Caspase inhibitors reduce severity of cartilage lesions in experimental osteoarthritis.
ABSTRACT To examine the therapeutic efficacy of caspase inhibitors in experimental osteoarthritis (OA).
Experimental OA was induced in rabbits by anterior cruciate ligament transection (ACLT). Rabbits were treated with intraarticular (i.a.) injections of caspase inhibitors 3 times per week starting 1 week postoperatively. Animals were killed 9 weeks after ACLT, for macroscopic, histologic, and immunohistochemical assessment of the knee joints.
I.a. administration of the pan-caspase inhibitor Z-VAD-FMK significantly reduced cartilage degradation, as assessed by macroscopic and microscopic criteria. Untreated knees showed large numbers of chondrocytes with active caspase 3 and the p85 fragment of poly(ADP-ribose) polymerase (PARP p85) that is generated during apoptosis. The frequency of cells positive for PARP p85 and active caspase 3 was reduced in Z-VAD-FMK-treated knees. Inhibitors specific for caspase 3 or caspase 8 showed no significant efficacy. Caspase 1 inhibitor and the combination of caspase 3 and caspase 8 inhibitors reduced OA pathology.
These results provide direct support for a role of cell death in OA pathogenesis. Caspase inhibitors reduced the severity of cartilage lesions in experimental OA, suggesting that they may have disease-modifying activity in human OA.
- SourceAvailable from: David Sadigursky[Show abstract] [Hide abstract]
ABSTRACT: OBJETIVO: Avaliar se a injeção intra-articular de Plasma rico em plaquetas (PRP) pode reduzir a apoptose pós-traumática de condrócitos. MÉTODOS: Foi desenvolvido um estudo experimental duplo-cego com quatro joelhos de coelhos adultos. Após a anestesia, os animais foram submetidos à contusão padronizada dos joelhos. Depois foi injetado 1ml de PRP humano nos dois joelhos esquerdos e 1ml de solução fisiológica (SF) nos dois joelhos direitos. Os dois coelhos foram mantidos no mesmo ambiente sob controle de temperatura, de atividades diárias e de alimentação. A eutanásia dos animais ocorreu dez dias após a intervenção e foram realizadas biópsias da cartilagem de cada joelho. As peças foram preparadas para análise em microscopia eletrônica (ME). RESULTADOS: Quatro preparados para ME foram obtidos, cada um correspondendo a um joelho. Os joelhos-PRP apresentaram as taxas de apoptose de 47,62% (50/105) e de 48,36% (59/122), respectivamente. Nos joelhos-SF as taxas de apoptose foram, respectivamente, 56,67% (17/30) e 70,40% (88/125). A diferença do índice de apoptose nos joelhos-PRP (48,02%) e nos joelhos-SF (67,74%) foi significante (p<0,001) e OR=0,439 (IC95%=0.287-0.673). CONCLUSÃO: A injeção intra-articular de PRP imediatamente ao trauma, reduz as taxas de apoptose (pós-traumática) de condrócitos de coelhos.Acta Ortopédica Brasileira 04/2011; 19(2):102-105. · 0.16 Impact Factor
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ABSTRACT: Study the impact of intra-articular interleukin-1 receptor antagonist (IL-1 ra) treatment on lubricin biosynthesis following anterior cruciate ligament transection (ACLT) in the rat and evaluate the effect of combined IL-1 ra and recombinant human lubricin (rhPRG4) treatments on chondrocyte apoptosis and MMP-13 expression.Osteoarthritis and Cartilage 09/2014; · 4.66 Impact Factor
