Intraarticular injection of heparin‐binding insulin‐like growth factor 1 sustains delivery of insulin‐like growth factor 1 to cartilage through binding to chondroitin sulfate

Objective

Insulin‐like growth factor 1 (IGF‐1) stimulates cartilage repair but is not a practical therapy due to its short half‐life. We have previously modified IGF‐1 by adding a heparin‐binding domain and have shown that this fusion protein (HB‐IGF‐1) stimulates sustained proteoglycan synthesis in cartilage. This study was undertaken to examine the mechanism by which HB‐IGF‐1 is retained in cartilage and to test whether HB‐IGF‐1 provides sustained growth factor delivery to cartilage in vivo and to human cartilage explants.

Methods

Retention of HB‐IGF‐1 and IGF‐1 was analyzed by Western blotting. The necessity of heparan sulfate (HS) or chondroitin sulfate (CS) glycosaminoglycans (GAGs) for binding was tested using enzymatic removal and cells with genetic deficiency of HS. Binding affinities of HB‐IGF‐1 and IGF‐1 proteins for isolated GAGs were examined by surface plasmon resonance and enzyme‐linked immunosorbent assay.

Results

In cartilage explants, chondroitinase treatment decreased binding of HB‐IGF‐1, whereas heparitinase had no effect. Furthermore, HS was not necessary for HB‐IGF‐1 retention on cell monolayers. Binding assays showed that HB‐IGF‐1 bound both CS and HS, whereas IGF‐1 did not bind either. After intraarticular injection in rat knees, HB‐IGF‐1 was retained in articular and meniscal cartilage, but not in tendon, consistent with enhanced delivery to CS‐rich cartilage. Finally, HB‐IGF‐1 was retained in human cartilage explants but IGF‐1 was not.

Conclusion

Our findings indicate that after intraarticular injection in rats, HB‐IGF‐1 is specifically retained in cartilage through its high abundance of CS. Modification of growth factors with heparin‐binding domains may be a new strategy for sustained and specific local delivery to cartilage.

     

Synthesis of insulin‐like growth factor binding protein 3 in vitro in human articular cartilage cultures

Objective

To quantify the rate of synthesis of insulin‐like growth factor binding protein 3 (IGFBP‐3) and insulin‐like growth factor 1 (IGF‐1) by in vitro cultures of normal and osteoarthritic (OA) human articular cartilage.

Methods

Levels of IGF‐1 and IGFBP‐3 in media from in vitro cultures of human cartilage were determined by radioimmunoassay (RIA). IGFBPs were characterized by immunoblots and ligand blots. Ultrafiltration and RIA analysis of synovial fluid (SF) samples and washings of cartilage samples ex vivo were used to calculate partition coefficients and to estimate the amount of IGF‐1 and IGFBP‐3 in cartilage in vivo.

Results

OA cartilage synthesized 150 ng of IGFBP‐3 per gm of cartilage per day, compared with 50 ng synthesized by normal cartilage. The surface zone of normal cartilage produced more IGFBP‐3 than did the deep zone. Immunoblots and ligand blots confirmed the presence of IGFBP‐3. IGFBP‐3 synthesis was stimulated by exogenous IGF‐1. No freshly synthesized IGF‐1 was detected. The quantities of IGF‐1 and IGFBP‐3 present ex vivo were 11.3 and 78.7 ng/gm of cartilage in normal cartilage and 21.6 and 225.4 ng/gm in OA cartilage.

Conclusion

The results show that while IGFBP‐3 is synthesized in explant cultures, IGF‐1 is not. The rate of IGFBP‐3 synthesis is 3 times higher in OA than in normal cartilage. Both IGFBP‐3 and IGF‐1 penetrate into cartilage from SF in vivo. We estimate that the quantities of IGFBP‐3 produced in culture by human cartilage are small compared with the amount supplied in the form of “small complexes” from the circulation. The high value of the partition coefficient of IGFBP‐3 implies binding to the matrix.

     

Basic fibroblast growth factor inhibits the anabolic activity of insulin‐like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes

Objective

To determine the effects of basic fibroblast growth factor (bFGF) on the chondrocyte anabolic activity promoted by insulin‐like growth factor 1 (IGF‐1) and osteogenic protein 1 (OP‐1).

