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Cannabidiol causes endothelium-dependent
vasorelaxation of human mesenteric arteries
via CB
1
activation
Christopher P. Stanley, William H. Hind, Cristina Tufarelli, and Saoirse E. O’Sullivan*
School of Medicine, University of Nottingham, Royal Derby Hospital, Derby DE22 3DT, UK
Received 4 September 2014; revised 20 May 2015; accepted 12 June 2015
Time for primary review: 41 days
Aims The protective effects of cannabidiol (CBD) have been widely shown in preclinical models and have translated into
medicines for the treatment of multiple sclerosis and epilepsy. However, the direct vascular effects of CBD in humans
are unknown.
Methods
and results
Using wire myography, the vascular effects of CBD were assessed in human mesenteric arteries, and the mechanisms of
action probed pharmacologically. CBD-induced intracellular signalling was characterizedusing human aortic endothelial
cells (HAECs). CBD caused acute, non-recoverable vasorelaxation of human mesenteric arteries with an R
max
of
40%. This was inhibited by cannabinoid receptor 1 (CB
1
) receptor antagonists, desensitization of transient receptor
potential channels using capsaicin, removal of the endothelium, and inhibition of potassium efflux. There was no role for
cannabinoid receptor-2 (CB
2
) receptor, peroxisome proliferator activated receptor (PPAR)g, the novel endothelial
cannabinoid receptor (CB
e
), or cyclooxygenase. CBD-induced vasorelaxation was blunted in males, and in patients
with type 2 diabetes or hypercholesterolemia. In HAECs, CBD significantly reduced phosphorylated JNK, NFkB,
p70s6 K and STAT5, and significantly increased phosphorylated CREB, ERK1/2, and Akt levels. CBD also increased
phosphorylated eNOS (ser1177), which was correlated with increased levels of ERK1/2 and Akt levels. CB
1
receptor
antagonism prevented the increase in eNOS phosphorylation.
Conclusion This study shows, for the first time, that CBD causes vasorelaxation of human mesenteric arteries via activation of CB
1
and TRP channels, and is endothelium- and nitric oxide-dependent.
-----------------------------------------------------------------------------------------------------------------------------------------------------------
Keywords Vasorelaxation †Human †Cannabidiol †Cannabinoid †Endothelium
1. Introduction
Numerous studies have shown that endogenous, synthetic, and plant-
derived cannabinoids cause vasorelaxation of a range of animal and
human arterial beds.
1,2
The extent of cannabinoid-induced vasorelaxa-
tion and the mechanisms involved often differs between the cannabin-
oid compound studied, the arterial bed used, and the species
employed. These mechanisms include activation of cannabinoid recep-
tor one (CB
1
), cannabinoid receptor two (CB
2
), transient receptor
potential vanilloid one (TRPV1), peroxisome proliferator activated re-
ceptor gamma (PPARg), and an as yet unidentified endothelial-bound
cannabinoid receptor (CB
e
).
1,2
Vasorelaxant mediators implicated in
cannabinoid-induced vasorelaxation include nitric oxide production,
prostaglandin production, metabolite production, and ion channel
modulation, some of which have been shown to be coupled to recep-
tor activation.
1,2
Cannabidiol (CBD) is a naturally occurring molecule found in the plant
Cannabis sativa. Unlike the related molecule D
9
-tetrahydrocannabinol
(THC), it does not activate CB
1
receptors in the brain, and is devoid
of the psychotropic actions of THC. Indeed, CBD may antagonize
the psychoses associated with cannabis abuse.
3
Other receptor sites
implicated in the actions of CBD include the orphan G-protein-coupled
receptor GPR55, the putative endothelial cannabinoid receptor (CB
e
),
the transient receptor potential vanilloid 1 (TRPV1) receptor,
a1-adrenoceptors, mopioid receptors and 5-HT
1A
receptors,
4,5
A
CBD/THC combination (1 : 1 ratio, Sativex/Nabiximols) is currently
licensed internationally in more than 20 countries for the treatment
of spasticity in multiple sclerosis, and an as yet unlicensed CBD alone
*Corresponding author. Email: mbzso@nottingham.ac.uk
&The Author 2015. Published by Oxford University Press on behalf of the European Society of Cardiology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
Cardiovascular Research
doi:10.1093/cvr/cvv179
Cardiovascular Research Advance Access published June 30, 2015
by guest on July 7, 2015Downloaded from
product (Epidiolex) has entered an expanded access programme
in children with intractable epilepsies. CBD has also received orphan
designation status in treating newborn children with neonatal
hypoxic-ischaemic encephalopathy.
In addition to the licensed indications, preclinical evidence suggests
CBD has therapeutic potential in diseases associated with inflamma-
tion, oxidative stress, gastrointestinal disturbances, neurodegeneration,
cancer, diabetes, and nociception.
6–10
In the cardiovascular system,
CBD treatment in vivo reduces endothelial and cardiac dysfunction in
cardiomyopathy associated with diabetes.
11,12
CBD also reduces vas-
cular inflammation associated with endotoxic shock,
13
has a protective
role in diabetic retinopathy,
14
and is cardioprotective after coronary ar-
tery ligation.
15
Furthermore, CBD reduces infarct size and increases
cerebral blood flow in a mouse model of stroke when delivered either
pre- or post-ischaemia through activation of 5-HT
1A
receptors.
