Stephanie M. Wang ‘13 and Weng-Lang Yang
The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA
Brain ischemia is the underlying cause of neuron death during stroke and brain trauma. In addition to necrosis, neural cells exposed to the condition of oxygen depletion during ischemia can also undergo apoptosis, which significantly contributes to brain injury. Adrenomedullin (AM), a multifunctional hormone, in combination with its enhancing binding protein, AMBP-1, has been shown to effectively reduce tissue damage under hemorrhage and ischemia/reperfusion in animal models. To evaluate a beneficial effect of AM/AMBP-1 administration in brain ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human neuroblastoma SH-SY5Y cells. After exposure to 1% O2 for 20 h, the neural cells were injury with a reduction of the cellular ATP levels and an increase of lactate dehydrogenase released in culture medium. Pre-administration of AM/AMBP-1 significantly reduced the hypoxia-induced cell injury. Moreover, AM/AMBP-1 treatment reduced the number of TUNEL-positive cells and activation of caspase-3 in comparison to those cells exposed to hypoxia alone. AM/AMBP-1 prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after hypoxia exposure. Correspondingly, treatment of forskolin, a stimulator of cAMP production, also protected neural cells from hypoxia-induced injury. Inhibition of PKA, a downstream target of cAMP, by KT5720 abolished the protective effect of AM/AMBP-1 on hypoxia-induced apoptosis. These results indicate that AM/AMBP-1 elevates cAMP levels, followed by activating PKA activity, to protect neural cells from the injury caused by hypoxia. This study suggests that AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from brain ischemia.
Cerebral ischemia results from an insufficient supply of blood and oxygen to the brain after injuries. It is the underlying cause of neuron death during stroke and brain trauma, two leading perpetrators of death in the United States . According to the National Center of Injury Prevention and Control, about 1.4 million Americans sustain traumatic and ischemic brain injuries annually. More than 10% of those injured are either severely disabled or die, and patients surviving from this disease require massive sending on long-term rehabilitations such as speech, occupational, and physical therapies . So far, very limited treatments are available for patients with stroke and brain trauma. Although tissue plasminogen activator has been approved by the FDA, it is only beneficial for a small number of patients . Development of new therapeutics is imperative in this field.
During the brain damage, two major processes that lead to cell death: necrosis and apoptosis. Within the core of the ischemic area, where blood flow is most severely restricted, necrotic cell death is dominant and occurs within a few days after damage. In the periphery of the ischemic area, where collateral blood flow can buffer the full effects of the damage, which may start several days after transient ischemia, is mainly apoptosis [3,4]. In principle, the apoptotic cascades during brain damage are reversible, which can be the major target of therapeutic interventions [5-7].
Adrenomedullin (AM), a potent vasoactive peptide was discovered by Kitamura et al. . This peptide, consisting of 52 amino acids, acts as a circulating hormone to induce various biological activities in a paracrine or autocrine manner . AM is multifunctional in nature and can regulate the proliferation, differentiation, and migration of a number of different cell types as well as exert its regulatory abilities on blood pressure, water, and electrolyte balance . A specific AM binding protein (i.e., AMBP-1) has been identified in mammalian blood  and further purified and characterized by Pio et al. as human complement factor H . AMBP-1 can facilitate AM to confine in the interstitial space for the accession to AM receptors and modulate AM biological activity. Moreover, AMBP-1 can prolong half-life of AM in the circulation by protecting it from protease degradation (reviewed by Zudaire et al.). Administration of AM combined with AMBP-1 has been shown to have protective effects on tissue damage caused by low oxygen and blood supply conditions, such as gut ischemia-reperfusion  and hemorrhagic shock  in animal models. These studies prompt us to evaluate the effectiveness of AM/AMBP-1 on protecting brain damage under ischemic condition.
Here, we used an in vitro cell model to study neuron injury caused by oxygen depletion. Human neuroblastoma SH-SY5Y cells were treated with retinoic acid to differentiate into neuron-like cell type and exposed to hypoxic condition at 1% O2. We first identified the injury and apoptosis of differentiated SH-SY5Y cells after hypoxia exposure. We then examined the effect of AM in combined with AMBP-1 on hypoxia-induced cell injury and apoptosis. We further elucidated the signaling pathway mediated by AM/AMBP-1 in regulating hypoxia-induced apoptosis in differentiated SH-SY5Y cells.
