Fig 2: Anti-VEGF therapy led to decreased infiltration of CD68+ macrophages in human GBM A–GAnti-CD68 (A), anti-CSFR1 (B), and anti-CD206 (C) immunohistochemistry of patient samples derived from treatment-naive GBM (n = 24), post-radiochemotherapy (S/RTx/CTx) (n = 7), and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) (n = 29) therapy are shown. Corresponding quantification of tumor-infiltrating cells is displayed (anti-CD68, D), (anti-CSF1R, E), and (anti-CD206, F). Ratio of CD206+ versus CD68+ cells (G).HAmount of CD206+ perivascular cells per blood vessel.IDouble staining of Ang-2 (brown) and CD206 (red) of a S/RTx/CTx/Bev patient, arrowheads indicating perivascular CD206+ cells around Ang-2+ vessels. Arrows pointing on parenchymal CD206+ cells.J CD206+ cells in relation to the percentage of hypoxic (CAIX positive) tumor area.Data information: Statistical analysis: Kruskal–Wallis (Dunn's post-test). *P < 0.05, ***P < 0.005; data are mean ± SEM. Scale bars (A–C, I): 100 µm.

Fig 3: Bevacizumab therapy leads to reduced vessel density and increased Ang-2 expression A–EImmunohistochemistry stainings with antibodies directed against CD31 (A) and Ang-2 (B) in treatment-naive GBM, post-radiochemotherapy (S/RTx/CTx), and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) therapy are displayed. Quantitative analyses of microvessel densities and Ang-2+ vessels are shown in (C) and (D). The ratio of Ang-2+ versus CD31+ vessels was determined in (E). Scale bars: 200 µm (A), 100 µm (B).FWestern Blot and corresponding quantification of CD31 and Ang-2 in control and aflibercept-treated mice (n = 5 each).G–I CAIX staining of treatment-naive GBM and S/RTx/CTx/Bev patients. Analyses of hypoxic (CAIX +) area with regard to the whole tumor in human GBM (H). Tumor-infiltrating lymphocytes (CD8/CD3) in human patients (I). Scale bar: 100 µm.Data information: Kruskal–Wallis (C–E, H–I). *P < 0.05, **P < 0.01, ***P < 0.005; treatment-naive GBM n = 26, S/RTx/CTx n = 7, S/RTx/CTx/Bev n = 29; t-test (F); data are mean ± SEM.

Fig 4: Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas A, B ELISA displaying human Ang-2 (A) and Ang-1 (B) level in serum of healthy volunteers (Ang-2 n = 4; Ang-1 n = 21) or patients with low-grade diffuse glioma (WHO II) (n = 5), anaplastic astrocytoma (WHO III) (n = 7), or glioblastoma (WHO IV) (n = 39) are shown.CThe TCGA database was accessed to obtain gene expression level for Ang-1, Ang-2, Tie2, and Tie1 in GBM in comparison with normal brain (box-and whisker plot showing median, 25–75th percentile, upper and lower quartile including outliers).DAng-2 expression and quantitative analysis of Ang-2 in healthy human brain (n = 3), low-grade diffuse glioma (n = 14), anaplastic astrocytoma (n = 12), or glioblastoma (n = 11) are shown. Scale bar: 100 µm.ECo-staining of Ang-2 and vWF (endothelial cells), aSMA (mural cells), and IBA1 (microglia) in different glioblastoma specimen. Normal brain tissue was used to assess Ang-2 staining specificity. Scale bar: 20 µm.FAng-2 predicts survival of glioblastoma patients.Data information: If not indicated differently, Kruskal–Wallis test (Dunn's post-test) was applied, ***P < 0.005; data are mean ± SEM.Source data are available online for this figure.

Fig 5: Effects of dabigatran on endothelium function based on selected biomarkers of endothelial dysfunction in BALB/c mice injected with 4T1 breast cancer cells. Mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in Section 2.5, injected with 4T1 breast cancer cells, and euthanized at 24 h, 2 days, and 7 days after 4T1 breast cancer cell injection. The panel of selected biomarkers of endothelial dysfunction (Ang-1 (A), Ang-2 (B), sTie-2 (C), sFLT-1 (D), SDC-1 (E), sE-sel (F), sICAM-1 (G), vWF (H), PAI-1 (I), t-PA (J), sP-sel (K), and THBS-1 (L)) was measured in the plasma using the microLC/MS-MRM method as described in Section 2. The data are presented as the median ± IQR. Statistical analysis was performed with two-way ANOVA (A,C–H,J–L) or Kruskal-Wallis (B,I) tests followed by appropriate post hoc tests. The symbols *, **, and *** denote statistical significance at p < .05, p < .01, and p < .001, respectively.