draft · draft chronic intermittent hypobaric hypoxia provides vascular protection in aorta of...

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Chronic intermittent hypobaric hypoxia provides vascular

protection in aorta of two-kidney one clip rat model of

hypertension

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2017-0356.R2

Manuscript Type: Article

Date Submitted by the Author: 30-Sep-2017

Complete List of Authors: Congrui, Fu; Hebei Medical University

Na, Li; Medical College, Hebei University, Department of Physiology Yujia, Yuan; Hebei Medical University, Department of Physiology Ri, Wang; Hebei Medical University, Department of Physiology Jinting, Chen; Hebei Medical University, Department of Physiology Jing, Yang; Hebei Medical University, Department of Physiology Zan, Guo; Hebei Medical University, Department of Physiology Sheng, Wang; Hebei Medical University, Department of Physiology; Hebei Collaborative Innovation Center for Cardio-cerebrovascular Disease Yi, Zhang; Hebei Medical University, Department of Physiology; Hebei Collaborative Innovation Center for Cardio-cerebrovascular Disease yixian, Liu; Hebei Medical University; Hebei Collaborative Innovation Center for Cardio-cerebrovascular Disease

Jinghui, Dong; Hebei Medical University, Department of Physiology

Keyword: 2K1C rats, chronic intermittent hypobaric hypoxia, endothelium-dependent relaxation, p38, BMP-4

Is the invited manuscript for consideration in a Special

Issue?: N/A

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Title page

Chronic intermittent hypobaric hypoxia provides

vascular protection in aorta of two-kidney one clip rat

model of hypertension

Congrui Fu1, Na Li

2, Yujia Yuan

1, Ri Wang

1, Jinting Chen

1, Jing Yang

1 ,

Zan Guo 1

, Sheng Wang

1, 3, Yi Zhang

1, 3, Yixian Liu

1, 3*, Jinghui Dong

1*

1Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China,

2Department of Physiology, Medical College, Hebei University, Baoding, Hebei, Chi-

na and 3Hebei Collaborative Innovation Center for Cardio-Cerebrovascular Disease,

Shijiazhuang, Hebei, China.

*Corresponding author: Yixian Liu, Department of Physiology, Hebei Medical Uni-

versity, 361 East Zhongshan Road, Shijiazhuang, Hebei, 050017, China. Email: phy-

[email protected]. Tel: +86-0311-86265643; Fax: +86-0311-86261183

*Jinghui Dong, Department of Physiology, Hebei Medical University, 361 East

Zhongshan Road, Shijiazhuang, Hebei, 050017, China. Email: [email protected].

Tel: +86-0311-86265643; Fax: +86-0311-86261183

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Abstract

Many studies have demonstrated that chronic intermittent hypobaric

hypoxia (CIHH) can reduce blood pressure in spontaneously hypertensive

rats and renovascular hypertensive (RVH) rats in which endothelial

dysfunction is determined as a critical factor. However, whether CIHH

can regulate vasodilation of the aorta in RVH rats remains unknown. The

purpose of this study was to investigate the effect of CIHH on impaired

relaxation of the aorta in the two-kidney one-clip (2K1C) RVH rat model.

The results showed CIHH improved the impaired endothelium-dependent

relaxation in the 2K1C rat aorta. The endothelial dysfunction was

prevented by the p38 antagonist SB203580, but not by the ERK1/2

antagonist PD98059 or JNK antagonist SP600125. Furthermore, the

expression of p-eNOS, HIF-1ɑ and HIF-2ɑ increased while that of p-p38

and BMP-4 decreased in CIHH-treated aortas from 2K1C rats. Finally,

the p-eNOS expression was up-regulated and the p-p38 expression was

down-regulated by pre-incubation of SB203580 or the BMP-4 antagonist

Noggin with the aorta. In conclusion, CIHH ameliorated the impairment

of endothelium-dependent relaxation through up-regulating the

expression of p-eNOS which may be mediated by the inhibition of BMP-

4 / p-p38 MAPK and up-regulating the expression of HIFs in the 2K1C

rat aorta.

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Key words: 2K1C rats, chronic intermittent hypobaric hypoxia,

endothelium-dependent relaxation, p38, BMP-4

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Introduction

Hypertension is caused by pathological changes in renal and vascular

structures (Curb et al. 1996). Accumulated studies have shown that

endothelial dysfunction could contribute to elevated blood pressure in

hypertension (Carlisle et al. 2016; Statsenko et al. 2015).

CIHH is a programmed hypobaric hypoxia created by simulating

altitude in a hypobaric chamber, which yields numerous beneficial effects

on the body. Previous studies demonstrated that CIHH could protect the

cardiovascular system and brain against ischemia/reperfusion injury

(Zhang et al. 2016; Zhang et al. 2012). Importantly, CIHH was shown to

decrease the arterial blood pressure in patients with essential hypertension

and in rats with spontaneously hypertension (Aleshin et al. 1993;

Manukhina et al. 2000). Recent research suggested that CIHH treatment

improved the reduced vasorelaxation of mesenteric arteries through

activating BKCa channels in rats with renovascular hypertension (RVH)

(Guan et al. 2016). However, whether CIHH modulates vascular tension

in the aorta of RVH rats remains unclear.

Hypoxia inducible factors (HIFs) are oxygen-sensitive transcriptional

activators related to hypoxia. HIFs are comprised of an O2-regulated

HIF-1α or HIF-2α subunit and form a complex with HIF-1β (Prabhakar et

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al. 2012). Many of the above beneficial effects of CIHH are mediated by

HIFs. However, whether CIHH provides effective vascular protection in

hypertension through the HIF pathway is still unknown.

Conventional mitogen-activated protein kinases (MAPKs) include the

extracellular signal-regulated kinases (ERKs), c-Jun amino (N)-terminal

kinases (JNKs) and p38 isoforms (α, β, γ and δ) (Hadwiger et al. 2011).