Materials and Methods
Cell culture and materials
The human neuroblastoma cell line SH-SY5Y was obtained from the American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in Dulbecco’s Modified Eagle Medium/F12 Medium (1:1, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM L-glutamine. SH-SY5Y cells were maintained at 37°C in a humidified incubator containing 95% air and 5% CO2. SH-SY5Y cells were treated with retinoic acid (RA, 10 µM, Sigma, St. Louis, MO) for 5 days to differentiate into neuron-like cell type . Typical morphology of the differentiated SH-SY5Y cells is shown in Figure 1B. Differentiated SH-SY5Y cells were plated into 96-, 12-, or 24-well plates 72 h before experiments. AM and AM receptor antagonist (AM 22-52) were obtained from Phoenix Pharmaceuticals (Belmont, CA) and AMBP-1 was obtained from Cortex Biochem (San Leandro, CA). Forskolin and protein kinase A (PKA) inhibitor, KT5720 were from Tocris Bioscience (Ellisville, MO). Anti-cleaved caspase-3 polyclonal rabbit antibody (Cat. No. 9661) was from Cell Signaling (Danvers, MA) and has been applied to other study for Western blotting . Anti-actin antibody was from Sigma.
Hypoxia was produced using a sealed chamber containing 1% O2, 5% CO2, and 94% N2, and placed in an incubator at 37°C. Different agents were added to the differentiated SH-SY5Y cells 30 min before exposure to hypoxia. After 20 h incubation in the hypoxic chamber, culture media and cells were collected for further analyses.
Adenosine triphosphate (ATP) detection assay
ATP levels in the differentiated SH-SY5Y cells were determined by ATPlite kit from PerkinElmer (Boston, MA), according to the manufacturer’s instructions. Briefly, after hypoxia, 50 µl of cell lysis solution was added into cells cultured in a 96-well plate with 100 µl of medium per well, followed by 5 min shaking. Subsequently, 50 µl of the substrate buffer solution (luciferase/lucerin) was added and shaken for another 5 min. The reaction was developed in the dark for 10 min and measured on a luminescence plate reader. Protein levels in the cell lysate were determined by a DC protein assay kit from Bio-Rad (Hercules, CA). ATP levels were expressed as a ratio of ATP in light units/mg protein.
Lactate dehydrogenase (LDH) assay
The release of LDH in the cell culture supernatant was measured according to the protocol provided by the manufacturer (Pointe Scientific; Canton, MI). The higher LDH activity detected in the cell supernatant, the more numbers of dead and dying cells. The LDH activity was normalized to protein concentration.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
SH-SY5Y cells were grown on a chamber slide. After 20 h in hypoxia, cells were fixed with 4% paraformaldehyde for 1 h at room temperature. Cells were washed with PBS (phosphate buffer saline; pH 7.2) and permealized on ice for 2 min with 0.1% Triton X-100/0.1% sodium citrate. After washing with PBS, cells were incubated with a TUNEL reaction mixture from Roche Applied Science (Indianapolis, IN) at 37°C for 1 h. The slides were sealed with Vectashield mounting medium containing propidium iodide (PI) from Vector Labs (Burlingham, CA). The green fluorescent, TUNEL-positive cells were counted under a fluorescent microscope. Total cell number in the field was determined by PI staining.
Western blot analysis for cleaved caspase-3
SH-SY5Y cells were lysed in cell lysis buffer containing a protease inhibitor cocktail (Roche Applied Science; Indianapolis, IN). Total protein (25 µg) from cell lysate was loaded on 4-12% Bis-Tris gels (Invitrogen) and subjected to electrophoresis using MES-SDS running buffer (Invitrogen). After electrophoresis, gels were transferred to 0.2-µm nitrocellulose membranes (Invitrogen) and blocked with 5% nonfat dry milk in 10 mM Tris-HCl with 0.1% Tween-20, pH 7.5 (TBST). The membranes were incubated with anti-cleaved caspase-3 polyclonal rabbit antibody or anti-actin antibody overnight at 4°C. Afterwards, the membranes were washed with TBST and incubated with HRP-linked anti-rabbit IgG (Southern Biotech, Birmingham, AL) for 1 h at room temperature and detected using chemiluminescence and autoradiography. Intensity of the band was analyzed by Bio-Rad GS-800 Calibrated Densitometer.
Measurement of 3’-5’-cyclic adenosine monophosphate (cAMP) and PKA activity
An aliquot of 100 µl of cell lysate was used for each cAMP measurement. Intracellular cAMP content was measured using a cAMP Biotrak enzyme-immunoassay system (GE Healthcare; Buckinghamshire, UK), according to the manufacturer’s instructions. A standard curve was performed to calculate the concentration of cAMP in cell lysate. To determine PKA activity, cells were grown on 24-well plates, exposed to hypoxia, lysed in cell lysis buffer and applied to PKA activity assay kit from Stressgen (Ann Arbor, MI), according to the manufacturer’s instructions. The cAMP levels and PKA activity were normalized to protein concentration.
