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Breast cancer cells stimulate osteoprotegerin (OPG) production by endothelial cells through direct cell contact.

Reid PE, Brown NJ, Holen I - Mol. Cancer (2009)

Bottom Line: In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor.In contrast, the pro-angiogenic factors VEGF, bFGF and TGFbeta had no effect on HuDMEC OPG levels.These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Academic Units of Clinical Oncology and Surgical Oncology, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK. p.reid@sheffield.ac.uk

ABSTRACT

Background: Angiogenesis, the sprouting of capillaries from existing blood vessels, is central to tumour growth and progression, however the molecular regulation of this process remains to be fully elucidated. The secreted glycoprotein osteoprotegerin (OPG) is one potential pro-angiogenic factor, and clinical studies have demonstrated endothelial cells within a number of tumour types to express high levels of OPG compared to those in normal tissue. Additionally, OPG can increase endothelial cell survival, proliferation and migration, as well as induce endothelial cell tube formation in vitro. This study aims to elucidate the processes involved in the pro-angiogenic effects of OPG in vitro, and also how OPG levels may be regulated within the tumour microenvironment.

Results: It has previously been demonstrated that OPG can induce tube formation on growth factor reduced matrigel. In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor. We also show that cell contact between HuDMECs and the T47D breast cancer cell line increases endothelial cell OPG mRNA and protein secretion levels in in vitro co-cultures. These increases in endothelial cell OPG secretion were dependent on alphanubeta3 ligation and NFkappaB activation. In contrast, the pro-angiogenic factors VEGF, bFGF and TGFbeta had no effect on HuDMEC OPG levels.

Conclusion: These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro. Additionally, we show that breast cancer cells cause increased levels of OPG expression by endothelial cells, and that direct contact between endothelial cells and tumour cells is required in order to increase endothelial OPG expression and secretion. Stimulation of OPG secretion was shown to involve alphanubeta3 ligation and NFkappaB activation.

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Effect of VEGF in combination with OPG on endothelial cell tube formation. HuDMECs were cultured on growth factor reduced matrigel for 8 hours and branch point number counted as described in materials and methods. (a) Untreated control, (b) OPG 10 ng/ml, (c) VEGF 1 ng/ml, (d) OPG 10 ng/ml and VEGF 1 ng/ml. (e) Quantification of tube formation through measurement of branch point number. Data represented as mean ± S.E.M. from three independent experiments performed in triplicate. ***, p < 0.001 compared to VEGF (1 ng/ml), OPG (10 ng/ml) or untreated control; +++, p < 0.001 compared to untreated control only.
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Figure 1: Effect of VEGF in combination with OPG on endothelial cell tube formation. HuDMECs were cultured on growth factor reduced matrigel for 8 hours and branch point number counted as described in materials and methods. (a) Untreated control, (b) OPG 10 ng/ml, (c) VEGF 1 ng/ml, (d) OPG 10 ng/ml and VEGF 1 ng/ml. (e) Quantification of tube formation through measurement of branch point number. Data represented as mean ± S.E.M. from three independent experiments performed in triplicate. ***, p < 0.001 compared to VEGF (1 ng/ml), OPG (10 ng/ml) or untreated control; +++, p < 0.001 compared to untreated control only.

Mentions: It has previously been shown that OPG can induce endothelial cell tube formation on growth factor reduced matrigel [3]. However, in the tumour microenvironment it is possible that OPG also enhances vessel formation induced by other pro-angiogenic factors such as VEGF. Therefore, to further elucidate how OPG may affect tube formation, endothelial cells were treated with a combination of OPG and VEGF to establish whether the two have a synergistic effect on tube formation in vitro. As demonstrated in figure 1, OPG or VEGF when administered alone significantly increased tube formation, almost doubling the number of branch points (p < 0.001). When OPG and VEGF were added together, tube formation was significantly increased by 20% compared to VEGF alone (p < 0.001) and 26% compared to OPG alone (p < 0.001). This suggests that within the tumour microenvironment OPG is able to act in concert with other pro-angiogenic factors such as VEGF to further enhance angiogenesis and additionally, that OPG and VEGF act via different pathways to induce endothelial cell tube formation.


