Toxicology and Applied Pharmacology 279 (2014) 141–149

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Chronic infusion of lisinopril into hypothalamic paraventricular nucleus modulates cytokines and attenuates oxidative stress in rostral ventrolateral medulla in hypertension Hong-Bao Li a,1, Da-Nian Qin b,⁎,1, Le Ma c,1, Yu-Wang Miao a, Dong-Mei Zhang e, Yan Lu d, Xin-Ai Song a, Guo-Qing Zhu f, Yu-Ming Kang a,⁎⁎,1 a

Department of Physiology and Pathophysiology, Xi'an Jiaotong University Cardiovascular Research Center, Xi'an Jiaotong University School of Medicine, Xi'an 710061, China Department of Physiology, Shantou University Medical College, Shantou 515041, China c Department of Public Health, Xi'an Jiaotong University School of Medicine, Xi'an 710061, China d Department of Clinical Laboratory, Sanaitang Hospital, Lanzhou 730030, China e Department of Physiology, Dalian Medical University, Dalian 116044, China f Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, Nanjing 210029, China b

a r t i c l e

i n f o

Article history: Received 2 February 2014 Revised 7 May 2014 Accepted 6 June 2014 Available online 14 June 2014 Keywords: Hypertension Rostral ventrolateral medulla Hypothalamic paraventricular nucleus Angiotensin-converting enzyme Cytokines Oxidative stress

a b s t r a c t The hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) play a critical role in the generation and maintenance of sympathetic nerve activity. The renin–angiotensin system (RAS) in the brain is involved in the pathogenesis of hypertension. This study was designed to determine whether inhibition of the angiotensin-converting enzyme (ACE) in the PVN modulates cytokines and attenuates oxidative stress (ROS) in the RVLM, and decreases the blood pressure and sympathetic activity in renovascular hypertensive rats. Renovascular hypertension was induced in male Sprague–Dawley rats by the two-kidney one-clip (2K1C) method. Renovascular hypertensive rats received bilateral PVN infusion with ACE inhibitor lisinopril (LSP, 10 μg/h) or vehicle via osmotic minipump for 4 weeks. Mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), and plasma proinflammatory cytokines (PICs) were significantly increased in renovascular hypertensive rats. The renovascular hypertensive rats also had higher levels of ACE in the PVN, and lower level of interleukin-10 (IL-10) in the RVLM. In addition, the levels of PICs, the chemokine MCP-1, the subunit of NAD(P)H oxidase (gp91phox) and ROS in the RVLM were increased in hypertensive rats. PVN treatment with LSP attenuated those changes occurring in renovascular hypertensive rats. Our findings suggest that the beneficial effects of ACE inhibition in the PVN in renovascular hypertension are partly due to modulation cytokines and attenuation oxidative stress in the RVLM. © 2014 Elsevier Inc. All rights reserved.

Introduction Both hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) play important roles in the regulation of sympathetic drive and cardiovascular activity. The number of PVN neurons projecting to the RVLM (PVN–RVLM neurons) is as much as sevenfold as the number of PVN neurons projecting to the spinal cord (Agarwal et al., 2011; Kumagai et al., 2012), and the PVN–RVLM pathway contributes to the change in sympathetic nerve activity (SNA) observed after activation of the PVN. The RVLM also contains presympathetic neurons that have spontaneous activity and directly

⁎ Corresponding author. ⁎⁎ Correspondence to: Y.-M. Kang, Department of Physiology & Pathophysiology, Xi'an Jiaotong University School of Medicine, Xi'an 710061, China. Fax: +86 2982657677. E-mail addresses: [email protected] (D.-N. Qin), [email protected] (Y.-M. Kang). 1 These authors contributed equally to this study.

http://dx.doi.org/10.1016/j.taap.2014.06.004 0041-008X/© 2014 Elsevier Inc. All rights reserved.

