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BASIC SCIENCE
Calcitriol protects renovascular function in
hypertension by down-regulating angiotensin II
type 1 receptors and reducing oxidative stress
Jinghui Dong
1,2,3
, Siu Ling Wong
1,2
*
, Chi Wai Lau
1,2
, Hung Kay Lee
4
,
Chi Fai Ng
5
, Lihong Zhang
2
, Xiaoqiang Yao
1,2
, Zhen Yu Chen
6
,
Paul M. Vanhoutte
7,8
, and Yu Huang
1,2
*
1
Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Hong Kong, China;
2
School of Biomedical Sciences, Chinese University of
Hong Kong, Hong Kong, China;
3
Department of Physiology, Hebei Medical University, Shijiazhuang, China;
4
Department of Chemistry, Chinese University of Hong Kong, Hong Kong,
China;
5
Department of Surgery, Chinese University of Hong Kong, Hong Kong, China;
6
School of Life Sciences, Chinese University of Hong Kong, Hong Kong, China;
7
Department of
Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; and
8
Department 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 renovascular
dysfunction in hypertension and, if so, whether or not such protection alters the expression of key proteins involved
in that dysfunction.
Methods
and 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 uorescence revealed an ele-
vated level of reactive oxygen species (ROS) in these arteries which was reduced by calcitriol. Immunouorescence
showed that calcitriol treatment reduced the expression of AT
1
R, NOX-2, NOX-4, and p67
phox
and increased that
of superoxide dismutase (SOD)-1. Twelve-hour exposure to calcitriol prevented angiotensin (Ang) II-induced increases
in ROS and the over-expression of NOX-2, NOX-4, and p67
phox
in renal arteries fromnormotensive patients. Aspecic
antagonist of the human vitamin Dreceptor (VDR), TEI-9647, abolished these effects of calcitriol. Both in vitro exposure
to 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 AT
1
R, NAD(P)Hoxidase 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 of
AT
1
R and radical generating and scavenging enzymes and thus preventing ROS over-production. The present ndings
suggest that calcitriol is effective in preserving endothelial function in hypertension.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Keywords Calcitriol Vitamin D Oxidative stress Endothelial dysfunction Hypertension
Introduction
Calcitriol (1,25-dihydroxyvitamin D3) is the major active form of
vitamin D. Besides its classical function of regulating calcium and
phosphate 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 most
of the effects of calcitriol.
1,2
Stimulation of VDR activates in turn
retinoid X receptors and recruits various co-factors to form a
transcriptional complex, by which vitamin D modulates, directly
or indirectly, 3% of the gene transcriptions of the body.
An inverse correlation exists between the vitamin D level in the
blood and the incidence of heart failure, cardiovascular mortality,
and elevation of arterial blood pressure.
35
Patients with chronic
kidney disease receiving vitamin Dshowa reduction in cardiovascular
mortality
6
and a lower relative risk of pre-dialysis mortality.
7
In the
Health Professionals Follow-up Study, subjects with low circulating
25-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 Journal
doi:10.1093/eurheartj/ehr459
European Heart Journal Advance Access published January 19, 2012

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infarction.
8
Since vitamin Ddirectly inhibits renintranscription, several
studies have focused on its effects on the cardiovascular system in
disease states with hyperactive reninangiotensin system (RAS).
9,10
Thus, arterial blood pressure is elevated in VDR knockout mice
9
and in mice decient in 1a-hydroxylase,
11
an enzyme transforming in-
active calcidiol tobioactivecalcitriol, indicating a housekeeping role for
vitamin D in cardiovascular health. In vivo and in vitro administration of
calcitriol suppresses endothelium-dependent contractions in
the aortae of the spontaneously hypertensive rats (SHR).
12,13
Vitamin D also possesses anti-inammatory, anti-proliferative, and
anti-hypertrophic properties
14
which may offer additional
cardiovascular benets. Vitamin D reduces cardiac hypertrophy in
the SHR.
