Angiotensin II induces interleukin-6 expression in astrocytes: Role of reactive oxygen species and NF-κB

Molecular and Cellular Endocrinology 437 (2016) 130e141

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Angiotensin II induces interleukin-6 expression in astrocytes: Role of reactive oxygen species and NF-kB Yugandhar V. Gowrisankar 1, Michelle A. Clark* Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328, United States

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 March 2016 Received in revised form 21 June 2016 Accepted 8 August 2016 Available online 15 August 2016

Previously, we showed that the bio-peptide angiotensin (Ang) II induces interleukin-6 (IL-6) in cultured astrocytes; however, the mechanism(s) involved in this effect were unknown. In the current study, we determined in brainstem and cerebellum astrocytes from the spontaneously hypertensive rat (SHR), the effect of Ang II to induce IL-6 as well as reactive oxygen species (ROS) generation. Results from this study showed that Ang II significantly induced the differential expression of IL-6 mRNA and protein levels in astrocytes from both regions of Wistar and SHRs. There were differences in the ability of Ang II to induce IL-6 mRNA and protein levels, but these differences were not apparent at all time points examined. Ang II also induced ROS generation, but there were no significant differences between ROS generation in SHR samples as compared to the Wistar samples. Ang II-induced IL-6 levels were mediated via the AT1/Nuclear Factor Kappa beta/ROS pathway. Overall, our findings suggest that there may be dysregulation in IL6 production from astrocytes, contributing to differences observed in SHRs versus its normotensive control. Elucidating the mechanisms involved in Ang II pro-inflammatory effects in the central nervous system may lead to the development of novel therapeutic strategies that can be harnessed not just to treat hypertension, but other Ang II-mediated diseases as well. © 2016 Published by Elsevier Ireland Ltd.

Keywords: Astrocytes Angiotensin II Interleukin-6 Reactive oxygen species NF-kB

1. Introduction The pathophysiological mechanisms underlying regulation of blood pressure are complex; however, it is well recognized that the peptides produced by the renin-angiotensin system (RAS) greatly contribute to maintenance of blood pressure (Mehta and Griendling, 2007). Ang II is the most well-known peptide produced by this system. This peptide is a potent biologically active

Abbreviations: AAALAC, Association for Assessment and Accreditation of Laboratory Animal Care International; ACE, Angiotensin Converting Enzyme; Ang, Angiotensin; AT1R, Ang type 1 receptor; BCA, Bicinchoninic acid; DMEM/F12, Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12; FBS, Fetal Bovine Serum; GFAP, Glial Fibrillary Acidic Protein; H2O2, Hydrogen Peroxide; NaCl, Sodium chloride; NaF, Sodium fluoride; NF-kB, Nuclear Factor Kappa beta; NaVO4, Sodium orthovanadate; PMSF, Phenylmethylsulfonyl fluoride; qPCR, quantitative PCR; RAS, Renin Angiotensin System; SHR, spontaneously hypertensive rat. * Corresponding author. College of Pharmacy, Department of Pharmaceutical Sciences, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328, United States. E-mail address: [email protected] (M.A. Clark). 1 Present address: Institute for Neuro Immune Medicine, College of Osteopathic Medicine, Nova Southeastern University, United States. http://dx.doi.org/10.1016/j.mce.2016.08.013 0303-7207/© 2016 Published by Elsevier Ireland Ltd.

octapeptide with multiple activities on host cells and tissues. Ang II has emerged as a critical hormone that affects the functions of virtually all organs including the heart, kidneys, the vasculature, and the brain. Given its diverse functions and its potency in affecting cardiovascular physiology, it is not surprising that dysregulation of Ang II effects is tied to numerous cardiovascular diseases including hypertension, congestive heart failure, and others (Mehta and Griendling, 2007; Veerasingham and Raizada, 2003). Indeed, agents that target the actions of Ang II receptors (Ang receptor blockers, example Losartan), and agents that target the synthesis of Ang II (ACE inhibitors, example Lisinopril, and renin inhibitors, example Aliskiren) are popular blood pressure lowering medications. Ang II exerts most of its actions through Ang type 1 (AT1) receptors, and this receptor mediates effects of Ang II including systemic vasoconstriction and cell proliferation (Mehta and Griendling, 2007; Murphy et al., 1991; Mukoyama et al., 1993; Kambayashi et al., 1993). It is this receptor that is blocked by Ang receptor blockers such as Losartan. Alternatively, Ang II can also act on AT2 receptors to cause vasodilation and apoptosis (Griendling et al., 1997; Tallant et al., 1991; Hunyady and Catt, 2006). Hyperactivity of the brain RAS has been implicated in the development of

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hypertension in several types of experimental and genetic hypertension animal models. In addition, a substantial proportion of Ang II's other cardiovascular actions, e.g. heart failure, results from Ang II actions in the brain (Morimoto and Sigmund, 2002) (Matsukawa et al., 1991). Thus, it is essential to understand how Ang II works in the brain, and determine central Ang II signaling pathways that are dysregulated leading to hypertension. Identification of such signaling processes is essential for understanding the mechanisms that regulate physiological activities elicited by Ang II. These intracellular pathways may malfunction, leading to pathological consequences such as hypertension, and thus present targets for manipulation for disease prevention. Reactive Oxygen Species (ROS) consist of a number of substances that under normal physiological conditions play a pivotal role in innate immunity, cell signaling, and regulation of vascular integrity (Bayir, 2005). These molecules consist of biologically active oxygen species/radicals namely, hydrogen peroxide (H2O2), hypochlorus acid, fatty acid hydroperoxidase, reactive aldehydes and singlet oxygen which are generated at low levels in a constant fashion as byproducts of normal cellular metabolism (Bayir, 2005). H2O2 is considered to be the most important ROS that has clinical significance, and is the key molecule induced by Ang II (Touyz, 2004). Imbalances between the production and clearance rates of ROS lead to oxidative stress, resulting in several pathophysiological processes in humans. ROS have been linked to, neurodegenerative diseases, cancer, inflammatory conditions, and diabetic mellitus (Valko et al., 2007; Sayre et al., 2001). Supporting data from many studies conducted in different cell types, such as vascular smooth muscle cells, endothelial cells, adventitial cells, and mesangial cells also suggest that Ang II induces ROS (Dhalla et al., 2000; Pagano et al., 1997; Jaimes et al., 1998; Touyz and Schiffrin, 1999; Harrison, 1997) leading to hypertension (Rajagopalan et al., 1996; Laursen et al., 1997; Touyz and Schiffrin, 2000). In the brain, ROS are best known for their role in the pathogenesis of primary neurodegenerative diseases, such as amyotrophic lateral sclerosis (Deng et al., 1993) and Alzheimer's disease (Smith et al., 1997). It has been postulated that neuronal death in these diseases may be mediated by oxidative stress caused by the aberrant metabolism of superoxides (Schulz et al., 1995). Despite their well-known role in neurodegeneration, very little is understood about ROS as second messengers in normal neural processes in the brain, and even less is known about the role of redox mechanisms in CNS-mediated regulation of cardiovascular functions. Findings from Petterson et al. (Peterson et al., 2006) showed that excessive production of ROS in the brain plays a crucial role in the pathogenesis of Ang II-dependent hypertension. Furthermore, excessive brain ROS production has been found in various animal models of hypertension (Zimmerman and Davisson, 2004). In spontaneously hypertensive rats (SHRs) it has been shown that ROS may contribute to maintaining hypertension in these animals (Schnackenberg et al., 1998; Schnackenberg and Wilcox, 1999; Suzuki et al., 1998; Suzuki et al., 1995; Yoshioka et al., 1985; Nakazono et al., 1991). However, the data supporting the role of ROS in this hypertension model are derived from peripheral cell systems including mesenteric arteries and the kidneys (Schnackenberg et al., 1998; Tschudi et al., 1996). Given the evidence for superoxide-generating and scavenging systems throughout the brain (Lindenau et al., 2000; Shimohama et al., 2000) and the importance of ROS signaling in a wide range of Ang II-regulated cellular processes, one of the objectives of this study was to determine the role of Ang II mediated ROS generation in brain astrocytes isolated from SHRs. Interleukin-6 (IL-6) is a multifunctional cytokine known to mediate inflammatory processes in the body (Akira and Kishimoto, 1992; Loppnow and Libby, 1990). Ang II and ROS induce IL-6 protein

