Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

González, Marcelo; Rojas, Susana; Avila, Pía; Cabrera, Lissette; Villalobos, Rodrigo; Palma, Carlos; Aguayo, Claudio; Peña, Emilio Duro; Gallardo, Victoria; Guzmán-Gutiérrez, Enrique; Sáez, Tamara; Salsoso, Rocío; Sanhueza, Carlos; Pardo, Fabián; Leiva, Andrea; Sobrevía, Luis

doi:10.1371/journal.pone.0122398

Cited by 52 publications

(60 citation statements)

References 44 publications

(98 reference statements)

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“…In addition, ROS levels and nitric oxide bioavailability, both key factors of oxidative stress, are related to eNOS activity. 38 ROS produced during endothelial dysfunction can promote eNOS uncoupling. 39 In our in vitro model, the sole interaction of DF with cell membrane, even in the absence of significant internalization, was capable of restoring eNOS levels in front of oxidative stress.…”

Section: Discussionmentioning

confidence: 99%

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

Palomo

Mir

Rovira

et al. 2016

Blood

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Key Points• Specific interaction of DF with EC membranes is followed by its internalization mainly through macropinocytic mechanisms.• DF attachment to the cell membrane is sufficient to perform its antiinflammatory and antioxidant effects on the endothelium.Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization.Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations. (Blood. 2016;127(13):1719-1727 Introduction Defibrotide (DF) is a mixture of 90% single-stranded phosphodiester oligonucleotides (length, 9-80 mer; average molecular mass, 16.5 6 2.5 KDa) and 10% double-stranded phosphodiester oligonucleotides, derived from the controlled depolymerization of porcine intestinal mucosal DNA.1-3 Several functions, specially related to hemostasis, have been ascribed to DF. 4 In this regard, our group has demonstrated the protective effect of DF on the endothelium, by preventing the endothelial damage associated with hematopoietic cell transplantation (HCT) conditions, 5,6 and with the deleterious effect of immunosuppresants. 7 In our in vitro endothelial activation model, DF has exhibited reproducible effects on endothelial cells (ECs) from different origins. DF demonstrates antiinflammatory, antithrombotic, and antiapoptotic properties. However, although its effects are increasingly better understood, its precise mechanism of action remains to be elucidated.There is limited knowledge about DF pharmacokinetics, pharmacodynamics, and mechanisms of action. [8][9][10] However, 2 distinct properties of ...

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Section: Discussionmentioning

confidence: 99%

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

Palomo

Mir

Rovira

et al. 2016

Blood

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“…Insulin-induced vasodilation is known to be impaired whereby superoxide reacted with NO and eliminated the action of NO-induced vasodilation. [27][28][29] In some cases, NOS inhibition paradoxically promoted vasodilation, whereby excess amounts of superoxide caused the oxidation of NOS cofactors, tetrahydrobiopterin (BH 4 ). This previously reported observation may account for our finding that insulin decreased the NO formation in endothelial cells cultured under high glucose conditions.…”

Section: Insulin-induced Nitric Oxide In Retinal Microvesselsmentioning

confidence: 99%

NADPH Oxidase–Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature

Oku

Horie

Matsuo

et al. 2015

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. To determine whether insulin induces nitric oxide (NO) formation in retinal microvessels and to examine the effects of high glucose on the formation of NO. METHODS.Freshly isolated rat retinal microvessels were incubated in normal (5.5 mM) or high (20 mM) glucose with or without insulin (100 nM). The levels of insulin-induced NO and reactive oxygen species (ROS) in the retinal microvessels were determined semiquantitatively using fluorescent probes, 4,5-diaminofluorescein diacetate, and hydroethidine, respectively, and a laser scanning confocal microscope. The insulin-induced changes of NO in rat retinal endothelial cells and pericytes cultured at different glucose concentrations (5.5 and 25 mM) were determined using flow cytometry. Nitric oxide synthase (NOS) protein levels were determined by Western blot analysis; intracellular levels of ROS were determined using fluorescence-activated cell sorting (FACS) analysis of ethidium fluorescence; and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RNA expression was quantified using realtime PCR.RESULTS. Exposure of microvessels to insulin under normal glucose conditions led to a significant increase in NO levels; however, this increase was significantly suppressed when the microvessels were incubated under high glucose conditions. Intracellular levels of ROS were significantly increased in both retinal microvessels and cultured microvascular cells under high glucose conditions. The expression of NOS and NADPH oxidase were significantly increased in endothelial cells and pericytes under high glucose conditions. CONCLUSIONS. The increased formation of NO by insulin and its suppression by high glucose conditions suggests that ROS production mediated by NADPH oxidase is important by insulin's effect on the retinal microvasculature.Keywords: insulin, nitric oxide, ROS, high glucose, retinal microvessel N itric oxide (NO) is an endothelium-derived vasodilator, which plays a role in the autoregulation of ocular circulation. 1 Insulin stimulates the formation of NO and increases ocular blood flow. 2,3 Both insulin and NO are involved in the pathogenesis of diabetic retinopathy. Insulin treatment is related to the severity of diabetic retinopathy, [4][5][6] and care must be taken because insulin therapy often causes a temporary worsening of the diabetic retinopathy. Because increased levels of NO are associated with the breakdown of the blood-retinal barrier 7 and formation of diabetic macular edema, 8 the detrimental effects associated with the use of insulin in patients with diabetic retinopathy may be the result of changes in the bioavailability of insulin-induced NO.Hyperglycemia is the primary cause of macro-and microvascular complications in individuals with diabetes, but this is still a debated topic.9-11 Reactive oxygen species (ROS), including superoxides (O 2 À ), are produced endogenously by endothelial cells in response to cytokines, growth factors, and/ or shear stress. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the prima...