project to the spinal cord (Kumagai et al., 2012). The basal activity of presympathetic neurons in the RVLM is a major mechanism responsible for the generation of resting blood pressure (BP) and sympathetic outflow (Kumagai et al., 2012; Zhang et al., 2008). The renin–angiotensin system (RAS) plays an important role in the pathophysiology of cardiovascular disease (Veerasingham and Raizada, 2003). Angiotensin II (ANG II) is a principal and biologically active component of the RAS, exerts its actions mainly via interaction with the angiotensin II type 1 receptor (AT1-R), thereby contributing to sympathoexcitation and hypertensive response (Sriramula et al., 2011; Kasal and Schiffrin, 2012; Kumagai et al., 2012). AT1 receptors are widely distributed in the central nervous system from the forebrain to the brain stem (Xu et al., 1998). A growing body of evidence indicates that the main peptide of the RAS, ANG II, induces inflammatory molecules and contributes to the pathophysiology of cardiovascular diseases (Kang et al., 2009a,b; Qi et al., 2013). Recent studies also suggest that cytokines and RAS can contribute to these changes (Qi

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et al., 2013; Sriramula et al., 2013). Furthermore, data from our laboratory suggest that ANG II infusion increases proinflammatory cytokine (PIC) levels, such as tumor necrosis factor alpha-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in plasma and PVN (Kang et al., 2009a,b), and ANG II blockade decreases PIC levels in plasma and PVN in cardiovascular disease (Kang et al., 2009a,b; Qi et al., 2013). It is well known that angiotensin-converting enzyme (ACE) promotes ANG II production through acting on the blood-born angiotensin I (ANG I). In addition, inhibition of ACE results in the reduction of PICs in the hearts of the spontaneously hypertensive rats (Miguel-Carrasco et al., 2010). However, whether inhibition of ACE in the PVN modulates cytokines in the RVLM and decreases the blood pressure and sympathetic activity during renovascular hypertension is still unclear. Reactive oxygen species (ROS) in the RVLM is increased and contributes to the neural mechanisms of hypertension in strokeprone spontaneously hypertensive rats (SHRSPs) (Kishi and Hirooka, 2012; Kishi et al., 2008) and spontaneously hypertensive rats (SHRs) (Koga et al., 2008). Our laboratory and others have shown that chronic peripheral ANG II infusion in rat induces hypertension, which is accompanied by superoxide accumulation in the RVLM or the PVN and increased sympathetic activity (Kang et al., 2009a,b; Zimmerman et al., 2004). Increasing evidence demonstrates that NAD(P)H oxidase derived ROS are important mediators of ANG II signaling (Iwanami et al., 2009). ANG II not only augments ROS formation, increases the oxidase activity, but also upregulates mRNA and protein expression of most NAD(P)H oxidase subunits in vitro and in vivo (Liu et al., 2008; Peng et al., 2013). In renovascular hypertensive rats, chronic oxidative stress within the RVLM is a major mechanism leading to chronic sympathoexcitation and high blood pressure (Oliveira-Sales et al., 2009). Considering that the 2K1C model of arterial hypertension has a strong neurogenic component and central oxidative stress involved in the maintenance of high blood pressure, we tested the hypothesis that inhibition of ACE in the PVN attenuates 2K1C hypertensive responses and sympathoexcitation by the preferential reduction of oxidative stress and modulation of cytokines within the RVLM using lisinopril (LSP), an anti-hypertensive drug and potent reversible ACE inhibitor (Sharma and Singh, 2012). Materials and methods Animal. Experiments were performed on seven-week-old male Sprague–Dawley rats weighing between 275 g and 300 g. The rats were housed in a climate-controlled room with a 12 h light–dark cycle and allowed access to standard rat chow and tap water ad libitum (Kang et al., 2004; Yang et al., 1998). These experiments were approved by the Animal Care and Use Committee of Xi'an Jiaotong University and conformed to the Guidelines for the Care and Use of Experimental Animals of the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996). All the following surgeries were performed under anesthesia and aseptic conditions. General experimental protocol. After undergoing subcutaneously implantation of bilateral PVN cannulae, the rats were allowed a week for recovery. Measurement of baseline blood pressure (BP) was continuous for 3 days, and then renovascular hypertension was induced in male Sprague–Dawley rats for 4 weeks by the two-kidney one-clip (2K1C) method as previously reported (Han et al., 2011; Zhu et al., 2009). The osmotic minipumps (Alzet Model 1004, Durect Corporation, Cupertino, CA) were connected to the PVN cannulae for the continuous infusion of the ACE inhibitor lisinopril (LSP, 10 μg/h) or vehicle (artificial cerebrospinal fluid, aCSF) directly into the bilateral PVN over 4 weeks (Kang et al., 2009a,b). At the end of 4 weeks, rats were anesthetized with a ketamine (80 mg/kg) and xylazine (10 mg/kg) mixture (ip) and euthanized to collect blood and brain tissue for molecular and immunohistochemical studies. Some rats were anesthetized for terminal electrophysiological studies.

