Gene deletion of either interleukin‐1β, interleukin‐1β–converting enzyme,inducible nitric oxide synthase,or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transection of the medial collateral ligament and partial medial meniscectomy

Objective

To investigate the development of osteoarthritis (OA) after transection of the medial collateral ligament and partial medial meniscectomy in mice in which genes encoding either interleukin‐1β (IL‐1β), IL‐1β–converting enzyme (ICE), stromelysin 1, or inducible nitric oxide synthase (iNOS) were deleted.

Methods

Sectioning of the medial collateral ligament and partial medial meniscectomy were performed on right knee joints of wild‐type and knockout mice. Left joints served as unoperated controls. Serial histologic sections were obtained from throughout the whole joint of both knees 4 days or 1, 2, 3, or 4 weeks after surgery. Sections were graded for OA lesions on a scale of 0–6 and were assessed for breakdown of tibial cartilage matrix proteoglycan (aggrecan) and type II collagen by matrix metalloproteinases (MMPs) and aggrecanases with immunohistochemistry studies using anti‐VDIPEN, anti‐NITEGE, and Col2‐3/4C_short neoepitope antibodies. Proteoglycan depletion was assessed by Alcian blue staining and chondrocyte cell death, with the TUNEL technique.

Results

All knockout mice showed accelerated development of OA lesions in the medial tibial cartilage after surgery, compared with wild‐type mice. ICE‐, iNOS‐, and particularly IL‐1β–knockout mice developed OA lesions in the lateral cartilage of unoperated limbs. Development of focal histopathologic lesions was accompanied by increased levels of MMP‐, aggrecanase‐, and collagenase‐generated cleavage neoepitopes in areas around lesions, while nonlesional areas showed no change in immunostaining. Extensive cell death was also detected by TUNEL staining in focal areas around lesions.

Conclusion

We postulate that deletion of each of these genes, which encode molecules capable of producing degenerative changes in cartilage, leads to changes in the homeostatic controls regulating the balance between anabolism and catabolism, favoring accelerated cartilage degeneration. These observations suggest that these genes may play important regulatory roles in maintaining normal homeostasis in articular cartilage matrix turnover.

     

Pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, reduces the progression of experimental osteoarthritis in guinea pigs

OBJECTIVE: To evaluate the in vivo therapeutic effect of pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, on the development of lesions in a guinea pig model of osteoarthritis (OA), and to determine the influence of pioglitazone on the synthesis of matrix metalloproteinase 13 (MMP-13) and interleukin-1beta (IL-1beta) in articular cartilage. METHODS: The OA model was created by partial medial meniscectomy of the right knee joint. The guinea pigs were divided into 4 treatment groups: unoperated animals that received no treatment (normal), operated animals (OA guinea pigs) that received placebo, OA guinea pigs that received oral pioglitazone at 2 mg/kg/day, and OA guinea pigs that received oral pioglitazone at 20 mg/kg/day. The animals began receiving medication 1 day after surgery and were killed 4 weeks later. Macroscopic and histologic analyses were performed on the cartilage. The levels of MMP-13 and IL-1beta in OA cartilage chondrocytes were evaluated by immunohistochemistry. RESULTS: OA guinea pigs treated with the highest dosages of pioglitazone showed a significant decrease, compared with the OA placebo group, in the surface area (size) and grade (depth) of cartilage macroscopic lesions on the tibial plateaus. The histologic severity of cartilage lesions was also reduced. A significantly higher percentage of chondrocytes in the middle and deep layers stained positive for MMP-13 and IL-1beta in cartilage from placebo-treated OA guinea pigs compared with normal controls. Guinea pigs treated with the highest dosage of pioglitazone demonstrated a significant reduction in the levels of both MMP-13 and IL-1beta in OA cartilage. CONCLUSION: This is the first in vivo study demonstrating that a PPARgamma agonist, pioglitazone, could reduce the severity of experimental OA. This effect was associated with a reduction in the levels of MMP-13 and IL-1beta, which are known to play an important role in the pathophysiology of OA lesions.     

