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 2

Expression of NCL increases during astrocytic tumor progression and NCL is expressed in blood vessels in low- and high-grade gliomas in vivo.

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Expression of NCL increases during astrocytic tumor progression and NCL ...
(A–F) NCL (brown) expression increases during tumor progression of human astrocytomas from WHO grade I (A, n = 20), II (B, n = 28), III (C, n = 53), to IV (= glioblastoma, D, n = 46). In low-grade astrocytomas (WHO grades I and II, n = 48), NCL expression is significantly higher than in the normal brain parenchyma (E and F, n = 8) but significantly lower as compared with high-grade astrocytomas (E and F, WHO grades III and IV, n = 96). NCL expression slightly decreases in recurrent WHO grade IV tumors (E, n = 49). (G) NCL expression positively correlates with the expression of the proliferation marker Ki-67 in gliomas (n = 167). (H) Glioblastoma cell lines (LN-229, LN-18, and GBM-1) proliferation was significantly decreased upon NCL knockdown n = 4). (I) qPCR revealing a significant downregulation of about 70% of NCL mRNA expression upon siRNA-targeted NCL knockdown (siNCL, n = 3). (J) Western blot showing NCL downregulation in LN-229 cells transfected with siRNA against NCL (siNCL) (n = 3). No NCL downregulation was observed in LN-229 transfected with the control siRNA (siControl) (n = 3). (K) Quantification of Western blot revealing a significant downregulation of NCL protein expression by siRNA-targeted NCL knockdown as compared with control cells in LN-229 cells (n = 3). (L–O) NCL expression in tumor blood vessels. Note the increasing expression of NCL in the blood vessel wall (arrows) as well as in perivascular cells (arrowheads) in astrocytomas of higher grades. Data represent mean ± SEM. For statistical analysis, 1-way ANOVA with Tukey’s post hoc test (E and F), Pearson correlation analysis (G), and 2-tailed unpaired Student’s t test (H, I, and K) were performed. *P < 0.05, **P < 0.01, ****P < 0.0001. Scale bars: 100 μm (A–D, upper panel), 50 μm (A–D, lower panel, and G–J).