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ABSTRACT: When endoplasmic reticulum (ER) stress, i.e. the excessive accumulation of unfolded proteins in ER, endangers homeostasis, apoptosis is induced by C/EBP homologous protein (Chop). In osteoarthritis (OA) cartilage, Chop expression and apoptosis increase as degeneration progresses. We investigated the role of Chop in murine chondrocyte apoptosis and in the progression of cartilage degeneration. We induced experimental OA in Chop-knockout (Chop(-/-)) mice by medial collateral ligament transection and menisectomy and compared cartilage degeneration, apoptosis, and ER stress in Chop(-/-)- and wild-type (Chop(+/+)) mice. In our in vitro experiments we treated murine Chop(-/-) chondrocytes with the ER stress inducer tunicamycin (TM) and evaluated apoptosis, ER stress, and chondrocyte function. In vivo, the degree of ER stress was similar in Chop(-/-)- and Chop(+/+) mice. However, in Chop(-/-) mice apoptosis and cartilage degeneration were lower by 26.4% and 42.4% at 4 weeks, by 26.8% and 44.9% at 8 weeks, and by 26.9% and 32.3% at 12 weeks after surgery than Chop(+/+) mice, respectively. In vitro, the degree of ER stress induction by TM was similar in Chop(-/-)- and Chop(+/+) chondrocytes. On the other hand, apoptosis was 55.3% lower and the suppression of collagen type II and aggrecan mRNA was 21.0% and 23.3% less, and the increase of matrix metalloproteinase-13 mRNA was 20.0% less in Chop(-/-)- than Chop(+/+) chondrocytes. Our results indicate that Chop plays a direct role in chondrocyte apoptosis and that Chop-mediated apoptosis contributes to the progression of cartilage degeneration in mice.Osteoarthritis and Cartilage 04/2014; · 4.26 Impact Factor
ARTHRITIS & RHEUMATISM
Vol. 54, No. 6, June 2006, pp 1814–1821
© 2006, American College of Rheumatology
Caspase Inhibitors Reduce Severity of
Cartilage Lesions in Experimental Osteoarthritis
Darryl D’Lima,1Juan Hermida,1Sanshiro Hashimoto,2Clifford Colwell,1and Martin Lotz2
Objective. To examine the therapeutic efficacy of
caspase inhibitors in experimental osteoarthritis (OA).
Methods. Experimental OA was induced in rab-
bits by anterior cruciate ligament transection (ACLT).
Rabbits were treated with intraarticular (IA) injections
of caspase inhibitors 3 times per week starting 1 week
postoperatively. Animals were killed 9 weeks after
ACLT, for macroscopic, histologic, and immunohisto-
chemical assessment of the knee joints.
Results. IA administration of the pan-caspase
inhibitor Z-VAD-FMK significantly reduced cartilage
degradation, as assessed by macroscopic and micro-
scopic criteria. Untreated knees showed large numbers
of chondrocytes with active caspase 3 and the p85
fragment of poly(ADP-ribose) polymerase (PARP p85)
that is generated during apoptosis. The frequency of
cells positive for PARP p85 and active caspase 3 was
reduced in Z-VAD-FMK–treated knees. Inhibitors spe-
cific for caspase 3 or caspase 8 showed no significant
efficacy. Caspase 1 inhibitor and the combination of
caspase 3 and caspase 8 inhibitors reduced OA pa-
Conclusion. These results provide direct support
for a role of cell death in OA pathogenesis. Caspase
inhibitors reduced the severity of cartilage lesions in
experimental OA, suggesting that they may have
disease-modifying activity in human OA.
Osteoarthritis (OA) is the most prevalent joint
disease, but therapies that interfere with the progressive
destruction of articular cartilage are not available (1,2).
Previous concepts regarding OA pathogenesis empha-
sized the role of enzymes that degrade cartilage extra-
cellular matrix components (3). Cytokines such as
interleukin-1 (IL-1) are important stimuli of matrix-
degrading enzymes and of the inflammatory response in
OA-affected joints (4). Recently, increased cell death
has been documented as a feature of OA cartilage in
humans and in animal models (5–10). Cell density is
reduced in aging and OA cartilage (11,12).
