Article

Cannabinoid 1 receptor activation contributes to vascular inflammation and cell death in a mouse model of diabetic retinopathy and a human retinal cell line

Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, GA, USA.
Diabetologia (Impact Factor: 6.67). 03/2011; 54(6):1567-78. DOI: 10.1007/s00125-011-2061-4
Source: PubMed

ABSTRACT

Recent studies have demonstrated that cannabinoid-1 (CB(1)) receptor blockade ameliorated inflammation, endothelial and/or cardiac dysfunction, and cell death in models of nephropathy, atherosclerosis and cardiomyopathy. However the role of CB(1) receptor signalling in diabetic retinopathy remains unexplored. Using genetic deletion or pharmacological inhibition of the CB(1) receptor with SR141716 (rimonabant) in a rodent model of diabetic retinopathy or in human primary retinal endothelial cells (HREC) exposed to high glucose, we explored the role of CB(1) receptors in the pathogenesis of diabetic retinopathy.
Diabetes was induced using streptozotocin in C57BL/6J Cb(1) (also known as Cnr1)(+/+) and Cb(1)(-/-) mice aged 8 to 12 weeks. Samples from mice retina or HREC were used to determine: (1) apoptosis; (2) activity of nuclear factor kappa B, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), poly (ADP-ribose) polymerase and caspase-3; (3) content of 3-nitrotyrosine and reactive oxygen species; and (4) activation of p38/Jun N-terminal kinase/mitogen-activated protein kinase (MAPK).
Deletion of CB(1) receptor or treatment of diabetic mice with CB(1) receptor antagonist SR141716 prevented retinal cell death. Treatment of diabetic mice or HREC cells exposed to high glucose with SR141716 attenuated the oxidative and nitrative stress, and reduced levels of nuclear factor κB, ICAM-1 and VCAM-1. In addition, SR141716 attenuated the diabetes- or high glucose-induced pro-apoptotic activation of MAPK and retinal vascular cell death.
Activation of CB(1) receptors may play an important role in the pathogenesis of diabetic retinopathy by facilitating MAPK activation, oxidative stress and inflammatory signalling. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of this devastating complication of diabetes.

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ARTICLE
Cannabinoid 1 receptor activation contributes to vascular
inflammation and cell death in a mouse model of diabetic
retinopathy and a human retinal cell line
A. B. El-Remessy & M. Rajesh & P. Mukhopadhyay &
B. Horváth & V. Patel & M. M. H. Al-Gayyar &
B. A. Pillai & P. Pacher
Received: 30 November 2010 /Accepted: 30 December 2010 /Published online: 4 March 2011
#
Springer-Verlag (outside the USA) 2011
Abstract
Aims/hypothesis Recent studies have demonstrated that
cannabinoid-1 (CB
1
) receptor blockade ameliorated inflam-
mation, endothelial and/or cardiac dysfunction, and cell
death i n m odels of nep hropat hy, athe roscler osis and
cardiomyopathy. However the role of CB
1
receptor signalling
in diabetic retinopathy remains unexplored. Using genetic
deletion or pharmacological inhibition of the CB
1
receptor
with SR141716 (rimonabant) in a rodent model of diabetic
retinopathy or in human primary retinal endothelial cells
(HREC) exposed to high glucose, we explored the role of
CB
1
receptors in the pathogenesis of diabetic retinopathy.
Methods Diabetes was induced using streptozotocin in
C57BL/6J Cb
1
(also known as Cnr1)
+/+
and Cb
1
/
mice aged
8to12weeks.SamplesfrommiceretinaorHRECwereused
to determine: (1) apoptosis; (2) activity of nuclear factor kappa
B, intercellular adhesion molecule 1 (ICAM-1), vascular cell
adhesion molecule 1 (VCAM-1), poly (ADP-ribose) polymer-
ase and caspase-3; (3) content of 3-nitrotyrosine and reactive
oxygen species; and (4) activation of p38/Jun N-terminal
kinase/mitogen-activated protein kinase (MAPK).
Results Deletio n of CB
1
receptor or treatment of diabetic
mice with CB
1
receptor antagonist SR141716 prevented
retinal cell death. Treatment of diabetic mice or HREC cells
exposed to high glucose with SR141716 attenuated the
oxidative and nitrative stress, and reduced levels of nuclear
factor κB, ICAM-1 and VCAM-1. In addition, SR141716
attenuated the diabetes- or high glucose-induced pro-
apoptotic activation of MAPK and retinal vascular cell death.
Conclusions/interpretation Activation of CB
1
receptors
may play an important role in the pathogenesis of diabetic
retinopathy by facilitating MAPK activation, oxidative
stress and inflammatory signalling. Conversely, CB
1
recep-
tor inhibition may be beneficial in the treatment of this
devastating complication of diabetes.
Keywords Apoptosis
.
Cannabinoid
.
CB
1
.
ICAM-1
.
NFκB
.
Rimonabant
.
SR141716
.
VCAM-1
Abbreviations
CB
1
Cannabinoid 1
DCF 2,7-Dichlorofluorescein
DHDCF 2,7-Dichlorodihydro-fluorescein diacetate
GFAP Glial fibrillary acidic protein
HREC Human primary retinal endothelial cell s
ICAM-1 Intercellular adhesion molecule 1
JNK Jun N-terminal kinase
A. B. El-Remessy and M. Rajesh contributed equally to this study.
Electronic supplementary material The online version of this article
(doi:10.1007/s00125-011-2061-4) contains supplementary material,
which is available to authorised users.
