©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Components of the Protein Synthesis and Folding Machinery Are Induced in Vascular Smooth Muscle Cells by Hypertrophic and Hyperplastic Agents
IDENTIFICATION BY COMPARATIVE PROTEIN PHENOTYPING AND MICROSEQUENCING (*)

(Received for publication, May 17, 1995; and in revised form, July 5, 1995)

Wayne F. Patton (§) Hediye Erdjument-Bromage Andrew R. Marks (1)(¶) Paul Tempst (**) Mark B. Taubman (1)(**)(§§)

From the Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Cardiovascular Institute and Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Vascular smooth muscle cells (VSMC) are the principal cellular component of the blood vessel wall. Atherosclerosis, hypertension, and angiogenesis are associated with abnormal VSMC growth. Angiotensin II is hypertrophic for cultured adult rat aortic VSMC, whereas platelet-derived growth factor and serum are hyperplastic. To identify changes in specific proteins associated with either hyperplastic or hypertrophic growth, high resolution two-dimensional gel electrophoresis was performed on extracts from quiescent rat aortic VSMC and from VSMC exposed for 24 h to growth factors (10% fetal calf serum, platelet-derived growth factor, or angiotensin II). 12 proteins were up-regulated and 5 down-regulated by treatment with growth factors. Eight of the up-regulated and one of the down-regulated proteins were identified by internal protein microsequencing from electroblotted two-dimensional gels or by co-electrophoresis of purified proteins in two-dimensional gels. Four of the proteins up-regulated by growth factors were identified as mediators of protein folding. These were heat shock proteins, HSP-60 and HSP-70, protein disulfide isomerase, and protein disulfide isomerase isozyme Q-2. Additional proteins were identified as elongation factor EF-1beta, a component of the protein synthesis apparatus, and calreticulin, another putative molecular chaperone. Vimentin and actin were also up-regulated, whereas an isoform of myosin heavy chain was down-regulated. Hyperplastic and hypertrophic growth were accompanied by similar changes in protein expression, suggesting that both types of growth require up-regulation of the protein synthesis and folding machinery.


INTRODUCTION

Growth and migration of VSMC (^1)are considered to be key events in the pathogenesis of atherosclerosis, hypertension, and angiogenesis(1) . VSMC display two distinct growth responses: hyperplasia, characterized by increased DNA and protein synthesis as well as cell division, and hypertrophy, characterized by increased cell size and protein content without DNA synthesis or cell division(2) . Atherogenesis is characterized by the hyperplastic response, involving the migration of VSMC from the vessel media to the intima and the proliferation of medial and intimal VSMC(1) . Chronic hypertension involves predominantly enlargement of preexisting VSMC (hypertrophy) within the media of the blood vessel as well as proliferation(2) . In cell culture, the nature of the growth stimulus, rather than intrinsic differences in growth responsiveness of distinct cell subpopulations, appears to determine whether VSMC undergo hyperplasia or hypertrophy(2) . PDGF and serum mediate a hyperplastic response in adult rat aortic VSMC(3) . In the same cells, Ang, arginine vasopressin, and thrombin more typically mediate a hypertrophic response(3, 4, 5, 6, 7, 8, 9) .

Considerable progress has been made in elucidating the molecular events associated with hypertrophic and hyperplastic VSMC growth. Many studies have focused on examining mRNAs induced by growth factor activation of VSMC. These have identified mRNAs encoding a variety of proto-oncogenes, proteins involved in the cell cycle, components of the extracellular matrix, cytokines, and growth factors(9, 10, 11, 12, 13, 14, 15) . Such studies were designed to identify growth factor-responsive proteins based upon differences in mRNA accumulation and would not identify proteins whose levels were altered predominantly by changes in translation. Studies based upon alterations in protein levels have been limited by the difficulty of displaying and identifying the large number of proteins expressed by cultured cells. We have utilized high resolution two-dimensional gel electrophoresis and protein microsequencing to examine differences in protein expression among serum-deprived cultured rat aortic VSMC and VSMC subjected to hyperplastic (PDGF, 10% serum) and hypertrophic (Ang) growth stimuli. This has led to the identification of nine proteins whose levels were either increased (eight) or decreased (one) after a 24-h exposure to growth agonists. The majority of these proteins are different from those previously identified using mRNA screening strategies and include proteins involved in the protein synthesis and folding machinery as well as components of the cytoskeleton.


