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Shed syndecan-2 inhibits angiogenesis.

De Rossi G, Evans AR, Kay E, Woodfin A, McKay TR, Nourshargh S, Whiteford JR - J. Cell. Sci. (2014)

Bottom Line: The heparan sulphate proteoglycan syndecan-2 is expressed on mesenchymal cells in the vasculature and, like the other members of its family, can be shed from the cell surface resulting in the release of its extracellular core protein.We demonstrate that shed syndecan-2 regulates angiogenesis by inhibiting endothelial cell migration in human and rodent models and, as a result, reduces tumour growth.Furthermore, our findings show that these effects are mediated by the protein tyrosine phosphatase receptor CD148 (also known as PTPRJ) and this interaction corresponds with a decrease in active β1 integrin.

View Article: PubMed Central - PubMed

Affiliation: William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK.

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Constitutively released syndecan-2 extracellular core protein from HEK293t cells inhibits angiogenesis in a xenograft tumour model. (A) Schematic diagram of syndecan-2 lacking both cytoplasmic and transmembrane domains (eS2ED), which is constitutively released from HEK293t cells. (B) Dot blot of conditioned media from cells transfected with either empty vector (EV) or eS2ED constructs. Dot blots were probed with the anti-HA antibody, and a strong signal is obtained from cells transfected with eS2ED. (C) Constitutively released syndecan-2 inhibits tumour growth. Micrograph of tumours from SCID-SHO mice injected with either empty vector cells or eS2ED cells. Scale bar: 1 cm. (D) Tumours derived from eS2ED cells have smaller diameter. Error bars represent the s.e.m. **P<0.01 (n = 5 tumours per condition, Student's t-test. (E) Empty vector cells and S2ED cells have the same growth kinetics over 96 h in culture. (F) Immunofluorescence staining of tumour sections derived from empty vector cells and eS2ED-transfected cells for the endothelial cell marker CD31 (red; tumour cells are GFP positive). Considerably more CD31-positive structures are evident in tumours derived from cells transfected with empty vector as compared the tumours in which syndecan-2 is constitutively released. Scale bar: 200 µm. (G) Quantification of the area of micrographs positive for CD31 was performed and expressed as a percentage of the GFP-positive tumour cells. Data are the mean from three separate images from sections of three tumours per group. Error bars represent the s.e.m. ****P<0.0001 (unpaired Student's t-test).
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f01: Constitutively released syndecan-2 extracellular core protein from HEK293t cells inhibits angiogenesis in a xenograft tumour model. (A) Schematic diagram of syndecan-2 lacking both cytoplasmic and transmembrane domains (eS2ED), which is constitutively released from HEK293t cells. (B) Dot blot of conditioned media from cells transfected with either empty vector (EV) or eS2ED constructs. Dot blots were probed with the anti-HA antibody, and a strong signal is obtained from cells transfected with eS2ED. (C) Constitutively released syndecan-2 inhibits tumour growth. Micrograph of tumours from SCID-SHO mice injected with either empty vector cells or eS2ED cells. Scale bar: 1 cm. (D) Tumours derived from eS2ED cells have smaller diameter. Error bars represent the s.e.m. **P<0.01 (n = 5 tumours per condition, Student's t-test. (E) Empty vector cells and S2ED cells have the same growth kinetics over 96 h in culture. (F) Immunofluorescence staining of tumour sections derived from empty vector cells and eS2ED-transfected cells for the endothelial cell marker CD31 (red; tumour cells are GFP positive). Considerably more CD31-positive structures are evident in tumours derived from cells transfected with empty vector as compared the tumours in which syndecan-2 is constitutively released. Scale bar: 200 µm. (G) Quantification of the area of micrographs positive for CD31 was performed and expressed as a percentage of the GFP-positive tumour cells. Data are the mean from three separate images from sections of three tumours per group. Error bars represent the s.e.m. ****P<0.0001 (unpaired Student's t-test).

Mentions: To investigate the potential effects of shed syndecan-2 on angiogenesis we established a mammalian expression system in which a constitutively released form of the molecule (lacking both transmembrane and cytoplasmic domains, M1–F141, eS2ED) was expressed in HEK293t cells (Fig. 1A). The HA epitope was also incorporated into eS2ED between D27 and K28 enabling us to readily detect eS2ED in conditioned media as compared to controls transfected with empty vector (Fig. 1B; supplementary material Fig. S1). Using these cell lines, we investigated the anti-angiogenic properties of shed syndecan-2 in a xenograft tumour model. Specifically, SCID-SHO mice were injected with empty vector cells or cells that constitutively released syndecan-2 (eS2ED) and tumour sizes were quantified at 21 days. Mice injected with control (empty vector) cells developed vascularised tumours that were significantly larger both in terms of diameter and weight as compared to tumours generated in mice injected with the eS2ED cells (Fig. 1C,D; supplementary material Fig. S2). We compared the growth kinetics in culture between empty vector cells and eS2ED-transfected cell lines and found no differences, indicating that cell proliferation defects were not the reason tumours derived from eS2ED cells failed to develop (Fig. 1E). Immunofluorescence staining of tumour sections for the endothelial cell marker CD31 (also known as PECAM1) revealed that tumours derived from empty vector control cells contained CD31-positive structures consistent with blood vessels. CD31-positive cells were greatly reduced in tumour sections derived from cells constitutively releasing syndecan-2 (Fig. 1F,G). These findings suggest that constitutive release of the syndecan-2 ectodomain in this model is inhibitory to angiogenesis.