A. B. El-Remessy
:
M. M. H. Al-Gayyar
:
B. A. Pillai
Program in Clinical and Experimental Therapeutics,
College of Pharmacy, University of Georgia,
Augusta, GA, USA
A. B. El-Remessy
:
M. M. H. Al-Gayyar
:
B. A. Pillai
Charlie Norwood VA Medical Center,
Augusta, GA, USA
M. Rajesh
:
P. Mukhopadhyay
:
B. Horváth
:
V. Patel
:
P. Pacher (*)
Section on Oxidative Stress Tissue Injury,
Laboratory of Physiological Studies,
National Institutes of Health/National Institute on Alcohol Abuse
and Alcoholism,
5625 Fishers Lane, MSC-9413,
Bethesda, MD 20892-9413, USA
e-mail: pacher@mail.nih.gov
B. Horváth
Institute of Human Physiology and Clinical Experimental
Research, Semmelweis University,
Budapest, Hungary
Diabetologia (2011) 54:15671578
DOI 10.1007/s00125-011-2061-4
Page 1
MAPK Mitogen-activated protein kinase
NFκB Nuclear factor κB
PARP Poly(ADP-ribose) polymeras e
ROS Reactive oxygen species
VCAM-1 Vascular cell adhesion molecule 1
Introduction
Vascular inflammation and endothelial cell death are
characteristic features of diabetic retinopathy [1, 2]. The
early stages of the inflammatory reaction are characterised
by leucocyte adhesion to the vessel wall, leading to altered
vessel reactivity and subsequent activation of transcription
factors including nuclear factor κB (NFκB), which ulti-
mately results in capillary endothelial cell apoptosis and
vascular cell loss in the diabetic retina [3, 4]. A critical role
of increased oxidative and nitrative stress in medi ating
vascular inflammation and cell death is supported by
previous studies [4, 5] and a review [6]. Based on the
evidence that NFκB has a well-conserved cysteine residue,
NFκB activity is tightly linked with its redox regulation [ 7 ].
Prior studies have shown that oxidative stress can induce
production of inflammatory cytokines and adhesion molecules
via activation of NFκB[8, 9]. Therefore, devising treatments
that target oxidative stress and inflammation could be of
great clinical significance for diabetic retinopathy.
The recent ly discovered endocannabinoid system, which
consists of the endocanna binoids, their metabolising
enzymes and the main cannabinoid 1 (CB
1
) and canna-
binoid 2 (and perhaps other yet not determined) receptors,
has been implicated as an important factor in regulation of
energy balance, food intake, metabolism and inflammation
in health and disease [10, 11]. While cannabinoid 2
receptors are predominantly localised on immune cells,
CB
1
receptor is mostly found in the central nervous system
and the retina [12]; however, both receptors are also present
in cardi ovascular and virtually all other tissues, albeit at
much lower levels [10]. It has been observed that: (1) the
CB
1
receptors [12 ] and endocannabinoids [13, 14] are
present within the retina; (2) endocannabinoid anandamide
level is elevated in the retina of patients with diabetic
retinopathy [14]; and (3) CB
1
receptor activation in
coronary endothelial [15] and inflammatory [16, 17]cellsby
endocannabinoids or synthetic CB
1
ligands mediates mitogen-
activated protein kinase (MAPK) activation, reactive oxygen
species (ROS) generation and inflammatory response [1518],
as well as promoting atherosclerosis [19]. These observations,
coupled with the multiple beneficial effects of the CB
1
receptor antagonist rimonabant (SR141716) on inflammatory
markers as observed in obese and/or type 2 diabetic patients,
and in various preclinical disease models [10, 11], and with
the recently reported attenuation of albuminuria by CB
1
receptor blockade in an experimental model of diabetic
nephropathy [20], raise the possibility of a direct effect of
CB
1
receptor signalling in pro-inflammatory and pro-
apoptotic response in retinal endothelial cells. Besides
regulating photoreception and neurotransmission in the retina,
the endocannabinoid system affects intraocular pressure and
ocular blood vessels [21, 22], and plant-d eriv ed canna-
binoids such as cannabidiol and tetr ahydroc annabin ol
exert neuroprotective effects against retinal neurotoxicity
[23], presumably by their a ntioxidant properties, indepen-
dently of conventional cannabinoid receptors.
To assess the potential role of the CB
1
receptor in the
pathogenesis of retinal vascular injury in diabetes, we
evaluated the effects of the selective CB
1
receptor inhibitor,
SR141716/rimonabant or of genetic deletion of CB
1
receptors
in a mouse model of diabetic retinopathy and in human
primary retinal endothelial cells (HREC) exposed to high
glucose. Our s tudy demonstrates that pharmacologi cal
inhibition or genetic deletion of CB
1
attenuates retinal
oxidative stress, release of pro-inflammatory mediators and
activation of p38/Jun N-terminal kinase (JNK) MAPK in
streptozocin-induced diabetic mice, as well as in HREC
exposed to high glucose.
Methods
Animals and treatment
The animal procedures adhered to the National Institutes of
Health (NIH) guide lines and were approved by the
Institutional Animal Care and Use Committee of the
National Institute on Alcohol Abuse and Alcoholism
(NIAAA)/NIH. Diabetes was induced in 8- to 12-week-
old male C57/BL6J or in CB
1
receptor knockout (Cb
1
[also
known as Cnr1]
/
) and wild-type (Cb
1
+/+
) male mice (23
25 g; Jackson Laboratories, Bar Harbor, ME, USA) by
multiple intra-peritoneal injection of streptozotocin as previ-
ously described [24]. After 1 week, blood glucose levels
were measured using a glucometer (Ascensia Counter; Bayer
Healthcare, NY, USA) by mandi bular puncture blood
sampling. Mice with blood sugar values >14 mmol/l (ap-
proximately 250 mg/dl) were used for the study. Diabetes
was allowed to develop further for 1 additional week before
animals were treated for 11 weeks with the selective CB
1
receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-4-methyl-3-pyrazole carboxamide
(SR141716A/rimonabant; 10 mg/kg daily, i.p.; NIDA Drug
Supply Program, Research Triangle Park, NC, USA).
