Fig 2: The syndecan-2 extracellular core protein inhibits angiogenesis. (A) Schematic diagram of S2ED consisting of GST fused to the syndecan-2 ectodomain core protein. (B) Angiogenesis is inhibited by S2ED when incorporated into Matrigel. C57BL/6 mice were given sub-dermal injections of Matrigel supplemented with VEGF and FGF containing either 0.5 µM of S2ED or GST. Excised sections of Matrigel plugs were immunostained for CD31 (endothelial cells) and DRAQ5 (all cell nuclei) and analysed by confocal microscopy, and the percentage of cells positive for CD31 calculated. Less endothelial cells migrate into Matrigel plugs containing S2ED. Error bars represent the s.e.m. of four separate images. *P<0.05 (Mann–Whitney t-test). (C) Photographs of plugs after excision. (D) The anti-angiogenic effects of S2ED are dose dependant. Rat aortic rings were seeded in collagen I containing the indicated concentrations of GST or S2ED and the angiogenic sprouts counted after 4 days. Data were obtained from at least 10 rings from four separate rats per condition. *P<0.05, **P<0.01 (one-way ANOVA comparing to untreated controls using the Tukey's comparisons test). (E) S2ED inhibits tubule formation in a HUVEC co-culture system. Using the V2a kit, HUVECs and fibroblasts were co-cultured in the presence of VEGF, Suramin, S2ED and GST. Micrographs, showing fewer tubules, were obtained in the presence of S2ED and these were shorter (F) and less branched (G; branch points denoted by arrows). Scale bar: 500 µm. Error bars represent the s.e.m. of four separate images. *P<0.05, **P<0.01, ****P<0.0001 (Mann–Whitney t-test).

Fig 3: The anti-angiogenic properties of S2ED are due to inhibition of endothelial cell migration and are mediated by amino acids P124–F141. (A) Diagram of the mutant proteins used in this study. Full-length syndecan-2 extracellular core protein S2ED comprising E19–F141, a truncated form of S2ED lacking the adhesion regulatory domain, S2ED?P124–F141 (green), and a mutant containing only the GAG attachment domain and the C-terminal adhesion regulatory domain, S2ED?L73–G123. (B) The adhesion regulatory domain of syndecan-2 inhibits angiogenic sprout formation. Rat aortas were seeded in collagen I matrices containing 0.5 µM of GST, S2ED or the mutant forms of S2ED. Sprouts from eight rings per condition were counted and the mean calculated. Error bars represent the s.e.m. ***P<0.005, ****P<0.0001 (one-way ANOVA with Bonferroni multiple comparison). (C,D) Scratch wound migration assays were performed in the presence of 0.5 µM of the indicated fusion proteins on brain endothelial cells. (D) Endothelial cell migration speed is reduced in the presence of S2ED proteins containing the 18-amino-acid regulatory domain. Cell speed data represents mean±s.e.m. measurements from at least 25 individual cells at the leading edge per treatment. (E) S2ED inhibits endothelial cell invasion into collagen I matrices. GST or S2ED (0.5 µM) were incorporated into collagen I matrices in transwells. After 6 h, cells that had migrated were counted and the data represents the mean±s.e.m. of triplicate assays. *P<0.05 (by Student's t-test). (F) Cell proliferation is unaffected by S2ED. Confluent monolayers of sEND cells were grown in the presence of 0.5 µM of the proteins indicated and mean proliferation measurements were calculated from four wells per condition.

Fig 4: Engagement of CD148 by shed syndecan-2 corresponds with a reduction in active ß1 integrin in endothelial cells leading to inhibition of cell migration and less angiogenesis.

Fig 5: Expression of the Syndecan Family Members in Quiescent and Activated Adult NSCs(A and B) (A) Variations in the expression of Sdc1, Sdc2, and Sdc4 in freshly sorted LeXbright, LeX+EGFR+, and total SVZ cells were confirmed by (A) qRT-PCR (0: undetectable) and (B) immunostaining (scale bar: 5 µm).(C) Quantification of SDC1-, SDC2-, and SDC4-positive cells reveals higher expression of SDC1 by actively proliferating LeX+EGFR+ cells.Data are represented as the mean ± SD from four independent experiments of three or four mice. *p < 0.05.