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Proteomic analysis of articular cartilage vesicles from normal and osteoarthritic cartilage.

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ARTHRITIS & RHEUMATISM
Vol. 63, No. 2, February 2011, pp 401–411
DOI 10.1002/art.30120
© 2011, American College of Rheumatology
Proteomic Analysis of Articular Cartilage Vesicles From
Normal and Osteoarthritic Cartilage
Ann K. Rosenthal, Claudia M. Gohr, James Ninomiya, and Bassam T. Wakim
protein ␤ig-H3, DEL-1, vitronectin, and serine protease
HtrA1 (P < 0.01).
Conclusion. These findings lend support to the
concept of ACVs as physiologic structures in articular
cartilage. Changes in OA ACVs are largely quantitative
and reflect an altered matrix and the presence of
inflammation, rather than revealing fundamental
changes in composition.
Objective. Articular cartilage vesicles (ACVs) are
extracellular organelles found in normal articular cartilage. While they were initially defined by their ability
to generate pathologic calcium crystals in cartilage of
osteoarthritis (OA) patients, they can also alter the
phenotype of normal chondrocytes through the transfer
of RNA and protein. The purpose of this study was to
analyze the proteome of ACVs from normal and OA
human cartilage.
Methods. ACVs were isolated from cartilage samples from 10 normal controls and 10 OA patients. We
identified the ACV proteomes using in-gel trypsin digestion, nanospray liquid chromatography tandem mass
spectrometry analysis of tryptic peptides, followed by
searching an appropriate subset of the Uniprot database. We further differentiated between normal and OA
ACVs by Holm-Sidak analysis for multiple comparison
testing.
Results. More than 1,700 proteins were identified
in ACVs. Approximately 170 proteins satisfied our stringent criteria of having >1 representative peptide per
protein present, and a false discovery rate of <5%.
These proteins included extracellular matrix components, phospholipid binding proteins, enzymes, and
cytoskeletal components, including actin. While few
proteins were seen exclusively in normal or OA ACVs,
immunoglobulins and complement components were
present only in OA ACVs. Compared to normal ACVs,
OA ACVs displayed decreases in matrix proteoglycans
and increases in transforming growth factor ␤–induced
Articular cartilage vesicles (ACVs) are 50–
150-nm membrane-bound extracellular organelles that
are found in normal articular cartilage (1). They were
initially characterized in reference to their role in the
pathologic mineralization of cartilage, in studies which
mirrored studies of matrix vesicles derived from growth
plate cartilage and other normally mineralizing tissues
(2). ACVs concentrate enzymes, ions, and substrates
necessary for mineral formation (1). Isolated ACVs
generate pathologic calcium-containing crystals identical
to those found in arthritic human joints (1,3). Articular
cartilage, however, does not typically undergo matrix
mineralization, except in pathologic conditions such as
osteoarthritis (OA) (4).
While a primary role for ACVs in pathologic
mineralization seems plausible, the high quantity of
ACVs in normal healthy articular cartilage remains
puzzling (5). Few structures in nature have only a single
pathologic function, and the energy expenditure required for the formation of ACVs is unlikely to be
wasted. It has been postulated that in growth plate
cartilage, matrix vesicles may participate in matrix repair, in addition to matrix mineralization (6). We recently demonstrated that ACVs contain RNA (7), like
other types of extracellular vesicles (8). ACVs specifically transfer their labeled RNA and protein to intact
naive primary chondrocytes via simple coculture. Importantly, exposure of normal chondrocytes to small quantities of intact ACVs induces markers of chondrocyte
hypertrophy, such as those seen in OA cartilage (7).
Dr. Rosenthal’s work was supported by a grant from the NIH
(R01-AR-056215).
Ann K. Rosenthal, MD, Claudia M. Gohr, BS, James Ninomiya, MD, Bassam T. Wakim, PhD: Medical College of Wisconsin,
Milwaukee.
Address correspondence to Ann K. Rosenthal, MD, Division
of Rheumatology, cc-111W, Zablocki VA Medical Center, 5000 W.
National Avenue, Milwaukee, WI 53295-1000. E-mail:
ann.rosenthal@va.gov.
Submitted for publication April 26, 2010; accepted in revised
form October 21, 2010.
401
402
ROSENTHAL ET AL
Table 1. The articular cartilage vesicle proteome*
Accession no.