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secretion from various cell types (Beasley, 1997). It has been shown in retinal pigment epithelial cells that Ang II increases the activities of intracellular pathways such as, ERK1/2 Extracellular signal regulated kinase mitogen activated protein (MAP) kinase, the Janus MAP kinase, and Nuclear Factor Kappa beta (NF-kB) leading to an increase in ROS and IL-6 production (Wu et al., 2010). NF-kB is a transcription factor that controls DNA transcription, cell survival and cytokine production (Gilmore, 2006; Brasier, 2006). The intracellular signaling pathways by which Ang II induces ROS and IL-6 production in astrocytes are unknown. However, previous studies suggest that both Ang II and ROS may utilize similar pathways to induce IL-6 levels. In IL-6 knockout mice, Ang II-mediated hypertension decreased, suggesting a role for this cytokine and neuroinflammatory processes generated by the cytokines in this type of hypertension (Brands et al., 2010). Previously, we have shown that Ang II induces IL-6 mRNA and protein levels from astrocytes isolated from normotensive rats but the role of ROS, NF-kB and other pathways in this effect was not tested (Kandalam and Clark, 2010; Kandalam et al., 2012). Further, whether Ang II mediates ROS and IL-6 production in astrocytes isolated from a hypertensive rat model, the SHR, is unknown and is a focus of the current study. In this study, we used brainstem and cerebellum astrocytes isolated from Wistar and SHRs to determine whether ROS and IL-6 levels are dysregulated in the SHR and established a link between Ang II-mediated ROS generation and IL-6 production. The interrelationship between Ang II, ROS, and IL-6 production through the NF-kB signaling pathway was also determined using SHR as a genetic hypertensive animal model. The SHR was selected for these studies as it is a widely used and accepted animal model to study hypertension due to similarities exhibited in these animals as compared to humans (Okamoto and Aoki, 1963). Ang II plays a crucial role in the development and establishment of the hypertensive state in this rat (Harrap et al., 1990; Wu and Berecek, 1993). Most importantly, centrally-produced Ang II mediates hypertension in this animal (Greenwood et al., 1963). Further, astrocytes perform immune functions (Alarcon et al., 2005; Constantinescu et al., 2005; Fontana et al., 1984; Hull et al., 2006), synthesize and release neurotrophic factors and are involved in the formation of neural scars following injury (Fawcett and Asher, 1999; Silver and Miller, 2004). Thus, the findings from this study are essential in validating the immunomodulatory effects of astrocytes as these cells secrete IL-6, a function that may be different in astrocytes isolated from a hypertensive model as compared to the normotensive control. Brainstem and cerebellum areas of the brain were selected as astrocytes isolated from these regions are known to contain RAS components (Tallant and Higson, 1997). Most importantly, the brainstem is known to be involved in cardiovascular regulation. Cerebellum astrocytes were also included in these studies as astrocytes from this area of the brain was shown to express RAS components albeit at low levels (Tallant and Higson, 1997). In our laboratory, we routinely study the effects of Ang II and other peptides in both brainstem and cerebellum astrocytes (Clark and Gonzalez, 2007a,b; Clark et al., 2008). 2. Materials and methods 2.1. Reagents and test substances Ang II was obtained from Bachem (Torrance, CA). PD123319, the selective AT2 receptor blocker was obtained from Sigma (St. Louis, MO), and Losartan (the AT1 receptor blocker) was kindly provided by Du Pont Merck (Wilmington, DE). The NF-kB inhibitor BAY117082 was purchased from Santa Cruz Biotechnology (Dallas, Texas), and YCG063 (ROS inhibitor) was bought from EMD Millipore

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(Billerica, MA). The rat IL-6 and the b-actin antibodies were purchased from ABBIOTEC (San Diego, CA), and Sigma (St Louis, MO), respectively. The IL-6 Quantikine ELISA kit was purchased from R&D Systems (McKinley, MN). Western blotting supplies were purchased from Biorad Laboratories (Hercules, CA) or from VWR International (Suwannee, GA). Quantitative PCR (qPCR) products including the primer sets for IL-6, were obtained from Applied Biosystems (Foster City, CA). All other chemicals, tissue culture supplies, and general laboratory supplies were purchased from either VWR International (Suwannee, GA), Fischer Scientific (Waltham, MA) or Sigma (St. Louis, MO).

plus Real Time PCR system from Applied Biosystems. The widely accepted comparative Ct (threshold cycle) method was used to perform relative quantification of qPCR results (Livak and Schmittgen, 2001). An arithmetic formula (fold difference ¼ 2DDCt) was used to calculate the relative IL-6 mRNA expression in Ang II-stimulated astrocyte cultures as compared to the unstimulated controls, after normalization to the levels of the housekeeping control gene b-actin (Rn00667869) which was also supplied by Applied Biosystems. Data are thus expressed as fold change in IL-6 mRNA expression as compared to basal IL-6 mRNA expression in unstimulated astrocytes.

2.2. Astrocyte preparation

2.4. Cell treatments and intracellular IL-6 protein measurement

Timed pregnant Wistar and SHRs were obtained from Charles River Laboratories (Wilmington, MA) and maintained in the AAALAC-accredited animal facility of Nova Southeastern University. All animal protocols were approved by the University Institutional Animal Care and Use Committee. During all aspects of the astrocyte isolation procedure, care was taken to minimize pain and discomfort to the animals. Primary cultures of astrocytes were prepared from the cerebellum and the brainstem of 2e3 days old neonatal rat pups by physical dissociation as previously described (Tallant and Higson, 1997). Cells were maintained in DMEM/F12 containing 10 mM HEPES pH 7.5 with 10% FBS, 10,000 I.U/mL penicillin, 10,000 mg/mL streptomycin and 25 mg/mL amphotericin B (complete media), at 37
C in a humidified incubator (5% CO2 and 95% air). Cultures were fed every 3e4 days until confluent. After astrocytes reached confluency, they were fed with complete media and shaken overnight to remove oligodendrocytes, and other cells. The debris was removed by two washes with complete media and trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA) was used to detach the cells. This method yields an astroglial enriched culture system which consists mostly of reactive astrocytes. Astrocytes were subsequently plated at a ratio of 1e10, and grown to confluency before treatments. Isolated cells showed a positive immunoreactivity with an antibody against glial fibrillary acidic protein (GFAP) and negative immunoreactivity with markers for neurons, or oligodendrocytes. Prior to all treatments, cells were incubated in serum-free media for 48 h to make them quiescent; all subsequent cell treatments were conducted in serum-free media.

