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 10

NCL positively regulates endothelial glucose metabolism via glycolytic enzymes, including HK2, but does not affect fatty acid oxidation in human brain endothelial cells.

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NCL positively regulates endothelial glucose metabolism via glycolytic e...
(A–C) Metabolic assays of HCMECs, upon NCL downregulation with siRNA targeting NCL. NCL knockdown decreased the glycolytic flux (A, n = 3), glucose uptake (B, n = 4), and Lac production (C, n = 4) in HCMECs as compared with the tested controls. (D) qPCR revealing a significant downregulation of about 30% of hexokinase-2 (HK2) mRNA expression by siRNA-targeted NCL knockdown. PFKFB3 expression showed slight but no significant increase (n = 3). (E and F) Western blot using antibodies against PFKFB3 revealed no significant regulation of PFKFB3 expression by NCL knockdown (n = 3). (G–J) HCMECs were stained for NCL (green), PFKFB3 (red), and the general nuclear marker DAPI (blue). No difference in PFKFB3 expression could be seen between NCL knockdown HCMECs (G and H) and the HCMECs treated with a control siRNA (I and J). (K) NCL knockdown in HCMECs did not affect fatty acid oxidation (n = 3). (L and M) Western blot using antibodies against carnitine palmitoyltransferase 1A (CPT1A) showed no difference in CPT1A protein expression between NCL knockdown HCMECs and the control condition (n = 3). (N) qPCR showed no significant regulation of CPT1A mRNA expression upon siRNA-targeted NCL knockdown (n = 3). Data represent mean ± SEM. Two-tailed unpaired Student’s t test were performed. *P < 0.05, **P < 0.01. Scale bars: 20 μm in G–J.