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.

     

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.

     

Inhibition of insulin‐like growth factor binding protein 5 proteolysis in articular cartilage and joint fluid results in enhanced concentrations of insulin‐like growth factor 1 and is associated with improved osteoarthritis

Objective

The complement component C1s is present in dog joint fluid in an activated state. Since C1s degrades insulin‐like growth factor binding protein 5 (IGFBP‐5), we undertook to determine whether inhibiting C1s in joint fluid would result in an increase in the amount of intact IGFBP‐5 and IGF‐1 in cartilage and joint fluid, and whether C1s inhibition would be associated with a reduction in cartilage destruction during the development of osteoarthritis (OA).

Methods

Twenty‐two dogs were randomized to 3 treatment groups. All dogs underwent anterior cruciate ligament transection and were exercised. Dogs received 1 of 3 treatments: buffer alone (controls; n = 6); PB‐145, a peptide derived from the sequence of antithrombin III (n = 9); and pentosan polysulfate (PPS; n = 7). PB‐145 or saline was injected into the joint space 3 times per week for 3 weeks. PPS was injected intramuscularly weekly for 3 weeks.

Results

Joint histology showed preservation of chondrocytes and a smooth joint surface in the animals treated with PB‐145 and PPS. Mankin scoring showed statistically significant reductions in joint destruction with PB‐145 and PPS treatments (P < 0.01) compared with buffer control. Mean active collagenase concentrations were decreased by these two treatments. Immunoblotting of joint fluid showed that both treatments increased concentrations of intact IGFBP‐5. Direct analysis of IGFBP‐3 and IGFBP‐5 protease activity showed that IGFBP‐5 was degraded more rapidly and that PB‐145 and PPS inhibited the degradation of both proteins. Total IGF‐1 concentrations in joint fluid were increased 5.6–5.8‐fold by these two treatments. Analysis showed that C1s was being activated in joint fluid and that its activation was inhibited by the addition of PB‐145 or PPS.

Conclusion

The findings suggest that direct inhibition of the serine protease C1s results in increased concentrations of intact IGFBP‐5 and that proteolysis of IGFBP‐3 is also inhibited, probably by the inhibition of some other protease. This increase in concentrations of intact IGFBP‐3 and IGFBP‐5 leads to an increase in IGF‐1 which is associated with an improvement in joint architecture during the development of OA.

     

The growth hormone insulin‐like growth factor 1 axis in children and adolescents with inflammatory bowel disease and growth retardation

Context There is scarce knowledge about the growth hormone (GH) insulin‐like growth factor‐1 (IGF1) axis in children & adolescents with inflammatory bowel disease (IBD) and growth retardation. Objective To describe the pattern of GH and IGF1 secretion in children & adolescents with IBD. Design A retrospective review of 28 patients (23 M) of IBD (25 Crohn’s Disease and three Ulcerative Colitis) and growth retardation who had investigation of the GH/IGF‐1 axis. Height velocity (HV) and serum IGF1 were converted to standard deviation score (SDS); to account for delayed puberty in girls over 11 years and boys over 12 years, HV and serum IGF1 SDS were adjusted for bone age. Results Median (range) age and Ht SDS at the time of endocrine evaluation was 14·3 years (7·7,17·0) and ?2·0(?3·6,?0·9), respectively. Median HVSDS over the prior 12 months was ?2·2(?7·7,2·8). Median peak serum GH on insulin tolerance test (ITT) was 5·8 mcg/l (1·3, 24·0), and median serum IGF1 SDS was ?0·9(?3·1, 0·1). Five of 28 (18%) had a peak serum GH of >12 mcg/l. Overall, four had biochemical evidence of functional GH deficiency (peak GH < 3 mcg/l and IGF1 SDS < 0) and 11 children had biochemical evidence suggesting GH resistance (peak GH > 6 mcg/l and IGF1 SDS < 0). However, only one child had a peak serum GH > 6 mcg/l and a very low IGF1 SDS of r = ?0·49, P = 0·008), but there was no association with HV and there was no association between IGF1 SDS and Ht or HV SDS. IGF1 SDS showed a negative association with erythrocyte sedimentation rate (r = ?0·41, P = 0·04). Conclusion Growth retardation in children and adolescents with IBD is commonly associated with a range of biochemical abnormalities ranging from functional GH deficiency to GH resistance. In these children, poor relationship between systemic markers of growth and height velocity point to an important role of growth factors at the target organ level in modulating growth in children with IBD. The value of assessing the GH/IGF‐1 axis and whether it predicts subsequent response to growth‐promoting therapy requires further exploration.     