Cytoskeletal
P60709
P63261
P63267
P68032
P68133
P62736
P68104
P26038
P06396
O00560
P04264
P35241
Q05639
P07437
P04350
Q13885
Q9BVA1
P68371
P15311
Q13509
P02760
Q9BQL6
Q9UPN3
Cytoplasmic enzymes
P61626
Q06830
P04406
P06733
P14618
P00338
P32119
P00558
P62937
P12724
P60174
Q9UKU9
P18669
Q8N0Y7
Q9UKZ9
P50148
O95837
P34096
P13929
P09104
P04899
P06744
Q05524
P28330
Q6UXX5
Q96PE3
P17858
P13637
P05023
Extracellular matrix
P10915
O15335
P21810
Description
Peptide
count
Scan count
% coverage
pI
␤-actin
␥-actin
␣-actin-3
␣-cardiac actin
␣-actin-1
␣-actin-2
Elongation factor 1-alpha 1
Moesin
Gelsolin precursor
Syntenin 1
Cytokeratin 1
Radixin
Elongation factor 1-alpha 2
Tubulin ␤ chain
Tubulin ␤-4 chain
Tubulin ␤-2A chain
Tubulin ␤-2B chain
Tubulin ␤-2C chain
Ezrin
Tubulin ␤-3 chain
␣1-microglobulin/bikunin precursor
Kindlin 1
Microtubule-actin crosslinking factor 1
10
10
8
8
8
7
4
7
6
2
3
4
3
2
2
2
2
2
3
2
2
2
2
104
104
55
59
59
54
79
124
60
13
10
73
78
2
2
2
2
2
72
2
2
2
7
28.000
28.000
25.266
20.424
20.424
17.507
12.554
12.326
11.765
8.389
7.776
7.376
7.343
4.505
4.505
4.494
4.494
4.494
4.444
4.444
3.693
2.954
0.773
5.315
5.332
5.332
5.25
5.25
5.256
8.81
6.131
5.954
7.042
7.792
6.085
8.817
4.788
4.788
4.788
4.788
4.8
6.002
4.835
5.98
5.966
5.284
Lysozyme C precursor (EC 3.2.1.17)
Peroxiredoxin-1 (EC 1.11.1.15)
GAPDH (EC 1.2.1.12)
␣-enolase (EC 4.2.1.11)
M1/M2 isozymes of pyruvate kinase
(EC 2.7.1.40)
L-lactate dehydrogenase A chain (EC 1.1.1.27)
Peroxiredoxin-2 (EC 1.11.1.15)
Phosphoglycerate kinase 1 (EC 2.7.2.3)
Peptidyl-prolyl cis-trans isomerase A (EC 5.2.1.8)
Eosinophil cationic protein precursor
(EC 3.1.27.-)
Triosephosphate isomerase (EC 5.3.1.1)
Angiopoietin-like 2
Phosphoglycerate mutase 1 (EC 5.4.2.1)
Probable phosphoglycerate mutase 4
(EC 5.4.2.1)
Procollagen C–proteinase enhancer 2
Guanine nucleotide–binding protein G(q)
G-protein ␣ subunit 14
Ribonuclease 4 precursor (EC 3.1.27.-)
␤-enolase (EC 4.2.1.11)
␥-enolase (EC 4.2.1.11)
Adenylate cyclase–inhibiting G ␣ protein
Glucose-6-phosphate isomerase (EC 5.3.1.9)
␣-enolase, lung specific (EC 4.2.1.11)
Long-chain acyl-CoA dehydrogenase (EC
1.3.99.13)
Inter-␣ inhibitor H5–like protein
Type I inositol-3,4-bisphosphate 4-phosphatase
(EC 3.1.3.66)
6-phosphofructokinase, liver type (EC 2.7.1.11)
Na⫹/K⫹ ATPase 3 (EC 3.6.3.9)
Na⫹/K⫹ ATPase 1 (EC 3.6.3.9)
6
6
5
8
11
339
113
54
136
147
45.946
30.151
28.443
27.483
23.962
8.944
7.793
8.21
6.933
7.505
7
4
6
2
2
58
60
77
30
3
22.356
20.812
19.952
18.293
16.875
8.021
5.713
7.804
7.49
10.142
3
7
2
2
36
48
62
62
16.532
14.402
12.648
12.598
6.541
7.137
6.803
6.263
4
2
2
2
2
2
2
2
2
2
67
2
20
12
9
9
37
21
71
2
11.325
10.198
10.141
9.524
8.295
8.295
7.627
5.745
4.803
4.651
8.241
5.612
5.842
8.801
7.318
4.916
5.362
8.081
5.832
7.202
6
2
71
2
4.646
4.606
8.821
6.579
2
2
2
2
7
7
4.108
2.863
2.835
7.017
5.241
5.347
Proteoglycan link protein
Chondroadherin precursor
Biglycan precursor
26
21
19
1,110
530
368
70.339
52.368
50.000
7.003
9.203
7.087
PROTEOMIC ANALYSIS OF CARTILAGE VESICLES IN OSTEOARTHRITIS
403
Table 1. (Cont’d)
Accession no.
Q15582
P12109
P20774
P51888
P12111
O75339
P49747
Q92743
P12110
O43854
P08493
P02751
P04004
P02649
P51884
P07585
P01009
Q8IUL8
P02671
Q92954
P16112
P01023
P02458
Q14055
P00488
P07996
P20849
Q03692
P35443
P24821
P98160
P20908
Q96QB0
P13611
P35555
Growth factors
Q16674
P55107
O75888
Q8NI99
Inflammatory
components
P01834
P02743
P01857
P02647
P02748
P60827
P01859
P05090
P06727
P04220
P01871
P01031
P01860
P01861
P01877
P01876
P07357
Description
Kerato-epithelin
Collagen ␣1(VI) chain precursor
Mimecan precursor (osteoglycin)
Prolargin precursor
Collagen ␣3(VI) chain precursor
Cartilage intermediate-layer protein 1 precursor
Cartilage oligomeric matrix protein precursor
Serine protease HtrA1 precursor (EC 3.4.21.-)
Collagen ␣2(VI) chain precursor
Integrin-binding protein DEL-1
Matrix Gla protein precursor
Fibronectin precursor
Vitronectin precursor
Apolipoprotein E precursor
Lumican precursor
Decorin precursor
␣1-antitrypsin precursor
Cartilage intermediate-layer protein 2
precursor
Fibrinogen ␣-chain precursor
Lubricin
Aggrecan core protein precursor
␣2-macroglobulin precursor
Collagen ␣1(II) chain precursor
Collagen ␣2(IX) chain precursor
Coagulation factor XIIIA chain precursor (EC
2.3.2.13)
Thrombospondin 1 precursor
Collagen ␣1(IX) chain precursor
Collagen ␣1(X) chain precursor
Thrombospondin 4 precursor
Tenascin precursor (TN)
Perlecan
Collagen ␣1(V) chain precursor
