Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature
Marc Schwab, Ignazio de Trizio, Moheb Ghobrial, Jau-Ye Shiu, Oguzkan Sürücü, Francesco Girolamo, Mariella Errede, Murat Yilmaz, Johannes Haybaeck, Alessandro Moiraghi, Philippe P. Monnier, Sean E. Lawler, Jeffrey P. Greenfield, Ivan Radovanovic, Karl Frei, Ralph Schlapbach, Viola Vogel, Daniela Virgintino, Katrien De Bock, Thomas Wälchli
Marc Schwab, Ignazio de Trizio, Moheb Ghobrial, Jau-Ye Shiu, Oguzkan Sürücü, Francesco Girolamo, Mariella Errede, Murat Yilmaz, Johannes Haybaeck, Alessandro Moiraghi, Philippe P. Monnier, Sean E. Lawler, Jeffrey P. Greenfield, Ivan Radovanovic, Karl Frei, Ralph Schlapbach, Viola Vogel, Daniela Virgintino, Katrien De Bock, Thomas Wälchli
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Research Article Angiogenesis Neuroscience

Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature

Abstract

Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.

Authors

Marc Schwab, Ignazio de Trizio, Moheb Ghobrial, Jau-Ye Shiu, Oguzkan Sürücü, Francesco Girolamo, Mariella Errede, Murat Yilmaz, Johannes Haybaeck, Alessandro Moiraghi, Philippe P. Monnier, Sean E. Lawler, Jeffrey P. Greenfield, Ivan Radovanovic, Karl Frei, Ralph Schlapbach, Viola Vogel, Daniela Virgintino, Katrien De Bock, Thomas Wälchli

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Figure 1

Nucleolin is expressed in endothelial and perivascular cells during human brain development, is downregulated in the adult brain, and is reactivated in glial brain tumors in vivo.

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Nucleolin is expressed in endothelial and perivascular cells during huma...
Sections (20 μm) of human fetal (GW 18-22) and adult brains as well as sections from human GBMs were stained for Nucleolin, the vascular endothelial cell markers CD31 and CD105, the astrocytic marker GFAP, the pericyte marker NG2, and TO-PRO-3 nuclear counterstaining. (A–C and P) Nucleolin (green) is highly expressed in the nuclei of the developing human fetal brain (A) and of human brain tumors (C), but it shows a significant downregulation in the adult normal/healthy brain (B and P). (D–I, Q, and R) Nucleolin (green) is highly expressed in CD31+ blood vessel endothelial cells (red) in the human fetal (D and Q) and pathological brain (F and Q), but it is significantly downregulated in endothelial cell of the quiescent adult brain (E and Q). (G–I and R) Nucleolin shows a high expression in CD105+ activated endothelial cells (red) in the human fetal brain (G and R) and in glioblastoma (I and R) but is significantly downregulated in the quiescent adult normal brain (H and R), with a very low number of CD105+ endothelial cells in the quiescent adult brain (H and R). (J–L and S) Nucleolin (green) is highly expressed in GFAP+ neural precursors cells (red) in the fetal brain (J) and in tumoral astrocytes in glioblastoma (L), but it is significantly downregulated in adult normal brain (K and S). (M–O and T) In human fetal and adult brains, NG2+ pericytes (red) partially express low levels of Nucleolin (M and N); Nucleolin expression is highly upregulated in human brain tumor NG2+ pericytes (O and T). Data represent mean ± SEM of 2–3 patients (2–3 sections per patient, based on tissue availability). For statistical analysis, 1-way ANOVA with Tukey’s post hoc test were performed. *P < 0.05, **P < 0.01, ****P < 0.0001. Scale bars: 25 μm in A–O, left panels; and 15 μm in A–O, right panels.