close

Вход

Забыли?

вход по аккаунту

?

Large-scale gene expression profiling reveals major pathogenetic pathways of cartilage degeneration in osteoarthritis.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 54, No. 11, November 2006, pp 3533–3544
DOI 10.1002/art.22174
© 2006, American College of Rheumatology
Large-Scale Gene Expression Profiling Reveals
Major Pathogenetic Pathways of
Cartilage Degeneration in Osteoarthritis
Thomas Aigner,1 Katrin Fundel,2 Joachim Saas,3 Pia M. Gebhard,1 Jochen Haag,1 Tilo Weiss,3
Alexander Zien,4 Franz Obermayr,5 Ralf Zimmer,2 and Eckart Bartnik3
set on gene alterations in OA cartilage and, importantly,
indicate major mechanisms underlying central cell biologic alterations that occur during the OA disease
process. These results identify molecular targets that
can be further investigated in the search for therapeutic
interventions.
Objective. Despite many research efforts in recent
decades, the major pathogenetic mechanisms of osteoarthritis (OA), including gene alterations occurring
during OA cartilage degeneration, are poorly understood, and there is no disease-modifying treatment
approach. The present study was therefore initiated in
order to identify differentially expressed disease-related
genes and potential therapeutic targets.
Methods. This investigation consisted of a large
gene expression profiling study performed based on 78
normal and disease samples, using a custom-made
complementary DNA array covering >4,000 genes.
Results. Many differentially expressed genes were
identified, including the expected up-regulation of anabolic and catabolic matrix genes. In particular, the
down-regulation of important oxidative defense genes,
i.e., the genes for superoxide dismutases 2 and 3 and
glutathione peroxidase 3, was prominent. This indicates
that continuous oxidative stress to the cells and the
matrix is one major underlying pathogenetic mechanism in OA. Also, genes that are involved in the
phenotypic stability of cells, a feature that is greatly
reduced in OA cartilage, appeared to be suppressed.
Conclusion. Our findings provide a reference data
Osteoarthritis (OA) is one of the most common
disabling conditions, affecting many parts of the joint,
including bone, synovium, ligaments, and articular cartilage. Although OA is mainly characterized by functional loss of the articular cartilage matrix covering the
joint surfaces, it is obvious that cells are active players
during the disease process. In many laboratories, smallscale expression analyses have been performed on normal and OA cartilage specimens. These analyses have
revealed activation (1,2) as well as phenotypic instability
(3–5) of articular chondrocytes. A gene expression profile of ⬃1,200 different genes and some 20 samples
provided an initial set of interesting data on differentially expressed genes, but was limited due to the commercial array used, which focused on cancer-relevant
genes (6). Since then, a few more studies on gene
expression profiles in articular cartilage have been reported (for review, see ref. 7). In addition, gene association studies have identified numerous genes that might
confer susceptibility to OA (for review, see ref. 8).
However, knowledge about changes in OA cartilage remains limited. A broader gene expression profile
of OA chondrocytes needs to be established using
modern screening technologies, in order to better characterize the cellular events and regulatory pathways
directly involved in cartilage destruction. In this study,
we used extensive gene expression profiling to investigate molecular alterations that would provide clues
regarding the major pathogenetic factors in the OA
disease process.
Supported by the German Ministry of Research (BMBF
grants 01GG9823 and 01GG9824).
1
Thomas Aigner, MD, DSc, Pia M. Gebhard, MSc, Jochen
Haag, PhD: University of Leipzig, Leipzig, Germany; 2Katrin Fundel,
MSc, Ralf Zimmer, PhD: University of Munich, Munich, Germany;
3
Joachim Saas, PhD, Tilo Weiss, PhD, Eckart Bartnik, PhD: SanofiAventis Deutschland GmbH, Frankfurt, Germany; 4Alexander Zien,
PhD: Max Planck Institut for Biological Cybernetics, Tuebingen,
Germany; 5Franz Obermayr, PhD: GPC Biotech, Munich, Germany.
Address correspondence and reprint requests to Thomas
Aigner, MD, DSc, Osteoarticular and Arthritis Research, Institute of
Pathology, University of Leipzig, Liebigstrasse 26, D-04103 Leipzig,
Germany. E-mail: thomas.aigner@medizin.uni-leipzig.de.
Submitted for publication January 30, 2006; accepted in
revised form July 24, 2006.
3533
3534
AIGNER ET AL
MATERIALS AND METHODS
Clinical cases and RNA isolation. For study of messenger RNA (mRNA) expression levels by complementary
DNA (cDNA) array and quantitative polymerase chain reaction (PCR) techniques, cartilage from human femoral condyles
was processed as described previously (6). Normal articular
cartilage (18 specimens, from subjects ages 45–88 years) and
cartilage with early degeneration (20 specimens, from subjects
ages 43–91 years) were obtained at autopsy, within 48 hours of
death. OA cartilage was obtained at the time of total knee
replacement (21 samples with mild OA according to the
Mankin scale [9], from patients ages 61–84 years; 19 samples
with moderate or severe OA, from patients ages 61–84 years).
Cartilage was considered to be normal if it showed no significant macroscopic softening or surface fibrillation and had a
Mankin grade of ⬍3. Early degenerative cartilage was defined
as cartilage that showed moderate fibrillation and softening
but no advanced erosion of the articular cartilage, corresponding to a Mankin grade of 3–6. Cartilage from patients with
rheumatoid arthritis was excluded from the study. Only primary degenerated cartilage was used; regenerative cartilage
(osteophytic tissue) was not studied. RNA from cartilage tissue
was isolated as described previously (10).
Oligonucleotide fingerprinting. Two cDNA libraries
were constructed from 2 separate pools of mRNA, using
chondrocytes that were originated from OA and normal cartilage (starting from 1 mg total RNA each) and transfected
into Escherichia coli. Bacterial colonies were plated, and
200,000 clones per library were placed into microtiter plates
and subjected to oligonucleotide fingerprinting as described
previously (11). Briefly, the inserts of all selected clones were
PCR amplified and spotted onto nylon filters. One hundred
ninety-five short oligonucleotides were radioactively labeled
and hybridized to these filters. Hybridizations were analyzed
with phosphorimagers and automated detection software and
clustered by the k-means algorithm (12). Approximately
330,000 cDNA clones had sufficient hybridization information
to be subjected to clustering, which resulted in 8,821 different
clusters. The accuracy of the clustering was controlled by
hybridization of randomly selected cDNA probes onto the
same nylon filters.
