Prothrombin gene expression in articular cartilage with a putative role in cartilage degeneration secondary to joint immobility

OBJECTIVE: To test the hypothesis that thrombin is expressed by chondrocytes from human and animal articular cartilage and to monitor its levels of expression during cartilage degeneration induced by joint immobility in a rat model. METHODS: Rat knees were immobilized for periods of 2 or 4 weeks, after which the articular cartilage was harvested, total RNA extracted, and the differential display (ddPCR) protocol applied to identify differentially expressed genes. One differentially expressed fragment showed 100% homology with the prothrombin gene. Results were verified by RT-PCR, Northern and Western blot analysis, and immunohistochemistry in human, rat, and rabbit articular cartilage. RESULTS: In our rat model of cartilage degeneration induced by joint immobilization, increases in the levels of prothrombin mRNA, thrombin protein, and fibrin deposition were observed. Expression of the prothrombin gene by chondrocytes was confirmed by ddPCR (rat), RT-PCR (rat and human), and by Northern blot analysis (rabbit). In addition, thrombin-like immunoreactivity was increased in chondrocytes after a 4 week immobilization period compared with rat knees receiving sham surgery. Thrombin activity was reflected by the presence of fibrin immunoreactivity in operated rat knee joints. CONCLUSION: Articular chondrocytes express the prothrombin gene and its local expression in joints is translated into thrombin protein. Prothrombin expression is increased in response to joint immobility. Our results support generation of thrombin locally in joints and an upregulation of thrombin expression in cartilage degeneration secondary to immobility. These results may provide information on the source of increased thrombin activity in various animal models and in clinical forms of arthritis.     

Light‐activated gene transduction enhances adeno‐associated virus vector–mediated gene expression in human articular chondrocytes

Objective

To evaluate the effects of ultraviolet (UV) light as an adjuvant for recombinant adeno‐associated virus (rAAV) transduction in human articular chondrocytes.

Methods

Primary articular chondrocytes and immortalized chondrocytes (tsT/AC62) were exposed to various doses of UV light (0–1,000 J/m²) and infected at various multiplicities of infection (MOIs) with rAAV containing the enhanced green fluorescent protein (EGFP) gene. Cells were analyzed for viability and EGFP expression by fluorescence‐activated cell sorting on days 2, 4, and 8 following infection. To evaluate the transduction efficiency in intact articular cartilage, full‐thickness explants were exposed to UV light (0–200 J/m²), infected with rAAV‐eGFP, and analyzed for transduction via immunohistochemistry.

Results

Toxicity from UV exposure was observed at doses ≥500 J/m² and ≥200 J/m² in primary and immortalized chondrocyte cultures, respectively. Transduction efficiency was dependent on the UV dose, MOI, and time. In the cell line, the adjuvant effect of UV on the percentage of cells transduced was modest, but 100 J/m² increased the mean fluorescence intensity (MFI) of the transduced cells 4‐fold. In contrast, UV treatment had a profound effect on the transduction efficiency of primary chondrocytes, which reached ∼100% after exposure to 100 J/m² of UV light and 10³ MOIs for 8 days. Under the same conditions, 200 J/m² of UV light enhanced the MFI 7‐fold. In cartilage explants, there was no difference in the number of transduced chondrocytes at the edge of the explants in the superficial, intermediate, or basal zones; however, 200 J/m² of UV light increased the transduction efficiency 2‐fold at a low MOI. In the center of the explants, the superficial chondrocytes were efficiently transduced; those in the intermediate and basal zones could not be efficiently transduced under any condition. In the superficial chondrocytes, a low MOI and 200 J/m² of UV light increased the transduction efficiency 3‐fold (to 100%).

Conclusion

UV light at doses of up to 200 J/m² (which do not significantly affect cell viability) significantly enhances the transduction efficiency and expression of the transduced gene in cultures of rAAV‐infected primary chondrocytes and in chondrocytes in the superficial zone of intact articular cartilage. These findings support the concept that UV‐activated gene transduction could be used as an adjuvant for in vivo rAAV articular cartilage gene therapy with low viral titers to prevent and/or treat arthritis.

