MODULATION OF ENDOTHELIAL ACTIVATION AND CEREBRAL ANGIOGENESIS BY TNF FAMILY LIGANDS AND RESVERATROL: AN IN VITRO STUDY

Abstract

Vascular endothelial cell activation and apoptosis (programmed cell death) are critical in inflammation and angiogenesis (the formation of new blood vessels). Tumor necrosis factor (TNF) is a pro-inflammatory cytokine known for its ability to induce endothelial cell activation and apoptosis. However, the ability of two death ligands in the TNF superfamily: TRAIL (TNF-Related Apoptosis-Inducing Ligand) and Fas ligand (FasL), to activate vascular endothelium is less well defined, and forms the basis of this work. We find that in the human endothelial cell line EA.hy926, TRAIL induces endothelial cell activation (activation of the transcription factor NF-?B with increased expression of the adhesion protein ICAM-1 and adhesion of human neutrophils) when it concurrently induces apoptosis. In addition, angiogenesis is implicated in diseases of the central nervous system, and its modulation represents an attractive therapeutic strategy. We investigated the modulatory potential of the two endogenous molecules TRAIL and FasL as well as an exogenous molecule resveratrol, a phytochemical present in red wine, in angiogenesis. We modeled cerebral angiogenesis with the human brain endothelial cell line hCMEC/D3 and primary bovine brain endothelial cells. Resveratrol inhibited several parameters of angiogenesis (proliferation, migration and tube formation) in human umbilical vein endothelial cells, however, neither TRAIL nor FasL had an effect on this model. By contrast, in hCMEC/D3 cells both resveratrol and TRAIL inhibited all parameters while FasL had minimal effects. Resveratrol did not induce apoptosis in hCMEC/D3 but arrested cell cycle progression to G2/M phase and inhibited phosphorylation of Akt/PKB, a key cell survival protein kinase. This leads to a reduction in cell growth, endothelial migration and tube formation, hence, inhibition of in vitro angiogenesis. TRAIL induced anti-angiogenic effects in hCMEC/D3 due to apoptosis. The data suggests that TRAIL primarily influences angiogenesis through induction of vascular endothelial apoptosis while resveratrol induces cell cycle arrest, both of which may lead to vessel regression. These are the first studies to report the modulation of different aspects of endothelial cell activation by TRAIL and resveratrol in several endothelial cell culture models, with a particular focus on the central nervous system.