Time-Dependent Vascular Effects of Endocannabinoids Mediated by Peroxisome Proliferator-Activated Receptor Gamma (PPARγ)

The aim of the present study was to examine whether endocannabinoids cause PPARγ-mediated vascular actions. Functional vascular studies were carried out in rat aortae. Anandamide and N-arachidonoyl-dopamine (NADA), but not palmitoylethanolamide, caused significant vasorelaxation over time (2 hours). Vasorelaxation to NADA, but not anandamide, was inhibited by CB1 receptor antagonism (AM251, 1 μM), and vasorelaxation to both anandamide and NADA was inhibited by PPARγ antagonism (GW9662, 1 μM). Pharmacological inhibition of de novo protein synthesis, nitric oxide synthase, and super oxide dismutase abolished the responses to anandamide and NADA. Removal of the endothelium partly inhibited the vasorelaxant responses to anandamide and NADA. Inhibition of fatty acid amide hydrolase (URB597, 1 μM) inhibited the vasorelaxant response to NADA, but not anandamide. These data indicate that endocannabinoids cause time-dependent, PPARγ-mediated vasorelaxation. Activation of PPARγ in the vasculature may represent a novel mechanism by which endocannabinoids are involved in vascular regulation

The activity of the endocannabinoid metabolising enzyme fatty acid amide hydrolase in subcutaneous adipocytes correlates with BMI in metabolically healthy humans

<p>Abstract</p> <p>Background</p> <p>The endocannabinoid system (ECS) is a ubiquitously expressed signalling system, with involvement in lipid metabolism and obesity. There are reported changes in obesity of blood concentrations of the endocannabinoids anandamide (AEA) and 2-arachidonoylglcyerol (2-AG), and of adipose tissue expression levels of the two key catabolic enzymes of the ECS, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Surprisingly, however, the activities of these enzymes have not been assayed in conditions of increasing adiposity. The aim of the current study was to investigate whether FAAH and MGL activities in human subcutaneous adipocytes are affected by body mass index (BMI), or other markers of adiposity and metabolism.</p> <p>Methods</p> <p>Subcutaneous abdominal mature adipocytes, fasting blood samples and anthropometric measurements were obtained from 28 metabolically healthy subjects representing a range of BMIs. FAAH and MGL activities were assayed in mature adipocytes using radiolabelled substrates. Serum glucose, insulin and adipokines were determined using ELISAs.</p> <p>Results</p> <p>MGL activity showed no relationship with BMI or other adiposity indices, metabolic markers (fasting serum insulin or glucose) or serum adipokine levels (adiponectin, leptin or resistin). In contrast, FAAH activity in subcutaneous adipocytes correlated positively with BMI and waist circumference, but not with skinfold thickness, metabolic markers or serum adipokine levels.</p> <p>Conclusions</p> <p>In this study, novel evidence is provided that FAAH activity in subcutaneous mature adipocytes increases with BMI, whereas MGL activity does not. These findings support the hypothesis that some components of the ECS are upregulated with increasing adiposity in humans, and that AEA and 2-AG may be regulated differently.</p

