ISSN: 1524-4571 Copyright © 2010 American Heart Association. All rights reserved. Print ISSN: 0009-7330. Online

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DOI: 10.1161/CIRCRESAHA.110.222794 2010;107;984-991; originally published online Aug 19, 2010; Circ. Res.

and Yu Huang TipoeKay Lee, Chi Fai Ng, Aimin Xu, Xiaoqiang Yao, Paul M. Vanhoutte, George L.

Wing Tak Wong, Xiao Yu Tian, Yangchao Chen, Fung Ping Leung, Limei Liu, Hung

Stress�Dependent Cyclooxygenase-2 Upregulation: Implications on HypertensionBone Morphogenic Protein-4 Impairs Endothelial Function Through Oxidative

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Bone Morphogenic Protein-4 Impairs EndothelialFunction Through Oxidative Stress–Dependent

Cyclooxygenase-2 UpregulationImplications on Hypertension

Wing Tak Wong,* Xiao Yu Tian,* Yangchao Chen, Fung Ping Leung, Limei Liu, Hung Kay Lee,Chi Fai Ng, Aimin Xu, Xiaoqiang Yao, Paul M. Vanhoutte, George L. Tipoe, Yu Huang

Rationale: Bone morphogenic protein (BMP)4 can stimulate superoxide production and exert proinflammatoryeffects on the endothelium. The underlying mechanisms of how BMP4 mediates endothelial dysfunction andhypertension remain elusive.

Objective: To elucidate the cellular pathways by which BMP4-induced endothelial dysfunction is mediatedthrough oxidative stress–dependent upregulation of cyclooxygenase (COX)-2.

Methods and Results: Impaired endothelium-dependent relaxations, exaggerated endothelium-dependent con-tractions, and reactive oxygen species (ROS) production were observed in BMP4-treated mouse aortae, whichwere prevented by the BMP4 antagonist noggin. Pharmacological inhibition with thromboxane prostanoidreceptor antagonist or COX-2 but not COX-1 inhibitor prevented BMP4-induced endothelial dysfunction, whichwas further confirmed with the use of COX-1�/� or COX-2�/� mice. Noggin and knockdown of BMP receptor1A abolished endothelium-dependent contractions and COX-2 upregulation in BMP4-treated aortae. Apocyninand tempol treatment were effective in restoring endothelium-dependent relaxations, preventing endothelium-dependent contractions and eliminating ROS overproduction and COX-2 overexpression in BMP4-treatedaortae. BMP4 increased p38 mitogen-activated protein kinase (MAPK) activity through a ROS-sensitivemechanism and p38 MAPK inhibitor prevented BMP4-induced endothelial dysfunction. COX-2 inhibitionblocked the effect of BMP4 without affecting BMP4-induced ROS overproduction and COX-2 upregulation.Importantly, renal arteries from hypertensive rats and humans showed higher levels of COX-2 and BMP4accompanied by endothelial dysfunction.

Conclusions: We show for the first time that ROS serve as a pathological link between BMP4 stimulation and thedownstream COX-2 upregulation in endothelial cells, leading to endothelial dysfunction through ROS-dependentp38 MAPK activation. This BMP4/ROS/COX-2 cascade is important in the maintenance of endothelialdysfunction in hypertension. (Circ Res. 2010;107:984-991.)

Key Words: bone morphogenic protein 4 � cyclooxygenase-2 � reactive oxygen species � endothelial dysfunction� endothelium-dependent contractions

Bone morphogenic protein (BMP)4 belongs to the BMPfamily, 6 of which (BMP2 to BMP7) fall into the

transforming growth factor-� superfamily. BMP4 was origi-nally discovered to participate in embryonic development andbone and cartilage formation.1–3 BMP4 is upregulated incalcified atherosclerotic plaques4,5 and reduces endothelium-dependent relaxations (EDRs).6

BMP4 is a mechanosensitive and proinflammatory gene;disturbed flow increases BMP4 production in cultured endo-thelial cells.7 BMP4 promotes the expression of intracellularadhesion molecules and monocyte adhesion in a reactiveoxygen species (ROS)-dependent manner.7–9 Overexpressionof BMP4 in endothelial cells enhances pulmonary vascularremodeling in pulmonary hypertension.10 The infusion of

Original received April 23, 2010; revision received August 5, 2010; accepted August 6, 2010. In July 2010, the average time from submission to firstdecision for all original research papers submitted to Circulation Research was 12.9 days.

From the Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, School of Biomedical Sciences (W.T.W., X.Y.T., Y.C., F.P.L., L.L.,X.Y., Y.H.); and Departments of Chemistry (H.K.L.) and Surgery (C.F.N.), Chinese University of Hong Kong; and Departments of Medicine (A.X.),Pharmacology (A.X., P.M.V.), and Anatomy (G.L.T.), University of Hong Kong, China.

*These authors contributed equally to this work.Correspondence to Yu Huang or Wing Tak Wong, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, NT, Hong Kong, China.

E-mail yu-huang@cuhk.edu.hk or jack_wong@cuhk.edu.hk© 2010 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.222794

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BMP4 induces hypertension in mice partly through stimulat-ing the expression and activity of vascular NADPH oxidasesand the subsequent overproduction of ROS, which disturbsendothelial function.11

However, the exact underlying mechanisms of BMP4-induced oxidative stress and endothelial dysfunction remainelusive. COX-2 could be involved because ROS triggers therelease of prostanoids via the action of COX-2.12 COX-2 isupregulated in atherosclerotic lesions13 and catalyzes theproduction of the majority of vascular prostanoids in humanatherosclerotic areas.14 COX-2 inhibition improves endothe-lial function in patients with hypertension and coronary heartdisease.15,16 COX-2 can be also expressed constitutively in ratand human vascular endothelial cells,17,18 and its expressionis upregulated with aging in hamster aortae,19 suggesting thatCOX-2 plays an important role in both the physiological andpathological regulation of vascular function, depending onthe level of its expression and activity.

The present study hypothesizes that upregulated COX-2 inendothelial cells plays a critical role in BMP4-inducedendothelial dysfunction. Mouse, rat, and human arteries wereused to investigate whether or not BMP4 impairs EDRs andfacilitates endothelium-dependent contractions (EDCs) andwhether oxidative stress serves as a link between BMP4stimulation and downstream COX-2 upregulation. Finally,the possible clinical relevance of this BMP4/COX-2 pathwayin hypertension was examined.

MethodsAn expanded Methods section is available in the Online DataSupplement at http://circres.ahajournals.org.