Bilateral PVN cannulae implantation for chronic infusion studies. The method for implantation of bilateral PVN cannulae has been described previously (Qi et al., 2013). Briefly, after the rat was anesthetized with a ketamine (80 mg/kg) and xylazine (10 mg/kg) mixture (ip), the head was placed into a stereotaxic apparatus. The skull was then opened, and a stainless steel double cannula was implanted into the PVN according to stereotaxic coordinates. The cannula was fixed to the cranium using dental acrylic and two stainless steel screws. Rats received buprenorphine (0.01 mg/kg, sc) immediately following surgery and 12 h postoperatively. The histological identification was made to verify each injection site. The success rate of bilateral PVN cannulation is 64%, and only animals with verifiable bilateral PVN injection sites were used in the final analysis. Mean arterial pressure (MAP) measurement. Blood pressure was determined by a tail−cuff occlusion and acute experiment method. Unanesthetized rats were warmed to an ambient temperature of 32 °C by placing rats in a holding device mounted on a thermostatically controlled warming plate. Rats were allowed to habituate to this procedure for 3 days prior to each experiment. Blood pressure values were averaged from seven consecutive cycles per day obtained from each rat. At the end of the 4th week, rats were anesthetized. The femoral artery was cannulated with polyethylene catheters for the measurement of arterial blood pressure (BP). The catheters were prior filled with 0.1 ml heparinized saline (50 units/ml) and connected to a pressure transducer attached to a digital BP monitor and a polygraph. MAP and heart rate (HR) data were collected for 30 min and averaged. Electrophysiological recordings. Rats were anesthetized with a ketamine (80 mg/kg) and xylazine (10 mg/kg) mixture (ip). A retroperitoneal incision was made and the left renal sympathetic nerve was isolated. The renal nerve was placed on a platinum electrode which is connected with the recording system and immersed in warm mineral oil. Maximum renal sympathetic nerve activity (RSNA) was detected using an intravenous bolus administration of sodium nitroprusside (SNP, 10 μg). At the end of the experiment, the background noise, defined as the signal-recorded postmortem, was subtracted from actual RSNA and subsequently expressed as percent of maximum (in response to SNP). The general methods for recording and data analysis have been described previously (Guggilam et al., 2007; Kang et al., 2008, 2010). Collection of blood and tissue samples. Rats were decapitated under anesthesia with a ketamine (80 mg/kg) and xylazine (10 mg/kg) mixture (ip) to collect blood and tissue samples at the end of the 4th week of the experiment. Trunk blood samples were collected in chilled ethylenediaminetetraacetic acid tubes. Plasma samples were separated and stored at −80 °C until assayed for cytokines. Tissue microdissection. Microdissection procedure was used to isolate the PVN and the RVLM as previously described (Gao et al., 2005). The tissues were collected from both sides of the PVN and the RVLM of individual rat. Biochemical assays. The levels of IL-1β and IL-6 in plasma and tissue were quantified using commercially available rat ELISA kits (Biosource International Inc., Camarillo, California) according to the manufacturer's instructions. TNF-α in tissue was measured using a high sensitivity kit (GenStar Biosolutions Co., Beijing, China). According to the manufacturer's descriptions, the standards or sample diluents were added and incubated in the appropriate well of specific antibody precoated microtiter plate. Conjugate was added and incubated for 1 h at 37 °C and then washed. The reactions were stopped with stop

H.-B. Li et al. / Toxicology and Applied Pharmacology 279 (2014) 141–149

2K1C+PVN LSP (n=7)

180

2K1C+PVN vehicle (n=7)

160

SHAM+PVN vehicle (n=7)

MAP (mmHg)

SHAM+PVN LSP (n=7)

*

*

*†

*†

*†

22

25

28

*

*

*

*

*†

*†

13

16

19

* 140

* 120 100 80 C1

C2

1

4

7

10

Time (days)

143

Santa Cruz Biotechnology, Santa Cruz, CA) in the RVLM, and ACE (sc-20791, Santa Cruz Biotechnology, Santa Cruz, CA) in the PVN as previously described (Agarwal et al., 2011). Protein loading was controlled by probing all blots with β-actin antibody (Thermo Scientific, USA) and normalizing their protein intensities to that of β-actin. Band densities were analyzed with NIH ImageJ software.