Complementary DNA array production and expression analysis. Complementary DNA arrays were produced
with 7,808 complementary DNAs spotted using a custom-made
needle printer. More than 700 identical arrays were produced
for the gene expression studies. For probe synthesis (i.e.,
cDNA synthesis by reverse transcription), 1 ␮g of total RNA
was used. Using a 2-split protocol, 500 ng total RNA each was
labeled in 2 independent reactions using 33P and Superscript II
(Invitrogen Life Technologies, Paisley, UK), and subsequently
pooled and purified. The reaction mixtures were primed using
random hexamers. Between 0.7 ⫻ 108 and 1 ⫻ 108 counts were
hybridized onto 4 identical arrays using semiautomated hybridization machines. Hybridizations were carried out in plastic
boxes in a volume of 60 ml formamide-based buffer, overnight
at 50°C. After washing, filters were exposed for 5 days to
phosphorimager screens and then scanned using a Fuji (Tokyo,
Japan) BAS 5000 system. Images were then processed using
Image Split software (GPC Biotech, Munich, Germany) to
generate images of the individual filters. Using Consolen Batch
software (GPC Biotech), grids were automatically set onto the
images. Spot intensities were then determined, and data were
automatically transferred into the database. Visual grid–based
software (GPC Biotech) was used for image analysis to determine the raw spot intensities. Local background was determined and subtracted from the target spot intensities. Duplicate spots on an array that showed a large difference in
expression were considered outliers and eliminated from further processing.
To consolidate replicate data, the mean and SEM for
each target spot were calculated. By comparison of the mean of
the target replicate values with the distribution of the background values, the probability of significant expression was
estimated for each target. If individual filters in an experiment
showed an uneven distribution of the signal or other abnormalities, the experiment was repeated. Correlation factors
within and between runs were determined; the mean withinrun and between-run correlations were 0.979 and 0.965, respectively. Various possible methods for data processing were
compared, and those yielding the most robust outcome (i.e.,
differentially regulated genes) were chosen.
Complementary DNA array normalization and outlier
detection. Data were normalized by median absolute deviation
(MAD) scale normalization (13) to fix the median and MAD
(a measure of the variability within the data) of each sample to
a common level. The overall expression value distributions of
all samples were thereby rendered similar. The normalized
data include negative values due to background correction of
the original data. For further analysis, the general background
level was estimated to be 0.01, and expression values ⬍0.01
were set to 0.01.
The individual hybridizations were checked for data
consistency using cluster analysis, principal components analysis, and analysis of data distributions. We found 5 of the
original 83 samples to be outliers in at least 2 of the 3 types
of analysis and removed them prior to further processing
(for details, see supplementary text, available online at http://
www.mrw.interscience.wiley.com/suppmat/0004-3591/suppmat/).
Detection of differential expression. Within the given
microarrays the number of spots per gene varies, and the
experimental technique of spotting different clones for a given
gene results in high variability of the spot intensities representing the same gene. This necessitates use of a robust combination method. Differentially expressed genes were detected
according to the following procedure. First, the 2-sided Wilcoxon rank sum test was applied to calculate P values for spots.
Spot P values were combined into gene P values by Stouffer’s
method (14), in which P values are transformed to Z scores that
are combined under consideration of the direction of regulation,
and finally the Z scores are transformed back to P values.
The gene P values are converted into q values by use of
the R-library q value (15). The q value quantifies the false
discovery rate, i.e., a q value of 0.01 indicates that when the
subset of all genes having a q value of ⱕ0.01 is used to define
the group of genes with significant differential expression, 1%
of the selected genes have to be expected to be false-positive.
The false discovery rate was used to rank differentially expressed genes. We applied cutoff criteria for false discovery
rate and fold change to select genes for detailed analysis. To
MOLECULAR PHENOTYPING OF OA CHONDROCYTES
Figure 1. Gene expression patterns of a, collagens and b, noncollagenous matrix proteins in cartilage samples with early degeneration
(deg.) and with late-stage osteoarthritis (OA). Values are the mean
fold difference from normal. ⴱ ⫽ q (false discovery rate; see Materials
and Methods) ⬍ 0.05; ⴱⴱ ⫽ q ⬍ 0.01; ⴱⴱⴱ ⫽ q ⬍ 0.001.
estimate the overall fold change for each gene, we determined
individual spot fold changes (separately for each pair of
samples from the groups to be compared); the overall gene
fold change is estimated as the mean of the corresponding spot
fold changes.
3535
Clustering. Two different clustering protocols were
performed. For both, data were log2-transformed and subsequently transformed to Z scores so that, for all genes, the mean
is 0 with a standard deviation of 1.
In the first protocol, clustering was done on neutrally
preselected genes, i.e., without using our knowledge of sampleto-disease assignment (“unsupervised” clustering). Genes were
selected by significant expression and significant variance
between the analyzed samples. Clustering was done on this
gene subset and the entire sample set by applying Spearman’s
rank correlation and average linkage.
In the second protocol, clustering was done on genes
that were preselected by disease group (“supervised” clustering): the top 50 genes were selected on the basis of their fold
change and the P value between groups (normal versus late
OA and normal versus early OA). The genes preselected for
significant differences between the normal and the late OA
samples were clustered against all samples. The genes preselected for significant differences between the normal and the
early OA samples were clustered against the normal and early
OA samples. Clustering was done by applying Spearman’s rank
correlation and complete linkage.
Complementary DNA synthesis and real-time PCR.
First-strand cDNA was synthesized and real-time PCR (TaqMan) was performed for 10 selected genes (Agg, btg2,
COL1A1, COL2A1, COL3A1, GAPDH, GPX3, SOD2, SOX9,
and tob1) as described previously (16,17) (for a more detailed
description as well as sequence information on the primers and
probes used, see supplementary Table 1 and text, available
online at http://www.mrw.interscience.wiley.com/suppmat/
0004-3591/suppmat/).
General screening strategy. In order to achieve the
largest amount of data possible for analysis (18), a large series
of nonpooled samples was profiled (4 independent hybridization experiments each). For analysis of the expression data we
used a 4-step strategy, as follows: 1) The primary data were
normalized. 2) The expression levels of gene groups of known
relevance to chondrocyte anabolism were examined, to validate the data obtained. 3) Differences in expression levels
Table 1. Genes up- or down-regulated in early degenerative cartilage lesions compared with normal samples*
RefSeq
Annotation
NM_002669
NM_005252
NM_002026
Pleiotropic regulator 1 (PRL1 homolog, Arabidopsis) (PLRG1)
v-Fos FBJ murine osteosarcoma viral oncogene homolog (FOS)
Fibronectin 1 (FN1)
NM_003068
NM_198057
NM_006854
NM_003479
NM_001109
NM_004960
NM_004161
NM_006833
NM_153425
NM_015692
NM_016732
NM_006423
Snail homolog 2 (Drosophila) (SNAI2)
Delta sleep–inducing peptide, immunoreactor (DSIPI)
KDEL endoplasmic reticulum protein retention receptor 2
Protein tyrosine phosphatase type IVA, member 2 (PTP4A2)
A disintegrin and metalloproteinase domain 8 (ADAM8)
Fusion, derived from t(12;16) malignant liposarcoma (FUS)
RAB1A, member RAS oncogene family (RAB1A)
COP9 subunit 6 (MOV34 homolog, 34 kd) (COPS6)
Tumor necrosis factor receptor I–associated death domain (TRADD)
␣2-macroglobulin family protein VIP (VIP)
RNA binding protein (hnRNP-associated with lethal yellow) (RALY)
Rab acceptor 1 (prenylated) (RABAC1)
Normal
mean
Fold change
q
P
0.10
0.12
1.69
1.92
1.88
1.58
0.035
0.000
0.000
0.000
0.000
0.000
0.28
0.57
0.27
2.74
1.70
9.40
3.87
12.46
1.62
0.24
0.50
0.11
0.47
0.55
0.55
0.55
0.55
0.55
0.57
0.57
0.58
0.59
0.63
0.65
0.021
0.035
0.028
0.021
0.021
0.016
0.029
0.000
0.026
0.003
0.003
0.003
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
* Only genes that were expressed at a level of ⬎0.1 (assigned by the program used) in at least 1 sample group are shown. Up- or down-regulation
is defined as a fold change of ⬎1.5 or ⬍0.66 in early degenerative versus normal cartilage, and a false discovery rate (q) of ⬍0.05 (i.e., 5% of the
selected genes have to be expected to be false-positive).
