aliquots ofDA conditioned medium (Fig. 9). Shedding of Elustin-binding Proteins and Tropoelustin Molecular Weight-The production of the 52-kDa tropoelastin product could not be linked directly to a reduction of elastinbinding proteins on vascular SMC surfaces.When aorta SMC cultures were exposed for 5 days to chondroitin sulfate to induce shedding of elastin-binding proteins from the cell surfaces and accumulation of tropoelastin, they failed to produce a 52-kDa species (Fig.10).
1412
52-kDa Tropoelastin Product Impaired andElastin Assembly
lated. There may therefore be unique features of the intracel- ments in which chondroitin sulfate was added to aorta SMC lular environment of DA SMC which favor post-translational to strip elastin-binding proteins from cell surfaces did not, processing or degradation of tropoelastin resulting in a 52- however, result in the production of a 52-kDa tropoelastin kDa product. Alternatively, the DA SMC might generate a product. Thisdoes not exclude the possibility that the tropoemay complex intracellularly with the number of 68-70-kDa isoforms as the productsof alternative lastin in aorta SMC splicing of tropoelastin mRNA, and one of these isoforms, elastin-binding protein (Hinek and Mecham, 1990), and this which is not produced by the aorta to the same extent, may may protect it during transport through the cell membrane, be particularly susceptible to intracellular cleavage, yielding whereas in DA, SMC that are deficient in elastin-binding proteins (Hinek et al. 1991), a proportion of the tropoelastin a 52-kDa protein.This is furthersupported by ourdata showing the early appearanceof large amounts of the 52-kDa is unprotected and easily cleaved. These results also suggest species in DA cells and by our inability to detect a DA-specific that stripping of elastin receptors from the cell surface does extracellular proteolytic enzyme capable of degrading the 68- not induce a signalling mechanism which might be directly kDa tropoelastin produced by aorta cells. linked toprocessing of a 52-kDa tropoelastinproduct. The functional significance of impaired elastin assembly The degradationof nonassembled tropoelastin observed in of large amounts of 52-kDa copper deficiency states associated with decreased lysyl oxi- associated with the accumulation dase activity and impaired cross-linking results in multiple tropoelastin product in the DA is of particular interest. The low molecular weight tropoelastin-derived peptides (Sandberg DA develops intimal cushions in late gestation, and these and Wolt, 1982; Romero et al., 1986). Discrete 66-, 61-, 56-, structures are formed by migration of SMC into the suben52-, and 45-kDa soluble elastin fragments lacking the car- dothelium (Boudreau and Rabinovitch,1991; Boudreau et al., boxyl-terminal region were also detectedin longterm cultures 1991). Impaired assembly of elastin as thick laminae would of neonatal rat aortic SMC (Franzblau et al., 1989). In the also seem to facilitate the migration of SMC by removing a DA, there is no evidence of decreased lysyl oxidase activity physical barrier to which they might attach. Moreover, since (Zhu et al. 1993). Moreover, the 52-kDa protein observed in tropoelastin and its related peptides were described as cheDA was the only species lower than 68-kDa molecular mass motactic factors for numerous cell types (Senior et al., 1980, immunoprecipitated by the anti-tropoelastin antibody, sug- 1984; Mecham et al., 1984; Wrenn and Mecham, 1986), one gesting that it represents the only cleavage product and is can speculate thatlocal accumulation of relatively stable but relatively resistant to furtherproteolytic degradation. This is still soluble elastin product canserve as a potent chemoattracsignificant since the tropoelastin antibodywe used will react tant for SMC. with lower molecular weight degradation products of bovine The developmentally regulated or tissue-specific heterogetropoelastin (data not shown).Moreover, the 52-kDa tropoe- neity of tropoelastin isoforms, as well as their possible postlastin product can be harvested from DA SMC culture me- translational modifications, may