In vivo effects of interleukin-1 on articular cartilage. Prolongation of proteoglycan metabolic disturbances in old mice

We investigated the effects of intraarticular injections of interleukin-1 alpha (IL-1 alpha) into the knee joints of young (3-month-old) and old (18-month-old) C57Bl/10 mice. In this in vivo study, 35S-sulfate incorporation and release were used to compare the effects of IL-1 on patellar cartilage proteoglycan metabolism. IL-1-induced stimulation of proteoglycan degradation was confined to the first 24 hours after injection in both young and old animals, and was only slightly lower in old cartilage than in young. In old patellar cartilage, IL-1-induced suppression of proteoglycan synthesis appeared to be more prolonged. Also, the amount of time needed to restore the cartilage matrix, characterized by proteoglycan synthesis above normal levels, was longer in old animals. Histologic analysis confirmed the retarded recovery in the cartilage of old mice. Autoradiography showed that the chondrocytes of the medial side of the femorotibial area were most vulnerable to IL-1-induced suppression of proteoglycan synthesis, especially the medial tibial plateau. As with the patellar cartilage, IL-1-induced suppression of proteoglycan synthesis in this area of articular cartilage was more prolonged in old animals. Our data indicated that the impact of IL-1 on articular cartilage is higher in old mice and that, consistent with certain loci found to be at risk in experimental osteoarthritis, there are sites at which IL-1 is more likely to act.     

The in situ up-regulation of chondrocyte interleukin-1-converting enzyme and interleukin-18 levels in experimental osteoarthritis is mediated by nitric oxide

OBJECTIVE: To investigate in situ the relationship between 2 key mediators implicated in osteoarthritic (OA) cartilage: nitric oxide (NO) and interleukin-1-converting enzyme (ICE). Interleukin-18 (IL-18) was also studied and served as reference for the effects of ICE. METHODS: An OA model was created in dogs by sectioning (stab wound) the anterior cruciate ligament of the right stifle joint. Three experimental groups were studied: unoperated untreated dogs, operated untreated dogs (OA), and OA dogs treated with oral N-iminoethyl-L-lysine (L-NIL), a specific inhibitor of inducible nitric oxide synthase (iNOS) (10 mg/kg twice a day starting immediately after surgery). At 12 weeks after surgery, cartilage from the femoral condyles and tibial plateaus were processed for immunohistochemistry for ICE, IL-18, and protease inhibitor 9 (PI-9), a natural inhibitor of ICE, followed by morphometric analysis. Cartilage specimens from the femoral condyles of untreated OA dogs were dissected and incubated with specific inhibitors of different signaling pathways likely to be involved in the OA process: SB 202190 (10 microM; a p38 mitogen-activated protein kinase [MAPK] inhibitor), PD 98059 (100 microM; a MAPK kinase 1/2 [MEK-1/2] inhibitor), NS-398 (10 ng/ml; a specific cyclooxygenase 2 [COX-2] inhibitor), and L-NIL (50 microM). RESULTS: Both ICE and IL-18 were present in situ in the canine cartilage, with a significant increase in the level of these 2 proteins in OA cartilage. In contrast, the level of PI-9 was lower in OA than in normal cartilage (difference not statistically significant). Compared with untreated OA cartilage, oral treatment with L-NIL significantly decreased ICE and IL-18 levels in cartilage from the femoral condyles and tibial plateaus, to values similar to those in normal dogs. L-NIL also increased the PI-9 level in normal dogs compared with OA dogs, reaching statistical significance for femoral condyle cartilage. Interestingly, in vitro experiments demonstrated significant inhibition of ICE levels by p38, MEK-1/2, and COX-2 inhibitors, but not by the iNOS inhibitor. CONCLUSION: This study demonstrated that in situ in OA cartilage, the stimulation of chondrocytes by NO is at least partly responsible for the up-regulation of ICE and IL-18 synthesis while decreasing the level of the ICE inhibitor PI-9. The ICE level is controlled by the activation of at least 2 MAPK pathways, p38 and MEK-1/2. Interestingly, it appears that ICE synthesis is not regulated by the endogenous production of NO. These data highlight the role played by iNOS in regulating the synthesis of major catabolic factors involved in OA cartilage degradation.     

