Nociceptive tolerance is improved by bradykinin receptor B1 antagonism and joint morphology is protected by both endothelin type A and bradykinin receptor B1 antagonism in a surgical model of osteoarthritis
© Kaufman et al.; licensee BioMed Central Ltd. 2011
Received: 28 July 2010
Accepted: 16 May 2011
Published: 16 May 2011
Endothelin-1, a vasoconstrictor peptide, influences cartilage metabolism mainly via endothelin receptor type A (ETA). Along with the inflammatory nonapeptide vasodilator bradykinin (BK), which acts via bradykinin receptor B1 (BKB1) in chronic inflammatory conditions, these vasoactive factors potentiate joint pain and inflammation. We describe a preclinical study of the efficacy of treatment of surgically induced osteoarthritis with ETA and/or BKB1 specific peptide antagonists. We hypothesize that antagonism of both receptors will diminish osteoarthritis progress and articular nociception in a synergistic manner.
Osteoarthritis was surgically induced in male rats by transection of the right anterior cruciate ligament. Animals were subsequently treated with weekly intra-articular injections of specific peptide antagonists of ETA and/or BKB1. Hind limb nociception was measured by static weight bearing biweekly for two months post-operatively. Post-mortem, right knee joints were analyzed radiologically by X-ray and magnetic resonance, and histologically by the OARSI histopathology assessment system.
Single local BKB1 antagonist treatment diminished overall hind limb nociception, and accelerated post-operative recovery after disease induction. Both ETA and/or BKB1 antagonist treatments protected joint radiomorphology and histomorphology. Dual ETA/BKB1 antagonism was slightly more protective, as measured by radiology and histology.
BKB1 antagonism improves nociceptive tolerance, and both ETA and/or BKB1 antagonism prevents joint cartilage degradation in a surgical model of osteoarthritis. Therefore, they represent a novel therapeutic strategy: specific receptor antagonism may prove beneficial in disease management.
Osteoarthritis (OA) is characterized by a progressive destruction of articular cartilage accompanied by subchondral bone remodeling, osteophyte formation, and synovial membrane inflammation . Clinically, this disease progresses slowly and principally affects the hands and large weight-bearing joints. Pain is the primary complaint of patients with OA. Its etiology is multifactorial: subchondral bone can have micro-fractures, osteophytes can cause stretching of peri-osteal nerve endings, ligaments may be stretched, the joint capsule can be inflamed or distended, the synovium may be inflamed, and muscles may spasm . Furthermore, neo-innervation of joint tissue concurrent with angiogenesis [3, 4] may contribute to deep joint pain. Further understanding of the molecular mechanisms behind these effects should provide avenues towards targeted disease-modifying or -slowing treatments [5, 6].
We have previously shown that endothelin-1 (ET-1), a 21-amino-acid potent vasoconstrictor peptide, plays a major role in OA pathogenesis. It reduces cartilage anabolism by inhibiting collagen and proteoglycan synthesis . It causes matrix metalloproteinases one and thirteen to be synthesized and activated in OA cartilage . ET-1 also causes excessive production of nitric oxide, which is generated as the result of an increase in inducible nitric oxide synthase levels . These effects occur mainly via endothelin receptor type A (ETA) : it is expressed in articular tissue by chondrocytes, synoviocytes, and endothelial cells, where it plays a significant role in cartilage and bone metabolism [11, 12]; ETA also potentiates inflammatory joint pain induced by ET-1 [13, 14].
ET-1 affects vascular homeostasis via the renin-angiotensin-aldosterone system . Through cross-talk with the kallikrein-kinin system , it can also mediate kinin-induced pain and inflammation. Bradykinin (BK), the inflammatory nonapeptide vasodilator, has also been implicated in OA pain and inflammation. It is generated in OA synovium, as in all inflamed tissue; it also is released due to the increased vascular pressure in subchondral bone . BK binds two receptors, bradykinin receptor B1 (BKB1) and bradykinin receptor B2 (BKB2). The effects of BK in OA occur largely via BKB1, a receptor implicated in articular nociception [18, 19] and pro-inflammatory reactions . BKB1 also potentiates the effects of other pro-inflammatory mediators such as cytokines and prostaglandins. BKB2, though it has been implicated in nociceptor sensitization in OA [17, 19], may be less relevant as a therapeutic target in the context of a chronic inflammatory response. It is constitutively expressed to a large extent, and is primarily involved in the acute phase of inflammation [21, 22]. In contrast, BKB1 is up-regulated in chronic inflammatory conditions, its expression often induced secondary to inflammatory mediator release [22–24].