Methods

Human articular chondrocytes were cultured in alginate beads or as cartilage explants in serum‐free medium with or without IGF‐1 (100 ng/ml), OP‐1 (100 ng/ml), or bFGF (0–100 ng/ml). Cell survival, proliferation, proteoglycan synthesis, and total proteoglycan accumulation were measured after 21 days of culture in alginate beads, and proteoglycan synthesis was measured in explants.

Results

Cell survival was not altered by bFGF at any dose, and chondrocyte proliferation was stimulated only at doses above 1 ng/ml. When combined with IGF‐1, 1 ng/ml of bFGF stimulated proliferation to 170% of control, but when combined with IGF‐1 and OP‐1, proliferation increased to 373% of control. Doses of bFGF of 100 ng/ml decreased total proteoglycan levels accumulated per cell by 60% compared with control and also inhibited the ability of IGF‐1 or OP‐1 to increase proteoglycan production. Likewise, sulfate incorporation in response to IGF‐1 and OP‐1 alone or together was completely inhibited by 50 ng/ml bFGF in both alginate and explant cultures.

Conclusion

The anabolic activity of IGF‐1 and OP‐1, alone and in combination, is significantly inhibited by bFGF. The results suggest that excessive release of bFGF from the cartilage matrix during injury, with loading, or in arthritis could contribute to increased proliferation and reduced anabolic activity in articular cartilage.

     

Genetic inhibition of fibroblast growth factor receptor 1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice

Objective

Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR‐1) in the development of osteoarthritis (OA) and its underlying mechanisms.

Methods

FGFR‐1 was deleted from the articular chondrocytes of adult mice in a cartilage‐specific and tamoxifen‐inducible manner. Two OA models (aging‐associated spontaneous OA, and destabilization‐induced OA), as well as an antigen‐induced arthritis (AIA) model, were established and tested in Fgfr1‐deficient and wild‐type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR‐1 inhibitor on human articular chondrocytes was examined.

Results

The gross morphologic features of Fgfr1‐deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12‐month‐old Fgfr1‐deficient mice displayed greater aggrecan staining compared to 12‐month‐old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR‐1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP‐13) and up‐regulation of FGFR‐3 in mouse and human articular chondrocytes.

Conclusion

Disruption of FGFR‐1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down‐regulation of MMP‐13 expression and up‐regulation of FGFR‐3 levels may contribute to the phenotypic changes observed in Fgfr1‐deficient mice.

     

One of two chondrocyte‐expressed isoforms of cartilage intermediate‐layer protein functions as an insulin‐like growth factor 1 antagonist

Objective

Aging and osteoarthritic (OA) cartilage commonly demonstrate enhanced expression of the large, transforming growth factor β (TGFβ)–inducible glycoprotein cartilage intermediate‐layer protein (CILP) as well as enhanced extracellular inorganic pyrophosphate (PPi) that promotes the deposition of calcium pyrophosphate dihydrate crystals. In normal chondrocytes, TGFβ induces elevated chondrocyte extracellular PPi. Insulin‐like growth factor 1 (IGF‐1) normally blocks this response and reduces extracellular PPi. However, chondrocyte resistance to IGF‐1 is observed in OA and aging. Because CILP was reported to chromatographically fractionate with PPi‐generating nucleotide pyrophosphatase phosphodiesterase (NPP) activity, it has been broadly assumed that CILP itself has NPP activity. Our objective was to directly define CILP functions and their relationship to IGF‐1 in chondrocytes.

Methods

Using primary cultures of articular chondrocytes from the knee, we defined the function of the previously described CILP (CILP‐1) and of a recently described 50.6% identical protein that we designated the CILP‐2 isoform.

Results

Both CILP isoforms were constitutively expressed by primary cultured articular chondrocytes, but only CILP‐1 expression was detectable in cultured knee meniscal cartilage cells. Neither CILP isoform had intrinsic NPP activity. But CILP‐1 blocked the ability of IGF‐1 to decrease extracellular PPi, an activity specific for the CILP‐1 N‐terminal domain. The CILP‐1 N‐terminal domain also suppressed IGF‐1–induced (but not TGFβ‐induced) proliferation and sulfated proteoglycan synthesis, and it inhibited ligand‐induced IGF‐1 receptor autophosphorylation.