Statistical analysis. Data were expressed as mean ± SEM. All data were analyzed by ANOVA followed by a post-hoc Tukey test. Blood pressure data were analyzed by repeated measures ANOVA. A P value of b 0.05 was considered significantly different.

Results

Fig. 1. Effects of PVN LSP or vehicle on mean arterial pressure (MAP) of 2K1C rats and SHAM rats. 2K1C induced an increase in MAP compared with SHAM. Bilateral PVN infusions of LSP attenuated 2K1C-induced hypertensive response. *P b 0.05 versus SHAM groups (SHAM + PVN LSP or SHAM + PVN vehicle); †P b 0.05 2K1C + PVN LSP versus 2K1C + PVN vehicle.

solution and read at 450 nm for IL-1β, TNF-α and IL-6 measurements using a microtiter plate reader (MK3, Thermo Fisher Scientific, USA). Immunohistochemical and immunofluorescence studies. The PVN were counted manually in two representative 18 μm transverse sections at about 1.80 mm from bregma, and an average value was reported. The RVLM was identified as the region extending caudally 500–700 μm from the caudal pole of the facial nucleus. This distance corresponded to 12.4–12.6 mm from the bregma as described previously (Nishi et al., 2013; Paxinos et al., 1980). Immunohistochemical and immunofluorescence stainings were performed in floating sections as described previously (Kang et al., 2006, 2009a,b) to identify IL-1β (sc-1251, Santa Cruz Biotechnology, Santa Cruz, CA), IL-10 (sc-1783, Santa Cruz Biotechnology, Santa Cruz, CA), gp91phox (sc-20782, Santa Cruz Biotechnology, Santa Cruz, CA) in the RVLM and angiotensin-converting enzyme (ACE; sc-20791, Santa Cruz Biotechnology, Santa Cruz, CA) expressions in the PVN. For each animal, the positive neurons within the bilateral borders of the PVN or RVLM were manually counted in three consecutive sections and an average value was reported. To measure the production of superoxide anion, dihydroethidium (DHE; Molecular Probes) stock (15 mM) was made in DMSO, and dilutions of the stock were used only on the experimental day. Slices containing the RVLM were incubated in DHE for 30 min at 4 °C and protected from light. DAPI for nuclear staining was from Molecular Probes. Western blot. The tissue homogenate from the PVN and RVLM were subjected to Western blot analysis for determination of protein levels of MCP-1 (FL-148, Santa Cruz Biotechnology, Santa Cruz, CA), IL-1β (sc-1251, Santa Cruz Biotechnology, Santa Cruz, CA), IL-10 (sc-1783, Santa Cruz Biotechnology, Santa Cruz, CA), gp91phox (sc-20782,

Mean arterial blood pressure (MAP) measurement Bilateral PVN infusions of LSP attenuated renovascular hypertension (Fig. 1). By observing the results of acute experiment, MAP in 2K1C rats was significantly higher than that in SHAM rats. There were no significant differences in the body weight and HR between SHAM rats and 2K1C rats (Table 1). Rats were trained by MAP measurement daily for at least 14 days before 2K1C or sham operation to minimize stress induced MAP fluctuation. The MAP was measured every three days in conscious state by using a noninvasive computerized tail cuff system. 2K1C induced a significant increase in MAP compared with the controls; and MAP remained elevated throughout 28 days (Fig. 1).

Renal sympathetic nerve activity (RSNA) Conscious RSNA was measured 5 h after rats recovered from anesthesia. 2K1C rats exhibited higher RSNA (% of max) when compared to SHAM rats. Bilateral PVN infusion of LSP attenuated RSNA in 2K1C rats (Fig. 2).

Expression of ACE in the PVN As shown in Figs. 3 and 4A, the expression of ACE in the PVN was significantly upregulated in 2K1C rats compared with SHAM rats. 4 weeks bilateral infusions of LSP into the PVN decreased the expression of ACE in the PVN of 2K1C rats (Figs. 3 and 4A).

