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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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GSDCs expressing YKL-40 stabilize endothelial cell vessels in a manner dependent on VE-cadherin and N-cadherin activityA. HMVECs and either control or YKL-40 shRNA GSDCs were pre-stained with Calcein AM (green) and Calcein red, respectively, and plated together on Matrigel. Tube formation was analyzed over a 64-hour time course and representative images were shown at 16, 24, 40, and 64 hr. White arrows demonstrated breaks in the tube networks, while black arrows on the phase contrast images depicted gaps in the corresponding networks. Bars: 100 μm. B. Quantification of the tubules formed by HMVECs plus control or YKL-40 shRNA GSDCs. N=3, *P≤0.05 compared to controls. C. Same condition as described in A was set up in the presence of recombinant VEGF (10 ng/ml), an anti-VEGF (100 ng/ml), VE-cadherin (20 μg/ml), or N-cadherin antibody (50 μg/ml). 24 hr following incubation, tubules with fluorescence were analyzed and quantified. N=3, *P≤0.05 compared to mIgG.
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Figure 7: GSDCs expressing YKL-40 stabilize endothelial cell vessels in a manner dependent on VE-cadherin and N-cadherin activityA. HMVECs and either control or YKL-40 shRNA GSDCs were pre-stained with Calcein AM (green) and Calcein red, respectively, and plated together on Matrigel. Tube formation was analyzed over a 64-hour time course and representative images were shown at 16, 24, 40, and 64 hr. White arrows demonstrated breaks in the tube networks, while black arrows on the phase contrast images depicted gaps in the corresponding networks. Bars: 100 μm. B. Quantification of the tubules formed by HMVECs plus control or YKL-40 shRNA GSDCs. N=3, *P≤0.05 compared to controls. C. Same condition as described in A was set up in the presence of recombinant VEGF (10 ng/ml), an anti-VEGF (100 ng/ml), VE-cadherin (20 μg/ml), or N-cadherin antibody (50 μg/ml). 24 hr following incubation, tubules with fluorescence were analyzed and quantified. N=3, *P≤0.05 compared to mIgG.

Mentions: In an attempt to assess if GSDCs indeed act as mural cells to stabilize endothelial cell vessels, we employed a tube formation assay on Matrigel by co-culturing both HMVECs and GSDCs. HMVECs pre-labeled with Calcein AM (green fluorescence) were mixed with either control or YKL-40 shRNA GSDCs pre-labeled with Calcein red (red fluorescence). Stability of tubules formed by HMVECs and GSDCs was monitored over a 64-hr time course. As shown in Fig. 7A, HMVECs co-cultured with control GSDCs maintained tubules longer than did HMVECs in the presence of YKL-40 shRNA GSDCs. The breakdowns and gaps in the tube network, indicated by arrows, were significantly less in HMVECs with control GSDCs at 24 hours and 40 hours than corresponding HMVECs co-cultured with YKL-40 shRNA GSDCs (Fig. 7A & 7B). HMVECs alone maintained their own tubules only between 24–36 hr (data not shown). To further validate the individual role of VE-cadherin and N-cadherin in vascular stability, we treated this co-culture system with a VE-cadherin or N-cadherin neutralizing antibody. When HMVECs co-cultured with control GSDCs were treated with either cadherin antibody, tubule stability was decreased to the level seen in the co-culture of HMVECs and YKL-40 shRNA GSDCs (Fig. 7C & Supplemental Fig. 5). As expected in the system of HMVECs and YKL-40 shRNA GSDCs, anti-VE-cadherin and N-cadherin antibodies were unable to influence the stability because of impaired activity of VE-cadherin and N-cadherin by YKL-40 gene knockdown. To determine a role of VEGF in the co-culture system, we treated HMVECs and control GSDCs with recombinant VEGF, and we found that the addition of VEGF to the YKL-40-expressing system developed more stabilized tubules than did control counterparts (Fig. 7C & Supplemental Fig. 5). The phenotype of elevated tubes is probably due to the synergistic cooperation of these two strong angiogenic factors for the tube generation and stabilization, as the vessel-destabilized property of VEGF may be notably minimized in the presence of YKL-40. An anti-VEGF antibody partially rescued the tube stability formed by HMVECs and YKL-40 shRNA cells that express a high level of VEGF. Failure of full tubule recovery in the presence of the anti-VEGF antibody is attributed to impaired N-cad function, even when VEGF is inhibited. Thus, these data suggest that the ability of GSDCs to stabilize endothelial cell-based vasculature is reliant on YKL-40 expression that regulates VEGF, VE- and N-cadherin activity. Altogether, in coordination with tumor vasculature found in vivo, the in vitro system identifying cell-cell contacts/adhesion, permeability, and stability of vascular wall cells have provided the critical mechanisms strengthening our conclusion that YKL-40 plays a central role in mural cell-mediated tumor angiogenesis via autocrine and paracrine loops.