AnimalsC57BL/6J mice, spontaneously hypertensive rats (SHR), and Wistar–Kyoto (WKY) rats were supplied by Chinese University of HongKong Laboratory Animal Center, whereas COX-1�/� or COX-2�/�

mice were supplied by University of Hong Kong. All of theexperiments were conducted under our institutional guidelines forthe humane treatment of laboratory animals.

Human Renal Arteries SpecimensThe present study was approved by the Joint Chinese University ofHong Kong–New Territories East Cluster Clinical Research EthicsCommittee. Human renal arteries were harvested from nephrectomyspecimens from normotensive and hypertensive patients after obtain-ing informed consent. The mean age of patients was 62.5 years(range, 44 to 70 years). The indications for surgery included tumor(5 patients) and poorly functioning kidney (1 patient) in each group.History of hypertension was defined as having persistent elevatedblood pressure, systolic pressure of �140 mm Hg, or diastolicpressure of �90 mm Hg and requiring medical therapy.

Blood Vessel PreparationAdult male mice and rats were euthanized by CO2 suffocation, andmouse aortae or rat intralobar renal arteries were removed and placedin ice-cold Krebs solution (mmol/L): 119 NaCl, 4.7 KCl, 2.5 CaCl2,1 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 11 D-glucose. Arteries werecleaned of adhering adipose tissue and cut into ring segments of2 mm in length. As described previously,20,21 mouse aortic ringswere incubated for 12 hours in DMEM (Gibco, Grand Island, NY)culture media with 10% FBS (Gibco), 100 IU of penicillin, and 100�g/mL streptomycin and were placed in a CO2 incubator with 95%O2 plus 5% CO2 with and without BMP4. After 12 hours ofincubation, rings were suspended in a myograph (Danish Myo

Technology, Aarhus, Denmark) for recording of changes in isometrictension. Briefly, 2 steel wires (40 �m in diameter) were insertedthrough the lumen of the vessel, and each wire was fixed to thejaws built in the myograph. The organ chamber was filled with 5mL of Krebs solution and gassed by 95% O2/5% CO2 at 37°C(pH�7.4). Each ring was stretched to 3 mN, an optimal tension,and then allowed to stabilize for 90 minutes before the start ofeach experiment.

Functional StudiesSome arterial rings were exposed to BMP4 in control solution or inthe presence of one of the following inhibitors: noggin (BMP4antagonist, 100 ng/mL), apocynin (NADPH oxidase inhibitor,100 �mol/L), tempol (SOD mimetic, 100 �mol/L), or SB202190(p38 mitogen-activated protein kinase [MAPK] inhibitor; 10 �mol/L). After incubation with BMP4 for 12 hours, rings were suspendedin a myograph and subjected to 30-minute exposure to S18886(thromboxane prostanoid receptor antagonist; 100 nmol/L), cele-coxib (3 �mol/L), or sc-560 (COX-1 inhibitor, 0.3 �mol/L). Theconcentration of these individual inhibitors is known to be specificagainst the respective target.19,22 The first series of experimentsexamined the alterations in EDRs. Rings were contracted withphenylephrine (1 �mol/L) to establish a stable tension and acetyl-choline (ACh) was then added cumulatively (1 nmol/L to 10 �mol/L). ACh-induced relaxations were abolished by 100 �mol/L NG-nitro-L-arginine methyl ester (L-NAME) (NOS inhibitor), or byendothelium removal. The second set of experiments examinedEDCs. Aortic rings were first treated for 30 minutes with100 �mol/L L-NAME to eliminate the interference of endothelium-derived nitric oxide (NO), a procedure commonly adopted to unmaskACh-induced EDCs,19,23 and then contractions were elicited by ACh(0.1 to 30 �mol/L). EDC was expressed as active tension by dividingthe peak contraction (mN) by 2� vessel length in millimeters.Intralobar renal arteries were dissected from SHR and WKY rats andsubjected to 12-hour organ culture in DMEM in control and in thepresence of 100 ng/mL noggin or 3 �mol/L celecoxib. Both EDRsand EDCs were studied and compared in different treatment groups.Human arteries were treated using the same protocol as for rat renalarteries.

ROS Detection by Electron ParamagneticResonance Spin TrappingTo measure ROS released from arterial tissues, electron paramag-netic resonance (EPR) was performed with 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine hydrochloride (TEMPONE-H, Alexis)and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, Alexis) as spin trapagents. All EPR samples were placed in 100-�L glass tubes and

Non-standard Abbreviations and Acronyms

ACh acetylcholine

BMP bone morphogenic protein

BMPR bone morphogenic protein receptor

COX cyclooxygenase

EDC endothelium-dependent contraction

EDR endothelium-dependent relaxation

EPR electron paramagnetic resonance

L-NAME NG-nitro-L-arginine methyl ester

MAPK mitogen-activated protein kinase

ROS reactive oxygen species

SHR spontaneously hypertensive rat

shRNA short hairpin RNA

WKY Wistar–Kyoto rat

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suspended in Krebs solution. To inhibit reactions catalyzed bytransition metals, DTPA (0.2 mmol/L) was added. X-band EPRspectra were measured at room temperature using an EMX EPRspectrometer (Bruker). The EPR settings were as follows: fieldcenter, 3480 G; field sweep, 100 G, microwave frequency, 9.746GHz; microwave power, 10 mW; modulation frequency, 100 kHz;modulation amplitude, 0.3 G; conversion time, 1024 ms; timeconstant, 640 ms.

Constructs, Lentivirus Production,and TransductionWe have designed 2 short hairpin (sh)RNAs targeting mouse BMPreceptor 1a: shRNA1 (5�-GCT GTT AAA TTC AAC AGT GACACA AAT G-3�) and shRNA2 (5�-TCT CTC TAT GAC TTC CTGAAA TGT GCC A-3�); and 1 shRNA targeting firefly luciferase asa control: 5�-TGC GCT GCT GGT GCC AAC CCT ATT CT-3�.DNA fragments containing shRNAs sequence were synthesized andcloned into lentiviral RNA interference vector pLUNIG after anneal-ing as described previously.24,25

The VSV-G–pseudotyped lentiviruses were produced by cotrans-fecting 293T cells with the transfer vector and 3 packaging vectors(pMDLg/pRRE, pRSV-REV, and pCMV-VSVG) as described pre-viously. Subsequent purification was performed using ultracentrifu-gation. Mouse blood vessels were cultured in 24-well plates andwere transduced with lentivirus and 8 �g/mL polybrene (Sigma).

