The endocannabinoid anandamide causes endothelium-dependent vasorelaxation in human mesenteric arteries.

The endocannabinoid anandamide (AEA) causes vasorelaxation in animal studies. Although circulating AEA levels are increased in many pathologies, little is known about its vascular effects in humans. The aim of this work was to characterise the effects of AEA in human arteries. Ethical approval was granted to obtain mesenteric arteries from patients (n=31) undergoing bowel resection. Wire myography was used to probe the effects and mechanisms of action of AEA. RT-PCR was used to confirm the presence of receptor mRNA in human aortic endothelial cells (HAECs) and intracellular signalling proteins were measured using multiplex technology. AEA caused vasorelaxation of precontracted human mesenteric arteries with an Rmax of ∼30%. A synthetic CB1 agonist (CP55940) caused greater vasorelaxation (Rmax ∼60%) while a CB2 receptor agonist (HU308) had no effect on vascular tone. AEA-induced vasorelaxation was inhibited by removing the endothelium, inhibition of nitric oxide (NO) synthase, antagonising the CB1 receptor and antagonising the proposed novel endothelial cannabinoid receptor (CBe). AEA-induced vasorelaxation was not affected by CB2 antagonism, by depleting sensory neurotransmitters, or inhibiting cyclooxygenase activity. RT-PCR showed CB1 but not CB2 receptors were present in HAECs, and AEA and CP55940 had similar profiles in HAECs (increased phosphorylation of JNK, NFκB, ERK, Akt, p70s6K, STAT3 and STAT5). Post hoc analysis of the data set showed that overweight patients and those taking paracetamol had reduced vasorelaxant responses to AEA. These data show that AEA causes moderate endothelium-dependent, NO-dependent vasorelaxation in human mesenteric arteries via activation of CB1 receptors.

Cannabidiol protects an in vitro model of the blood-brain barrier from oxygen-glucose deprivation via PPARγ and 5-HT1A receptors.

BACKGROUND AND PURPOSE: In vivo and in vitro studies have demonstrated a protective effect of cannabidiol (CBD) in reducing infarct size in stroke models and against epithelial barrier damage in numerous disease models. We aimed to investigate whether CBD also affects blood-brain barrier (BBB) permeability following ischaemia. EXPERIMENTAL APPROACH: Human brain microvascular endothelial cell (HBMEC) and human astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen-glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance. KEY RESULTS: CBD (10 µM) prevented the increase in permeability caused by 4 h OGD. CBD was most effective when administered before the OGD, but protective effects were observed up to 2 h into reperfusion. This protective effect was inhibited by a PPARγ antagonist and partly reduced by a 5-HT1A receptor antagonist, but was unaffected by antagonists of cannabinoid CB1 or CB2 receptors, TRPV1 channels or adenosine A2A receptors. CBD also reduced cell damage, as measured by LDH release and by markers of cellular adhesion, such as the adhesion molecule VCAM-1. In HBMEC monocultures, CBD decreased VCAM-1 and increased VEGF levels, effects which were inhibited by PPARγ antagonism. CONCLUSIONS AND IMPLICATIONS: These data suggest that preventing permeability changes at the BBB could represent an as yet unrecognized mechanism of CBD-induced neuroprotection in ischaemic stroke, a mechanism mediated by activation of PPARγ and 5-HT1A receptors.

Cannabidiol causes endothelium-dependent vasorelaxation of human mesenteric arteries via CB1 activation.

AIMS: The protective effects of cannabidiol (CBD) have been widely shown in preclinical models and have translated into medicines for the treatment of multiple sclerosis and epilepsy. However, the direct vascular effects of CBD in humans are unknown. METHODS AND RESULTS: Using wire myography, the vascular effects of CBD were assessed in human mesenteric arteries, and the mechanisms of action probed pharmacologically. CBD-induced intracellular signalling was characterized using human aortic endothelial cells (HAECs). CBD caused acute, non-recoverable vasorelaxation of human mesenteric arteries with an Rmax of ∼ 40%. This was inhibited by cannabinoid receptor 1 (CB1) receptor antagonists, desensitization of transient receptor potential channels using capsaicin, removal of the endothelium, and inhibition of potassium efflux. There was no role for cannabinoid receptor-2 (CB2) receptor, peroxisome proliferator activated receptor (PPAR)γ, the novel endothelial cannabinoid receptor (CBe), or cyclooxygenase. CBD-induced vasorelaxation was blunted in males, and in patients with type 2 diabetes or hypercholesterolemia. In HAECs, CBD significantly reduced phosphorylated JNK, NFκB, p70s6 K and STAT5, and significantly increased phosphorylated CREB, ERK1/2, and Akt levels. CBD also increased phosphorylated eNOS (ser1177), which was correlated with increased levels of ERK1/2 and Akt levels. CB1 receptor antagonism prevented the increase in eNOS phosphorylation. CONCLUSION: This study shows, for the first time, that CBD causes vasorelaxation of human mesenteric arteries via activation of CB1 and TRP channels, and is endothelium- and nitric oxide-dependent.

