cause vasoconstriction or vasodilation by producing a number of vasoactive substances such as endothelin (ET), endothelium-derived contracting factor (EDCF), prostaglandin H

2 (PGH

2 ), thromboxane A

2 (TXA

2 ),

prostacyclin (PGI 2 ), endothelium-derived relaxing factor

(EDRF), and endothelium-derived hyperpolarizing fac-tor (EDHF) [8]. Functional alteration of endothelium is associated with cardiovascular diseases, especially hypertension. Elevated vascular resistance in hyperten-sion is related to imbalance of action of vasodilators, such as NO and PGI

2 , and vasoconstrictors, such as

ET-1 and TXA 2 . Endothelial cells are a major source of

angiotensin-converting enzyme (ACE) [9], producing angiotensin II and degrading bradykinin. These factors play important regulatory roles in the maintenance of blood pressure, electrolyte and fl uid balance, and in the development and remodeling of the cardiovascular sys-tem via the renin – angiotensin system and the kallikrein – kinin system. Inhibition of ACE is also an important approach to the treatment of hypertension [10]. In this study, we tested our hypothesis that hydrogen peroxide may upregulate ACE expression in human umbilical vein endothelial cells (HUVECs) and investigated the under-lying mechanisms. The results revealed a new link between oxidative stress and hypertension.

Hydrogen peroxide induces overexpression of angiotensin-converting enzyme in human umbilical vein endothelial cells Xiaoqin Mu , Kaiwen He , Hui Sun , Xin Zhou , Lingling Chang , Xin Li , Wenfeng Chu , Guofen Qiao & Yanjie Lu

Department of Pharmacology, Harbin Medical University, Harbin, P. R. China

Abstract Oxidative stress has been linked to endothelial dysfunction in atherosclerosis and hypertension. The present study was designed to inves-tigate the eff ect of hydrogen peroxide (H

2 O

2 ) on angiotensin-converting enzyme (ACE), a key regulator of the renin – angiotensin system,

and the mechanisms underlying ACE regulation in human umbilical vein endothelial cells (HUVECs). We used Tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, enzyme-linked immunosorbent assay (ELISA) for cAMP measurement, real-time PCR for mRNA detection, and Western blot for protein analysis in the study. Our results demonstrated that H

2 O

2 (50 – 1000 μ M) decreased HUVECs viability by inducing apoptosis. Notably, H

2 O

2

upregulated ACE expression in a concentration-dependent manner. H 2 O

2 100 μ M signifi cantly enhanced cyclic adenosine monophosphate

(cAMP) expression by 1.48-fold ( P � 0.05). Additionally, forskolin 10 μ M, a cAMP agonist, was also found to enhance ACE expression by 1.78-fold ( P � 0.05); in contrast, H-89 10 μ M, a protein kinase A (PKA) inhibitor, abolished H

2 O

2 -induced ACE expression and prevented

the enhancing eff ect of forskolin-induced ACE expression. Similar eff ects on ACE mRNA were also observed. cAMP-response element-specifi c decoy oligodeoxynucleotides (CRE-dODN) containing binding sites for cAMP-response element-binding protein (CREB) inhibited ACE expression at both the mRNA and protein levels. Negative control CRE-dODN had no eff ect on ACE expression. We conclude that H

2 O

2 upregulates the expression of ACE through the activation of cAMP/PKA/CREB signal pathway in HUVECs, indicating a role of

oxidative stress in the pathophysiology of hypertension.

Keywords: oxidative stress , ACE , cAMP , hypertension , HUVECs

Introduction

Oxidative stress represents an imbalance between oxidation and antioxidation in vivo , and generates a large number of oxidation intermediates such as superoxide anion, hydroxyl radicals, and hydrogen peroxide. It is involved in the development and progression of cardiovas-cular diseases, including hypertension [1], atherosclerosis [2], and cardiac hypertrophy [3]. Many studies have shown that there is a close relationship between oxidative stress and elevated blood pressure. De Champlain et al. demonstrated that reactive oxygen species (ROS) enhance production of inositol triphosphate while reducing cyclic GMP in vascular smooth muscle cells, resulting in vasoconstriction [4]. As well, superoxide can induce vasoconstriction by suppressing the expression of nitric oxide (NO), the most important endogenous vasodilator [5,6]. In addition, higher production of hydrogen peroxide (H