MATERIALS AND METHODS

Cell Culture

VSMC were isolated from the thoracic aortas of 250-350-g male Sprague-Dawley rats by enzymatic dissociation and grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated calf serum (CS), as previously described(16) . To produce quiescence, cells (passages 2-5) were incubated for 48 h in a ``nongrowth'' medium containing insulin, transferrin, and ascorbate(17, 18) . For growth factor stimulation, cells were washed twice with phosphate-buffered saline and then treated for 24 h with 5 half-maximal units/ml of human natural PDGF (Collaborative Biomedical Products, Bedford, MA), 10M Ang (Sigma), or 10% CS in fresh nongrowth medium. Cells were then processed for electrophoresis as previously described(19) .

To measure DNA and protein synthesis, VSMC were plated in 12-well dishes. At approx25% confluence, cultures were incubated in nongrowth medium for 48 h and then treated for 24 h with either PDGF or Ang in the presence of [^3H]thymidine or [^3H]leucine (1 µCi/well). Triplicate experiments were performed using 3 wells per treatment per experiment. Incorporation of the radiolabeled material was determined by liquid scintillation spectrometry of the trichloroacetic acid-precipitable material as previously described(5) .

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis employed large format polyacrylamide gels, with modifications of the method of O'Farrell (20) for the Millipore Investigator two-dimensional Electrophoresis System(21) . 20-100 µg of protein solubilized in two-dimensional sample buffer (8 M urea, 2% Nonidet P-40, 2% ampholytes, 100 mM dithiothreitol, 0.1% SDS, 12.5 mM Tris, pH 8.0) were loaded on analytical gels (up to 1 mg for preparative gels). Protein concentration was determined using a solution-based protein assay (Bio-Rad). Isoelectric focusing was performed for 18,000 V-h in a 4% T, 2.6% C polyacrylamide tube gel (18 cm long and 1-mm internal diameter) containing 9.5 M urea, 2.0% (v/v) Nonidet P-40, and 2.0% (v/v) Millipore two-dimensional optimized carrier ampholytes(21) . The second dimension (1 mm 22 cm 22 cm) employed a 12% T, 2.6% C high tensile strength acrylamide-based matrix (Duracryl, Millipore) without a stacking gel run at a constant power of 16 watts(21, 22) . Gels were fixed for 2-12 h in 50% methanol, 10% acetic acid and silver stained as previously reported (21) . For use as co-electrophoresis standards, actin, vimentin, myosin light chain, calmodulin, and smooth muscle tropomyosins were obtained from Sigma. Rat HSP-70 and hamster recombinant GRP78 were obtained from Stressgen Biotechnologies Corp. (Victoria, British Columbia).

Image Analysis and Computerized Databasing

Images were obtained by digitizing gels at 1024 1024 pixels resolution with 256 gray scale levels using a BioImage® 110S computerized imaging system (BioImage Products, Ann Arbor, MI) as described(21, 23) . This UNIX-based system is capable of comparing 14 two-dimensional gels simultaneously and determining isoelectric point, molecular weight, integrated optical density, and spot shape data for each protein. A typical question asked the data base was: ``Find polypeptides reproducibly expressed in the Ang-stimulated cell pattern that differ by 2.5-fold or more from those reproducibly expressed in the quiescent cell patterns, that also have integrated optical densities greater than 0.3, high spot shape quality, molecular weights in the range of 17,000-75,000 daltons, and isoelectric points in the pH range of 4.0-7.2.'' Gels were also visually inspected using a light box.

Protein Sequence Analysis

Internal amino acid sequence analysis was performed as described(24, 25) . Total cell lysates were separated on preparative two-dimensional gels and transferred to nitrocellulose membranes (Schleicher & Schull) using a large format (38 46 20 cm) electroblotter (Polytech Inc., Somerville, MA), which allowed for as many as six two-dimensional gels to be transferred simultaneously(26, 27) . Transfer was performed for 5 h at a constant 66 V; the initial power was 45 watts and rose to 55 watts during the run. Proteins were visualized with 0.1% amido black (Serva, Paramus, NJ) in 10% acetic acid.

