Biomedical subjects

R F Loeser

Publications and source records attributed to R F Loeser.

At least 19 recordsLinked to original sources

PubMed2002

CD44 and integrin matrix receptors participate in cartilage homeostasis.

Articular chondrocytes express the matrix receptors CD44 and integrins. Both of these receptors exhibit interactions with adjacent extracellular matrix macromolecules. In addition, both integrins and CD44 have the capacity for signal transduction as well as modulated interactions with the actin cytoskeleton. As such, both receptor families provide the chondrocytes a means to detect changes in matrix composition or to function as mechanotransducers. Disruption of CD44 or integrin-mediated cell-matrix interactions, either experimentally induced or when present in osteoarthritis, have profound effects on cartilage metabolism. Thus, CD44 and integrin receptors play a critical role in maintaining cartilage homeostasis.

Animals↗

PubMed2001

Modifiers of change in physical functioning in older adults with knee pain: the Observational Arthritis Study in Seniors (OASIS).

OBJECTIVE: To ascertain predictors of decline in physical functioning among older adults reporting knee pain. METHODS: The Observational Arthritis Study in Seniors was a longitudinal study of 480 adults over 65 years of age. Measurements of strength, sociodemographic characteristics, disease burden (including radiographic knee osteoarthritis [OA]), self-reported disability, and functional limitations were obtained on participants at baseline and at 15 and 30 months. RESULTS: Radiographic evidence of OA at baseline was moderately associated with an increased decline in both transfer (P = 0.06) and ambulatory-based performance tasks (P = 0.04) but not in self-reported disability. This effect disappeared after accounting for baseline levels of knee pain intensity and knee strength. CONCLUSION: Knee pain intensity and knee strength may mediate the relationship between radiographic evidence of knee OA and change in performance. Although it is not clear whether joint disease precedes or follows a decline in muscular strength, these results may help to identify subpopulations of older persons with knee OA who may benefit from interventions aimed at slowing the progression of disability related to transfer and ambulatory-based tasks.

Activities of Daily Living↗

PubMed2001

Mechanisms of chondrocyte adhesion to cartilage: role of beta1-integrins, CD44, and annexin V.

The initial adhesion of transplanted chondrocytes to surrounding host cartilage may be important in the repair of articular defects. Adhesion may position cells to secrete molecules that fill the defect and integrate repair tissue with host tissue. While chondrocytes are known to become increasingly adherent to cartilage with time, the molecular basis for this is unknown. The objective of this study was to investigate the role of beta1-integrin, CD44, and annexin V receptors in chondrocyte adhesion to cartilage. Chondrocytes were cultured in high density monolayer, released with trypsin, and allowed to recover in suspension for 2 h at 37 degrees C. Under these conditions, flow cytometry analysis showed that chondrocytes expressed beta1-integrins, CD44, and annexin V. In a rapid screening assay to assess chondrocyte adhesion to cartilage, cell detachment decreased from 79% at 10 min following transplantation to 10% at 320 min. Treatment of cells with a monoclonal antibody to block beta1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls. Similarly, results from a parallel-plate shear flow adhesion assay showed that blocking beta1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls at each level of applied shear (0-70 Pa). In both assays, treatment of cells with reagents that block CD44 (hyaluronan oligosaccharides or monoclonal Ab IM7) or annexin V (polyclonal Ab #8958) had no detectable effect on adhesion. With cartilage treated with chondroitinase ABC, blocking beta1-integrins also increased chondrocyte detachment, while blocking CD44 and annexin V also had no detectable effect. Under the conditions studied here, beta1-integrins appear to mediate chondrocyte adhesion to a cut cartilage surface. Delineation of the mechanisms of adhesion may have clinical implications by allowing cell manipulations or matrix treatments to enhance chondrocyte adhesion and retention at a defect site.

Animals↗

PubMed2000

Autocrine stimulation by insulin-like growth factor 1 and insulin-like growth factor 2 mediates chondrocyte survival in vitro.