Antagonism of ETA and/or BKB1 may represent a novel therapeutic option to alleviate, and perhaps prevent or reverse, the pain, inflammation, and tissue damage that occur as OA progresses from an acute to a chronic state. We hypothesize that ETA and BKB1 antagonism will diminish OA progress in a synergistic manner. In the present work, we describe a preclinical study of the efficacy of treatment of surgically induced OA with ETA and/or BKB1 peptide antagonists, using an established rat model of the disease. We found that BKB1 antagonist treatment diminished hind limb nociception, and both ETA and/or BKB1 antagonism protected joint radiomorphology and histomorphology. This demonstrates that ETA and BKB1 receptor expression is involved in OA pathogenesis, and that specific receptor antagonism may prove beneficial in OA disease management.
Materials and methods
Rat model of osteoarthritis
Eight-week-old male Lewis rats were purchased from Charles River Canada (Saint-Constant, Quebec) and housed under standard conditions. All procedures were approved by the Sainte-Justine Hospital Research Centre animal ethics committee and conformed to Canadian Council on Animal Care guidelines .
OA was induced by surgical transection of the right anterior cruciate ligament. The procedure was modified from previously published reports [26–29], and is described in detail in Additional file 1. Briefly, animals were anaesthetized and subjected to either anterior cruciate ligament transection or sham surgery. One group of animals, kept as negative controls, were not operated upon.
Over the course of two months post-operatively, animals were treated by weekly intra-articular injections of ETA and/or BKB1 specific peptide antagonists: BQ-123 (ETA antagonist; Sigma-Aldrich, Oakville, Ontario) [30, 31], R-954 (BKB1 antagonist; a kind gift from Pierre Sirois, IPS Thérapeutique, Sherbrooke, Quebec) [32, 33], both, or saline vehicle, was injected into the right knee at a dose of 30 nmol in a volume of 50 μL. Injections were performed under isoflurane anaesthesia, using a 28G needle; the procedure is described in detail in Additional file 1. Chemical structures of the antagonists are depicted in Additional file 2. Doses were based upon previously published reports [14, 19].
Static weight bearing
Over the course of the study, animal nociception was evaluated biweekly by the static weight bearing test. A static weight bearing apparatus was reverse-engineered from previously published reports [34–36], designed, and machined by Usinage FB (Le Gardeur, Quebec). Design diagrams and photos are appended in Additional files 3 and 4.
All values are given as mean ± standard deviation (SD) per experimental group.
Static weight bearing data were analyzed by repeated measures analysis of variance (ANOVA), which compares the global differences between groups of response profiles measured on the same subjects repeatedly over the course of the study [38, 39]. Test values were taken as the dependent variable and treatment group as the independent variable, with the animal as the grouping factor. Sphericity was confirmed with Mauchly's W test. Tukey multiple comparisons testing was used to establish significance in between groups, with directionality taken from the sign of the mean difference. P-values less than 0.05 were considered statistically significant. Analyses were conducted using R (version 2.12.1) .
Euthanasia and sample preparation
At four or eight weeks post-surgery, animals were sacrificed by cardiac puncture under deep isoflurane anaesthesia. The right knee was dissected, and 40-mm-long samples were cut and stored in phosphate-buffered saline until imaged by digital micro-X-ray (DX) and/or micro-magnetic resonance (MR). Samples were dissected the same day as the radiological scans.
All knee samples were X-rayed using a Faxitron MX-20 specimen X-ray system (Faxitron X-Ray Corporation, Lincolnshire, IL). Anteroposterior and lateral views were acquired at 5 × magnification (10 × 10 μm pixel size) using a dose of 26 kV for 6 seconds. Images were analyzed using OsiriX software (version 3.7.1) . Radiological evidence of joint degradation was scored by two blinded examiners using an OA radiological score modified from Clark et al.  and Esser et al. . Bone demineralization, subchondral bone erosion, and heterotopic ossification were all scored on a scale from zero (normal) to three (marked degenerative changes). Total scores were calculated by summing the individual scores for each index, with a maximum possible score of nine.
