Cannabidiol Attenuates Cardiac Dysfunction, Oxidative Stress

doi:10.1016/j.jacc.2010.07.033 2010;56;2115-2125 J. Am. Coll. Cardiol.

Raphael Mechoulam, and Pál Pacher Lauren Becker, György Haskó, Lucas Liaudet, David A. Wink, Aristidis Veves,Shingo Matsumoto, Yoshihiro Kashiwaya, Béla Horváth, Bani Mukhopadhyay,

Mohanraj Rajesh, Partha Mukhopadhyay, Sándor Bátkai, Vivek Patel, Keita Saito,

Inflammatory and Cell Death Signaling Pathways in Diabetic CardiomyopathyCannabidiol Attenuates Cardiac Dysfunction, Oxidative Stress, Fibrosis, and

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Journal of the American College of Cardiology Vol. 56, No. 25, 2010© 2010 by the American College of Cardiology Foundation ISSN 0735-1097/$36.00P

PRE-CLINICAL RESEARCH

Cannabidiol Attenuates Cardiac Dysfunction,Oxidative Stress, Fibrosis, and Inflammatory andCell Death Signaling Pathways in Diabetic CardiomyopathyMohanraj Rajesh, PHD,* Partha Mukhopadhyay, PHD,* Sandor Batkai, MD, PHD,* Vivek Patel,*Keita Saito, PHD,‡ Shingo Matsumoto, PHD,‡ Yoshihiro Kashiwaya, MD, PHD,†Béla Horvath, MD, PHD,* Bani Mukhopadhyay, PHD,* Lauren Becker,* György Hasko, MD, PHD,§Lucas Liaudet, MD,� David A. Wink, PHD,‡ Aristidis Veves, MD,¶ Raphael Mechoulam, PHD,#Pal Pacher, MD, PHD*

Bethesda, Maryland; Newark, New Jersey; Lausanne, Switzerland; Boston, Massachusetts;and Jerusalem, Israel

Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation,oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabeticcardiomyopathy and primary human cardiomyocytes exposed to high glucose.

Background Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis inhumans.

Methods Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosismarkers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy,and flow cytometry.

Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associatedwith increased oxidative-nitrative stress, nuclear factor-�B and mitogen-activated protein kinase (c-Jun N-terminal ki-nase, p-38, p38�) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascularcell adhesion molecule-1), tumor necrosis factor-�, markers of fibrosis (transforming growth factor-�, connective tis-sue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 andpoly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyltransferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardialdysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways.Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclearfactor-�B activation, and cell death in primary human cardiomyocytes.

Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, stronglysuggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhapsother cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death andfibrosis. (J Am Coll Cardiol 2010;56:2115–25) © 2010 by the American College of Cardiology Foundation

ublished by Elsevier Inc. doi:10.1016/j.jacc.2010.07.033

Jerusalem, Ein Kerem, Jerusalem, Israel. Drs. Rajesh and Partha Mukhopadhyaycontributed equally to this article. This study was supported by the IntramuralResearch Program of the NIH/NIAAA (to Dr. Pacher) and NIDA Grant DA9789(to Dr. Mechoulam). Dr. Horvath is a recipient of a Hungarian Research CouncilScientific Research Fund Fellowship (NKTH-OTKA-EU, MB08-80238). Dr. Vevesreceives funding from Novartis for an investigator-initiated research grant, unrelatedto this study. Dr. Mechoulam is a consultant for GW Pharmaceuticals, UnitedKingdom, which is not involved in this publication and is unaware of it. All otherauthors have reported that they have no relationships to disclose.

rom the *Laboratory of Physiological Studies, National Institute on Alcoholbuse and Alcoholism (NIAAA), National Institutes of Health, Bethesda,aryland; †Laboratory of Metabolic Control, NIAAA, National Institutes ofealth, Bethesda, Maryland; ‡Radiation Biology Branch, National Cancer

nstitute, National Institutes of Health, Bethesda, Maryland; §Department ofurgery, University of Medicine and Dentistry, New Jersey–New Jersey Medicalchool, Newark, New Jersey; �Department of Intensive Care Medicine, Universityospital, Lausanne, Switzerland; ¶Microcirculation Laboratory and Joslin-Beth

srael Deaconess Foot Center, Beth Israel Deaconess Medical Center, Harvard
edical School, Boston, Massachusetts; and the #Department for Medicinalhemistry and Natural Products, Faculty of Medicine, Hebrew University of
Manuscript received May 15, 2010; revised manuscript received July 5, 2010,accepted July 6, 2010.



