Cerebral Ischemia Enhances Vascular Angiotensin AT1 Receptor–Mediated Contraction in Rats
Background and Purpose— The aim of the study was to examine how focal cerebral ischemia affects the expression and function of vascular angiotensin II receptors.
Materials and Methods— We used an intraluminal filament occlusion technique to occlude the right middle cerebral artery (MCA) of the rat. Myographs were used for functional studies of the MCA and real-time polymerase chain reaction, for determination of relative mRNA levels.
Results— The contractile responses to angiotensin II were stronger in the right occluded MCA compared with the left MCA and the MCA from sham-operated rats 48 hours after MCA occlusion (P<0.05). The angiotensin II type 1 (AT1) receptor antagonists candesartan and losartan abolished the enhanced responses to angiotensin II (P<0.05), whereas the AT2 receptor antagonist PD123319 had no effect. The amount of AT1 receptor mRNA was lower in the occluded MCAs compared with nonoccluded MCAs 48 hours after occlusion (P<0.05), whereas the mRNA levels of angiotensin converting enzyme (ACE) were higher in the occluded arteries. The mRNA levels of the AT2 receptor and nuclear factor-κB were unchanged.
Conclusions— Focal cerebral ischemia in the rat upregulated the contractile responses to angiotensin II in the ipsilateral MCA, and this contraction was mediated by AT1 receptors. Real-time polymerase chain reaction revealed decreased AT1 receptor mRNA levels in the occluded MCA, whereas the amount of ACE mRNA was increased, suggesting locally enhanced angiotensin II production. These results support a role for AT1 receptors in cerebral ischemia, and we think that AT1 receptors might be a future therapeutic target in ischemic stroke.
Angiotensin II (Ang II) is an octapeptide that acts at different sites in the brain (eg, the vascular, neuroendocrine, and behavioral systems). Angiotensin converting enzyme (ACE), which converts Ang I to Ang II, is expressed in cerebral microvessels of the rat1 suggesting local formation of Ang II in addition to circulating Ang II. In cerebral vessels, Ang II has been found to induce vasoconstriction2,3 and vasodilatation,4 depending on the species studied and methods used. The responses to Ang II are predominantly mediated by the Ang II type 1 (AT1) and type 2 (AT2) receptor subtypes, of which the AT1 receptor subtype mediates contraction of cerebral arteries in adult rats.5 The present study is based on the hypothesis that cerebral ischemia induces a change in local vascular receptor expression and function, which might influence the ischemia and have a role in the development of the penumbral zone. In a previous study, we revealed an upregulation of contractile endothelin type B (ETB) receptors with enhanced transcription in the middle cerebral artery (MCA) of the rat 48 hours after temporal MCA occlusion.6 In the present study we used the same experimental model. The functional responses to Ang II in the MCA were examined by myographs, and the relative mRNA levels of AT1, AT2, ACE, and the transcription factor nuclear factor-κB (NF-κB) were quantified by real-time polymerase chain reaction (PCR). NF-κB mRNA levels were determined because this transcription factor is activated by inflammatory cytokines, which might be involved in receptor regulation in cerebral ischemia. According to the transfac database,7 there exist possible binding sites for NF-κB in the AT1 receptor promoter (GenBank accession number S66402). This study examined the impact of focal cerebral ischemia on the vascular angiotensin system.
Materials and Methods
Middle Cerebral Artery Occlusion
Focal cerebral ischemia was induced by the intraluminal occlusion technique described by Memezawa et al.8 Male Wistar-Hannover rats (Møllegaard Breeding Center, Copenhagen, Denmark) weighing 350 to 420 g were used for the study. The experimental procedures were approved by the Ethics Committee for Laboratory Animal Experiments at the University of Lund (M217-00). The animals were fasted overnight with access to water. Anesthesia was induced by inhalation through a mask with 4.5% halothane in N2O:O2 (70:30, vol/vol). The rats were kept anesthetized with 1% to 1.5% halothane in N2O:O2 (70:30, vol/vol) during the surgical procedure. The right MCA was occluded by inserting a filament into the internal carotid artery, which was advanced further until it closed the origin of the MCA. When the surgical procedures were finished, anesthesia was discontinued and the animals were allowed to recuperate. Two hours after MCA occlusion, the animals were briefly reanesthetized to allow withdrawal of the filament to achieve reperfusion. Sham-operated rats underwent the same surgical procedures as the MCA-occluded rats except that the filament was immediately withdrawn after insertion. These animals were also reanesthetized for 15 minutes 2 hours after operation.