OBJECTIVE: To determine the role of autocrine stimulation by insulin-like growth factor 1 (IGF-1) and IGF-2 in mediating chondrocyte survival and to determine whether chondrocytes from older individuals are more susceptible to cell death when IGF action is blocked. METHODS: Survival was assessed in human and monkey chondrocytes cultured in suspension in alginate under serum-free conditions. The role of IGFs in mediating survival was determined by treating cultures with neutralizing antibodies to IGF-1 and IGF-2, an antibody that blocks the type 1 IGF receptor, and antisense oligonucleotides to inhibit IGF-1 production. Survival was measured in chondrocyte cultures from young and old adult monkeys in the presence and absence of the IGF receptor blocking antibody and ceramide to induce cell death. RESULTS: Cell survival of >90% was noted when chondrocytes were cultured for as long as 107 days in alginate in a supplemented serum-free medium. Compared with controls, survival was significantly reduced by treatment with neutralizing antibodies to IGF-1 (25% cell death), neutralizing antibodies to IGF-2 (18% cell death), antibody to the IGF receptor (45% cell death), and IGF-1 antisense oligonucleotides (28% cell death). Cell death from inhibition of the type 1 IGF receptor was associated with an increase in caspase 3 activity and with positive DNA fragmentation, consistent with apoptotic cell death. Chondrocytes from old adult monkeys were more susceptible to cell death than were those from young adult monkeys when the IGF receptor was blocked and cell death was further stimulated by ceramide. CONCLUSION: Autocrine production of IGFs helps to maintain chondrocyte survival in vitro and could play a similar role in vivo. With aging, chondrocytes may become more susceptible to factors that induce cell death.

Animals↗

PubMed2000

Functional estrogen receptors in adult articular cartilage: estrogen replacement therapy increases chondrocyte synthesis of proteoglycans and insulin-like growth factor binding protein 2.

OBJECTIVE: Epidemiologic studies suggest a protective effect of estrogen replacement therapy (ERT) against the development of knee and hip osteoarthritis, but a potential mechanism for this effect is not known. The present study was done to determine if functional estrogen receptors (ERs) are present in adult articular cartilage and to determine if ERT in vivo affects the production of insulin-like growth factor binding proteins (IGFBPs). METHODS: Reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry were used to measure messenger RNA (mRNA) and protein for ERs in adult monkey articular cartilage. Cultured chondrocytes transfected with a reporter construct containing the estrogen response element (ERE/luciferase) were stimulated with estrogen in vitro to determine functional activity of the ERs. IGFBP production was measured by ligand and immunoblotting of conditioned media of cells cultured from control and estrogen-treated surgically menopausal monkeys. Proteoglycan (PG) synthesis was estimated by measurement of 35SO4 incorporation. RESULTS: ERa and ERbeta mRNA were present in adult monkey articular cartilage, and ER protein was demonstrated by immunoblotting and immunohistochemistry. Estrogen treatment in vitro of cells transfected with the ERE/luciferase construct resulted in a 2.87-fold increase (P = 0.0163) in reporter production over that of untreated cells. Compared with untreated controls, IGFBP-2 production was significantly increased (P < 0.008) in conditioned media of chondrocytes cultured from monkeys that had received ERT in vivo. Increased IGFBP-2 in these cultures was associated with a 1.41-fold increase (P = 0.02) in the level of sulfate incorporation. CONCLUSION: Transcriptionally functional ER are present in adult articular cartilage, and ERT increases the production of IGFBP-2 and the synthesis of PGs by chondrocytes from surgically menopausal monkeys. These results indicate that estrogen can have a direct effect on adult articular cartilage.

Adult↗

PubMed2000

Reduction in the chondrocyte response to insulin-like growth factor 1 in aging and osteoarthritis: studies in a non-human primate model of naturally occurring disease.

