# No effectiveness of synthetic anti-inflammatory tetrapeptides in a mouse model of ischemic stroke

Heleen M den Hertog, Karen Gertz, Wim A Dik, Dirk J Duncker, Nisar A Khan, Matthias Endres, Robbert Benner, Peter J Koudstaal

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Heleen M den Hertog, Karen Gertz, Wim A Dik, Dirk J Duncker, Nisar A Khan, et al.. No effectiveness of synthetic anti-inflammatory tetrapeptides in a mouse model of ischemic stroke. Journal of Experimental Stroke & Translational Medicine, 2010. hal-04363814

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# No effectiveness of synthetic anti-inflammatory tetrapeptides in a mouse model of ischemic stroke

Heleen M den Hertog¹, Karen Gertz²,³, Wim A Dik⁴, Dirk J Duncker⁵, Nisar A Khan⁴, Matthias Endres²,³, Robbert Benner⁴ and Peter J Koudstaal¹

Departments of ¹ Neurology, ⁴ Immunology, and ⁵ Cardiology, Erasmus University Medical Center, Rotterdam, The Netherlands

² Department of Neurology, ³ Center for Stroke Research Berlin, Charité Universitätsmedizin Berlin, Germany

## Abstract

**Background.** Inflammation plays an important role in the pathophysiology of ischemic stroke and may contribute to secondary damage following ischemic stroke. Recently, it has been shown that synthetic oligopeptides related to human chorionic gonadotropin (hCG) and various other synthetic oligopeptides may have immunomodulatory effects.

We aimed to investigate the effects of two promising synthetic anti-inflammatory tetrapeptides, namely the hCG-related peptide AQGV and the p53-related peptide EPPE on infarct volume and on inflammatory gene expression in a mouse model of focal cerebral ischemia.

**Methods.** In a randomized single-blinded fashion, mice received two intravenous injections of either AQGV (30 mg/kg bodyweight) or EPPE (30mg/kg bodyweight) or phosphate buffered saline. The first dose was administered directly before 90-minutes middle cerebral artery occlusion, and the second dose was injected directly after reperfusion. Infarct volume and gene expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), E-selectin and intercellular adhesion molecule-1 (ICAM-1) were measured 24 hours after reperfusion.

**Results.** We found no significant effect of either AQGV (108.7 +/- 12.1 mm³ versus PBS 114.9 +/- 23.9 mm³; P=0.7) or EPPE (131.8 +/-12.8 mm³ versus PBS 114.9 +/- 23.9 mm³; P=0.1) on direct lesion volume. All three experimental groups displayed similar mRNA expression levels of TNF-α, IL-6, E-selectin and ICAM-1 at 24 hours after the ischemic insult.

**Conclusion.** AQGV and EPPE did not show any beneficial effects in this mouse model of acute ischemic stroke. Further studies are needed to investigate the nature and dynamics of the immunomodulatory effects of synthetic oligopeptides in acute ischemic stroke.

**Keywords:** Ischemic stroke, inflammation, immunomodulation

## 1. Introduction

Inflammation plays a central role in the pathophysiology of ischemic stroke. In the early phase of ischemia and reperfusion, an inflammatory cascade is set off in the ischemic core and in the penumbra, where perfusion is compromised but tissue is still viable (Del Zoppo et al, 2001). Numerous pro-inflammatory genes are upregulated, including those for transcription factors, heat shock proteins, cytokines, chemokines and adhesion molecules. The classical pro-inflammatory cytokines interleukin-6 (IL-6), and tumor

necrosis factor-α (TNF-α), produced by microglia, astrocytes, endothelial cells, and neurons, mediate the inflammatory processes (Wang et al, 2007). They activate leucocytes, stimulate the release of chemokines, and increase adhesion molecule expression on cerebral microvessels and circulating leukocytes, including intercellular adhesion molecule (ICAM-1) and E-selectin (Wang et al, 2007). The early inflammatory phase is followed by a tissue-remodelling phase during which tissue-remodelling factors like MMP9, MMP2 and TGF-β are expressed and is associated with healing of the ischemic brain tissue. The inflam-

<footnote>* **Correspondence should be sent to:**
Heleen M den Hertog, M.D., Ph.D.; Department of Neurology, Erasmus University Medical Center, PO Box 2040, 3000 CA Rotterdam,
The Netherlands; Email address: m.denhertog@erasmusmc.nl; Telephone: +31107040704; Fax: +31107044721.</footnote>
matory processes start within 2 hours after stroke onset and sustain for several days. Besides its beneficial effect, inflammation may contribute to the secondary progression of ischemic brain injury. The acute inflammatory phase is believed to be detrimental, while the chronic phase inflammation may be essential for repair and regeneration (Del Zoppo et al, 2001; Barone et al, 1999; Bowen et al, 2006). This is a concept that holds promise for new therapeutic interventions.

