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BASIC SCIENCE

Calcitriol protects renovascular function inhypertension by down-regulating angiotensin IItype 1 receptors and reducing oxidative stressJinghui Dong1,2,3, Siu Ling Wong1,2*, Chi Wai Lau1,2, Hung Kay Lee4,Chi Fai Ng5, Lihong Zhang2, Xiaoqiang Yao1,2, Zhen Yu Chen6,Paul M. Vanhoutte7,8, and Yu Huang1,2*

1Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Hong Kong, China; 2School of Biomedical Sciences, Chinese University ofHong Kong, Hong Kong, China; 3Department of Physiology, Hebei Medical University, Shijiazhuang, China; 4Department of Chemistry, Chinese University of Hong Kong, Hong Kong,China; 5Department of Surgery, Chinese University of Hong Kong, Hong Kong, China; 6School of Life Sciences, Chinese University of Hong Kong, Hong Kong, China; 7Department ofPharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; and 8Department of Pharmacy, King Saud University, Riyadh, Saudi Arabia

Received 6 May 2011; revised 11 September 2011; accepted 17 November 2011

Aims The present study investigated whether or not calcitriol, an active form of vitamin D, protects against renovasculardysfunction in hypertension and, if so, whether or not such protection alters the expression of key proteins involvedin that dysfunction.

Methodsand results

Changes in isometric tension showed that the impaired endothelium-dependent relaxations in renal arteries of hyper-tensive patients were enhanced by 12 h in vitro treatment with calcitriol. Dihydroethidium fluorescence revealed an ele-vated level of reactive oxygen species (ROS) in these arteries which was reduced by calcitriol. Immunofluorescenceshowed that calcitriol treatment reduced the expression of AT1R, NOX-2, NOX-4, and p67phox and increased thatof superoxide dismutase (SOD)-1. Twelve-hour exposure to calcitriol prevented angiotensin (Ang) II-induced increasesin ROS and the over-expression of NOX-2, NOX-4, and p67phox in renal arteries from normotensive patients. A specificantagonist of the human vitamin D receptor (VDR), TEI-9647, abolished these effects of calcitriol. Both in vitro exposureto and chronic in vivo administration of calcitriol enhanced relaxations to acetylcholine and abolished exaggerated endo-thelium-dependent contractions in renal arteries of normotensive rats pre-exposed to Ang II or harvested from spon-taneously hypertensive rats (SHR). Reactive oxygen species levels and expressions of AT1R, NAD(P)H oxidase subunits,SOD-1, and SOD-2 in SHR arteries were normalized by the chronic treatment with calcitriol.

Conclusion In vivo and in vitro activation of VDR with calcitriol improves endothelial function by normalizing the expressions ofAT1R and radical generating and scavenging enzymes and thus preventing ROS over-production. The present findingssuggest that calcitriol is effective in preserving endothelial function in hypertension.

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -Keywords Calcitriol † Vitamin D † Oxidative stress † Endothelial dysfunction † Hypertension

IntroductionCalcitriol (1,25-dihydroxyvitamin D3) is the major active form ofvitamin D. Besides its classical function of regulating calcium andphosphate homeostasis, it also acts on the cardiovascular,immune and endocrine systems, illustrating the wide tissue distri-bution of the vitamin D receptor (VDR), which mediates mostof the effects of calcitriol.1,2 Stimulation of VDR activates in turnretinoid X receptors and recruits various co-factors to form a

transcriptional complex, by which vitamin D modulates, directlyor indirectly, �3% of the gene transcriptions of the body.

An inverse correlation exists between the vitamin D level in theblood and the incidence of heart failure, cardiovascular mortality,and elevation of arterial blood pressure.3–5 Patients with chronickidney disease receiving vitamin D show a reduction in cardiovascularmortality6 and a lower relative risk of pre-dialysis mortality.7 In theHealth Professionals Follow-up Study, subjects with low circulating25-hydroxyvitamin D concentrations had a higher risk of myocardial

* Corresponding author. Tel: +852 2609 6787, Fax: +852 2603 5022, Email: yu-huang@cuhk.edu.hk (Y.H.); christine.slwong.cuhk@gmail.com (S.L.W.)

Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2012. For permissions please email: journals.permissions@oup.com

European Heart Journaldoi:10.1093/eurheartj/ehr459

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infarction.8 Since vitamin D directly inhibits renin transcription, severalstudies have focused on its effects on the cardiovascular system indisease states with hyperactive renin–angiotensin system (RAS).9,10

Thus, arterial blood pressure is elevated in VDR knockout mice9

and in mice deficient in 1a-hydroxylase,11 an enzyme transforming in-active calcidiol tobioactive calcitriol, indicating a housekeeping role forvitamin D in cardiovascular health. In vivo and in vitro administration ofcalcitriol suppresses endothelium-dependent contractions inthe aortae of the spontaneously hypertensive rats (SHR).12,13

Vitamin D also possesses anti-inflammatory, anti-proliferative, andanti-hypertrophic properties14 which may offer additionalcardiovascular benefits. Vitamin D reduces cardiac hypertrophy inthe SHR.15 It also directly acts on endothelial and vascular smoothmuscle cells, increasing re-endothelialization and fibrinolysis while de-creasing coagulation.14

Endothelial dysfunction, resulting from an imbalance between therelease of endothelium-derived relaxing and contracting factors, is apredictor of impaired cardiovascular function.16,17 Hypertension isassociated with the over-production of vasoconstrictor mediators18

and renal insufficiency is common in hypertensive patients.19

Acetylcholine-induced relaxations are attenuated in renal arteriesfrom hypertensive patients.20 However, it is uncertain whether or

not vitamin D supplementation can improve endothelial functionin the renal vasculature of hypertensive animals and patients. Thepresent experiments determined whether or not calcitriol protectsrenovascular function in hypertensive humans. When this appearedto be the case, the molecular mechanisms involved were analysedusing renal arteries of hypertensive animals.

MethodsThe use of human renal arteries was approved by the Joint ChineseUniversity of Hong Kong–New Territories East Cluster ClinicalResearch Ethics Committee. The animal experiments were approvedby the CUHK Animal Experimentation Ethics Committee andconformed to the Guide for the Care and Use of LaboratoryAnimals published by the US National Institute of Health (NIHPublication No. 85-23, revised 1996). The detailed Methods sectionis available as a Supplementary material online.

Preparation of human arteriesHuman renal arteries were incubated for 12 h in the presence or absenceof calcitriol (100 nmol/L) and/or angiotensin II (Ang II, 1 mmol/L). Thevitamin D receptor antagonist TEI-9647 (1 mmol/L) was added 30 minbefore calcitriol. Losartan (AT1R antagonist, 3 mmol/L),

Figure 1 In vitro exposure to calcitriol for 12 h improves vascular function in renal arteries with endothelium from hypertensive patients (HT).(A) Acetylcholine (ACh)-induced relaxations were enhanced by calcitriol (100 nmol/L) treatment; this effect was prevented by a specific antag-onist against human vitamin D receptor, TEI-9647 (TEI, 1 mmol/L). Acute (30-min) exposure of the arteries to losartan (3 mmol/L) or diphe-nyleneiodonium (DPI, 100 nmol/L) partially prevented the impairment. The graph shows means+ SEM of three to six experiments on samplesfrom different patients, with relaxations expressed as percentage of the stable contraction to phenylephrine (Phe). ***P , 0.001 vs. normoten-sive patients (NT); ###P , 0.001 vs. control from hypertensive patients (control) and †††P , 0.001 vs. hypertensive patients, calcitriol. (B) Dihy-droethidium fluorescence of reactive oxygen species showing that calcitriol reduced the reactive oxygen species level in the renal arteries fromhypertensive patients, an effect antagonized by TEI-9647. Acute (30-min) treatment with losartan, DPI or tempol (100 mmol/L) also decreasedthe reactive oxygen species levels in these arteries.

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diphenyleneiodonium [DPI, inhibitor of NAD(P)H oxidase, 100 nmol/L],or tempol [scavenger of reactive oxygen species (ROS), 100 mmol/L]were administered 30 min before obtaining contractions to phenylephrineor measuring ROS levels by dihydroethidium fluorescence microscopy.

Animals and treatment protocolsSix-month-old male SHR and age-matched Wistar–Kyoto rats (WKY)were assigned to one of the following three groups: (1) WKY control,(2) SHR treated with vehicle dimethyl sulfoxide (DMSO) (SHR +vehicle), and (3) SHR treated with calcitriol at 150 ng/kg per day (SHR +calcitriol). Calcitriol was administered by oral gavage for 4.5 months.

Culture of human aortic endothelial cellsand primary rat aortic endothelial cellsHuman aortic endothelial cells (HAEC) and primary rat aortic endo-thelial cells were cultured in Medium 200 supplemented with lowserum growth supplement and RPMI supplemented with 10% foetalbovine serum, respectively, as described.21,22

Immunofluorescence microscopy, westernblotting, and reverse transcriptase-polymerase chain reactionHuman renal arteries were processed for immunofluorescencemicroscopy for the detection of AT1R, NOX-2, NOX-4, p67phox,and superoxide dismutase (SOD)-1. Human and/or rat renal arteriesand aortic endothelial cells were probed for NOX-2, NOX-4,p67phox, nitrotyrosine, AT1R, AT2R, SOD-1, and SOD-2 by westernblotting. The effect of calcitriol on AT1R transcription was examinedby reverse transcriptase-polymerase chain reaction.

