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Angiopoietin-1-induced PI3-kinase activation prevents neuronal apoptosis¹

Université de Caen, UMR 6551-CNRS, IFR 47, Centre Cyceron, Bd H. Becquerel, BP 5229, 14074 Caen cedex, France

²Correspondence: Université de Caen, UMR 6551-CNRS, Bd H. Becquerel, BP 5229, F-14074 Caen cedex, France. E-mail: e.petit{at}neuro.unicaen.fr

SPECIFIC AIM

Although angiopoietin-1 (Ang-1) is best characterized as an endothelial growth factor, its presence in the brain after an ischemic event suggested that Ang-1 could play a role in neuronal death. To examine a potential neuroprotective effect of Ang-1, we have investigated, on murine primary neuronal cultures, whether Ang-1 could protect against necrotic or apoptotic neuronal death and studied the neuroprotective molecular mechanisms, focusing on the phosphatidylinositol 3-kinase (PI3-K) signaling pathway through the functional neuronal Tie-2 receptor.

PRINCIPAL FINDINGS

1. The angiopoietin receptor Tie-2 is present in neurons and is functional
In endothelial cells, Ang-1 and Ang-2 are the principal ligands identified for the Tie-2 receptor. Although both bind to Tie-2 with a similar affinity, Ang-1 induces autophosphorylation of Tie-2 whereas Ang-2 does not. Accordingly, Ang-2 is regarded as the physiological antagonist for Tie-2. In the present study, we characterized the presence of Tie-2 in murine primary cultured neurons (Fig. 1 A). To assess the functionality of the neuronal Tie-2 receptor, neurons were treated with either Ang-1 TFD (a slightly modified version of Ang-1, kindly provided by Dr. G. D. Yancopoulos, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA) or Ang-2, and the phosphorylation of Tie-2 was studied by immunoblotting. Our results showed that Ang-1 TFD (200 ng/mL) induces a significant increase by threefold (n=3, P<0.001) in Tie-2 phosphorylation (Fig. 1B ). As with endothelial cells, no induction was detected in neurons treated with Ang-2 (Fig. 1C ), even at elevated concentrations (800 ng/mL). The presence of an active Tie-2 receptor in neurons suggests that Ang-1 could exert a biological effect on these cells.

View larger version (52K): [in this window] [in a new window]   Figure 1. Neuronal Tie-2 is functional. A) Immunoprecipitation of whole cell extracts showing that neurons (500 µg), as RBE4 cells (200 µg), express Tie-2. Neuronal extracts were first immunoprecipitated (IP) with Tie-2 antibodies, then separated by SDS-PAGE, and transferred on membranes. Immunoblots (IB) were revealed with Tie-2 antibodies. NoAb represents a control where the antibody for immunoprecipitation was omitted. B, C) Effects of Ang-1 and Ang-2 on Tie-2 phosphorylation in neurons. After 15 min of application with Ang-1 TFD (200 ng/mL), Ang-2 (800 ng/mL), or vehicle (C) into the bathing media, cellular extracts from neuronal cultures were immunoprecipitated with antibodies raised against Tie-2, followed by western-immunoblotting analysis with antibodies against P-tyr (results are representative of 3 separate experiments). Ang-1 TFD is a slightly modified version of Ang-1. Recombinant human Ang-2 (rhAng-2) was purchased from R&D Systems (Minneapolis, MN, USA).

2. Ang-1 protects neurons from apoptosis
Ischemia-induced neuronal injury is thought to result from an excessive release of excitatory amino acids such as glutamate. Various studies have reported that neuronal damage has both a necrotic and an apoptotic component. Therefore, to investigate the potential neuroprotective effect of Ang-1, we submitted primary cultures of neurons to NMDA (N-methyl-D-aspartate) or to serum deprivation (SD) exposition in the presence or not of increasing concentrations of Ang-1. When neurons were treated or pretreated with Ang-1 TFD, whatever the concentrations studied (50 to 200 ng/mL), Ang-1 TFD did not prevent neuronal death induced by the excitotoxic stress. However, when Ang-1 TFD (200 ng/mL) was applied to neurons cultured in the absence of serum (Fig. 2 A), this cytokine exhibited a neuroprotective effect of 40% of cells (n=12, P<0.005). These results show that Ang-1 protects neurons from apoptotic stress but not from excitotoxic necrotic stress.