Statistical AnalysisResults represent means�SEM from different animals or humans.Concentration–response curves were analyzed by nonlinear regres-sion curve fitting using GraphPad Prism software (Version 4.0, SanDiego, Calif). The protein expression was quantified by densitometer(FluorChem, Alpha Innotech, San Leandro, Calif) and normalized toGAPDH and then compared with control. Statistical significance wasdetermined by 2-tailed Student’s t test or 1-way ANOVA followedby the Bonferroni post hoc test when more than 2 treatments werecompared. P�0.05 indicates statistically significant difference.

ResultsBMP4 Impairs EDRsACh-induced EDRs in mouse aortae were impaired in aconcentration-dependent manner by 12 hours of exposure toBMP4 at 10, 20, and 80 ng/mL (Figure 1 A). BMP4 exposurealso reduced EDRs in a time-dependent manner at 12, 18, and24 hours (Figure 1B). The BMP4-treated aortae contracted inresponse to concentrations of ACh of �1 �mol/L, as shownin the representative traces (Figure 1C). BMP4 antagonistnoggin at 100 ng/mL prevented BMP4-induced reduction ofEDRs (Figure 1D). By contrast, endothelium-independentrelaxations to sodium nitroprusside were unaffected byBMP4 (Figure 1E).

BMP4-Induced Endothelial Dysfunction IsMediated Through COX-2Thirty-minute treatment with COX-2 inhibitor celecoxib(3 �mol/L) or thromboxane prostanoid receptor antagonistS18886 (0.1 �mol/L) prevented the impaired EDRs inBMP4-treated aortae, whereas COX-1 inhibitor sc-560(0.3 �mol/L) had no effect (Figure 2A). BMP4 failed toimpair EDRs of aortae from COX-2�/� mice as comparedwith those from wild-type or COX-1�/� mice (Figure 2B and2C). Celecoxib or S18886 also abolished BMP4-inducedACh-mediated EDCs in the presence of nitric oxide synthaseinhibitor L-NAME (100 �mol/L), whereas sc-560 had lesseffect (Figure 2D). Similarly, the ability of BMP4 to enhance

EDC response was also abolished in aortae from COX-2�/�

mice, as compared with those from wild-type or COX-1�/�

mice (Figure 2E and 2F).

BMP4-Induced COX-2 Upregulation Is MediatedThrough BMP Receptor 1ABMP4 antagonist noggin prevented EDCs (Figure 3A) andCOX-2 upregulation (Figure 3B) in BMP4-treated aortae.Knocking down BMP receptor (BMPR)1A by shRNA alsoabolished EDCs (Figure 3C). Knockdown of BMPR1A re-duced BMPR1a expression and COX-2 upregulation inBMP4-treated mouse aortae (Figure 3D). BMP4-inducedCOX-2 upregulation in mouse aortae was mainly confined toendothelial cells as the removal of endothelium significantlyreduced the COX-2 expression showed by Western blotting(Figure 3E). Immunostaining also showed a similar increaseof COX-2 expression in the endothelium triggered by BMP4in mouse aortae (Online Figure I).

BMP4 Upregulates COX-2 Through ROSand MAPKCotreatment of NADPH oxidases inhibitor apocynin(100 �mol/L) or ROS scavenger tempol (100 �mol/L)significantly improved EDRs in BMP4-treated aortae (Figure4A). Cotreatment with specific p38 MAPK inhibitor

Figure 1. BMP4 impairs EDRs in mouse aortae. A,Concentration-dependent effect of BMP4 (10, 20, 80 ng/mL;12-hour incubation) on ACh-induced EDRs in C57BL/6J mouseaortae precontracted by phenylephrine (Phe). B, Time-dependent effect of BMP4 (12, 18, and 24 hours) on EDRs. C,Representative traces showing BMP4-reduced EDRs and theappearance of contraction caused by ACh at concentration of�1 �mol/L. D, Effect of noggin (100 ng/mL, 12 hours) on EDRsimpaired by BMP4 (20 ng/mL). E, Endothelium-independentrelaxations induced by sodium nitroprusside (SNP) in controland BMP4-treated mouse aortae. All experiments were per-formed on C57BL/6J mouse aortae. Data are means�SEM of 6to 8 mice. *P�0.05 vs control; #P�0.05 vs BMP4.

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SB202190 at 10 �mol/L also improved the reduced EDRs(Figure 4B). Likewise, apocynin, tempol, or SB202190 pre-vented EDCs in BMP4-treated aortae (Figure 4C and 4D).Western blotting demonstrated that COX-2 upregulation byBMP4 was reduced by apocynin, tempol, or SB202190, butnot by extracellular signal-regulated kinase inhibitorPD98059 (10 �mol/L) or c-Jun N-terminal kinase inhibitorSP600125 (10 �mol/L) (Figure 4E and 4F). In primaryculture of murine aortic endothelial cells, BMP4-inducedCOX-2 upregulation and p38 MAPK phosphorylation wasabolished by noggin, tempol, apocynin, or SB202190,whereas the COX-1 expression remained unaffected (Figure5A and 5B).

BMP4 Increases ROS ProductionBMP4 significantly elevated ROS production in ACh-stimulated (10 �mol/L) aortae, as determined by EPR spec-troscopy (Figure 6A and 6B). Noggin, apocynin, tempol, orremoval of endothelium significantly inhibited the ROSproduction in BMP4-treated mouse aortae, whereasSB202190, sc-560, or celecoxib had no effect. In aortae fromCOX-1�/� or COX-2�/� mice, BMP4 induced a similarincrease of ROS production as compared with aortae fromwild-type mice (Figure 6C).

Association Between BMP4 and COX-2 inHypertensive Rats and Human SubjectsNoggin or celecoxib reversed the impaired EDRs in SHRrenal arteries as compared with those from WKY (Figure 7A

and 7B). Noggin or celecoxib abolished the enhanced EDCsin SHR renal arteries as compared with WKY rat arteries(Figure 7C and 7D). Noggin also prevented the increasedexpression of both BMP4 and COX-2 and inhibited theincreased phosphorylation of p38 MAPK in SHR renalarteries (Figure 7E through 7G). In addition, SB202190inhibited the increased COX-2 expression and p38 phosphor-ylation in SHR renal arteries (Figure 7H and 7I).

In renal arteries from hypertensive patients, noggin orcelecoxib significantly enhanced ACh-induced EDRs (Figure8A and 8B). The expression levels of both BMP4 and COX-2were higher in arteries from hypertensive patients than inthose from normotensive subjects (Figure 8C). Noggin treat-ment reduced BMP4 and COX-2 expression in renal arteriesfrom hypertensive patients (Figure 8D). In renal arteries fromnormotensive patients, BMP4 reduced the ACh-inducedEDRs, and caused COX-2 upregulation, which was preventedby noggin (Figure 8E and 8F).