Spots of interest were excised from the nitrocellulose membrane and subjected to in situ proteolytic cleavage for 3 h at 37 °C using 0.5 µg of trypsin (sequencing grade from Boehringer Mannheim) in 50 µl of 100 mM NH(4)HCO(3), supplemented with 0.3% Tween 80 (Calbiochem). The resulting peptide mixture was reduced and S-alkylated with 0.1% beta-mercaptoethanol (Bio-Rad) and 0.3% 4-vinylpyridine (Aldrich), respectively, and fractionated by narrow-bore reversed phase high performance liquid chromatography (HPLC) using a 2.1-mm Vydac C4 (214TP54) column (The Separations Group, Hesperia, CA). HPLC system configuration and solvents were as described (28) ; a flow rate of 100 µl/min was used. Fractions were collected by hand and stored at -70 °C before sequence analysis. Trypsin blanks were analyzed as controls.

Selected peak fractions were analyzed by automated Edman degradation using an Applied Biosystems model 477A sequencer. Stepwise liberated phenylthiohydantoin-amino acids were identified using an ``on-line'' 120A HPLC system (Applied Biosystems) equipped with a phenylthiohydantoin C18 (2.1 220 mm; 5-micron particle size) column (Applied Biosystems). The standard AB method was optimized for sub-pmole phenylthiohydantoin-amino acid analysis as described(25, 29) . After storage, column fractions were supplemented with neat trifluoroacetic acid (10% final concentration) before loading onto the sequencing disc. Peptide sequences were compared to data base entries (PIR, SwissProt, GenBank).


RESULTS

Differences in Growth in Response to Ang and PDGF

To verify that PDGF and Ang produced different growth responses in the early passaged adult rat aortic VSMC, DNA and protein synthesis were measured in response to 24 h of treatment with each agonist. In agreement with previous reports(6, 7) , Ang did not significantly stimulate DNA synthesis but did cause a marked increase in protein synthesis, typical of the hypertrophic response (Fig. 1). In contrast, PDGF produced a hyperplastic response, stimulating both DNA and protein synthesis. The response to PDGF was associated with an increase in cell number, with a doubling time of approx36 h (data not shown).


Figure 1: DNA and protein synthesis in rat aortic VSMC. Quiescent VSMC (incubated in nongrowth medium for 48 h) were incubated in fresh nongrowth medium or treated with 1 µM Ang or 5 half-maximal units/ml of PDGF for 24 h in the presence of either [^3H]thymidine (A) or [^3H]leucine (B). Incorporation of the radiolabel into DNA or protein was determined by liquid scintillation spectrometry of trichloroacetic acid precipitates as described(5) . Data are expressed as percentage increase relative to quiescent VSMC and represents the average (±S.E.) of three experiments, each performed on triplicate wells. Experiments shown were performed at passage 5. Essentially identical results have been found using cells from passages 3-13 (data not shown).



Changes in Protein Expression in Response to Growth Agonists

Changes in polypeptide expression in response to Ang, PDGF, and CS were monitored by high resolution two-dimensional gel electrophoresis. approx1000 polypeptides were resolved on 12% gels, approx800 of which had integrated optical densities greater than 0.3. Of these proteins, matched across all of the gels used in this study, the expression of approx2% of the polypeptides was reproducibly altered by stimulation with each of the growth agonists (Ang, PDGF, and CS). In response to CS, levels of 12 polypeptides were significantly increased (>2.5-fold higher than untreated controls), whereas levels of 5 polypeptides were significantly decreased (>2.5-fold lower than controls). The principal polypeptides found to be regulated by growth agonists in VSMC are listed in Table 1. A representative two-dimensional gel showing the location of these polypeptides is shown in Fig. 2. A magnified region from an Ang-stimulated and unstimulated VSMC gel is shown in Fig. 3. Four of the five down-regulated polypeptides were found in this region of the gel.




