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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 mediates the interaction of VE-cadherin/β-catenin/actin and cell-cell adhesion in HMVECsA. Western blot analysis of VE-cad (VE-cad), N-cad, β-cate, and actin protein expression from the lysates of HMVECs treated with either control or YKL-40 shRNA GSDC-conditioned media for 24 hours. B. Immunoprecipitation of VE-cad from the HMVEC lysates treated for 24 hours with conditioned media from control, YKL-40 shRNA, or YKL-40 shRNA cells plus an anti-VEGF Ab (100 ng/ml) followed by immunoblotting of β-cate. IgG levels were used as a control. Immunoprecipitation of β-cate followed by immunoblotting against actin was similarly performed in HMVEC lysates. *P<0.05 compared with control. n=3. C. Double staining of VE-cad (red) with β-cate (green) in HMVECs treated with conditioned media from control, YKL-40 shRNA, or YKL-40 shRNA cells plus an anti-VEGF Ab to determine the extent of co-localization (yellow) indicated by arrows. Nuclei (blue) were stained by DAPI. A bar: 20 μm. D. Quantification of the immunocytochemistry images in part C, normalized to cell number. N=3, *P≤0.05 compared to control. +P≤0.05 compared to control and YKL-40 shRNA. E. HMVEC aggregation was measured and quantified in the presence of GSDC control or YKL-40 shRNA media with an anti-VE-cad Ab (50 μg/ml). N=3, *P≤0.05 compared to control. F. HMVECs were treated with GSDC control medium in the presence of mAY or mIgG (10 μg/ml) overnight. Cell lystates were subjected to immunoprecipitation with an anti-VE-cadherin antibody followed by immunoblotting against β-cate. G. HMVECs treated with GSDC control medium in the presence of mAY or mIgG (10 μg/ml) were measured for cell aggregation. N=3, *P≤0.05 compared to control.
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Figure 3: YKL-40 mediates the interaction of VE-cadherin/β-catenin/actin and cell-cell adhesion in HMVECsA. Western blot analysis of VE-cad (VE-cad), N-cad, β-cate, and actin protein expression from the lysates of HMVECs treated with either control or YKL-40 shRNA GSDC-conditioned media for 24 hours. B. Immunoprecipitation of VE-cad from the HMVEC lysates treated for 24 hours with conditioned media from control, YKL-40 shRNA, or YKL-40 shRNA cells plus an anti-VEGF Ab (100 ng/ml) followed by immunoblotting of β-cate. IgG levels were used as a control. Immunoprecipitation of β-cate followed by immunoblotting against actin was similarly performed in HMVEC lysates. *P<0.05 compared with control. n=3. C. Double staining of VE-cad (red) with β-cate (green) in HMVECs treated with conditioned media from control, YKL-40 shRNA, or YKL-40 shRNA cells plus an anti-VEGF Ab to determine the extent of co-localization (yellow) indicated by arrows. Nuclei (blue) were stained by DAPI. A bar: 20 μm. D. Quantification of the immunocytochemistry images in part C, normalized to cell number. N=3, *P≤0.05 compared to control. +P≤0.05 compared to control and YKL-40 shRNA. E. HMVEC aggregation was measured and quantified in the presence of GSDC control or YKL-40 shRNA media with an anti-VE-cad Ab (50 μg/ml). N=3, *P≤0.05 compared to control. F. HMVECs were treated with GSDC control medium in the presence of mAY or mIgG (10 μg/ml) overnight. Cell lystates were subjected to immunoprecipitation with an anti-VE-cadherin antibody followed by immunoblotting against β-cate. G. HMVECs treated with GSDC control medium in the presence of mAY or mIgG (10 μg/ml) were measured for cell aggregation. N=3, *P≤0.05 compared to control.