ROS detection by dihydroethidiumfluorescence and electron paramagneticresonanceROS levels in human and rat renal arteries and HAEC were deter-mined by dihydroethidium fluorescence using confocal microscopyand by electron paramagnetic resonance (EPR) using 1-hydroxy-

Figure 2 Renal arteries from hypertensive patients exhibit altered expression levels of the oxidative stress-related proteins which are allnormalized by calcitriol incubation as detected by immunofluorescence microscopy. Yellowish-green autofluorescence indicated the elastinof the internal and external elastic laminae, of which the former delineated the vessel wall into the luminal endothelium and the medialsmooth muscle layer while the latter separated the smooth muscle layer from the adventitia. Signals from Alexa Fluor 546-conjugated second-ary antibodies attached to primary antibodies against AT1R, NOX-2, NOX-4, p67phox, and SOD-1 appeared reddish orange. High levels ofarterial AT1R, NOX-2, NOX-4, and p67phox signified by the intense reddish orange colour were reduced by calcitriol, while the low levelof SOD-1 was up-regulated. The calcitriol-induced modulation was prevented by TEI-9647. Photomicrographs are representative imagesfrom experiments performed on samples from four to five different patients. The scale bar indicates length (100 mm). E, endothelium; SM,vascular smooth muscle. *P , 0.05, **P , 0.01, ***P , 0.001 vs. control; #P , 0.05, ##P , 0.01, ###P , 0.001 vs. calcitriol.

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2,2,6,6-tetramethyl-4-oxo-piperidine hydrochloride (TEMPONE-H,Alexis Biochemical Corp., San Diego, CA, USA) as the spin trappingagent.23

Statistical analysisThe data are presented as means+ SEM of four to eight experiments.pD2 is the negative logarithm of the acetylcholine concentrationneeded to cause 50% of the maximum relaxation and Emax denotesthe maximum response to either constrictor or dilator. The changesafter treatment are presented as fold change of the value (equalledto 1) obtained in the absence of treatment in tissue or cells of thesame source. Statistical significance was determined by one-wayANOVA followed by Bonferroni post hoc tests whenever appropriate(GraphPad Software, San Diego, CA). P values ,0.05 were acceptedto indicate statistically significant differences.

Results

In vitro exposure to calcitriol enhancesrelaxations in renal arteries fromhypertensive patientsThe relaxations to the endothelium-dependent dilator acetylcho-line were attenuated significantly in renal arteries obtained fromhypertensive patients (pD2: 6.19+ 0.55, Emax: 15.3+6.7%, n ¼ 6in hypertensive patients vs. pD2: 6.96+0.05, Emax: 87.1+ 2.6%,n ¼ 4 in normotensive patients; Figure 1A). Twelve hour in vitrotreatment with calcitriol (100 nmol/L) enhanced the relaxations(pD2: 6.34+ 0.20, Emax: 54.1+4.5%, n ¼ 4), an effect prevented

by the human VDR antagonist, TEI-9647 (1 mmol/L; Figure 1A).Dihydroethidium fluorescence showed that the high ROS level inthe vascular wall of renal arteries from hypertensive patients wasreduced by calcitriol, an effect also antagonized by TEI-9647(Figure 1B). Acute exposure of arteries from hypertensive patientsto an AT1R antagonist (losartan, 3 mmol/L), an NAD(P)H oxidaseinhibitor (DPI, 100 nmol/L) or an ROS scavenger (tempol,100 mmol/L) also partially restored the acetylcholine-inducedrelaxations (Figure 1A) and decreased the ROS level (Figure 1B).

Calcitriol treatment normalizes thealtered expression of enzymes relatedto oxidative stressImmunofluorescence measuring reddish orange Alexa Fluor 546signals from respective antibodies demonstrated that the expres-sion of AT1R, NOX-2, NOX-4, and p67phox in renal arteriesfrom hypertensive patients were diminished by 12 h exposureto calcitriol, while that of SOD-1 was enhanced. The effects ofcalcitriol were antagonized by TEI-9647 (Figure 2).

Calcitriol reduces the augmentedproduction of reactive oxygen speciesinduced by angiotensin II in human renalarteries and endothelial cellsTwelve-hour incubation of renal arteries from normotensivepatients showed that Ang II at 0.3 mmol/L caused significantimpairment in acetylcholine-induced relaxations; the impairment

Figure 3 In vitro exposure to calcitriol prevents the angiotensin (Ang) II-induced reactive oxygen species production and up-regulation ofNAD(P)H subunits in renal arteries from normotensive patients. Dihydroethidium fluorescence and western blotting, respectively, showedthat angiotensin II (1 mmol/L, 12 h) increased (A) the arterial reactive oxygen species level and (B) the expression of NOX-2, NOX-4, andp67phox, all of which were reduced by calcitriol treatment. (A and B) TEI-9647 abolished the effects of calcitriol. Photomicrographs andblots are representative images from experiments performed on samples from four different patients.

