Triiodothyronine stimulates45Ca-accumulation via synthesis of insulin-like growth factor-I in cultures of osteoblast-like cells

We examined the effects of triiodothyronine (T₃) on⁴⁵Ca-accumulation into extracellular matrix in osteoblast-like MC3T3-El cells cultured for 7-week period, which was considered to be long enough to develop calcification. T₃ increased⁴⁵Ca-accumulation into sodium dodecyl sulfate-insoluble, EDTA-extractable materials cultures of MC3T3-El cells in a dose-dependent manner in the range between 10 pM and 10 nM. Contrary to the stimulatory effect on⁴⁵Ca-accumulation, T₃ inhibited DNA synthesis in MC3T3-El cells dose dependently between 10 pM and 10 nM. T₃ stimulated the secretion of insulin-like growth factor-I (IGF-I) dose dependently between 0.1 nM and 10 nM in MC3T3-El cells cultured for 7 weeks. Antibodies to IGF-I suppressed the⁴⁵Ca-accumulation induced by T₃ almost to the control level. These results strongly suggest that T₃ stimulates⁴⁵Ca-accumulation via synthesis of IGF-I in osteoblast-like cells.     

Effects of fibronectin on articular cartilage chondrocyte proteoglycan synthesis and response to insulin-like growth factor-I

Fibronectin, a ubiquitous glycoprotein of the extracellular matrix, serves as a substrate for cell attachment. Binding to fibronectin through cell-surface receptors promotes a flattened cell shape, stimulates the phosphorylation of intracellular protein, and changes the pattern of gene expression. Although fibronectin is abundant in normal articular cartilage, its effects on chondrocytes are not well understood. Proteolytic fragments of fibronectin stimulate the catabolism of matrix in articular cartilage and may promote the degeneration of cartilage in osteoarthritis; however, intact fibronectin may regulate other aspects of matrix metabolism, including matrix synthesis. To determine whether intact fibronectin affects the synthetic activity of chondrocytes, as well as to determine the responses of chondrocytes to the anabolic growth factor.insulin-like growth factor-I, we compared the incorporation of [³⁵S] by articular chondrocytes of the rat culture.din the presence and absence of commercially prepared cellular fibronectin and 0, 10, or 100 ng/ml recomBinanf human insulin-like growth factor-I. Monolayer and alginate suspension cultures were compared to determine whether responses differed under conditions in which fibronectin promoted a flattened cell shape (monolayer culture) and under those in which cells maintained a spherical shape (alginate culture). In alginate cultures, fibronectin alone stimulated the incorporation of [³⁵S]. Fibronectin with 10 ng/ml insulin-like growth factor-I had additive effects in alginate culture, producing the maximum incorporation of [³⁵S]. In monolayer cultures, fibronectin did not stimulate incorporation and blocked stimulation by 100 ng/ml insulin-like growth factor-I. The cells from the monolayer culture were much less active synthetically (at all doses of the growth factor) than those culture in alginate. Thus, fibronectin enhanced proteoglycan synthesis and the response to insulin-like growth factor-I in alginate but inhibited the response to the growth factor in monolayers. These observations suggest intact fibronectin may contribute to the maintenance or repair of the matrix of articular cartilage by stimulating proteoglycan synthesis.     

Cyclic AMP stimulates45Ca-accumulation via synthesis of insulin-like growth factor-I in cultures of osteoblast-like cells

Summary The effects of cAMP on insulin-like growth factor-I (IGF-I) secretion and⁴⁵Ca-accumulation into extracellular matrix were investigated in cloned osteoblast-like MC3T3-E1 cells cultured for 7 weeks, which was considered to be long enough to develop calcification. Dibutyryl cAMP stimulated IGF-I secretion dose dependently in the range between 0.1 mM and 3 mM. Dibutyryl cAMP also stimulated accumulation of⁴⁵Ca into sodium dodecyl sulfate-insoluble, EDTA-extractable materials in cultures of these cells dose dependently between 0.1 mM and 3 mM. The patterns of both stimulations were similar. The dibutyryl cAMP-stimulated IGF-I secretion preceded the⁴⁵Ca-accumulation. Antibodies to IGF-I suppressed the dibutyryl cAMP-stimulated⁴⁵Ca-accumulation almost to the control level. Taking account of our previous report that exogenous IGF-I stimulates⁴⁵Ca-accumulation in cultures of these cells, the present results suggest that cAMP stimulates⁴⁵Ca-accumulation via synthesis of IGF-I in osteoblast-like cells.     