3536
AIGNER ET AL
Table 2. Genes up- or down-regulated in cartilage lesions with moderate/severe late-stage OA compared with normal samples*
RefSeq
Annotation
Normal
mean
Fold
change
q
P
NM_000088
NM_170746
NM_002160
NM_000090
NM_033150
NM_000093
NM_173343
NM_002026
NM_002775
NM_000089
NM_003118
NM_018058
NM_058175
NM_003613
NM_080630
NM_001848
NM_002023
NM_032977
NM_004000
NM_003254
Collagen, type I, ␣1 (COL1A1)
Selenoprotein H (SELH)
Tenascin C (hexabrachion) (TNC)
Collagen, type III, ␣1 (COL3A1)
Collagen, type II, ␣1 (COL2A1)
Collagen, type V, ␣1 (COL5A1)
Interleukin-1 receptor, type II (IL1R2)
Fibronectin 1 (FN1)
Protease, serine, 11 (IGF binding) (PRSS11)
Collagen, type I, ␣2 (COL1A2)
Secreted protein, acidic, cysteine-rich (osteonectin)
Cartilage acidic protein 1 (CRTAC1)
Collagen, type VI, ␣2 (COL6A2)C2
Cartilage intermediate-layer protein (CILP)
Collagen, type XI, ␣1 (COL11A1)
Collagen, type VI, ␣1 (COL6A1)
Fibromodulin (FMOD)
Caspase 10, apoptosis-related cysteine
Chitinase 3–like 2 (CHI3L2)
Tissue inhibitor of metalloproteinases 1 (TIMP1)
0.09
0.10
0.05
0.07
0.14
0.06
0.08
1.69
0.09
0.06
0.19
0.34
0.28
0.28
0.04
0.38
1.49
0.37
0.10
0.36
9.55
9.27
8.06
7.37
6.07
5.81
5.34
4.69
4.17
3.41
3.34
3.01
2.96
2.72
2.66
2.58
2.56
2.53
2.11
2.05
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
NM_002084
NM_005195
NM_006472
NM_002422
NM_024906
NM_005412
NM_175617
NM_004417
NM_018659
NM_006763
NM_005950
NM_002178
NM_005952
NM_005749
NM_002065
NM_006169
NM_000636
Glutathione peroxidase 3 (plasma) (GPX3)
CCAAT/enhancer binding protein (C/EBP), delta
Thioredoxin-interacting protein (TXNIP)
Stromelysin 1 (MMP3)
Stearoyl-CoA desaturase 4 (SCD4)
Serine hydroxymethyltransferase 2 (mitochondrial)
Metallothionein 1E (functional) (MT1E)
Dual-specificity phosphatase 1 (DUSP1)
Cytokine-like protein C17 (C17)
BTG family, member 2 (BTG2)
Metallothionein 1G (MT1G)
Insulin-like growth factor binding protein 6 (IGFBP6)
Metallothionein 1X (MT1X)
Transducer of ERBB2, 1 (TOB1)
Glutamate-ammonia ligase (glutamine synthase) (GLUL)
Nicotinamide N-methyltransferase (NNMT)
Superoxide dismutase 2, mitochondrial (SOD2)
3.99
0.57
0.69
0.38
0.77
0.16
0.57
0.79
0.77
0.17
0.20
0.44
0.63
0.21
0.38
0.15
0.22
0.12
0.15
0.29
0.29
0.31
0.32
0.33
0.33
0.35
0.36
0.38
0.43
0.43
0.45
0.48
0.48
0.48
0.000
0.000
0.000
0.000
0.000
0.007
0.000
0.000
0.000
0.007
0.000
0.004
0.000
0.000
0.016
0.000
0.005
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.002
0.000
0.001
* Only a selection of genes that were expressed at a level of ⬎0.1 (assigned by the program used) in at least 1 sample group is shown. Up- or
down-regulation is defined in this table as a fold change of ⬎2 or ⬍0.5 in cartilage with moderate/severe late-stage osteoarthritis (OA) versus normal
cartilage, and a false discovery rate (q) of ⬍0.05 (i.e., 5% of the selected genes have to be expected to be false-positive). For the complete list of
genes with a fold change of ⬎1.5 or ⬍0.66, see supplementary Table 3.
between the different sample groups of interest (normal versus
early degenerative disease, early degenerative disease versus
mild late-stage OA, mild versus moderate or severe late-stage
OA) were analyzed. 4) Finally, a clustering analysis was
performed for all samples based on their gene expression
profiles. All data are available online at http://www.mrw.
interscience.wiley.com/suppmat/0004-3591/suppmat/.
Statistical analysis. For in vivo investigations, the
significance of differences in expression levels was evaluated
with the nonparametric Wilcoxon/Mann-Whitney test. This
nonparametric test is more likely to be appropriate than, for
example, the t-test, because it is not based on assumptions
regarding the distribution of expression values (e.g., normal
distribution). For the in vitro probes, the t-test for pairwise
comparison was used, due to the limited number of comparisons
being made. P values less than 0.05 were considered significant.
RESULTS
Findings of oligonucleotide fingerprinting and
array generation. To select OA-relevant cDNA clones,
extensive screening based on OliCode technology (11)
was performed (11). For this analysis 400,000 clones,
derived from 2 cDNA libraries prepared from normal
and OA tissue, were hybridized with 195 oligonucleotides and cDNA clones grouped into 8,821 clusters and
36,000 singletons. A total of 5,280 clusters with ⬎3 hits
were selected for expression profiling. Additionally,
⬎1,000 genes of interest with regard to cartilage/
chondrocyte biology were spotted on the arrays (for the
MOLECULAR PHENOTYPING OF OA CHONDROCYTES
full gene list, see http://www.bio.ifi.lmu.de/publications/
OA_cDNA_AR2006/).