Many studies have shown changes in the activity of MAPKs along with

hypoxia. Hypoxia-induced pulmonary arterial hypertension was found to

be improved by reducing the activity of the p38 MAPK signaling

pathway (Awad et al. 2016; Yan et al. 2016). The expression of ERK

increased after the rats were exposed to chronic intermittent hypoxia for 5

weeks (Micova et al. 2016). Chronic intermittent hypoxia-activated

MAPK family members, ERK, JNK and p38, rely on the O2

concentration in the rat obstructive sleep apnea model (Wang et al. 2016).

However, whether MAPKs participate in the effect of CIHH on vascular

tension in RVH rats remains elusive. In this study, antagonists of

ERK1/2, JNK and p38 were used to verify their roles in the protective

effect of CIHH.

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Bone morphogenic protein 4 (BMP4), belonging to the BMP family,

was originally discovered to participate in embryonic development and

bone and cartilage formation (Li et al. 2001; Massague 2000). Up-

regulation of BMP-4 has been linked to hypertension in the systemic

circulation, whereas the disruption of BMP-4 signaling is associated with

the development of pulmonary hypertension (Csiszar et al. 2008). BMP4

stimulation was shown to lead to endothelial dysfunction through reactive

oxygen species (ROS)-dependent p38 MAPK activation (Kamali et al.

2016).

Thus, the current study investigated the potential protective effects of

CIHH against endothelial dysfunction in the two-kidney one-clip (2K1C)

hypertensive rat model and discussed the mechanism involved.

Materials and methods

Animals and group

Fifty-two young male Sprague-Dawley rats (170–190 g, supplied by

the Experimental Animal Center of Hebei Medical University) were

randomly divided into eight groups: (1) Sham group: rats in this group

were treated with a sham operation; (2) CIHH group: rats in this group

were subjected to a sham operation and then treated with CIHH, which

was performed to simulate a 5000 m altitude (Pb = 404 mmHg) for 6 h

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daily over 28 days in a hypobaric chamber, while a normal oxygen

environment was provided the rest of the time; (3) 2K1C group: rats

received a 2K1C operation but without CIHH treatment. In the 2K1C

operation, the left renal artery was clipped with a small silver clip to

produce hypertension; (4) 2K1C + CIHH group: rats received a 2K1C

operation and CIHH treatment; (5) 2K1C + SB203580 group: aortas were

isolated from 2K1C rats and then incubated with SB203580 for 30 min

before use; (6) 2K1C + PD98059 group: the protocol was the same as that

in group 5, except that PD98059 was used for the incubation; (7) 2K1C +

SP600125 group: the protocol was the same as at for group 5, except that

SP600125 was used for the incubation; (8) 2K1C + Noggin group: the

protocol was the same as that for group 5, except that Noggin was used

for the incubation. The animals were housed at constant temperature (21

± 1°C) under a 12-h light/dark cycle and had free access to chow diet and

water. The animals were cared for in accordance with the Guide to the

Care and Use of Experimental Animals (Vol. 1, 2nd ed., 1993, and Vol. 2,

1984, available from the Canadian Council on Animal Care). All animal

procedures were approved by the Institutional Animal Care and Use

Committee of Hebei Medical University.

Chemicals and antibodies

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Protease inhibitor and phosphoric acid protease inhibitors were

purchased from Roche Mannheim (Mannheim, Germany). Mouse

polyclonal anti-HIF-1α and anti-HIF-2α primary antibodies, rabbit

polyclonal primary antibodies to p-eNOS, eNOS, p-p38, p38, p-ERK,

ERK, p-JNK, JNK and BMP-4 were from Abcam (Cambridge, UK). The

rabbit polyclonal primary antibody to β-actin primary antibody was from

Cell Signaling Technology (Danvers, MA, USA). The horseradish

peroxidase labeled anti-rabbit and anti-mouse secondary antibodies for

Western blot were from KPL Scaffold Inc (La Mirada, CA, USA) and

Earthox Life Sciences (Millbrae, California, USA), respectively. All

secondary antibodies for immunofluorescence staining were from

Jackson Laboratories Inc (West Grove, PA, USA). All general chemicals

were purchased from Sigma (St. Louis, MO, USA) unless stated

otherwise. Polyvinylidene difluoride (PVDF) membranes and the

enhanced chemiluminescence detecting reagents were from Millipore

Corporation (Billerica, MA, USA).

Preparation of renovascular hypertension rat model

Renovascular hypertension was induced by the Goldblatt 2K1C

method as described in a previous study (Guo et al. 2015). Briefly, the

rats were anesthetized with sodium pentobarbital (35 mg / kg, ip), and a

retroperitoneal flank incision was performed. The left renal artery was

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exposed and partially occluded by a U-shaped silver clip with an internal

diameter of 0.20 mm. After the operation, all animals received penicillin

(100000 units) and an analgesic ketorolac (0.6 mg / kg, ip) injection to

prevent infection and pain. The rats were maintained four weeks for full

recovery from the operation. Blood pressure was recorded by using the

tail-cuff electrosphygmomanometer system (AD Instruments, Sydney,

Australia) after the rats were stabilized and remained quiescent. An

average of three readings was taken as the systolic blood pressure for

each rat. Only 2K1C rats with a systolic arterial blood pressure > 150

mmHg were used in the experiment.