OA radiological scores were statistically analyzed by one-way ANOVA, with total scores taken as the dependent variable and treatment group as the independent variable. Pairwise post-hoc testing with Holm correction was used to establish significance in between treatment groups. P-values less than 0.05 were considered statistically significant. Analyses were conducted using R (version 2.12.1) .
Micro-magnetic resonance imaging
A subset of animals were sacrificed four weeks post-operatively and their right knees were imaged by micro-MR. Imaging was performed using a Bruker PharmaScan (Ettlingen, Germany) 7 Tesla MR scanner at the McGill University Small Animal Imaging Lab (Montreal, Quebec). Knee samples were placed in a custom-made support inside a 15-mL centrifuge tube, which was then filled with the MR-inert buffer FC-770 (3M Fluorinert Electronic Liquid). Samples were introduced into a 1H mouse brain radio frequency (RF) coil (inner diameter 22 mm), and centered in the magnet. The RF coil was tuned and matched to the sample, and the magnet was then shimmed. The system was controlled via Bruker ParaVision software (version 5.0).
Positioning was confirmed with a tri-pilot rapid scan, which was then used to place 14 coronal slices for two-dimensional anatomical scanning of the joint using a rapid acquisition with relaxation enhancement (RARE) multiecho spin echo pulse sequence (TurboRARE). Scan parameters were as follows: repetition time (TR) = 3500 milliseconds (ms), echo time (TE) = 36 ms, echo train length (ETL) = 8, slice thickness = 500 μm, acquisition matrix = 384 × 384, and number of averages = 4. Voxel size was . These scans were then repeated in the sagittal projection.
Once these scans were acquired, one 1-mm-thick axial slice was placed in the center of the knee joint in order to scan the articular cartilage with a series of multislice multiecho (MSME) T2-weighted pulse sequences. Scan parameters were TR = 3500 ms, ETL = 1, acquisition matrix = 192 × 256, with voxel size of 156.25 × 156.25 × 1000 μm. 16 different TE were used: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, and 160 ms.
Total scan time was roughly 1 hour per sample. Scan sequences were based on previously published reports .
Image processing and analysis
where signal intensity S is defined as a function of echo time TE, and is related to the spin density M0 and the transverse relaxation time T2. The equation was solved for the mean T2 value over the 16-image stack by using least-squares single-exponential curve-fitting, with initial guesses of M0 = signal intensity at 10 ms and T2 = 30 ms, in order to guarantee rapid convergence . OsiriX then generated a T2 fit map graph with regression line and values for T2 and M0.
After radiological examination, knee samples were fixed in 10% neutral buffered formalin for two weeks, decalcified with RDO Rapid Decalcifier (Apex Engineering Products, Aurora, Illinois) for three days, circulated, and embedded in paraffin. Five-micron sagittal sections were acquired from the middle of the knee joint. Histomorphological staining was performed as previously described : slides were deparaffinized, rehydrated, stained with Safranin O (which colors proteoglycans red), counterstained with Fast Green FCF (which colors proteins green) and with Weigert's hematoxylin (which colors nuclei black), dehydrated, cleared, and mounted in Permount. Representative digital photomicrographs were acquired with a Leica DM R microscope (Wetzlar, Germany) fitted with a QImaging Retiga 1300 B camera (Surrey, British Columbia), controlled by QCapture software (version 2.95.0). Images were captured at 50 × (low-power) or 200 × (high-power) magnification, and subsequently color-matched and balanced using Adobe Photoshop CS3.
Four slides from each condition were scored by two blinded examiners using the Osteoarthritis Research Society International (OARSI) histopathology assessment system , which assigns numeric values to grade, or depth progression into cartilage (0-6), and stage, or extent of joint involvement (0-4); multiplying grade and stage yields a total OA score with a maximum value of 24. Scores were averaged in between the two examiners; inter-examiner variation was within ± 5%.
OARSI scores were statistically analyzed by one-way ANOVA, with total scores taken as the dependent variable and treatment group as the independent variable. Pairwise post-hoc testing with Holm correction was used to establish significance in between treatment groups. P-values less than 0.05 were considered statistically significant. Analyses were conducted using R (version 2.12.1) .
