Discussion

We have previously shown that the rapid induction of endothelial cell apoptosis by TGF-β1 is mediated by the autocrine or paracrine activation of VEGFR2 by endothelial cell VEGF (Ferrari et al., 2006). This finding raised a fundamental question: Does VEGF-mediated apoptosis promote or inhibit TGF-β1 induction of angiogenesis? The data reported here show that VEGF-mediated apoptosis is an early event required for induction of blood vessel formation by TGF-β1. Therefore, endothelial cell apoptosis is not only necessary for pruning the forming vascular network during the late stages of angiogenesis; it also occurs in the initial steps of, and is required for angiogenesis to proceed.

Because of the multiple inhibitory effects of TGF- β1 on endothelial cells, it has been proposed that TGF- β1 induces angiogenesis in vivo indirectly through its action on immune response cells. However, a variety of findings indicate that TGF- β1 has important, direct effects on angiogenesis in vivo. Half of the mice genetically deficient in TGF-β1 die in utero and show defective vasculogenesis and angiogenesis, a phenotype consistent with abundant TGF-β1 expression in endothelial precursors. Importantly, during mouse embryogenesis ALK1 (TGF-β receptor I) expression is mainly confined to endothelial cells, and is upregulated at sites of active angiogenesis. Mice genetically deficient in ALK1 die from defects in angiogenesis, as do mice deficient in either ALK5 (TGF-β receptor I) or TGF-β receptor II (Dickson et al., 1995; Li et al., 1999; Oshima et al., 1996). In humans mutations of ALK1, the endothelial cell TGF-β1 receptor, cause hereditary hemorrhagic teleangiectasia, a condition characterized by absence of capillary bed (angiogenesis) in certain vascular districts. In addition, in adult animals TGF- β1 induces endothelial cell apoptosis, a process required for glomerular capillary formation (Fierlbeck et al., 2003).

Our finding that TGF-β1-induced endothelial cell apoptosis occurs rapidly and is followed by a long period in which the cells are refractory to the apoptotic effect of TGF-β1 can be explained by several, non-mutually exclusive mechanisms, including proteasomal degradation of phosphorylated Smad2 (Lo and Massague, 1999; Zhang et al., 2001), downregulation of TGF-β receptor (TGF-βR) and/or VEGFR2 expression (Anders et al., 1997; Mandriota et al., 1996; Minami et al., 2001). Our observation is also consistent with the finding that induction of endothelial cell death is followed by increased proliferation and resistance to apoptosis (Sakao et al., 2005). A variety of tumors contain high levels of both TGF-β1 and VEGF, which do not result in massive endothelial cell apoptosis. Our finding of the transient nature of the apoptotic effect of TGF-β1-induced apoptosis, and the known effect of TGF-β1 on TGF-β receptor and VEGFR2 expression can explain the apparent lack of endothelial cell apoptosis in these tumors.

Under our experimental conditions siRNA-mediated downregulation of VEGFR2 expression did not result in endothelial cell apoptosis in vitro for at least 72 h; similarly, inhibition of VEGF did not cause rapid endothelial cell apoptosis in vivo. These findings are seemingly in contrast with the current concept that VEGF/VEGFR2 signaling is required for endothelial cell survival in vitro and in vivo (Gerber et al., 1998a; Gerber et al., 1998b; Lee et al., 2007; Sweeney et al., 2002). However, several reports have shown that inhibition of VEGF – VEGFR2 signaling by antibodies or chemical inhibitors of the receptor does not cause rapid apoptosis of cultured endothelial cells in the absence of a pro-apoptotic stimulus (Geng et al., 2001; Lu et al., 2005; Sakao et al., 2007). The genetic deficiency of VEGF in cultured endothelial cells results in apoptosis only after 72 h incubation in serum-free medium, a pro-apoptotic culture condition (Gerber et al., 1998a; Gerber et al., 1998b; Lee et al., 2007). Our observation that downregulation of VEGFR2 expression does not provide an apoptotic stimulus per se is also consistent with the recent finding that Notch-1 downregulates VEGFR2 expression without inducing endothelial cell apoptosis (Shawber et al., 2007). In addition, to the best of our knowledge, our previous (Ferrari et al., 2006) and present work is the first that tested the effect of siRNA-mediated downregulation of VEGFR2 expression on endothelial cell apoptosis. The specificity of the effect of the VEGFR2 siRNAs we used is shown by their lack of downregulation of VEGFR1 expression. Their functional efficacy was shown by our previous results showing that transfection with VEGFR2 siRNAs abolish MAPK activation in response to VEGF (Ferrari et al., 2006). Our finding that inhibition of VEGF - VEGFR2 signaling does not induce rapid endothelial cell apoptosis is also supported by our analysis of endothelial cell apoptosis in the CAM. In these experiments TGF-β1 induced endothelial cell apoptosis as rapidly (6 h) as in vitro. Inhibition of VEGF by neutralizing antibody did not increase endothelial cell apoptosis in the presence of TGF-β1; on the contrary, it blocked the apoptotic effect of TGF-β1. Based on our data we cannot exclude that inhibition of VEGF or VEGFR2 expression for a longer time would result in apoptosis in the absence of TGF-β1. However, our results show that the rapid apoptotic effect of TGF-β1 on endothelial cells requires VEGF signaling and is necessary for the progression of the angiogenic process. Thus, VEGF-VEGFR2 signaling not only provides endothelial cells with pro-survival and proliferative/migratory stimuli required for vessel formation but also mediates the initial apoptotic effect necessary for TGF-β1 induction of angiogenesis.