16 –19
Unlike other cannabinoids, the direct vascular effects of CBD have
not been fully investigated in either animal or human studies.
1
Jarai
et al.
20
showed that CBD (10 mmol/L) had no effect on vascular
tone in the perfused mesenteric arterial bed of mice. However, Offer-
taler et al.
21
reported that CBD caused a concentration-dependent
near maximal vasorelaxation of isolated rat mesenteric arteries, but
no mechanisms of action were probed. In the rat isolated aorta, we
showed that CBD causes a time-dependant vasorelaxant response
that was inhibited by antagonism of the PPARgreceptor and inhibition
of superoxide dismutase.
22
In light of the increasing evidence that CBD has beneficial effects on
the cardiovascular system, and since the vascular effects of CBD remain
to be characterized in human vasculature, the aim of the present study
was to establish the acute vascular effects of CBD in human arteries and
to underpin the pharmacology behind any potential response.
2. Methods
Ethical approval was granted by the Derbyshire Research Ethics Committee
and Derbyshire Hospitals Trust Research and Development to take mesen-
teric tissue from patients (27 males, 10 females) undergoing colorectal sur-
gery. Informed consent was gained in accordance with the Declaration of
Helsinki. Mesenteric arteries have been extensively used to characterize
the pharmacological effects of cannabinoids.
1
Excised mesenteric tissue
was placed in physiological saline solution (PSS) and transported back to
the lab. Arteries (701 +42 mmdiameter,mean+SEM) were dissected
from mesenteric tissue, cleaned of any adherent fatty and connective tissue
and cut into 2 mm segments. Artery segments were either used fresh or
after overnight storage in PSS at 48C. Overnight storage had no significant
effect on the contractile or relaxation responses of mesenteric arteries (see
Supplementary material online, Figure S1). Artery segments were mounted
on tungsten wires on a Mulvany-Halpern myograph (Danish Myo Technol-
ogy, Denmark) at 378C in PSS solution and gassed with 5% CO
2
in O
2
. Ten-
sion was measured using isometric force displacement transducers and
recorded using Chart 5 Pro (ADinstruments, Oxfordshire, UK). Using nor-
malization software, arteries were set to an internal diameter producing
90 mmHg pressure. To establish artery viability, the ability of arteries to
contract to high potassium PSS (KPSS) (composition, mmol/L: NaCl 0,
KCl 124, CaCl
2
.2H
2
O2.5,MgSO
4
.7H
2
O1.17,NaHCO
3
25, KH
2
PO
4
1.18, C
10
H
16
N
2
O
8
0.027, C
6
H
12
O
6
5.5 all dissolved in triple distilled water)
or to contract to U46619 (.5 mM), and to relax to 10 mmol/L bradykinin
(.70% relaxation) was measured.
2.1 Experimental protocol
Viable arteries were contracted using a combination of U46619 (50–
250 nmol/L) and Endothelin-1 (1– 3 nmol/L). Once a stable contraction
.5 mN was achieved, cumulative concentration –response curves were
constructed to CBD. CBD was added in 5-min intervals with measure-
ments taken in the final minute of each concentration addition and ex-
pressed as percentage relaxation of pre-imposed tone. Responses were
compared with ethanol-treated vehicle controls carried out in adjacent
arterial segments from the same patient. To characterize mechanisms
underpinning CBD-induced vasorelaxation, all interventions were com-
pared with a CBD control–response carried out in adjacent arteries
fromthesamepatient.Insomeexperiments,theendotheliumwasre-
moved by abrasion using a human hair. A role for the involvement of nitric
oxide was investigated using NG-nitro-L-arginine methyl ester (L-NAME,
300 mmol/L, present throughout). A role for cyclooxygenase (COX) was
assessed using indomethacin (10 mmol/L, present throughout). A potential
role for potassium channel hyperpolarization was investigated by carrying
out concentration – response curves to CBD in arteries contracted using
KPSS to inhibit potassium efflux. Potential cannabinoid receptor involve-
ment in CBD-induced vasorelaxation was assessed with CB
1
antagonist
(AM251, 100 nmol/L or LY320135 1 mmol/L), CB
2
receptor antagonist
AM630 (100 nmol/L), or proposed endothelial cannabinoid receptor
(CB
e
, O1918, 10 mmol/L). Desensitization of sensory nerves was achieved
via incubation (1 h) with capsaicin (10 mmol/L) followed by three washouts
in PSS. In experiments to establish the potential location of the CB
1
recep-
tor, the effects of AM251 in endothelial-denuded arteries were compared
with arteries that were endothelial denuded only, arteries treated with
AM251 only and CBD control arteries. In each of these protocols, there
was no significant difference in the level of contraction immediately before
the CBD concentration response curve.