Thoracic aortic rings preparation and measurement of vascular

relaxation

The rats were killed by anesthetization with sodium pentobarbital (50

mg / kg, ip). The thoracic aorta was dissected, and surrounding

connective tissues were carefully removed. The arterial segment was cut

into 3–4 mm rings. The rings were then suspended between stainless steel

triangles in an organ bath filled with Krebs-Henseleit (K-H) solution of

the following composition (mmol / L): NaCl 119.0; KCl 4.7; CaCl2 2.5;

MgCl2 1.0; NaHCO3 25.0; KH2PO4 1.2; glucose 11.0; pH 7.4. The bath

solution was constantly gassed with a mixture of 95% O2 and 5% CO2

and maintained at 37°C. Before the experiment, the aortic rings were

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allowed to equilibrate for 60 min under a resting tension of 2.5 g, during

which the K-H solution was replaced every 15 min. The aortic ring was

connected to a force transducer, and the isometric force was recorded

using a biological signal recording system. After equilibration for 1 h,

phenylephrine (PE, 1 µmol / L) was given to induce contractions. When

the contraction was steady, acetylcholine (ACh, from 1 nmol / L to 10

µmol / L) or sodium nitroprusside (SNP, 1 nmol / L to 1 µmol / L) was

added in a cumulative manner to record the endothelial-dependent

relaxation or endothelial-independent relaxation response curve. The

rings with Ach (10 µmol / L)-induced relaxation of more than 80% was

used as the intact endothelium specimen. The extent of relaxation was

expressed as the percentage of relaxation from a submaximal

phenylephrine-induced constriction. Vasocontraction was determined in

relative values as the percentage of 60 mmol / L KCl contraction. Each

of the antagonists, when used, was applied for a 30-min incubation period

prior to the addition of Ach. The experiments were designed to determine

whether p38, ERK1/2, JNK and BMP-4 had effects on Ach-induced

vasorelaxation in the rat aorta by exposure to the p38 antagonist

SB203580 (10 µmol / L), ERK1/2 antagonist PD98059 (10 µmol / L),

JNK antagonist SP600125 (10 µmol / L) or BMP-4 antagonist Noggin

(100 ng / ml) before Ach treatment.

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Western blotting

The thoracic aortic rings were homogenized in ice-cold RIPA lysis

buffer containing protease inhibitor and phosphoric acid protease

inhibitors. The homogenates were incubated on ice for 20 min and then

centrifuged. The supernatant was collected, and the protein concentration

was determined using the bicinchoninic acid (BCA) method (Generay

biotechnology, Shanghai, China) according to the manufacturer's

instructions. Briefly, 20 µl of different concentrations of standard protein

solutions, samples to be measured or blank control were added into the

wells of 96-well plates, and then 200 µl of working fluid was added to

each well and mixed slightly. The reagents were incubated for 30 min at

37°C, cooled to room temperature and then read at 562 nm in a

spectrophotometer. A standard protein curve was drawn, and the

concentrations of the samples were calculated through the standard curve

regression formula. Equal amounts of protein samples (10 µg) were

electrophoresed through a 10% SDS-polyacrylamide gel and then

transferred onto a PVDF membrane. Non-specific binding sites were

blocked by 5% non-fat milk or 1% BSA in 0.05% Tween-20 phosphate-

buffered saline (PBST), and then incubated overnight at 4°C with primary

antibodies against p-eNOS and eNOS, HIF-1α and HIF-2α, p-p38, p38, p-

ERK1/2, ERK1/2, p-JNK, JNK, BMP-4 and β-actin. After being washed

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three times with Tris buffered saline with Tween 20 (TBST), the

membranes were incubated with appropriate horseradish peroxidase-

labeled secondary antibodies for 1 h at room temperature. After washing

again, the signals were developed with an enhanced chemiluminescence

system. Band images were quantified, and the ratio of the integrated

optical density (IOD) of the target band to that of β-actin or of the

phosphorylated protein to that of the total protein was determined.

Immunofluorescence staining

The thoracic aortic rings were dissected and stored in 4%

paraformaldehyde at 4°C for 48 h. The ring was embedded in paraffin

and sectioned (5 µm). After deparaffinization and hydration, the sections

were treated with 3% H2O2, heated in citrate buffer and permeabilized

with 0.25% Triton X-100 in turn. After incubation with normal goat

serum for 1 h at 37°C, the sections were incubated at 4°C overnight with

primary antibody against HIF-1α and HIF-2α. After rinsing, the sections

were incubated with matched secondary antibodies tagged with

fluorescence reporters for 1 h at 37°C. Finally sections were washed, air-

dried and mounted on slides with Vectashield Antifade Mounting

Medium. Phosphate-buffered saline was used as negative controls in

place of primary antibodies. Sections were imaged using a DM6000B

fluorescence microscope (Leica Microsystems , Germany).

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Statistical analysis

Results are presented as means ± SEM from different groups.

Concentration-response curves were analyzed by nonlinear regression

curve fitting using GraphPad Prism software. Statistical significance was

determined by one-way ANOVA or two-way ANOVA followed by the

Tukey post hoc test when two treatments were compared (GraphPad

Software, San Diego, CA, USA). P < 0.05 indicated a statistically

significant difference.

Results

Differences in body weight and blood pressure between different

groups

The body weight was significantly lower in the 2K1C group than in

the Sham, CIHH and 2K1C + CIHH groups from the fourth week after

the 2K1C operation (Table 1).

The systolic blood pressure was significantly higher in the 2K1C

group than in the Sham group from the first week after the 2K1C

operation. CIHH treatment for 4 weeks significantly decreased the

systolic blood pressure in the 2K1C + CIHH group from the fifth week

compared with the 2K1C group (Table 2).

Relaxation effects of Ach and SNP on aortic rings

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Ach induced relaxation of aortic rings in a concentration-dependent

manner (1 nmol / L-10 µmol / L). The relaxation was obviously impaired

in 2K1C rats compared with Sham rats. CIHH treatment for 4 weeks had

no obvious effect on the relaxation of aortic rings compared with the

Sham group, but CIHH treatment significantly improved the relaxation in

the 2K1C+CIHH group compared with the 2K1C group (Figure 1A). In

addition, Western blot results demonstrated that the expression of p-

eNOS was decreased in the 2K1C group compared with the Sham group,

and strikingly increased in the 2K1C + CIHH group compared with the

2K1C group (Figure 1C). However, no significant change was observed

in the expression of eNOS in each group. On the other hand, the

relaxation induced by SNP showed no difference among these groups

(Figure 1B). These results demonstrated that CIHH improved the

impaired endothelium-dependent relaxation in aortas isolated from 2K1C

rats by promoting the expression of p-eNOS.

