Original Contribution

Angiotensin II Peptide Vaccine Protects Ischemic Brain Through Reducing Oxidative Stress

Kouji Wakayama, Munehisa Shimamura, Jun-ichi Suzuki, Ryo Watanabe, Hiroshi Koriyama, Hiroshi Akazawa, Hironori Nakagami, Hideki Mochizuki, Mitsuaki Isobe, Ryuichi Morishita
https://doi.org/10.1161/STROKEAHA.116.016269
Stroke. 2017;48:1362-1368
Originally published March 31, 2017

Abstract

Background and Purpose—Medication nonadherence is one of major risk factors for the poor outcome in ischemic stroke. Vaccination is expected to solve such a problem because of its long-lasting effects, but its effect on ischemic brain damage is still unknown. Here, we focused on vaccination for renin–angiotensin system and examined the effects of angiotensin II (Ang II) peptide vaccine in permanent middle cerebral artery occlusion model in rats.

Methods—Male Wistar rats were exposed to permanent middle cerebral artery occlusion after 3× injections of Ang II peptide vaccine, and the serum or brain level of anti–Ang II antibody was examined. The effects of the vaccine were evaluated by differences in infarction volume, brain renin–angiotensin system components, and markers for neurodegeneration and oxidative stress.

Results—Ang II vaccination successfully produced anti–Ang II antibodies in serum without concomitant change in blood pressure. Sufficient production of serum anti–Ang II antibody led to reduction of infarct volume and induced the penetration of anti–Ang II antibody in ischemic hemisphere, with suppressed expression of Ang II type 1 receptor mRNA. Vaccinated rats with sufficient antibody production showed the reduction of Fluoro-Jade B–positive cells, spectrin fragmentation, 4-hydroxynonenal-positive cells, and Nox 2 mRNA expression.

Conclusions—Our findings indicate that Ang II vaccination exerts neuroprotective and antioxidative effects in cerebral ischemia, with renin–angiotensin system blockade by penetration of anti–Ang II antibodies into ischemic brain lesion. Ang II peptide vaccination could be a promising approach to treat ischemic stroke.

Introduction

Inhibition of renin–angiotensin system (RAS) and control of blood pressure (BP) by angiotensin-converting enzyme inhibitors (ACE-Is) or angiotensin II (Ang II) receptor blockers (ARBs) are important for in the primary1 and secondary2 prevention of ischemic stroke. The protective effect of ACE-Is and ARBs has been attributed to their inhibitory effect on the expression of proinflammatory cytokines and oxidative stress in postischemic brain3–6 or their direct neuroprotective effects.7 However, recent studies have revealed inconsistent drug intake in >60% patients 1 year after hospitalization.8 Nonadherence to secondary prevention medication is a major risk factor for the recurrence of ischemic stroke.9 In addition, economic burden is a concern pertaining to long-term treatment. To alleviate the compliance concerns and improve preventive outcomes, we assessed the effects of the recently developed Ang II peptide vaccine10 in preventing ischemic stroke.

Although vaccines have traditionally been used to prevent infectious diseases, their therapeutical use has been recently expanded against the adult common diseases, such as hypertension,11 Alzheimer’s disease,12–14 and so on, by targeting self-antigens. Immunization is a cost-effective intervention compared with conventional therapy owing to its long-lasting effects and does not require daily intake of medication.15 However, there are only few reports regarding the development of a vaccine to treat ischemic stroke.

A vaccine targeting NR1 subunit of N-methyl-d-asparate receptor in rats with ischemic stroke has been reported earlier16; however, clinical trials for the same have not been conducted. The association of auto-antibodies to N-methyl-d-asparate receptor NR1 subunit with neuropsychiatric systemic lupus erythematosus17 and anti-N-methyl-d-asparate receptor encephalitis18 is noteworthy. Selection of appropriate target molecules is a key challenge for therapeutic vaccination in patients with ischemic stroke owing to safety concerns.19

Therefore, here we focused on developing a vaccine against RAS because the inhibition of RAS by ACE-Is and ARBs has been widely accepted as a safe and effective treatment for the primary and secondary prevention of ischemic stroke. Because previous reports demonstrated that Ang II peptide vaccine or Ang II DNA vaccine was free from anti–Ang II autoimmune response and exerted a long-lasting antihypertensive effect in animal experiments10,20 and human clinical trials,20 Ang II peptide vaccine is a promising therapeutic modality for the treatment of hypertension. However, it is still unclear whether the protective effect of Ang II peptide vaccine against ischemic damages is over and above its antihypertensive effects. To clarify this important question, we examined its efficacy in preventing ischemic damage using a permanent focal ischemia model of normotensive rats.

