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Tumor-derived mural-like cells coordinate with endothelial cells: role of YKL-40 in mural cell-mediated angiogenesis.

Francescone R, Ngernyuang N, Yan W, Bentley B, Shao R - Oncogene (2013)

Bottom Line: YKL-40 expressed by GSDCs was associated with increased interaction of neural cadherin/β-catenin/smooth muscle alpha actin; thus, mediating cell-cell adhesion and permeability.In cell co-culture systems, YKL-40 enhanced both GSDC and HMVEC contacts, restricted vascular leakage, and stabilized vascular networks.Collectively, the data inform new mechanistic insights into the cooperation of mural cells with endothelial cells induced by YKL-40 during tumor angiogenesis, and also enhance our understanding of YKL-40 in both mural and endothelial cell biology.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, Morrill Science Center, University of Massachusetts, Amherst, MA, USA.

ABSTRACT
Tumor neo-vasculature is characterized by spatial coordination of endothelial cells with mural cells, which delivers oxygen and nutrients. Here, we explored a key role of the secreted glycoprotein YKL-40, a mesenchymal marker, in the interaction between endothelial cells and mesenchymal mural-like cells for tumor angiogenesis. Xenotransplantation of tumor-derived mural-like cells (GSDCs) expressing YKL-40 in mice developed extensive and stable blood vessels covered with more GSDCs than those in YKL-40 gene knockdown tumors. YKL-40 expressed by GSDCs was associated with increased interaction of neural cadherin/β-catenin/smooth muscle alpha actin; thus, mediating cell-cell adhesion and permeability. YKL-40 also induced the interaction of vascular endothelial cadherin/β-catenin/actin in endothelial cells (HMVECs). In cell co-culture systems, YKL-40 enhanced both GSDC and HMVEC contacts, restricted vascular leakage, and stabilized vascular networks. Collectively, the data inform new mechanistic insights into the cooperation of mural cells with endothelial cells induced by YKL-40 during tumor angiogenesis, and also enhance our understanding of YKL-40 in both mural and endothelial cell biology.

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YKL-40 decreases permeability of HMVECs, GSDCs, and their combination in a manner dependent on VE-cad or N-cad activityA. HMVECs were plated on inserts and allowed to attach and spread. Cells were treated overnight with either GSDC control conditioned media (CM, 24 hr serum-free media) or YKL-40 shRNA CM (Top), control or YKL-40 shRNA CM with mouse IgG or an anti-VE-cad Ab (20 μg/ml) (Middle), or anti-VEGF Ab (100 ng/ml) (Bottom). Cell permeability was then measured using FITC-Dextran as described in the Methods. B. GSDC control or YKL-40 shRNA cells were used for the same permeability assay (Top) as described in (A), in the presence of an anti-N-cad Ab (50 μg/ml) (Middle) or VEGF Ab (100 ng/ml) (Bottom). C. GSDC control or YKL-40 shRNA cells were first plated on the insert. 2 hr following attaching and spreading, HMVECs were plated on the top of the GSDCs to form a second layer and allowed to attach and spread in the presence of mIgG or an anti-VEGF Ab (100 ng/ml). The same permeability assay was performed on the next day. D. GSDCs were pre-treated with mIgG or an anti-N-cad Ab (50 μg/ml) overnight to exclude the possibility that the top layer of HMVECs prevents the antibody from access to the bottom layer of GSDCs. Then, GSDCs and HMVECs were set up as described in C in the presence of mIgG or an anti-VE-cad Ab (20 μg/ml). The permeability was measured. N=6, *P≤0.05 compared to corresponding controls at the same time points.
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Figure 6: YKL-40 decreases permeability of HMVECs, GSDCs, and their combination in a manner dependent on VE-cad or N-cad activityA. HMVECs were plated on inserts and allowed to attach and spread. Cells were treated overnight with either GSDC control conditioned media (CM, 24 hr serum-free media) or YKL-40 shRNA CM (Top), control or YKL-40 shRNA CM with mouse IgG or an anti-VE-cad Ab (20 μg/ml) (Middle), or anti-VEGF Ab (100 ng/ml) (Bottom). Cell permeability was then measured using FITC-Dextran as described in the Methods. B. GSDC control or YKL-40 shRNA cells were used for the same permeability assay (Top) as described in (A), in the presence of an anti-N-cad Ab (50 μg/ml) (Middle) or VEGF Ab (100 ng/ml) (Bottom). C. GSDC control or YKL-40 shRNA cells were first plated on the insert. 2 hr following attaching and spreading, HMVECs were plated on the top of the GSDCs to form a second layer and allowed to attach and spread in the presence of mIgG or an anti-VEGF Ab (100 ng/ml). The same permeability assay was performed on the next day. D. GSDCs were pre-treated with mIgG or an anti-N-cad Ab (50 μg/ml) overnight to exclude the possibility that the top layer of HMVECs prevents the antibody from access to the bottom layer of GSDCs. Then, GSDCs and HMVECs were set up as described in C in the presence of mIgG or an anti-VE-cad Ab (20 μg/ml). The permeability was measured. N=6, *P≤0.05 compared to corresponding controls at the same time points.

