The Impact of Semicarbazide Sensitive Amine Oxidase Activity on Rat Aortic Vascular Smooth Muscle Cells.

Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble- and membrane-bound transmembrane protein expressed in the vascular endothelial and in smooth muscle cells. In vascular endothelial cells, SSAO contributes to the development of atherosclerosis by mediating a leukocyte adhesion cascade; however, its contributory role in the development of atherosclerosis in VSMCs has not yet been fully explored. This study investigates SSAO enzymatic activity in VSMCs using methylamine and aminoacetone as model substrates. The study also addresses the mechanism by which SSAO catalytic activity causes vascular damage, and further evaluates the contribution of SSAO in oxidative stress formation in the vascular wall. SSAO demonstrated higher affinity for aminoacetone when compared to methylamine (Km = 12.08 µM vs. 65.35 µM). Aminoacetone- and methylamine-induced VSMCs death at concentrations of 50 & 1000 µM, and their cytotoxic effect, was reversed with 100 µM of the irreversible SSAO inhibitor MDL72527, which completely abolished cell death. Cytotoxic effects were also observed after 24 h of exposure to formaldehyde, methylglyoxal and H2O2. Enhanced cytotoxicity was detected after the simultaneous addition of formaldehyde and H2O2, as well as methylglyoxal and H2O2. The highest ROS production was observed in aminoacetone- and benzylamine-treated cells. MDL72527 abolished ROS in benzylamine-, methylamine- and aminoacetone-treated cells (**** p < 0.0001), while ßAPN demonstrated inhibitory potential only in benzylamine-treated cells (* p < 0.05). Treatment with benzylamine, methylamine and aminoacetone reduced the total GSH levels (**** p < 0.0001); the addition of MDL72527 and ßAPN failed to reverse this effect. Overall, a cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs where SSAO was identified as a key mediator in ROS formation. These findings could potentially associate SSAO activity with the early developing stages of atherosclerosis through oxidative stress formation and vascular damage.

Semicarbazide-Sensitive Amine Oxidase (SSAO) and Lysyl Oxidase (LOX) Association in Rat Aortic Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. ßAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (## p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (**** p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). ßAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (#### p < 0.0001 for Si6 vs. Sictrl and ### p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (# p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.

PI3Kδ inhibition prevents IL33, ILC2s and inflammatory eosinophils in persistent airway inflammation.

BACKGROUND: Phosphoinositide-3-kinase-delta (PI3Kδ) inhibition is a promising therapeutic approach for inflammatory conditions due to its role in leucocyte proliferation, migration and activation. However, the effect of PI3Kδ inhibition on group 2 innate lymphoid cells (ILC2s) and inflammatory eosinophils remains unknown. Using a murine model exhibiting persistent airway inflammation we sought to understand the effect of PI3Kδ inhibition, montelukast and anti-IL5 antibody treatment on IL33 expression, group-2-innate lymphoid cells, inflammatory eosinophils, and goblet cell metaplasia. RESULTS: Mice were sensitised to house dust mite and after allowing inflammation to resolve, were re-challenged with house dust mite to re-initiate airway inflammation. ILC2s were found to persist in the airways following house dust mite sensitisation and after re-challenge their numbers increased further along with accumulation of inflammatory eosinophils. In contrast to montelukast or anti-IL5 antibody treatment, PI3Kδ inhibition ablated IL33 expression and prevented group-2-innate lymphoid cell accumulation. Only PI3Kδ inhibition and IL5 neutralization reduced the infiltration of inflammatory eosinophils. Moreover, PI3Kδ inhibition reduced goblet cell metaplasia. CONCLUSIONS: Hence, we show that PI3Kδ inhibition dampens allergic inflammatory responses by ablating key cell types and cytokines involved in T-helper-2-driven inflammatory responses.

Co-Incubation with PPARß/δ Agonists and Antagonists Modeled Using Computational Chemistry: Effect on LPS Induced Inflammatory Markers in Pulmonary Artery.