Endocannabinoids in aqueous humour of patients with or without diabetes

Objective The primary aim was to determine endocannabinoid (EC) concentrations of 2-arachidonoylglycerol (2-AG), oleoylethanolamine (OEA), palmitoylethanolamine (PEA) and anandamide (AEA) in the aqueous humour of patients, and to investigate any differences in gender and diabetic or ocular disease status.Methods and Analysis Adult participants (age >18 years) listed for a routine cataract surgery were recruited. For patients with diabetes, results from their most recent retinopathy grading were recorded. A sample of aqueous humour was removed from the anterior chamber of the patients and snap-frozen in liquid nitrogen. Levels of 2-AG, PEA, OEA and AEA were measured by liquid chromatography-tandem mass spectrometry.Results Aqueous humour samples were taken from 93 patients (female:male=58:35), with a mean age±SD of 72.7±9.5 years. Following gender-specific analysis, the mean aqueous concentration of AEA in female patients without diabetes was significantly higher than in female patients with diabetes (0.20±0.03 nM vs 0.07±0.02 nM, p=0.001). Among female patients with diabetes, the aqueous concentration of 2-AG was higher in those with diabetic retinopathy compared with those with no retinopathy (0.30+0.16 nM vs 0.04±0.01 nM, p=0.0025). The aqueous level of the sum of EC was higher in those with ocular comorbidity (2.49±0.73 vs 1.44±0.17, p=0.0002).Conclusion There were gender, diabetes status and comorbidity differences in aqueous humour EC levels. Since EC receptors are present in ocular tissues, including the retina (neurons, glia and endothelial cells), differential levels of ECs in the aqueous humour of patients with and without diabetes may provide a novel therapeutic target for diabetic retinopathy

Human vascular cell responses to the circulating bone hormone osteocalcin

The purpose of this study was to characterize the direct effects of uncarboxylated osteocalcin (ucOCN) on vascular cell biology in vitro, to assess its potential function in pathophysiological conditions such as atherosclerosis. Human aortic endothelial cells (HAECs) and smooth muscle cells (HASMCs) were treated with ucOCN (0.1–50 ng/ml) and changes in phosphorylation of intracellular signaling proteins, angiogenesis, proliferation, migration, monolayer permeability, and protein secretion were measured. In HAECs, phosphorylated JNK and CREB were decreased with ucOCN (p

Osteokines and the vasculature: a review of the in vitro effects of osteocalcin, fibroblast growth factor-23 and lipocalin-2

Bone-derived factors that demonstrate extra-skeletal functions, also termed osteokines, are fast becoming a highly interesting and focused area of cross-disciplinary endocrine research. Osteocalcin (OCN), fibroblast growth factor-23 (FGF23) and lipocalin-2 (LCN-2), produced in bone, comprise an important endocrine system that is finely tuned with other organs to ensure homeostatic balance and health. This review aims to evaluate in vitro evidence of the direct involvement of these proteins in vascular cells and whether any causal roles in cardiovascular disease or inflammation can be supported. PubMed, Medline, Embase and Google Scholar were searched for relevant research articles investigating the exogenous addition of OCN, FGF23 or LCN-2 to vascular smooth muscle or endothelial cells. Overall, these osteokines are directly vasoactive across a range of human and animal vascular cells. Both OCN and FGF23 have anti-apoptotic properties and increase eNOS phosphorylation and nitric oxide production through Akt signalling in human endothelial cells. OCN improves intracellular insulin signalling and demonstrates protective effects against endoplasmic reticulum stress in murine and human endothelial cells. OCN may be involved in calcification but further research is warranted, while there is no evidence for a pro-calcific effect of FGF23 in vitro. FGF23 and LCN-2 increase proliferation in some cell types and increase and decrease reactive oxygen species generation, respectively. LCN-2 also has anti-apoptotic effects but may increase endoplasmic reticulum stress as well as have pro-inflammatory and pro-angiogenic properties in human vascular endothelial and smooth muscle cells. There is no strong evidence to support a pathological role of OCN or FGF23 in the vasculature based on these findings. In contrast, they may in fact support normal endothelial functioning, vascular homeostasis and vasodilation. No studies examined whether OCN or FGF23 may have a role in vascular inflammation. Limited studies with LCN-2 indicate a pro-inflammatory and possible pathological role in the vasculature but further mechanistic data is required. Overall, these osteokines pose intriguing functions which should be investigated comprehensively to assess their relevance to cardiovascular disease and health in humans

The endogenous cannabinoid anandamide increases human airway epithelial cell permeability through an arachidonic acid metabolite