Comparison of HIF-1α and HIF-2α expression between groups

The expression of HIF-1α (green) and HIF-2α (red) was examined by

an immunofluorescence assay. The results showed that HIF-1α and HIF-

2α expressed in the layers of blood vessels, especially in endothelial cells

after CIHH treatment. Compared with the Sham group, the fluorescent

intensities of HIF-1α and HIF-2α in endothelial cells were significantly

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increased in the CIHH group. Similarly, they were also higher in the

2K1C + CIHH group than in the 2K1C group (Figure 2A). The

expression levels of these proteins were also studied by Western blotting.

After the CIHH treatment, the expression levels of both HIF-1α and HIF-

2α were increased clearly compared with the Sham group. No significant

change was observed in the expression of the two HIF subunits in the

2K1C group compared with the Sham group, while CIHH treatment up-

regulated their expression levels in the 2K1C + CIHH group compared

with the 2K1C group (Figure 2B). The results suggested CIHH could up-

regulate the expression of HIF-1α and HIF-2α in the aortic ring with or

without hypertension.

Effects of SB203580, PD98059 and SP600125 on the impaired

endothelium-dependent relaxation in 2K1C rat aorta

In order to examine whether MAPKs participated in the impaired

endothelium-dependent relaxation in the 2K1C rat aorta, antagonists of

the three MAPKs were used. The impaired endothelium-dependent

relaxation in the 2K1C aortic rings was rescued in the presence of p38

antagonist SB203580, but not by ERK1/2 antagonist PD98059 or JNK

antagonist SP600125 (Figure 3A). The results indicated that the impaired

endothelium-dependent relaxation in the 2K1C rat aorta was mediated by

p38, but not by ERK1/2 or JNK.

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Comparisons of expression of p-p38 / p38, p-ERK1/2 / ERK1/2 and

p-JNK / JNK between groups

Whether CIHH treatment could affect the expression of various

MAPK members was investigated by observing their protein levels. As

shown in Figure 3B and 3C, minimal changes were seen in the expression

of p-ERK1/2/ ERK1/2 and p-JNK/JNK among the different groups.

Similarly, no obvious change of the p38 expression was found among

these groups. CIHH treatment itself had no obvious effect on the

expression of p-p38 compared with the Sham group. However, the

expression of the p-p38 protein was strikingly augmented in the 2K1C

group compared with the Sham group. Furthermore, CIHH treatment

reduced the augmented expression of p-p38 in the 2K1C + CIHH group

compared with the 2K1C group. The results suggested that CIHH

treatment might rescue the impaired endothelium-dependent relaxation in

aortic rings of 2K1C rats by inhibiting the augmented expression of p-p38

(Figure 3A).

Effect of Noggin on impaired endothelium-dependent relaxation of

aortic rings isolated from 2K1C group

We observed the effect of Noggin, an antagonist of BMP-4, on the

impaired endothelium-dependent relaxation in the 2K1C rat aorta. The

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results showed Noggin ameliorated the impaired endothelium-dependent

relaxation in 2K1C aortic rings (Figure 4A), which suggested that BMP-4

mediated the endothelium dysfunction in renovascular hypertension.

Effect of CIHH on expression of BMP-4

In order to examine whether CIHH treatment could regulate the

expression of BMP-4, we observed changes in the level of this protein in

different groups. As with the expression of p-p38, the expression of

BMP-4 in the 2K1C group was higher than that in the Sham group, and it

was obviously declined in 2K1C + CIHH rats compared with 2K1C rats

(Figure 4B, C). The changes of these proteins, together with the rescuing

effects of Noggin on endothelium-dependent relaxation of aortic rings of

2K1C rats indicated a relationship of endothelial dysfunction with

enhanced BMP-4.

Effects of SB203580 and Noggin on expression of p-eNOS / eNOS and

p-p38 / p38

The expression of p-eNOS in the aorta of 2K1C rats was less than that

in the Sham group. After preincubation of the p38 antagonist SB203580

or BMP-4 antagonist Noggin, the expression of p-eNOS was remarkably

increased in the 2K1C + SB203580 or 2K1C + Noggin group compared

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with the 2K1C group (Figure 5A, B). No significant change was

observed in the expression of eNOS in each group.

By contrast, the expression of p-p38 in 2K1C rats was much greater

than that in the Sham group. After application of the p38 antagonist

SB203580 or BMP-4 antagonist Noggin, the amount of p-p38 was

reduced compared with the 2K1C group. No obvious change was found

in the expression of p38 in each group (Figure 5A, B). The results

illustrated that the decreased expression of p-eNOS was concomitant with

the increased expression of p-p38 in aortas isolated from 2K1C rats.

Furthermore, the expression of p-p38 could be inhibited by giving the

BMP-4 antagonist Noggin, suggesting that BMP-4 is part of the upstream

cascade of p-p38.

Discussion

The present study tested the effects of CIHH on endothelial

dysfunction in the 2K1C rat aorta and explored the underlying

mechanisms. The findings showed that CIHH could improve the impaired

endothelium-dependent relaxation in the 2K1C rat aorta and increase the

expression of p-eNOS, HIF-1ɑ and HIF-2ɑ. The blockage of p-p38/p38

and BMP-4 rescued the impaired endothelium-dependent relaxation in the

2K1C rat aorta. Furthermore, CIHH reduced the increased expression of

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p-p38/p38 and BMP-4 in the 2K1C + CIHH group compared with the

2K1C group. Finally, we showed that the blockage of p38 or BMP-4 was

concomitant with the increased expression of p-eNOS. Thus, we can

conclude that CIHH improved the impaired endothelium-dependent

relaxation in the 2K1C rat aorta through inhibiting BMP4 and p-p38 and

up-regulating the expression of HIFs, consequently resulting in higher

eNOS activity.

