Calcium signaling leads to mitochondrial depolarization in impact-induced chondrocyte death in equine articular cartilage explants

OBJECTIVE: Chondrocyte apoptosis is an important factor in the progression of osteoarthritis. This study aimed to elucidate the mechanisms involved upstream of caspase 9 activation and, in particular, calcium signaling and mitochondrial depolarization. METHODS: Articular cartilage explants obtained from healthy horses were subjected to a single impact load (500-gm weight dropped from a height of 50 mm) and cultured in vitro for up to 48 hours. Chondrocyte death was quantified by the TUNEL method. Release of proteoglycans was determined by the dimethylmethylene blue assay. Weight change was measured, and mitochondrial depolarization was determined using JC-1 staining. To assess the role of calcium signaling in impact-induced chondrocyte death, explants were preincubated in culture medium containing various concentrations of calcium. Inhibitors were used to assess the role of individual signaling components in impact-induced chondrocyte death. RESULTS: Calcium quenching, inhibitors of calpains, calcium/calmodulin-regulated kinase II (CaMKII), and mitochondrial depolarization reduced impact-induced chondrocyte death after 48 hours in culture. Transient mitochondrial depolarization was observed 3-6 hours following a single impact load. Mitochondrial depolarization was prevented by calcium quenching, inhibitors of calpain, CaMKII, permeability transition pore formation, ryanodine receptor, and the mitochondrial uniport transporter. Cathepsin B did not appear to be involved in impact-induced chondrocyte death. The calpain inhibitor prevented proteoglycan loss, but the percentage weight gain and proteoglycan loss were unaffected by all treatments used. CONCLUSION: Following a single impact load, calcium is released from the endoplasmic reticulum via the ryanodine receptor and is taken up by the mitochondria via the uniport transporter, causing mitochondrial depolarization and caspase 9 activation. In addition, calpains and CaMKII play important roles in causing mitochondrial depolarization.     

Effect of nalidixic acid, pipemidic acid and cinoxacin on chondrocyte metabolism in explants of articular cartilage

Explants of immature bovine articular cartilage were exposed to nalidixic acid, pipemidic acid and cinoxacin at one and ten times the human therapeutic plasma level for 7 days. Only nalidixic acid had significant effects on the chondrocyte metabolism. 20 micrograms/ml nalidixic acid caused an increase of 35S-sulfate incorporation into glycosaminoglycans at day 7. Two hundred micrograms/ml nalidixic acid inhibited the incorporation of 3H-thymidine into DNA. The incorporation of 35S-sulfate into glycosaminoglycans was decreased at day 0, while at day 7 the incorporation had returned to the control value. Pipemidic acid and cinoxacin had no significant effects on either the 3H-thymidine or the 35S-sulfate incorporation.     

Aging,articular cartilage chondrocyte senescence and osteoarthritis

The incidence of osteoarthritis (OA), the disease characterized by joint pain and loss of joint form and function due to articular cartilage degeneration, is directly correlated with age. The strong association between age and increasing incidence of osteoarthritis (OA) marks OA as an age related disease. Yet, like many other age related diseases, OA is not an inevitable consequence of aging; instead, aging increases the risk of OA. Articular cartilage aging changes that may lead to articular cartilage degeneration include fraying and softening of the articular surface, decreased size and aggregation of proteoglycan aggrecans and loss of matrix tensile strength and stiffness. These changes most likely are the result of an age related decrease in the ability of chondrocytes to maintain and repair the tissue manifested by decreased mitotic and synthetic activity, decreased responsiveness to anabolic growth factors and synthesis of smaller less uniform aggrecans and less functional link proteins. Our recent work suggests that progressive chondrocyte senescence marked by expression of the senescence associated enzyme beta-galactosidase, erosion of chondrocyte telomere length and mitochondrial degeneration due to oxidative damage causes the age related loss of chondrocyte function. New efforts to prevent the development and progression of OA might include strategies that slow the progression of chondrocyte senescence or replace senescent cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Calcium signaling leads to mitochondrial depolarization in impact‐induced chondrocyte death in equine articular cartilage explants

Objective

Chondrocyte apoptosis is an important factor in the progression of osteoarthritis. This study aimed to elucidate the mechanisms involved upstream of caspase 9 activation and, in particular, calcium signaling and mitochondrial depolarization.

Methods

Articular cartilage explants obtained from healthy horses were subjected to a single impact load (500‐gm weight dropped from a height of 50 mm) and cultured in vitro for up to 48 hours. Chondrocyte death was quantified by the TUNEL method. Release of proteoglycans was determined by the dimethylmethylene blue assay. Weight change was measured, and mitochondrial depolarization was determined using JC‐1 staining. To assess the role of calcium signaling in impact‐induced chondrocyte death, explants were preincubated in culture medium containing various concentrations of calcium. Inhibitors were used to assess the role of individual signaling components in impact‐induced chondrocyte death.