Cell death and matrix degradation in OA carti-
lage correlate with one another (7,13) and are mecha-
nistically linked, since matrix degradation results in the
loss of survival mechanisms (14), and cell death can
contribute to matrix degradation (15) and calcification
(16). Potential inducers of chondrocyte death in OA
include oxygen radicals (17,18), 15-deoxy-?12,14-
prostaglandin J2(19), death receptor activation (20,21),
mitochondrial dysfunction (22), and mechanical stress
Cell death in OA cartilage has certain features of
apoptosis, or programmed cell death (5). Apoptosis is
mediated by a cascade of aspartate-specific cysteine
proteases or caspases (27), and increased caspase 3
expression has been noted to correlate with reduced cell
density in human OA cartilage (9). Pharmacologic in-
hibitors of these enzymes have been explored as poten-
tial therapeutic agents in experimental models of dis-
eases that are associated with increased cell death
(28–30). In vitro studies of chondrocytes showed that
caspase inhibitors can prevent cell death and maintain
chondrocyte function (31,32).
While inhibiting apoptosis may increase cell via-
bility in vitro, it has yet to be demonstrated whether this
occurs in vivo and protects against cartilage lesions in
animal models of OA. The objective of this study was to
test the hypothesis that the inhibition of apoptosis by
intraarticular (IA) injection of caspase inhibitors re-
duces cartilage degradation in experimental OA.
Supported by the NIH (grants AG-07996 and AR-46990) and
a Skaggs Clinical Scholarship.
1Darryl D’Lima, MD, Juan Hermida, MD, Clifford Colwell,
MD: The Scripps Research Institute and the Scripps Clinic, La Jolla,
California;2Sanshiro Hashimoto, MD, Martin Lotz, MD: The Scripps
Research Institute, La Jolla, California.
Address correspondence and reprint requests to Martin Lotz,
MD, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, CA 92037. E-mail: email@example.com.
Submitted for publication June 24, 2005; accepted in revised
form February 15, 2006.
MATERIALS AND METHODS
Experimental osteoarthritis. NZW rabbits (Western
Oregon Rabbit Co., Philomath, OR), weighing 3.5–4.5 kg, and
with closed epiphyses, were used in all experiments. All studies
were approved by the institutional animal care and use com-
mittee. Anterior cruciate ligament transection (ACLT) was
performed using lateral arthrotomy (33). The patella was
dislocated medially, and the ACL was transected with a sharp
blade. Complete transection was confirmed by a manual
anterior drawer test. The knee joints were irrigated with sterile
saline and closed in layers with sutures. All animals were
maintained in individual cages and were allowed unlimited
activity. The animals were killed 9 weeks after the surgery. The
effects of ACLT in rabbits have been previously characterized
(34), and the majority of rabbits that underwent ACLT devel-
oped cartilage degeneration by 8 weeks.
Treatment groups. Caspase inhibitors tested in this
study were the pan-caspase inhibitor Z-VAD-FMK, Z-DEVD-
FMK for caspase 3, Ac-IETD-CHO for caspase 8, and Ac-
YVAD-CHO for caspase 1 (or IL-1?–converting enzyme). All
inhibitors were purchased from Calbiochem (La Jolla, CA).
The rabbit knee joints were injected with 100 ?l of caspase
inhibitor at 25 ?g/ml (0.54 mM) in saline or with 100 ?l saline
The dosage of caspase inhibitor was chosen based on in
vitro experiments that showed efficacy of caspase inhibition
using 100 ?M concentrations. A concentration of 540 ?M of
agent in a 0.1-ml injection would result in a final concentration
of ?100 ?M in 0.4 ml rabbit joint synovial fluid. The synovial
fluid volume of 0.4 ml was an estimate based on joint effusions
that were sampled in pilot experiments with ACLT.