Methods

Animals

Male Sprague–Dawley rats (3 weeks old) obtained from CLEA Japan, Inc (Tokyo, Japan) were housed under a 12-hour light/12-hour dark cycle with free access to food and water under temperature- and humidity-controlled conditions. Female rats were not used to avoid any influences of sex steroids. The experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Tokyo.

Peptide Syntheses

To induce sufficient immune response, keyhole limpet hemocyanin (KLH; Wako Pure Chemical Industries, Osaka, Japan) carrier protein was conjugated to the N-terminus of Ang II using glutaraldehyde (Peptide Institute Inc, Osaka, Japan) as previously described.10

Ang II Peptide Vaccine Immunization

Immunization was performed at the age of 4, 6, and 7 weeks. Detailed procedure is described in the online-only Data Supplement.

Study Design

Detailed information is described in the online-only Data Supplement.

Surgical Procedure

Permanent middle cerebral artery occlusion (pMCAo; n=92) or sham (n=16) surgery was performed at 1 week or 15 days after the third vaccination or saline injection. Detailed information is described in the online-only Data Supplement.

Measurement of BP and Sample Collection

Systolic BP was recorded using the tail-cuff system (BP-98A; SOFTRON Co., Tokyo, Japan) at 0, 14, 21, and 28 days after the first vaccination and at 24 hours after pMCAo. Detailed procedures are described in the online-only Data Supplement.

Histological Analysis

To identify the infarct area, cresyl violet staining was performed and infarction volume was calculated. Neurodegeneration and oxidative stress in the brain was evaluated using Fluoro-Jade B staining and immunohistochemistry for Ang II type 1 receptor (AT1R) and 4-hydroxynonenal (HNE). NeuroTrace 530/615 Red Fluorescent Nissl Stain was used to label neurons. Detailed procedure is described in the online-only Data Supplement.

Western Blot

Protein expression of spectrin alpha chain or 4-HNE was evaluated using Western blot. Detailed procedure is described in the online-only Data Supplement.

Quantification of Serum Anti–Ang II Antibody Titer by Enzyme-Linked Immunosorbent Assay

Serum anti–Ang II antibody titer was quantified using enzyme-linked immunosorbent assay. The anti-Ang II specific antibody titer was defined as the serum dilution that exhibited half-maximal optical density (OD) at 450 nm (OD50%). Detailed procedure is described in the online-only Data Supplement.

Anti–Ang II Antibody Penetration Into Brain Parenchyma

Anti–Ang II antibody penetration into brain parenchyma was measured by enzyme-linked immunosorbent assay using the brain homogenate. Detailed procedure is described in the online-only Data Supplement.

Quantification of Ang II Protein Expression in Plasma and Brain

Detailed procedure for sample processing is described in the online-only Data Supplement. Ang II concentration in brain homogenate or plasma samples was measured using standard radioimmunoassay by an external laboratory (SRL, Tokyo, Japan).

Real-Time Polymerase Chain Reaction

The mRNA levels of AT1R, angiotensinogen, NADPH oxidase 2, and 18s ribosomal RNA (endogenous control) were measured by real-time polymerase chain reaction. For more detail, please see online-only Data Supplement.

Statistical Analysis

Data are expressed as mean±standard deviation. Data were analyzed using Statview for windows version 5 (SAS Institute Inc, Tokyo, Japan). Detailed information is described in the online-only Data Supplement.