2 O

2 ) has been observed in hypertensive subjects, with

a signifi cant correlation between H 2 O

2 and systolic blood

pressure [7]. Vascular endothelial cells (VECs) play important

roles in the regulations of platelet function, plasma procoagulant factor activation, clearance of activated clotting factor, fi brinolysis, and vascular tone. VECs can

Correspondence: Yanjie Lu, Department of Pharmacology, Harbin Medical University, 157 Baojian Road, Harbin, Heilongjiang province, 150081, P. R. China. Tel: � 86-451-86671354. Fax: � 86-451-86671354. E-mail: yjlu86@gmail.com

(Received date: 17 September 2012 ; Accepted date: 12 November 2012; Published online: 10 December 2012)

Free Radical Research, February 2013; 47(2): 116–122© 2013 Informa UK, Ltd.ISSN 1071-5762 print/ISSN 1029-2470 onlineDOI: 10.3109/10715762.2012.749987

ORIGINAL ARTICLE

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H 2 O 2 upregulates angiotensin converting enzyme 117

Material and methods

Chemicals

Hydrogen peroxide solution (H 2 O

2 ) was purchased from

shanghai shuangjianjingxi chemical CO. Ltd (shanghai, China). Phosphate-buff ered saline (PBS) was purchased from Solarbio Bioscience & Technology Company (Beijing, China). Forskolin and N-[2-(p-Bromocinnam-ylamino)ethyl]-5-isoquinolinesulfonamide • 2HCl hydrate (H-89) were obtained from Beyotime institute of Biotech-nology (shanghai, China). cAMP-response element-decoy oligodeoxynucleotides (CRE-dODN) was synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd (Shanghai, China).

Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in Dulbecco ’ s Modifi ed Eagle ’ s Medium/Nutrient Ham ’ s Mixture F-12 (DMEM/F-12; Hyclone, Logan, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, USA) under standard culture conditions (37 ° C, 95% humidifi ed air and 5% CO

2 ).

MTT cell viability assay

HUVECs were seeded in 96-well culture plates with 1 � 10 4 cells/well, and incubated at 37 ° C with 5% CO

2 .

After treatment of the HUVECs with diff erent concentra-tions of H

2 O

2 or PBS as control for 12 h, MTT assay

(Amresco, Solon, USA) was performed. Briefl y, 20 μ l of MTT solution (5 mg/ml) was added to each well, and the cells were continuously incubated for 4 h. Formazan crys-tals were then dissolved in 150 μ l DMSO. The optical density (OD) of the wells was measured with a microplate reader (BioTek, Richmond, USA) at 490 nm. Cell survival (CS) was calculated by the following equation: CS � ( mean OD treated wells / mean OD control wells ) � 100%.

TUNEL assay

HUVECs were seeded on cover slips and exposed to diff erent concentrations of H

2 O

2 or PBS as control. After

12 h treatment, apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay using a kit methodology (Roche, Nutley, USA). Briefl y, cells were washed once with PBS and fi xed in 4% paraformaldehyde for 1 h at 15 – 25 ° C. Cells were then washed with PBS and exposed to a block-ing solution (3% H

2 O

2 in methanol) for 10 min at 15 – 25 ° C.

Cells were next washed with PBS and incubated in per-meabilization solution (0.1% triton X-100 in 0.1% sodium citrate) for 2 min on ice. They were then washed twice with PBS and treated with TUNEL reaction mixture for 60 min at 37 ° C in the dark in a humidifi ed atmosphere. Finally, the cells were washed three times with PBS and

analyzed under fl uorescence microscope (Olympus, Tokyo, Japan), using an excitation wavelength in the range of 450 – 500 nm and a detection wavelength in the range of 515 – 565 nm (green). Apoptotic index was calculated as percentage of TUNEL-positive cells by means of the fol-lowing equation: percentage of apoptotic cells � ( number of TUNEL-positive cells / total number of cells ) � 100%.

