R E S E A R C H A R T I C L E
Liraglutide mitigates TNF
‐α induced pro‐atherogenic changes
and microvesicle release in HUVEC from diabetic women
Pamela Di Tomo
1,2,3|
Paola Lanuti
1,2,3|
Natalia Di Pietro
2,3|
Maria Pompea Antonia Baldassarre
1,2,3|
Marco Marchisio
1,2,3|
Assunta Pandolfi
2,3|
Agostino Consoli
1,2,3|
Gloria Formoso
1,2,31
Department of Medicine and Aging Sciences, G. d'Annunzio University, Chieti, Italy
2
Department of Medical, Oral, and Biotechnological Sciences,“G. d'Annunzio” University, Chieti, Italy
3
Aging and Translational Medicine Research Center, CeSI‐Met, “G. d'Annunzio” University, Chieti, Italy
Correspondence
Gloria Formoso, Department of Medicine and Aging Sciences, Edificio CeSi‐Met, Room 271, G. d'Annunzio University, Via Polacchi, 13, 66100 Chieti, Italy.
Email: [email protected] Funding information
Ministry of University and Research Govern-ment grant, Grant/Award Number: 20123BJ89E_003; research grant from Novo Nordisk, Grant/Award Number: NA
Abstract
Background:
To evaluate whether exposure to GLP‐1 receptor agonist Liraglutide could modulate pro‐atherogenic alterations previously observed in endothelial cells obtained by women affected by gestational diabetes (GD), thus exposed in vivo to hyperglycemia, oxidative stress, and inflammation and to evaluate endothelial microvesicle (EMV) release, a new reliable bio-marker of vascular stress/damage.Methods:
We studied Liraglutide effects and its plausible molecular mechanisms on monocyte cell adhesion and adhesion molecule expression and membrane exposure in control (C‐) human umbilical vein endothelial cells (HUVEC) as well as in HUVEC of women affected by GD exposed in vitro to TNF‐α. In the same model, we also investigated Liraglutide effects on EMV release.Results:
In response to TNF‐α, endothelial monocyte adhesion and VCAM‐1 and ICAM‐1 expression and exposure on plasma membrane was greater in GD‐HUVEC than C‐HUVEC. This was the case also for EMV release. In GD‐HUVEC, Liraglutide exposure significantly reduced TNF‐α induced endothelial monocyte adhesion as well as VCAM‐1 and ICAM‐1 expression and exposure on plasma membrane. In the same cells, Liraglutide exposure also reduced MAPK/NF‐kB activation, peroxynitrite levels, and EMV release.Conclusions:
TNF‐α induced pro‐atherogenic alterations are amplified in endothelial cells chronically exposed to hyperglycemia in vivo. Liraglutide mitigates TNF‐α effects and reduces cell stress/damage indicators, such as endothelial microvesicle (EMV) release. These results foster the notion that Liraglutide could exert a protective effect against hyperglycemia and inflammation triggered endothelial dysfunction.K E Y W O R D S
diabetes mellitus, endothelial dysfunction, GLP‐1 receptor agonists
Abbreviations: AMPK, 5‐AMP‐activated protein kinase; BSA, bovine serum albumin; CD, cluster domain; C‐HUVEC, control umbilical vein endothelial cell; CREB, cAMP response element binding protein; EMV, endothelium derived microvesicles; FACS, fluorescent‐activated cell sorting; FITC, fluorescein isothiocyanate; FMO, fluorescence minus one; FSC, forward scatter; GD‐HUVEC, umbilical vein endothelial cells obtained by women with gestational diabetes; GLP‐1, glucagone like peptide‐1; GLP‐1Rx, glucagone like peptide‐1 receptor; GW, gestational week; HUVEC, human umbilical vein endothelial cell; ICAM‐1, intercellular cell adhesion molecule‐1; LEADER, liraglutide effect and action in diabetes: evaluation of cardiovascular outcome results; MAPK, mitogen‐activated protein kinase; MFI, mean fluorescence intensity; MV, microvesicles; NF‐kB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; PAI‐1, plasminogen activator inhibitor‐1; PE, phycoerythrin; PE‐Cy7, R‐phycrythrin‐cyanine 7; PFC, polychromatic flow cytometry; PKA, protein kinase A; PMT, photomultiplier; SSC, side scatter; TNF‐α, tumour necrosis factor‐α; VCAM‐1, vascular cell adhesion molecule‐1
DOI: 10.1002/dmrr.2925
Diabetes Metab Res Rev. 2017;33:e2925. https://doi.org/10.1002/dmrr.2925
Copyright © 2017 John Wiley & Sons, Ltd.