Recombinant human insulin-like growth factor-I stimulates in vitro matrix synthesis and cell proliferation in rabbit flexor tendon

Flexor tendons have an intrinsic ability for repair, with a capacity to metabolize matrix components and to proliferate. To identify factors with the potential of affecting those abilities, the effects of recombinant human insulin-like growth factor (rhIGF-I), insulin and fetal calf serum (FCS) on the synthesis of proteoglycan, collagen, and non-collagen protein and cell proliferation were investigated in short-term explant cultures of the deep flexor tendon of the rabbit. Matrix synthesis and cell proliferation were stimulated dose dependently by rhIGF-I at doses between 10 and 250 and at 10-100 ng/ml, respectively, by insulin at 250-5,000 ng/ml, and by FCS at 2-15%. Estimated maximal stimulation (Emax) of up to three times the control value was observed with rhIGF-I at 250 ng/ml. Maximal stimulation was observed at 5,000 ng/ml with insulin, and FCS at 15%. rhIGF-I was more potent than insulin in stimulating protein synthesis and cell proliferation. The Emax of stimulation of proteoglycan and collagen synthesis by rhIGF-I were two times that of FCS, and the Emax of cell proliferation by FCS was twice that of rhIGF-I. Growth factors thus have the ability to stimulate matrix synthesis and cell proliferation in rabbit flexor tendon. This provides a rationale for further studies on the role of growth factors in flexor tendon healing in humans.     

Growth hormone and insulin-like growth factor-I and mesangial matrix in uremic rats

Combined growth hormone (GH) and insulin-like growth factor-I (IGF-I) therapy has been advocated for clinical use to minimize the diabetogenic effect of GH and enhance their anabolic effects. However, GH has been shown to accelerate the development of glomerular sclerosis in experimental animals and IGF-I mediates the renal effects of GH. The purpose of this study was therefore to examine morphometrically the effects of GH (1 mg intraperitoneally three times a week), IGF-I (50 g/kg body weight subcutaneously twice a day), and combined GH/IGF-I treatments in vivo on mesangial matrix at 3–20 days after 5/6 nephrectomy in 140- to 150-g rats. There were no significant changes in growth and renal function after GH and/or IGF-I treatment. The effects of GH and IGF-I on glomerular size were additive, which were more prominent in juxtamedullary glomeruli. GH induced proportional increases in mesangial area (MA) and glomerular area (GA), whereas IGF-I induced a similar increase in GA without a corresponding change in MA. When compared with GH treatment alone, combined GH/IGF-I treatment resulted in a lesser degree of mesangial expansion despite an enhanced glomerular size. While additional studies are needed to examine the long-term effects of these findings, our results suggest a potentially beneficial effect of combined GH/IGF-I therapy during uremia.     

Regional regenerative potential of meniscal cartilage exposed to recombinant insulin-like growth factor-I in vitro

It is well recognised that meniscal tears situated within the inner, avascular region do not heal. We investigated the potential effect of insulin-like growth factor-I (IGF-I) in promoting regeneration of meniscal tissue in the inner, middle and outer zones of the meniscus. Sheep menisci were harvested and monolayer cell cultures prepared. Various concentrations of IGF-I were used in the presence or absence of 10% fetal calf serum (FCS). We measured the uptake of radioactive thymidine, sulphur, and proline to assess cell proliferation and formation of extracellular matrix (ECM). IGF-I, in the presence or absence of FCS, increased the formation of DNA and ECM in all meniscal zones. However, the response of the cells from the avascular zone was greater than that from the vascular zone. Our findings indicate that fibrochondrocytes cultured from avascular meniscal tissue have the ability to regenerate when exposed to anabolic cytokines such as IGF-I.     