Peroxisome proliferator activated receptor beta/delta (PPARß/δ) is a nuclear receptor ubiquitously expressed in cells, whose signaling controls inflammation. There are large discrepancies in understanding the complex role of PPARß/δ in disease, having both anti- and pro-effects on inflammation. After ligand activation, PPARß/δ regulates genes by two different mechanisms; induction and transrepression, the effects of which are difficult to differentiate directly. We studied the PPARß/δ-regulation of lipopolysaccharide (LPS) induced inflammation (indicated by release of nitrite and IL-6) of rat pulmonary artery, using different combinations of agonists (GW0742 or L-165402) and antagonists (GSK3787 or GSK0660). LPS induced release of NO and IL-6 is not significantly reduced by incubation with PPARß/δ ligands (either agonist or antagonist), however, co-incubation with an agonist and antagonist significantly reduces LPS-induced nitrite production and Nos2 mRNA expression. In contrast, incubation with LPS and PPARß/δ agonists leads to a significant increase in Pdk-4 and Angptl-4 mRNA expression, which is significantly decreased in the presence of PPARß/δ antagonists. Docking using computational chemistry methods indicates that PPARß/δ agonists form polar bonds with His287, His413 and Tyr437, while antagonists are more promiscuous about which amino acids they bind to, although they are very prone to bind Thr252 and Asn307. Dual binding in the PPARß/δ binding pocket indicates the ligands retain similar binding energies, which suggests that co-incubation with both agonist and antagonist does not prevent the specific binding of each other to the large PPARß/δ binding pocket. To our knowledge, this is the first time that the possibility of binding two ligands simultaneously into the PPARß/δ binding pocket has been explored. Agonist binding followed by antagonist simultaneously switches the PPARß/δ mode of action from induction to transrepression, which is linked with an increase in Nos2 mRNA expression and nitrite production.

Dysregulation of TRPV4, eNOS and caveolin-1 contribute to endothelial dysfunction in the streptozotocin rat model of diabetes.

Endothelial dysfunction is a common complication in diabetes in which endothelium-dependent vasorelaxation is impaired. The aim of this study was to examine the involvement of the TRPV4 ion channel in type 1 diabetic endothelial dysfunction and the possible association of endothelial dysfunction with reduced expression of TRPV4, endothelial nitric oxide synthase (eNOS) and caveolin-1. Male Wistar rats (350-450 g) were injected with 65 mg/kg i.p. streptozotocin (STZ) or vehicle. Endothelial function was investigated in aortic rings and mesenteric arteries using organ bath and myograph, respectively. TRPV4 function was studied with fura-2 calcium imaging in endothelial cells cultured from aortas from control and STZ treated rats. TRPV4, caveolin-1 and eNOS expression was investigated in these cells using immunohistochemistry. STZ-treated diabetic rats showed significant endothelial dysfunction characterised by impaired muscarinic-induced vasorelaxation (aortic rings: STZ-diabetics: Emax = 29.6 ± 9.3%; control: Emax = 77.2 ± 2.5% P˂0.001), as well as significant impairment in TRPV4-induced vasorelaxation (aortic rings, 4αPDD STZ-diabetics: Emax = 56.0 ± 5.5%; control: Emax = 81.1 ± 2.1% P˂0.001). Furthermore, STZ-diabetic primary aortic endothelial cells showed a significant reduction in TRPV4-induced intracellular calcium elevation (P˂0.05) compared with the control group. This was associated with significantly lower expression of TRPV4, caveolin-1 and eNOS and this was reversed by insulin treatment of the endothelial cultures from STZ -diabetic rats. Taken together, these data are consistent with the hypothesis that signalling through TRPV4, caveolin-1, and eNOS is downregulated in STZ-diabetic aortic endothelial cells and restored by insulin treatment.

Preclinical Neuropathic Pain Assessment; the Importance of Translatability and Bidirectional Research.

