Abstract
Background Apoptosis of osteoblasts and osteoclasts regulates bone homeostasis. Skeletal injury in humans results in angiogenic responses primarily mediated by vascular endothelial growth factor(VEGF), a protein essential for bone repair in animal models. Osteoblasts release VEGF in response to a number of stimuli and express receptors for VEGF in a differentiation dependent manner. This study investigates the putative role of VEGF in regulating the lifespan of primary human osteoblasts(PHOB) in vitro.
Methods PHOB were examined for VEGF receptors. Cultures were supplemented with VEGF(0–50ng/mL), a neutralising antibody to VEGF, mAB VEGF(0.3ug/mL) and Placental Growth Factor (PlGF), an Flt-1 receptor-specific VEGF ligand(0–100 ng/mL) to examine their effects on mineralised nodule assay, alkaline phosphatase assay and apoptosis.. The role of the VEGF specific antiapoptotic gene target BCl2 in apoptosis was determined.
Results PHOB expressed functional VEGF receptors. VEGF 10 and 25 ng/mL increased nodule formation 2.3- and 3.16-fold and alkaline phosphatase release 2.6 and 4.1-fold respectively while 0.3ug/mL of mAB VEGF resulted in approx 40% reductions in both. PlGF 50ng/mL had greater effects on alkaline phosphatase release (103% increase) than on nodule formation (57% increase). 10ng/mL of VEGF inhibited spontaneous and pathological apoptosis by 83.6% and 71% respectively, while PlGF had no significant effect. Pretreatment with mAB VEGF, in the absence of exogenous VEGF resulted in a significant increase in apoptosis (14 vs 3%). BCl2 transfection gave a 0.9% apoptotic rate. VEGF 10 ng/mL increased BCl2 expression 4 fold while mAB VEGF decreased it by over 50%.
Conclusions VEGF is a potent regulator of osteoblast lifespan in vitro. This autocrine feedback regulates survival of these cells, mediated via the KDR receptor and expression of BCl2 antiapoptotic gene.
Theses abstracts were prepared by Professor Roger Lemaire. Correspondence should be addressed to EFORT Central Office, Freihofstrasse 22, CH-8700 Küsnacht, Switzerland.
