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The expression of angiotensin-converting enzyme 2–angiotensin-(1–7)–Mas receptor axis are upregulated after acute cerebral ischemic stroke in rats
Neuropeptides 47 (2013) 289–295
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The expression of angiotensin-converting enzyme 2–angiotensin-(1–7)–Mas receptor axis are upregulated after acute cerebral ischemic stroke in rats Jie Lu b, Teng Jiang a, Liang Wu a, Li Gao b, Yao Wang b, Feng Zhou a, Shugang Zhang b, Yingdong Zhang a,⇑ a b
Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing, PR China Department of Neurology, Nanjing Brain Hospital, Nanjing Medical University, Nanjing, PR China
a r t i c l e
i n f o
Article history: Received 1 May 2013 Accepted 9 September 2013 Available online 18 September 2013 Keywords: Ischemic cerebrovascular diseases Renin–Angiotensin system Angiotensin-(1–7) Angiotensin-converting enzyme 2 (ACE2) Mas
a b s t r a c t There is now unequivocal evidence that the angiotensin-converting enzyme 2(ACE2)–Ang-(1–7)–Mas axis is a key component of the renin–angiotensin system (RAS) cascade, which is closely correlated with ischemic insult occurrence. Our previous studies demonstrated that the Ang-(1–7), was an active member of the brain RAS. However, the ACE2–Ang-(1–7)–Mas axis expression after cerebral ischemic injury are currently unclear. In the present study, we investigated the time course of ACE2–Ang-(1–7) and Mas receptor expression in the acute stage of cerebral ischemic stroke. The content of Ang-(1–7) in ischemic tissues and blood serum was measured by specific EIA kits. Real-time PCR and western blot were used to determine messenger RNA (mRNA) and protein levels of the ACE2 and Mas. The cerebral ischemic lesion resulted in a significant increase of regional cerebral and circulating Ang-(1–7) at 6–48 h compared with sham operation group following focal ischemic stroke (12 h: 7.276 ± 0.320 ng/ml vs. 2.466 ± 0.410 ng/ml, serum; 1.024 ± 0.056 ng/mg vs. 0.499 ± 0.032, brain) (P < 0.05). Both ACE2 and Mas expression were markedly enhanced compared to the control in the ischemic tissues (P < 0.05). Mas immunopositive neurons were also seen stronger expression in the ischemic cortex (19.167 ± 2.858 vs. 7.833 ± 2.483) (P < 0.05). The evidence collected in our present study will indicate that, ACE2–Ang-(1–7)–Mas axis are upregulated after acute ischemic stroke and would play a pivotal role in the regulation of acute neuron injury in ischemic cerebrovascular diseases. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction Ischemic stroke, the third leading cause of death in the world, is a severe complication of hypertension and arteriosclerosis (Bos et al., 2012; Danielyan et al., 2012). High blood pressure is present in 70–80% of patients with acute ischemic stroke and is associated independently with a poor functional outcome (Olindo et al., 2003). Manipulation of the renin–angiotensin system has emerged as an effective strategy to prevent stroke in patients at risk. Angiotensin II (Ang II) was discovered as a circulating pro-hypertensive peptide. Circulating Ang II, through activation of its physiological AT1 receptors (AT1R) (Tan et al., 2007), was characterized as a major regulator of vascular tone and fluid metabolism. Activation of the RAS predisposes to atherosclerosis and thromboembolic events (Verdecchia et al., 2012) and is involved in ischemic brain injury. Ang II exerts blood vessel damaging actions which are mediated mainly by the Ang II AT1R. It is well-established that the ACE-Ang II-AT1R axis would exacerbate cerebral ischemic damage (Olindo et al., 2003; Tan et al., 2007; Verdecchia et al., 2012).
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However, there are additional pathways within the RAS that may functionally antagonize an activated ACE-Ang II-AT1R pathway (Fraga-Silva et al., 2012; Santos et al., 2012). Ang-(1–7) is another biologically active component of the RAS, of which the actions are often opposite to those attributed to Ang II. Ang II promotes cell proliferation, vasoconstriction and prothrombotic activity, whereas Ang-(1–7) has antiproliferative, vasodilator and antithrombotic actions, suggesting that it has great potential to treat cerebrovascular diseases (Verdecchia et al., 2012; Fraga-Silva et al., 2012; Santos et al., 2012). Indeed, we have previously shown that this heptapeptide exerts important beneficial effects in the brain after ischemic stroke, such as upregulating the endothelial NO synthase (eNOS) expression (Zhang et al., 2008), increasing bradykinin release (Lu et al., 2008), reducing oxidative stress and attenuating neuronal apoptosis (Jiang et al., 2012). These effects are generally mediated by the activation of the G protein–coupled receptor Mas, which was identified as an endogenous binding site for Ang-(1–7). Thus, Ang-(1–7), in conjunction with its receptor Mas and ACE2, the main enzyme involved in its formation, represents a neuroprotective axis within the RAS, of which its actions balance the ACE-Ang II-AT1R effects. Evidence suggests that the ACE2–Ang-(1–7)–Mas receptor axis opposes the local actions of
E-mail addresses: [email protected], [email protected] (Y. Zhang). 0143-4179/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.npep.2013.09.002
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the ACE-Ang II-AT1R axis in liver (Pate Bilman et al., 2012), heart (Li et al., 2012; Patel et al., 2012), and kidney (Ferrario and Varagic, 2010). The Ang-(1–7) Mas receptor is thought to counteract AT1Rmediated effects and to exert protective effects against ischemia (Iwai et al., 2012; Ferreira et al., 2001). Recent studies indicate that the ACE2–Ang-(1–7)–Mas receptor axis could be involved in cerebrovascular disease development and exert important neuroprotective effects (Zhang et al., 2008; Lu et al., 2008; Jiang et al., 2012; Mecca et al., 2011). However, no data to date are available about ACE2–Ang-(1–7)–Mas axis expression after cerebral ischemic damage. In the present study, we investigated the time course of the brain and circulating ACE2–Ang-(1–7) and Mas receptor expression in vivo in the acute stage of cerebral ischemic stroke in rats. 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (280–320 g) were purchased from National Rodent Laboratory Animal Resources Shanghai branch of China. The animals were housed in a standard animal room with a 12 h light/dark cycle and given free access to food and water. All experimental protocols were performed in accordance with the guidelines for the human use of laboratory animals of our Institute and approved by the Nanjing Medical University Experimental Animal Care. 2.2. pMCAO model Rats were subjected to middle cerebral artery occlusion (MCAO) by intraluminal MCAO as described previously (Jiang et al., 2012). In brief, they were anesthetized with 10% chloral hydrate (0.35 mL100 g1). A 3.0 cm length of monofilament nylon suture (U 0.26 mm), with its tip rounded by heating near a flame, was inserted from the right external carotid artery (ECA) into the lumen of internal carotid artery (ICA), then advanced until resistance was felt (1.8–2.0 cm from the bifurcation). The reduction of the middle cerebral artery blood flow was confirmed by a laser Doppler perfusion monitor (moor VMS-LDF-1; moor instruments Inc., UK) immediately after the occlusion and the filament remained there until the rat was sacrificed. Throughout the procedure, body temperature was maintained at 37 ± 0.5 °C with a thermostatically-controlled infrared lamp. The sham-operated rats were treated identically, except that the middle cerebral artery (MCAs) were not occluded after the neck incision. 2.3. Tissue collection Rats were sacrificed by decapitation at 6, 12, 24, and 48 h after focal ischemic stroke and samples of cerebral ischemic cortices and blood were collected, weighed and immediately frozen in liquid nitrogen, and stored at 80 °C for further analysis. The ischemic core and penumbra was dissected according to a well-established method (Jiang et al., 2012) in the rat model of MCAO. Serum was obtained after centrifugation (3200 rpm for 10 min at 4 °C), frozen, and stored at 80 °C until analysis. 2.4. Elisa We examined the levels of Ang-(1–7) in cerebral ischemic tissues and blood serum at 6, 12, 24, and 48 h (n = 6) after focal ischemic stroke by using a commercially available EIA kit(Peninsula Laboratories, San Carlos, CA). The rationale for time points was se-
lected based on the previous studies of our group (Lu et al., 2008; Jiang et al., 2012). Protein samples of cerebral ischemic cortices were prepared for assay using CytoBuster™ Protein Extraction Reagent (Novagen). The total protein concentration of each sample was analyzed using a modified Bradford assay. The supernatant was collected and stored at 80 °C until use. The levels of Ang(1–7) were measured according to the manufacturer’s instructions. All assays were done in duplicate. The average absorbance values (A650) for each set of reference standards, control and samples were calculated and a standard curve was constructed. Using the mean absorbance value for each sample, the corresponding concentration of it from the standard curve was determined. 2.5. RNA extraction and real-time PCR Total RNA extraction and cDNA synthesis were performed as described previously (Lu et al., 2008). Levels of ACE2 and Mas mRNA were determined by real-time quantitative PCR using a SYBRÒ Premix Ex Taq™ Kit (Takara, Dalian, China) according to the manufacturer’s instructions. The cDNA amplification of a specific sequence of rat ACE2, Mas or GAPDH was performed by PCR using the following primers: sense 50 -TTCTCAGCCTTGTTGCTG TTGCTA-30 and antisense 50 -TCACTGTTTTGGAATCCTGCGT-30 for ACE2, sense 50 -CCTGCATACTGGGAAGACCA-30 and antisense 50 TCCCTTCCTGTTTCTCATGG-30 for Mas,and sense 50 -GAAAAGCTG TGGCGTGAT-30 and antisense 50 -AAGGTGGAAGAATGGGAGTT-30 for rat GAPDH. The PCR reaction was conducted at 95 °C for 30 s and followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s in the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The qRT-PCR results were analyzed and expressed as relative mRNA expression of CT (threshold cycle) value, which was then converted to fold changes. Quantitative real-time RTPCR assay was performed to detect GAPDH expression that was used to normalize the amount of cDNA for each sample. 