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2116 Rajesh et al. JACC Vol. 56, No. 25, 2010Cannabidiol for Diabetic Complications December 14/21, 2010:2115–25

Cardiovascular complications arethe leading cause of morbidityand mortality in diabetic pa-tients. Diabetic cardiomyopathycharacterized by myocardial leftventricular dysfunction (both di-astolic and later systolic), inde-pendent of atherosclerosis andcoronary artery disease, has beenwell documented in both humansand animals (1–3). The mecha-nism of diabetic cardiac dysfunc-tion is complex and involves in-creased oxidative/nitrative stress(4–7), activation of various down-stream transcription factors, pro-inflammatory and cell death path-ways such as nuclear factor (NF)-�B(8,9), poly(adenosine diphosphate[ADP]-ribose) polymerase (PARP)(10), and mitogen-activated pro-tein kinase (MAPK) (11,12), inac-tivation of pro-survival pathwayssuch as Akt (13), eventually culmi-nating in cell death (14) andchanges in the composition of ex-tracellular matrix with enhancedcardiac fibrosis and increased in-flammation (15).

Various components of theCannabis sativa (marijuana) plant,termed cannabinoids (e.g., themost characterized active ingredient,the delta 9-tetrahydrocannabinol[THC]), exert potent analgesiceffects through the activation ofclassic CB1 receptors located inthe central nervous system and anti-inflammatory properties throughthe activation of CB2 cannabi-noid receptors on immune cells(16). However, the major limita-tion of the therapeutic utility of

HC is the development of centrally mediated CB1-ependent psychoactive effects (16). Furthermore, the CB1eceptor activation in the cardiovascular system by endocan-abinoids may also contribute to the pathophysiology ofultiple cardiovascular diseases, including heart failure

nd atherosclerosis (17). In contrast to THC, cannabidiolCBD), the most abundant cannabinoid of Cannabisativa, which has been approved for the treatment ofnflammation, pain, and spasticity associated with multi-le sclerosis in humans since 2005 in Canada (18), doesot bind to these receptors (19); therefore, it is devoid ofsychoactive properties and has no potential to cause

Abbreviationsand Acronyms

ADP � adenosinediphosphate

CBD � cannabidiol

HCM � humancardiomyocytes

HG � high glucose

HNE � hydroxynonenal

ICAM � intercellularadhesion molecule

I�B-� � inhibitor of nucleartranscription factor nuclearfactor-�B

iNOS � inducible nitricoxide synthase

JNK � c-Jun N-terminalkinase

MAPK � mitogen-activatedprotein kinase

MMP � matrixmetalloproteinase

NADPH � nicotinamideadenine dinucleotidephosphate

NF-�B � nuclear factorkappa B

NT � nitrotyrosine

PARP � poly(ADP-ribose)polymerase

ROS � reactive oxygenspecies

SOD � superoxidedismutase

THC � delta9-tetrahydrocannabinol

TNF � tumor necrosisfactor

TUNEL � terminaldeoxynucleotidyl transferasedUTP nick end labeling

VCAM � vascular celladhesion molecule

dverse cardiac toxicity (20). Importantly, CBD is well ncontent.onlinejacc.orDownloaded from

olerated without side effects when chronically adminis-ered to humans (21,22).

