META060 inhibits osteoclastogenesis and matrix metalloproteinases in vitro and reduces bone and cartilage degradation in a mouse model of rheumatoid arthritis

Objective

The multikinase inhibitor META060 has been shown to inhibit NF‐κB activation and expression of markers of inflammation. This study was undertaken to investigate the effect of META060 on biomarkers associated with bone and cartilage degradation in vitro and its antiinflammatory efficacy in vivo in both acute and chronic inflammation models.

Methods

Glycogen synthase kinase 3β (GSK3β)–dependent β‐catenin phosphorylation was evaluated in RAW 264.7 macrophages to assess kinase inhibition. The inhibition of osteoclastogenesis and tartrate‐resistant acid phosphatase (TRAP) activity was evaluated in RANKL‐treated RAW 264.7 cells. The inhibition of interleukin‐1β (IL‐1β)–mediated markers of inflammation was analyzed in human rheumatoid arthritis synovial fibroblasts (RASFs). Mice with carrageenan‐induced acute inflammation and collagen‐induced arthritis (CIA) were used to assess efficacy.

Results

META060 inhibited the activity of kinases (spleen tyrosine kinase [Syk], Bruton's tyrosine kinase [Btk], phosphatidylinositol 3‐kinase [PI 3‐kinase], and GSK3) associated with RA and inhibited β‐catenin phosphorylation. META060 inhibited osteoclastogenesis, as indicated by decreased transformation of RAW 264.7 cells to osteoclasts and reduced TRAP activity, and inhibited IL‐1β–activated prostaglandin E₂, matrix metalloproteinase 3, IL‐6, IL‐8, and monocyte chemotactic protein 1 in RASFs. In mice with acute inflammation, oral administration of META060 reduced paw swelling similar to the effect of aspirin. In mice with CIA, META060 significantly reduced the arthritis index and decreased bone, joint, and cartilage degradation. Serum IL‐6 concentrations in these mice were inhibited in a dose‐dependent manner.

Conclusion

Our findings indicate that META060 reduces swelling in a model of acute inflammation and inhibits bone and cartilage destruction in a model of chronic inflammation. Its efficacy is associated with the inhibition of multiple protein kinases, including Syk, Btk, PI 3‐kinase, and GSK3. These results warrant further clinical testing of META060 for its therapeutic potential in the treatment of inflammatory diseases.

     

Ascorbic acid increases the severity of spontaneous knee osteoarthritis in a guinea pig model

OBJECTIVE: To determine whether ascorbic acid might be of benefit for the treatment of spontaneous osteoarthritis (OA) when administered over a long period of time. METHODS: We investigated the effects of 8 months' exposure to low, medium, and high doses of ascorbic acid on the in vivo development of histologic knee OA in the male Hartley guinea pig. The low dose represented the minimum amount needed to prevent scurvy. The medium dose was the amount present in standard laboratory guinea pig chow and resulted in plasma levels comparable with those achieved in a person consuming 200 mg/day (5 fruits and vegetables daily). The high dose was the amount shown in a previous study of the guinea pig to slow the progression of surgically induced OA. RESULTS: We found an association between ascorbic acid supplementation and increased cartilage collagen content but, in contrast to findings in a previous study of surgically induced OA in the guinea pig, ascorbic acid worsened the severity of spontaneous OA. Active transforming growth factor beta (TGF beta) was expressed in marginal osteophytes, whose size and number were significantly increased with increasing intake of ascorbic acid. Synovial fluid levels of cartilage oligomeric matrix protein, a biomarker of cartilage turnover, corroborated the histologic findings. CONCLUSION: Ascorbic acid has been shown to activate latent TGF beta. Prolonged intraarticular exposure to TGF beta has been shown to cause OA-like changes. We found expression of active TGF beta in osteophytes, a prominent feature of the joint histology seen in association with ascorbic acid treatment. Thus, the deleterious effects of prolonged ascorbic acid exposure may be mediated in part by TGF beta. This worsening of OA with ascorbic acid supplementation suggests that ascorbic acid intake should not be supplemented above the currently recommended dietary allowance (90 mg/day for men and 75 mg/day for women).     

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.     

Alterations in a cartilage matrix glycoprotein in canine osteoarthritis

A number of biochemical abnormalities have been described in osteoarthritic (OA) cartilage, but little study has been devoted to changes in the noncollagenous, nonproteoglycan proteins of cartilage in this condition. Using a canine model of OA produced by transection of the anterior cruciate ligament of the knee, we have demonstrated that distinct alterations occur in a 550,000-dalton cartilage matrix glycoprotein in OA canine cartilage. This protein is a major protein constituent of normal articular cartilage. Fragments which are immunologically cross-reactive with the 550,000-dalton protein were more abundant in OA cartilage than in normal articular cartilage. Immunofluorescence studies revealed that staining with specific antiserum to the protein was absent in OA cartilage. This was the only noncollagenous, nonproteoglycan protein noted to undergo significant changes in this model of OA.     