All data were expressed as the means ± SE (standard error) of at least four independent experiments and were compared by one-way analyses of variance (ANOVA) and the Student-Newman Keuls’ test. Differences in values were considered significant if p<0.05.
Effect of AM/AMBP-1 on cellular damage under hypoxia
To assess the cellular damage of SH-SY5Y cells under hypoxia (1% O2), we measured the ATP levels in these cells. The correlation between ATP levels and cell death has been well established in neural cells . Oxygen depletion for 20 h resulted in a significant decrease of ATP levels in SH-SY5Y cells by 32.6% in comparison to normoxia (Figure 2). We then examined the effect of AM/AMBP-1 on hypoxia-induced cell injury. It has been reported that an administration of AM at 100 nM exert an optimal effect on inhibiting cytokine release in macrophage stimulated with lipopolysaccharide . Furthermore, AM co-administered with AMBP-1 at ratio of 2:1 has better outcomes than AM treatment alone . Therefore, we selected two doses of AM/AMBP-1 (100/50 and 200/100 nM) to examine whether these molecules could protect SH-SY5Y cells from hypoxia-induced cell injury. As shown in Figure 2, AM alone and AMBP-1 alone at 100 nM and 50 nM, respectively, didn’t change the ATP levels of cells exposed to hypoxia. When AM was administered at 200 nM, there was a slight increase of ATP levels in hypoxia-treated cells, but it didn’t reach a statistical significance. However, when AM co-administered with AMBP-1 at 100/50 nM, the ATP levels of hypoxia-treated cells had an 83.4% increase in comparison to hypoxia controls (Figure 2). Similar results were also observed at 200/100 nM of AM/AMBP-1 (Figure 2). Thus, the AM/AMBP-1 at 100/50 nM was applied to the following experiments.
To confirm the protective effect of AM/AMBP-1 on hypoxia-induced injury, we analyzed the release of LDH in the supernatant, another marker for measuring cell injury and death. LDH activity was increased by 41.4% under the condition of hypoxia in comparison to the normoxia controls (Figure 3A). When adding 100/50 nM of AM/AMBP-1, the LDH activity of the hypoxia-treated SH-SY5Y cells reduced to the level similar to that of the normoxia controls (Figure 3A). Correspondingly, the ATP levels of AM/AMBP-1 administered cells were significantly higher than those of cells exposed to hypoxia alone (Figure 3B).
Effects of AM/AMBP-1 on hypoxia-induced apoptosis
We next examined whether the protective effect of AM/AMBP-1 on hypoxia-induced cell injury was associated with apoptosis. After hypoxia, SH-SY5Y cells were subjected to TUNEL assay to detect the DNA fragmentation of apoptotic cells. There were very few green fluorescence cells in normoxia condition (Figure 4A). Hypoxia resulted in an 8.5-fold increase of TUNEL positive cells in comparison to cells grown in normoxia condition (Figure 4B). In the presence of AM/AMBP-1, the number of TUNEL positive cells was 80.2% less than that in the cells exposed to hypoxia alone (Figure 4B). To further confirm the occurrence of apoptosis in SH-SY5Y cells after hypoxia, we used Western blot analysis to determine the activation of caspase-3, a critical executioner of cell apoptosis . As shown in Figure 4C, cleaved caspase-3 at 19 kDa was barely detected in the normoxia controls, while its intensity in the cells exposed to hypoxia was increased by 68.4% in comparison to the normoxia controls. In contrast, the level of cleaved caspase-3 in the cells exposed to hypoxia in the presence of AM/AMBP-1 became similar to that in the normoxia controls (Figure 4C). Taken together, these results indicate that AM/AMBMP-1 can protect SH-SY5Y cells from apoptosis induced by hypoxia.