Three separate experiments were performed: experi-
ment 1, in which 12 rabbits were divided into 2 groups of 6
each, with rabbits in each group injected with either saline or
pan-caspase inhibitor; experiment 2, in which a total of 36 knees
in 18 rabbits were injected with either saline, pan-caspase inhib-
itor, caspase 3 inhibitor, or caspase 8 inhibitor, resulting in 9
knees per group; and experiment 3, in which a total of 36 knees
in 18 rabbits were injected with either saline, pan-caspase
inhibitor, caspase 1 inhibitor, or a combination of caspase 3
inhibitor and caspase 8 inhibitor, resulting in 9 knees per group.
Gross morphologic assessment of articular cartilage.
The distal femur and proximal tibia were harvested, keeping
the 3.5–4-cm shaft of the bones. The articular cartilage surface
of each specimen was covered with a solution of India ink
(Eberhard Faber, Lewisburg, TN) in phosphate buffered saline
(PBS) (1:5 ratio). Excess ink solution was removed by gently
blotting with a tissue that was premoistened with PBS. Subse-
quently, all joints were photographed, and digital images were
Articular cartilage was assessed using the following
grading scale: grade 1 (intact surface), surface is normal in
appearance and does not retain India ink; grade 2 (minimal
fibrillation), surface retains India ink as elongated specks or
light gray patches; grade 3 (overt fibrillation), areas are velvety
in appearance and retain India ink as intense black patches;
and grade 4 (erosion), loss of cartilage, exposing the underlying
Each harvested rabbit femur and tibia was placed in a
specimen container labeled with a unique rabbit number and
the side (right or left leg). The investigator who performed the
morphologic analysis was not part of the surgical team and had
no prior access to the treatment code.
Surface area of lesions. Articular surfaces of femoral
condyles and tibial plateaus were gently blotted dry and
cleaned of loose tissue. A previously described technique was
used to quantify the cartilage lesion area (35). Each femoral
shaft was clamped to an optical bench. An image (resolution 60
pixels per mm; on-screen magnification 20?) of the femoral
condyles was obtained using an EOS D30 digital camera
(Canon, Lake Success, NY) with a 100-mm macro lens at a
distance of ?12 cm. A millimeter scale was included in the
photograph to accurately scale the image. The scaled image
was then projected onto a 3-dimensional (3-D) model of the
femoral condyles. The 3-D surface area of the lesion was
measured by interactively plotting the margins of the lesion. A
digital image of the articular surface of the tibia was obtained
as described above. No 3-D projection was used, since the
tibial surface was relatively flat and 2-dimensional measure-
ments did not vary significantly from 3-D measurements.
Histologic grading. Distal femur and proximal tibia
from the rabbit knee joints were fixed in 10% buffered
formalin, decalcified in TBD-2 decalcifier (ThermoShandon,
Pittsburg, CA), and embedded in paraffin blocks. Sagittal
sections of lateral and medial femoral condyles and coronal
sections of tibial plateaus were used for histologic analysis.
Quantitative assessment of sulfated glycosaminoglycan
(GAG) content was performed after the tissue sections were
stained with Safranin O–fast green. Digital images were ac-
quired from tibial and femoral condylar articular cartilage at
10? magnification. The intensity of the Safranin O stain was
quantitated using the method described by Martin et al (36) to
yield a normalized red value, which was used as a GAG
content score (intensity of Safranin O stain) for each knee.
Tissue preparation for immunohistochemistry. Se-
lected sections of the knee joints were deparaffinized in 3
changes of Hemo-De (Fisher Scientific, Pittsburgh, PA) and
rehydrated in graded ethanol and water. For horseradish
peroxidase (HRP)–conjugated antibody, endogenous peroxi-
dase was blocked by incubating the sections with 3% H2O2for
5 minutes at room temperature. Endogenous biotin or avidin
binding sites were blocked by sequential incubation for 15
minutes with avidin and biotin (Vector, Burlingame, CA).