Expression of cytokines in the RVLM 2K1C rats had higher levels of IL-1β (Figs. 4B and 5) and MCP-1 (Fig. 4B), and lower level of IL-10 (Figs. 4B and 5) in the RVLM than those of SHAM rats. PVN treatment with LSP attenuated the increases in IL-1β and MCP-1, and a decrease in IL-10 in the RVLM of 2K1C rats (Figs. 4B and 5).

Table 1 Changes of body weight, MAP and HR at the end of the 4th week of the experiment. Parameters

Body weight, g MAP, mm Hg HR, bpm

SHAM

2K1C

Vehicle

LSP

Vehicle

LSP

365 ± 7 (n = 10) 101 ± 4 (n = 7) 352 ± 5 (n = 7)

358 ± 9 (n = 9) 98 ± 3 (n = 8) 360 ± 10 (n = 8)

351 ± 8 (n = 11) 155 ± 5 (n = 7)⁎ 358 ± 7 (n = 7)

345 ± 10 (n = 10) 127 ± 4 (n = 8)⁎† 355 ± 6 (n = 8)

The values shown are the mean ± SEM. 2K1C, 2-kidney, 1-clip; MAP, mean arterial pressure; HR, heart rate. ⁎ Pb 0.05 versus SHAM groups (SHAM + PVN LSP or SHAM + PVN vehicle). † Pb 0.05 2K1C + PVN LSP versus 2K1C + PVN vehicle.

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A 2K1C+PVN LSP 2K1C+PVN vehicle SHAM+PVN LSP SHAM+PVN vehicle

2K1C+PVN LSP (n=7)

B

2K1C+PVN vehicle (n=7)

100

% of max

80 60

SHAM+PVN LSP (n=7)

*

*†

SHAM+PVN vehicle (n=7)

40 20 0

RNSA

Fig. 2. Effects of PVN LSP or vehicle on renal sympathetic nerve activity (RSNA) of 2K1C rats and SHAM rats. RSNA was increased in 2K1C rats compared with SHAM rats. Treatment with PVN LSP for 4 weeks attenuated RSNA of 2K1C rats. *P b 0.05 versus SHAM groups (SHAM + PVN LSP or SHAM + PVN vehicle); †P b 0.05 2K1C + PVN LSP versus 2K1C + PVN vehicle.

Superoxide and NAD(P)H oxidase in the RVLM

Co-expression of IL-1β and gp91phox in the RVLM

Immunofluorescence revealed that 2K1C rats had more superoxide in the RVLM, as determined by fluorescent labeled dihydroethidium (DHE) and the NAD(P)H oxidase subunit gp91phox, when compared with SHAM rats (Fig. 6). Bilateral PVN infusion of LSP for 4 weeks decreased gp91phox and DHE in the RVLM of 2K1C rats (Fig. 6).

Double labeling using confocal microscopy revealed that 52.6% of the gp91 phox positive neurons are also positive for IL-1β in 2K1C rats (Fig. 7). Only 27.6% of gp91 phox positive neurons were positive for IL-1β in 2K1C rats treated with PVN infusion of LSP (Fig. 7).

A

2K1C+PVN LSP

2K1C+PVN vehicle

SHAM+PVN LSP SHAM+PVN vehicle

B

number of positive neurons (cells per 2х105 μm2)

ACE

30

20

* *†

2K1C+PVN LSP (n=7) 2K1C+PVN vehicle (n=7) SHAM+PVN LSP (n=7˅ SHAM+PVN vehicle (n=7)

10

0

ACE in PVN

Fig. 3. Immunofluorescence for angiotensin-converting enzyme (ACE) expression in the PVN. ACE expression in the PVN was lower in the LSP-treated 2K1C rats than in vehicle-treated 2K1C rats. *P b 0.05 versus SHAM groups (SHAM + PVN LSP or SHAM + PVN vehicle); †P b 0.05 2K1C + PVN LSP versus 2K1C + PVN vehicle.

