Journal of Molecular and Cellular Cardiology 52 (2012) 237–244

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Journal of Molecular and Cellular Cardiology

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Original article

Bone morphogenic protein-4 induces endothelial cell apoptosis through oxidativestress-dependent p38MAPK and JNK pathway

Xiao Yu Tian a,1, Lai Hang Yung a,1, Wing Tak Wong a, Jian Liu a, Fung Ping Leung a, Limei Liu a,Yangchao Chen a, Siu Kai Kong b, Kin Ming Kwan b, Siu Man Ng c, Paul B.S. Lai c, Lai Ming Yung a,Xiaoqiang Yao a, Yu Huang a,⁎a Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, School of Biomedical Sciences, Chinese University of Hong Kong; Hong Kong SAR, Chinab School of Life Sciences, Chinese University of Hong Kong; Hong Kong SAR, Chinac Department of Surgery, Chinese University of Hong Kong; Hong Kong SAR, China

⁎ Corresponding author at: School of Biomedical ScHong Kong, Shatin, NT, Hong Kong, China. Tel.: +852 26

E-mail address: yu-huang@cuhk.edu.hk (Y. Huang).1 Contributed equally to this work.

0022-2828/$ – see front matter © 2011 Elsevier Ltd. Alldoi:10.1016/j.yjmcc.2011.10.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 August 2011Received in revised form 16 September 2011Accepted 17 October 2011Available online 25 October 2011

Keywords:Bone morphogenic protein-4Reactive oxygen speciesMitogen-activated protein kinaseApoptosisEndothelial cells

The expression of bone morphogenic protein 4 (BMP4), a new pro-inflammatory marker, is increased by dis-turbed flow in endothelial cells (ECs). BMP4 stimulates production of reactive oxygen species (ROS) andcauses endothelial cell dysfunction. The present study examined BMP4-induced apoptosis in ECs and isolatedarteries from rat, mouse, and human, and the signaling pathways mediating BMP4-induced apoptosis. Apo-ptosis was assessed by flow cytometry to detect Annexin-V positive cells, and terminal deoxynucleotidyltransferase dUTP nick end (TUNEL) labeling. The superoxide production was measured by dihydroethidiumfluorescence. BMP4 induced EC apoptosis in human mesenteric arteries, mouse aortic endothelium, rat pri-mary ECs, and human ECs. BMP4-induced EC apoptosis was mediated through ROS production by activationof NADPH oxidase, which led to cleaved caspase-3 expression. BMP4 also induced sequential activation ofp38 MAPK and JNK which was upstream of caspase 3 activation. Knockdown of BMP receptor 1A by lentiviralshRNA or NOX4 siRNA transfection inhibited BMP4-induced ROS production, p38 and JNK phosphorylation,and caspase-3 activation in ECs. JNK siRNA inhibited BMP4-induced JNK phosphorylation and caspase-3 acti-vation. The present study delineates that BMP4 causes EC apoptosis through activation of caspase-3 in a ROS/p38MAPK/JNK-dependent signaling cascade.

© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Bone morphogenic protein-4 (BMP4), originally discovered as abone growth factor [1] exerts an inflammatory effect in blood vessels.BMP4 and other members of the BMP family of ligands are detectablein atherosclerotic plaques [2–4]. BMP4 induces monocyte adhesion inresponse to oscillatory shear stress through NAD(P)H oxidase-dependent ROS [5] and impairs endothelial function through activa-tion of NAD(P)H oxidase [6,7] although the underlying mechanismsof BMP4-induced vascular inflammation remain incompletely known.

Apoptosis is essential for maintaining normal development ofmulti-cellular organisms through elimination of unwanted cells [8].In the cardiovascular system, however, elevated vascular cell apopto-sis is associated with increased incidence of failing heart [9], ad-vanced and unstable atherosclerotic plaques [10], hypertension [11],and diabetes [12]. Endothelial cell (EC) apoptosis contributes to thedevelopment of atherosclerosis through an increased permeability

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of endothelial monolayer and subsequent uptake of lipids in the vas-cular wall [13]. Clinical studies suggest a contributory role of EC apo-ptosis in plaque destabilization and thrombosis [14].