15
It also directly acts on endothelial and vascular smooth
muscle cells, increasing re-endothelialization and brinolysis while de-
creasing coagulation.
14
Endothelial dysfunction, resulting from an imbalance between the
release of endothelium-derived relaxing and contracting factors, is a
predictor of impaired cardiovascular function.
16,17
Hypertension is
associated with the over-production of vasoconstrictor mediators
18
and renal insufciency is common in hypertensive patients.
19
Acetylcholine-induced relaxations are attenuated in renal arteries
from hypertensive patients.
20
However, it is uncertain whether or
not vitamin D supplementation can improve endothelial function
in the renal vasculature of hypertensive animals and patients. The
present experiments determined whether or not calcitriol protects
renovascular function in hypertensive humans. When this appeared
to be the case, the molecular mechanisms involved were analysed
using renal arteries of hypertensive animals.
Methods
The use of human renal arteries was approved by the Joint Chinese
University of Hong KongNew Territories East Cluster Clinical
Research Ethics Committee. The animal experiments were approved
by the CUHK Animal Experimentation Ethics Committee and
conformed to the Guide for the Care and Use of Laboratory
Animals published by the US National Institute of Health (NIH
Publication No. 85-23, revised 1996). The detailed Methods section
is available as a Supplementary material online.
Preparation of human arteries
Human renal arteries were incubated for 12 h in the presence or absence
of calcitriol (100 nmol/L) and/or angiotensin II (Ang II, 1 mmol/L). The
vitamin D receptor antagonist TEI-9647 (1 mmol/L) was added 30 min
before calcitriol. Losartan (AT
1
R 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 specic 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 samples
from 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 uorescence of reactive oxygen species showing that calcitriol reduced the reactive oxygen species level in the renal arteries from
hypertensive patients, an effect antagonized by TEI-9647. Acute (30-min) treatment with losartan, DPI or tempol (100 mmol/L) also decreased
the reactive oxygen species levels in these arteries.
J. Dong et al. Page 2 of 11

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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 administered30 min before obtaining contractions tophenylephrine
or measuring ROS levels by dihydroethidium uorescence microscopy.
Animals and treatment protocols
Six-month-old male SHR and age-matched WistarKyoto 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 cells
and primary rat aortic endothelial cells
Human aortic endothelial cells (HAEC) and primary rat aortic endo-
thelial cells were cultured in Medium 200 supplemented with low
serum growth supplement and RPMI supplemented with 10% foetal
bovine serum, respectively, as described.
21,22
Immunouorescence microscopy, western
blotting, and reverse transcriptase-
polymerase chain reaction
Human renal arteries were processed for immunouorescence
microscopy for the detection of AT
1
R, NOX-2, NOX-4, p67
phox
,
and superoxide dismutase (SOD)-1. Human and/or rat renal arteries
and aortic endothelial cells were probed for NOX-2, NOX-4,
p67
phox
, nitrotyrosine, AT
1
R, AT
2
R, SOD-1, and SOD-2 by western
blotting. The effect of calcitriol on AT
1
R transcription was examined
by reverse transcriptase-polymerase chain reaction.
ROS detection by dihydroethidium
uorescence and electron paramagnetic
resonance
ROS levels in human and rat renal arteries and HAEC were deter-
mined by dihydroethidium uorescence using confocal microscopy
and 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 all
normalized by calcitriol incubation as detected by immunouorescence microscopy. Yellowish-green autouorescence indicated the elastin
of the internal and external elastic laminae, of which the former delineated the vessel wall into the luminal endothelium and the medial
smooth 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 AT
1
R, NOX-2, NOX-4, p67
phox
, and SOD-1 appeared reddish orange. High levels of
arterial AT
1
R, NOX-2, NOX-4, and p67
phox
signied by the intense reddish orange colour were reduced by calcitriol, while the low level
of SOD-1 was up-regulated. The calcitriol-induced modulation was prevented by TEI-9647. Photomicrographs are representative images
from experiments performed on samples from four to ve 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.
Calcitriol protects renovascular function Page 3 of 11

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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 trapping
agent.