Protein expression in PVN

2K1C 2K1C SHAM + + + PVN LSP PVN vehicle PVN LSP

SHAM + PVN vehicle

ACE β-actin

Protein expression in PVN (Arbitrary units)

A

1.5

Protein expression in RVLM (Arbitrary units)

H.-B. Li et al. / Toxicology and Applied Pharmacology 279 (2014) 141–149

1.5

B Protein expression in RVLM 2K1C 2K1C SHAM + + + PVN LSP PVN vehicle PVN LSP

SHAM + PVN vehicle

MCP-1 IL-1β IL-10 gp91phox β-actin

2K1C+PVN LSP (n=7) 2K1C+PVN vehicle (n=7) SHAM+PVN LSP (n=7) SHAM+PVN vehicle (n=7)

*

*†

1.0

145

0.5

0.0

ACE

* 1.0

*†

*†

*

*†

*†

*

*

0.5

0.0

MCP-1 IL-1β

IL-10 gp91phox

Fig. 4. Western blot of MCP-1, IL-1β, IL-10 and gp91phox in the RVLM and ACE in the PVN. (A). 2K1C rats had higher levels of ACE in the PVN when compared with SHAM rats. PVN treatment with LSP decreased expression of ACE in the PVN of 2K1C rats. (B). 2K1C rats had higher levels of MCP-1, IL-1β and gp91phox, and lower level of IL-10 in the RVLM when compared with SHAM rats. PVN treatment with LSP decreased expression of MCP-1, IL-1β and gp91phox, and increased IL-10 expression in the RVLM of 2K1C rats. *P b 0.05 versus SHAM groups (SHAM + PVN LSP or SHAM + PVN vehicle); †P b 0.05 2K1C + PVN LSP versus 2K1C + PVN vehicle.

A

2K1C+PVN LSP

2K1C+PVN vehicle

SHAM+PVN LSP

SHAM+PVN vehicle

IL-1 β

IL-10

*

20

*†

2K1C+PVN LSP (n=7) 2K1C+PVN vehicle (n=7) SHAM+PVN LSP (n=7) SHAM+PVN vehicle (n=7)

10

0

IL-1β in RVLM

C

number of positive neurons (cells per 2х105 μm2)

30

number of positive neurons (cells per 2х105 μm2)

B

100 80 60 40 20 0

*† * IL-10 in RVLM

Fig. 5. RVLM levels of interleukin-1β (IL-1β) and interleukin-10 (IL-10) immunoreactivity in 2K1C rats and SHAM rats. (A and B) RVLM levels of IL-1β immunoreactivity (green) in 2K1C rats were increased than in SHAM rats. PVN treatment with LSP decreased the IL-1β immunoreactivity in the RVLM of 2K1C rats. (A and C) RVLM levels of IL-10 immunoreactivity in 2K1C rats were lower than in SHAM rats. PVN treatment with LSP increased the IL-10 immunoreactivity in the RVLM of 2K1C rats. *P b 0.05 versus SHAM groups (SHAM + PVN LSP or SHAM + PVN vehicle); †P b 0.05 2K1C + PVN LSP versus 2K1C + PVN vehicle.

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A

2K1C+PVN LSP

2K1C+PVN vehicle

SHAM+PVN LSP SHAM+PVN vehicle

gp91phox

25

*

20 15 10

*†

2K1C+PVN LSP (n=7) 2K1C+PVN vehicle (n=7) SHAM+PVN LSP (n=7) SHAM+PVN vehicle (n=7)

5 0

gp91phox in RVLM

C fluorescent intensity

B

number of positive neurons (cells per 2х105 μm2)

DHE

*

80 60

*†

40 20 0

DHE in RVLM

Fig. 6. Superoxide and NAD(P)H oxidase in the RVLM. (A). Immunofluorescence for the NAD(P)H oxidase subunit gp91phox (red) and superoxide as determined by fluorescent-labeled dihydroethidium (DHE) in the RVLM in different groups. (B) Comparison of gp91phox positive neurons in the RVLM in different groups. (C) Immunofluorescent intensity of DHE in the RVLM of different groups of rats. *P b 0.05 versus SHAM groups (SHAM + PVN LSP or SHAM + PVN vehicle); †P b 0.05 2K1C + PVN LSP versus 2K1C + PVN vehicle.

