Angiopoietin-1-induced PI3-kinase activation prevents neuronal apoptosis.

Although angiopoietin-1 (Ang-1) is recognized as an endothelial growth factor, its presence in brain following an ischemic event suggests a role in the evolution of neuronal damage. Using primary neuronal cultures, we showed that neurons express Ang-1 and possess the functional angiopoietin-receptor Tie-2, which is phosphorylated in the presence of Ang-1. We further investigated in vitro whether Ang-1 could protect neurons against either excitotoxic necrosis or apoptosis induced by serum deprivation (SD). A neuroprotective effect for Ang-1 was detected exclusively in the apoptotic paradigm. Treatment of cells with the phosphatidyl-inositol 3-kinase (PI3-K) inhibitor, LY294002, inhibited Ang-1-induced phosphorylation of Akt, restored the cleavage of the effector caspase-3, and reduced the protective effect of Ang-1 against SD-induced toxicity. These findings suggest that Ang-1 has a neuroprotective effect against apoptotic stress and that this effect is dependent on the PI3-K/Akt pathway and inhibition of caspase-3 cleavage. This study provides evidence that Ang-1 is not just angiogenic but also neuroprotective. The understanding of neuroprotective mechanisms induced by Ang-1 may promote strategies based on the pleiotropic effects of angiogenic factors. Such approaches could be useful for the treatment of brain diseases in which both neuronal death and angiogenesis are involved.

Neurons and astrocytes express EPO mRNA: oxygen-sensing mechanisms that involve the redox-state of the brain.

Erythropoietin (Epo), the major hormone controlling the hypoxia-induced increase in the number of erythrocytes, has also a functional role in the brain. However, few data exist as to the cellular source of brain-derived Epo as well as to the molecular mechanisms that control Epo expression in the central nervous system. Using patch-clamp and RT-PCR methods, we provide direct evidence that, besides astrocytes, neurons are a source of Epo in the brain. Both the astrocytic and neuronal expression of Epo mRNA are induced not only by hypoxia, but also by desferrioxamine (DFX) and cobalt chloride (CoCl(2)), two agents known to mimic the hypoxic induction of Epo in hepatoma cells. This induction is blocked by cycloheximide suggesting that de novo protein synthesis is required. Furthermore, the addition of H(2)O(2) decreases the hypoxia-induced Epo mRNA levels. These data indicate that, following hypoxia, a common oxygen sensing and signaling pathway leads to increased Epo gene expression in both nervous and hepatoma cells; this pathway would be dependent on the redox-state of the brain. Furthermore, we show that the in vivo administration of CoCl(2) and DFX to mice induces an increased Epo mRNA level in the neocortex. As Epo protects the brain against ischemia, our in vivo experiments suggest that the use of molecules such as CoCl(2) or DFX, that provoke an increased Epo gene expression in the brain, could be useful in the development of potential therapeutic strategies for the treatment of hypoxic or ischemic brain injury.

Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia.

We investigated the hypothesis that hypoxia induces angiogenesis and thereby may counteract the detrimental neurological effects associated with stroke. Forty-eight to seventy-two hours after permanent middle cerebral artery occlusion we found a strong increase in the number of newly formed vessels at the border of the infarction. Using the hypoxia marker nitroimidazole EF5, we detected hypoxic cells in the ischemic border of the neocortex. Expression of vascular endothelial growth factor (VEGF), which is the main regulator of angiogenesis and is inducible by hypoxia, was strongly up-regulated in the ischemic border, at times between 6 and 24 hours after occlusion. In addition, both VEGF receptors (VEGFRs) were up-regulated at the border after 48 hours and later in the ischemic core. Finally, the two transcription factors, hypoxia-inducible factor-1 (HIF-1) and HIF-2, known to be involved in the regulation of VEGF and VEGFR gene expression, were increased in the ischemic border after 72 hours, suggesting a regulatory function for these factors. These results strongly suggest that the VEGF/VEGFR system, induced by hypoxia, leads to the growth of new vessels after cerebral ischemia. Exogenous support of this natural protective mechanism might lead to enhanced survival after stroke.