In atherosclerotic plaques, ECs undergoing apoptosis produce highlevels of apoptotic blebs containing active oxidized phospholipid thatstimulate adhesion of monocytes to ECs [15–17]. Enhanced produc-tion of NAD(P)H oxidases-derived ROS, a hallmark in hypertensionand atherosclerosis, is found in ECs as a result of disturbed flow [18]and ROS released by pro-inflammatory cytokines and oxidized lipo-proteins promote EC apoptosis [19]. However, it is unclear whetherROS production induced by BMP4 can initiate a cascade of cellularevents leading to EC apoptosis.

Apart from the classical intracellular pathway initiated by BMPswhich involves receptor-mediated activation of Smad, existing evi-dence shows that BMPs activate MAPKs in various cell types [20]such as lung fibroblasts [20] and pulmonary arterial myocytes [21].MAPKs serve as the downstream targets in H2O2-induced apoptosisof rat VSMCs [22]. However, it remains unclear whether MAPKs areinvolved in the signaling cascade triggered by BMP4 in ECs.

BMP4 can induce apoptosis in both vascular smooth muscle cells[23] and ECs [24,25]. However, BMP4 also possesses anti-apoptotic ef-fects [26–28]. In the present study, we investigated the pro-apoptotic

Fig. 1. BMP4 induced EC apoptosis in human arteries. (A) BMP4 caused DNA fragmenta-tion as assessed by TUNEL staining. (B) BMP4 induced cleaved caspase-3 expression asassessed by immunofluorescence imaging in ECs of humanmesenteric arteries. Represen-tative images shown are from 4 independent experiments from different subjects. Barsrepresent 100 μm.

Fig. 2. BMP4 induced EC apoptosis in mouse and rat. BMP4 (10 ng/mL, 24 h)-induced cleavedmouse aortae and (B)Western blotting of aortic tissue. Native endothelial cells were labeledData are mean±SEM (n=6). ⁎Pb0.05 compared with control; #Pb0.05 compared with BMapoptosis through ROS-dependent caspase-3 activation. (C) BMP4 treatment for 24 h caused(D) BMP4 induced cleaved caspase-3 expression in RAECs as compared with H2O2. ⁎Pb0.05

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effect of BMP4 in ECs and arteries from different species and demon-strated that BMP4-induced caspase-3 activation is mediated throughthe sequential activation of BMPR1A, NADPH oxidase, and downstreamp38 MAPK and JNK.

2. Materials and methods

2.1. Isolation and primary culture of rat aortic endothelial cells (RAECs)

The experimental protocols were approved by the institutionalanimal care and use committee and were consistent with the Guidefor the Care and Use of Laboratory Animals published by the NationalInstitutes of Health. RAECs were isolated from the thoracic aorta ofmale Sprague–Dawley rats (260–280 g) using an enzymatic digestionmethod [29]. The aorta was incubated in phosphate-buffered-saline(PBS) containing 0.2% collagenase with shaking for 15 min at 37 °C,then centrifuged for 5 min at 800 g. The cells were suspended inRPMI medium 1640 (Gibco, Grand Island, NY, USA) containing 10%fetal bovine serum (Gibco) and 1% penicillin/streptomycin and set-tled for 1 h. Culture medium was changed afterwards. The identityof the RAECs was confirmed by a positive staining of PECAM-1(Santa Cruz, CA, USA), and used within the first two passages. For

caspase-3 expression as assayed by (A) immunofluorescence of en face endothelium ofwith PECAM-1 (red) and cleaved caspase-3 (green), which was localized in the nucleus.P4. Photos are representative for samples from 4 different mice. BMP4 induces RAECa concentration-dependent apoptotic changes in RAECs as assessed by TUNEL staining.compared with control from different rats.

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transfection experiment, RAECs were transfected with NOX4 siRNApool (SMARTpools, Thermo Scientific, Lafayette, CO, USA) or nontar-geting siRNA as control by electroporation using Nucleofector II ma-chine (Amaxa/Lonza, Walkersville, MD, USA) according to themanufacturer's instruction.

2.2. Human endothelial cell culture and transfection

Human umbilical vein endothelial cells (HUVECs, Lonza, Basel,Switzerland, No.CC-2517) were grown in EGM (Clonetics, San Diego,USA) supplemented with bovine brain extract (BBE, Clonetics), penicil-lin (100 μg/mL) and streptomycin (100 μg/mL) in gelatin-coated flasksand maintained at 37 °C in a 95% O2 plus 5% CO2 condition. The cellsat passage 4–6 were used when at ~80–90% confluency. For the knock-down experiment, 80% confluent cells were transfected with prede-signed JNK siRNA (Invitrogen, Carlsbad, CA, USA), with LipofectamineRNAiMAX (Invitrogen) in Opti-MEM for 24 h. After transfection, Opti-MEM medium was replaced with EBM without phenol red for furthertreatment.