Effect of oral glucosamine on cartilage degradation in a rabbit model of osteoarthritis

OBJECTIVE: To determine whether oral glucosamine alleviates cartilage degradation in an animal model of osteoarthritis (OA). METHODS: The effect of 8 weeks of daily oral glucosamine hydrochloride on degeneration of articular cartilage was evaluated in rabbits in which anterior cruciate ligament transection (ACLT) was performed to induce OA. Animals were treated with glucosamine (n = 16) or a placebo (n = 16) and necropsied at 11 weeks. Seven unoperated rabbits served as controls. The articular cartilage was evaluated macroscopically and histologically and analyzed for total type II collagen and glycosaminoglycan (GAG) content. RESULTS: Histologic analysis revealed that loss of proteoglycan, based on Safranin O-fast green staining, was significantly reduced in the lateral tibial plateau cartilage of ACL-transected limbs in the glucosamine group compared with ACL-transected limbs in the placebo group, with a similar, but not significant, trend for the lateral femoral condylar cartilage. Likewise, macroscopic analysis of cartilage showed that the lateral tibial plateau alone had a significantly lower rate of disease in the glucosamine group, which was consistent with the results of the independent histologic assessment. However, no significant treatment effect was detected when composite histologic scores were analyzed. A significant reduction in GAG content was observed in the femoral condyles of placebo-treated ACL-transected joints, but not in the same region of glucosamine-treated ACL-transected joints, compared with their respective contralateral unoperated joints. CONCLUSION: Oral administration of glucosamine had a detectable, site-specific, partial disease-modifying effect in this model of OA. From a clinical perspective, the administration of glucosamine did not prevent fibrillation and/or erosions of the articular cartilage in all of the treated animals, and no effects were detected in the medial joint compartments.     

Pioglitazone,a peroxisome proliferator–activated receptor γ agonist,reduces the progression of experimental osteoarthritis in guinea pigs

Objective

To evaluate the in vivo therapeutic effect of pioglitazone, a peroxisome proliferator–activated receptor γ (PPARγ) agonist, on the development of lesions in a guinea pig model of osteoarthritis (OA), and to determine the influence of pioglitazone on the synthesis of matrix metalloproteinase 13 (MMP‐13) and interleukin‐1β (IL‐1β) in articular cartilage.

Methods

The OA model was created by partial medial meniscectomy of the right knee joint. The guinea pigs were divided into 4 treatment groups: unoperated animals that received no treatment (normal), operated animals (OA guinea pigs) that received placebo, OA guinea pigs that received oral pioglitazone at 2 mg/kg/day, and OA guinea pigs that received oral pioglitazone at 20 mg/kg/day. The animals began receiving medication 1 day after surgery and were killed 4 weeks later. Macroscopic and histologic analyses were performed on the cartilage. The levels of MMP‐13 and IL‐1β in OA cartilage chondrocytes were evaluated by immunohistochemistry.

Results

OA guinea pigs treated with the highest dosages of pioglitazone showed a significant decrease, compared with the OA placebo group, in the surface area (size) and grade (depth) of cartilage macroscopic lesions on the tibial plateaus. The histologic severity of cartilage lesions was also reduced. A significantly higher percentage of chondrocytes in the middle and deep layers stained positive for MMP‐13 and IL‐1β in cartilage from placebo‐treated OA guinea pigs compared with normal controls. Guinea pigs treated with the highest dosage of pioglitazone demonstrated a significant reduction in the levels of both MMP‐13 and IL‐1β in OA cartilage.

Conclusion

This is the first in vivo study demonstrating that a PPARγ agonist, pioglitazone, could reduce the severity of experimental OA. This effect was associated with a reduction in the levels of MMP‐13 and IL‐1β, which are known to play an important role in the pathophysiology of OA lesions.