The adaptations of the immune system during pregnancy are striking. The maternal immune system is under tight control to prevent rejection of the foetal allograft. This suggests that a specific hormonal environment is responsible for modulating the immune system during pregnancy. Human chorionic gonadotropin (hCG) is secreted by placental syncytiotrophoblasts, but is also produced by the pituitary gland and leucocytes in non-pregnant females and males (Benner et al, 2005; Birken et al, 1996; Yoshimoto et al, 1979). HCG consists of an α- and a β-chain. In human pregnancy urine, hCG occurs in a variety of forms, including oligopeptidic breakdown products of the αβ-chain (Benner et al, 2005). HCG preparations have been recognized to exert immunomodulatory activities that are not due to the native molecule nor to its α- and β-chain, but resided in specific peptide fractions (Benner et al, 2005). Based on known preferential cleavage sites, several oligopeptides from β-hCG residues have been synthesized. These oligopeptides including AQGV have shown promise in the treatment of ischemia-reperfusion injury such as septic shock (Khan et al, 2002), hemorrhagic shock (van den Berg et al, 2009) and renal ischemia-reperfusion injury (Khan et al, 2009). Several regulatory oligopeptides based on the primary sequence of other proteins have also been generated and proven biologically active in different models tested. One of these regulatory oligopeptides is the p53-related tetrapeptide EPPE.

In this study, as a first step in assessing potential beneficial effects of synthetic anti-inflammatory tetrapeptides in acute ischemic stroke, we investigated the effects of the β-hCG related synthetic tetrapeptide AQGV and the p53-related synthetic tetrapeptide EPPE on infarct volume and on inflammatory gene expression in an animal model of focal cerebral ischemia.

## 2. Methods

### 2.1 Animals

Experiments were performed in male 129S6/SvEv mice, weighing 20-30g, in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and with approval of the local Animal Care Committee.

### 2.2 Synthetic anti-inflammatory tetrapeptides

β-hCG-related AQGV showed the most powerful protection in a model of renal ischemia-reperfusion injury (Khan et al, 2009). Since there are many similarities in the pathophysiology of acute cerebral ischemia-reperfusion injury and acute renal ischemia-reperfusion injury, we decided to study the effects of AQGV, and another promising p53-related tetrapeptide EPPE in our model of focal cerebral ischemia.

AQGV and EPPE were synthesized (Ansynth Service BV Roosendaal, the Netherlands) using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based methodology with a 2-chlorotritylchloride resin as the solid support and dissolved in 0.9% sodium chloride at a concentration of 30 mg/ml.

### 2.3 Model of focal cerebral ischemia

Anesthesia was induced by 1.5% halothane and maintained with 1% halothane in 70% N₂O and 30% O₂. Ischemia was induced by filamentous occlusion of the left middle cerebral artery for 90 minutes as described previously (Gertz et al, 2003). In brief, brain ischemia was induced with an 8.0 nylon monofilament coated with a silicone resin/hardener mixture (Xantopren M Mucosa and Activator NF Optosil Xantopren, Haereus Kulzer, Germany). The filament was introduced into the left common carotid artery up to the anterior cerebral artery. Thereby the middle cerebral artery and anterior choroidal arteries were occluded. Filaments were withdrawn after 90 minutes to allow reperfusion. The core temperature of the mice during the procedure was maintained at 36.5°C using a feedback temperature control unit.

### 2.4 Administration of synthetic anti-inflammatory tetrapeptides

A previous dose-escalating study of AQGV in a model of renal ischemia-reperfusion injury showed that AQGV was effective in doses above 0.3 mg/kg bodyweight (Khan et al, 2009). For this study, we determined the effect of the highest tested dose in the dose escalating study, namely 30mg/kg bodyweight.