2.3. Human mesenteric artery specimens

The present study was approved by the Joint Chinese University ofHong Kong—New Territories East Cluster Clinical Research Ethics Com-mittee. Human smallmesenteric arterieswere harvested during surger-y from 4 colon cancer patients, after obtaining their informed consent.

Fig. 3. BMP4 induces RAEC apoptosis through ROS-dependent caspase-3 activation. (A) N(100 μmol/L) suppressed the increased apoptotic rate in RAECs in response to BMP4 (10 nO2− in RAECs after 30-min treatment, which was blocked by noggin, apocynin, or T+D. (C

RAECs was also inhibited by noggin, apocynin, or T+D pre-treatment. Data are mean±SEM

Arteries were dissected in PBS, and incubated with BMP4 (30 ng/mL,24 h) in DMEM.

2.4. Chemical treatment

The ECs were treated with recombinant BMP4 (10, 30 and 100 ng/mL, dissolved in 4 mM HCl with 0.1% BSA, R&D Systems, Minneapolis,MN, USA) for 24 h and collected for apoptosis and protein assay. Forother drug treatments, ECswere treatedwith one of the following: nog-gin (BMP4 antagonist, 100 ng/mL, R&D Systems), apocynin (NADPHox-idase inhibitor, 100 μmol/L, Sigma, St. Louis, MO, USA), tiron (1 mmol/L,Sigma) plus DETCA (100 μmol/L, Sigma) (T+D, ROS scavengers),SP600125 (JNK inhibitor, 10 μmol/L, Tocris, Bristol, UK), PD98059(ERK inhibitor, 20 μmol/L, Tocris), and SB202190 (p38 MAPK inhibitor,10 μmol/L, Tocris) for 30 min prior to BMP4 exposure.

2.5. Western blotting

After treatment, cells were trypsinized and homogenized in lysisbuffer containing complete protease and phosphatase inhibitor cocktail(RocheDiagnostics, Indianapolis, IN, USA), followed by centrifugation at20,000 g for 20 min to collect supernatants. The protein samples (20 μg)were separated with 12.5% SDS-polyacrylamide gel and transferred toan Immobilon-P polyvinylidene difluoride membrane. Membraneswere blocked with 1% bovine serum albumin. Primary antibodiesagainst phospho-p38 MAPK, p38 MAPK, phospho-SAPK/JNK, SAPK/

oggin (100 ng/mL), apocynin (100 μmol/L) or tiron (1 mmol/L) plus DETCA (T+D)g/mL) as assayed by TUNEL staining. (B) BMP4 increased intra-cellular generation of) Western blot analysis showed that BMP4-induced increase of cleaved caspase-3 in(n=5). #Pb0.05 in compared with control; ⁎Pb0.05 compared with BMP4.

Fig. 4. ROS mediates p38 MAPK and JNK activation in BMP4-induced apoptosis inRAECs. (A) The up-regulation of cleaved caspase-3 in response to BMP4 was reversedby SB202190 or SP600125. (B) Increased level of phospho-p38 MAPK in response toBMP4 (10 ng/mL) for 2 h was prevented by treatment with noggin (100 ng/mL), apoc-ynin (100 μmol/L), or tiron (1 mmol/L) plus DETCA (100 μmol/L) (T+D) without af-fecting total p38 MAPK. (C) The elevated phospho-JNK level stimulated by 4 hexposure to BMP4 was abolished by noggin, apocynin and T+D without affectingtotal JNK. (D) Western blot analysis showed that activation of JNK following 4-hourtreatment with BMP4 in RAECs was reduced by MAPK inhibitor SB202190 (10 μmol/L) and JNK inhibitor SP600125 (10 μmol/L). (E and F) BMP4 (10 ng/mL, 24 h) inducedphosphorylations of p38 and JNK in mouse aortae, which were inhibited by noggin(100 ng/mL, 24 h).Data are expressed as mean±SEM (n=4) from different rats ormice. #Pb0.05 compared with control; ⁎Pb0.05 compared with BMP4.

