Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice
Sulochana S. Bhandarkar, … , Hellmut G. Augustin, Jack L. Arbiser
Sulochana S. Bhandarkar, … , Hellmut G. Augustin, Jack L. Arbiser
Published July 13, 2009
Citation Information: J Clin Invest. 2009;119(8):2359-2365. https://doi.org/10.1172/JCI33877.
View: Text | PDF
Research Article Dermatology

Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice

Abstract

Hemangiomas are the most common type of tumor in infants. As they are endothelial cell–derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T–transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/–, and Ang2–/– mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/– cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/– cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function.

Authors

Sulochana S. Bhandarkar, Marisa Jaconi, Levi E. Fried, Michael Y. Bonner, Benjamin Lefkove, Baskaran Govindarajan, Betsy N. Perry, Ravi Parhar, Jamie Mackelfresh, Allie Sohn, Michael Stouffs, Ulla Knaus, George Yancopoulos, Yvonne Reiss, Andrew V. Benest, Hellmut G. Augustin, Jack L. Arbiser

×

Figure 1

Differential tumor growth in endotheliomas derived from Ang2 wild-type, heterozygous, and knockout mice.

Options: View larger image (or click on image) Download as PowerPoint
Differential tumor growth in endotheliomas derived from Ang2 wild-type, ...
Tumor formation in Ang2–/– cells and Ang2+/– cells in vivo (A). Six mice were injected with 2.5 × 105Ang–/– or Ang+/– cells. Animals were euthanized on day 30, secondary to tumor burden in the Ang–/– animals. The figure depicts the average tumor volume (mm3) in both experimental groups. Error bar represents SEM. P < 0.05 versus control. Morphological analysis revealed typical cavernous hemangiomas in wild-type endothelioma–derived hemangiomas (B) and in Ang2+/– endothelioma–derived hemangiomas (C) compared with the solid growth of Ang2–/– endothelioma–derived sarcomas (D). (E–G) CD31 immunohistochemistry of Ang2+/+, Ang2+/–, and Ang2–/– tumors, respectively, with positive staining in all 3 tumors, but decreased differentiation in the Ang2–/– tumors. (H–J) Immunocytochemistry of CD31, with pink staining representing CD31 and blue DAPI staining representing nuclei. (J) RT-PCR of Ang2+/+, Ang2+/–, and Ang2–/– cells for CD31, with GAPDH as a loading control. Original magnification, ×40. (K) Represented is RT-PCR for CD31 in all 3 phenotypes of endothelial cells. Note presence of the endothelial marker CD31 in all 3 types of cells. GAPDH represents a loading control. PCR was performed using QIAGEN reagents per manufacturer’s protocol and 35 cycles performed at an annealing temperature of 55°C. To confirm expression of the endothelial marker CD31, PCR was performed using the following primer sequences: forward, 5′-GTGAAGGTGCATGGCGTATC-3′; reverse 5′-CACAAAGTTCTCGTTGGAGGT-3′, producing a 192-bp product. PCR for GAPDH was used as a control: forward, 5′-AGGTCGGTGTGAACGGATTT-3′; reverse, 5′-CTCCTGGAAGATGGTGATGG-3′, resulting in a 224-bp band.