DiscussionThe present study demonstrates that upregulated expressionof COX-2 plays an essential role in BMP4-induced endothe-

Figure 2. BMP4-induced endothelial dysfunction is mediatedthrough COX-2. Effects of 30-minute treatment with 3 �mol/Lcelecoxib, 0.3 �mol/L sc-560, or 0.1 �mol/L S18886 on theimpaired EDRs (A) and augmented EDCs in the presence ofL-NAME (D) in mouse aortae treated with BMP4 (20 ng/mL, 12hours). B, Effect of BMP4 on EDRs in aortae from COX-2�/�

mice compared with those from wild type or COX-1�/� (C). E,Effect of BMP4 on EDCs in aortae from COX-2�/� mice com-pared with those from wild type or COX-1�/� (F). Data aremeans�SEM of 5 to 7 mice. *P�0.05 vs control; #P�0.05vs BMP4.

Figure 3. BMP4-induced COX-2 upregulation is mediatedthrough BMPR1A. A, Noggin prevented BMP4-induced EDCsin mouse aortae. B, Noggin reduced BMP4-induced COX-2(72 kDa) upregulation in mouse aortae. *P�0.05 vs control;#P�0.05 vs BMP4. C, Effect of BMPR1A shRNA on BMP4-induced EDCs compared with scramble control. D, Effect ofBMPR1A shRNA on BMPR1a expression and BMP4-inducedCOX-2 upregulation. *P�0.05 vs scramble control. E, Effect ofendothelium removal on the COX-2 expression in control andBMP4-treated mouse aortae. �endo indicates endotheliumremoval. *P�0.05 vs �endo control; #P�0.05 vs �endo control.Data are means�SEM of 6 mice. Protein expressions were nor-malized to GAPDH and compared to control.

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lial dysfunction. We demonstrate for the first time that theimpaired EDRs and exaggerated EDCs in the BMP4-treatedmouse aortae can be abolished by a BMP4 antagonist, COX-2inhibitor, and ROS scavengers and are absent in COX-2�/�

mice. We also show that BMP4 upregulates COX-2 expres-sion through a BMP4/BMPR1A/ROS/p38 MAPK signalingpathway. Furthermore, the present study provides novelevidence for a significant contribution of BMP4 and COX-2to endothelial dysfunction in spontaneously hypertensive ratsand its relevance to human hypertension. Collectively, thepresent findings clearly support that BMP4 is an upstreamactivator which triggers overexpression of COX-2 in endo-thelial cells as an important downstream target enzymeresponsible for the initiation and maintenance of endothelialdysfunction. A possible pathophysiological significance ofBMP4 in hypertension is thus revealed.

Earlier works by others demonstrated the involvement ofBMP4 in atherosclerosis and hypertension and in mediatinginflammatory responses of endothelial cells induced by shearstress via a ROS-dependent mechanism involving Nox1-based NADPH oxidase.7,8 In mouse, the infusion of BMP4caused hypertension and impaired ACh-induced aortic relax-ations through stimulation of NADPH oxidase,11 suggestingthat BMP4 could serve as a potential novel predictor ofvascular dysfunction. Indeed, the present results show thatBMP4 directly impaired EDRs in blood vessels from 3different species. The data are in line with BMP4-induced

endothelial dysfunction in rat carotid arteries.6 The harmfuleffect of BMP4 on endothelial cells is confirmed by the useof noggin. Like other members of the transforming growthfactor-� superfamily, BMPs exert their cellular actions viamembrane receptor complex (BMPR1a, -1b, and -2).26,27

Existing evidence suggests that BMPR1a plays an importantrole in the formation of blood vessels and of the atrioventric-ular valves.28,29 The present study used shRNA to knockdown BMPR1a in the mouse aortae and shows that thedetrimental vascular action of BMP4 is mediated through theBMP receptor.

The present study also demonstrates that BMP4 facilitatesEDCs, which is a pathophysiological response seen in hyper-tension. Similar to the previous demonstration of the pivotalrole of COX-2 in the appearance of EDCs in hamster aortae,19

BMP4-induced EDC is abolished by COX-2 inhibitor andthromboxane prostanoid receptor antagonist, suggesting thatCOX-2–dependent arachidonic acid metabolites act as endo-thelium-derived contracting factors in BMP4-treated arteries.By contrast, COX-1 mediated EDCs in the aortae of agedmice and spontaneously hypertensive rats,22,30 suggesting asignificant difference in the role of COX isoforms, dependingon the pathological initiators and species. If the vasculareffect of BMP4 depends on the activation of COX-2, then thisenzyme could be the target responding to BMP4-inducedoxidative stress. The critical role of COX-2 in BMP4-induced

Figure 4. BMP4 upregulates COX-2 through ROS and MAPKin mouse aortae. A and C, Effects of apocynin (100 �mol/L, 12hours) and tempol (100 �mol/L, 12 hours) on impaired EDRs (A)and augmented EDCs (C) induced by BMP4 (20 ng/mL, 12hours). B and D, Effect of SB202190 (10 �mol/L, 12 hours) onimpaired EDRs (B) and augmented EDCs (D) induced by BMP4(20 ng/mL, 12 hours). E, Effects of apocynin and tempol onBMP4-induced COX-2 upregulation. F, Effects of SB202190,PD98059, and SP600125 (10 �mol/L each, 12 hours) on BMP4-induced COX-2 upregulation. Data are means�SEM of 4 to 6mice. *P�0.05 vs control; #P�0.05 vs BMP4.

Figure 5. BMP4 increases p38 MAPK phosphorylation andCOX-2 expression in murine aortic endothelial cells. A andB, Effects of noggin, tempol, apocynin, and SB202190 onBMP4-induced (20 ng/mL) p38 MAPK phosphorylation (A) andCOX-2 upregulation (B) in primary cultured murine aortic endo-thelial cells. B, BMP4 had no effect on COX-1 expression. Pro-tein expressions were normalized to GAPDH and compared tocontrol. Data are means�SEM of 4 to 6 mice. *P�0.05 vs con-trol; #P�0.05 vs BMP4.