23
Statistical analysis
The data are presented as means +SEM of four to eight experiments.
pD
2
is the negative logarithm of the acetylcholine concentration
needed to cause 50% of the maximum relaxation and E
max
denotes
the maximum response to either constrictor or dilator. The changes
after treatment are presented as fold change of the value (equalled
to 1) obtained in the absence of treatment in tissue or cells of the
same source. Statistical signicance was determined by one-way
ANOVA followed by Bonferroni post hoc tests whenever appropriate
(GraphPad Software, San Diego, CA). P values ,0.05 were accepted
to indicate statistically signicant differences.
Results
In vitro exposure to calcitriol enhances
relaxations in renal arteries from
hypertensive patients
The relaxations to the endothelium-dependent dilator acetylcho-
line were attenuated signicantly in renal arteries obtained from
hypertensive patients (pD
2
: 6.19+0.55, E
max
: 15.3+6.7%, n 6
in hypertensive patients vs. pD
2
: 6.96+0.05, E
max
: 87.1+2.6%,
n 4 in normotensive patients; Figure 1A). Twelve hour in vitro
treatment with calcitriol (100 nmol/L) enhanced the relaxations
(pD
2
: 6.34+0.20, E
max
: 54.1+4.5%, n 4), an effect prevented
by the human VDR antagonist, TEI-9647 (1 mmol/L; Figure 1A).
Dihydroethidium uorescence showed that the high ROS level in
the vascular wall of renal arteries from hypertensive patients was
reduced by calcitriol, an effect also antagonized by TEI-9647
(Figure 1B). Acute exposure of arteries from hypertensive patients
to an AT
1
R antagonist (losartan, 3 mmol/L), an NAD(P)H oxidase
inhibitor (DPI, 100 nmol/L) or an ROS scavenger (tempol,
100 mmol/L) also partially restored the acetylcholine-induced
relaxations (Figure 1A) and decreased the ROS level (Figure 1B).
Calcitriol treatment normalizes the
altered expression of enzymes related
to oxidative stress
Immunouorescence measuring reddish orange Alexa Fluor 546
signals from respective antibodies demonstrated that the expres-
sion of AT
1
R, NOX-2, NOX-4, and p67
phox
in renal arteries
from hypertensive patients were diminished by 12 h exposure
to calcitriol, while that of SOD-1 was enhanced. The effects of
calcitriol were antagonized by TEI-9647 (Figure 2).
Calcitriol reduces the augmented
production of reactive oxygen species
induced by angiotensin II in human renal
arteries and endothelial cells
Twelve-hour incubation of renal arteries from normotensive
patients showed that Ang II at 0.3 mmol/L caused signicant
impairment 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 of
NAD(P)H subunits in renal arteries from normotensive patients. Dihydroethidium uorescence and western blotting, respectively, showed
that angiotensin II (1 mmol/L, 12 h) increased (A) the arterial reactive oxygen species level and (B) the expression of NOX-2, NOX-4, and
p67
phox
, all of which were reduced by calcitriol treatment. (A and B) TEI-9647 abolished the effects of calcitriol. Photomicrographs and
blots are representative images from experiments performed on samples from four different patients.
J. Dong et al. Page 4 of 11

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was more pronounced at 1 mmol/L (pD
2
: 6.96+0.05, E
max
:
87.13+2.58% in control; pD
2
: 6.61+0.10, E
max
: 70.00+4.53%
at 0.3 mmol/L Ang II; pD
2
: 6.69+0.05, E
max
: 34.26 +7.36% at
1 mmol/L Ang II; n 4; see Supplementary material online, Figure
S1). Hence, 1 mmol/L of Ang II was used in subsequent experi-
ments. Angiotensin II increased the ROS level (Figure 3A) and
up-regulated the expression of NOX-2, NOX-4, and p67
phox
(Figure 3B) in renal arteries from normotensive patients. The
increases in protein expressions and ROS level were prevented
by co-incubation with calcitriol, an effect antagonized by
TEI-9647 (Figure 3). TEI-9647 alone did not affect the ROS level
(Figure 3A). Likewise, HAEC incubated with Ang II for 12 h
exhibited an increased ROS level, which was attenuated by
co-incubation with calcitriol or 100 mmol/L tempol. TEI-9647
prevented the effect of calcitriol without modifying that of
tempol (Figure 4).