Removal of the MCA and Evaluation of Ischemic Damage in Rats
After 22 or 46 hours of recovery, the animals were reanesthetized and decapitated. The brain was quickly removed and chilled in bicarbonate buffer solution (see next paragraph for composition). The right and left MCAs were removed, cut into segments, and immediately denuded of the endothelium for mounting in myographs or snap-frozen at −80°C for examination by real-time PCR. The ischemic brain damage was confirmed in all animals by staining coronal slices of the brains with 1% 2,3,5-triphenyltetrazolium chloride dissolved in saline solution at 37°C for 20 minutes.9
In Vitro Pharmacology and Myograph Tissue Bath
The MCAs were cut into cylindrical segments and the endothelium was removed mechanically by inserting a thin thread and rubbing off the endothelium. The vessel segments were then threaded onto two 40-μm-diameter stainless steel wires and mounted in a Mulvany-Halpern myograph (Danish Myo Technology A/S) for recording of isometric tension.10,11 One wire was connected to a force-displacement transducer attached to an analog-to-digital converter unit (ADInstruments). The other wire was attached to a movable displacement device, thus allowing fine adjustments of vascular tension by varying the distance between the wires. The experiments were recorded on a computer with Chart software (ADInstruments).
The segments were immersed in temperature-controlled (37°C) tissue baths containing a bicarbonate buffer solution of the following composition (mmol/L): NaCl 119, NaHCO3 15, KCl 4.6, MgCl2 1.2, NaH2PO4 1.2, CaCl2 1.5, and glucose 5.5. The solution was continuously gassed with 5% CO2 in O2, resulting in a pH of 7.4. The vessels were given an initial tension of 1.2 mN and were adjusted to this level of tension for 1 hour. The contractile capacity was determined by exposure to a potassium-rich (63.5 mmol/L) buffer solution with the same composition as the bicarbonate buffer solution except that NaCl was exchanged for KCl. The contraction to 63.5 mmol/L potassium was used as a reference for the contractile capacity. Removal of the endothelium was confirmed by monitoring responses to 10−5 mol/L acetylcholine (ACh) after precontraction with 5-hydroxytryptamine (5-HT). Concentration-response curves for Ang II (10−12 to 10−6.5 mol/L) were obtained by cumulative application. To characterize the Ang II receptor type responsible for the responses, 2 different AT1 receptor antagonists, candesartan (10−7 mol/L) and losartan (10−7 mol/L), and 1 AT2 receptor antagonist, PD123319 (10−6 mol/L), were added to the buffer solution 30 minutes before application of Ang II (in separate experiments).
Total cellular RNA was extracted using the FastRNA kit GREEN (BIO 101) and following the supplier’s instructions. The resulting pellet was finally washed with 75% ethanol, air-dried, and redissolved in 40 μL diethylpyrocarbonate-treated water.
Reverse transcription of total RNA to cDNA was carried out using the GeneAmp RNA PCR kit (Perkin-Elmer Applied Biosystems) in a Perkin-Elmer DNA thermal cycler. First-strand cDNA was synthesized from total RNA in a 40-μL reaction volume with random hexamers as primers. The reaction mixture was incubated at 25°C for 10 minutes, heated to 42°C for 15 minutes, heated further to 99°C for 5 minutes, and chilled to 5°C for 5 minutes. Real-time PCR was performed in a GeneAmp 5700 sequence detection system using the GeneAmp SYBR GREEN kit (Perkin-Elmer Applied Biosystems) with the previously synthesized cDNA as the template in a 50-μL reaction volume. A no-template control was included in all experiments. The GeneAmp 5700 sequence detection system monitored the growth of DNA in real time by using an optical imaging system that monitored the binding of a fluorescent dye to double-stranded DNA. Specific primers for the AT1 and AT2 receptors, ACE, and NF-κB were designed as follows: for the AT1 receptor, forward primer 5′-GGATGGTTCTCAGAGAGAGTACAT-3′ and reverse primer 5′-CCTGCCCTCTTGTACCTGTTG -3′; for the AT2 receptor, forward primer 5′-TCTGTTAGTGGGATGCATGTAATCA-3′ and reverse primer 5′-TGTGGGCCTCCAAACCATT-3′; for ACE, forward primer 5′-CCCGGAAATACGAAGAATTGC-3′ and reverse primer 5′-GGCTCTCCCCACCTTGTCTC-3′ and for NF-κB, forward primer 5′-GAGAGCCAGTAGCACGCATG-3′ and reverse primer 5′-CCTGGGTTCGTGGAATGAGT-3′.
Elongation factor-1 (EF-1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs were used as endogenous standards, and the primers were designed as follows: for EF-1, forward primer 5′-GCAAGCCCATGTGTGTTGAA-3′ and reverse primer 5′-TGATGACACCCACAGCAACTG-3′ and for GAPDH, forward primer 5′-GGCCTTCCGTGTTCCTACC-3′ and reverse primer 5′-CGGCATGTCAGATCCACAAC-3′.