2.2 Cell culture
Human aortic endothelial cells (PromoCell, Germany, passage 4) were
grown in PromoCell Endothelial Cell Growth medium to confluence on
6-well plates and treated for 10 min with increasing concentrations of
CBD, after which time the medium was removed and the cells collected
in cell lysis buffer (RIPA buffer, SigmaAldrich) with phosphatase and prote-
ase inhibitors (Roche). Some experiments were performed in the presence
of AM251 or capsazepine. The protein concentration of the cell lysate was
measured using a BCA assay (BCA-1KT, SigmaAldrich). The levels of phos-
phorylated ERK/MAP kinase 1/2 (Thr185/Tyr187), Akt (Ser473), STAT3
(Ser727), JNK (Thr183/Tyr185), p70 S6 kinase (Thr412), NFkB (Ser536),
STAT5A/B (Tyr694/699), CREB (Ser133), and p38 (Thr180/Tyr182)
were measured in cell lysates using the LuminexwxMAP
w
technology using
a commercially available panel (MilliplexTM, 48-680MAG, Merck Millipore),
and normalized to total protein content. eNOS phosphorylation was mea-
sured using a PathScan Phospho-eNOS (ser1177) sandwich ELISA accord-
ing to the manufacturer’s instructions (Cell Signaling Technology, USA), and
was normalized to total protein content.
2.3 Reverse transcription-polymerase chain
reaction
The presence of target sites of action was investigated at the mRNA level
using reverse transcription followed by polymerase chain reaction
(RT-PCR) under control conditions, and in the presence of a high glucose
(25 mM) or high insulin (500 nM) medium for 96 h. Human astrocytes
(HAs) were used as a positive control known to express all the target sites
of action of interest.
23
Total RNA was extracted from HAs and HAECs
using Allprep DNA/RNA kit with on column DNaseI treatment (Qiagen,
Germany). Reverse transcription with and without reverse transcriptase
was performed in 30 ml final volume using 3 mg of total RNA and random
primers with the High Capacity cDNA Reverse Transcription Kit (Life
Technologies, UK) according to the manufacturer’s instructions. PCRs
were carried out in a final volume of 25 ml with Zymotaq (ZymoResearch,
USA) using 2 ml of reverse transcription product as the template. Primer
pairs used to amplify 128 bp of the control house-keeping gene
C.P. Stanley et al.Page 2 of 11
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Hypoxanthine-guanine PhosphoRibosylTransferase (HPRT) were from ref.
24; those for 99 bp PPARaand 87 bp PPARgwere from ref. 25; those for
303 bp CB1R and 365 bp CB2R were from ref. 26; those for 511 bp TRPV1
were from ref. 27; and finally the 380 bp calcitonin gene-related peptide
(CGRP) receptor (CGRPR) cDNA fragment was amplified using the pri-
mers reported in ref. 28. After 5 min at 958C, PCRs were performed for
40 cycles except those for CB2R that was carried out for 50 cycles. The
cycles included 30 s at 958C, 30 s at the annealing temperature that was op-
timal for each primer pair (568C for CB1R and CB2R; 608C for all others)
and a final extension step of 30 s at 728C. Amplification products were se-
parated by gel electrophoresis through ethidium bromide stained 2% agar-
ose (CB1R, CB2R, TRPV1, CGRPR) and 3% NuSieve 3:1 (PPARa, PPARg
and HPRT) and visualized using a Biorad Chemidoc.
2.4 Statistical analysis
Graphs represent mean percentage relaxations, with error bars represent-
ing the standard error of the mean (SEM) fit to non-linear Regression
(Curve Fit) (Prism Version 6; GraphPad Software, CA, USA). nrepresents
the number of arteries from patients. Comparisons between intervention
and control artery segments from the same patient were made using R
max
(the calculated maximal response to CBD) and EC
50
(potency of CBD)
compared by Student’s t-test. In experiments to assess the location of
the CB
1
receptor, comparisons were made between artery segments
from the same patient using one way analysis of variance (ANOVA) with
Dunnetts post-hoc analysis. Significance was determined as P,0.05.
2.5 Chemicals
All salts, L-NAME, indomethacin and bradykinin were supplied by Sigma
Chemical Co. (Poole, UK). AM251, LY 320135, AM630, and capsaicin
were purchased from Tocris (Bristol, UK). CBD was a kind gift from GW
Pharmaceuticals (Wiltshire, UK). L-NAME and indomethacin were dis-
solved in PSS solution. CBD, bradykinin, and capsaicin were all dissolved
in ethanol at 10 mM with further dilutions made in distilled water.
AM251, LY320135, and AM630 were dissolved in DMSO at 10 mmol/L
with further dilutions made in distilled water.
3. Results
Thirty-four patients (24 males and 10 females) were recruited for this
study. Twenty-seven had cancer and 7 had inflammatory bowel
disorder. A summary of patient characteristics, medical history, and
medications is presented in Table 1.
CBD caused vasorelaxation of pre-constricted human mesenteric
arteries with an R
max
of around 40% vasorelaxation (R
max
P,0.0001
compared with vehicle control, n¼12, Figure 1Aand C,Table 2). For
comparison, the vasorelaxant response to 10 mmol/L bradykinin
(83 +3(mean+SEM) % relaxation) in the same patients is repre-
sented in Figure 1C. When added to un-contracted arteries, CBD had
no effect on baseline tone (n¼6, representative raw trace shown in
Figure 1A). In time-dependent experiments, a single concentration of
10 mmol/L CBD caused an initial vasorelaxation of 57 +4% relaxation
at 15 min, developing to 78 +7% at 120 min (P,0.001, n¼6,
Figure 1D).