Calcitriol prevents angiotensin II-induced
vascular dysfunction in WistarKyoto
rats renal arteries
Twelve-hour incubation with Ang II (100 nmol/L) of renal arteries
from normotensive WKY resulted in impaired acetylcholine-
induced relaxations (pD
2
: 6.95+0.03, E
max
: 77.2+1.2% in control,
n 4; pD
2
: 6.07+0.36 vs. E
max
: 41.2+7.5% in Ang II-treated rings,
n 5; P ,0.05; Figure 5A and C) and unmasked endothelium-
dependent contractions (E
max:
0.35+0.35% in control, n 4 vs.
E
max
: 73.8+4.3% in Ang II-treated rings, n 6; P ,0.05; Figure 5B
and D). Pre-treatment with calcitriol before exposure to Ang II signi-
cantly prevented the attenuation in endothelium-dependent relaxa-
tions caused by the peptide (pD
2
: 7.00+0.08, E
max
: 69.6+1.8%,
n 5,) and abolished the endothelium-dependent contractions
(Figure 5AD). Pre-incubationwith losartan, DPI, or tempol prevented
the Ang II-induced impairment of the relaxation to acetylcholine and
reduced the enhanced endothelium-dependent contraction
(Figure 5CE).
Angiotensin II (100 nmol/L, 12 h) augmented the expressions of
NOX-2 (see Supplementary material online, Figure S2A) and
NOX-4 (see Supplementary material online, Figure S2B) in WKY
renal arteries. The NOX-2 and NOX-4 over-expression caused
by the peptide was prevented by incubation with either calcitriol
or losartan (see Supplementary material online, Figure S2). Like-
wise, the ROS level of primary cultured WKY aortic endothelial
cells was elevated by 12 h exposure to Ang II and this was pre-
vented by calcitriol, losartan, tempol, or DPI (see Supplementary
material 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 aortic
endothelial cells. Dihydroethidium uorescence showed that the reactive oxygen species level was reduced by calcitriol (Cal), and tempol, but
only 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.
Calcitriol protects renovascular function Page 5 of 11

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Calcitriol abolishes endothelium-
dependent contractions and corrects
protein expressions in SHR arteries
in vitro
The endothelium-dependent contractions of SHR renal arteries to
acetylcholine were abolished by 12 h exposure to either calcitriol
or losartan (Figure 6A and B). Actinomycin-D (2 mmol/L, an RNA
synthesis inhibitor) reversed the inhibitory effect of calcitriol on
the contractions (Figure 6B). Western blot analysis showed that
the tissue levels of AT
1
R (Figure 6C), NOX-2 (Figure 6D), and
NOX-4 (Figure 6E) were reduced in calcitriol-treated SHR arteries.
RT-PCRresults showed that AT
1
RmRNAlevel was reduced by 12 h
calcitriol exposure (see Supplementary material online, Figure S4).
Dihydroethidium uorescence 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. By
contrast, acute calcitriol exposure for 30 min did not reduce
ROS levels (Figure 7A). Electron paramagnetic resonance measure-
ments in homogenized renal arteries conrmed that SHR renal ar-
teries exhibited a higher ROS level, which was reduced by 12 h of
exposure to calcitriol (Figure 7B).
In a cell-free radical-generating system [hypoxanthine
(100 mmol/L) plus xanthine oxidase (9 mU/mL, HXXO)], calcitriol
did not affect the HXXO-induced EPR signal which is indicative of
ROS generation (see Supplementary material online, Figure S5B and
C), whereas this signal was abolished by the xanthine oxidase
inhibitor oxypurinol (100 mmol/L, see Supplementary material
online, Figure S5D).