Conclusion

Two CILP isoforms are differentially expressed by chondrocytes. Neither CILP isoform exhibits PPi‐generating NPP activity. But, increased expression of CILP‐1, via N‐terminal domain–mediated inhibitory effects of CILP‐1 on chondrocyte IGF‐1 responsiveness, could impair chondrocyte growth and matrix repair and indirectly promote PPi supersaturation in aging and OA cartilage.

     

Detection of nitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin‐1β and with chondrocyte resistance to insulin‐like growth factor 1

Objective

To determine whether oxidative damage to cartilage proteins can be detected in aging and osteoarthritic (OA) cartilage, and to correlate the results with the local production of interleukin‐1β (IL‐1β) and the responsiveness of isolated chondrocytes to stimulation with insulin‐like growth factor 1 (IGF‐1).

Methods

The presence of nitrotyrosine was used as a measure of oxidative damage. Histologic sections of knee articular cartilage, obtained from young adult and old adult cynomolgus monkeys, which develop age‐related, naturally occurring OA, were evaluated. Each cartilage section was graded histologically on a scale of 0–7 for the presence of OA‐like changes, and serial sections were immunostained using antibodies to nitrotyrosine and IL‐1β. Chondrocytes isolated and cultured from cartilage adjacent to the sections used for immunostaining were tested for their response to IGF‐1 stimulation by measuring sulfate incorporation in alginate cultures. For comparison with the monkey tissues, cartilage sections from human tissue donors and from tissue removed at the time of OA‐related joint replacement surgery were also immunostained for nitrotyrosine and IL‐1β.

Results

The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans. All sections that were highly positive for IL‐1β also showed staining for nitrotyrosine. However, in a few sections from older adult monkeys and humans, nitrotyrosine was present but IL‐1β was absent, suggesting that some age‐related oxidative damage is independent of IL‐1β. In chondrocytes that were isolated from monkey cartilage positive for nitrotyrosine or IL‐1β, the response to stimulation with IGF‐1 was significantly reduced. In some samples from older adult monkeys, IGF‐1 resistance was seen in cells isolated from tissue that did not stain for nitrotyrosine or IL‐1β.

Conclusion

Oxidative damage due to the concomitant overproduction of nitric oxide and other reactive oxygen species is present in both aging and OA cartilage. This damage can contribute to the resistance of chondrocytes to IGF‐1 stimulation, but it is unlikely to be the sole cause of IGF‐1 resistance in these chondrocytes.

     

Effects of antiinflammatory drugs on arthritic cartilage: A high‐frequency quantitative ultrasound study in rats

Objective

To evaluate the ability of 55‐MHz quantitative ultrasound (US) to detect the in vivo effects of experimental arthritis, as well as those of two antiinflammatory drugs, naproxen (NPX) and dexamethasone (DEX), on cartilage and subchondral bone.

Methods

Arthritis was induced in both knees of 108 rats by intraarticular injection of zymosan (ZYM). Two groups of arthritic rats (n = 36 per group) were treated daily with either NPX (10 mg/kg/day) or DEX (0.1 mg/kg/day). Using a 3‐dimensional US microscope, patellae were explored in vitro on days 5, 14, and 21 after injections. US assessment included the analysis of quantitative indices of local modifications involving cartilage and bone: integrated reflection coefficient (IRC) from the cartilage surface and apparent integrated backscatter from the cartilage internal structure (cartilage matrix) (AIB_cartilage) and the cartilage–bone interface (AIB_bone).

Results

ZYM induced articular surface fibrillation that resulted in a decrease in IRC at all times (P < 0.02) and in an increase in AIB_bone on days 5 and 14 (P < 0.005). Fibrillation was not changed by NPX administration, while it disappeared following DEX treatment. Cartilage–bone interface alterations were prevented by DEX and partially compensated for by NPX. Cartilage matrix echogenicity decreased with time in all groups due to maturation (P < 0.05), except in DEX‐treated rats.

Conclusion

Quantitative 55 MHz US allowed detection of early cartilage and bone lesions due to experimental arthritis, and also allowed detection of the effects of antiinflammatory drugs. NPX seemed to have an effect on subchondral bone lesions, but not on cartilage. DEX appeared to repair articular surface and bone, but prevented animal growth and cartilage maturation.

