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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 21, 2005 as doi:10.1096/fj.04-2960fje. |
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,1
* Center for Cardiovascular Research/Institute of Pharmacology and Toxicology, and
Department of Experimental Neurology, Charité-University Medicine Berlin; and
Institute of Pharmacology, University Hospital of Schleswig-Holstein, Campus Kiel, Christian-Albrechts-University of Kiel, Germany
2Correspondence: Center for Cardiovascular Research (CCR)/Institute of Pharmacology und Toxicology Charité-Universitätsmedizin Berlin, Hessische Strasse 3-4, Berlin 10115 , Germany. E-mail: thomas.unger{at}charite.de
SPECIFIC AIMS
The aim of the study was to investigate whether cerebral AT2 receptors play a neuroprotective role in cerebral ischemia-induced brain injury.
PRINCIPAL FINDINGS
1. AT2 receptors are up-regulated exclusively in neurons in response to focal cerebral ischemia
We examined regulation of angiotensin AT1 and AT2 receptors in response to cerebral ischemia. Real-time RT-PCR analysis showed that cerebral AT2 receptor mRNA in the peri-infarct zone increased 2.2-fold 2 days after unilateral medial cerebral artery occlusion (MCAO) compared with sham operation (P<0.01). Western blot analysis confirmed a 2.1-fold up-regulation of AT2 receptor protein after MCAO (P<0.01); there was no significant change of cerebral AT1 receptors at the mRNA and protein levels. Immunofluorescence staining on brain sections revealed that increased AT2 receptor immunostained cells were mainly detected in the ischemic vs. the contralateral hemisphere and were abundantly distributed in brain regions adjacent to the infarct area including cerebral frontal cortex, piriform cortex, striatum, and hippocampus. In contrast, AT1 receptor immunostained cells found in brain regions such as cerebral frontal cortex, cingulum, and striatum remained unchanged between ischemic and contralateral side.
Double immunofluorescence staining confirmed that AT2 receptors were located exclusively in neurons; colocalization of AT2 receptors with GFAP (astrocyte marker) was not detected. In contrast, AT1 receptors were mainly localized in astrocytes and only sparsely in cortical neurons (data not shown).
The AT2 receptor overexpressing neurons in the striatum adjacent to the ischemic area showed a more intense neurite staining, as revealed by MAP2 immunofluorescence labeling, than other neurons without or with weak AT2 receptor expression (Fig. 1
).
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2. AT2 receptors protect brain tissue after focal cerebral ischemia
We investigated the role of brain AT2 receptors after cerebral infarction induced by MCAO by analyzing infarct volume, brain edema, and functional outcome after selective and exclusive blockade of brain AT1 and AT2 receptors via chronic intracerebroventricular infusion (AT1 blockade: irbesartan, 2 nM/h; AT2 blockade: PD123317, 1 nM/h) before and up to 24 h after cerebral ischemia. One day after the infarct, unilateral injury was clearly detected as an area of pallor sharply demarcated from the adjacent tissue (cresyl violet staining). Ischemic lesions comprised parts of the frontal, parietal, and temporal cortices together with the lateral segments of the caudate nucleus. The total volume of injury (cortical+subcortical areas) was reduced by 43% (P<0.05 vs. vehicle group) in rats treated with the AT1 receptor antagonist irbesartan (Fig. 2
A). Blockade of central AT1 receptors reduced ischemia-induced brain edema (vehicle: 61±9 mm3; irbesartan: 33±7 mm3; P<0.01 vs. vehicle group).
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Central cotreatment with the AT2 receptor antagonist PD123177 abolished the effects of irbesartan on infarct volume (P>0.05 vs. vehicle group) and brain edema (irbesartan+PD123177: 53±8 mm3, P>0.05 vs. vehicle group).
To determine whether a reduction in infarct volume was associated with a better functional outcome, neurological deficits produced by MCAO were evaluated in rats treated with irbesartan alone or irbesartan plus PD123177. Neurological deficits were significantly improved after irbesartan treatment 1 day after ischemia, but when PD123177 was given together with irbesartan, the neuroprotective effect of irbesartan was abolished (Fig. 2B
).
To exclude a potential drug effect on peripheral vascular angiotensin receptors and cerebral blood flow (CBF), pressor responses to i.v. angiotensin II infusions and changes in regional CBF were examined. Irbesartan and its combination with PD123177 did not affect pressor responses and regional CBF.
