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Polyethylene glycol-conjugated human adrenomedullin as a possible treatment for vascular dementia
Peptides 121 (2019) 170133
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Polyethylene glycol-conjugated human adrenomedullin as a possible treatment for vascular dementia
T
⁎
Sayaka Nagata , Motoo Yamasaki, Kazuo Kitamura Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Adrenomedullin PEGylated Ischemic brain injury Four-vessel occlusion
Adrenomedullin (AM) is a multifunctional bioactive peptide. Recent studies have shown that AM has protective effects against ischemic brain damage. We recently prepared a long-acting human AM derivative that was conjugated with a 60 kDa polyethylene glycol (PEG-AM), which had an effect similar to that of native AM. In this study, we examined the effect of PEG-AM on four-vessel occlusion model rats, which exhibit vascular dementia. From day 10 to day 14 after surgery, the learning and memory abilities of the rats were examined using a Morris water maze. The rats were treated with a single subcutaneous injection of 1.0 or 10.0 nmol/kg of PEG-AM. PEGAM treatment reduced the escape latency in the hidden platform test. Furthermore, the treatment increased the time spent in the platform quadrant in the probe test. The data showed that PEG-AM injection prevented memory loss and learning disorders in dose-dependent manner. On day 14, the immunoreactive AM concentration in plasma was 9.749 ± 2.167 pM in the high-dose group (10.0 nmol/kg) and 0.334 ± 0.073 pM in the low-dose group (1.0 nmol/kg). However, even in the low-dose group, a significant effect was observed in both tests. The present data indicate that PEG-AM is a possible therapeutic agent for the treatment of ischemic brain injury or vascular dementia.
1. Introduction
It displayed decreased acute hypotensive activity and prolonged the plasma half-life of hAM. A single subcutaneous injection of PEG-AM improved IBD in a dextran sulfate sodium (DSS)-induced colitis mouse model [8]. We speculated that, similar to native hAM, PEG-AM treatment may be effective for treatment of other diseases. In the present study, we evaluated the effect of PEG-AM on cerebral ischemia using a four-vessel occlusion model, which is often used as a model of memory and learning disorders [9].
Human adrenomedullin (hAM) is a 52 amino acid peptide with an amidated C-terminus and a disulfide bond. hAM was originally isolated from a human pheochromocytoma in 1993 [1] and is widely distributed among various organs and tissues, including normal adrenal medulla, heart, blood vessel, kidney, lung, pancreas and the central nervous system [2]. In the brain, the highest concentrations of hAM are detected at in the thalamus and hypothalamus [3]. In addition, hAM is also expressed in pathological conditions such as neoplasms or ischemic injury [4]. hAM has shown several therapeutic effects in experimental models of various diseases, including ischemic heart disease, inflammatory bowel disease (IBD) and stroke [5–7]. However, in these studies, longterm intravenous injection of hAM was necessary because hAM is rapidly cleared from the blood. Therefore, in a previous study [8], we developed a human adrenomedullin derivative that was conjugated with a 60 kDa polyethylene glycol at the N terminus (PEG-AM). PEG-AM stimulated cAMP production in cultured human embryonic kidney cells expressing AM receptors. PEG-AM was detectable in the blood at 10 days after injection.
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2. Materials and methods 2.1. Induction of ischemic brain injury in rats and PEG-AM treatment 8-week-old male Wistar rats were purchased from Japan SLC, Inc. (Shizuoka, Japan) and housed under a 12-h light/12-h dark cycle in specific pathogen-free conditions with a normal diet. The present study was performed in accordance with the Animal Welfare Act and with the approval of the LSI Medience Corporation Institutional Animal Care and Use Committee (2015-0629). PEG-AM (1.0 or 10.0 nmol/kg) or saline (control) was administered to the rats (n = 10 per group) via a single subcutaneous injection before vertebral artery coagulation. The rats
Corresponding author at: Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. E-mail address: [email protected] (S. Nagata).
https://doi.org/10.1016/j.peptides.2019.170133 Received 25 June 2019; Received in revised form 15 August 2019; Accepted 19 August 2019 Available online 23 August 2019 0196-9781/ © 2019 Elsevier Inc. All rights reserved.