For patients suffering with chronic neuropathic pain the need for suitable novel therapies is imperative. Over recent years a contributing factor for the lack of development of new analgesics for neuropathic pain has been the mismatch of primary neuropathic pain assessment endpoints in preclinical vs. clinical trials. Despite continuous forward translation failures across diverse mechanisms, reflexive quantitative sensory testing remains the primary assessment endpoint for neuropathic pain and analgesia in animals. Restricting preclinical evaluation of pain and analgesia to exclusively reflexive outcomes is over simplified and can be argued not clinically relevant due to the continued lack of forward translation and failures in the clinic. The key to developing new analgesic treatments for neuropathic pain therefore lies in the development of clinically relevant endpoints that can translate preclinical animal results to human clinical trials. In this review we discuss this mismatch of primary neuropathic pain assessment endpoints, together with clinical and preclinical evidence that supports how bidirectional research is helping to validate new clinically relevant neuropathic pain assessment endpoints. Ethological behavioral endpoints such as burrowing and facial grimacing and objective measures such as electroencephalography provide improved translatability potential together with currently used quantitative sensory testing endpoints. By tailoring objective and subjective measures of neuropathic pain the translatability of new medicines for patients suffering with neuropathic pain will hopefully be improved.

Impact of the UK Psychoactive Substances Act on awareness, use, experiences and knowledge of potential associated health risks of novel psychoactive substances.

AIMS: The risk of potential harms prompted the UK government to introduce the Psychoactive Substances Act in 2016. The aim of the present study was to evaluate the impact and effectiveness of this new legislation on patterns of novel psychoactive substance (NPS) awareness, use, experiences and risk awareness in a self-selected sample of UK consumers to inform education and policy. METHODS: The Bristol Online Survey was advertised on the Bluelight drug forum and social media Facebook pages and University email between 7 January and 7 February 2015 (168 responses) and 9 March to 18 September 2017 (726 responses). UK country of residence responses were extracted for analysis (SPSS). RESULTS: In a predominantly university-educated, young (< 25 years) self-selecting sample, 1 year after introduction of the legislation, NPS use (in males, under 18s, those educated to school/college level, P < .001) has increased, whilst health risk awareness has not changed and remains poor. Users are switching to sourcing NPSs via street dealers (49%) and the darknet (31%) and showing an increase in preference for the herbal NPS Salvia divinorum (P < .05). The main reasons for NPS use remain the influence of friends (69%) in a social setting and to get high (76%) usually in combination with alcohol, cannabis or ecstasy. CONCLUSION: Regulation alone, so far, has not impacted on health risk awareness, NPS drug demand and culture in our UK survey sample. Alongside regulation, NPS health promotion education (particularly in schools, colleges) is needed that addresses resilience and both the risks and beneficial effects of NPS.

Dysfunction in nitric oxide synthesis in streptozotocin treated rat aorta and role of methylglyoxal.

Diabetic vascular dysfunction is a major complication of diabetes. Methylglyoxal (MGO) is a dicarbonyl metabolite elevated in diabetic plasma that reacts with interstitial molecules to form advanced glycation end products (AGE). We investigated whether MGO affects the release of nitric oxide (NO) from rat aortic smooth muscle cells (ASMCs), and if L-arginine can prevent these effects of MGO. MGO was significantly elevated in serum from streptozotocin (STZ)-treated rats (121 ±â€¯11.2 µM) compared with vehicle control rats (27.5 ±â€¯9.2 µM). The pathological concentration of MGO (100 µM) was then applied to investigate its effect on inducible nitric oxide synthase (iNOS) expression and NO release on interferon-gamma (IFN-γ) (100 IU/ml) and lipopolysaccharide (LPS) (100 µg/ml)-stimulated control ASMCs. MGO (100 µM) inhibited IFN-γ and LPS-stimulated iNOS expression through inhibiting Akt phosphorylation and inhibition of iNOS expression was prevented by L-arginine (100 µM) co-treatment. These findings show for the first time that MGO inhibits IFN-γ and LPS-stimulated iNOS expression in ASMCs, in addition to inhibiting IFN-γ and LPS-induced Akt phosphorylation. The actions of MGO might contribute to the vascular dysfunction induced by MGO in diabetes.

Phytochemical characterization of Tabernanthe iboga root bark and its effects on dysfunctional metabolism and cognitive performance in high-fat-fed C57BL/6J mice.