Results

Anti–Ang II Antibody in Immunized Rats

Normal rats were immunized using 3 doses of Ang II peptide; the antibody titers against Ang II and the concentration of Ang II in plasma and brain homogenates were measured at 28 days after first immunization (Figure 1A). Consistent with a previous report stating that BP was not altered in normotensive Ang II peptide vaccinated mice,10 no differences in BP were observed between the groups (Figure I in the online-only Data Supplement). Although anti–Ang II antibody was not observed in saline-injected rats (controls), serum titer of anti–Ang II antibody was significantly increased in the vaccinated rats (Figure 1B). The concentration of Ang II in plasma was significantly increased in the vaccinated rats (Figure IIA in the online-only Data Supplement). A significant positive correlation between serum anti–Ang II antibody titer and plasma Ang II level was observed in the vaccinated rats (Figure IIB in the online-only Data Supplement; r=0.669, P=0.0153). To investigate whether vaccination affected the brain Ang II concentration, the concentration of Ang II in the brain was examined. However, no significant differences were observed between the groups (Figure 1C). To examine whether anti–Ang II antibodies penetrate from systemic circulation to nonischemic brain tissue, anti–Ang II antibody level was quantified by enzyme-linked immunosorbent assay using brain homogenate. There was no significant change in anti–Ang II antibody levels in the intact brain tissue between vaccinated and control rats (Figure 1D).

Figure 1.
Figure 1.

Anti-angiotensin (Ang) II antibody production and Ang II level in nonischemic vaccinated rats. A, Immunization schedule: Narrow downward facing arrows, subcutaneous injection of Ang II peptide vaccine or saline; void upward facing arrows, blood pressure measurement; thick upward facing arrow, sample collection. B, Serum anti–Ang II antibody titer of Ang II vaccinated (V) and saline-treated (S) rats at 28 days after first injection. C, No significant difference in brain Ang II concentration was observed between groups. D, No significant difference in brain parenchymal anti–Ang II antibody was observed between groups. The titer is expressed as the dilution of serum giving half-maximal binding (optical density: OD50%) ±SD of the mean. **P=0.006 vs S. Each group included n=8 in B, n=6 in C, and n=9 (S) or n=11 (V) in D.

Effect of Ang II Peptide Vaccine in the pMCAo Rats

Next, we examined whether Ang II peptide vaccine affected ischemic brain damage. Ang II–vaccinated rats and control rats were subjected to pMCAo at 1 week after the third vaccination (Figure 2A). Rats with high titer of serum anti–Ang II antibody showed smaller infarct volume (Figure 2B; r=−0.759, P=0.006) at 24 hours of pMCAo. Based on the result, we divided the rats into 2 groups: low titer group (OD50% <6000, VL group) and high titer group (OD50% ≥6000, VH group). Representative images of the cresyl violet–stained sections from each group showed a reduced infarct volume in VH rats (Figure III in the online-only Data Supplement). On quantitative analysis, VH rats showed a significant reduction in the infarct volume compared with the VL and control rats (Figure 2C). These results indicate that high titers of serum anti–Ang II antibody may be necessary to demonstrate the protective effects against ischemic brain. Next, we examined whether the protective effects of Ang II vaccine could last 15 days after the vaccination. As expected, infarct volume was less in VH rats when the rats were exposed to pMCAo at 15 days after third vaccination (Figure IV in the online-only Data Supplement). Because no differences were observed in BP between VH and control rats (Figure V in the online-only Data Supplement), the protective effects of Ang II vaccine were not because of its actions on BP.

Figure 2.
Figure 2.

Effect of angiotensin II (Ang II) peptide vaccine on cerebral infarct volume. A, Cerebral infarct volume was evaluated at 24 hours after permanent middle cerebral artery occlusion (pMCAo) surgery. B, A significant negative correlation was observed between the infarct volume and the serum anti–Ang II antibody titer (r=−0.759, P=0.006). C, Infarct volume was reduced in VH rats whose serum anti–Ang II antibody titer was OD50% ≥6000 compared with that in VL rats whose serum anti–Ang II antibody titer was OD50% <6000 and saline-treated (S) rats. *P<0.05 vs S, #P<0.05 vs VL. Each group included n=5 (sham), n=5 (VH), n=6 (VL), or n=8 (S) in C.