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JNK (1:500, Cell signaling Technology, Beverly, MA, USA), cleavedcaspase-3 (1:2000 for Western blotting; 1:200 for immunofluores-cence, Cell signaling Technology), caspase-3 (1:2000, Calbiochem, SanDiego, CA, USA), NOX4 (Abcam, Cambridge, UK), BMPR1A (Santa Cruz,CA, USA), housekeeping GAPDH (1:50000, Ambion, Austin, TX, USA),and β-actin (1:4000, Sigma, MO, USA) were used.

2.6. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay

The TUNEL assay was used to detect DNA fragmentation in situusing the ApopTag apoptosis detection kit (Chemicon, Temecula, CA,USA). Briefly, cells or tissue sections were fixed with 4% paraformal-dehyde, washed and stained according to the manufacturer's instruc-tions. The samples were then counterstained with hematoxylin. Photoswere taken under a Leica DMRBE microscope.

2.7. Flow cytometry

For analyses of cell apoptosis, the apoptotic rate wasmeasured by PI(propidium iodide) and annexin-V staining with flow cytometryaccording to manufacturer's instructions (BD Biosciences, CA, USA).Briefly, after treatments, cells were trypsinized and collected by centri-fugation. The cells were washed and re-suspended in binding buffer at1×106 cells/mL. 1×105 cells were stained with PI and Annexin-V FITCfor 15 min before analysis by FACSort flow cytometer (BD Biosciences).The instrument was set to collect 1×105 cells and the profile was ana-lyzed using CellQuest software.

2.8. Dihydroethidium (DHE) fluorescence imaging

After treatment of the RAECs with 10 ng/mL BMP4 and inhibitors,cells were rinsed with normal physiological saline solution (NPSS), andincubated with DHE (5 μmol/L) at 37 °C in dark for 15 min, then washedtwice with NPSS. Fluorescence was observed by confocal microscope(515-nm excitation; 585-nm long pass filter; Olympus FV1000, Tokyo,Japan). DHE fluorescence intensity was analyzed by Fluoview (version1.5). For each section, a square region with an area of 80 μm×80 μmwas selected for analysis. The summarized data was expressed as com-pared with control to indicate the fold change in fluorescence intensityamong different treatments.

2.9. Immunofluorescence staining

Expression of cleaved caspase-3 in human mesenteric artery wasdetected. The human artery samples were then fixed with 4% parafor-maldehyde for overnight, cut into two segments, and kept in frozenand paraffin blocks. Both frozen and paraffin sections were preparedfor immunostaining. Paraffin sections were heated in citrate buffer forantigen retrieval. Frozen sections were dried and rinsed with 0.01% Tri-ton in PBS once for 30 s. Samples were blocked by 5% normal donkeyserum for 30 min. Then, sections were incubated with cleavedcaspase-3 primary antibodies at 4 °C overnight. Afterwards, slideswere washed with PBS and incubated with the secondary antibodies(AlexaFluor546, Molecular Probes, Eugene, OR. USA) for 1 h at roomtemperature. After washing twice with PBS, fluorescence was observedby fluorescence microscope.

2.10. Immunofluorescence staining of en face endothelium frommouse aorta

Aortic rings (2 mm length segment from C57BL/6 J mouse aorta)were treated with BMP4 (10 ng/mL) and noggin (100 ng/mL) in DMEMfor 24 h, and fixedwith 4%paraformaldehyde, thenwashedwith PBS, fol-lowed by the same procedure as frozen section. After being incubatedwith secondary antibody (AlexaFluor546, AlexaFluor488), aortic ringswere cut open and the endothelium side was placed upside down on

the coverslip, with another coverslip placed on the top for mounting,then observed under confocal microscope (Olympus FV1000, Tokyo,Japan).

2.11. Constructs, lentivirus production and transduction

Two shRNAs (short hairpin RNAs) targeting mouse Bmpr1a weredesigned: shRNA1: 5′- GCT GTT AAA TTC AAC AGT GAC ACA AAT G -3′;shRNA2: 5′- TCT CTC TAT GAC TTC CTG AAA TGT GCC -3′, and onescramble shRNA as a control [30]. DNA fragments containing shRNAssequence were synthesized and cloned into lentiviral RNAi (RNA inter-ference) vector pLUNIG after annealing as described [31]. The VSV-G-pseudotyped lentiviruses were produced by cotransfecting 293Tcells with the transfer vector and three packaging vectors: pMDLg/pRRE, pRSV-REV, and pCMV-VSVG. Subsequent purification was

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performed using ultracentrifugation. RAECs were cultured in 12-wellplates and transfected with lentivirus and 8 μg/mL polybrene (Sigma).