To investigate the time-dependent effect of Ang II on IL-6 intracellular protein expression, quiescent brainstem and cerebellum astrocytes from Wistar and the SHRs were treated with 100 nM Ang II and the cells were harvested at 1, 4, 8, 12, 16, 24 and 48 h after the addition of Ang II. Quiescent brainstem and cerebellum astrocytes from Wistar and SHRs were also treated with 100 nM Ang II in the presence and absence of the selective AT1 receptor blocker (Losartan, 10 mM), or the selective AT2 receptor blocker (PD123319, 10 mM), or the ROS inhibitor (YCG063, 2 mM) or the NF-kB inhibitor (BAY11-7082, 10 mM) for 4 h. Unstimulated brainstem and cerebellum astrocytes were used as the controls. Immediately following treatments, cell lysates were prepared by washing cells with PBS containing 0.01 mM NaVO4 followed by solubilization in supplemented lysis buffer (100 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% Triton X-100, 50 mM Tris-HCl, 0.01 mM NaVO4, 0.1 mM PMSF and 0.6 mM leupeptin, pH 7.4). The supernatant was clarified by centrifugation (12,000  g for 10 min, at 4
C) and the protein concentrations of the cell lysates were measured by the Bicinchoninic acid (BCA) method as described by the manufacturer. Cell lysates were stored at 80
C until western blot analysis were done. Solubilized proteins were separated in 10% polyacrylamide gels and transferred on to the nitrocellulose membranes. Nonspecific binding to the membranes was prevented by incubation with 5% Blotto (5% nonfat dry milk, 0.05% Tween- 20 in Tris-buffered saline). Subsequently, membranes were incubated with an IL-6 antibody (1:1000 in Tris buffered saline containing 5% milk) overnight at 4
C. Subsequently, the membranes were probed with goat antirabbit antibody coupled to horseradish peroxidase. The immunoreactive bands were visualized using enhanced chemiluminescence and normalized using a b-actin antibody (Sigma - St. Louis, MO). Data was quantified by densitometric analysis using the Image-J software. Data are thus expressed as fold change in IL-6 protein expression as compared to basal IL-6 protein expression in the unstimulated astrocytes.

2.3. Cell treatments and measurement of IL-6 mRNA expression To investigate the effect of Ang II on IL-6 mRNA expression levels, quiescent brainstem and cerebellum astrocytes from Wistar and SHRs were treated with 100 nM Ang II and the cells were harvested at 1, 2, 4, 8, 12, 16 and 24 h after the addition of Ang II. Quiescent brainstem and cerebellum astrocytes from Wistar and SHRs were also treated with 100 nM Ang II in the presence and absence of the selective AT1 receptor blocker (Losartan, 10 mM), or the selective AT2 receptor blocker (PD123319, 10 mM), or the ROS inhibitor (YCG063, 2 mM) or the NF-kB inhibitor (BAY11-7082, 10 mM) for 4 h. Unstimulated brainstem and cerebellum astrocytes were used as the controls. After the treatments, total RNA was extracted from astrocytes using the trizol method. Total RNA concentrations were determined using Bio-Rad SmartSpecTM spectrophotometer. Two micrograms of total RNA from each sample were reverse transcribed into the complementary DNA (cDNA) using a high capacity reverse transcription reagent kit (Applied Biosystems, Foster City, CA). qPCR was performed using the TaqMan Universal master mix, and the TaqMan gene expression assay for rat IL-6 (Rn01410330) which were all supplied by Applied Biosystems (Foster City, CA). Samples were assayed in triplicates in 96 well plates using the StepOneTM

2.5. Cell treatment and secreted IL-6 protein measurement To investigate the time-dependent effect of Ang II on IL-6 secreted protein expression, quiescent brainstem and cerebellum astrocytes from Wistar and the SHRs were treated with 100 nM Ang II and the cells were harvested at 1, 4, 8, 12, 16, 24, and 48 h after the addition of Ang II. Angiotensin receptor blockers and inhibitors for ROS and NF-kB were also used in this study to identify the Ang receptor subtypes involved in this Ang II effect and to determine intracellular pathways involved in this Ang II effect. Quiescent brainstem and cerebellum astrocytes from Wistar and SHRs were treated with 100 nM Ang II in the presence and absence of the selective AT1 receptor blocker (Losartan, 10 mM), or the selective AT2 receptor blocker (PD123319, 10 mM), or the ROS inhibitor (YCG063, 2 mM) or the NF-kB inhibitor (BAY11-7082, 10 mM) for 4 h.

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Unstimulated brainstem and cerebellum astrocytes were used as the controls. The concentrations of Ang II, Losartan, and PD123319 were selected based on our previous studies (Clark and Gonzalez, 2007a,b; Clark et al., 2008). The concentrations of BAY11-7082 and YCG063 selected were similar to those used by other investigators (Nookala and Kumar, 2014; Yu et al., 2013). After the incubation times, the medium was collected and centrifuged at 12,000 rpm for 1 min at 4
C. The supernatant was stored at 80
C prior to analysis. IL-6 in the media was measured by ELISA and was performed according to the manufacturer's protocol (R&D Systems, Minneapolis, MN). 2.6. DCFDA measurement of intracellular ROS levels To investigate the time-dependent effect of Ang II on intracellular ROS levels, quiescent brainstem and cerebellum astrocytes isolated from Wistar and SHRs were treated with 100 nM Ang II for 0.5, 1 2, 4 and 6 h and the intracellular ROS production was measured using the 20 ,70 -dichlorofluorescein diacetate (DCFDA) fluorescent dye. Basal ROS levels were obtained from astrocytes that were not treated with Ang II. DCFDA is a membrane-permeable dye that gets trapped inside cells upon hydrolysis by intracellular esterases; it is then subsequently oxidized to the fluorescent product dichlorofluorescein (DCF) by ROS and was measured as previously described (Ding et al., 2007). For measurement of ROS, cells were grown in 96 well plates. Quiescent cells were washed twice with PBS and incubated for 30 min with 25uM DCFDA diluted in DMEM/F-12 medium. Cells were subsequently treated with Ang II for 0.5, 1, 2, 4, and 6 h to determine the time-dependent effect of Ang II on ROS production. Cells were also treated in the presence or absence of Ang receptor blockers (10 mM Losartan or 10 mM PD123319), or the ROS inhibitor (YCG063, 2 mM) or the NF-kB inhibitor (BAY11-7082, 10 mM), for 4 h to ascertain the Ang receptors involved, and if this Ang II effect was mediated by ROS/NF-kB pathways. At the end of the incubation periods, cells were washed twice with PBS and the relative fluorescence of DCF was measured using a Biotek multiplate reader at excitation and emission wavelengths of 485 and 530 nm, respectively. The data was expressed as fold change in basal DCF values (Ding et al., 2007). 2.7. Statistical analysis All data are expressed as the mean ± SEM of 5 or more experiments, as indicated. T-tests or repeated measures one-way analysis of variance (ANOVA) with Dunnett's post-test was used to compare treatment groups with control. Wistar verses SHR sample comparisons were made using 2-way ANOVA followed by a Tukey test. PRISM 6 (graph Pad) was used for all statistical analysis. The criterion for statistical significance was p < 0.05. 3. Results 3.1. Effect of time on Ang II mediated IL-6 mRNA expression Previously, we showed that Ang II induces IL-6 mRNA expression in normotensive Sprague Dawley (SD) rat astrocytes (Kandalam et al., 2012). Compared to basal levels, Ang II treatment significantly induced IL-6 mRNA expression in both SHR and Wistar brainstem and cerebellum samples at most time points examined (Fig. 1). The induction patterns were similar in Wistar cerebellum and brainstem samples. Further in these samples, at the shorter time points, Ang II-induced IL-6 mRNA expression was much greater than the Ang II induction observed at longer time points. In contrast, in SHR astrocytes the trend was for a greater increase in

Fig. 1. Effect of time on Ang II-mediated IL-6 mRNA expression in brainstem and cerebellum astrocytes. Quiescent monolayers of brainstem (Panel A) and cerebellum (Panel B) astrocytes from Wistar and SHRs were incubated with 100 nM Ang II for 1 h to 24 h. IL-6 mRNA expression was analyzed by quantitative PCR as described. Ang II mediated IL-6 mRNA expression was expressed as the fold-increase over basal IL-6 levels. Each value represents the mean ± SEM of preparations of astrocytes isolated from six or more litters of neonatal rat pups. * denotes p < 0.05 as compared with basal levels for IL-6 mRNA expression in astrocytes prepared from Wistar astrocytes. # denotes p < 0.05 as compared with basal levels for IL-6 mRNA expression in astrocytes prepared from the SHR samples. ˆdenotes p < 0.05 as compared with corresponding Wistar samples by two-way analysis of variance.