Injury to the bronchial epithelium in respiratory diseases such as asthma and COPD results in the lossof barrier function and an elevated sensitivity to environmental insults. An increased release of theendogenous cannabinoid, anandamide in response to inhalation of allergen in asthmatic patients hasbeen reported. The aim of this study was, therefore, to determine the effects of endocannabinoids onbronchial epithelial cell permeability and to investigate the mechanisms involved.Calu-3 human bronchial epithelial cells were cultured at air–liquid interface to allow developmentof tight junctions. Changes in Transepithelial Electrical Resistance (TEER), a reflection of epithelial per-meability, were measured at various time points post-treatment, and expression of the tight junctionproteins, occludin and ZO-1, were determined using Western immunoblotting.Anandamide produced a significant reduction in TEER, which was unaffected by cannabinoid receptorantagonists, but attenuated by URB597, an inhibitor of fatty acid amide hydrolase, and by a combinationof cyclooxygenase (COX) and lipoxygenase (LOX) blockade. The anandamide metabolite, arachidonicacid, showed similar TEER decrease that was also prevented in the presence of COX and LOX inhibitor.Expression of occludin and ZO-1 were also reduced by anandamide.These findings indicate a pro-inflammatory-like effect of anandamide on bronchial epithelial per-meability, mediated by cyclooxygenase and lipoxygenase metabolites, and suggest that inhibition ofanandamide degradation might provide a novel approach to treat airway inflammation

The effects of acute and sustained cannabidiol dosing for seven days on the haemodynamics in healthy men: A randomised controlled trial

© 2020 The British Pharmacological Society Background: In vivo studies show that cannabidiol (CBD) acutely reduces blood pressure (BP) in men. The aim of this study was to assess the effects of repeated CBD dosing on haemodynamics. Methods: Twenty-six healthy males were given CBD (600 mg) or placebo orally for seven days in a randomised, placebo-controlled, double-blind, parallel study (n = 13/group). Cardiovascular parameters were assessed at rest and in response to isometric exercise after acute and repeated dosing using Finometer®, Vicorder® and Duplex ultrasound. Results: Compared to placebo, CBD significantly reduced resting mean arterial pressure (P =.04, two-way ANOVA, mean difference (MD) –2 mmHg, 95% CI -3.6 to −0.3) after acute dosing, but not after repeated dosing. In response to stress, volunteers who had taken CBD had lower systolic BP after acute (P =.001, two-way ANOVA, MD −6 mmHg, 95% CI –10 to −1) and repeated (P =.02, two-way ANOVA, MD −5.7 mmHg, 95% CI –10 to −1) dosing. Seven days of CBD increased internal carotid artery diameter (MD +0.55 mm, P =.01). Within the CBD group, repeated dosing reduced arterial stiffness by day 7 (pulse wave velocity; MD −0.44 m/s, P =.05) and improved endothelial function (flow mediation dilatation, MD +3.5%, P =.02, n = 6 per group), compared to day 1. Conclusion: CBD reduces BP at rest after a single dose but the effect is lost after seven days of treatment (tolerance); however, BP reduction during stress persists. The reduction in arterial stiffness and improvements in endothelial function after repeated CBD dosing are findings that warrant further investigation in populations with vascular diseases

Osteocalcin does not influence acute or chronic inflammation in human vascular cells

Some human observational studies have suggested an anti-inflammatory role of osteocalcin (OCN). An inflammatory protocol using IFN-γ and TNF-α (10 ng/mL) was employed to examine the acute (24 hr) and chronic (144 hr) effects of uncarboxylated OCN (ucOCN) in commercial, primary, sub-cultured human aortic endothelial (HAEC) and smooth muscle cells (HASMCs). The inflammatory protocol increased phosphorylation of intracellular signalling proteins (CREB, JNK, p38, ERK, AKT, STAT3, STAT5) and increased secretion of adhesion markers (VCAM-1, ICAM-1, MCP-1) and pro-inflammatory cytokines (IL-6, IL-8). After acute inflammation, there were no additive or reductive effects of ucOCN in either cell type. Following chronic inflammation, ucOCN did not affect cell responses, nor did it appear to have any pro- or anti-inflammatory effects when administered acutely or chronically on its own in either cell type. Additionally, ucOCN did not affect lipopolysaccharide (LPS) induced acute inflammation in HAECs or HASMCs. The findings of this study do not support a causal role for OCN within the models of vascular inflammation chosen. Further confirmatory studies are warranted