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endothelial dysfunction is supported by the observations thattreatment with celecoxib prevented the BMP4-induced endo-thelial dysfunction. More importantly, BMP4 lost its abilityto impair endothelial function in COX-2�/� mice. ROSproduced by Nox1-based NADPH oxidase is the majordownstream target of BMP4 to mediate inflammatory vascu-lar responses.8,31 In the present study, BMP4 induces ROSproduction in cultured endothelial cells and in isolated ratarteries, which is in line with previous reports.8,31 Moreover,the use of EPR spectroscopy spin trap to assess the ROSproduction in mouse aortae in situ permitted the confirmationof the endothelial origin of ROS production induced byBMP4 both in the presence and absence of ACh stimulation.The ROS production was not reduced by a selective COX-2inhibitor and was present in the aortae of COX-2�/� mice.ROS removal by scavengers annulled BMP4-induced COX-2overexpression. Taken together, these findings imply thatROS induced by BMP4 is responsible for BMP4-mediatedupregulation of COX-2, which is constitutively expressed inmouse endothelial cells. By contrast, a specific COX-1inhibitor did not influence the vascular effect of BMP4.Finally, BMP4 did not alter the expression of COX-1 inmouse aortae and cultured mouse aortic endothelial cells.Collectively, our study provides evidence showing thatCOX-2 is most likely to serve as the link between BMP4 andendothelial dysfunction.

The present study indicates that p38 MAPK activationparticipates in the regulation of COX-2 expression because aMAPK inhibitor attenuated COX-2 upregulation and im-proved endothelial function in BMP4-treated mouse aortae.ROS enhances the activity of p38 MAPK in vascular tis-

sues.32 BMP4 can activate MAPK (p38 and p44/42) inendothelial cells and myocytes.31,33 The present findings thussuggest that MAPK activation is functionally coupled toCOX-2 upregulation, COX-2–dependent EDCs, and im-paired EDRs in response to BMP4. In addition, ROS scav-engers prevented the BMP4-induced COX-2 upregulationand phosphorylation of p38 MAPK in cultured mouse endo-thelial cells, thus confirming that endothelial cells are theprimary site of action for BMP4 and also supporting anessential role of ROS-dependent p38 MAPK activation in theharmful action of BMP4.

To further elucidate the pathophysiological significance ofBMP4 and COX-2 in endothelial dysfunction, the presentstudy examined the vascular protective effect of the BMP4antagonist noggin or the COX-2 inhibitor celecoxib in spon-taneously hypertensive rats. Treatment of SHR renal arterieswith either noggin or celecoxib normalized relaxations to thelevel in WKY rat arteries and abolished EDCs. Although theinfusion of BMP4 causes hypertension in healthy mice,11 itwas unclear whether or not BMP4 is a biomarker or is

Figure 6. BMP4 increases ROS production. A, RepresentativeEPR spectra showing that the markedly increased amplitude ofROS signal in BMP4-treated mouse aortae in response to10 �mol/L ACh for 5 minutes. B, Summarized data of ROS pro-duction measured by EPR spectroscopy in the presence of nog-gin, apocynin, tempol, SB202190, and sc-560 in BMP4-treatedmouse aortae. Tempone only indicates tempone without mouseaorta; �endo, endothelium removal. C, BMP4-induced ROSproduction in response to ACh in aortae from wild-type (WT),COX-1�/�, and COX-2�/� mice. Data are means�SEM of 4 to 6mice. *P�0.05 vs control; #P�0.05 vs BMP4.

Figure 7. Roles of BMP4 and COX-2 in endothelial dysfunc-tion in spontaneously hypertensive rats. A and C, Treatment(12 hours) with noggin (Nog) improved ACh-induced EDRs (A)and abolished EDCs (C) in intralobar renal arteries of SHR butnot of WKY rats. B and D, Effects of celecoxib incubation onEDRs (B) and EDCs (D) in SHR renal arteries E through G,Effects of noggin on BMP4 expression (E), COX-2 expression(F), and p38 phosphorylation (G) in SHR renal arteries. H andI, Effects of SB202190 (SB) on p38 phosphorylation (H) andCOX-2 expression (I) in SHR renal arteries. Data aremeans�SEM of 6 to 8 rats. *P�0.05 vs WKY; #P�0.05 vs SHR.

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actually linked to essential hypertension. The present studyshows a marked increase of BMP4 and COX-2 in hyperten-sive animals, and both were attenuated by noggin, thussuggesting an association between BMP4 and COX-2 inhypertension. In addition, ROS scavengers also improvedEDRs and attenuated EDCs in renal arteries from SHRs. ROSscavengers also reduced COX-2 expression and p38 MAPKactivation without affecting BMP4 expression in renal arter-ies from SHRs, further confirming the positive role of ROS inmediating BMP4-induced COX-2 upregulation in hyperten-sion (Online Figure II). Our data also reveal such associationin renal arteries from hypertensive patients. However, thepresent study cannot rule out partial involvement of BMP2 inendothelial dysfunction in hypertension because BMP2 isupregulated in a rat model of hypertension and BMP4antagonist can also neutralize BMP2.33

In conclusion, the present study demonstrates a critical roleof endothelial COX-2 in BMP4-induced endothelial dysfunc-tion. BMP4-induced oxidative stress upregulates COX-2 inendothelial cells. In addition, the BMP4 antagonist nogginprotects endothelial function in hypertension. Because both

BMP4 and COX-2 are expressed in hypertensive human renalarteries, the present data may be relevant to cardiovasculardisease. Indeed, the present findings in mouse, rat, and humanarteries supports an important role of BMP4-dependentCOX-2 in endothelial dysfunction in hypertension. Our re-sults also suggest that the BMP4 signaling cascade could bea potential target for pharmacological intervention to preventCOX-2–dependent vascular dysfunction.

Sources of FundingThis work was supported by the Hong Kong Research Grant Council(CUHK465308, CUHK466110, and HKU 2/07C) and CUHK Fo-cused Investment Scheme.

DisclosuresNone.

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Figure 8. Roles of BMP4 and COX-2 in endothelial dysfunc-tion in hypertensive patients. A and B, Effects of noggin (A) orcelecoxib (B) on ACh-induced relaxations in renal arteries fromhypertensive patients. C, Increased expressions of BMP4 andCOX-2 in renal arteries from hypertensive patients comparedwith normotensive subjects. D, Effect of noggin on BMP4 andCOX-2 expressions in renal arteries from hypertensive patients.E, Effect of BMP4 on ACh-induced relaxations in renal arteriesfrom normotensive subjects. F, Effect of noggin on BMP4-induced COX-2 expression in renal arteries from normotensivesubjects. Data are means�SEM of 3 to 5 subjects. *P�0.05 vscontrol; #P�0.05 vs BMP4. HT indicates hypertension.