Validation and evaluation of expression analysis
of marker genes of cellular differentiation and anabolic
activity. For examination of the chondrocyte gene expression profile we first focused on the main function of
this cell type, preservation and turnover of the cartilage
matrix. Genes involved in cartilage anabolism were first
investigated. Types II and III collagen were strongly
up-regulated in peripheral and central OA cartilage,
which is consistent with the findings of previous studies
(1,4). Type I collagen (COL1A1, COL1A2) was also
up-regulated (Figure 1a). This is not surprising and does
not necessarily indicate a major shift in the cellular
phenotype of chondrocytes (19), but reflects the general
metabolic activation of OA chondrocytes, as has been
shown by quantitative PCR (17). Collagen types VI
(COL6A1, COL6A2, COL6A3), IX (COL9A2,
COL9A3), and XI (COL11A1, COL11A2) were also
found to be significantly up-regulated, but to a much
lesser degree. Similar results with regard to COL6 have
been reported previously (20). Interestingly, expression
of other collagens (COL5A1, COL15A1), so far not
known to be expressed in adult articular chondrocytes,
was also detectable.
In contrast to the collagens, noncollagenous matrix proteins were generally less up-regulated in OA
chondrocytes, except for fibromodulin, cartilage
intermediate-layer protein, fibronectin, tenascin, and
osteonectin/secreted protein, acidic and rich in cysteine
(Figure 1b). Another interesting phenomenon was that
most of the noncollagenous proteins were much more
strongly expressed in normal articular cartilage than
were the collagens. This supports the notion of a rather
high turnover of the noncollagenous cartilage matrix
compared with the collagenous compartment. These
observations are consistent with previous data showing
almost no overall regulation of aggrecan and decorin
(6,17,21), in contrast to the strong up-regulation of
fibronectin, tenascin, and osteonectin (6,22,23), in OA
chondrocytes.
Expression of SOX9, the major transcription
factor known to be relevant to chondrocyte phenotypic
stability (24,25), was significantly down-regulated. This
finding was also in accordance with previously described
data (26).
Quantitative PCR analysis confirmed the upregulation of the matrix proteins type I collagen (fold
change observed by quantitative PCR [fold changeqPCR]
12.6; P ⬍ 0.005), type II collagen (fold changeqPCR 17.6;
P ⬍ 0.0001), and type III collagen (fold change 36.4; P ⬍
3537
0.0001), as well as the down-regulation of SOX9 (fold
changeqPCR 0.4; P ⬍ 0.01) in OA cartilage samples.
Aggrecan was not significantly regulated in diseased tissue
compared with normal tissue (fold changeqPCR 0.7).
Comparison of genes that were differentially
expressed in normal and early degenerative cartilage.
Table 1 summarizes the genes that were significantly
(q ⬍ 0.05) differentially regulated between normal and
early degenerative cartilage. Overall, only 15 genes were
significantly up- or down-regulated, even if less stringent
criteria were applied (regulation ⬎50%, q ⬍ 0.05); this
indicated that normal and early degenerative cartilage
lesions are similar in terms of general cell biology. The
alternative interpretation that early lesions within the
joint cartilage are mostly focal could largely be excluded
because samples were obtained only from the lesional
areas, in which the expression of the genes was altered
by 2-fold at most. Thus, the difference between normal
and early degenerative cartilage was only minor, in terms
of both the number of regulated genes and the intensity
of gene regulation.
One of the up-regulated genes was fibronectin
(fold change 1.58; q ⬍ 0.001), a gene that is well known
to be up-regulated early in cartilage degeneration (27).
However, the increase of fibronectin might be ambiguous since its degradation products have been shown to
be detrimental to cartilage homeostasis (28,29). Regardless, however, this finding further emphasizes the potential importance of fibronectin in maintenance and degradation of articular cartilage.
Comparison of genes that were differentially
expressed in normal cartilage and severe late-stage OA
cartilage. In the next step, we analyzed the genes that
were significantly differentially regulated between normal and late-stage OA chondrocytes. This revealed a
significantly higher number of differentially expressed
genes than in the other comparisons (Table 2 and
supplementary Tables 2 and 3, available online at http://
www.mrw.interscience.wiley.com/suppmat/0004-3591/
suppmat/). An ontology analysis based on Geneontology
(www.geneontology.org) revealed a broad spectrum of
regulated genes (Figure 2) and emphasized the upregulation of numerous genes involved in extracellular
matrix formation, as indicated above. In contrast, many
genes involved in oxidative damage defense, namely
GPX3 (glutathione peroxidase 3) (fold change 0.12; q ⬍
0.001), SOD2 (superoxide dismutase) (fold change 0.48;
q ⬍ 0.01), SOD3 (fold change 0.62; q ⬍ 0.001), and
TXNIP (thioredoxin-interacting protein) (fold change
0.29; q ⬍ 0.001), appeared to be down-regulated. Findings with regard to GPX3 and SOD2 expression were
3538
Figure 2. Functional role of genes that were significantly (P ⱕ 0.01,
log2 fold change ⬎1) down-regulated (a) or up-regulated (b) in the
comparison of normal versus late-stage osteoarthritis cartilage. The
area in each chart assigned to a functional category corresponds to the
negative logarithm of the P value determined for the category by
hypergeometric distribution, i.e., the larger an area, the more significant the overrepresentation of the category.
confirmed by quantitative PCR (GPX3 fold changeqPCR
0.08 [P ⬍ 0.0001]; SOD2 fold changeqPCR 0.08 [P ⬍
0.06]).
Other findings of interest pertained to genes of
the transducer of ERBB2 (tob)/B cell translocation gene
(btg) family, namely tob1 (fold change 0.39; q ⬍ 0.001),
btg1 (fold change 0.53; q ⬍ 0.001), and btg2 (fold change
0.36; q ⬍ 0.05). For tob1 and btg2, the findings were
confirmed by quantitative PCR (tob1 fold changeqPCR
0.13 [P ⬍ 0.001]; btg2 fold changeqPCR 0.17 [P ⬍ 0.05]).
AIGNER ET AL
A third group of regulated genes included numerous genes for cytokines or genes involved in cytokine
signaling. One interesting finding was that many genes
related to the interleukin-1 (IL-1) pathway were, in
contrast to our expectation, not up-regulated, but rather,
down-regulated, in OA chondrocytes. This included
IL1B (fold change 0.5; q ⬍ 0.001) itself as well as IL6,
IL-8, and LIF (leukemia inhibitory factor). Additionally,
functional antagonist/scavenger receptor type II was
down-regulated (fold change 5.3; q ⬍ 0.001). However,
the expression levels of relevant genes were very low in
all samples, which warrants a caveat about data reliability despite statistical significance. Thus, additional investigations are necessary in order to evaluate the importance of the IL-1␤ pathway in OA joint disease.
Comparison of genes that were differentially
expressed in mild late-stage and moderate or severe
late-stage OA cartilage. Only 14 genes fulfilled the
(rather nonstringently defined) criteria of differential
expression (regulation ⬎50%; q ⬍ 0.05) when specimens
of cartilage with mild versus moderate or severe latestage OA were compared (Table 3). The results were in
accordance with the cluster analysis findings discussed
below, showing the close similarity of gene expression in
mild and moderate/high late-stage OA cartilage.
