Effects of vascular endothelial growth factor on the lymphocyte-endothelium interactions: Identification of caveolin-1 and nitric oxide as control points of endothelial cell anergy

Tumors may evade immune responses at multiple levels, including through a defect in the lymphocyte-vessel wall interactions. The angiogenic nature of endothelial cells (EC) lining tumor blood vessels may account for such anergy. In this study, we examined whether mechanisms other than down-regulation of adhesion molecules could be involved, particularly signaling pathways dependent on the caveolae platforms. To mimic the influence of the tumor microenvironment, EC were exposed to TNF-alpha and the proangiogenic vascular endothelial growth factor (VEGF). We identified a dramatic inhibition of lymphocyte adhesion on activated EC following either short or long VEGF pretreatments. We further documented that VEGF did not influence the abundance of major adhesion molecules, but was associated with a defect in ICAM-1 and VCAM-I clustering at the EC surface. We also found that overexpression of the caveolar structural protein, caveolin-1, overcame the VEGF-mediated inhibition of adhesion and restored ICAM-1 clustering. Conversely, EC transduction with a caveolin-1 small interfering RNA reduced the TNF-alpha-dependent increase in adhesion. Finally, we identified. VEGF-induced NO production by the endothelial NO synthase as the main target of the changes in caveolin-1 abundance. We found that the NO synthase inhibitor N-nitro-L-arginine methyl ester could reverse the inhibitory effects of VEGF on lymphocyte adhesion and EC cytoskeleton rearrangement. Symmetrically, a NO donor was shown to prevent the ICAM clustering-mediated lymphocyte adhesion, thereby recapitulating the effects of VEGF. In conclusion. this study provides new insights on the mechanisms leading to the tumor EC anergy vs immune cells and opens new perspectives for the use of antiangiogenic strategies as adjuvant approaches to cancer immunotherapy.