Endocannabinoids and Stroke Prevention: Review of Clinical Studies

Abstract

The societal burden of ischemic stroke suggests a need for additional therapeutic categories in stroke prevention. Modulation of the endocannabinoid system (ECS) is a rational target for stroke prevention because of its effects on inflammation, vascular tone, and metabolic balance, all well-described stroke risk factors. In this article, we summarize the existing ECS clinical studies in human subjects' research as they relate to conventional vascular risk factors associated with ischemic stroke. To date, 2-arachidonoylglycerol (2-AG) derivative endocannabinoids are consistently reported to be elevated in insulin resistance, whereas the N-arachidonoylethanolamine (AEA) endocannabinoid derivatives are elevated in obesity. The ECS role in metabolic health should examine the effects of 2-AG reduction and AEA augmentation as a means of stroke risk reduction. Cannabinoid receptors are reported on macrophages within atherosclerotic plaques and suggest a role for immunomodulation as a therapeutic for atherosclerosis through both peripheral immune cell CB1 antagonism and/or CB2 agonist. The effects of ECS on hypertension, smoking, physical activity, obstructive sleep apnea, heart failure, and atrial fibrillation are incompletely described and deserve further study. A limitation to ECS research is significant overlap with noncannabinoid molecular targets. Further exploration of the ECS needs to include the larger metabolomics context for a greater understanding of its therapeutic potential. Clinical translational studies in stroke prevention should be directed at ECS in metabolic balance and atherosclerosis.

Introduction

The societal burden of ischemic stroke is tremendous. With 15 million new cases worldwide annually, ischemic stroke is a major component of worldwide mortality and a leading cause of permanent disability. Effective prevention surpasses even the best management of an acute cerebral ischemia. Therefore, stroke prevention has been the subject of extensive clinical research efforts.

Stroke prevention encompasses a host of medical and surgical strategies directed at lessening the impact of conventional vascular risk factors on a population scale either before an index event or to lower the probability of stroke recurrence. The medical therapy mainstays of stroke prevention are antithrombotics, cholesterol-lowering agents, glucose regulation, antihypertensive treatment, physical activity, and smoking cessation. Therefore, optimal stroke prevention in the clinical setting results from the modification of these myriad cardiovascular and metabolic risk factors. The current review will explore clinical studies of endocannabinoids in each of these established ischemic stroke risk factors.

Two major stroke risk factors, atherosclerosis and metabolic syndrome, are driven by chronic inflammation. Currently, no stroke prevention therapy is directed at reducing systemic inflammation. The endocannabinoid system (ECS) has therapeutic potential for stroke prevention by altering levels of chronic inflammation.

Endocannabinoid System

The ECS is a distributed system of multiple phospholipid-derived bioactive fatty acid amides, G-coupled protein receptors, nuclear receptors, ligand-gated ion channels, and a synthetic and degrading enzymes.¹ The ECS is a subset of a larger milieu of bioactive lipids with affinity for cannabinoid receptors. Endocannabinoid molecules have activity at nonendocannabinoid receptors. These molecules are expressed in both constitutive and facultative manners throughout human physiology. Endocannabinoids have gained attention for key roles in reproductive biology, neurotransmission, metabolic regulation, vascular regulation, and immune cell function.

The most widely known endocannabinoids are of two derivatives: N-acylethanolamines (NEAs), of which the most studied is N-arachidonoylethanolamine (AEA), or anandamide; and 2-acyl glycerol derivatives, the most common of which is 2-arachidonoylglycerol (2-AG). These two “flagship” molecules are derived by separate metabolic pathways and have affinity for both cannabinoid and noncannabinoid receptors. In addition to the classically described cannabinoid receptors, CB1 and CB2; AEA and 2-AG crossreact with many additional noncannabinoid receptors, such as vanilloid receptors, orphan G-protein-coupled receptors, calcium channel receptors, and the peroxisome proliferator-activated receptors (PPARs). NEAs and 2-AG derivative molecules are synthesized by diacylglycerol lipases and are degraded by fatty acid amide hydrolases (FAAHs) and monoacylglycerol lipases (MAGLs), respectively.