Results

Calcium quenching, inhibitors of calpains, calcium/calmodulin‐regulated kinase II (CaMKII), and mitochondrial depolarization reduced impact‐induced chondrocyte death after 48 hours in culture. Transient mitochondrial depolarization was observed 3–6 hours following a single impact load. Mitochondrial depolarization was prevented by calcium quenching, inhibitors of calpain, CaMKII, permeability transition pore formation, ryanodine receptor, and the mitochondrial uniport transporter. Cathepsin B did not appear to be involved in impact‐induced chondrocyte death. The calpain inhibitor prevented proteoglycan loss, but the percentage weight gain and proteoglycan loss were unaffected by all treatments used.

Conclusion

Following a single impact load, calcium is released from the endoplasmic reticulum via the ryanodine receptor and is taken up by the mitochondria via the uniport transporter, causing mitochondrial depolarization and caspase 9 activation. In addition, calpains and CaMKII play important roles in causing mitochondrial depolarization.

     

Chondrocyte-derived apoptotic bodies and calcification of articular cartilage

Chondrocytes exposed to nitric oxide (NO) or antibody to Fas undergo cell death by apoptosis. This study examines structural and functional properties of chondrocyte-derived apoptotic bodies. In NO treated cartilage, the dense pericellular matrix that normally surrounds the cells is degraded and apoptotic bodies accumulate within and in the vicinity of the chondrocyte lacunae. Functional analysis shows that apoptotic bodies isolated from NO-treated chondrocytes or cartilage produce pyrophosphate. The levels of pyrophosphate produced by apoptotic bodies are increased by pretreatment of the chondrocytes with transforming growth factor β and decreased by interleukin 1. Apoptotic bodies contain alkaline phosphatase and NTP pyrophosphohydrolase activities and can precipitate calcium. These results suggest that chondrocyte-derived apoptotic bodies express functional properties that may contribute to the pathologic cartilage calcification observed in aging and osteoarthritis.     

New developments in the pathogenesis of articular cartilage calcification

Articular cartilage, unlike growth plate cartilage, is specialized to not undergo matrix calcification. However, articular cartilage mineralization, in the form of CPPD (chondrocalcinosis) and hydroxyapatite crystals, frequently accompanies and complicates osteoarthritis and aging. Recent work has demonstrated that certain features of growth cartilage development and mineralization are shared in degenerative cartilage. These include chondrocyte proliferation, hypertrophy and increased apoptosis. Moreover, parathyroid hormone related protein (PTHrP), one of the central mediators of endochondral development, is abundant in osteoarthritic cartilage. Cartilage PPi elaboration and cytosolic transglutaminase activity are markedly increased with aging. Only recently have the molecular identities been defined for the chondrocyte inorganic pyrophosphate (PPi)-generating isozymes of the phosphodiesterase nucleotide pyrophosphatase (PDNP) family (including PC-1 and B10), and for transglutaminase in articular cartilage. This review focuses on the evolving understanding of the potential roles, in articular cartilage calcification, of PTHrP, PDNP family enzymes, PPi metabolism, and transglutaminase activity.     

Relationship between chondrocyte apoptosis and matrix depletion in human articular cartilage

OBJECTIVE: Chondrocyte apoptosis has been described in studies of the pathogenesis of various arthritides. Since matrix degradation is important in the pathology of arthritis, we investigated the effect of matrix depletion on chondrocyte apoptosis. METHODS: Consecutive 1 mm sections of child's rib cartilage were cultured under 4 sets of conditions: (1) control; and treatment with (2) hyaluronidase, (3) collagenase, (4) hyaluronidase/collagenase alternately. Sections were harvested on Days 5, 8, and 11, and the proportion of apoptotic cells was measured by propidium iodide staining and flow cytometry. Changes of Fas and Fas ligand expression were verified by immunohistochemistry and Western blot. RESULTS: Collagenase treatment led to an increase of apoptosis, which began on Day 8, whereas hyaluronidase treatment had no effect on chondrocyte viability up to Day 11. Alternating hyaluronidase and collagenase treatment induced chondrocyte death earlier, but the difference in apoptotic rate was not significant on Days 8 or 11 compared to collagenase treatment alone. Immunohistochemistry showed the expression of Fas ligand in all enzymatically treated specimens. Expression of Fas receptor was ubiquitous in both control and treated specimens. CONCLUSION: The collagen framework is important in the maintenance of chondrocyte survival in human cartilage. Upregulation of Fas ligand in matrix depleted specimens suggested that the Fas pathway may have a role in apoptosis induced by matrix depletion.     

