Glucocorticoids inhibit vascular endothelial growth factor expression in growth plate chondrocytes
Introduction
Longitudinal bone growth depends on the proliferation, differentiation and subsequent ossification of growth plate cartilage. The rates of chondrocyte proliferation and differentiation to the hypertrophic phenotype are determined by a complex interplay between many signaling molecules. Also the ensuing process of endochondral ossification is tightly regulated, and involves the interrelated processes of extracellular matrix degradation, apoptosis of terminal hypertrophic chondrocytes and angiogenesis: the invasion of the growth plate by new blood vessels from the metaphysis. The molecular mechanisms involved in chondrocyte differentiation and endochondral bone formation have been reviewed by Stevens and Williams (1999). Signals that direct the polarity of the growth plate, angiogenesis and bone formation originate in the growth plate itself, as was elegantly demonstrated by excising, inverting and reimplanting rabbit growth plates (Abad et al., 1999). While the polarity of the cartilage was maintained, the epiphyseal bone, which became adjacent to the hypertrophic zone of the growth plate, became the site of blood vessel and bone cell invasion and longitudinal bone growth.
Angiogenesis can be induced by basic fibroblast growth factor (Baron et al., 1994), transferrin (Carlevaro et al., 1997) and other factors (Alini et al., 1996), but vascular endothelial growth factor (VEGF) is the quintessential angiogenic factor in this respect, as was demonstrated by treating growing mice with a soluble antagonist (Gerber et al., 1999). This treatment resulted in suppression of blood vessel invasion, accompanied by impaired resorption of terminal chondrocytes and formation of the primary spongiosa (trabeculae of the metaphyseal bone). VEGF is expressed by many tissues and cell types, including the growth plate cartilage (Gerber et al., 1999, Horner et al., 1999, Carlevaro et al., 2000, Garcia-Ramirez et al., 2000). Its expression can be induced by many factors, including hypoxia (Shweiki et al., 1992), estrogen (Cullinan-Bove and Koos, 1993), osteogenic protein-1/BMP-7 (Yeh and Lee, 1999), PDGF (Nauck et al., 1998) and IGF-I (Goad et al., 1996, Warren et al., 1996, Bermont et al., 2000, Miele et al., 2000, Garcia-Ramirez et al., 2000).
Given its pivotal role in maintaining the normal structure of the growth plate (Gerber et al., 1999), VEGF could be a potential target for agents, which disturb longitudinal growth. Glucocorticoid hormones are well known to induce growth retardation (Blodgett et al., 1956, Allen, 1996, Rooman et al., 1999) as well as osteoporosis (Manelli and Giustina, 2000). Direct local actions of glucocorticoids on the growth plate have been demonstrated in vivo (Baron et al., 1992), which likely occur through interaction with the glucocorticoid receptor (GR) (Silvestrini et al., 1999). Although many of the inhibitory actions of pharmacological doses of glucocorticoids on skeletal growth appear to be mediated by interference with the GH/insulin-like growth factor-I axis (Jux et al., 1998), their effects on angiogenesis and in particular VEGF expression in the growth plate are worth investigating. Preliminary data from our laboratory indicate that prednisolone treatment of juvenile piglets leads to a severely disturbed invasion of blood vessels to the growth plate.1 Previously, we have used cultured rabbit costal chondrocytes to study the effects of dexamethasone on the expression of IGF binding proteins (Koedam et al., 2000). In this study, we set out to characterize the effects of pharmacological doses of dexamethasone and other glucocorticoids on VEGF expression by cultured porcine growth plate chondrocytes.
Section snippets
Materials
Recombinant human IGF-I was kindly provided by Eli Lilly & Co., Indianapolis, IN. Cortisol (hydrocortisone, Solu-Cortef) was from Pharmacia & Upjohn B.V. (Woerden, The Netherlands). Prednisolone (Di-Adreson-F) was from N.V. Organon (Oss, The Netherlands). The GR antagonist Org34116 was kindly provided by Dr. M. de Gooyer (N.V. Organon, Oss, The Netherlands). Dexamethasone (as dexamethasone disodium phosphate) was from Merck Sharpe & Dohme (Haarlem, The Netherlands). Platelet-derived growth
Characterization of growth plate chondrocytes
The chondrocyte phenotype of the cultured porcine epiphyseal cells was maintained, as shown in Fig. 1. Typical chondrocytic markers were expressed, as assessed by Northern blotting of total RNA isolated from the cells. Specifically, transcripts were detected for collagen II (5.4 kb), aggrecan (8.0 kb) and the cartilage-specific gene CD-RAP (0.8 kb) (Fig. 1A). CD-RAP could be amplified from cDNA obtained from these cells (Fig. 1B), further illustrating the phenotype. Collagen I mRNA, specific
Discussion
In this study, we have examined the effects of glucocorticoids, in particular dexamethasone, on the expression of VEGF. We cultured primary chondrocytes from the epiphyseal growth plates of neonatal piglets. This model system was used earlier in the study of dexamethasone effects on the expression of IGF-binding proteins (Smink et al., 2002a). In many ways, the phenotype of these cells resembles that of the rabbit costal chondrocytes, which we described earlier to study the expression of IGFBPs
Acknowledgements
We thank Ms. C.M.P.C.D. Peeters and Dr. F. Groenendaal for making the tibia of neonatal piglets available to us.
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