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 8

NCL induces regulation of angiogenic pathways and promotes phosphorylation of VEGFR2 in HCMECs.

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NCL induces regulation of angiogenic pathways and promotes phosphorylati...
(A and B) Heatmap showing the expression of the top 15 genes driving the enrichment of “angiogenesis” pathways in HCMECNCL KD and the top 50 genes driving the enrichment of “oxidative phosphorylation and glycolysis” pathways in HCMECControl KD. (C–E) VEGFA expression was not differentially regulated, and VEGFR2 expression was upregulated (C) in HCMECsNCL KD as compared with HCMECsControl KD. NCL knockdown induced a significant upregulation of the Dll4-Jagged-Notch signaling pathway, including in HES1, NOTCH1, NOTCH4, and Jagged1 (JAG1), while HES2 and DLL4 were not differentially regulated upon NCL knockdown (D). siNCL treatment caused a significant downregulation of the YAP-TAZ gene YAP1 as well as the YAP-TAZ downstream effector gene CTGF but not of the YAP-TAZ downstream effector gene CYR61 (E). (F–O) HCMECs were stained for NCL (gray), F-actin (green, stained with phalloidin), VEGFR2 (red, in F–I), or p-VEGFR2 (red, in K–N) and the general nuclear marker DAPI (blue). VEGFR2 expression was not regulated (J, n = 3), but pVEGFR2 expression was significantly downregulated (O, n = 3) upon siRNA-mediated NCL knockdown in HCMECs as compared with the control condition. Data represent mean ± SEM. Wald test corrected for multiple testing using the Benjamini-Hochberg method (C–E) and 2-tailed unpaired Student’s t test (J and O) were performed. **P < 0.01, ***P < 0.001. The boxed areas (white box) in F, G, K, and L are zoomed-in images shown in H, I, M, and N, respectively. Scale bars: 70 μm in F, G, K, and L; 20 μm in H, I, M, and N.