Richard W. Simpson

A long-acting glucagon-like peptide-1 analogue attenuates induction of plasminogen activator inhibitor type-1 and vascular adhesion molecules

Glucagon-like peptide-1 (GLP-1) administration attenuates endothelial cell dysfunction in diabetic patients and inhibits tumour necrosis factor alpha (TNF)-mediated plasminogen activator inhibitor type-1 (PAI-1) induction in human vascular endothelial cells. The short half-life of GLP-1 mediated via degradation by the enzyme dipeptidyl peptidase 4 mandates the clinical use of long-acting GLP-1 analogues. The effects of a long-acting GLP-1 analogue on PAI-1 and vascular adhesion molecule expression in vascular endothelial cells are unknown. In this report, we demonstrate for the first time that the treatment with liraglutide, a long-acting GLP-1 analogue, inhibited TNF or hyperglycaemia-mediated induction of PAI-1, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA and protein expression in a human vascular endothelial cell line. In addition, treatment attenuated TNF- or hyperglycaemia-mediated induction of the orphan nuclear receptor Nur77 mRNA expression. Taken together, these observations indicate that liraglutide inhibits TNF- or glucose-mediated induction of PAI-1 and vascular adhesion molecule expression, and this effect may involve the modulation of NUR77. These effects suggest that liraglutide may potentially improve the endothelial cell dysfunction associated with premature atherosclerosis identified in type 2 diabetic patients.

A GLP-1 receptor agonist liraglutide inhibits endothelial cell dysfunction and vascular adhesion molecule expression in an ApoE-/- mouse model.

The glucagon like peptide-1 receptor (GLP-1R) agonist liraglutide attenuates induction of plasminogen activator inhibitor type-1 (PAI-1) and vascular adhesion molecule (VAM) expression in human vascular endothelial cells (hVECs) in vitro and may afford protection against endothelial cell dysfunction (ECD), an early abnormality in diabetic vascular disease. Our study aimed to establish the dependence of the in vitro effects of liraglutide on the GLP-1R and characterise its in vivo effects in a mouse model of ECD. In vitro studies utilised the human vascular endothelial cell line C11-STH and enzyme-linked immunosorbent assays (ELISA) for determination of PAI-1 and VAM expression. In vivo studies of vascular reactivity and immunohistochemical analysis were performed in the ApoE-/- mouse model. In vitro studies demonstrated GLP-1R-dependent liraglutide-mediated inhibition of stimulated PAI-1 and VAM expression. In vivo studies demonstrated significant improvement in endothelial function in liraglutide treated mice, a GLP-1R dependent effect. Liraglutide treatment also increased endothelial nitric oxide synthase (eNOS) and reduced intercellular adhesion molecule-1 (ICAM-1) expression in aortic endothelium, an effect again dependent on the GLP-1R. Together these studies identify in vivo protection, by the GLP-1R agonist liraglutide, against ECD and provide a potential molecular mechanism responsible for these effects.

Glucagon-like peptide-1 attenuates tumour necrosis factor-α-mediated induction of plasmogen activator inhibitor-1 expression

Glucagon-like peptide-1 (GLP-1) has been proposed as a target for treatment of type 2 diabetes. GLP-1 has also been demonstrated to improve endothelial cell dysfunction in diabetic patients. Elevated plasminogen activator inhibitor type-1 [corrected] (PAI-1) levels have been implicated in endothelial cell dysfunction. The effect of GLP-1 on PAI-1 expression in vascular endothelial cells has not been explored. In a spontaneously transformed human umbilical vein endothelial cell (HUVEC) line, C11-spontaneously transformed HUVEC (STH) and primary HUVEC cells, GLP-1 treatment, in the presence of a dipeptidyl peptidase IV inhibitor, attenuated induction of PAI-1 protein and mRNA expression by tumour necrosis factor-alpha (TNF-alpha). GLP-1 also inhibited the effect of TNF-alpha on a reporter gene construct harbouring the proximal PAI-1 promoter. In addition, GLP-1 attenuated TNF-alpha-mediated induction of Nur77 mRNA and TNF-alpha-mediated binding of nuclear proteins (NPs) to the PAI-1, Nur77, cis-acting response element nerve growth factor induced clone B response element (NBRE). GLP-1 treatment also inhibited TNF-alpha-mediated induction of Akt phosphorylation. Taken together, these observations suggest that GLP-1 inhibits TNF-alpha-mediated PAI-1 induction in vascular endothelial cells, and this effect may involve Akt-mediated signalling events and the modulation of Nur77 expression and NP binding to the PAI-1 NBRE.