990 Circulation Research October 15, 2010

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21. Wang Y, Huang Y, Lam KS, Li Y, Wong WT, Ye H, Lau CW, VanhouttePM, Xu A. Berberine prevents hyperglycemia-induced endothelial injuryand enhances vasodilatation via adenosine monophosphate-activatedprotein kinase and endothelial nitric oxide synthase. Cardiovasc Res.2009;82:484–492.

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Novelty and Significance

What Is Known?

● Oxidative stress contributes to the vascular complications ofhypertension.

● Bone morphogenic protein (BMP)4 activates NADPH oxidase and isassociated with vascular inflammation.

● Infusion of BMP4 in mice induces hypertension and causes endothe-lial dysfunction

What New Information Does This Article Contribute?

● BMP4 impairs endothelium-dependent relaxations and induces en-dothelium-dependent contractions in the mouse aorta by activat-ing BMP receptor 1A.

● BMP4-induced endothelial dysfunction is mediated by oxidativestress–dependent upregulation of cyclooxygenase (COX)-2.

● The expression of COX-2 and BMP4 were increased in renal arteriesfrom hypertensive patients compared with those from normoten-sives subjects.

BMP4 is a proinflammatory gene induced by disturbed flow inendothelial cells. The limited data available indicate that BMP4reduces endothelium-dependent relaxations and induces hyper-

tension by activating NADPH oxidase. However, it is unknownwhether or not COX-2, another mediator of vascular inflamma-tion, contributes to BMP4-induced endothelial dysfunction inhypertension. The present study demonstrates that BMP4 ex-aggerates endothelium-dependent contractions in mouse aorta,whereas silencing of BMP receptor 1A prevents the harmfuleffects of BMP4. In mouse arteries and endothelial cells, BMP4increases the production of reactive oxygen species, which inturn activate p38 MAPK and contribute to the BMP4-inducedCOX-2 upregulation and the altered vascular reactivity. BMP4-induced endothelial dysfunction is absent in COX-2–deficientmice, supporting a close association between the 2 proinflam-matory factors. Finally, we confirm the role of BMP4 and COX-2in endothelial dysfunction in hypertensive rats and humans. TheBMP4 antagonist noggin improves endothelial function andreduces BMP4 and COX-2 expression in renal arteries fromhypertensive rats and patients. Thus, BMP4 and its downstreamoxidative stress–dependent upregulation of the expression andactivity of COX-2 are important in the development of hyperten-sion. These findings suggest that strategies specifically targetingthis signaling cascade could be potentially useful in treatingvascular dysfunction related to human hypertension.

Wong et al BMP4, COX-2, and Endothelial Dysfunction 991

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Bone Morphogenic Protein-4 Impairs Endothelial Function Through Oxidative Stress-dependent Cyclooxygenase-2 Upregulation: Implications on Hypertension

Wing Tak Wong, PhD; Xiao Yu Tian, MPhil; Yangchao Chen, PhD; Fung Ping Leung, PhD; Limei Liu, MPhil; Hung Kay Lee, PhD; Chi Fai Ng, MD; Aimin Xu, PhD; Xiaoqiang Yao, PhD; Paul M Vanhoutte, MD, PhD; George L Tipoe, PhD; and Yu Huang, PhD

From Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, School of Biomedical Sciences (W.T.W., X.Y.T., F.P.L., Y.C., L.L., X.Y., Y.H.); Departments of Chemistry (H.K.L.) and Surgery (C.F.N.), Chinese University of Hong Kong, China; Departments of Medicine (A.X.), Pharmacology (A.X., P.M.V.) and Anatomy (G.L.T.), University of Hong Kong, Hong Kong, China First author’s surname: Wong Short title: BMP4, COX-2 and endothelial dysfunction Complete address of corresponding author: Yu Huang, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, NT, Hong Kong, China. Tel: (852) 26096787; Fax: (852) 26035022; e-mail: yu-huang@cuhk.edu.hk; and Wing Tak Wong, email: jack_wong@cuhk.edu.hk Journal subject code: [95] Endothelium/vascular type/nitric oxide; [130] Animal models of human disease; [149] Hypertension - basic studies

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SUPPLEMENTAL MATERIAL DETAILED METHODS Animals C57BL/6J mice, spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were supplied by CUHK Laboratory Animal Center, while COX-1-/- or COX-2-/- mice were supplied by University of Hong Kong. All of the experiments were conducted under our institutional guidelines for the humane treatment of laboratory animals. Patients The present study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. Human renal arteries were harvested from nephrectomy specimens from normotensive and hypertensive patients, after obtaining their informed consent. The mean age of patients was 62.5 (range 44-70) years old. The indications of surgery include tumor (five patients) and poorly-functioning kidney (one patient) in each group. History of hypertension was defined as having persistent elevated blood pressure, systolic pressure over 140 mmHg or diastolic pressure over 90 mmHg, and requiring medical therapy. Blood vessel preparation Adult male mice and rats were sacrificed by CO2 suffocation and mouse aortae or rat intralobal renal arteries were removed and placed in ice-cold Krebs solution (mmol/L): 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 11 D-glucose. Arteries were cleaned of adhering adipose tissue and cut into ring segments of 2 mm in length. As described 1, 2, mouse aortic rings were incubated for 12 hours in Dulbeco’s Modified Eagle’s Media (DMEM, Gibco, Grand Island, NY, USA) culture media with 10% fetal bovine serum (FBS, Gibco), 100 IU penicillin and 100 μg/ml streptomycin, and placed in a CO2 incubator with 95% O2 plus 5% CO2 with and without BMP4. After 12-h incubation, rings were suspended in a myograph (Danish Myo Technology, Aarhus, Denmark) for recording of changes in isometric tension. Briefly, two steel wires (40-µm in diameter) were inserted through vessel's lumen, and each wire was fixed to the jaws built in the myograph. The organ chamber was filled with 5 mL Krebs solution and gassed by 95% O2-5% CO2 at 37°C (pH ~7.4). Each ring was stretched to 3 mN, an optimal tension, and then allowed to stabilize for 90 min before the start of each experiment. Functional studies Some arterial rings were exposed to BMP4 in control solution or in the presence of one of the following inhibitors: noggin (BMP4 antagonist, 100 ng/mL), apocynin (NADPH oxidase inhibitor, 100 μmol/L), tempol (SOD mimetic, 100 μmol/L), or SB202190 (p38 MAPK inhibitor, 10 μmol/L). After incubation with BMP4 for 12 hours, rings were suspended in a myograph and subjected to 30-min exposure to S18886 (TP receptor antagonist, 100 nmol/L), celecoxib (3 μmol/L) or sc-560 (COX-1 inhibitor, 0.3 μmol/L). The concentration of these individual inhibitors is known to be specific against the respective target 3. The first series of experiments examined the alterations in endothelium-dependent relaxations (EDR). Rings were contracted with phenylephrine (1 μmol/L) to establish a stable tension and acetylcholine (ACh) was then added cumulatively (1 nmol/L–10 μmol/L). ACh-induced relaxations were abolished by 100 μmol/L NG-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), or by endothelium-removal. The second set of experiments examined endothelium-dependent contractions (EDC). Aortic rings were first treated for 30 min with 100 μmol/L L-NAME to eliminate the interference of endothelium-derived nitric oxide (NO), a procedure commonly adopted to unmask ACh-induced EDC 4, and then contractions were elicited by ACh (0.1-30 μmol/L). EDC was expressed as active tension by dividing the peak contraction (mN) by 2x vessel length in mm. Intralobal renal arteries were dissected from SHR and WKY rats and subjected to 12-hour organ culture in DMEM in control and in the presence of 100 ng/mL noggin or 3 μmol/L celecoxib. Both EDR and EDC were studied and compared in different