Removal of the endothelium significantly reduced the potency
(EC
50
) of CBD (P,0.0001, Figure 2A,Table 2). The maximum vasore-
laxation to CBD also correlated positively with the endothelium-
dependent bradykinin response in patients (r¼0.394, P¼0.0158,
Figure 2B). Inhibition of COX activity using indomethacin had no effect
on the CBD-induced vasorelaxation (n¼6, Figure 2C). In arteries
....................................................................................
Table 1 Patient characteristics, diagnosis, and
medications
Characteristic Range Mean +++++ SEM
Ethnicity 34 UK white
Male 24
Female 10
Age 36 –82 65 +2.1
Weight (kg) 52 –126 76 +3
BMI (kg/m
2
) 17.5 –36.4 27.1 +0.7
Vessel size (mm) 346– 1372 701 +42
Bradykinin response (% relaxation) 70 –109 85 +1.4
Smoking habits
Non-smokers 28
0–10 CPD 3
10– 20 CPD 3
Drinking habits
,10 units p/w 23
10– 20 units p/w 7
.20 units p/w 4
Operation
Right hemicolectomy 10
Left hemicolectomy 7
Sigmoid colectomy 5
Anterior resection 10
Abdominoperineal resection 1
Total colectomy 1
Reason for surgery
Cancer 27
Inflammatory bowel disorder 7
Dukes Staging
Dukes A 10
Dukes B 9
Dukes C 8
Dukes D 0
Systolic blood pressure (mm/Hg) 110 –188 143+3
Diastolic blood pressure (mm/Hg) 62 –101 82 +1
Diabetic 10
Heart disease 9
Heart failure 0
Hypercholesterolemia 15
Hypertensive 16
a-1-adrenoceptor antagonist 3
ACE inhibitors 7
AT1 receptor antagonists 2
Beta-blockers 6
Calcium-channel blocker 3
Digoxin 2
Diuretics 3
GTN 3
Hypoglycaemic medication 6
Nsaid medication 14
Statin 14
Thiazolidinedione 1
CBD Induced vasorelaxation of human arteries Page 3 of 11
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contracted using high potassium physiological salt solution (KPSS),
CBD-induced vasorelaxation was significantly inhibited (R
max
P,0.001, n¼5Figure 2D). Although incubation with L-NAME did
not significantly affect the concentration– response curve to CBD
(Figure 2B,Table 2), a trend for a reduction in the vasorelaxant effect
of CBD was seen. Therefore, in cultured endothelial cells, we tested
whether CBD affects eNOS activation and found that CBD
(10 mmol/L, 10 min) significantly increased eNOS phosphorylation at
ser1177 (P,0.05, n¼9, Figure 2F). Neither endothelium-denudation,
L-NAME, or KPSS contraction affected control vasorelaxant responses
(see Supplementary material online, Figure S2).
Antagonism of the CB
1
receptor using AM251 (100 nmol/L) signifi-
cantly inhibited CBD-induced vasorelaxation (R
max
P,0.001, n¼9,
Figure 3A,Table 2). To confirm this result, a second, structurally differ-
ent antagonist LY320135 was used, which also significantly reduced the
maximal response to CBD (CBD R
max
45 +3.5; CBD&LY R
max
30 +
5.4, P,0.05, Table 2). Antagonism of the CB
2
receptor using AM630
(100 nmol/L) had no effect on CBD-induced vasorelaxation (n¼8,
Figure 3C). Desensitization of TRP channels using capsaicin (10 mmol/
L) reduced CBD-induced vasorelaxation (P,0.0001, n¼7,
Figure 3B). Antagonism of the proposed CB
e
receptor using O-1918
(10 mmol/L, n¼7, Figure 3D) had no effect on the CBD-induced vasor-
elaxation. In the presence of the PPARgantagonist GW9662, neither
the immediate nor the time-dependent vasorelaxation was inhibited
(n¼5, representative raw trace shown in Figure 1B). Neither
AM251, LY320135, or capsaicin pre-treatment affected control vasor-
elaxant responses (see Supplementary material online, Figure S2).
In experiments to determine the location of the CB
1
receptor,
AM251, and endothelial denudation were compared in combination
and individually against control CBD responses, obtained from adjacent
segments of artery from the same patients (n¼6, Figure 4A). AM251
alone, and AM251 plus denudation, resulted in a significant reduction
in the maximal response (R
max
)toCBDtosimilarextent(P,0.05,
Figure 4C). However, when looking at the entire concentration re-
sponse curve to CBD (AUC values), the combination of AM251 and
endothelial denudation had a more significant (P,0.01) reduction
than AM251 alone (P,0.05, Figure 4B).
Across the 37 patients tested, considerable variability of control
responses to CBD was observed among patients (the maximal re-
sponse to CBD ranged from 2 to 75% relaxation), so post hoc analysis
was carried out to establish any relationships between CBD responses
and patient characteristics (see Supplementary material online, Table S1
and Figures 3and 4). CBD responses were slightly reduced in males
compared with females (P¼0.0166), but were not affected by age,
BMI, or smoking status. Looking at concurrent diseases, CBD re-
sponses were reduced in patients with type-2 diabetes (P,0.0001),
Figure 1 CBD relaxes human mesenteric arteries. Typical trace data showing the acute (A) and time-dependent (B) vasorelaxant effects of CBD (also
in the presence of the PPARgamma antagonist GW9662) in the human mesenteric artery. (C) Mean (+SEM, n¼12) concentration-response curves to
CBD compared with vehicle controls carried out in adjacent segments of mesenteric artery from the same patient. The vasorelaxant response to
10 mmol/L bradykinin in the same patients is shown for comparison. (D) Mean time-dependent vasorelaxant response to a single concentration of
CBD (10 mmol/L) compared with vehicle controls carried out in adjacent segments of mesenteric artery (n¼6). R
max
and EC
50
values were compared
by paired Students t-test, *P,0.05, ****P,0.0001.