Peptides 121 (2019) 170133
S. Nagata, et al.
were then anesthetized with 2% isoflurane in a mixture of 30% O2 and 70% N2O during surgery. Both sides of the vertebral artery were electro-coagulated by an electrode needle. The next day, both sides of the common carotid artery were temporarily occluded with suture thread. Thirty minutes after induction of ischemia, reperfusion was initiated by removal of the suture thread. PEG-AM was prepared as described previously [8]. 2.2. Morris water maze test The spatial learning and memory performances of rats were evaluated using the Morris water maze test. In brief, rats were required to locate a hidden platform (12 cm in diameter) immediately below the surface (1 cm) of the water in a circular pool (150 cm in diameter and 30 cm in depth, maintained at 23 ± 1 °C) that was located in a room with indirect lighting. The test included two phases: a hidden platform test and a probe test. At the beginning of the trial, rats were placed into the pool in one of the three quadrants that did not contain the platform. From day 10 to day 14 after surgery, rats performed four trials per day in the Morris water maze. The trials were initiated from different positions in the pool. The platform was consistently located in the target quadrant, and rats were given 90 s. to locate the platform. We measured the arrival time at the platform (escape latency). Animals that failed to find the escape platform in the time provided were placed on the platform for 30 s. A probe test was conducted on day 14 at 1 h after the final trial to examine spatial reference memory. The platform was removed from the pool, and rats were allowed to swim freely for 60 s. Performances were videotaped and analyzed using image tracking software, which calculated the time required to locate the platform (Smart, Panlab), and the time that rats remained in the quadrant with the platform was calculated as follows: Time spent in the platform quadrant (%) = (time spent in the quadrant with the platform (sec)/60 s)×100
Fig. 1. Effect of PEG-AM on the hidden platform test results in rats with vascular dementia (n = 10). The results are shown as the means ± SEM. * P < 0.05 vs. control.
increased the time spent in the platform quadrant in the probe test. The percentages of time spent in the platform quadrant (%) for the control and low and high-dose PEG-AM (1.0 and 10.0 nmol/kg) groups were 33.0 ± 4.6, 48.3 ± 1.6 and 52.4 ± 3.0%, respectively. The time spent in the platform quadrant was significantly increased in rats treated with low and high doses of PEG-AM compared with the control group. No difference was observed between low- and high-dose groups (Fig. 2). 3.3. Plasma concentration of immunoreactive AM in rats after the trial We examined the hAM blood concentrations of rats in different groups after the trial. The immunoreactive hAM concentrations in the 1.0 and 10.0 nmol/kg PEG-AM groups were 0.334 ± 0.073 and 9.749 ± 2.167 pM, respectively (n = 10) (Fig. 3). Immunoreactive hAM was not detectable in the control group.
2.3. Plasma concentration of immunoreactive hAM in rats after the trial Peripheral blood samples were collected via the abdominal aorta in tubes containing heparin sodium, and plasma samples were obtained by centrifugation at 1800×g. Immunoreactive hAM was measured in rat plasma using a fluorescence enzyme immunoassay as previously described [10].
4. Discussion The present study showed that a single subcutaneous injection of PEG-AM prevented memory disturbances and learning disabilities in rats with ischemic brain injury. In the case of AM treatment, long-term intravenous injection of AM is necessary because AM is rapidly cleared from the blood. In the present study, we collected blood samples and then determined the blood
2.4. Statistical analysis All data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). Multiple comparisons were evaluated by two-way and one-way ANOVA followed by Tukey’s multiple comparison test. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. Effects of PEG-AM on the hidden platform test results In rats with ischemic brain injury, treatment with PEG-AM shortened the escape latency in a dose-dependent manner. On day 11 after surgery, the escape latency was significantly shorter in rats treated with high-dose PEG-AM (10.0 nmol/kg) than in the control group. On days 12 and 13 after surgery, the escape latency was significantly shorter in rats treated with low and high doses of PEG-AM (1.0 and 10.0 nmol/kg) than in the control group (Fig. 1).
Fig. 2. Effect of the PEG-AM on the probe test results in rats with vascular dementia (n = 10). The results are shown as the means ± SEM. ** P < 0.01 vs. control.