Penetration of Anti–Ang II Antibody Into Brain Parenchyma

To clarify the mechanism of preventive effects of Ang II peptide vaccine on cerebral infarction, we first measured brain anti–Ang II antibody level at 24 hours after pMCAo in the brain homogenate samples. Anti–Ang II antibody in the brain tissue of VH rats was significantly increased by 56-fold compared with that in the brain tissue of control rats, whereas brain anti–Ang II antibody was also significantly increased in VH rats compared with that in the VL rats (Figure 3A). These findings indicate that anti–Ang II antibody penetrated into ischemic lesion. There was a strong positive correlation between serum anti–Ang II antibody titer and brain parenchymal anti–Ang II antibody change (Figure VI in the online-only Data Supplement; r=0.747, P=0.0003). Temporal profile of brain anti–Ang II antibody showed the penetration of anti–Ang II antibody into ischemic lesion as early as 12 hours after cerebral ischemia (Figure 3B).

Figure 3.
Figure 3.

Anti-angiotensin (Ang II) antibody penetration into ischemic lesion in vaccinated rats. A, Rats with high titers of serum anti–Ang II antibody (VH, OD50% ≥6000) showed a significant increase in anti–Ang II antibody in ischemic brain tissue as compared with that in rats with low titers of serum anti–Ang II antibody (VL, OD50% <6000) and saline-treated (S) rats. B, Anti-Ang II antibody in ischemic brain had increased as early as 12 hours after pMCAo in VH rats as compared with that in sham-operated VH rats. **P<0.01 vs VL, ##P<0.01 vs S, †P<0.05 vs VH (sham). Each group included n=7 (S), n=7 (VH), n=10 (VL) in A and n=5 (VH [sham]), n=7 (VH [6 h]), n=6 (VH [12 h]) in B.

Subsequently, we examined the effects of Ang II peptide vaccine on the brain RAS components. Because VH rats showed preventive effects in terms of the infarct volume and penetration of anti–Ang II antibody into the parenchyma, we compared the expression of RAS components between VH and control rats at each time point. In sham-operated rats, the expression of AT1R or angiotensinogen mRNA was not altered by Ang II peptide vaccination (Figure 4A and 4B). In comparison with sham-operated rats, AT1R mRNA was significantly increased at 24 hours after pMCAo in ischemic hemisphere of nonvaccinated rats, whereas the increased expression was significantly inhibited in VH rats (Figure 4A). Although angiotensinogen mRNA was decreased in the ischemic hemisphere at 24 hours after pMCAo in nonvaccinated control rats, its expression was attenuated in VH rats (Figure 4B).

Figure 4.
Figure 4.

Effect of angiotensin (Ang II) peptide vaccination on gene expression of brain renin–angiotensin system in ischemic lesion. Ang II type 1 receptor (AT1R) and angiotensinogen mRNA expressions were not affected by vaccination in sham-operated rats. A, Reduced AT1R mRNA expression was observed in ischemic hemisphere of serum anti–Ang II high titer (VH) rats at 24 hours after pMCAo as compared with that in saline-treated (S) rats. B, S rats had significantly decreased expression of angiotensinogen mRNA in ischemic hemisphere as compared with that in saline-treated sham rats at 24 hours after pMCAo. **P<0.01 vs saline-treated sham, ##P<0.01 vs S (24 h), †P<0.05 vs saline-treated sham. Each group included n=6 except for n=7 (6 h [VH]), n=5 (12 h [S]) in A and n=6 except for n=7 (12 h [VH]), n=5 (12 h [S]) in B.

Effect of Ang II Peptide Vaccine on Neurodegeneration and Oxidative Stress

Finally, we examined the molecular mechanisms of the protective effects of Ang II peptide vaccine against ischemic brain damage. The number of Fluoro-Jade B–positive cells, which are degenerated neurons in the early phase of cerebral ischemia,21 was significantly decreased in VH rats compared with control rats (Figure 5A). In addition, Western blotting of spectrin breakdown products, reliable markers for neurodegeneration,22 revealed a significant reduction in 150/145 kDa fragments of VH rats (Figure VIIA in the online-only Data Supplement). These results suggest that the reduction in the infarct volume by Ang II peptide vaccine is because of the prevention of neurodegeneration after pMCAo.

Figure 5.
Figure 5.

