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Increased apoptosis in human osteoarthritic cartilage corresponds to reduced cell density and expression of caspase-3.

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Vol. 50, No. 2, February 2004, pp 507–515
DOI 10.1002/art.20020
© 2004, American College of Rheumatology
Increased Apoptosis in Human Osteoarthritic Cartilage
Corresponds to Reduced Cell Density
and Expression of Caspase-3
Mohammed Sharif, Anne Whitehouse, Patrick Sharman, Mark Perry, and Mike Adams
analysis of variance showed that the differences between
groups for both TUNEL-positive cells and expression of
caspase-3 were statistically significant (P < 0.0001).
There was a significant positive correlation between
TUNEL-positive cells and expression of caspase-3 (r ⴝ
0.654, P< 0.01).
Conclusion. The data suggest that apoptosis is
increased, on average, 2–4-fold in OA cartilage. Considering that OA develops over many years, such an
increase in the rate of apoptosis in the articular cartilage could play an important role in the disease process.
Objective. Chondrocyte apoptosis has been described in both human and experimentally induced
osteoarthritis (OA), but its importance in the etiopathogenesis of OA is uncertain. The aims of this study were
to determine the rate of chondrocyte apoptosis using
different methods, and to investigate the relationship
between this process and cartilage cellularity, expression of proapoptotic molecules, and expression of antiapoptotic molecules in articular cartilage obtained from
patients with OA and from nonarthritic controls.
Methods. We examined the extent of apoptosis in
OA and nonarthritic control cartilage using expression
of caspase-3, an enzyme that mediates the final stage of
cell death by apoptosis, as well as the TUNEL method.
We used immunohistochemistry to analyze the expression of a panel of proapoptotic and antiapoptotic molecules that regulate apoptosis in articular cartilage, in
order to determine whether the rate of apoptosis is
associated with the expression of these molecules.
Results. The median (range) percentage of
TUNEL-positive chondrocytes in knee OA cartilage
(n ⴝ 10 specimens), hip OA cartilage (n ⴝ 9), and
control cartilage (n ⴝ 7) was 3.11 (1.67–3.67), 1.86
(1.22–2.89), and 0.39 (0.00–1.78), respectively. When all
cartilage samples were pooled, apoptosis showed a
strong inverse correlation with cellularity (r ⴝ ⴚ0.74,
P < 0.0001). The percentage (range) of cells expressing
caspase-3 in the 3 groups was 15.70 (7.40–20.50), 15.77
(7.42–20.5), and 7.40 (5.90–8.00), respectively. One-way
Osteoarthritis (OA) is the most common joint
disease in the elderly population (1), causing significant
pain and disability. The etiology of OA is complex and
diverse, consisting of a range of biomechanical, biochemical, and genetic factors (2) that converge in a final
common pathway (3) characterized by a progressive
focal loss of articular cartilage. Normal cartilage homeostasis and structural integrity depend on chondrocytes, which account for only ⬃5% of the total cartilage
volume (4). Chondrocytes maintain the dynamic equilibrium between production of the extracellular matrix
and its enzymatic degradation. Loss of this balance in
favor of catabolic events results in the loss of articular
cartilage seen in OA. Thus, chondrocyte viability is
essential for maintaining the integrity of articular cartilage, and reduced cellularity (attributable to either
necrosis or apoptosis) may predispose the aging individual to matrix degeneration and may be associated with
the onset and/or progression of OA.
Necrosis is a pathologic form of cell death and is
associated with acute cell injury. Apoptosis (programmed cell death) is a physiologic process that is vital
in normal development, cell turnover, and removal of
damaged or potentially carcinogenic cells (5). Chondrocyte apoptosis has been described in both human (6,7)
and experimentally induced (8) OA, but its importance
Dr. Sharif is a recipient of a Career Scientist Award from the
NHS Executive South West R&D Directorate, Bristol, UK.
Mohammed Sharif, PhD, Anne Whitehouse, BSc, Patrick
Sharman, BSc, Mark Perry, PhD, Mike Adams, PhD: University of
Bristol, Bristol, UK.
