Fig. 4

Upregulation of IGF1, CXCL10, EDN1 and ANG contributes to IL30-driven endothelial cell proliferation. A, B Elisa assays of IGF1, CXCL10 and EDN1 release by untreated (0 ng/ml) or rhIL30 treated (50-100 ng/ml) HUVEC (A) and HAEC (B). ANOVA: p < 0.01. *p < 0.01, Tukey HSD Test compared with untreated cells. **p < 0.01, Tukey HSD Test compared with cells treated with 50 ng/ml or untreated. Results are expressed as mean ± SD. C Elisa assay of ANG by untreated (0 ng/ml) or rhIL30 treated (50-100 ng/ml) HUVEC. ANOVA: p < 0.0001. *p < 0.01, Tukey HSD test compared with untreated cells. Results are expressed as mean ± SD. D Cytofluorimetric analyses of IGF1R, CXCR3, EDNRA and EDNRB expression in HUVEC (top) and HAEC (bottom). Red lines: isotype control. Experiments were performed in triplicate. E, F MTT assay of HUVEC (E) and HAEC (F), untreated (CTRL) or treated with rhIGF1, rhCXCL10 (5–50 ng/ml) or rhEDN1 (5–100 ng/ml). ANOVA: p < 0.001. *p < 0.05, Tukey HSD Test compared with 0 ng/ml. **p < 0.05, Tukey HSD Test compared with 0 and 5 ng/ml. Experiments were performed in triplicate and results are expressed as mean ± SD. G, H MTT assay of HUVEC (G) and HAEC (H), untreated (CTRL) or treated with anti-IGF1, anti-CXCL10 or anti-EDN1 Abs (0.5–5 μg/ml—48 h). ANOVA: p < 0.001. *p < 0.01, Tukey HSD Test compared with 0.0 µg/ml. **p < 0.05, Tukey HSD Test compared with 0.0, 0.5, 1.0 and 2.5 µg/ml. ***p < 0.05, Tukey HSD Test compared with 0.0, 0.5 and 1.0 µg/ml. Experiments were performed in triplicate and results are expressed as mean ± SD. I, J MTT assay of HUVEC untreated or treated with rhANG (5–50 ng/ml) (I) or with anti-ANG Abs (0.5–5 μg/ml) (J). ANOVA: p < 0.0001. *p < 0.05, Tukey HSD Test compared with 0 ng/ml or 0.0 µg/ml. **p < 0.01, Tukey HSD Test compared with 0, 0.5, 1.0 and 2.5 µg/ml. Experiments were performed in triplicate and results are expressed as mean ± SD. K, L MTT assay of HUVEC untreated (CTRL) or treated with rhIL30 (50 ng/ml), anti-IGF1 (K) or anti-CXCL10 (L) Abs (5 µg/ml), and rhIL30 + anti-IGF1 (K) or rhIL30 + anti-CXCL10 (L) Abs. ANOVA: p < 0.0001. *p < 0.01, Tukey HSD Test compared with CTRL. **p < 0.01, Tukey HSD Test compared with CTRL and cells treated with anti-IGF1 Abs. ***p < 0.01, Tukey HSD Test compared with CTRL and cells treated with rhIL30. Experiments were performed in triplicate and results are expressed as mean ± SD. M, N MTT assay of HUVEC untreated (CTRL) or treated with rhIL30 (50 ng/ml), anti-EDN1 (M) or anti-ANG (N) Abs (5 µg/ml), and rhIL30 + anti-EDN1 (M) or rhIL30 + anti-ANG (N) Abs. ANOVA: p < 0.0001. *p < 0.01, Tukey HSD Test compared with CTRL. **p < 0.01, Tukey HSD Test compared with CTRL and cells treated with rhIL30. Experiments were performed in triplicate and results are expressed as mean ± SD. O, P, Q MTT assay of HAEC untreated (CTRL) or treated with rhIL30 (50 ng/ml), anti-IGF1 (O) or anti-CXCL10 (P) or anti-EDN1 (Q) Abs (5 µg/ml), and rhIL30 + anti-IGF1 (O) or rhIL30 + anti-CXCL10 (P) or rhIL30 + anti-EDN1 (Q) Abs. ANOVA: p < 0.0001. *p < 0.01, Tukey HSD Test compared with CTRL. **p < 0.01, Tukey HSD Test compared with CTRL and cells treated with anti-IGF1 Abs. ***p < 0.01, Tukey HSD Test compared with CTRL and cells treated with rhIL30. Experiments were performed in triplicate and results are expressed as mean ± SD. R Immunohistochemistry shows that endothelin-1 (EDN1) expression is scanty in wild type DU145 tumors (a) and its endothelial network (arrowheads), while it is distinct in IL30 overexpressing DU145 tumors (b) and especially marked in the walls of its vascular network (arrowheads). Results from EV-tumors were comparable to wild type tumors. Magnification: X400. S, T Immunohistochemical features of IL30 knockout and overexpressing PC3 tumors versus wild type tumors (S, a-c) showing a different proliferation (S, d-f), vascularization (T, a-c) and expression of IGF1 (T, d-f, vessels indicated by arrows). IGF1 expression in the vascular endothelium was moderate in the wild type tumor, almost absent in the IL30KO tumor and increased in the IL30 overexpressing tumor, as indicated by the arrows. Results from EV-transfected and NTgRNA-tumors were comparable to wild type tumors. Magnification: X400; Ta-c, X200