IL-6 mRNA expression after a longer time of exposure to the peptide. A comparison of the normalized cycle threshold (Ct) values showed that the basal mRNA expression of IL-6 in SHR samples were significantly lower (brainstem: 33.0 ± 0.44; cerebellum: 32.6 ± 0.29) as compared to Wistar astrocytes (brainstem: 30.7 ± 0.47; cerebellum: 30.2 ± 0.68). This differential pattern of effect of Ang II on IL-6 mRNA expression is also reflected in differences observed in SHR versus Wistar astrocytes. Compared to the Wistar samples, Ang II-mediated IL-6 mRNA levels were significantly lower at 1, 2, 12 and 16 h in brainstem SHR astrocytes (Fig. 1A). A similar differential effect of Ang II was also observed at the 1, 2, 4 and 24 h time points in cerebellum SHR astrocytes compared to Wistar samples as well (Fig. 1B). These findings suggests that Ang II affects both these brain regions in the generation of IL-6 mRNA expression and that Ang II's ability to induce this cytokine differs somewhat in the SHR astrocytes versus the Wistar astrocytes.

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The Ang receptor(s) mediating this Ang II effect was also investigated. Results of our study showed that incubation of cultured astrocytes with the AT1 receptor blocker or the AT2 receptor blocker alone had no effect on IL-6 mRNA expression (Table 1). However, pretreatment of astrocytes with Losartan prevented Ang II mediated upregulation in IL-6 mRNA expression (Table 1). These results demonstrated that the upregulation of IL-6 mRNA by Ang II was mediated by the AT1 receptor.

Similar to our previous findings (Kandalam and Clark, 2010; Kandalam et al., 2012), pretreatment of both brainstem and cerebellum astrocytes with Losartan prevented Ang II-mediated IL-6 protein secretion (Table 3). The AT2 receptor blocker was ineffective in preventing this Ang II effect. However, treatment with the inhibitor alone had no effect on IL-6 secretion from these cells. These findings suggest that Ang II-induced IL-6 secretion also occurred through interaction with the AT1 receptor.

3.2. Effect of time on Ang II mediated IL-6 intracellular protein expression

3.4. Effect of time on Ang II-mediated intracellular ROS levels

Compared to the basal levels, Ang II treatment induced IL-6 intracellular protein expression in both SHR and Wistar brainstem and cerebellum samples in a similar manner (Fig. 2). Compared to the Wistar controls, Ang II significantly induced IL-6 intracellular protein expression to a greater extent in brainstem and cerebellum samples only at the 48 h and 16 h time points, respectively. There was no differences in the basal levels of IL-6 intracellular proteins (data not shown). To determine the Ang receptors involved in this effect, Ang receptor blockers were also used as described before. As shown in Table 2, treatment with the receptor blockers alone had no effect on IL-6 intracellular protein expression. However, pretreatment with Losartan, the AT1 receptor blocker, prevented Ang II-induced IL-6 intracellular protein expression. The AT2 receptor blocker, PD123319, was ineffective in preventing this effect of Ang II. The findings of this study also suggest that Ang II's effect on intracellular IL-6 protein expression was an AT1 receptor-mediated event.

Compared to the basal levels, Ang II significantly induced intracellular ROS levels in most of the time points examined in Wistar and SHR brainstem and cerebellum samples (Fig. 4). The actions of Ang II to induce ROS production in these cells were similar in both SHR and Wistar astrocytes isolated from both regions. Also, the basal ROS production levels were similar in astrocytes isolated from both rats (data not shown). Further, the region from where the astrocytes were isolated did not have a profound effect on the ability of Ang II to induce ROS production in these cells. The role of Ang receptors and ROS involved in this effect was also determined. Incubation of astrocytes with the inhibitors alone had no effect on ROS production (Tables 4 and 5). Both Losartan and YCG063 prevented Ang II-induced ROS production. In contrast, the AT2 receptor blocker, PD123319, had no significant effect on Ang IImediated ROS production in SHR and Wistar astrocytes from both regions (Table 4). These studies demonstrated that the peptide induced ROS production through the Ang AT1 receptor and that it had a direct effect on ROS production.

3.3. Effect of time on Ang II mediated secreted IL-6 protein levels Ang II treatment also significantly increased IL-6 protein secretion in both SHR and Wistar brainstem and cerebellum astrocytes at all the time points examined (Fig. 3). In the Wistar brainstem samples (Fig. 3A), the Ang II induction was more variable with peak effects occurring at 12 and 24 h. In SHR brainstem astrocytes, the peak effect occurred early at 8 h (Fig. 3A). Further, at the 12, 16 and 24 h time points, SHR IL-6 secreted protein levels were significantly different from their Wistar counterparts (Fig. 3A). In Wistar cerebellum astrocytes (Fig. 3B), the peak effect occurred at 16 h while this effect occurred at 12 h in the SHR cerebellum samples. There was a significant difference in Ang IImediated IL-6 secretion in SHR cerebellum astrocytes as compare to the Wistar samples at the 24 and 48 h time points (Fig. 3B). The basal secretion of IL-6 was not different in the SHR samples as compared to the Wistar samples (data not shown). The findings of this study suggested that the pattern of Ang II induction of IL-6 secretion was different in SHR astrocytes compared to the Wistar cells.

3.5. The effect of ROS inhibition on Ang II-mediated IL-6 mRNA and protein generation In these studies, we established that Ang II induced ROS generation as well as IL-6 mRNA and IL-6 protein production (Figs. 1e4). To determine whether ROS generation occurred before the Ang II-mediated IL-6 effect, quiescent astrocytes were treated with 100 nM Ang II with or without YCG063 (2 mM, the ROS inhibitor) for 4 h. The IL-6 mRNA levels as well as the intracellular IL-6 protein, and the secreted IL-6 protein were measured. The basal expression of IL-6 mRNA and protein levels were measured in unstimulated cells. Pretreatment of astrocytes with YCG063 alone had no effect on the basal levels of IL-6 mRNA and protein levels (Tables 6e8). The ROS inhibitor prevented Ang II-mediated IL-6 mRNA levels (Table 6), as well as Ang II-induced IL-6 intracellular (Table 7), and secreted protein levels (Table 8) in both brainstem and cerebellum astrocytes from the two species of rats. These findings suggest that Ang II generated ROS is an upstream event of IL-6 production.