2.6. Western blotting The methods used for immunoblotting have been described previously (Lu et al., 2008). Extract proteins were subjected to 6% SDS–PAGE, and proteins were transferred to onto nitrocellulose membranes (Sigma) and incubated with primary rabbit polyclonal antibodies against Mas1 (1:200 dilution, Santa Cruz Biotechnology; USA), ACE2 (1:400 dilution, Santa Cruz Biotechnology; USA) and bactin (internal control) (1:1000 dilution, Cell Signaling Beverly; USA) overnight at 4 °C, respectively. Goat anti-rabbit IgG conjugated with peroxidase (1:5000) was used as a secondary antibody. Membranes were incubated with secondary antibodies for 2 h at room temperature and were then revealed using ECL-plus Western blotting detection reagent. Band intensities were quantified by densitometric scanning with the Multi-Analyst software (Bio-Rad). 2.7. Immunohistochemistry Immunohistochemistry was carried out as previously described (Jiang et al., 2012) with some modifications to determine the brain localization of Mas receptors. Rats were euthanized at 24 h after pMCAO and perfused transcardially with 0.9% saline (pH 7.4) followed by a fixative solution containing 4% paraformaldehyde in 0.1 M PBS (pH 7.4), then the brains were removed, fixed for 12 h at 4 °C and embedded in paraffin. The paraffin-embedded sections (6 lm) received deparaffinization and rehydration treatments while endogenous peroxidase activity was blocked with 3% H2O2 for 30 min. The sections were blocked with 5% normal goat serum for 30 min, incubated with the primary antibody (1:100; anti-Mas1 receptor rabbit polyclonal antibodies; Santa Cruz Biotechnology) overnight at 4 °C and then treated with biotinylated goat anti-
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rabbit IgG (1:400 dilution; Zhongshan Inc., China) at room temperature for 1 h. Immunoreactivity was tested with the avidinbiotinperoxidase technique, using diaminobenzidine (DAB) as the chromogen. Sections were dried, dehydrated in graded alcohols and mounted on the slides. Quantification of the staining was done on a microscopy equipped with a charge-coupled device (CCD) camera. Cell counting was performed on five randomly selected non-overlapping fields in ischemic penumbra region per slide by 3 independent observers who were blinded to the experimental groups. Data obtained in every field were added together to make a final data for each slide and expressed as percentage of total cell number. 2.8. Statistical analysis Data were represented as mean ± S.D. (standard deviation) and were examined for the homogeneity of variance. Student’s t-test (two-tailed) was performed to analyze the data using SPSS 16.0 software. For real-time PCR, the CT value of the target gene of a sample was first corrected for the CT value of GAPDH, before being statistically analyzed. P < 0.05 was considered statistically significant. 3. Results 3.1. The Ang-(1–7) concentration in serum and cerebral ischemic tissues
Fig. 1. Ang-(1–7) concentrations were measured in sera(A) and supernatants of brain homogenate (B) by using specific ELISA kits. The bars illustrate mean ± S.D. data from 6 independent experiments. Cerebral infarction resulted in a significant increase of Ang-(1–7) at 6, 12, 24 and 48 h after pMCAO compared with shamoperated group in rats. Ang-(1–7) formation reached a maximum of 12 h after cerebral ischemic stroke. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated at each time point.
In order to investigate the changes of Ang-(1–7), its levels in the rat blood serum and ischemic brain cortex were measured by ELISA at 6, 12, 24, and 48 h after ischemic stroke. Cerebral infarction resulted in a significant increase of Ang-(1–7) compared with shamoperated group in rats (P < 0.05). Ang-(1–7) formation reached a maximum of 12 h (7.276 ± 0.320 ng/ml, serum; 1.024 ± 0.056 ng/ mg, brain) and were markedly elevated by 2.951-fold and 2.052fold compared with sham-operated group (2.466 ± 0.410 ng/ml, serum; 0.499 ± 0.032 ng/mg, brain) (n = 6, P < 0.05), respectively, after pMCAO (Fig. 1). 3.2. Real-time analysis for the Mas1 and ACE2 mRNA expression Fig. 2 shows a representative qRT-PCR analysis of the Mas1 and ACE2 mRNA expression in the ischemic brain cortex after ischemic stroke at each time point, respectively. The cDNA for GAPDH was used as an internal control. Data are presented as fold-changes in the pMCAO groups relative to Sham groups (mean ± S.D.). The Mas1 mRNA expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, 24, and 48 h after pMCAO (P < 0.05). The Mas1 receptors mRNA expression reached a maximum of 24 h (5.828 ± 0.370) and were markedly elevated by 5.828-fold compared with sham-operated group (n = 6, P < 0.05), respectively, after pMCAO (Fig. 2(A)). The ACE2 mRNA showed increased expression in the groups of pMCAO in rats at 6, 12, and 24 h after cerebral ischemic stroke compared with the sham-operated groups. The ACE2 mRNA expression reached a maximum of 12 h (5.560 ± 0.786) and were markedly elevated by 5.560-fold compared with sham-operated group (n = 6, P < 0.05), respectively, after pMCAO. There was no significant difference at 48 h after cerebral ischemic stroke (P > 0.05) (Fig. 2(B)). 3.3. Western blot analysis for the Mas1 and ACE2 protein expression Fig. 3 shows a representative western blot analysis of the Mas1 and ACE2 protein expression in the ischemic brain cortex after ischemic stroke at each time point, respectively. Protein b-actin
Fig. 2. (A) Real-time PCR analysis of Mas1 and GAPDH mRNA in the ischemic core and penumbra of the pMCAO and Sham rats. Gene GAPDH was used as an internal control. Data are presented as fold-changes in the pMCAO groups relative to Sham groups (mean ± S.D.). The Mas1 mRNA expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, 24, and 48 h after pMCAO. Mas1 receptors mRNA expression reached a maximum of 24 h after cerebral ischemic stroke. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated. (B) Realtime PCR analysis of ACE2 and GAPDH mRNA in the ischemic core and penumbra of the pMCAO and Sham rats. Gene GAPDH was used as an internal control. Data are presented as fold-changes in the pMCAO groups relative to Sham groups (mean ± S.D.). The ACE2 mRNA expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, and 24 h after pMCAO. The ACE2 mRNA expression reached a maximum of 12 h after cerebral ischemic stroke. There was no significant difference at 48 h after stroke. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated.
was adopted as internal control. The Mas1 protein expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, 24, and 48 h after pMCAO. The Mas1 protein expression reached a maximum of 24 h (0.660 ± 0.033) and were markedly elevated by 2.933-fold compared with sham-operated group (0.225 ± 0.030) (n = 6, P < 0.05), respectively, after pMCAO (Fig. 3(A)). The ACE2 protein showed increased expression
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Fig. 3. (A) Representative Western blot analysis of Mas1 and b-actin protein in the ischemic core and penumbra of cerebral ischemic stroke (pMCAO) rats and sham rats at each time point. Quantification of Mas1 receptor protein after the expression levels were normalized to b-actin. The Mas1 protein expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, 24, and 48 h after pMCAO. The Mas1 protein expression reached a maximum of 24 h after cerebral ischemic stroke. All results are given as mean ± S.D., n = 6 in each group. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated. (B) Representative Western blot analysis of ACE2 and b-actin protein in the ischemic core and penumbra of cerebral ischemic stroke (pMCAO) rats and control rats at each time point. Quantification of ACE2 protein after the expression levels were normalized to b-actin. The ACE2 protein expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, and 24 h after pMCAO. The ACE2 protein expression reached a maximum of 12 h after cerebral ischemic stroke. There was no significant difference at 48 h after stroke. All results are given as mean ± S.D., n = 6 in each group. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated.
in the groups of pMCAO in rats at 6, 12, and 24 h after cerebral ischemic stroke compared with the sham-operated groups. The ACE2 protein expression reached a maximum of 12 h (0.853 ± 0.061) and were markedly elevated by 2.057-fold and 2.204-fold compared with sham-operated group (0.387 ± 0.055)
(n = 6, P < 0.05), respectively, after pMCAO. There was no significant difference at 48 h after cerebral ischemic stroke (P > 0.05) (Fig. 3(B)). These data showed that the expression of both the ACE2 and Mas1 was induced by acute cerebral ischemia injury.
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Fig. 4. (A) Representative images showing the immunohistochemical staining of Mas1 receptor in cerebral cortices of pMCAO and Sham rats at 6, 12, 24, and 48 h after stroke. Images are shown at 400 magnification; red arrows indicate positive staining in cerebral tissues. (B) Representative images and bar charts showing the quantification of Mas receptor in brain are shown for each group. The Mas1 receptor expression showed a significant increase in pMCAO groups (19.167 ± 2.858) compared with sham-operated group (7.833 ± 2.483) (P < 0.05). The Mas1 receptor expression showed a significant increase in 24 h after pMCAO compared with 48 h after pMCAO group (P < 0.016). There was no significant difference between other time point in pMCAO group (P > 0.05). Data are shown as mean ± S.D., n = 6 in each group. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated.