In vivo treatment with calcitriol
ameliorates renovascular dysfunction
in spontaneously hypertensive rats
Systolic arterial blood pressurewas reduced signicantly in SHRafter
4.5-month treatment with calcitriol (194.6+4.9 mmHg before
treatment vs. 169.9+4.9 mmHg after treatment; see Supplemen-
tary material online, Figure S6). Pronounced acetylcholine-induced
contractions were observed in renal arteries of vehicle-treated
SHR (E
max
: 88.5+5.3% in SHR + vehicle, n 9) compared with
WKY (E
max
: 13.7+10.9% in WKY, n 10; Figure 8A and B). The
contractions were absent in rings without endothelium (Figure 8C).
Chronic oral treatment with calcitriol (150 ng/kg/day for 4.5
months) attenuated the contractions (E
max
: 40.9+10.6% in
SHR-receiving calcitriol, n 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 same
extent as seen with chronic treatment with calcitriol, by the acute
treatment with losartan, DPI, and tempol (Figure 8E).
The AT
1
R expression was elevated in SHR renal arteries
(Figure 8F, see Supplementary material online, Figure S7A) while
that of AT
2
R was not different from control (see Supplementary
material online, Figure S7B). The over-expression of AT
1
R was
Figure 5 Angiotensin (Ang) II induces vascular dysfunction in renal arteries from normotensive WistarKyoto rats (WKY). Exposure to
angiotensin 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 WistarKyoto rats. (AC) Combined exposure to calcitriol (100 nmol/L) enhanced
the relaxations and reduced the contractions. (CE) Losartan (3 mmol/L), diphenyleneiodonium (DPI, 100 nmol/L), and tempol (100 mmol/L)
reversed endothelial dysfunction in angiotensin II-treated WistarKyoto rat renal arteries. Data are means +SEM of four to ve 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, see
Supplementary material online, Figure S7A). Renal arteries of SHR
exhibited augmented contractions to Ang II and the augmentation
was prevented by a 12 h treatment with 100 nmol/L calcitriol (see
Supplementary material online, Figure S7C).
The level of oxidative stress as reected by the nitrotyrosine
content (Figure 8F, see Supplementary material online, Figure S8A)
was augmented in SHR renal arteries, as were the expressions
of NOX-2 and p67
phox
(Figure 8G, see Supplementary material
online, 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 corrected
the over-expression of NOX-2, p67
phox
, and nitrotyrosine, and
enhanced the expression of both SOD isoforms.
Renal arteries from SHR treated with vehicle exhibited an
increased ROS accumulation across their wall compared with
WKY preparations (Figure 9A). The elevated ROS level was
reduced in arteries from calcitriol-treated SHR (Figure 9A). By
contrast, 30-min calcitriol treatment did not reduce the arterial
ROS level, whereas it was acutely decreased by DPI, tempol, and
losartan (Figure 9B).
Discussion
The present study provides evidence of the protective effect of cal-
citriol, an active form of vitamin D, on human renovascular function
in hypertension. The major novel ndings include: (1) the impaired
endothelium-dependent relaxations in renal arteries fromhyperten-
sive patients can be partially restoredby a 12 h in vitro incubation with
calcitriol; (2) this improvement is accompanied by the normalization
of oxidative stress-related proteins including NOX-2, NOX-4,
p67
phox
, and SOD-1 and a resulting reduction in ROS levels; (3) cal-
citriol prevents the up-regulation of NOX-2, NOX-4, and p67
phox
and the increase in ROS levels induced by Ang II in renal arteries
fromnormotensive patients and in HAEC; (4) the effects of calcitriol
are antagonized by the specic human VDR antagonist, TEI-9647,
pinpointing the positive role of this receptor in the protective
effects of calcitriol; (5) the ndings in human arteries are supported
by similar results obtained in isolated renal arteries harvested from
SHR and in those from WKY exposed to Ang II; (6) substantiating
the in vitro ndings, chronic in vivo oral administration of calcitriol
reverses renovascular dysfunction in the SHR as reected by the
reduction in endothelium-dependent contractions which most
likely result from the down-regulation of AT
1
R, NOX-2, and
p67
phox
, and the up-regulation of SOD-1 and SOD-2; and (7) calci-
triol per se does not possess radical scavenging activity but reduces
oxidative stress by transcriptional regulation of the radical generating
and scavenging enzymes.