Mentions: To explore effects of YKL-40 expressed by GSDCs on intercellular junctions of vascular endothelial cells via a paracrine manner, we used conditioned media from control or YKL-40 shRNA GSDCs in the culture of human microvascular endothelial cells (HMVECs) and measured intercellular contacts of VE-cadherin and β-catenin. HMVECs expressed a stronger level of VE-cadherin than N-cadherin, suggestive of a main role of VE-cadherin in cell to cell contacts (Fig. 3A). The media from control and YKL-40 shRNA cells did not alter expression of VE-cadherin, N-cadherin, or β-catenin (Fig. 3A & Supplemental Fig. 3A). However, like GSDCs, co-immunoprecipitation studies showed that media from YKL-40 shRNA cells inhibited the interaction of VE-cadherin with β-catenin, and β-catenin with actin in HMVECs (Fig. 3B & Supplemental Fig. 3B). A minimal level of N-cadherin/β-catenin association was detected in HMVECs treated with conditioned media from either control or YKL-40 shRNA cells (data not shown). Because VEGF secretion was significantly elevated in the conditioned media from YKL-40 shRNA cells (Fig. 2A), the increased VEGF may contribute mainly to the reduced VE-cadherin/β-catenin interaction. To test this possibility, we used an anti-VEGF neutralizing antibody in the co-immunoprecipitation assay of HMVECs. VEGF blockade restored the association of VE-cadherin with β-catenin (Fig. 3B). Consistent with the co-immunoprecipitation data, HMVECs treated with conditioned medium of YKL-40 shRNA cells displayed reduced co-localization of VE-cadherin and β-catenin to 13% relative to control cell medium, and treatment with an anti-VEGF neutralizing antibody recovered the co-localization to approximately 63% of the control levels (Fig. 3C & 3D). In addition, the cell aggregation analysis unveiled the similar inhibition of cell to cell adhesion (by 46–54%) by conditioned media from YKL-40 shRNA, control cells treated with a VE-cadherin neutralizing antibody, or YKL-40 shRNA cells treated a VE-cadherin antibody (Fig. 3E). To confirm the key role played by YKL-40 in endothelial cell to cell interaction, we also treated conditioned medium of the control GSDCs expressing YKL-40 with a neutralizing YKL-40 antibody (mAY) (40). The conditioned medium containing mAY abolished the interaction between VE-cadherin and β-catenin relative to the control mIgG medium (Fig. 3F). Accordingly, the control GSDC medium in the presence of mAY suppressed HMVEC aggregation by 60% compared to the aggregation in the presence of mIgG (Fig. 3G). While neither the neutralizing anti-VEGF antibody nor mAY altered expression of VE-cadherin, N-cadherin and β-catenin in HMVECs (data not shown), treatment of control GSDCs with mAY inhibited YKL-40 expression; in contrast, the anti-VEGF antibody in YKL-40 shRNA GSDCs induced VEGF (Supplemental Fig. 3C), suggestive of distinct responses of mural cells to individual inhibitors. Collectively, these data suggest that YKL-40 may induce the VEcadherin/β-catenin/actin pathway and cell to cell adhesion in endothelial cells, and that YKL-40 blockade inhibits these effects, which is largely dependent on VEGF.


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 mediates the interaction of VE-cadherin/β-catenin/actin and cell-cell adhesion in HMVECsA. Western blot analysis of VE-cad (VE-cad), N-cad, β-cate, and actin protein expression from the lysates of HMVECs treated with either control or YKL-40 shRNA GSDC-conditioned media for 24 hours. B. Immunoprecipitation of VE-cad from the HMVEC lysates treated for 24 hours with conditioned media from control, YKL-40 shRNA, or YKL-40 shRNA cells plus an anti-VEGF Ab (100 ng/ml) followed by immunoblotting of β-cate. IgG levels were used as a control. Immunoprecipitation of β-cate followed by immunoblotting against actin was similarly performed in HMVEC lysates. *P<0.05 compared with control. n=3. C. Double staining of VE-cad (red) with β-cate (green) in HMVECs treated with conditioned media from control, YKL-40 shRNA, or YKL-40 shRNA cells plus an anti-VEGF Ab to determine the extent of co-localization (yellow) indicated by arrows. Nuclei (blue) were stained by DAPI. A bar: 20 μm. D. Quantification of the immunocytochemistry images in part C, normalized to cell number. N=3, *P≤0.05 compared to control. +P≤0.05 compared to control and YKL-40 shRNA. E. HMVEC aggregation was measured and quantified in the presence of GSDC control or YKL-40 shRNA media with an anti-VE-cad Ab (50 μg/ml). N=3, *P≤0.05 compared to control. F. HMVECs were treated with GSDC control medium in the presence of mAY or mIgG (10 μg/ml) overnight. Cell lystates were subjected to immunoprecipitation with an anti-VE-cadherin antibody followed by immunoblotting against β-cate. G. HMVECs treated with GSDC control medium in the presence of mAY or mIgG (10 μg/ml) were measured for cell aggregation. N=3, *P≤0.05 compared to control.