Mentions: To further evaluate permeability of HMVECs and GSDCs, one of the key vascular functions, we utilized a permeability method that assays the ability of cells to be permeable to Dextran conjugated with FITC. First, we treated HMVECs with either conditioned media from control or YKL-40 shRNA GSDCs, and we found that control medium-treated HMVECs restricted the permeability that allowed FITC-Dextran to cross through the cells 30% less than did YKL-40 shRNA medium-treated cells (Fig. 6A, top graph). In order to verify the functional role of VE-cadherin in HMVEC permeability, we added a VE-cadherin neutralizing antibody to the media. While mIgG, as a control, did not have an impact in the permeability treated with control or YKL-40 shRNA media, a VE-cadherin antibody increased cell permeability in control medium-treated HMVECs (Fig. 6A, middle graph). However, this VE-cadherin neutralization in YKL-40 shRNA medium-treated cells failed to enhance the permeability induced by YKL-40 shRNA, consistent with HMVEC-HMVEC adhesion found earlier (Fig. 3E). This implies that VE-cadherin plays a key role in the elevated permeability of HMVECs treated with YKL-40 shRNA media. In order to determine the effect of VEGF on permeability, as VEGF mediated the disassociation of β-catenin from VE-cadherin (Fig. 3B–3D), we treated the HMVECs with an anti-VEGF neutralizing antibody. The anti-VEGF antibody fully reversed the GSDC YKL-40 shRNA medium-induced permeability to the level treated with GSDC control media (Fig. 6A, bottom graph). As expected, the anti-VEGF antibody did not have effects on the permeability of control medium-treated HMVECs because of the considerably lower level of VEGF in the control media. To validate this endothelial cell permeability restrained by YKL-40, we treated HMVECs with mAY in the presence of GSDC control medium and found that mAY induced HMVEC permeability by 51% (Supplemental Fig. 4A). These results suggest that YKL-40 maintains HMVEC permeability and that YKL-40 blockade destabilizes the permeability probably through VE-cadherin activation.


Tumor-derived mural-like cells coordinate with endothelial cells: role of YKL-40 in mural cell-mediated angiogenesis.

Francescone R, Ngernyuang N, Yan W, Bentley B, Shao R - Oncogene (2013)

YKL-40 decreases permeability of HMVECs, GSDCs, and their combination in a manner dependent on VE-cad or N-cad activityA. HMVECs were plated on inserts and allowed to attach and spread. Cells were treated overnight with either GSDC control conditioned media (CM, 24 hr serum-free media) or YKL-40 shRNA CM (Top), control or YKL-40 shRNA CM with mouse IgG or an anti-VE-cad Ab (20 μg/ml) (Middle), or anti-VEGF Ab (100 ng/ml) (Bottom). Cell permeability was then measured using FITC-Dextran as described in the Methods. B. GSDC control or YKL-40 shRNA cells were used for the same permeability assay (Top) as described in (A), in the presence of an anti-N-cad Ab (50 μg/ml) (Middle) or VEGF Ab (100 ng/ml) (Bottom). C. GSDC control or YKL-40 shRNA cells were first plated on the insert. 2 hr following attaching and spreading, HMVECs were plated on the top of the GSDCs to form a second layer and allowed to attach and spread in the presence of mIgG or an anti-VEGF Ab (100 ng/ml). The same permeability assay was performed on the next day. D. GSDCs were pre-treated with mIgG or an anti-N-cad Ab (50 μg/ml) overnight to exclude the possibility that the top layer of HMVECs prevents the antibody from access to the bottom layer of GSDCs. Then, GSDCs and HMVECs were set up as described in C in the presence of mIgG or an anti-VE-cad Ab (20 μg/ml). The permeability was measured. N=6, *P≤0.05 compared to corresponding controls at the same time points.
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Related In: Results  -  Collection