The constitutive expression of these endocannabinoids has been referred to as endocannabinoid “tone” and varies across disease states. The tone is hypothesized to be an inherent response to physiologic stress and a marker of disease severity. Given its role in neurotransmission, inflammation, metabolic balance, and vascular tone, the ECS system provides opportunities for the exploration of stroke prevention therapeutic targets.

The complexity of the ECS system has made it difficult to study. Species-specific characteristics, contradictory findings in preclinical studies, and overlapping pharmacological targets have contributed to this complexity. Furthermore, preclinical studies of stroke prevention have been plagued by lack of translation from animal models to human studies. Therefore, the following review summarizes the existing evidence for a relationship between conventional ischemic stroke risk factors and endocannabinoid signaling, focusing on AEA and 2-AG expression in clinical studies. Interpretation of the current science of endocannabinoid signaling in clinical studies may present a more straightforward path to therapeutic translation.

Arterial Hypertension

Hypertension remains the single most important modifiable risk factor in ischemic stroke. AEA is observed to be elevated in patients with arterial hypertension compared with nonhypertensive controls.² Circulating AEA was positively correlated with both systolic and diastolic blood pressure in normotensive depressed women compared with controls, although differences in body mass index (BMI) between groups (depressed 31.5 vs. control 25.4 kg/m²) may explain this difference.³ Acute psychosocial stress in nonobese healthy volunteers was associated with concomitant elevations of blood pressure, cortisol, and circulating AEA in white American subjects but not in African American or Asian American subjects.⁴ Despite preclinical studies suggesting a role in vasodilation, currently no clear relationship between arterial hypertension and ECS signaling in humans exist at this time; the topic deserves further study.

Obesity and Insulin Resistance

Insulin resistance is an inflammatory state associated with increased risk of ischemic stroke hypothesized to be attributable to increased platelet thrombogenicity and accelerated atherosclerosis. The main endocannabinoids, AEA and 2-AG, have been observed to be elevated in overweight and obese subjects compared with normal weight controls.^2,5,6 Furthermore, BMI is positively correlated with plasma AEA levels but not 2-AG for individuals with a BMI >30 kg/m².^7,8 However, 2-AG is strongly correlated with the components of the metabolic syndrome, including waist circumference, intraabdominal fat area, low high density lipoprotein (HDL), elevated triglycerides, and fasting insulin.⁹

In nondiabetic obese postmenopausal women, levels of 2-AG were significantly higher for insulin-resistant women compared with insulin-sensitive controls. AEA levels were higher in insulin-sensitive women both at baseline and after 6 months of weight loss (mean loss of 4.9 kg) when compared with insulin-resistant controls; however, with no significant differences in fasting endocannabinoid levels between baseline and 6 months of follow-up despite weight loss. That is weight loss did not seem to affect levels of AEA.¹⁰ Overweight women with polycystic ovarian syndrome and greater insulin resistance, demonstrated by the homeostatic model of insulin resistance (HOMA-IR), in which higher levels are associated with greater insulin resistance, had elevated levels of both AEA and 2-AG compared with controls (HOMA-IR 2.7 vs. 1.0).

Subcutaneous adipose tissue in diabetic obese patients was observed to have higher levels of AEA compared with both nondiabetic obese and control subjects. The same study also observed lower levels of 2-AG in subcutaneous adipose tissue in controls leading to the hypothesis that diabetic subcutaneous fat may differ from visceral adipose.¹¹ The effect of glucose administration on endocannabinoid expression was investigated in obese and normal weight controls using the oral glucose tolerance test and hyperinsulinemic clamp. AEA was substantially higher in the obese compared with control (16 vs. 4.5 pmol/mL) and both groups decreased slightly after oral glucose challenge or insulin infusion.¹² This difference may relate to the doubling of subcutaneous expression of ECS-metabolizing enzyme, FAAH, observed in lean controls, a further example of the dynamic nature of the ECS.^13,14 The location of adiposity seems to play a role in endocannabinoid signaling, as visceral obesity is associated with higher 2-AG, supporting the hypothesis of the inflammation of the insulin-resistant state.¹⁵