Address correspondence and reprint requests to Mohammed
Sharif, MD, Department of Anatomy, Southwell Street, University of
Bristol, Bristol BS2 8EJ, UK. E-mail:
Submitted for publication April 29, 2003; accepted in revised
form October 1, 2003.
in the etiopathogenesis of OA is uncertain. In a recent
study of OA cartilage, 6% apoptosis was observed using
the TUNEL method (6), while other investigators (9)
using a similar method observed ⬍1% chondrocyte
death. Previous studies using the TUNEL method found
apoptosis rates of ⬍1% to 28% in OA cartilage, and 0%
to 7% in nonarthritic cartilage (7,9–11). Not surprisingly, authors of studies in which high levels of apoptosis
were found concluded that the process is more important in the etiopathogenesis of OA than did the authors
of studies in which a very low percentage of apoptotic
chondrocytes in OA was observed (9,12).
Recent investigations have questioned the true
extent of apoptosis in OA cartilage and the specificity of
the widely used TUNEL method itself (9,13). Therefore,
we determined the extent of apoptosis in OA cartilage
using the TUNEL method, as well as expression of one
of the key enzymes, caspase-3, that mediate the final
stage of cell death by apoptosis (14). In addition, we
used immunohistochemical analysis to examine the balance between panels of proapoptotic and antiapoptotic
molecules in articular cartilage obtained from both
patients with OA and nonarthritic controls.
This study was approved by the regional ethics committee as part of a study titled “Cartilage Destruction in
Osteoarthritis.” A written information sheet and a consent
form were presented to patients prior to surgery, and only
tissue samples obtained from patients who consented were
used in the study.
Collection and preparation of cartilage samples. Osteoarthritic cartilage on bone was obtained from patients
undergoing elective total knee replacement surgery (n ⫽ 10) or
total hip replacement surgery (n ⫽ 9) at Avon Orthopaedic
Centre, Bristol, UK. Nonarthritic tissue was obtained from the
hip joints of 7 patients undergoing surgery for osteoporotic
femoral neck fracture at Bristol Royal Infirmary, Bristol, UK.
The mean ⫾ SD age of patients was 67 ⫾ 11 years for those
with knee OA and 68 ⫾ 8 years for those with hip OA. The
mean ⫾ SD age of nonarthritic controls was 68 ⫾ 14 years. The
male-to-female ratio in the 3 groups was also very similar.
All OA cartilage was obtained ⬃5 mm from the site of
the OA lesions. In order to minimize the topographic variations in apoptosis and expression of the proapoptotic and
antiapoptotic molecules, control tissue was obtained from sites
similar to those from which the OA samples were obtained.
Tissue samples were stored under sterile conditions in a
refrigerator and processed in the laboratory within 24 hours of
surgery. Full-depth samples of cartilage were cut from the
underlying subchondral bone, snap frozen in liquid nitrogen,
and then stored at ⫺70°C until required for sectioning.
Frozen cartilage specimens were embedded in CryoMatrix (Shandon, Pittsburgh, PA), and then consecutive 7-␮m
sections were cut (at ⫺24°C), using a cryostat (Bright Instru-
ment Company, Cambridgeshire, UK). For each patient, 3
sections were mounted onto each of 12 labeled SuperFrost
Plus microscope slides (BDH Chemicals, Poole, UK). The
sections were air dried at room temperature for 2 hours and
then wrapped with silica in tin foil and stored at ⫺70°C until
required for TUNEL or immunohistochemical staining. One
set of slides from each patient was used for toluidine blue
staining, for the assessment of general cartilage condition and
cell counts.
Toluidine blue staining protocol. Toluidine blue stains
proteoglycan blue and enables a general assessment of cartilage condition. Toluidine blue staining was performed with a
standard protocol, and the stained sections were used for
determination of cellularity, empty lacunae counts, and the
presence of fibrillation.
TUNEL method. TUNEL is a specific immunohistochemical technique that enables sensitive and specific staining
of the high concentrations of DNA 3⬘-OH ends that are
localized in apoptotic bodies (15,16). Apoptotic nuclei were
stained as described in the protocol provided by the manufacturer (ApopTag Plus Peroxidase In Situ Apoptosis Detection
Kit; Intergen, Oxford, UK).