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was more pronounced at 1 mmol/L (pD2: 6.96+ 0.05, Emax:87.13+ 2.58% in control; pD2: 6.61+0.10, Emax: 70.00+ 4.53%at 0.3 mmol/L Ang II; pD2: 6.69+0.05, Emax: 34.26+7.36% at1 mmol/L Ang II; n ¼ 4; see Supplementary material online, FigureS1). Hence, 1 mmol/L of Ang II was used in subsequent experi-ments. Angiotensin II increased the ROS level (Figure 3A) andup-regulated the expression of NOX-2, NOX-4, and p67phox

(Figure 3B) in renal arteries from normotensive patients. Theincreases in protein expressions and ROS level were preventedby co-incubation with calcitriol, an effect antagonized byTEI-9647 (Figure 3). TEI-9647 alone did not affect the ROS level(Figure 3A). Likewise, HAEC incubated with Ang II for 12 hexhibited an increased ROS level, which was attenuated byco-incubation with calcitriol or 100 mmol/L tempol. TEI-9647prevented the effect of calcitriol without modifying that oftempol (Figure 4).

Calcitriol prevents angiotensin II-inducedvascular dysfunction in Wistar–Kyotorats renal arteriesTwelve-hour incubation with Ang II (100 nmol/L) of renal arteriesfrom normotensive WKY resulted in impaired acetylcholine-

induced relaxations (pD2: 6.95+0.03, Emax: 77.2+1.2% in control,n ¼ 4; pD2: 6.07+0.36 vs. Emax: 41.2+7.5% in Ang II-treated rings,n ¼ 5; P , 0.05; Figure 5A and C) and unmasked endothelium-dependent contractions (Emax: 0.35+0.35% in control, n ¼ 4 vs.Emax: 73.8+4.3% in Ang II-treated rings, n ¼ 6; P , 0.05; Figure 5Band D). Pre-treatment with calcitriol before exposure to Ang II signifi-cantly prevented the attenuation in endothelium-dependent relaxa-tions caused by the peptide (pD2: 7.00+0.08, Emax: 69.6+1.8%,n ¼ 5,) and abolished the endothelium-dependent contractions(Figure 5A–D). Pre-incubation with losartan, DPI, or tempol preventedthe Ang II-induced impairment of the relaxation to acetylcholine andreduced the enhanced endothelium-dependent contraction(Figure 5C–E).

Angiotensin II (100 nmol/L, 12 h) augmented the expressions ofNOX-2 (see Supplementary material online, Figure S2A) andNOX-4 (see Supplementary material online, Figure S2B) in WKYrenal arteries. The NOX-2 and NOX-4 over-expression causedby the peptide was prevented by incubation with either calcitriolor losartan (see Supplementary material online, Figure S2). Like-wise, the ROS level of primary cultured WKY aortic endothelialcells was elevated by 12 h exposure to Ang II and this was pre-vented by calcitriol, losartan, tempol, or DPI (see Supplementarymaterial online, Figure S3).

Figure 4 Twelve-hour incubation with angiotensin II (Ang II, 1 mmol/L) augmented the reactive oxygen species level in cultured human aorticendothelial cells. Dihydroethidium fluorescence showed that the reactive oxygen species level was reduced by calcitriol (Cal), and tempol, butonly the effect of calcitriol was antagonized by TEI-9647 (TEI; 1 mmol/L). The bar graph represents means+ SEM of four experiments.***P , 0.001 vs. control; ###P , 0.001 vs. angiotensin II; ††P , 0.01 vs. combined treatment with angiotensin II plus calcitriol.

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Calcitriol abolishes endothelium-dependent contractions and correctsprotein expressions in SHR arteriesin vitroThe endothelium-dependent contractions of SHR renal arteries toacetylcholine were abolished by 12 h exposure to either calcitriolor losartan (Figure 6A and B). Actinomycin-D (2 mmol/L, an RNAsynthesis inhibitor) reversed the inhibitory effect of calcitriol onthe contractions (Figure 6B). Western blot analysis showed thatthe tissue levels of AT1R (Figure 6C), NOX-2 (Figure 6D), andNOX-4 (Figure 6E) were reduced in calcitriol-treated SHR arteries.RT-PCR results showed that AT1R mRNA level was reduced by 12 hcalcitriol exposure (see Supplementary material online, Figure S4).