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Figure 6: YKL-40 decreases permeability of HMVECs, GSDCs, and their combination in a manner dependent on VE-cad or N-cad activityA. HMVECs were plated on inserts and allowed to attach and spread. Cells were treated overnight with either GSDC control conditioned media (CM, 24 hr serum-free media) or YKL-40 shRNA CM (Top), control or YKL-40 shRNA CM with mouse IgG or an anti-VE-cad Ab (20 μg/ml) (Middle), or anti-VEGF Ab (100 ng/ml) (Bottom). Cell permeability was then measured using FITC-Dextran as described in the Methods. B. GSDC control or YKL-40 shRNA cells were used for the same permeability assay (Top) as described in (A), in the presence of an anti-N-cad Ab (50 μg/ml) (Middle) or VEGF Ab (100 ng/ml) (Bottom). C. GSDC control or YKL-40 shRNA cells were first plated on the insert. 2 hr following attaching and spreading, HMVECs were plated on the top of the GSDCs to form a second layer and allowed to attach and spread in the presence of mIgG or an anti-VEGF Ab (100 ng/ml). The same permeability assay was performed on the next day. D. GSDCs were pre-treated with mIgG or an anti-N-cad Ab (50 μg/ml) overnight to exclude the possibility that the top layer of HMVECs prevents the antibody from access to the bottom layer of GSDCs. Then, GSDCs and HMVECs were set up as described in C in the presence of mIgG or an anti-VE-cad Ab (20 μg/ml). The permeability was measured. N=6, *P≤0.05 compared to corresponding controls at the same time points.
Mentions: To further evaluate permeability of HMVECs and GSDCs, one of the key vascular functions, we utilized a permeability method that assays the ability of cells to be permeable to Dextran conjugated with FITC. First, we treated HMVECs with either conditioned media from control or YKL-40 shRNA GSDCs, and we found that control medium-treated HMVECs restricted the permeability that allowed FITC-Dextran to cross through the cells 30% less than did YKL-40 shRNA medium-treated cells (Fig. 6A, top graph). In order to verify the functional role of VE-cadherin in HMVEC permeability, we added a VE-cadherin neutralizing antibody to the media. While mIgG, as a control, did not have an impact in the permeability treated with control or YKL-40 shRNA media, a VE-cadherin antibody increased cell permeability in control medium-treated HMVECs (Fig. 6A, middle graph). However, this VE-cadherin neutralization in YKL-40 shRNA medium-treated cells failed to enhance the permeability induced by YKL-40 shRNA, consistent with HMVEC-HMVEC adhesion found earlier (Fig. 3E). This implies that VE-cadherin plays a key role in the elevated permeability of HMVECs treated with YKL-40 shRNA media. In order to determine the effect of VEGF on permeability, as VEGF mediated the disassociation of β-catenin from VE-cadherin (Fig. 3B–3D), we treated the HMVECs with an anti-VEGF neutralizing antibody. The anti-VEGF antibody fully reversed the GSDC YKL-40 shRNA medium-induced permeability to the level treated with GSDC control media (Fig. 6A, bottom graph). As expected, the anti-VEGF antibody did not have effects on the permeability of control medium-treated HMVECs because of the considerably lower level of VEGF in the control media. To validate this endothelial cell permeability restrained by YKL-40, we treated HMVECs with mAY in the presence of GSDC control medium and found that mAY induced HMVEC permeability by 51% (Supplemental Fig. 4A). These results suggest that YKL-40 maintains HMVEC permeability and that YKL-40 blockade destabilizes the permeability probably through VE-cadherin activation.

Bottom Line: YKL-40 expressed by GSDCs was associated with increased interaction of neural cadherin/β-catenin/smooth muscle alpha actin; thus, mediating cell-cell adhesion and permeability.In cell co-culture systems, YKL-40 enhanced both GSDC and HMVEC contacts, restricted vascular leakage, and stabilized vascular networks.Collectively, the data inform new mechanistic insights into the cooperation of mural cells with endothelial cells induced by YKL-40 during tumor angiogenesis, and also enhance our understanding of YKL-40 in both mural and endothelial cell biology.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, Morrill Science Center, University of Massachusetts, Amherst, MA, USA.

ABSTRACT
Tumor neo-vasculature is characterized by spatial coordination of endothelial cells with mural cells, which delivers oxygen and nutrients. Here, we explored a key role of the secreted glycoprotein YKL-40, a mesenchymal marker, in the interaction between endothelial cells and mesenchymal mural-like cells for tumor angiogenesis. Xenotransplantation of tumor-derived mural-like cells (GSDCs) expressing YKL-40 in mice developed extensive and stable blood vessels covered with more GSDCs than those in YKL-40 gene knockdown tumors. YKL-40 expressed by GSDCs was associated with increased interaction of neural cadherin/β-catenin/smooth muscle alpha actin; thus, mediating cell-cell adhesion and permeability. YKL-40 also induced the interaction of vascular endothelial cadherin/β-catenin/actin in endothelial cells (HMVECs). In cell co-culture systems, YKL-40 enhanced both GSDC and HMVEC contacts, restricted vascular leakage, and stabilized vascular networks. Collectively, the data inform new mechanistic insights into the cooperation of mural cells with endothelial cells induced by YKL-40 during tumor angiogenesis, and also enhance our understanding of YKL-40 in both mural and endothelial cell biology.

Show MeSH
Related in: MedlinePlus