Cell culture All human cell line experiments were approved
by the NIH Office of Human Subjects Research. HREC were
obtained from Cell Applications (San Diego, CA, USA) and
1568 Diabetologia (2011) 54:15671578
Page 2
grown in HREC growth medium (Cell Applications) in
culture dishes coated with 0.2% (wt/wt) gelatin (Sigma, St
Louis, MO, USA). HREC were used for the experiments in
the passages 3 to 6. Cells were maintained in normal glucose
(5 mmol/l) or high glucose (30 mmol/l) for 48 h. For osmotic
controls, cells were maintained in either
L-glucose (30 mmol/l)
or mannitol (30 mmol/l) for the same duration.
Determination of retinal cell death in flat-mounted re tina
TUNEL assay was performed using immunoperoxidas e
staining (ApopTag-Peroxidase; Roche Applied Science,
Indiannapolis, IN, USA) in whole-mount retina as
described previously [25, 26]. After permeabilisation,
TUNEL-horseradish peroxidise staining with 3-amino-9-
ethylcarbazole was performed following the manufac-
turer s instructions. The total number of cells positive for
TUNEL-horseradish peroxidise was counted in each retina
using light microscopy. TUNEL was also performed in 10 μm
eye sections frozen to optimal cutting temperature, using a kit
(ApopTAG in situ cell death detection kit; TUNEL-FITC) as
described previously [27].
Determination of apoptosis in HREC b y flow cytometry
After various treatments, apoptosis/necrosis was determined
with flow cytometry as described previously [15, 24].
Determination of glial activation and immunolocalisation
studies Retinal sections were fixed using 2% (wt/wt)
paraformaldehyde in PBS and allowed to react overnight
with polycl onal anti-glial fibrillary acidic protein (GFAP)
antibody for glial activation (Affinity BioReagents, Rockford,
IL, USA) , monoclonal anti-NFκB p 65 (BD Bioscience
Pharmingen, San Diego, CA, USA) and monoclonal anti-
vascular cell adhesion molecule 1 (VCAM-1) (R&D Systems,
Minneapolis, MN, USA), followed by Texas Red- or Oregon
Green-conjugated goat anti-mouse antibodies (Invitrogen,
Carlsbad, CA, USA). Data (three fields per retina, n=4 in
each group) were analysed using AxioObserver.Z1 Micro-
scope (Carl Zeiss, Thornwood, NY, USA) and Axio-software
to quantify the density of immunostaining.
Determination of cell surface intercellular adhesion molecule 1
and VCAM-1 levels Measurements of cell surface intercel-
lular adhesion molecule 1 (ICAM-1) and VCAM-1 levels in
HRECs were done by in situ ELISA as described
previously [28].
Western blot analysis Total lysates from retinas or cells
were extracted with RIPA buffer as described before
[15, 26 ]. Blots were probed with polyclonal anti-phospho-
p38, p38, phospho-JNK/JNK (Cell Signaling, Danvers,
MA, USA), monoclonal anti-NFκB p65 (BD Bioscience),
polyclonal anti-VCAM-1 (R&D Systems) and monoclonal
anti-3-nitrotyrosine (Cayman Chemicals, Ann Arbor, MI,
USA). Membranes were reprobed with β-actin (M illipore,
Billerica, MA, USA) to confirm equal loading. The
primary antibody was detected using appropriate horse-
radish peroxi dase-c onjugated antibodies (GE Healthcare,
Piscataway, NJ, U SA) and enhanced chemilumin esc ence .
The films were scanned, and band intensit y was quanti fied
using densitometry software (BioRad, Hercules, CA,
USA) and expressed as relative optical density.
Determination of 3-nitrotyrosine content Quantification
of 3-nitrotyrosine levels in retinal extracts was performed
using slot blot analysis as described [25, 26]. 3-Nitrotyrosine
content in HREC extracts was determined using an ELISA
kit (Hycult Biotech nology, Uden, t he Netherlands) as
described previously [29].
Determination of poly (ADP-ribose) polymerase and
caspase 3/7 activi ties Poly (ADP-ribose) polymerase
(PARP) and caspase 3/7 activities in the HREC extracts
were performed using kits (Trevigen, Gaithersburg, MD,
USA, and Promega, Madison, WI, USA, respectively) as
described previously by our group [29].
Determination of ROS generation in retina and HREC 2
,7-
Dichlorofluorescein ( DCF) is the oxidation product of
2,7-dichlorodihyd ro-fluorescein diacetate (DHDCF)
(Invitroge n), a marker of cellu lar ox idat ion by hydroge n
peroxide, peroxynitrite and hydroxy radicals. For HREC,
DCF was detected using flow cytometery techniques as
described before [15]. Briefly, cells were incubated for
15 min with 5 μmol/l DHDCF at 37°C then measured at
excitation of 488 nm with standard settings using a flow
cytometer (FACS Calibur; Becton Dickinson, San Jose,
CA, USA). For retina lysate, DCF was measured as
described previously [30]. Briefly, equal volumes of
retinal lysates were in cubated with DHDCF (10 μmol/l)
for 60 min at 37°C then measured at excitation 450 nm
using a plate reader (BioTek, Winooski, VT, USA).
Statistical analysis The results were expressed as mean ±
SEM. Differences among experimental groups were evaluated
by ANOVA and the significance of differences between groups
wasassessedbyTukeys post-hoc test. The analysis was
performed using a statistical software package (GraphPad-
Prism 5; GraphPad, La Jolla, CA, USA). Significance was
defined as p<0.05.
Results
Metabolic variables Induction of diabetes by multiple low
doses of streptozotocin led to marked reduction in the
Diabetologia (2011) 54:15671578 1569
Page 3
bodyweight with concomitant increase in blood glucose
levels in wild-type (Cb
1
+/+
) and Cb
1
/
mice respectively
(Electronic supplementary material [ESM] Fig. 1). However,
blood glucose levels were unaffected during the 12 week
study period in Cb
1
/
mice compared with wild-type mice
(ESM Fig. 1). Similarly treatment of diabetic mice with SR
141716A for 11 weeks did not significantly alter body
weight or blood glucose levels (ESM Fig. 2).