Nonspecific staining was blocked by incubation of sections with
10% normal serum or bovine serum albumin in PBS. Sections
were digested in 2 mg/ml hyaluronidase for 30 minutes and
permeabilized in 0.2% Triton X-100/PBS for 5 minutes at
Immunohistochemistry for poly(ADP-ribose) polymer-
ase (PARP) cleavage and caspase activation. Polyclonal rabbit
antibody specific for the p85 fragment of PARP (PARP p85)
(catalog no. G7341; Promega, Madison, WI) and polyclonal
antibody to the active form of caspase 3 (catalog no. 3015-100;
BioVision, Mountain View, CA) were used. Primary antibody
was applied at a dilution of 1:100 and incubated overnight at
4°C. The negative control was rabbit IgG at 1 mg/ml. The
following day, the sections were washed, blocked with 3%
H2O2for 5 minutes, washed again, and incubated with diluted
secondary antibody (HRP–anti-rabbit IgG) for 1 hour. Slides
were washed and sections were incubated for 4–10 minutes in
diaminobenzidine substrate. The slides were rinsed in tap
DISEASE-MODIFYING ROLE OF CASPASE INHIBITORS IN OA1815
water, counterstained with methyl green, rehydrated in
1-butanol, cleared with Hemo-De, and mounted in Refrax
mounting medium (Anatech, Battle Creek, MI).
Cartilage cellularity. To determine whether the OA
changes were associated with a loss of chondrocytes, cartilage
cellularity was quantified by counting the number of chondro-
cytes (4?,6-diamidino-2-phenylindole–stained nuclei) in a mi-
croscope field. Digital images were captured at 20? magnifi-
cation of the full thickness of articular cartilage in a sagittal
section through the middle of each femoral condyle and
through a coronal section across the tibia. The total number of
chondrocytes was evaluated in each picture. Cell density was
expressed as the number of chondrocytes per mm2.
Statistical analysis. Statistically significant differences
between control and treatment groups were determined using
paired t-tests for numerical data (such as surface area of lesion,
number of cells positive for active caspase 3 or PARP p85), and
using the Mann-Whitney U test for grades (gross and histo-
Figure 1. A and B, Gross grading of control and Z-VAD-FMK–treated knees. Knee joints were harvested 9 weeks after anterior cruciate ligament
transection (ACLT). Photographs were taken after India ink staining. Representative examples of untreated (A) and Z-VAD-FMK–treated (B) knees are
shown. Areas that are stained with India ink represent fibrillated cartilage. Arrowheads indicate areas with complete loss of articular cartilage and exposure
of subchondral bone. C and D, Morphologic grading of knee joints from rabbits with bilateral ACLT treated with intraarticular (IA) injections of caspase
inhibitors or saline. A total of 12 rabbits underwent bilateral ACLT. Six rabbits received IA injections of caspase inhibitors 3 times per week for 9 weeks
starting 1 week postoperatively. The animals were injected with 100 ?l of caspase inhibitor at 25 ?g/ml (0.54 mM) in saline. A control group of 6 rabbits
received the same number of IA injections of 100 ?l saline. Morphologic assessment of the knee joints (C) and digital image analysis of the lesion size (D)
were performed as described in Materials and Methods. Values are the mean and SEM.
1816 D’LIMA ET AL
logic grading). The sample sizes were chosen based on prior
studies of this animal model, in which differences in grades of
1.5 could be detected with a power of 80% in a sample size of
8. P values less than 0.05 were considered significant.
IA injection of a pan-caspase inhibitor reduced
the severity of experimental OA lesions. To investigate
the effect of caspase inhibition in experimental OA, the
pan-caspase inhibitor Z-VAD-FMK was injected IA into
the knee joints of rabbits that had undergone ACLT.