C.P. Stanley et al.Page 4 of 11
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hypercholesterolemia (P¼0.0320), but not different in patients with
cancer, heart disease, or hypertension (Supplementary material online,
Figure S4). CBD responses were reduced in those taking statins
(P¼0.0042), hypoglycaemic medication (P,0.0001) and beta-blockers
(P¼0.0094), but not those taking ACE inhibitors or NSAIDs (Supple-
mentary material online, Figure S4).
To establish the intracellular mechanisms activated by CBD, human
aortic endothelial cells were treated for 10 min with increasing concen-
trations of CBD. This led to a significant reduction in phosphorylated
JNK (Figure 5B), NFkB(Figure 5C), p70s6 K (Figure 5G), and STAT5
(Figure 5I). CBD also significantly increased phosphorylated CREB
(only at 30 mM, Figure 5A), ERK1/2 (Figure 5E), and Akt (Figure 5F). In
the presence of the CB
1
receptor antagonist AM251 (100 nM) or the
TRPV1 antagonist capsazepine (1 mM), CBD no longer significantly in-
creased phosphorylated ERK1/2 (Figure 6A). The increase in phos-
phorylated Akt was only inhibited by AM251 (Figure 6B). The levels
of phosphorylated ERK1/2 (P¼0.0379, R ¼0.3639) and Akt (P¼
0.0343, R ¼0.3749), but none of the other intracellular signalling path-
ways, were positively correlated with the increase in phosphorylated
eNOS levels (Figure 6C). In the presence of AM251, the increase in
phosphorylated eNOS was no longer significant (Figure 6D).
As the CBD vasorelaxant responses were blunted in patients with
type-2 diabetes, we carried out RT-PCR in human aortic endothelial
cells (HAECs) to establish the effects of a high glucose (25 mM) or
high insulin (500 nM) environment on the expression of the relevant
target sites at the RNA level. Human astrocytes were used a positive
control for these target sites.
23
In HAECs, all targets (PPARaand g,
CB1R, CB2R, TRPV1, and CGRPR) were found to be present in control
conditions (see Figure 7). After 96 h in either a high insulin or high
glucose medium, the expression of CB2R appeared increased, and
the expression of TRPV1 and CGRPR appeared decreased (see
Figure 7).
4. Discussion
This is the first study to show that CBD-induces vasorelaxation in
human mesenteric arteries which is dependent on CB
1
and TRP recep-
tor activation, the endothelium, nitric oxide, and potassium channel
modulation. CBD-induced vasorelaxation is reduced in males, and in
patients with type-2 diabetes, hypercholesterolemia and in patients
taking statins, beta blockers and hypoglycaemic medication.
We found that CBD causes half-maximal vasorelaxation with a
pEC
50
in the mid-micromolar range. Similar findings have been re-
ported in the rat mesenteric artery, where CBD causes vasorelaxa-
tion with mid-micromolar potency, however, in the rat model CBD
caused near maximal vasorelaxation. This might suggest that the
efficacy of CBD is reduced in human vasculature. However, it should
be noted that the present studies were performed in older patients
with a variety of comorbidities and medications, while animal studies
are performed in same gender, young homogenous populations. As
we observed that some diseases and medications were associated
with lower responses to CBD, this might account for the apparent
reduced efficacy in humans. As no mechanistic studies with CBD in
animal tissue have yet been reported, we cannot compare the
mechanisms of action established in the present study with that
from animal tissue.
The endothelium mediates vasorelaxation of the CBD analogue
Abn-CBD, and this vasorelaxation is associated with activation of
the CB
e
receptor which is antagonized using O-1918.
21,29
We also
found that removal of the endothelium reduced responses to CBD
and that CBD vasorelaxant responses correlated with bradykinin re-
sponses, indicating an endothelial site of action for CBD. However, in
the presence of O-1918, CBD-induced vasorelaxation is unaltered,
suggesting that the endothelial component is not CB
e
.Wealsofound
that CBD responses tended to be reduced in the presence of
L-NAME. To explore this further, we found that CBD significantly in-
creased the phosphorylation of eNOS in human aortic endothelial
cells, suggesting that production of NO a least partially underlies
the endothelium-dependent vasorelaxant effect of CBD. The
present study also reports that CBD-induced vasorelaxation is
significantly inhibited in arteries contracted using high potassium
solution, as has been shown for the vascular response to many can-
nabinoids. This suggests a predominant mechanism of CBD-induced
vasorelaxation is activation of potassium channels and subsequent
hyperpolarization. Given the extent of inhibition caused by KPSS, it
is unlikely that potassium channel involvement is exclusive to the
endothelium.
Activation of CB
1
and CB
2
receptor has been implicated in
cannabinoid-induced vasorelaxation.