OBJECTIVE: Although the development of osteoarthritis (OA) is closely associated with aging, the mechanism for this association is not clear. This study was designed to determine the effects of aging and OA on the chondrocyte response to stimulation with insulin-like growth factor 1 (IGF-1) in a non-human primate model of naturally occurring OA. METHODS: Chondrocytes were isolated from cartilage removed separately from the medial and lateral femoral condyles and tibial plateaus of cynomolgus monkeys at the time of necropsy. Each joint site was scored histologically on a scale of 0-7 for OA pathologic changes. Isolated chondrocytes were cultured in alginate in serum-free medium and stimulated with IGF-1 or des(1-3) IGF-1, which has a much lower affinity for IGF binding proteins (IGFBP) than IGF-1. Response was measured as the ability to stimulate sulfate and proline incorporation. RESULTS: Cartilage samples from 34 monkeys ranging in age from 6.7 years to 27 years and with histologic scores ranging from 0 to 7 were analyzed. A significant decline in the response to IGF-1 was noted with both increasing age and increasing OA score. Controlling for the OA score, the estimated effect of age on IGF-1 response, measured by total sulfate incorporation, was a decline of 3.81% per year (P = 0.0001), or a 75% decline over 20 years as a monkey ages from young to older adult. Controlling for age, the effect of OA score was significant only for proline incorporation in the alginate matrix (estimated slope coefficient +/-standard error -15.9 +/- 7.2; P = 0.03), suggesting a negative effect of OA on retention of 3H-proline-labeled proteins in the matrix. There was a significantly reduced response to des(1-3) IGF-1 with increasing age, but no effect of OA score on response to des(1-3) IGF-1. There was no effect of age on cell viability. CONCLUSION: These results demonstrate a significant age-related decline in the chondrocyte response to IGF-1. The finding that increasing OA score was associated with a reduced response to intact IGF-1 but not des(1-3) IGF-1 suggests a role of increased production of inhibitory IGFBP in OA. Since the cells from older animals had a reduced response to both forms of IGF-1, the mechanism of the reduced response with age cannot be attributed to changes in IGFBP. Age-related changes in IGF receptors or, more likely, age-related alterations in intracellular signal transduction may also be involved.

Aging↗

PubMed2000

Aging and the etiopathogenesis and treatment of osteoarthritis.

Knee OA in the older adult is often the result of multiple factors, some of which are intrinsic to the joint tissues and related to aging and others, such as obesity, which are extrinsic and influence the rate of development and severity of the disease. Obesity and quadricep weakness are factors that may be modifiable. The focus in managing patients with knee OA should not only be centered on the improvement of pain but also improvement in function. A combination of simple analgesics and nonpharmacologic measures, including exercises to strengthen the knee extensors, should be employed. Patients also can benefit from aerobic exercises, such as a walking or swimming program and strategies for joint protection. When medications are needed, the least toxic drugs possible should be used given the increased susceptibility of older adults to unwanted side effects.

Aged↗

PubMed2000

Integrin expression by primary and immortalized human chondrocytes: evidence of a differential role for alpha1beta1 and alpha2beta1 integrins in mediating chondrocyte adhesion to types II and VI collagen.