3. AT2 receptors support survival and neurite outgrowth in primacy cortical neurons
We next focused on the role of AT2 receptors on neuronal viability and neurite outgrowth in primary cultured cortical neurons to explore mechanisms underlying AT2 receptor-mediated neuroprotection. First, we confirmed by immunofluorescence staining with AT1 or AT2 receptor-specific antibodies that AT1 and AT2 receptors were expressed in primary cultured neurons after 48 h in vitro culture. After examining the dose dependency of the effects of angiotensin II on neuronal viability (calcein AM assay) and mean neurite length/neuron (stained with MAP2) at concentrations ranging from of 5–100 nM, we chose 10 nM as the effective concentration. When stimulated with exogenous angiotensin II alone (10 nM), neuronal viability and mean neurite length/neuron increased by 136% (P<0.01) and 143% (P<0.05), respectively, compared with control. The AT2 receptor antagonist PD123319 (200 nM) reversed the effects of angiotensin II on neuronal viability (136–73%, P<0.01) and mean neurite length/neuron (163–104 µm, P<0.01). In contrast, blocking AT1 receptors with irbesartan (200 nM) further enhanced the effects of angiotensin II on neuronal viability (136–185%, P<0.01) and mean neurite length/neuron (63–256 µm, P<0.01) vs. that of angiotensin II alone. Irbesartan (200 nM) or PD123319 (200 nM) in the absence of angiotensin II did not significantly influence neuronal viability and mean neurite length/neuron.
CONCLUSIONS AND SIGNIFICANCE
After transient cerebral ischemia by MCAO in the rat, AT2 receptors were up-regulated exclusively in neuronal cells of the peri-ischemic brain. Our data demonstrate that up-regulated cerebral AT2 receptors were associated with neurite outgrowth in the ischemic brain and were required for the cerebroprotective effects of central AT1 receptor blockade in vivo. These findings of a cerebroprotective role of the AT2 receptor were further supported by the fact that in primary neuronal cells from embryonic rat brain, which harbor AT1 and AT2 receptors, stimulation of AT2 receptors but not AT1 receptors prolonged neuronal survival and promoted neurite growth. (Fig. 3
).
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The renin-angiotensin system is an important player in controlling cardiovascular homeostasis. Most peripheral and central actions of angiotensin II, such as blood pressure and osmotic control, are mediated by AT1 receptors. AT2 receptors have been shown to be involved in the control of cell proliferation, cell differentiation, tissue regeneration, and apoptosis in various cell types. In the adult brain, AT2 receptors have been located predominantly in regions involving motor and sensory functions, such as the cerebral cortex.
After ischemia-induced brain injury, expression of AT2 receptors was increased mainly in the cerebral frontal cortex, piriform cortex, hippocampus, and striatum and localized exclusively in neurons. Expression of cerebral AT1 receptors, mainly distributed in astrocytes, remained unaltered after ischemia. The differential localization of AT1 and AT2 receptors in the brain agrees with an in vitro report showing that AT1 and AT2 receptors exist mainly in primary cultured astrocytes and neurons, respectively.
Increased AT2 receptors and decreased AT1 receptors after cerebral ischemia have been reported based on autoradiography. With regard to the AT1 receptors, these findings are intriguing, given that exclusive blockade of central (nonvascular) AT1 receptors can markedly reduce infarct size, improve postischemic neurological outcome, and suppress the ischemia-induced stimulation of AP-1 transcription factors in the brain. On the other hand, AT1 receptors are dominant compared with AT2 receptors in most brain areas; AT1-mediated postischemic events may occur without further up-regulation. In contrast, up-regulation of the suppressed AT2 receptor speaks in favor of a general role of this receptor in the repair of injured tissue.
By central administration of an AT1 receptor antagonist alone or in combination with an AT2 receptor antagonist in vivo, we demonstrated that AT1 receptor blockade can reduce infarct size and cerebral edema and improve neurological outcome only if the AT2 receptor is intact (i.e., unopposed).
Since it has been clearly shown that AT1 receptors mediate apoptosis in myocytes, endothelial cells in vitro, and rat blood vessels in vivo, we cannot exclude that a direct inhibition of AT1 receptor-mediated apoptosis in astrocytes may play a role in the neuroprotective effects of the AT1 receptor antagonist.
Little is known about mechanisms involved in AT2 receptor-mediated neuroprotection. Our previous studies have shown that AT2 receptors may contribute to Schwann cell-mediated myelination and the neuroregenerative responses of dorsal root ganglia that occur after sciatic nerve transection in adult rats. In accordance, the present study for the first time presents ex vivo data revealing that increased AT2 receptors are associated with intense neurite outgrowth in neurons of the postischemic striatum. Corroborating these findings, stimulation of AT2 receptors prolonged neuronal survival and promoted neurite outgrowth in primary cortical neurons in culture. The present data, together with earlier findings, favor the view that cerebral AT2 receptors may exert neuroprotective effects by supporting neuronal survival and neurite outgrowth in response to cerebral ischemia-induced neuronal injury.
From the present study, we can delineate that cerebral ischemia induces overexpression of AT2 receptors in neurons of the ischemic brain, which may mediate cellular adaptation to hypoxia through mechanisms involving neuronal survival and neurite outgrowth. Treatment with AT1 receptor blockers may result in increased production of brain angiotensin peptides, which may lead to activation of up-regulated, unopposed AT2 receptors in order to maintain or recover neuronal function. Further experiments are needed to clarify the signaling mechanisms responsible for the AT2 receptor-mediated neuroprotection.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2960fje; doi: 10.1096/fj.04-2960fje
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