1
Since human vascular smooth
muscle and endothelial cells express these receptors,
30 –35
and CBD
has been shown to bind to these receptors at low micromolar concen-
trations,
36,37
they were considered as potential mechanisms underpin-
ning CBD-induced vasorelaxation. Antagonism of the CB
1
receptor in
two separate experiments using AM251 (see Figures 3and 4) revealed
inhibition of CBD-induced vasorelaxation, suggesting CB
1
is a target for
CBD. A second structurally different antagonist, LY320135, was also
found to inhibit the vasorelaxant response to CBD, further implicating
CB
1
receptor activation. Other authors have suggested that CBD may
....................................................................................
Table 2 The maximal vasorelaxant responses and
potency of CBD in human mesenteric arteries
Vehicle CBD n
R
max
10.2 +3.5 39.2 +4.0 ****
EC
50
24.98 +0.87 25.14 +0.21 12
Control CBD Intervention n
Minus endothelium R
max
51.6 +2.8 44.6 +3.8
EC
50
25.84 +0.18 25.21 +0.18 **** 8
L-NAME R
max
51.4 +4.9 39.1 +6.6
EC
50
25.39 +0.26 25.24 +0.35 6
Indomethacin R
max
50.4 +4.0 55.2 +4.6
EC
50
25.82 +0.26 25.26 +0.20 6
KPSS contracted R
max
49.7 +5.8 8.9+2.4 ***
EC
50
25.45 +0.30 25.59 +0.73 5
AM251 R
max
53.9 +3.7 24.2 +4.9 ***
EC
50
25.57 +0.19 25.53 +0.49 9
LY320135 R
max
45.0 +3.5 30.2 +5.4 *
EC
50
25.83 +0.24 25.88 +0.54 6
AM630 R
max
58.7 +3.9 59.5 +5.5
EC
50
25.56 +0.17 25.48 +0.23 8
Capsaicin
pre-treatment
R
max
47.7 +2.4 21.3 +3.9 ****
EC
50
25.92 +0.15 25.85 +0.39 7
O-1918 R
max
51.8 +2.8 43.8 +3.9
EC
50
25.68 +0.16 25.61 +0.26 7
Sigmoidal concentration-response curves to CBD were fitted using Prism and R
max
and
EC
50
values were compared by Student’s t test (with Welch’s correction for groups
with unequal standard deviations).
CBD Induced vasorelaxation of human arteries Page 5 of 11
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have indirect actions at CB
1
through inhibition of FAAH activity or
transport,
30
rather than direct activation. However, we have previously
shown that CBD is a more efficacious vasorelaxant of human mesen-
teric arteries that anandamide
38
and that the mechanisms of action
of CBD presented in the present study are different to those revealed
recently in our laboratory for the endocannabinoid 2-AG.
39
Despite
this, CBD has low affinity for CB
1
receptors so the possibility still exists
that some of the actions of CBD are through inhibition of endocanna-
binoid degradation. Antagonism of the CB
2
receptor using AM630 did
not inhibit CBD-induced vasorelaxation. This was unsurprising as CB
2
receptor activation is not commonly found to underpin the vasorelax-
ant effects of cannabinoids.
1
The CB
1
receptor is expressed in both human endothelial cells and
vascular smooth muscle cells.
32,35
In order to establish the location of
the CB
1
receptor mediated the vasorelaxant response to CBD, we
compared responses with CBD in arteries both denuded and treated
with AM251 to either intervention alone. Although the reduction in
the maximal response to CBD was similar in arteries treated with
AM251 alone as to both interventions, the entire responseto CBD (re-
presented by the AUC data) was more significantly reduced by the
combination of both interventions. We take this data to suggest that
CBD acts at CB
1
located on both the endothelium and smooth muscle.
CB
1
activation has been shown to be coupled to the release of NO.
40
In
support of this, we found that in human endothelial cells, CBD
increased the phosphorylation of eNOS, the mRNA of CB1R was pre-
sent, and in the presence of AM251, the increase in eNOS phosphor-
ylation by CBD was no longer significant.
Plant-derived cannabinoids are good activators of the TRPV channel
family
41
and CBD induces cancer cell apoptosis
42
and anti-hyperalgaesic
responses to inflammatory pain
43,44
through activation of TRPV chan-
nels. In the present study, desensitization of TRP channels by exposure
to the TRPV1 agonist capsaicin inhibited CBD-induced vasorelaxation,
implicating TRP activation. In the rat mesenteric artery, vasorelaxation
to two chemically closely related cannabinoids, THC and cannabinol,
are also inhibited by capsaicin pre-treatment, acting via the release of
the vasoactive neuropeptide calcitonin gene-related peptide
(CGRP).
45
Recent work showed that CGRP vasorelaxant responses
in human arteries are endothelium-independent,
46
suggesting the re-
sidual relaxation to CBD observed after endothelium-denudation is
probably the TRP component of this response. However, we also ob-
served that the increase in ERK caused by CBD in human endothelial
cells was inhibited by TRPV1 antagonism, indicating that TRP activation
on both the endothelium and smooth muscle cells could mediate some
of the effects of CBD.