OBJECTIVE: Chondrocytes have been shown to express beta1-containing integrins both in vitro and in situ, but their role in regulating chondrocyte function is poorly understood. The objective of this study was to determine how the relative expression of different integrins may be modulated in relation to the differentiated state and proliferative capacity of the chondrocyte. DESIGN: Integrin expression by four different cell lines of human chondrocytes immortalized with Simian virus 40 large T-antigen (SV40-TAg) was studied and compared to primary chondrocytes. Differences in alpha1 and alpha2 integrin subunit expression were utilized to further study the role of these integrins in mediating adhesion to types II and VI collagen. RESULTS: The overall cell-surface levels of beta1-containing integrins were higher on all four immortalized cell lines which expressed over 10-fold higher levels of alpha2 and alpha3 integrin subunits compared to primary cells. However, primary cells expressed higher levels of the alpha1 integrin subunit which was not expressed by T/C28a4 cells and expressed at variable and lower levels in the other lines. Levels of the alpha3 integrin subunit were significantly greater on the highly proliferative juvenile costal chondrocyte lines (T/C-28a4, C-2812, and C-20a4) compared to primary articular chondrocytes and tsT/AC-62 cells which were derived from adult articular chondrocytes. Expression of alpha5 was similar among primary cells and cell lines except on C-20/A4 cells which had an average of over 4-fold higher levels. None of the primary or immortalized chondrocytes tested expressed significant levels of alpha4. Cell adhesion assays revealed that both alpha1beta1 and alpha2beta1 could serve as chondrocyte adhesion receptors for types II and VI collagen. In cell lines expressing both integrins, alpha1beta1 was the preferential receptor for type VI collagen while alpha2beta1 was the preferential receptor for type II collagen. Rather than inhibiting adhesion, incubation with the alpha3 blocking antibody P1B5 increased adhesion of C-28/12 cells to both fibronectin and type II collagen by 67% and 100% respectively. CONCLUSIONS: Immortalization with SV40-TAg results in altered integrin expression by chondrocytes. Changes in the relative levels of alpha1, alpha2, and alpha3 subunits may significantly alter the manner in which chondrocytes interact with types II and VI collagen in the extracellular matrix.

Adult↗

PubMed2000

Exercise and weight loss in obese older adults with knee osteoarthritis: a preliminary study.

OBJECTIVE: The purposes of this pilot study were to determine if a combined dietary and exercise intervention would result in significant weight loss in older obese adults with knee osteoarthritis, and to compare the effects of exercise plus dietary therapy with exercise alone on gait, strength, knee pain, biomarkers of cartilage degradation, and physical function. DESIGN: Single-blind, two-arm, randomized clinical trial conducted for 24 weeks. SETTING: A university health and exercise science center. PARTICIPANTS: Twenty-four community-dwelling obese older adults aged > or = 60 years, body mass index > or = 28, knee pain, radiographic evidence of knee osteoarthritis, and self-reported physical disability. INTERVENTION: Randomization into two groups: exercise and diet (E&D) and exercise alone (E). Exercise consisted of a combined weight training and walking program for 1 hour three times per week. The dietary intervention included weekly sessions with a nutritionist utilizing cognitive-behavior modification to change dietary habits to reach a group goal of an average weight loss of 15 lb (6.8 kg) over 6 months. MEASUREMENTS: All measurements were conducted at baseline and 3 and 6 months, except for synovial fluid analysis, which was obtained only at baseline and 6 months. In addition, weight was measured weekly in the E&D group. Physical disability and knee pain were measured by self-report and physical performance was measured using the 6-minute walk and stair climb tasks. Biomechanical testing included kinetic and kinematic analysis of gait and isokinetic strength testing. Synovial fluid was analyzed for levels of total proteoglycan, keratan sulfate, and interleukin-1 beta. RESULTS: Twenty-one of the 24 participants completed the study, with one dropout in the E&D group and two in the E group. The E&D group lost a mean of 18.8 lb (8.5 kg) at 6 months compared with 4.0 lb (1.8 kg) in the E group (P = .01). Significant improvements were noted in both groups in self-reported disability and knee pain intensity and frequency as well as in physical performance measures. However, no statistical differences were found between the two groups at 6 months in knee pain scores or self-reported performance measures of physical function. There was no difference in knee strength between the groups, with both groups showing modest improvements from baseline to 6 months. At 6 months, the E&D group had a significantly greater loading rate (P = .03) and maximum braking force (P = .01) during gait. There were no significant between-group differences in the other biomechanical measures. Synovial fluid samples were obtainable at both baseline and 6 months in eight participants (four per group). The level of keratan sulfate decreased similarly in both groups from an average baseline of 96.8 +/- 37.1 to 71.5 +/- 23 ng/microg total proteoglycan. The level of IL-1 decreased from 25.3 +/- 9.8 at baseline to 8.3 +/- 6.1 pg/mL. The decrease in IL-1 correlated with the change in pain frequency (r = -0.77, P = .043). CONCLUSIONS: Weight loss can be achieved and sustained over a 6-month period in a cohort of older obese persons with osteoarthritis of the knee through a dietary and exercise intervention. Both exercise and combined weight loss and exercise regimens lead to improvements in pain, disability, and performance. Moreover, the trends in the biomechanical data suggest that exercise combined with diet may have an additional benefit in improved gait compared with exercise alone. A larger study is indicated to determine if weight loss provides additional benefits to exercise alone in this patient population.