View larger version (38K): [in this window] [in a new window]   Figure 2. Inhibition of PI3-kinase blocks the Ang-1-mediated neuroprotection following serum deprivation toxicity. A) To induce apoptosis, near-pure cortical neuronal cultures (DIV 7) were transferred to a serum-deprived medium. Neuronal survival was assessed by cell counting following trypan blue staining after 24 h in the presence or not of the PI3-K inhibitor, LY 294002 (10 µM). Inhibition of PI3-kinase by LY 294002 blocks the Ang-1-mediated neuroprotection after serum deprivation toxicity (mean±SE, n=12).*P < 0.005 vs. control, # P < 0.005 vs. Ang-1 TFD alone by ANOVA with Bonferronni-Dunn’s test. B, C) Total cellular extracts from neuronal cultures treated or not with Ang-1 TFD (200 ng/mL), LY 294002, or both were electrophoretically separated on SDS-PAGE gels. Blots were probed with antibodies to P-Akt, Akt, active caspase-3, and actin.

3. Inhibition of PI3-kinase prevents Ang-1-induced neuroprotection against serum deprivation
Several studies have indicated that neurotrophic factors could protect neurons against ischemic insults via the PI3-K/Akt signaling pathways. It has been demonstrated in endothelial cells that Ang-1 plays an anti-apoptotic function by activating this pathway. To determine whether Ang-1 regulates the activity of Akt in cultured neurons, we studied the effect of Ang-1 on cells cultured in control conditions and then on cells submitted to apoptotic stress. In control conditions, we showed that addition of Ang-1 resulted in a rapid increase in the phosphorylation of Akt (5 min), which reached a maximum at 10 min and thereafter slowly decreased (30 min). We have next characterized the intracellular signaling cascade initiated by the Ang-1/Tie-2 interaction that leads to neuroprotection based on the PI3-K/Akt pathway. During serum withdrawal, neurons were incubated with Ang-1 TFD (200 ng/mL) in the presence or not of the PI3-K inhibitor LY294002. SD-induced neuronal death is accompanied by a reduction of Akt phosphorylation and an enhanced active caspase-3 expression (Fig. 2A, B ). The neuroprotective effects of Ang-1 TFD were abolished by LY294002 (n=12, P<0.005), whereas LY294002 alone failed to modify SD-induced cell death (Fig. 2A ). Concurrently, Western blot analyses performed after 24 h of treatment showed that the application of Ang-1 TFD restored the phosphorylation of Akt (Fig. 2B ) and inhibited the activation of caspase-3 (Fig. 2C ); these two effects were reversed by the presence of LY294002 (Fig. 2B, C ). Taken together, these results demonstrate that the activation of PI3-K/Akt is prerequisite for the protective actions of Ang-1 on apoptotic-induced neuronal death involving caspase-3 activation.

CONCLUSIONS

Until now, Ang-1 has been shown to both stabilize newly formed vasculature and inhibit vascular permeability in diverse animal models. Although the angiopoietin receptor Tie-2 has so far been described strictly in endothelial cells, in this study, using mouse neuronal cortical cultures, we reported that neurons express the angiopoietin receptor Tie-2 necessary for the transduction of Ang-1 signaling. This was confirmed by showing that phosphorylation of the receptor was induced by application of Ang-1 but not of Ang-2, the physiological antagonist. Based on these results, it might be proposed that, in brain, Ang-1 could act in both an autocrine and a paracrine fashion.

Ischemia-induced neuronal injury is thought to result from an excessive release of excitatory amino acids such as glutamate. Until recently, cerebral ischemic damage was generally described to result only from necrosis, but there is now evidence that a delayed apoptosis contributes to cell death. Recent studies have described modulations in the expression of VEGF and angiopoietins in response to cerebral ischemia that could be related to the delayed onset of angiogenesis, but also to a neuroprotective effect of these molecules on neuronal death. In accordance with this latter hypothesis, a delayed application of VEGF has been described to reduce ischemic damage and enhance angiogenesis in the ischemic area with improving neurological recovery. Based on the above and on our present findings, we postulated that the delayed expression of Ang-1 described in response to cerebral ischemia could contribute to the development of angiogenesis and concomitantly to control delayed neuronal apoptosis.

