Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS.

The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other.

Angiotensin AT2-receptor stimulation improves survival and neurological outcome after experimental stroke in mice.

This study investigated the effect of post-stroke, direct AT2-receptor (AT2R) stimulation with the non-peptide AT2R-agonist compound 21 (C21) on infarct size, survival and neurological outcome after middle cerebral artery occlusion (MCAO) in mice and looked for potential underlying mechanisms. C57/BL6J or AT2R-knockout mice (AT2-KO) underwent MCAO for 30 min followed by reperfusion. Starting 45 min after MCAO, mice were treated once daily for 4 days with either vehicle or C21 (0.03 mg/kg ip). Neurological deficits were scored daily. Infarct volumes were measured 96 h post-stroke by MRI. C21 significantly improved survival after MCAO when compared to vehicle-treated mice. C21 treatment had no impact on infarct size, but significantly attenuated neurological deficits. Expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB) (receptor for BDNF) and growth-associated protein 43 (GAP-43) were significantly increased in the peri-infarct cortex of C21-treated mice when compared to vehicle-treated mice. Furthermore, the number of apoptotic neurons was significantly decreased in the peri-infarct cortex in mice treated with C21 compared to controls. There were no effects of C21 on neurological outcome, infarct size and expression of BDNF or GAP-43 in AT2-KO mice. From these data, it can be concluded that AT2R stimulation attenuates early mortality and neurological deficits after experimental stroke through neuroprotective mechanisms in an AT2R-specific way. Key message • AT2R stimulation after MCAO in mice reduces mortality and neurological deficits.• AT2R stimulation increases BDNF synthesis and protects neurons from apoptosis.• The AT2R-agonist C21 acts protectively when applied post-stroke and peripherally.

Direct angiotensin type 2 receptor (AT2R) stimulation attenuates T-cell and microglia activation and prevents demyelination in experimental autoimmune encephalomyelitis in mice.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.

AT2-receptor stimulation enhances axonal plasticity after spinal cord injury by upregulating BDNF expression.

It is widely accepted that the angiotensin AT2-receptor (AT2R) has neuroprotective features. In the present study we tested pharmacological AT2R-stimulation as a therapeutic approach in a model of spinal cord compression injury (SCI) in mice using the novel non-peptide AT2R-agonist, Compound 21 (C21). Complementary experiments in primary neurons and organotypic cultures served to identify underlying mechanisms. Functional recovery and plasticity of corticospinal tract (CST) fibers following SCI were monitored after application of C21 (0.3mg/kg/dayi.p.) or vehicle for 4 weeks. Organotypic co-culture of GFP-positive entorhinal cortices with hippocampal target tissue served to evaluate the impact of C21 on reinnervation. Neuronal differentiation, apoptosis and expression of neurotrophins were investigated in primary murine astrocytes and neuronal cells. C21 significantly improved functional recovery after SCI compared to controls, and this significantly correlated with the increased number of CST fibers caudal to the lesion site. In vitro, C21 significantly promoted reinnervation in organotypic brain slice co-cultures (+50%) and neurite outgrowth of primary neurons (+25%). C21-induced neurite outgrowth was absent in neurons derived from AT2R-KO mice. In primary neurons, treatment with C21 further induced RNA expression of anti-apoptotic Bcl-2 (+75.7%), brain-derived neurotrophic factor (BDNF) (+53.7%), the neurotrophin receptors TrkA (+57.4%) and TrkB (+67.9%) and a marker for neurite growth, GAP43 (+103%), but not TrkC. Our data suggest that selective AT2R-stimulation improves functional recovery in experimental spinal cord injury through promotion of axonal plasticity and through neuroprotective and anti-apoptotic mechanisms. Thus, AT2R-stimulation may be considered for the development of a novel therapeutic approach for the treatment of spinal cord injury.

Direct angiotensin II type 2 receptor stimulation in Nω-nitro-L-arginine-methyl ester-induced hypertension: the effect on pulse wave velocity and aortic remodeling.

Pulse wave velocity (PWV), a direct marker of arterial stiffness, is an independent cardiovascular risk factor. Although the angiotensin II type 1 receptor blockade belongs to major antihypertensive and cardioprotective therapies, less is known about the effects of long-term stimulation of the angiotensin II type 2 receptor. Previously, compound 21, a selective nonpeptide angiotensin II type 2 receptor agonist improved the outcome of myocardial infarction in rats along with anti-inflammatory properties. We investigated whether compound 21 alone or in combination with angiotensin II type 1 receptor blockade by olmesartan medoxomil could prevent PWV increase and aortic remodeling in N(ω)-nitro-L-arginine-methyl ester (L-NAME)-induced hypertension. Male adult Wistar rats (n=65) were randomly assigned to control, L-NAME, L-NAME+compound-21, L-NAME+olmesartan, and L-NAME+olmesartan+compound-21 groups and treated for 6 weeks. We observed that L-NAME hypertension was accompanied by enhanced PWV, increased wall thickness, and stiffness of the aorta, along with elevated hydroxyproline concentration. Olmesartan completely prevented hypertension, PWV and wall thickness increase, and the increase of aortic stiffness and partly prevented hydroxyproline accumulation. Compound 21 partly prevented all of these alterations, yet without concomitant prevention of blood pressure rise. Although the combination therapy with olmesartan and compound 21 led to blood pressure levels, PWV, and wall thickness comparable to olmesartan-alone-treated rats, only in the combination group was complete prevention of increased hydroxyproline deposition achieved, resulting in even more pronounced stiffness reduction. We conclude that chronic angiotensin II type 2 receptor stimulation prevented aortic stiffening and collagen accumulation without preventing hypertension in rats with inhibited NO synthase. These effects were additive to angiotensin II type 1 receptor blockade, yet without additional blood pressure-lowering effect, and they seem to be NO and blood pressure independent.