     

The insulin‐like growth factor binding proteins in uncultured human cartilage: Increases in insulin‐like growth factor binding protein 3 during osteoarthritis

Objective

To assess changes in the insulin‐like growth factor binding proteins (IGFBPs) in uncultured cartilage during stages of osteoarthritis (OA), and to determine if OA cartilage is capable of autocrine secretion of IGFBPs.

Methods

Articular cartilage was dissected from fibrillated and nonfibrillated sites of 11 human femoral heads, and extracted in buffer containing 8M urea. IGFBPs were identified by immunoprecipitation and subsequent analysis by ¹²⁵I–IGF‐2 Western ligand blotting (WLB), radioimmunoassay, or 2‐site immunoradiometric assay (IRMA). IGFBPs were assessed in cartilage extracts by WLB. IGFBP‐3 content was determined by IRMA and synthesis by metabolic labeling with ³⁵S‐cysteine in organ cultures.

Results

Sample grouping into 3 distinct OA strata was supported by gross pathology of the femoral heads, histologic grading of cartilage slices, and biochemical analysis of the glycosaminoglycan and protein content of the extracts. Group I was normal/mild OA, group II was intermediate OA, and group III was severe OA. IGFBP‐2 was present in all samples, IGFBP‐4 in sporadic samples, and BP‐3 in group II–III samples. By IRMA, group I had a mean ± SD of 6.26 ± 2.6 ng IGFBP‐3/mg soluble protein (IGFBP‐3) (n = 6), group II had a mean ± SD 14 ± 7.5 IGFBP‐3 (n = 10), and group III had a mean ± SD 17.03 ± 8.94 IGFBP‐3 (n = 6). Analysis of variance showed group differences (F[3,19] = 3.84, P = 0.04), and post hoc tests revealed that IGFBP‐3 levels were higher for group III versus group I (P = 0.04). OA cartilage synthesized IGFBP‐3.

Conclusion

Increases in net cartilage content of IGFBP‐3 occurred in intact OA cartilage, reaching statistically significant elevation in severe disease. There was autocrine IGFBP‐3 production in OA cartilage.

     

Effects of a stress‐reduction program on psychological function,pain, and physical function of systemic lupus erythematosus patients: A randomized controlled trial

Objective

To assess the effects of a stress‐reduction program on pain, psychological function, and physical function in persons with systemic lupus erythematosus (SLE) who experience pain.

Methods

Ninety‐two SLE patients were assigned randomly to receive either biofeedback‐assisted cognitive‐behavioral treatment (BF/CBT), a symptom‐monitoring support (SMS) intervention, or usual medical care (UC) alone.

Results

BF/CBT participants had significantly greater reductions in pain and psychological dysfunction compared with the SMS group (pain, P = 0.044; psychological functioning, P < 0.001) and the UC group (pain, P = 0.028; psychological functioning, P < 0.001). BF/CBT had significantly greater improvement in perceived physical function compared with UC (P = 0.035), and improvement relative to SMS was marginally significant (P = 0.097). At a 9‐month followup evaluation, BF/CBT continued to exhibit relative benefit compared with UC in psychological functioning (P = 0.023).

Conclusion

This study supports the utility of a brief stress management program for short‐term improvement in pain, psychological function, and perceived physical function among persons with SLE who experience pain.

     

Clinical optical coherence tomography of early articular cartilage degeneration in patients with degenerative meniscal tears

Objective

Quantitative and nondestructive methods for clinical diagnosis and staging of articular cartilage degeneration are important to the evaluation of potential disease‐modifying treatments in osteoarthritis (OA). Optical coherence tomography (OCT) is a novel imaging technology that can generate microscopic‐resolution cross‐sectional images of articular cartilage in near real‐time. This study tested the hypotheses that OCT can be used clinically to identify early cartilage degeneration and that OCT findings correlate with magnetic resonance imaging (MRI) T2 values and arthroscopy results.

Methods

Patients undergoing arthroscopy for degenerative meniscal tears were recruited under Institutional Review Board–approved protocols. Thirty consecutive subjects completing preoperative 3.0T MRI, arthroscopy, and intraoperative OCT comprised the study group. Qualitative and quantitative OCT results and MRI T2 values were compared with modified Outerbridge cartilage degeneration scores (0–4 scale) assigned at arthroscopy.