3.4. Immunodetection of the Mas1 receptor expression Fig. 4 shows representative images showing the immunohistochemical staining of Mas1 receptor in cerebral cortices of pMCAO and Sham rats at 24 h after stroke. Images are shown at 400 magnification; red arrows indicate positive staining in cerebral tissues. Representative images and bar charts showing the quantification of Mas receptor in brain are shown for each group. The Mas1 receptor expression showed a significant increase in pMCAO groups (19.167 ± 2.858) compared with sham-operated group (7.833 ± 2.483) (n = 6, P < 0.05) (Fig. 4). 4. Discussion Blood pressure (BP) is the major risk factor for ischemic stroke (Ovbiagele et al., 2011) with a continuous association between both systolic and diastolic blood pressures. It has been revealed that antihypertensive treatment is the most important therapy to reduce the risk of stroke by approximately 30–40% (Olindo et al., 2003; Verdecchia et al., 2012; Ovbiagele et al., 2011). All stroke patients and patients with transient ischemic attack (TIA) have to be regarded as very high-risk patients. Hypertension increases the risk of recurrent strokes. During the last decade research has shown that reducing the activity of the ACE-Ang II-AT1R axis may have beneficial effects beyond the lowering of blood pressure (Olindo et al., 2003; Verdecchia et al., 2012; Ovbiagele et al., 2011). There is growing evidence of cerebroprotective effects for medica-
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tion influencing the RAS, such as angiotensin receptor antagonists (ARB) or ACE inhibitors (ACEI) (Nguyen Dinh Cat and Touyz, 2011). Both ACEI and ARB could increase the plasma levels of Ang-(1–7), suggesting that part of the beneficial effects of them could be mediated by Ang-(1–7) (Ferrario et al., 1997; Ferreira and Santos, 2005; Trask and Ferrario, 2007). Renin converts angiotensinogen into Angiotensin I (Ang I), and ACE converts Ang I into Ang II. ACE2, a homologue of ACE, catalyzes the conversion of Ang II into the Ang-(1–7) peptide (Ferreira et al., 2012) that binds to the Mas receptor to stimulate vasodilation and plays an important role in controlling cardiovascular function (Ferreira et al., 2012; Ye et al., 2012; Xiao et al., 2011; Zheng et al., 2011). ACE2 is expressed in many tissues, including the brain, heart, kidney, liver, lung, and pancreas (Ferreira et al., 2012; Ye et al., 2012; Xiao et al., 2011; Zheng et al., 2011; Silva et al., 2012; Shahid et al., 20112; Wang et al., 2012). The brain possesses the same RAS as the systemic circulation. Recent experimental studies have shown that the brain RAS plays an important role in stroke (Arnold et al., 2012). Ang II is a key mediator in the mechanism of hypertension and plays a pathophysiological role for the development of ischemic stroke. In addition to Ang II, Ang-(1–7) may also have important biological activities in the brain (Nautiyal et al., 2012; Cerrato et al., 2012; Freund et al., 2012; Cheng et al., 2012). A growing body of evidence suggests that the ACE2–Ang-(1–7)–Mas receptor axis opposes the actions of the ACE-Ang II-AT1R system in many tissues, including the heart, liver, and kidney (Pate Bilman et al., 2012; Li et al., 2012; Patel et al., 2012; Ferrario and Varagic, 2010). Previous work suggested that Ang-(1–7) exert its stroke-protective effects through stimulation of Mas receptor (Jiang et al., 2012). The presence of ACE2 in brain regions involved in the central regulation of cardiovascular function, suggests that ACE2 is part of the brain RAS. Indeed, evidence show that ACE2 interacts with RAS components (Xiao et al., 2011; Zheng et al., 2011; Silva et al., 2012) in the central nervous system (CNS). Overexpression of ACE2 in the brain decreases BP (Yamazato et al., 2007) and prevents the Ang II-mediated development of hypertension (Sriramula et al., 2011). In our previous studies in rats demonstrated that the Ang-(1–7), was an active member of the brain RAS (Zhang et al., 2008; Lu et al., 2008; Jiang et al., 2012). However, the ACE2–Ang-(1–7)–Mas axis expression after cerebral ischemic injury are currently unclear. This study characterized the time course of Ang-(1–7), ACE2 and Mas expression in cerebral tissues and blood serum within 48 h of stroke onset. In the present work, we found that cerebral ischemic injury resulted in a significant increase of cerebral and circulating Ang-(1–7) at 6–48 h following focal ischemic stroke compared to the control. Both Mas1 and ACE2 expression in the ischemic tissues and blood serum were markedly enhanced as a result of the acute ischemic insult. The Mas immunopositive neurons were also seen stronger expression in the ischemic cortex. Our findings indicate that regional cerebral and circulating levels of Ang-(1–7), ACE2, and Mas receptor are upregulated after acute cerebral ischemic stroke in rats. The beneficial effects of ARB and ACEI are not only limited to reduction of vasoconstriction, but also include a significant decrease in vascular and endorgan inflammation (Dendorfer et al., 2005), the result of excessive AT1 receptor activation and a major participant in the pathogenesis of hypertension. The ARB and ACEI have been showed to upregulate of the ACE2–Ang-(1–7)–Mas axis expression during treatments, in hypertensive rats and normotensive rats (Ferrario et al., 1997; Ferreira and Santos, 2005; Trask and Ferrario, 2007). Elsewhere we reported that, in male rats, the Ang(1–7) exerts neuroprotective effects against cerebral ischemia (Jiang et al., 2012); it upregulates eNOS (Zhang et al., 2008) and potentiates bradykinin (Lu et al., 2008). The protective effect of ACE2–Ang-(1–7)–Mas pathway on ischemic stroke has been re-
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lated to blood pressure control (Nautiyal et al., 2012) and improvement of cerebral blood supply (Iyer et al., 1998). However, the benefits can go beyond those. The activation of the Mas receptor for Ang-(1–7) inhibits Ang II-induced vasoconstriction in human internal mammary arteries and forearm resistance vessels (Roks et al., 1999). Other studies suggest that Mas receptor activation by Ang-(1–7) improves endothelial function by activating eNOS and facilitating NO release (Zhang et al., 2008; Cerrato et al., 2012; Cheng et al., 2012). The orally active Mas receptor agonist AVE0991 exerts effects similar to Ang-(1–7) (Ferreira et al., 2007), suggesting that drugs that activate the Mas receptor may have a benefit as cardiovascular therapeutic agents. ACE2–Ang-(1–7)–Mas axis plays a broad role in vascular regulation (Dantas and Sandberg, 2005), inflammation (El-Hashim et al., 2012), oxidative stress (Raffai et al., 2011), and apoptosis (Jiang et al., 2012). ACE2–Ang-(1–7)–Mas axis could exert its stroke-protective effects through stimulation of Mas receptors. We show for the first time that ACE2–Ang-(1–7)–Mas axis expression was upregulated in vivo following acute ischemic stroke in rats. The cerebral acute ischemic neuron injury is associated with upregulation of the ACE2–Ang-(1–7)–Mas axis and promotes increased circulating Ang-(1–7). These results suggest that the ACE2–Ang-(1– 7)–Mas pathway located in the neuron is involved in ischemic damage. The evidence collected so far indicate that, ACE2–Ang(1–7)–Mas receptor axis should be viewed as a promising therapeutic tool for the treatment of cerebrovascular diseases. Further insights in this regard would be obtained by studies aimed to clarify the significant variation in prevention and functional outcome after ischemic stroke. Conflict of interest None. Acknowledgments This work was supported in part by grants from the Medical Science and Technology Foundation for Young Scientists of Nanjing City (Grant No. QYK09186), Science and Technology Foundation of Nanjing Medical University (Grant No. 09NJMUMl25) and the Natural Science Foundation of Jiangsu Province (Grant No. BK2010116). References Arnold, A.C., Gallagher, P.E., Diz, D.I., 2012. Brain renin–angiotensin system in the nexus of hypertension and aging. Hypertens. Res. 36, 5–13. Bos, D., van der Rijk, M.J., Geeraedts, T.E., Hofman, A., Krestin, G.P., Witteman, J.C., van der Lugt, A., Ikram, M.A., Vernooij, M.W., 2012. Intracranial carotid artery atherosclerosis: prevalence and risk factors in the general population. Stroke 43, 1878–1884. Cerrato, B.D., Frasch, A.P., Nakagawa, P., Longo-Carbajosa, N., Peña, C., Höcht, C., Gironacci, M.M., 2012. Angiotensin-(1–7) upregulates central nitric oxide synthase in spontaneously hypertensive rats. Brain Res. 1453, 1–7. Cheng, W.H., Lu, P.J., Hsiao, M., Hsiao, C.H., Ho, W.Y., Cheng, P.W., Lin, C.T., Hong, L.Z., Tseng, C.J., 2012. Renin activates PI3K-Akt-eNOS signalling through the angiotensin AT1 and Mas receptors to modulate central blood pressure control in the nucleus tractus solitarii. Br. J. Pharmacol. 166, 2024–2035. Danielyan, K.E., Oganesyn, H.M., Nahapetyan, K.M., Kevorkian, G.A., Vladimir, D., Galoyan, A.A., Grigorian, G.S., 2012. Stroke burden in adults in Armenia. Int. J. Stroke 7, 248–249. Dantas, A.P., Sandberg, K., 2005. Regulation of ACE2 and ANG-(1–7) in the aorta: new insights into the renin–angiotensin system in the control of vascular function. Am. J. Physiol. Heart Circ. Physiol. 289, H980–981. Dendorfer, A., Dominiak, P., Schunkert, H., 2005. ACE inhibitors and angiotensin II receptor antagonists. Handb. Exp. Pharmacol. 170, 407–442. El-Hashim, A.Z., Renno, W.M., Raghupathy, R., Abduo, H.T., Akhtar, S., Benter, I.F., 2012. Angiotensin-(1–7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-jB-dependent pathways. Br. J. Pharmacol. 166, 1964–1976. Ferrario, C.M., Chappell, M.C., Tallant, E.A., Brosnihan, K.B., Diz, D.I., 1997. Counterregulatory actions of angiotensin-(1–7). Hypertension 30, 535–541.