Immunohistochemical analysis. The slides were defrosted for at least 30 minutes, and immunostaining was
performed at room temperature. A standard indirect immunohistochemistry method was used to stain for caspase-3,
proapoptotic molecules (p53 and Bag-1), antiapoptotic molecules (Bcl-2, Bcl-x, and Bax), and a chondrocyte proliferation
marker, Ki-67. All of these antibodies are monoclonal antibodies to the human protein raised in mice. Ki-67 was purchased
from Becton Dickinson (Oxford, UK), and all other monoclonal antibodies were obtained from Neomarker (Fremont, CA).
For each patient, 8 slides were used (1 each for the 7
antibodies, and 1 as a negative control). To obtain the dilutions
required for optimal staining, each primary antibody was
tested at a range of concentrations in phosphate buffered
saline. The concentrations (2 ␮g/ml for the majority of the
primary antibodies) at which positive cells were sufficiently
stained to be clearly visible but at which nonspecific background staining was minimized were used for all subsequent
staining of the cartilage sections.
Microscopic examination and scoring of cartilage sections. All slides were prepared and examined in a blinded
manner, in order to reduce bias during scoring. Each patient
was given a code letter (upper case). The different antibodies
for each patient were then randomly allocated a code letter
(lower case). There was no intentional relationship between
the codes allocated for a given antibody in different patients.
Slides were observed using a DMRB light microscope (Leica,
Milton Keynes, UK) at ⫻20 or ⫻40 magnification. Images of
sections were digitally captured using Optimas software (MediaCybernetics, Silver Spring, MD). The toluidine blue–
stained sections were scored as follows: for quality of section,
0 (very poor) to 3 (good); for fibrillation, 0 (none) to 4
(fissured); for overall cellularity and cellularity of each cartilage zone, 0 (no cells) to 4 (abundant); and for empty lacunae,
as a percentage of all lacunae counted. Following immunohistochemical staining, sections were scored for intensity of
staining on a scale of 0–3 (0 ⫽ none, 3 ⫽ strong) and for the
percentage of positive cells in each zone. The TUNEL sections
Figure 1. A, Femoral head from a patient who underwent hemiathroplasty following osteoporotic subcapital fracture of the
femoral neck, representative of the gross appearance of typical nonarthritic control cartilage used in this study. B, Section of
cartilage obtained from femoral head shown in A, stained with toluidine blue. C, Femoral head from a patient with
osteoarthritis (OA) who underwent total hip replacement, representative of the gross appearance of typical OA cartilage used
in this study. D, Section of cartilage obtained from femoral head shown in C, stained with toluidine blue.
were scored for the percentage of cells positive for TUNEL in
each zone.
Validation of counting of positively stained cells and
empty lacunae. To ensure that assessment of the proportion of
cells staining positive for apoptotic and proliferative markers
was consistent with methods used in previous work, a second
observer recounted a random selection of 10 slides from 10
different individuals. The proportion of cells staining positive
was reassessed, and the first and second readings were compared.
Statistical analysis. The data generated in this study
are nonparametric. Therefore, the Kruskal-Wallis test was
used, followed by Dunn’s test (for the posttest statistic).
One-way analysis of variance (ANOVA) was used to investi-
gate the differences between the 3 specimen groups for
cellularity and the percentage of apoptosis. Spearman’s rank
correlation coefficient was calculated for correlations between
the percentage of apoptosis and cartilage fibrillation, caspase-3
expression, and proapoptotic and antiapoptotic molecule expression for each zone, as well as for the cartilage as a whole.
The gross appearance of typical control and OA
cartilage specimens and the corresponding histologic
sections stained with toluidine blue, as used in the study,
are shown in Figure 1. Safranin O staining showed loss
Table 1.
Fibrillation, cellularity, and empty lacunae in articular cartilage*
Cartilage type
Fibrillation score
(range 0–4)
Cellularity score
(range 0–3)
% empty
Knee osteoarthritis
Hip osteoarthritis
Nonarthritic control
1.6 ⫾ 0.71
1.60 ⫾ 0.46
0.43 ⫾ 0.53†
1.61 ⫾ 0.18
1.50 ⫾ 0.20
2.86 ⫾ 0.66†
35 ⫾ 20
31 ⫾ 23
25 ⫾ 13
* Values are the mean ⫾ SD.