CB
1
receptor plays a role in diabetes-induced retinal cell
death The CB
1
receptor is highl y abundant in the inner and
outer plexiform layers of the retina [12]. However, the exact
role of CB
1
receptor in modulating retinal function in
response to diabetes is not fully understood. Diabetes
induced greater than sevenfold increases in retinal cell
death as indicated by quantitative analysis of TUNEL-
positive cells in flat-mounted retina (Fig. 1a, c). Deletion of
Cb
1
completely protected diabetic animals from retinal cell
death, suggesting a potential role of CB
1
receptor activation
in mediating cell death. Co-localisati on studies in diabetic
retinal sections demonstrated that several TUNEL-posi tive
cells were located within endothelial cells as indicated by
iso-lectin B4 (Fig. 1b). We next evaluated the effect of the
CB
1
receptor antagonist SR 141716A in diabetic animals.
As shown in Fig. 1d, treatment of diabetic animals with SR
141716A significantly reduced TUNEL-posi tive cells com-
pared with diabetic animals treated with vehicle.
CB
1
receptor inhibition attenuates diabetes-induced oxida-
tive and nitrative stress in vivo To explore the possible
GCL
IPL
INL
ONL
GCL
IPL
INL
ONL
Cb
1
+/+
diabetes Cb
1
/
diabetes
Cb
1
+/+
diabetes Cb
1
+/+
diabetes
a
b
Isolectin B4 Isolectin B4+TUNEL
0
4
8
16
12
Veh Diab
*
0
4
8
14
16
20
*
cd
Veh
Veh+SR1
Diab
Diab+SR1
TUNEL-positive
cells/retina area
TUNEL-positive
cells/retina area
Cb
1
+/+
Cb
1
+/+
Cb
1
/
Cb
1
/
Fig. 1 CB
1
receptor plays a role in diabetes-induced retinal cell death.
a Representative images (×200 magnification, scale bars 25 μm) and
(c) statistical analysis showing that diabetes induced significant increases in
retinal cell death as indicated by quantitative analysis of TUNEL-positive
cells in flat-mounted retina. Deletion of Cb
1
completely protected diabetic
animals from retinal cell death (n=6). Arrows (a) indicate TUNEL-
positive cells in retinal flat mounts. b Co-localisation studies in diabetic
retinal sections demonstrated that several TUNEL-positive cells (green)
are located within ganglion layer (GCL) and localised with endothelial
cells as indicated by isolectin B4 (red); ×200 magnification; scale bars
25 μm. IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer
nuclear layer. d Statistical analysis showing that treatment of diabetic
animals (Diab) with SR 141716A (SR1) significantly reduced TUNEL-
positive cells compared with diabetic animals treated with vehicle (Veh,
n=6). *p<0.05 vs vehicle group;
p<0.05 vs wild-type diabetes
Veh Veh+SR1 Diab Diab+SR1
0
1
2
Veh Veh+SR1 Diab Diab+SR1
Veh Veh+SR1 Diab Diab+SR1
a
0
100
200
b
*
*
Relative DCF fluorescence ROD of nitrotyrosine
Fig. 2 CB
1
receptor inhibition attenuates diabetes-induced oxidative
and nitrative stress in vivo. a Slot blot and statistical analysis of mouse
retinal lysate showing twofold increase in 3-nitrotyrosine formation in
diabetic mice as compared with controls (n=56). b Statistical
analysis showing twofold increase in ROS formation as indicated by
DCF fluorescence in diabetic mice as compared with controls (n=6).
*p<0.05 vs vehicle group; p<0.05 vs diabetes. Diab, diabetic
animals; SR1, SR 141716A; Veh, vehicle-treated
1570 Diabetologia (2011) 54:15671578
Page 4
mechanism of the protective effects of CB
1
receptor
blockade, we next examined its effects on the well
established phenomenon of diabetes-induced oxidative and
nitrative stress. As shown in Fig. 2a, b, mice retinal lysate
showed twofold increase in 3-nitrotyrosine formation
measured by slot blot and twofold incr ease in ROS
formation as indicated by DCF fluorescence in diabetic
retina as compared with controls. Treatmen t of dia-
betic animals with SR 141716A significantly reduced
3-nitrotyrosine and ROS formation but did not alter control
levels.
CB
1
receptor inhibition attenuates high glucose-induced
oxidative stress in HREC As shown in Fig. 3ag , flow
cytometry of DCF showed 2.4-fold increase in ROS.
ELISA assay of nitrotyrosine showed a fivefold increase
in 3-nitrotyrosine generation in HREC cells as compared
with those maintained in normal glucose (Fig. 3h). Treat-
ment of the cells with CB
1
antagonist, SR 141716A
(2 μmol/l) blocked high glucose-induced increase in
oxidative and nitrative stress. Interestingly, treatment of
HREC with SR 141716A alone or osmotic controls did not
alter ROS or 3-nitrotyrosine formati on.
CB
1
receptor inhibition atte nuated diabetes-induced glial
activation and NFκB production As shown in Fig. 4a,
diabetes enhanced glial activation as indicated by increases
in the intensity of GFAP immunoreactivity in the filaments
of Müller cells that extend from the nerve fibre and inner
plexiform layers into the outer nuclear layer of retina as
10
0
10
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2
10
3
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4
FL1-H
10
0
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2
10
3
10
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FL1-H
10
0
10
1
10
2
10
3
10
4
FL1-H
10
0
10
1
10
2
10
3
10
4
FL1-H
10
0
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1
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2
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3
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4
FL1-H
10
0
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1
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2
10
3
10
4
FL1-H
0
20
40
60
80
100
Cells (n)
0
20
40
60
80
100
Cells (n)
0
20
40
60
80
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Cells (n)
0
20
40
60
80
100
Cells (n)
0
20
40
60
80
100
Cells (n)
0
20
40
60
80
100
Cells (n)
13.5
31.6 20.9
16.0
13.9
0
10
20
30
40
DCFDA mean intensity
0
2
4
6
3-NT (fold change)
abc
def
hg
*
*
SR1
HG+SR1
HG
30 mmol/l (
L-glucose)
5 mmol/l (
D-glucose)
30 mmol/l (mannitol)
SR1
HG+SR1
HG
30 mmol/l (
L-glucose)
5 mmol/l (
D-glucose)
30 mmol/l (mannitol)
11.5
Fig. 3 CB
1
receptor inhibition
attenuates high glucose-induced
oxidative stress in HREC.
