RESUMEN
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)-kappaB 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-kappaB and triggered a biphasic ERK 1/2 activation. By using the respective ERK 1/2 and NF-kappaB 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-kappaB. 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-kappaB activation and iNOS induction by ePBEF/NAMPT/visfatin, while the NAMPT inhibitor APO866 prevented the effects of ePBEF/NAMPT/visfatin on iNOS and NF-kappaB. 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.
Asunto(s)
Citocinas/farmacología , Citocinas/fisiología , Músculo Liso Vascular/fisiología , Nicotinamida Fosforribosiltransferasa/metabolismo , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Técnicas de Cultivo de Célula , Diabetes Mellitus/sangre , Diabetes Mellitus/fisiopatología , Flavonoides/farmacología , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/fisiología , Cinética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Nicotinamida Fosforribosiltransferasa/farmacología , Nicotinamida Fosforribosiltransferasa/fisiología , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Obesidad/sangre , Obesidad/fisiopatología , Transducción de SeñalRESUMEN
BACKGROUND AND PURPOSE: Diabetes mellitus is prevalent in the elderly population. It is also a disease causing tissue damage through several different mechanisms. Some of these mechanisms are also activated by ageing and this overlap raises questions about how diabetes induces damage in the elderly. Early products of non-enzymatic glycation of proteins (Amadori adducts), and the ageing process share the capacity to induce oxidative stress and inflammation in human peritoneal mesothelial cells (HPMCs). We have evaluated the interactions between the age of the donor of the HPMCs and the pro-inflammatory effects of Amadori adducts in those cells. EXPERIMENTAL APPROACH: HPMCs were isolated from 20 individuals (age range 21-81 years) and grown in culture. Using different experimental approaches we determined NF-kappaB dependent transcriptional activity and different NF-kappaB-related pro-inflammatory gene and protein expressions in basal (or non-stimulated) conditions and after stimulation with two Amadori adducts; highly-glycated haemoglobin and glycated bovine serum albumin. KEY RESULTS: Amadori-induced effects on NF-kappaB dependent-transcription and on the activity of NOS, COX and several NF-kappaB-related pro-inflammatory genes (iNOS, COX-2, TNF-alpha, IL-1beta, and IL6) diminished as the donor's age increased, being practically absent in cells from donors more than 65 years old. Such decreased effects were inversely correlated with an increased basal expression and activity of these pro-inflammatory markers with age. CONCLUSIONS AND IMPLICATIONS: Pro-inflammatory effects of Amadori-adducts in HPMCs were strongly dependent on cell donor's age. This may have significant implications for the mechanisms underlying diabetes-induced tissue damage in patients of different ages.