Collagen ␣2(V) chain precursor
Versican core protein precursor
Fibrillin 1 precursor
Melanoma-derived growth regulatory protein
precursor
Bone morphogenetic protein 3b precursor
CD256 antigen
Angiopoietin-like 6
Ig␬ chain C region
Serum amyloid P–component precursor
Ig␥ 1 chain C region
Apolipoprotein A-I precursor
Complement component C9 precursor
Complement C1q TNF-related protein 8
precursor
Ig␥ 2 chain C region
Apolipoprotein D precursor
Apolipoprotein A-IV precursor
Ig␮ heavy chain disease protein
Ig␮ chain C region
Complement C5 precursor
Ig␥ 3 chain C region
Ig␥ 4 chain C region
Ig␣ 2 chain C region
Ig␣ 1 chain C region
Complement component C8 ␣ chain precursor
Peptide
count
Scan count
% coverage
pI
20
47
9
15
108
41
18
11
36
9
2
36
9
5
4
2
2
8
499
5,609
91
1,001
8,071
1,422
1,686
175
6,976
83
9
764
262
88
18
3
5
118
39.092
37.549
33.221
32.461
30.069
28.041
26.288
25.625
25.613
25.000
23.301
21.500
18.619
15.773
14.793
9.471
8.852
7.872
7.35
5.311
5.478
9.189
6.458
8.156
4.352
7.525
5.836
6.917
9.497
5.48
5.587
5.659
6.226
8.296
5.401
7.992
6
8
25
5
8
3
2
127
165
5,529
15
231
41
4
7.390
7.265
7.081
6.920
5.078
4.064
3.967
5.745
9.298
4.11
6.067
8.227
8.976
5.798
4
2
2
2
2
6
2
2
2
2
42
5
23
656
3
60
2
5
16
8
3.333
2.932
2.647
2.393
2.181
2.164
1.469
1.334
0.972
0.557
4.728
8.488
9.492
4.451
4.8
6.131
4.948
6.123
4.436
4.817
3
17
28.244
8.564
2
2
2
6
9
2
5.021
4.800
2.766
9.329
9.445
8.281
3
12
6
4
5
2
23
397
54
12
32
5
48.113
34.978
23.939
21.348
12.522
11.450
5.627
6.161
7.952
5.59
5.448
9.667
3
2
4
2
2
8
2
2
2
2
2
6
6
10
5
5
29
4
3
6
6
3
11.043
9.524
8.586
7.928
6.828
6.623
5.862
5.810
5.000
4.816
4.795
7.299
5.062
5.297
5.139
6.403
6.172
7.296
7.037
5.773
6.143
6.118
404
ROSENTHAL ET AL
Table 1. (Cont’d)
Accession no.
Q9BXJ3
P13671
Membrane
P07355
P04083
Q08431
P08758
P14555
P04271
P09525
P06702
P08133
P63104
P10909
P21589
P27105
P60903
O15232
Q9HCJ1
O75131
P50995
P43007
Q96FN4
Nuclear
P62805
P84243
P28001
O60814
Q00056
Other
P62988
O60687
O75340
O43293
Q7Z7G0
P08582
Q9H4G0
Q9Y2E4
Signaling
P62834
P61224
P29992
P17252
Q9UJ30
Peptide
count
Description
Complement C1q tumor necrosis factor–related
protein 4 precursor
Complement component C6 precursor
Scan count
% coverage
pI
2
5
4.559
7.933
2
5
2.677
6.407
26
17
17
433
391
1,001
68.343
57.391
54.264
7.302
6.726
7.877
18
9
420
97
48.903
48.611
4.932
9.031
3
7
2
11
2
6
6
3
2
5
2
3
2
2
2
85
62
7
86
4
379
22
29
3
96
2
39
13
7
17
45.055
33.333
28.070
23.512
20.000
16.481
16.376
14.634
14.433
12.757
10.976
6.331
6.139
4.511
4.380
4.523
5.873
5.78
5.44
4.728
5.945
6.631
7.589
6.827
6.312
7.558
5.631
7.254
5.938
5.761
Histone H4
Histone H3.3
Histone H2A (10 subtypes)
Histone H2B (11 subtypes)
Homeobox protein Hox-A4 (Hox-1D) (Hox-1.4)
7
2
3
2
2
65
5
23
8
55
56.863
28.889
21.705
17.600
6.250
11.762
11.649
11.202
10.134
9.764
Ubiquitin
Sushi repeat–containing protein SRPX2
precursor
Probable calcium-binding protein ALG-2
Death-associated protein kinase 3 (EC 2.7.1.37)
Target of Nesh-SH3 precursor (Tarsh)
Melanotransferrin precursor
Band 4.1–like protein 1 (neuronal protein 4.1)
Disco-interacting protein 2 homolog C
2
7
73
81
32.895
20.215
6.603
6.888
3
2
5
3
2
2
80
4
25
38
10
3
17.801
5.947
5.395
4.607
2.951
1.928
5.17
6.501
9.252
5.715
5.455
7.023
Ras-related protein Rap-1A precursor
Ras-related protein Rap-1B precursor
Guanine nucleotide–binding protein ␣ 11 subunit
Protein kinase C ␣ type (EC 2.7.1.37)
Protein kinase C ␤ type (EC 2.7.11.13)
3
3
3
5
2
11
11
51
14
2
13.587
13.587
10.028
9.687
3.577
6.321
5.639
5.538
6.672
6.613
Annexin II
Annexin I
Lactadherin precursor (milk fat globule/
endothelial growth factor VIII)
Annexin V
Phospholipase A2, membrane precursor (EC
3.1.1.4)
S100 protein, ␤ chain
Annexin IV
S100 A9 protein
Annexin VI
14-3-3 protein ␨/␦
Clusterin precursor
5⬘-nucleotidase precursor (EC 3.1.3.5)
Protein 7.2b
S100 A10 protein
Matrilin 3 precursor
Progressive ankylosis protein homolog (ANK)
Copine III
Annexin XI
Neutral amino acid transporter A (SATT)
Copine II
* This table represents a subset of the identified proteins that were represented by at least 2 peptides and had a false discovery rate of ⱕ5%. Peptide
count refers to the number of peptides unique to the protein. Scan count is the number of times that one or more of the peptides unique to the
protein were counted. Peptide coverage is the percentage of the protein covered by the peptides present. pI refers to the isoelectric point of the
protein.