† P ⬍ 0.001 versus knee osteoarthritis and hip osteoarthritis.
of glycosaminoglycan from all OA sections compared
with controls (data not shown). Validation studies for
the toluidine blue and immunohistochemical staining
showed minimal intraobserver and interobserver variation. All correlation coefficients for intraobserver and
interobserver variation for cell count, percentage of
apoptosis, and positive immunohistochemical staining
were between 0.89 and 0.96 (P ⬍ 0.0001). However,
although both observers found fewer empty lacunae in
nonarthritic cartilage compared with OA cartilage, the
correlation coefficients for intraobserver and interobserver variations for empty lacunae counts were low.
The results for cartilage fibrillation, cellularity, and
empty lacunae counts are shown in Table 1.
Fibrillation scores for knee and hip OA were very
similar to each other but were greater than the score for
nonarthritic control cartilage. OA cartilage was hypocellular compared with nonarthritic control cartilage.
There appeared to be more empty lacunae in OA
cartilage than in nonarthritic control cartilage, but this
difference did not reach statistical significance.
Apoptosis (according to the TUNEL reaction)
was more frequent in knee and hip OA cartilage than in
nonarthritic control cartilage (Figure 2). One-way
Figure 2. Mean and SEM percentage of apoptosis in cartilage obtained from 10 patients with knee osteoarthritis (OA), 9 patients with
hip OA, and 7 nonarthritic (NA) control subjects.
ANOVA showed the differences between groups to be
highly significant (P ⬍ 0.0001). However, the difference
between knee OA and hip OA was not statistically
significant (P ⬎ 0.05). There were also significant differences in the percentage of apoptosis in different
zones within each group and between groups (Table 2).
Within each of the cartilage groups, the percentage of
apoptosis was highest in the superficial zone and lowest
in the deep zone. In both knee and hip OA cartilage, the
percentage of apoptosis was significantly higher (P ⬍
0.01) in the superficial zone compared with the deep
zone. In all 3 zones, knee OA cartilage showed higher
cell death by apoptosis compared with hip OA cartilage
and nonarthritic control cartilage.
Spearman’s correlation coefficient for the correlation between overall cellularity and apoptosis for all
cartilage specimens used in the study was ⫺0.74 (95%
confidence interval ⫺0.88, ⫺0.48) (P ⬍ 0.0001) (Figure
3). Correlations between cellularity scores and apoptosis
within each group of specimens (knee OA, hip OA, and
nonarthritis) were ⫺0.537, ⫺0.509, and ⫺0.721, respectively. However, these correlations did not reach statistical significance.
A typical OA cartilage section immunostained
for caspase-3, and the corresponding negative control
(all reagents except the primary antibody to caspase-3)
are shown in Figure 4. The percentage of chondrocytes
positive for caspase-3 in nonarthritic and OA cartilage
and the zonal changes in the expression of the enzyme
are shown in Figure 5. Because there were no significant
differences between either zonal or overall expression of
caspase-3 between knee OA and hip OA cartilage, the 2
OA groups were pooled in Figure 5. There were significantly (P ⬍ 0.001) more cells expressing caspase-3 in
both knee and hip OA cartilage compared with nonarthritic control cartilage, and the expression of caspase-3
correlated with the mean percentage of apoptosis in all
cartilage groups (r ⫽ 0.654, P ⬍ 0.01). The percentage of
chondrocytes staining for caspase-3 in different zones of
the cartilage was highest in the superficial zone and
lowest in the middle zone in all 3 cartilage groups
Table 2.
Apoptosis in each zone in articular cartilage*
Cartilage zone
Cartilage type
Knee osteoarthritis
Hip osteoarthritis
Nonarthritic control
4.12 (2.67–5.33)
3.34 (1.67–3.67)
1.00 (0.00–3.33)†
3.00 (0.17–4.33)
1.50 (0.67–4.00)
0.17 (0.00–2.00)†
2.34 (0.17–3.00)
0.75 (0.33–1.67)
0.00 (0.00–00)†
3.11 (1.67–3.67)
1.86 (1.22–2.89)
0.39 (0.00–1.78)
* Values are the median (range) percent.
† P ⬍ 0.001 versus knee osteoarthritis and hip osteoarthritis.
(Figure 5). Expression of caspase-3 in the superficial
zone was significantly higher than that in the middle and
deep zones in all 3 groups (P ⬍ 0.001). However, the
difference between the deep and middle zones was
significant only in the nonarthritis group (P ⬍ 0.05).