Different protocols for inducing intermittent hypoxia produce diverse

responses. For example, a long-cycle intermittent hypobaric hypoxia (6 h

a day for 28–42 days) showed a cardiac protective effect (Peng et al. 2012)

and also an anti-hypertensive effect (Li et al. 2016). However,

normobaric hypoxia (20–50 s hypoxia, 20–50 s normoxia alternately,

10–12 h a day) was shown to cause an increase in arterial blood pressure

and deleterious myocardial infarction induced by cardiac

ischemia/reperfusion (Ramond et al. 2007). The short-cycle intermittent

normobaric hypoxia in the obstructive sleep apnea patients may cause

hypertension (Lesske et al. 1997). CIHH as used in our study is a

programmed intermittent hypoxia with a long cycle which has many

beneficial effects on the body. This work represents our first efforts

focusing on the protective effect of CIHH on aortic endothelial

dysfunction in renovascular hypertension. Results of this study confirmed

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our hypothesis that CIHH could rescue the dysfunction of the

endothelium in 2K1C rats.

Under hypoxia, HIF-α subunits are stabilized and translocated to the

nucleus where they heterodimerize with the arylhydrocarbon receptor

nuclear translocator and bind to hypoxia response elements (HREs)

located within regulatory elements of HIF target genes. There are three

types of HIF-α subunits, HIF-1α, HIF-2α, and HIF-3α. At present, HIF-

1α and HIF-2α have gained increased attention (Liu et al. 2016).

Interestingly, these two subunits show different patterns of tissue

distributions. HIF-1α is ubiquitously expressed in the body, but HIF-2α

expression is restricted to specific tissues, such as blood vessels (Ema et

al. 1997). Many studies have identified NOS as one of the target proteins

of HIFs with HREs in its regulatory elements (Coulet et al. 2003; Yuan et

al. 2016). In the present study, when the rats were exposed to CIHH, the

expression levels of HIF-1α and HIF-2α in endothelial cells were both

increased, so do the protein expressions. Correspondingly, the expression

of p-eNOS was also increased. The results suggested that CIHH treatment

increased the expression of HIF-1α and HIF-2α in normal and

hypertensive rats, which counterbalanced the decrease in eNOS activity

in hypertensive rats.

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MAPKs are relevant to the expression of eNOS. For example,

advanced glycation end-products is known to cause endothelial

dysfunction by a mechanism associated with decreased eNOS expression

in human coronary artery endothelial cells through activation of p38 and

ERK1/2 (Ren et al. 2017). Adenosine was shown to be required for

ERK1/2 activation by statins, which resulted in Akt and

eNOS phosphorylation (Merla et al. 2007). Thus, we examined whether

CIHH could increase eNOS by modulating the MAPK pathway. In this

study, the impaired endothelium-dependent relaxation in 2K1C aortic

rings was restored by the p38 antagonist SB203580, but it was not altered

by the ERK1/2 antagonist PD98059 or JNK antagonist SP600125. The

results suggested that p38, but not ERK1/2 or JNK mediated the

endothelial dysfunction in the aorta in hypertension. Accordingly, the

expression of p-p38 was also remarkably increased in the 2K1C rat aorta.

CIHH treatment reversed the abnormal expression of p-p38 MAPK in the

2K1C rat aorta, while the expression of p-ERK and p-JNK basically

remained in a stable state in each group. Thus, we can draw a conclusion

that CIHH improved the relaxation of aortas in 2K1C rats partly through

inhibiting the activity of p38.

Previous studies demonstrated that BMP4 increased phosphorylation

of p38 MAPK to promote chondrogenesis and exert proinflammatory

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effects on endothelial dysfunction (Kamali et al. 2016; Kim et al. 2016).

Therefore, we investigated whether CIHH could inhibit the activity of p-

p38 via down-regulating the expression of BMP-4 to exert a protective

effect. In our study, expression levels of p-p38 and BMP-4 both

decreased by CIHH treatment in the 2K1C + CIHH group compared with

the 2K1C group. In addition, BMP-4 was determined to be in the

upstream cascade of p-p38, as the expression of p-p38 was inhibited by

giving the BMP-4 antagonist Noggin. Furthermore, the expression of p-

eNOS was strikingly augmented and p-p38 remarkably reduced by the

incubation of SB203580 or Noggin with 2K1C rat aortas. These results

indicated that CIHH treatment restored p-eNOS expression in aortas

isolated from 2K1C rats through inhibiting BMP-4 and in turn inhibiting

the p38 MAPK activity.

In this study, the observed vascular protection was explained by

“short-term” mechanisms such as phosphorylation of eNOS and p38

proteins. Longer-term mechanisms for the anti-hypertensive effect of

CIHH have also been described, such as the potentiating effect on

baroreflex (Gao et al. 2012) or enhancement of the relaxation of

mesenteric arteries (Guan et al. 2016). Furthermore, CIHH has been

demonstrated to induce a cardiovascular protective effect for at least two

weeks and a neuroprotective effect for at least one week (Wang et al.

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2017; Zhang et al. 2000). Determining the length of time that the

antihypertensive effect can be maintained will require further observation.

Together, results of our study demonstrated that CIHH could improve

the impaired endothelium-dependent relaxation in 2K1C hypertensive rat

aortic rings. The mechanism may be that CIHH inhibits the expression of

BMP-4 and the activity of p38, thus increasing the expression of p-eNOS.

Furthermore, CIHH may up-regulate the expression of HIF-1α and HIF-

2α in endothelial cells, and then increase the expression of the product of

the target genes, p-eNOS. Thus, CIHH may potentially be developed as a

therapeutic to ameliorate endothelial dysfunction in hypertension.

Acknowledgements

This work was supported by the University Science and Technology Re-

search Project of Hebei Province, China (ZD2017052).

Disclosure: The authors declare no conflict of interest in this study.

