Efecto de altas concentraciones de D-Glucosa sobre la adhesión leucocitaria en células endoteliales humanas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina. Departamento Farmacología y Terpéutica. Fecha de lectura: 15 de Enero de 2009

Inflammation Determines the Pro-Adhesive Properties of High Extracellular D-Glucose in Human Endothelial Cells In Vitro and Rat Microvessels In Vivo

Background: Hyperglycemia is acknowledged as an independent risk factor for developing diabetes-associated atherosclerosis. At present, most therapeutic approaches are targeted at a tight glycemic control in diabetic patients, although this fails to prevent macrovascular complications of the disease. Indeed, it remains highly controversial whether or not the mere elevation of extracellular D-glucose can directly promote vascular inflammation, which favors early proatherosclerotic events. Methods and Findings: In the present work, increasing extracellular D-glucose from 5.5 to 22 mmol/L was neither sufficient to induce intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, analyzed by flow cytometry, nor to promote leukocyte adhesion to human umbilical vein endothelial cells (HUVEC) in vitro, measured by flow chamber assays. Interestingly, the elevation of D-glucose levels potentiated ICAM-1 and VCAM-1 expression and leukocyte adhesion induced by a pro-inflammatory stimulus, such as interleukin (IL)-1b (5 ng/mL). In HUVEC, high D-glucose augmented the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and nuclear transcription factor-kB (NF-kB) elicited by IL-1b, measured by Western blot and electromobility shift assay (EMSA), respectively, but had no effect by itself. Both ERK 1/2 and NF-kB were necessary for VCAM-1 expression, but not for ICAM-1 expression. In vivo, leukocyte trafficking was evaluated in the rat mesenteric microcirculation by intravital microscopy. In accordance with the in vitro data, the acute intraperitoneal injection of D-glucose increased leukocyte rolling flux, adhesion and migration, but only when IL-1b was coadministered. Conclusions: These results indicate that the elevation of extracellular D-glucose levels is not sufficient to promote vascular inflammation, and they highlight the pivotal role of a pro-inflammatory environment in diabetes, as a critical factor conditioning the early pro-atherosclerotic actions of hyperglycemia

Glycosylated human oxyhaemoglobin activates nuclear factor-jB and activator protein-1 in cultured human aortic smooth muscle

1 Diabetic vessels undergo structural changes that are linked to a high incidence of cardiovascular diseases. Reactive oxygen species (ROS) mediate cell signalling in the vasculature, where they can promote cell growth and activate redox-regulated transcription factors, like activator protein-1 (AP-1) or nuclear factor-kB (NF-kB), which are involved in remodelling and inflammation processes. Amadori adducts, formed through nonenzymatic glycosylation, can contribute to ROS formation in diabetes. 2 In this study, we analysed whether Amadori-modified human oxyhaemoglobin, glycosylated at either normal (N-Hb) or elevated (E-Hb) levels, can induce cell growth and activate AP-1 and NF-kB in cultured human aortic smooth muscle cells (HASMC). 3 E-Hb (1 nM–1 mM), but not N-Hb, promoted a concentration-dependent increase in cell size from nanomolar concentrations, although it failed to stimulate HASMC proliferation. At 10 nM, E-Hb stimulated both AP-1 and NF-kB activity, as assessed by transient transfection, electromobility shift assays or immunofluorescence staining. The effects of E-Hb resembled those of the proinflammatory cytokine tumour necrosis factor-a (TNF-a). E-Hb enhanced intracellular superoxide anions content and its effects on HASMC were abolished by different ROS scavengers. 4 In conclusion, E-Hb stimulates growth and activates AP-1 and NF-kB in human vascular smooth muscle by redox-sensitive pathways, thus suggesting a possible direct role for Amadori adducts in diabetic vasculopathy.Ministerio de Ciencia y Tecnología (España)Comunidad de MadridFondo de Investigaciones SanitariasInstituto de Salud Carlos IIIDepto. de FisiologíaFac. de FarmaciaTRUEpu

Extracellular PBEF/NAMPT/visfatin activates pro-inflammatory signalling in human vascular smooth muscle cells through nicotinamide phosphoribosyltransferase activity

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 athero-thrombotic diseases linked to metabolic disorders.Ministerio de Educación y CienciaInstituto de Salud Carlos IIIComunidad de Madrid-Universidad Autónoma de MadridFundación de Investigación Médica Mutua MadrileñaInstituto DanoneSociedad Española de Farmacología/Laboratorios AlmirallDepto. de FisiologíaFac. de FarmaciaTRUEpu

Inflammation, glucose, and vascular cell damage:The role of the pentose phosphate pathway

Additional file 1. Supplementary figures

Amadori adducts activate nuclear factor-kB-related proinflammatory genes in cultured human peritoneal mesothelial cells

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 38.7+/-16 years) were exposed to different Amadori adducts, that is, highly glycated haemoglobin (10 nM) and glycated bovine serum albumin (0.25 mg/ml-1), 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-alpha, IL-1beta 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.Fondo de Investigaciones SanitariasComunidad de MadridMinisterio de Educación y Ciencia (España)Instituto de Salud Carlos IIIDepto. de FisiologíaFac. de FarmaciaTRUEpu

Complete blockade of the vasorelaxant effects of Ang-(1-7) and bradykinin in murine microvessels by antagonists of the receptor Mas.

The heptapeptide angiotensin-(1-7) is a biologically active metabolite of angiotensin II, the predominant peptide of the renin-angiotensin system. Recently, we have shown that the receptor Mas is associated with angiotensin-(1-7)-induced signalling and mediates, at least in part, the vasodilatory properties of angiotensin-(1-7). However, it remained controversial whether an additional receptor could account for angiotensin-(1-7)-induced vasorelaxation. Here, we used two different angiotensin-(1-7) antagonists, A779 and d-Pro-angiotensin-(1-7), to address this question and also to study their influence on the vasodilatation induced by bradykinin. Isolated mesenteric microvessels from both wild-type and Mas-deficient C57Bl/6 mice were precontracted with noradrenaline, and vascular reactivity to angiotensin-(1-7) and bradykinin was subsequently studied using a small-vessel myograph. Furthermore, mechanisms for Mas effects were investigated in primary human umbilical vein endothelial cells. Both angiotensin-(1-7) and bradykinin triggered a concentration-dependent vasodilatation in wild-type microvessels, which was absent in the presence of a nitric oxide synthase inhibitor. In these vessels, the pre-incubation with the Mas antagonists A779 or d-Pro-angiotensin-(1-7) totally abolished the vasodilatory capacity of both angiotensin-(1-7) and bradykinin, which was nitric oxide mediated. Accordingly, Mas-deficient microvessels lacked the capacity to relax in response to either angiotensin-(1-7) or bradykinin. Pre-incubation of human umbilical vein endothelial cells with A779 prevented bradykinin-mediated NO generation and NO synthase phosphorylation at serine 1177. The angiotensin-(1-7) antagonists A779 and d-Pro-angiotensin-(1-7) equally block Mas, which completely controls the angiotensin-(1-7)-induced vasodilatation in mesenteric microvessels. Importantly, Mas also appears to be a critical player in NO-mediated vasodilatation induced by renin-angiotensin system-independent agonists by altering phosphorylation of NO synthase