Preparations of the root bark of Tabernanthe iboga have long been used in Central and West African traditional medicine to combat fatigue, as a neuro-stimulant in rituals, and for treatment of diabetes. The principal alkaloid of T. iboga, ibogaine, has attracted attention in many countries around the world for providing relief for opioid craving in drug addicts. Using a plant metabolomics approach, we detected five phenolic compounds, including 3-O-caffeoylquinic acid, and 30 alkaloids, seven of which were previously reported from T. iboga root bark. Following a report that iboga extracts contain insulinotropic agents, we aimed to determine the potential alleviating effects of the water extract of iboga root bark on high-fat diet (HFD)-induced hyperglycemia as well as its effects on cognitive function in male C57BL/6J mice. Feeding a HFD to mice for 10 weeks produced manifestations of metabolic syndrome such as increased body weight and increased plasma levels of glucose, triacylglycerols, total cholesterol, LDL-cholesterol, insulin, leptin, and pro-inflammatory mediators (IL-6, MCP-1, ICAM-1), as compared to mice fed a low-fat diet (LFD). Supplementation of HFD with iboga extract at ibogaine doses of 0.83 (low) and 2.07 (high) mg/kg/day did not improve these HFD-induced metabolic effects except for a reduction of plasma MCP-1 in the low dose group, indicative of an anti-inflammatory effect. When the HFD mice were tested in the water maze, the high-dose iboga extract caused hippocampus-dependent impairments in spatial learning and memory, as compared to mice receiving only a HFD.

Diabetic dyslipidaemia is associated with alterations in eNOS, caveolin-1, and endothelial dysfunction in streptozotocin treated rats.

BACKGROUND: Diabetes is a complex progressive disease characterized by chronic hyperglycaemia and dyslipidaemia associated with endothelial dysfunction. Oxidized LDL (Ox-LDL) is elevated in diabetes and may contribute to endothelial dysfunction. The aim of this study was to relate the serum levels of Ox-LDL with endothelial dysfunction in streptozotocin (STZ)-diabetic rats and to further explore the changes in endothelial nitric oxide synthase (eNOS) and caveolin-1 (CAV-1) expression in primary aortic endothelial cells. METHODS: Diabetes was induced with a single intraperitoneal injection of STZ in male Wistar rats. During the hyperglycaemic diabetes state serum lipid markers, aortic relaxation and aortic endothelial cell eNOS and CAV-1 protein expressions were measured. RESULTS: Elevated serum Ox-LDL (STZ 1486 ± 78.1 pg/mL vs control 732.6 ± 160.6 pg/mL, P < .05) was associated with hyperglycaemia (STZ 29 ± 0.9 mmol/L vs control: 7.2 ± 0.2 mmol/L, P < .001) and hypertriglyceridaemia (STZ 9.0 ± 1.5 mmol/L vs control: 3.0 ± 0.3 mmol/L, P < .01) in diabetic rats. A significant reduction was observed in STZ-diabetic aortic endothelial cell eNOS and CAV-1 of 40% and 30%, respectively, accompanied by a compromised STZ-diabetic carbachol-induced vasodilation (STZ 29.6 ± 9.3% vs control 77.2 ± 2.5%, P < .001). CONCLUSIONS: The elevated serum Ox-LDL in hyperglycaemic STZ-diabetic rats may contribute to diabetic endothelial dysfunction, possibly through downregulation of endothelial CAV-1 and eNOS.

The Non-Genomic Effects of the PPARßγ Agonist GW0742 on Streptozotocin Treated Rat Aorta.