Additional 5-micron sections were processed for immunohistochemical detection of type II collagen. Slides were deparaffinized, rehydrated, and washed in phosphate-buffered saline (PBS). Sections were incubated in 2 mg/mL hyaluronidase for 30 minutes at 37°C, followed by permeabilization with 0.3% Triton X-100 for 30 minutes at room temperature. Endogenous peroxidase activity was then quenched with 2% hydrogen peroxide in PBS for 15 minutes. Sections were blocked with normal mouse serum (Vector Laboratories, Burlingame, California) for 1 hour, after which they were blotted and then incubated with monoclonal mouse anti-rat type II collagen (clone SPM239; Spring Bioscience, Pleasanton, California) for 18 hours at 4°C. Sections were then washed in PBS, incubated with biotinylated anti-mouse IgG (Vector) for 1 hour at room temperature, and stained using the avidin-biotin complex method (Vectastain ABC kit; Vector). Color was developed using 3,3'-diaminobenzidine (Dako Diagnostics, Mississauga, Ontario) containing hydrogen peroxide. Slides were counterstained with Harris modified hematoxylin, dehydrated, cleared, mounted, and examined by light microscopy as described above.
ETA and BKB1 antagonism ameliorates OA nociceptive tolerance
Static weight bearing post-hoc tests
None/Saline vs Sham/Saline
Sham/Saline vs ACLT/Saline
ACLT/BQ-123 vs ACLT/Saline
ACLT/R-954 vs ACLT/Saline
ACLT/BQ-123+R-954 vs ACLT/Saline
Antagonist treatment improved radiological indices of OA
OA radiological scores
Mean total radiological score
Cartilage mean T2 values
Mean T 2 value (ms)
Antagonism protects joint histomorphology
OARSI histopathology scores
Mean OARSI score
Type II collagen, the major structural collagen of cartilage, was detected by immunohistochemistry (Figure 4 right column). ACLT saline-treated animals displayed significant losses of articular surface type II collagen (Figure 4i) with some localization in the deep zones of cartilage, reflecting cartilage remodeling processes. Animals treated with ETA and/or BKB1 antagonists (Figure 4l,o,r) displayed varying degrees of protection, retaining some type II collagen staining. Neither sham surgery nor intra-articular injection of saline vehicle negatively affected joint type II collagen expression (Figures 4c and 4f): protein was localized in the superficial zone of articular cartilage, indicating functional joint tissue.
In the present study, we investigated whether antagonism of ETA and/or BKB1 could slow and/or prevent osteoarthritic cartilage degradation and joint nociception in a rat surgical model of OA. We provide several lines of evidence that suggest protective effects of ETA and/or BKB1 antagonism in vivo: BKB1 antagonist treatment improved hind limb nociceptive tolerance, and both ETA and/or BKB1 antagonist treatment ameliorated radiological indices of disease, and protected articular cartilage and bone histomorphometry.
The most interesting finding of our study is that nociceptive tolerance was augmented in our model after BKB1 antagonist treatment, with faster post-operative recovery than vehicle-treated controls. These results are consistent with other reports , where local treatment with BKB1 receptor antagonists reduced overt acute joint nociception. We extend this finding to the dual antagonist treatment approach to male animals in a model of chronic pain, as well as relating it to measures of joint integrity by radiology and histology. Low-grade joint pain is the most common reason for patient presentation, and is often the major debilitating factor in OA cases [47, 48]. Thus, the anti-nociceptive effects of BKB1 antagonism make this treatment strategy attractive. Surprisingly, single ETA antagonism was relatively ineffective at diminishing joint nociception in our model. This finding, which contradicts our initial hypothesis, suggests that ETA potentiation of ET-1-induced joint pain  may not be direct, especially in a chronic inflammatory state.