Synthesis of proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer cultures, and resurfaced cartilage cultures

OBJECTIVE: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems. METHODS: Bovine calf articular cartilage discs or isolated cells were used in 1 of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells. RESULTS: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from approximately 25 microg/cm(2)/day on days 0-1 to approximately 3 microg/cm(2)/day on days 5-9), while increasing in monolayer culture (from approximately 1 pg/cell/day on days 0-1 to approximately 7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching approximately 2 microg/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells. CONCLUSION: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.     

The effect of glycosaminoglycan loss on chondrocyte viability: a study on porcine cartilage explants

OBJECTIVE: Loss of glycosaminoglycan (GAG) is an early event in osteoarthritis. Recent findings showed increased cell death in arthritic cartilage and linkage with extracellular matrix degradation. The aim of this study was to analyze the direct effect of GAG loss on chondrocyte survival and cell death following mechanical injury. METHODS: In full-thickness cartilage explants from porcine knee joints, GAG was depleted by digestion with chondroitinase ABC. Explants were subjected to single-impact mechanical injury. Cell viability and the types of cell death were analyzed by Live/Dead cell assay, staining for active caspase 3, and sensitivity to caspase inhibitor. RESULTS: GAG depletion did not directly lead to increased cell death. In chondroitinase ABC-treated explants, but not in control explants, mechanical injury caused an immediate reduction in cell viability (from 84.6% to 71.0%); the reduction was prominent in the superficial zone. This immediate cell death was not inhibited by the pancaspase inhibitor Z-VAD-FMK, suggesting cell necrosis. During subsequent culture, viability in these explants decreased further, to 50.5% on day 3. The second wave of cell death was reduced by the addition of Z-VAD-FMK in chondroitinase ABC-treated explants and was also associated with activation of caspase 3, suggesting apoptotic mechanisms of cell death. CONCLUSION: These results indicate that GAG loss alone does not directly lead to chondrocyte death. In response to mechanical injury, there is an immediate induction of necrotic cell death that is seen only in GAG-depleted explants and primarily in the superficial zone. During subsequent culture, cell death spreads via apoptotic mechanisms.     

Statin prevents chondrocyte aging and degeneration of articular cartilage in osteoarthritis (OA)

Recent reports have shown that statin (HMG-CoA reductase inhibitors) may have the potential to inhibit inflammatory arthritis. More recently, the idea that chondrocyte aging is closely associated with the progression of cartilage degeneration has been promulgated. Here, we demonstrate the potential of statin as protective agents against chondrocyte aging and degeneration of articular cartilage during the progression of osteoarthritis (OA), both in vitro and in vivo. The OA-related catabolic factor, IL-1β induced marked downregulation of cellular activity, expression of a senescent biomarker, specific senescence-associated β-galactosidase activity and shortening of the cellular lifespan in chondrocytes. In contrast, treatment with statin inhibited the IL-1β-induced production of cartilage matrix degrading .enzymes (metalloprotease-1 and -13) and cellular senescence in of chondrocytes in vitro. In addition, this statin accelerated the production of cartilage matrix proteoglycan in chondrocytes. The in vivo study was performed on the STR/OrtCrlj mouse, an experimental model which spontaneously develops an osteoarthritic process. In this mouse model, treatment with statin significantly reduced the degeneration of articular cartilage, while the control knee joints showed progressive cartilage degeneration over time. These findings suggest that statin may have the potential to prevent the catabolic stress-induced chondrocyte disability and aging observed in articular cartilage. Our results indicate that statin are potential therapeutic agents for protection of articular cartilage against the progression of OA.     

软骨细胞凋亡与骨关节病退行性变关系的研究进展

为进一步探讨大骨节病软骨细胞坏死的发病机理，对生理性软骨细胞凋亡、骨关节病的病理性软骨细胞凋亡、引起软骨细胞凋亡的因素、软骨细胞凋亡的意义、软骨细胞凋亡与骨关节病的关系5个方面的研究现况进行了综述，结果发现，软骨细胞凋亡与骨关节病的发病机理密切相关，进而为研究大骨节病的分子发病机理提供了思路。     

Articular cartilage repair using dedifferentiated articular chondrocytes and bone morphogenetic protein 4 in a rabbit model of articular cartilage defects