3.2. Effects of PEG-AM on the probe test results In rats with ischemic brain injury, treatment with PEG-AM 2
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concentration of AM is not understood, increased AM levels may protect the brain similar to increased atrial natriuretic peptide in heart failure. However, the human brain tissue level of proAM protein (14-kDa) was decreased in parallel with microtuble dismantlement in patients with frontotemporal lobar degeneration that presents with primary tauopathy, when compared to non-demented control patients [23]. Therefore, further detailed studies should be performed to clarify the roles of AM in dementia. AM exerts its actions through a combination of the calcitonin receptor-like receptor (CLR) and either receptor activity modifying protein 2 (RAMP2) or RAMP3. CLR/RAMP2 is the main receptor of AM and is highly expressed in the brain [24]. In one study, RAMP2 knockout mice required more time to solve a maze [24]. PEG-AM stimulated cAMP production in cultured human embryonic kidney cells expressing CLR/RAMP2 receptor [8]. The increase in cAMP levels leads to activation of protein kinase A (PKA). PKA phosphorylates eNOS at serine 633, and NO is released in the endothelium [25]. Thus, the vasodilator action may contribute the maintenance of cerebral blood flow. Furthermore, the neuroprotective mechanisms of AM after ischemic brain injury may be mediated by both suppression of apoptosis and promotion of angiogenesis [7]. In addition, AM increases the blood flow in the brain and spinal cord [26]. We hypothesized that PEG-AM could protect the brain via these effects. PEG-AM is thought to act directly on the AM receptor in the brain. The blood-brain barrier (BBB) typically does not allow large molecules such as PEG-AM to pass. We speculate that PEGAM can reach the brain tissue because the BBB is disrupted in this model. In the present study, PEG-AM was injected before surgery. Further studies should focus on PEG-AM treatment during the post-ischemic time period. In addition, we did not evaluate the tissue or the mechanism of action of a single injection of PEG-AM. In future studies, we will investigate the tissue to determine the mechanism of action of PEGAM. In conclusion, we demonstrated that PEG-AM exerts neuroprotective actions in the ischemic brain. The present data suggest the potential of PEG-AM as a novel therapeutic tool, without the need for longterm infusion, for treatment of ischemic brain injury. However, further studies are necessary to confirm these hypotheses.
Fig. 3. Plasma concentrations of PEG-AM after the trial. The results are shown as the means ± SEM. * P < 0.0001 vs. 1.0 nmol/kg PEG-AM.
concentration of PEG-AM after the trial. In the low-dose group (1.0 nmol/kg), the PEG-AM blood concentration was very low at 2 weeks after administration. However, in the high-dose group (10.0 nmol/kg), effective blood concentrations of PEG-AM were still observed for 2 weeks after a single subcutaneous injection. These results are consistent with the half-life of PEG-AM described in our previous study [8]. In our previous study, we demonstrated that the acute hypotension caused by native AM was decreased by PEGylation [10]. This effect is beneficial for the treatment of stroke using PEG-AM because the acute decrease in blood pressure exacerbates stroke. In addition, the pharmacological effects of PEG-AM were comparable in the low- and highdose groups. In other studies, injection of native AM at a high concentration (1.0 μg/kg/min) improved ischemic brain injury [11]. PEGAM had a neuroprotective effect, even when it was injected in a much lower dose than native AM. It should be noted that treatment can be performed with a very low dose. In some studies, injection of an AM resulted in exacerbation of brain damage [4]. Knockout (KO) mice lacking AM in the endothelium exhibited a smaller infarct volume after permanent focal cerebral ischemia [12]. In other reports, normal aging resulted in increased expression of AM in the brain, and AM ablation prevented Tau phosphorylation in female mice and favored memory preservation in advanced age [13]. However, several studies reported that AM administration is beneficial in models of stroke [7] and vascular dementia [14,11]. Transgenic (Tg) mice that overproduce AM showed reduced oxidative stress, apoptosis and infarct volume after ischemic brain injury compared with wild type mice [15]. Furthermore, experiments examining acute [12] and chronic [13] ischemia in the brain using AM KO mice showed increased tissue damage in these mice compared with wild type mice. These data suggest that AM may be an important therapeutic agent for brain ischemic injury [16]. The four-vessel occlusion model, which results in transient global brain ischemia, was used in this study. Other models that more accurately represent stroke and Alzheimer’s disease exist. In AAPosk-Tg mice, an E693Δ amyloid precursor protein mutation