Aims/hypothesis Extracellular pre-B cell colony-enhancing factor/nicotinamide phosphoribosyltransferase/visfatin (ePBEF/NAMPT/visfatin) is an adipocytokine, whose circulating levels are enhanced in metabolic disorders, such as diabetes mellitus and obesity. Here, we explored the ability of ePBEF/NAMPT/visfatin to promote vascular inflammation, as a condition closely related to atherothrombotic diseases. We specifically studied the ability of PBEF/ NAMPT/visfatin to directly activate pathways leading to inducible nitric oxide synthase (iNOS) induction in cultured human aortic smooth muscle cells, as well as the mechanisms involved. Methods iNOS levels and extracellular signal-regulated kinase (ERK) 1/2 activity were determined by western blotting. Nuclear factor (NF)-κB activity was assessed by electrophoretic mobility shift assay. Results ePBEF/NAMPT/visfatin (10-250 ng/ml) induced iNOS in a concentration-dependent manner. At a submaximal concentration (100 ng/ml), ePBEF/NAMPT/visfatin time-dependently enhanced iNOS levels up to 18 h after stimulation. Over this time period, ePBEF/NAMPT/visfatin elicited a sustained activation of NF-κB and triggered a biphasic ERK 1/2 activation. By using the respective ERK 1/2 and NF-κB inhibitors, PD98059 and pyrrolidine dithiocarbamate, we established that iNOS induction by ePBEF/NAMPT/visfatin required the consecutive upstream activation of ERK 1/2 and NF-κB. The pro-inflammatory action of ePBEF/NAMPT/visfatin was not prevented by insulin receptor blockade. However, exogenous nicotinamide mononucleotide, the product of NAMPT activity, mimicked NF-κB activation and iNOS induction by ePBEF/NAMPT/visfatin, while the NAMPT inhibitor APO866 prevented the effects of ePBEF/NAMPT/visfatin on iNOS and NF-κB. Conclusions/interpretation Through its intrinsic NAMPT activity, ePBEF/NAMPT/visfatin appears to be a direct contributor to vascular inflammation, a key feature of atherothrombotic diseases linked to metabolic disorders.

1 Alterations of the vessel structure, which is mainly determined by smooth muscle cells through cell growth and/or cell death mechanisms, are characteristic of diabetes complications. We analysed the in¯uence of high glucose (22 mM) on cultured human aortic smooth muscle cell growth and death, as hyperglycaemia is considered one of the main factors involved in diabetic vasculopathy. 2 Growth curves were performed over 96 h in medium containing 0.5% foetal calf serum. Cell number increased by 2 ± 4 fold over the culture period in the presence of 5.5 mM (low) glucose, while a 20% reduction in ®nal cell number was observed with high glucose. Under serum-free conditions, cell number remained constant in low glucose cultures, but a 40% decrease was observed in high glucose cultures, suggesting that high glucose may induce increased cell death rather than reduced proliferation. Reduced ®nal cell number induced by high glucose was also observed after stimulation with 5 or 10% foetal calf serum. 3 The possible participation of oxidative stress was investigated by co-incubating high glucose with di erent reactive oxygen species scavengers. Only catalase reversed the e ect of high glucose. Intracellular H 2 O 2 content, visualized with 2',7'-dichloro¯uorescein and quanti®ed by¯ow cytometry, was increased after high glucose treatment. 4 To investigate the cell death mechanism induced by high glucose, apoptosis and necrosis were quanti®ed. No di erences were observed regarding the apoptotic index between low and high glucose cultures, but lactate dehydrogenase activity was increased in high glucose cultures. 5 In conclusion, high glucose promotes necrotic cell death through H 2 O 2 formation, which may participate in the development of diabetic vasculopathy. British Journal of Pharmacology (2001) 133, 967 ± 974

Visfatin, also known as extracellular pre–B-cell colony–enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is an adipocytokine whose circulating levels are enhanced in metabolic disorders, such as type 2 diabetes mellitus and obesity. Circulating visfatin levels have been positively associated with vascular damage and endothelial dysfunction. Here, we investigated the ability of visfatin to directly impair vascular reactivity in mesenteric microvessels from both male Sprague-Dawley rats and patients undergoing non-urgent, non-septic abdominal surgery. The pre-incubation of rat microvessels with visfatin (50 and 100 ng/mL) did not modify the contractile response to noradrenaline (1 pmol/L to 30 µmol/L), as determined using a small vessel myograph. However, visfatin (10 to 100 ng/mL) concentration-dependently impaired the relaxation to acetylcholine (ACh; 100 pmol/L to 3 µmol/L), without interfering with the endothelium-independent relaxation to sodium nitroprusside (1 nmol/L to 3 µmol/L). In both cultured human umbilical vein endothelial cells and rat microvascular preparations, visfatin (50 ng/mL) stimulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as determined by lucigenin-derived chemiluminiscence. The relaxation to ACh impaired by visfatin was restored by the NADPH oxidase inhibitor apocynin (10 µmol/L). Additionally, the Nampt inhibitor APO866 (10 mmol/L to 10 µmol/L), but not an insulin receptor-blocking antibody, also prevented the stimulation of NADPH oxidase and the relaxation impairment elicited by visfatin. Accordingly, the product of Nampt activity nicotinamide mononucleotide (100 nmol/L to 1 mmol/L) stimulated endothelial NADPH oxidase activity and concentration-dependently impaired ACh-induced vasorelaxation. In human mesenteric microvessels pre-contracted with 35 mmol/L potassium chloride, the endothelium-dependent vasodilation to bradykinin (1 nmol/L to 3 µmol/L) was equally impaired by visfatin and restored upon co-incubation with APO866. In conclusion, visfatin impairs endothelium-dependent relaxation through a mechanism involving NADPH oxidase stimulation and relying on Nampt enzymatic activity, and therefore arises as a potential new player in the development of endothelial dysfunction.

1 Diabetes mellitus leads to a high incidence of several so-called complications, sharing similar pathophysiological features in several territories. Previous reports points at early nonenzymatic glycosylation products (Amadori adducts) as mediators of diabetic vascular complications. In the present study, we analysed a possible role for Amadori adducts as stimulators of proinflammatory pathways in human peritoneal mesothelial cells (HPMCs). 2 Cultured HPMCs isolated from 13 different patients (mean age 38.7716 years) were exposed to different Amadori adducts, that is, highly glycated haemoglobin (10 nM) and glycated bovine serum albumin (0.25 mg ml À1 ), as well as to their respective low glycosylation controls. Amadori adducts, but not their respective controls, elicited a marked increase of NF-kB activation, as determined by electromobility shift assays and transient transfection experiments. 3 Additionally, Amadori adducts significantly increased the production of NF-kB-related proinflammatory molecules, including cytokines, such as TNF-a, IL-1b or IL-6, and enzymes, such as cyclooxygenase-2 and inducible nitric oxide (NO) synthase, this latter leading to the release of NO by HPMCs. 4 The effects of Amadori adducts were mediated by different reactive oxygen and nitrosative species (e.g. superoxide anions, hydroxyl radicals, and peroxynitrite), as they were blunted by coincubation with the appropriate scavengers. Furthermore, NO generated upon exposure to Amadori adducts further stimulated NF-kB activation, either directly or after combination with superoxide anions to form peroxynitrite. 5 We conclude that Amadori adducts can favour peritoneal inflammation by exacerbating changes in NO synthesis pathway and triggering NF-kB-related proinflammatory signals in human mesothelial cells.