     

Esculetin (dihydroxycoumarin) inhibits the production of matrix metalloproteinases in cartilage explants, and oral administration of its prodrug, CPA-926, suppresses cartilage destruction in rabbit experimental osteoarthritis

OBJECTIVE: To investigate the in vitro effects of 6,7-dihydroxycoumarin (esculetin) on the production of matrix metalloproteinases (MMP) in rabbit articular cartilage, and the in vivo effects of orally administered CPA-926, a prodrug of esculetin, on cartilage destruction in rabbit experimental osteoarthritis (OA). METHODS: In vitro studies were performed using rabbit articular cartilage explants. Esculetin 10-100 microM was added to cartilage explants in the presence or absence of interleukin 1alpha (IL-1alpha). Effects of esculetin on cartilage metabolism were assessed. Proteoglycan release into medium was determined by dye precipitation with 1,9-dimethylmethylene blue, synthesis of proMMP-1 (interstitial procollagenase) and proMMP-3 (prostromelysin 1) by Western blotting, and collagen degradation activity using FITC labeled collagen. In vivo experimental OA was induced in the knee joints of 15 Japanese adult white rabbits by partial lateral meniscectomy. Ten rabbits were orally administered 200 or 400 mg/kg/day of CPA-926 from the day of surgery for 14 days. The size of the macroscopic erosive area on the femoral condyle and tibial plateau was measured, and cartilage destruction was histologically evaluated. Collagenolytic activities in synovial fluid were measured using FITC labeled collagen as a substrate. RESULTS: In vitro, esculetin inhibited the IL-1alpha induced release of proteoglycan into the medium in a dose dependent manner. The collagenolytic activities in cartilage explant medium induced by IL-1alpha were also suppressed with the addition of 33-100 microM esculetin (p = 0.0209 at 33 and 100 microM, p = 0.0202 at 66 microM). Western blotting of cartilage explant medium showed a decrease in the levels of proMMP-1 and proMMP-3 in the medium by treatment with esculetin. In vivo: At 14 days after surgery, the femoral condyle and tibial plateau in the control group showed macroscopic erosions of cartilage. Compared with the control group, the rabbits treated with CPA-926 at the dose of 400 mg/kg exhibited reduction of the size of the erosive area on the tibial plateau (p = 0.009). Histological evaluation indicated protection against the development of destructive changes in the tibial plateau cartilage at a dose of 200 mg/kg (p = 0.0442) and 400 mg/kg (p = 0.0446) of CPA-926. CONCLUSION: These results indicate that esculetin inhibits matrix degradation in rabbit joint cartilage explants through the suppression of MMP synthesis, secretion, or activity. Prophylactic administration of its prodrug, CPA-926, appears to provide some protection against cartilage destruction in a short term rabbit experimental OA model.     

Osteoprotegerin inhibits cartilage degradation through an effect on trabecular bone in murine experimental osteoarthritis

OBJECTIVE: To characterize bone microarchitectural changes and to test the hypothesis that disrupting local cytokine equilibrium could modify cartilage degradation in a murine model of experimental osteoarthritis (OA). METHODS: Ten-week-old male C57BL/6 mice underwent medial meniscectomy of their right knees and a sham operation of their left knees. The mice received intraperitoneal injections of osteoprotegerin (OPG) (10 mg/kg), interleukin-1 receptor antagonist (IL-1Ra) (100 mg/kg), or phosphate buffered saline for 6 weeks. The microarchitecture of the trabecular bone, the OA score, and expression of ADAMTS-4 and ADAMTS-5 were assessed. Proteoglycan release was measured in cartilage explant cultures in the presence of IL-1Ra and OPG. RESULTS: In the meniscectomized knees, bone volume/tissue volume (BV/TV) was lower, whereas trabecular separation, the OA score, and aggrecanase expression were higher than in the sham-operated knees. After treatment with OPG, BV/TV was significantly increased and trabecular separation was reduced in the knees that underwent meniscectomy. The OA score and the number of ADAMTS-positive cells were significantly decreased by treatment with OPG but were not affected by IL-1Ra. Moreover, OPG did not directly reduce the release of proteoglycans from cartilage explant cultures. CONCLUSION: In an experimental model of OA, meniscectomy induced bone loss and cartilage degradation at 6 weeks. Systemic administration of OPG prevented bone and cartilage degradation in vivo but had no effect on cartilage in vitro. These data collectively indicate that bone could be a contributor in the early stages of OA pathogenesis. They further suggest that disruption of RANKL/OPG balance might result in the degradation of cartilage subjected to mechanical loading. Specific targeting of the bone cytokine network might help to prevent OA.     

Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice.

OBJECTIVE: To map aggrecan cleavage by matrix metalloproteinases (MMPs) and aggrecanases in normal murine tibial articular cartilage (CBA strain) and in the development of spontaneous osteoarthritis (OA) in the STR/ort mouse and to assess the influence of sex hormone status on these conditions in gonadectomized STR/ort mice. METHODS: The distributions of neoepitopes of aggrecan generated by MMP (VDIPEN) and aggrecanase (NITEGE) cleavage were investigated by immunohistochemistry. RESULTS: VDIPEN neoepitope was detected mainly in the pericellular matrix of deep-zone chondrocytes in normal tibial cartilage from STR/ort and CBA mice. In early OA, VDIPEN immunostaining also localized to the pericellular matrix of chondrocytes at the site of the lesion. With increasing severity of OA lesions, VDIPEN immunostaining was also detected in the interterritorial matrix, close to the site of the lesion. In contrast, NITEGE mapped most strongly to the pericellular matrix of upper-zone chondrocytes in normal tibial cartilage. As with VDIPEN, NITEGE was strongly expressed in the pericellular matrix at the site of early OA lesions. With advancing OA, NITEGE colocalized with VDIPEN in both the pericellular and interterritorial matrices of chondrocytes adjacent to OA lesions and in those of the deep zones. Hormone status did not appear to influence the development of OA or the distribution of aggrecan neoepitopes in STR/ort mice. CONCLUSION: MMP- and aggrecanase-generated neoepitopes map predominantly to different regions in normal murine tibial cartilage. However, both groups of enzymes generate increased amounts of neoepitopes in pericellular and interterritorial matrix adjacent to histopathologic lesions of OA. Aggrecan degradation and the development of OA appear to be independent of sex hormone status in this model.     

Serum levels of resistin and interleukin-17 are associated with increased cartilage defects and bone marrow lesions in patients with knee osteoarthritis

Objectives: To investigate cross-sectional associations between serum levels of resistin and interleukin-17 (IL-17) and cartilage defects and bone marrow lesions (BMLs) in patients with knee symptomatic osteoarthritis (OA).

Methods: One hundred and ninety-four consecutively-selected patients with knee symptomatic OA (mean 55.4 years, range 34–74, 87% females) were included in Anhui Osteoarthritis (AHOA) Study. Knee cartilage defects and BMLs were determined at different sites using T2-weighted fat-suppressed fast spin echo MRI. Serum resistin, IL-17, and high-sensitivity C-reactive protein (hs-CRP) levels were measured using ELISA.

Results: In multivariable analyses, serum resistin was positively associated with cartilage defects at lateral femoral, lateral tibial, and medial tibial (all p?p?2.45?pg/ml), IL-17 was positively and significantly associated with cartilage defect score at nearly all sites (ORs: 1.33–1.44, all p?p?Conclusions: Serum levels of resistin were positively and independently associated with cartilage defects and BMLs in patients with knee OA. Serum IL-17 was significantly associated with cartilage defects and BMLs in patients with an increased inflammatory status. These suggest that metabolic and inflammatory mechanisms may have a role to play in knee OA.     