Effect of resveratrol on cartilage protection and apoptosis inhibition in experimental osteoarthritis of rabbit

To observe the effect of resveratol on cartilage,chondrocyte apoptosis, and nitric oxide in experimental osteoarthritis (OA) of rabbit and to study the mechanism of resveratol in the treatment of osteoarthritis. Thirty New Zealand rabbits were randomly divided into 5 groups: group A (normal control group), group B (model control group), group C (resveratrol intervention high-dosage group), group D (resveratrol intervention middle dosage group), and group E (resveratrol intervention low-dosage group). The model of OA of the knee was established using Hulth technique in groups B, C, D, and E. After 4?weeks, group A and group B rabbits were administered daily a knees injection of dimethylsulfoxide (DMSO), whereas groups C, D, and E were administered daily a knees injection of resveratrol in DMSO in different dosages for 2?weeks. Daily dosage for rabbits of groups C, D, and E was fixed at 50, 20, and 10?μmol/kg, respectively. Then, the rabbits were killed, and the lateral cartilage sections of right femoral medial condyle were evaluated using a histological scoring system (H&E and safranin-O staining) and analyzed by TUNEL for apoptosis. Nitric oxide (NO) in synovial fluid was measured by nitrate reduction method. Histological evaluation of cartilage tissue revealed a significantly reduced cartilage destruction; the evaluation also revealed the loss of matrix proteoglycan content in cartilage in resveratrol intervention groups compared to the model control. Resveratrol reduced the apoptosis rate of chondrocyte and level of NO in the synovial fluid. In the above experiments of OA rabbits, the protective effects of resveratrol were enhanced with increased resveratrol dosage. Resveratrol controls the progression of experimental OA. One of the mechanism(s) responsible for this effect would include lowering of the apoptosis rate of chondrocyte and reducing the production of NO in experimental OA.     

Differential expression patterns of matrix metalloproteinases and their inhibitors during development of osteoarthritis in a transgenic mouse model

Objective: To characterise the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during degeneration of articular cartilage in a transgenic Del1 mouse model for osteoarthritis.

Methods: Northern analysis was used to measure mRNA levels of MMP-2, -3, -8, -9, -13, and -14, and TIMP-1, -2, and -3 in total RNA extracted from knee joints of transgenic Del1 mice, harbouring a 15 amino acid deletion in the triple helical domain of the α1(II) collagen chain, using their non-transgenic littermates as controls. Immunohistochemistry was used to study the presence of cleavage products (neoepitopes) of type II collagen, and the distribution of MMP-13 and TIMP-1 in degenerating cartilage.

Results: Each of the MMP and TIMP mRNAs analysed exhibited distinct expression patterns during development and osteoarthritic degeneration of the knee joint. The most striking change was up regulation of MMP-13 mRNA expression in the knee joints of Del1 mice at the onset of cartilage degeneration. However, the strongest immunostaining for MMP-13 and its inhibitor TIMP-1 was not seen in the degenerating articular cartilage but in synovial tissue, deep calcified cartilage, and subchondral bone. The localisation of type II collagen neoepitopes in chondrocytes and their pericellular matrix followed a similar pattern; they were not seen in cartilage fibrillations, but in adjacent unaffected cartilage.

Conclusion: The primary localisation of MMP-13 and TIMP-1 in hyperplastic synovial tissue, subchondral bone, and calcified cartilage suggests that up regulation of MMP-13 expression during early degeneration of articular cartilage is a secondary response to cartilage erosion. This interpretation is supported by the distribution of type II collagen neoepitopes. Synovial production of MMP-13 may be related to removal of tissue debris released from articular cartilage. In the deep calcified cartilage and adjacent subchondral bone, MMP-13 probably participates in tissue remodelling.

     

Effect of montelukast in a guinea pig model of cough variant asthma

Cough variant asthma is known as a major cause of chronic cough. Fundamental features of cough variant asthma are prolonged non-productive cough responding to bronchodilator therapy, no history of wheezing or dyspnea attack, normal cough sensitivity and slightly increased bronchial responsiveness. Recently, we reported the animal model of cough variant asthma. The aim of this study was to clarify the involvement of cysteinyl leukotrienes (cysLTs) in this model by using a specific leukotriene receptor antagonist, montelukast. Cough number and specific airway resistance (sRaw) were measured during the antigen inhalation (1.5 min) and following 18.5 min, which was carried out 72 h after the first antigen inhalation in actively sensitized guinea pigs, and then total cell number and cell differentials in bronchoalveolar lavage fluid (BALF) were measured. Montelukast significantly reduced the antigen re-inhalation-induced cough, increase in sRaw, and increase in total cell number in BALF. In conclusion, cysLTs may play an important part in antigen-induced cough associated with bronchoconstriction and airway inflammation in cough variant asthma.     

Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis

Objective

Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA).

Methods

Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low‐density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real‐time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis/N‐terminal sequencing, while its ability to activate proteinase‐activated receptor 2 (PAR‐2) was determined using a synovial perfusion assay in mice.

Results

Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP‐1 and processed proMMP‐3 to its fully active form. Exogenous matriptase significantly enhanced cytokine‐stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase‐dependent. Matriptase also induced MMP‐1, MMP‐3, and MMP‐13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR‐2, and we demonstrated that matriptase‐dependent enhancement of collagenolysis from OA cartilage is blocked by PAR‐2 inhibition.

Conclusion

Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR‐2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.

     

Effect of pregnancy on gastric motility in vivo and in vitro in the guinea pig

Experiments were designed to examine the effects of pregnancy on gastric emptying and on the in vitro contractile response of gastric smooth muscle to cholinergic stimulation. Gastric emptying of a radio-labeled liquid marker (51Cr in saline) was determined at regular intervals (5, 15, 30, and 60 min) in nonpregnant and pregnant (third trimester) guinea pigs. Gastric emptying after 30 min also was determined for animals 1 and 4 days postpartum. The in vitro contractile responses of muscle strips from the fundus, corpus, and antrum of nonpregnant and pregnant animals after stimulation with acetylcholine were analyzed with respect to the maximal force developed and the pD2 value (negative logarithm of the dose that gives a one-half maximal response). The results can be summarized as follows. Pregnancy was characterized by an increase in the amount of marker retained within the stomach at each time point. For nonpregnant animals the time to one-half emptying was approximately 10 min; in pregnant animals this was prolonged to 30 min. Gastric emptying was reduced in animals 1-day postpartum but returned to normal by 4 days after delivery. In contrast, pregnancy had no effect on the maximal contractile response to acetylcholine of muscle strips from the orad and caudad stomach or on their respective pD2 values. The results suggest that pregnancy is associated with decreased gastric emptying of liquids in the guinea pig, but that the delay cannot be attributed to alterations in the maximal force-generating capacity of gastric smooth muscle.     

Role of synovial membrane inflammation in cartilage matrix breakdown in the Pond-Nuki dog model of osteoarthritis

Using the Pond-Nuki procedure, osteoarthritis was induced in 26 crossbred dogs. Their right knees were subjected to anterior cruciate ligament sectioning, and their left knees received a sham operation. The unoperated knees of 7 additional dogs served as controls. Cartilage and synovial membranes were excised 2, 4, and 8 weeks after surgery. Collagenolytic activity, determined by direct tissue assay, was higher at all times tested in osteoarthritic cartilage and synovia than in sham operated and control specimens. The increased collagenolytic activity of the cartilage did not correlate with the collagenolytic activity of the synovium, but it did correlate (r = 0.57) with the degree of synovial inflammation, which was graded histologically. Treatment for 4 weeks with prednisone (0.20-0.25 mg/kg/day) blocked the increased collagenolytic activity of the cartilage. Our results indicate that stimulating factors may, but collagenolytic enzymes probably to not, diffuse from the synovium to the cartilage and modulate tissue breakdown. Prednisone may also suppress cartilage breakdown by either acting at the level of the synthesis or by acting on the release/action of the stimulating factors.     

Collagenolytic activity and collagen matrix breakdown of the articular cartilage in the Pond-Nuki dog model of osteoarthritis

Recent reports on the Pond-Nuki model of osteoarthritis in the dog have provided evidence for partial disruption of the collagen network. The possibility of collagenase involvement in these localized changes was studied. Animals were killed 2, 4, 8, and 12 weeks after surgery. The left knee served as a sham-operated control. Cartilages from femoral condyles were processed for light and electron microscopy and assayed for collagenolytic activity by a direct tissue assay, based on the measurement of digestion of endogenous cartilage collagen. Animals killed at 2 and 4 weeks showed fibrillation and mild erosion of femoral condyles, which usually progressed to ulceration by 8 and 12 weeks. Electron microscopy demonstrated fiber disruption of the mid-zone perilacunar collagen as early as 2 weeks after the operation. Total collagenolytic activity, measured after activation by aminophenylmercuric acetate, was significantly higher in decreased cartilage than in controls of 2, 4, and 8 weeks; the peak value was at 4 weeks. Collagenase was shown, by its specific action on type I collagen, to be present at 2 and 4 weeks; however, other metalloproteases may also contribute to the digestion. The correlation between increased collagenolytic activity and the early osteoarthritic changes in cartilage suggests a role of this enzyme activity in the disease process.     