Putative Determinants of Arterial Wall Compliance in NIDDM

Aortoiliac arterial wall compliance was measured in apparently healthy subjects and in patients with noninsulin-dependent diabetes (NIDDM), on diet alone, who had no clinical evidence of peripheral arterial disease. Compliance was significantly lower in patients with diabetes. The two clinical groups were combined to provide as wide a range of blood glucose values as possible. After allowing for the influence of age, there were significant negative correlations between compliance and free fatty acid and insulin levels. These were almost completely accounted for by differences in blood glucose levels. Therefore, arterial compliance was best predicted on the basis of age and the area under the blood glucose curve.

GLP-1-dependent and independent effects and molecular mechanisms of a dipeptidyl peptidase 4 inhibitor in vascular endothelial cells.

The potential atheroprotective effects of glucagon-like peptide-1 (GLP-1), long-acting GLP-1 analogues and inhibitors of the enzyme dipeptidyl peptidase 4 (DPP-4) are currently the subject of intense research. Recent evidence suggests the effects of DPP-IV inhibitors, may, in-part, be mediated by GLP-1 independent molecular mechanisms. In this report we demonstrate that treatment of human vascular endothelial cells with the DPP-IV inhibitor sitagliptin inhibited tumour necrosis factor alpha (TNFα) induction of plasminogen activator inhibitor type-1 (PAI-1), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein expression and that this effect was observed to be both GLP-1-dependent and independent. Importantly we identify a molecular mechanism involving sitagliptin-mediated attenuation of TNFα-mediated induction of NFκB and orphan nuclear receptor NUR77 mRNA expression, also able to be reproduced, in part, independent of GLP-1. Taken together these observations may serve to provide a molecular explanation, involving transcriptional regulation of gene expression, for recent in vivo studies suggesting DPP-IV inhibitors may have novel, GLP-1 independent, effects in acting to attenuate endothelial cell dysfunction and atherogenesis.

Exenatide in acute ischemic stroke.

Poststroke hyperglycemia is associated with worse outcome, but trials using intravenous insulin have shown inconsistent results (1). Exenatide, a synthetic glucagon-like peptide-1 receptor (GLP1R) agonist, reduces blood glucose without causing hypoglycemia (2). Moreover, in vitro and animal studies indicate that it may have a neuroprotective action (3). Eleven patients (eight males, three females, median age 80 years, 49–84 years, two with history of diabetes, eight received tissue plasminogen activator) were recruited prospectively in a nonrandomized pilot study between 2010 and 2011. Exenatide, five-micrograms, was administered subcutaneously within 12 h of ischemic stroke onset and then twice daily for the duration of their hospital stay. Adverse events and finger-prick glucose levels were assessed during the hospital stay, followed by a three-month modified Rankin Scale. Exenatide was started at a median time of nine-hours (4.17–11.5 h) after stroke onset and was continued for a median duration of six-days (one-eight days). There were no serious adverse events or deaths related to exenatide. Mild nausea (n = 6) and vomiting (n = 5) were common adverse events but were always successfully controlled within the first 24 h of starting antiemetics and did not lead to any other complications. There was no symptomatic hypoglycemia and the rate of hyperglycemia (defined as 8.6 mmol/l) was low (4.9%) (Fig. 1). This is the first study of a GLP-1R agonist in human acute stroke patients. Exenatide, when administered with prompt antiemetic therapy, was safe and tolerable in acute ischemic stroke patients and did not worsen any patient’s neurological or functional outcome. We recommend prophylactic antiemetic therapy when exenatide is given in the setting of acute stroke. Further trials are indicated to confirm the effectiveness of exenatide in controlling poststroke hyperglycemia.

Molecular and cellular mechanisms of glucagon-like peptide-1 receptor agonist-mediated attenuation of cardiac fibrosis.

Background: Glucagon-like peptide-1 receptor agonists may have a role in modulation of cardiac fibrosis. Our study aimed to determine the effect of the glucagon-like peptide-1 receptor agonist liraglutide in obesity, hypertension and age-induced murine models of cardiac fibrosis and identify associated molecular mechanisms. Methods: C57Bl/6J mice on a high-fat diet and C57Bl/6J mice on a normal chow diet treated with angiotensin II were used to induce obesity and hypertension-mediated cardiac fibrosis, respectively. C57Bl/6J mice 20 months old were used to study age-induced cardiac fibrosis. Liraglutide treatment of 30 µg/kg/day–300 µg/kg s.c. twice daily was administered for 4 weeks. Results: Liraglutide treatment attenuated obesity, hypertension and age-induced increases in interstitial cardiac fibrosis and expression of inflammatory and oxidative stress markers. Conclusions: These observations identify a potential role for liraglutide in the prevention of cardiac fibrosis and identify molecular mechanisms associated with these effects.