Risk factors for symptomatic knee osteoarthritis fifteen to twenty-two years after meniscectomy

OBJECTIVE: To evaluate the influence of age, sex, body mass index (BMI), extent of meniscal resection, cartilage status, and knee load on the development of radiographically evident osteoarthritis (OA) of the knee and knee symptoms after meniscal resection. METHODS: We evaluated 317 patients with no cruciate ligament injury (mean +/- SD age 54 +/- 11 years) who had undergone meniscal resection 15-22 years earlier (followup rate 70%), with radiographic and clinical examination. The Knee injury and Osteoarthritis Outcome Score was used to quantify knee-related symptoms. Sixty-eight unoperated subjects identified from national population records were included as a reference group. RESULTS: Symptomatic radiographic OA (corresponding to Kellgren/Lawrence grade > or =2) was present in 83 of 305 operated knees (27%) and 7 of 68 control knees (10%) (relative risk 2.6, 95% confidence interval [95% CI] 1.3-6.1). Patients who had undergone total meniscectomy and subjects with obesity (BMI > or =30) had a greater likelihood of tibiofemoral radiographic OA than those who had undergone partial meniscal resection and those with a BMI <25, respectively. Furthermore, degenerative meniscal tear, intraoperative cartilage changes, and lateral meniscectomy were associated with radiographic OA more frequently than were longitudinal tear, absence of cartilage changes, and medial meniscectomy, respectively. Symptomatic tibiofemoral or patellofemoral radiographic OA was associated with obesity, female sex, and degenerative meniscal tear. CONCLUSION: Contributing risk factors for OA development after meniscal resection are similar to risk factors for common knee OA. Systemic factors and local biomechanical factors interact. Obesity, female sex, and preexisting early-stage OA are features associated with poor self-reported and radiographic outcome. Partial meniscal resection is associated with less radiographic OA over time than is total meniscectomy.     

Prostromelysin and procollagenase genes are differentially UP-regulated in chondrocytes from the knees of rabbits with experimental osteoarthritis

Objective. To determine the relative expressions of matrix metalloprotease (MMP) genes pro-MMP1 and pro-MMP3 in the cartilage of rabbits with experimentally induced osteoarthritis (OA), and to assess the role of the chondrocyte in this process. Methods. OA was induced in rabbits after partial medial meniscectomy. Rabbits were killed at 4 weeks or 8 weeks, and total cellular RNA was prepared from cartilage and probed by Northern blotting with pro-MMP ³²P-labeled complementary DNA. Monolayer chondrocytes were used to assess MMP-inducing activity of chondrocyte factor(s). Results. Pro-MMP messenger RNAs (mRNAs) were up-regulated in experimental OA cartilage; pro-MMP3 mRNA expression exceeded that of pro-MMP1. Conditioned medium from OA-derived chondrocytes up-regulated pro-MMP mRNAs in normal chondrocytes. Conclusion. Up-regulation of MMP genes in this OA model may contribute to cartilage degradation. Chondrocytes up-regulate MMP genes via an autocrine pathway.     

Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice

Objective

To map aggrecan cleavage by matrix metalloproteinases (MMPs) and aggrecanases in normal murine tibial articular cartilage (CBA strain) and in the development of spontaneous osteoarthritis (OA) in the STR/ort mouse and to assess the influence of sex hormone status on these conditions in gonadectomized STR/ort mice.

Methods

The distributions of neoepitopes of aggrecan generated by MMP (VDIPEN) and aggrecanase (NITEGE) cleavage were investigated by immunohistochemistry.

Results

VDIPEN neoepitope was detected mainly in the pericellular matrix of deep‐zone chondrocytes in normal tibial cartilage from STR/ort and CBA mice. In early OA, VDIPEN immunostaining also localized to the pericellular matrix of chondrocytes at the site of the lesion. With increasing severity of OA lesions, VDIPEN immunostaining was also detected in the interterritorial matrix, close to the site of the lesion. In contrast, NITEGE mapped most strongly to the pericellular matrix of upper‐zone chondrocytes in normal tibial cartilage. As with VDIPEN, NITEGE was strongly expressed in the pericellular matrix at the site of early OA lesions. With advancing OA, NITEGE colocalized with VDIPEN in both the pericellular and interterritorial matrices of chondrocytes adjacent to OA lesions and in those of the deep zones. Hormone status did not appear to influence the development of OA or the distribution of aggrecan neoepitopes in STR/ort mice.

