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 5

NCL affects HCMEC actin cytoskeleton orientation in vitro.

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NCL affects HCMEC actin cytoskeleton orientation in vitro. (A–F) HCMEC t...
(A–F) HCMEC treated with control or NCL siRNA were left to spread on fibronectin-coated glass substrate and stained for F-actin (green) and DAPI (blue), seen in A and D. (G) Schematic illustration of actin fiber orientation characterization. (H) Schematic illustration of circularity index indicating the reference circular index (circle = 1). (I and J) NCL knockdown decreased HCMEC cell spreading, as measured by cell circularity and cell area measurements. NCL knockdown HCMECs had a significantly less elongated shape (I, n = 3). HCMEC spreading was significantly decreased upon NCL knockdown (J, n = 3). (K) Phalloidin actin fibers (green) were more randomly organized in the HCMECNCL KD (E and F) as compared with the control (B and C). The distribution of actin orientation shows a clear classical peak close to 0 degrees in the control HCMECControl KD (gray curve). In HCMECNCL KD, the classical peak of actin orientation was lost and HCMEC actin orientation was more randomly distributed (black curve) (K). Data represent mean ± SEM. Two-tailed unpaired Student’s t test were performed. **P < 0.01. Scale bars: 20 μm in A–F.