212 Increase in the Gene Expression of MMP 13 and BMP 2 in Human Arthrotic Cartilage Cells After the Treatment with 5 Aza Deoxy Cytidine (original) (raw)
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Growth-related oncogene ? induction of apoptosis in osteoarthritis chondrocytes
Arthritis & Rheumatism, 2002
Objective. To evaluate the apoptotic effect of the chemokine growth-related oncogene ␣ (GRO␣), which we recently reported to be up-regulated in osteoarthritis (OA) chondrocytes. Chondrocyte apoptosis is considered to be a major determinant of cartilage damage in OA, a disease resulting from the aberrant production of inflammatory mediators (cytokines and chemokines) and effectors (matrix metalloproteinases and reactive oxygen and nitrogen species) by chondrocytes.
Injurious Mechanical Compression of Bovine Articular Cartilage Induces Chondrocyte Apoptosis
Archives of Biochemistry and Biophysics, 2000
A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glyocosaminoglycan loss and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa, and increased with peak stress in a dose dependent manner. This increase in apoptosis was maximal by 24 hours after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells appeared to apoptose. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, though this trend was not significant until 24 MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after threshold injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7-12 MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13 MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6 MPa peak stress. Nitrite levels were significantly increased above controls at 20 MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death along with a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at threshold stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes.
In vitro model for the study of necrosis and apoptosis in native cartilage
Journal of Pathology, 2002
Apoptosis plays a role in everything from early development to ageing and in a host of disease states. Studying this important process in the in vivo state is critical, to understand its varied role and to open further avenues of therapeutic intervention. The present paper presents an ex vivo bovine articular cartilage model to study apoptotic and necrotic processes following acute injury. Ex vivo bovine articular cartilage was assessed 1, 3 and 6 days following holmium : YAG laser treatment (780 mJ). Markers to visualize cell viability, caspase-3 activity, changes in mitochondrial membrane potential and the degree of DNA fragmentation (TUNEL assay) were used alone or in various combinations. Standard histology and transmission electron microscopy (TEM) were also performed for a more comprehensive assessment. A significant progression (p < 0.05) of ethidium/caspase-3-positive signal depth at day 3 preceded a significant increase (p < 0.05) in TUNEL signal depth by day 6. The mitochondrial matrix marker CMXRos was shown to provide an alternative to calcein-AM for assessing cell viability. The identification of chondrocyte apoptosis morphology by TEM was not conclusive. Nevertheless, TEM revealed that cells which were clearly necrotic also stained positively for TUNEL, thus indicating the risk of using TUNEL alone for the assessment of apoptosis. The model described here allows the rapid, spatial and temporal determination of cell viability and of apoptotic and necrotic processes in whole-tissue specimens after acute injury, and permits study of the balance between these events. The assessment of healthy and diseased cartilage and of the effects of surgical, pharmaceutical or in vitro intervention are immediate applications of these protocols. Moreover, this model may be useful for the study of key mechanisms involved in apoptosis or for the establishment of other markers of apoptosis. Copyright © 2002 John Wiley & Sons, Ltd.
Arthritis & Rheumatism, 2004
Methods. Bovine articular cartilage disks were obtained from the femoropatellar groove of animals ages 0.5-23 months and placed in culture. Cartilage disks were preincubated overnight with the cellpermeable superoxide dismutase (SOD) mimetic Mn(III) porphyrin (0-12.5 M) or ␣-tocopherol (0-50 M) and then injured by a single unconfined compression to a final strain of 50% at a velocity of 1 mm/second. After 4 days of additional incubation, the disks were fixed and embedded for light and electron microscopy. Apoptotic cells were quantified morphologically by the appearance of nuclear blebbing on light microscopy. Biosynthetic activity was demonstrated by incorporation of radiolabeled proline. The antioxidative action of the SOD mimetic was confirmed by histologic examination of cartilage after incubation with nitroblue tetrazolium.
The Journal of rheumatology, 2003
We have reported that articular cartilage showed early stage degeneration at 7 and 14 days after immobilization, moderate degeneration at 28 days, and severe degeneration at 42 days in rabbits. To test whether apoptosis occurs in association with p53 expression in chondrocytes during the process of articular cartilage degeneration, we investigated the degree of cartilage degeneration, the frequency of apoptotic cells, and the levels of p53 mRNA in rabbits and mice after knee immobilization. Right knees of male Japanese white rabbits were immobilized in full extension with fiberglass casts for up to 42 days. Similarly, right knees of male p53 wild-type [p53 (+/+)] and p53 null [p53 (-/-)] mice were immobilized in full extension with bandage tape for up to 84 days. Apoptotic cells were confirmed by TUNEL staining on the sections of knee joints. Total RNA of articular chondrocytes obtained from Day 0 or immobilized knees was analyzed semiquantitatively by RT-PCR using specific primers ...
Oxidant conditioning protects cartilage from mechanically induced damage
Journal of Orthopaedic Research, 2010
Articular cartilage degeneration in osteoarthritis has been linked to abnormal mechanical stresses that are known to cause chondrocyte apoptosis and metabolic derangement in in vitro models. Evidence implicating oxidative damage as the immediate cause of these harmful effects suggests that the anti-oxidant defenses of chondrocytes might influence their tolerance for mechanical injury. Based on evidence that anti-oxidant defenses in many cell types are stimulated by moderate oxidant exposure, we hypothesized that oxidant pre-conditioning would reduce acute chondrocyte death and proteoglycan depletion in cartilage explants after exposure to abnormal mechanical stresses. Porcine cartilage explants were treated every 48 hours with tert-butyl hydrogen peroxide (tBHP) at non-lethal concentrations (25, 100, 250, 500 µM) for a varying number of times (1, 2 or 4) prior to a bout of unconfined axial compression (5 MPa, 1 Hz, 1800 cycles). When compared with untreated controls, tBHP had significant positive effects on post-compression viability, lactate production, and proteoglycan losses. Overall, the most effective regime was 100 µM tBHP applied 4 times. RNA analysis revealed significant effects of 100 µM tBHP on gene expression. Catalase, hypoxia-inducible factor-1alpha (HIF-1α), and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) were significantly increased relative to untreated controls in explants treated 4 times with 100 µM tBHP, a regime that also resulted in a significant decrease in matrix metalloproteinase-3 (MMP-3) expression. These findings demonstrate that repeated exposure of cartilage to sub-lethal concentrations of peroxide can moderate the acute effects of mechanical stress, a conclusion supported by evidence of peroxide-induced changes in gene expression that could render chondrocytes more resistant to oxidative damage.