Figure 2 Mechanisms of CBD-induced relaxation of human mesenteric arteries. Mean (+SEM) CBD-induced vasorelaxation of human mesenteric
arteries after removal of the endothelium (n¼8, A), in arteries incubated with L-NAME (300 mmol/L, n¼6, B), in the presence of the non-selective
COX inhibitor indomethacin (10 mmol/L, n¼6, D) or in arteries contracted using a high potassium (KPSS) Krebs (n¼5, E). (C) Maximal responses
to CBD correlated with the vasorelaxant response to the endothelium-dependent vasorelaxant bradykinin. (F) In cultured human aortic endothelial cells,
CBD (10 mmol/L, 10 min) increased eNOS phosphorylation at ser1177 (n¼9). Control responses to CBD and interventions were carried out in ad-
jacent segments of mesenteric artery from the same patient. R
max
and EC
50
values were compared by paired Students t-test, *P,0.05, **P,0.01,
***P,0.001, ****P,0.0001.
C.P. Stanley et al.Page 6 of 11
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Figure 4 Location of the CB
1
receptor. Mean CBD-induced vasorelaxation in control arteries, endothelial denuded arteries, in arteries incubated with
the CB
1
antagonist AM251 or in arteries that are endothelial denuded and incubated with AM251 (A) and the corresponding R
max
(B) and AUC (C) values
within each patient (n¼6). Control responses to CBD and the three interventions were carried out in adjacent segments of mesenteric artery from the
same patient. Data were compared using one way analysis of variance (ANOVA) with Dunnett’s post hoc analysis comparing against the CBD control data.
*P,0.05, **P,0.01.
Figure 3 Target sites of action for CBD-induced relaxation of human mesenteric arteries. CBD-induced vasorelaxation of human mesenteric arteries
after 10 min incubation (pre-contraction) with the CB
1
antagonist AM251 (100 nmol/L, n¼9, A), the CB
2
antagonist AM630 (100 nmol/L, n¼8, C), the
proposed endothelial receptor (CB
e
) antagonist O-1918 (10 mmol/L, n¼7, D), or after desensitization of sensory nerves by 1 h pre-treatment with the
TRPV1 agonist capsaicin (10 mmol/L, n¼7, B). Control responses to CBD and interventions were carried out in adjacent segments of mesenteric artery
from the same patient. R
max
and EC
50
values were compared by paired Students t-test ,*P,0.05, **P,0.01, ***P,0.001, ****P,0.0001.
CBD Induced vasorelaxation of human arteries Page 7 of 11
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In the rat aortae, CBD causes time-dependent vasorelaxation that
can be inhibited by PPARgantagonism.
22
In human small mesenteric
arteries, we found that CBD-induced vasorelaxation also gradually in-
creases with time, but this effect was not inhibited by PPARgantagon-
ism. However, we previously observed in rats that PPARgmediated
time-dependent vasorelaxant responses to cannabinoids were only
observed in conduit arteries such as the superior mesenteric artery
and aorta, but not in third-order mesenteric arteries.
47
Thus the
lack of PPARg-mediated vasorelaxation seen to CBD may be due to
the size of the arteries in the present study. An interesting observation
was that the vasorelaxant response to CBD was non-recoverable,
persisting up to 2 h post-administration. This is in contrast to our
previous observations with THC
47
where tone recovered. However,
the mechanisms of action (CB
1
, NO, and the endothelium) of CBD
reported in the present study are very different to that reported
for THC.
48
Figure 5 Signal transduction by CBD in human endothelial cells. Levels of phosphorylated CREB (A), JNK (B), NFkB(C), p38 (D), ERK/MAP kinase 1/2
(E), Akt (F), p70 S6 kinase (G), STAT3 (H), and STAT5A/B (I) were measured in human aortic endothelial cell lysates after 10 min treatment with in-
creasing concentrations of CBD using the Luminex
w
xMAP
w
technology and normalized to total protein content. MFI, median fluorescent intensity. Data
are presented as mean +SEM (n¼6) and were analysed by ANOVA with Dunnett’s post-hoc analysis against the vehicle control response. *P,0.05,
**P,0.01, ***P,0.001, ****P,0.0001.
C.P. Stanley et al.Page 8 of 11
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Human endothelial cell-based studies showed that CBD causes a
range of intracellular signalling pathwaysto be altered at concentrations
from 100 nM, but not in a classical concentration-dependent manner.
This non-classical concentration – response, particularly for ERK and
Akt activation, may be a result of activation of multiple targets by
CBD. Indeed the ERK activation appeared to be inhibited by antagonists
of both CB
1
and TRPV1. Bell-shaped response curves to CBD are also
commonly observed.
49,50
The observed phosphorylation of ERK and
Akt is consistent with known CB
1
-mediated signal transduction, and
CB
1
-mediated activation of ERK has been observed in human umbilical
vein endothelial cells.
35
Indeed, we found that CB
1
antagonism pre-
vented this increase in ERK. Cannabinoid activation of both MAPK
and Akt in the vasculature has also been suggested to be via non-CB
1
/
CB
2
mechanisms such as CB
e
.
51,52
However, given our response to
CBD was not antagonized by O-1918, it is unlikely that CBD acts
through this site. Vasorelaxation to many compounds is mediated by
activation of ERK and Akt, thus the CBD-induced increased in both
ERK and Akt and therefore both may represent the intracellular signal-
ling mechanisms underpinning the vasorelaxant effects of CBD, as sug-
gested by the positive correlation with eNOS phosphorylation and the
inhibition of eNOS phosphorylation by AM251.