Effects of AM/AMBP-1 on cellular cAMP levels after hypoxia
It has been indicated that AM can transmit its signal through the receptors to regulate cellular cAMP levels for executing its biological activities . To identify the involvement of AM receptors in regulating hypoxia-induced injury, 1 µM of AM 22-52, an AM receptor antagonist, was added with AM/AMBP-1 to SH-SY5Y cells before exposure to hypoxia. After hypoxia, ATP levels in the cells treated with AM 22-52 and AM/AMBP-1 were 56% less than those in the cells treated with AM/AMBP-1 alone (0.56±0.044 vs. 1.00±0.11, P<0.05). We then examined whether cAMP could mediate AM/AMBP-1 in regulating cellular responses to hypoxia. As shown in Figure 5, cAMP levels in SH-SY5Y cells exposed to hypoxia were decreased 41.9% in comparison to the normoxia controls, while administration of AM/AMBP-1 prevented the reduction of cAMP levels after hypoxia. To further examine the effect of cellular levels of cAMP on hypoxia-induced cell injury, SH-SY5Y cells were pre-treated with forskolin before hypoxia. Forskolin directly activates adenylate cyclase and raises cAMP levels in a wide variety of cell types, including neurons . In the presence of 1 µM of forskolin, ATP levels in hypoxia-treated cells were significantly higher than those in cells exposed to hypoxia alone (Figure 6A). We also observed that cells pre-treated with forskolin resulted in a significant decrease of cleaved caspase-3 levels after hypoxia, as demonstrated by Western blotting (Figure 6B). These results indicate that the protective effect of AM/AMBP-1 on neural cells under the hypoxia condition may be due to their ability of elevating intracellular cAMP levels through activation of AM receptors.
Regulation of PKA by AM/AMBP-1 under hypoxia
It is well known that cAMP can activate PKA . After observing the changes of cAMP levels, we then assessed the PKA activity in SH-SY5Y cells. Under hypoxia, there was a 21% reduction of PKA activity in comparison to the normoxia controls (Figure 7). In the presence of AM/AMBP-1, the reduction of PKA activity after hypoxia was prevented (Figure 7). To further confirm the involvement of PKA activity in the protection of hypoxia-induced injury by AM/AMBP-1, we treated SH-SY5Y cells with 1 µM of KT5720, a PKA inhibitor. As shown in Figure 8, the cleaved capase-3 levels in the cells treated with KT5720 and AM/AMBP-1 were the same as those treated with vehicle alone after hypoxia. In contrast, administration of AM/AMBP-1 could effectively prevent the cleavage of caspase-3 in cells exposed to hypoxia (Figure 4C). These results indicate that blocking PKA activity can diminish the protective effect of AM/AMBP-1 on hypoxia-inducing apoptosis.
In this study, we used human neuroblastoma SH-SY5Y cells differentiated to neuron-like cell type, followed by hypoxia (1% O2) exposure, to simulate brain ischemia. We first demonstrated that hypoxia caused damage of SH-SY5Y cells shown a reduction of cellular ATP levels and an increase of LDH released into supernatant. By administration of AM/AMBP-1, the cell injury induced by hypoxia was significantly alleviated. The cellular ATP levels and release of LDH in the AM/AMBP-1-treated cells exposed to hypoxia were comparable to those in cells grown in normal condition. Although we doubled amount of AM/AMBP-1 administration from 100/50 nM to 200/100 nM, we didn’t observe further improvement of cell injury, indicating that AM/AMBP-1 at 100/50 nM was an optimal dose for treating SH-SY5Y cells to protect them from hypoxia-induced injury. Furthermore, AM co-administered with AMBP-1 had a better protective effect on hypoxia-induced injury than administration of AM alone in SH-SY5Y cells, which is agreed with a previous study shown an enhancement of AM activity in combined with AMBP-1 . Administration of AMBP-1 alone had no protective effect on the cell injury induced by hypoxia.
Although the small intestine is the major source of AM production under both physiological and pathophysiological conditions , AM is also produced in the central nervous system (CNS) at a relative low amount [23, 24]. AM plays important roles in the maintenance of homeostasis through central mechanisms . The primary biosynthesis site of AMBP-1 is the liver ; however, AMBP-1 can be detected in the rat brain by immunohistochemical staining . Under inflammation and ischemia/reperfusion injury in the gut, the expression of AMBP-1 is down-regulated [13, 28]. Administration of AM/AMBP-1 has been shown to prevent tissue damage in animal models of intestinal and hepatic ischemia/reperfusion injury [13, 29]. In view of that, the observation of the protective effect of AM/AMBP-1 in cultured neural cells exposed to hypoxia provides a rational strategy of using AM/AMBP-1 to treat brain damage caused by ischemia. There is a concern in crossing the blood-brain barrier (BBB) for AM/AMBP-1 to reach the therapeutic targets. It is well known that stroke can disrupt the BBB [30,31]. A previous study has demonstrated that distribution of AM in the brain increases significantly in sepsis , suggesting that brain permeability increases under disease conditions. In this scenario, we expect that BBB permeability to human AM and AMBP-1 will be markedly enhanced after ischemic stroke.