ROS are involved in the impairment of vascular function and
the development of hypertension.
24
They impair endothelium-
dependent relaxations and facilitate endothelium-dependent
contractions.
20,2527
These radicals are generated by various
enzymes, of which NAD(P)H oxidase represents the major
source. 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 oxidative
stress-related proteins. (A and B) Tissue culture with calcitriol (100 nmol/L) or losartan (3 mmol/L) for 12 h reduced contractions of quiescent
spontaneously 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) AT
1
R, (D) NOX-2
and (E) NOX-4. Data are means +SEM of four to ve experiments. **P , 0.01, ***P ,0.001 vs. WistarKyoto rats;
##
P , 0.01,
###
P , 0.001
vs. 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 ROS
production, the activity of the local RAS is increased in the vascular
tissue of hypertensive patients, which helps to explain why AT
1
R
blockers represent a major class of anti-hypertensive drugs.
29,30
The present study shows that losartan and DPI restore the
impaired endothelium-dependent relaxations and reduce the
ROS levels in renal arteries from hypertensive patients, thus
conrming the critical role of AT
1
R activation and ROS over-
production in the vascular dysfunction of human hypertension.
Vitamin D is a potent endogenous suppressor of the RAS
because it inhibits renin transcription both in vitro and in vivo.
31
Chronic treatment with calcitriol or other active forms of
vitamin D prevents the development of cardiac hypertrophy in
the SHR by suppressing the cardiac RAS.
32
The present ndings reveal the regulatory role of vitamin D on
the RAS and on ROS production in renal arteries of both hyper-
tensive humans and animals. First, in vitro calcitriol corrects the
abnormal expression of AT
1
R, the radical generating [NAD(P)H
oxidase with its subunits NOX-2, NOX-4, and p67
phox
] and scav-
enging (SOD-1) enzymes and the resulting augmented ROS levels
in the renal arteries from hypertensive patients, in Ang II-treated
renal 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 in
vitro and in vivo but also those treatments abolish the exaggerated
endothelium-dependent contractions in renal arteries from SHR
and WKY, the latter after incubation with Ang II. The reductions
in endothelium-dependent relaxations and the unmasking of
endothelium-dependent contractions are sensitive to an AT
1
R
antagonist, an inhibitor of NAD(P)H oxidase and ROS scavengers,
indicative of a pathogenic involvement of AT
1
R and NAD(P)H
oxidase. Third, the western blot analysis shows an up-regulation
of AT
1
R and NAD(P)H oxidase subunits, NOX-2 and p67
phox
,
and a down-regulation of SOD-1 and SOD-2 in SHR arteries com-
pared with those of WKY, and these alterations are normalized by
calcitriol. The reductions in the mRNA and protein levels of AT
1
R
may account for a diminished contraction in response to Ang II.
Fourth, ROS detection by dihydroethidium uorescence and EPR
in aortic endothelial cells and SHR renal arteries conrms an
ROS overproduction, which can be reduced by both in vitro and
chronic in vivo calcitriol treatment. Taken in conjunction, the
present results strongly suggest a regulatory role of vitamin D
on RAS activity and ROS production in the vascular wall, which
becomes particularly obvious in hypertension.