Three groups of mice (10 mice per group) received, in a randomized single-blinded fashion, either AQGV (30 mg/kg bodyweight) or EPPE (30mg/kg bodyweight) or 1 ml phosphate buffered saline (PBS), which was administered intravenously in two slow injections (0.1 ml over 5 minutes). Administration of the first dose was started directly before middle cerebral artery occlusion (MCAO). The second dose was administered directly after reperfusion.

### 2.5 Measurement of ischemic lesion size

The animals were sacrificed 24 hours after reperfusion. The brains were snap-frozen in isopentane for cryostat sectioning. Infarct areas were quantitated by
use of sigma 4.0 software (sigmaScan Pro 4.0, Jan-
del scientific) on 20-µm hematoxylin-and eosin-
stained cryostat sections without knowledge of treat-
ment. Direct infarct volumes were calculated by
summing the lesion volumes of each section (we
used direct lesion size) (mm³).

## 2.6 Evaluation of mRNA levels by real-time quantita-
tive polymerase chain reaction

RNA was isolated from 20-µm cryostat sections with
a Qiagen RNeasy kit (QIAGEN, Hilden, Germany)
according to the manufacturer's instructions, and re-
verse transcribed into cDNA. We determined gene
expression levels of TNF-α, IL-6, E-selectin and
ICAM-1 with Applied Biosystems 7700 PCR machine
(Foster City, California), as described previously. The
expression levels of these genes were quantified by
normalization against the mRNA levels of the house-
hold gene ABL.

<figure><img src="image_2.png" /><figcaption>Fig. 1. Effects of treatment with AQGV and EPPE versus PBS on mean direct ischemic lesion volume (mm3) +/- SD.</figcaption></figure>

## 3.2 AQGV and EPPE do not influence mRNA tran-
scription levels of TNF-α, IL-6, ICAM-1 and E-selectin

All three experimental groups displayed comparable
mRNA expression levels of TNF-α, IL-6, ICAM-1 and

E-selectin at 24 hours after the ischemic insult (Fig.
2A and 2B).

## 2.7 Data analysis and presentation

Statistical analyses were performed using SPSS ver-
sion 11 software (SPSS, inc., Chicago III). Data are
presented as mean values +/- SD of the ten mice per
group. Comparisons were made by one-way of va-
riance (ANOVA), followed by Duncan's multiple range
test.

## 3. Results

Of the total 30 mice entered in the study, two mice in
the EPPE group and one mouse in the AQGV group
died, because of technical failure during surgery.

### 3.1 AQGV and EPPE do not alter infarct volume

The direct infarct volume was not significantly differ-
ent between AQGV-treated mice and mice treated
with PBS (108.7 +/- 12.1 mm³ versus 114.9 +/- 23.9
mm³; P=0.7) and between EPPE-treated and mice
treated with PBS (131.8 +/-12.8 mm³ versus 114.9
+/- 23.9 mm³; P=0.1) (Fig.1).
<figure><img src="image_3.png" /><figcaption>Fig 2. Relative expression of mRNA transcription levels of pro-inflammatory cytokines TNF-α and IL-6 (A) and adhesion molecules E-selectin and ICAM-1 (B) in the brain 24 hours after reperfusion. Transcription levels are normalized to the expression level of the household gene ABL. Data are presented as the mean of per group +/- SD.</figcaption></figure>

*Neither AQGV nor EPPE caused significant differences in the transcription levels of TNF-α, IL-6, E-selectin and ICAM-1 as compared with PBS.

4. Discussion

In this study, we found that in an established mouse model of ischemic stroke, treatment with two synthetic anti-inflammatory tetrapeptides had no effect on infarct volume or on mRNA expression levels of adhesion molecules and pro-inflammatory cytokines.

Our results deviate from the beneficial effects of treatment with synthetic anti-inflammatory oligopeptides in various other *in vivo* models of ischemia-reperfusion injury (Khan et al, 2002; van den Berg et al, 2009; Khan et al, 2009). Treatment with such synthetic anti-inflammatory oligopeptides has been shown to inhibit renal ischemia-reperfusion injury and to increase survival rates by reducing systemic inflammation and adhesion molecule expression in the kidney (Khan et al, 2009). These peptides also atte-

nuated inflammation and liver damage after hemorrhagic shock (van den Berg et al, 2009) and decreased morbidity and mortality associated with lipopolysaccharide injection (Khan et al, 2002).