Dihydroethidium fluorescence showed that the excessive pro-duction of ROS in SHR arteries was alleviated after 12 h of incuba-tion with calcitriol, an effect prevented by actinomycin-D. Bycontrast, acute calcitriol exposure for 30 min did not reduceROS levels (Figure 7A). Electron paramagnetic resonance measure-ments in homogenized renal arteries confirmed that SHR renal ar-teries exhibited a higher ROS level, which was reduced by 12 h ofexposure to calcitriol (Figure 7B).

In a cell-free radical-generating system [hypoxanthine(100 mmol/L) plus xanthine oxidase (9 mU/mL, HXXO)], calcitrioldid not affect the HXXO-induced EPR signal which is indicative ofROS generation (see Supplementary material online, Figure S5B andC ), whereas this signal was abolished by the xanthine oxidase

inhibitor oxypurinol (100 mmol/L, see Supplementary materialonline, Figure S5D).

In vivo treatment with calcitriolameliorates renovascular dysfunctionin spontaneously hypertensive ratsSystolic arterial blood pressurewas reduced significantly in SHR after4.5-month treatment with calcitriol (194.6+ 4.9 mmHg beforetreatment vs. 169.9+ 4.9 mmHg after treatment; see Supplemen-tary material online, Figure S6). Pronounced acetylcholine-inducedcontractions were observed in renal arteries of vehicle-treatedSHR (Emax: 88.5+ 5.3% in SHR + vehicle, n ¼ 9) compared withWKY (Emax: 13.7+ 10.9% in WKY, n ¼ 10; Figure 8A and B). Thecontractions were absent in rings without endothelium (Figure 8C).Chronic oral treatment with calcitriol (150 ng/kg/day for 4.5months) attenuated the contractions (Emax: 40.9+10.6% inSHR-receiving calcitriol, n ¼ 8; Figure 8A and B). Acute (30-min) ex-posure of renal arteries from SHR-receiving vehicle to calcitriol(100 nmol/L) did not modify the contractions (Figure 8D). By con-trast, the contractions in these arteries were reduced, to the sameextent as seen with chronic treatment with calcitriol, by the acutetreatment with losartan, DPI, and tempol (Figure 8E).

The AT1R expression was elevated in SHR renal arteries(Figure 8F, see Supplementary material online, Figure S7A) whilethat of AT2R was not different from control (see Supplementarymaterial online, Figure S7B). The over-expression of AT1R was

Figure 5 Angiotensin (Ang) II induces vascular dysfunction in renal arteries from normotensive Wistar–Kyoto rats (WKY). Exposure toangiotensin II (100 nmol/L, 12 h) (A) impaired relaxations to acetylcholine (ACh) and (B) unmasked contractions to the muscarinic agonist(B) in renal arteries with endothelium from normotensive Wistar–Kyoto rats. (A–C) Combined exposure to calcitriol (100 nmol/L) enhancedthe relaxations and reduced the contractions. (C–E) Losartan (3 mmol/L), diphenyleneiodonium (DPI, 100 nmol/L), and tempol (100 mmol/L)reversed endothelial dysfunction in angiotensin II-treated Wistar–Kyoto rat renal arteries. Data are means+ SEM of four to five experiments.***P , 0.001 vs. control; ###P , 0.001 vs. angiotensin II.

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normalized by the chronic treatment with calcitriol (Figure 8F, seeSupplementary material online, Figure S7A). Renal arteries of SHRexhibited augmented contractions to Ang II and the augmentationwas prevented by a 12 h treatment with 100 nmol/L calcitriol (seeSupplementary material online, Figure S7C ).

The level of oxidative stress as reflected by the nitrotyrosinecontent (Figure 8F, see Supplementary material online, Figure S8A)was augmented in SHR renal arteries, as were the expressionsof NOX-2 and p67phox (Figure 8G, see Supplementary materialonline, Figure S8B and C), while the levels of SOD-1 and SOD-2(Figure 8H, see Supplementary material online, Figure S8D and E)were diminished. Chronic treatment with calcitriol correctedthe over-expression of NOX-2, p67phox, and nitrotyrosine, andenhanced the expression of both SOD isoforms.

Renal arteries from SHR treated with vehicle exhibited anincreased ROS accumulation across their wall compared withWKY preparations (Figure 9A). The elevated ROS level wasreduced in arteries from calcitriol-treated SHR (Figure 9A). Bycontrast, 30-min calcitriol treatment did not reduce the arterialROS level, whereas it was acutely decreased by DPI, tempol, andlosartan (Figure 9B).