BACKGROUND: The ubiquitous nuclear receptor PPARß/δ is increasingly being studied in regards to numerous diseases including diabetes following on the finding that PPARß/δ agonist GW0742 controls Type 1 Diabetes in rats. Studies have shown that GW0742 has off target, non- PPARß/δ effects in the cell although there are some key questions that remain to be addressed in respect to the significance of this control on vascular tone. METHODS: Using isometric organ baths, rat aorta rings were exposed to ROCK inhibitors and the changes in contraction and dilation measured. RESULTS: Our data shows that the PPARß/δ agonist GW0742 (10-7M) inhibits contractile responses to U46619 and phenylephrine, and that these responses are similar in normal and Streptozotocin (STZ) diabetic rat aorta. ROCK inhibitors Fasudil and Y27632 significantly reduced GW0742 mediated dilation of naïve rat aorta, but Fasudil had no effect on GW0742 dilation in STZ diabetic rat aorta. In contrast, STZ diabetic rat aorta pre-contracted with high [K+] Krebs lacked a dilatory response to GW0742, which taken together indicates that the mechanism of action of GW0742 mediated dilation changes in the diabetic state compared to non-diabetic state. CONCLUSION: This is the first direct evidence demonstrating the non- PPARß/δ effect of GW0742 on contraction is irrespective to the diabetic state, and that GW0742 has the potential to induce vasodilation via multiple off-target mechanisms.

An international survey on the awareness, use, preference, and health perception of novel psychoactive substances (NPS).

OBJECTIVE: This survey investigated the level of public awareness, preference, and motivation of novel psychoactive substances (NPS) use as well as knowledge of potential associated health risks. METHODS: A Bristol Online Survey was advertised through social media and a drug forum "Bluelight" between January 7 and February 7, 2015. RESULTS: Responses were received from 17 countries, mainly from Europe. Most responses (83%) came from university educated students. Two-thirds (65%) of the 168 respondents were aware of NPS. Awareness was significantly increased in those with bisexual or homosexual orientation (p < .05) and those in employment (p < .05). Fourteen percent of the 168 respondents were users of NPS, and use was significantly affected by age and employment (p < .01) but unaffected by level of education (p > .05). Nearly half of the NPS users perceived NPS to carry either a low risk to health (20%) or did not know whether or not they posed a health risk (29%). CONCLUSIONS: These survey data indicate that awareness of NPS and, importantly, perception of the potential health risks associated with NPS use is lacking. NPS awareness and use is higher in those in employment but is unaffected by level of education. This highlights the need for targeted drugs education intervention by policy-makers in schools and universities.

Methylglyoxal, A Metabolite Increased in Diabetes is Associated with Insulin Resistance, Vascular Dysfunction and Neuropathies.

BACKGROUND: Diabetes mellitus (DM) is a pandemic metabolic disease characterized by a chronically elevated blood glucose concentration (hyperglycemia) due to insulin dysfunction. Approximately 50% of diabetics show diabetes complications by the time they are diagnosed. Vascular dysfunction, nephropathy and neuropathic pain are common diabetes complications. Chronic hyperglycemia contributes to reactive oxygen species (ROS) generation such as methylglyoxal (MGO). METHODS: Peer reviewed research papers were studied through bibliographic databases searching focused on review questions and inclusion/exclusion criteria. The reviewed papers were appraised according to the searching focus. The characteristics of screened papers were described, and a deductive qualitative content analysis methodology was applied to the included studies using a conceptual framework to yield this comprehensive systematic review. RESULTS: Sixty-six papers were included in this review. Eleven papers related methylglyoxal generation to carbohydrates metabolism, ten papers related lipid metabolism to methylglyoxal and 5 papers showed the proteolytic pathways that contribute to methylglyoxal generation. Methylglyoxal metabolism was derived from 7 papers. Descriptive figure 1 was drawn to explain methylglyoxal sources and how diabetes increases methylglyoxal generation. Furthermore, twenty-six papers related methylglyoxal to diabetes complications from which 9 papers showed methylglyoxal ability to induce insulin dysfunction, an effect which was described in schematic figure 2. Additionally, fifteen papers revealed methylglyoxal contribution to vascular dysfunction and 3 papers showed methylglyoxal to cause neuropathic pain. Methylglyoxal-induced vascular dysfunction was drawn in a comprehensive figure 3. This review correlated methylglyoxal with diabetes and diabetes complications which were summarised in table 1. CONCLUSION: The findings of this review suggesting methylglyoxal as an essential therapeutic target for managing diabetes in the future.