Table 1 Effects of Losartan and PD123319 on Ang II-mediated brainstem and cerebellum IL-6 mRNA levels. Astrocyte treatment

Wistar (Fold change)

100 nM Ang II 10 mM Losartan 10 mM Losartan þ 100 nM Ang II 10 mM PD123319 10 mM PD123319 þ 100 nM Ang II

3.50 1.10 1.20 1.20 3.45

Brainstem ± ± ± ± ±

0.8* 0.3 0.4 0.2 0.7*

SHR (Fold change) Cerebellum 2.20 1.30 1.40 1.10 2.20

± ± ± ± ±

0.6* 0.3 0.2 0.2 0.3*

Brainstem 4.70 1.10 1.30 1.10 4.30

± ± ± ± ±

0.6* 0.2 0.3 0.2 0.4*

Cerebellum 2.90 1.10 1.10 1.10 2.90

± ± ± ± ±

0.7* 0.2 0.3 0.2 0.6*

Brainstem and cerebellum astrocytes were treated with 100 nM Ang II in the presence and absence of the inhibitors for 4 h. Astrocytes were also treated with the inhibitors alone for 4 h. IL-6 mRNA expression was analyzed by quantitative PCR as described. * Denotes p < 0.05 as compared to basal IL-6 mRNA expression. Values are calculated based on the individual experiments of 5 or more preparation of astrocytes.

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astrocytes, growth arrested astrocytes from both Wistar and SHRs were treated with the NF-kB inhibitor BAY11-7082 (10 mM) in the presence or absence of 100 nM Ang II for 4 h. IL-6 mRNA, IL-6 intracellular, and IL-6 secreted protein levels as well as ROS generation were measured as described. Astrocytes that were not treated with the peptide or the inhibitors were used as the controls. Pretreatment with BAY11-7082 alone had no effect on the basal levels of ROS production (Table 5), IL-6 mRNA (Table 6), IL-6 intracellular protein (Table 7), and IL-6 secreted protein (Table 8). However the inhibitor prevented Ang II-mediated effects on ROS production (Table 5), IL-6 mRNA (Table 6) and IL-6 protein production (Tables 7 and 8) in astrocytes from both brain regions and in both Wistar and SHRs. Overall the findings of our studies suggest that Ang II acting on the AT1 receptor induces NF-kB leading to ROS generation and subsequently IL-6 production. 4. Discussion

Fig. 2. Effect of time on Ang II-mediated IL-6 intracellular protein expression in brainstem and cerebellum astrocytes. Quiescent monolayers of brainstem (Panel A) and cerebellum (Panel B) astrocytes from Wistar and SHRs were incubated with 100 nM Ang II for 1 h to 48 h. Intracellular IL-6 protein expression was analyzed by western blot technique as described. Ang II-mediated intracellular IL-6 protein levels are expressed as the fold-increase over basal intracellular IL-6 protein levels. Each value represents the mean ± SEM of preparations of astrocytes isolated from six or more litters of neonatal rat pups. * denotes p < 0.05 as compared with basal levels for intracellular IL-6 protein expression in astrocytes prepared from the Wistar samples. # denotes p < 0.05 as compared with basal levels for intracellular IL-6 protein expression in astrocytes prepared from the SHR astrocytes. ˆdenotes p < 0.05 as compared with corresponding Wistar samples by two-way analysis of variance.

3.6. The role of NF-kB in Ang II-mediated IL-6 and ROS generation Previous studies have established the NF-kB pathway as an intracellular signal in Ang II-mediated ROS and IL-6 production in the periphery (Wu et al., 2010). To determine if this pathway is important in Ang II-mediated ROS and IL-6 generation in

It is well recognized that a localized RAS exists in the central nervous system (CNS) (Baltatu and Bader, 2003). All the functional components of the RAS are expressed in the major cell types in the brain (Thomas and Mendelsohn, 2003; Lenkei et al., 1996). Injection of Ang II into specific brain regions elicited both cardiovascular and dipsogenic effects (Parsons and Coffman, 2007), indicating the importance of the brain RAS in regulating both neurological and cardiovascular functions. Astrocytes contain most components of the RAS and are the major source of angiotensinogen, the precursor molecule for Ang II in the brain pointing to a key role for astrocytes in the central effects of Ang II (Saavedra, 2005). It addition to its vasoactive properties, Ang II also serves as an immunomodulatory agent regulating cell growth, inflammation, apoptosis and several other cellular processes (Chae et al., 2001; Kannel et al., 1987; Kiechl et al., 2001; Ruiz-Ortega et al., 2002). Indeed, many signaling pathways including MAP kinases, protein kinase C, and tyrosine kinases are reported to be activated by treatment of astrocytes with Ang II (Clark and Gonzalez, 2007a,b; Clark et al., 2008; Tallant and Higson, 1997). Physiological effects of Ang II in astrocytes include growth effects, prostacyclin secretion, and early response genes induction (Tallant and Higson, 1997; Clark and Gonzalez, 2007a,b; Clark et al., 2008; Delaney et al., 2008). Despite its importance in central cardiovascular and volume homeostasis, the precise pathways and signaling mechanisms used by Ang II in the brain are incompletely understood. Recently, a novel signaling mechanism for Ang II involving superoxide and other ROS has been identified in peripheral tissues. One of the main oxidative pathways involved in the production of ROS is represented by NAD(P)H oxidase (Ishikawa et al., 2004; Kazama et al., 2004). Given the evidence for superoxide-generating and scavenging systems throughout the brain (Lindenau et al., 2000; Shimohama et al., 2000), and the importance of ROS signaling in a wide range of Ang II-regulated cellular processes, we sought to establish a proinflammatory role for Ang II in the CNS using astrocytes as a model system. Ang II also enhances the expression of the inflammatory cytokines TNF-a and IL-6 in peripheral cells (Ruiz-Ortega et al., 2002). Interestingly, IL-6 levels are elevated in patients with certain forms of hypertension, stroke and heart attack (Chae et al., 2001; Kannel et al., 1987; Kiechl et al., 2001; Kuller et al., 1991; Kuller et al., 1996; Ershler et al., 1994), and Ang II vasoactive effects are compromised in IL-6 knockout mice (Lee et al., 2006). These findings suggest that IL-6 is an important cytokine involved in Ang II effects. IL-6 is considered to be a key cytokine involved in the production of Creactive protein (CRP) (Abeywardena et al., 2009), which is an important risk factor for myocardial infarction and other diseases (Kiechl et al., 2001; Ridker et al., 1997). Elevated concentrations of

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Table 2 Effects of Losartan and PD123319 on Ang II-mediated brainstem and cerebellum intracellular IL-6 protein levels. Astrocyte treatment

Wistar (Fold change) Brainstem

100 nM Ang II 10 mM Losartan 10 mM Losartan þ 100 nM Ang II 10 mM PD123319 10 mM PD123319 þ 100 nM Ang II

1.70 1.02 1.08 1.10 1.73

± ± ± ± ±

0.09* 0.16 0.12 0.10 0.26*

SHR (Fold change) Cerebellum 1.60 0.94 0.90 0.90 1.72

± ± ± ± ±

0.1* 0.23 0.08 0.12 0.25*

Brainstem 1.80 1.10 0.91 1.01 1.73

± ± ± ± ±

0.12* 0.1 0.15 0.11 0.22*

Cerebellum 1.64 1.02 0.93 1.04 1.50

± ± ± ± ±

0.13* 0.21 0.08 0.16 0.1*

Brainstem and cerebellum samples were treated with 100 nM Ang II in the presence and absence of the inhibitors for 4 h. Astrocytes were also treated with the inhibitors alone for 4 h. IL-6 protein expression was analyzed by western blot technique as described. * Denotes p < 0.05 as compared to basal IL-6 intracellular protein expression. Values are calculated based on the individual experiments of 5 or more preparation of astrocytes.

