Cardiovascular Complications

Accurate glucose, blood pressure, and plasma lipid controls, as well as preventive care practices, are effective in reducing the number of complications in certain patient cohorts with diabetes; however, they have their own limitations. For example, although intensive glucose-lowering therapy reduces glycated hemoglobin levels, it increases 5-year mortality compared with standard therapy (ACCORD trial).48 The understanding of the pathogeneses of microvascular and macrovascular complications in diabetes is paramount for the development of new therapeutic targets. Recently, several studies highlighted the important role of the ECS in the regulation of vascular inflammation, oxidative stress, and atherosclerosis,49 suggesting that the modulation of the ECS and the administration of plant-derived cannabinoids with antioxidant and anti-inflammatory properties might be beneficial in the treatment of cardiovascular complications associated with diabetes.

Both CB1 and CB2 receptors are expressed in the cells of the cardiovascular system, including cardiomyocytes, fibroblasts, endothelial and vascular smooth muscle cells, and infiltrating immune cells.49 CB1 receptor activation by AEA or synthetic agonists induces ROS production, MAPK activation, and cell death in human coronary endothelial cells,50 mimicking the pathogenesis of diabetes-induced endothelial dysfunction.21 The activation of MAPK is only partially mediated by ROS, suggesting direct CB1receptor–mediated MAPK activation. Furthermore, activation of CB1 receptors leads to increased angiotensin-1 receptor expression and nicotinamide adenine dinucleotide phosphate oxidase activity, which contribute to ROS production.51 In contrast to CB1 receptor activation, the activation of the CB2 receptor is coupled with decreased endothelial cell activation, monocyte-endothelial adhesion, and transendothelial monocyte migration after TNF-α or lipopolysaccharide stimulation,52 hallmarks of the development of atherosclerosis. Activation of the CB2 receptor also attenuates TNF-α–triggered activation of both NF-κB and Rho, up-regulation of adhesion molecules, and increased expression levels of monocyte chemoattractant protein-1 (MCP-1) in endothelial cells. CB1receptor activation by ECs and/or synthetic ligands also promotes ROS generation, MAPK activation, and inflammatory responses in macrophages and neutrophils,53,54 whereas CB2 receptor activation exerts opposing functions.4

Vascular smooth muscle proliferation and migration are also key events in the pathogenesis of atherosclerosis and, therefore, in all macrovascular complications of diabetes. CB1 receptors were suggested to play an important role in this event because receptor blockade was able to inhibit vascular smooth muscle proliferation and migration in response to platelet-derived growth factor stimulation by inhibiting Ras and extracellular signal–regulated kinase 1/2 activation.55 The application of a CB2 receptor agonist showed a similar efficacy in the attenuation of vascular smooth muscle proliferation,56 indicating an opposing role of the cannabinoid receptors in both endothelial cell activation and vascular smooth muscle proliferation.

The first direct evidence that cannabinoid receptors play a key role in the pathogenesis of atherosclerosis came from an ApoE−/−mouse model.57 THC treatment reduced the development of atherosclerotic plaques and macrophage content through the activation of CB2 receptors. The antiatherosclerotic properties of THC were associated with a reduction of the TH1 response and an inhibition of monocyte/macrophage migration to the site of inflammation. Later, it was shown that the CB1 receptor antagonist RIO was also able to inhibit atherosclerosis in mouse models.58,59Its beneficial effects paralleled an improved metabolic profile and a decreased inflammatory cytokine level, decreasing thioglycollate-induced macrophage recruitment.

The relevance of these described findings in metabolic syndrome was investigated by long-term RIO treatment in obese Zucker rats.60 RIO was able to attenuate increased systolic blood pressure and metabolic abnormalities without altering endothelium-dependent relaxation and restored vascular contraction induced by α-adrenergic agonists. RIO also increased cyclooxygenase 2 expression and prostacyclin production in the aortas of obese Zucker rats.61 In a rat model of prediabetic metabolic syndrome, long-term RIO treatment did not alter macrovascular response, as shown by the unaltered endothelial function of aortic rings and the incidence of ischemic myocardial lesions, but it diminished microvascular complications, reducing the albumin-creatinine ratio, an index of renal vascular function, and the fraction of sclerotic glomeruli.62