Real-time PCR was carried out with the following profile: 50°C for 2 minutes, followed by 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute.
Candesartan, losartan, and PD123319 were generous gifts from Dr P. Morsing, Astra-Zeneca, Molndal, Sweden. Ang II, 5-HT, and ACh (Sigma-Aldrich) were dissolved in 0.9% saline.
In Vitro Pharmacology and Myograph Tissue Bath
Forty-eight hours after cerebral ischemia, contractile experiments were performed with 5 to 8 rats per group (1 or 2 segments from each vessel). For experiments with Ang II receptor blockers, 3 occluded MCAs were treated with losartan and PD123319, and 2 occluded MCAs were treated with candesartan. Contractile experiments 24 hours after ischemia were performed on MCA-occluded rats with 4 animals per group (1 or 2 segments from each vessel). The Emax values refer to the maximum contraction calculated as a percentage of the contractile capacity of 63.5 mmol/L K+. Data are expressed as mean±SEM. Statistical analyses were performed with 1-way ANOVA followed by Dunnett’s multiple comparison test (3 groups) or Students’ t test (2 groups). P<0.05 was considered significant.
PCR experiments on vessels extracted 48 hours after ischemia were performed on 5 or 6 MCA-occluded rats, and experiments on vessels extracted 24 hours after ischemia were performed on 4 MCA-occluded rats. The samples were analyzed in duplicates. The amount of mRNA was calculated relative to the amount of EF-1 and GAPDH mRNAs (48 hours of ischemia) or GAPDH mRNA (24 hours of ischemia) in the same sample by the formula X0/R0=2CtR−CtX, where X0 is the original amount of target mRNA, R0 is the original amount of EF-1/GAPDH mRNAs, CtR is the CT value for EF-1/GAPDH mRNAs, and CTX is the CT value for the target. CT values refer to the number of PCR cycles performed per gene product in 1 sample at a specific point of time. Data are expressed as mean±SEM. Statistical analyses were performed with unpaired Students’ t test, and P<0.05 was considered significant.
Immediately before MCA occlusion, blood pH, Pao2, Paco2, and blood glucose levels were measured and were within normal limits, with no significant differences between the groups (data not shown). When coronal slices of the brains were stained with 2,3,5-triphenyltetrazolium chloride, ischemic damage could be observed in the right cerebral hemispheres from MCA-occluded rats. The amount of brain damage was compared with the results of previous studies by Memezawa et al12 and Kiyota et al,13 in which the same occlusion technique was used. In the artery-occluded animals used for the current study, the brain damage extended over the lateral caudoputamen and most of the overlying cortex in the distribution of the right MCA. There were no signs of ischemia in the brains from the sham-operated rats (data not shown).
In Vitro Pharmacology
K+-induced contractions did not differ significantly between the groups (ie, 48 hours after MCA occlusion, the Emax was 1.56±0.18 mN for the right, occluded MCA; 1.18±0.34 mN for the left, nonoccluded MCA; and 1.45±0.43 mN for the right MCA from sham-operated rats; P>0.05). Twenty-four hours after MCA occlusion, the Emax was 1.58±0.27 mN for the right, occluded MCA and 1.23±0.26 mN for the left, nonoccluded MCA (P>0.05). The relaxant responses to ACh after 5-HT precontraction were totally abolished, which indicated a properly removed endothelium. In vessels extracted 48 hours after ischemia, Ang II induced a significantly stronger contraction in the occluded, right MCA compared with the nonoccluded, left MCA and the right MCA from sham-operated rats (Emax was 65±15% in the occluded MCA, 15±8% in the nonoccluded MCA, and 17±6% in the right MCA from sham-operated rats; P<0.05; Figure 1). The 2 AT1 receptor blockers candesartan (10−7 mol/L) and losartan (10−7 mol//L) had no effect by themselves, but they totally inhibited the responses to Ang II in occluded MCAs (Emax was 0±0% for both; P<0.05; Figure 2), whereas the AT2 receptor blocker PD123319 (10−6 mol//L) had no inhibitory effect (Emax was 51±20%), suggesting that the AT1 receptors are responsible for the upregulated responses induced by cerebral ischemia. The contractile responses to Ang II were not increased after 24 hours of ischemia (Emax=8±4% in the occluded MCA and 14±4% in the nonoccluded MCA), which demonstrate that the changes occurred between 24 and 48 hours after the onset of cerebral ischemia.