Effect of angiotensin (Ang) II peptide vaccine on neurodegeneration and oxidative stress in ischemic lesion. A, Schematic illustration of a coronal brain section. The shaded area represents the ischemic lesion, and the boxed area was the observed ischemic core region. B, Twenty-four hours after pMCAo, Fluoro-Jade B (FJB)–positive cells (arrows) were significantly decreased in serum anti–Ang II high titer (VH) rats as compared with that in saline-treated control (S) rats. C, More 4-hydroxynonenal (4-HNE)-positive cells (arrows) were observed in lesions of S rats as compared with that in VH rats. Densitometric analysis of Western blot showed significantly decreased 4-HNE expression in VH rats as compared with that in S rats. Bar=20 μm, ##P=0.0067 vs S, **P<0.01 vs sham, #P<0.05 vs S. Each group included n=5 in A and n=8 (sham), n=6 (S), n=6 (VH) in B.

Because the pharmacological inhibition of RAS using ACE-Is or ARBs has been reported to exert its neuroprotective effects via its antioxidative property,23 we further examined whether the neuroprotective effect of Ang II peptide vaccine may be related to the antioxidative mechanism in neurons. First, we checked whether neurons expressed AT1R and 4-HNE, oxidative stress marker in the ischemic brain. Immunohistochemistry showed that the AT1R-positive cells were increased mainly in neurons in ischemic core and peri-infarct region (Figure VIIIB in the online-only Data Supplement). Also, most 4-HNE-positive cells showed increased expression of AT1R in ischemic hemisphere (Figure VIIIC in the online-only Data Supplement) and 4-HNE-positive cells were predominantly composed of neurons (Figure VIIID in the online-only Data Supplement). These results indicate that increased expression of AT1R was associated with increased oxidative stress in neurons in the ischemic hemisphere. In the vaccinated rats with Ang II peptide vaccine, the expression of 4-HNE was markedly decreased (Figure 5C), suggesting that Ang II vaccine suppressed oxidative stress through AT1R signalings in neurons. Western blot assay also showed that 4-HNE formation was significantly increased in the ischemic hemisphere of nonimmunized rats, whereas its expression was significantly decreased by Ang II vaccination. Additionally, the expression of NADPH oxidase 2 mRNA, which is the most critical NADPH oxidase in the ischemic brain,24 was significantly decreased in vaccinated rats (Figure VIIB in the online-only Data Supplement). Thus, Ang II vaccine inhibited oxidative stress in the ischemic brain.

Discussion

In the present study, immunization with Ang II peptide vaccine significantly prevented the exacerbation of ischemic brain damage by the inhibition of neurodegeneration and antioxidative effects. Although anti–Ang II antibody does not seem to penetrate into the intact brain, our data revealed that anti–Ang II antibody may penetrate the brain once the blood–brain barrier has been broken down as a result of ischemic damage. This observation was confirmed by the significant positive correlation in the amount of anti–Ang II antibody between serum and brain. Circulating high titer of anti–Ang II antibody may cross blood–brain barrier and increase its expression in the ischemic brain tissue and prevent further ischemic damage. Increased expression of plasma Ang II in the vaccinated rat might be explained by diminished suppression of feedback on renin secretion via blockade of systemic RAS as previously reported.25

Independent of systemic RAS, the brain has been the center of interest as an important site for the production of Ang II from tissue angiotensinogen by renin and ACE.26 Similar to the previous reports that showed increased AT1R mRNA expression at 24 hours after middle cerebral artery occlusion,27 this change was observed in nonimmunized rats. Inhibition of the upregulation of AT1R expression by vaccination may prevent the activation of brain RAS. In contrast, angiotensinogen mRNA expression in ischemic brain tissue was shown to increase rapidly at 1 to 2 hours followed by return to baseline level at 6 hours after middle cerebral artery occlusion.5 In the present study, angiotensinogen mRNA expression was decreased in the ischemic hemisphere in the control group after 24 hours of pMCAo, whereas Ang II vaccination attenuated this decrease in the present study. Decreased expression of angiotensinogen mRNA in nonimmunized rats is probably because of negative regulation by the activation of AT1R signaling pathway induced by Ang II in astrocytes.28