We found that single and dual ETA/BKB1 antagonist treatments decreased radiological disease indices, in terms of osteophyte formation, cartilage thinning, and subchondral bone remodeling, with dual antagonism being most protective. As well, cartilage T2, increased in ACLT animals, was decreased by antagonist treatment, which indicates a cartilage-preserving effect. Longer cartilage transverse relaxation times are an indicator of cartilage degradation; this MR parameter is indicative of cartilage composition and integrity [49–51]. Radiographic evidence is the main criterion for OA diagnosis and progression [52, 53]. The most common clinical diagnostic test is via X-ray of the affected joint: joint space narrowing as measured on X-ray is often used as a longitudinal marker of disease evolution. It is difficult to directly compare radiological parameters between human and rat knees due to the quadrupedal nature of the animal and the markedly different radiological anatomy that this entails . However, we were able to detect radiological evidence of OA progression in ACLT animals, as has been described in similar studies [44, 55].
OA induction in rat knees leads to a rapid decrease in cartilage proteoglycan staining, along with articular surface disruption and osteophyte formation [26, 27]. ETA/BKB1 antagonist treatment protected the proteoglycan content of the joint and preserved articular surface integrity. Furthermore, there was some protection of type II collagen protein expression. This allowed the joint cartilage to retain its normal biophysical properties, as cartilage proteoglycans are responsible, along with collagen, for retaining water in the tissue, which provides spring and resilience [56, 57]. These findings likely suggest that the protection of cartilage proteoglycans, collagens, and articular surface histomorphology may be one explanation for the increased pain tolerance observed in antagonist-treated animals; our results concur with those of other reports, which correlated the preservation of articular cartilage proteoglycan staining with pain tolerance behavior .
The ET-1 and BK systems are involved in joint tissue inflammation and nociception, concomitant with pro-inflammatory mediators. However, exploration of potential therapeutic targets in these systems has been modest: the main classes of disease-modifying osteoarthritis drugs currently in development include cytokine and matrix metalloproteinase inhibitors, anti-resorptives, and growth factors . To our knowledge, the only clinical trial of a drug targeting a vasoactive factor in OA is the bradykinin receptor B2 antagonist Icatibant, by Sanofi-Aventis . This drug is no longer in clinical development , due to mixed results: while it provided local analgesia in knee OA, no anti-inflammatory effect could be detected . Our results suggest that ETA and BKB1 represent novel therapeutic targets in OA. Specific receptor antagonists could be tested in clinical trials for OA pain and tissue damage.
Using a rat surgically induced model of OA, we demonstrated that local treatment with specific peptide antagonists of ETA and/or BKB1 may slow or stabilize the development of radiomorphological and histomorphological changes occurring in OA pathogenesis. Furthermore, we showed that BKB1 antagonist treatment accelerated recovery of, and improved longitudinally, nociceptive tolerance in ACLT animals. Taken together, our results indicate that blocking ETA and BKB1 improves OA prognostic indices, which implies that defective signaling might play a role in chronic OA pain. Our results also raise the possibility of targeted receptor antagonism as a relevant therapeutic option. Further studies are required to understand the mechanisms underlying the exact nature of receptor cross-regulation and synergism.
anterior cruciate ligament transection
analysis of variance
bradykinin receptor B1
bradykinin receptor B2
endothelin receptor type A
echo train length
Osteoarthritis Research Society International
rapid acquisition with relaxation enhancement
We thank Archana Sangole for valuable help with the static weight bearing apparatus design. We thank Saadallah Bouhanik of the Viscogliosi Laboratory for Molecular Genetics of Musculoskeletal Disorders (Montreal, Quebec) for providing access to and help with the Faxitron micro-X-ray system, as well as Jason Cakiroglu (MR engineer) and Barry J. Bedell (director) of the Small Animal Imaging Lab at McGill University (Montreal, Quebec) for providing access to their 7 Tesla micro-MR scanner. We thank Stéphane Faubert and Serge Nadeau for their surgical instruction, as well as Denise Carrier (director) and the staff of the Sainte-Justine Hospital Research Centre animal facility for their technical assistance. Finally, we thank Kessen Patten for his writing and editing suggestions.
This work was supported by operating grants from The Arthritis Society (William T. Holland Arthritis Research Grant, RG05/084) and the Canadian Institutes of Health Research (IMH-94011). Publication costs were payed by the Yvon Roberge publication support fund of the Faculty of Dentistry, Université de Montréal. GNK held a Sainte-Justine Hospital Foundation/Foundation of the Stars bursary.
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