affect the way in which dium even in the absence of proteinase inhibitors without elastin is finally assembled intofibers and laminae ina given observing further lower molecular weight products. It can be tissue. This may be of particular pathophysiological signifiextracted from DA tissue by 0.5 M acetic acid, suggestingthat cance in conditions in which impaired assembly of elastic it belongs to thesoluble compartment of the matrix and that fibers has been reported in blood vessels and in skin (for i t is not an in vitro artifact associated with incubation under reviews see Sandberg, 1976; Sandberg et al., 1981; Rosenserum-free conditions. bloom, 1984; Indik et al., 1989). Further studies will be necOur observation that the 52-kDa protein does not react essaryto show whether a 52-kDatropoelastinproductis with the specific antibody recognizing the carboxyl terminus observed in pathological conditions such as pulmonary hyperof tropoelastin molecule suggests that this particular fragmenttension andatherosclerosis, in which there is impaired assemis missing. This was supported by sequence data confirming bly of newly synthesizedtropoelastin associated withinthat the amino terminus was present. Our amino acid analysis creased SMC migration and proliferation. of 52-kDa tropoelastin suggests that this protein is at least AcknowZedgrnents-We acknowledge Dr. Fred Keeley for construc157 aminoacids shorter than theoriginal tropoelastin. These features may explain why the 52-kDa protein remains soluble, tive discussion and help with regard to sequencing of the peptide. We as has also been shown by the pulse-chase experiments. The thank Dr. Robert Mecham and Dr. Joel Rosenbloom for the gifts of and Dr. Joel Rosenbloom for a critical reading of the proper alignment of tropoelastin molecules among microfi- antibodies manuscript. We also thank Satti Beharry, Mary Tang Kong, Michel brils seems to be necessary for elastin fiber assembly. The Paquet, and Moriake Kosakebe forhelp in preparingthe monoclonal particular role of the carboxyl-terminalregion of tropoelastin antibody. Wearegrateful to Joan Jowlabar and Susy Taylor for in complexing tropoelastin molecules with microfibrils has excellent secretarial assistance in preparing the manuscript. been suggested by Wrenn et al. (1987) and by Franzblau et al. REFERENCES (1989). It is possible that the cysteines of the carboxyl terBoudreau, N., and Rabinovitch, M. (1991) Lab. Inuest. 6 4 , 187-199 minus participate in anchoring tropoelastin to the microfi- Boudreau, N., Turley, E., and Rabinovitch, M. (1991) Deu. Biol. 1 4 3 , 235-247 brillar scaffold. The lack of a carboxyl terminus (and associ- Buchanan, J. W. (1978) Birth Defects 1 2 4 , 349-360 Burnett, W., Eichner, R., and Rosenbloom, J. (1980) Biochemistry 1 9 , 1106ated cysteines) on the 52-kDa tropoelastin product might, 1111 therefore, help explain why, in the fetal DA, elastin fiber Chinman. S. D.. Faris. B.. Barone. L. M.. Pratt. C. A., and Franzblau, c. (1985) J: BioL’Chem. 260; 12780-12785 assembly is impaired relative to that in the aorta. Cicilia, G., May, M., Ornstein-Goldstein, N., Indik, Z., Morrow, S., Yeh, H. S., In previous studies we have shown that the 67-kDa elastin- Rosenbloom. J. C.. Bovd. C.. Rosenbloom, J.. and Yoon, K. (1985) Biochemistry 24,3075-3080 binding protein plays a critical role in tropoelastin transport Davidson, J. M., Leslie, B., Wolt, T., Crystal, R. G., and Sandberg, L. B. (1982a) Arch. Biochem. Biophys. 218,31-37 and assembly (Hinek et al., 1988; Mecham et al., 1989; Hinek Davidson, J. M.,Smith, K., Shibabara, S., Tolstoshev, P., and Crystal, R. G. and Mecham, 1990). The relative deficiencyin elastin-binding (1982b) J. Bid. Chem. 257,747-754 proteinsin DA comparedwith aortaSMC suggested the Davidson, J. M., Shibahara, S., Schafer, M. P., Harrison, M., Leach, C., P., and Crystal, R. G. (1984) Biochem. J. 220,643-652 possibility that when tropoelastinaccumulates because it DeTolstoshev, Reeder, E. G., Girard, N., Munsteren, J. C., Patterson, D. F., and Gittenberger-de-Groot,A. C. (1988) Am. 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6.;