2.12. Statistical analysis

Results represent means±SEM of n experiments and data ana-lyzed by Student t test. Statistical significance was determined bytwo-tailed Student's t-test or one-way ANOVA followed by Bonferronipost-hoc tests when more than two treatments were compared.Pb0.05 was considered statistically significant.

3. Results

3.1. BMP4 induced apoptosis in the endothelial cells from human, rat,and mouse

First, we assessed the pro-apoptotic effect of BMP4 in isolated ar-teries from human and mouse. BMP4 (30 ng/mL) induced DNA frag-mentation as assessed by TUNEL staining (Fig. 1A) and increased thelevel of cleaved caspase-3 as assessed by immunofluorescence imag-ing (Fig. 1B) in endothelium of human mesenteric arteries after 24 htreatment with BMP4 (30 ng/mL). Expression of cleaved caspase-3was also observed in isolated mouse aortae after BMP4 (10 ng/mL,24 h) treatment, which was inhibited by co-incubation with noggin(100 ng/mL), as assessed by immunofluorescence staining of en face

Fig. 5. BMPR1A knockdown prevents BMP4-induced caspase-3 activation. (A) BMPR1A knocfluorescence. Both shRNAs targeting BMPR1A inhibited p38 phosphorylation (B) and casp⁎Pb0.05 compared with BMP4.

endothelium from mouse aorta (Fig. 2A) and Western blot from aor-tic tissue (Fig. 2B).

To further investigate the underlying mechanisms of BMP4-induced EC apoptosis, we verified the pro-apoptotic effects of BMP4in primary ECs from rat aorta (RAECs) and HUVECs. BMP4 caused ap-optosis as measured by TUNEL staining in RAECs (Fig. 2C), and byflow cytometry for detection of annexin-V positive cells in HUVECs(Supplemental Fig. 1A and B) in a concentration-dependent manner.BMP4 (24 h) also induced cleaved caspase-3 in RAECs as comparedto H2O2 in RAECs (BMP4 10 ng/mL, Fig. 2D) and in HUVECs (BMP430 ng/mL, Supplemental Fig. 1B and E).

3.2. BMP4-induced EC apoptosis through ROS-dependent caspase-3activation

After showing the pro-apoptotic effects of BMP4 in ECs fromdifferent species, we studied whether such effects were mediatedby oxidative stress. Noggin (100 ng/mL), apocynin (100 μmol/L), ortiron (1 mmol/L) plus DETCA (100 μmol/L) suppressed the increasedapoptotic rate in RAECs in response to BMP4 (10 ng/mL) (Fig. 3A).BMP4 significantly increased intra-cellular generation of O2

− in RAECsafter 30 min treatment, which was blocked by noggin, apocynin, ortiron plusDETCA (Fig. 3B).Western blot analysis revealed that BMP4 in-duced cleaved caspase-3 in RAECswas also inhibited by treatmentwithnoggin, apocynin, or tiron plus DETCA treatment (Fig. 3C). Similar pro-

kdown by two shRNA inhibited ROS production induced by BMP4 as measured by DHEase-3 activation (C). Data are mean±SEM (n=4). #Pb0.05 compared with control;

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apoptotic effect of BMP4 was also observed in HUVECs (SupplementalFig. 1C and E).

3.3. ROS mediated p38 MAPK and JNK activation in BMP4-induced ECapoptosis

To further elucidate the signaling cascade leading to EC apoptosisactivated by BMP4-induced ROS, we studied whether p38 MAPKand/or JNK were involved. Caspase-3 activation induced by BMP4was inhibited by SB202190 (10 μmol/L, p38 MAPK inhibitor) orSP600125 (10 μmol/L, JNK inhibitor) in RAECs (Fig. 4A). Similar resultswere also found by TUNEL staining in RAECs (Supplemental Fig. 2A)and also in HUVECs (Supplemental Fig. 1D and F). Phosphorylationof p38 MAPK in response to BMP4 for 2 h was prevented by treatmentwith noggin, apocynin, or tiron plus DETCAwithout affecting total p38(Fig. 4B). Likewise, phosphorylation of JNK stimulated by 4-h expo-sure to BMP4 was also abolished by noggin, apocynin, and tiron plusDETCA without affecting total JNK (Fig. 4C).

