The role of vascular endothelial growth factor in glucocorticoid-induced bone loss: evaluation in a minipig model

Vascular endothelial growth factor (VEGF) has been recently shown to play an important role during endochondral bone formation in hypertrophic cartilage remodeling, ossification, and angiogenesis. To our knowledge there are no previous studies investigating the role of VEGF in osteoporosis. We hypothesized that VEGF expression in bone would be reduced under glucocorticoid (GC) treatment and tested this in a minipig model. As part of a larger study, 17 primiparous sows (Göttingen minipig) were allocated to two experimental groups when they were 15 months old: a control group (n = 9) and a group receiving GC treatment for 15 months (n = 8). All animals were fed a semisynthetic diet until they were sacrificed. The GC group received prednisolone orally at a daily dose of 1 mg/kg body wt for 8 weeks and thereafter 0.5 mg/kg body wt. VEGF levels in lumbar vertebrae were measured by enzyme-linked immunosorbent assay (ELISA) and verified by Western blot analysis. VEGF and its receptors (VEGFR) were localized by immunohistochemistry. Expression of VEGF-mRNA was analyzed by reverse transcription-polymerase chain reaction. VEGF protein was quantified in supernatants of cultivated osteoblasts by ELISA. Spinal bone mineral density was assessed in vivo by quantitative computed tomography. Expression of cyclooxygenase-2 (COX-2) protein was investigated by immunohistochemistry. High VEGF concentrations were measured in normal lumbar vertebrae whereas VEGF concentrations were 60% lower (P < 0.0001) in GC-treated minipigs. VEGF levels were associated (r = 0.7) with rates of spinal trabecular bone loss, which differed significantly (P < 0.0013) between controls (-0.47 +/- 2.2% SEM) and GC-treated minipigs (-12.8 +/- 2.3% SEM). Osteoblasts were immunopositive for VEGF. VEGF receptors VEGFR-2 (KDR, flk-1) and VEGFR-1 (flt-1) could be immunostained on osteoclasts and osteoblasts. VEGF-mRNA and protein were detectable in the lumbar vertebrae of all animals. The expression of COX-2 protein was decreased in GC-treated animals. VEGF is produced in osteoblasts and its concentration is decreased in GC-treated animals as well as in osteoblasts exposed to GC. Since reductions in VEGF concentrations correlate with parallel measurement of bone mineral density in GC-treated minipigs we hypothesize that VEGF may be an important modulating factor for bone remodeling, specifically in GC-induced osteoporosis. GC inhibit COX-2 and hence prostaglandin E2 (PGE2) production. Since PGE2 is able to increase VEGF synthesis, this may be the link between GC and VEGF decrease.