Activities of Daily Living↗

PubMed2000

Chondrocyte integrin expression and function.

The extracellular matrix (ECM) is an "information rich" environment and interactions between the chondrocyte and ECM regulate many biological processes important to cartilage homeostasis and repair including cell attachment, growth, differentiation, and survival. The integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these processes. Chondrocytes have been found to express several members of the integrin family which can serve as receptors for fibronectin (alpha 5 beta 1), types II and VI collagen (alpha 1 beta 1, alpha 2 beta 1, alpha 10 beta 1), laminin (alpha 6 beta 1), and vitronectin and osteopontin (alpha V beta 3). Integrin expression can be regulated by growth factors including IGF-I and TGF-beta. By providing a link between the ECM and the cytoskeleton, integrins may be important transducers of mechanical stimuli. Integrin binding stimulates intracellular signaling which can affect gene expression and regulate chondrocyte function. Further studies are needed to more clearly define the role of integrins in cartilage.

Animals↗

PubMed1999

Cell surface receptors transmit sufficient force to bend collagen fibrils.

To better understand the dynamic interaction of cells with their surrounding extracellular matrix, chondrocytes and rat embryo fibroblasts were overlaid with individual collagen fibrils and observed with high-resolution video-enhanced differential interference contrast microscopy. Although the cells had a polygonal shape characteristic of nonmotile cells, they used processes usually associated with cell locomotion to acquire the collagen fibrils. Instead of being transported in a retrograde direction, fibrils on the dorsal cell surface were bent, and regions of the bent fibrils were shifted in diverse directions. A blocking antibody to the beta1 integrin subunit significantly inhibited collagen fibril acquisition and bending. Enhanced actin assembly was only occasionally associated with fibrils undergoing rearrangement. Considering that the relatively stiff collagen fibrils require the application of force to be bent, this study shows that cells with a polygonal morphology (as opposed to a polarized, motile shape) are capable of exerting force through the beta1 integrins on the dorsal surface of the cell. Analysis of the bending patterns indicates that fibril buckling was induced by retrograde force combined with regions held stationary and/or the fibrils were bent by forces acting in opposing directions.

Actins↗

PubMed1999

Estrogen replacement therapy modulation of the insulin-like growth factor system in monkey knee joints.