A previous study has demonstrated cardiac protection byBD in myocardial ischemic reperfusion injury (23); there-

ore, we have investigated the potential protective effects ofBD in diabetic hearts and in primary human cardiomyo-

ytes exposed to high glucose. Our findings underscore theotential of CBD for the prevention/treatment of diabeticomplications.

ethods

nimals and treatment. All the animal protocols con-ormed to the National Institutes of Health (NIH) guide-ines and were approved by the Institutional Animal Carend Use Committee of National Institute on Alcohol Abusend Alcoholism (NIAAA)-NIH. Diabetes mellitus wasnduced in male C57/BL6J mice 8 to 12 weeks old,eighing 23 to 25 g (Jackson Laboratories, Bar Harbor,aine) by intraperitoneal injection of streptozotocin

Sigma, St. Louis, Missouri) at the dose of 50 mg/kgissolved in 100 mM citrate buffer pH 4.5 for 5 consecutiveays. After 1 week, blood glucose levels were measuredsing Ascensia Coutour Glucometer (Bayer HealthCare,arrytown, New York) by mandibular vein puncture blood

ampling. Mice that had blood sugar values �250 mg/dlere used for the study. In the first set of experiments-week diabetic mice were treated with CBD (1, 10, or 20g/kg intraperitoneally) or vehicle for 11 weeks (Onlineig. 1). In another set of experiments, 8-week diabetic miceere treated with CBD or vehicle for 4 weeks (Online Fig. 2).he CBD was isolated as described earlier (24). The

orresponding control groups were treated with eitherehicle or CBD alone for the same duration. All the animalsere provided with food and water ad libitum.emodynamic measurements in mice. Left ventricular

erformance was measured in mice anesthetized with 2%soflurane as previously described (25,26).

etermination of superoxide dismutase activity, malon-ialdehyde, reduced glutathione, oxidized glutathione,-HNE, and protein carbonyl content. The superoxideismutase (SOD) activities, and reduced glutathione andxidized glutathione, malondialdehyde, 4-HNE, and pro-ein carbonyl levels in the myocardial tissues were deter-ined as described in the Online Appendix.Determination of myocardial reactive oxygen species

ROS) by electron paramagnetic resonance spectrometer isescribed in the Online Appendix.everse transcription and real-time polymerase chain

eaction. Preparation of samples and reverse transcriptionnd real-time polymerase chain reaction (PCR) experimentsrom heart tissues and the primers are described in the

nline Appendix and Online Table 1.etermination of PARP, caspase 3/7 activities, chromatin

ragmentation, TUNEL, and 3-NT content. The PARPnd caspase 3/7 activities, chromatin fragmentation, termi-
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2117JACC Vol. 56, No. 25, 2010 Rajesh et al.December 14/21, 2010:2115–25 Cannabidiol for Diabetic Complications

TUNEL), and 3-nitrotyrosine (3-NT) content in the heartomogenates and/or human cardiomyocyte extracts areescribed in the Online Appendix.

estern immunoblot analysis. Sample preparations,estern immunoblot analysis, and sources of antibodies are

escribed in the Online Appendix.mmunohistochemistry. The immunohistochemistry/taining from frozen or formalin-fixed myocardial tissuesnitrotyrosine, TUNEL, Sirius red) is described in thenline Appendix.ell culture studies. Human cardiomyocytes (HCM) alongith the culture medium were purchased from ScienCellesearch Laboratories (Carlsbad, California) and were main-

ained and treated as described in the Online Appendix.imultaneous determination of cytosolic and mitochondrialOS generation and apoptosis by flow cytometry. Mi-

ochondrial superoxide/ROS generation and cell death were

Figure 1 Cannabidiol Attenuates Diabetes-Induced Left Ventric

(A) Representative pressure-volume (P-V) loops at different preloads after inferiorend-diastolic P-V relations (EDPVR) in control (Co) and diabetic mice treated with vand EDPVR in diabetic mice indicates decreased systolic and diastolic functions, w11 weeks. (B) Twelve weeks of diabetes was associated with decrease in left venrespect to time (�dP/dt), stroke work, ejection fraction, cardiac output, and load-iend-diastolic volume relation [dP/dt-EDV], and end-systolic pressure-volume relatio(LVEDP) and prolongation of relaxation time constants (� Weiss and Glantz), which11 weeks. Results are mean � SEM of 8 to 11 per group. *p � 0.05 versus vehi