Tenocyte responses to mechanical loading in vivo: A role for local insulin‐like growth factor 1 signaling in early tendinosis in rats

Objective

To investigate tenocyte regulatory events during the development of overuse supraspinatus tendinosis in rats.

Methods

Supraspinatus tendinosis was induced by running rats downhill at 1 km/hour for 1 hour a day. Tendons were harvested at 4, 8, 12, and 16 weeks and processed for brightfield, polarized light, or transmission electron microscopy. The development of tendinosis was assessed semiquantitatively using a modified Bonar histopathologic scale. Apoptosis and proliferation were examined using antibodies against fragmented DNA or proliferating cell nuclear antigen, respectively. Insulin‐like growth factor 1 (IGF‐1) expression was determined by computer‐assisted quantification of immunohistochemical reaction. Local IGF‐1 signaling was probed using antibodies to phosphorylated insulin receptor substrate 1 (IRS‐1) and ERK‐1/2.

Results

Tendinosis was present after 12 weeks of downhill running and was characterized by tenocyte rounding and proliferation as well as by glycosaminoglycan accumulation and collagen fragmentation. The proliferation index was elevated in CD90+ tenocytes in association with tendinosis and correlated with increased local IGF‐1 expression by tenocytes and phosphorylation of IRS‐1 and ERK‐1/2. Both apoptosis and cellular inflammation were absent at all time points.

Conclusion

In this animal model, early tendinosis was associated with local stimulation of tenocytes rather than with extrinsic inflammation or apoptosis. Our data suggest a role for IGF‐1 in the load‐induced tenocyte responses during the pathogenesis of overuse tendon disorders.

     

The circulating insulin‐like growth factor system in children with coeliac disease: an additional marker for disease activity

Background

Chronic undernutrition resulting from coeliac disease (CD) could be associated with changes in the circulating insulin‐like growth factor (IGF) system, which may participate in the pathogenesis of growth retardation occurring in these patients.

Methods

We performed a cross‐sectional study in CD subjects attempting to (1) document the pattern of serum IGF‐I and IGF binding protein (IGFBP) 1 and 3 at diagnosis and (2) assess the response of circulating IGF system to dietary treatments, in comparison with the response of clinical and laboratory findings utilized for the diagnosis of CD. Thirty‐two prepubertal CD children were divided into three groups based on the dietetic treatment: at diagnosis (D, n=18); on gluten‐free diet for at least 6 months (GFD, n=7); and on gluten challenge for at least 3 months (CH, n=7). Six postpubertal CD patients were also studied at diagnosis.

Results

In prepubertal children IGF‐I levels were significantly reduced (by 29%) in D vs sex‐ and age‐matched normal control (NC) subjects, with reductions being more pronounced before 3 years of age. Likewise, serum IGFBP‐3 concentrations were decreased by 22%, whereas circulating IGFBP‐1 levels were increased by 60%, compared with NC, with more marked IGFBP changes in older children. Similar alterations were observed in postpubertal patients. Changes in the circulating IGF system disappeared in GFD subjects and reappeared in CH children, as positivity of disease‐specific antibodies. Body mass index (BMI) also improved in GFD subjects, but did not decrease in CH children. Changes in IGF‐I and IGFBPs did not correlate with each other. Levels of IGF‐I, but not of IGFBPs, maintained the relation with age and correlated significantly with BMI and positivity of antibodies.