Real time PCR technique

The total RNA was isolated from HUVECs by TRIzol (Invitrogen, CA, USA). The extracted RNA was treated with RNase-free DNase prior to the reverse transcription step and then it was reversely transcribed into complemen-tary DNA using High Capacity cDNA Reverse Transcrip-tion Kit (Ambion Inc., Austin TX, USA). Real-time quantitative reverse transcription PCR (real-time qRT-PCR) was carried out using ACE or GAPDH primers: ACE-forward: 5 ′ -TGGTGACTGATGAGGCTGAG-3 ′ , ACE-reverse: 5 ′ -TCTTGCTGGTCTCTGTGGTG-3 ′ ; GAPDH-forward: 5 ′ -AAGAAGGTGGTGAAGCAGGC-3 ′ , GAPDH-reverse: 5 ′ -TCCACCACCCAGTTGCTGTA-3 ′ . Real-time qRT-PCR was performed using SYBR ® Green PCR Master Mix (Ambion Inc.), according to the manufacturer ’ s protocol. DNA was amplifi ed in an ABI 7500 sequence detection system (Applied Biosystem, Foster City, CA, USA), using the same cycling parameters as follows: 95 ° C for 10 min followed by 40 cycles of a three-stage temperature profi le of 95 ° C for 15 sec and 60 ° C for 15 sec, then 72 ° C for 30 sec. All real-time qRT-PCR analyses were conducted using the 2 delta – delta Ct variant (2 � ∆ ∆ CT ) method with the expression level of GAPDH as an internal control. All reactions were per-formed in duplicate.

Western blotting

HUVECs were prepared in lysis buff er and proteinase inhibitors (Beyotime, Shanghai, China). Cellular protein (150 μ g) from each sample was denatured in 4 � SDS-PAGE sample buff er and resolved on 8% Tris-glycine gels. The separated proteins were transferred to a nitrocellulose membrane followed by blocking with 5% non-fat milk powder (w/v) in PBS for 3 h at room temperature. After blocking, the membranes were probed with ACE primary antibodies (Abcam, Cambridge, USA) overnight at 4 ° C. This was followed by incubation with appropriate infra-red-fl uorescent labeled secondary antibody for 1 h at room temperature and fl uorescent signals were detected by an Odyssey ® infrared scanner (LI-COR, Lincoln, USA).

ELISA assay

cAMP Assay Kit was purchased from BioVision (Mountain View, USA). The content of cAMP was deter-mined according to the manufacturer ’ s protocol. The kit utilizes recombinant Protein G-coated 96-well plate to effi ciently anchor cAMP polyclonal antibodies onto the plate. cAMP-HRP conjugate directly competes with cAMP

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118 X. Mu et al.

from sample binding to the cAMP antibody on the plate. After incubation and washing, the amount of cAMP-HRP bound to the plate can be determined by reading HRP activity at OD450 nm. The intensity of OD450 nm is inversely proportional to the cAMP concentration in sam-ples. Experiments were performed at room temperature and data were relative to the control group.

Preparation and transfection of decoy ODNs

cAMP-response element-decoy oligodeoxynucleotides (CRE-dODN) containing the CRE cis -element TGACGTCA compete with CRE enhancers for binding transcription factors and inhibit CRE-directed trans-cription in vivo studies. Single-stranded CRE-dODN 5 ¢ -TGACGTCATGACGTCATGACGTCA-3 ¢ and negative control CRE-dODN 5 ¢ -TGTGGTCATGTGGTCATGTGGTCA-3 ¢ were transfected with X-tremeGENE (Roche, Nutley, USA).

Statistical analysis

All data were expressed as mean � SD. Statistical analy-sis was performed by ANOVA and the Dunnett- t test. All statistical comparisons were performed by Prism v5 (GraphPad, USA). A two-tailed value of P � 0.05 was taken to indicate statistical signifi cance.

Results

Eff ects of H 2 O 2 on HUVECs viability

Cell viability was assessed by MTT assay to detect injury of human umbilical vein endothelial cells (HUVECs) by hydrogen peroxide (H

2 O

2 ). Cell survival was slightly

decreased by 7.5%, 15.0% in 50 and 100 μ M H 2 O

2 treat-

ment groups, respectively, and markedly decreased by 24.5%, 32.9%, 43.5%, and 55.5% in 200, 400, 800, and 1000 μ M H

2 O

2 groups, respectively (Figure 1). The results revealed

that HUVECs were injured after 12 h exposure to H 2 O

2

in a concentration-dependent manner. It was also observed that when the H

2 O

2 concentration reached 200 μ M, the

morphology of the cells began to change. With increase in H

2 O

2 concentration, more spherical cells were observed.