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etermined as described (27) and are detailed in the Onlineppendix.tatistical analysis. Results are expressed as mean � SEM.tatistical comparisons were made by 1-way analysis of vari-nce followed by Newman-Keuls post-hoc analysis usingraphPad Prism 5 software (San Diego, California). When

eterogeneity of variance was present, analysis of variance waserformed after logarithmic transformation of the data. Prob-bility values of p � 0.05 were considered significant.

esults

lood glucose levels, pancreas insulin content, and bodyeights. Diabetic animals exhibited increased blood glu-ose levels (Online Figs. 1A and 2A) with the decrease inhe body weight (Online Fig. 1D). Diabetic animals alsoad increased glycosylated hemoglobin (HbA1c) levels

ysfunction

ava occlusion, showing differences in the end-systolic P-V relations (ESPVR) andor cannabidiol (CBD). The shift of P-V loops right and changed slope of ESPVRere less pronounced in diabetic mice treated with CBD (20 mg/kg daily) for

r systolic pressure (LVSP), maximum first derivative of ventricular pressure withndent indexes of contractility (pre-load–recruitable stroke work [PRSW], dP/dt–ax], respectively), and an increase in left ventricular end-diastolic pressurelargely attenuated by CBD treatment (20 mg/kg daily intraperitoneally) forntrol/CBD alone; #p � 0.05 versus diabetes (D).

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ith concomitant decline in the pancreas insulin contentOnline Fig. 1B and 2B). The CBD or vehicle treatment1, 10, or 20 mg/kg intraperitoneally) for 11 or 4 weeksid not significantly alter the body weight, blood glucose

evel, or pancreas insulin content in either control oriabetic animals (Online Figs. 1 and 2).

Figure 2 CBD Attenuates Diabetes-Induced Myocardial Oxidati

Oxidative stress in the myocardial tissues were determined by measuring (A) malospecies (ROS) levels by electron paramagnetic resonance spectrometer, as descri(NADPH) oxidase subunits messenger ribonucleic acid (mRNA) expression by real-t(reduced glutathione [GSH] and oxidized glutathione [GSSG]) content, and (G) supdiol (CBD) alone; #p � 0.05 versus diabetes (D), n � 6 to 9 per group.


BD treatment attenuates diabetes-induced hemody-amic alterations. Twelve weeks of established diabetes wasssociated with impaired diastolic and systolic left ventricularunction, which was largely attenuated by the treatment withBD for 11 weeks (starting 1 week after the establishment ofiabetes) (Fig. 1). The CBD treatment also improved the

ress

ehyde (MDA), (B) 4-HNE, (C) protein carbonyls content, and (D) reactive oxygenthe Methods section, and the (E) nicotinamide adenine dinucleotide phosphateverse transcriptase-polymerase chain reaction, (F) endogenous antioxidantsdismutase (SOD) activity. *p � 0.05 versus vehicle control (Co) and cannabi-

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iabetes-induced myocardial dysfunction when it was given forweeks in 8-week diabetic mice (Online Fig. 3).BD treatment attenuates diabetes-induced myocardial

xidative stress. There was increased accumulation ofipid peroxides (Figs. 2A and 2B), protein carbonyls (Fig. 2C),OS generation (Fig. 2D), expression of messenger ribo-ucleic acid of various ROS-generating nicotinamide ade-ine dinucleotide phosphate (NADPH) oxidases (p22phox,67phox, gp91phox) (Fig. 2E) with concordant decrease ofeduced/oxidized glutathione ratio (Fig. 2F) and attenuatedctivity of the superoxide-eliminating enzyme, the SODFig. 2G), in hearts of diabetic mice. These changes werettenuated when mice were treated with CBD for 11 weeksuring the course of the diabetes (Figs. 2A to 2G).BD treatment attenuates diabetes-induced myocardialuclear factor-�B activation and inflammation. Ashown in Figure 3A, there was a marked inhibitor ofuclear transcription factor NF-�B (I�B-�) degradation