a Representative histograms of
DCFDA fluorescence with
D-glucose (5 mmol/l), (b)SR
141716A (SR1), (c)
L-glucose
(30 mmol/l), (d) mannitol
(30 mmol/l), (e) high glucose
(HG;
D-glucose 30 mmol/l) and
(f) high glucose + SR1. Mean
fluorescence intensityheight at
~525 nm (FL1-H) (a) 13.5,
(b) 11.5, (c) 13.9, (d) 16.0,
(e) 31.6 and (f) 20.9. g Summary
data of DCFDA fluorescence
from the indicated representative
treatment condition ( n=46).
High glucose (30 mmol/l
D-glucose) induced an approxi-
mately 2.4-fold increase in DCF
fluorescence in HREC cells.
h Statistical analysis showing
approximately fivefold increase
in 3-nitrotyrosine (NT) forma-
tion in HREC cells maintained
in high glucose as compared
with those maintained in normal
glucose. Treatment of the cells
with CB
1
antagonist SR
141716A (2 μmol/l) almost
completely prevented high
glucose-induced increase in
oxidative and nitrative stress.
Treatment of HREC with SR1
alone or osmotic controls did
not alter ROS or 3-nitrotyrosine
formation. *p<0.05 vs vehicle
group;
p<0.05 vs diabetes
Diabetologia (2011) 54:15671578 1571
Page 5
compared with c ontrols. In addition, diabetes enhanced
NFκB activation (twofold), which was mainly localised in
the vascular layers of the retina, compared with controls
(Fig. 4b, c). Additional studies with isolectin B4, a marker
of endothelial cells confirmed that NFκB was colocalised
with en dothelial cells (data not shown). Treatment of
diabetic animals with SR 141716A blocked these effects
but did not alter control levels.
CB
1
r eceptor inhibition attenuated diabetes-induced adhesion
molecule pr oduction in vivo We next examined levels of the
adhesion molecules ICAM-1 and VCAM-1 in rat retinas. As
shown in Fig. 5a, c, diabetes enhanced VCAM-1 production
(twofold), which was mainly localised in retinal capillaries,
compared with controls. This effect was paralleled by
significant increases (1.9-fold) in ICAM-1 production in
diabetic animals (Fig. 5b, d). Treatment of diabetic animals
a
b
c
GCL
IPL
INL
ONL
GCL
IPL
INL
ONL
Veh Veh+SR1 Diab Diab+SR1
Veh Veh+SR1 Diab Diab+SR1
0
3
2
1
Diab+SR1
ROD of retinal NFκB
*
Veh
Veh+SR1
Diab
Fig. 4 CB
1
receptor inh ibition attenuated diabetes-induced glial
activation and NFκB production. a Representative images showing a
substantial increase in the intensity of GFAP immunoreactivity in the
filaments of Müller cells in diabetes (Diab) vs vehicle (Veh). This
increase extended from the nerve fibre layer and inner plexiform layer
(IPL) into the outer nuclear layer (ONL) of retina as compared with
controls. b Representative images of NFκB in mouse retinal sections
showing an approximately 2.5-fold increase in diabetic mice as
compared with the controls. NFκB was mainly localised within retinal
capillaries (n=6). Magnification (a, b) ×200; scale bars 25 μm. GCL,
ganglion cell layer; INL, inner nuclear layer; SR1, SR 141716A.
c Statistical analysis of above findings (b); *p<0.05 vs vehicle group;
p<0.05 vs diabetes. ROD, relative optic density
1572 Diabetologia (2011) 54:15671578
Page 6
with SR 141716A blocked these effects but did not alter
control levels.
CB
1
receptor inhibition attenuated high glucose-induced
ICAM-1 and VCAM-1 production in HREC As shown in
Fig. 6 , treatment of HRECs with high glucose significantly
increased levels of ICAM-1 and VCAM-1 by 2.5- to 3.5-
fold, respectively, compared with normal glucose. When
the cells were incubated with SR 141716A, high glucose-
induced adhesion molecules levels were significantly
reduced, but not completely blocked by SR 141716A.
Treatment of HREC with SR 141716A alone or osmotic
controls did not alter levels of adhesion molecules.
CB
1
r eceptor inhibition attenuates diabetes-induced proapop-
totic MAPK activation in vivo and in HREC Activation of
proapoptotic MAPK pathways, including p38 MAPK and
JNK, is a known downstream target of oxidative stress and
inflammatory mediators [24]. In comparison to controls,
diabetes increased activation of p38 MAPK (2.4-fold) , this
increase being mitigated by SR 141716A treatment (Fig. 7a, b).
Moreover , treatment of HRECs with high glucose significant-
a
b
c d
GCL
IPL
INL
ONL
Veh Veh+SR1 Diab Diab+SR1
Actin
ICAM-1
Veh Veh+SR1 Diab Diab+SR1
0
1
2
0
1
2
Veh
Veh+SR1
Diab
Diab+SR1
ROD of retinal VCAM-1
*
*
Veh
Veh+SR1
Diab
Diab+SR1
Relative ICAM-1 level
Fig. 5 CB
1
receptor inhibition attenuated diabetes-induced adhesion
molecule production in vivo. a Representative images of VCAM-1 in
mice retinal sections showing an approximately twofold increase in
diabetic (Diab) mice as compared with vehicle (Veh)-treated controls.
