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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 expression in GSDC-transplanted tumors is associated with vascular stability, mural cell coverage, angiogenesis, and tumor growthA. Representative immunofluorescent images of control and YKL-40 shRNA GSDC brain tumor sections from SCID/Beige mice depicted single staining of CD31 (red) (a, b) and double staining of CD31 (red) with either SMa (green) (c, d) or fibrinogen (green) (e, f). DAPI (blue) was used to stain the nuclei. B. Quantification of CD31 vessel density and vessel diameter from A (a, b) as described in the Methods. The latter was an average of individual luminal diameters. C. Quantification of percent mural cell coverage of CD31 vessels from A (c, d). The data were derived from the ratio of SMa density to CD31 density. D. Quantification of the ratio of fibrinogen vs. CD31 for vessel leakiness from A (e, f), in which the ratio of fibrinogen density to CD31 density in the control tumors was set as 1 unit. E. Representative control and YKL-40 shRNA GSDC tumor staining images of the proliferation marker Ki67. F. Percentage of Ki67 positive cells with brown nuclear staining was quantified. G. Cell proliferation in culture using MTS assay. N=12. H. Kaplan-Myer Survival curve of SCID/Beige mice bearing control or YKL-40 shRNA tumors. N=5. *P≤0.05 compared to corresponding controls. Bars: 100 μm.
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Figure 1: YKL-40 expression in GSDC-transplanted tumors is associated with vascular stability, mural cell coverage, angiogenesis, and tumor growthA. Representative immunofluorescent images of control and YKL-40 shRNA GSDC brain tumor sections from SCID/Beige mice depicted single staining of CD31 (red) (a, b) and double staining of CD31 (red) with either SMa (green) (c, d) or fibrinogen (green) (e, f). DAPI (blue) was used to stain the nuclei. B. Quantification of CD31 vessel density and vessel diameter from A (a, b) as described in the Methods. The latter was an average of individual luminal diameters. C. Quantification of percent mural cell coverage of CD31 vessels from A (c, d). The data were derived from the ratio of SMa density to CD31 density. D. Quantification of the ratio of fibrinogen vs. CD31 for vessel leakiness from A (e, f), in which the ratio of fibrinogen density to CD31 density in the control tumors was set as 1 unit. E. Representative control and YKL-40 shRNA GSDC tumor staining images of the proliferation marker Ki67. F. Percentage of Ki67 positive cells with brown nuclear staining was quantified. G. Cell proliferation in culture using MTS assay. N=12. H. Kaplan-Myer Survival curve of SCID/Beige mice bearing control or YKL-40 shRNA tumors. N=5. *P≤0.05 compared to corresponding controls. Bars: 100 μm.

Mentions: To investigate a potential role of YKL-40 in tumor vascular stability, permeability, and angiogenesis in vivo, we engaged an orthotopic xenografted tumor model by injecting GSDCs expressing scramble RNA or one of YKL-40 shRNAs (shRNA 1) into the brains of SCID/Beige mice for a 5-month observation period. After the mice were sacrificed, we examined tumor sections for angiogenesis by staining CD31, an endothelial cell marker. GSDC control tumors revealed a strong vascularized phenotype as an intense CD31-positive vessel density was found throughout the entire tumor region (Fig. 1A-a). In contrast, YKL-40 shRNA tumors displayed a significant reduction of the vessel density by approximately 60% (Fig. 1A-b & 1B). In addition, most of the vessels in the control tumors contained a visible lumen, whereas vessels in the YKL-40 shRNA tumors were collapsed and vessel diameters diminished to 30% relative to the control ones (Fig. 1A-a, b, & 1B). An analysis of mural cell coverage by co-staining CD31 and SMa indicated that over 90% of endothelial cell-based vessels in the control tumors were covered with mural cells, in comparison with less than 40% of vessels lined with mural cells in the YKL-40 shRNA tumors (Fig. 1A–c, d, & 1C). To distinguish vessels covered by the tumor-derived GSDCs from those by host-derived mural cells, we similarly injected GSDCs carrying green fluorescent protein (GFP) into different mice. The majority of endothelial cell vessels (CD31) were surrounded by GFP-positive GSDCs at the abluminal site where GFP and SMa were co-localized, while a few vessels were stained with CD31 only (Supplemental Fig. 2A–2F), suggesting that tumor-derived mural-like cells make a significant contribution to tumor vessel coverage. Consistent with this finding, YKL-40 was expressed by SMa-positive mural cells in addition to tumor cells (Supplemental Fig. 2H & 2I). Vessel permeability was measured by diffusion of fibrinogen from the blood circulation. A limited amount of fibrinogen was identified to be diffused out of capillaries in the control tumors, contrary to that in the YKL-40 shRNA tumors which contained more than 6-fold greater diffusion of fibrinogen, indicative of leakier vessels (Fig. 1A–e, f, & Fig. 1D). These in vivo data suggest that YKL-40 expressed by GSDCs mediates vascular mural cell coverage, stability, and angiogenesis.


