Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium–derived factor

Abstract

Natural inhibitors of angiogenesis are able to block pathological neovascularization without harming the preexisting vasculature. Here we show that two such inhibitors, thrombospondin-1 and pigment epithelium-derived factor, derive specificity for remodeling vessels from their dependence on Fas/Fas ligand (FasL)-mediated apoptosis to block angiogenesis. Both inhibitors upregulated FasL on endothelial cells. Expression of the essential partner of FasL, Fas/CD95 receptor, was low on quiescent endothelial cells and vessels but greatly enhanced by inducers of angiogenesis, thereby specifically sensitizing the stimulated cells to apoptosis by inhibitor-generated FasL. The anti-angiogenic activity of thrombospondin-1 and pigment epithelium-derived factor both in vitro and in vivo was dependent on this dual induction of Fas and FasL and the resulting apoptosis. This example of cooperation between pro- and anti-angiogenic factors in the inhibition of angiogenesis provides one explanation for the ability of inhibitors to select remodeling capillaries for destruction.

In vivo upregulation of CD95 and CD95L causes synergistic inhibition of angiogenesis by TSP1 peptide and metronomic doxorubicin treatment

“…Neutralizing antibody against TSP1 receptor CD36 were previously shown to inhibit TSPmediated induction of CD95L and apoptosis. 10,11 The same antibody completely abrogated endothelial cell apoptosis and antiangiogenesis by DI-TSP/DXR combination (Figure 6a-c). Since CD95 upregulation by DXR is p53-dependent 18 while apoptosis by DI-TSP is not, 10 we used Pifithrin-a (PFT-a), an inhibitor of p53-dependent transcription, 28 in order to eliminate DXR induction of CD95 mRNA and protein.…”

“…18 DI-TSP upregulated CD95L expression and induced HMVECs apoptosis in synergy with DXR TSP1 increases mRNA and surface CD95L in the endothelial cells via signaling cascade mediated by its antiangiogenic receptor, CD36. 10,11,25 Since HUVECs are CD36 negative, 6 we examined the ability of DI-TSP, a short peptide derivative of TSP1, to induce similar events only in the CD36-positive HMVECs. 5,6 Treatment (24 h) with DI-TSP at antiangiogenic, proapoptotic concentration (10 nM) resulted in pronounced upregulation of CD95L that was preserved in the presence of DXR (Figure 2a).…”

“…Detection of DXR-dependent increase in CD95/ Fas proved difficult, as the plugs were supplemented with the excess of exogenous proangiogenic factors (VEGF or bFGF), which are known to induce endothelial CD95. 11 Seeking conditions that would allow unequivocal demonstration of in vivo CD95 upregulation by the chosen DXR dose, we performed the assay using Matrigel plugs lacking exogenous VEGF and heparin. We were able to detect at least a 10-fold increase in CD95 staining in the presence of DXR (0.2 mg/kg) (Figure 3a,b).…”

“…10 TSP1 selectively destroys proliferating, but not quiescent endothelium: our recent studies attribute this selectivity to the CD36-dependent upregulation of the cell surface CD95L, a ligand for the death receptor CD95/Fas. 11 Binding of CD95, a member of the TNF receptor superfamily to its ligand [12][13][14][15][16] triggers apoptotic response via an exhaustively studied pathway that involves formation of the intracellular death-inducing signaling complexes (DISCs). 17 CD95 itself is constitutively low on the resting endothelial cells and its surface expression is induced by proangiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or interleukin-8 (IL-8).…”

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“…31 VEGF also promotes proliferation of vascular endothelial cells and these activated endothelial cells upregulate surface Fas, thereby becoming sensitive to apoptosis when the Fas ligand is activated by inhibitors such as PEDF. 38 Fas-mediated signaling mainly induces procaspase-8 auto-proteolytic cleavage, although it may subsequently cause activation of procaspase-9. PEDF increases the expression and transcriptional activity of peroxisome proliferator-activated receptor-g (PPARg) in human umbilical vein endothelial cells and PEDF's action on PPARg results in apoptosis of endothelial cells perhaps mediated through p53.…”