The animals received 3 injections per week for 8 weeks
starting 1 week after ligament transection. Control rab-
bits that had undergone ACLT and received saline
injections showed cartilage lesions on femoral condyles
(Figure 1A), lateral tibial plateaus, and posteromedial
tibial plateaus, ranging from grade 3 to grade 4 full-
thickness cartilage defects (Figure 1C). In Z-VAD-
Figure 2. Histology of rabbit knee joints. Knee joints were harvested
from A–C, untreated or D–F, Z-VAD-FMK–treated rabbits 9 weeks
after anterior cruciate ligament transection (ACLT). Sections were
prepared and stained with Safranin O. Representative images of
femoral condyles from ACLT knees that were injected with saline
(untreated) or Z-VAD-FMK are shown. A, Center of the cartilage
lesion in the saline-injected knee. B and C, Higher magnifications of
the area shown in A. D, Center of the cartilage lesion on the femoral
condyle in the Z-VAD-FMK–treated knee. E and F, Higher magnifi-
cations of the area shown in D. (Original magnification ? 10 in A and
D; ? 20 in B and E; ? 40 in C and F.)
Figure 3. Cell density and glycosaminoglycan (GAG) staining at lesion sites. Knee joints were harvested from untreated rabbits (n ? 6) 9 weeks
after anterior cruciate ligament transection. A and B, Full-thickness cartilage sections, including the center of the lesion (left part of the images) to
the adjacent areas with partial erosion and intact articular cartilage surface, were prepared and stained with Safranin O–fast green to visualize GAGs
(A) and with 4?,6-diamidino-2-phenylindole (DAPI) to identify cell nuclei (B). C and D, Quantitative analysis of GAG staining intensity (C) and cell
density (D). Cell density was calculated as the number of DAPI-positive cells divided by the cartilage area. Values are the mean and SEM.
DISEASE-MODIFYING ROLE OF CASPASE INHIBITORS IN OA 1817
FMK–treated rabbits, lesion severity was reduced (Fig-
ure 1B). The grades ranged from 2 to 3, with only 1
rabbit having a grade 4 lesion, but the difference in
lesion grade was not statistically significant (P ? 0.1). A
significant reduction (41%) in mean total surface area of
the lesions (Figure 1D) was found between the control
and Z-VAD-FMK groups (P ? 0.001).
Histopathology of knee joints. Microscopic exam-
ination of the knee joints showed that Z-VAD-FMK
preserved cartilage integrity in the weight-bearing areas
of the femoral condyles that were most severely affected
in the untreated animals. In the saline-injected knees,
lesions varied in size of full-thickness defects with expo-
sure of subchondral bone. With increasing distance from
the center of the lesion, cartilage architecture gradually
changed to partial-thickness lesions, surface fibrillations,
and normal appearance (Figure 2). Loss of GAGs
followed a similar pattern (Figure 3A). In the fibrillated
lesion areas, partial or complete loss of chondrocytes
could be noted (Figure 3B). There was a correlation
between cell density, Safranin O staining intensity, and
structural integrity. In addition to reducing lesion size,
Z-VAD-FMK increased cartilage cellularity by 31%
(P ? 0.02) compared with controls (Figure 4), which
supports the theory that caspase inhibition affects carti-
lage structural changes by preventing the loss of chon-
Reduced caspase 3 activation and PARP cleavage
after IA injection of Z-VAD-FMK. Cartilage sections
were examined for activation of caspase 3, a key regu-
lator of most apoptosis pathways, and for cleavage of
PARP to generate the p85 fragment, a feature and
marker of apoptosis. In the untreated knees, chondro-
cytes within and adjacent to the cartilage lesions were
positive for these 2 apoptosis markers (Figure 5). In
Z-VAD-FMK–treated knees, analysis of the same carti-
lage regions that are most susceptible to ACLT-induced
degradation showed a reduced number of cells with
active caspase 3 and PARP p85 (Figure 5). These results
support the notion that caspase-mediated apoptosis
pathways contribute to cartilage pathology in this animal
model and suggest that they are effectively modulated by
IA injection of Z-VAD-FMK.
Comparison of specific caspase inhibitors with
Z-VAD-FMK. In the first series of experiments with the
OA animal model, Z-VAD-FMK treatment led to sig-
Figure 4. Cartilage cellularity in control and Z-VAD-FMK–treated
knee joints. Data shown represent total cell count (4?,6-diamidino-2-
phenylindole–positive cells) of the entire histologic section (6 rabbits
per group). Cartilage cellularity was increased by 31% in Z-VAD-
FMK–treated knee joints compared with controls. Values are the
mean and SEM.