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 expression in GSDC-transplanted tumors is associated with vascular stability, mural cell coverage, angiogenesis, and tumor growthA. Representative immunofluorescent images of control and YKL-40 shRNA GSDC brain tumor sections from SCID/Beige mice depicted single staining of CD31 (red) (a, b) and double staining of CD31 (red) with either SMa (green) (c, d) or fibrinogen (green) (e, f). DAPI (blue) was used to stain the nuclei. B. Quantification of CD31 vessel density and vessel diameter from A (a, b) as described in the Methods. The latter was an average of individual luminal diameters. C. Quantification of percent mural cell coverage of CD31 vessels from A (c, d). The data were derived from the ratio of SMa density to CD31 density. D. Quantification of the ratio of fibrinogen vs. CD31 for vessel leakiness from A (e, f), in which the ratio of fibrinogen density to CD31 density in the control tumors was set as 1 unit. E. Representative control and YKL-40 shRNA GSDC tumor staining images of the proliferation marker Ki67. F. Percentage of Ki67 positive cells with brown nuclear staining was quantified. G. Cell proliferation in culture using MTS assay. N=12. H. Kaplan-Myer Survival curve of SCID/Beige mice bearing control or YKL-40 shRNA tumors. N=5. *P≤0.05 compared to corresponding controls. Bars: 100 μm.
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Related In: Results  -  Collection

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Figure 1: YKL-40 expression in GSDC-transplanted tumors is associated with vascular stability, mural cell coverage, angiogenesis, and tumor growthA. Representative immunofluorescent images of control and YKL-40 shRNA GSDC brain tumor sections from SCID/Beige mice depicted single staining of CD31 (red) (a, b) and double staining of CD31 (red) with either SMa (green) (c, d) or fibrinogen (green) (e, f). DAPI (blue) was used to stain the nuclei. B. Quantification of CD31 vessel density and vessel diameter from A (a, b) as described in the Methods. The latter was an average of individual luminal diameters. C. Quantification of percent mural cell coverage of CD31 vessels from A (c, d). The data were derived from the ratio of SMa density to CD31 density. D. Quantification of the ratio of fibrinogen vs. CD31 for vessel leakiness from A (e, f), in which the ratio of fibrinogen density to CD31 density in the control tumors was set as 1 unit. E. Representative control and YKL-40 shRNA GSDC tumor staining images of the proliferation marker Ki67. F. Percentage of Ki67 positive cells with brown nuclear staining was quantified. G. Cell proliferation in culture using MTS assay. N=12. H. Kaplan-Myer Survival curve of SCID/Beige mice bearing control or YKL-40 shRNA tumors. N=5. *P≤0.05 compared to corresponding controls. Bars: 100 μm.
Mentions: To investigate a potential role of YKL-40 in tumor vascular stability, permeability, and angiogenesis in vivo, we engaged an orthotopic xenografted tumor model by injecting GSDCs expressing scramble RNA or one of YKL-40 shRNAs (shRNA 1) into the brains of SCID/Beige mice for a 5-month observation period. After the mice were sacrificed, we examined tumor sections for angiogenesis by staining CD31, an endothelial cell marker. GSDC control tumors revealed a strong vascularized phenotype as an intense CD31-positive vessel density was found throughout the entire tumor region (Fig. 1A-a). In contrast, YKL-40 shRNA tumors displayed a significant reduction of the vessel density by approximately 60% (Fig. 1A-b & 1B). In addition, most of the vessels in the control tumors contained a visible lumen, whereas vessels in the YKL-40 shRNA tumors were collapsed and vessel diameters diminished to 30% relative to the control ones (Fig. 1A-a, b, & 1B). An analysis of mural cell coverage by co-staining CD31 and SMa indicated that over 90% of endothelial cell-based vessels in the control tumors were covered with mural cells, in comparison with less than 40% of vessels lined with mural cells in the YKL-40 shRNA tumors (Fig. 1A–c, d, & 1C). To distinguish vessels covered by the tumor-derived GSDCs from those by host-derived mural cells, we similarly injected GSDCs carrying green fluorescent protein (GFP) into different mice. The majority of endothelial cell vessels (CD31) were surrounded by GFP-positive GSDCs at the abluminal site where GFP and SMa were co-localized, while a few vessels were stained with CD31 only (Supplemental Fig. 2A–2F), suggesting that tumor-derived mural-like cells make a significant contribution to tumor vessel coverage. Consistent with this finding, YKL-40 was expressed by SMa-positive mural cells in addition to tumor cells (Supplemental Fig. 2H & 2I). Vessel permeability was measured by diffusion of fibrinogen from the blood circulation. A limited amount of fibrinogen was identified to be diffused out of capillaries in the control tumors, contrary to that in the YKL-40 shRNA tumors which contained more than 6-fold greater diffusion of fibrinogen, indicative of leakier vessels (Fig. 1A–e, f, & Fig. 1D). These in vivo data suggest that YKL-40 expressed by GSDCs mediates vascular mural cell coverage, stability, and angiogenesis.

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