When the concentration of H 2 O

2 reached 2000 μ M, the

cells became detached from the bottom of the culture dish and fl oated in the solution, indicating cell death.

Cell apoptosis induced by H 2 O 2

After treatment of HUVECs with 50, 100, 200, 400, 800, and 1000 μ M H

2 O

2 for 12 h, the cells were fi xed and

TUNEL assay was performed. The number of apoptotic cells was increased with increasing H

2 O

2 concentration in

the tested range shown in Figure 2. Based on the above-mentioned experiments, 100 μ M hydrogen peroxide caused signifi cant changes in cell viability and apoptosis. We decided to use 100 μ M H

2 O

2 in all subsequent exper-

iments. At H 2 O

2 concentration of 1000 μ M, about half of

the cells (54.0%) exhibited apoptotic death.

Upregulation of ACE expression by H 2 O 2

After treatment of HUVECs with 0, 50, 100, 200, 400, 800, and 1000 μ M H

2 O

2 for 12 h, the protein level of

angiotensin-converting enzyme (ACE) was increased in a concentration-dependent manner (Figure 3). H

2 O

2 at a

concentration of 100 μ M resulted in a 69.9% increase in ACE level ( P � 0.05 vs control); while H

2 O

2 at a higher

concentration of 1000 μ M induced a 179.7% increase in ACE level ( P � 0.01 vs control). Consistently, a H

2 O

2

concentration of 100 μ M also caused signifi cant eff ects on HUVECs viability and apoptosis.

Involvement of cAMP/PKA/CREB pathway in H 2 O 2 -induced ACE expression

As shown in Figure 4, when HUVECs were cultured with 100 μ M of H

2 O

2 for 12 h, cAMP content was elevated by

1.48-fold ( P � 0.05 vs control). To test whether the cAMP/PKA/CREB signaling pathway is involved in the regula-tion of ACE expression induced by H

2 O

2 at mRNA or

protein levels, further experiments were performed. After HUVECs were treated with H

2 O

2 (100 μ M, 12 h), forsko-

lin (10 μ M, 12 h), a cAMP activator, or H-89 (10 μ M, 12 h), a protein kinase A (PKA) inhibitor, total RNA was isolated and ACE mRNA was measured using qRT-PCR, with results normalized to ACE expression for non-treatment control. ACE expression was elevated by 1.8-fold by forskolin ( P � 0.05 vs control), similar to the level of H

2 O

2 -induced ACE expression (1.7-fold, P � .05

vs control). H-89 abolished the forskolin-induced increase of ACE expression and also prevented elevated eff ect of ACE by H

2 O

2 at mRNA level (Figure 5A).

To further confi rm mRNA data, Western blot analysis was employed to evaluate ACE expression at protein level. Both H

2 O

2 (100 μ M, 12 h) and forskolin (10 μ M, 12 h)

induced similar enhancements in ACE expression, 1.7-fold

Figure 1. Eff ects of H 2 O

2 on cell viability in HUVECs. After

treatment of the cells with diff erent concentrations of H 2 O

2 , cell

viability was analyzed by MTT assay. The data are expressed as mean � SD ( n � 6 batches of cells in each group), * P � 0.05, * * P � 0.05 vs . control.

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H 2 O 2 upregulates angiotensin converting enzyme 119

increase in H 2 O

2 group and 1.8-fold in forskolin group,

respectively. As expected, the eff ects of H 2 O

2 or forskolin

were reversed in the presence of H-89 (Figure 5B). To test the involvement of CREB in ACE expression

induced by H 2 O

2 , HUVECs were pre-transfected with

cAMP-response element-decoy oligodeoxynucleotide (CRE-dODN), containing perfect binding sites for CREB, for 36 h. Then H

2 O

2 or forskolin was added to the medium

for an additional 12 h. As shown in Figure 5C and 5D CRE-dODN not only reversed the eff ects of both H

2 O

2

and forskolin on ACE expression at mRNA and protein levels, but also further decreased ACE expression level compared to control values. Negative control CRE-dODN failed to reverse the elevated expression induced by H

2 O

2

or forskolin.