Clustering of sample probes. In order to investigate differences and overlaps in the expression of genes
within the different sample groups, clustering analysis
was performed. This analysis revealed 2 primary clusters
(Figure 3), with a clear separation between the normal
and early degenerative cartilage samples versus the
peripheral and central OA cartilage samples. The normal and early degenerative cartilage samples could not
be clearly distinguished from one another, nor could the
peripheral and central OA samples. This provides evidence in support of the hypothesis that differences
between normal and early degenerative cartilage, as well
as between peripheral and central OA cartilage, are
relatively minor, but the combined normal and early
degenerative cartilage group is clearly different from the
combined peripheral and central OA group.
Figure 4 shows the biased cluster analysis of
genes that were strongly preselected (the 50 most highly
differentially expressed genes) in the analysis of normal
versus late OA cartilage and in the analysis of normal
versus early degenerative cartilage. These can be seen as
marker genes to distinguish between the groups at the
gene expression level. Figure 4a shows perfect separation of the normal samples versus the late OA samples
based on the selected genes. Interestingly, Figure 4b
shows that the samples from early degenerative cartilage
MOLECULAR PHENOTYPING OF OA CHONDROCYTES
3539
Table 3. Genes up- or down-regulated in cartilage lesions with moderate/severe late-stage OA compared with mild late-stage OA*
RefSeq
Annotation
Low-grade
OA mean
Fold change
q
P
NM_000214
NM_153498
NM_015913
NM_002026
NM_000088
NM_002775
NM_006025
NM_002160
NM_001823
NM_018058
NM_000093
NM_000701
Jagged 1 (Alagille syndrome) (JAG1)
CamKI-like protein kinase (CKLiK)
Endoplasmic reticulum thioredoxin superfamily member, 18 kd
Fibronectin 1 (FN1)
Collagen, type I ␣1 (COL1A1)
Protease, serine, 11 (IGF binding) (PRSS11)
26 serine protease (P11)
Tenascin C (hexabrachion) (TNC)
Creatine kinase, brain (CKB)
Cartilage acidic protein 1 (CRTAC1)
Collagen, type V ␣1 (COL5A1)
ATPase, Na⫹/K⫹ transporting, ␣1 polypeptide (ATP1A1)
0.79
0.13
0.07
3.92
0.67
0.23
0.14
0.26
0.07
0.59
0.23
0.10
2.52
2.41
2.18
2.14
2.13
1.83
1.82
1.80
1.78
1.77
1.67
1.61
0.004
0.022
0.022
0.000
0.000
0.000
0.015
0.000
0.042
0.000
0.000
0.026
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
NM_005752
NM_018659
C-type lectin, superfamily member 1 (cartilage-derived)
Cytokine-like protein C17 (C17)
0.42
0.41
0.61
0.61
0.000
0.010
0.000
0.000
* Only genes that were expressed at a level of ⬎0.1 (assigned by the program used) in at least 1 sample group are shown. Up- or down-regulation
is defined as a fold change of ⬎1.5 or ⬍0.66 in cartilage with moderate/severe late-stage osteoarthritis (OA) versus cartilage with mild late-stage
OA, and a false discovery rate (q) of ⬍0.05 (i.e., 5% of the selected genes have to be expected to be false-positive).
again clustered, at least in part, with the normal samples
despite the selection of maximally discriminating genes.
This again supports the notion that the separation of
normal versus early degenerative cartilage is less clear,
even with highly preselected genes. However, there was
a distinction between the groups at the gene expression
level (see Table 1).
Comparative analysis of differential expression
levels of susceptibility genes associated with incidence
of knee OA. A number of genes have been previously
described to be linked to the development and/or progression of OA of the knee (30–34). Expression levels of
these genes in articular cartilage varied and were increased in some cases and decreased in others; most
were not significantly up- or down-regulated (Table 4).
This is not surprising since the involvement of these
genes in the development of OA might entail modification of developmental processes or other indirect effects, such as promotion of obesity and/or and increased
pain (in)sensitivity. Also, the present data set is not very
suitable for evaluating the importance of expression
levels of these genes with regard to OA development,
because a longitudinal database would be needed for
assessment of predisposing factors. Thus, if, e.g., reduced expression of certain genes increases the risk for
development of OA, then OA-susceptible normal samples should show reduced levels of these genes; however,
expression levels of these genes would not necessarily be
increased or reduced during the development of OA.
DISCUSSION
This work provides the first extensive gene expression profile of normal, early degenerative, and mild
and moderate/severe late-stage OA chondrocytes. The
most striking feature in the data from all 4 groups is the
high variability of gene expression levels between the
different donors, for many genes. Whereas this might be
expected for the disease samples for various reasons
(e.g., different stages), the finding of a comparable
variability among normal donors is very surprising. Since
no rhythms (e.g., circadian) in articular cartilage tissue
have been reported to date, this indicates that a wide
range of gene expression levels is compatible with normal functioning of the tissue (as well as tissue dysfunction). Thus, networks of molecules, rather than single
components, appear to determine the normal state, an
issue that is particularly relevant with regard to development of therapeutic interventions.
The second important result of our study is that
macroscopically less damaged or relatively normalappearing cartilage from joints with late-stage OA cannot be considered normal, nor is it similar to early
degenerative lesions. This is in accordance with previous
studies showing that cartilage that is obtained from
joints with advanced OA but has a lower Mankin grade
is metabolically activated and shows matrix alterations
similar to those in cartilage with moderate/high Mankin
grades (1,35–38). Our present analysis indicates that
these rather normal-looking areas are severely changed
3540
AIGNER ET AL
Figure 3. Dendrogram and heat map of all samples and genes preselected for significant expression and variance. The displayed genes were
preselected based on significant expression and significant variance between the analyzed samples. Clustering was done by applying Spearman’s rank
correlation and average linkage. A good separation between the normal (N) and early degenerative (E) versus the peripheral (P) and central (C)
osteoarthritis (OA) cartilage samples is seen. Only 1 of the early degenerative cartilage samples is clustered closer to the peripheral and central OA
samples than to the other normal and early degenerative cartilage samples. The normal and early degenerative cartilage samples cannot be
distinguished from one another, as is also the case for the peripheral and central samples. This provides evidence in support of the hypothesis that
normal and early degenerative cartilages are similar and peripheral and central OA cartilages are also similar, but the 2 combined groups are clearly
different.
in terms of their gene expression profile. Also, the
similarity between the peripheral and central areas
provides evidence against the notion that peripheral OA
cartilage is a good model for early OA (39), for which it
is often used, and it further suggests that there is no
major shift between the 2 tissue and cell types, as
MOLECULAR PHENOTYPING OF OA CHONDROCYTES
3541
Figure 4. Dendrogram and heat map of a, genes selected from the analysis of normal versus late-stage OA cartilage and b, genes selected from the
analysis of normal versus early degenerative cartilage. For both analyses, the top 50 genes were selected based on their fold change and P value
between the groups considered. Clustering was done by applying Spearman’s rank correlation and complete linkage. Thus, the selected genes can
be seen as marker genes to distinguish between the corresponding groups at the gene expression level. a, A perfect separation between the normal
samples and the late-stage OA samples is seen. Interestingly, the early degenerative cartilage samples again clustered with the normal samples, even
though they were not considered in the preselection of genes for this analysis. b, The separation between normal and early degenerative cartilage
is not perfect, and several samples are misclassified. This provides further evidence in support of the notion of a lack of clear distinction between
normal and early degenerative cartilage, even in an analysis using highly preselected genes. There is, however, a distinction between the groups at
the gene expression level. See Figure 3 for definitions.