VCAM-1 was mainly localised within retinal capillaries; n=5, ×200
magnification, scale bar 25 μm. GCL, gang lion cell lay er; IPL,
inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear
layer. c Statistical analysis of above (a) find ings, expressed as
relative optical density (ROD). b Western blot analysis showing
increase in adhesion molecule ICAM-1 levels in diabetic animals as
compared with the controls (n =56). Treatment of diabetic an imals
with SR 141 716A (SR1) blocked these effects, but did not alter
control levels. d Statistical analysis of above findings (b)showing
1.9-fold increase in diabetic animals.*p<0.05 vs vehicle group;
p<0.05 vs diabetes
Diabetologia (2011) 54:15671578 1573
Page 7
ly increased MAPK (p38 and JNK) activation as compared
with cells maintained in normal glucose. This was attenuated
upon treatment with SR 141716A (Fig. 7c, d).
CB
1
receptor inhibition mitigates high glucose-induced
apoptosis in HREC HREC cell s maintained in high glucose
for 48 h showed significant increases in the activity of cell
death markers PARP and cleaved caspase-3, as compared
with those maintained in normal glucose (Fig. 8a, b). In
addition, HREC cells maintained in high glucose showed
significant increase in apoptosis as indicated by Annexin V
using flow cytometry; this increase was attenuated by SR
141716A (Fig. 8c, d).
Discussion
The main findings of the current study are that: (1) deletion
or pharmacological inhibition of the CB
1
receptor with
SR141716 prevents diabetes-induced retinal vascular cell
death; and (2) CB
1
inhibition ameliorates diabetes-induced
retinal oxidative stress, cellular adhesion molecule produc-
tion and inflammation. Although pharmacological inhibi-
tion of CB
1
receptor has been associated with numerous
cytoprotective and anti-inflammatory effects in models of
ischaemiareperfusion injury, cardiomyopathy, nephropathy
and atherosclerosis [17 19 , 29 , 31 33 ], a potential role of
the CB
1
receptor in the pathogenesis of diabetic retinopathy
has not been explored previously.
Diabetes-induced retinal oxidative and nitrative stress
have been well documented in patients and animals, and
have been positively correlated with vascular cell death
[3436]. The biochemical mechanisms involved in promot-
ing oxidative stress are complex and include activation of
several cellular pathways, as reviewed by Caldwell at al.
[37]. Recent studies have demonstrated that activation of
CB
1
receptors with endocannabinoids or synthetic ligands
can promote oxidative stress, inflammation, cell death and/
or organ dysfunction in models of cardiomyopathy [17, 18,
38], atherosclerosis [19] and nephropathy [20, 29, 32], and
likewise in macrophages [16], neutrophils [ 17 ], murine or
human cardiomyocytes [18, 38], and human coronary artery
endothelial cells [15]. In agreement with the above-
mentioned studies, our results demonstrate that pharmaco-
logical inhibition of CB
1
confers marked protection against
diabetes or high glucose-induced oxidative/nitrative stress
in retinas or in cultured HREC.
Diabetic retinopat hy has been perceived as an inflam-
matory disease, in the pathogenesis of which adhesion
molecules may be involved [39, 40]. Previous studies have
shown that oxidative stress can ind uce produc tion of
inflammatory cytokines and adhesion molecules via activa-
tion of the redox-regulated transcription factor NFκB[8, 9].
Upon activation, NFκB translocates to the nucleus, where it
regulates the expression of a large number of genes
including those encoding cellular adhesion molecules such
as ICAM-1 and VCAM-1 [41]. In agreement with this, our
results show significant increases in levels o f NFκB,
ICAM-1 and VCAM-1 in the retina of diabetic mice and
in HREC cells maintained in high glucose. These results
lend further support to previous reports showing enhanced
NFκB p65 in diabetic rats [42] and enhanced levels of
ICAM-1 and VCAM-1 in endothelial cells cultured in high
glucose [3]. The notion that CB
1
activation may promote
inflammation and tissue injury is supported by several
studies showing that genetic delet ion or pharmacological
inhibition of CB
1
consistently exerts beneficial effects on
the inflammation and oxidative/nitrative stress cell death
0
1
2
3
4
0
1
2
3
4
5
VCAM-1 (fold change)
*
*
a
b
ICAM-1 (fold change)
30 mmol/l (L-glucose)
5 mmol/l (
D-glucose)
30 mmol/l (mannitol)
SR1
HG+SR1
HG
30 mmol/l (
L-glucose)
5 mmol/l (
D-glucose)
30 mmol/l (mannitol)
SR1
HG+SR1
HG
Fig. 6 CB
1
receptor inhibition attenuated high glucose-induced
ICAM-1 and VCAM-1 production in HREC. a Statistical analysis of
ICAM-1 and (b) VCAM-1 levels measured by ELISA and showing
2.5- and 3.5-fold increases, respectively in HREC cells maintained in
high glucose (HG; 30 mmol/l
D-glucose) as compared with those
maintained in normal glucose. When the cells were incubated with SR
141716A (SR1; 2 μmol/l), high glucose-induced adhesion molecule
production was significantly reduced. Treatment of HREC with SR
141716A alone or osmotic controls did not alter levels of adhesion
molecules. *p<0.05 vs vehicle group;
p<0.05 vs diabetes
1574 Diabetologia (2011) 54:15671578
Page 8
cascade [17, 18, 3133, 43, 44]. In agreement, chronic
treatment of our diabetic animals with SR141716 almost
completely blocked the increases in retinal activation of
NFκB and production of ICAM-1 and VCAM-1, and similar
findings were obtained in HREC maintained in high glucose.
