Visfatin Impairs Endothelium-Dependent Relaxation in Rat and Human Mesenteric Microvessels through Nicotinamide Phosphoribosyltransferase Activity

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

The angiotensin-(1-7)/mas axis counteracts angiotensin II-dependent and -independent pro-inflammatory signaling in human vascular smooth muscle cells

Background and Aims: Targeting inflammation is nowadays considered as a challenging pharmacological strategy to prevent or delay the development of vascular diseases. Angiotensin-(1-7) is a member of the renin-angiotensin system (RAS) that binds Mas receptors and has gained growing attention in the last years as a regulator of vascular homeostasis. Here, we explored the capacity of Ang-(1-7) to counteract human aortic smooth muscle cell (HASMC) inflammation triggered by RAS-dependent and -independent stimuli, such as Ang II or interleukin (IL)-1β. Methods and Results: In cultured HASMC, the expression of inducible nitric oxide synthase (iNOS) and the release of nitric oxide were stimulated by both Ang II and IL-1β, as determined by Western blot and indirect immunofluorescence or the Griess method, respectively. iNOS induction was inhibited by Ang-(1-7) in a concentration-dependent manner. This effect was equally blocked by two different Mas receptor antagonists, A779 and D-Pro7-Ang-(1-7), suggesting the participation of a unique Mas receptor subtype. Using pharmacological inhibitors, the induction of iNOS was proven to rely on the consecutive upstream activation of NADPH oxidase and nuclear factor (NF)-κB. Indeed, Ang-(1-7) markedly inhibited the activation of the NADPH oxidase and subsequently of NF-κB, as determined by lucigenin-derived chemiluminescence and electromobility shift assay, respectively. Conclusion: Ang-(1-7) can act as a counter-regulator of the inflammation of vascular smooth muscle cells triggered by Ang II, but also by other stimuli beyond the RAS. Activating or mimicking the Ang-(1-7)/Mas axis may represent a pharmacological opportunity to attenuate the pro-inflammatory environment that promotes and sustains the development of vascular diseases.This work was funded by grants from Ministerio de Economía y Competitividad (SAF2014-52762-R

Visfatin as a novel mediator released by inflamed human endothelial cells.

BACKGROUND:Visfatin is a multifaceted adipokine whose circulating levels are enhanced in different metabolic diseases. Extracellular visfatin can exert various deleterious effects on vascular cells, including inflammation and proliferation. Limited evidence exists, however, on the capacity of human vascular cells to synthesize and release visfatin by themselves, under basal or pro-inflammatory conditions. METHODS AND RESULTS:Intracellular visfatin was detected by Western blot in non-stimulated human umbilical vein endothelial cells (HUVEC). However, exposing HUVEC for 18 h to a series of pro-inflammatory stimulus, such as interleukin (IL)-1β (1 to 10 ng/mL), tumor necrosis factor-α (1 to 10 ng/mL) or angiotensin II (10 pmol/L to 1 μmol/L) markedly enhanced intracellular visfatin content. Using IL-1β (10 ng/mL; 18 h), it was determined that the increase in intracellular visfatin, which was paralleled by enhanced visfatin mRNA levels, relied on a signalling mechanism involving both nuclear factor-κB and poly (ADP ribose) polymerase-1 activation. Moreover, IL-1β modified the sub-cellular localization of visfatin; while in non-stimulated HUVEC immunoreactive visfatin predominantly showed an intra-nuclear granular pattern, in IL-1β-inflamed cells an extra-nuclear filamentous staining, co-localising with F-actin fibers and suggesting a secretory pattern, was mainly found. Indeed, IL-1β promoted visfatin secretion, as determined by both ELISA and immunocytochemistry. CONCLUSIONS:Human endothelial cells synthesize and release visfatin, particularly in response to inflammation. We suggest that the inflamed endothelium can be a source of visfatin, which arises as a local inflammatory mediator and a potential therapeutic target to interfere with vascular inflammation

Inflammation enhances intracellular visfatin levels in HUVEC.