Figure 2: Two-dimensional electrophoretic protein map of serum-stimulated rat aortic smooth muscle cells. Large format gels were used, and proteins were visualized by silver staining (see ``Materials and Methods''). Proteins that were either up- or down-regulated by treatment with serum, Ang, or PDGF are marked with the spotnumber (coordinates and additional information are listed in Table 1). Additional marker proteins (``landmarks'' on the map) were located by co-electrophoresis of purified proteins (labeled with name). Three of the annotated proteins (and actin) were identified by co-electrophoresis and five by internal microsequencing.




Figure 3: Detailed display of two-dimensional protein patterns of rat aortic smooth muscle cells grown in defined medium or stimulated with Ang. Magnified, matching regions of computerized gel images (full size in Fig. 2) are shown. Four of the down-regulated and one of the up-regulated proteins appear in this section of the gels (listed in Table 1). Spots 111 and 109 represent HSP-60 and a fragment of myosin heavy chain, respectively; the others are unidentified.



Identification of Proteins by Co-electrophoresis

Several cytoplasmic proteins known to be present in VSMC were identified by co-electrophoresis of cell homogenates with commercially available purified proteins (the positions of these proteins have been indicated on Fig. 2). These included isoforms of actin, vimentin, myosin light chain, smooth muscle tropomyosin, HSP-70 and calmodulin. This approach identified three polypeptides up-regulated in response to growth stimulation: HSP-70, vimentin, and total cellular actin (multiple isoforms). In addition, we had previously identified calreticulin (Ro/SS-A antigen) in the rat WT2 fibroblast cell line by N-terminal sequence analysis and many other cytoskeletal proteins by immunoblotting in a variety of other cell lines(19, 22, 30) . The identification of calreticulin as an up-regulated molecule was based upon its comigration with rat WT2 fibroblast lysates. Although total actin content doubled in growth-stimulated cells, the amounts of actin-associated proteins (myosin light chain and VSMC-specific tropomyosin isoforms) remained unaltered.

Identification of Proteins by Microsequencing

Five proteins altered by CS, PDGF, and Ang were identified by protein microsequencing. Spots of interest were cut from amido black-stained nitrocellulose blots (4 blots total), proteins digested in situ, and peptides separated by HPLC. Selected peak fractions were sequenced and the results compared to entries in GenBank, PIR, and SwissProt data bases. The results of the internal protein sequence analysis and identification are summarized in Table 2. Four up-regulated proteins included elongation factor 1-beta, protein disulfide isomerase, a putative protein disulfide isomerase (isozyme Q-2, ERp61, GRP58), and HSP-60. Protein disulfide isomerase Q-2 always had two more acidic isoforms associated with it. These isoforms were also up-regulated by Ang, PDGF, and CS. Microsequencing also identified one protein down-regulated by all growth agonists as a myosin heavy chain isoform. The position of the protein in the two-dimensional gels indicates that it probably represents a proteolytic fragment of myosin heavy chain. It was not possible to determine based upon the available sequence whether the myosin heavy chain was a VSMC-specific or nonmuscle isoform due to the high sequence conservation in the myosin heavy chain family.



Quantitative Changes in Protein Expression

Quantitative information concerning the relative abundance of the nine identified polypeptides is presented in Fig. 4. All three growth agonists produced similar responses, although Ang treatment tended to cause a greater induction of the growth-stimulated polypeptides than either PDGF or CS.


Figure 4: Quantitative analysis of the nine identified polypeptides regulated by serum, PDGF, and Ang. Passage 2-5 rat aortic VSMC were made quiescent for 48 h and then treated for 24 h with 1) fresh ``nongrowth'' medium or fresh medium containing 2) 1 µM Ang, 3) PDGF (5 half maximal units/ml), or 4) 10% CS. Each bar represents the average (±S.D.) of 3-4 different two-dimensional gels. The y axis is presented in integrated optical density units and provides a measure of each protein's relative abundance.



Unidentified Proteins Regulated by Growth Factors

In addition to the nine proteins identified above, four polypeptides up-regulated by treatment with growth agonists and four down-regulated by agonist treatment were not sufficiently abundant to identify by protein microsequencing. These are listed in Table 1.