References

Aleshin IA, Kots Ia I, Tverdokhlib VP, Galiautdinov GS, Vdovenko LG, Zabirov MR, et al. 1993. The nondrug treatment of hypertension patients by their adaptation to periodic hypoxia in a barochamber. Ter Arkh. 65(8): 23-29 Awad KS, West JD, de Jesus Perez V, MacLean M. 2016. Novel signaling pathways in pulmonary arterial hypertension (2015 Grover Conference Series). Pulm Circ. 6(3): 285-294. doi:10.1086/688034.

Page 23 of 36



Draft

23

23

Carlisle RE, Werner KE, Yum V, Lu C, Tat V, Memon M, et al. 2016. Endoplasmic reticulum stress inhibition reduces hypertension through the preservation of

resistance blood vessel structure and function. J Hypertens. 34(8): 1556-1569. doi:10.1097/HJH.0000000000000943.

Coulet F, Nadaud S, Agrapart M, Soubrier F. 2003. Identification of hypoxia-response element in the human endothelial nitric-oxide synthase gene promoter. J Biol Chem. 278(47): 46230-46240. doi:10.1074/jbc.M305420200.

Csiszar A, Labinskyy N, Jo H, Ballabh P, Ungvari Z. 2008. Differential proinflammatory and prooxidant effects of bone morphogenetic protein-4 in coronary and pulmonary arterial endothelial cells. Am J Physiol Heart Circ Physiol. 295(2): H569-577. doi:10.1152/ajpheart.00180.2008. Curb JD, Pressel SL, Cutler JA, Savage PJ, Applegate WB, Black H, et al. 1996. Effect of diuretic-based antihypertensive treatment on cardiovascular disease risk in older

diabetic patients with isolated systolic hypertension. Systolic Hypertension in the Elderly Program Cooperative Research Group. JAMA. 276(23): 1886-1892 Ema M, Taya S, Yokotani N, Sogawa K, Matsuda Y, Fujii-Kuriyama Y. 1997. A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular

development. Proc Natl Acad Sci U S A. 94(9): 4273-4278 Gao L, Guan Y, Cui F, Liu YX, Zhou ZN, Zhang Y. 2012. Facilitation of chronic intermittent hypobaric hypoxia on carotid sinus baroreflex in anesthetized rats. Chin J

Physiol. 55(1): 62-70. doi:10.4077/CJP.2012.AMM076. Guan Y, Li N, Tian YM, Zhang L, Ma HJ, Maslov LN, et al. 2016. Chronic intermittent hypobaric hypoxia antagonizes renal vascular hypertension by enhancement of

vasorelaxation via activating BKCa. Life Sci. 157: 74-81. doi:10.1016/j.lfs.2016.05.028.

Guo Z, Liu YX, Yuan F, Ma HJ, Maslov L, Zhang Y. 2015. Enhanced vasorelaxation effect of endogenous anandamide on thoracic aorta in renal vascular hypertension rats. Clin Exp Pharmacol Physiol. doi:10.1111/1440-1681.12450.

Hadwiger JA, Nguyen HN. 2011. MAPKs in development: insights from Dictyostelium signaling pathways. Biomol Concepts. 2(1-2): 39-46. doi:10.1515/BMC.2011.004. Kamali M, Khodadoost M, Tavakoli H, Kamalinejad M, Gachkar L, Adibi P, et al. 2016.

The Role of Syndrome Differentiation in the Clinical Efficacy of Punica Granatum on Patients with Ulcerative Colitis. Iran J Med Sci. 41(3 Suppl): S15 Kim H, Sonn JK. 2016. Rac1 promotes chondrogenesis by regulating STAT3 signaling

pathway. Cell Biol Int. 40(9): 976-983. doi:10.1002/cbin.10635. Lesske J, Fletcher EC, Bao G, Unger T. 1997. Hypertension caused by chronic intermittent hypoxia--influence of chemoreceptors and sympathetic nervous system. J Hypertens. 15(12 Pt 2): 1593-1603 Li N, Guan Y, Zhang L, Tian Y, Zhang Y, Wang S. 2016. Depressive Effects of Chronic

Intermittent Hypobaric Hypoxia on Renal Vascular Hypertension through Enhancing Baroreflex. Chin J Physiol. 59(4): 210-217. doi:10.4077/CJP.2016.BAF444. Li RH, Wozney JM. 2001. Delivering on the promise of bone morphogenetic proteins.

Trends Biotechnol. 19(7): 255-265 Liu YM, Ying SP, Huang YR, Pan Y, Chen WJ, Ni LQ, et al. 2016. Expression of HIF-1alpha and HIF-2alpha correlates to biological and clinical significance in papillary

thyroid carcinoma. World J Surg Oncol. 14(1): 30. doi:10.1186/s12957-016-0785-9. Manukhina EB, Mashina S, Smirin BV, Lyamina NP, Senchikhin VN, Vanin AF, et al. 2000. Role of nitric oxide in adaptation to hypoxia and adaptive defense. Physiol Res. 49(1): 89-97

Page 24 of 36



Draft

24

24

Massague J. 2000. How cells read TGF-beta signals. Nat Rev Mol Cell Biol. 1(3): 169-178. doi:10.1038/35043051.

Merla R, Ye Y, Lin Y, Manickavasagam S, Huang MH, Perez-Polo RJ, et al. 2007. The central role of adenosine in statin-induced ERK1/2, Akt, and eNOS phosphorylation.

Am J Physiol Heart Circ Physiol. 293(3): H1918-1928. doi:10.1152/ajpheart.00416.2007. Micova P, Hahnova K, Hlavackova M, Elsnicova B, Chytilova A, Holzerova K, et al.