CBD also significantly decreased the level of phosphorylated JNK and
NFkB, key pro-inflammatory pathways, in human endothelial cells. This is
consistent with previous studies showing CBD can attenuate the increase
in JNK and NFkB caused by hepatic ischemia/reperfusion injury,
53
diabet-
ic cardiomyopathy,
11
and hyperglycaemia.
12
Our data suggest that reduc-
tions in these inflammatory pathways in endothelial cells may underpin
some of the protective effects of CBD observed in the vasculature.
5
Previous studies have shown a decrease in the phosphorylation of
p70s6K, an mTOR substrate, in response to synthetic CB
1/2
agonist
54
or THC
55
in cancer cells linked to autophagy pathways. STAT5 is
also crucial in the regulation of cell fate, and its activation is key in angio-
genesis.
56
The reduction in the levels of phosphorylated p70s6K and
STAT5 in human endothelial cells in response to CBD in the present
study may represent the intracellular signalling mechanisms underpin-
ning the anti-angiogenic effects of CBD reported by Solinas et al.
57
in
human umbilical vein endothelial cells.
Given the variability of the responses seen to CBD, post hoc analysis
of patient medical notes was undertaken. We found that CBD-induced
Figure 7 The effects of high insulin and glucose on the expression
of cannabinoid targets in HAECs. RT-PCR showing the presence of
PPARaand g,CB
1
,CB
2
, TRPV1, CGRP receptors, and a house-
keeping gene hypoxanthine-guanine phosphoribosyltransferase
(HPRT) in human aortic endothelial cells (HAECs) grown in control
conditions (first column) or a high insulin (500 nM, second column) or
high glucose (25 mM, third column) environment for 96 h. Human as-
trocytes (HA) are shown as a positive control for cannabinoid targets.
Figure 6 Signal transduction by CBD in human endothelial cells. Levels of phosphorylated ERK/MAP kinase 1/2 (A) and Akt (B) measured in human
aortic endothelial cell lysates after 10 min treatment with CBD in the presence of the CB
1
antagonist AM251 (100 nM) or the TRPV1 antagonist capza-
sepine (1 mM). (C) Correlation of levels of phosphorylated ERK1/2 and Akt with levels of phosphorylated eNOS in human aortic endothelial cell lysates
after 10 min treatment with CBD. MFI, median fluorescent intensity. (D) The effects of the CB
1
receptor antagonist AM251 on CBD-stimulated eNOS
phosphorylation. Data are presented as mean +SEM (n¼6) and were analysed by ANOVA with Sidak’s multiple comparison test of selected pairs.
**P,0.01, ***P,0.001.
CBD Induced vasorelaxation of human arteries Page 9 of 11
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vasorelaxation was enhanced in females compared with males. The en-
hanced vasorelaxation observed in female patient arteries compared
with males may be due to protective effects of oestrogen on endothe-
lial function.
58
It has also been shown that CB
1
receptor expression was
increased in the leucocyte cells of females when compared with
males.
59
CBD responses were also reduced in those with increased
cholesterol or diabetes. In rats fed high cholesterol diets, CB
1
receptor
expression is reduced.
60
Similarly, CB
1
expression is reduced (and as-
sociated vasorelaxant responses to anandamide) in obese rats.
60
To
test whether this might also be true in human aortic endothelial cells,
we carried out RT-PCR on the major targets for cannabinoid in control
condition and after prolonged exposure to a high-glucose or high-
insulin environment. We did not find a reduction in CB1R expression;
however, CB2R expression did appear to be up-regulated, which is in
agreement with numerous studies showing that CB
2
is up-regulated in
vasculature pathologies.
61
We did however also observe a decrease in
the expression of TRPV1 and CGRPR in response to both a high-
glucose or high-insulin medium, which is consistent with reports show-
ing that TRPV1 vasorelaxant responses and receptor coupling to nitric
oxide is disrupted in diabetes.
62
Interestingly, the TRPV1 receptor has a
cholesterol binding site which reduces its function.
63
Thus the blunted
CBD response in diabetic and hypercholesteraemia patients may be as
a result of reduced TRPV1 expression and/or function, which warrants
further investigation. Several medications (beta blockers, statins, and
oral hypoglycaemics) were also associated with reduced vasorelaxant
responses to CBD, but it is not yet clear whether this represents a
drug – drug interaction, or whether it is a result of the pathology for
which the medication is being taken.
In conclusion, this study reports that CBD causes vasorelaxation of
the human mesenteric artery. This vasorelaxation is mediated through
CB
1
, TRP channels, the endothelium and potassium channel activation.
CBD also causes time-dependent vasorelaxation of human mesenteric
arteries, but this was not due to PPARgactivation. The vasorelaxant
effects of CBD are reduced in patients with hypercholesterolemia
and type-2 diabetes, which may be a result of a reduced TRPV1 com-
ponent. In human endothelial cells, CBD causes alterations in the phos-
phorylation of many intracellular proteins that might explain the
vasorelaxant (eNOS, ERK, and Akt), anti-inflammatory (JNK and
NFkB) and anti-angiogenic effects of CBD (p70s6K and STAT5).
Supplementary material
Supplementary Material is available at Cardiovascular Research online.
Funding
This work was supported by the British Heart Foundation (FS/09/061).
Funding to pay the Open Access publication charges for this article was
provided by the British Heart Foundation.
Conflict of interest: none declared.
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