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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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Involvement of the VEGF receptor VEGFR2 in OPG mediated endothelial 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) VEGF 10 ng/ml, (c) VEGF 10 ng/ml and VEGFR2 neutralising antibody (1 μg/ml), (d) OPG 100 ng/ml, (e) OPG 100 ng/ml and VEGFR2 neutralising antibody (1 μg/ml). (f) 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 in combination with VEGFR2 antibody; NS, no significant difference.
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Figure 2: Involvement of the VEGF receptor VEGFR2 in OPG mediated endothelial 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) VEGF 10 ng/ml, (c) VEGF 10 ng/ml and VEGFR2 neutralising antibody (1 μg/ml), (d) OPG 100 ng/ml, (e) OPG 100 ng/ml and VEGFR2 neutralising antibody (1 μg/ml). (f) 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 in combination with VEGFR2 antibody; NS, no significant difference.

Mentions: To confirm this, the levels of HuDMEC tube formation induced by either OPG or VEGF were determined in the presence or absence of a VEGFR2 neutralising antibody. As demonstrated in figure 2, both VEGF and OPG stimulated HuDMEC tube formation on growth factor reduced matrigel compared to control. VEGF-induced tube formation was inhibited in the presence of the anti-VEGFR2 antibody as observed by a 60% decrease in branchpoint number (p < 0.001). In contrast, administration of the anti-VEGFR2 antibody had no effect on OPG-induced tube formation, suggesting that OPG does not induce tubule formation through interacting with the VEGFR2 receptor (Figure 2).


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

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

Involvement of the VEGF receptor VEGFR2 in OPG mediated endothelial 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) VEGF 10 ng/ml, (c) VEGF 10 ng/ml and VEGFR2 neutralising antibody (1 μg/ml), (d) OPG 100 ng/ml, (e) OPG 100 ng/ml and VEGFR2 neutralising antibody (1 μg/ml). (f) 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 in combination with VEGFR2 antibody; NS, no significant difference.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Involvement of the VEGF receptor VEGFR2 in OPG mediated endothelial 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) VEGF 10 ng/ml, (c) VEGF 10 ng/ml and VEGFR2 neutralising antibody (1 μg/ml), (d) OPG 100 ng/ml, (e) OPG 100 ng/ml and VEGFR2 neutralising antibody (1 μg/ml). (f) 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 in combination with VEGFR2 antibody; NS, no significant difference.
Mentions: To confirm this, the levels of HuDMEC tube formation induced by either OPG or VEGF were determined in the presence or absence of a VEGFR2 neutralising antibody. As demonstrated in figure 2, both VEGF and OPG stimulated HuDMEC tube formation on growth factor reduced matrigel compared to control. VEGF-induced tube formation was inhibited in the presence of the anti-VEGFR2 antibody as observed by a 60% decrease in branchpoint number (p < 0.001). In contrast, administration of the anti-VEGFR2 antibody had no effect on OPG-induced tube formation, suggesting that OPG does not induce tubule formation through interacting with the VEGFR2 receptor (Figure 2).

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