Ferrario, C.M., Varagic, J., 2010. The ANG-(1–7)/ACE2/Mas axis in the regulation of nephron function. Am. J. Physiol. Renal Physiol. 298, F1297–1305. Ferreira, A.J., Santos, R.A., Almeida, A.P., 2001. Angiotensin-(1–7): cardioprotective effect in myocardial ischemia/reperfusion. Hypertension 38, 665–668. Ferreira, A.J., Santos, R.A., 2005. Cardiovascular actions of angiotensin-(1–7). Braz. J. Med. Biol. Res. 38, 499–507. Ferreira, A.J., Jacoby, B.A., Araújo, C.A., Macedo, F.A., Silva, G.A., Almeida, A.P., Caliari, M.V., Santos, R.A., 2007. The nonpeptide angiotensin-(1–7) receptor Mas agonist AVE-0991 attenuates heart failure induced by myocardial infarction. Am. J. Physiol. Heart Circ. Physiol. 292, H1113–1119. Ferreira, A.J., Bader, M., Santos, R.A., 2012. Therapeutic targeting of the angiotensinconverting enzyme 2/angiotensin-(1–7)/Mas cascade in the renin–angiotensin system: a patent review. Expert Opin. Ther. Pat. 22, 567–574. Fraga-Silva, R.A., Da Silva, D.G., Montecucco, F., Mach, F., Stergiopulos, N., da Silva, R.F., Santos, R.A., 2012. The angiotensin-converting enzyme 2/angiotensin-(1– 7)/Mas receptor axis: a potential target for treating thrombotic diseases. Thromb. Haemost. 108, 1089–1096. Freund, M., Walther, T., von Bohlen und Halbach, O., 2012. Immunohistochemical localization of the angiotensin-(1–7) receptor Mas in the murine forebrain. Cell Tissue Res. 348, 29–35. Iwai, M., Nakaoka, H., Senba, I., Kanno, H., Moritani, T., Horiuchi, M., 2012. Possible involvement of angiotensin-converting enzyme 2 and Mas activation in inhibitory effects of angiotensin II Type 1 receptor blockade on vascular remodeling. Hypertension 60, 137–144. Iyer, S.N., Chappell, M.C., Averill, D.B., Diz, D.I., Ferrario, C.M., 1998. Vasodepressor actions of angiotensin-(1–7) unmasked during combined treatment with lisinopril and losartan. Hypertension 31, 699–705. Jiang, T., Gao, L., Shi, J., Lu, J., Wang, Y., Zhang, Y., 2012a. Angiotensin-(1–7) modulates renin–angiotensin system associated with reducing oxidative stress and attenuating neuronal apoptosis in the brain of hypertensive rats. Pharmacol. Res. 67, 84–93. Jiang, T., Gao, L., Guo, J., Lu, J., Wang, Y., Zhang, Y., 2012b. Suppressing inflammation by inhibiting the NF-jB pathway contributes to the neuroprotective effect of angiotensin-(1–7) in rats with permanent cerebral ischaemia. Br. J. Pharmacol. 67, 1520–1532. Li, P., Zhang, F., Zhou, Y.B., Cui, B.P., Han, Y., 2012. Superoxide anions modulate the effects of angiotensin-(1–7) in the rostral ventrolateral medulla on cardiac sympathetic afferent reflex and sympathetic activity in rats. Neuroscience 223, 388–398. Lu, J., Zhang, Y., Shi, J., 2008. Effects of intracerebroventricular infusion of angiotensin-(1–7) on bradykinin formation and the kinin receptor expression after focal cerebral ischemia-reperfusion in rats. Brain Res. 1219, 127–135. Mecca, A.P., Regenhardt, R.W., O’Connor, T.E., Joseph, J.P., Raizada, M.K., Katovich, M.J., Sumners, C., 2011. Cerebroprotection by angiotensin-(1–7) in endothelin1-induced ischaemic stroke. Exp. Physiol. 96, 1084–1096. Nautiyal, M., Shaltout, H.A., de Lima, D.C., do Nascimento, K., Chappell, M.C., Diz, D.I., 2012. Central angiotensin-(1–7) improves vagal function independent of blood pressure in hypertensive (mRen2)27 rats. Hypertension 60, 1257–1265. Nguyen Dinh Cat, A., Touyz, R.M., 2011. A new look at the renin–angiotensin system – focusing on the vascular system. Peptides 32, 2141–2150. Olindo, S., Cabre, P., Deschamps, R., Chatot-Henry, C., René-Corail, P., Fournerie, P., Saint-Vil, M., May, F., Smadja, D., 2003. Acute stroke in the very elderly: epidemiological features, stroke subtypes, management, and outcome in Martinique, French West Indies. Stroke 34, 1593–1597. Ovbiagele, B., Diener, H.C., Yusuf, S., Martin, R.H., Cotton, D., Vinisko, R., Donnan, G.A., Bath, P.M., 2011. PROFESS investigators (2011) level of systolic blood pressure within the normal range and risk of recurrent stroke. JAMA 306, 2137– 2144. Pate Bilman, V., Mares-Guia, L., Nadu, A.P., Bader, M., Campagnole-Santos, M.J., Santos, R.A., Santos, S.H., 2012. Decreased hepatic gluconeogenesis in transgenic rats with increased circulating angiotensin-(1–7). Peptides 37, 247–251. Patel, V.B., Bodiga, S., Fan, D., Das, S.K., Wang, Z., Wang, W., Basu, R., Zhong, J., Kassiri, Z., Oudit, G.Y., 2012. Cardioprotective effects mediated by angiotensin II type 1 receptor blockade and enhancing angiotensin 1–7 in experimental heart failure in angiotensin-converting enzyme 2-null mice. Hypertension 59 (6), 1195–1203. Raffai, G., Durand, M.J., Lombard, J.H., 2011. Acute and chronic angiotensin-(1–7) restores vasodilation and reduces oxidative stress in mesenteric arteries of saltfed rats. Am. J. Physiol. Heart Circ. Physiol. 301, H1341–1352. Roks, A.J., Van Geel, P.P., Pinto, Y.M., Buikema, H., Henning, R.H., De Zeeuw, D., Van Gilst, W.H., 1999. Angiotensin-(1–7) is a modulator of the human renin– angiotensin system. Hypertension 34, 296–301. Santos, R.A., Ferreira, A.J., Verano-Braga, T., Bader, M., 2012. Angiotensin-converting enzyme 2, angiotensin-(1–7) and Mas: new players of the renin angiotensin system. J. Endocrinol. 216, R1–R17. Shahid, M., 2011. The angiotensin-converting enzyme 2-angiotensin-(1–7) axis: the other side of the renin–angiotensin system. Exp. Physiol. 96, 987–988. Silva, R.A., Chu, Y., Miller, J.D., Mitchell, I.J., Penninger, J.M., Faraci, F.M., Heistad, D.D., 2012. Impact of ACE2 deficiency and oxidative stress on cerebrovascular function with aging. Stroke 43, 3358–3363. Sriramula, S., Cardinale, J.P., Lazartigues, E., Francis, J., 2011. ACE2 overexpression in the paraventricular nucleus attenuates angiotensin II-induced hypertension. Cardiovasc. Res. 92, 401–408. Tan, P.S., Killinger, S., Horiuchi, J., Dampney, R.A., 2007. Baroreceptor reflex modulation by circulating angiotensin II is mediated by AT1 receptors in the
J. Lu et al. / Neuropeptides 47 (2013) 289–295 nucleus tractus solitarius. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R2267–R2278. Trask, A.J., Ferrario, C.M., 2007. Angiotensin-(1–7): pharmacology and new perspectives in cardiovascular treatments. Cardiovasc. Drug Rev. 25, 162–174. Verdecchia, P., Gentile, G., Angeli, F., Reboldi, G., 2012. Beyond blood pressure: evidence for cardiovascular, cerebrovascular, and renal protective effects of renin–angiotensin system blockers. Ther. Adv. Cardiovasc. Dis. 6, 81–91. Wang, Y., Wang, J., Liu, R., Qi, H., Wen, Y., Sun, F., Yin, C., 2012. Severe acute pancreatitis is associated with upregulation of the ACE2–angiotensin-(1–7)– Mas axis and promotes increased circulating angiotensin-(1–7). Pancreatology 12, 451–457. Xiao, L., Gao, L., Lazartigues, E., Zucker, I.H., 2011. Brain-selective overexpression of angiotensin-converting enzyme 2 attenuates sympathetic nerve activity and enhances baroreflex function in chronic heart failure. Hypertension 58, 1057–1065. Yamazato, M., Yamazato, Y., Sun, C., Diez-Freire, C., Raizada, M.K., 2007. Overexpression of angiotensin-converting enzyme 2 in the rostral
295
ventrolateral medulla causes long-term decrease in blood pressure in the spontaneously hypertensive rats. Hypertension 49 (4), 926–931. Ye, M., Wysocki, J., Gonzalez-Pacheco, F.R., Salem, M., Evora, K., Garcia-Halpin, L., Poglitsch, M., Schuster, M., Batlle, D., 2012. Murine recombinant angiotensinconverting enzyme 2: effect on angiotensin II-dependent hypertension and distinctive angiotensin-converting enzyme 2 inhibitor characteristics on rodent and human angiotensin-converting enzyme 2. Hypertension 60, 730– 740. Zhang, Y., Lu, J., Shi, J., Lin, X., Dong, J., Zhang, S., Liu, Y., Tong, Q., 2008. Central administration of angiotensin-(1–7) stimulates nitric oxide release and upregulates the endothelial nitric oxide synthase expression following focal cerebral ischemia/reperfusion in rats. Neuropeptides 42, 593–600. Zheng, H., Liu, X., Patel, K.P., 2011. Angiotensin-converting enzyme 2 overexpression improves central nitric oxide-mediated sympathetic outflow in chronic heart failure. Am. J. Physiol. Heart Circ. Physiol. 301, H2402–H2412.