IL-6 have also been found in brain tissue, cerebrospinal fluid, and blood of patients with Alzheimer's disease, stroke, Parkinson disease, CNS infections and other brain conditions, suggesting an important role for IL-6 in central-mediated diseases (Terreni and De

Fig. 3. Effect of time on Ang II-mediated IL-6 secreted protein expression in brainstem and cerebellum astrocytes. Quiescent monolayers of brainstem (Panel A) and cerebellum (Panel B) astrocytes from Wistar and SHRs were incubated with 100 nM Ang II for 1 h to 48 h. Secreted IL-6 protein levels were measured by an ELISA method as described. Ang II-mediated secreted IL-6 protein levels were expressed as the fold-increase over basal secreted IL-6 protein levels. Each value represents the mean ± SEM of preparations of astrocytes isolated from six or more litters of neonatal rat pups. * denotes p < 0.05 as compared with basal levels for secreted IL-6 protein expression in astrocytes prepared from Wistar astrocytes. # denotes p < 0.05 as compared with basal levels for secreted IL-6 protein levels in astrocytes prepared from the SHR samples. ˆ denotes p < 0.05 as compared with corresponding Wistar samples by two-way analysis of variance.

Simoni, 1998). Lee et al (Lee et al., 2006) tested the role of endogenous IL-6 in hypertension caused by Ang II. In the present study, we showed that Ang II differentially induced IL-6 mRNA and protein (intracellular and secreted) expression in a time-dependent manner in primary cultures of astrocytes isolated from the brainstem and cerebellum regions of SHRs and normotensive Wistar rats. The induced levels of IL-6 mRNA by Ang II translated into an increase in IL-6 intracellular and secreted protein. Further our data also showed that the increase in IL-6 protein was temporal with greater increases observed first in the intracellular IL-6 protein levels, then subsequent increases were observed in the secreted IL6 protein levels. Surprisingly, there was also a pattern of higher Ang II-induced IL-6 protein levels in Wistar astrocytes or no differences between Wistar and SHR samples at most time points examined. The lower basal expression of IL-6 mRNA in SHR astrocytes may correlate with the significant differences in the ability of the peptide to induce IL-6 mRNA at the earlier time points in these samples. Further, basal IL-6 (intracellular and secreted) protein levels in SHRs versus Wistar rats were similar, while basal IL-6 mRNA expression levels were decreased by exposure to Ang II in both cerebellum and brainstem samples. There are presumably several reasons for the poor correlations generally reported in the literature between the levels of mRNA and the level of protein, and these may not be mutually exclusive. First, there are many complicated and varied post-translational mechanisms involved in turning mRNA into protein that are not yet sufficiently well-defined to be able to compute protein concentrations from mRNA; and second, proteins may differ substantially in their in vivo half-lives (Greenbaum et al., 2003; Baldi and Long, 2001; Szallasi, 1999). The Ang II-mediated induction pattern of IL-6 in SHR samples were also different from the pattern observed in Wistar samples. This difference was particularly more noticeable in the effect of Ang II on the IL-6 mRNA levels and the Ang II-mediated IL-6 secreted protein levels. Both brainstem and cerebellum Wistar astrocytes were initially more responsive to Ang II at the earlier time points, an effect which was not sustained throughout the time of exposure to the peptide. On the other hand, the effect of Ang II on IL-6 protein secretion was similar initially but a more robust effect of the peptide was observed in the Wistar samples at later time points. This trend may reflect the lower basal levels of IL-6 mRNA in the SHR samples (accounts for lower effect observed in initial IL-6 mRNA levels). Since the basal levels of IL-6 secreted protein were similar in SHR versus Wistar samples, it is plausible that the initial effects of Ang II on the cytokine secretion would be similar but as the system is challenged for a longer time with Ang II the pattern changes to reflect that other pathways that mediate Ang II effects on IL-6 secretion may be differentially expressed and affected differently by Ang II in the SHR model. Previous studies point to Ang II-mediated pro-inflammatory properties (Benigni et al., 2010) and a role of IL-6 in Ang II-mediated

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Table 3 Effects of Losartan and PD123319 on Ang II-mediated brainstem and cerebellum secreted IL-6 protein levels. Astrocyte treatment

Wistar (Fold change) Brainstem

100 nM Ang II 10 mM Losartan 10 mM Losartan þ 100 nM Ang II 10 mM PD123319 10 mM PD123319 þ 100 nM Ang II

1.40 1.10 1.10 1.10 1.72

± ± ± ± ±

0.1* 0.1 0.1 0.1 0.2*

SHR (Fold change) Cerebellum 1.60 0.90 1.00 0.90 1.74

± ± ± ± ±

0.2* 0.04 0.05 0.5 0.3*

Brainstem 1.40 1.10 0.90 1.10 1.62

± ± ± ± ±

0.2* 0.1 0.1 0.03 0.3*

Cerebellum 1.30 0.92 1.00 1.00 1.42

± ± ± ± ±

0.1* 0.04 0.3 0.1 0.2*

Brainstem and cerebellum samples were treated with 100 nM Ang II in the presence and absence of the inhibitors for 4 h. Astrocytes were also treated with the inhibitors alone for 4 h. Secreted IL-6 protein levels were analyzed by an ELISA method as described. * Denotes p < 0.05 as compared to basal IL-6 protein secretion. Values are calculated based on the individual experiments of 5 or more preparation of astrocytes.

hypertension (Brands et al., 2010). In our current study the unexpected findings that IL-6 levels are for the most part reduced in SHR samples are a bit paradoxical. Astrocytes are just one cell type in the brain in which IL-6 is produced. Further studies to measure IL-6 levels in other brain cells are needed to elucidate the cellular patterns of IL-6 production and whether the expression of the cytokine differ in the hypertensive condition in these cells. Further an interplay does exist between neurons and other glial cells and a

Fig. 4. Effect of time on Ang II-mediated ROS levels in brainstem and cerebellum astrocytes. Quiescent monolayers of astrocytes from Wistar and SHRs brainstem (Panel A) and cerebellum (Panel B) astrocytes were incubated with 100 nM Ang II for 0.5 h to 6 h. Intracellular ROS levels were measured by DCF fluorescence assay method as described. Ang II-mediated intracellular ROS levels are expressed as the foldincrease over basal intracellular ROS levels. Each value represents the mean ± SEM of preparations of astrocytes isolated from six or more litters of neonatal rat pups. * denotes p < 0.05 as compared with basal levels for intracellular ROS in astrocytes prepared from the Wistar samples. #denotes p < 0.05 as compared with basal levels for secreted IL-6 protein levels in astrocytes prepared from the SHR astrocytes.