Results

Arthroscopic grades showed cartilage abnormality in 23 of the 30 patients. OCT grades were abnormal in 28 of the 30 patients. Both qualitative and quantitative OCT strongly correlated with the arthroscopy results (P = 0.004 and P = 0.0002, respectively, by Kruskal‐Wallis test). Neither the superficial nor the deep cartilage T2 values correlated with the arthroscopy results. The quantitative OCT results correlated with the T2 values in the superficial cartilage (Pearson's r = 0.39, P = 0.03).

Conclusion

These data show that OCT can be used clinically to provide qualitative and quantitative assessments of early articular cartilage degeneration that strongly correlate with arthroscopy results. The correlation between the quantitative OCT values and T2 values for the superficial cartilage further supports the utility of OCT as a clinical research tool, providing quantifiable microscopic resolution data on the articular cartilage structure. New technologies for nondestructive quantitative assessment of human articular cartilage degeneration may facilitate the development of strategies to delay or prevent the onset of OA.

     

The combination of insulin‐like growth factor 1 and osteogenic protein 1 promotes increased survival of and matrix synthesis by normal and osteoarthritic human articular chondrocytes

Objective

Although growth factor therapy could be an attractive method for stimulating the repair of damaged cartilage matrix, there is evidence that with aging and/or with the development of osteoarthritis (OA), articular chondrocytes may become unresponsive to growth factor stimulation. The aim of the current study was to compare the ability of insulin‐like growth factor+(IGF‐1) and osteogenic protein+(OP‐1), alone and in combination, to stimulate human normal and OA chondrocytes in culture.

Methods

Chondrocytes isolated by enzymatic digestion of cartilage obtained from subjects undergoing knee replacement for OA (n = 6) or from normal ankle joints of tissue donors (n = 7) were cultured in alginate beads in serum‐free medium and treated for 21 days with 100 ng/ml IGF‐1, 100 ng/ml OP‐1, or both. Controls were treated with vehicle alone. The cultures were evaluated for cell survival, cell number by DNA analysis, matrix production by particle exclusion assay, and level of accumulated proteoglycan by dimethylmethylene blue assay.

Results

After 21 days in serum‐free alginate culture, survival of cells from OA cartilage was 65 ± 2% (mean ± SEM), while survival of cells from normal cartilage was significantly greater (82 ± 3%). Treatment with either IGF‐1 or OP‐1 alone minimally improved survival, while the combination IGF +OP significantly improved survival, to 87 ± 2% for OA cells and 95±1% for normal cells. Cell proliferation was noted only in the IGF+OP group; this was significant for both normal and OA cells (∼2‐fold increase in DNA levels). Matrix production, assessed by particle exclusion and by proteoglycan accumulation, was greatest in the cells treated with IGF + OP in both normal and OA cultures. When proteoglycan levels were corrected for cell numbers (mg proteoglycan/ng DNA), a significant increase over control was noted with OP‐1 alone and IGF IGF‐1 alone, in both normal and OA cultures, with the greatest levels in the combination group (3‐fold increase over control).

Conclusion

OP‐1 was more potent than IGF‐1 in stimulating proteoglycan production in both normal and OA cells. However, the best results were obtained with the combination, suggesting that combined therapy with IGF‐1 and OP‐1 may be an effective strategy for treating OA cartilage damage.

     

Increased apoptosis in human osteoarthritic cartilage corresponds to reduced cell density and expression of caspase‐3

Objective

Chondrocyte apoptosis has been described in both human and experimentally induced osteoarthritis (OA), but its importance in the etiopathogenesis of OA is uncertain. The aims of this study were to determine the rate of chondrocyte apoptosis using different methods, and to investigate the relationship between this process and cartilage cellularity, expression of proapoptotic molecules, and expression of antiapoptotic molecules in articular cartilage obtained from patients with OA and from nonarthritic controls.

Methods

We examined the extent of apoptosis in OA and nonarthritic control cartilage using expression of caspase‐3, an enzyme that mediates the final stage of cell death by apoptosis, as well as the TUNEL method. We used immunohistochemistry to analyze the expression of a panel of proapoptotic and antiapoptotic molecules that regulate apoptosis in articular cartilage, in order to determine whether the rate of apoptosis is associated with the expression of these molecules.