The present study, in demonstrating the inhibitory effect of a
specic antagonist against human VDR TEI-9647,
33
establishes a
crucial 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 patients
were partially rescued by 12 h incubation with calcitriol, and this
was 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) renal
arteries. (A) Dihydroethidium uorescence showed that spontaneously hypertensive rats renal arteries exhibited elevated reactive oxygen
species level compared with WistarKyoto rat (WKY) arteries. Twelve-hour calcitriol treatment reduced the reactive oxygen species level
and this effect was abolished by actinomycin-D (AD; 2 mmol/L). In contrast, acute exposure (30 min) of spontaneously hypertensive rat
renal arteries to calcitriol (100 nmol/L) did not reduce reactive oxygen species level. (B) The reactive oxygen species level measured by
EPR was greater in renal arteries from spontaneously hypertensive rats compared with those from WistarKyoto rats and calcitriol
reduced the elevated reactive oxygen species level in spontaneously hypertensive rat renal arteries. Data are means +SEM of four experiments.
***P ,0.001 vs. WistarKyoto rats;
###
P ,0.001 vs. spontaneously hypertensive rat without treatments.
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calcitriol exerts its benecial effects via VDR activation. Consist-
ently, 12 h incubation with Ang II increases the ROS level in
HAEC, and this is prevented by both calcitriol and the anti-oxidant
tempol. However, only the effect of calcitriol, but not that of
tempol, is antagonized by TEI-9647, demonstrating that the
protective effect of calcitriol can indeed be attributed to VDR
activation.
The vascular protective effect of calcitriol observed in the
present study cannot be explained by direct scavenging of ROS
as 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-dependent
contractions reported in the SHR aortae, which is non-genomic
in nature and has been attributed to inhibition of endothelial
calcium inux.
13
Instead, calcitriol exerts its effect through
genomic regulation. This conclusion is prompted by the present
measurements of the protein expression of radical generating
and scavenging enzymes, with calcitriol reducing the expression
of the former [NAD(P)H oxidase and its subunits] but augmenting
that of the latter (SOD-1 and SOD-2). The genomic impact of cal-
citriol is conrmed by the experiments with the mRNA synthesis
inhibitor, actinomycin-D, which abolishes its protective effect
against endothelium-dependent contractions and increases in
ROS levels. A genomic action is consistent with the conclusion
that the effects of calcitriol reported in the present study are
due to VDR activation. Indeed, upon binding of and activation by
vitamin D, VDR forms a heterodimer complex with the retinoid
X receptors (RXR). The VDRRXR complex can bind to
specic DNA sequences, termed vitamin D responsive elements,
located in the promoter regions of various vitamin-D-dependent
genes.
34
Because of the limited supply of renal arteries from patients
after informed consent, a limitation of the present study is the
small sample size of the human specimens available with an un-
avoidable variability due to the differences in disease progression
between the donors. However, since treated preparations were
compared systematically with untreated tissues from the same
patient, consistent results were obtained, conrming the critical
role of RAS and ROS in hypertension-associated vascular dysfunc-
tion and demonstrating the protective effects of calcitriol and its
antagonism by the vitamin D receptor blocker (TEI-9647). The
conclusions reached were comforted by experiments on cultured
human endothelial cells. They were fully supported by the studies
on animal blood vessels, where the source of the studied
material is adequately controlled. Thus, it seems reasonable to
accept that the current data on human blood vessels indeed
demonstrate the vascular protective effects of calcitriol in human
hypertension.
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) concentrationcon-
traction curves showing the inhibitory effect of calcitriol on acetylcholine (ACh)-induced contractions, which were endothelium dependent as
reected by the absence of contractions in rings without endothelium (-Endo, C). Endothelium-dependent contractions were unaffected by
acute exposure (30-min) to calcitriol (100 nmol/L) (D), but attenuated by losartan (3 mmol/L), diphenyleneiodonium (DPI, 100 nmol/L), and
tempol (100 mmol/L) (E). Data are means +SEM of ve experiments. ***P ,0.001 vs. WistarKyoto rat, +Endo, or spontaneously hyper-
tensive rat control;
###
P ,0.001 vs. spontaneously hypertensive rat treated with vehicle. Representative blots showing that calcitriol
reduced the over-expression of AT
1
R, nitrotyrosine (F), NOX-2, p67
phox
(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 ndings substantiate the capability of chronic treat-
ment with vitamin D to reduce ROS level in rat arteries
12
but
provide novel insights into the possible mechanisms by which it
regulates radical homeostasis. Calcitriol is a potent suppressor of
the RAS.