2016. Chronic intermittent hypoxia affects the cytosolic phospholipase A2alpha/cyclooxygenase 2 pathway via beta2-adrenoceptor-mediated ERK/p38 stimulation. Mol Cell Biochem. 423(1-2): 151-163. doi:10.1007/s11010-016-2833-8. Peng YJ, Nanduri J, Zhang X, Wang N, Raghuraman G, Seagard J, et al. 2012. Endothelin-1 mediates attenuated carotid baroreceptor activity by intermittent hypoxia. J Appl Physiol (1985). 112(1): 187-196.

doi:10.1152/japplphysiol.00529.2011. Prabhakar NR, Semenza GL. 2012. Adaptive and maladaptive cardiorespiratory

responses to continuous and intermittent hypoxia mediated by hypoxia-inducible factors 1 and 2. Physiol Rev. 92(3): 967-1003. doi:10.1152/physrev.00030.2011. Ramond A, Ribuot C, Levy P, Joyeux-Faure M. 2007. Deleterious myocardial

consequences induced by intermittent hypoxia are reversed by erythropoietin. Respir Physiol Neurobiol. 156(3): 362-369. doi:10.1016/j.resp.2006.10.011. Ren X, Ren L, Wei Q, Shao H, Chen L, Liu N. 2017. Advanced glycation end-products

decreases expression of endothelial nitric oxide synthase through oxidative stress in human coronary artery endothelial cells. Cardiovasc Diabetol. 16(1): 52. doi:10.1186/s12933-017-0531-9.

Statsenko ME, Derevianchenko MV. 2015. Possibilities of Correction of Endothelial Dysfunction at the Background of Combined Antihypertensive Therapy in Patients

With Arterial Hypertension and Type 2 Diabetes. Kardiologiia. 55(3): 17-20 Wang J, Zhang S, Ma H, Yang S, Liu Z, Wu X, et al. 2017. Chronic Intermittent Hypobaric Hypoxia Pretreatment Ameliorates Ischemia-Induced Cognitive

Dysfunction Through Activation of ERK1/2-CREB-BDNF Pathway in Anesthetized Mice. Neurochem Res. 42(2): 501-512. doi:10.1007/s11064-016-2097-4. Wang Y, Hai B, Niu X, Ai L, Cao Y, Li R, et al. 2016. Chronic intermittent hypoxia

disturbs insulin secretion and causes pancreatic injury via MAPK signaling pathway. Biochem Cell Biol. doi:10.1139/bcb-2016-0167. Yan S, Wang Y, Liu P, Chen A, Chen M, Yao D, et al. 2016. Baicalin Attenuates

Hypoxia-Induced Pulmonary Arterial Hypertension to Improve Hypoxic Cor Pulmonale by Reducing the Activity of the p38 MAPK Signaling Pathway and MMP-9. Evid Based Complement Alternat Med. 2016: 2546402. doi:10.1155/2016/2546402. Yuan XH, Fan YY, Yang CR, Gao XR, Zhang LL, Hu Y, et al. 2016. Progesterone amplifies oxidative stress signal and promotes NO production via H2O2 in mouse

kidney arterial endothelial cells. J Steroid Biochem Mol Biol. 155(Pt A): 104-111. doi:10.1016/j.jsbmb.2015.09.029. Zhang S, Guo Z, Yang S, Ma H, Fu C, Wang S, et al. 2016. Chronic intermittent

hybobaric hypoxia protects against cerebral ischemia via modulation of mitoKATP. Neurosci Lett. 635: 8-16. doi:10.1016/j.neulet.2016.10.025. Zhang Y, Zhong N, Zhu HF, Zhou ZN. 2000. Antiarrhythmic and antioxidative effects

of intermittent hypoxia exposure on rat myocardium. Acta Physiol. Sin. . 52: 4 Zhang Y, Zhou ZN. 2012. [Beneficial effects of intermittent hypobaric hypoxia on the body]. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 28(6): 504-509

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Figure captions

Figure 1 Effect of CIHH on vasodilation and expression of p-eNOS /

eNOS on aortic rings. A, CIHH improved the impaired Ach (1 nmol / L

to 10 µmol / L)-induced relaxation on aortic rings in the 2K1C group. B,

No change of SNP (1 nmol / L to 1 µmol / L)-induced relaxation was

observed on aortic rings in the 2K1C group compared with the Sham

group, and CIHH had no protective effect on the endothelial relaxation. C,

CIHH reversed the decreased expression of p-eNOS / eNOS on aortic

rings in the 2K1C group. All data are expressed as the mean ± SEM, n =

6 for Sham, CIHH and 2K1C+ CIHH groups, n = 5 for 2K1C group. **P

< 0.01 vs. Sham group; †P < 0.05 and

††P < 0.01 vs. 2K1C group.

Figure 2 Effects of CIHH treatment on expression of HIF-1α and HIF-2α

in aortic rings. A, Immunofluorescence staining showed the fluorescent

intensities of HIF-1α (green) and HIF-2α (red) in endothelial cells were

significantly increased in the CIHH group, compared with the Sham

group. Similarly, they were also higher in the 2K1C + CIHH group

compared with 2K1C group. B, Western blot showed that HIF-1α and

HIF-2α were both up-regulated in CIHH and 2K1C + CIHH groups. All

data are expressed as the mean ± SEM, n = 5 for each group. Scale bar =

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50 μm. **P < 0.01 vs. Sham group;

†P < 0.05 and

††P < 0.01 vs. 2K1C

group.

Figure 3 Effects of antagonists of MAPKs on the endothelial vasodilation

of aortic rings in the 2K1C group and effect of CIHH on expression of

MAPKs. A, Impaired vasodilation was rescued by preincubation of p38

antagonist SB203580 (10 µmol / L), but not by ERK1/2 antagonist

PD98059 (10 µmol / L) or JNK antagonist SP600125 (10 µmol / L). B, C,

Expression of p-p38 / p38 was increased obviously in the 2K1C group,

but it was decreased by CIHH treatment in the 2K1C + CIHH group. The

expression of p-ERK1/2 / ERK1/2 and p-JNK / JNK showed no obvious

changes with or without CIHH treatment in the 2K1C group. All data are

expressed as the mean ± SEM, n = 5 for each group. **P < 0.01 vs. Sham

group; ††P < 0.01 vs. 2K1C group.