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Neuropeptides journal homepage: www.elsevier.com/locate/npep
The expression of angiotensin-converting enzyme 2–angiotensin-(1–7)–Mas receptor axis are upregulated after acute cerebral ischemic stroke in rats Jie Lu b, Teng Jiang a, Liang Wu a, Li Gao b, Yao Wang b, Feng Zhou a, Shugang Zhang b, Yingdong Zhang a,⇑ a b
Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing, PR China Department of Neurology, Nanjing Brain Hospital, Nanjing Medical University, Nanjing, PR China
a r t i c l e
i n f o
Article history: Received 1 May 2013 Accepted 9 September 2013 Available online 18 September 2013 Keywords: Ischemic cerebrovascular diseases Renin–Angiotensin system Angiotensin-(1–7) Angiotensin-converting enzyme 2 (ACE2) Mas
a b s t r a c t There is now unequivocal evidence that the angiotensin-converting enzyme 2(ACE2)–Ang-(1–7)–Mas axis is a key component of the renin–angiotensin system (RAS) cascade, which is closely correlated with ischemic insult occurrence. Our previous studies demonstrated that the Ang-(1–7), was an active member of the brain RAS. However, the ACE2–Ang-(1–7)–Mas axis expression after cerebral ischemic injury are currently unclear. In the present study, we investigated the time course of ACE2–Ang-(1–7) and Mas receptor expression in the acute stage of cerebral ischemic stroke. The content of Ang-(1–7) in ischemic tissues and blood serum was measured by specific EIA kits. Real-time PCR and western blot were used to determine messenger RNA (mRNA) and protein levels of the ACE2 and Mas. The cerebral ischemic lesion resulted in a significant increase of regional cerebral and circulating Ang-(1–7) at 6–48 h compared with sham operation group following focal ischemic stroke (12 h: 7.276 ± 0.320 ng/ml vs. 2.466 ± 0.410 ng/ml, serum; 1.024 ± 0.056 ng/mg vs. 0.499 ± 0.032, brain) (P < 0.05). Both ACE2 and Mas expression were markedly enhanced compared to the control in the ischemic tissues (P < 0.05). Mas immunopositive neurons were also seen stronger expression in the ischemic cortex (19.167 ± 2.858 vs. 7.833 ± 2.483) (P < 0.05). The evidence collected in our present study will indicate that, ACE2–Ang-(1–7)–Mas axis are upregulated after acute ischemic stroke and would play a pivotal role in the regulation of acute neuron injury in ischemic cerebrovascular diseases. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction Ischemic stroke, the third leading cause of death in the world, is a severe complication of hypertension and arteriosclerosis (Bos et al., 2012; Danielyan et al., 2012). High blood pressure is present in 70–80% of patients with acute ischemic stroke and is associated independently with a poor functional outcome (Olindo et al., 2003). Manipulation of the renin–angiotensin system has emerged as an effective strategy to prevent stroke in patients at risk. Angiotensin II (Ang II) was discovered as a circulating pro-hypertensive peptide. Circulating Ang II, through activation of its physiological AT1 receptors (AT1R) (Tan et al., 2007), was characterized as a major regulator of vascular tone and fluid metabolism. Activation of the RAS predisposes to atherosclerosis and thromboembolic events (Verdecchia et al., 2012) and is involved in ischemic brain injury. Ang II exerts blood vessel damaging actions which are mediated mainly by the Ang II AT1R. It is well-established that the ACE-Ang II-AT1R axis would exacerbate cerebral ischemic damage (Olindo et al., 2003; Tan et al., 2007; Verdecchia et al., 2012).
⇑ Corresponding author. Tel.: +86 02552271000.
However, there are additional pathways within the RAS that may functionally antagonize an activated ACE-Ang II-AT1R pathway (Fraga-Silva et al., 2012; Santos et al., 2012). Ang-(1–7) is another biologically active component of the RAS, of which the actions are often opposite to those attributed to Ang II. Ang II promotes cell proliferation, vasoconstriction and prothrombotic activity, whereas Ang-(1–7) has antiproliferative, vasodilator and antithrombotic actions, suggesting that it has great potential to treat cerebrovascular diseases (Verdecchia et al., 2012; Fraga-Silva et al., 2012; Santos et al., 2012). Indeed, we have previously shown that this heptapeptide exerts important beneficial effects in the brain after ischemic stroke, such as upregulating the endothelial NO synthase (eNOS) expression (Zhang et al., 2008), increasing bradykinin release (Lu et al., 2008), reducing oxidative stress and attenuating neuronal apoptosis (Jiang et al., 2012). These effects are generally mediated by the activation of the G protein–coupled receptor Mas, which was identified as an endogenous binding site for Ang-(1–7). Thus, Ang-(1–7), in conjunction with its receptor Mas and ACE2, the main enzyme involved in its formation, represents a neuroprotective axis within the RAS, of which its actions balance the ACE-Ang II-AT1R effects. Evidence suggests that the ACE2–Ang-(1–7)–Mas receptor axis opposes the local actions of
E-mail addresses: [email protected], [email protected] (Y. Zhang). 0143-4179/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.npep.2013.09.002
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the ACE-Ang II-AT1R axis in liver (Pate Bilman et al., 2012), heart (Li et al., 2012; Patel et al., 2012), and kidney (Ferrario and Varagic, 2010). The Ang-(1–7) Mas receptor is thought to counteract AT1Rmediated effects and to exert protective effects against ischemia (Iwai et al., 2012; Ferreira et al., 2001). Recent studies indicate that the ACE2–Ang-(1–7)–Mas receptor axis could be involved in cerebrovascular disease development and exert important neuroprotective effects (Zhang et al., 2008; Lu et al., 2008; Jiang et al., 2012; Mecca et al., 2011). However, no data to date are available about ACE2–Ang-(1–7)–Mas axis expression after cerebral ischemic damage. In the present study, we investigated the time course of the brain and circulating ACE2–Ang-(1–7) and Mas receptor expression in vivo in the acute stage of cerebral ischemic stroke in rats. 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (280–320 g) were purchased from National Rodent Laboratory Animal Resources Shanghai branch of China. The animals were housed in a standard animal room with a 12 h light/dark cycle and given free access to food and water. All experimental protocols were performed in accordance with the guidelines for the human use of laboratory animals of our Institute and approved by the Nanjing Medical University Experimental Animal Care. 2.2. pMCAO model Rats were subjected to middle cerebral artery occlusion (MCAO) by intraluminal MCAO as described previously (Jiang et al., 2012). In brief, they were anesthetized with 10% chloral hydrate (0.35 mL100 g1). A 3.0 cm length of monofilament nylon suture (U 0.26 mm), with its tip rounded by heating near a flame, was inserted from the right external carotid artery (ECA) into the lumen of internal carotid artery (ICA), then advanced until resistance was felt (1.8–2.0 cm from the bifurcation). The reduction of the middle cerebral artery blood flow was confirmed by a laser Doppler perfusion monitor (moor VMS-LDF-1; moor instruments Inc., UK) immediately after the occlusion and the filament remained there until the rat was sacrificed. Throughout the procedure, body temperature was maintained at 37 ± 0.5 °C with a thermostatically-controlled infrared lamp. The sham-operated rats were treated identically, except that the middle cerebral artery (MCAs) were not occluded after the neck incision. 2.3. Tissue collection Rats were sacrificed by decapitation at 6, 12, 24, and 48 h after focal ischemic stroke and samples of cerebral ischemic cortices and blood were collected, weighed and immediately frozen in liquid nitrogen, and stored at 80 °C for further analysis. The ischemic core and penumbra was dissected according to a well-established method (Jiang et al., 2012) in the rat model of MCAO. Serum was obtained after centrifugation (3200 rpm for 10 min at 4 °C), frozen, and stored at 80 °C until analysis. 2.4. Elisa We examined the levels of Ang-(1–7) in cerebral ischemic tissues and blood serum at 6, 12, 24, and 48 h (n = 6) after focal ischemic stroke by using a commercially available EIA kit(Peninsula Laboratories, San Carlos, CA). The rationale for time points was se-
lected based on the previous studies of our group (Lu et al., 2008; Jiang et al., 2012). Protein samples of cerebral ischemic cortices were prepared for assay using CytoBuster™ Protein Extraction Reagent (Novagen). The total protein concentration of each sample was analyzed using a modified Bradford assay. The supernatant was collected and stored at 80 °C until use. The levels of Ang(1–7) were measured according to the manufacturer’s instructions. All assays were done in duplicate. The average absorbance values (A650) for each set of reference standards, control and samples were calculated and a standard curve was constructed. Using the mean absorbance value for each sample, the corresponding concentration of it from the standard curve was determined. 2.5. RNA extraction and real-time PCR Total RNA extraction and cDNA synthesis were performed as described previously (Lu et al., 2008). Levels of ACE2 and Mas mRNA were determined by real-time quantitative PCR using a SYBRÒ Premix Ex Taq™ Kit (Takara, Dalian, China) according to the manufacturer’s instructions. The cDNA amplification of a specific sequence of rat ACE2, Mas or GAPDH was performed by PCR using the following primers: sense 50 -TTCTCAGCCTTGTTGCTG TTGCTA-30 and antisense 50 -TCACTGTTTTGGAATCCTGCGT-30 for ACE2, sense 50 -CCTGCATACTGGGAAGACCA-30 and antisense 50 TCCCTTCCTGTTTCTCATGG-30 for Mas,and sense 50 -GAAAAGCTG TGGCGTGAT-30 and antisense 50 -AAGGTGGAAGAATGGGAGTT-30 for rat GAPDH. The PCR reaction was conducted at 95 °C for 30 s and followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s in the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The qRT-PCR results were analyzed and expressed as relative mRNA expression of CT (threshold cycle) value, which was then converted to fold changes. Quantitative real-time RTPCR assay was performed to detect GAPDH expression that was used to normalize the amount of cDNA for each sample. 