DiscussionThe present study provides evidence of the protective effect of cal-citriol, an active form of vitamin D, on human renovascular functionin hypertension. The major novel findings include: (1) the impaired

endothelium-dependent relaxations in renal arteries from hyperten-sive patients can be partially restored by a 12 h in vitro incubation withcalcitriol; (2) this improvement is accompanied by the normalizationof oxidative stress-related proteins including NOX-2, NOX-4,p67phox, and SOD-1 and a resulting reduction in ROS levels; (3) cal-citriol prevents the up-regulation of NOX-2, NOX-4, and p67phox

and the increase in ROS levels induced by Ang II in renal arteriesfrom normotensive patients and in HAEC; (4) the effects of calcitriolare antagonized by the specific human VDR antagonist, TEI-9647,pinpointing the positive role of this receptor in the protectiveeffects of calcitriol; (5) the findings in human arteries are supportedby similar results obtained in isolated renal arteries harvested fromSHR and in those from WKY exposed to Ang II; (6) substantiatingthe in vitro findings, chronic in vivo oral administration of calcitriolreverses renovascular dysfunction in the SHR as reflected by thereduction in endothelium-dependent contractions which mostlikely result from the down-regulation of AT1R, NOX-2, andp67phox, and the up-regulation of SOD-1 and SOD-2; and (7) calci-triol per se does not possess radical scavenging activity but reducesoxidative stress by transcriptional regulation of the radical generatingand scavenging enzymes.

ROS are involved in the impairment of vascular function andthe development of hypertension.24 They impair endothelium-dependent relaxations and facilitate endothelium-dependentcontractions.20,25– 27 These radicals are generated by variousenzymes, of which NAD(P)H oxidase represents the majorsource. Reactive oxygen species scavengers and inhibition of the

Figure 6 In vitro exposure to calcitriol attenuates endothelium-dependent contractions and reduces the exaggerated expression of oxidativestress-related proteins. (A and B) Tissue culture with calcitriol (100 nmol/L) or losartan (3 mmol/L) for 12 h reduced contractions of quiescentspontaneously hypertensive rat (SHR) renal arteries with endothelium to acetylcholine (ACh). (B) The effect of calcitriol was abolished by com-bined incubation with actinomycin-D (2 mmol/L). Data are means+ SEM of four experiments. ***P , 0.001 vs. control; ###P , 0.001 vs. calci-triol. Twelve-hour incubation with calcitriol in spontaneously hypertensive rat renal arteries reduced the expressions of (C) AT1R, (D) NOX-2and (E) NOX-4. Data are means+ SEM of four to five experiments. **P , 0.01, ***P , 0.001 vs. Wistar–Kyoto rats; ##P , 0.01, ###P , 0.001vs. spontaneously hypertensive rats control.

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hyper-active NAD(P)H oxidase prevent the development of endo-thelial dysfunction in the SHR.20,28 In addition to augmented ROSproduction, the activity of the local RAS is increased in the vasculartissue of hypertensive patients, which helps to explain why AT1Rblockers represent a major class of anti-hypertensive drugs.29,30

The present study shows that losartan and DPI restore theimpaired endothelium-dependent relaxations and reduce theROS levels in renal arteries from hypertensive patients, thusconfirming the critical role of AT1R activation and ROS over-production in the vascular dysfunction of human hypertension.Vitamin D is a potent endogenous suppressor of the RASbecause it inhibits renin transcription both in vitro and in vivo.31

Chronic treatment with calcitriol or other active forms ofvitamin D prevents the development of cardiac hypertrophy inthe SHR by suppressing the cardiac RAS.32

The present findings reveal the regulatory role of vitamin D onthe RAS and on ROS production in renal arteries of both hyper-tensive humans and animals. First, in vitro calcitriol corrects theabnormal expression of AT1R, the radical generating [NAD(P)Hoxidase with its subunits NOX-2, NOX-4, and p67phox] and scav-enging (SOD-1) enzymes and the resulting augmented ROS levelsin the renal arteries from hypertensive patients, in Ang II-treatedrenal arteries from normotensive subjects and in HAEC. Second,in terms of vascular reactivity, not only are endothelium-dependent relaxations improved by exposure to calcitriol both invitro and in vivo but also those treatments abolish the exaggerated

endothelium-dependent contractions in renal arteries from SHRand WKY, the latter after incubation with Ang II. The reductionsin endothelium-dependent relaxations and the unmasking ofendothelium-dependent contractions are sensitive to an AT1Rantagonist, an inhibitor of NAD(P)H oxidase and ROS scavengers,indicative of a pathogenic involvement of AT1R and NAD(P)Hoxidase. Third, the western blot analysis shows an up-regulationof AT1R and NAD(P)H oxidase subunits, NOX-2 and p67phox,and a down-regulation of SOD-1 and SOD-2 in SHR arteries com-pared with those of WKY, and these alterations are normalized bycalcitriol. The reductions in the mRNA and protein levels of AT1Rmay account for a diminished contraction in response to Ang II.Fourth, ROS detection by dihydroethidium fluorescence and EPRin aortic endothelial cells and SHR renal arteries confirms anROS overproduction, which can be reduced by both in vitro andchronic in vivo calcitriol treatment. Taken in conjunction, thepresent results strongly suggest a regulatory role of vitamin Don RAS activity and ROS production in the vascular wall, whichbecomes particularly obvious in hypertension.