The mRNA levels for arteries extracted 48 hours after occlusion were compared by using 2 different endogenous standards, GAPDH and EF-1, and the results were almost identical. This suggests that the standards in all probability have an unchanged expression in cerebral vessels after ischemia and therefore are suitable for these experiments. In PCR experiments 24 hours after MCA occlusion, GAPDH was used as an endogenous standard. In vessels extracted 48 hours after MCA occlusion, the AT1 receptor mRNA levels were significantly lower in the right, occluded MCA compared with the left, nonoccluded MCA (P<0.05; Figure 3 and the Table), whereas the ACE mRNA levels were higher in the occluded MCA (P<0.05 when compared with EF-1, P=0.25 when compared with GAPDH; Figure 3 and the Table). There were no differences in the amount of mRNA coding for the AT2 receptor or NF-κB (the Table). In vessels extracted 24 hours after MCA occlusion, there were no differences in the mRNA levels for any of the products (the Table).
This is the first study to show that experimental focal cerebral ischemia results in an enhanced contractile response to Ang II in the ipsilateral MCA. The contraction was totally abolished by either of the AT1 receptor blockers studied, candesartan or losartan, whereas the AT2 receptor blocker PD123319 had no inhibitory effect, thus demonstrating that the response were mediated via AT1 receptors. The increased contractile responses to Ang II were significant 48 hours after the MCA occlusion. There were no increased contractile responses in the occluded arteries 24 hours after MCA occlusion, suggesting that the changes occurred between 24 and 48 hours after induction of ischemia. Interestingly, real-time PCR revealed a lower level of AT1 receptor mRNA in the occluded MCA compared with the nonoccluded MCA 48 hours after occlusion, whereas the level of ACE mRNA was higher on the ipsilateral side. The AT1 receptor and ACE mRNA levels did not differ between the occluded and nonoccluded MCA 24 hours after induction of ischemia.
Previous studies have shown that the AT1 receptor antagonist candesartan decreases the reduction in brain blood flow and the size of brain damage after cerebral ischemia in genetically hypertensive rats.14 However, in those studies, pretreatment with the drug for several days was necessary to achieve a neuroprotective effect. This phenomenon was considered due to inhibition of the hypertension-induced arterial remodeling and the normalization of cerebrovascular autoregulation, putatively linked to normalization of vascular endothelial nitric oxide synthase and inducible nitric oxide synthase levels.15 The same positive influence on cerebral arteries might explain the effects of the AT1 receptor inhibitors losartan and candesartan, which protect against stroke in hypertensive patients (the LIFE16 and SCOPE17 studies, respectively). On the other hand, a newly presented study (the ACCESS study18) has demonstrated that treatment with the AT1 receptor inhibitor candesartan in the acute phase of cerebral ischemia has a beneficial effect on vascular events (combination of cerebrovascular and cardiovascular events) during the subsequent 12 months. These results were blood pressure independent, thus suggesting that there might be additional explanations for the beneficial effects of AT1 receptor blockers.
Enhanced AT1 receptor–mediated responses in cerebral arteries after an ischemic event might be harmful if they imply reduced perfusion in the marginal zone of the infarct and impaired collateral blood flow. The contradictory fact that the AT1 receptor mRNA levels were lower in the occluded MCA compared with the nonoccluded MCA 48 hours after MCA occlusion might depend on an enhanced translation of mRNA or a posttranslational modification without a corresponding rise in transcription of the gene. The amount of NF-κB mRNA was not altered in the experiment, which further supports this hypothesis, because possible binding sites for this transcription factor are found in the AT1 receptor promoter7 (GenBank accession number S66402). The decreased AT1 receptor mRNA level might also be due to a negative-feedback situation, wherein enhanced levels of Ang II inhibit further transcription of the receptors. The rise in ACE mRNA levels 48 hours after MCA occlusion might be one explanation for the results of previous studies, which have demonstrated that ACE inhibitors improve neurologic outcome in normotensive rats19 and decrease the size of brain damage in genetically hypertensive rats20 after cerebral ischemia. In the study with normotensive rats, the ACE inhibitor was added 30 minutes before the ischemic period,19 which suggests that the effects are not due to long-term effects on the vessel wall of the cerebral arteries. In addition, a study in humans has shown that the ACE inhibitor ramipril reduces the incidence of fatal and nonfatal stroke in a group of patients with relatively normal blood pressure but at high risk of stroke (the HOPE study).21
In conclusion, the present study showed that focal cerebral ischemia induced in the rat increased contractile AT1 receptor–mediated responses in the ipsilateral MCA 48 hours after MCA occlusion, whereas the levels of AT1 receptor mRNA were lower in the same artery. The relative amount of ACE mRNA was higher in the occluded MCA, suggesting a locally enhanced production of Ang II after cerebral ischemia. The results provide a possible explanation of the beneficial effects of AT1 receptor blockers and ACE inhibitors in cerebral ischemia.
This study was supported by a grant from the Swedish Research Council (grant No. 5958) and the Swedish Heart Lung Foundation.
- Received June 30, 2003.
- Revision received November 27, 2003.
- Accepted December 29, 2003.
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