OBJECTIVE: To observe redifferentiation of dedifferentiated chondrocytes after transplantation into the joint, and to evaluate the ability of dedifferentiated chondrocytes transduced with adenovirus containing bone morphogenetic protein 4 (BMP-4) to redifferentiate in vitro and in vivo in a rabbit model of articular cartilage defects. METHODS: Monolayer and pellet culture systems were used to evaluate the redifferentiation of dedifferentiated chondrocytes transduced with BMP-4. A rabbit model of partial-thickness articular cartilage defects was used to evaluate cartilage repair macroscopically and histologically, 6 and 12 weeks after transplantation with first-passage, fifth-passage, or transduced fifth-passage chondrocytes. Histologic grading of the repaired tissue was performed. Expression of BMP-4 and the ability of transplanted cells to recover a chondrocytic phenotype were also assessed. RESULTS: BMP-4--expressing dedifferentiated chondrocytes recovered a chondrocytic phenotype in vitro. After transplantation into the joint, some of the dedifferentiated chondrocytes in the defect sites could undergo redifferentiation and formed matrix that displayed positive toluidine blue staining for glycosaminoglycans. Histologic scores of the regenerative tissue revealed significantly better cartilage repair in rabbits transplanted with BMP-4--expressing cells than in the other treatment groups. Staining with toluidine blue revealed expression of BMP-4 in the cells and in the matrix surrounding the cells. CONCLUSION: Some dedifferentiated chondrocytes can redifferentiate after transplantation into the load-bearing joint. BMP-4 can be used to induce redifferentiation of dedifferentiated chondrocytes in vitro and in vivo, which could help enhance articular cartilage repair.     

Type x collagen synthesis in human osteoarthritic cartilage. indication of chondrocyte hypertrophy

Objective. To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker. Methods. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with ³H-proline, immunoprecipitation, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically. Results. Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen. Conclusion. Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage.     

Age related changes in the turnover of proteoglycans from explants of bovine articular cartilage

Articular cartilage in explant culture synthesized 2 proteoglycan types of different size. The proportion of 35S-sulfate incorporated into the small proteoglycan was higher in mature (17%) than in immature cartilage (11%). The chondroitin sulfate chains of both proteoglycans, synthesized by mature cartilage were shorter than those of immature cartilage, with a higher ratio of 6-sulfated over 4-sulfated disaccharides. Radiolabelled macromolecules from tissue of both ages were released in 2 phases: an initial fast release followed by a period of slow release. The half-lives of both proteoglycan populations were shorter in mature cartilage than in immature tissue (-45%). The amount of proteoglycans released during the initial short phase increases with age, from 23-24% in immature cartilage to 28-29% in mature tissue.     

Apoptotic chondrocytes from osteoarthrotic human articular cartilage and abnormal calcification of subchondral bone

OBJECTIVE: Osteoarthrosis (OA) is accompanied by altered subchondral bone remodeling. We investigated the role of chondrocytes in the mechanism of abnormal cartilage calcification. METHODS: Knee articular cartilage samples from OA and normal tissue were studied. Macroscopic and microscopic observations, alkaline phosphatase staining for light and electron microscopy (bright and dark fields). TUNEL technique, electron diffraction, and x-ray microanalyses were performed. RESULTS: Chondrocytes from patients displayed a morphology of apoptosis and showed abundant alkaline phosphatase (ALP)-rich matrix vesicles (MV) budding from the plasma membrane with hydroxyapatite microcrystals on their surface. Farther from the cells, hydroxyapatite crystals were detected on the MV surface and increased as they approached the subchondral bone. The concentration of Ca and P and their ratio increased inside the ALP-rich MV in relation to the proximity to subchondral bone. In the OA subchondral bone the ratio Ca/P varied from 3.936 to 0.974. In normal tissue the ratio was very homogeneous (maximum 1.973, minimum 1.781). CONCLUSION: In situ, apoptotic chondrocytes correlate with factors known to be involved in the calcification of the extracellular matrix. This suggests that apoptosis is involved in the abnormal calcification of OA cartilage, and consequently in the altered remodeling of the subchondral bone.     

Experimental hydroxyapatite synovial and articular cartilage calcification. light and electron microscopic studies

Intraarticular calciphylaxis with cartilage and synovial calcification was produced in rabbits by oral administration of a single dose of dehydrotachysterol followed by an intraarticular injection of ferrous chloride. Synovial membrane apatite deposits were seen in the interstitium, on collagen fibers, and within cell vacuoles. In contrast, long-term administration of dehydrotachysterol alone induced only articular cartilage calcification. Such calcification was limited to the mid and deep zone of cartilage. Most calcium deposits were apatite-like crystals. Acute crystal-associated inflammation was not demonstrated in these specimens with crystals sequestered in synovial or cartilage tissue. Further studies on these models will examine the relation of apatite to joint disease.