Elena Cercas

High glucose induces cell death of cultured human aortic smooth muscle cells through the formation of hydrogen peroxide

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 influence 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. 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 final 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 final cell number induced by high glucose was also observed after stimulation with 5 or 10% foetal calf serum. The possible participation of oxidative stress was investigated by co‐incubating high glucose with different reactive oxygen species scavengers. Only catalase reversed the effect of high glucose. Intracellular H2O2 content, visualized with 2′,7′‐dichlorofluorescein and quantified by flow cytometry, was increased after high glucose treatment. To investigate the cell death mechanism induced by high glucose, apoptosis and necrosis were quantified. No differences were observed regarding the apoptotic index between low and high glucose cultures, but lactate dehydrogenase activity was increased in high glucose cultures. In conclusion, high glucose promotes necrotic cell death through H2O2 formation, which may participate in the development of diabetic vasculopathy.

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.

Evidence for sodium azide as an artifact mediating the modulation of inducible nitric oxide synthase by C-reactive protein.

C-reactive protein (CRP) is an acute-phase protein identified as a cardiovascular risk marker. In recent years, an increasing number of studies have investigated the possible direct effects of CRP on the vasculature, using mainly commercial CRP. In the present work, a potential role for CRP as a modulator of inducible nitric oxide synthase (iNOS) induction was explored. Cultured human aortic vascular smooth muscle cells (HASMC) were stimulated for 18 hours with 10 ng/mL interleukin-1β (IL-1β), resulting in a marked increase of iNOS levels and NO production, as determined by Western blotting and nitrite measurement, respectively. Commercial CRP (1 to 100 μg/mL) concentration-dependently inhibited the effects elicited by IL-1β. Unexpectedly, similar results were observed when the commercial CRP solution was replaced by the corresponding vehicle medium containing growing concentrations of sodium azide. The inhibitory effects of commercial CRP or vehicle medium were lost on sodium azide removal by dialysis. In conclusion, sodium azide from the commercial CRP solution, but not CRP itself, mainly accounts for the inhibitory effect on IL-1β-evoked iNOS induction and NO release. Care should be taken before attributing any biologic role to commercial CRP containing sodium azide.

Prevention of endothelial dysfunction in streptozotocin-induced diabetic rats by gliclazide treatment.

The aim of the present work was to analyze whether the oral hypoglycemic drug gliclazide affects diabetic endothelial dysfunction in streptozotocin-induced diabetic rats. Gliclazide was compared with glibenclamide, ascorbic acid, and aminoguanidine. An insulin-dependent model of diabetes was selected to exclude insulin-releasing effects of the drugs. Both in isolated aortic segments and mesenteric microvessels, endothelium-dependent relaxation evoked by acetylcholine (ACh, 1 nM to 10 microM) was significantly reduced in vessels from diabetic animals. This impairment was reversed when the segments were previously incubated with 100 U/ml superoxide dismutase. When streptozotocin-induced diabetic rats were orally treated from the time of diabetes induction with gliclazide (10 mg/kg) or ascorbic acid (250 mg/kg), ACh-induced endothelium-dependent relaxation was well preserved both in aortic segments and mesenteric microvessels. In addition, the impaired vasodilatation to exogenous nitric oxide (NO) in aortic segments was also improved in gliclazide-treated diabetic rats. On the other hand, oral treatment with glibenclamide (1 and 10 mg/kg) or aminoguanidine (250 mg/kg) did not produce significant improvements in diabetic endothelial dysfunction. We conclude that gliclazide reverses the endothelial dysfunction associated with diabetes. This effect appears to be due not to the metabolic actions of the drug but rather to its antioxidant properties, as it can be mimicked by other antioxidants. We propose that the mechanism involved is the inactivation of reactive oxygen species, which are increased in diabetes probably as a result of increased early protein glycosylation products, such as glycosylated hemoglobin (HbA(1c)). These effects of gliclazide are not shared by other oral hypoglycemic agent such as glibenclamide, or by blockade of advanced glycosylation end product (AGE) generation with aminoguanidine.

Amadori adducts activate nuclear factor‐κB‐related proinflammatory genes in cultured human peritoneal mesothelial cells

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). 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‐κB activation, as determined by electromobility shift assays and transient transfection experiments. Additionally, Amadori adducts significantly increased the production of NF‐κB‐related proinflammatory molecules, including cytokines, such as TNF‐α, IL‐1β 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. 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‐κB activation, either directly or after combination with superoxide anions to form peroxynitrite. We conclude that Amadori adducts can favour peritoneal inflammation by exacerbating changes in NO synthesis pathway and triggering NF‐κB‐related proinflammatory signals in human mesothelial cells.

Glycosylated human oxyhaemoglobin activates nuclear factor‐κB and activator protein‐1 in cultured human aortic smooth muscle

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‐κB (NF‐κB), which are involved in remodelling and inflammation processes. Amadori adducts, formed through nonenzymatic glycosylation, can contribute to ROS formation in diabetes. 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‐κB in cultured human aortic smooth muscle cells (HASMC). E‐Hb (1 nM–1 μM), 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‐κB 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‐α (TNF‐α). E‐Hb enhanced intracellular superoxide anions content and its effects on HASMC were abolished by different ROS scavengers. In conclusion, E‐Hb stimulates growth and activates AP‐1 and NF‐κB in human vascular smooth muscle by redox‐sensitive pathways, thus suggesting a possible direct role for Amadori adducts in diabetic vasculopathy.