causes Alzheimer’s-type dementia because of enhanced formation of synaptotoxic amyloid β oligomerization, and these mice are used as a model for Alzheimer’s disease [17]. In addition, SJLB mice, which express human tau cDNA with an N279 K mutation, are used to model human dementia [18]. We plan to examine the effects of PEG-AM in this model of dementia in a future study, even though the results in the present study were clear. Clinical studies indicate that high AM levels or increasing AM from the first to the second day post-stroke predict a poor outcome in stroke patients [19–21]. In addition, midregional proAM also increases in Alzheimer's disease [22]. Consequently, the AM level is related to the severity of tissue damage. Although the reason for the increased plasma
Declaration of Competing Interest The authors disclose that Nagata S., Yamasaki M. and Kitamura K. own stock in the Himuka AM Pharma Corporation. Acknowledgements This work was supported in part by a Scientific Research Grant for Creating Start-ups from the Advanced Research and Technology (START Program) from the Japan Science and Technology Agency (grant number: ST262010WV). References [1] K. Kangawa, K. Kitamura, M. Kawamoto, Y. Ichiki, S. Nakamura, H. Matsuo, T. Eto, Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma, Biochem. Biophys. Res. Commun. 192 (1993) 553–560. [2] Y. Ichiki, K. Kitamura, K. Kangawa, M. Kawamoto, H. Matsuo, T. Eto, Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma, FEBS Lett. 338 (1994) 6–10. [3] F. Satoh, K. Takahashi, O. Murakami, K. Totsune, M. Sone, M. Ohneda, K. Abe, Y. Miura, Y. Hayashi, H. Sasano, et al., Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma, J. Clin. Endocrinol. Metab. 80 (1995) 1750–1752. [4] X. Wang, T.L. Yue, F.C. Barone, R.F. White, R.K. Clark, R.N. Willette, A.C. Sulpizio, N.V. Aiyar, R.R. Ruffolo Jr, G.Z. Feuerstein, Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage, Proc Natl Acad Sci U S A 92 (1995) 11480–11484. [5] S. Nagata, T. Hikosaka, K. Kitamura, Effect of adrenomedullin administration in two rat models of experimental inflammatory bowel disease, Am. J. Life Sci. 3 (2015)
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[17] T. Tomiyama, T. Nagata, H. Shimada, R. Teraoka, A. Fukushima, H. Kanemitsu, H. Takuma, R. Kuwano, M. Imagawa, S. Ataka, Y. Wada, E. Yoshioka, T. Nishizaki, Y. Watanabe, H. Mori, A new amyloid beta variant favoring oligomerization in Alzheimer’s-type dementia, Ann. Neurol. 63 (2008) 377–387. [18] T. Taniguchi, N. Doe, S. Matsuyama, Y. Kitamura, H. Mori, N. Saito, C. Tanaka, Transgenic mice expressing mutant (N279K) human tau show mutation dependent cognitive deficits without neurofibrillary tangle formation, FEBS Lett. 579 (2005) 5704–5712. [19] M. Serrano-Ponz, C. Rodrigo-Gasque, E. Siles, E. Martinez-Lara, L. Ochoa-Callejero, A. Martinez, Temporal profiles of blood pressure, circulating nitric oxide, and adrenomedullin as predictors of clinical outcome in acute ischemic stroke patients, Mol. Med. Rep. 13 (2016) 3724–3734. [20] H. Zhang, B. Tang, C.G. Yin, Y. Chen, Q.L. Meng, L. Jiang, W.P. Wang, G.Z. Niu, Plasma adrenomedullin levels are associated with long-term outcomes of acute ischemic stroke, Peptides 52 (2014) 44–48. [21] J. Liu, J. Yan, J.M. Greer, S.J. Read, R.D. Henderson, S.E. Rose, A. Coulthard, P.A. McCombe, Correlation of adrenomedullin gene expression in peripheral blood leukocytes with severity of ischemic stroke, Int. J. Neurosci. 124 (2014) 271–280. [22] K. Buerger, O. Uspenskaya, O. Hartmann, O. Hansson, L. Minthon, K. Blennow, H.J. Moeller, S.J. Teipel, A. Ernst, A. Bergmann, H. Hampel, Prediction of Alzheimer’s disease using midregional proadrenomedullin and midregional proatrial natriuretic peptide: a retrospective analysis of 134 patients with mild cognitive impairment, J. Clin. Psychiatry 72 (2011) 556–563. [23] H. Ferrero, I.M. Larrayoz, M. Solas, A. Martinez, M.J. Ramirez, F.J. Gil-Bea, Reduced adrenomedullin parallels microtubule dismantlement in frontotemporal lobar degeneration, Mol. Neurobiol. 55 (2018) 9328–9333. [24] K. Igarashi, T. Sakurai, A. Kamiyoshi, Y. Ichikawa-Shindo, H. Kawate, A. Yamauchi, Y. Toriyama, M. Tanaka, T. Liu, X. Xian, A. Imai, L. Zhai, S. Owa, T. Koyama, R. Uetake, M. Ihara, T. Shindo, Pathophysiological roles of adrenomedullin-RAMP2 system in acute and chronic cerebral ischemia, Peptides 62 (2014) 21–31. [25] A. Iring, Y.J. Jin, J. Albarran-Juarez, M. Siragusa, S. Wang, P.T. Dancs, A. Nakayama, S. Tonack, M. Chen, C. Kunne, A.M. Sokol, S. Gunther, A. Martinez, I. Fleming, N. Wettschureck, J. Graumann, L.S. Weinstein, S. Offermanns, Shear stress-induced endothelial adrenomedullin signaling regulates vascular tone and blood pressure, J. Clin. Invest. 130 (2019) 2775–2791. [26] K. Nakamura, H. Toda, K. Terasako, M. Kakuyama, Y. Hatano, K. Mori, K. Kangawa, Vasodilative effect of adrenomedullin in isolated arteries of the dog, Jpn. J. Pharmacol. 67 (1995) 259–262.