The present study, in demonstrating the inhibitory effect of aspecific antagonist against human VDR TEI-9647,33 establishes acrucial role of VDR in the calcitriol-induced restoration of renovas-cular function in hypertension. Indeed, the impaired endothelium-dependent relaxations of renal arteries from hypertensive patientswere partially rescued by 12 h incubation with calcitriol, and thiswas antagonized by the VDR antagonist, strongly suggesting that

Figure 7 In vitro exposure to calcitriol normalizes the reactive oxygen species (ROS) level in spontaneously hypertensive rat (SHR) renalarteries. (A) Dihydroethidium fluorescence showed that spontaneously hypertensive rats renal arteries exhibited elevated reactive oxygenspecies level compared with Wistar–Kyoto rat (WKY) arteries. Twelve-hour calcitriol treatment reduced the reactive oxygen species leveland this effect was abolished by actinomycin-D (AD; 2 mmol/L). In contrast, acute exposure (30 min) of spontaneously hypertensive ratrenal arteries to calcitriol (100 nmol/L) did not reduce reactive oxygen species level. (B) The reactive oxygen species level measured byEPR was greater in renal arteries from spontaneously hypertensive rats compared with those from Wistar–Kyoto rats and calcitriolreduced the elevated reactive oxygen species level in spontaneously hypertensive rat renal arteries. Data are means+ SEM of four experiments.***P , 0.001 vs. Wistar–Kyoto rats; ###P , 0.001 vs. spontaneously hypertensive rat without treatments.

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calcitriol exerts its beneficial effects via VDR activation. Consist-ently, 12 h incubation with Ang II increases the ROS level inHAEC, and this is prevented by both calcitriol and the anti-oxidanttempol. However, only the effect of calcitriol, but not that oftempol, is antagonized by TEI-9647, demonstrating that theprotective effect of calcitriol can indeed be attributed to VDRactivation.

The vascular protective effect of calcitriol observed in thepresent study cannot be explained by direct scavenging of ROSas demonstrated by the lack of direct effect of calcitriol on gener-ation of free radicals by the cell-free HXXO reaction. It is not im-mediate, in contrast to the inhibition of endothelium-dependentcontractions reported in the SHR aortae, which is non-genomicin nature and has been attributed to inhibition of endothelialcalcium influx.13 Instead, calcitriol exerts its effect throughgenomic regulation. This conclusion is prompted by the presentmeasurements of the protein expression of radical generatingand scavenging enzymes, with calcitriol reducing the expressionof the former [NAD(P)H oxidase and its subunits] but augmentingthat of the latter (SOD-1 and SOD-2). The genomic impact of cal-citriol is confirmed by the experiments with the mRNA synthesisinhibitor, actinomycin-D, which abolishes its protective effectagainst endothelium-dependent contractions and increases inROS levels. A genomic action is consistent with the conclusion

that the effects of calcitriol reported in the present study aredue to VDR activation. Indeed, upon binding of and activation byvitamin D, VDR forms a heterodimer complex with the retinoidX receptors (RXR). The VDR–RXR complex can bind tospecific DNA sequences, termed vitamin D responsive elements,located in the promoter regions of various vitamin-D-dependentgenes.34

Because of the limited supply of renal arteries from patientsafter informed consent, a limitation of the present study is thesmall sample size of the human specimens available with an un-avoidable variability due to the differences in disease progressionbetween the donors. However, since treated preparations werecompared systematically with untreated tissues from the samepatient, consistent results were obtained, confirming the criticalrole of RAS and ROS in hypertension-associated vascular dysfunc-tion and demonstrating the protective effects of calcitriol and itsantagonism by the vitamin D receptor blocker (TEI-9647). Theconclusions reached were comforted by experiments on culturedhuman endothelial cells. They were fully supported by the studieson animal blood vessels, where the source of the studiedmaterial is adequately controlled. Thus, it seems reasonable toaccept that the current data on human blood vessels indeeddemonstrate the vascular protective effects of calcitriol in humanhypertension.

Figure 8 Chronic in vivo treatment with calcitriol reduces the augmented endothelium-dependent contractions and normalizes the expres-sion of oxidative stress-related proteins in spontaneously hypertensive rat renal arteries. (A) Representative traces and (B) concentration–con-traction curves showing the inhibitory effect of calcitriol on acetylcholine (ACh)-induced contractions, which were endothelium dependent asreflected by the absence of contractions in rings without endothelium (-Endo, C ). Endothelium-dependent contractions were unaffected byacute exposure (30-min) to calcitriol (100 nmol/L) (D), but attenuated by losartan (3 mmol/L), diphenyleneiodonium (DPI, 100 nmol/L), andtempol (100 mmol/L) (E). Data are means+ SEM of five experiments. ***P , 0.001 vs. Wistar–Kyoto rat, +Endo, or spontaneously hyper-tensive rat control; ###P , 0.001 vs. spontaneously hypertensive rat treated with vehicle. Representative blots showing that calcitriolreduced the over-expression of AT1R, nitrotyrosine (F), NOX-2, p67phox (G) and up-regulated the levels of SOD-1 and SOD-2 (H ). W,WKY; SV, SHR-treated with vehicle; SC, SHR-treated with calcitriol.