There is increasing recognition that CB
1
receptor
activation may promote activation of stress signalling
pathways including p38 and JNK MAPKs, leading to cell
death [15, 18, 29, 45]. In agreem ent with this, we found
marked increases (tenfold) in TUNEL-positive cell s in
retinal flat-mounts and sections of diabetic animals; these
increases were largely attenuated by CB
1
receptor deletion
or treatment with SR141716, illustrating a causal role of
CB
1
receptors in mediating retinal cell death. This is also
consistent with the elevated endocann abinoid anandamide
(endogenous ligand for CB
1
receptors) levels observed in
retinas of patients with diabetic retinopathy [14]. Immuno-
localisation studies using isolectin-B4, a marker for
endothelial cells, showed colocalisation of several of
TUNEL-positive cells within vascular endothelial cells,
lending further support to previous r eports showing
apoptosis of retinal capillaries as early as 12 week s of
diabetes duration [46, 47]. Apoptosis of retinal capillary
cells begins early in diabetes and is likely to contribute to
the capillary obliteration that is an important feature of
diabetic retinopathy. The increase in cell death was
associated with increases in oxidative markers in vivo and
in vitro. It is well known that oxidative and nitrative stress
may also lead to activation of p38 and JNKMAPKs,
promoting cell death. Our results showed that SR141716
significantly reduced activation of p38 and JNKMAPKs
in diabetic mice and in retinal endothelial cells maintained in
high glucose. In agreement with this, activation of p38 MAPK
hasbeenreportedindiabeticretinas[5, 16, 25, 26, 48].
Previous studies have demonstrated a pro-apoptotic role of
JNK activation in inducing vascular cell death in vivo
0
0. 5
1.0
1. 5
*
phospho-JNK/JNK
phospho-P38MAPK/
P38MAPK
0
0.4
0.8
1.2
1.6
*
30 mmol/l (
L
-glucose)
5 mmol/l (
D
-glucose)
30 mmol/l (mannitol)
SR1
HG+SR1
HG
30 mmol/l (
L
-glucose)
5 mmol/l (
D
-glucose)
30 mmol/l (mannitol)
SR1
HG+SR1
HG
Actin
JNK
phospho-JNK
p38MAPK
phospho-p38MAPK
30 mmol/l (
L
-glucose)
5 mmol/l (
D
-glucose)
30 mmol/l (mannitol)
SR1
HG+SR1
HG
p38 MAPK
phospho-p38 MAPK
Veh Veh+SR1 Diab Diab+SR1
0
0.6
1.2
1.8
he
V
1RS+h
e
V
b
ai
D
1R
S+ba
i
D
*
K
PAM8
3
p/
K
PAM83p-ohpsohp
b
de
ca
Fig. 7 CB
1
receptor inhibition attenuates diabetes-induced proapop-
totic MAPK activation in vivo and in HREC. a Western blot of mice
retinal lysate showing an increase in the activation of p38MAPK in
diabetic (Diab) as compared with the vehicle (Veh)-treated controls;
the increase was mitigated by SR 141716A (SR1) treatment (n=46).
b Statistical analysis of above results (a), indicating a 2.4-fold
increase. c Western blot of HREC cell lysate showing significant
increases in activation of p38MAPK and JNK in cells maintained in
high glucose (HG; 30 mmol/l
D-glucose) as compared with cells
maintained in normal glucose. These increases were mitigated upon
treatment with SR 141716A (2 μmol/l). d, e Statistical analysis of
above (c) findings for MAPK and JNK respectively. *p<0.05 vs
vehicle group;
p<0.05 vs diabetes
Diabetologia (2011) 54:15671578 1575
Page 9
andinvitro[15, 49, 50]. Cell death of retinal capillaries
was further confirmed by significant increases in activity
of c leaved caspase-3 and PARP in response to high
glucose. Treatment with SR141716 only partially attenuated
cell death, suggesting that activation of CB
1
receptor is not
the only player mediating high glucose-induced cell demise.
In summary, our results demonstrate that pharmacolog-
ical blockade and/or genetic deletion of the CB
1
receptors
ameliorate diabetes-induced retinal oxidative stress and
production of cellular adhesion molecules, and prevent cell
death, strongly supporting an important role for activation
of CB
1
receptor in the pathogenesis of diabetic retinopathy.
Acknowledgements This work was supported by intramural
research grants to NIH-NIAAA to P. Pacher and grants from JDRF
(2-2008-149) and Vision Discovery Institute to A. B. El-Remessy.
B. Horváth was supported by an NKTH-OTKA-EU fellowship (MB08-
A-80238). The authors are indebted to G. Kunos (Scientific Director of
NIH-NIAAA) for providing key resources f or the completion of this study .
Duality of interest The authors declare that there is no duality of
interest associated with this manuscript.