DISCUSSION

This study reports the use of high resolution two-dimensional gel electrophoresis to identify proteins regulated by growth factors in cultured rat aortic VSMC. Among the proteins identified were two classes of proteins known to assist in the protein folding process, molecular chaperones and protein disulfide isomerases. These proteins have not been previously shown to be regulated by growth factors in cultured VSMC and have not been identified in VSMC utilizing RNA-based screening strategies.

Post-translational or co-translational folding is a necessary aspect of manufacturing new proteins (reviewed in (31) ). Molecular chaperones interact with a wide array of polypeptides from the onset of translation until attainment of the final folded state(32, 33, 34) . The two families of heat shock proteins most intimately associated with general protein folding are HSP-60 and HSP-70(35) . HSP-70 has been shown to be inducible by serum mitogens in several cell types(36, 37) . HSP-70 mRNA has also been found to be induced in intact aorta after treatment with sympathomimetic or hypertensive agents and in cultured aortic VSMC by hypertrophic stimuli(38, 39, 40, 41, 42) . Yeast strains lacking a class of cytoplasmic HSP-70 show defects in protein synthesis(33, 34) . The growth defect can be overcome by overproduction of a protein related to the translation elongation factor, EF-1alpha. The rate of synthesis of EF-1alpha increases 6-fold following mitogenic stimulation (43) . The present study would not have detected changes in EF-1alpha levels because this protein has an apparent isoelectric point of 10.0 (44) , well outside the pH range (pH 4.0-7.5) of the two-dimensional gels. However, another component of the elongation factor complex, EF-1beta, was found to be up-regulated by Ang, PDGF, and CS.

Protein disulfide isomerases are capable of catalyzing a wide range of protein disulfide oxidoreduction reactions(45, 46) . Protein disulfide isomerases bind newly synthesized proteins in the process of folding in the endoplasmic reticulum and are also found in stable association with misfolded molecules in this compartment(45, 47) . The enzyme has also been shown to facilitate the oxidative refolding of lysozyme in vitro, provided it is present in amounts comparable to that found in the endoplasmic reticulum(48) . At least one protein disulfide isomerase isoform has been found to be expressed at high levels in normal cells engaged in the synthesis of large amounts of secreted proteins(49) .

Calreticulin acts as a major Ca-binding protein in the lumen of the endoplasmic reticulum(50) . Calreticulin shares significant homology to calnexin, an 88-kDa endoplasmic reticulum-associated protein determined to be a novel molecular chaperone participating in the assembly of murine class I histocompatibility molecules(47, 51) . Calreticulin has been found in the nucleus and recently has been shown to modulate gene expression by binding to the glucocorticoid receptor(52, 53) , further suggesting it serves as a nuclear chaperone. We have recently found that antisense oligonucleotides to calreticulin mRNA inhibited PDGF-mediated growth of cultured rat aortic VSMC by approx80%, suggesting that calreticulin may play a critical role in the growth response of these cells(69) . Several similarities between the 5`-flanking regions of calreticulin, protein disulfide isomerase, GRP78, and GRP94 suggest that they may be similarly regulated(54) . GRP78 is approximately 60% homologous to HSP-70. No growth-induced changes in GRP78 were observed in this study.

A variety of cytoskeleton proteins have been previously identified as being growth factor-responsive in VSMC using RNA-based strategies(10, 55, 56, 57, 58) . These include phospholamban, smooth muscle isoforms of alpha-actin and -actin, CHIP28 (a channel protein), alpha-calponin, SM22alpha (a member of the calponin family), tropoelastin, vinculin, and smooth muscle isoforms of myosin heavy chain. The approach taken in the current study was similar to a recently published two-dimensional gel study examining the hypertrophic effects of Ang and arginine vasopressin on rat aortic VSMC(59) . In that report, a 2-3-fold increase in actin content, a 2.5-7-fold increase in vimentin content, and a 3-6-fold increase in tropomyosin content were observed upon stimulation of quiescent VSMC with hypertrophic agents for 96 h. We found similar increases in total actin and vimentin content in response to 24 h of either hyperplastic or hypertrophic stimuli. In contrast, total VSMC-specific tropomyosin and myosin light chain content were altered little by 24 h of stimulation (data not shown).