Discussion

In the current study, we demonstrated that H 2 O

2 induces

the expression of ACE in HUVECs in a concentration-dependent manner; the cAMP/PKA/CREB signaling path-way is involved in H

2 O

2 -induced ACE expression. Our

results provide novel evidence that oxidative stress is a major regulator of ACE expression, and elucidate the cAMP/PKA/CREB signaling pathway involved in the regulation of ACE expression by H

2 O

2 .

Accumulating evidence demonstrates that reactive oxy-gen species (ROS) play an important role in the develop-ment and maintenance of hypertension by directly altering vascular function and tone by decreasing nitric oxide (NO) bioavailability and/or signaling [11,12], or by pro-moting vascular cell proliferation, infl ammation, apopto-sis, and extracellular matrix alterations [13,14]. The endothelium is not only a selective barrier to prevent the diff usion of macromolecules from the vascular lumen into the interstitial space, but also plays crucial roles in the regulation of vascular tone, infl ammation, vascular growth, platelet aggregation, and coagulation [15,16]. The increased ROS contribute to hypertension by damaging

Figure 2. Eff ects of H 2 O

2 on apoptosis determined by TUNEL assay in HUVECs. (A) Respective fl uorescence microscopic images (10 � )

showing the number of apoptotic cells in diff erent concentrations of H 2 O

2 . (B) Averaged data of apoptotic cells with positive TUNEL

staining ( n � 6 batches of cells in each group), * P � 0.05, * * P � 0.05 vs . control.

Figure 3. Eff ects of H 2 O

2 on the expression of ACE in HUVECs.

Upper panel, example of Western blot bands; lower panel, mean data of ACE expression in diff erent groups of H

2 O

2 ( n � 6 batches

of cells in each group). * P � 0.05, * * P � 0.05 vs . control.

Figure 4. Eff ects of H 2 O

2 on the content of cAMP. cAMP level

was measured in the HUVECs in the absence or presence of H

2 O

2 (100 μ M, 12 h). ( n � 6 batches of cells in each group),

* P � 0.05 vs . control.

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120 X. Mu et al.

endothelial function in several ways. ROS react with NO directly and reduce its bioavailability [17,18]; ROS pro-duction in the media and adventitia of vessel walls may impair NO signaling within vascular tissues [19,20]; ROS may impair the activity of a number of other pathways, by modifying responsiveness to endothelium-independent vasodilators [21,22]. In the vasculature, endothelium is the major source of ACE. Our results demonstrate that H

2 O

2 displayed damaging eff ects on

HUVECs by decreasing the viability of HUVECs by inducing cell apoptosis. It has been reported that hydro-gen peroxide can obviously induce upregulations of urokinase-type plasminogen activator receptor (u-PAR) [23], platelet-activating factor (PAF) [24], and tumor necrosis factor alpha (TNF alpha) [25]. Interestingly, in the present study hydrogen peroxide signifi cantly upreg-ulated ACE expression in HUVECs. Endothelial cells are a major source of ACE, which is an essential element of

Figure 5. Involvement of the cAMP-PKA-CREB signaling pathway in the regulation of ACE induced by H 2 O

2 in HUVECs. (A) ACE mRNA

expression in diff erent groups of H 2 O

2 (100 μ M, 12 h), forskolin (10 μ M, 12 h), H

2 O

2 � H-89 (10 μ M, 12 h), and forskolin � H-89,

respectively ( n � 5 – 6 batches of cells in each group). (B) Protein level of ACE with the same treatments as (A), Upper panel, examples of Western blot bands; lower panel, averaged data of ACE levels ( n � 6 batches of cells in each group). (C) ACE mRNA expression in diff erent groups of H

2 O

2 (100 μ M, 12 h), forskolin (10 μ M, 12 h), H

2 O

2 � CRE-dODN (150 nM, 48 h), forskolin � CRE-dODN, and NC (150 nM,

48 h), respectively ( n � 4 – 6 batches of cells in each group). NC: negative control CRE-dODN. (D) Protein levels of ACE expression were measured in HUVECs treated the same as (C). Upper panel, examples of Western blot bands; lower panel, averaged data of ACE levels ( n � 6 batches of cells in each group). * P � 0.05 vs . control, # P � 0.05, ## P � 0.01 vs . H

2 O

2 , � P � 0.05, � � P � 0.01 vs. forskolin.