Interaction of exogenous liposomal insulin-like growth factor-I cDNA gene transfer with growth factors on collagen expression in acute wounds

Growth factors have been shown to modulate the complex cascade of wound healing, however, interaction between different growth factors during dermal and epidermal regeneration is still not entirely defined. We have recently shown that exogenous liposomal gene transfer of cDNA results in physiologic expression and response in an acute wound. In the present study we determined the interaction between insulin-like growth factor-I (IGF-I), a mesenchymal growth factor, administered as liposomal cDNA, with other dermal and epidermal growth factors on collagen synthesis in an acute wound. Sprague-Dawley rats were given a scald burn to inflict an acute wound and divided into two groups to receive weekly subcutaneous injections of liposomes plus a beta-galactosidase containing plasmid (Lac Z [0.2 microg, vehicle]), or liposomes plus the IGF-I cDNA containing plasmid (2.2 microg) and Lac Z (0.2 microg). Immunological assays, histological and immunohistochemical techniques were used to determine growth factor concentration and different types of collagen (I, III, and IV) after IGF-I cDNA gene transfer. IGF-I cDNA transfer accelerated reepithelization and was associated with increased levels of IGF-I, fibroblast growth factor, keratinocyte growth factor, vascular endothelial cell growth factor, and platelet-derived growth factor protein expression. IGF-I cDNA had no effect on transforming growth factor-beta. IGF-I cDNA significantly increased type IV collagen while it had no effect on types I and III collagen. Exogenously administered IGF-I cDNA increased protein concentrations of keratinocyte growth factor, fibroblast growth factor, platelet-derived growth factor, and type IV collagen. We conclude that liposomal IGF-I gene transfer can accelerate wound healing without causing an increase in types I and III collagen expression.     

The effect of dynamic compression on the response of articular cartilage to insulin-like growth factor-I.

Articular cartilage is routinely subjected to mechanical forces and to cell-regulatory molecules. Previous studies have shown that mechanical stimuli can influence articular chondrocyte metabolic activity, and biochemical studies have shown that growth factors and cytokines control many of the same cell functions. Little is known, however, of the relationships or interplay, if any, between these two key components of the articular environment. This study investigated the comparative and interactive effects of low amplitude, sinusoidal, dynamic compression and insulin-like growth factor-I (IGF-I), a polypeptide in synovial fluid that is anabolic for cartilage. In bovine patellofemoral cartilage explants, IGF-I increased protein and proteoglycan synthesis 90% and 120%, respectively while dynamic compression increased protein and proteoglycan synthesis 40% and 90%, respectively. Stimulation by IGF-I was significantly greater than by dynamic compression for both protein and proteoglycan synthesis. When applied together, the two stimuli enhanced protein and proteoglycan synthesis by 180% and 290%, respectively, a degree greater than that achieved by either stimulus alone. IGF-I augmented protein synthesis with a time constant of 12.2 h. Dynamic compression increased protein synthesis with a time constant of 2.9 h, a rate significantly faster than that of IGF-I, suggesting that these signals act via distinct cell activation pathways. When used together, dynamic compression and IGF-I acted with a time constant of 5.6 h. Thus, dynamic compression accelerated the biosynthetic response to IGF-I and increased transport of IGF-I into the articular cartilage matrix, suggesting that, in addition to independently stimulating articular chondrocytes, cyclic compression may improve the access of soluble growth factors to these relatively isolated cells.     

