Cannabidiol is a non-psychoactive phytocannabinoid, which is well tolerated in humans and already on the market as part of a licensed treatment for spasticity in multiple sclerosis (Sativex^® GW Pharmaceuticals, Cambridge, UK). CBD alone (Epidiolex^®, GW Pharmaceuticals, Cambridge, UK) is in Phase 3 clinical trials in children with intractable epilepsies (Dravet Syndrome and Lennox-Gastaut Syndrome). Epidiolex has also received orphan designation status in the United States for the treatment of neonatal hypoxia-ischaemic encephalopathy. CBD is the focus of much research because of its potential in a number of other therapeutic areas. This is due to its anti-inflammatory, anti-convulsant, anti-oxidant, anxiolytic, anti-nausea, anti-tumoural and anti-psychotic properties (Mechoulam et al., 2002; Campos et al., 2016; Fasinu et al., 2016; Leo et al., 2016; Rohleder et al., 2016). A number of preclinical studies have also shown beneficial effects of CBD in a range of disorders of the cardiovascular system (Stanley et al., 2013a).

In diabetes, in vivo treatment with CBD is anti-inflammatory and can prevent, and delay, the onset of type 1 diabetes in non-obese diabetic prone mice (Weiss et al., 2006), and decreases myocardial dysfunction, cardiac fibrosis and oxidative stress in diabetic cardiomyopathy (Rajesh et al., 2010). CBD treatment also blocks the increases in iCAM-1 and VEGF in the retina from streptozotocin-induced diabetic rats (El-Remessy et al., 2006). CBD has anti-nociceptive effects against diabetic peripheral neuropathy in mice (Toth et al., 2010) and Sativex is reported to be beneficial in patients with neuropathic pain associated with diabetes or allodynia, although not an official indication for the drug (Hoggart et al., 2015). Together, this suggests that CBD may be effective against a number of diabetes related complications.

Cannabidiol causes vasorelaxation in isolated arteries of rats and in humans (O’Sullivan et al., 2009; Stanley et al., 2015). We have also shown that 2h incubation in vitro with CBD can enhance aortic and femoral artery vasorelaxation to ACh (Stanley et al., 2013b). In femoral arteries, this mechanism of endothelium-dependent vasorelaxant enhancement by CBD was mediated via CB₂ cannabinoid receptors, raised superoxide dismutase activity, elevated COX activity, and activation of vasodilatory EP4 prostanoid receptors (Wheal et al., 2014). Using ZDF rats as a model of type 2 diabetes, the aim of the present study was to determine if chronic in vivo treatment with CBD would similarly improve endothelial function across a variety of arteries.

Prior to, and following, 7 days of treatment, ZDF rats were heavier than ZDF lean rats (see Table 1). There was no difference in the weight gain over 7 days in the ZDF Lean or diabetic rats treated with CBD compared to vehicle (Figures 1A,B). However, the vehicle-treated ZDF rats had a significant increase in weight over time (P < 0.05 at day 8) that was not observed in the CBD-treated ZDF rats (see Figure 1B). Post-mortem blood glucose levels were not different between groups treated with vehicle versus CBD, but were higher in ZDF rats compared to ZDF Lean rats (P < 0.0001, Table 1).

The aim of this study was to establish if in vivo treatment with CBD could improve vascular function, and we have demonstrated that just 7 days treatment with CBD enhanced endothelium-dependent vasorelaxation in mesenteric arteries from ZDF rats due to COX- and NO-mediated mechanisms. Seven days treatment with CBD was also associated with a positive change in the profile of circulating metabolic and cardiovascular proteins. These data support the growing evidence that cannabinoids, and specifically CBD, maybe be a beneficial treatment of diabetes (Sidney, 2016).

We previously showed that 2 h incubation of arteries in vitro with CBD can enhance aortic and femoral artery vasorelaxation to ACh (Stanley et al., 2013b; Wheal et al., 2014). We have now extended this work to show that 7 days treatment in vivo with CBD also improves vasorelaxation to ACh. This enhanced vasorelaxant response after CBD treatment was inhibited in the presence of indomethacin or L-NAME, suggesting that this is both COX- and NO-dependent, which is in agreement with our previous findings of COX- (Wheal et al., 2014) and NO- (Stanley et al., 2015) dependent mechanisms of action for CBD in the vasculature. Daily treatment with CBD did not affect vasorelaxation to ACh in mesenteric arteries from the ZDF lean control rats, suggesting that the positive vascular effects of CBD are revealed when vascular dysfunction is present. This is consistent with the known pro-homeostatic properties of cannabinoids, where they only affect the functioning of a perturbed system, but not a healthy one.