Effect of gastrin-releasing peptide (GRP) on guinea pig gallbladder contrction in vitro

Few studies have reported the effects of gastrin-releasing peptide (GRP)/bombesin on the guineas pig gallbladder, and the results are contradictory. Because such contradictory results may, in part, be due to technical factors, we investigated the effect of GRP on guinea pig gallbaladder smooth muscle, using a improved horizontal organ bath. The guinea pigs were killed and the gallbladder was removed. Four longitudinal uscle strips (2×12mm) were suspended in Krebs-Ringer solution at 37°C and aerated with 95% O₂ and 5% CO₂. The mechanical activity of the strips was recorded isotonically by displacement-voltage transducers. via L-arms, to which a piezoelectric element with a frequency of 100Hz and movement of 50μm was applied. GRP contracted gallbladder muscle strips dose dependently, but the calculated maximal response was 22.4% and 20.1% of the acetylcholine-and cholecystokinin octapeptide (CCK8)-induced responses, respectively. The GRP-induced contraction was unaffected by the muscarinic blocker, atropine, or by the CCK receptor antagonist, loxiglumide. It is concluded that GRP weakly, but apparently directly, stimulates guinea pig gallbladder contraction.     

软骨寡聚基质蛋白对骨关节炎软骨破坏早期诊断价值的研究

目的 探讨软骨寡聚基质蛋白(COMP)对骨关节炎软骨破坏早期诊断价值.方法 兔右后膝伸直位石膏管型固定法制作骨关节炎模型;形态学方法观察造模不同时期关节病理切片;免疫组织化学方法检测软骨内COMP水平;酶联免疫吸附试验(ELISA)方法检测血清COMP水平.应用t检验,Pearson相关性分析.结果 ①伸直位石膏管型制动2周,模型关节呈现早期骨关节炎改变,制动6周呈现典型的中晚期骨关节炎特征;②造模前、模型2周、模型6周血清COMP含量分别是[(3.35±0.20)、(3.64±0.18)、(3.96±0.44)μg/L,P均＜0.05];③未造模、模型2周、模型6周关节软骨内COMP表达强度分别是[(2.7±1.8)%,(5.7±0.7)%,(7.6±0.7)%,P均＜0.05];④模型2周血清COMP水平与模型2周组、模型6周组OA病理评分存在线性相关关系(r均＞0.770,P均＜0.05).结论 骨关节炎血清COMP检测对早期诊断骨关节炎软骨破坏具有重要的意义.

Abstract：

Objective To study the diagnostic value of cartilage oligomeric matrix protein for early cartilage destruction in osteoarthritis and assess its value in the prediction of the disease progression.Methods The osteoarthritis animal models were developed by immobilizing the right knees of 18 rabbits in full extension position using plaster East.Knee joint pathological changes at week 2 and 6 were examined for pathological severity evaluation of osteoarthritis.ELISA sandwich method was used to measure the levels of cartilage oligomeric matrix protein(COMP) in serum before and after modeling(at week 2 and 6 respectively) and immunohistolgy method was used to examine the levels of COMP in knee articular cartilage of osteoarthritis animal models.Correlation analysis was performed to demonstrate the relationship between the levels of COMP in the serum and the pathological severity of osteoarthritis.Pearson's test and t-test were used for correlation analysis.Results ①) Osteoarthritis animal models could be successfully developed by immobilizing the right knees of rabbits in full extension position using plaster east for 2 weeks.Early histopathological changes in the articular cartilage could be observed,At week 6,the typical histopathological characteristics could be seen.②With the extension of modeling time,serum COMP levels persistently increased.The serum COMP levels before modeling,at modeling week 2,week 6 were (3.35±0.20),(3.64±0.18),(3.96±0.44) μg/L respectively,the difference was significant (P＜0.05).③ The level of COMP in the articular cartilage of non-osteoarthritis animal models,models at week 2,week 6 were (2.7±1.8 )％ ,(5.7±0.7)％,(7.6±0.7)％ respectively (P＜0.05 for all).④ The level of COMP in the serum was linearily correlated with the pathological severity of osteoarthritis(r＞0.770 for all,and P＜0.05 for all).Conclusion Levels of COMP in the serum can help to make early diagnosis of osteoarthritis,and elevated COMP level can predict the progression of osteoarthritis.