To study the putative neuroprotective effects of Ang-1, we compared the action of this cytokine on cortical neurons exposed to either excitotoxic necrosis or apoptotic stress. Our results showed that Ang-1 failed to prevent neuronal death induced by a NMDA treatment. However, the application of Ang-1 rescued neurons from death induced by serum withdrawal. These findings provide for the first time evidence that Ang-1 could exert, as in endothelial cells and with a similar range of concentrations, anti-apoptotic effects on neurons. Like VEGF, Ang-1 is an angiogenic factor, which is neuroprotective in vitro.

We further investigated the intracellular signaling pathways involved in the neuroprotective effect of Ang-1. The anti-apoptotic effect of Ang-1 in endothelial cells has been ascribed to the PI3-K/Akt pathway. Using an inhibitor of this pathway (LY 294002), we blocked the neuroprotective effect of Ang-1. We showed that the phosphorylation of Tie-2 in neurons could activate Akt and thereby protect cortical neurons from serum deprivation. We observed a concomitant decrease in the level of activated caspase-3, which was also reversed by the PI3-K inhibitor. Consequently, it can be postulated that caspase-3 may be an intracellular component downstream of the neuronal Akt activation induced by Ang-1. This latter result is in accordance with a recent study that found that the anti-apoptotic effect of Ang-1 in endothelial cells is mediated by an inhibition of the activation of caspase-9, -7, and -3. Caspases are activated in cascades in which upstream (activator) caspases lead to activation of downstream (effector) caspases. One of the most studied cascades is that of caspase-9, which leads to activation of caspase-3 and -7. In humans, the phosphorylation of caspase-9 by Akt leads to a reduction of its protease activity and consequently to a reduction of the effector caspsase-3 activation. In the mouse, active Akt is ineffective at phosphorylating caspase-9. Overall, our results propose that Ang-1 prevents neuronal apoptosis by activating Akt, which may execute anti-apoptotic pathway leading to an inhibition of caspase-3 but independent of caspase-9 phosphorylation. The inhibitors of apoptosis (IAP) are also able to inhibit the caspase activation. Human XIAP has been described to inhibit caspase-9 as well as downstream effector caspases, caspase-3 and -7. Recently, it has been demonstrated that Ang-1 prevents human endothelial cell apoptosis not only by activating Akt, which may execute anti-apoptotic pathway through caspase-9 phosphorylation, but also by up-regulating the IAP survivin. Our results suggest that Ang-1 could protect neurons from apoptosis by activating Akt and inhibiting caspase-3 activation, but we cannot rule out the possibility that Ang-1 could also increase IAP expression in neurons (Fig. 3 ). Among the IAPs, the neuronal apoptosis inhibitory protein (NAIP) could be a potential candidate. Indeed, NAIP has been characterized as a protective molecule on cortical neurons, which act as a direct inhibitor of caspase-3 and -7.

View larger version (25K): [in this window] [in a new window]   Figure 3. Angiopoietin-1-induced PI3-kinase activation prevents neuronal apoptosis. In neurons, interaction of Ang-1 with its receptor Tie-2 induces an activation of the PI3-K/Akt pathway with a concomitant decrease in the level of activated caspase-3 leading to neuroprotection against serum deprivation toxicity.

Stroke is caused (at least initially) by a disruption of blood flow to the brain and establishing early reperfusion can reduce the magnitude and extent of tissue injury. It is therefore likely that strategies that enhance both early reperfusion and neuroprotection will be investigated. Angiogenic factors are of interest. Based on our in vitro data and in accordance with recent in vivo data, it could be speculated that Ang-1, in addition to its angiogenic effect, could also play an important protective role in cerebral ischemia by acting directly on neuronal cells. Ang-1 is described as an anti-permeability and anti-inflammatory agent. These observations raise the possibility that in stroke, administration of Ang-1 could be used early not only to attenuate both the neuronal damage and blood–brain barrier leakage, but also to stimulate angiogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0372fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0372fje

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This Article Full Text (PDF) All Versions of this Article: 17/3/443 02-0372fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by VALABLE, S. Articles by PETIT, E. Search for Related Content PubMed PubMed Citation Articles by VALABLE, S. Articles by PETIT, E.