Figure 4 Effects of BMP-4 antagonist Noggin on the impaired

endothelium-dependent relaxation in 2K1C aortic rings and effect of

CIHH on BMP4 expression. A, The impaired Ach-induced vasodilation

was rescued by incubation of BMP4 antagonist Noggin (100 ng / mL). B,

CIHH treatment inhibited BMP4 expression in the 2K1C + CIHH group.

All data are expressed as the mean ± SEM, n = 4 for 2K1C + Noggin

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group, n = 5 for other groups. **P < 0.01 vs. Sham group;

††P < 0.01 vs.

2K1C group.

Figure 5 Effects of p38 antagonist SB203580 and BMP-4 antagonist

Noggin on expression of p-eNOS / eNOS and p-p38 / p38. The

expression of p-eNOS was up-regulated and the expression of p-p38 was

down-regulated by preincubation of the two antagonists compared with

the 2K1C group. All data are expressed as the mean ± SEM, n = 5 for

each group. **P < 0.01 vs. Sham group;

††P < 0.01 vs. 2K1C group.

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Table 1. The changes of bodyweight in rats on CIHH and/or 2K1C treatment

0 week(g) 1 week(g) 2 week(g) 3 week(g) 4 week(g) 5 week(g) 6 week(g) 7 week(g) 8 week(g)

Sham 182.5±2.0 184.3±3.1 193.7±3.6 206.8±5.2 229.3±5.8 250.0±7.0 277.8±8.1 319.2±9.2 354.8±9.3

CIHH 182.3±2.3 188.2±2.8 193.8±3.7 204.8±4.9 222.0±6.3† 247.0±5.3

†† 271.8±7.6

†† 318.2±8.9

†† 345.5±9.4

††

2K1C 178.7±1.9 185.3±2.8 190.3±3.7 196.7±4.8 212.3±5.6** 216.2±5.9** 226.7±6.1** 255.0±7.5** 278.0±9.5**

2K1C+CIHH 181.2±1.8 187.2±3.0 191.7±3.8 201.3±5.0 220.8±5.5† 249.7±6.0

† 265.0±7.7

†† 298.2±8.0

†† 328.8±10.3

††

All data are expressed as mean ± SEM, n=6 for each group. *P < 0.05 and

**P < 0.01 vs. Sham group;

†P < 0.05 and

††P <

0.01 vs. 2K1C group. CIHH treatment was given from the fifth week in CIHH and 2K1C+CIHH group.

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Table 2. The changes of systolic blood pressure in rats on CIHH and/or 2K1C treatment

Group

0 week

(mmHg)

1 week

(mmHg)

2 week

(mmHg)

3 week

(mmHg)

4 week

(mmHg)

5 week

(mmHg)

6 week

(mmHg)

7 week

(mmHg)

8 week

(mmHg)

Sham 107.5±2.3 110.0±2.0 112.3±2.5 110.0±3.2 116.2±2.9 111.0±3.4 111.3±4.6 114.7±4.1 115.0±5.4

CIHH 108.5±1.9 108.3±2.2†† 112.7±2.6

†† 114.2±3.7

†† 112.8±3.3

†† 115.2±5.2

†† 113.5±4.1

†† 115.8±4.2

†† 116.0±4.7

††

2K1C 108.2±3.0 123.3±2.8** 166.8±3.5** 169.2±2.4** 169.0±2.9** 168.2±2.5** 172.2±6.4** 168.2±5.5** 169.0±4.7**

2K1C+CIHH 108.2±4.3 117.7±3.2 166.3±3.6 164.3±3.0 164.2±5.4 147.7±3.2†† 143.7±3.1

†† 139.3±4.6

†† 140.3±3.9

††

All data are expressed as mean ± SEM, n=6 for each group. *P < 0.05 and

**P < 0.01 vs. Sham group;

†P < 0.05 and

††P <

0.01 vs. 2K1C group. CIHH treatment was given from the fifth week in CIHH and 2K1C+CIHH group.

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Table 3. Primary and secondary antibodies

Antibody Source concentration

HIF-1α Abcam 1:500

HIF-2α Abcam 1:500

p-eNOS Abcam 1:500

eNOS Abcam 1:500

p-p38 Abcam 1:1000

p38 Abcam 1:1000

p-ERK Abcam 1:1000

ERK Abcam 1:1000

p-JNK Abcam 1:1000

JNK Abcam 1:1000

BMP-4 Abcam 1:1000

β-actin Cell Signaling Technology 1:5000

HRP anti-rabbit IgG KPL Scaffold Inc 1:500

HRP anti-mouse IgG Earthox life sciences 1:500

FITC goat anti-mouse Jackson Laboratories Inc. 1:200

Cy3 goat anti-mouse Jackson Laboratories Inc. 1:200

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Figure 1 Effect of CIHH on vasodilation and expression of p-eNOS / eNOS on aortic rings.

86x65mm (300 x 300 DPI)

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Figure 2 Effects of CIHH treatment on expression of HIF-1α and HIF-2α in aortic rings.

86x58mm (300 x 300 DPI)

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Figure 3 Effects of antagonists of MAPKs on the endothelial vasodilation of aortic rings in the 2K1C group and effect of CIHH on expression of MAPKs.

86x60mm (300 x 300 DPI)

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Figure 4 Effects of BMP-4 antagonist Noggin on the impaired endothelium-dependent relaxation in 2K1C aortic rings and effect of CIHH on BMP4 expression.

86x60mm (300 x 300 DPI)

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Figure 5 Effects of p38 antagonist SB203580 and BMP-4 antagonist Noggin on expression of p-eNOS / eNOS and p-p38 / p38.

86x66mm (300 x 300 DPI)

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