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“…Cell viability estimated by Trypan blue exclusion was higher than 97% (not shown). Sixteen hours prior, the experimental incubation medium was changed to sera-free M199 (González et al, 2015). …”

Section: Methodsmentioning

confidence: 99%

“…Overall L-arginine transport (2 μCi/ml L-[ 3 H]arginine (NEN, Dreieich, FRG), 0–250 μM L-arginine, 1 min, 37°C) was measured as previously described (González et al, 2015). Briefly, transport assays were performed in Krebs [in mM: 131 NaCl, 5.6 KCl, 25 NaHCO 3 , 1 NaH 2 PO 4 , 20 Hepes, 2.5 CaCl 2 , 1 MgCl 2 (pH 7.4, 37°C)] in cells preincubated (12 h) with M199 in the absence (control) or presence (1–30 min) of insulin (1 nM).…”

Section: Methodsmentioning

confidence: 99%

“…Specifically, hydrogen peroxide (H 2 O 2 ) and peroxynitrite (ONOO − ) induce rapid and transient contraction or relaxation in human placental chorionic plate arteries (Mills et al, 2009). In addition, H 2 O 2 and ONOO − have higher stability than their respective precursors (Beckman and Koppenol, 1996), and may cause deleterious effects in vascular beds when insufficiently buffered/neutralized (González et al, 2011, 2015). In this regard, several reports have shown that ROS reduces NO availability and vascular relaxation in human placenta vasculature, during either healthy or pathological conditions such as preeclampsia (PE; Bernardi et al, 2008; Catarino et al, 2012), intrauterine growth restriction (IUGR; Takagi et al, 2004), and gestational diabetes mellitus (GDM; Coughlan et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Insulin Induces Relaxation and Decreases Hydrogen Peroxide-Induced Vasoconstriction in Human Placental Vascular Bed in a Mechanism Mediated by Calcium-Activated Potassium Channels and L-Arginine/Nitric Oxide Pathways

et al. 2016

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HIGHLIGHTS Short-term incubation with insulin increases the L-arginine transport in HUVECs.Short-term incubation with insulin increases the NO synthesis in HUVECs.Insulin induces relaxation in human placental vascular bed.Insulin attenuates the constriction induced by hydrogen peroxide in human placenta.The relaxation induced by insulin is dependent on BKCa channels activity in human placenta. Insulin induces relaxation in umbilical veins, increasing the expression of human amino acid transporter 1 (hCAT-1) and nitric oxide synthesis (NO) in human umbilical vein endothelial cells (HUVECs). Short-term effects of insulin on vasculature have been reported in healthy subjects and cell cultures; however, its mechanisms remain unknown. The aim of this study was to characterize the effect of acute incubation with insulin on the regulation of vascular tone of placental vasculature. HUVECs and chorionic vein rings were isolated from normal pregnancies. The effect of insulin on NO synthesis, L-arginine transport, and hCAT-1 abundance was measured in HUVECs. Isometric tension induced by U46619 (thromboxane A2 analog) or hydrogen peroxide (H2O2) were measured in vessels previously incubated 30 min with insulin and/or the following pharmacological inhibitors: tetraethylammonium (KCa channels), iberiotoxin (BKCa channels), genistein (tyrosine kinases), and wortmannin (phosphatidylinositol 3-kinase). Insulin increases L-arginine transport and NO synthesis in HUVECs. In the placenta, this hormone caused relaxation of the chorionic vein, and reduced perfusion pressure in placental cotyledons. In vessels pre-incubated with insulin, the constriction evoked by H2O2 and U46619 was attenuated and the effect on H2O2-induced constriction was blocked with tetraethylammonium and iberiotoxin, but not with genistein, or wortmannin. Insulin rapidly dilates the placental vasculature through a mechanism involving activity of BKCa channels and L-arginine/NO pathway in endothelial cells. This phenomenon is related to quick increases of hCAT-1 abundance and higher capacity of endothelial cells to take up L-arginine and generate NO.

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Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

Cited by 52 publications

References 44 publications

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

NADPH Oxidase–Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature

Insulin Induces Relaxation and Decreases Hydrogen Peroxide-Induced Vasoconstriction in Human Placental Vascular Bed in a Mechanism Mediated by Calcium-Activated Potassium Channels and L-Arginine/Nitric Oxide Pathways

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