Thrombospondin-I (TSP-I) treatment of dermal microvascular endothelial cells (MvEC) has been shown to upregulate Fas ligand (FasL) and to induce apoptosis by a mechanism that requires caspase-8 activity. We have examined the potential anti-angiogenic effects of TSP-I on primary human brain MvEC. The addition of TSP-I to primary human brain MvEC cultured as monolayers on type I collagen, induced cell death and apoptosis (evidenced by caspase-3 cleavage) in a dose- (5-30 nM) and time-dependent (maximal at 17 h) manner. TSP-I treatment for 17 h induced caspase-3 cleavage that required caspase-8 activity and the tumor necrosis factor receptor I (TNF-RI). We did not find a requirement for Fas, or the tumor necrosis-related apoptosis-inducing ligand receptors (TRAIL-R) I and 2. We confirmed the findings using caspase inhibitors, blocking antibodies and small interfering RNA (siRNA). Further analysis indicated that the TSP-I induction of caspase-3 cleavage of primary human brain MvEC adherent to collagen required the synthesis of new message and protein, and that TSP-I induced the expression of TNFα mRNA and protein. Consistent with these findings, when the primary human brain MvEC were propagated on collagen gels mAb anti-TNF-RI reversed the inhibitory effect, in part, of TSP-I on tube formation and branching. These data identify a novel mechanism whereby TSP-I can inhibit angiogenesis-through induction of apoptosis in a process mediated by TNF-RI. © 2008 Wiley-Liss, Inc.