Fig. 8 CB
1
receptor inhibition mitigates high glucoseinduced cell
death in HREC. a Western blot statistical analysis of HREC cell lysate
showing significant increases in the activity of PARP and (b) caspase-3
in cells maintained in high glucose (HG) as compared with cells
maintained in normal glucose. These increases were mitigated upon
treatment with SR 141716A (SR1; 2 μmol/l). c Flow cytometric
analysis of cell death with
D-glucose (5 mmol/l), (d) SR 141716A
(SR1), (e)
L-glucose (30 mmol/l), (f) mannitol (30 mmol/l), (g) high
glucose (HG;
D-glucose 30 mmol/l) and (h) high glucose + SR1. Cells
were maintained in different media as indicated and treated with CB1
antagonists for 1 h, followed by incubation with different media for
48 h in the continuous presence of CB1 antagonists. ch Sytox green
(y-axes), x-axes: annexin V-APC. i Summary of the results showing
significant increase in apoptosis in HREC cells maintained in high
glucose (30 mmol/l
D-glucose) compared with those maintained in
normal glucose. When the cells were incubated with SR 141716A
(2 μmol/l), the high glucose-induced apoptosis was significantly
reduced. Treatment of HREC with SR 141716A alone or osmotic
controls did not alter cell death. n=46; *p<0.05 vs vehicle group;
p<0.05 vs diabetes. FL4-H, fluorescence intensityheight at ~675 nm
1576 Diabetologia (2011) 54:15671578
Page 10
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  • Source
    • "Therefore, our results suggest that CB2 activation reduces oxidative stress which is in accordance with the previous studies [17, 23]. Conversely, CB1 receptor activation is related to the augmentation of oxidative stress as well as other depressive effects [49–52]. In fact, selective CB1 antagonists have been speculated as a potential tool for the treatment of cardiovascular disease [32, 49]. "
    [Show abstract] [Hide abstract] ABSTRACT: Hypercholesterolemia is one of the most important risk factors for erectile dysfunction, mostly due to the impairment of oxidative stress and endothelial function in the penis. The cannabinoid system might regulate peripheral mechanisms of sexual function; however, its role is still poorly understood. We investigated the effects of CB2 activation on oxidative stress and fibrosis within the corpus cavernosum of hypercholesterolemic mice. Apolipoprotein-E-knockout mice were fed with a western-type diet for 11 weeks and treated with JWH-133 (selective CB2 agonist) or vehicle during the last 3 weeks. CB2 receptor expression, total collagen content, and reactive oxygen species (ROS) production within the penis were assessed. In vitro corpus cavernosum strips preparation was performed to evaluate the nitric oxide (NO) bioavailability. CB2 protein expression was shown in cavernosal endothelial and smooth muscle cells of wild type and hypercholesterolemic mice. Treatment with JWH-133 reduced ROS production and NADPH-oxidase expression in hypercholesterolemic mice penis. Furthermore, JWH-133 increased endothelial NO synthase expression in the corpus cavernosum and augmented NO bioavailability. The decrease in oxidative stress levels was accompanied with a reduction in corpus cavernosum collagen content. In summary, CB2 activation decreased histological features, which were associated with erectile dysfunction in hypercholesterolemic mice.
    Full-text · Article · Nov 2013 · Clinical and Developmental Immunology
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    • "Different pathophysiological stimuli are involved in the development of diabetic cardiomyopathy (DCM) and mediate tissue injury, leading to left ventricular (LV) systolic and diastolic dysfunction. The mechanisms of DCM are multifaceted, involving modified action potential, Ca 2+ transient and Ca 2+ sensitivity of contractile elements345, increased oxidative stress678, activation of various pro-inflammatory and apoptotic signaling pathways9101112, decreased autophagy131415 and the accumulation of advanced glycation end products [16,17] among many others. Numerous enzymes that contribute to myocardial injury have been documented to be abnormally expressed in the diabetic myocardium [8,18,19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Heme oxygenase-1 (HO-1) has been implicated in cardiac dysfunction, oxidative stress, inflammation, apoptosis and autophagy associated with heart failure, and atherosclerosis, in addition to its recognized role in metabolic syndrome and diabetes. Numerous studies have presented contradictory findings about the role of HO-1 in diabetic cardiomyopathy (DCM). In this study, we explored the role of HO-1 in myocardial dysfunction, myofibril structure, oxidative stress, inflammation, apoptosis and autophagy using a streptozotocin (STZ)-induced diabetes model in mice systemically overexpressing HO-1 (Tg-HO-1) or mutant HO-1 (Tg-mutHO-1). The diabetic mouse model was induced by multiple peritoneal injections of STZ. Two months after injection, left ventricular (LV) function was measured by echocardiography. In addition, molecular biomarkers related to oxidative stress, inflammation, apoptosis and autophagy were evaluated using classical molecular biological/biochemical techniques. Mice with DCM exhibited severe LV dysfunction, myofibril structure disarray, aberrant cardiac oxidative stress, inflammation, apoptosis, autophagy and increased levels of HO-1. In addition, we determined that systemic overexpression of HO-1 ameliorated left ventricular dysfunction, myofibril structure disarray, oxidative stress, inflammation, apoptosis and autophagy in DCM mice. Furthermore, serine/threonine-specific protein kinase (Akt) and AMP-activated protein kinase (AMPK) phosphorylation is normally inhibited in DCM, but overexpression of the HO-1 gene restored the phosphorylation of these kinases to normal levels. In contrast, the functions of HO-1 in DCM were significantly reversed by overexpression of mutant HO-1. This study underlines the unique roles of HO-1, including the inhibition of oxidative stress, inflammation and apoptosis and the enhancement of autophagy, in the pathogenesis of DCM.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "In a cell culture model of hyperglycemia and hypoxia, phosphorylation of c-Jun N-terminal kinases (JNK) and p38 MAPK is induced, leading to overproduction of ROS and disruption of tight junctions in ARPE-19 cells [47]. In streptozotocin-induced diabetic mice, activation of cannabinoid-1 receptor contributes to DR by increasing MAPK activation, oxidative stress, and inflammatory signaling [48]. In addition, PARP and nuclear factor-kappa B (NFκB) are also downstream effectors of oxidative stress. "
    [Show abstract] [Hide abstract] ABSTRACT: Oxidative stress plays a crucial role in the pathogenesis of retinal ischemia/hypoxia, a complication of ocular diseases such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Oxidative stress refers to the imbalance between the production of reactive oxygen species (ROS) and the ability to scavenge these ROS by endogenous antioxidative systems. Free radicals and ROS are implicated in the irreversible damage to cell membrane, DNA, and other cellular structures by oxidizing lipids, proteins, and nucleic acids. Anti-oxidants that can inhibit the oxidative processes can protect retinal cells from ischemic/hypoxic insults. In particular, treatment using anti-oxidants such as vitamin E and lutein, inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or related signaling pathways, and administration of catalase and superoxide dismutase (SOD) are possible therapeutic regimens for DR, ROP, and other retinal ischemic diseases. The role of oxidative stress in the pathogenesis of DR and ROP as well as the underlying mechanisms involved in the hypoxia/ischemia-induced oxidative damage is discussed. The information provided will be beneficial in understanding the underlying mechanisms involved in the pathogenesis of the diseases as well as in developing effective therapeutic interventions to treat oxidative stress-induced damages.
    Full-text · Article · Oct 2012 · Oxidative Medicine and Cellular Longevity
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