n the cytosol of diabetic hearts, with increased phos-

Figure 3 CBD Attenuates Diabetes-Induced Myocardial NF-�B A

(A) Western blot analysis demonstrates inhibitor of nuclear transcription factor NFnuclear translocation of p65 nuclear factor (NF)-�B in the nuclear fraction of the h(D) The messenger ribonucleic acid (mRNA) expression of ICAM (intercellular adhefactor (TNF)-� in the respective groups, as indicated. (F) Western blot analysis forsues. *p � 0.05 versus vehicle control (Co) and cannabidiol (CBD) alone; #p � 0


horylation of I�B-� leading to release of active p65F-�B, which subsequently translocates to the nucleus to

nduce the inflammatory and apoptotic gene expressionsFig. 3B). Gel shift assay also confirmed the NF-�Bctivation in diabetic hearts (Fig. 3C). The CBD treatment ofiabetic mice inhibited the I�B-� and subsequent65NF-�B nuclear translocation (Figs. 3A to 3C). TheBD treatment also inhibited the NF-�B–dependentRNA and/or protein expression of intercellular adhesionolecule (ICAM)-1 and vascular cell adhesion molecule

VCAM)-1 (Figs. 3D and 3F) and pro-inflammatory cyto-ine tumor necrosis factor (TNF)-� (Figs. 3E and 3G),espectively, in the diabetic myocardial tissues.

BD treatments attenuates diabetes-induced nitrativetress. There was significant increase in inducible nitricxide synthase (iNOS) expression (Fig. 4A) and 3-NTccumulation (Figs. 4B to 4E) in hearts of diabetic miceompared to vehicle or CBD alone treated mice. TheBD treatment attenuated the diabetes-induced iNOS

tion

B-�) expression and its phosphorylation in the cytosolic fraction and (B) thesue homogenates. (C) The gel shift assay demonstrates NF-�B activation.olecule)-1 and VCAM (vascular cell adhesion molecule)-1. (E) Tumor necrosis

otein expression of ICAM-1/VCAM-1, and (G) TNF-� protein in the myocardial tis-rsus diabetes (D), n � 6 to 9 per group.

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xpression and 3-NT accumulation (marker of nitrativetress) (Figs. 4B to 4E).BD treatment attenuates diabetes-induced MAPK

ctivation and apoptosis. There was marked increase inhe p38MAPK (Fig. 5A) and c-Jun N-terminal kinaseJNK) (Fig. 5B) activation in the myocardial tissues ofiabetic mice. In addition, there was marked activation38�MAPK (Fig. 5C) and slightly diminished p38�MAPKFig. 5C) in the diabetic myocardium. There was also activa-ion of MAPKAPK-2 in the diabetic heart (Fig. 5D). CBDreatment for 11 weeks significantly mitigated p38MAPK,NK, p38�MAPK, MAPKAPK-2 activation, while itas not effective in restoring the p38�MAPK levels. In

ddition, Akt activation was also significantly hamperedn the diabetic myocardium, which was attenuated withBD treatment (Fig. 5E). In diabetic myocardium, thereas marked increase in caspase 3 cleavage, caspase 3/7

ctivity (Figs. 6A and 6B), chromatin fragmentation, andARP activity (Figs. 6C and 6D), and enhanced apopto-is (Figs. 6E and 7); all these changes in diabetes werettenuated by CBD treatment.BD treatment attenuates diabetes-associated myocardialbrosis. Real-time reverse transcriptase-polymerase chaineaction analysis revealed significant increases in the pro-brotic gene expressions (Fig. 8A) and in collagen deposi-ion (Fig. 8B) in diabetic hearts, and these were attenuatedy CBD (Fig. 8).BD post-treatment after the establishment of diabetic