Thus, during early OA, ACVs may be released from the
matrix by matrix-degrading enzymes and interact directly with chondrocytes to promote chondrocyte hypertrophy.
The contents and functions of ACVs, however,
remain poorly elucidated. It is not known whether
ACVs, like growth plate matrix vesicles, are formed
through zeiotic blebbing (9). It has also been suggested
that ACVs are products of stressed or apoptotic cells
(10), and would thus be significantly altered in OA
cartilage. Proteomic analysis of exosomes (11) and several types of growth plate matrix vesicles (9,12) revealed
PROTEOMIC ANALYSIS OF CARTILAGE VESICLES IN OSTEOARTHRITIS
important information relevant to the functions and
mechanisms of formation of these vesicles. In this study,
we characterized the ACV proteome and compared the
proteomes of ACVs derived from OA and normal
human articular cartilage.
MATERIALS AND METHODS
Cartilage. Human OA hyaline articular cartilage was
obtained from deidentified, discarded pathologic specimens at
the time of surgery for total knee replacement for OA (n ⫽
10). None of the specimens contained visible crystal deposits in
the cartilage. Snap-frozen normal adult human cartilage from
knees of adult donors with no clinical joint disease (n ⫽ 10)
was obtained from the National Disease Research Interchange
and the Musculoskeletal Transplant Foundation. All visible
cartilage was cleaned of adherent bone and stored at –70°C
until use. Previous work has demonstrated that there are no
significant differences between ACVs derived from fresh or
from frozen cartilage (13). All experiments with human tissue
were approved by the Institutional Review Boards of the
Zablocki Veteran Affairs Medical Center, Milwaukee and the
Medical College of Wisconsin.
ACV isolation. ACVs were isolated from whole cartilage as previously described (1). Briefly, hyaline articular
cartilage was minced and weighed under sterile conditions.
Cartilage pieces were incubated in Dulbecco’s modified Eagle’s medium with 0.1% hyaluronidase (1 ml/gm wet weight
cartilage) for 5 minutes to remove surface hyaluronate, and for
10 minutes with 0.5% trypsin (1 ml/gm cartilage). Trypsin
inhibitor (0.2% soybean trypsin inhibitor, 1 ml/gm cartilage)
was added to inactivate any remaining trypsin. All incubations
were performed at 37°C with 5% CO2 with stirring.
After washing, cartilage pieces were incubated for 45
minutes with 0.2% bacterial collagenase type II (2.5 ml/gm
cartilage; Worthington). Additional media was added so that
the final collagenase concentration was 0.05% in a total of 10
ml media/gm cartilage, and cartilage was incubated overnight
with stirring. The mixture was filtered and centrifuged at 500g
for 15 minutes to remove cells, then at 37,000g for 15 minutes
to remove large cell fragments and organelles. The supernatant was then centrifuged at 120,000g for 60 minutes to pellet
the ACV fraction. ACVs were resuspended in Hanks’ balanced
salt solution and protein concentrations were determined by
Lowry assay (14). Electron microscopy of ACVs isolated in this
manner has revealed a biphasic population of membranebound vesicles, 50–150 nm in diameter, that are morphologically distinguishable from cellular debris (15), and are identical
to ACVs that have spontaneously elaborated into the conditioned media of healthy primary chondrocyte monolayers (16).
Their small size and lack of DNA (7) distinguish them from
apoptotic bodies.
Proteome analysis. Protein from each ACV preparation (50 ␮g) was polymerized into a gel in the cap of an
Eppendorf tube by adding 100 ␮l acrylamide/bis (30% acrylamide:2.67% bis-acrylamide), 2 ␮l of 10% ammonium persulfate, and 2 ␮l TEMED (17). Gel pieces were transferred to the
corresponding Eppendorf tubes in 1 ml of 40% methanol and
7% acetic acid, and incubated for 30 minutes. Gel pieces were
405
washed in 50% acetonitrile in water, followed by 50% acetonitrile in 100 mM ammonium bicarbonate (pH 8.0). Gels were
then dried under vacuum using a Savant SpeedVac. Two
hundred microliters of ammonium bicarbonate (100 mM) (pH
8.0) containing 1 ␮g trypsin (Promega) was added to each gel
piece and incubated overnight at 37°C. Each gel piece was then
extracted twice with 70% acetonitrile in 0.1% formic acid, and
the extracts of each gel were pooled together and dried.
Twenty microliters of GuHCl (6M) in 5 mM potassium phosphate and 1 mM dithiothreitol (pH 6.5) was added to each
dried sample, sonicated, and peptides were extracted using a
C18 ZipTip (Millipore). The extracted peptides were collected
and dried. Five microliters of formic acid (0.1%) in MS-grade
water containing 5% acetonitrile was added to each sample.
Samples were subjected to nanospray liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis
using an LTQ mass spectrometer (Thermo-Fisher), coupled to
a Surveyor high-performance liquid chromatography system
equipped with a Micro AS auto sampler (Thermo-Fisher). The
instrument was interfaced with an Aquasil, C18 PicoFrit
capillary column (75 ␮m ⫻ 10 cm; New Objective). The mobile
phases consisted of 1) 0.1% formic acid containing 5% acetonitrile (solvent A) and 2) 0.1% formic acid in 95% acetonitrile
(solvent B). A 180-minute linear gradient was used. The ions
eluted from the column were electrosprayed at a voltage of
1.75 kV. The respective proteomes were determined from
searching the MS/MS data against the human subset of the
Uniprot database.