Figure 5. Immunohistochemistry of the p85 fragment of poly(ADP-
ribose) polymerase (PARP p85) and active caspase 3. Knee joints were
harvested from saline-injected (untreated) (A and B) or Z-VAD-
FMK–treated (C and D) rabbits 9 weeks after anterior cruciate
ligament transection. Sections were stained with antibody to the p85
cleaved form of PARP or with antibody to the active form of caspase
3. (Original magnification ? 10 in A and C; ? 20 in B and D.)
1818 D’LIMA ET AL
nificant reduction in OA severity. Z-VAD-FMK is a
broad-spectrum inhibitor, with activity predominantly
against caspases 1, 3, and 4. Two additional and separate
animal studies were performed with specific inhibitors
(Z-DEVD-FMK for caspase 3, Ac-IETD-CHO for
caspase 8, and Ac-YVAD-CHO for caspase 1). These
experiments also included Z-VAD-FMK. All inhibitors
were administered according to the same schedule as
Z-VAD-FMK in the first experiment. Analysis of rabbit
knee joints 9 weeks after ACLT showed that treatment
with the caspase 3 or caspase 8 inhibitor did not signif-
icantly reduce the severity of cartilage lesions (Figure 6).
The caspase 1–specific inhibitor and the combination of
caspase 3 and caspase 8 inhibitors were effective, as
indicated by a significant reduction in lesion surface area
This study addressed the hypothesis that inhibi-
tion of chondrocyte death by IA injection of caspase
inhibitors protects against cartilage degradation in ex-
perimental OA. The rationale for the present study was
based on the occurrence of apoptosis in OA cartilage (5)
and the ability of caspase inhibitors to prevent chondro-
cyte death in vitro (32).
The results show that the broad-spectrum
caspase inhibitor Z-VAD-FMK reduces the size of OA
cartilage lesions. Although the grade of the cartilage
lesions was not significantly improved by treatment with
Z-VAD-FMK, the reduction in lesion size represents a
structure-modifying effect. This effect of Z-VAD-FMK
is associated with reduced PARP cleavage and caspase 3
activation and increased cellularity in OA-affected car-
tilage. Collectively, these observations support the con-
clusion that cell death in this experimental model con-
tributes to OA pathogenesis and is mediated, at least in
part, by apoptotic mechanisms.
Experimental OA induced by ACLT in rabbits is
one of the most widely used models to examine disease-
modifying OA therapies (35,37–40). ACLT results in
abnormal knee biomechanics, including increased ante-
rior drawer at extension and at 90° of flexion, as well as
an increased internal rotation similar to that observed in
human OA knees. The most severe areas of cartilage
degeneration in rabbits that have undergone ACLT
occur in medial femoral condyles, followed by lateral
femoral condyles. In the tibial plateaus, ACLT causes
mild to moderate OA lesions in the areas not covered by
the menisci (41). The primary mechanism in this model
is mechanical stress, which mediates its effects on patho-
genesis via changes in gene expression protein synthesis
and cell survival (42,43). The observed therapeutic ben-
efit of Z-VAD-FMK supports the notion that cell death
has a role in this model and is consistent with a recent
study that showed that Z-VAD-FMK inhibited chondro-
cyte apoptosis in a model of acute osteochondral injury
The major therapeutic effect observed in the
present study was a reduction in lesion size in response
to injection of Z-VAD-FMK. The general quality of
lesions in saline-injected and drug-treated knees was
similar. At the center of the primary lesion sites on
femoral condyles, defects were most severe and could
represent complete loss of articular cartilage. With
increasing distance from the lesion center, architectural
damage decreased to partial-thickness degradation, fi-
brillation only on the articular surface, and eventually
normal surface. Loss of GAGs and cell density followed
a similar pattern, and the number of cells positive for
active caspase 3 and PARP p85 was greatest in areas
adjacent to the center of the lesion. While Z-VAD-FMK
had significant effects on lesion size, there was no
statistically significant effect on lesion grade, although a
trend was noted. This difference reflects the fact that the
grading system measures depth of lesion, which can vary
independently of lesion area.