2.6. Western blotting The methods used for immunoblotting have been described previously (Lu et al., 2008). Extract proteins were subjected to 6% SDS–PAGE, and proteins were transferred to onto nitrocellulose membranes (Sigma) and incubated with primary rabbit polyclonal antibodies against Mas1 (1:200 dilution, Santa Cruz Biotechnology; USA), ACE2 (1:400 dilution, Santa Cruz Biotechnology; USA) and bactin (internal control) (1:1000 dilution, Cell Signaling Beverly; USA) overnight at 4 °C, respectively. Goat anti-rabbit IgG conjugated with peroxidase (1:5000) was used as a secondary antibody. Membranes were incubated with secondary antibodies for 2 h at room temperature and were then revealed using ECL-plus Western blotting detection reagent. Band intensities were quantified by densitometric scanning with the Multi-Analyst software (Bio-Rad). 2.7. Immunohistochemistry Immunohistochemistry was carried out as previously described (Jiang et al., 2012) with some modifications to determine the brain localization of Mas receptors. Rats were euthanized at 24 h after pMCAO and perfused transcardially with 0.9% saline (pH 7.4) followed by a fixative solution containing 4% paraformaldehyde in 0.1 M PBS (pH 7.4), then the brains were removed, fixed for 12 h at 4 °C and embedded in paraffin. The paraffin-embedded sections (6 lm) received deparaffinization and rehydration treatments while endogenous peroxidase activity was blocked with 3% H2O2 for 30 min. The sections were blocked with 5% normal goat serum for 30 min, incubated with the primary antibody (1:100; anti-Mas1 receptor rabbit polyclonal antibodies; Santa Cruz Biotechnology) overnight at 4 °C and then treated with biotinylated goat anti-
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rabbit IgG (1:400 dilution; Zhongshan Inc., China) at room temperature for 1 h. Immunoreactivity was tested with the avidinbiotinperoxidase technique, using diaminobenzidine (DAB) as the chromogen. Sections were dried, dehydrated in graded alcohols and mounted on the slides. Quantification of the staining was done on a microscopy equipped with a charge-coupled device (CCD) camera. Cell counting was performed on five randomly selected non-overlapping fields in ischemic penumbra region per slide by 3 independent observers who were blinded to the experimental groups. Data obtained in every field were added together to make a final data for each slide and expressed as percentage of total cell number. 2.8. Statistical analysis Data were represented as mean ± S.D. (standard deviation) and were examined for the homogeneity of variance. Student’s t-test (two-tailed) was performed to analyze the data using SPSS 16.0 software. For real-time PCR, the CT value of the target gene of a sample was first corrected for the CT value of GAPDH, before being statistically analyzed. P < 0.05 was considered statistically significant. 3. Results 3.1. The Ang-(1–7) concentration in serum and cerebral ischemic tissues
Fig. 1. Ang-(1–7) concentrations were measured in sera(A) and supernatants of brain homogenate (B) by using specific ELISA kits. The bars illustrate mean ± S.D. data from 6 independent experiments. Cerebral infarction resulted in a significant increase of Ang-(1–7) at 6, 12, 24 and 48 h after pMCAO compared with shamoperated group in rats. Ang-(1–7) formation reached a maximum of 12 h after cerebral ischemic stroke. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated at each time point.
In order to investigate the changes of Ang-(1–7), its levels in the rat blood serum and ischemic brain cortex were measured by ELISA at 6, 12, 24, and 48 h after ischemic stroke. Cerebral infarction resulted in a significant increase of Ang-(1–7) compared with shamoperated group in rats (P < 0.05). Ang-(1–7) formation reached a maximum of 12 h (7.276 ± 0.320 ng/ml, serum; 1.024 ± 0.056 ng/ mg, brain) and were markedly elevated by 2.951-fold and 2.052fold compared with sham-operated group (2.466 ± 0.410 ng/ml, serum; 0.499 ± 0.032 ng/mg, brain) (n = 6, P < 0.05), respectively, after pMCAO (Fig. 1). 3.2. Real-time analysis for the Mas1 and ACE2 mRNA expression Fig. 2 shows a representative qRT-PCR analysis of the Mas1 and ACE2 mRNA expression in the ischemic brain cortex after ischemic stroke at each time point, respectively. The cDNA for GAPDH was used as an internal control. Data are presented as fold-changes in the pMCAO groups relative to Sham groups (mean ± S.D.). The Mas1 mRNA expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, 24, and 48 h after pMCAO (P < 0.05). The Mas1 receptors mRNA expression reached a maximum of 24 h (5.828 ± 0.370) and were markedly elevated by 5.828-fold compared with sham-operated group (n = 6, P < 0.05), respectively, after pMCAO (Fig. 2(A)). The ACE2 mRNA showed increased expression in the groups of pMCAO in rats at 6, 12, and 24 h after cerebral ischemic stroke compared with the sham-operated groups. The ACE2 mRNA expression reached a maximum of 12 h (5.560 ± 0.786) and were markedly elevated by 5.560-fold compared with sham-operated group (n = 6, P < 0.05), respectively, after pMCAO. There was no significant difference at 48 h after cerebral ischemic stroke (P > 0.05) (Fig. 2(B)). 3.3. Western blot analysis for the Mas1 and ACE2 protein expression Fig. 3 shows a representative western blot analysis of the Mas1 and ACE2 protein expression in the ischemic brain cortex after ischemic stroke at each time point, respectively. Protein b-actin
Fig. 2. (A) Real-time PCR analysis of Mas1 and GAPDH mRNA in the ischemic core and penumbra of the pMCAO and Sham rats. Gene GAPDH was used as an internal control. Data are presented as fold-changes in the pMCAO groups relative to Sham groups (mean ± S.D.). The Mas1 mRNA expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, 24, and 48 h after pMCAO. Mas1 receptors mRNA expression reached a maximum of 24 h after cerebral ischemic stroke. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated. (B) Realtime PCR analysis of ACE2 and GAPDH mRNA in the ischemic core and penumbra of the pMCAO and Sham rats. Gene GAPDH was used as an internal control. Data are presented as fold-changes in the pMCAO groups relative to Sham groups (mean ± S.D.). The ACE2 mRNA expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, and 24 h after pMCAO. The ACE2 mRNA expression reached a maximum of 12 h after cerebral ischemic stroke. There was no significant difference at 48 h after stroke. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated.
was adopted as internal control. The Mas1 protein expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, 24, and 48 h after pMCAO. The Mas1 protein expression reached a maximum of 24 h (0.660 ± 0.033) and were markedly elevated by 2.933-fold compared with sham-operated group (0.225 ± 0.030) (n = 6, P < 0.05), respectively, after pMCAO (Fig. 3(A)). The ACE2 protein showed increased expression
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Fig. 3. (A) Representative Western blot analysis of Mas1 and b-actin protein in the ischemic core and penumbra of cerebral ischemic stroke (pMCAO) rats and sham rats at each time point. Quantification of Mas1 receptor protein after the expression levels were normalized to b-actin. The Mas1 protein expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, 24, and 48 h after pMCAO. The Mas1 protein expression reached a maximum of 24 h after cerebral ischemic stroke. All results are given as mean ± S.D., n = 6 in each group. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated. (B) Representative Western blot analysis of ACE2 and b-actin protein in the ischemic core and penumbra of cerebral ischemic stroke (pMCAO) rats and control rats at each time point. Quantification of ACE2 protein after the expression levels were normalized to b-actin. The ACE2 protein expression showed a significant increase in pMCAO groups compared with control groups at 6, 12, and 24 h after pMCAO. The ACE2 protein expression reached a maximum of 12 h after cerebral ischemic stroke. There was no significant difference at 48 h after stroke. All results are given as mean ± S.D., n = 6 in each group. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated.
in the groups of pMCAO in rats at 6, 12, and 24 h after cerebral ischemic stroke compared with the sham-operated groups. The ACE2 protein expression reached a maximum of 12 h (0.853 ± 0.061) and were markedly elevated by 2.057-fold and 2.204-fold compared with sham-operated group (0.387 ± 0.055)
(n = 6, P < 0.05), respectively, after pMCAO. There was no significant difference at 48 h after cerebral ischemic stroke (P > 0.05) (Fig. 3(B)). These data showed that the expression of both the ACE2 and Mas1 was induced by acute cerebral ischemia injury.
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Fig. 4. (A) Representative images showing the immunohistochemical staining of Mas1 receptor in cerebral cortices of pMCAO and Sham rats at 6, 12, 24, and 48 h after stroke. Images are shown at 400 magnification; red arrows indicate positive staining in cerebral tissues. (B) Representative images and bar charts showing the quantification of Mas receptor in brain are shown for each group. The Mas1 receptor expression showed a significant increase in pMCAO groups (19.167 ± 2.858) compared with sham-operated group (7.833 ± 2.483) (P < 0.05). The Mas1 receptor expression showed a significant increase in 24 h after pMCAO compared with 48 h after pMCAO group (P < 0.016). There was no significant difference between other time point in pMCAO group (P > 0.05). Data are shown as mean ± S.D., n = 6 in each group. ⁄P < 0.05: groups of pMCAO vs. groups of sham-operated.