Breast cancer cells stimulate osteoprotegerin (OPG) production by endothelial cells through direct cell contact.

Reid PE, Brown NJ, Holen I - Mol. Cancer (2009)

Effect of VEGF in combination with OPG on endothelial cell tube formation. HuDMECs were cultured on growth factor reduced matrigel for 8 hours and branch point number counted as described in materials and methods. (a) Untreated control, (b) OPG 10 ng/ml, (c) VEGF 1 ng/ml, (d) OPG 10 ng/ml and VEGF 1 ng/ml. (e) Quantification of tube formation through measurement of branch point number. Data represented as mean ± S.E.M. from three independent experiments performed in triplicate. ***, p < 0.001 compared to VEGF (1 ng/ml), OPG (10 ng/ml) or untreated control; +++, p < 0.001 compared to untreated control only.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719583&req=5

Figure 1: Effect of VEGF in combination with OPG on endothelial cell tube formation. HuDMECs were cultured on growth factor reduced matrigel for 8 hours and branch point number counted as described in materials and methods. (a) Untreated control, (b) OPG 10 ng/ml, (c) VEGF 1 ng/ml, (d) OPG 10 ng/ml and VEGF 1 ng/ml. (e) Quantification of tube formation through measurement of branch point number. Data represented as mean ± S.E.M. from three independent experiments performed in triplicate. ***, p < 0.001 compared to VEGF (1 ng/ml), OPG (10 ng/ml) or untreated control; +++, p < 0.001 compared to untreated control only.
Mentions: It has previously been shown that OPG can induce endothelial cell tube formation on growth factor reduced matrigel [3]. However, in the tumour microenvironment it is possible that OPG also enhances vessel formation induced by other pro-angiogenic factors such as VEGF. Therefore, to further elucidate how OPG may affect tube formation, endothelial cells were treated with a combination of OPG and VEGF to establish whether the two have a synergistic effect on tube formation in vitro. As demonstrated in figure 1, OPG or VEGF when administered alone significantly increased tube formation, almost doubling the number of branch points (p < 0.001). When OPG and VEGF were added together, tube formation was significantly increased by 20% compared to VEGF alone (p < 0.001) and 26% compared to OPG alone (p < 0.001). This suggests that within the tumour microenvironment OPG is able to act in concert with other pro-angiogenic factors such as VEGF to further enhance angiogenesis and additionally, that OPG and VEGF act via different pathways to induce endothelial cell tube formation.

Bottom Line: In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor.In contrast, the pro-angiogenic factors VEGF, bFGF and TGFbeta had no effect on HuDMEC OPG levels.These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Academic Units of Clinical Oncology and Surgical Oncology, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK. p.reid@sheffield.ac.uk

ABSTRACT

Background: Angiogenesis, the sprouting of capillaries from existing blood vessels, is central to tumour growth and progression, however the molecular regulation of this process remains to be fully elucidated. The secreted glycoprotein osteoprotegerin (OPG) is one potential pro-angiogenic factor, and clinical studies have demonstrated endothelial cells within a number of tumour types to express high levels of OPG compared to those in normal tissue. Additionally, OPG can increase endothelial cell survival, proliferation and migration, as well as induce endothelial cell tube formation in vitro. This study aims to elucidate the processes involved in the pro-angiogenic effects of OPG in vitro, and also how OPG levels may be regulated within the tumour microenvironment.

Results: It has previously been demonstrated that OPG can induce tube formation on growth factor reduced matrigel. In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor. We also show that cell contact between HuDMECs and the T47D breast cancer cell line increases endothelial cell OPG mRNA and protein secretion levels in in vitro co-cultures. These increases in endothelial cell OPG secretion were dependent on alphanubeta3 ligation and NFkappaB activation. In contrast, the pro-angiogenic factors VEGF, bFGF and TGFbeta had no effect on HuDMEC OPG levels.

Conclusion: These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro. Additionally, we show that breast cancer cells cause increased levels of OPG expression by endothelial cells, and that direct contact between endothelial cells and tumour cells is required in order to increase endothelial OPG expression and secretion. Stimulation of OPG secretion was shown to involve alphanubeta3 ligation and NFkappaB activation.

Show MeSH
Related in: MedlinePlus