Short-term glucocorticoid treatment of piglets causes changes in growth plate morphology and angiogenesis.
OBJECTIVE
Glucocorticoid treatment of children often leads to growth retardation, and the precise target(s) in the growth plate responsible for this effect are unknown. Angiogenesis is an important part of the endochondral ossification process, and VEGF expressed in the growth plate is essential for proper angiogenesis to occur. Since glucocorticoid treatment down-regulates VEGF expression in cultured chondrocytes, we hypothesized that in vivo glucocorticoid treatment could result in VEGF down-regulation in the growth plate and disturbed angiogenesis, thus contributing to the growth retardation.


METHODS
We treated 6-week-old prepubertal piglets (10 kg) for 5 days with prednisolone (50 mg/day). Tibial growth plate sections were studied for apoptosis and the expression of VEGF protein and mRNA and MMP-9 protein. Capillaries in the metaphysis were visualized by CD31 immunostaining. Growth plate morphology (width of various zones) was determined by interactive measurements on hematoxylin/eosin stained sections and apoptotic cells were detected by TUNEL assay.


RESULTS
In the prednisolone-treated animals, the total width of the growth plate decreased to 81% of controls (P<0.02), which was explained by a decrease of the width of the proliferative zone to 73% (P<0.05). The treatment had no effect on the orderly organization of the chondrocyte columns. In the growth plates of control animals, apoptosis was shown in 5.8% of the hypertrophic chondrocytes and was limited to the terminal hypertrophic chondrocytes. In prednisolone-treated animals, 40.5% of the hypertrophic chondrocytes was apoptotic (P<0.02), with apoptotic chondrocytes also appearing higher in the hypertrophic zone. We observed fewer capillaries and loss of their parallel organization in the metaphysis in the prednisolone-treated animals. The capillaries were shorter and chaotic in appearance. In contrast to controls, in prednisolone-treated animals VEGF mRNA and protein could not be detected in the hypertrophic zone of the growth plate. Trabecular bone length in the primary spongiosa was also diminished by the treatment. No changes were observed in the expression pattern of MMP-9, a matrix metalloproteinase, which is also important for angiogenesis and bone formation.


CONCLUSIONS
These results indicate that short-term glucocorticoid treatment of growing piglets severely disturbs the width of the growth plate, apoptosis of chondrocytes, VEGF expression by hypertrophic chondrocytes, the normal invasion of blood vessels from the metaphysis to the growth plate and bone formation at the chondro-osseous junction. These effects could alter the dynamics of endochondral ossification and thus contribute to glucocorticoid-induced growth retardation.
Smink
J J
JJ
Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, Utrecht, The Netherlands.
Buchholz
I M
IM
Hamers
N
N
van Tilburg
C M
CM
Christis
C
C
Sakkers
R J B
RJ
de Meer
K
K
van Buul-Offers
S C
SC
Koedam
J A
JA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002196:Capillaries; D005260:Female; D005938:Glucocorticoids / Q000494:pharmacology*; D006132:Growth Plate / Q000033:anatomy & histology / Q000187:drug effects* / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D020780:Matrix Metalloproteinase 9 / Q000032:analysis; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D011239:Prednisolone / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D013552:Swine; D013977:Tibia; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis*
D005938:Glucocorticoids; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D011239:Prednisolone; D020780:Matrix Metalloproteinase 9