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Tumor-derived microvesicles induce proangiogenic phenotype in endothelial cells via endocytosis.

Kawamoto T, Ohga N, Akiyama K, Hirata N, Kitahara S, Maishi N, Osawa T, Yamamoto K, Kondoh M, Shindoh M, Hida Y, Hida K - PLoS ONE (2012)

Bottom Line: In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC.Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore.Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Vascular Biology, Graduate School of Dental Medicine, University of Hokkaido, Sapporo, Hokkaido, Japan.

ABSTRACT

Background: Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear.

Methodology/principal findings: Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis.

Conclusion: We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment.

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Related in: MedlinePlus

Characterization of isolated normal endothelial cells (NEC) from mice.(A) Representative flow cytometric analysis of NEC showing the expression of the endothelial markers CD31, CD105, CD144, and BS1-B4 lectin (mouse endothelial marker) (white area). Control levels with normal isotype IgG are shown in the gray area. Binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 revealed the high purity of the isolated NEC by FACS. (B) RT-PCR revealed that NEC expressed CD31, CD105, CD144, vascular endothelial growth factor receptor-1, 2 (VEGFR1 and VEGFR2) and not the monocyte marker CD11b and the hematopoietic marker CD45. To demonstrate that NEC were purified without any blood cell contamination, we also performed RT-PCR in mouse peripheral blood mononuclear cells (PBMC). Negative control was performed with template-free samples. (C) A375-SM cultured medium are analyzed by flow cytometry. The figure shows the forward and side scatter plot of the medium. A375-SM cells were cultured for 48 h. (D) Isolated TMV were observed by electron scanning microscopy. For sample preparation, TMV were coated on a glass cover for 4 h. (E, F) Flow cytometric analysis of TMV showing expression of HLA and phosphatidylserine (white area). (G) Western blotting analysis of TMV showing expression of HLA and β-actin. Same amount of proteins were loaded. These results indicate that some TMV might be shedding microvesicles derived from tumor plasma membrane.
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pone-0034045-g001: Characterization of isolated normal endothelial cells (NEC) from mice.(A) Representative flow cytometric analysis of NEC showing the expression of the endothelial markers CD31, CD105, CD144, and BS1-B4 lectin (mouse endothelial marker) (white area). Control levels with normal isotype IgG are shown in the gray area. Binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 revealed the high purity of the isolated NEC by FACS. (B) RT-PCR revealed that NEC expressed CD31, CD105, CD144, vascular endothelial growth factor receptor-1, 2 (VEGFR1 and VEGFR2) and not the monocyte marker CD11b and the hematopoietic marker CD45. To demonstrate that NEC were purified without any blood cell contamination, we also performed RT-PCR in mouse peripheral blood mononuclear cells (PBMC). Negative control was performed with template-free samples. (C) A375-SM cultured medium are analyzed by flow cytometry. The figure shows the forward and side scatter plot of the medium. A375-SM cells were cultured for 48 h. (D) Isolated TMV were observed by electron scanning microscopy. For sample preparation, TMV were coated on a glass cover for 4 h. (E, F) Flow cytometric analysis of TMV showing expression of HLA and phosphatidylserine (white area). (G) Western blotting analysis of TMV showing expression of HLA and β-actin. Same amount of proteins were loaded. These results indicate that some TMV might be shedding microvesicles derived from tumor plasma membrane.

Mentions: NEC were isolated from mouse dermal tissue using a magnetic cell sorting system as described above. Flow cytometric analysis revealed the binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 in NEC (Figure 1A). RT-PCR revealed that NEC expressed CD31, CD105, CD144, VEGFR1, and VEGFR2, indicating that EC characteristics were present even during culture. Isolated EC were negative for the monocyte marker CD11b and hematopoietic marker CD45 by RT-PCR. As the negative controls, template-free samples were used (Figure 1B). These results showed a high purity of isolated NEC.