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Figure 3: YKL-40 mediates the interaction of VE-cadherin/β-catenin/actin and cell-cell adhesion in HMVECsA. Western blot analysis of VE-cad (VE-cad), N-cad, β-cate, and actin protein expression from the lysates of HMVECs treated with either control or YKL-40 shRNA GSDC-conditioned media for 24 hours. B. Immunoprecipitation of VE-cad from the HMVEC lysates treated for 24 hours with conditioned media from control, YKL-40 shRNA, or YKL-40 shRNA cells plus an anti-VEGF Ab (100 ng/ml) followed by immunoblotting of β-cate. IgG levels were used as a control. Immunoprecipitation of β-cate followed by immunoblotting against actin was similarly performed in HMVEC lysates. *P<0.05 compared with control. n=3. C. Double staining of VE-cad (red) with β-cate (green) in HMVECs treated with conditioned media from control, YKL-40 shRNA, or YKL-40 shRNA cells plus an anti-VEGF Ab to determine the extent of co-localization (yellow) indicated by arrows. Nuclei (blue) were stained by DAPI. A bar: 20 μm. D. Quantification of the immunocytochemistry images in part C, normalized to cell number. N=3, *P≤0.05 compared to control. +P≤0.05 compared to control and YKL-40 shRNA. E. HMVEC aggregation was measured and quantified in the presence of GSDC control or YKL-40 shRNA media with an anti-VE-cad Ab (50 μg/ml). N=3, *P≤0.05 compared to control. F. HMVECs were treated with GSDC control medium in the presence of mAY or mIgG (10 μg/ml) overnight. Cell lystates were subjected to immunoprecipitation with an anti-VE-cadherin antibody followed by immunoblotting against β-cate. G. HMVECs treated with GSDC control medium in the presence of mAY or mIgG (10 μg/ml) were measured for cell aggregation. N=3, *P≤0.05 compared to control.
Mentions: To explore effects of YKL-40 expressed by GSDCs on intercellular junctions of vascular endothelial cells via a paracrine manner, we used conditioned media from control or YKL-40 shRNA GSDCs in the culture of human microvascular endothelial cells (HMVECs) and measured intercellular contacts of VE-cadherin and β-catenin. HMVECs expressed a stronger level of VE-cadherin than N-cadherin, suggestive of a main role of VE-cadherin in cell to cell contacts (Fig. 3A). The media from control and YKL-40 shRNA cells did not alter expression of VE-cadherin, N-cadherin, or β-catenin (Fig. 3A & Supplemental Fig. 3A). However, like GSDCs, co-immunoprecipitation studies showed that media from YKL-40 shRNA cells inhibited the interaction of VE-cadherin with β-catenin, and β-catenin with actin in HMVECs (Fig. 3B & Supplemental Fig. 3B). A minimal level of N-cadherin/β-catenin association was detected in HMVECs treated with conditioned media from either control or YKL-40 shRNA cells (data not shown). Because VEGF secretion was significantly elevated in the conditioned media from YKL-40 shRNA cells (Fig. 2A), the increased VEGF may contribute mainly to the reduced VE-cadherin/β-catenin interaction. To test this possibility, we used an anti-VEGF neutralizing antibody in the co-immunoprecipitation assay of HMVECs. VEGF blockade restored the association of VE-cadherin with β-catenin (Fig. 3B). Consistent with the co-immunoprecipitation data, HMVECs treated with conditioned medium of YKL-40 shRNA cells displayed reduced co-localization of VE-cadherin and β-catenin to 13% relative to control cell medium, and treatment with an anti-VEGF neutralizing antibody recovered the co-localization to approximately 63% of the control levels (Fig. 3C & 3D). In addition, the cell aggregation analysis unveiled the similar inhibition of cell to cell adhesion (by 46–54%) by conditioned media from YKL-40 shRNA, control cells treated with a VE-cadherin neutralizing antibody, or YKL-40 shRNA cells treated a VE-cadherin antibody (Fig. 3E). To confirm the key role played by YKL-40 in endothelial cell to cell interaction, we also treated conditioned medium of the control GSDCs expressing YKL-40 with a neutralizing YKL-40 antibody (mAY) (40). The conditioned medium containing mAY abolished the interaction between VE-cadherin and β-catenin relative to the control mIgG medium (Fig. 3F). Accordingly, the control GSDC medium in the presence of mAY suppressed HMVEC aggregation by 60% compared to the aggregation in the presence of mIgG (Fig. 3G). While neither the neutralizing anti-VEGF antibody nor mAY altered expression of VE-cadherin, N-cadherin and β-catenin in HMVECs (data not shown), treatment of control GSDCs with mAY inhibited YKL-40 expression; in contrast, the anti-VEGF antibody in YKL-40 shRNA GSDCs induced VEGF (Supplemental Fig. 3C), suggestive of distinct responses of mural cells to individual inhibitors. Collectively, these data suggest that YKL-40 may induce the VEcadherin/β-catenin/actin pathway and cell to cell adhesion in endothelial cells, and that YKL-40 blockade inhibits these effects, which is largely dependent on VEGF.

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