ardiomyopathy attenuates diabetes-induced myocardialxidative/nitrative stress, cell death, and fibrosis. Re-arkably, CBD 20 (mg/kg) treatment also attenuated the

iabetes-induced increased myocardial nitrative stress, celleath (Online Fig. 4) and fibrosis (Online Fig. 5) when itas given for 4 weeks in 8-week diabetic mice.BD treatment attenuates high glucose-induced cytosolic

nd mitochondrial ROS generation and 3-NT formation inCM. High glucose (HG) treatment of HCM for 48 harkedly increased cytosolic (Online Fig. 6A) and mito-

hondrial (Online Fig. 6B) ROS/superoxide generationompared with cells treated with either D-glucose 5 mM,-glucose 30 mM, or CBD (4 �M) alone for the sameuration. The CBD markedly attenuated the HG-inducedncreased ROS generation (Online Figs. 6A and 6B) and-NT accumulation in HCM (Online Fig. 6C).BD mitigates HG-induced NF-�B activation and

poptosis in HCM. The HG treatment induced NF-�Bctivation (Online Figs. 7A and 7B) and increased apoptosisnd PARP-dependent cell death in cardiomyocytes (Onlineigs. 8A and 8B); the antiapoptotic activity of CBD wasediated, at least in part, by its ability to modulate Akt

ctivity (Online Fig. 8A).

iscussion

ccumulating evidence suggests that increased oxidative/

Figure 4 CBD Inhibits Diabetes-Induced Myocardial,iNOS Expression, and 3-NT Accumulation

(A) Expression of inducible nitric oxide synthase (iNOS) was determined byWestern immunoblot in the heart tissues. (B) Levels of 3-nitrotyrosine(3-NT) in the heart samples were quantitatively determined by enzyme-linkedimmunosorbent assay with indicated cannabidiol (CBD) concentration(mg/kg body weight), respectively. (C) Representative gel indicates thenitrated proteins analyzed by immunoprecipitation (I.P) with 3-NT specificantibody. (D) Representative images for the histochemical staining for 3-NTaccumulation in the formalin-fixed myocardial tissues (400� magnification).(E) Immunofluorescence staining for 3-NT from frozen sections as describedin Methods (400� magnification). *p � 0.05 versus vehicle control (Co)and CBD alone; #p � 0.05 versus diabetes (D), n � 6 to 8 per group.

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tream pro-inflammatory and cell death pathways playivotal roles in the development of complex biochemical,echanical, and structural alterations associated with dia-

etic cardiomyopathy (3,4,6,11,12,14,15). However, in spitef the accumulating knowledge obtained during the pastecades, the treatment of diabetic cardiomyopathy stillemains poor and largely symptomatic (1,2).

Cannabidiol, a nonpsychoactive component of marijuana,as been shown to exert anti-inflammatory and antioxidantffects both in vitro and in various preclinical models ofeurodegeneration and inflammatory disorders, independentrom classical CB1 and CB2 receptors (20). Furthermore, CBDas recently been reported to lower the incidence of diabetesmong nonobese diabetic mice (28) and to preserve thelood-retinal barrier in experimental diabetes (29).

In the present study, we have evaluated the effects ofBD treatment (for 11 weeks administered after the de-

truction of pancreatic beta cells and development of frankype 1 diabetes mellitus, as well as in 8-week diabetic

Figure 5 CBD Mitigates Diabetes-Induced Myocardial Activatio

Western blot analysis shows the (A) p38 mitogen-activated protein kinase (MA(E) Akt activation in the myocardial tissues. *p � 0.05 versus vehicle control

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ion, oxidative/nitrative stress, cell death, and interrelatedignaling pathways, using a mouse model of type I diabeticardiomyopathy or primary human cardiomyocytes exposedo HG. Because significant cardiac dysfunction in thisodel starts to develop from 4 weeks of established diabetes