39–42. [6] R. Nakamura, J. Kato, K. Kitamura, H. Onitsuka, T. Imamura, Y. Cao, K. Marutsuka, Y. Asada, K. Kangawa, T. Eto, Adrenomedullin administration immediately after myocardial infarction ameliorates progression of heart failure in rats, Circulation 110 (2004) 426–431. [7] C.F. Xia, H. Yin, C.V. Borlongan, J. Chao, L. Chao, Postischemic infusion of adrenomedullin protects against ischemic stroke by inhibiting apoptosis and promoting angiogenesis, Exp. Neurol. 197 (2006) 521–530. [8] S. Nagata, M. Yamasaki, K. Kitamura, Anti-inflammatory effects of PEGylated human adrenomedullin in a mouse DSS-induced colitis model, Drug Dev. Res. 78 (2017) 129–134. [9] H.H. Zhou, L. Zhang, H.X. Zhang, J.P. Zhang, W.H. Ge, Chimeric peptide Tat-HANR2B9c improves regenerative repair after transient global ischemia, Front. Neurol. 8 (2017) 509. [10] K. Kuwasako, K. Kubo, M. Tokashiki, M. Tamura, S. Tsuda, S. Kubo, K. YoshizawaKumagaye, J. Kato, K. Kitamura, Biological properties of adrenomedullin conjugated with polyethylene glycol, Peptides 57 (2014) 118–121. [11] A. Dogan, Y. Suzuki, N. Koketsu, K. Osuka, K. Saito, M. Takayasu, M. Shibuya, J. Yoshida, Intravenous infusion of adrenomedullin and increase in regional cerebral blood flow and prevention of ischemic brain injury after middle cerebral artery occlusion in rats, J. Cereb. Blood Flow Metab. 17 (1997) 19–25. [12] L. Ochoa-Callejero, A. Pozo-Rodrigalvarez, R. Martinez-Murillo, A. Martinez, Lack of adrenomedullin in mouse endothelial cells results in defective angiogenesis, enhanced vascular permeability, less metastasis, and more brain damage, Sci. Rep. 6 (2016) 33495. [13] I.M. Larrayoz, H. Ferrero, E. Martisova, F.J. Gil-Bea, M.J. Ramirez, A. Martinez, Adrenomedullin contributes to age-related memory loss in mice and is elevated in aging human brains, Front. Mol. Neurosci. 10 (2017) 384. [14] K. Watanabe, M. Takayasu, A. Noda, M. Hara, T. Takagi, Y. Suzuki, J. Yoshia, Adrenomedullin reduces ischemic brain injury after transient middle cerebral artery occlusion in rats, Acta Neurochir. (Wien) 143 (2001) 1157–1161. [15] K. Miyashita, H. Itoh, H. Arai, T. Suganami, N. Sawada, Y. Fukunaga, M. Sone, K. Yamahara, T. Yurugi-Kobayashi, K. Park, N. Oyamada, N. Sawada, D. Taura, H. Tsujimoto, T.H. Chao, N. Tamura, M. Mukoyama, K. Nakao, The neuroprotective and vasculo-neuro-regenerative roles of adrenomedullin in ischemic brain and its therapeutic potential, Endocrinology 147 (2006) 1642–1653. [16] K. Sugio, N. Horigome, T. Sakaguchi, M. Goto, A model of bilateral hemispheric ischemia–modified four-vessel occlusion in rats, Stroke 19 (1988) 922.
4
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Polyethylene glycol-conjugated human adrenomedullin as a possible treatment for vascular dementia
T
⁎
Sayaka Nagata , Motoo Yamasaki, Kazuo Kitamura Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Adrenomedullin PEGylated Ischemic brain injury Four-vessel occlusion
Adrenomedullin (AM) is a multifunctional bioactive peptide. Recent studies have shown that AM has protective effects against ischemic brain damage. We recently prepared a long-acting human AM derivative that was conjugated with a 60 kDa polyethylene glycol (PEG-AM), which had an effect similar to that of native AM. In this study, we examined the effect of PEG-AM on four-vessel occlusion model rats, which exhibit vascular dementia. From day 10 to day 14 after surgery, the learning and memory abilities of the rats were examined using a Morris water maze. The rats were treated with a single subcutaneous injection of 1.0 or 10.0 nmol/kg of PEG-AM. PEGAM treatment reduced the escape latency in the hidden platform test. Furthermore, the treatment increased the time spent in the platform quadrant in the probe test. The data showed that PEG-AM injection prevented memory loss and learning disorders in dose-dependent manner. On day 14, the immunoreactive AM concentration in plasma was 9.749 ± 2.167 pM in the high-dose group (10.0 nmol/kg) and 0.334 ± 0.073 pM in the low-dose group (1.0 nmol/kg). However, even in the low-dose group, a significant effect was observed in both tests. The present data indicate that PEG-AM is a possible therapeutic agent for the treatment of ischemic brain injury or vascular dementia.