1
|I N T R O D U C T I O N
When compared with non‐diabetic controls, type 2 diabetic (T2D) patients present a 2 to 4‐fold increase in the risk of developing cardio-vascular disease (CVD).1Both hyperglycemia and insulin resistance are
likely to play a role for this increased risk, by fostering inflammation and endothelial dysfunction, 2 processes strictly intertwined in the vascular wall.2
In the last 10 years, glucagon like peptide‐1 (GLP‐1) receptor (GLP‐1Rx) agonists have been introduced among the therapeutic options for type 2 diabetes, due to their ability to stimulate insulin secretion and suppress glucagon release in a glucose‐dependent man-ner.3 GLP
‐1 receptor agonists have also a host of extra‐pancreatic actions. As recently demonstrated by the Liraglutide Effect and Action in Diabetes: Evaluation of Cardiovascular Outcome Results (LEADER) study, use of the GLP‐1Rx agonist Liraglutide is associated reduced risk of cardiovascular events in high risk T2D patients,4and very similar
results have been obtained with the use of semaglutide.5However, the mechanism responsible for these results has not been completely clarified.
GLP‐1Rx protein has been detected in human coronary artery endothelial cells (HCAEC) as well as in human umbilical vein endothe-lial cells (HUVEC),6,7suggesting a possible direct GLP
‐1Rx agonists effects on the vessels wall. Indeed, GLP‐1Rx agonist's treatment has been shown to exert several potentially protective actions in preclini-cal models of cardiovascular dysfunction.8 Liraglutide seems to increase endothelial nitric oxide synthase phosphorylation and nitric oxide (NO) production by the 5‐AMP‐activated protein kinase (AMPK)‐dependent pathway in cultured HCAEC.9,10Moreover, studies
in cultured endothelial cells demonstrated that Liraglutide treatment prevents the increase in plasminogen activator inhibitor‐1 (PAI‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) protein levels following exposure to tumour necrosis factor‐α (TNF‐α) or high glucose.11
Human umbilical cord endothelial cells (HUVEC) obtained from mother affected by gestational diabetes (GD‐HUVEC) represent a valid cellular model of initial endothelial damage in diabetes. Recently, we reported that these cells, which were exposed in vivo, albeit tran-siently, to hyperglycemia, oxidative stress and inflammation, exhibit persisting pro‐atherogenic modifications in vitro.12Among such pro‐
atherogenic modifications, we observed that, upon exposure to TNF‐α, GD‐HUVEC presented increased leucocyte adhesion molecules (VCAM‐1 and intercellular adhesion molecule‐1) expression and expo-sure on the cell surface, and increased leukocytes adhesion to the endothelial surface.12 In this cellular model, we also observed enhanced release of microvesicles in the culture medium (Formoso et al, unpublished observations). Endothelium derived microvesicles (EMV) have been recently identified as new markers of vascular dam-age. EMV, as detected by surface antigens (vascular endothelial cadherin), which are endothelial cell‐type specific transmembrane adhesion molecules, derive selectively from human endothelial cells. Their plasma levels could thus be interpreted as a specific marker of endothelial dysfunction. Circulating levels of EMV might represent an index of cell activation and/or tissue degeneration occurring during pathophysiological events in vivo.13EMV levels indeed are increased in a wide range of athero‐thrombotic disorders, with an interesting
relationship between EMV levels and disease pathophysiology, activity, or progression.14-16
Based on this background, in the present study, we evaluated whether exposure to the GLP‐1 receptor agonist Liraglutide could modulate the pro‐atherogenic alterations we previously observed in endothelial cells exposed in vivo to hyperglycemia, oxidative stress, and inflammation. Specifically, we explored whether, in this cellular model, following exposure to TNF‐α, Liraglutide affects adhesion mol-ecules expression and membrane exposure, and, consequently, mono-cyte adhesion to the cultured cells; we also investigated the plausible molecular mechanisms involved.