     

Immature articular cartilage is more susceptible to blood‐induced damage than mature articular cartilage: An in vivo animal study

Objective

Cartilage of young but skeletally mature dogs is more susceptible to blood‐induced damage than that of old dogs. The aim of the present study was to investigate whether cartilage of skeletally immature individuals is even more adversely affected by exposure to blood than that of mature individuals, as suggested by clinical practice experience with humans.

Methods

Right knees of 3 groups of 6 beagle dogs (skeletally immature, young mature, and old animals) were injected with autologous blood on days 0 and 2. On day 4, cartilage matrix proteoglycan turnover (content, synthesis, and release), synovial inflammation, and cartilage‐destructive properties of the synovial tissue were determined and compared with those of the left uninjected control knees.

Results

Subsequent to intraarticular bleeding, cartilage proteoglycan content decreased in an age‐dependent manner, with the largest decrease occurring in cartilage of immature animals. Proteoglycan synthesis per cell also decreased in an age‐dependent manner, with the largest decrease occurring in the immature animals. Cartilage proteoglycan release increased in all 3 groups, but the decrease was not age dependent. Interestingly, immature animals showed a large increase in cartilage DNA content upon exposure to blood, whereas mature animals did not. Histologic analysis showed a mild synovitis in animals of all ages, but catabolic inflammatory activity was found only in immature animals.

Conclusion

Joints of skeletally immature dogs appeared to be more susceptible than joints of mature dogs to the adverse effects of a joint hemorrhage. These data suggest that for humans, specifically young children are at risk for joint damage after a joint hemorrhage.

     

Integrin‐mediated adhesion of human articular chondrocytes to cartilage

Objective

To determine 1) the kinetics and strength of adhesion of human articular chondrocytes to a cut cartilage surface, and 2) the role of specific integrins in mediating such adhesion, using an in vitro model.

Methods

Human articular chondrocytes isolated from cadaveric donors (mean ± SD age 38 ± 13 years) were cultured in high‐density or low‐density monolayer. Following release from culture with trypsin and a 2–2.5‐hour recovery period, chondrocytes were analyzed either for adhesion to cartilage or for integrin expression by flow cytometry.

Results

Following culture in monolayer, adhesion of chondrocytes to cartilage increased with time, from 6–16% at 10 minutes to a maximum of 59–82% at 80–320 minutes. After 80 minutes of adhesion, the resistance of cells to flow‐induced shear stress (50% detachment) was ∼21 Pa. Chondrocyte adhesion to cartilage decreased with pretreatment of cells with monoclonal antibodies that bound to and blocked certain integrins. After an 80‐minute incubation time, adhesion of chondrocytes cultured in high‐density monolayer decreased from the value of IgG1‐treated controls (55%) with blocking of the β1 integrin subunit (to 23%) or with blocking of α5β1 (to 36%). Following expansion of chondrocytes in low‐density monolayer, the mechanisms of adhesion to cartilage were generally similar. After an 80‐minute incubation time, adhesion of chondrocytes cultured in low‐density monolayer decreased from the value of IgG1‐treated controls (62%) with blocking of the β1 integrin subunit (to 30%) or with blocking of α5β1 (to 44%). Additionally, adhesion of these cells decreased to 46% by blocking of αvβ5, with a similar trend in effect for chondrocytes cultured in high‐density monolayer. Blocking of the α1 or α3 integrin subunits or αvβ3 had no detectable effect on adhesion, even though these receptors were detected by flow cytometry.

Conclusion

Under the culture and seeding conditions studied, β1, α5β1, and αvβ5 integrins mediate human chondrocyte adhesion to cartilage. These chondrocyte integrins have a potential role in the initial adhesion and retention of chondrocytes at a cartilage defect site following clinical procedures of chondrocyte transplantation.