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H 2 O 2 upregulates angiotensin converting enzyme 121

the renin – angiotensin system, transforming angiotensin I to angiotensin II, which controls blood pressure. Enhanced ACE expression results in angiotensin II formation, con-sequently causing the constriction of blood vessels and the elevation of blood pressure. Therefore, ACE inhibitors are used as eff ective antihypertensive agents that suppress angiotensin II production by inhibiting ACE activity.

It has been reported that nicotine, nicotine metabolites, and low-density lipoproteins can cause overexpression of ACE in HUVECs [26,27]. Several signaling pathways are involved in regulating ACE expression, such as the cAMP-PKA pathway [28], protein kinase C (PKC) path-way [29], and tyrosine kinase-MAPK pathway [30]. Xavier reported that ACE promoter stimulation by ISO and isobutyl methylxanthine is blocked by protein kinase A inhibitors, implicating that β -adrenergic stimulation of the ACE gene depends on phosphorylation of protein kinase A targets [28]. Villard et al. demonstrated that PMA-activated PKC strongly upregulates ACE mRNA level in HUVECs [29]. Saijonmaa et al. also reported that intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were likely involved in ACE induction by VEGF [30]. In cultured cells, the expression of ACE is also modulated by hor-mones [31 – 33] and calcium ionophore [34]. The above-mentioned studies indicate that some signaling pathways are involved in the regulation of ACE expression.

In the present study, we show that H 2 O

2 potentially

induces the expression of ACE in HUVECs. To our knowledge, this is the fi rst report demonstrating that oxi-dative stress stimulates ACE expression. Cyclic adenos-ine 3 ¢ , 5 ¢ -monophosphate (cAMP) is a typical second messenger regulated by hormone and some extracellular signal molecules in mammalian cells. cAMP regulate cells physical activity and metabolism by phosphorylat-ing protein kinase A (PKA) [35]. The activated PKA can be translocated into the nucleus where it subsequently activates the respective transcription factors. These in turn cause activation or repression of gene transcription [36,37]. In our study, H

2 O

2 markedly induced an increase

in cAMP expression in HUVECs. Furthermore, forsko-lin, a cAMP agonist, enhanced the expression of ACE; however, H-89, a PKA inhibitor, completely abolished the eff ects of forskolin- and H

2 O

2 -induced ACE expres-

sion. Our results showed that CRE-dODN induced more down-regulation of ACE level compared with control level, presumably by silencing endogenous CREB. By using decoy oligodeoxynucleotide (dODN) technique, we demonstrated that CREB is potentially involved in the regulation of ACE expression induced by H

2 O

2 in

HUVECs. Our results indicate that the cAMP/PKA/CREB signaling pathway is potentially involved in the regulation of ACE expression induced by H

2 O

2 in

HUVECs. However, we cannot exclude the participation of other signaling pathways in the mediation of ACE expression by H

2 O

2 in HUVECs.

There are limitations to the current study. First, we used H

2 O

2 as a model of oxidative stress. Many previous reports

used this in vitro model to mimic the pathophysiological

condition of oxidative stress observed in infl ammatory lung diseases, including COPD and asthma. In the current study, we used 100 μ M H

2 O

2 to observe its eff ect on ACE

expression in HUVECs and this concentration is within the range of previous reports [38,39]. Second, we used HUVECs in the study that may have characteristics diff er-ent from those of endothelial cells from other resources, such as endothelial cells from microvasculature, pulmo-nary artery and vein, and aorta [40]. Responses of diff erent types of endothelial cells to H

2 O

2 may be altered. How-

ever, we should be careful when extrapolating the fi ndings obtained in this in vitro model to the “ real ” pathophysio-logical conditions in oxidative stress condition.

Conclusion

The present study demonstrates that H 2 O

2 upregulates

ACE expression through the cAMP/PKA/CREB signal-ing pathway in HUVECs. These findings provide new insight into understanding the regulation of ACE expression under oxidative stress conditions, and new evidence in support of oxidative stress involvement in hypertension pathophysiology.

Declaration of interest

The authors report no confl icts of interest. The authors alone are responsible for the content and writing of the article.

This study was supported in part by the National Nature Science Foundation of China (30971252 and 81170219), the Funds for Creative Research Groups of the National Natural Science Foundation of China (81121003), and Major Program of National Natural Science Foundation of China (81130088).

No confl ict of interest exits in the submission of this manuscript.

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