3.4. Immunodetection of the Mas1 receptor expression Fig. 4 shows representative images showing the immunohistochemical staining of Mas1 receptor in cerebral cortices of pMCAO and Sham rats at 24 h after stroke. Images are shown at 400 magnification; red arrows indicate positive staining in cerebral tissues. Representative images and bar charts showing the quantification of Mas receptor in brain are shown for each group. The Mas1 receptor expression showed a significant increase in pMCAO groups (19.167 ± 2.858) compared with sham-operated group (7.833 ± 2.483) (n = 6, P < 0.05) (Fig. 4). 4. Discussion Blood pressure (BP) is the major risk factor for ischemic stroke (Ovbiagele et al., 2011) with a continuous association between both systolic and diastolic blood pressures. It has been revealed that antihypertensive treatment is the most important therapy to reduce the risk of stroke by approximately 30–40% (Olindo et al., 2003; Verdecchia et al., 2012; Ovbiagele et al., 2011). All stroke patients and patients with transient ischemic attack (TIA) have to be regarded as very high-risk patients. Hypertension increases the risk of recurrent strokes. During the last decade research has shown that reducing the activity of the ACE-Ang II-AT1R axis may have beneficial effects beyond the lowering of blood pressure (Olindo et al., 2003; Verdecchia et al., 2012; Ovbiagele et al., 2011). There is growing evidence of cerebroprotective effects for medica-
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tion influencing the RAS, such as angiotensin receptor antagonists (ARB) or ACE inhibitors (ACEI) (Nguyen Dinh Cat and Touyz, 2011). Both ACEI and ARB could increase the plasma levels of Ang-(1–7), suggesting that part of the beneficial effects of them could be mediated by Ang-(1–7) (Ferrario et al., 1997; Ferreira and Santos, 2005; Trask and Ferrario, 2007). Renin converts angiotensinogen into Angiotensin I (Ang I), and ACE converts Ang I into Ang II. ACE2, a homologue of ACE, catalyzes the conversion of Ang II into the Ang-(1–7) peptide (Ferreira et al., 2012) that binds to the Mas receptor to stimulate vasodilation and plays an important role in controlling cardiovascular function (Ferreira et al., 2012; Ye et al., 2012; Xiao et al., 2011; Zheng et al., 2011). ACE2 is expressed in many tissues, including the brain, heart, kidney, liver, lung, and pancreas (Ferreira et al., 2012; Ye et al., 2012; Xiao et al., 2011; Zheng et al., 2011; Silva et al., 2012; Shahid et al., 20112; Wang et al., 2012). The brain possesses the same RAS as the systemic circulation. Recent experimental studies have shown that the brain RAS plays an important role in stroke (Arnold et al., 2012). Ang II is a key mediator in the mechanism of hypertension and plays a pathophysiological role for the development of ischemic stroke. In addition to Ang II, Ang-(1–7) may also have important biological activities in the brain (Nautiyal et al., 2012; Cerrato et al., 2012; Freund et al., 2012; Cheng et al., 2012). A growing body of evidence suggests that the ACE2–Ang-(1–7)–Mas receptor axis opposes the actions of the ACE-Ang II-AT1R system in many tissues, including the heart, liver, and kidney (Pate Bilman et al., 2012; Li et al., 2012; Patel et al., 2012; Ferrario and Varagic, 2010). Previous work suggested that Ang-(1–7) exert its stroke-protective effects through stimulation of Mas receptor (Jiang et al., 2012). The presence of ACE2 in brain regions involved in the central regulation of cardiovascular function, suggests that ACE2 is part of the brain RAS. Indeed, evidence show that ACE2 interacts with RAS components (Xiao et al., 2011; Zheng et al., 2011; Silva et al., 2012) in the central nervous system (CNS). Overexpression of ACE2 in the brain decreases BP (Yamazato et al., 2007) and prevents the Ang II-mediated development of hypertension (Sriramula et al., 2011). In our previous studies in rats demonstrated that the Ang-(1–7), was an active member of the brain RAS (Zhang et al., 2008; Lu et al., 2008; Jiang et al., 2012). However, the ACE2–Ang-(1–7)–Mas axis expression after cerebral ischemic injury are currently unclear. This study characterized the time course of Ang-(1–7), ACE2 and Mas expression in cerebral tissues and blood serum within 48 h of stroke onset. In the present work, we found that cerebral ischemic injury resulted in a significant increase of cerebral and circulating Ang-(1–7) at 6–48 h following focal ischemic stroke compared to the control. Both Mas1 and ACE2 expression in the ischemic tissues and blood serum were markedly enhanced as a result of the acute ischemic insult. The Mas immunopositive neurons were also seen stronger expression in the ischemic cortex. Our findings indicate that regional cerebral and circulating levels of Ang-(1–7), ACE2, and Mas receptor are upregulated after acute cerebral ischemic stroke in rats. The beneficial effects of ARB and ACEI are not only limited to reduction of vasoconstriction, but also include a significant decrease in vascular and endorgan inflammation (Dendorfer et al., 2005), the result of excessive AT1 receptor activation and a major participant in the pathogenesis of hypertension. The ARB and ACEI have been showed to upregulate of the ACE2–Ang-(1–7)–Mas axis expression during treatments, in hypertensive rats and normotensive rats (Ferrario et al., 1997; Ferreira and Santos, 2005; Trask and Ferrario, 2007). Elsewhere we reported that, in male rats, the Ang(1–7) exerts neuroprotective effects against cerebral ischemia (Jiang et al., 2012); it upregulates eNOS (Zhang et al., 2008) and potentiates bradykinin (Lu et al., 2008). The protective effect of ACE2–Ang-(1–7)–Mas pathway on ischemic stroke has been re-
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lated to blood pressure control (Nautiyal et al., 2012) and improvement of cerebral blood supply (Iyer et al., 1998). However, the benefits can go beyond those. The activation of the Mas receptor for Ang-(1–7) inhibits Ang II-induced vasoconstriction in human internal mammary arteries and forearm resistance vessels (Roks et al., 1999). Other studies suggest that Mas receptor activation by Ang-(1–7) improves endothelial function by activating eNOS and facilitating NO release (Zhang et al., 2008; Cerrato et al., 2012; Cheng et al., 2012). The orally active Mas receptor agonist AVE0991 exerts effects similar to Ang-(1–7) (Ferreira et al., 2007), suggesting that drugs that activate the Mas receptor may have a benefit as cardiovascular therapeutic agents. ACE2–Ang-(1–7)–Mas axis plays a broad role in vascular regulation (Dantas and Sandberg, 2005), inflammation (El-Hashim et al., 2012), oxidative stress (Raffai et al., 2011), and apoptosis (Jiang et al., 2012). ACE2–Ang-(1–7)–Mas axis could exert its stroke-protective effects through stimulation of Mas receptors. We show for the first time that ACE2–Ang-(1–7)–Mas axis expression was upregulated in vivo following acute ischemic stroke in rats. The cerebral acute ischemic neuron injury is associated with upregulation of the ACE2–Ang-(1–7)–Mas axis and promotes increased circulating Ang-(1–7). These results suggest that the ACE2–Ang-(1– 7)–Mas pathway located in the neuron is involved in ischemic damage. The evidence collected so far indicate that, ACE2–Ang(1–7)–Mas receptor axis should be viewed as a promising therapeutic tool for the treatment of cerebrovascular diseases. Further insights in this regard would be obtained by studies aimed to clarify the significant variation in prevention and functional outcome after ischemic stroke. Conflict of interest None. Acknowledgments This work was supported in part by grants from the Medical Science and Technology Foundation for Young Scientists of Nanjing City (Grant No. QYK09186), Science and Technology Foundation of Nanjing Medical University (Grant No. 09NJMUMl25) and the Natural Science Foundation of Jiangsu Province (Grant No. BK2010116). References Arnold, A.C., Gallagher, P.E., Diz, D.I., 2012. Brain renin–angiotensin system in the nexus of hypertension and aging. Hypertens. Res. 36, 5–13. Bos, D., van der Rijk, M.J., Geeraedts, T.E., Hofman, A., Krestin, G.P., Witteman, J.C., van der Lugt, A., Ikram, M.A., Vernooij, M.W., 2012. Intracranial carotid artery atherosclerosis: prevalence and risk factors in the general population. Stroke 43, 1878–1884. Cerrato, B.D., Frasch, A.P., Nakagawa, P., Longo-Carbajosa, N., Peña, C., Höcht, C., Gironacci, M.M., 2012. Angiotensin-(1–7) upregulates central nitric oxide synthase in spontaneously hypertensive rats. Brain Res. 1453, 1–7. Cheng, W.H., Lu, P.J., Hsiao, M., Hsiao, C.H., Ho, W.Y., Cheng, P.W., Lin, C.T., Hong, L.Z., Tseng, C.J., 2012. Renin activates PI3K-Akt-eNOS signalling through the angiotensin AT1 and Mas receptors to modulate central blood pressure control in the nucleus tractus solitarii. Br. J. Pharmacol. 166, 2024–2035. Danielyan, K.E., Oganesyn, H.M., Nahapetyan, K.M., Kevorkian, G.A., Vladimir, D., Galoyan, A.A., Grigorian, G.S., 2012. Stroke burden in adults in Armenia. Int. J. Stroke 7, 248–249. Dantas, A.P., Sandberg, K., 2005. Regulation of ACE2 and ANG-(1–7) in the aorta: new insights into the renin–angiotensin system in the control of vascular function. Am. J. Physiol. Heart Circ. Physiol. 289, H980–981. Dendorfer, A., Dominiak, P., Schunkert, H., 2005. ACE inhibitors and angiotensin II receptor antagonists. Handb. Exp. Pharmacol. 170, 407–442. El-Hashim, A.Z., Renno, W.M., Raghupathy, R., Abduo, H.T., Akhtar, S., Benter, I.F., 2012. Angiotensin-(1–7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-jB-dependent pathways. Br. J. Pharmacol. 166, 1964–1976. Ferrario, C.M., Chappell, M.C., Tallant, E.A., Brosnihan, K.B., Diz, D.I., 1997. Counterregulatory actions of angiotensin-(1–7). Hypertension 30, 535–541.