3542
AIGNER ET AL
Table 4. Detected differential expression levels of candidate genes for knee osteoarthritis, which were suggested in the literature*
Late OA vs. normal
Gene
RefSeq
Annotation
Ref.
Normal
mean
Fold
change
q
P
AACT
ANKH
BLP2
BMP2
CILP
CIRBP
COX2
DUSP1
EIF4A1
ESR1
GPRK6
H3F3B
HIST2H2AA
IL1R1
NCOR2
OPG
RHOB
S100B
SUI1
TNFAIP6
NM_001085
NM_054027
NM_025141
NM_001200
NM_003613
NM_001280
NM_000963
NM_004417
NM_001416
NM_000125
NM_002082
NM_005324
NM_003516
NM_000877
NM_006312
NM_002546
NM_004040
NM_006272
NM_005801
NM_007115
Serine (or cysteine) proteinase inhibitor, clade A
Ankylosis, progressive homolog
BBP-like protein 2 (BLP-2), transcript variant 2
Bone morphogenetic protein 2
Cartilage intermediate-layer protein
Cold-inducible RNA binding protein
Prostaglandin-endoperoxide synthase 2
Dual-specificity phosphatase 1
Eukaryotic translation initiation factor 4A, isoform 1
Estrogen receptor 1
G protein–coupled receptor kinase 6
H3 histone, family 3B
Histone 2, H2aa (HIST2H2AA)
Interleukin-1 receptor type I
Nuclear receptor corepressor 2
Osteoprotegerin
Ras homolog gene family, member B (ARHB)
S100 calcium-binding protein, ␤ (neural)
Putative translation initiation factor
Tumor necrosis factor ␣–induced protein 6
32
55
34
32
32
34
32
34
34
32
34
34
34
56
32
32
34
34
34
32
1.13
0.16
0.09
0.02
0.28
0.18
0.04
0.79
0.14
0.06
0.03
1.20
0.13
0.07
0.15
0.05
0.77
0.27
0.38
0.30
0.90
1.59
0.97
1.32
2.70
0.59
1.56
0.30
0.94
1.20
1.64
0.43
0.81
1.00
0.94
1.07
0.29
0.97
0.61
0.92
0.285
0.000
0.659
0.314
0.000
0.000
0.274
0.000
0.504
0.490
0.122
0.000
0.163
0.618
0.562
0.590
0.000
0.605
0.000
0.691
0.141
0.000
0.769
0.170
0.000
0.000
0.129
0.000
0.440
0.414
0.037
0.000
0.056
0.679
0.560
0.619
0.000
0.652
0.000
0.858
* Other “hot candidate” molecules, such as asporin (57), LRCH1 (30), and FRZB (31), were not represented on the arrays.
recently suggested (40). Obviously, the most severely
damaged areas with no or little cartilage remaining are
difficult to assess with the methods as used in our study,
and were therefore not investigated. However, these
areas are of little clinical relevance since they represent
a stage of the disease at which therapy will not be of
benefit.
Certainly, great caution would be required if
early degenerative cartilage from any donor were to be
studied as a substitute for early OA cartilage (40). A
significant portion of it might not represent progressive
disease. Still, these early lesions were shown to be
biochemically similar to early lesions of OA (41). Also,
we cannot rule out the possibility that gene expression
levels might be altered during the (short) time delay
between death and autopsy, which is nearly unavoidable
in studies of human samples (40). Obviously, this time
has to be kept as short as possible, and further studies
are needed in order to evaluate more directly its influence on changes in gene expression. However, extensive
studies on mRNA expression levels in human brain
tissue have shown no significant changes in the gene
expression pattern even several days postmortem
(42,43). Also, a comparative analysis of changes in gene
expression levels in relation to time since death did not
reveal any evidence of significant changes occurring
during the time frame investigated (up to 48 hours); in
particular, no hypoxia-induced genes appeared to be
up-regulated (Aigner T, et al: unpublished observations). Cartilage is, in this respect, a unique tissue in that
chondrocytes remain viable for a long time (44).
The similarity of mild and moderate/severe latestage OA specimens presumably reflects the fact that all
cartilage areas in a joint are exposed to the same
synovial factors, i.e., potent cytokines and growth factors. These are secreted by activated synoviocytes (45)
and diffuse into the cartilage from the synovial joint
space.
Another interesting finding is the identification
of gene groups that are most differentially expressed
between OA and normal chondrocytes. In this respect,
the strong up-regulation of matrix constituents was to be
expected (1,2,9), and thus represented an excellent
internal positive control.
The strong down-regulation of major components of oxidative cellular defense is a new and surprising observation, given that oxidative stress is increased in
OA chondrocytes (for review, see ref. 46). The dramatic
down-regulation of enzymes that are key in cellular
oxidative defense is presumably one important reason
for the increased accumulation of oxidatively damaged
molecules within the cells. Thus, even a reduction of the
gene dose of SOD2 of 50% in heterozygous knockout
mice leads to significantly increased oxidative stress and
cell damage (47). Also, the down-regulation of the
extracellular isoform, SOD3, and of GPX3 might in-
MOLECULAR PHENOTYPING OF OA CHONDROCYTES
crease reactive oxygen species in the extracellular space
and might enhance cartilage matrix breakdown in this
way (48).
Also, the down-regulation of a third group of
genes, for members of the tob/btg group of proteins, fits
very well into the scenario of cell biologic changes
occurring in OA cartilage (49,50). This might represent
an important clue for understanding of the cell biology
of OA: the molecules of the btg/tob family are thought
to be involved in phenotype stabilization of cells and
inhibition of proliferative activity, both of which are
reversed in OA chondrocytes.
A fourth group of proteins of interest found in
our study comprised proteins from the IL-1 signaling
pathway, which appear to be down-regulated rather than
activated in OA cartilage. This is somewhat in contrast
to many previous assumptions (for review, see ref. 51),
but is in accordance with recent in vitro and in vivo data.