The first clinical trial investigating the potential benefit of long-term CB1 receptor blockade with RIO on the progression of atherosclerosis in obese patients with coronary artery disease did not have a clear conclusion (STRADIVARIUS trial).63 Although RIO failed to alter disease progression for the primary end point (ie, atheroma volume), it showed a favorable effect on the secondary end point (ie, total atheroma volume). Additional post hoc exploratory analyses revealed that the changes in mean maximum atheroma thickness were favorably affected by RIO. However, changes in atheroma volume in the most diseased 10-mm subsegments showed no significant difference between treatments. To clarify whether this secondary end point result can be translated into a clinical benefit (eg, myocardial infarction, stroke, and cardiovascular death reduction), the CRESCENDO trial was launched.29 The trial, however, was prematurely terminated because of increasing concerns related to increased anxiety and suicide rates in the RIO treatment group. Additional trials are needed to clarify whether modification of the ECS can lead to a clinically relevant decrease in macrovascular complications of diabetes, as soon as an effective peripheral CB1 receptor antagonist30 or a CB2 receptor agonist4 reaches the clinical phase of development.

Independent from macrovascular complications, diabetic cardiomyopathy is a distinct primary disease process that leads to heart failure in diabetic patients. Diabetic cardiomyopathy is characterized by left ventricular hypertrophy and diastolic dysfunction due to myocardial collagen and advanced glycation end product deposition.64 ROS have been implicated in all stages of the development of heart failure, including cardiac hypertrophy, fibrosis, and contractile dysfunction.65 The role of the ECS in diabetic cardiomyopathy has not been investigated in detail, even though several studies have shown the involvement of cannabinoid receptors in oxidative stress–related cardiac dysfunction. CB1receptors can mediate oxidative stress and cell death in doxorubicin-induced cardiomyopathy models and in human cardiomyocytes66,67; this damage is enhanced in mice deficient in the main EC, AEA-metabolizing enzyme, FAAH.54 CB1 receptor inhibition by RIO was also shown to be protective in a myocardial infarction model in which mice were fed a standard or high-fat diet.68 In contrast, the activation of CB2 receptors showed cardioprotective effects,69 which were mediated by three different mechanisms: reduced superoxide production, increased levels of extracellular signal–regulated kinase 1/2 and signal transducer and activator of transcription-3 phosphorylation, and inhibited neutrophil recruitment. Although direct involvement of the ECS has not yet been proven in diabetic cardiomyopathy, the plant-derived cannabinoid CBD attenuates inflammation, oxidative stress, cell death, myocardial dysfunction, and fibrosis in a diabetic cardiomyopathy model.14 These beneficial effects involve the attenuation of diabetes-induced myocardial NF-κB and MAPK activation and the promotion of survival mechanisms (eg, AKT/protein kinase B activation).

Diabetic Nephropathy

Diabetes is a leading cause of renal failure, accounting for 44% of all new cases in 2008.18 Hyperglycemia stimulates ROS generation, which ultimately leads (via diverse pathways) to diabetic nephropathy characterized by mesangial expansion, thickening of the glomerular basement membrane, and glomerular sclerosis.70There is strong evidence that both the synthetic and degradative pathways of the ECS are present in the kidney,71 and the CB1receptor is expressed in both glomeruli and tubular epithelial cells.72 In intrarenal arteries, the CB1 receptor is present in the endothelium,72 and the CB2 receptor is present in mesangial cells.71 Cannabinoid receptors play opposing roles in the regulation of oxidative stress in the kidney, as observed in a murine nephropathy model induced by cisplatin. The CB1 receptor promotes inflammation, oxidative/nitrative stress, and cell death through the activation of the p38-MAPK pathway.73 In contrast, CB2 receptor agonists limit damage after cisplatin administration by reducing oxidative stress, inflammation, and apoptosis.74 For therapeutic purposes, it is important that plant-derived CBD is also able to ameliorate cisplatin-induced nephrotoxicity.75

The first direct indication that the ECS plays an important role in the pathogenesis of diabetic nephropathy came from a murine model of metabolic syndrome.72 Blockade of CB1 receptors by RIO prolonged the lifespan of obese Zucker rats, even at a dose that did not influence the development of obesity. This effect was concurrent with a delay in the progression of renal failure as shown by the prevention of the development of proteinuria, improved creatinine clearance, and reduction of glomerular injury and renal hypertrophy compared with vehicle-treated rats. Similarly, RIO was also able to reduce the albumin-creatinine ratio and glomerular sclerosis in a prediabetic rat model of metabolic syndrome.62Definitive proof for the direct involvement of CB1 receptors in diabetic nephropathy arose from a type 1 diabetic model in which metabolic effects did not confound the outcome.76 The CB1receptor was found to be overexpressed by glomerular podocytes after streptozotocin treatment. The selective CB1 antagonist AM-251 reduced proteinuria by preventing a decrease in the mRNA and protein levels of the slit diaphragm molecules nephrin, podocin, and zonula occludens-1 in diabetic kidneys.76 Similar to the cisplatin-induced nephropathy model, an opposing protective effect of CB2receptor activation was demonstrated in the type 1 diabetic nephropathy model. CB2 agonists ameliorated albuminuria, podocyte protein down-regulation, and glomerular monocyte infiltration without affecting early markers of fibrosis and reduced chemokine receptor-2 expression in both the renal cortex and cultured podocytes, suggesting that CB2 receptor activation may interfere with the deleterious effects of MCP-1 signaling.77Podocytes express the CB2 receptor both in vitro and in vivo. The CB2 receptor was down-regulated in kidney biopsy specimens from patients with advanced diabetic nephropathy, and renal levels of the CB2 ligand 2-AG were reduced in diabetic mice, suggesting impaired CB2 signaling.77