The most effective inhibitor in the present study
was Z-VAD-FMK. This is a broad-spectrum inhibitor
with activity predominantly against caspases 1, 3, and 4.
Figure 6. Gross grading of control and caspase inhibitor–treated knees.
Rabbits underwent anterior cruciate ligament transection, and 6 animals
per group received intraarticular injections of caspase inhibitors for 8
weeks starting 1 week postoperatively. Digital image analysis of the lesion
and Methods. The surface area of the lesions was normalized to that of
the lesions on the contralateral control knee. Connecting lines show
statistically significant comparisons (P ? 0.05). C1 Inh ? caspase 1
inhibitor. Values are the mean and SEM.
DISEASE-MODIFYING ROLE OF CASPASE INHIBITORS IN OA1819
In addition to inhibiting caspases in apoptosis pathways,
Z-VAD-FMK inhibits caspase 1, which is needed to
process inactive proIL-1? and proIL-18 into biologically
active forms (45,46). IL-1 and IL-18 promote joint
inflammation and cartilage degradation (47,48). An
orally administered nonpeptide inhibitor of caspase 1
reduced joint damage in 2 murine models of OA (49). In
the present study we tested a caspase 1–selective peptide
inhibitor and found that it had disease-modifying activ-
ity, but less so than Z-VAD-FMK. We also tested
inhibitors of caspase 3, an executioner caspase involved
in apoptosis pathways that can be activated by different
upstream and extracellular triggers, and of caspase 8,
which is linked to death receptor activation (50). With
the caspase 3 inhibitor there was a trend toward in-
creased lesion size and the caspase 8 inhibitor showed no
efficacy when injected alone. However, the combination
of the 2 inhibitors was effective. These findings suggest
that other apoptosis pathways besides death receptors
may be involved in this animal model.
Concentrations of caspase inhibitors injected into
the rabbit joints were in the millimolar range, which
raises concerns about potential nonspecific effects on
other pathways. While this cannot be excluded, the
relative potency of the different caspase inhibitors that
are similar in structure corresponds to the spectrum of
caspases they inhibit, with the pan-caspase inhibitor
being more effective compared with individual inhibi-
tors, and the combination of caspase 3 and 8 inhibitors
showing additive effects.
The present study provides proof-of-concept for
caspase inhibition as a therapeutic strategy for cartilage
injury and OA. ACL tears in humans are associated with
cartilage injury and predispose to the development of
posttraumatic OA (51). Immediately following joint
injury there is a degradation of cartilage extracellular
matrix (52,53), elevation of levels of matrix-degrading
enzymes (54), and chondrocyte death in the affected
areas (55). IA treatment with caspase inhibitors during
the initial time period following joint injury has the
potential to rescue cells from apoptosis, thus limiting the
extent of cartilage lesion and, through maintaining cel-
lularity, enhancing lesion repair. In established idio-
pathic OA, the progression of cartilage lesions is related
to continued loss of chondrocytes, which suggests poten-
tial efficacy of caspase inhibitors.
The magnitude of the caspase inhibitor effects
observed in the present study was comparable with
published data on other OA drug candidates with
disease-modifying potential, such as hyaluronans (39),
N-acetylglucosamine (35), or glucosamine (40), in the
same animal model. The inhibitors used in the present
study are small peptides with a short IA half-life. Ther-
apeutic efficacy of caspase inhibition can be improved in
future studies by using different chemical entities and
formulations to extend IA half-life.
We thank Lilo Creighton and Bao Nguyen for assis-
tance in histology and image analysis.
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