RVLM levels of proinflammatory cytokines RVLM levels of TNF-α (Fig. 8A) and IL-6 (Fig. 8B) were higher in 2K1C rats than those in SHAM rats. PVN treatment with LSP reduced the levels of TNF-α and IL-6 in the RVLM of 2K1C rats (Figs. 8A and B). Plasma humoral factors Plasma levels of IL-1β (Fig. 8C) and IL-6 (Fig. 8D) in 2K1C rats were higher than those in SHAM rats. 2K1C rats treated with LSP had significantly lower levels of plasma IL-1β and IL-6 than those in 2K1C rats treated with vehicle, but these values remained higher than those in SHAM rats (Figs. 8C and D). Discussion The novel findings of this study are that: (i) 2K1C induced sympathoexcitation and hypertensive responses are associated with the levels of cytokines and oxidative stress in the RVLM; and (ii) chronic inhibition of ACE in the PVN attenuates sympathoexcitation and ROS and modulates cytokines in the RVLM in renovascular hypertension. The PVN is an important integrative site in the control of sympathetic outflow and cardiovascular function, particularly via its projections to principal centers of sympathetic drive, the RVLM, and the intermediolateral (IML) cell column of the spinal cord (Kumagai et al., 2012; Xu et al., 2012; Zhang et al., 2008). It is well known that baseline sympathetic outflow mediated by the RVLM is mainly dependent on the spontaneous activity of preautonomic neurons (Gao et al., 2008). Previous studies (Chen et al., 2010; Kang

et al., 2009a,b) have demonstrated that PVN axons have terminal sites closely associated with spinally projecting RVLM neurons, many of which are likely to terminate at sympathetic preganglionic neurons. The mechanism by which the sympathoexcitatory is enhanced in the hypertensive state has been a topic of intense investigation for many years. There is increasing evidence that ANG II binding to neuronal AT1 receptors upregulates hypertensive response by acting on the cardiovascular centers of the central nervous system and increasing sympathetic activity (Qi et al., 2013). A growing body of evidence indicates that cytokine mediators, including TNF-α, IL-10, IL-1β and IL-6, are capable of regulating various RAS components in a variety of mammalian tissues, including the heart and the brain (Kang et al., 2010; Sriramula et al., 2013). ACE is responsible for ANG II production. Lisinopril (LSP) is a potent reversible and long-acting ACE inhibitor compared with other ACE inhibitors. LSP produced significant falls in both systolic and diastolic blood pressure in hypertensive patient during the long-term therapy. The dose of LSP selected in this study was found to be effective in chronic studies in hypertensive rats (Ongali et al., 2003). Suppression of central ACE has been found to down-regulate the proinflammatory cytokines (PICs), such as TNF-α, IL-1β and IL-6 in the heart of the spontaneously hypertensive rat, and also normalizes the exaggerated sympathetic activity of the heart failure rats (MiguelCarrasco et al., 2010). The present study also proved that ACE inhibition modulates cytokines in the RVLM and plasma. Our results are consistent with the finding that peripheral ANG II induces production of PIC through activating immune cells (Ruiz-Ortega et al., 2002). In addition, both in vitro and in vivo studies have demonstrated the existence of cross-talk between the RAS and TNF-α (Kang et al., 2008; Mariappan et al., 2012).

H.-B. Li et al. / Toxicology and Applied Pharmacology 279 (2014) 141–149

2K1C+PVN LSP

2K1C+PVN vehicle

SHAM+PVN LSP

147

SHAM+PVN vehicle

IL-1β

gp91phox

Merge

Fig. 7. Co-localization of double labeling for gp91phox positive neurons and IL-1β positive neurons in the RVLM of 2K1C rats and SHAM rats. Double labeling using confocal microscopy confirmed that 52.6% of the gp91phox positive neurons are also positive for IL-1β in 2K1C rats. Only 27.6% of gp91phox positive neurons were positive for IL-1β in 2K1C rats treated with PVN infusion of LSP.