Thus, investigators at our laboratory have shown clearly
reduced responsiveness of OA chondrocytes to stimulation with IL-1 (52). Also, activation of IL-6 and LIF
expression in OA cartilage, as would be expected after
stimulation with IL-1␤, was not observed (53). Although
attenuation of the IL-1␤ pathway might at first appear to
be a potentially beneficial treatment avenue, providing
protection of articular cartilage from catabolic stimulation, recent data suggest that IL-1 activity might be
important for cartilage tissue homeostasis (54).
Overall, besides providing an extensive primary
data set, the present study offers significant information
on the types of samples important for studying normal,
degenerative, or OA cartilage, which is clearly an area
that holds great potential in terms of gene expression
profiling analyses. At the same time, we have identified
foci of gene alterations, confirming known alterations
(matrix synthesis, proliferation, phenotypic instability of
cells) as well as identifying innovative new ones (oxidative defense). Most importantly, the results provide not
only general direction, but also details on specific targets, in terms of therapeutic intervention: the tob1, btg2,
SOD2, SOD3, and GPX3 genes should be the subject of
further research.
ACKNOWLEDGMENTS
We would like to acknowledge Drs. G. Zeiler and W.
Eger (Orthopedic Hospital, Rummelsberg, Germany) for providing OA cartilage samples, Dr. Stephan Söder (Erlangen,
Germany) for help in acquisition of normal cartilage tissue,
and Freya Boggasch, Anke Nehlen, and Brigitte Bau for expert
technical assistance.
3543
REFERENCES
1. Aigner T, Stoss H, Weseloh G, Zeiler G, von der Mark K.
Activation of collagen type II expression in OA and rheumatoid
cartilage. Virchows Arch B Cell Pathol Incl Mol Pathol 1992;62:
337–45.
2. Lippiello L, Hall MD, Mankin HJ. Collagen synthesis in normal
and osteoarthritic human cartilage. J Clin Invest 1977;59:593–600.
3. Girkontaite I, Frischholz S, Lammi P, Wagner K, Swoboda B,
Aigner T, et al. Immunolocalization of type X collagen in normal
fetal and adult OA cartilage with monoclonal antibodies. Matrix
Biol 1996;15:231–8.
4. Aigner T, Bertling W, Stoss H, Weseloh G, von der Mark K.
Independent expression of fibril-forming collagens I, II, and III in
chondrocytes of human OA cartilage. J Clin Invest 1993;91:
829–37.
5. Aigner T, Zhu Y, Chanksy HH, Matsen FA, Maloney WJ, Sandell
LJ. Reexpression of type IIA procollagen by adult articular
chondrocytes in OA cartilage. Arthritis Rheum 1999;42:1443–50.
6. Aigner T, Zien A, Gehrsitz A, Gebhard PM, McKenna LA.
Anabolic and catabolic gene expression pattern analysis in normal
versus OA cartilage using complementary DNA–array technology.
Arthritis Rheum 2001;44:2777–89.
7. Aigner T, Gebhard PM, Kueffner R, Zhang H, Marshall KW.
cDNA arrays in degenerative arthritis research. Future Rheumatol
2006;1:101–9.
8. Loughlin J. The genetic epidemiology of human primary OA:
current status. Expert Rev Mol Med 2005;7:1–12.
9. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and
metabolic abnormalities in articular cartilage from osteo-arthritic
human hips. II. Correlation of morphology with biochemical and
metabolic data. J Bone Joint Surg Am 1971;53:523–37.
10. McKenna LA, Gehrsitz A, Soeder S, Eger W, Kirchner T, Aigner
T. Effective isolation of high quality total RNA from human adult
articular cartilage. Anal Biochem 2000;286:80–5.
11. Meier-Ewert S, Lange J, Gerst R, Schmitt A, Freund J, Eige T, et
al. Comparative gene expression profiling by oligonucleotide
fingerprinting. Nucleic Acids Res 1998;26:2216–23.
12. Herwig R, Poustka AJ, Muller C, Bull C, Lehrach H, O’Brien J.
Large-scale clustering of cDNA-fingerprinting data. Genome Res
1999;9:1093–105.
13. Dudoit S, Yang YH. Bioconductor R packages for exploratory
analysis and normalization of cDNA microarray data. In: Parmigiani G, Garett ES, Irizarry RA, Zeger SL, editors. The analysis of
gene expression data. New York: Springer; 2003. p. 73–101.
14. Rosenthal R. Meta-analytic procedures for social sciences. Beverly
Hills: Sage Publications; 1984.
15. Storey JD, Tibshirani R. Statistical significance for genomewide
studies. Proc Natl Acad Sci U S A 2003;100:9440–5.
16. Bau B, Gebhard PM, Haag J, Knorr T, Bartnik E, Aigner T.
Relative messenger RNA expression profiling of collagenases and
aggrecanases in human articular chondrocytes in vivo and in vitro.
Arthritis Rheum 2002;46:2648–57.
17. Gebhard PM, Gehrsitz A, Bau B, Soder S, Eger W, Aigner T.
Quantification of expression levels of cellular differentiation
markers does not support a general shift in the cellular phenotype
of OA chondrocytes. J Orthop Res 2003;21:96–101.
18. Kendziorski C, Irizarry RA, Chen KS, Haag JD, Gould MN. On
the utility of pooling biological samples in microarray experiments.
Proc Natl Acad Sci U S A 2005;102:4252–7.
19. Lefebvre V, Peeters-Joris C, Vaes G. Production of collagens,
collagenase and collagenase inhibitor during the dedifferentiation
of articular chondrocytes by serial subcultures. Biochim Biophys
Acta 1990;1051:266–75.
20. Hambach L, Neureiter D, Zeiler G, Kirchner T, Aigner T. Severe
disturbance of the distribution and expression of type VI collagen
chains in OA articular cartilage. Arthritis Rheum 1998;41:986–96.
3544
21. Poole AR, Rosenberg LC, Reiner A, Toneseu M, Bogoch E,
Roughley PJ. Contents and distributions of the proteoglycans
decorin and biglycan in normal and osteoarthritric human articular
cartilage. J Orthop Res 1996;14:681–9.
22. Salter DM. Tenascin is increased in cartilage and synovium from
OA knees. Br J Rheumatol 1993;32:780–6.
23. Burton-Wurster N, Lust G. Synthesis of fibronectin in normal and
OA articular cartilage. Biochim Biophys Acta 1984;800:52–8.
24. Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B.
Sox9 is required for cartilage formation. Nat Genet 1999;22:85–9.
25. Zhao Q, Eberspaecher H, Lefebvre V, de Crombrugghe B.
Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis. Dev Dyn 1997;209:377–86.
26. Aigner T, Gebhard PM, Schmid E, Bau B, Harley V, Poschl E.
SOX9 expression does not correlate with type II collagen expression in adult articular chondrocytes. Matrix Biol 2003;22:363–72.
27. Lorenzo P, Bayliss MT, Heinegard D. Altered patterns and
synthesis of extracellular matrix macromolecules in early OA.
Matrix Biol 2004;23:381–91.