Bioinformatic analysis. Data were analyzed using Visualize software (written by Dr. Brian Halligan, Medical
College of Wisconsin, Milwaukee). Proteins were considered
definitely present if the search results showed at least 2
matched peptides per protein and a false discovery rate (FDR)
of ⱕ5%. Proteins that satisfied the above criteria were further
subjected to Holm-Sidak analysis for multiple comparison
testing in order to detect those present at significantly different
levels between groups. P values less than 0.05 were considered
significant.
RESULTS
More than 1,700 different gene products were
identified in one or more of the 20 ACV samples.
Approximately 170 proteins were represented by ⬎1
peptide and having an FDR of ⱕ5%, which satisfied our
stringent criteria. We eliminated several proteins that
resulted from blood contamination (including hemoglobin and unique red cell proteins), which is unavoidable
in these specimens. Table 1 lists the remaining proteins,
including their accession numbers, number of peptides
identified per protein, scan count, protein coverage, and
the pI of the proteins.
The ACV proteome. As shown in Table 1, ACV
proteins fell into several categories, as delineated by
cellular location and function. Extracellular matrix
(ECM) proteins, including collagens, proteoglycans, and
other matrix proteins, were prominent among the types
406
ROSENTHAL ET AL
Table 2. Comparison of the proteomes of articular cartilage vesicles derived from normal and osteoarthritic cartilage*
Accession no.
OA only
P02671
P02649
P01857
P01031
P60174
P01834
P21589
P00738
P06727
Normal
only
Q9UKU9
P68104
Q14055
Q16674
P13611
P57053
Up-regulated
in OA
P02768
Q15582
O43854
P02743
P04004
Q92743
P12111
Description
Fibrinogen ␣-chain
precursor
Apolipoprotein E
precursor
Ig ␥ 1 chain C
region
Complement C5
precursor
Triosephosphate
isomerase (EC
5.3.1.1)
Ig ␬ chain C
region
5⬘-nucleotidase
precursor (EC
3.1.3.5)
Haptoglobin
precursor
Apolipoprotein
A-IV precursor
Angiopoietinrelated protein 2
precursor
Elongation factor
1-alpha 1
Collagen ␣2(IX)
chain precursor
Melanoma-derived
growth
regulatory
protein
precursor
Versican core
protein
precursor
Histone H2B (11
subtypes)
Serum albumin
precursor
Transforming
growth factor
␤–induced
protein ig-h3
precursor
Integrin-binding
protein
DEL-1
Serum amyloid
P–component
precursor
Vitronectin
precursor
Serine protease
HtrA1 precursor
(EC 3.4.21.-)
Collagen ␣3(VI)
chain precursor
Total
peptides
Total
coverage
Normal
run
fraction
OA run
fraction
Normalized
ratio
Log
normalized
ratio
Normalized P
H-S
5
5.08
0
40
–
–
2.29 ⫻ 10⫺33
0
5
15.77
0
60
–
–
2.87 ⫻ 10⫺24
0
6
23.94
0
50
–
–
4.89 ⫻ 10⫺13
6.16 ⫻ 10⫺11
8
6.62
0
20
–
–
3.20 ⫻ 10⫺7
3.65 ⫻ 10⫺5
3
16.47
0
40
–
–
2.03 ⫻ 10⫺6
2.24 ⫻ 10⫺4
3
48.11
0
30
–
–
3.78 ⫻ 10⫺6
4.08 ⫻ 10⫺4
5
11.32
0
30
–
–
3.01 ⫻ 10⫺4
3.00 ⫻ 10⫺2
3
6.40
0
20
–
–
3.01 ⫻ 10⫺4
3.03 ⫻ 10⫺2
4
8.59
0
20
–
–
5.69 ⫻ 10⫺4
4.89 ⫻ 10⫺2
–
–
6
11.56
60
0
–
–
5.51 ⫻ 10⫺15
7.11 ⫻ 10⫺13
4
12.55
20
0
–
–
2.10 ⫻ 10⫺6
2.29 ⫻ 10⫺4
3
4.06
10
0
–
–
6.10 ⫻ 10⫺5
6.45 ⫻ 10⫺3
3
28.24
30
0
–
–
1.43 ⫻ 10⫺4
1.47 ⫻ 10⫺2
2
0.97
20
0
–
–
3.36 ⫻ 10⫺4
3.31 ⫻ 10⫺2
2
17.60
10
0
–
–
3.36 ⫻ 10⫺4
2.95 ⫻ 10⫺2
–
–
8
16.58
10
60
16.21
4.02
3.98 ⫻ 10⫺9
4.70 ⫻ 10⫺7
20
39.09
50
100
13.93
3.80
8.94 ⫻ 10⫺86
0
9
25.00
50
70
4.18
2.06
5.75 ⫻ 10⫺8
6.67 ⫻ 10⫺6
12
34.98
40
100
3.30
1.72
2.83 ⫻ 10⫺25
0
9
18.62
80
90
2.81
1.49
5.93 ⫻ 10⫺14
7.53 ⫻ 10⫺12
11
25.63
70
90
2.30
1.20
4.84 ⫻ 10⫺7
5.42 ⫻ 10⫺5
108
30.07
100
100
2.00
1.00
1.59 ⫻ 10⫺189
0
PROTEOMIC ANALYSIS OF CARTILAGE VESICLES IN OSTEOARTHRITIS
407
Table 2. (Cont’d)
Accession no.