pro-inflammatory state can be influenced by this interaction. For example, Min et al. (Min et al., 2006) examined whether astrocytes modulate microglial inflammatory responses through decreasing microglial ROS level. They demonstrated that astrocyte culture conditioned media induced nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), increased the antioxidant response element binding activity of Nrf2, and enhanced heme oxygenase-1 expression and activity in microglia. These responses might lead to decreased microglial intracellular ROS levels and suppression of interferon-g (IFN-g)-induced inflammatory responses. These findings indicated that excessive microglial brain inflammation and subsequent tissue damage might be prevented by interactions between microglia and astrocytes (Min et al., 2006). Further, it is appreciated that the soluble IL-6 receptor (sIL-6R) plays a pivotal role in determining the levels of IL-6 expressed by astrocytes in the CNS and furthermore influences IL-6 function in the CNS (Van Wagoner et al., 1999). In addition, Nuclear Factor-IL-6 (NF-IL-6) is believed to participate in transcriptional regulation of the IL-6 promoter by proinflammatory cytokines as well (Akira, 1992; Hu et al., 1998). In the current study, we did not measure sIL-6R and NF-IL-6 and differences in these two modulators of IL-6 may be contributing to the differences we observed in SHR samples as well. Recent studies showed that treatment with Ang receptor blockers can reduce the circulating levels of some inflammatory mediators, such as IL-6, TNF-a, MCP-1 and CRP (Fliser et al., 2004; Schieffer et al., 2004). Hence, the role of Ang receptors involved in Ang II-induced IL-6 expression was also tested using specific Ang receptor blockers. Results from this study showed that, Ang II induced IL-6 mRNA and protein (intracellular and secreted) expressions were mediated via the AT1 receptor. These findings are similar to the previous studies reported in cerebellar astrocytes and vascular smooth muscle cells (Kandalam et al., 2012; Funakoshi et al., 1999; Sanz-Rosa et al., 2005; Cheng et al., 2005). This effect could be due to direct activation of signaling pathways by Ang II or mediated by indirect mechanisms such as ROS generation (Reid et al., 2005; Schieffer et al., 2000). Recent studies have demonstrated increased oxidative stress in the brain of the SHR and the stroke prone SHR (SHRSP) (Tai et al., 2005; Lee et al., 2004; Kishi et al., 2004). Using the electron spin resonance technique, levels of oxidative stress in whole SHR brains were found to be higher than in controls, and even higher in SHRSP (Lee et al., 2004). In addition, total superoxide dismutase (SOD) levels were decreased in the Rostral Ventro-Lateral Medulla (RVLM) of the SHRSP (Kishi et al., 2004), suggesting that increased brain ROS in SHRSP may arise from an imbalance of endogenous ROSscavenging mechanisms. However, the possibility of dysregulation of an enzymatic source of brain ROS in these animals has not been extensively studied. Another intriguing possibility is that brain ROS in SHR are generated by increased sensitivity to Ang II, as these rats show increased neuronal activation to both central and

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Table 4 Effects of Losartan and PD123319 on Ang II-mediated brainstem and cerebellum intracellular ROS levels. Astrocyte treatment

Wistar (Fold change) Brainstem

100 nM Ang II 10 mM Losartan 10 mM Losartan þ 100 nM Ang II 10 mM PD123319 10 mM PD123319 þ 100 nM Ang II

1.40 1.03 0.96 1.10 1.58

± ± ± ± ±

0.1* 0.1 0.1 0.04 0.2*

SHR (Fold change) Cerebellum 1.40 0.96 1.10 1.10 1.60

± ± ± ± ±

0.2* 0.2 0.2 0.2 0.2*

Brainstem 1.30 1.02 1.10 1.10 1.34

± ± ± ± ±

0.03* 0.1 0.2 0.1 0.3*

Cerebellum 1.70 0.97 0.91 0.92 1.67

± ± ± ± ±

0.1* 0.1 0.3 0.4 0.1*

Brainstem and cerebellum samples were treated with 100 nM Ang II in the presence and absence of the inhibitors for 4 h. Astrocytes were also treated with the inhibitors alone for 4 h. ROS levels were analyzed by DCF fluorescence assay method as described. * Denotes p < 0.05 as compared to basal DCF fluorescence. Values are calculated based on the individual experiments of 5 or more preparation of astrocytes.

Table 5 Effects of YCG063 and BAY11-7082on Ang II-mediated brainstem and cerebellum intracellular ROS levels. Treatment

Wistar (Fold change)

SHR (Fold change)

Brainstem

Cerebellum

100 nM Ang II 2 mM YCG063 2 mM YCG063 þ 100 nM Ang II 10 mM BAY11-7082 10 mM BAY11-7082 þ 100 nM Ang II

1.4 ± 0.1* 1.03 ± 0.1 0.92 ± 0.1 1.10 ± 0.1 1.03 ± 0.1

1.41 1.10 0.90 0.93 0.96

± ± ± ± ±

0.2* 0.3 0.1 0.1 0.1

Brainstem 1.30 1.10 0.96 1.10 1.00

± ± ± ± ±

0.03* 0.1 0.2 0.1 0.1

Cerebellum 1.70 1.10 1.10 0.90 1.10

± ± ± ± ±

0.1* 0.2 0.2 0.2 0.2

Brainstem and cerebellum samples were treated with 100 nM Ang II in the presence and absence of the inhibitors for 4 h. Astrocytes were also treated with the inhibitors alone for 4 h. ROS levels were analyzed by DCF fluorescence assay method as described. * Denotes p < 0.05 as compared to basal DCF fluorescence. Values are calculated based on the individual experiments of 5 or more preparation of astrocytes.

peripheral Ang II infusions (Blume et al., 1999; Blume et al., 1999), as well as Ang II receptor upregulation in the Nucleus Tractus Solitarii (NTS) (Chan et al., 2002). It has become clear that, besides the Ang II-dependent hypertension, increased ROS production in the brain is also common in various animal models of hypertension. In the current study, we showed that astrocytes isolated from both

SHRs and Wistar rats were equally sensitive to Ang II-induced ROS generation. Basal ROS generation was also similar. Since Ang II is known to primarily induce H2O2 and the method we used measures multiple intracellular ROS, it is plausible that our assay may not be sensitive enough to detect changes in just H2O2 levels in our samples. Further, although the substrate DCF-DA and its related

Table 6 Effects of YCG063 and BAY11-7082 on Ang II-mediated brainstem and cerebellum IL-6 mRNA levels. Astrocyte treatment

Wistar (Fold change)

100 nM Ang II 2 mM YCG063 2 mM YCG063 þ 100 nM Ang II 10 mM BAY11-7082 10 mM BAY11-7082 þ 100 nM Ang II

2.50 1.10 1.04 0.97 0.98

Brainstem ± ± ± ± ±

0.5* 0.1 0.1 0.1 0.1

SHR (Fold chnage) Cerebellum 2.10 1.10 1.10 1.00 0.96

± ± ± ± ±

0.3* 0.1 0.1 0.1 0.1

Brainstem 2.30 1.10 0.98 1.00 0.99

± ± ± ± ±

0.2* 0.1 0.1 0.5 0.1

Cerebellum 2.80 1.10 1.10 1.01 1.10

± ± ± ± ±

0.7* 0.14 0.13 0.2 0.3

Brainstem and cerebellum astrocytes were treated with 100 nM Ang II in the presence and absence of the inhibitors for 4 h. Astrocytes were also treated with the inhibitors alone for 4 h. IL-6 mRNA expression was analyzed by quantitative PCR as described. * Denotes p < 0.05 as compared to basal IL-6 mRNA expression. Values are calculated based on the individual experiments of 5 or more preparation of astrocytes.

