PDF file - 3357K, Figure S1. DnMAML-mediated EC-specific Notch blockade inhibits larger vessel function. Figure S2. Flow cytometry analysis of PDGFRβ in LLC-VEGFA tumors of control and double Tg. Figure S3. NO production in the microvasculature of LLC-VEGFA tumors in response to EC-specific Notch inhibition. Figure S4 Effect of the NO-independent soluble guanylyl cyclase agonist BAY41-2272 on tumor growth in EC-specific Notch-inhibited mice. Figure S3. NO production in the microvasculature of LLC-VEGFA tumors in response to EC-specific Notch inhibition. Figure S5. dnMAML-mediated EC Notch blockade inhibits growth, functional blood vessels and eNOS activation of B16F10-VEGFA tumors. Figure S6. Expression analysis of vasodilatory genes Nts and Adm in response to EC-specific Notch inhibition in tumors. Table S1. RT-PCR analysis of Ace2, Edn2, Agt and Ednra in bulk and sorted endothelial cells (EC) from LLC-VEGFA tumors grown in control and double Tg VE-cadherin-tTA x TetOS-dnMAML-GFP.
ARTICLE 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. Cancer Res; 74(9); 2402–11. ©2014 AACR.