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)

GSDCs expressing YKL-40 stabilize endothelial cell vessels in a manner dependent on VE-cadherin and N-cadherin activityA. HMVECs and either control or YKL-40 shRNA GSDCs were pre-stained with Calcein AM (green) and Calcein red, respectively, and plated together on Matrigel. Tube formation was analyzed over a 64-hour time course and representative images were shown at 16, 24, 40, and 64 hr. White arrows demonstrated breaks in the tube networks, while black arrows on the phase contrast images depicted gaps in the corresponding networks. Bars: 100 μm. B. Quantification of the tubules formed by HMVECs plus control or YKL-40 shRNA GSDCs. N=3, *P≤0.05 compared to controls. C. Same condition as described in A was set up in the presence of recombinant VEGF (10 ng/ml), an anti-VEGF (100 ng/ml), VE-cadherin (20 μg/ml), or N-cadherin antibody (50 μg/ml). 24 hr following incubation, tubules with fluorescence were analyzed and quantified. N=3, *P≤0.05 compared to mIgG.
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Related In: Results  -  Collection

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Figure 7: GSDCs expressing YKL-40 stabilize endothelial cell vessels in a manner dependent on VE-cadherin and N-cadherin activityA. HMVECs and either control or YKL-40 shRNA GSDCs were pre-stained with Calcein AM (green) and Calcein red, respectively, and plated together on Matrigel. Tube formation was analyzed over a 64-hour time course and representative images were shown at 16, 24, 40, and 64 hr. White arrows demonstrated breaks in the tube networks, while black arrows on the phase contrast images depicted gaps in the corresponding networks. Bars: 100 μm. B. Quantification of the tubules formed by HMVECs plus control or YKL-40 shRNA GSDCs. N=3, *P≤0.05 compared to controls. C. Same condition as described in A was set up in the presence of recombinant VEGF (10 ng/ml), an anti-VEGF (100 ng/ml), VE-cadherin (20 μg/ml), or N-cadherin antibody (50 μg/ml). 24 hr following incubation, tubules with fluorescence were analyzed and quantified. N=3, *P≤0.05 compared to mIgG.
Mentions: In an attempt to assess if GSDCs indeed act as mural cells to stabilize endothelial cell vessels, we employed a tube formation assay on Matrigel by co-culturing both HMVECs and GSDCs. HMVECs pre-labeled with Calcein AM (green fluorescence) were mixed with either control or YKL-40 shRNA GSDCs pre-labeled with Calcein red (red fluorescence). Stability of tubules formed by HMVECs and GSDCs was monitored over a 64-hr time course. As shown in Fig. 7A, HMVECs co-cultured with control GSDCs maintained tubules longer than did HMVECs in the presence of YKL-40 shRNA GSDCs. The breakdowns and gaps in the tube network, indicated by arrows, were significantly less in HMVECs with control GSDCs at 24 hours and 40 hours than corresponding HMVECs co-cultured with YKL-40 shRNA GSDCs (Fig. 7A & 7B). HMVECs alone maintained their own tubules only between 24–36 hr (data not shown). To further validate the individual role of VE-cadherin and N-cadherin in vascular stability, we treated this co-culture system with a VE-cadherin or N-cadherin neutralizing antibody. When HMVECs co-cultured with control GSDCs were treated with either cadherin antibody, tubule stability was decreased to the level seen in the co-culture of HMVECs and YKL-40 shRNA GSDCs (Fig. 7C & Supplemental Fig. 5). As expected in the system of HMVECs and YKL-40 shRNA GSDCs, anti-VE-cadherin and N-cadherin antibodies were unable to influence the stability because of impaired activity of VE-cadherin and N-cadherin by YKL-40 gene knockdown. To determine a role of VEGF in the co-culture system, we treated HMVECs and control GSDCs with recombinant VEGF, and we found that the addition of VEGF to the YKL-40-expressing system developed more stabilized tubules than did control counterparts (Fig. 7C & Supplemental Fig. 5). The phenotype of elevated tubes is probably due to the synergistic cooperation of these two strong angiogenic factors for the tube generation and stabilization, as the vessel-destabilized property of VEGF may be notably minimized in the presence of YKL-40. An anti-VEGF antibody partially rescued the tube stability formed by HMVECs and YKL-40 shRNA cells that express a high level of VEGF. Failure of full tubule recovery in the presence of the anti-VEGF antibody is attributed to impaired N-cad function, even when VEGF is inhibited. Thus, these data suggest that the ability of GSDCs to stabilize endothelial cell-based vasculature is reliant on YKL-40 expression that regulates VEGF, VE- and N-cadherin activity. Altogether, in coordination with tumor vasculature found in vivo, the in vitro system identifying cell-cell contacts/adhesion, permeability, and stability of vascular wall cells have provided the critical mechanisms strengthening our conclusion that YKL-40 plays a central role in mural cell-mediated tumor angiogenesis via autocrine and paracrine loops.

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