Activation of p38 and JNK by BMP4 (10 ng/mL, 24 h) was also ob-served in isolated mouse aortae, which were inhibited by co-treatmentwith noggin (100 ng/mL) (Figs. 4E and F). To further elucidate the cross-talk between p38 MAPK and JNK in BMP4-induced EC apoptosis,we found that p38 inhibitor SB202190 (10 μmol/L) inhibited JNKphosphorylation induced by BMP4 (Fig. 4D), while JNK inhibitorSP600125 (10 μmol/L) did not inhibit p38 phosphorylation induced byBMP4 (Supplemental Fig. 2B).

Fig. 6. NOX4 mediates BMP4-induced ROS and caspase-3 activation. (A and B) NOX4inhibition also reduced BMP4-induced ROS production as measured by DHE fluores-cence. NOX4 siRNA inhibited BMP4-induced cleaved p38 phosphorylation (C), JNKphosphorylation (D), and caspase 3 expression (E and F). Data are mean±SEM(n=4). ⁎Pb0.05 compared with control; #Pb0.05 compared with BMP4.

3.4. BMP4-induced EC apoptosis is mediated through BMPR1A

Since BMP4 have several BMP receptors including BMPR1A, BMPR1B,and BMPR2, we studied whether BMPR1A is involved in BMP4-inducedEC apoptosis. BMPR1A knockdown by lentiviral shRNA transfectioninhibited BMP4-induced ROS production as measured by DHE fluores-cence (Fig. 5A). Moreover, BMPR1A knockdown reduced p38 phosphor-ylation induced by BMP4 (Fig. 5B). In addition, BMP4-induced caspase-3activation was also reduced (Fig. 5C). Efficiency of transfection was ver-ified by the reduced expression of BMPR1A by both shRNAs compared toscramble control (Supplemental Fig. 3A).

3.5. NADPH oxidase-derived ROS is required for BMP4-induced apoptosis

Previous studies indicated that NADPH oxidase is the major sourceof BMP4-induced ROS [6,32]. We studied whether NOX4 is involved inBMP4-induced ROS production and apoptosis. NOX4 siRNA reducedthe NOX4 expression in RAECs (Supplemental Fig. 3B). ROS productioninduced by BMP4 also reduced after NOX4 siRNA transfection (Figs. 6Aand B). In addition, NOX4 inhibition reduced phosphorylations of p38(Fig. 6C) and JNK (Fig. 6D) in response to BMP4. BMP4-induced cleavedcaspase-3 expression was also inhibited (Figs. 6E and F).

3.6. Knockdown of JNK inhibits BMP4-induced caspase-3 activation

Based on the results that BMP4 induced JNK phosphorylation andJNK inhibitor inhibited BMP4-induced EC apoptosis, we studied wheth-er knockdown of JNK by siRNA could inhibit BMP4-induced caspase-3

Fig. 7. Knockdown of JNK prevents caspase-3 activation. (A) JNK siRNA suppressed boththe BMP4- and ROS-induced EC apoptosis by flow cytometry. (B) Treatment with JNKsiRNA reduced the level of cleaved caspase-3 in response to BMP4 or ROS in HUVECs.Data are expressed asmean±SEM (n=4). #Pb0.05 comparedwith control (with lipofec-tamine RNAiMAX only). N.S. refers to no statistical difference compared with the controlgroup. ROS was generated using HXXO (100 μmol/L hypoxanthine+0.01 u/mL xanthineoxidase or H2O2 (50 μmol/L)).

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activation. Treatment with 20 nmol/L JNK siRNA for 24 h reduced theJNK expression in HUVECs (Supplemental Fig. 3C). Under the samecondition, JNK siRNA suppressed both the BMP4 (30 ng/mL)- andROS [both H2O2 (100 μmol/L) and hypoxanthine (100 μmol/L) plusxanthine oxidase (0.01 u/mL) (HXXO)]-induced EC apoptosis as deter-mined by flow cytometry (Fig. 7A). Treatment with JNK siRNA reducedthe elevated expression of cleaved caspase-3 in response to BMP4 orROS in HUVECs (Fig. 7B).

4. Discussion

The present study demonstrated BMP4-induced EC apoptosis inhuman, rat, and mouse. We showed that BMP4 activates caspase-3in ECs and this action is mediated through ROS-dependent p38 andJNK activation. Interfering with the BMP4 signaling by knockdownof BMPR1A, NADPH oxidase subtype NOX4, or JNK prevented BMP4-induced EC apoptosis.