The effects of obesity, diabetes and metabolic syndrome on the hydrolytic enzymes of the endocannabinoid system in animal and human adipocytes

Background: Circulating endocannabinoid levels are increased in obesity and diabetes. We have shown that fatty acid amide hydrolase (FAAH, an endocannabinoid hydrolysing enzyme) in subcutaneous adipose tissue positively correlates with BMI in healthy volunteers. The aim of the present study was to investigate whether the hydrolytic enzymes of the endocannabinoid system are affected by diabetes or metabolic syndrome in obesity. Methods. Using radiolabelled substrates, FAAH and monoacylglycerol lipase (MGL) activities were assessed in adipocytes from various adipose depots in Zucker rats (n = 22, subcutaneous abdominal, visceral and epididymal) and bariatric patients (n = 28, subcutaneous abdominal and omental). Results: FAAH activity was significantly increased in adipocytes of obese (Zucker Fatty) compared to Zucker lean rats (P < 0.05) but was not raised in the Zucker Diabetic Fatty rats (ZDF). MGL activity was raised in both Zucker Fatty (P < 0.001-0.01) and ZDF rats (P < 0.05) and was positively correlated with body weight and plasma glucose levels (P < 0.01). In bariatric patients (BMI range 37-58 kg.m2), there was a trend for MGL activity to correlate positively with BMI, reaching significance when type 2 diabetic patients were removed. FAAH and MGL activities in obese humans were not correlated with blood pressure, skinfold thicknesses, fasting glucose, insulin, HbA1c, triglycerides or cholesterol levels. Conclusions: FAAH in adipocytes is differentially altered in animal models of obesity and diabetes, while MGL activity is increased by both. However, in obese humans, FAAH or MGL activity in adipocytes is not affected by diabetes, dyslipidaemia or other markers of metabolic dysfunction. This suggests increased circulating levels of endocannabinoids are not a result of altered degradation in adipose tissue. © 2014 Cable et al.; licensee BioMed Central Ltd

Remote effects of acute kidney injury in a porcine model

Background: Acute Kidney Injury (AKI) is a common and serious disease with no specific treatment. An episode of AKI may affect organs distant to the kidney, further increasing the morbidity associated with AKI. The mechanism of organ cross-talk after AKI is unclear. The renal and immune systems of pigs and humans are alike. Using a preclinical animal (porcine) model, we test the hypothesis that early effects of AKI on distant organs is by immune cell infiltration leading to inflammatory cytokine production, extravasation and edema. Study Design: In 29 pigs exposed to either sham-surgery or renal ischemia-reperfusion (control, n=12; AKI, n=17) we assessed remote organ (liver, lung, brain) effects in the short-(from 2 to 48h reperfusion) and longer-term (5 weeks later) using immunofluorescence (for leucocyte infiltration, apoptosis), a cytokine array, tissue elemental analysis (electrolytes), blood hematology and chemistry (e.g. liver enzymes) and PCR (for inflammatory markers). Results: AKI elicited significant, short-term (~24h) increments in enzymes indicative of acute liver damage (e.g. AST:ALT ratio; P=0.02) and influenced tissue biochemistry in some remote organs (e.g. lung tissue [Ca++] increased; P=0.04). These effects largely resolved after 48h and no further histopathology, edema, apoptosis or immune cell infiltration was noted in liver, lung or hippocampus in the short- and longer-term. Conclusions: AKI has subtle biochemical effects on remote organs in the short-term including a transient increment in markers of acute liver damage. These effects resolved by 48h and no further remote organ histopathology, apoptosis, edema or immune cell infiltration was noted