OBJECTIVE: Epidemiologic studies have suggested that estrogen replacement therapy may lower the risk of osteoarthritis in women, but the mechanism of this effect is unknown. Since estrogen acts in other tissues in part through regulation of the insulin-like growth factor (IGF) system as well as cytokines including interleukin-6 (IL-6), we determined whether estrogen replacement regulates the levels of these factors in synovial fluid (SF). METHODS: Levels of IGF-1, IGF-2, IGF binding proteins (IGFBP) 1-3, and IL-6 were measured in SF samples obtained from 67 female adult cynomolgus monkeys that had been ovariectomized and treated for 30 months in 1 of 3 groups. Group 1 (n = 24) had no estrogen replacement (control), group 2 (n = 22) received estrogen (Premarin) at the human equivalent of 0.625 mg/day, and group 3 (n = 21) received estrogen at the same dose as group 2, plus progesterone (Provera) at the equivalent of 2.5 mg/day. RESULTS: Compared with controls, estrogen-treated monkeys had 2-fold higher SF levels of IGF-1 (P < 0.001), 1.7-fold higher IGF-2 (P < 0.006), 5.9-fold higher IGFBP-1 (P < 0.02), and 2.5-fold higher IGFBP-3 (P < 0.001). Estrogen plus progesterone-treated monkeys had SF levels of IGF-1, IGF-2, IGFBP-1, and IGFBP-3 that were intermediate between the levels in the control and estrogen groups, except that the level of IGFBP-3 was significantly greater than that in the control group (P < 0.001). SF levels of IGFBP-2 and IL-6 did not differ by treatment group. Treatment group did not affect the serum levels of IGF-1 and IL-6, but IGF-2 and IGFBP-3 were increased by 1.6- and 1.8-fold, respectively, in the estrogen group (P < 0.001). There was no correlation between changes in serum and SF levels of IGF components, except for a weak correlation for IGFBP-3 levels from control (r = 0.464, P = 0.04) and estrogen-treated (r = 0.577, P = 0.008) animals. CONCLUSION: This study demonstrates a significant effect of estrogen replacement on IGF system components in synovial fluid, of which at least some are distinct from any systemic changes observed. The results indicate a potential stimulatory effect of estrogen on joint tissues in vivo.

Animals↗

PubMed1998

Evaluation of musculoskeletal complaints in the older adult.

During the evaluation of older adults with musculoskeletal complaints, it is necessary to differentiate periarticular conditions from intraarticular or arthritic conditions. It also is useful to classify the symptoms as acute or chronic and as mono-, oligo-, or polyarticular. Close attention should be paid to the history and physical exam findings, while laboratory tests and radiographs should only be used as supporting evidence of a clinical diagnosis. A search for the presence of joint inflammation is particularly important in the diagnostic process. Urgent evaluation is necessary in a patient with an acutely warm and swollen joint to rule out the presence of infection.

Aged↗

PubMed1997

Growth factor regulation of chondrocyte integrins. Differential effects of insulin-like growth factor 1 and transforming growth factor beta on alpha 1 beta 1 integrin expression and chondrocyte adhesion to type VI collagen.

OBJECTIVE: The ability of growth factors to modulate integrin expression may be important with regard to processes involved in tissue repair and remodeling. This study was undertaken to determine the effect of transforming growth factor beta (TGF beta) and insulin-like growth factor 1 (IGF-1) on chondrocyte beta 1 integrin expression and integrin-mediated adhesion to extracellular matrix proteins. METHODS: Chondrocytes obtained from normal bovine articular cartilage were cultured in the presence or absence of 10% fetal bovine serum, 100 pM IGF-1, or 100 pM TGF beta. Integrin expression and function were measured by protein blotting of immunoprecipitated integrins, Northern blot analysis, and cell adhesion assays. RESULTS: Immunoprecipitation with an anti-beta 1 integrin antibody coprecipitated the alpha 1 integrin subunit and a band representing alpha 3 and alpha 5, as previously reported. Compared with serum-free cultures, the use of serum resulted in an average 10-fold increase in the alpha 1 band and a 12-fold increase in the alpha 3/alpha 5 band. IGF-1 increased alpha 1 and alpha 3/alpha 5 by an average of 3-fold and 4-fold, respectively. TGF beta also increased alpha 3/alpha 5 by > 5-fold but decreased alpha 1 to an average of 24% of that found in serum-free controls. Northern blot analysis revealed that TGF beta significantly increased alpha 5 integrin subunit RNA levels. IGF-1 did not have a significant effect on alpha 5 or alpha 1 integrin subunit RNA levels, suggesting that its effects on integrins are posttranscriptional. In cell adhesion assays, TGF beta treatment resulted in a 50% decrease in the adhesion of chondrocytes to type VI collagen, while adhesion to type II collagen and fibronectin was stimulated. IGF-1 stimulated adhesion to all 3 proteins. An alpha 1 integrin blocking antibody inhibited up to 75% of the adhesion of human chondrocytes to type VI collagen. CONCLUSION: Both IGF-1 and TGF beta stimulate chondrocyte cell surface expression of the alpha 3/alpha 5 integrin subunit band and stimulate adhesion of chondrocytes to fibronectin and type II collagen. The 2 growth factors have opposite effects on expression of alpha 1 beta 1, with IGF-1 increasing and TGF beta decreasing cell surface levels of this integrin. TGF beta-treated cells also have decreased adhesion to type VI collagen. The opposing effects of IGF-1 and TGF beta on chondrocyte expression of alpha 1 beta 1 and on adhesion to type VI collagen suggest that alpha 1 beta 1 mediates chondrocyte adhesion to type VI collagen. This was confirmed by using an antibody to the alpha 1 integrin subunit to block adhesion of chondrocytes to type VI collagen.