4,10), with gradually increasing fibrosis thereafter (12,15)peaking around 8 weeks of established diabetes), in the firstreatment protocol (Online Fig. 1A.), we aimed to study ifBD treatment can prevent the development of character-

stic alterations of type I diabetic cardiomyopathy; in theecond treatment protocol (Online Fig. 2A), we sought toetermine if it is able to reverse these changes once theyave already developed.Consistent with previous reports, diabetic cardiomyopa-

hy was characterized by declined diastolic and systolicyocardial performance associated with enhanced myocar-

ial expression of NADPH oxidase isoforms p22phox,67phox, gp91phox, attenuated antioxidant defense (de-reased glutathione content and SOD activity) coupled with

MAPKs and Augments Akt Activation

B) c-Jun N- terminal kinase (JNK), (C) p38�/�MAPK, (D) MAPKAPK-2, andnd cannabidiol (CBD) alone; #p � 0.05 versus diabetes (D), n � 6 per group.

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ion (4,6,10,12,15). The HG-induced ROS generation inddition to inducing lipid peroxidation may also initiatectivation of various stress signaling pathways (e.g., jun

Figure 6 CBD Mitigates Diabetes-Induced Myocardial Apoptosi

(A) Western blot analysis for the cleaved (Clvd) caspase (Casp) 3 and (B) caspas(PARP) activation and (E) quantitative terminal deoxynucleotidyl transferase dUTPversus vehicle control (Co) and cannabidiol (CBD) alone; #p � 0.05 versus diabet

Figure 7 CBD Mitigates Apoptosis in the Diabetic Myocardium

Shown are the representative terminal deoxynucleotidyl transferase dUTP nick endthat were treated with cannabidiol (CBD) for 11 weeks. For details, see Online App


-terminal kinase and p38MAPK). Our results are also ingreement with previous studies demonstrating enhancedctivation of p38MAPK and its downstream effector

Cell Death

activity, (C) chromatin fragmentation, and (D) poly(ADP-ribose) polymerased labeling (TUNEL) assay were performed, as described in Methods. *p � 0.05n � 6 to 9 per group.

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p38MAPKAPK-2) in diabetic cardiomyopathy models andemonstrating that pharmacological inhibition of38MAPK signaling attenuates the expression of cardiacnflammatory markers, such as TNF-�, interleukin-1�,nd interleukin-6, and collagen content associated withiabetic cardiomyopathy (11,12). Likewise, with recentvidence supporting an emerging role of p38� activationn diabetic cardiomyopathy (12), in addition to its alreadystablished role in mediating cell death during myocar-ial ischemic-reperfusion injury. The HG-induced ROSeneration also impairs important pro-survival signaling

Figure 8 CBD Attenuates Diabetes-Induced Cardiac Fibrosis

(A) Messenger ribonucleic acid (mRNA) expression of the profibrotic genes in thealone; #p � 0.05 versus diabetes (D), n � 9 per group. (B) Sirius red staining indare representative from 4 independent experiments. *p � 0.05 versus vehicle congroup. CTGF � connective tissue growth factor; MMP � matrix metalloproteinase;

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ro-inflammatory and cell death pathways such as NF-�B8,9) and nuclear enzyme poly(ADP)-ribose polymerase 110), which in turn regulate expression of importantro-inflammatory cytokines, cell adhesion molecules, andNOS. The latter results in increased nitrosative/nitrativetress, which is also implicated in cardiovascular compli-ations of diabetes (5). A recent study has also suggestedhat the NF-�B activation may induce increased oxidativetress and contributes to mitochondrial and cardiac dysfunc-ion in type II diabetes (9). Importantly, the oxidative-itrative stress, stress signaling, and inflammatory pathways

rdial tissues. *p � 0.05 versus vehicle control (Co) and cannabidiol (CBD)g collagen deposition and implying the extent of cardiac fibrosis. Images showno) and cannabidiol (CBD) alone; #p � 0.05 versus diabetes (D), n � 4 to 6 pertransforming growth factor.

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ually promoting the development of myocardial fibrosis3,9,11,15,30).