Inhibition of VEGF signaling – which blocks TGF-β1 induction of apoptosis - blocked endothelial cell cord formation in vitro with no apparent morphological changes in the cell monolayer. Conversely, inhibition of FGF-2 - which induces apoptosis - resulted in few scattered and disrupted cord-like structures. Thus, both inhibition of, and excess apoptosis block angiogenesis, indicating that an optimal level of apoptosis is required for vessel formation.

A previous report has shown that TGF-β1/ALK1 (TGF-β receptor I) signaling in endothelial cells stimulates angiogenesis through Smad activation (Goumans et al., 2002). Our data are not in contrast with this finding as our results do not imply that apoptosis is Smad-independent. Indeed, Smad signaling could act upstream of VEGF (e.g. by promoting VEGF expression). Several papers have shown that TGF-β1 induction of VEGF in a variety of cell types other than endothelial cells is Smad-dependent. One article (Bostrom et al., 2004) reported that overexpression of, or stimulation of ALK-1 results in Smad activation and upregulation of VEGF expression in endothelial cells. However, the possibility that in these cells TGF-β1 induction of VEGF expression is mediated by a Smad-independent mechanism (e.g. p38^MAPK) was not explored. We are currently investigating this hypothesis.

Our experiments on endothelial cell differentiation in embryoid bodies provide genetic evidence for a role of VEGF signaling in TGF-β1 induction of vasculogenesis. Consistent with previous observations (Feraud et al., 2001; Gualandris et al., 2000b), both VEGF and TGF-β1 increased endothelial cell differentiation and organization in embryoid bodies. However, we found that TGF-β1 has no such effect on embryoid bodies genetically deficient in VEGF. A previous report has shown that TGF-β1 increases endothelial cell differentiation in a VEGF-independent manner and inhibits endothelial tube formation in embryoid bodies (Mallet et al., 2006). Two reasons can explain this discrepancy with our results. The independence of TGF-β1 action from VEGF was shown by using a neutralizing antibody added a day 0 of differentiation; in contrast, we used cells genetically deficient in VEGF or VEGF receptors. In addition, the authors used PECAM as a marker of endothelial cell differentiation, whereas we used ICAM-2. These endothelial cell markers have different expression patterns during embryoid body differentiation. In differentiating embryoid bodies PECAM+ cells can be found that are not organized into vessels, suggesting that PECAM+ cells are endothelial cell precursors; conversely, ICAM-2+ cells are confined to patent vasculature (Bautch et al., 2000).

The genetic deficiency of VEGFR2 caused a dramatic decrease in endothelial cell differentiation, which was not increased by either VEGF or TGF-β1. Thus, the very low number of endothelial cells in VEGFR2-deficient embryoid bodies did not allow any conclusion as to the role of VEGFR2 in TGF-β1 induction of vascular structures in embryoid bodies. However, our data obtained with ES cells genetically deficient in VEGF or VEGFR1 are consistent with our results obtained with mature endothelial cells, showing the requirement of VEGF-VEGFR2 signaling for TGF-β1 induction of angiogenesis in vitro and in vivo. This mechanism can therefore play a significant role in the control of endothelial stem cell differentiation during angiogenesis in vivo.

Our findings that TGF-β1 induces apoptosis and angiogenesis in vitro and in vivo through the autocrine or paracrine activation of VEGFR2 by VEGF indicate a potential pharmacological approach to anti-angiogenesis therapy. High levels of VEGF, VEGFR2 and TGF-β1 are present in many tumors. Crosstalk between the signaling pathways activated by these growth factors controls endothelial cell apoptosis (Ferrari et al., 2006) and angiogenesis. Understanding the mechanism(s) that modulate this crosstalk can permit the development of novel pharmacological tools to convert VEGF, a potent angiogenesis inducer and survival factor, into an inducer of uncontrolled endothelial cell apoptosis, and therefore into a potent anti-angiogenesis factor.

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Supplementary Material

Supp Fig s1

Supplemental Figure 1. In vitro angiogenesis. Confluent BCE cells grown in gelatin-coated dishes were incubated in the absence (upper panel) or presence of TGF-β1 (1 ng/ml; lower panel) in medium supplemented with 0.5% calf serum. The cultures were photographed after 72 h incubation. Shown are enlarged images of panel a and b of Fig. 2.

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ACKNOWLEDGEMENTS

We are grateful to Drs. A. Joyner, Andras Nagy, Janet Rossant and Guo-Hua Fong for their generous gifts of wt and mutant ES cells. This work was supported by grants NIH R01 HL070203 and R01 HL070203-03S1 to P.M., and by funds from the Department of Cardiothoracic Surgery of NYU School of Medicine.

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