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treatment groups. Human arteries were treated using the same protocol as for rat renal arteries. Primary culture of murine aortic endothelial cells The method was modified from Kobayashi et al. as previously reported 5. Briefly, male mice were anaethesized with an intraperitoneal injection of pentobarbital sodium. 0.5 mL PBS containing 1,000 U/ml of heparin was perfused into the circulation from the left ventricle. The abdominal aortae were rapidly removed and placed in 20% FBS-DMEM, cleaned off surrounding tissues and then cut open. The aortae were incubated with collagenase type II for 10 min at 37 oC. Endothelial cells were collected by centrifugation at 3000 rpm for 5 min. The pellet was gently re-suspended by pipette with 5 mL of 20% FBS-DMEM and cultured in a 25 cm2 tissue culture flask. After 1-h incubation at 37 oC, the medium was removed, changed to EGM (endothelial cell growth medium supplemented with bovine brain extract, Lonza, Walkersville, MD). The identity of endothelial cells was confirmed by immunohistochemical staining against eNOS, while there was no positive staining of α-smooth muscle actin (αSMA) for detecting smooth muscle cell and prolyl-4-hydroxylase (P4H) for detecting fibroblast. ROS detection by EPR spin trapping To measure ROS released from arterial tissues, electron paramagnetic resonance (EPR) was performed with 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine hydrochloride (TEMPONE-H, Alexis) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, Alexis) as spin trap agents. All EPR samples were placed in 100 μL glass tubes and suspended in Krebs solution. In order to inhibit reactions catalyzed by transition metals, DTPA (0.2 mmol/L) was added. X-band EPR spectra were measured at room temperature using an EMX EPR spectrometer (Bruker). The EPR settings were as follows: field center, 3480 G; field sweep, 100 G, microwave frequency, 9.746 GHz; microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.3 G; conversion time, 1024 msec; time constant, 640 msec. Constructs, lentivirus production and transduction We have designed two shRNAs (short hairpin RNA) targeting mouse BMP receptor 1a: shRNA1 (5'- GCT GTT AAA TTC AAC AGT GAC ACA AAT G -3') and shRNA2 (5'- TCT CTC TAT GAC TTC CTG AAA TGT GCC A -3'); and one shRNA targeting firefly luciferase: 5'-TGC GCT GCT GGT GCC AAC CCT ATT CT-3' as a control. DNA fragments containing shRNAs sequence were synthesized and cloned into lentiviral RNAi (RNA interference) vector pLUNIG after annealing as previously described 6, 7. The VSV-G-pseudotyped lentiviruses were produced by cotransfecting 293T cells with the transfer vector and three packaging vectors: pMDLg/pRRE, pRSV-REV, and pCMV-VSVG as previously described. Subsequent purification was performed using ultracentrifugation. Mouse blood vessels were cultured in 24-well plates and were transduced with lentivirus and 8 μg/ml polybrene (Sigma). Western blotting Isolated arteries were subjected to similar procedures as in functional studies, frozen in liquid nitrogen, and homogenized in an ice-cold RIPA lysis buffer that contained 1 µg/mL leupeptin, 5 µg/mL aprotinin, 100 µg/mL PMSF, 1 mmol/L sodium orthovanadate, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L sodium fluoride, and 2 µg/mL β-glycerolphosphate. Tissue or cell lysates were centrifuged at 20,000 ×g for 20 min. The supernatants were collected and protein concentrations were analyzed using the Lowry method (Bio-rad, Hercules, CA, USA) 8. Protein samples were electrophoresed on a 10% SDS-poly-acrylamide gel and transferred onto an immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Nonspecific binding sites were blocked by 5% non-fat milk in 0.05% Tween-20 phosphate-buffered saline (PBST), then incubated overnight at 4 °C with primary antibodies including COX-2 or COX-1 (Cayman, Ann Arbor, MI, USA), p38 MAPK, phospho-p38 MAPK (Cell Signaling Technology, Beverly, MA, USA), BMPR1A (Santa Cruz, CA, USA) or BMP4 (Sigma-Aldrich, St. Louis, MO, USA), followed by a HRP-conjugated swine anti-rabbit or anti-mouse IgG (DakoCytomation, Carpinteria, CA, USA), developed with an enhanced chemiluminescence detection system (ECL reagents, Amersham Pharmacia), and finally exposed to X-ray films. Equal protein loading