Oxidative stress plays an important role in cellular events includingapoptosis in ECs [33,34]. Oscillatory shear stress up-regulates the BMP4production, leading to inflammatory responses such as increasedmonocyte adhesion, through NOX1-based ROS and NF-κB activation[35,36]. BMP4 plays a crucial role in hypertension [37] and atherosclero-sis [38–40]. BMP4 infusion in mice induces hypertension through acti-vation of NADPH oxidase [37]. In ECs, the apoptosis level increaseswith inflammation, and the apoptotic blebs are able to stimulate the at-tachment of monocytes towards ECs, thus exaggerating vascular in-flammation [41–43]. In the present study, ROS scavengers or NOX4siRNA inhibited BMP4-induced EC apoptosis, suggesting that this pro-cess is alsomediated throughNADPH oxidase-derived ROS. This findingis in line with previous studies showing that NADPH oxidase subunits,NOX1, NOX2, and NOX4 contribute to EC apoptosis [44–47]. MAPKsplay an important role in the regulation of different cellular activitiesand there are three major signaling components, e.g., p38 MAPK, JNK/SAPK and ERK. p38 MAPK and JNK/SAPK contribute to the regulationof cell apoptosis upon stress stimuli [48–50]. Depending on stimuli,p38 MAPK and JNK/SAPK also have a role in cell proliferation and sur-vival [50]. On the other hand, ERK is involved in cell proliferation, differ-entiation, and survival. MAPKs are regulated by ROS in ECs to expressproinflammatory phenotype [51–53]. The present study shows thatBMP4-induced EC apoptosis is associated with p38 and JNK activationbased on the following observations: (1) BMP4 increases p38 and JNKphosphorylation; (2) inhibition of p38 and JNK reduce BMP4-inducedapoptosis; and (3) knockout of JNK inhibits BMP4-induced EC apopto-sis. JNK signaling contributes to EC apoptosis triggered by other stimuli,such as oxidized LDL, TNFα, or high glucose [12,54–56]. The present re-sults also suggest that BMP4-induced EC apoptosis is partly mediatedthrough JNK activation.

BMP ligands exert both pro- and anti-apoptotic effects in ECs in dif-ferent conditions. BMP2 and BMP4 stimulate cell proliferation and an-giogenesis in pulmonary artery ECs [57], human microvascular ECs[58], and endothelial precursor cells [28]. However, BMP4 can alsocause apoptosis in human ECs including HUVECs [24,25], suggestingthat the effect of BMP4 may vary depending on EC types and cultureconditions. BMPR1A is involved in apoptosis during development inmouse [59–61]. In addition, Smad1/5 signaling mediates BMP4-induced apoptosis while Smad6/7 protects ECs from apoptosis in dif-ferent ECs [24,61]. In the present study, we found that BMPR1A-mediated the pro-apoptotic effect of BMP4 in ECs is inhibited byknockdown of BMPR1A using shRNA. More importantly, BMP4-induced Smad activation was unaffected by ROS scavengers, p38 orJNK inhibitor, or knockdown of NOX4 (Supplemental Figure S4B).And ROS-generating agents such as H2O2 or HXXO did not increaseSmad phosphorylation (Supplemental Figure S4B), suggesting thatBMP4 and BMPR1A-induced caspase-3 activation in ECs is most likelyto be mediated through oxidative stress rather than the Smad path-way, which differs from the previous reported findings.

In summary, BMP4 causes EC apoptosis in cultured ECs and arteriesfrom human, mouse, and rat. The pro-apoptotic effect of BMP4 is medi-ated through BMPR1A. BMP4-induced caspase-3 activation is mediatedthrough NADPH oxidase-derived ROS and downstream activation ofp38 and JNK. These findings extend our understanding of the positiverole of BMP4 signaling in EC apoptosis and associated vascular dysfunc-tion under pathological situations such as hypertension.

Disclosures

None.

Acknowledgments

This study was supported by Hong Kong General Research Fund(CUHK 465308, 466110, and 465611), National Basic Research Programof China (2012CB517805), and CUHK Focused Investment Scheme.

Appendix A. Supplementary data

Supplementary data to this article can be found online at doi:10.1016/j.yjmcc.2011.10.013.

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