     

Efficient gene delivery to articular cartilage using electroporation

Effective in vivo gene transfer into articular cartilage has not yet been established. Since chondrocytes are embedded within a rich extracellular matrix, various gene transfer methods have failed to introduce genes into deeper layers of the articular cartilage. In this study, we developed new superfine pointed needle electrodes for in situ electroporation (EP), and investigated the efficiency of gene transfer into articular cartilage with different degrees of degeneration. Full-thickness articular cartilage slices were obtained from the knee joint of a 3–4-month-old rabbit. The cartilage tissues were treated briefly with trypsin to partly remove matrix proteoglycan. Human articular cartilage with different grades of degeneration was also used. For EP, the articular cartilage surface was soaked in a solution containing green fluorescent protein (GFP) plasmid. Then, the superfine pointed 7-needle electrodes were gently stabbed into the surface layer of the articular cartilage and the gene was transfected by an electroporator. GFP expression was examined by immunohistochemical analysis. Cartilage tissue was successfully transfected with the GFP gene by the electrodes and EP. Transfection efficiency was enhanced by depleting the matrix proteoglycan in rabbit articular cartilage. Chondrocytes in the deeper layer of the articular cartilage were also transfected and expressed GFP. In human osteoarthritic cartilage, ca. 30% of the cells in the deeper layer were transfected by selecting optimal EP conditions. No adverse effects of EP on damaged articular cartilage were obvious from histological analysis or TUNEL staining. The results indicated that EP-mediated in vivo gene transfer into articular cartilage may provide a useful therapeutic strategy to treat cartilage degeneration.     

Efficient gene delivery to articular cartilage using electroporation

Abstract

Effective in vivo gene transfer into articular cartilage has not yet been established. Since chondrocytes are embedded within a rich extracellular matrix, various gene transfer methods have failed to introduce genes into deeper layers of the articular cartilage. In this study, we developed new superfine pointed needle electrodes for in situ electroporation (EP), and investigated the efficiency of gene transfer into articular cartilage with different degrees of degeneration. Full-thickness articular cartilage slices were obtained from the knee joint of a 3–4-month-old rabbit. The cartilage tissues were treated briefly with trypsin to partly remove matrix proteoglycan. Human articular cartilage with different grades of degeneration was also used. For EP, the articular cartilage surface was soaked in a solution containing green fluorescent protein (GFP) plasmid. Then, the superfine pointed 7-needle electrodes were gently stabbed into the surface layer of the articular cartilage and the gene was transfected by an electroporator. GFP expression was examined by immunohistochemical analysis. Cartilage tissue was successfully transfected with the GFP gene by the electrodes and EP. Transfection efficiency was enhanced by depleting the matrix proteoglycan in rabbit articular cartilage. Chondrocytes in the deeper layer of the articular cartilage were also transfected and expressed GFP. In human osteoarthritic cartilage, ca. 30% of the cells in the deeper layer were transfected by selecting optimal EP conditions. No adverse effects of EP on damaged articular cartilage were obvious from histological analysis or TUNEL staining. The results indicated that EP-mediated in vivo gene transfer into articular cartilage may provide a useful therapeutic strategy to treat cartilage degeneration.     

Subtype‐specific peripheral blood gene expression profiles in recent‐onset juvenile idiopathic arthritis

Objective

To identify differences in peripheral blood gene expression between patients with different subclasses of juvenile idiopathic arthritis (JIA) and healthy controls in a multicenter study of patients with recent‐onset JIA prior to treatment with disease‐modifying antirheumatic drugs (DMARDs) or biologic agents.

Methods

Peripheral blood mononuclear cells (PBMCs) from 59 healthy children and 136 patients with JIA (28 with enthesitis‐related arthritis [ERA], 42 with persistent oligoarthritis, 45 with rheumatoid factor [RF]–negative polyarthritis, and 21 with systemic disease) were isolated from whole blood. Poly(A) RNA was labeled using a commercial RNA amplification and labeling system (NuGEN Ovation), and gene expression profiles were obtained using commercial expression microarrays (Affymetrix HG‐U133 Plus 2.0).