P12109
P12110
Downregulated
in OA
P10915
Q08431
P02458
P10909
P26038
P21810
P51888
O75339
P16112
O15335
Q92954
Q8IUL8
O60687
P49747
P02751
P35443
P35241
O15232
P29992
P15311
P07996
P98160
Description
Total
peptides
Total
coverage
Normal
run
fraction
OA run
fraction
Normalized
ratio
Log
normalized
ratio
Normalized P
⫺121
H-S
Collagen ␣1(VI)
chain precursor
Collagen ␣2(VI)
chain precursor
47
39.01
100
100
1.94
0.95
2.58 ⫻ 10
0
36
28.36
100
100
1.79
0.84
5.72 ⫻ 10⫺119
0
Proteoglycan link
protein
Lactadherin
precursor (MFGE8)
Collagen ␣1(II) chain
precursor
Clusterin precursor
Moesin
Biglycan precursor
Prolargin precursor
Cartilage
intermediate-layer
protein 1 precursor
Aggrecan core
protein precursor
Chondroadherin
precursor
Lubricin
Cartilage
intermediate-layer
protein 2 precursor
Sushi repeat–
containing protein
SRPX2 precursor
Cartilage oligomeric
matrix protein
precursor
Fibronectin precursor
Thrombospondin 4
precursor
Radixin
Matrilin 3 precursor
Guanine nucleotide–
binding protein
␣ 11 subunit
Ezrin
Thrombospondin 1
precursor
Perlecan
26
70.34
100
100
0.68
⫺0.56
8.25 ⫻ 10⫺11
1.01 ⫻ 10⫺8
17
54.26
100
100
0.64
⫺0.65
1.43 ⫻ 10⫺12
1.78 ⫻ 10⫺10
8
4.84
80
50
0.56
⫺0.85
1.21 ⫻ 10⫺5
1.29 ⫻ 10⫺3
6
7
19
15
41
16.48
12.31
50.00
32.46
28.04
100
80
100
100
100
80
90
80
90
100
0.52
0.50
0.48
0.43
0.41
⫺0.95
⫺1.01
⫺1.06
⫺1.21
⫺1.29
3.38 ⫻ 10⫺10
1.45 ⫻ 10⫺4
4.17 ⫻ 10⫺12
1.11 ⫻ 10⫺38
3.17 ⫻ 10⫺61
4.09 ⫻ 10⫺8
1.49 ⫻ 10⫺2
5.17 ⫻ 10⫺10
0
0
25
7.08
100
100
0.40
⫺1.33
2.08 ⫻ 10⫺243
0
21
55.43
100
100
0.39
⫺1.36
4.52 ⫻ 10⫺26
0
8
8
7.26
7.87
100
70
50
60
0.33
0.33
⫺1.58
⫺1.62
2.45 ⫻ 10⫺11
2.49 ⫻ 10⫺8
3.01 ⫻ 10⫺9
2.91 ⫻ 10⫺6
7
20.22
60
30
0.32
⫺1.65
1.37 ⫻ 10⫺6
1.52 ⫻ 10⫺4
18
26.29
100
90
0.27
⫺1.90
5.21 ⫻ 10⫺142
0
36
2
21.50
2.39
100
100
100
90
0.24
0.21
⫺2.08
⫺2.26
1.88 ⫻ 10⫺75
1.67 ⫻ 10⫺73
0
0
4
5
3
7.38
12.76
10.03
60
80
40
30
20
10
0.19
0.14
0.14
⫺2.42
⫺2.87
⫺2.89
2.00 ⫻ 10⫺7
8.07 ⫻ 10⫺16
9.34 ⫻ 10⫺5
2.30 ⫻ 10⫺5
1.00 ⫻ 10⫺13
9.76 ⫻ 10⫺3
3
4
4.44
3.33
60
60
20
10
0.10
0.09
⫺3.27
⫺3.42
9.34 ⫻ 10⫺10
3.32 ⫻ 10⫺7
1.11 ⫻ 10⫺7
3.75 ⫻ 10⫺5
6
2.16
50
10
0.09
⫺3.51
7.08 ⫻ 10⫺10
8.50 ⫻ 10⫺8
* Proteins that were significantly different between groups by Holm-Sidak analysis for multiple comparisons are included. Total peptides refers to
the number of unique peptides belonging to any one protein. Total coverage refers to the percent of the protein covered by the peptides. Normal
run fraction is the number of runs that contained peptides characteristic of that protein in the normal articular cartilage vesicles (ACVs).
Osteoarthritis (OA) run fraction is the number of runs that contained peptides characteristic of that protein in OA ACVs. The normalized ratio is
the ratio of total scan count in the OA group:normal group, taking into account the number of runs for each specimen group. H-S is the P value
for the normalized Holm-Sidek analysis for multiple comparisons.
of proteins present. The presence of multiple chains of
type VI collagen and several members of the small
leucine-rich proteoglycan family confirmed the pericellular location of ACVs. Other ECM proteins, such as
DEL-1, are of particular interest, as their function in
cartilage has not been thoroughly investigated and their
association with ACVs may have important implications
for understanding their roles in cartilage. Key chondro-
408
cyte growth factors, including bone morphogenetic protein 3 (BMP-3), angiopoietin-related protein, and melanoma inhibitory protein (cartilage-derived retinoic
acid–sensitive protein [CD-RAP]), were also present in
ACVs.
The presence of phospholipid-binding, integral
membrane proteins and some signaling pathway components supports the notion of the membrane-rich composition of ACVs. The annexin family of proteins is
particularly prominent in the ACV proteome, and is also
well represented in the growth plate matrix vesicle
proteome (9,18). ANK, a putative pyrophosphate transporter, is present in ACVs. Milk fat globule/endothelial
growth factor VIII, also known as lactadherin, is also a
prominent component of exosomal vesicles (19). It has
been observed in articular cartilage (20), but lacks an
assigned function. Clusterin participates in membrane
recycling and cell adhesion (21), and copine III is a
ubiquitously distributed protein that may also have a
kinase activity (22). Cell-signaling proteins, such as the
Ras proteins, are common to several types of extracellular vesicles (11). Isoforms of protein kinase C were
also present in ACVs.