Table 7 Effects of YCG063 and BAY11-7082 on Ang II-mediated brainstem and cerebellum intracellular IL-6 protein levels. Astrocyte treatment

Wistar (Fold change) Brainstem

100 nM Ang II 2 mM YCG063 2 mM YCG063 þ 100 nM Ang II 10 mM BAY11-7082 10 mM BAY11-7082 þ 100 nM Ang II

1.53 1.10 1.01 1.10 1.10

± ± ± ± ±

0.1* 0.1 0.3 0.2 0.2

SHR (Fold change) Cerebellum

Brainstem

Cerebellum

± ± ± ± ±

2.0 ± 0.2*ˆ 1.10 ± 0.1 0.90 ± 0.2 0.99 ± 0.1 0.97 ± 0.14

1.91 0.94 1.13 1.00 0.94

1.54 1.00 0.95 1.00 1.10

0.1* 0.1 0.1 0.1 0.2

± ± ± ± ±

0.2* 0.2 0.2 0.3 0.2

Brainstem and cerebellum samples were treated with 100 nM Ang II in the presence and absence of the inhibitors for 4 h. Astrocytes were also treated with the inhibitors alone for 4 h. IL-6 protein expression was analyzed by western blot technique as described. * Denotes p < 0.05 as compared to basal IL-6 intracellular protein expression. ˆ Denotes <0.05 as compared with corresponding Wistar samples by two-way analysis of variance. Values are calculated based on the individual experiments of 5 or more preparation of astrocytes.

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Table 8 Effects of YCG063 and BAY11-7082 on Ang II-mediated brainstem and cerebellum secreted IL-6 protein levels. Astrocyte treatment

Wistar (Fold change) Brainstem

100 nM Ang II 2 mM YCG063 2 mM YCG063 þ 100 nM Ang II 10 mM BAY11-7082 10 mM BAY11-7082 þ 100 nM Ang II

1.50 1.10 0.82 1.10 0.98

± ± ± ± ±

0.2* 0.1 0.1 0.2 0.2

SHR (Fold change) Cerebellum 1.54 1.00 1.10 1.00 1.10

± ± ± ± ±

0.1* 0.2 0.2 0.1 0.1

Brainstem 1.30 1.10 0.98 0.98 0.94

± ± ± ± ±

0.2* 0.03 0.1 0.02 0.1

Cerebellum 1.27 1.00 1.10 1.10 0.99

± ± ± ± ±

0.1* 0.1 0.1 0.2 0.2

Brainstem and cerebellum samples were treated with 100 nM Ang II in the presence and absence of the inhibitors for 4 h. Astrocytes were also treated with the inhibitors alone for 4 h. Secreted IL-6 protein levels were analyzed by an ELISA method as described. * Denotes p < 0.05 as compared to basal IL-6 protein secretion. Values are calculated based on the individual experiments of 5 or more preparation of astrocytes.

compounds have been used extensively there is substantial evidence that these molecules can generate ROS and thus artifacts (Dikalov et al., 2007). The exact molecular pathways involved in Ang II-mediated IL-6 production in astrocytes are not known. Han et al (Han et al., 1999) showed that Ang II induced IL-6 production in rat vascular smooth cells, which is mediated by the universal and pleiotropic transcription factor NF-kB. Recent in vivo studies also examined the role of NF-kB in the pathogenesis of hypertension. Increased expression of NF-kB subunits has been demonstrated in the thoracic aorta and cardiac tissue of SHRs, and these levels are normalized by treatments targeting the RAS (Gupta et al., 2005). Importantly, early inhibition of NF-kB prevents hypertension in SHRs (RodriguezIturbe et al., 2005), and also prevents cardiac hypertrophy independent of its pressure-reducing effects (Gupta et al., 2005). Given that the etiology of hypertension in SHR may involve Ang IIinduced oxidative stress in the brain (Kishi et al., 2004; Polizio and Pena, 2005), one intriguing possibility is that hypertension in these animals also involves redox-dependent activation of NF-kB within the cardiovascular nuclei. In the brain, NF-kB is known to play a role in a number of inflammatory processes, including ischemia, seizures, and acute trauma, as well as in a number of neurodegenerative diseases, especially Alzheimer's disease (Mattson and Camandola, 2001). Ang II-induced ROS production may also mediate gene transcription through activation of NF-kB as well. NF-kB activity is known to be redox-regulated (Turpaev, 2002; Dalton et al., 1999), with redox-sensitive MAP kinase pathways, especially those regulating JNK and p38 MAPK, being involved in NF-kB activation. Furthermore, increased levels of oxidized thioredoxin activate NFkB (Turpaev, 2002). Ang II, activates NF-kB via the high affinity type 1 angiotensin receptor (AT1A) in vascular tissues and hepatocytes in a heterotrimeric G protein-dependent manner (Mehta and Griendling, 2007; Zhai et al., 2005). Wu et al. showed that NF-kB pathway as an intracellular signal in Ang II-mediated ROS and IL-6 production in retinal pigment epithelial cells (Wu et al., 2010). In the current study, we determined whether NF-kB mediates Ang II ROS and IL-6 mRNA and protein levels. Our findings showed that pretreatment with the NF-kB inhibitor, BAY11-7082, inhibited both Ang II-mediated ROS production and IL-6 mRNA and protein (intracellular and secreted) levels. We also showed that inhibition of ROS generation also inhibits IL-6 mRNA and protein levels as well. Taken together, these findings suggest that Ang II acting on the Ang AT1 receptor induces NF-kB leading to ROS generation and subsequently IL-6 production in both brain regions from the Wistar and SHRs.

5. Conclusion Progress in this field continues to reveal the increasing

complexity of the molecular mechanisms involved in Ang IImediated central control of blood pressure, and it is becoming apparent that perturbations in brain ROS and neuro-inflammatory processes have a profound effect on cardiovascular function. The challenge remains in defining the precise mechanisms by which these mediators participate in the central regulation of blood pressure under acute and chronic conditions. Results from the current study sought to give new insights into the proinflammatory nature of Ang II through its ability to induce the inflammatory cytokine IL-6. Our data established Ang II-mediated IL-6 production through that AT1/NF-kB/ROS pathway as one of the key signaling pathways in astrocytes from the hypertensive SHR model. Although some of the findings of this study was unexpected, the differences observed in SHRs as compared to the Wistar controls may lead to the identification of key neuroinflammation factors governing the hypertensive condition in this rat model. We acknowledge that these studies were performed under specific conditions in astrocytes and that additional studies are needed using in vivo systems, co-cultures, tissue slices, and using other conditions to fully translate our findings into the creation of products for therapeutic interventions for hypertension. However, the novelty of our studies lies in the data that was provided that may lead to a better understanding of the molecular underpinnings of neuroinflammatory processes, in particular, ROS-mediated long term changes in neuro-cardiovascular regulation has the potential to fundamentally advance our understanding of the mechanisms linking the CNS with cardiovascular diseases. This will ultimately lead to the development of novel therapeutic strategies for the treatment of neurogenic hypertension and possibly other diseases as well. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgement This work was supported by a President's Faculty Research and Development Grant (#335889) from Nova Southeastern University. References Abeywardena, M.Y., Leifert, W.R., Warnes, K.E., Varghese, J.N., Head, R.J., 2009. Cardiovascular biology of interleukin-6. Curr. Pharm. Des. 15, 1809e1821. Akira, S., 1992. NF-IL6 and gene regulation. Res. Immunol. 143, 734e736. Akira, S., Kishimoto, T., 1992. IL-6 and NF-IL6 in acute-phase response and viral infection. Immunol. Rev. 127, 25e50. Alarcon, R., Fuenzalida, C., Santibanez, M., von Bernhardi, R., 2005. Expression of scavenger receptors in glial cells. Comparing the adhesion of astrocytes and microglia from neonatal rats to surface-bound beta-amyloid. J. Biol. Chem. 280, 30406e30415. Baldi, P., Long, A.D., 2001. A Bayesian framework for the analysis of microarray expression data: regularized t -test and statistical inferences of gene changes.

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