The percentage of chondrocytes positive for the
proliferative marker Ki-67 was higher in both knee OA
and hip OA compared with nonarthritic control cartilage. However, the difference was significant only between knee OA and nonarthritic cartilage (Table 3).
There were no significant differences in the expression
of Ki-67 between the zones within knee OA, hip OA, or
nonarthritic cartilage. Chondrocytes in both nonarthritic
and OA cartilage showed positive staining for the proapoptotic molecules (p53 and Bax) and the antiapoptotic
molecules (Bcl-2, Bcl-x, and Bag-1), but no significant
differences were observed in the expression of these
molecules between the cartilage groups (Table 3). However, in all cartilage samples, the percentage of cells
expressing p53 was higher in the superficial zone than in
the middle and deep zones (P ⬍ 0.01), as shown in
Figure 3. Association between cellularity and the mean percentage of
apoptosis in articular cartilage obtained from nonarthritic (NA) control subjects, patients with knee osteoarthritis (kOA), and patients with
hip OA (hOA).
Figure 6. The percentage of cells expressing Bax and
Bcl-2 also appeared to be higher in the superficial zone
compared with the middle and deep zones, but the
differences were not significant. Spearman’s correlations
between the expression of these molecules in different
Figure 4. A, Representative osteoarthritis cartilage section immunostained for caspase-3. B, Corresponding negative control. (Original
magnification ⫻ 200 in A; ⫻ 20 in B.)
Figure 5. Percentage of chondrocytes expressing caspase-3 in nonarthritic (NA) and osteoarthritic (OA) cartilage. Bars show the mean
and SEM.
zones of the cartilage and the presence of apoptosis
showed that only Bcl-2 was significantly associated with
apoptosis in the superficial zone (r ⫽ ⫺0.526, P ⬍ 0.05).
The data obtained in this study show that apoptosis (TUNEL-positive chondrocytes) in OA cartilage
was, on average, ⬃4-fold higher than that in nonarthritic
control cartilage. Expression of caspase-3 was ⬃2-fold
higher in OA cartilage and correlated with apoptosis as
determined by the TUNEL method. Moreover, cellularity was inversely proportional to apoptosis when all
specimens were pooled. Taken together, these results
suggest that a small but significant proportion of chondrocytes die by apoptosis in OA cartilage compared with
normal tissue, leaving the tissue with a reduced density
of living cells.
To date, there have been several reports on cell
death in articular cartilage, and most investigators agree
that there is increased cell death by apoptosis in OA
cartilage compared with nonarthritic tissue. What is
debatable is the extent of apoptosis and its role in the
etiopathogenesis of OA. Using in situ TUNEL staining
of OA knee cartilage, Blanco and colleagues (6) found,
on average, 6% cell death, while similar studies by
Hashimoto et al (7) and Heraud et al (11) showed 22.3%
and 18%, respectively. These discrepancies may be
attributable to topographic variations in apoptosis as
well as slight differences in the TUNEL protocols used.
Indeed, a higher rate of apoptosis has been reported at
the site of OA lesions when compared with a nonlesion
site within the same OA knee joint (10).
The relatively low apoptosis rates in the current
study compared with those in several previous studies
could be attributable to the sampling of the cartilage ⬃5
mm from the site of focal lesions. In a recent study (9),
following careful characterization of a TUNEL detection kit (Oncor, Gaithersburg, MD) and using fetal
growth plate cartilage as positive control, Aigner et al
observed a very low rate of apoptosis in OA cartilage.
The TUNEL kit used in the current study (ApopTag
Plus Peroxidase In Situ Apoptosis Detection) had been
characterized by the manufacturer in a manner similar
to that in the latter study and was used exactly as
suggested in the manufacturer’s protocol. Low rates of
apoptosis appear to be more realistic, because a high
level of chondrocyte death would rapidly lead to OA
cartilage becoming acellular. However, it is not known
how much time is required for a chondrocyte to die by
apoptosis and for its remains to be cleared from the
cartilage matrix.
