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 4

NCL promotes brain vascular endothelial cell sprouting and proliferation in vitro and can be targeted with the NCL-specific aptamer AS1411.

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NCL promotes brain vascular endothelial cell sprouting and proliferation...
(A–D) HCMECs were stained for NCL (green), F-actin (stained with Phalloidin, red), and the general nuclear marker DAPI (blue). NCL expression was decreased and restricted to nucleoli upon siRNA-mediated knockdown in HCMECs (C and D) as compared with control-siRNA treated HCMECs (A and B). (E) Western blot showing NCL downregulation in HCMECs transfected with siRNA against NCL (siNCL) as compared with control siRNA–treated HCMECs (n = 3). (F) Quantification of Western blot revealing a significant downregulation of 70% NCL protein expression by siRNA-targeted NCL knockdown as compared with control cells (n = 3). (G) qPCR revealing a significant downregulation of more than 80% of NCL mRNA expression by siRNA-targeted NCL knockdown (n = 3). (H–K) HCMEC sprout formation (number of sprouts per spheroid) was decreased upon siRNA-mediated NCL knockdown (J and K) as compared with the control group (H and I). The boxed areas in H and J are enlarged in I and K, respectively. (L and M) HCMEC sprout formation and total sprout length were significantly reduced upon NCL knockdown as compared with the control group (L and M) (n = 3). (N) HCMEC proliferation was significantly decreased upon NCL knockdown (n = 3). (O) HCMEC cell proliferation was dose-dependently decreased upon treatment with the NCL-specific aptamer AS1411. Cells were treated for 96 hours with 1.25 μM, 5 μM, and 10 μM of the NCL targeting aptamer AS1411 or the control aptamer CRO (n = 3). Data represent mean ± SEM. For statistical analysis, 2-tailed unpaired Student’s t test (F–G and L–N) and 2-way ANOVA Tukey’s multiple-comparison test comparing treatment columns (O) were performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars: 20 μm in A–D; 150 μm in H and J; and 50 μm in I and K.