28. Homandberg GA, Davis G, Maniglia C, Shirikhande A. Cartilage
chondrolysis by fibronectin fragments causes cleavage of aggrecan
at the same site as found in OA cartilage. OA Cartilage 1997;5:
450–3.
29. Yasuda T, Poole AR. A fibronectin fragment induces type II
collagen degradation by collagenase through an interleukin1–mediated pathway. Arthritis Rheum 2002;46:138–48.
30. Spector TD, Reneland RH, Mah S, Valdes AM, Hart DJ, Kammerer S, et al. Association between a variation in LRCH1 and
knee OA: a genome-wide single-nucleotide polymorphism association study using DNA pooling. Arthritis Rheum 2006;54:524–32.
31. Loughlin J, Dowling B, Chapman K, Marcelline L, Mustafa Z,
Southam L, et al. Functional variants within the secreted frizzledrelated protein 3 gene are associated with hip OA in females. Proc
Natl Acad Sci U S A 2004;101:9757–62.
32. Valdes AM, Van Oene M, Hart DJ, Surdulescu GL, Loughlin J,
Doherty M, et al. Reproducible genetic associations between
candidate genes and clinical knee OA in men and women.
Arthritis Rheum 2006;54:533–9.
33. Valdes AM, Hart DJ, Jones KA, Surdulescu G, Swarbrick P, Doyle
DV, et al. Association study of candidate genes for the prevalence
and progression of knee OA. Arthritis Rheum 2004;50:2497–507.
34. Mahr S, Burmester GR, Hilke D, Gobel U, Grutzkau A, Haupl T,
et al. Cis- and trans-acting gene regulation is associated with OA.
Am J Hum Genet 2006;78:793–803.
35. Poole AR. Can serum biomarker assays measure the progression
of cartilage degeneration in OA? [editorial]. Arthritis Rheum
2002;46:2549–52.
36. Aigner T, Vornehm SI, Zeiler G, Dudhia J, von der Mark K,
Bayliss MT. Suppression of cartilage matrix gene expression in
upper zone chondrocytes of OA cartilage. Arthritis Rheum 1997;
40:562–9.
37. Sweet BM, Thonar EJ, Immelman AR, Solomon L. Biochemical
changes in progressive osteoarthrosis. Ann Rheum Dis 1977;36:
387–98.
38. Wang J, Verdonk P, Elewaut D, Veys EM, Verbruggen G.
Homeostasis of the extracellular matrix of normal and OA human
articular cartilage chondrocytes in vitro. OA Cartilage 2003;11:
801–9.
39. Horton WE Jr, Yagi R, Laverty D, Weiner S. Overview of studies
comparing human normal cartilage with minimal and advanced
OA cartilage. Clin Exp Rheumatol 2005;23:103–12.
40. Yagi R, McBurney D, Laverty D, Weiner S, Horton WE Jr.
Intrajoint comparisons of gene expression patterns in human OA
AIGNER ET AL
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
suggest a change in chondrocyte phenotype. J Orthop Res 2005;
23:1128–38.
Squires GR, Okouneff S, Ionescu M, Poole AR. The pathobiology
of focal lesion development in aging human articular cartilage and
molecular matrix changes characteristic of OA. Arthritis Rheum
2003;48:1261–70.
Preece P, Cairns NJ. Quantifying mRNA in postmortem human
brain: influence of gender, age at death, postmortem interval,
brain pH, agonal state and inter-lobe mRNA variance. Brain Res
Mol Brain Res 2003;118:60–71.
Preece P, Virley DJ, Costandi M, Coombes R, Moss SJ, Mudge
AW, et al. An optimistic view for quantifying mRNA in postmortem human brain. Brain Res Mol Brain Res 2003;116:7–16.
Lasczkowski GE, Aigner T, Gamerdinger U, Weiler G, Bratzke H.
Visualization of postmortem chondrocyte damage by vital staining
and confocal laser scanning 3D microscopy. J Forensic Sci 2002;
47:663–6.
Oehler S, Neureiter D, Meyer-Scholten C, Aigner T. Subtyping of
OA synoviopathy. Clin Exp Rheumatol 2002;20:633–40.
Henrotin Y, Kurz B, Aigner T. Oxygen and reactive oxygen species
in cartilage degradation: friends or foes? OA Cartilage 2005;13:
643–54.
Strassburger M, Bloch W, Sulyok S, Schuller J, Keist AF, Schmidt
A, et al. Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous
apoptosis in murine myocardium in vivo. Free Radic Biol Med
2005;38:1458–70.
Tiku ML, Gupta S, Deshmukh DR. Aggrecan degradation in
chondrocytes is mediated by reactive oxygen species and protected
by antioxidants. Free Radic Res 1999;30:395–405.
Matsuda S, Rouault J, Magaud J, Berthet C. In search of a
function for the TIS21/PC3/BTG1/TOB family [review]. FEBS
Lett 2001;497:67–72.
Gebauer M, Saas J, Haag J, Dietz U, Takigawa M, Bartnik E, et al.
Repression of anti-proliferative factor Tob1 in OA cartilage.
Arthritis Res Ther 2005;7:R274–84.
Goldring MB. OA and cartilage: the role of cytokines. Curr
Rheumatol Rep 2000;2:459–65.
Fan Z, Bau B, Yang H, Soeder S, Aigner T. Freshly isolated OA
chondrocytes are catabolically more active than than normal
chondrocytes, but less responsive to catabolic stimulation with
interleukin-1␤. Arthritis Rheum 2005;52:136–43.
Fan Z, Bau B, Yang H, Aigner T. Il-␤ induction of Il-6 and LIF in
normal articular human chondrocytes involves the ERK, p38 and
NFkB signaling pathways. Cytokine 2004;28:17–24.
Clements KM, Price JS, Chambers MG, Visco DM, Poole AR,
Mason RM. Gene deletion of either interleukin-1␤, interleukin1␤–converting enzyme, inducible nitric oxide synthase, or stromelysin 1 accelerates the development of knee OA in mice after
surgical transection of the medial collateral ligament and partial
medial meniscectomy. Arthritis Rheum 2003;48:3452–63.
Pendleton A, Johnson MD, Hughes A, Gurley KA, Ho AM,
Doherty M, et al. Mutations in ANKH cause chondrocalcinosis.
Am J Hum Genet 2002;714:933–40.
Smith AJ, Keen LJ, Billingham MJ, Perry MJ, Elson CJ, Kirwan
JR, et al. Extended haplotypes and linkage disequilibrium in the
IL1R1-IL1A-IL1B-IL1RN gene cluster: association with knee
osteoarthritis. Genes Immun 2004;5:451–60.
Kizawa H, Kou I, Iida A, Sudo A, Miyamoto Y, Fukuda A, et al.
An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to OA. Nat Genet 2005;37:
138–44.
Документ
Категория
Без категории
Просмотров
3
Размер файла
917 Кб
Теги
expressions, major, scala, large, degeneration, pathogenetic, profiling, osteoarthritis, genes, cartilage, reveal, pathways
1/--страниц
Пожаловаться на содержимое документа