Conclusions

These results demonstrate that CD patients show significant changes in serum IGF‐I, in younger children, and IGFBPs (particularly IGFBP‐1), in older children and adolescents, correlating with clinical course and response to dietary treatments. The alteration in the circulating IGF system could be implicated in the pathogenesis of growth retardation occurring in CD and may provide an additional tool in monitoring of the disease. Copyright © 1999 John Wiley & Sons, Ltd.

     

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.

     

Effects of chronic growth hormone and insulin‐like growth factor 1 deficiency on osteoarthritis severity in rat knee joints

Objective

To determine the effects of chronic deficiency of growth hormone (GH) and insulin‐like growth factor 1 (IGF‐1) on osteoarthritis (OA) severity.

Methods

Thirty‐five rats were divided into 4 treatment groups at 4 weeks of age: 1 control group (normal GH/IGF‐1 levels [heterozygous]) and 3 groups of dwarves with a genetic mutation that results in GH deficiency. The first dwarf group received GH for 64 weeks (GH replete) and the second received GH until 14 weeks of age, followed by saline for 50 weeks (adult‐onset GH/IGF‐1 deficiency [AO‐GHD]). The third dwarf group received saline injections only (lifetime GH deficient [GHD]). Sections of the medial knee joint compartment were graded and measured histologically; data were summarized using factor analysis, and treatment effects were assessed using analysis of variance and adjusting for body weight.

Results

Terminal IGF‐1 levels and body weights were significantly affected by treatment (P = 0.002 and P < 0.001, respectively). Factor analysis yielded a total of 5 factors, the first 3 of which were not significantly affected by treatment. Factor 4 (weighted by medial tibial plateau articular cartilage width and area) was significantly affected by treatment (P < 0.012), with larger values in the AO‐GHD group than in the GHD group (P < 0.05). Factor 5 (weighted primarily by articular cartilage structure and loss of toluidine blue staining scores) also was significantly affected by treatment (P < 0.001), and was significantly lower (less severe lesions) in the GH replete group than in all other treatment groups (P < 0.05). Despite the presence of cartilage lesions, osteophytes and subchondral sclerosis were not observed in GH/IGF‐1–deficient animals.

Conclusion

These results indicate that chronic GH/IGF‐1 deficiency causes an increased severity of articular cartilage lesions of OA without the bony lesions normally seen in this disease.

     

The insulin‐like growth factor binding protein 3 ternary complex is reduced in cirrhosis

Abstract: Background/Aims: In healthy adults, serum insulin‐like growth factor I (IGF‐I), IGF binding protein 3 (IGFBP‐3) and acid labile subunit (ALS) form a 150‐kDa ternary complex under the control of growth hormone (GH). Approximately 80–90% of circulating IGF‐I is bound to the ternary complex. In cirrhosis the GH/IGF axis is severely disturbed and the individual components of the ternary complex are reduced. However, the degree of ternary complex formation in cirrhosis has not previously been described. Methods: Serum IGF‐I, IGFBP‐3, ALS, the 150‐kDa ternary complex and IGFBP‐3 proteolysis were all measured in six compensated and six decompensated cirrhotic patients and compared to six healthy controls. Results: Patients with compensated cirrhosis had decreased levels of IGF‐I (55%), IGFBP‐3 (64%) and ALS (53%), and in the decompensated patients these levels were decreased even further: IGF‐I (32%), IGFBP‐3 (37%) and ALS (27%) compared to healthy controls. The levels of the ternary complex followed this pattern, with low levels seen in the compensated patients (66%) and a further reduction in the decompensated patients (27%). Ternary complex levels correlated negatively with the Child–Pugh score. No increase in IGFBP‐3 proteolysis was found in cirrhotic patients compared to healthy controls. Conclusion: Cirrhosis is associated with reduced levels of the 150‐kDa ternary IGFBP‐3 complex correlating with the degree of liver disease.