<p>(A) Concentration-dependent effect of IL-1β (1 to 10 ng/mL; 18 h) on cellular visfatin content determined by Western blotting. (B) Time course of visfatin induction by IL-1β (10 ng/mL) over 48 h. Visfatin content was also determined in HUVEC challenged for 18 h with either (C) TNF-α (1 to 10 ng/mL) or (D) Ang II (10 pmol/L to 1 µmol/L). Data are the mean±SEM of five independent experiments. *<i>P</i><0.05 vs non-stimulated cells. Representative gels are shown on the top.</p

Inflammation promotes visfatin secretion by HUVEC.

<p>(A) Representative microphotographs of visfatin release from HUVEC grown on Immobilon-P membranes and treated with or without IL-1β, (10 ng/mL; 18 h) in the absence or the presence of the PARP-1 inhibitor PJ34 (10 µmol/L). Secreted visfatin appears as a diffuse halo of extracellular positive immunostaining (black arrows). Magnification 400x. (B) Visfatin content determined by ELISA in cell supernanants treated with or without IL-1β (10 ng/mL; 18 h). Data are the mean±SEM for four independent experiments. *<i>P</i><0.05 vs basal. </p

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

The activation of NADPH oxidase and the impaired relaxation to ACh induced by visfatin in rat microvessels relies on Nampt activity.

<p>(A) NADPH oxidase activity was measured in cultured human umbilical vein endothelial cells (HUVEC) stimulated for 20–30 min with visfatin (50 ng/mL), the Nampt inhibitor APO866 (10 µmol/L) or both compounds together, as well as with the product of the Nampt reaction NMN (10 and 100 µmol/L). Results are expressed as mean ± SEM of 5 experiments performed in triplicate. *<i>P</i><0.05 vs control cultures; †<i>P</i><0.05 vs visfatin (B) Effect of visfatin (50 ng/mL) and APO866 (10 µmol/L), alone or in combination, on NADPH oxidase activity in rat microvascular preparations. Results are expressed as mean ± SEM of 8 segments obtained from 4 animals. *<i>P</i><0.05 vs control cultures; †<i>P</i><0.05 vs visfatin. (C) Rat microvessels were pre-incubated with visfatin (50 ng/mL), alone or in the presence of APO866 (10 nmol/L to 10 µmol/L), subsequently contracted with 1 µmol/L NA and then exposed to increasing concentrations of ACh (100 pmol/L to 3 µmol/L). Results are expressed as mean ± SEM of 58 segments obtained from 11 animals. *<i>P</i><0.05 vs control curve; †<i>P</i><0.05 vs visfatin alone. (D) Correlation between the APO866 concentrations used for pre-incubation and the pD₂ values for ACh. (E) The vessels were pre-incubated with NMN (100 nmol/L to 1 mmol/L), contracted with 1 µmol/L NA and then relaxed with cumulative concentrations of ACh (100 pmol/L to 3 µmol/L). Results represent the mean ± SEM of 32 segments obtained from 5 animals. *<i>P</i><0.05 vs control curve. (F) Correlation between the NMN concentrations used for pre-incubation and the pD2 values for ACh.</p

NADPH oxidase mediates the impaired vasodilation to ACh induced by visfatin in rat mesenteric microvessels.

<p>(A) Cultured human umbilical vein endothelial cells were stimulated with increasing concentrations of visfatin (10 to 100 ng/mL) for 20–30 min and the generation of superoxide anions by NADPH oxidase was determined lucigenin-derived chemiluminiscence. Results are expressed as mean ± SEM obtained from 6 experiments performed in triplicate. *<i>P</i><0.05 vs control cultures without visfatin. (B) Rat microvessels were pre-incubated with visfatin (50 ng/mL) and/or apocynin (10 µmol/L), contracted with 1 µmol/L NA and then exposed to cumulative concentrations of ACh (100 pmol/L to 3 µmol/L). Results are expressed as mean ± SEM of 35 segments from 5 animals. *<i>P</i><0.05 vs control curve; †<i>P</i><0.05 vs visfatin.</p

The impaired relaxation to ACh induced by visfatin in rat microvessels does not depend on the insulin receptor.

<p>The microvessels were pre-incubated with visfatin (50 ng/mL) alone or combined with either an antibody blocking the activation of the insulin receptor (InsR Ab; 2 µg/mL) and then contracted with 1 µmol/L NA and exposed to increasing concentrations of ACh (100 pmol/L to 3 µmol/L). Results are expressed as mean ± SEM of 20 segments obtained from 4 animals. *<i>P</i><0.05 vs control curve.</p