Overall, it would appear that AEA levels are elevated in otherwise metabolically healthy obese individuals, whereas circulating 2-AG seems to be correlated with metabolic syndrome and insulin resistance. 2-AG, elevated in the inflammatory state of insulin resistance, activates human platelets in vitro possibly contributing to thrombotic risk.¹⁶ Given the current observation of the “obesity paradox,” the epidemiologically observed protective effect of obesity on stroke and heart disease, might suggest that higher levels of AEA may partially explain this finding. Further investigation is certainly warranted. Clinical studies determining whether 2-AG and AEA can distinguish healthy and nonhealthy obesity are needed.

Obstructive Sleep Apnea

Obstructive sleep apnea when moderate to severe increases the risk of hypertension, nocturnal atrial arrhythmia, and ischemic stroke. AEA¹⁷ and 2-AG¹⁸ exhibit diurnal variation. The potential role of endocannabinoids was investigated in obstructive sleep apnea in 20 nondiabetic dyslipidemic obese subjects compared with controls and found no differences in plasma AEA between sleep apnea and controls (1.8 pmol/mL vs. 2.1 pmmol). However, the severity of obstructive sleep apnea is positively correlated with AEA expression. When adjusted for age, BMI, HOMA-IR, and abdominal circumference, the apnea–hypopnea index, a marker of apnea severity where higher levels indicate worse disease, were 11.5, 20.8, and 46.7 events per hour compared with circulating AEA levels of 2.65, 3.18, and 3.61 nmol/L (r=0.36, p<0.01); the most severe of which were elevated compared with overweight controls, 2.53 nmol/L.

Of note, the HOMA-IR was positively correlated with severity of obstructive sleep apnea in this study, but the correlation remained significant after controlling for this factor.¹⁹ In addition to apnea severity, both AEA and 2-AG appear strongly correlated with blood pressure elevations in obese diabetics with severe sleep apnea as indicated by increases in the respiratory disturbance index. Notably, levels of 2-AG were markedly elevated in both obstructive sleep apnea groups (obese diabetic and nondiabetic) compared with obese controls.²⁰ Given these findings, ECS dysregulation in moderate-to-severe sleep apnea may be independent of known covariates such as obesity and diabetes.

Physical Activity

Physical exercise doubled levels of AEA in healthy volunteers completing an exercise protocol.²¹ In 10 healthy nonobese men and women, AEA was observed to substantially increase after a 30-min treadmill test maintaining 72% and 82% of calculated maximal heart rate respectively, although this marked increase in plasma AEA was not seen in the high-intensity exercise group who maintained 92% maximal heart rate for 30 min.²² Overall circulating endocannabinoid levels were higher in the 72% maximal heart rate group compared with the remaining groups. Acute increases in AEA were not observed with resistance training in young type 1 diabetics compared with baseline (0.09 vs. 0.11 pmol/mL).²³ AEA levels were elevated in healthy male cyclists at 75% maximal exertion and during the recovery period.²⁴ These data might suggest that transient AEA elevations are secondary to ischemic demand for oxidative metabolism at the threshold of anaerobic metabolism during high maximal heart rates. This finding would be consistent with the ischemic preconditioning therapeutic hypothesis of physical exercise.

Heart Failure

Systolic heart failure is associated with ischemic stroke when ejection fraction becomes depressed leading to ventricular stasis and thrombus formation. In 12 patients with dilated cardiomyopathy (mean age 43.6 years and ejection fraction of 21%), ventricular myocardial tissue samples demonstrated an 11-fold upregulation of CB2 receptor messenger RNA (mRNA) expression compared with younger control patients without heart failure. Additionally, circulating endocannabinoids were elevated compared with healthy controls with heart disease. The etiology of the heart failure was not reported, and healthy controls were not age matched, limiting its generalization.²⁵ Ten patients undergoing ventricular assist device pre- and postimplantation demonstrated substantially elevated plasma AEA and 2-AG compared with matched controls. No differences in AEA were observed but significantly lower 2-AG levels were observed postimplantation.²⁶ The postimplantation 2-AG reduction suggests causality, however, further study is warranted.

