Abstract
Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFA-driven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels.
Highlights
Nitric oxide (NO) is an important mediator of normal and pathologic vascular remodeling [1, 2]
We found that specific endothelial Notch activity in the context of increased VEGF signaling is required for endothelial NO synthase (eNOS) activation, and that the NO–soluble guanyl cyclase ligand–receptor pairing and activation is critical for permitting tumor perfusion in larger blood vessels but not in capillaries
To study the role of endothelial cell–specific Notch blockade on tumor angiogenesis, we used a tetracycline (Tet)based inducible system expressing a dominant-negative form of the Notch coactivator MAML fused to GFP. dnMAML has been used to assess the effect of pan-inhibition of Notch signaling used in various vascular models, and found to be inhibitory to Notch signaling [22,23,24,25,26], one cannot entirely rule out the possibility that some unknown pathway is impacted
Summary
Nitric oxide (NO) is an important mediator of normal and pathologic vascular remodeling [1, 2]. Genetic ablation of eNOS and pharmacologic approaches have shown that NO contributes to tumor progression by maintaining blood flow [5,6,7], inducing vascular hyperpermeability [7, 8], recruiting pericytes and promoting vessel morphogenesis [4], and reducing endothelial cell–leucocyte interactions [6]. ENOS is activated by phosphorylation of the Ser1117 residue, which is regulated by multiple signaling path-. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Ways [1, 2] In this context, the prototypical pro-angiogenic agent VEGFA was shown to activate eNOS in various studies [1, 2]. We have shown that Notch activates eNOS during embryonic heart development [9]
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