ResultsPrimary Human Osteoblasts express VEGF receptors(see Figure Figure1)1 ) Osteoblast rich cultures from trabecular bone explants demonstrated no significant differences in activity, receptor expression, cytokinerelease or response to angiogenic cytokines from the commercially available human osteoblast cell line. As shown, 97.8% of cells in the culture system expressed VEGF receptors. This biotinylated VEGF binding assay does not distinguish the VEGF receptor isotypes involved but rather indicates the functional biological activity of VEGF receptor expression in the cell population.Figure 1 Expression of functional VEGF receptors on Primary Human Osteoblasts. Mean Channel Fluorescence is measured using flow cytometric analysis of avidin- FITC labelling of osteoblast rich cultures treated with a biotinylated VEGF or negative control antibody (more ...)Exogenous VEGF induces alkaline phosphatase release (Figure (Figure22)The concentration of endogenously produced VEGF was measured in the cultures. The range was 0.8 – 2.25 ng/mL following 72 hours of incubation. This was within the range of exogenously administered VEGF that produced equivalent results. After 48 hours in culture, recombinant human VEGF 165 increased alkaline phosphatase release in a dose dependant manner. VEGF concentrations of 5, 10 and 25 ng/mL were sufficient to increase nodule formation 1.3-(not significant), 2.6 and 4.1-fold, over that of cultures replete of exogenous VEGF. Daily administration of 0.3 ug/mL of mAB VEGF again resulted in a significant decrease (39% reduction) in nodule formation in the cultures replete of exogenous VEGF, again highlighting the importance of this positive feedbackloop. PlGF was slightly more efficacious at 25 ng/mL (66% increase) and 50 ng/mL (103% increase) in its effects on alkaline phosphatase release. This data suggests that ligation and activation of the specific VEGF receptor types has differential effects on its various mitogenic activities.Figure 2 The effect of a neutralizing monoclonal antibody and of VEGF receptor-specific ligands on Primary Human Osteoblast alkaline phosphatase release in vitro. Bonenodule formation was assessed by von Kossa staining and alkaline phosphatase release by p-nitrophenyl (more ...)Exogenous VEGF induces bone nodule formation in primary human osteoblasts (Figure (Figure33 )After 18 days in culture, recombinant human VEGF 165 increased mineralized nodule formation in a dose dependant manner. VEGF concentrations of 5, 10 and 25 ng/mL weresufficient to increase nodule formation 1.6-, 2.3- and 3.16-fold respectively, over that of cultures replete of exogenous VEGF. Daily administration of 0.3 ug/mL of a neutralising antibody to VEGF (mAB VEGF) resulted in a significant decrease (44% reduction) in nodule formation in the cultures replete of exogenous VEGF. Addition of a control monoclonal antibody had no effect on the culture system. This data demonstrates that endogenously released VEGF is involved in a positive feedback loop that serves to stimulate osteoblast bone formation When compared to VEGF 165, Placental Growth Factor (PlGF) had only a minimal effect on mineralised nodule formation at 25 ng/mL (30% increase) and 50 ng/mL (57% increase) concentrations. This demonstrates that the Flt-1 receptor plays little role in the effects of VEGF on osteoblast formation of mineralised nodules..Figure 3 The effect of a neutralizing monoclonal antibody and of VEGF receptor-specific ligands on Primary Human Osteoblast Bone Nodule Formation in vitro. Bonenodule formation was assessed by von Kossa staining and alkaline phosphatase release by p-nitrophenyl (more ...)The effect of angiogenic cytokines on osteoblast apoptosis (Figure (Figure44)For this series of experiments serum free conditions were used in order to examine the effects of the various angiogenic cytokines in isolation. Control osteoblast apoptosis under these conditions was 25.6%, as compared to 3% in the presence of serum (see Figure Figure4).4 ). Cell cultures were treated with anti Fas-IgM (1000 ng/mL) and TNF alpha (100 ng/mL) to simulate 'pathological' apoptosis in conditions of excessive bone loss. This resulted in reproducible rates of programmed cell death of 68%.We found that TNF alpha, in the absence of Fas IgM, did not induce apoptosis of primaryhuman osteoblasts but rather served to increase expression of Fas receptor. Thus increasing concentrations of TNF alpha resulted in increased rates of 'pathological' apoptosis (Data not shown here). Addition of 40 ng/mL of IGF-1 inhibited spontaneous and pathological apoptosis by 73.6% and 53% respectively. Treatment of the cultures with 10 ng/mL of VEGF inhibited spontaneous and TNF/Fas induced programmed cell death by 83.6% and 71% respectively. Thus VEGF afforded significantly better protection from apoptosis under both normal and particularly pathological conditions than the 'gold standard' osteotropic cytokine IGF-1. PlGF had no significant effect on the rate of osteoblast apoptosis demonstrating that the Flt-1 receptor was not involved in the survival activity of VEGF. Both bFGF (46% reduction in spontaneous and 27% reductionin pathological apoptosis) and PDGF (34% reduction in spontaneous and 23.5% reduction in pathological apoptosis) at 25 ng/mL attenuated osteoblast apoptosis, but to a significantly lesser extent. These concentrations of VEGF, bFGF and PDGF were used, as they were found to be comparable in induction of endothelial cell proliferation (an in vitro measure of angiogenesis) in preliminary studies.Figure 4 The effects of osteotropic and angiogenic cytokines on Primary Human Osteoblast apoptosis in vitro. Osteoblast apoptosis was determined by Annexin V- Fluorescein Isothiocyanate labelling and hypodiploid DNA measurement as described in Materials and Methods. (more ...)Endogenous VEGF and BCl2 regulate osteoblast apoptosis. (Figure (Figure55)The percentage of cells staining positive for annexin V was analyzed by flow cytometry. Spontaneous apoptosis at 24 hours in the absence of serum is 25.6%. Pretreatment of the osteoblasts with VEGF 10 ng/mL (4.7% apoptosis rate) was almost as effective as culturein the presence of 10% serum (3% apoptosis rate) in inhibiting spontaneous programmed cell death.. Pretreatment of the cultures with a neutralising monoclonal antibody to VEGF(mAB VEGF), in the absence of exogenous VEGF resulted in a spontaneous apoptosis rate of 14%. This indicates that VEGF released in culture by
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