1. Introduction
It displayed decreased acute hypotensive activity and prolonged the plasma half-life of hAM. A single subcutaneous injection of PEG-AM improved IBD in a dextran sulfate sodium (DSS)-induced colitis mouse model [8]. We speculated that, similar to native hAM, PEG-AM treatment may be effective for treatment of other diseases. In the present study, we evaluated the effect of PEG-AM on cerebral ischemia using a four-vessel occlusion model, which is often used as a model of memory and learning disorders [9].
Human adrenomedullin (hAM) is a 52 amino acid peptide with an amidated C-terminus and a disulfide bond. hAM was originally isolated from a human pheochromocytoma in 1993 [1] and is widely distributed among various organs and tissues, including normal adrenal medulla, heart, blood vessel, kidney, lung, pancreas and the central nervous system [2]. In the brain, the highest concentrations of hAM are detected at in the thalamus and hypothalamus [3]. In addition, hAM is also expressed in pathological conditions such as neoplasms or ischemic injury [4]. hAM has shown several therapeutic effects in experimental models of various diseases, including ischemic heart disease, inflammatory bowel disease (IBD) and stroke [5–7]. However, in these studies, longterm intravenous injection of hAM was necessary because hAM is rapidly cleared from the blood. Therefore, in a previous study [8], we developed a human adrenomedullin derivative that was conjugated with a 60 kDa polyethylene glycol at the N terminus (PEG-AM). PEG-AM stimulated cAMP production in cultured human embryonic kidney cells expressing AM receptors. PEG-AM was detectable in the blood at 10 days after injection.
⁎
2. Materials and methods 2.1. Induction of ischemic brain injury in rats and PEG-AM treatment 8-week-old male Wistar rats were purchased from Japan SLC, Inc. (Shizuoka, Japan) and housed under a 12-h light/12-h dark cycle in specific pathogen-free conditions with a normal diet. The present study was performed in accordance with the Animal Welfare Act and with the approval of the LSI Medience Corporation Institutional Animal Care and Use Committee (2015-0629). PEG-AM (1.0 or 10.0 nmol/kg) or saline (control) was administered to the rats (n = 10 per group) via a single subcutaneous injection before vertebral artery coagulation. The rats
Corresponding author at: Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. E-mail address: [email protected] (S. Nagata).
https://doi.org/10.1016/j.peptides.2019.170133 Received 25 June 2019; Received in revised form 15 August 2019; Accepted 19 August 2019 Available online 23 August 2019 0196-9781/ © 2019 Elsevier Inc. All rights reserved.
Peptides 121 (2019) 170133
S. Nagata, et al.
were then anesthetized with 2% isoflurane in a mixture of 30% O2 and 70% N2O during surgery. Both sides of the vertebral artery were electro-coagulated by an electrode needle. The next day, both sides of the common carotid artery were temporarily occluded with suture thread. Thirty minutes after induction of ischemia, reperfusion was initiated by removal of the suture thread. PEG-AM was prepared as described previously [8]. 2.2. Morris water maze test The spatial learning and memory performances of rats were evaluated using the Morris water maze test. In brief, rats were required to locate a hidden platform (12 cm in diameter) immediately below the surface (1 cm) of the water in a circular pool (150 cm in diameter and 30 cm in depth, maintained at 23 ± 1 °C) that was located in a room with indirect lighting. The test included two phases: a hidden platform test and a probe test. At the beginning of the trial, rats were placed into the pool in one of the three quadrants that did not contain the platform. From day 10 to day 14 after surgery, rats performed four trials per day in the Morris water maze. The trials were initiated from different positions in the pool. The platform was consistently located in the target quadrant, and rats were given 90 s. to locate the platform. We measured the arrival time at the platform (escape latency). Animals that failed to find the escape platform in the time provided were placed on the platform for 30 s. A probe test was conducted on day 14 at 1 h after the final trial to examine spatial reference memory. The platform was removed from the pool, and rats were allowed to swim freely for 60 s. Performances were videotaped and analyzed using image tracking software, which calculated the time required to locate the platform (Smart, Panlab), and the time that rats remained in the quadrant with the platform was calculated as follows: Time spent in the platform quadrant (%) = (time spent in the quadrant with the platform (sec)/60 s)×100
Fig. 1. Effect of PEG-AM on the hidden platform test results in rats with vascular dementia (n = 10). The results are shown as the means ± SEM. * P < 0.05 vs. control.