Both PDGF and Ang were used as agonists to stimulate protein synthesis. As demonstrated in Fig. 1, Ang acts as a hypertrophic agent for adult rat aortic VSMC, whereas PDGF is hyperplastic(6, 7) . Ang and PDGF share a variety of intracellular signaling pathways, including the activation of phospholipase C, the induction of the mitogen-activated protein kinase system, and the activation of the Na-H antiporter (reviewed in (60) ). In addition, both agonists induce similar sets of early response genes, including c-fos and c-jun (reviewed in (13) ). Previous attempts by this laboratory to identify differences in gene expression in response to Ang and PDGF using differential screening (11) have been unsuccessful. Despite the relatively large number of proteins available for evaluation in the current study, no differences were seen in the 24-h expression of specific proteins in response to Ang or PDGF. This underscores the similarities in signaling between the two agonists and further suggests that there are a limited number of molecular events that distinguish the hypertrophic and hyperplastic responses of VSMC. In this regard, it should be noted that Dzau and co-workers (61) have provided evidence that the secretion of transforming growth factor-beta in response to Ang may be the critical event in converting a hyperplastic response to a hypertrophic one.

The present study describes a model system for analyzing approximately 1,000 proteins expressed in VSMC. Protein spots on two-dimensional gels must contain at least 15-20 pmol (combined from a maximum of 5 gels) of material to be suitable for identification by microsequencing. State-of-the-art approaches are required to allow for amino acid identification at the sub-pmol level(25, 28, 29) . We estimate that based on their abundance on two-dimensional gels, about 10% of these proteins can be identified by microsequencing. Indeed, eight proteins regulated by growth agonists in VSMC were present at levels below that necessary for even the most sensitive sequencing approaches currently available. These proteins could potentially be identified with specific antibodies or by comparison with the migration patterns of other known proteins on two-dimensional gels. Increasing the amount of total protein analyzed on two-dimensional gels should increase the percentage of proteins suitable for identification by sequencing. However, this usually results in very distorted gel patterns with minor spots being obscured by the more abundant proteins. Thus, pre-gel enrichment of the proteins of interest would be necessary.

In summary, high resolution two-dimensional gel electrophoresis and microsequencing has provided information on a group of proteins involved in protein synthesis and folding that had not previously been appreciated as growth factor-regulated molecules in VSMC. This underscores the power of this approach to identify classes of proteins whose regulation is not based on changes in steady state mRNA levels easily identifiable by mRNA- and cDNA-based screening strategies. Of note, this study did not identify the protein products of many of the early response genes, such as the proto-oncogenes or cell cycle-related genes found by others using analyses of mRNA. This is likely due in many cases to the relatively low concentration of these proteins in whole cell extracts and in some to the transient nature of the increase in protein levels. The two-dimensional gel approach should complement those involving mRNA and cDNA in providing a comprehensive analysis of molecular events associated with VSMC growth.


FOOTNOTES

*
The Sequencing Laboratory of Memorial Sloan-Kettering Cancer Center is supported by National Cancer Institute Core Grant 5 P30 CA08748-29. This work was supported in part by National Institutes of Health Grants RO1 HL43302 (to M. B. T.) and RO1 NS29814 (to A. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Microvascular Research Division, Boston University, Boston, MA 02215.

Bristol-Meyers Squibb Established Investigator of the American Heart Association.

**
Recipients of Career Scientist Awards from the Irma T. Hirschl-Monique Weill-Caulier Charitable Trusts.

§§
To whom correspondence should be addressed: Box 1269, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-0731; Fax: 212-860-7032.

(^1)
The abbreviations used are: VSMC, vascular smooth muscle cells; Ang, angiotensin II; CS, calf serum; HPLC, high performance liquid chromatography; PDGF, platelet-derived growth factor.


ACKNOWLEDGEMENTS

We thank Ai-Ru Chen, Lily Lam, Qin Su, and Mary Lui for assistance with two-dimensional gels and protein sequencing, Hong Zhang for maintaining smooth muscle cell cultures, and Dr. Ulrich Hartl for discussion.


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