Animals↗

PubMed1997

Human chondrocyte expression of growth-arrest-specific gene 6 and the tyrosine kinase receptor axl: potential role in autocrine signaling in cartilage.

OBJECTIVE: To determine if human articular chondrocytes express the axl tyrosine kinase receptor and its ligand Gas-6, a protein product of growth-arrest-specific gene 6, and to determine if Gas-6 and axl function in the regulation of chondrocyte growth and survival. METHODS: The presence of Gas-6 and axl was examined in situ in human articular cartilage by immunohistochemistry and in vitro in cell culture studies using primary human chondrocytes and immortalized human chondrocytes. The ability of recombinant Gas-6 to mediate adhesion of chondrocytes and to stimulate chondrocyte axl phosphorylation was determined. Studies of the role of Gas-6 and axl in cell proliferation and survival were also performed. RESULTS: Both Gas-6 and axl were detected in cartilage by immunohistochemical staining. Gas-6 and axl messenger RNA (mRNA) and protein were also detected in cultures of primary and immortalized human chondrocytes. Compared with cells cultured in medium containing 10% serum, Gas-6 mRNA levels were increased in immortalized chondrocytes cultured in serum-free medium, while axl expression decreased. Chondrocytes attached to Gas-6-coated plastic, and the attachment was blocked by a soluble Ig fusion protein containing the axl extracellular domain. Recombinant human Gas-6 and serum-free conditioned medium from primary and immortalized human chondrocyte cultures stimulated chondrocyte axl tyrosine phosphorylation. A mitogenic effect was noted both when immortalized chondrocytes were stimulated with recombinant Gas-6 or when they were made to overexpress axl by transfection. Addition of recombinant Gas-6 to serum-free medium resulted in increased survival of primary chondrocytes cultured at low density in agarose. CONCLUSION: These findings present evidence for an autocrine signaling pathway in cartilage involving Gas-6 and the axl tyrosine kinase adhesion receptor. Stimulation of axl by Gas-6 may play an important role in the control of chondrocyte growth and survival.

3T3 Cells↗

PubMed1996

Osteoarthritis in cynomolgus macaques. III: Effects of age, gender, and subchondral bone thickness on the severity of disease.

Knee joints from cynomolgus monkeys of both genders and a wide range of ages were examined to characterize further the natural history of osteoarthritis (OA) in these animals. The objectives of this study were to characterize better the subchondral bone changes previously noted in this disease, to determine whether the severity of OA in these animals is affected by age or weight, and to determine whether males and females are affected similarly. As had been seen in previous studies, the medial tibial plateau was the most severely affected site. The thickness of the subchondral plate in the medial tibial plateau increased with increasing severity of articular cartilage lesions in both males and females; however, in monkeys with subchondral plate thicknesses less than 400 microns, articular cartilage lesions were essentially absent. Subchondral plate thickness increased with increasing weight in both genders, but females had a higher subchondral plate thickness than males for a given body weight. There was no correlation between bone volume in the proximal tibial epiphysis and articular cartilage lesions of OA. The prevalence and severity of OA in the medial tibial plateau increased with increasing age, but were not affected by gender or weight. Although there was no correlation between articular cartilage lesions and body mass index or weight, the waist/hip circumference ratio and severity of articular cartilage lesions were correlated in the female monkeys. This work provides evidence that thickening of the subchondral bone plate may be more important than the volume of epiphyseal/metaphyseal cancellous bone in determining the biomechanical stresses in the joint and in influencing the development of articular cartilage lesions.