Treatment with acarbose may improve endothelial dysfunction in streptozotocin-induced diabetic rats.

We sought to determine whether a single reduction of hyperglycemia and those derivatives from nonenzymatic protein glycosylation may be effective in reducing the development of diabetic endothelial dysfunction. Therefore, we investigated how acarbose, an inhibitor of intestinal alpha-glucosidase that reduce hyperglycemia by lowering glucose absorption, may prevent the impairment of acetylcholine (ACh)-induced endothelium-dependent relaxations observed in isolated vascular segments from untreated streptozotocin-induced diabetic rats. When administered after diabetes induction, 10 mg/kg acarbose decreased modestly the enhancement of blood glucose and glycosylated hemoglobin (HbA1c) levels, but not those of advanced glycosylation end products (AGEs). This effect was linked to a partial improvement of ACh-induced responses both in conductance vessels, such as aortic segments, and resistance vasculature, like mesenteric microvessels. When acarbose was introduced after 6 weeks of untreated diabetes, blood glucose, HbA1c, and AGE levels were not affected and endothelial dysfunction remained unchanged in mesenteric microvessels, whereas a small improvement was observed in aortic segments. The addition of 100 U/ml superoxide dismutase enhanced the impaired relaxations to values similar to vessels from nondiabetic rats, indicating a main role for superoxide anions in diabetes-induced endothelial dysfunction. We conclude that hyperglycemia itself or elevated HbA1c, but not plasma AGEs, are related to enhanced oxidative stress and to the impairment of endothelium function associated to diabetes. This process can be partially prevented by reducing glucose absorption with acarbose.

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.

Vascular smooth muscle cell hypertrophy induced by glycosylated human oxyhaemoglobin

1 Nonenzymatic protein glycosylation is a possible mechanism contributing to oxidative stress and vascular disease in diabetes. In this work, the influence of 14%‐glycosylated human oxyhaemoglobin (GHHb), compared to the non‐glycosylated protein (HHb), was studied on several growth parameters of rat cultured vascular smooth muscle cells (VSMC). A role for reactive oxygen species was also analysed. 2 Treatment of VSMC for 48 h with GHHb, but not with HHb, increased planar cell surface area in a concentration dependent manner. The threshold concentration was 10 nM, which increased cell size from 7965±176 to 9411±392 μm2. Similarly, only GHHb enhanced protein content per well in VSMC cultures. 3 The planar surface area increase induced by 10 nM GHHb was abolished by superoxide dismutase (SOD; 50–200 u ml−1), deferoxamine (100 nM–100 μM), or dimethylthiourea (1 mM), while catalase (50–200 u ml−1) or mannitol (1 mM) resulted in a partial inhibition of cell size enhancement. 4 When a known source of oxygen free radicals was administered to VSMC, the xanthine/xanthine oxidase system, the results were analogous to those produced by GHHb. Indeed, enhancements of cell size were observed, which were inhibited by SOD, deferoxamine, or catalase. 5 These results indicate that, at low concentrations, GHHb induces hypertrophy in VSMC, this effect being mediated by superoxide anions, hydrogen peroxide, and/or hydroxyl radicals. Therefore, glycosylated proteins can have a role in the development of the structural vascular alterations associated to diabetes by enhancing oxidative stress.

Correction of glycosylated oxyhemoglobin-induced impairment of endothelium-dependent vasodilatation by gliclazide

We have investigated whether gliclazide, a second-generation sulfonylurea hypoglycemic agent, interferes with the impairment of endothelium-dependent nitric-oxide-mediated relaxation produced by 14%-glycosylated human oxyhemoglobin (GHHb). For comparative purposes, other agents, like glibenclamide, aminoguanidine, ascorbic acid or superoxide dismutase (SOD), were also tested. GHHb (10 nM) caused a reduction in endothelium-dependent relaxation induced by acetylcholine (1 nM to 10 microM) in both isolated aortic segments and mesenteric microvessels from normoglycemic nondiabetic rats. Preincubation of the vessels with gliclazide (100 nM to 10 microM) prevented the impairment of endothelial relaxation, the threshold concentration of gliclazide being 300 nM. In addition, 10 microM gliclazide also prevented the reduction by 10 nM GHHb of the relaxation induced by exogenous nitric oxide (NO, 10 nM to 100 microM). Determination of superoxide anion release measured by the reduction in ferricytochrome c indicated that GHHb produced significant amounts of these free radicals that were concentration-dependently inhibited by gliclazide. The impairment of endothelium-mediated responses was also prevented by 100 U/ml SOD or 10 microM ascorbic acid, but not by 10 microM glibenclamide or 100 microM aminoguanidine. We conclude that gliclazide can reduce the impairment of nitric-oxide-mediated endothelium-dependent relaxation produced by GHHb. This reduction is likely related to the antioxidant properties of the drug, a mechanism suggested by these studies which demonstrate the inactivation of superoxide anions produced by the glycosylated protein by gliclazide.