Zucker diabetic fatty rats used in this study were heavier, and had higher glucose levels, than the lean control rats. In ZDF diabetic rats, a contractile response to ACh was observed in mesenteric arteries at concentrations above 10 μM that was not observed in the CBD-treated diabetic animals or the ZDF lean control rats (see Figure 2B). This contractile response was also partly reduced in the presence of indomethacin (Figure 2D), thus it is tempting to suggest that this contractile response in the diabetic rats (not observed in the lean controls) is as a consequence of COX-derived vasoconstrictor production (Feletou et al., 2011; Matsumoto et al., 2015), and that CBD inhibits either the production, or action, of these COX-derived vasoconstrictors in diabetic rats.

In femoral arteries, treatment with CBD did not significantly improve vasorelaxation to ACh or SNP, but there was a trend for enhanced responses in both the lean and diabetic rats, which became significant for diabetic rats in the presence of indomethacin (Figure 3D). Similarly, although CBD treatment did not affect vasorelaxation to ACh in diabetic aortae, a significant effect of CBD was revealed in the presence of L-NAME (Figure 4F). The ability of CBD to augment endothelium-dependent vasorelaxation when either of the two of the main vasodilatory pathways are inhibited suggests that CBD may be enhancing vasorelaxation through another as yet unidentified mechanism. It also highlights the benefit of CBD in maintaining endothelium-dependent vasorelaxation in diabetic arteries when either vasodilator prostanoids or NO become more dysfunctional (Feletou et al., 2011; Prieto et al., 2014).

The ZDF rats used in this study had significantly raised circulating levels of c-peptide, insulin, leptin, vWF, A-PAI1 and iCAM, as would be expected from a model of type 2 diabetes. Treatment with CBD for 7 days reduced the levels of c-peptide, insulin, leptin and iCAM, such that they were no longer significantly different to the ZDF lean controls. Although these values were not significantly different from the vehicle-treated ZDF diabetic rats, we predict that more prolonged treatment with, or higher dose of, CBD might show a greater effect. Of note, the metabolic parameters were particularly affected by CBD, but without changes in blood glucose levels.

We recently performed a Phase II clinical trial assessing the effects of CBD on HDL-cholesterol in type 2 diabetic patients (Jadoon et al., 2016). A range of cardiovascular secondary and tertiary endpoints were included, although none were significantly affected by CBD, we suspect it was because the dose of CBD was too low. In the present study we used a dose of 10 mg/kg (equivalent to 800 mg per day in an 80 kg person), while in the clinical trial we used a dose of 100 mg, twice daily. In a recent study, we showed that a single dose of 600 mg CBD significantly reduced blood pressure and the blood pressure response to stress in healthy volunteers Please update (Jadoon et al., 2016). Further clinical studies with diabetic patients are required to examine doses of CBD more equitable to those observed in preclinical diabetes studies.

Of the biomarkers tested, circulating levels of VEGF and Endothelin-1 were raised by CBD treatment in the ZDF diabetic rats. We have previously found that CBD significantly increases VEGF secretion from endothelial cells (Hind et al., 2016), while El-Remessey and colleagues showed that CBD reduced retinal expression of VEGF in streptozotocin-treated rats (El-Remessy et al., 2006). We are not aware of any studies that have investigated the effects of CBD on Endothelin-1, although an increase in endothelin-1 is not consistent with an improvement in vascular function. Thus, the vascular significance of changes in VEGF or Endothelin-1 with CBD requires further investigation.

In conclusion, this study has shown that a short in vivo treatment protocol with CBD was associated with improvements in endothelium-dependent vasorelaxation in mesenteric arteries, and an improvement in the profile of cardiovascular and metabolic parameters. Further research is required to establish if this effect can be maintained and/or enhanced by more prolonged CBD treatment. The current study supports the growing evidence that CBD may be beneficial against a number of problems associated with diabetes including inflammation (Weiss et al., 2006, 2008; Lehmann et al., 2016), endothelial dysfunction (Rajesh et al., 2007), cardiomyopathy (Rajesh et al., 2010), retinal function (El-Remessy et al., 2006, 2010), and neuropathic pain (Toth et al., 2010).

AW: In vivo dosing, myography, serum Milliplex assays, experimental design and execution, data analysis and interpretation. Manuscript preparation and revision. KJ: Conducted Multiplex assays. Review of final manuscript. MR: Supervision of AW, experimental design, data analysis and interpretation, and manuscript revision. SO: Supervision of AW and KJ, conducted Multiplex assays, experimental design, data analysis and interpretation, and manuscript preparation and revision. ^∗∗∗P < 0.001.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.