Cytoskeletal components, including actins and
actin-capping factors, were present in ACVs, supporting
the theory that ACVs bud from chondrocyte microvilli
(9). Various forms of tubulin were also present. Enzymes in the ACV proteome included coagulation factor
XIIIA, a transglutaminase enzyme previously found in
ACVs, which may have a role in mineralization (23), and
peptidyl-prolyl cis-trans isomerase, which is involved in
protein folding, signal transduction, trafficking, assembly, and cell cycle regulation. Peroxiredoxin belongs to
the class of antioxidant enzymes (24).
While several nuclear histones were present, proteins from mitochondria and other intracellular organelles were not seen in the ACV proteome. Specifically, tetraspanins, which are characteristic markers of
multivesicular bodies from which exosomes are generated (11), were also absent from the ACV proteome.
Differences between OA and normal ACVs.
While the majority of proteins were shared by ACVs
from normal and OA cartilage, a few were seen exclusively in either OA ACVs or normal ACVs (Table 2). Six
proteins were found only in normal ACVs, while 9
proteins were seen exclusively in OA ACVs. Many of the
proteins that were exclusive to OA, such as fibrinogen,
complement, immunoglobulins, and apolipoproteins,
are typical markers of inflammation. Interestingly, chondrocytes are capable of generating apolipoprotein A-I,
and the N-terminal proteolytic product of this protein
ROSENTHAL ET AL
comprises the amyloid deposits found in aging knee
menisci (25). Immune complexes and complement have
been found in human OA cartilage (26) and in a rabbit
model of OA (27). While these inflammatory components could certainly originate from synovial fluid, chondrocytes are capable of synthesizing components of the
classical complement pathway, including C1, C2, and C4
(28).
A small group of proteins were present only in
normal ACVs. This heterogeneous group of proteins
included angiopoietin-related protein, a member of the
tumor necrosis factor ␣ family, which may play a role in
cartilage development (29). Elongation factor 1-alpha 1
participates in protein translation and microtubule formation. One chain of type IX collagen was also present.
Melanoma inhibitory protein (CD-RAP) was present
only in normal ACVs, as was versican, a large proteoglycan that is present in normal articular cartilage (30).
We used analytic methods specifically designed
to detect significant differences in protein levels between
groups in order to analyze quantitative differences between proteins in ACVs from normal and OA cartilage.
Proteins found in higher levels in normal ACVs than OA
ACVs included ECM proteins, such as type II collagen,
and several proteoglycans (including biglycan, proline/
arginine-rich end leucine-rich repeat protein, aggrecan,
and perlecan), as well as cartilage oligomeric matrix
protein, fibronectin, and thrombospondin. Interestingly,
lubricin levels were also significantly higher in normal
ACVs than in OA ACVs. This finding is consistent with
observed losses of lubricin in OA cartilage (31). Levels
of cartilage intermediate-layer proteins 1 and 2 were
also higher in normal ACVs than in OA ACVs. It is
expected that the matrix-destructive milieu of OA might
decrease quantities of these important proteins. Levels
of some members of the actin-binding family of proteins,
such as moesin, radixin, and ezrin, were also higher in
normal ACVs than in OA ACVs. These findings are less
readily explained.
Levels of 9 proteins were significantly increased
in OA ACVs. A dramatic increase in transforming
growth factor ␤ (TGF␤)–induced protein ␤ig-H3 was
noted, supporting the idea of a putative role for TGF␤ in
OA. Levels of DEL-1 were also increased in OA ACVs.
The functions of DEL-1 in cartilage and its involvement
in OA are largely unstudied. Vitronectin binds the ␣v␤3
integrin, and is present in the pericellular matrix of fetal
human articular cartilage. Levels of vitronectin were also
higher in OA ACVs. Some evidence points to a role for
ligands of the ␣v␤3 integrin in modulating interleukin-1␤
expression in chondrocytes (32). Serine protease HtrA1
PROTEOMIC ANALYSIS OF CARTILAGE VESICLES IN OSTEOARTHRITIS
levels were increased in OA ACVs, and this protease has
recently been implicated in aggrecan degradation in OA
(33). Increased levels of type VI collagen were observed
in OA ACVs, and have also been noted in OA cartilage
(34).
DISCUSSION
We characterized the proteome of human ACVs,
and found 170 proteins that satisfied our stringent
criteria for inclusion in the more closely examined
group. This group of proteins certainly does not represent an exhaustive list of ACV proteins. Several classes
of proteins known to be present in or on ACVs, such as
integrins, discoidin domain receptors (35), plasma cell
membrane glycoprotein 1, and alkaline phosphatase,
were represented in the full proteome, but were not
present in this select group. In general, integral membrane proteins are often underrepresented in proteomic
studies. One reason is the relative paucity of lysine and
arginine, which are the cleavage sites of trypsin (36).
Additional studies to confirm the presence or absence of
proteins on the full list will be necessary.
The ACV proteome reflects important structural
information about these organelles. ECM proteins represented the most prevalent protein type in ACVs. The
concentration of pericellular ECM proteins in ACVs
supports findings from histologic studies of their pericellular location (37). The presence of cell adhesion
proteins, such as fibronectin, vitronectin, and numerous
small leucine-rich proteoglycans, suggests that ACVs are
firmly rooted in matrix. The number of phospholipidbinding and membrane-associated proteins demonstrates the importance of membrane in this organelle.
The presence of actin and its associated cytoskeletal
components in the ACV proteome suggests that, similar
to matrix vesicles, ACVs are likely formed from zeiotic
blebbing of cell membrane microvilli (12).