Tumor-derived microvesicles induce proangiogenic phenotype in endothelial cells via endocytosis.

Kawamoto T, Ohga N, Akiyama K, Hirata N, Kitahara S, Maishi N, Osawa T, Yamamoto K, Kondoh M, Shindoh M, Hida Y, Hida K - PLoS ONE (2012)

Characterization of isolated normal endothelial cells (NEC) from mice.(A) Representative flow cytometric analysis of NEC showing the expression of the endothelial markers CD31, CD105, CD144, and BS1-B4 lectin (mouse endothelial marker) (white area). Control levels with normal isotype IgG are shown in the gray area. Binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 revealed the high purity of the isolated NEC by FACS. (B) RT-PCR revealed that NEC expressed CD31, CD105, CD144, vascular endothelial growth factor receptor-1, 2 (VEGFR1 and VEGFR2) and not the monocyte marker CD11b and the hematopoietic marker CD45. To demonstrate that NEC were purified without any blood cell contamination, we also performed RT-PCR in mouse peripheral blood mononuclear cells (PBMC). Negative control was performed with template-free samples. (C) A375-SM cultured medium are analyzed by flow cytometry. The figure shows the forward and side scatter plot of the medium. A375-SM cells were cultured for 48 h. (D) Isolated TMV were observed by electron scanning microscopy. For sample preparation, TMV were coated on a glass cover for 4 h. (E, F) Flow cytometric analysis of TMV showing expression of HLA and phosphatidylserine (white area). (G) Western blotting analysis of TMV showing expression of HLA and β-actin. Same amount of proteins were loaded. These results indicate that some TMV might be shedding microvesicles derived from tumor plasma membrane.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316594&req=5

pone-0034045-g001: Characterization of isolated normal endothelial cells (NEC) from mice.(A) Representative flow cytometric analysis of NEC showing the expression of the endothelial markers CD31, CD105, CD144, and BS1-B4 lectin (mouse endothelial marker) (white area). Control levels with normal isotype IgG are shown in the gray area. Binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 revealed the high purity of the isolated NEC by FACS. (B) RT-PCR revealed that NEC expressed CD31, CD105, CD144, vascular endothelial growth factor receptor-1, 2 (VEGFR1 and VEGFR2) and not the monocyte marker CD11b and the hematopoietic marker CD45. To demonstrate that NEC were purified without any blood cell contamination, we also performed RT-PCR in mouse peripheral blood mononuclear cells (PBMC). Negative control was performed with template-free samples. (C) A375-SM cultured medium are analyzed by flow cytometry. The figure shows the forward and side scatter plot of the medium. A375-SM cells were cultured for 48 h. (D) Isolated TMV were observed by electron scanning microscopy. For sample preparation, TMV were coated on a glass cover for 4 h. (E, F) Flow cytometric analysis of TMV showing expression of HLA and phosphatidylserine (white area). (G) Western blotting analysis of TMV showing expression of HLA and β-actin. Same amount of proteins were loaded. These results indicate that some TMV might be shedding microvesicles derived from tumor plasma membrane.
Mentions: NEC were isolated from mouse dermal tissue using a magnetic cell sorting system as described above. Flow cytometric analysis revealed the binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 in NEC (Figure 1A). RT-PCR revealed that NEC expressed CD31, CD105, CD144, VEGFR1, and VEGFR2, indicating that EC characteristics were present even during culture. Isolated EC were negative for the monocyte marker CD11b and hematopoietic marker CD45 by RT-PCR. As the negative controls, template-free samples were used (Figure 1B). These results showed a high purity of isolated NEC.

Bottom Line: In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC.Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore.Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Vascular Biology, Graduate School of Dental Medicine, University of Hokkaido, Sapporo, Hokkaido, Japan.

ABSTRACT

Background: Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear.

Methodology/principal findings: Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis.

Conclusion: We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment.

Show MeSH
Related in: MedlinePlus