Results

The median (range) percentage of TUNEL‐positive chondrocytes in knee OA cartilage (n = 10 specimens), hip OA cartilage (n = 9), and control cartilage (n = 7) was 3.11 (1.67–3.67), 1.86 (1.22–2.89), and 0.39 (0.00–1.78), respectively. When all cartilage samples were pooled, apoptosis showed a strong inverse correlation with cellularity (r = −0.74, P < 0.0001). The percentage (range) of cells expressing caspase‐3 in the 3 groups was 15.70 (7.40–20.50), 15.77 (7.42–20.5), and 7.40 (5.90–8.00), respectively. One‐way analysis of variance showed that the differences between groups for both TUNEL‐positive cells and expression of caspase‐3 were statistically significant (P < 0.0001). There was a significant positive correlation between TUNEL‐positive cells and expression of caspase‐3 (r = 0.654, P< 0.01).

Conclusion

The data suggest that apoptosis is increased, on average, 2–4‐fold in OA cartilage. Considering that OA develops over many years, such an increase in the rate of apoptosis in the articular cartilage could play an important role in the disease process.

     

Tonic activation of hypoxia‐inducible factor 1α in avascular articular cartilage and implications for metabolic homeostasis

Objective

To determine whether oxygen‐dependent activation patterns of hypoxia‐inducible factor 1α (HIF‐1α) observed in vascularized tissues are conserved within avascular and hypoxic articular cartilage and whether HIF‐1α affects cartilage matrix synthesis.

Methods

Explants of bovine articular cartilage and primary chondrocytes were exposed to normoxia (21% O₂), hypoxia (2% O₂), and simulated hypoxia (21% O₂ plus CoCl₂). Western blot and immunofluorescence analyses of HIF‐1α were performed to determine HIF‐1α activation patterns. To simulate cartilage loss from disease or injury, the top layers of cartilage were removed from osteochondral explants, and the residual cartilage was assessed for HIF‐1α immunolocalization and proteoglycan synthesis.

Results

We demonstrated continuous nuclear translocation of HIF‐1α in deeper layers of intact articular cartilage. HIF‐1α was not completely degraded in chondrocytes exposed to normoxia, but rather, colocalized to the Golgi complex, a finding not previously reported for any cell type. Following alteration of the oxygen gradient by removal of the top layers of cartilage, predominantly perinuclear HIF‐1α was found in the deeper layers. Restoration of intranuclear HIF‐1α to these areas was achieved by hypoxia and simulated hypoxia. Under conditions in which HIF‐1α was inactivated, matrix synthetic activity was altered (P < 0.0001) compared with control cartilage.

Conclusion

These findings demonstrate that hypoxia‐dependent activation of HIF‐1α is highly conserved and that changes in oxygen tensions following cartilage loss from injury or disease alter cartilage metabolism in part by changing HIF‐1α activity. The discovery of tonic activation of HIF‐1α within intact articular cartilage underscores its potential importance to cartilage homeostasis.

     

Delayed gadolinium‐enhanced magnetic resonance imaging of the meniscus: An index of meniscal tissue degeneration?

Objective

Much attention has been focused recently on the need for a better understanding of the mechanisms and natural progression of knee osteoarthritis (OA), particularly in its early stages. One technique that has been used to investigate early OA is delayed gadolinium‐enhanced magnetic resonance imaging of cartilage (dGEMRIC), where T1(Gd) (T1 value after penetration of the MRI contrast agent gadopentate dimeglumine [Gd‐DTPA²⁻]) is used as an index of the molecular status of articular cartilage. The goal of this study was to explore T1(Gd) in the meniscus and its relationship with articular cartilage T1(Gd) in knee dGEMRIC image data sets.

Methods

T1(Gd) maps of the meniscus and articular cartilage were made from knee dGEMRIC images obtained from prior studies of dGEMRIC of the knee in 21 asymptomatic subjects and 9 patients with self‐reported OA.

Results

T1(Gd) of the meniscus covered a range of values (247–515 msec) and patterns (homogeneous and focal variations). In addition, T1(Gd) of the meniscus correlated with that of articular cartilage (R = 0.38, P = 0.037; R = 0.57, P = 0.001 for correlations of the medial posterior meniscus with the medial femoral and tibial cartilage, respectively; T1[Gd] of the anterior meniscus and lateral compartments also correlated, with R > 0.38 and P < 0.037), potentially demonstrating parallel degradative processes in the knee.