Conclusion

MMP‐ and aggrecanase‐generated neoepitopes map predominantly to different regions in normal murine tibial cartilage. However, both groups of enzymes generate increased amounts of neoepitopes in pericellular and interterritorial matrix adjacent to histopathologic lesions of OA. Aggrecan degradation and the development of OA appear to be independent of sex hormone status in this model.

     

Inverse relationship between matrix remodeling and lipid metabolism during osteoarthritis progression in the STR/Ort mouse

OBJECTIVE: The biologic changes associated with osteoarthritis (OA) are incompletely understood. The aim of this study was to elucidate the molecular mechanisms underlying OA progression in an STR/Ort murine model of spontaneous disease. METHODS: Global patterns of gene expression were assessed using microarray analysis of articular cartilage/subchondral bone from the tibial plateaus of STR/Ort mice at 3, 9, and 12 months of age. The age-dependent severity of osteophyte formation and extent of cartilage damage were determined in the corresponding femurs using microfocal computed tomography and the Mankin histologic scoring system. Pathway analysis was used to identify the functions of genes associated with OA progression, and changes in gene expression were confirmed using immunohistochemistry. RESULTS: Six hundred twenty-one genes were associated with both osteophyte formation and cartilage damage in the STR/Ort joints. Genes involved in the development/function of connective tissue and in lipid metabolism were most significantly enriched and regulated during disease progression. Genes directly interacting with peroxisome proliferator-activated receptor alpha (PPARalpha)/PPARgamma were down-regulated, whereas those genes involved with connective tissue remodeling were up-regulated during disease progression. Associations of down-regulation of myotubularin-related phosphatase 1 (a phosphoinositide 3-phosphatase involved in lipid signaling) and up-regulation of biglycan (a member of the small leucine-rich protein family known to modulate osteoblast differentiation and matrix mineralization) with OA progression were confirmed by immunohistochemistry. CONCLUSION: Since adipogenesis and osteogenesis are inversely related in the developing skeletal tissue, these results suggest that a shift in the differentiation of mesenchymal cells from adipogenesis toward osteogenesis is a component of the OA pathophysiologic processes occurring in the tibial plateau joints of STR/Ort mice.     

Induction of cartilage damage by overexpression of T cell interleukin-17A in experimental arthritis in mice deficient in interleukin-1

OBJECTIVE: To examine the capacity of T cell interleukin-17A (IL-17A; referred to hereinafter as IL-17) to induce cartilage damage during experimental arthritis in the absence of IL-1. METHODS: Local IL-17 gene transfer was performed in the knee joint of IL-1-deficient mice and wild-type controls during streptococcal cell wall (SCW)-induced arthritis. Knee joints were isolated at various time points for histologic analysis of cartilage proteoglycan (PG) depletion. Expression of messenger RNA for inducible nitric oxide synthase, matrix metalloproteinases (MMPs) 3, 9, and 13, and ADAMTS-4 was determined by quantitative polymerase chain reaction analysis. VDIPEN staining was analyzed to study MMP-mediated cartilage damage. In addition, systemic anti-IL-1alpha/beta antibody treatment was performed in mice immunized with type II collagen and injected locally with an adenoviral vector expressing IL-17 or with control adenovirus. Knee joints were isolated and analyzed for cartilage PG depletion, chondrocyte death, and cartilage surface erosion. RESULTS: During SCW-induced arthritis, local T cell IL-17 gene transfer turned this acute, macrophage-driven joint inflammation into a severe, chronic arthritis accompanied by aggravated cartilage damage. Of high interest, the IL-1 dependency of cartilage PG depletion was fully abrogated when IL-17 was locally overexpressed in the joint. Moreover, local IL-17 gene transfer increased MMP expression without the need for IL-1, although IL-1 remained essential for part of the cartilage VDIPEN expression. Furthermore, when IL-17 was overexpressed in the knee joints of mice with collagen-induced arthritis, anti-IL-1 treatment did not reduce the degree of chondrocyte death or cartilage surface erosion. CONCLUSION: These data show the capacity of IL-17 to replace the catabolic function of IL-1 in cartilage damage during experimental arthritis.     