Differential effects of insulin-like growth factor-I on matrix and DNA synthesis in various regions and types of rabbit tendons

Tendon healing and integration of tendon grafts may be site or donor specific. To determine if differences exist in sensitivity to growth factors that have the potential to influence tendon repair, we compared the effects of recombinant human insulin-like growth factor-I on various types of tendon segments. The dose response effects on proteoglycan, collagen, noncollagen protein, and DNA synthesis were investigated in short-term explant cultures of intrasynovial intermediate and proximal segments of deep flexor tendons, extrasynovial segments of deep flexor tendons, and Achilles tendons of rabbits. The four different types of tendon segments cultured in media without recombinant human insulin-like growth factor-I synthesized similar amounts of each of the matrix components. Intrasynovial proximal segments synthesized 15 times less DNA than other tendon segments. Recombinant human insulin-like growth factor-I stimulated matrix and DNA synthesis of all tendon segments in a dose-dependent manner in intervals from 10 to 1,000 ng/ml. The potency (LogED₅₀) of the stimulation did not differ between the segments. The estimated maximal stimulation (E_max) of proteoglycan synthesis by recombinant human insulin-like growth factor-I was higher, and of collagen and noncollagen protein synthesis was lower, in intrasynovial proximal segments as compared with that of the other types of segments. In contrast, the estimated maximal stimulation of DNA synthesis by recombinant human insulin-like growth factor-I was 6-fold higher than controls in all types of tendons. These findings demonstrate differences in mitotic capacity between anatomical regions of tendons during culture without recombinant human insulin-like growth factor-I and in matrix synthesis after stimulation with it.     

Calcification of cartilage matrix in chondrocyte cultures derived from rachitic rat growth plate cartilage

Chondrocytes obtained from collagenase-digested epiphyseal growth plate cartilage of rachitic rats were grown in multilayer cultures. The cultured chondrocytes produced a metachromatic matrix and further electron microscopic examination revealed typical features of cartilage matrix collagen fibrils and matrix vesicles. The alkaline phosphatase activity in cultures was high during the entire 3-week culture period. Acid phosphatase showed a marked increase in activity during the first week of culture.The appearance of apatite crystals in the synthesized matrix was monitored by electron microscopy over a 3-week period. First crystals were consistently found to be associated with matrix vesicles, and in the older cultures calcification spread into the surrounding matrix. No collagen fibrils associated with mineralization were observed during the early culture period. This study clearly demonstrates that in chondrocyte cultures the first mineral crystals were found within or in close association with matrix vesicles. This gives further support to the hypothesis that matrix vesicles are the primary site of mineralization in cartilage. In addition to calcification studies it is suggested that this model is suitable for studying the effects of hormones or other agents on rachitic chondrocytes in vitro.     

Gene mediated insulin-like growth factor-I delivery to the synovium.

The feasibility of articular gene therapy using insulin-like growth factor-I transgene expression in synovial tissues was assessed in vitro by transfection of synovial explant and monolayer cultures. Synovial membrane was harvested from horses and distributed for explant culture in multiwell plates or digested for monolayer culture in multiwell plates and chamber slides. Synovial monolayers were cultured for 48 h after infection with 0, 100, 200, or 500 moi adenovirus-IGF-I (AdeIGF-I) to establish an optimum dose. Explants were then either infected with AdeIGF-I or adenoviral LacZ and cultured for 8 days, treated with 100 ng/ml recombinant IGF-I as a positive control, or remained as uninfected untreated culture controls. Expression of IGF-I in explants and monolayers was assessed by in situ hybridization and quantitative polymerase chain reaction (PCR), and translation confirmed by IGF-I radioimmunoassay (RIA) and tissue immunoreaction. Effects of IGF-I on synovial function was assessed by proteoglycan and hyaluronan assay, and northern blot assessment of decorin and collagen type I expression. Significant transgene expression in synovial cells was present for all AdeIGF-I concentrations. Similarly, medium IGF-I concentrations were significantly elevated in AdeIGF-I infected synovial monolayer and explant cultures at all time points. Peak IGF-I concentration of 246 +/- 43 ng/ml developed in explant cultures on day 4; IGF-I levels in control explant groups were unchanged over baseline values. In situ hybridization and immunolocalization for IGF-I indicated focal IGF-I expression in intimal and subintimal layers of infected explants, with diffuse immunoreaction throughout infected subintimal and fibrous layers. For monolayer cultures, intracellular immunoreaction to IGF-I was markedly higher in infected cells, and was most prominent at 100 moi. Effects of IGF-I on synoviocyte cultures were evident on northern blots, which showed decreased decorin expression and elevated type I collagen production in AdeIGF-I infected monolayers. Proteoglycan concentration in the medium from explant cultures rose over the initial 4 days but was similar between treatment groups. The concentration of hyaluronan in medium from explant cultures did not differ significantly within or between treated and control groups during the 8-day study period. These data indicate that IGF-I can be successfully introduced to synovial structures by adenoviral vectors and results in effective IGF-I ligand synthesis without untoward synovial morphologic effects.     