The ACV proteome also reflects potential functions of ACVs. Interestingly, the presence of several potent mineralization inhibitors, including matrix Gla protein, TGF␤-induced 68-kd protein/TGF␤-inducible gene
h3, and serine protease HtrA1, supports the hypothesis
that mineralization may not be the primary function of
ACVs. Several ACV-associated ECM proteins mediate
collagen fibril formation, and their presence may contribute to a matrix repair function for ACVs, such as that
proposed for growth plate matrix vesicles (6). ACVs also
possess some cell-signaling machinery, and could possibly respond to external signals. Growth factors, such as
BMP-3, and regulatory proteins, including CD-RAP and
409
factor XIIIa, may contribute to their ability to modulate
the chondrocyte phenotype (38).
The ACV proteome differs from that of other
extracellular membrane–limited particles and shares
some characteristics with the proteomes of matrix vesicles that have been derived from growth plate cartilage
(12). However, some unique proteins noted in the matrix
vesicle proteome, such as aquaporin, glycoprotein HT7,
and scavenger receptor type B, were not detected in the
ACV proteome. Interestingly, matrix vesicles derived
from an osteoblastic cell line had fewer proteins in
common with ACVs (9), suggesting that these immortalized bone cells may produce very different extracellular vesicles. The ACV proteome also shares fewer
similarities with exosomal proteomes. Lactadherin and
ubiquitin are common to both ACVs and exosomes (39).
The heterotrimeric G proteins, Hsp70 and Hsp90, and
members of the tetraspanin family that are particularly
characteristic of exosomal vesicles (11) were not seen in
ACVs.
We were able to compare and relatively quantify
proteins in ACVs derived from OA with those from
normal cartilage. Surprisingly, few proteins were unique
to one or the other type of ACV. These findings support
the hypothesis that ACVs are constitutively produced by
chondrocytes, and are not preferentially generated by
damaged or dying cells. ACVs are clearly affected by the
environment in which they reside. Differences in ECM
proteins in OA and normal ACVs largely reflect known
changes in OA ECM and increased catabolism. The
presence of complements and immunoglobulins in OA
ACVs warrants further consideration. While proteomic
techniques would seem a logical methodology to investigate important changes in cartilage proteins in OA,
cartilage is quite refractory to 2-dimensional gel-based
protein analysis (40). This is due to the high concentrations of strongly charged proteoglycans which run anomalously on sodium dodecyl sulfate polyacrylamide gels,
and to the highly crosslinked state of cartilage matrix,
which is typically not soluble in sample buffers (41).
Our studies are not without limitations. While all
the cartilage was from adult subjects, we were unable to
age-match the OA and normal samples, and agingrelated changes may confound the observed differences
between OA and normal ACVs. Further studies will be
necessary to address the effect of age on ACVs. Additionally, because of the stringent inclusion criteria, we
have only discussed a small group of the proteins found
in ACVs. Certainly, trypsin-resistant proteins, or proteins with low prevalence and/or low molecular weight,
may be underrepresented with these techniques (36). All
410
ROSENTHAL ET AL
of this work was done with collagenase-released ACVs,
and it is possible that the enzyme exposure required to
isolate ACVs from whole cartilage might alter their
protein profiles. For example, Xiao and colleagues
showed some decrease in ECM proteins in collagenasereleased matrix vesicles, compared to those that are
released into conditioned media (9). The ACVs we
studied exhibited multiple ECM proteins, but a direct
comparison of the proteome of ACVs released nonenzymatically into chondrocyte-conditioned media and
those obtained enzymatically from whole cartilage was
not possible, given the limitations of frozen cartilage
samples.
In conclusion, ACVs contain many proteins that
reflect their possible functions and mechanisms of production. The present results reinforce the physiologic
nature of ACVs. The demonstrated association of a few
proteins, such as DEL-1 and lactadherin, with ACVs
may have ramifications for understanding their role in
cartilage. The observed differences in OA ACVs, particularly the presence of inflammation markers, provide
additional support for a role of inflammation in OA.
Further analysis of the ACV proteome will lead to a
better understanding of the important roles of ACVs in
cartilage physiology and disease.
ACKNOWLEDGMENTS
We thank Dr. Brian Halligan for the Visualize software, and the Biotechnology and Bioengineering Center at the
Medical College of Wisconsin for access to their computer
cluster.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Rosenthal had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Rosenthal, Gohr, Ninomiya.
Acquisition of data. Rosenthal, Gohr, Ninomiya, Wakim.
Analysis and interpretation of data. Rosenthal, Gohr, Ninomiya,
Wakim.
18.
19.
20.
21.
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DOI 10.1002/art.27768
Clinical Images: Voluminous ectopic tumoral calcinosis of the spine in systemic sclerosis
The patient, a 62-year-old man with systemic sclerosis that had been diagnosed 3 years previously, had rapidly progressing diffuse
disease with a modified Rodnan skin score of 31, a pulmonary interstitial infiltrate seen on radiography, and diffusing capacity for
carbon monoxide of 44% of predicted. He was treated once monthly for 12 months with an intravenous bolus of 750 mg/m2
cyclophosphamide. Three years later, when the patient had a skin score of 19 and stable lung function, abdominal pain led to
serendipitous discovery of a voluminous abdominal calcinosis seen on radiography (A). Computed tomography showed a 68 ⫻ 58 ⫻
50–mm extensive ectopic tumoral calcinosis of the spine, focused on the L4/L5 and L5/S1 right posterior articular masses, invading
the spinal muscles with intracanalar extension up to the lateral recess, and entrapping the right L4 and L5 nerve roots (B). Results
of blood and urinary phosphocalcium analyses were normal. Apart from digital calcinosis, no other calcification was found on
full-body radiography. Results from 99mTc scintigraphy and 18F-fludeoxyglucose positron emission tomography were negative.
Cécile Durant, MD
Dominique Farge-Bancel, MD, PhD
Saint Louis Hospital
Paris, France
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