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was verified with use of a housekeeping anti-GAPDH antibody. Immunohistochemistry Aortic rings were fixed in 4% paraformaldehyde at 4ºC overnight, dehydrated, processed and embedded in paraffin. Cross sections at 5 μm were cut on microtome (Leica Microsystems, Germany). After rehydrated to water, sections were microwave boiled in 0.01 mol/L citrate buffer (pH 6.0) for 10 min for antigen retrieval, then incubated for 15 min with 3% H2O2 at room temperature to block endogenous peroxidase activity. After washed with phosphate buffer saline (PBS), sections were blocked in 5% normal donkey serum (Jackson Immunoresearch, West Grove, PA, USA) for 1 hour at room temperature. Primary antibody (goat polyclonal anti-COX-2, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA; rabbit polyclonal anti-eNOS, 1:100, Abcam, Cambridge, UK) diluted in normal serum were incubated overnight at 4ºC. The slides were washed with PBS three times (5 min each). Biotin-SP conjugated goat anti-rabbit secondary antibodies (1:500, Jackson Immunoresearch) diluted in PBS were added and incubated for 1 h at room temperature. Slides were washed with PBS three times (5 min each) and incubated for 30 min with streptavidin-HRP conjugate (1:500, Zymed laboratory, San Francisco, CA, USA) at room temperature, and washed. Positive staining was developed as brown precipitate by 3,3’-diamonobenzidine tetrachloride (DAB) chromogen substrate (Vector laboratory, Burlingame, CA, USA). Slides were rinsed with water and counterstained with hematoxylin. Pictures were taken under Leica DMRBE microscope with a SPOT-RT digital camera and SPOT Advanced software (Diagnostic Instruments, Sertling Heights, MI, USA. Drugs BMP4, acetylcholine, L-NAME, phenylephrine, noggin, tempol and sodium nitroprusside (SNP) were purchased from Sigma-Aldrich Chemical (St Louis, MO, USA). SB202190, PD98059 and SP600125 were from Tocris (Avonmouth, UK). Apocynin were from Calbiochem, EMD Biosciences (La Jolla, CA, USA). S18886 (3-[(6-amino-(4-chlorobenzensulphonyl) -2-methyl-5,6,7,8- tetrahydronapht] -1-yl) propionic acid) and sc-560 were kind gifts from the Institut de Recherches Servier (Suresnes, France). Celecoxib was from Pfizer. Acetylcholine, L-NAME, phenylephrine, tempol were prepared in distilled water and the others in DMSO (Sigma-Aldrich). Statistical analysis Results represent means±SEM from different animals or humans. Concentration-response curves were analyzed by non-linear regression curve fitting using GraphPad Prism software (Version 4.0, San Diego, CA). The protein expression was quantified by densitometer (FluorChem, Alpha Innotech, San Leandro, CA) and normalized to GAPDH and then compared with control. Statistical significance was determined by two-tailed Student’s t-test or one-way ANOVA followed by the Bonferroni post-hoc test when more than two treatments were compared. P<0.05 indicates statistically significant difference.

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DETAILED METHODS REFERENCES: 1. Wong WT, Tian X, Xu A, Ng CF, Lee HK, Chen ZY, Au CL, Yao X, Huang Y. Angiotensin II type

1 receptor-dependent oxidative stress mediates endothelial dysfunction in type 2 diabetic mice. Antioxid Redox Signal. 2010;13:757-768.

2. Wang Y, Huang Y, Lam KS, Li Y, Wong WT, Ye H, Lau CW, Vanhoutte PM, Xu A. Berberine prevents hyperglycemia-induced endothelial injury and enhances vasodilatation via adenosine monophosphate-activated protein kinase and endothelial nitric oxide synthase. Cardiovasc Res. 2009;82:484-492.

3. Yang D, Feletou M, Levens N, Zhang JN, Vanhoutte PM. A diffusible substance(s) mediates endothelium-dependent contractions in the aorta of SHR. Hypertension. 2003;41:143-148.

4. Tang EH, Leung FP, Huang Y, Feletou M, So KF, Man RY, Vanhoutte PM. Calcium and reactive oxygen species increase in endothelial cells in response to releasers of endothelium-derived contracting factor. Br J Pharmacol. 2007;151:15-23.

5. Kobayashi M, Inoue K, Warabi E, Minami T, Kodama T. A simple method of isolating mouse aortic endothelial cells. J Atheroscler Thromb. 2005;12:138-142.

6. Chen Y, Stamatoyannopoulos G, Song CZ. Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro. Cancer Res. 2003;63:4801-4804.

7. Chen Y, Lin MC, Yao H, Wang H, Zhang AQ, Yu J, Hui CK, Lau GK, He ML, Sung J, Kung HF. Lentivirus-mediated RNA interference targeting enhancer of zeste homolog 2 inhibits hepatocellular carcinoma growth through down-regulation of stathmin. Hepatology. 2007;46:200-208.

8. Chan YC, Leung FP, Wong WT, Tian XY, Yung LM, Lau CW, Tsang SY, Yao X, Chen ZY, Huang Y. Therapeutically relevant concentrations of raloxifene dilate pressurized rat resistance arteries via calcium-dependent endothelial nitric oxide synthase activation. Arterioscler Thromb Vasc Biol.30:992-999.

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Online Figure I Control BMP4

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eNOS

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Online Figure I. Representative pictures of immunohistochemistry showing COX-2 expression increased in the endothelium in mouse aortae after BMP4 treatment (20 ng/mL, 12h), while eNOS is used to label the presence of endothelium. In the negative control, slices were processed under the same procedure without the primary antibody. Arrows indicate the endothelial expressions of COX-2.

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7

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DH

(Com

pare

d to

WKY

)p-p38

p38

GAPDH

WKY SHR SHR0

1

2

Tempol - - +p-

p38/

p38

(Com

pare

d to

WKY

)

-8 -7 -6 -50

50

100

ControlTempol

SHR

ACh (log M)

Rel

axat

ion

(% P

he to

ne)

a b

c d e

* *

* * * #*

#

34303450

34703490

3510-4000-2000

020004000

Magnetic Field (Gauss)

dX''/d

B

WKY

SHR

Tempone only

34303450

34703490

3510-4000-2000

020004000

Magnetic Field (Gauss)

dX''/d

B

WKY

SHR

Tempone only

f g

Tempon

e only

WKY

SHR0

1000

2000

3000

EPR

Sig

nal I

nten

sity

(dX'

'/dB)

*

Tempon

e only

WKY

SHR0

1000

2000

3000

EPR

Sig

nal I

nten

sity

(dX'

'/dB)

*

Online Figure II. (a) Effect of tempol (100 μmol/L, 12 h incubation) on acetylcholine (ACh) -induced endothelium-dependent relaxations (EDR) in renal arteries from SHR precontracted with phenylephrine (Phe). (b) Effect of tempol (100 μmol/L, 12 h incubation) on ACh-induced endothelium-dependent contraction (EDC) in renal arteries from SHR in the presence of L-NAME. Data are means±SEM of 6 rats. * p<0.05 vs control. Effect of tempol (100 μmol/L, 12 h incubation) on the expressions of BMP4 (c), COX-2 (d), p38 MAPK phosphorylation (e) in renal arteries from SHR compared to those from WKY. Data are means±SEM of 4 rats. * p<0.05 vs WKY. # p<0.05 vs SHR. ROS production measured by EPR spectroscopy in renal arteries from SHR as showed in the representative EPR spectra (f) and summarized data (g). Data are means±SEM of 4 rats. * p<0.05 vs WKY.

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