Effects of acute 5-fluorouracil chemotherapy and insulin-like growth factor-I pretreatment on growth plate cartilage and metaphyseal bone in rats

With the intensified use of chemotherapy and improved survival rates for childhood malignancies, it has become increasingly apparent that some children or adult survivors show poor bone growth and develop osteoporosis. As a step to investigate underlying mechanisms, this project examined short-term effects in rats of chemotherapy agent 5-fluorouracil (5-FU) on cell proliferation, apoptosis, and bone formation at tibial growth plate cartilage and its adjacent bone-forming region metaphysis. In addition, since insulin-like growth factor (IGF-I) is important for bone growth, we examined whether IGF-I pretreatment would potentially protect growth plate cartilage and bone cells from chemotherapy damage. Two days after a single high dose of 5-FU injection, proliferation of growth plate chondrocytes and metaphyseal osteoblasts/preosteoblasts was dramatically suppressed, and apoptosis was induced among osteoblasts and preosteoblasts. As a result, there was a reduction in the chondrocyte number and zonal height at the proliferative zone and a decline in the number of osteoblasts and preosteoblasts on the metaphyseal trabecular bone surface. At day 2, no obvious deleterious effects were observed on the height of the growth plate hypertrophic zone and the bone volume fraction of the metaphyseal primary spongiosa trabeculae. At day 10, while cell proliferation and growth plate structure returned to normal, there were slight decreases in trabecular bone volume, body length increase, and tibial length. While pretreatment with 1-week IGF-I systemic infusion did not attenuate the suppressive effect of 5-FU on proliferation in both growth plate and metaphysis, it significantly diminished apoptotic induction in metaphysis. These results indicate that growth plate cartilage chondrocytes and metaphyseal bone cells are sensitive to chemotherapy drug 5-FU and that IGF-I pretreatment has some anti-apoptotic protective effects on metaphyseal bone osteoblasts and preosteoblasts.     

Dual transduction of insulin-like growth factor-I and interleukin-1 receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model.

This study evaluated the potential of gene induced synoviocyte expression of a combination of insulin-like growth factor-I (AdIGF-I) and interleukin-1 receptor antagonist protein (AdIL-1Ra) to control articular cartilage degradation in vitro. Cartilage explants and synovial membrane were harvested from young mature horses. Synovial monolayers were established and either (1) maintained as untransduced controls; (2) transduced with AdIGF-I at 200 MOI in 500 microl serum-free medium; (3) transduced with AdIL-1Ra at 100 MOI; or (4) transduced with a combination of AdIGF-I (200 MOI) and AdIL-1Ra (100 MOI). Following transduction, cartilage explants were exposed to the synovial monolayer medium using co-culture inserts. Cultures were maintained for 6 days in either serum-free medium or medium containing 10 ng/ml recombinant human interleukin-1beta. At termination, synovial cell RNA was isolated for real-time PCR analysis, and cartilage explants were collected for H&E and toluidine blue staining, immunohistochemistry for type II collagen and IGF-I, in situ localization of IGF-I and type II collagen gene expression, and biochemical assays. Synovial monolayers were readily transduced with both AdIGF-I and AdIL-1Ra. IGF-I and IL-1Ra protein were secreted at beneficial levels throughout the experiment, having peak concentrations of 94.6 ng/ml and 33.0 ng/ml, respectively. Transduction with IGF-I promoted cartilage production of proteoglycan and type II collagen, suggesting a beneficial role for healing injured cartilage. Transduction with IL-1Ra decreased the synovial expression of IL-1alpha and IL-1beta and matrix metalloproteinases, indicating a mechanism for prevention of matrix degradation. The beneficial effects of the combination of anabolic growth factors and catabolic blockers were evident in improved preservation of proteoglycan content of cartilage explants exposed to the depleting effects of IL-1. These results show that gene therapy combining anabolic growth factors to stimulate matrix synthesis and catabolic blockers to prevent matrix degradation by IL-1, protects and causes partial restoration of cartilage matrix, and suggest a potential benefit of combination gene therapy for cartilage healing.     