Ang II regulates reactive oxygen species production and oxidative stress in ischemic brain.29,30 Because the expression of 4-HNE, which was expressed in AT1R-positive neurons, and AT1R mRNA was reduced in the vaccinated rats, suppression of oxidative stress in damaged neurons through inhibition of AT1R signaling might be one of the mechanisms for the amelioration of ischemic injury. Considering that NADPH oxidase 2 is involved in the AT1R-reactive oxygen species axis in neurons31,32 and its expression was less in the vaccinated rats, the antioxidative stress effects might be through the inhibition of NADPH oxidase 2. These might be supported by the previous reports showing the neuroprotective and antioxidative stress effects of ARBs in the cultured neurons.33–35 Another possible mechanism of protective effects of vaccination is the inhibitory effect on calpain and caspase activities because the fragmentation of spectrins, cleaved forms from full spectrin (240 kDa) by caspase-3 and calpain, was significantly reduced. Ang II increased the intracellular Ca2+,36 and Ang II may have directly regulated the calpain activity in the brain, as reported in other types of tissues, such as aorta,37 kidney,38 and cardiomyocyte.39

In the present study, the cerebroprotective effects of Ang II vaccine lasted 15 days after the vaccination. Although we could not examine whether the effects were preserved >15 days because of the limitation of the stroke model, the preventive effects of Ang II peptide vaccine may have lasted up to several months because we observed the continuing existence of anti–Ang II antibody in serum at least ≤3 months after immunization.10 In addition, the previous clinical study using Ang II vaccine demonstrated the reversible antibody response against Ang II, with a half-life of ≈4 months after immunization.20 In clinical settings, this possible long-lasting efficacy would be promising compared with the standard inhibitor of the RAS because poor adherence to secondary preventive medication owing to poststroke dementia or other neurological deficits is a major risk of the recurrence of ischemic stroke.9

One may assume that excessive lowering of BP by Ang II peptide vaccine may be a risk factor for ischemic stroke.40 However, it is unlikely because no effects on BP were observed in the present study. This finding is compatible with the previous reports showing no BP lowering effects of Ang II peptide vaccine in normotensive mice,10 despite the reduction of BP in hypertensive rats.25 Therefore, the therapeutic efficacy of Ang II peptide vaccine for ischemic injury without a concomitant effect on BP seems to be a promising preventive approach for normotensive as well as hypertensive patients.

Because KLH itself is an immune activator and affects immune response under pathological conditions,10,41 KLH might have some influences on the production of anti–Ang II antibodies or the ischemic injury. However, in our previous study, we demonstrated that KLH itself did not produce the antibodies for Ang II.10 Also, in the present study, low Ang II titer level did not affect cerebral infarct size in the vaccinated rats. This indicated that nonspecific immune reaction by KLH did not affect the ischemic damages.

In the clinical application, one of the concerns is the high variability of antibody production. Although further study is necessary, we speculate that higher dose of the vaccine, improvement of the timing of vaccination, and optimization of the carrier protein or adjuvant could increase the antibody titer and reduce the variability. Alternatively, DNA vaccination might be another option because we recently found that DNA vaccine encoding Ang II was more effective than Ang II peptide vaccine in lowering BP in SHR rats.42

To summarize, prior immunization by Ang II peptide vaccine significantly ameliorated the neurodegenerative changes in cerebral infarction through the suppression of activated brain RAS and oxidative stress. The present study demonstrated the usefulness of Ang II peptide vaccination for the treatment of ischemic stroke.

Sources of Funding

This work was supported by grant-in-Aid for Young Scientists (B) Grant Number 24791487.

Disclosures

The Department of Clinical Gene Therapy is financially supported by AnGes MG, Novartis, Shionogi, Boeringher, and Rohto. The Department of Health Development and Medicine is financially supported by AnGes MG and Daicel. The Department of Advanced Clinical Science and Therapeutics is financially supported by AnGes MG. R. Morishita is a founder and stockholder of AnGes MG and a former board member. The other authors report no conflicts.

Footnotes

  • Received September 8, 2016.
  • Revision received January 21, 2017.
  • Accepted February 6, 2017.

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  • Angiotensin II Peptide Vaccine Protects Ischemic Brain Through Reducing Oxidative Stress
    Kouji Wakayama, Munehisa Shimamura, Jun-ichi Suzuki, Ryo Watanabe, Hiroshi Koriyama, Hiroshi Akazawa, Hironori Nakagami, Hideki Mochizuki, Mitsuaki Isobe and Ryuichi Morishita
    Stroke. 2017;48:1362-1368, originally published March 31, 2017
    https://doi.org/10.1161/STROKEAHA.116.016269
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