Atherosclerosis

Plasma AEA and 2-AG levels are higher in normal-weight coronary artery disease patients compared with controls (AEA 1.05 vs. 0.54 pmol/mL, 2-AG 13.3 vs. 7.67 pmol/mL, p<0.01). Furthermore, CB1 receptor mRNA expression is significantly elevated in lipid-rich atheroma (a marker of high-risk plaque) when compared with fibrous plaque, specifically found in CD68-expressing macrophages (foam cells). In vitro administration of CB1 receptor antagonist (1 μmol/L) to lipopolysaccharide-activated human macrophages decreased proinflammatory signaling by 25% through inhibition of phosphorylation of JNK signal transduction (interleukin [IL]-1β, IL-6, tumor necrosis factor-α, and matrix metalloproteinase [MMP]-9).²⁷ In contrast to coronary atherosclerosis, both CB1 receptor mRNA and protein were undetectable in both symptomatic and asymptomatic human carotid plaque samples and there was no difference in AEA or 2-AG between symptomatic and asymptomatic plaques. This finding needs to be replicated. However, CB2 receptor expression was decreased in downstream portions of symptomatic carotid plaques.²⁸

Arterial endocannabinoid levels sampled during coronary catheterization were reported in forty-three patients undergoing percutaneous coronary intervention for acute myocardial infarction and six patients with chronic stable angina. Interestingly, 25 of 43 patients demonstrated a marked elevation in arterial AEA observed in the infarct-related coronary artery compared with the aorta (401 pmol/mL vs. 35 pmol/mL), whereas serum AEA and 2-AG was undetectable in arterial sampling in patients with stable angina.²⁹

Thrombosis

In activated human platelet cell cultures, micromolar doses of AEA, cannabidiol (CBD), or WIN55,212-2 did not reduce P-selectin (a marker of platelet activity), nor did AEA, CBD, or WIN55,212-2 treatment alone activate platelets. Although extremely high levels of AEA (500–1000 μM) significantly reduced human platelet P-selectin expression from over 90% to 10–15%, the practical significance of this finding remains in doubt as intra-arterial levels of AEA have not been described to this concentration in vivo. Studies investigating the effects of ECS on thrombosis and coagulation are needed.

Therapeutic Potential

Despite overall limitations to our understanding of this physiology, the current public burden of stroke is great, and a successful translation of the ECS system would serve an immediate need. The existing clinical literature suggests possible roles for ECS modulation in stroke prevention through effects on atherosclerosis and metabolic disease. The following will briefly suggest avenues for clinical studies of ECS receptors and metabolites.

Atherosclerosis

Aortic and cervicocephalic atheroembolism is a major preventable cause of stroke. Plaque deposition is driven by inflammation, and an agent that reduces the driving force of atherosclerosis would be a potent therapeutic for stroke prevention by either stabilizing or regressing plaque. Reduction of proinflammatory cytokine formation through macrophage cannabinoid type 1 receptor blockade within atherosclerotic plaque suggests a role for CB1 antagonism. Of interest, this CB1 expression was not seen in human carotid plaques whether symptomatic or asymptomatic, yet CB2 receptors, also present on macrophages, were well described in carotid plaques. Notably, symptomatic carotid plaques had less collagen, increased MMP, and very limited CB2 receptor expression. Both studies need to be independently replicated to reconcile the apparent discrepancy. Preclinical studies have demonstrated that peripheral immune cell CB2 activation reduces inflammatory signaling, but this has not been shown in clinical studies to date. Therefore, a clinical study of simultaneous CB1 receptor antagonism and CB2 receptor agonism on plaque formation using agents restricted to the peripheral circulation to minimize off target central nervous system effects is a logical next step in the treatment of atherosclerosis.

