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CD40-Induced Signaling in Human Endothelial Cells Results in mTORC2- and Akt-Dependent Expression of Vascular Endothelial Growth Factor In Vitro and In Vivo¹

Transplantation Research Center and the Division of Nephrology, Department of Medicine, Children’s Hospital Boston, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115

We have examined CD40-dependent signals in endothelial cells (EC) mediating the expression of vascular endothelial growth factor (VEGF) and VEGF-induced angiogenesis. We treated confluent cultures of EC with soluble CD40L (sCD40L), and by Western blot found a marked increase in the phosphorylation of Akt, 4EBP-1, and S6K1, compared with untreated cells. EC were transfected with a full-length VEGF promoter-luciferase construct and cultured in the absence or presence of rapamycin and sCD40L. We found that rapamycin, which blocks mTORC1 and mTORC2 signaling, inhibited sCD40L-mediated transactivation of VEGF. In addition, by Western blot, we found that the transfection of EC with small interfering RNA (siRNA) to rictor (to inhibit mTORC2), and not raptor (to inhibit mTORC1), inhibited sCD40L-dependent protein expression of VEGF. In additions, we found that basal levels of phosphorylated Akt as well as VEGF were increased in EC transfected with the raptor siRNA. Also, rapamycin failed to inhibit VEGF promoter activation, as well as VEGF protein expression in EC transfected with a constitutively active construct of Akt, further demonstrating that mTORC1 is not necessary for CD40- and Akt-induced expression of VEGF. Finally, we injected human CD40L-transfected fibroblasts or mock transfectants into human skin on SCID mice. We found that the injection of CD40L transfectants, but not mock cells, resulted in VEGF expression and mediated a marked angiogenesis reaction, and this response was reduced in mice treated with rapamycin. Together, these observations indicate that mTORC2 and Akt facilitate CD40-inducible expression of VEGF in EC, which is of clinical importance in tumor growth and the progression of chronic inflammatory diseases.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant AI046756 (to D.M.B.). O.D. was supported by a Transplantation Fellowship from Swiss House for Advanced Research and Education-Novartis and the SICPA foundation.

² Current address: Department of Visceral Surgery, University Hospital Center, University of Lausanne, Lausanne, Switzerland.

³ Address correspondence and reprint requests to Dr. David M. Briscoe, Children’s Hospital Boston, Division of Nephrology, 300 Longwood Avenue, Boston, MA 02115. E-mail address: david.briscoe{at}childrens.harvard.edu

⁴ Abbreviations used in this paper: EC, endothelial cell; mTOR, mammalian target of rapamycin; mTORC, mammalian target of rapamycin complex; VEGF, vascular endothelial growth factor; CD40L, CD40 ligand; sCD40L soluble CD40L; DN, dominant negative.