Aging↗

PubMed1995

Osteoarthritis in cynomolgus macaques. II. Detection of modulated proteoglycan epitopes in cartilage and synovial fluid.

The purpose of the present study was to determine the usefulness of the monoclonal antibodies 7-D-4 and 3-B-3 as biomarkers of severity of naturally occurring osteoarthritis in the knee joints of adult cynomolgus macaques. The antibodies were used to immunolocate chondroitin sulfate proteoglycan epitopes in articular cartilage or synovial fluid from knee joints with a range in severity of osteoarthritis. The joints were examined radiographically, grossly, microradiographically, and histologically to characterize the severity of disease, and the results of three different methods of proteoglycan analysis (immunohistochemistry, enzyme-linked immunosorbent assay, and Western blot analysis) were compared. Subjectively, the degree of positive immunostaining for 7-D-4 was minimal in normal sites and increased as damage to articular cartilage increased. The scores for 7-D-4 immunostaining in the medial tibial plateau (the site most severely involved in this model) were correlated significantly with severity of damage to articular cartilage (p < 0.05, r2 = 0.50), thus supporting the subjective observations. The ratio of 7-D-4 to sulfated glycosaminoglycans in synovial fluid also was correlated with the score for 7-D-4 immunostaining in the medial tibial plateau (p < 0.05, r2 = 0.54) and with the score for 3-B-3 immunostaining in the medial femoral condyle (p < 0.05, r2 = 0.65). There were no significant correlations among scores for 3-B-3 immunostaining, severity scores, and the ratios of 3-B-3 to sulfated glycosaminoglycans in the synovial fluid. By Western blot analysis, both epitopes were sensitive markers of early cartilage damage in young adult monkeys but were less sensitive in older monkeys. This work provides evidence that measurement of the epitope recognized by 7-D-4 in synovial fluid or, by immunohistochemical or Western blot methods, in articular cartilage has potential use as a marker of severity of naturally occurring osteoarthritis.

Animals↗

PubMed1995

Expression of beta 1 integrins by cultured articular chondrocytes and in osteoarthritic cartilage.

Expression of beta 1 integrins was studied in vitro as articular chondrocytes reestablished a matrix in culture and in situ in a nonhuman primate model of osteoarthritis in order to investigate a potential role for integrins in mediating cell-extracellular matrix interactions in cartilage. Chondrocytes were found to express alpha 1 beta 1, alpha 3 beta 1, and alpha 5 beta 1 integrins both in vitro and in situ. Cell surface expression of beta 1 integrins increased as chondrocytes were maintained in cultured from 3 to 7 days. Increased beta 1 integrin expression was also observed in osteoarthritic cartilage compared with normal cartilage. The greatest relative increase in both systems was noted for the alpha 1 beta 1 integrin. The increase in chondrocyte beta 1 integrin expression in vitro was noted in both monolayer and alginate cultures and occurred prior to detectable changes in the differentiated phenotype of the chondrocyte. Disruption of the cytoskeleton with the drug dihydrocytochalasin B inhibited the cell culture induced increase in integrin expression, while treatment of cultured cells with TGF-beta resulted in increased expression of the alpha 5 beta 1 integrin. The modulation of beta 1 integrin expression noted in vitro and in situ indicates that chondrocytes are capable of regulated expression of beta 1 integrins and suggests that beta 1 integrins may play an important role in mediating chondrocyte-extracellular matrix interactions in cartilage.

Animals↗