increased the time spent in the platform quadrant in the probe test. The percentages of time spent in the platform quadrant (%) for the control and low and high-dose PEG-AM (1.0 and 10.0 nmol/kg) groups were 33.0 ± 4.6, 48.3 ± 1.6 and 52.4 ± 3.0%, respectively. The time spent in the platform quadrant was significantly increased in rats treated with low and high doses of PEG-AM compared with the control group. No difference was observed between low- and high-dose groups (Fig. 2). 3.3. Plasma concentration of immunoreactive AM in rats after the trial We examined the hAM blood concentrations of rats in different groups after the trial. The immunoreactive hAM concentrations in the 1.0 and 10.0 nmol/kg PEG-AM groups were 0.334 ± 0.073 and 9.749 ± 2.167 pM, respectively (n = 10) (Fig. 3). Immunoreactive hAM was not detectable in the control group.
2.3. Plasma concentration of immunoreactive hAM in rats after the trial Peripheral blood samples were collected via the abdominal aorta in tubes containing heparin sodium, and plasma samples were obtained by centrifugation at 1800×g. Immunoreactive hAM was measured in rat plasma using a fluorescence enzyme immunoassay as previously described [10].
4. Discussion The present study showed that a single subcutaneous injection of PEG-AM prevented memory disturbances and learning disabilities in rats with ischemic brain injury. In the case of AM treatment, long-term intravenous injection of AM is necessary because AM is rapidly cleared from the blood. In the present study, we collected blood samples and then determined the blood
2.4. Statistical analysis All data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). Multiple comparisons were evaluated by two-way and one-way ANOVA followed by Tukey’s multiple comparison test. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. Effects of PEG-AM on the hidden platform test results In rats with ischemic brain injury, treatment with PEG-AM shortened the escape latency in a dose-dependent manner. On day 11 after surgery, the escape latency was significantly shorter in rats treated with high-dose PEG-AM (10.0 nmol/kg) than in the control group. On days 12 and 13 after surgery, the escape latency was significantly shorter in rats treated with low and high doses of PEG-AM (1.0 and 10.0 nmol/kg) than in the control group (Fig. 1).
Fig. 2. Effect of the PEG-AM on the probe test results in rats with vascular dementia (n = 10). The results are shown as the means ± SEM. ** P < 0.01 vs. control.
3.2. Effects of PEG-AM on the probe test results In rats with ischemic brain injury, treatment with PEG-AM 2
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concentration of AM is not understood, increased AM levels may protect the brain similar to increased atrial natriuretic peptide in heart failure. However, the human brain tissue level of proAM protein (14-kDa) was decreased in parallel with microtuble dismantlement in patients with frontotemporal lobar degeneration that presents with primary tauopathy, when compared to non-demented control patients [23]. Therefore, further detailed studies should be performed to clarify the roles of AM in dementia. AM exerts its actions through a combination of the calcitonin receptor-like receptor (CLR) and either receptor activity modifying protein 2 (RAMP2) or RAMP3. CLR/RAMP2 is the main receptor of AM and is highly expressed in the brain [24]. In one study, RAMP2 knockout mice required more time to solve a maze [24]. PEG-AM stimulated cAMP production in cultured human embryonic kidney cells expressing CLR/RAMP2 receptor [8]. The increase in cAMP levels leads to activation of protein kinase A (PKA). PKA phosphorylates eNOS at serine 633, and NO is released in the endothelium [25]. Thus, the vasodilator action may contribute the maintenance of cerebral blood flow. Furthermore, the neuroprotective mechanisms of AM after ischemic brain injury may be mediated by both suppression of apoptosis and promotion of angiogenesis [7]. In addition, AM increases the blood flow in the brain and spinal cord [26]. We hypothesized that PEG-AM could protect the brain via these effects. PEG-AM is thought to act directly on the AM receptor in the brain. The blood-brain barrier (BBB) typically does not allow large molecules such as PEG-AM to pass. We speculate that PEGAM can reach the brain tissue because the BBB is disrupted in this model. In the present study, PEG-AM was injected before surgery. Further studies should focus on PEG-AM treatment during the post-ischemic time period. In addition, we did not evaluate the tissue or the mechanism of action of a single injection of PEG-AM. In future studies, we will investigate the tissue to determine the mechanism of action of PEGAM. In conclusion, we demonstrated that PEG-AM exerts neuroprotective actions in the ischemic brain. The present data suggest the potential of PEG-AM as a novel therapeutic tool, without the need for longterm infusion, for treatment of ischemic brain injury. However, further studies are necessary to confirm these hypotheses.