Labeling of human insulin-like growth factor-Ⅰ eukaryotic expression vector with green fluorescent protein

To label human insulin-like growth factor-Ⅰ (hIGF-Ⅰ) eukaryotic expression vector with green fluorescent protein (GFP) for the repair of articular cartilage defects. Methods: GFP cDNA was inserted into pcDNA3.1-hIGF-Ⅰ to construct the co-expression vector with two multiple cloning sites mammalian expression vector under two cytomegalovirus promoters/enhancers respectively. Recombinant pcGI was transfected into NIH 3T3 cells with the help of lipofectamine. Results : Enzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and hIGF-Ⅰ eDNA. Expression of hIGF-Ⅰ and GFP was confirmed in transfected NIH 3T3 cells by immunocytochemical analysis and fluorescence microscopy. Conclusions : hIGF-Ⅰ eukaryotic expression vector has been successfully labeled with GFP.     

Circulating insulin-like growth factor-I and benign prostatic hyperplasia--a prospective study.

OBJECTIVE: The aim of this study was to investigate the role of insulin-like growth factor-I (IGF-I), a strongly mitogenic and anti-apoptotic factor, in the development of benign prostatic hyperplasia (BPH). The bioactivity of IGF-I within tissues depends on circulating levels, as well as on the local production of IGF-I and the presence of IGF-binding proteins (IGFBPs). The IGFBPs regulate the efflux of IGF-I to the extravascular space and the bioavailability of IGF-I within tissues. MATERIAL AND METHODS: Within the Northern Sweden Health and Disease Study, 60 cases of BPH defined by a history of prostate resection were identified, and two controls per case were selected. IGF-I, IGFBP-1, IGFBP-3 and insulin were measured by immuno-radiometric assays in stored plasma samples drawn a mean of 3.2 years before surgery. RESULTS: The risk of BPH increased with increasing quartile levels of IGF-I adjusted for IGFBP-3 (p(trend) = 0.10) up to a relative risk of 2.16 (95% confidence interval 0.83-5.64) for the highest quartile. The risk decreased with increasing levels of IGFBP-1 (p(trend) = 0.10). CONCLUSIONS: Our results suggest that elevated IGF-I bioactivity may stimulate the development of BPH; however, they were not statistically significant and require confirmation from larger studies.     

Transgenic mice with osteoblast-targeted insulin-like growth factor-I show increased bone remodeling

To determine the effects of locally-expressed insulin-like growth factor (IGF-I) on bone remodeling, a transgene was produced in which murine IGF-I cDNA was cloned downstream of a gene fragment comprising 3.6 kb of 5' upstream regulatory sequence and most of the first intron of the rat Col1a1 gene. The construct was expressed at the mRNA and protein level in transfected osteoblasts. Five lines of transgenic mice were generated by embryo microinjection. Transgene mRNA levels were highest in calvaria, long bone and tendon, and lower in skin. Serum IGF-I and body weight were increased in males and females only in the highest expressing line. Histomorphometry showed that transgenic calvaria were wider and had greater marrow area and bone area. Transgenic calvaria had increased osteoclast number per bone surface. Percent collagen synthesis and cell replication were increased in transgenic calvaria. Femur length, cortical width and cross-sectional area were increased in transgenic femurs of the highest expressing line, while femoral trabecular bone volume was little affected. Thus, broad overexpression of IGF-I in cells of the osteoblast lineage increased indices of bone formation and resorption.