Ferrario, C.M., Varagic, J., 2010. The ANG-(1–7)/ACE2/Mas axis in the regulation of nephron function. Am. J. Physiol. Renal Physiol. 298, F1297–1305. Ferreira, A.J., Santos, R.A., Almeida, A.P., 2001. Angiotensin-(1–7): cardioprotective effect in myocardial ischemia/reperfusion. Hypertension 38, 665–668. Ferreira, A.J., Santos, R.A., 2005. Cardiovascular actions of angiotensin-(1–7). Braz. J. Med. Biol. Res. 38, 499–507. Ferreira, A.J., Jacoby, B.A., Araújo, C.A., Macedo, F.A., Silva, G.A., Almeida, A.P., Caliari, M.V., Santos, R.A., 2007. The nonpeptide angiotensin-(1–7) receptor Mas agonist AVE-0991 attenuates heart failure induced by myocardial infarction. Am. J. Physiol. Heart Circ. Physiol. 292, H1113–1119. Ferreira, A.J., Bader, M., Santos, R.A., 2012. Therapeutic targeting of the angiotensinconverting enzyme 2/angiotensin-(1–7)/Mas cascade in the renin–angiotensin system: a patent review. Expert Opin. Ther. Pat. 22, 567–574. Fraga-Silva, R.A., Da Silva, D.G., Montecucco, F., Mach, F., Stergiopulos, N., da Silva, R.F., Santos, R.A., 2012. The angiotensin-converting enzyme 2/angiotensin-(1– 7)/Mas receptor axis: a potential target for treating thrombotic diseases. Thromb. Haemost. 108, 1089–1096. Freund, M., Walther, T., von Bohlen und Halbach, O., 2012. Immunohistochemical localization of the angiotensin-(1–7) receptor Mas in the murine forebrain. Cell Tissue Res. 348, 29–35. Iwai, M., Nakaoka, H., Senba, I., Kanno, H., Moritani, T., Horiuchi, M., 2012. Possible involvement of angiotensin-converting enzyme 2 and Mas activation in inhibitory effects of angiotensin II Type 1 receptor blockade on vascular remodeling. Hypertension 60, 137–144. Iyer, S.N., Chappell, M.C., Averill, D.B., Diz, D.I., Ferrario, C.M., 1998. Vasodepressor actions of angiotensin-(1–7) unmasked during combined treatment with lisinopril and losartan. Hypertension 31, 699–705. Jiang, T., Gao, L., Shi, J., Lu, J., Wang, Y., Zhang, Y., 2012a. Angiotensin-(1–7) modulates renin–angiotensin system associated with reducing oxidative stress and attenuating neuronal apoptosis in the brain of hypertensive rats. Pharmacol. Res. 67, 84–93. Jiang, T., Gao, L., Guo, J., Lu, J., Wang, Y., Zhang, Y., 2012b. Suppressing inflammation by inhibiting the NF-jB pathway contributes to the neuroprotective effect of angiotensin-(1–7) in rats with permanent cerebral ischaemia. Br. J. Pharmacol. 67, 1520–1532. Li, P., Zhang, F., Zhou, Y.B., Cui, B.P., Han, Y., 2012. Superoxide anions modulate the effects of angiotensin-(1–7) in the rostral ventrolateral medulla on cardiac sympathetic afferent reflex and sympathetic activity in rats. Neuroscience 223, 388–398. Lu, J., Zhang, Y., Shi, J., 2008. Effects of intracerebroventricular infusion of angiotensin-(1–7) on bradykinin formation and the kinin receptor expression after focal cerebral ischemia-reperfusion in rats. Brain Res. 1219, 127–135. Mecca, A.P., Regenhardt, R.W., O’Connor, T.E., Joseph, J.P., Raizada, M.K., Katovich, M.J., Sumners, C., 2011. Cerebroprotection by angiotensin-(1–7) in endothelin1-induced ischaemic stroke. Exp. Physiol. 96, 1084–1096. Nautiyal, M., Shaltout, H.A., de Lima, D.C., do Nascimento, K., Chappell, M.C., Diz, D.I., 2012. Central angiotensin-(1–7) improves vagal function independent of blood pressure in hypertensive (mRen2)27 rats. Hypertension 60, 1257–1265. Nguyen Dinh Cat, A., Touyz, R.M., 2011. A new look at the renin–angiotensin system – focusing on the vascular system. Peptides 32, 2141–2150. Olindo, S., Cabre, P., Deschamps, R., Chatot-Henry, C., René-Corail, P., Fournerie, P., Saint-Vil, M., May, F., Smadja, D., 2003. Acute stroke in the very elderly: epidemiological features, stroke subtypes, management, and outcome in Martinique, French West Indies. Stroke 34, 1593–1597. Ovbiagele, B., Diener, H.C., Yusuf, S., Martin, R.H., Cotton, D., Vinisko, R., Donnan, G.A., Bath, P.M., 2011. PROFESS investigators (2011) level of systolic blood pressure within the normal range and risk of recurrent stroke. JAMA 306, 2137– 2144. Pate Bilman, V., Mares-Guia, L., Nadu, A.P., Bader, M., Campagnole-Santos, M.J., Santos, R.A., Santos, S.H., 2012. Decreased hepatic gluconeogenesis in transgenic rats with increased circulating angiotensin-(1–7). Peptides 37, 247–251. Patel, V.B., Bodiga, S., Fan, D., Das, S.K., Wang, Z., Wang, W., Basu, R., Zhong, J., Kassiri, Z., Oudit, G.Y., 2012. Cardioprotective effects mediated by angiotensin II type 1 receptor blockade and enhancing angiotensin 1–7 in experimental heart failure in angiotensin-converting enzyme 2-null mice. Hypertension 59 (6), 1195–1203. Raffai, G., Durand, M.J., Lombard, J.H., 2011. Acute and chronic angiotensin-(1–7) restores vasodilation and reduces oxidative stress in mesenteric arteries of saltfed rats. Am. J. Physiol. Heart Circ. Physiol. 301, H1341–1352. Roks, A.J., Van Geel, P.P., Pinto, Y.M., Buikema, H., Henning, R.H., De Zeeuw, D., Van Gilst, W.H., 1999. Angiotensin-(1–7) is a modulator of the human renin– angiotensin system. Hypertension 34, 296–301. Santos, R.A., Ferreira, A.J., Verano-Braga, T., Bader, M., 2012. Angiotensin-converting enzyme 2, angiotensin-(1–7) and Mas: new players of the renin angiotensin system. J. Endocrinol. 216, R1–R17. Shahid, M., 2011. The angiotensin-converting enzyme 2-angiotensin-(1–7) axis: the other side of the renin–angiotensin system. Exp. Physiol. 96, 987–988. Silva, R.A., Chu, Y., Miller, J.D., Mitchell, I.J., Penninger, J.M., Faraci, F.M., Heistad, D.D., 2012. Impact of ACE2 deficiency and oxidative stress on cerebrovascular function with aging. Stroke 43, 3358–3363. Sriramula, S., Cardinale, J.P., Lazartigues, E., Francis, J., 2011. ACE2 overexpression in the paraventricular nucleus attenuates angiotensin II-induced hypertension. Cardiovasc. Res. 92, 401–408. Tan, P.S., Killinger, S., Horiuchi, J., Dampney, R.A., 2007. Baroreceptor reflex modulation by circulating angiotensin II is mediated by AT1 receptors in the
J. Lu et al. / Neuropeptides 47 (2013) 289–295 nucleus tractus solitarius. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R2267–R2278. Trask, A.J., Ferrario, C.M., 2007. Angiotensin-(1–7): pharmacology and new perspectives in cardiovascular treatments. Cardiovasc. Drug Rev. 25, 162–174. Verdecchia, P., Gentile, G., Angeli, F., Reboldi, G., 2012. Beyond blood pressure: evidence for cardiovascular, cerebrovascular, and renal protective effects of renin–angiotensin system blockers. Ther. Adv. Cardiovasc. Dis. 6, 81–91. Wang, Y., Wang, J., Liu, R., Qi, H., Wen, Y., Sun, F., Yin, C., 2012. Severe acute pancreatitis is associated with upregulation of the ACE2–angiotensin-(1–7)– Mas axis and promotes increased circulating angiotensin-(1–7). Pancreatology 12, 451–457. Xiao, L., Gao, L., Lazartigues, E., Zucker, I.H., 2011. Brain-selective overexpression of angiotensin-converting enzyme 2 attenuates sympathetic nerve activity and enhances baroreflex function in chronic heart failure. Hypertension 58, 1057–1065. Yamazato, M., Yamazato, Y., Sun, C., Diez-Freire, C., Raizada, M.K., 2007. Overexpression of angiotensin-converting enzyme 2 in the rostral
295
ventrolateral medulla causes long-term decrease in blood pressure in the spontaneously hypertensive rats. Hypertension 49 (4), 926–931. Ye, M., Wysocki, J., Gonzalez-Pacheco, F.R., Salem, M., Evora, K., Garcia-Halpin, L., Poglitsch, M., Schuster, M., Batlle, D., 2012. Murine recombinant angiotensinconverting enzyme 2: effect on angiotensin II-dependent hypertension and distinctive angiotensin-converting enzyme 2 inhibitor characteristics on rodent and human angiotensin-converting enzyme 2. Hypertension 60, 730– 740. Zhang, Y., Lu, J., Shi, J., Lin, X., Dong, J., Zhang, S., Liu, Y., Tong, Q., 2008. Central administration of angiotensin-(1–7) stimulates nitric oxide release and upregulates the endothelial nitric oxide synthase expression following focal cerebral ischemia/reperfusion in rats. Neuropeptides 42, 593–600. Zheng, H., Liu, X., Patel, K.P., 2011. Angiotensin-converting enzyme 2 overexpression improves central nitric oxide-mediated sympathetic outflow in chronic heart failure. Am. J. Physiol. Heart Circ. Physiol. 301, H2402–H2412.