Shed syndecan-2 inhibits angiogenesis.

De Rossi G, Evans AR, Kay E, Woodfin A, McKay TR, Nourshargh S, Whiteford JR - J. Cell. Sci. (2014)

Constitutively released syndecan-2 extracellular core protein from HEK293t cells inhibits angiogenesis in a xenograft tumour model. (A) Schematic diagram of syndecan-2 lacking both cytoplasmic and transmembrane domains (eS2ED), which is constitutively released from HEK293t cells. (B) Dot blot of conditioned media from cells transfected with either empty vector (EV) or eS2ED constructs. Dot blots were probed with the anti-HA antibody, and a strong signal is obtained from cells transfected with eS2ED. (C) Constitutively released syndecan-2 inhibits tumour growth. Micrograph of tumours from SCID-SHO mice injected with either empty vector cells or eS2ED cells. Scale bar: 1 cm. (D) Tumours derived from eS2ED cells have smaller diameter. Error bars represent the s.e.m. **P<0.01 (n = 5 tumours per condition, Student's t-test. (E) Empty vector cells and S2ED cells have the same growth kinetics over 96 h in culture. (F) Immunofluorescence staining of tumour sections derived from empty vector cells and eS2ED-transfected cells for the endothelial cell marker CD31 (red; tumour cells are GFP positive). Considerably more CD31-positive structures are evident in tumours derived from cells transfected with empty vector as compared the tumours in which syndecan-2 is constitutively released. Scale bar: 200 µm. (G) Quantification of the area of micrographs positive for CD31 was performed and expressed as a percentage of the GFP-positive tumour cells. Data are the mean from three separate images from sections of three tumours per group. Error bars represent the s.e.m. ****P<0.0001 (unpaired Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215719&req=5

f01: Constitutively released syndecan-2 extracellular core protein from HEK293t cells inhibits angiogenesis in a xenograft tumour model. (A) Schematic diagram of syndecan-2 lacking both cytoplasmic and transmembrane domains (eS2ED), which is constitutively released from HEK293t cells. (B) Dot blot of conditioned media from cells transfected with either empty vector (EV) or eS2ED constructs. Dot blots were probed with the anti-HA antibody, and a strong signal is obtained from cells transfected with eS2ED. (C) Constitutively released syndecan-2 inhibits tumour growth. Micrograph of tumours from SCID-SHO mice injected with either empty vector cells or eS2ED cells. Scale bar: 1 cm. (D) Tumours derived from eS2ED cells have smaller diameter. Error bars represent the s.e.m. **P<0.01 (n = 5 tumours per condition, Student's t-test. (E) Empty vector cells and S2ED cells have the same growth kinetics over 96 h in culture. (F) Immunofluorescence staining of tumour sections derived from empty vector cells and eS2ED-transfected cells for the endothelial cell marker CD31 (red; tumour cells are GFP positive). Considerably more CD31-positive structures are evident in tumours derived from cells transfected with empty vector as compared the tumours in which syndecan-2 is constitutively released. Scale bar: 200 µm. (G) Quantification of the area of micrographs positive for CD31 was performed and expressed as a percentage of the GFP-positive tumour cells. Data are the mean from three separate images from sections of three tumours per group. Error bars represent the s.e.m. ****P<0.0001 (unpaired Student's t-test).
Mentions: To investigate the potential effects of shed syndecan-2 on angiogenesis we established a mammalian expression system in which a constitutively released form of the molecule (lacking both transmembrane and cytoplasmic domains, M1–F141, eS2ED) was expressed in HEK293t cells (Fig. 1A). The HA epitope was also incorporated into eS2ED between D27 and K28 enabling us to readily detect eS2ED in conditioned media as compared to controls transfected with empty vector (Fig. 1B; supplementary material Fig. S1). Using these cell lines, we investigated the anti-angiogenic properties of shed syndecan-2 in a xenograft tumour model. Specifically, SCID-SHO mice were injected with empty vector cells or cells that constitutively released syndecan-2 (eS2ED) and tumour sizes were quantified at 21 days. Mice injected with control (empty vector) cells developed vascularised tumours that were significantly larger both in terms of diameter and weight as compared to tumours generated in mice injected with the eS2ED cells (Fig. 1C,D; supplementary material Fig. S2). We compared the growth kinetics in culture between empty vector cells and eS2ED-transfected cell lines and found no differences, indicating that cell proliferation defects were not the reason tumours derived from eS2ED cells failed to develop (Fig. 1E). Immunofluorescence staining of tumour sections for the endothelial cell marker CD31 (also known as PECAM1) revealed that tumours derived from empty vector control cells contained CD31-positive structures consistent with blood vessels. CD31-positive cells were greatly reduced in tumour sections derived from cells constitutively releasing syndecan-2 (Fig. 1F,G). These findings suggest that constitutive release of the syndecan-2 ectodomain in this model is inhibitory to angiogenesis.

Bottom Line: The heparan sulphate proteoglycan syndecan-2 is expressed on mesenchymal cells in the vasculature and, like the other members of its family, can be shed from the cell surface resulting in the release of its extracellular core protein.We demonstrate that shed syndecan-2 regulates angiogenesis by inhibiting endothelial cell migration in human and rodent models and, as a result, reduces tumour growth.Furthermore, our findings show that these effects are mediated by the protein tyrosine phosphatase receptor CD148 (also known as PTPRJ) and this interaction corresponds with a decrease in active β1 integrin.

View Article: PubMed Central - PubMed

Affiliation: William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK.

Show MeSH
Related in: MedlinePlus