Endocannabinoids modulate human blood-brain barrier permeability in vitro.

BACKGROUND AND PURPOSE: Endocannabinoids alter permeability at various epithelial barriers, and cannabinoid receptors and endocannabinoid levels are elevated by stroke, with potential neuroprotective effects. We therefore explored the role of endocannabinoids in modulating blood-brain barrier (BBB) permeability in normal conditions and in an ischaemia/reperfusion model. EXPERIMENTAL APPROACH: Human brain microvascular endothelial cell and astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen-glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance. Endocannabinoids or endocannabinoid-like compounds were assessed for their ability to modulate baseline permeability or OGD-induced hyperpermeability. Target sites of action were investigated using receptor antagonists and subsequently identified with real-time PCR. KEY RESULTS: Anandamide (10 µM) and oleoylethanolamide (OEA, 10 µM) decreased BBB permeability (i.e. increased resistance). This was mediated by cannabinoid CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) channels, calcitonin gene-regulated peptide (CGRP) receptor (anandamide only) and PPARα (OEA only). Application of OEA, palmitoylethanolamide (both PPARα mediated) or virodhamine (all 10 µM) decreased the OGD-induced increase in permeability during reperfusion. 2-Arachidonoyl glycerol, noladin ether and oleamide did not affect BBB permeability in normal or OGD conditions. N-arachidonoyl-dopamine increased permeability through a cytotoxic mechanism. PPARα and γ, CB1 receptors, TRPV1 channels and CGRP receptors were expressed in both cell types, but mRNA for CB2 receptors was only present in astrocytes. CONCLUSION AND IMPLICATION: The endocannabinoids may play an important modulatory role in normal BBB physiology, and also afford protection to the BBB during ischaemic stroke, through a number of target sites.

Cannabinoids in experimental stroke: a systematic review and meta-analysis.

Cannabinoids (CBs) show promise as neuroprotectants with some agents already licensed in humans for other conditions. We systematically reviewed CBs in preclinical stroke to guide further experimental protocols. We selected controlled studies assessing acute administration of CBs for experimental stroke, identified through systematic searches. Data were extracted on lesion volume, outcome and quality, and analyzed using random effect models. Results are expressed as standardized mean difference (SMD) with 95% confidence intervals (CIs). In all, 144 experiments (34 publications) assessed CBs on infarct volume in 1,473 animals. Cannabinoids reduced infarct volume in transient (SMD -1.41 (95% CI -1.71), -1.11) P<0.00001) and permanent (-1.67 (-2.08, -1.27), P<0.00001) ischemia and in all subclasses: endocannabinoids (-1.72 (-2.62, -0.82), P=0.0002), CB1/CB2 ligands (-1.75 (-2.19, -1.31), P<0.00001), CB2 ligands (-1.65 (-2.09, -1.22), P<0.00001), cannabidiol (-1.20 (-1.63, -0.77), P<0.00001), Δ(9)-tetrahydrocannabinol (-1.43 (-2.01, -0.86), P<0.00001), and HU-211 (-2.90 (-4.24, -1.56), P<0.0001). Early and late neuroscores significantly improved with CB use (-1.27 (-1.58, -0.95), P<0.00001; -1.63 (-2.64, -0.62), P<0.002 respectively) and there was no effect on survival. Statistical heterogeneity and publication bias was present, median study quality was 4 (range 1 to 6/8). Overall, CBs significantly reduced infarct volume and improve functional outcome in experimental stroke. Further studies in aged, female and larger animals, with other co-morbidities are required.

Is the cardiovascular system a therapeutic target for cannabidiol?

Cannabidiol (CBD) has beneficial effects in disorders as wide ranging as diabetes, Huntington's disease, cancer and colitis. Accumulating evidence now also suggests that CBD is beneficial in the cardiovascular system. CBD has direct actions on isolated arteries, causing both acute and time-dependent vasorelaxation. In vitro incubation with CBD enhances the vasorelaxant responses in animal models of impaired endothelium-dependent vasorelaxation. CBD protects against the vascular damage caused by a high glucose environment, inflammation or the induction of type 2 diabetes in animal models and reduces the vascular hyperpermeability associated with such environments. A common theme throughout these studies is the anti-inflammatory and anti-oxidant effect of CBD. In the heart, in vivo CBD treatment protects against ischaemia-reperfusion damage and against cardiomyopathy associated with diabetes. Similarly, in a different model of ischaemia-reperfusion, CBD has been shown to reduce infarct size and increase blood flow in animal models of stroke, sensitive to 5HT(1A) receptor antagonism. Although acute or chronic CBD treatment seems to have little effect on haemodynamics, CBD reduces the cardiovascular response to models of stress, applied either systemically or intracranially, inhibited by a 5HT(1A) receptor antagonist. In blood, CBD influences the survival and death of white blood cells, white blood cell migration and platelet aggregation. Taken together, these preclinical data appear to support a positive role for CBD treatment in the heart, and in peripheral and cerebral vasculature. However, further work is required to strengthen this hypothesis, establish mechanisms of action and whether similar responses to CBD would be observed in humans.