The in vivo results were supported by in vitro findings that provided more mechanistic insight as to how the ECS influences the pathogenesis of renal failure in diabetes and the role of tubular processes in the effects of ECs during the development of diabetic kidney damage. In vitro, AEA significantly increases the hypertrophy of proximal tubular cells.78 CB1 antagonists reduced and CB2 antagonists increased the observed hypertrophy. In another study, the hyperlipidemia-induced tubular cell dysfunction observed in diabetic kidneys was modeled by palmitic acid–induced apoptosis in HK-2 cells.79 In this system, CB1 receptor overexpression was observed in a cyclooxygenase-dependent manner. Blockade of CB1 receptors was able to ameliorate palmitic acid–induced endoplasmic reticulum stress and the subsequent apoptosis. In rat mesangial cells, high glucose levels up-regulate CB1 mRNA expression levels and internalization in NF-κB– and cytosolic phospholipase A2–dependent manners.80 CB1 antagonism prevented high glucose concentration–induced apoptosis via the attenuation of endoplasmic reticulum stress, providing further evidence of the potential beneficial effects of CB1 receptor blockade in diabetic nephropathy.

Diabetic Retinopathy

Diabetes is the leading cause of new cases of blindness and preventable blindness among adults. Vascular inflammation and endothelial cell death caused by oxidative and nitrative stress are characteristics of diabetic retinopathy.21 In the early stages, retinopathy is characterized by microaneurysm formation and microvascular lesions and later by extensive intraretinal hemorrhage that culminates in proliferative diabetic retinopathy with neovascularization and either preretinal or vitreous hemorrhage.81

The ECS is present in the retina as shown by the presence of AEA, 2-AG, and the metabolizing enzymes FAAH and MAGL.82 CB1receptors are expressed in the layers of the retina, ciliary body, iris, and choroid, whereas CB2 receptors are localized to the retina.83 It has been shown that EC levels are elevated in the eyes of patients with diabetic retinopathy.84 2-AG levels are elevated in the iris, whereas AEA levels are increased in the cornea, ciliary body, retina, and the choroid. The role of such an increase gained importance when we received insight into the role of CB1 receptor activation in diabetic retinopathy. Deletion of the CB1 receptor or treatment with a CB1 receptor antagonist prevented retinal cell death in a murine diabetes model.85 Treatment of diabetic mice or human retinal cells with CB1 receptor antagonists after exposure to high glucose levels attenuated oxidative/nitrative stress, reduced NF-κB activation and adhesion molecule levels, and attenuated MAPK activation. These observations were supported by the fact that hyperglycemia up-regulated CB1 receptor expression and induced apoptosis in retina pigment epithelial cells, effects that were preventable with a CB1receptor antagonist.86 Interestingly, hyperglycemia also decreased FAAH expression, leading to a locally increased concentration of AEA and thereby increasing apoptosis via CB1 receptor signaling.

The effect of CBD was also examined in experimental diabetic retinopathy. CBD was able to reduce oxidative stress, inflammation, cell death, and vascular hyperpermeability associated with diabetes.12 Consistent with these findings, CBD also inhibited p38-MAPK signaling. Furthermore, CBD also attenuated high glucose–induced endothelial cell dysfunction, ROS generation, and barrier disruption in primary human coronary artery endothelial cells.52The protective effects of CBD on retinal cell death were, at least in part, due to the reduction of tyrosine nitration of glutamine synthase in macroglial cells, thereby preventing the accumulation and excitotoxicity of glutamine through N-methyl-d-aspartate receptors.87

We are indebted to Dr. George Kunos (National Institute of Alcohol Abuse and Alcoholism) for his continuous support and to Prof. Raphael Mechoulam for critically reading the paper and making valuable suggestions. We apologize to colleagues whose important work may not be covered due to the brief nature of this review.