Morphologic characteristics of developing osteoarthrotic lesions in the knee cartilage of STR/IN mice

The development of osteoarthrotic cartilage lesions in the knee joints of male STR/IN mice was studied with respect to their histologic appearance and their various localizations in the joint. Spontaneous articular cartilage degeneration on the medial portion of the tibial plateau was considered to be the initial event. Continued loss of cartilage subsequently led to a pronounced instability of the knee joint, with a varus deformity. This was followed by medial patellar luxation with corresponding osteoarthrotic lesions at the facies patellaris femoris. The most marked osteoarthrotic cartilage degeneration developed on the medial tibial condyle and at the facies patellaris femoris of the femoropatellar joint. Histologic examination of the osteoarthrotic defects in these two regions revealed distinct morphologic differences with respect to formation of chondrocyte clusters, tendency to regeneration, and proliferation reactions. Lectin binding experiments in normal articular cartilage revealed regional differences regarding the presence or absence of keratan sulfate in the extracellular matrix. The lack of keratan sulfate in tibial cartilage might reflect its tendency to degenerate spontaneously. It is therefore suggested that male STR/IN mice are particularly useful for studying two different types of osteoarthrosis, one due to a biomechanically induced instability (patellar luxation) and one due to biochemical changes (absence of keratan sulfate) of still unknown pathogenesis.     

Accelerated, aging-dependent development of osteoarthritis in alpha1 integrin-deficient mice

OBJECTIVE: Cell-matrix interactions regulate chondrocyte differentiation and survival. The alpha1beta1 integrin is a major collagen receptor that is expressed on chondrocytes. Mice with targeted inactivation of the integrin alpha1 gene (alpha1-KO mice) provide a model that can be used to address the role of cell-matrix interactions in cartilage homeostasis and osteoarthritis (OA) pathogenesis. METHODS: Knee joints from alpha1-KO and wild-type (WT) BALB/c mice were harvested at ages 4-15 months. Knee joint sections were examined for inflammation, cartilage degradation, and loss of glycosaminoglycans (by Safranin O staining). Immunohistochemistry was performed to detect the distribution of alpha1 integrin, matrix metalloproteinases (MMPs), and chondrocyte apoptosis. RESULTS: In WT mice, the alpha1 integrin subunit was detected in hypertrophic chondrocytes in the growth plate and in a subpopulation of cells in the deep zone of articular cartilage. There was a marked increase in alpha1-positive chondrocytes in the superficial and upper mid-zones in OA-affected areas in joints from old WT mice. The alpha1-KO mice showed more severe cartilage degradation, glycosaminoglycan depletion, and synovial hyperplasia as compared with the WT mice. MMP-2 and MMP-3 expression was increased in the OA-affected areas. In cartilage from alpha1-KO mice, the cellularity was reduced and the frequency of apoptotic cells was increased. These results suggest that the alpha1 integrin subunit is involved in the early remodeling process in OA cartilage. CONCLUSION: Deficiency in the alpha1 integrin subunit is associated with an earlier deregulation of cartilage homeostasis and an accelerated, aging-dependent development of OA.     

Pathogenesis of osteoarthritis-like changes in the joints of mice deficient in type IX collagen

OBJECTIVE: To examine the pathogenetic mechanisms of osteoarthritis (OA)-like changes in Col9a1-/- mice, which are deficient in type IX collagen. METHODS: Knee joints and temporomandibular joints (TMJs) from Col9a1-/- mice and their wild-type (Col9a1+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP-3) and MMP-13, degraded type II collagen, and the discoidin domain receptor 2 (DDR-2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle. RESULTS: Histologic analysis showed age-dependent OA-like changes in the knee and TMJs of Col9a1-/- mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP-13 and DDR-2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1-/- mice than in their wild-type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1-/- mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1-/- mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites. CONCLUSION: Lack of type IX collagen in Col9a1-/- mice results in age-dependent OA-like changes in the knee joints and TMJs.