In the current study, expression of caspase-3 was
Table 3. Chondrocytes positive for Ki-67, caspase-3, and proapoptotic and antiapoptotic molecules in
Cartilage type
Knee osteoarthritis
Hip osteoarthritis
0.00 (0.00–2.78)
7.40 (5.90–8.00)
6.67 (3.33–10.00)†
15.70 (7.40–20.50)†
1.12 (0.00–18.89)
15.77 (7.42–20.5)†
6.17 (3.33–11.67)
5.00 (0.00–8.33)
5.42 (0.00–11.11)
3.33 (0.00–13.33)
8.89 (2.22–11.11)
2.22 (0.00–3.33)
9.17 (6.67–18.89)
5.00 (0.00–7.22)
0.00 (0.00–2.20)
6.67 (4.40–23.33)
0.84 (0.00–10.00)
1.11 (0.00–6.67)
8.34 (3.33–16.67)
0.00 (0.00–3.33)
4.44 (1.11–10.00)
* Values are the median (range) percent.
† P ⬍ 0.05 versus nonarthritic cartilage.
Figure 6. Percentage of chondrocytes expressing p53 in different
zones of nonarthritic (NA) and osteoarthritic (OA) cartilage. Bars
show the mean and SEM.
highest in the superficial zone, where a greater number
of TUNEL-positive chondrocytes were also found.
There are several possible explanations for increased
chondrocyte death by apoptosis in the superficial zones
of the articular cartilage. First, cells in the superficial
zone undergo greater deformation than those found in
deeper zones (17,18). Second, the alignment of the
superficial zone collagen network parallel to the articular surface may be less protective, or may cause even
further damage, to superficial cells as compared with the
perpendicular alignment found in deeper zones (18).
As expected, cartilage fibrillation was significantly higher in all OA cartilage compared with nonarthritic control cartilage, but no association was found
between the degree of fibrillation and the number of
chondrocytes staining positive for apoptosis. Because
age-related decreases in cartilage cellularity (19) are
associated with an increased frequency of cartilage
fibrillation (20), it would seem that reduced cellularity
(due primarily to necrosis) is associated with cartilage
fibrillation, but that apoptosis is not. However, it is
worth noting that in other studies, investigators reported
increasing numbers of apoptotic cells with increasing
histologic grading (more fibrillation) of the OA cartilage
Reduced cellularity is a characteristic feature of
OA cartilage (22), and apoptosis has been proposed as
an underlying cause of the hypocellularity (20,23). In
this study, we found that the cellularity scores in both
knee and hip OA cartilage were markedly reduced
compared with those in nonarthritic control cartilage
(Table 1), and that cellularity scores correlated negatively with the percentage of apoptotic chondrocytes,
suggesting that reduced cellularity in OA cartilage may
be at least partially attributable to cell death by apoptosis. Empty lacunae were counted in order to provide
further evidence of hypocellularity in OA cartilage and
to determine any association with apoptosis. However,
although there appeared to be more empty lacunae in
OA cartilage compared with nonarthritic control cartilage, the differences were marginal, and no association
was found with apoptosis. Empty lacunae are often
difficult to count in thin sections, and the large number
found in the control cartilage (on average 25%) suggests
that many empty lacunae may represent artifactual loss
of cells to the neighboring sections.
In an attempt to explore the mechanisms of cell
death by apoptosis in human articular cartilage, expression of panels of proapoptotic molecules (p53 and Bax),
antiapoptotic molecules (Bcl-2, Bcl-x, and Bag-1), and a
chondrocyte proliferative marker (Ki-67) was analyzed
by immunohistochemistry. The data demonstrate large
variations (as indicated by the wide ranges of values
shown in Table 3) in expression of these molecules in all
3 cartilage groups, and there were no significant differences between groups. However, when expression of
these molecules was examined in relation to cartilage
zones, expression of the proapoptotic molecule p53 was
found to be significantly higher in the superficial zone in
all 3 groups. This is rather intriguing, because we also
found more apoptosis in the superficial zone. Nonetheless, there was no correlation between expression of p53
in the superficial zone and the percentage of apoptosis
in this zone. In a previous study (24), Yatsugi et al
observed increased expression of p53 in the superficial
zone, which correlated positively with apoptosis. In
addition, in the current study expression of the other
proapoptotic molecule, Bax, in OA cartilage appeared to
be considerably lower than that reported by Kim et al
(10) (0–13% versus 65%). These apparent differences in
the expression of p53 and Bax may be attributable to
topographic variations in the cartilage used and/or use of
different primary antibodies by the investigators.