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The present findings substantiate the capability of chronic treat-ment with vitamin D to reduce ROS level in rat arteries12 butprovide novel insights into the possible mechanisms by which itregulates radical homeostasis. Calcitriol is a potent suppressor ofthe RAS.31 Since the AT1R expression is decreased following calci-triol treatment, the modification of the NAD(P)H and SODexpressions and the reduction in ROS level observed uponchronic treatment with the vitamin may be due in part to alesser presence of AT1R. The latter is a key player in the pathogen-esis of hypertension and its activation serves as a primary triggerfor ROS production by NAD(P)H oxidase.35 Cyclooxygenases(COX) are the major source of endothelium-derived contractingfactors and ROS facilitate their production/action.25,26,36 Chronicadministration of calcitriol reduces the expression of COX-1 inthe rat aortae.12 Paricalcitriol, another active form of vitamin D,reduces excessive ROS production by down-regulatingpro-inflammatory factors such as inducible nitric oxide synthase,tumor necrosis factor-a, and COX-2.37 Endothelium-dependentcontractions of the SHR renal arteries are prevented by inhibitorsof cyclooxygenase.38 Thus, it is likely that the reduction in ROSproduction demonstrated in the present study leads to decreasedexpression/presence of endothelial cyclooxygenase(s) with in turnattenuation of endothelium-dependent contractions caused by theexposure to calcitriol.

The present study confirms that chronic oral administration ofcalcitriol reduces the elevated blood pressure of the SHR.12

Whether or not there is a relationship between low plasmalevels of vitamin D and elevated arterial blood pressure remains

controversial.3 Nevertheless, the present findings, together withthose from limited clinical and animal studies12,39,40 support theview that vitamin D supplementation can lower arterial bloodpressure. This may be due to indirect effects and not necessarilyto a direct genomic action on the blood vessel wall. However,the protective effect of calcitriol on renal arteries is not likely tobe secondary to the blood pressure reduction. Indeed, in thepresent study, the inhibition by calcitriol on the exaggeratedendothelium-dependent contractions of isolated SHR renal arter-ies was similar after a 12 h in vitro incubation and after chronicin vivo treatment, strongly suggesting that calcitriol improvesendothelial function directly.

In conclusion, chronic treatment with calcitriol protects againstrenovascular function in hypertension. The calcitriol-induced pro-tection is likely to be mediated by VDR activation leading to down-regulation of the expressions of AT1R, NAD(P)H subunits andup-regulation of SOD-1 and SOD-2. These in turn prevent theROS overproduction. The findings in human renal arteries andhuman endothelial cells are confirmed both by in vitro and in vivoresults in the animal. The present study suggests calcitriol/VDRactivation as a novel therapeutic strategy to amelioratehypertension-associated vascular dysfunction.

Supplementary materialSupplementary material is available at European Heart Journalonline.

Figure 9 Chronic in vivo treatment with calcitriol decreases the reactive oxygen species level in spontaneously hypertensive rat (SHR) renalarteries. (A) Representative images and summarized data showing that the elevated levels of reactive oxygen species in spontaneously hyper-tensive rat arteries are reduced by calcitriol. Data are means+ SEM of four to five experiments. *P , 0.05 vs. Wistar–Kyoto rats (WKY);#P , 0.05 vs. spontaneously hypertensive rats + vehicle. (B) Acute exposure (30-min) of spontaneously hypertensive rat renal arteries todiphenyleneiodonium (DPI, 100 nmol/L), tempol (100 mmol/L), and losartan (3 mmol/L) but not calcitriol (100 nmol/L) or dimethyl sulfoxidenormalized the reactive oxygen species level. Data are means+ SEM of four experiments. ***P , 0.001 vs. Wistar–Kyoto rats; ###P , 0.001vs. spontaneously hypertensive rat control.

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AcknowledgementsWe acknowledge the kind gift of TEI-9647 by Teijin PharmaLimited (Tokyo, Japan).

FundingThis work was supported by National Basic Research Program ofChina (2012CB517805), Hong Kong Research Grant Council(CUHK466110), and Focused Investment Scheme of the Chinese Uni-versity of Hong Kong.

Conflict of interest: none declared.

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