Fig. 3. Plasma concentrations of PEG-AM after the trial. The results are shown as the means ± SEM. * P < 0.0001 vs. 1.0 nmol/kg PEG-AM.
concentration of PEG-AM after the trial. In the low-dose group (1.0 nmol/kg), the PEG-AM blood concentration was very low at 2 weeks after administration. However, in the high-dose group (10.0 nmol/kg), effective blood concentrations of PEG-AM were still observed for 2 weeks after a single subcutaneous injection. These results are consistent with the half-life of PEG-AM described in our previous study [8]. In our previous study, we demonstrated that the acute hypotension caused by native AM was decreased by PEGylation [10]. This effect is beneficial for the treatment of stroke using PEG-AM because the acute decrease in blood pressure exacerbates stroke. In addition, the pharmacological effects of PEG-AM were comparable in the low- and highdose groups. In other studies, injection of native AM at a high concentration (1.0 μg/kg/min) improved ischemic brain injury [11]. PEGAM had a neuroprotective effect, even when it was injected in a much lower dose than native AM. It should be noted that treatment can be performed with a very low dose. In some studies, injection of an AM resulted in exacerbation of brain damage [4]. Knockout (KO) mice lacking AM in the endothelium exhibited a smaller infarct volume after permanent focal cerebral ischemia [12]. In other reports, normal aging resulted in increased expression of AM in the brain, and AM ablation prevented Tau phosphorylation in female mice and favored memory preservation in advanced age [13]. However, several studies reported that AM administration is beneficial in models of stroke [7] and vascular dementia [14,11]. Transgenic (Tg) mice that overproduce AM showed reduced oxidative stress, apoptosis and infarct volume after ischemic brain injury compared with wild type mice [15]. Furthermore, experiments examining acute [12] and chronic [13] ischemia in the brain using AM KO mice showed increased tissue damage in these mice compared with wild type mice. These data suggest that AM may be an important therapeutic agent for brain ischemic injury [16]. The four-vessel occlusion model, which results in transient global brain ischemia, was used in this study. Other models that more accurately represent stroke and Alzheimer’s disease exist. In AAPosk-Tg mice, an E693Δ amyloid precursor protein mutation causes Alzheimer’s-type dementia because of enhanced formation of synaptotoxic amyloid β oligomerization, and these mice are used as a model for Alzheimer’s disease [17]. In addition, SJLB mice, which express human tau cDNA with an N279 K mutation, are used to model human dementia [18]. We plan to examine the effects of PEG-AM in this model of dementia in a future study, even though the results in the present study were clear. Clinical studies indicate that high AM levels or increasing AM from the first to the second day post-stroke predict a poor outcome in stroke patients [19–21]. In addition, midregional proAM also increases in Alzheimer's disease [22]. Consequently, the AM level is related to the severity of tissue damage. Although the reason for the increased plasma
Declaration of Competing Interest The authors disclose that Nagata S., Yamasaki M. and Kitamura K. own stock in the Himuka AM Pharma Corporation. Acknowledgements This work was supported in part by a Scientific Research Grant for Creating Start-ups from the Advanced Research and Technology (START Program) from the Japan Science and Technology Agency (grant number: ST262010WV). References [1] K. Kangawa, K. Kitamura, M. Kawamoto, Y. Ichiki, S. Nakamura, H. Matsuo, T. Eto, Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma, Biochem. Biophys. Res. Commun. 192 (1993) 553–560. [2] Y. Ichiki, K. Kitamura, K. Kangawa, M. Kawamoto, H. Matsuo, T. Eto, Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma, FEBS Lett. 338 (1994) 6–10. [3] F. Satoh, K. Takahashi, O. Murakami, K. Totsune, M. Sone, M. Ohneda, K. Abe, Y. Miura, Y. Hayashi, H. Sasano, et al., Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma, J. Clin. Endocrinol. Metab. 80 (1995) 1750–1752. [4] X. Wang, T.L. Yue, F.C. Barone, R.F. White, R.K. Clark, R.N. Willette, A.C. Sulpizio, N.V. Aiyar, R.R. Ruffolo Jr, G.Z. Feuerstein, Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage, Proc Natl Acad Sci U S A 92 (1995) 11480–11484. [5] S. Nagata, T. Hikosaka, K. Kitamura, Effect of adrenomedullin administration in two rat models of experimental inflammatory bowel disease, Am. J. Life Sci. 3 (2015)
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