31
Since the AT
1
R expression is decreased following calci-
triol treatment, the modication of the NAD(P)H and SOD
expressions and the reduction in ROS level observed upon
chronic treatment with the vitamin may be due in part to a
lesser presence of AT
1
R. The latter is a key player in the pathogen-
esis of hypertension and its activation serves as a primary trigger
for ROS production by NAD(P)H oxidase.
35
Cyclooxygenases
(COX) are the major source of endothelium-derived contracting
factors and ROS facilitate their production/action.
25,26,36
Chronic
administration of calcitriol reduces the expression of COX-1 in
the rat aortae.
12
Paricalcitriol, another active form of vitamin D,
reduces excessive ROS production by down-regulating
pro-inammatory factors such as inducible nitric oxide synthase,
tumor necrosis factor-a, and COX-2.
37
Endothelium-dependent
contractions of the SHR renal arteries are prevented by inhibitors
of cyclooxygenase.
38
Thus, it is likely that the reduction in ROS
production demonstrated in the present study leads to decreased
expression/presence of endothelial cyclooxygenase(s) with in turn
attenuation of endothelium-dependent contractions caused by the
exposure to calcitriol.
The present study conrms that chronic oral administration of
calcitriol reduces the elevated blood pressure of the SHR.
12
Whether or not there is a relationship between low plasma
levels of vitamin D and elevated arterial blood pressure remains
controversial.
3
Nevertheless, the present ndings, together with
those from limited clinical and animal studies
12,39,40
support the
view that vitamin D supplementation can lower arterial blood
pressure. This may be due to indirect effects and not necessarily
to a direct genomic action on the blood vessel wall. However,
the protective effect of calcitriol on renal arteries is not likely to
be secondary to the blood pressure reduction. Indeed, in the
present study, the inhibition by calcitriol on the exaggerated
endothelium-dependent contractions of isolated SHR renal arter-
ies was similar after a 12 h in vitro incubation and after chronic
in vivo treatment, strongly suggesting that calcitriol improves
endothelial function directly.
In conclusion, chronic treatment with calcitriol protects against
renovascular function in hypertension. The calcitriol-induced pro-
tection is likely to be mediated by VDR activation leading to down-
regulation of the expressions of AT
1
R, NAD(P)H subunits and
up-regulation of SOD-1 and SOD-2. These in turn prevent the
ROS overproduction. The ndings in human renal arteries and
human endothelial cells are conrmed both by in vitro and in vivo
results in the animal. The present study suggests calcitriol/VDR
activation as a novel therapeutic strategy to ameliorate
hypertension-associated vascular dysfunction.
Supplementary material
Supplementary material is available at European Heart Journal
online.
Figure 9 Chronic in vivo treatment with calcitriol decreases the reactive oxygen species level in spontaneously hypertensive rat (SHR) renal
arteries. (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 ve experiments. *P ,0.05 vs. WistarKyoto rats (WKY);
#
P ,0.05 vs. spontaneously hypertensive rats + vehicle. (B) Acute exposure (30-min) of spontaneously hypertensive rat renal arteries to
diphenyleneiodonium (DPI, 100 nmol/L), tempol (100 mmol/L), and losartan (3 mmol/L) but not calcitriol (100 nmol/L) or dimethyl sulfoxide
normalized the reactive oxygen species level. Data are means +SEM of four experiments. ***P ,0.001 vs. WistarKyoto rats;
###
P ,0.001
vs. spontaneously hypertensive rat control.
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Acknowledgements
We acknowledge the kind gift of TEI-9647 by Teijin Pharma
Limited (Tokyo, Japan).
Funding
This work was supported by National Basic Research Program of
China (2012CB517805), Hong Kong Research Grant Council
(CUHK466110), and Focused Investment Scheme of the Chinese Uni-
versity of Hong Kong.
Conict of interest: none declared.
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