Conclusion

While the biophysical basis for the T1(Gd) results relative to meniscus molecular structure needs investigation, these findings introduce a potential means of examining the time course of meniscal tissue change in the development and progression of arthritis.

     

Role of apoptotic and matrix‐degrading genes in articular cartilage and meniscus of mature and aged rabbits during development of osteoarthritis

Objective

To determine expression patterns of apoptotic and matrix‐degrading genes during aging and development of osteoarthritis (OA), using a rabbit model of induced OA.

Methods

Six mature and 6 aged rabbits underwent anterior cruciate ligament transection and were killed 4 and 8 weeks after surgery, respectively, to create early‐grade and advanced‐grade OA. RNA from articular cartilage and menisci was examined for expression of the genes caspase 8, Fas, Fas ligand, p53, aggrecanase, matrix metalloproteinase 1 (MMP‐1), and MMP‐3. A second cohort of animals that had undergone no intervention in the joint was also killed. Parametric data were analyzed with analysis of variance and Student's t‐tests, while nonparametric data were assessed with the Mann‐Whitney U test.

Results

Expression levels of Fas, caspase 8, FasL, and MMP‐1 were significantly higher (>100%) in aged cartilage compared with mature cartilage (P < 0.05). After induction of OA, expression of apoptotic genes in aged rabbits remained high, while significant up‐regulation of Fas and caspase 8 (nearly 150% increase) was observed in mature rabbits (P < 0.05). No significant up‐regulation of these genes was observed in the menisci of aged or mature rabbits prior to or after induction of OA. Development of OA occurred more rapidly in aged cartilage compared with mature cartilage (P < 0.05).

Conclusion

Differential expression of apoptotic and matrix‐degrading genes occurs in aged compared with mature cartilage, both at baseline and during development of OA. This may be responsible for faster degradation of aged cartilage and its predisposition for developing OA.

     

Long‐term high‐physiological‐dose growth hormone reduces intra‐abdominal fat in HIV‐infected patients with a neutral effect on glucose metabolism

Objectives

The aim of the study was to investigate the effect of long‐term high‐physiological‐dose recombinant human growth hormone (rhGH) therapy on fat distribution and glucose metabolism in HIV‐infected patients.

Methods

Forty‐six HIV‐infected Caucasian men on highly active antiretroviral therapy (HAART), with an age range of 21–60 years and no significant comorbidity, were included in this randomized, placebo‐controlled, double‐blind, single‐centre trial. Twenty‐eight subjects were randomized to 0.7 mg/day rhGH, and 18 subjects to placebo, administered as daily subcutaneous injections between 1 and 3 pm for 40 weeks. Endpoints included changes in visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), limb fat mass, percentage of limb fat, plasma lipids, insulin resistance and glucose tolerance.

Results

VAT and trunk fat mass decreased significantly in the GH group compared with the placebo group [−19 cm² (−11%) vs. 12 cm² (6%), P=0.03, and −548 g (−9%) vs. 353 g (6%), P<0.01, respectively]. The beneficial fat redistribution in the GH group occurred without concomitant changes in subcutaneous fat at the abdomen or extremities. rhGH therapy was well tolerated. Insulin resistance, glucose tolerance, and total plasma cholesterol and triglycerides did not significantly change during intervention.

Conclusions

Daily 0.7 mg rhGH treatment for 40 weeks reduced abdominal visceral fat and trunk fat mass in HIV‐infected patients. This treatment appeared to be safe with respect to glucose tolerance and insulin sensitivity.

     

Increased collagen and aggrecan degradation with age in the joints of Timp3−/− mice

Objective

To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage.

Methods

Hind joints of Timp3^−/− and wild‐type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (… VDIPEN and … NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme‐linked immunosorbent assay.

Results

Articular cartilage from Timp3‐knockout animals (ages ≥6 months) showed reduced Safranin O staining and an increase in …VDIPEN content compared with cartilage from heterozygous and wild‐type animals. There was also a slight increase in … NVTEGE content in articular cartilage and menisci of Timp3^−/− animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3^−/− mice compared with wild‐type animals. This increase was significant in 15‐month‐old animals.

Conclusion

These results indicate that TIMP‐3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP‐3 may play a pathophysiologic role in the development of this disease.