Transdermal Buprenorphine Relieves Neuropathic Pain: A Randomized, Double-Blind, Parallel-Group, Placebo-Controlled Trial in Diabetic Peripheral Neuropathic Pain

OBJECTIVE To evaluate the efficacy and safety of transdermal buprenorphine in patients with diabetic peripheral neuropathic pain (DPNP). RESEARCH DESIGN AND METHODS This multicenter, randomized, double-blind, placebo-controlled, parallel-group trial enrolled patients with type 1 or type 2 diabetes and stable glycemic control who had been experiencing moderate to severe DPNP for at least 6 months on maximal tolerated conventional therapy. Patients were randomly assigned to receive buprenorphine (5 μg/h) or placebo patches. The dose was titrated to effect to a maximum of 40 μg/h. Paracetamol was available as rescue analgesia. The severity of pain and other symptoms of DPNP were assessed daily in a patient diary and at clinic visits. RESULTS One hundred eight-six patients were enrolled, with 93 randomized to either buprenorphine or placebo. A high proportion of patients did not complete the study (buprenorphine 37 of 93, placebo 24 of 93). The main reason for premature withdrawal in the buprenorphine group was adverse events commonly due to untreated nausea and/or vomiting. Among the per-protocol population, more patients in the buprenorphine group (86.3%) experienced a 30% reduction in average versus baseline pain at week 12 than those in the placebo group (56.6%, P < 0.001). A nonsignificant trend favored the buprenorphine group within the intention-to-treat analysis of the same end point (51.7% vs. 41.3%, P = 0.175). CONCLUSIONS Transdermal buprenorphine, when tolerated, is an effective therapy for DPNP and provides another option to manage this challenging painful condition. Nausea and constipation need to be managed proactively to optimize treatment outcomes.

PPARγ-independent thiazolidinedione-mediated inhibition of NUR77 expression in vascular endothelial cells.

The thiazolidinediones (TZDs) have been reported to reduce atherogenesis in preclinical models and atherosclerosis in clinical trials in pre-diabetic and diabetic patients. Although peroxisome proliferator-activated receptor γ (PPARγ)-mediated effects on gene expression have been thought responsible for this effect, a complete understanding of the molecular mechanisms responsible remains to be fully elucidated. We have previously reported PPARγ-independent modulation of NUR77 (also known as Nr4a1), an orphan nuclear receptor deemed important in the atherogenic process, in association with TZD-mediated inhibition of tumour necrosis factor α (TNFα) induction of plasminogen activator inhibitor type 1 expression. Here, we report NUR77 mRNA expression is increased in human vascular endothelial cells (HUVEC) stimulated by TNFα and that this effect is inhibited by a TZD in a PPARγ-independent manner. TZD treatment of HUVEC also inhibited the stimulatory effects of TNFα on NUR77 promoter activity, again in a PPARγ-independent manner, confirming the transcriptional nature of this effect. TZD treatment also attenuated the binding of nuclear proteins to the nuclear factor kappa B (NF-κB)-binding site of the NUR77 promoter in HUVEC in a PPARγ-independent manner. In addition, TZD treatment also inhibited TNFα-mediated induction of NF-κB1 mRNA expression. Our results suggest a potential PPARγ-independent molecular mechanism for the anti-atherogenic effects of TZDs involving NF-κB-mediated transcriptional inhibition of cytokine-mediated induction of the orphan nuclear receptor NUR77 in HUVEC.

Dapagliflozin attenuates human vascular endothelial cell activation and induces vasorelaxation: A potential mechanism for inhibition of atherogenesis:

Background: Sodium glucose transporter type 2 inhibitors may reduce cardiovascular events in type 2 diabetes. Our study aimed to determine the effect of the sodium glucose transporter type 2 inhibitor dapagliflozin on endothelial cell activation, vasoreactivity and atherogenesis using in vitro and in vivo models and identify associated molecular mechanisms. Methods: In vitro studies utilised human vascular endothelial cells stimulated with tumour necrosis factor α or hyperglycaemic conditions. In vivo studies were performed in C57Bl/6J mice to evaluate direct vasorelaxation responses evoked by acute dapagliflozin administration and acute vaso-protective effects of dapagliflozin on hyperglycaemia-induced endothelial dysfunction. Adult and aged Apolipoprotein E–deficient mice maintained on a high-fat diet were used to investigate endothelial-dependent vascular reactivity and atherogenesis. Dapagliflozin treatment (1.0 mg/kg/day) was administered for 4 weeks. Results: In vitro studies demonstrated dapagliflozin-mediated attenuation of tumour necrosis factor α- and hyperglycaemia-induced increases in intercellular adhesion molecule-1, vascular cell adhesion molecule-1, plasminogen activator inhibitor type 1 and NFκB expression. Acute dapagliflozin administration dose-dependently induced endothelium-independent vasorelaxation. Chronic dapagliflozin treatment improved endothelial function and significantly reduced in vivo vascular adhesion molecule and phospho-IκB expression together with macrophage vessel wall infiltration. Conclusion: These observations identify a potential role for dapagliflozin in the attenuation of atherogenesis and identify anti-inflammatory molecular mechanisms associated with these effects.