There are several explanations why no such beneficial effects of treatment with synthetic anti-inflammatory oligopeptides were found in our model of acute ischemic stroke. First, the role of inflammation in the acute phase of stroke is ambivalent. The inflammatory reaction following ischemic stroke triggers the removal of noxious agents and supports tissue cleaning and repair. Contrary to these beneficial effects, it may augment secondary damage by disruption of the blood-brain barrier, release of cytotoxic agents, edema resulting from endothelial cell injury and leucocyte-mediated injury, increased body temperature and microvascular thrombosis. Since the underlying mechanisms by which the synthetic anti-
inflammatory oligopeptides exert their effects are un-
clear, it cannot be excluded that they might also ne-
gate the positive effects of the inflammatory reaction.
On the other hand, synthetic anti-inflammatory oligo-
peptides did show beneficial effects in renal ische-
mia-reperfusion injury and there are many similarities
in the pathophysiology of acute cerebral ischemia-
reperfusion injury and acute renal ischemia-
reperfusion injury (Khan et al, 2009).

Furthermore, we did not compare the mRNA expres-
sion levels of adhesion molecules and pro-
inflammatory cytokines with those in brains of sham-
operated animals. Therefore, we do not know wheth-
er these pro-inflammatory genes were upregulated in
our model of ischemic stroke. However, there is sub-
stantial evidence from previous studies that these
genes are upregulated witin the first hours of stroke
onset (Del Zoppo et al, 2001; Wang et al, 2007; Ba-
rone et al, 1999).

It should also be noted that different synthetic anti-
inflammatory oligopeptides might have different mod-
es of action. This is supported by previous studies
that have identified different synthetic anti-inflamma-
tory oligopeptides as the most effective mediator
(Khan et al, 2002; van den Berg et al, 2009; Khan et
al, 2009).

Another explanation of the negative results of our
study is that it was not designed to investigate differ-
ent doses of AQGV or EPPE. Therefore, we cannot
rule out that there is a dose-dependent effect, as was
observed in previous studies (Khan et al, 2002; van
den Berg et al, 2009; Khan et al 2009). Also, the two
time-points of peptide administration chosen for the
current study may not have been optimal. Neither can
we exclude that repeated peptide administrations
during the study period could have been effective.

We have studied only the effects of AQGV and EPPE
on infarct volume and mRNA transcription levels at
24 hours after reperfusion. As the inflammatory reac-
tion is initiated within a few hours after stroke onset
and lasts for several days, it may be worthwhile to
monitor the early evolution of brain injury at different
points in time with MRI scanning (Hoehn et al, 2001).
The major advantage of MRI over histologic examina-
tions is its potential to non-invasively assess changes
in the brain in vivo. Finally, since no measurements
of brain concentrations of the administered tetrapep-
tides were performed we do not know whether the
oligopeptides underwent presystemic transformation
and actually crossed the blood-brain barrier. However,
a previous study showed that hCG crossed the
blood-brain barrier in an intact form in rats (Lukacs et
al, 1995). Furthermore, their small size as well as the
disruption of the blood-brain barrier following ischem-
ic stroke makes it likely that tetrapeptides did cross
the blood-brain barrier.

HCG is not the only protein source for regulatory oli-
gopeptides. Sequences of other proteins such as C-
reactive protein Ig heaving chain, phospholipase A2,
hemoglobin, b-defensins, lactoferrin and granulysin
possess anti-inflammatory activities with the function
being expressed upon the degradation of the parent
protein (King et al, 2002; King et al, 2003; King et al
2007; Miele et al 1988; Parish et al, 2001; Rawlings
et al, 2008; Robey et al, 1987). So far about 500 hu-
man genes encoding proteases/peptidases have
been identified. This might be related to regulation of
biological processes by peptidases and might imply
that virtually every protein within the body can serve
as a source of regulatory oligopeptides.

Our results do not negate the possibility that anti-
inflammatory oligopeptides might be useful for acute
stroke treatment. Further studies are needed to in-
vestigate the nature and dynamics of the immuno-
modulatory effects of synthetic anti-inflammatory oli-
gopeptides in acute ischemic stroke.

**5. Conclusion**

AQGV and EPPE did not show any beneficial effects
in this mouse model of acute ischemic stroke. Further
studies are needed to investigate the nature and dy-
namics of the immunomodulatory effects of synthetic
oligopeptides in acute ischemic stroke.

**Conflict of Interest**

None

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