After demonstrating that AM/AMBP-1 has neuroprotective properties under hypoxic conditions, we then elucidated the potential mechanism responsible for this novel protective effect. As it is widely accepted that hypoxia promotes cell apoptosis , we first tested whether AM/AMBP-1 can decrease hypoxia-induced neural cell apoptosis. By using TUNEL assay and detection of cleaved caspase-3 from Western blotting, we confirmed an induction of apoptosis in SH-SY5Y cells under hypoxic condition of 1% O2. When AM/AMBP-1 was administered, the number of apoptotic cells and levels of cleaved caspase-3 were significantly reduced in neural cells exposed to hypoxia. The protective effect of AM on apoptosis has also been observed in cultured rat endothelial cells under serum deprivation . Apoptosis has pathological consequences on immune and other cell function under various detrimental circulatory conditions such as ischemia/reperfusion injury [35, 36]. Moreover, inhibition of cell apoptosis has proven to be beneficial in reducing inflammation . Thus, an inhibition of neural cell apoptosis not only can directly reduce brain injury but also can prevent the damage caused by inflammation after ischemia.
AM and AMBP-1 are extracellular molecules. The next question is how intracellular apoptotic cascade in SH-SY5Y cells can be regulated by AM/AMBP-1 during hypoxia. AM mediates its activities via heterodimeric receptors that are composed of a seven transmembrane calcitonin-receptor-like receptor (CRLR)  and a receptor activity modifying protein (RAMP) [39, 40]. The RAMP family comprises three members (RAMP1, RAMP2 and RAMP3) [39, 40]. AM receptors are widely distributed in most cell populations, including neurons [39, 40]. In this study, we demonstrated that treatment of AM receptor antagonist diminished the protective effect of AM/AMBP-1 on hypoxia-induced injury, indicating that the interaction between AM and its receptors was required for this protection. Several signaling pathways can be stimulated by AM in various cell types  and the cAMP-PKA signaling pathway has been shown to mediate many of AM effects, including cell proliferation and migration [42, 43], cell protection , and cell regeneration . It has been demonstrated that activation of the cAMP-PKA pathway can protect neuronal cells against apoptosis and improve survival [46, 47]. Therefore, we examined whether the cAMP-PKA pathway could mediate AM/AMBP-1 in regulating hypoxia-induced apoptosis. We first demonstrated that cAMP levels were decreased in SH-SY5Y cells after hypoxia and AM/AMBP-1 treatment could effectively prevent the reduction of cAMP levels. Correspondingly, by adding forskolin, a stimulator of cAMP production, cell damage and activation of caspase-3 induced by hypoxia were reduced in SH-SY5Y cells. To further identify whether the protective effect by the elevated cAMP levels is PKA-dependent, we demonstrated that down-regulation of PKA activity was prevented by administration of AM/AMBP-1 after hypoxia. Furthermore, we inhibited the PKA activity by treating cells with KT5720, a pharmacological inhibitor of PKA. KT5720 overturned the protective effect of AM/AMBP-1 on hypoxia-induced injury and activated caspase-3 to the levels as same as hypoxia alone. These mechanistic studies involved in the regulation of hypoxia-induced apoptosis by AM/AMBP-1 in neural cells are summarized in Figure 9. This proposed model indicates that the beneficial effect of AM/AMBP-1 on protecting neural cells from hypoxia-induced cell injury is mediated by the cAMP-PKA pathway.
In summary, by using an in vitro cell culture system with differentiated human neuroblastoma cells, the novel neuroprotective effects of AM/AMBP-1 in hypoxia were discovered. This neuroprotection is mediated by a reduction of hypoxia-induced apoptosis. Furthermore, the attenuation of apoptosis by AM/AMBP-1 is through activation of the cAMP-PKA pathway in neural cells under hypoxia. These novel findings have considerable, potential biomedical implications for developing AM/AMBP-1 as therapeutic agents to reduce temporary and/or permanent brain damage caused by oxygen depletion. Further studies are underway to determine the efficacy of AM/AMBP-1 in animal models of stroke, brain trauma, and brain ischemia.
This work was supported by Faculty Practice Plan Research Fund of North Shore-Long Island Jewish Health System and funds from TheraSource. The authors greatly appreciate the constructive suggestions and comments from Allyson Weseley, Ph.D. We also thank Haiyun Deng, MD for his kind help with the experiment.
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