http://journals.sagepub.com/doi/full/10.1177/1759091415605114?utm_source=Adestra&utm_medium=email&
Abstract
Apelin is a peptide originally isolated from bovine stomach tissue extracts and identified as an endogenous ligand of the APJ receptor; recent work showed that apelin ameliorates the ischemic injury in the heart and the brain. Being an analogue to the angiotensin II receptor, the apelin/APJ signaling may mediate angiogenesis process. We explored the noninvasive intranasal brain delivery method and investigated therapeutic effects of apelin-13 in a focal ischemic stroke model of mice. Intranasal administration of apelin-13 (4 mg/kg) was given 30 min after the onset of stroke and repeated once daily. Three days after stroke, mice received apelin-13 had significantly reduced infarct volume and less neuronal death in the penumbra. Western blot analyses showed upregulated levels of apelin, apelin receptor APLNR, and Bcl-2 and decreased caspase-3 activation in the apelin-13-treated brain. The proinflammatory cytokines tumor necrosis factor-alpha, interleukin-1β, and chemokine monocyte chemoattractant protein-1 mRNA increased in the ischemic brain, which were significantly attenuated by apelin-13. Apelin-13 remarkably reduced microglia recruitment and activation in the penumbra according to morphological features of Iba-1-positive cells 3 days after ischemia. Apelin-13 significantly increased the expression of angiogenic factor vascular endothelial growth factor and matrix metalloproteinase-9 14 days after stroke. Angiogenesis illustrated by collagen IV + /5-bromo-2′-deoxyuridin + colabeled cells was significantly increased by the apelin-13 treatment 21 days after stroke. Finally, apelin-13 promoted the local cerebral blood flow restoration and long-term functional recovery. This study demonstrates a noninvasive intranasal delivery of apelin-13 after stroke, suggesting that the reduced inflammatory activities, decreased cell death, and increased angiogenesis contribute to the therapeutic benefits of apelin-13.
Introduction
Apelin was originally isolated from bovine stomach tissue extracts. It has been identified as an endogenous ligand of the APJ receptor, a G protein-coupled receptor related to angiotensin receptor AT1 (Lee et al., 2000a). Apelin is derived from a 77-amino acid length precursor peptide that can be cleaved by angiotensin-converting enzyme 2 into active apelins, including apelin-36 (42–77), apelin-17 (61–77), and apelin-13 (65–77; Lee et al., 2000b). Apelin-13 has completely conserved 13 C-terminal amino acids that are cross all species and exhibits the highest biological potency, including cardiomyocytes protection (Hosoya et al., 2000; Kleinz and Davenport, 2005; Simpkin et al., 2007). The active apelins are widely distributed in various organs and tissues, including the brain, lungs, testis, and uterus, and are highly expressed in the cardiovascular system. In the brain, apelins are widely expressed in neuronal cell bodies and fibers throughout the entire neuroaxis (Cheng et al., 2012). In neurological diseases, apelin level is significantly altered in the central nervous system. For example, apelin is significantly elevated in the epileptogenic temporal neocortex and absent in glial cells of temporal lobe epilepsy patients (Zhang et al., 2011).
Apelin receptor AGTRL1 was shown to associate with the development of ischemic stroke in the most recent genome-wide association study for ischemic stroke (Hata et al., 2011). As a neuropeptide, apelin exhibits neuroprotective function in both in vitro and in vivo studies. Pretreatment with apelin-13 or apelin-36 peptides, alone or in combination, increased hippocampal neuronal survival from 25% to 50% to 75% after HIV-induced excitotoxic injury (O’Donnell et al., 2007). Our previous in vitro study also showed that apelin-13 reduced serum deprivation-induced reactive oxygen species generation, mitochondria depolarization, cytochrome c release, and activation of caspase-3. We showed that apelin-13 could regulate cell survival kinases the protein kinase B (PKB, also known as AKT) and extracellular signal-regulated kinase (ERK)1/2 in cultured cortical neurons (Zeng et al., 2010). Most recently, apelin-13 was also demonstrated to protect brain from ischemia/reperfusion (IR) injury through activation of AKT and ERK1/2 signaling pathways in a mouse focal transient cerebral ischemia model (Yang et al., 2014). In a cerebral middle artery occlusion filament stroke model, apelin-36 reduced cell death and cerebral edema (Khaksari et al., 2012; Gu et al., 2013).
APJ has high-sequence homology with the angiotensin II type I receptor, but it binds to apelin instead of angiotensin II (O’Dowd et al., 1993; Lee et al., 2000a). Due to its similarity to the angiotensin II receptor, the functions of APJ have been widely studied on the cardiovascular system. Increasing evidence shows that the apelin/APJ signaling mediates the angiogenesis process. Overexpression of apelin increased Sirt3, vascular endothelial growth factor (VEGF)/VEGFR2, and angiopoietin-1 (Ang-1)/Tie-2 expression and the density of capillary and arteriole in the heart of diabetic mice (Zeng et al., 2014). Inhibition of apelin expression switched endothelial cells from proliferative to mature state in pathological retinal angiogenesis (Kasai et al., 2013). The proangiogenic role of apelin was also demonstrated in myocardial IR injury and murine hindlimb ischemia model. The loss of apelin impaired the angiogenesis and functional recovery, and exacerbated myocardial IR injury, while the elevation of apelin expression induced by adeno-associated virus transduction benefited the postischemic hindlimb perfusion (Qin et al., 2013; Wang et al., 2013). All the above evidence indicates the potential regenerative effects of apelin and a therapeutic application after ischemia. However, in all these in vivo studies, apelin was administered through lateral cerebral ventricle injection, which is highly invasive and less feasible in clinical conditions. As a potential protective drug for ischemic stroke treatment, it is important to seek for a noninvasive method to deliver apelin.
Intranasal administration is a noninvasive method to direct protein and peptide drugs into the brain by utilizing the olfactory neuronal distribution pathways in the cribriform plate, which leads to direct nose-to-brain drug distribution, bypasses the blood–brain barrier (BBB), and directly guides therapeutics to the brain (Hanson and Frey, 2008; Dhuria et al., 2010). Intranasal administration can directly transfer protein and peptides to the brain in similar or higher concentrations than that can be obtained by systemic administration (Scafidi et al., 2014). In this investigation, we tested the hypothesis that the neuroprotective effects of apelin-13 can be achieved by noninvasive intranasal delivery via reducing the infarct formation and inflammatory activities after ischemic stroke, leading to a long-term angiogenesis and functional recovery after stroke.
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