Induction of endothelial cell apoptosis by tgf-β1

Figure 2

Capillary cord formation in vitro

We then characterized the effect of neutralizing anti-VEGF and anti-FGF-2 antibodies on TGF-β1 induction of capillary cord formation. VEGF antibody blocks the apoptotic activity of TGF-β1 on endothelial cells, whereas antibody to FGF-2 induces apoptosis in the presence or absence of TGF-β1 (Ferrari et al., 2006; Mandriota and Pepper, 1997). Both antibodies blocked TGF-β1 induction of cord formation but with different effects. In the presence of VEGF antibody TGF-β1-treated cells (panel n) retained an intact monolayer. In contrast, addition of FGF-2 antibody to TGF-β1-treated cells caused massive cell death, with sparse, disrupted cord-like structures (panel k). This effect can be explained by previous findings that anti-FGF-2 antibody induces endothelial cell apoptosis by a VEGF-insensitive mechanism, and that FGF-2 ^−/−endothelial cells have higher apoptosis levels than wt cells (Ferrari et al., 2006; Mandriota and Pepper, 1997).

To analyze the role of apoptosis in TGF-β1 induction of angiogenesis we used z-VAD, a general caspase inhibitor that blocks apoptosis. z-VAD blocked induction of cord formation by TGF-β1 (Fig. 2, panel q) with an effect comparable to that of VEGF antibody. This result is consistent with previous reports showing that zVad and other apoptosis inhibitors block VEGF- and FGF-2-induced angiogenesis in other in vitro angiogenesis assays, as well as in vivo (Segura et al., 2002).

The inhibitory effect of z-VAD and of antibodies to FGF-2 or VEGF on capillary cord formation in vitro was rapidly reverted after removal of these reagents from the culture medium (Fig. 2, panels l, o, r). Similarly, removal of TGF-β1 from the culture medium caused rapid reversion of the cord-like structures into a monolayer (panels c, f, i), showing that the observed morphological changes did not reflect toxic effects of the reagents. Therefore, these results showed that endothelial cell apoptosis is an early event during induction of capillary cord formation by TGF-β1, and both upregulation and downregulation of apoptosis block the in vitro angiogenic activity of TGF-β1.

To characterize the role of VEGF receptors in TGF-β1 induction of angiogenesis we downregulated VEGFR1 and VEGFR2 by transient transfection of endothelial cells with specific siRNAs. These siRNAs effectively inhibit the expression of their respective proteins without cross-reacting, and abolish MAP kinase activation by VEGF (Ferrari et al., 2006). Transfection with VEGFR2 or VEGFR1 siRNA resulted in strong downregulation of the expression of the respective receptor for up to 96 h, without affecting the level of the other (Fig. 3 A and 3 B). Downregulation of VEGFR2 expression blocks TGF-β1 induction of endothelial cell apoptosis, whereas inhibition of VEGFR1 expression has no such effect (Ferrari et al., 2006). VEGFR2 siRNA-transfected cells did not undergo apoptosis although they were treated with TGF-β1 every 24 h for up to 72 h (Fig. 3 C), showing that downregulation of VEGFR2 expression did not provide an apoptotic stimulus per se. However, transfection with VEGFR2 siRNA abrogated both the apoptotic effect of TGF-β1 (Fig. 3 C) and TGF-β1 induction of cord formation in vitro (Fig. 3 D and E). In contrast, downregulation of VEGFR1 expression, which does not affect TGF-β1 induction of apoptosis (Ferrari et al., 2006), had no such effect (Fig. 3 D and E).

Fig. 3

Downregulation of VEGFR2 expression blocks TGF-β1 induction of capillary cord formation in vitro