Results

A total of 9,501 differentially expressed probe sets were identified among the JIA subtypes and controls (by analysis of variance; false discovery rate 5%). Specifically, 193, 1,036, 873, and 7,595 probe sets were different in PBMCs from the controls compared with those from the ERA, persistent oligoarthritis, RF‐negative polyarthritis, and systemic JIA patients, respectively. In patients with persistent oligoarthritis, RF‐negative polyarthritis, and systemic JIA subtypes, up‐regulation of genes associated with interleukin‐10 (IL‐10) signaling was prominent. A hemoglobin cluster was identified that was underexpressed in ERA patients but overexpressed in systemic JIA patients. The influence of JAK/STAT, ERK/MAPK, IL‐2, and B cell receptor signaling pathways was evident in patients with persistent oligoarthritis. In systemic JIA, up‐regulation of innate immune pathways, including IL‐6, Toll‐like receptor/IL‐1 receptor, and peroxisome proliferator–activated receptor signaling, were noted, along with down‐regulation of gene networks related to natural killer cells and T cells. Complement and coagulation pathways were up‐regulated in systemic JIA, with a subset of these genes being differentially expressed in other subtypes as well.

Conclusion

Expression analysis identified differentially expressed genes in PBMCs obtained early in the disease from patients with different subtypes of JIA and in healthy controls, providing evidence of immunobiologic differences between these forms of childhood arthritis.

     

Laser‐mediated microdissection facilitates analysis of area‐specific gene expression in rheumatoid synovium

Objective

Current approaches to analyzing gene expression in rheumatoid arthritis (RA) synovium are based on RNA isolated either from cultured synovial cells or from synovial biopsy specimens. This strategy does not, in general, allow distinction of specific gene expression between cells originating from different synovial areas, due to potential mixture of expression profiles. Therefore, we established the combination of laser‐mediated microdissection (LMM) and differential display to analyze profiles of gene expression in histologically defined areas of rheumatoid synovium. The present study was undertaken to establish parameters for this technique and assess its usefulness for gene expression analysis.

Methods

Cryosections derived from RA synovial tissues were used to obtain cell samples from synovial lining versus sublining, using a microbeam laser microscope. RNA was isolated and analyzed by nested RNA arbitrarily primed–polymerase chain reaction (RAP‐PCR) for differential display fingerprinting. Differentially expressed bands were cut out, and PCR products were eluted, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry analysis.

Results

Microdissected sections of RA synovial tissue containing ∼600 cells yielded enough RNA to produce a reproducible RNA fingerprint pattern. Several genes could be identified as being expressed differentially between the synovial lining and the sublining, and their expression could be confirmed at the messenger RNA and protein levels.

Conclusion

The combination of LMM and RAP‐PCR presents a valuable tool to obtain novel insights into the area‐dependent differential regulation of gene expression in RA synovium. Both known and previously unknown genes were revealed with this technique. This study is the first to demonstrate the potential of this analytic strategy in the investigation of a nonmalignant, multifactorial, inflammatory disease.

     

Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

Objective

To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis.

Methods

A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2‐inducible gene expression to prevent articular cartilage degeneration. Col2‐Cre;Smad3^fl/fl mice allowed study of the chondrocyte‐intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3^fl/fl mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP‐13) messenger RNA (mRNA) and protein expression.

Results

Chondrocyte‐specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2‐Cre;Smad3^fl/fl mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP‐13–mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2‐inducible MMP‐13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP‐13 expression.

Conclusion

Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2‐inducible MMP‐13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

     

Upregulation of insulin-like growth factor I gene expression in the lesions of osteoarthritic human articular cartilage.

Expression of insulin-like growth factor I (IGF-I)mRNA and IGF-I protein was studied in human osteoarthritic and young articular cartilage by in situ hybridisation and immunohistochemistry. In situ hybridisation showed that relatively low amounts of IGF-I mRNA signal were present in anatomically normal regions of osteoarthritic and young cartilage. In fibrillated osteoarthritic cartilage, however, the signal intensity was significantly higher than in non-fibrillated cartilage. Particularly high levels of IGF-I mRNA were present in the surface cell clones of more advanced lesions, the amount of signal being about four to five times greater than in anatomically normal cartilage. The amount of message varied with cartilage depth. In young cartilage there was less IGF-I mRNA in the superficial zone than in the middle and deep zones. In fibrillated regions of osteoarthritic joints the amount of message in surface cells was greater than in deeper regions. A specific human IGF-I antibody was used to show the presence intracellularly of IGF-I protein in osteoarthritic and young cartilage. Raised levels of IGF-I message in osteoarthritic chondrocytes may represent an attempt at increased matrix repair, operating by an autocrine/paracrine mechanism.