Recently, it was demonstrated that the proinflammatory cytokines TNF-α and IL-1β upregulated AT1-R density on rat cardiac fibroblasts (Elton, 2007). Acute application of IL-1β into the left ventricle or the PVN in normal rats results in a significant increase in mean arterial pressure (Shi et al., 2010a). Furthermore, microinjection of TNF-α or IL-1β in the PVN increases blood pressure and sympathetic outflow, which is partially dependent on the AT1-R (Shi et al., 2011). On the other hand, our previous studies have shown that PIC and AT1-R in the PVN regulate blood pressure and are involved in the sympathetic hyperactivity in heart failure (Kang et al., 2008). A recent study found that ANG II in the PVN facilitated production of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6. Moreover, the overexpression of IL-10, an anti-inflammatory cytokine, in the PVN also demonstrated anti-hypertensive effects (Shi et al., 2010a). Recent studies showed that the RVLM contains high concentrations of AT1 receptors and is a major site of the sympathoexcitatory action of central ANG II (Lenkei et al., 1998). An interesting question that arises from these observations is whether the inflammatory cytokines in the RVLM are involved in the modulation of the blood pressure and the sympathetic activity. In this study, we demonstrated that chronic inhibition of ACE in the PVN had significantly reduced MAP; RSNA decreased the levels of PIC (such as TNF-α, IL-1β, IL-6) and the chemokine MCP-1 and increased the levels of IL-10 in the RVLM. These results were consistent with the recent observation that ACE inhibition decreases PIC (IL-6 and TNF-α) and increases IL-10 in spontaneously hypertensive rats (Dalpiaz et al., 2013). NAD(P)H oxidase derived reactive oxygen species (ROS) acts as potent intra- and inter-cellular second messengers in signaling pathways causing hypertension (Hanna et al., 2002). It is well established that the stimulation of NAD(P)H oxidase derived ROS may increase sympathetic outflow (Knapp and Klann, 2002). Moreover,

ANG II is a potent stimulator of ROS (Zimmerman et al., 2002). Chan et al. (2005) demonstrated that tempol (an antioxidant) reduces the hypertensive responses and sympathoexcitation to microinjection of ANG II into the RVLM and the PVN in normal animals. Several studies also indicated that the intracerebroventricular administration of losartan (an AT1-R antagonist) inhibits the ANG II–induced increases in blood pressure, HR, and RSNA associated with the upregulation of AT1-R and NADPH oxidase subunits (p22phox, p47phox, and p67phox) in the RVLM of the chronic heart failure rabbits (Gao et al., 2004; Kang et al., 2009a,b). In the PVN and RVLM, the pro-inflammatory cytokines such as TNF-α and IL-1β are known to increase neuronal activity via activation of the transcription factor NF-κB (Shi et al., 2010b). Thus, it is possible that the inflammatory responses observed in the PVN and RVLM also contribute to sympathoexcitation in hypertension. Since these inflammatory molecules can induce ROS production, the functional roles of RVLM cytokines and their potential link to the ROS-related hyperactivity of RVLM neurons in hypertension (Hirooka, 2011) need to be elucidated. Finally, it will be of interest to assess whether infusion of ACE inhibitor LSP into the PVN is able to attenuate ROS generation within the RVLM of renovascular hypertensive rats. Our data demonstrated that inhibition of ACE in the PVN attenuates the MAP, RSNA, the subunit of NAD(P)H oxidase (gp91phox) and oxidative stress in the RVLM, indicating that 2K1C-induced hypertension may result from the augment of oxidative stress as indicated by the increased levels of gp91 phox within the RVLM. In summary, the present study found that both cytokines and oxidative stress in the RVLM contribute to the enhanced blood pressure and sympathetic activity in renovascular hypertension. ACE inhibition in the PVN may be beneficial to decrease sympathoexcitation in renovascular hypertension via interaction with cytokines and oxidative stress in the RVLM, which leads to a better understanding of the

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B 8 6 4

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80

IL-6 (pg/mg protein)

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20 0

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Fig. 8. Effects of PVN LSP or vehicle on the RVLM levels of TNF-α and IL-6 and the plasma levels of IL-1β and IL-6 of 2K1C rats and SHAM rats. (A and B) RVLM levels of TNF-α and IL-6 in 2K1C rats were higher than in SHAM rats. PVN treatment with LSP reduced the levels of TNF-α and IL-6 in the RVLM of 2K1C rats. (C and D) Plasma levels of IL-1β and IL-6 in 2K1C rats were higher than in SHAM rats. PVN treatment with LSP reduced the levels of IL-1β and IL-6 in the plasma of 2K1C rats. *P b 0.05 versus SHAM groups (SHAM + PVN LSP or SHAM + PVN vehicle); †P b 0.05 2K1C + PVN LSP versus 2K1C + PVN vehicle.

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