Novel therapy approach in primary stroke prevention: simultaneous inhibition of endothelin converting enzyme and neutral endopeptidase in spontaneously hypertensive, stroke-prone rats improves survival.

OBJECTIVES: Stroke, frequently a consequence of hypertension, is one of the leading causes of death and neurological disabilities worldwide. In the ischemic brain, levels of endothelin-1, one of the most potent vasoconstrictors, are raised. Anti-inflammatory and neuroprotective effects of endothelin antagonists after stroke have been described in literature. Based on these findings, we investigated the protective effect of the endothelin converting enzyme/neutral endopeptidase blocker, SLV 338, in salt-loaded, stroke-prone, spontaneously hypertensive rats. METHODS: Male, 8-week-old spontaneously hypertensive stroke-prone rats were put on a high salt diet and treated with either 30 mg/kg or 100 mg/kg SLV 338 or vehicle for 27 weeks. Blood pressure, neurological outcome, body weight, and mortality were investigated throughout treatment. In weeks 1 and 9, animals were housed in metabolic cages for collection of urinary and blood samples and assessment of salt water and food intake. In weeks 22 and 27, additional blood samples were taken. At the end of the study, all brains were analyzed using magnetic resonance imaging. RESULTS: SLV 338 was well tolerated in all animals. Neurological outcome and infarct size were similar in all groups. Albuminuria was considerably delayed and the incidence of stroke significantly lowered in treated animals. In spontaneously hypertensive stroke-prone rats, treatment with SLV 338 significantly (P = 0·01) improved survival in comparison to the vehicle treated group in a blood pressure-independent manner. DISCUSSION: Our data in spontaneously hypertensive stroke-prone rats demonstrate that combined endothelin converting enzyme/neutral endopeptidase inhibition could offer a new therapeutic approach for primary stroke prevention and improvement of mortality. The mechanism seems to be blood pressure-independent.

The promyelocytic leukemia zinc finger (PLZF) protein exerts neuroprotective effects in neuronal cells and is dysregulated in experimental stroke.

Stroke is one of the major medical burdens in industrialized countries. Animal experiments indicate that blockade of the angiotensin AT1 receptor (AT1R) improves neurological outcome after cerebral ischemia. These protective effects are partially mediated by the angiotensin AT2 receptor (AT2R). The transcription factor promyelocytic leukemia zinc finger (PLZF) was identified as a direct adapter protein of the AT2R. Furthermore, our group was able to demonstrate that PLZF also directly binds and mediates the effects of the human (pro)renin receptor [(P)RR] which is involved in brain development. Therefore, we hypothesized that PLZF is involved in neuroprotection. Here we show that PLZF and its receptors (P)RR and AT2R exhibited an ubiquitous expression pattern in different brain regions. Furthermore, stable PLZF overexpression in human neuronal cells was able to mediate neuroprotection in a glutamate toxicity model in vitro. Consistently, PLZF mRNA and protein were downregulated on the ipsilateral side in a stroke model in vivo, whereas the neurodetrimental PLZF target genes cyclin A2 and BID were upregulated under this condition. Further analyses indicated that the neuroprotective AT2R is upregulated upon stable PLZF overexpression in cultured neuronal cells. Finally, reporter gene assays demonstrated the functionality of (P)RR promoter polymorphisms regarding basal and PLZF-induced activity.

The beta-lactam antibiotic, ceftriaxone, dramatically improves survival, increases glutamate uptake and induces neurotrophins in stroke.

OBJECTIVE: Ceftriaxone has been reported to reduce neuronal damage in amyotrophic lateral sclerosis and in an in-vitro model of neuronal ischaemia through increased expression and activity of the glutamate transporter, GLT1. We tested the effects of ceftriaxone on mortality, neurological outcome, and infarct size in experimental stroke in rats and looked for underlying mechanisms. METHODS: Male normotensive Wistar rats received ceftriaxone (200 mg/kg intraperitoneal) as a single injection 90 min after middle cerebral artery occlusion (90 min with reperfusion). Forty-eight hours after middle cerebral artery occlusion, infarct size (MRI) and neurological deficits were estimated. GLT1 expression was determined by real time RT-PCR, immunoblotting and promoter reporter assay, astrocyte GLT1 activity by measuring glutamate uptake. Bacterial load in various organs was measured by real time RT-PCR, neurotrophins and IL-6 by immunoblotting. RESULTS: Ceftriaxone dramatically reduced early (24-h) mortality from 34.5% (vehicle treatment, n = 29) to 0% (P < 0.01, n = 19). In a subgroup, followed up for 4 weeks, mortality persisted at 0%. Ceftriaxone strongly tended to reduce infarct size, it significantly improved neuronal survival within the penumbra, reduced neurological deficits (P < 0.001) and led to an upregulation of neurotrophins (P < 0.01) in the peri-infarct zone. Ceftriaxone did not increase GLT1 expression, but increased GLT1 activity (P < 0.05). CONCLUSION: Ceftriaxone causes a significant reduction in acute stroke mortality in a poststroke treatment regimen in animal studies. Improved neurological performance and survival may be due to neuroprotection by activation of GLT1 and a stimulation of neurotrophins resulting in an increased number of surviving neurons in the penumbra.