Among the antiapoptotic molecules, expression
of Bcl-2 was generally higher than that of Bag-1 and
Bcl-x in all 3 groups (Table 3), and the expression
correlated negatively with apoptosis in the superficial
zone, suggesting that chondrocytes express more Bcl-2 in
an attempt to reduce the level of apoptosis in this zone.
In addition to its antiapoptotic function, Bcl-2 is also
thought to be involved in maintaining the differentiated
phenotype of chondrocytes (25). Higher expression is
observed in young cartilage (26), and because OA
cartilage is thought to behave in some ways like young
cartilage (27), up-regulation of Bcl-2 may be associated
with cartilage repair in OA. However, it is worth emphasizing that the interaction between the proapoptotic
molecules and various effector enzymes such as caspase3 that lead to cell death by apoptosis is very complex. In
the present study, large variations in the expression of
both proapoptotic and antiapoptotic molecules in a
relatively small number of cartilage specimens prevent
us from drawing any conclusions about the role of these
molecules in controlling apoptosis in human articular
The percentage of apoptotic cells increases with
age in the articular cartilage of skeletally mature animals
(19), but no sex-related differences have been reported
in animal or human tissue. The present study compares
apoptosis in cartilage from OA joints and nonarthritic
joints matched for sex and age. However, it was necessary for us to use nonarthritic hip cartilage as the control
for both OA knee and OA hip cartilage. Therefore,
some differences observed between OA knee and nonarthritic hip cartilage may be explained by underlying
differences between different joints rather than the
disease process itself. Nevertheless, in this study the data
showed no significant differences between knee OA and
hip OA cartilage. Indeed, for some of the immunohistochemical analyses (e.g., caspase-3), the data for knee
and hip OA were very similar. Another potential problem is that nonarthritic tissue from the hip was obtained
from patients with osteoporotic femoral neck fractures.
This is the usual control used in this type of research, but
it remains possible that osteoporotic changes in bone
could influence the overlying articular cartilage.
Recent studies have shown a positive correlation
between the degree of severity of OA and the number of
apoptotic chondrocytes in both experimentally induced
OA in rabbit cartilage (8) and human OA cartilage (7).
Cell death by apoptosis in articular cartilage may contribute to cartilage damage in and/or the pathogenesis of
OA via several mechanisms. First, an increased rate of
apoptosis in cartilage could lead to a reduction in
cartilage cellularity, and, thus, a reduced ability of the
cartilage cells to replace any damaged cartilage in the
early stages of OA. Second, accumulation of apoptotic
bodies and their removal from the tissue can cause
further degradation of the cartilage. Cartilage is avascular and contains no mononuclear phagocytes capable of
removing apoptotic bodies. However, it is likely that the
chondrocytes themselves are phagocytosing the apoptotic bodies, because chondrocytes with condensed mate-
rial in their cytoplasm have been observed by electron
microscopy (10). Apoptotic bodies in cartilage are structurally (28) and functionally (21) similar to matrix
vesicles associated with hydroxyapatite crystal deposition, and they are able to precipitate calcium from
solution in similar amounts to matrix vesicles (21) and
therefore could be responsible for abnormal cartilage
calcification in aging and OA. Also, apoptotic bodies
could release their contents (including proteases) into
the matrix, promoting enzymatic degradation. Destruction of matrix components, especially in the pericellular
region, may affect cell–matrix interactions, so that further apoptosis is triggered, and a vicious cycle is established.
In conclusion, the data presented in this report
show that at any point in time, 2–3% of chondrocytes in
OA cartilage are dying by apoptosis, compared with
0.6% in nonarthritic cartilage, and that an increased
level of apoptosis corresponds to reduced cellularity in
the cartilage matrix. The levels of apoptosis observed in
the current study are lower than those found in several
previous studies (6,7,11) but are consistent with results
of the recent (meticulous) study by Aigner et al (9). OA
usually develops over many years and in most patients
progresses rather slowly, so a low level of cell death by
apoptosis could play an important role in the disease
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expressions, caspase, correspond, increase, apoptosis, osteoarthritis, cartilage, human, reduced, density, cells
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