Metabolic syndrome of insulin resistance

A key component to developing metabolic ECS therapeutics will be determining the relationship of 2-AG and derivatives to the metabolic syndrome. It appears that circulating 2-AG is elevated in insulin resistance but not obesity. The 2-AG remains elevated despite weight loss. A key research question is to determine whether the endocannabinoid response is pathologic or therapeutic. Additionally, AEA appears elevated in obesity but not necessarily in insulin resistance. Furthermore, AEA has important noncannabinoid pharmacological targets relevant to metabolic health, the PPARs α and γ. PPAR-α regulates fatty acid oxidation and PPAR-γ regulates glucose metabolism, two nuclear receptors that appear to be affected by herbal cannabis.

The most pressing need is to determine the effects of increased ECS signaling in insulin resistance on metabolic stroke risk factors, lipid fractions, glucose, insulin, and visceral adiposity. A small placebo-controlled pilot study investigating the effects of combinations CBD and tetrahydrocannabivarin (THCV) on metabolic health parameters did not show a marked benefit after 12 weeks when compared with placebo.³⁰ Agents that target endocannabinoid degradation (i.e., FAAH/MAGL inhibitors), phytocannabinoid derivatives, or plasma stable ECS lipid congeners are all candidates for this role. The next step to translating potential therapeutic effects of the ECS would be a clinical trial to examine simultaneous AEA augmentation and 2-AG inhibition on biomarkers of metabolic health in patients with insulin resistance.

Limitations

The majority of the existing clinical studies have been single time point peripheral venous endocannabinoid measurements correlated to clinical and laboratory characteristics. Pooled peripheral venous blood levels may represent an “after-the-fact” estimation of endocannabinoid signaling and may not reflect local alterations of expression at the site of interest (e.g., neurovascular unit, adipocyte, atherosclerotic plaque, or myocardium).

The plasma half-life of ECS molecules is on the order of minutes, requiring substantial time considerations for research planning that may require greater than single time points. Furthermore, tissue-specific cannabinoid receptor expression in disease states requires greater exploration and understanding. Next, the primary role of ECS regulation in disease is still fundamentally not understood; whether it is a primary pathological dysregulation, response to disease, or irrelevant bystander. Finally, and most importantly, it should be recognized that ECS system overlaps substantially with noncannabinoid receptors and occupies a place in a much larger dynamic equilibrium of lipid physiology and noncannabinoid receptors. Instead of simply focusing on ECS, it may be more meaningful to describe an endocannabinoidome and study this system in the context of a larger metabolome as stroke prevention therapies are developed in this space.

Conclusion

Extensive and elegant work has been completed demonstrating key functions of the ECS system. Now, clinical studies should investigate the overall dynamics of these lipid-signaling molecules and role of AEA, 2-AG in metabolic disease and CB1/CB2 receptor modulation in atherosclerosis in the context of the larger metabolome. Given overall safety profile of cannabinoid agents and the pressing societal need, despite the complexity of ECS function, the time for clinical studies of ECS-modulating agents in stroke prevention is now.

Abbreviations Used

2-AG

2-arachidonoylglycerol

AEA

N-arachidonoylethanolamine

BMI

body mass index

CB1

type one cannabinoid receptor

CB2

type two cannabinoid receptor

CBD

cannabidiol

ECS

endocannabinoid system

FAAH

fatty acid amide hydrolase

HDL

high density lipoprotien

HOMA-IR

homeostatic model of insulin resistance

IL

interleukin

MAGL

monoacylglycerol lipase

MMP

matrix metalloproteinase

mRNA

messenger RNA

NEA

N-acylethanolamines

PPAR

peroxisome proliferator-activated receptor

THCV

tetrahydrocannabivarin

Author Disclosure Statement

No competing financial interests exist.

Cite this article as: Scharf EL, Ebbert JO (2020) Endocannabinoids and stroke prevention: review of clinical studies, Cannabis and Cannabinoid Research 5:1, 6–11, DOI: 10.1089/can.2018.0066.