Treatment with CBD (Supplemental Fig. 1) was able tottenuate the oxidative-nitrative stress (decreased the myo-ardial ROS generation and expression of p22phox, p67phox,p91phox, restored glutathione content and SOD activity,ecreased 3-NT formation) and alterations of the pro-urvival (Akt) and stress signaling (p38, p38�, JNK) path-ays in diabetic hearts. It also attenuated the NF-�B

ctivation, expression of iNOS, TNF�, and ICAM-1, celleath, and fibrosis in diabetic myocardium, and improvedhe associated characteristic functional alterations. Impor-antly, CBD treatment was able to attenuate/reverse (al-hough to a lesser extent) some of the discussed diabetes-nduced myocardial biochemical and functional changesfter the establishment of diabetic cardiomyopathy withbrosis (Online Figs. 2 to 5). The CBD treatment alsottenuated the HG-induced increased reactive oxygen anditrogen species generation, NF-�B activation, and celleath in primary human cardiomyocytes (Online Figs. 6 to 8).

The discussed beneficial effects of CBD could be explainedn part by its potent antioxidant properties, which was firstuggested by the Nobel Prize winner Dr. Julius Axelrod (31).n the Axelrod study, CBD was more protective againstlutamate-induced neurotoxicity than any of the well-knowntioxidants (e.g., ascorbate or �-tocopherol), indicating addi-ional cytoprotective effects of CBD beyond its potent antiox-dant properties (31). Indeed, our recent results suggest thatBD may exert potent effects on key pro-inflammatory path-ays such as NF-�B and on pro-survival signaling such as Akt

n vivo, which is most likely not related to its antioxidant effect.his is also supported by observations that CBD decreases

nflammation in models in which conventional antioxidants areot very effective (e.g., in arthritis [20,32]), as well as by recenttudies demonstrating that CBD is a potent inhibitor ofacterial lipopolysaccharide-activated NF-�B proinflamma-ory pathway in microglia cells (33). These results are also inupport of the emerging role of the inflammation in theevelopment and progression of diabetic cardiomyopathy9,11,15,30).

Collectively, our results strongly suggest that CBD mayave tremendous therapeutic potential in the treatment ofiabetic cardiovascular and other complications by attenu-ting diabetes-induced oxidative/nitrative stress, inflamma-ion, cell death, and fibrotic pathways.

cknowledgmentshe authors are indebted to Dr. Murali C. Krishna forenerously providing his resources and expertise with electronaramagnetic resonance spectrometer measurements, to Drs.ergey Dikalov and Kathy K. Griendling for sending thelectron paramagnetic resonance spectrometer probe duringhe time when it was not commercially available, and to Dr.eorge Kunos for providing key resources and support. Dr.acher dedicates this study to the 80th birthday of Professor
aphael Mechoulam and to Dr. Julius Axelrod.

eprint requests and correspondence: Dr. Pal Pacher, Sectionn Oxidative Stress Tissue Injury, Laboratory of Physiologicaltudies, National Institutes of Health/NIAAA, 5625 Fishersane, MSC-9413, Bethesda, Maryland 20892-9413. E-mail:[email protected].

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ey Words: cannabinoids y diabetic complications y inflammation yxidative stress.

APPENDIX

or a detailed discussion of the Methods, supplemental references,
able, and figures, please see the online version of this article.


doi:10.1016/j.jacc.2010.07.033 2010;56;2115-2125 J. Am. Coll. Cardiol.

Raphael Mechoulam, and Pál Pacher Lauren Becker, György Haskó, Lucas Liaudet, David A. Wink, Aristidis Veves,Shingo Matsumoto, Yoshihiro Kashiwaya, Béla Horváth, Bani Mukhopadhyay,

Mohanraj Rajesh, Partha Mukhopadhyay, Sándor Bátkai, Vivek Patel, Keita Saito,

Inflammatory and Cell Death Signaling Pathways in Diabetic CardiomyopathyCannabidiol Attenuates Cardiac Dysfunction, Oxidative Stress, Fibrosis, and

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