Moreover, we investigated whether Liraglutide treatment results in reduced cell stress/damage following exposure to TNF‐α, as indi-cated by reduced EMV release in culture media.
2
|M A T E R I A L S A N D M E T H O D S
2.1
|Material
Dulbecco's modified eagle medium (DMEM, CAT. D6046), M199 endothelial growth medium (CAT. M4530), L‐glutamine (CAT. G7513), Penicillin‐Streptomycin (CAT. P4333), Phosphate Buffered Saline (PBS, CAT. D8662), Exendin Fragment 9‐39 (CAT. E7269), Col-lagenase type 1A (CAT. C9891), human TNF‐α (CAT. T0157), Phorbol Myristate Acetate (PMA, CAT. P1585), and Ionomycin (CAT. I0634) were purchased from Sigma‐Aldrich (Saint Louis, USA). Foetal bovine serum (FBS, CAT. 41A0045K) was purchased from Gibco ‐LifeTechnol-ogies (Monza, Italy), 0.05% trypsin/0.02% EDTA (CAT. L11‐003) from Mascia Brunelli (Milan, Italy), and tissue‐culture disposables from Eppendorf (Hamburg, Germany). Anti‐mouse monoclonal nitrotyrosine antibody was from Upstate Biotechnology Inc. (MA, USA). PE‐labelled anti‐VCAM‐1 (CAT. 305806) and FITC‐labelled anti‐ICAM‐1 (CAT. 313104) antibodies were from BioLegend (San Diego, CA, USA). p44/42 Mitogen‐Activated Protein Kinase (MAPK, CAT. 9102), Phospho‐p44/42 MAPK (Thr202/Tyr204, CAT. 9101), NF‐kB p65 (CAT. 4764), Protein Kinase A (PKA) C‐α (CAT. 4782), Phospho‐PKA‐ C (Thr197, CAT. 4781), cAMP Response Element‐Binding protein (CREB, CAT. 9197), and Phospho‐CREB (Ser133, CAT. 9198) primary antibodies were purchased from Cell Signalling (Danvers, MA, USA). HKGreen‐4A probe it was synthesized by Prof. Dan Yang's lab. See Table 1.
2.2
|Cell cultures and experimental protocols
Umbilical cords were obtained from randomly selected healthy Cau-casian mothers (Control, C) and mothers with GD delivering at the Hospital of Chieti and Pescara (Italy). All procedures were in agree-ment with the ethical standards of the Institutional Committee on Human Experimentation (Reference Number: 1879/09COET) and with the Declaration of Helsinki Principles. After approval of the protocol by the Institutional Review Board, signed informed consent was obtained from each participating subject. Donor characteristics are described in Table 1 of our previous manuscript by Di Fulvio et al.12Briefly: normotensive C and GD women, matched for age and body mass index (BMI), underwent a 100‐g 3‐hour oral glucosetolerance test (OGTT) during 24th to 28th gestational week (gw) according to guidelines. Moreover, each woman performed a 7‐ point/day blood glucose self‐monitoring on 3 different days during the week at 34th to 36th gw to compare fasting and post‐prandial (either 1 or 2 hours after meal) capillary glucose levels. Umbilical cords for HUVEC explants were collected immediately after deliv-ery, which occurred between the 36th and the 40th gw at Chieti and Pescara Hospitals.
After perfusion of umbilical vein cords with 1 mg/mL Collagenase 1A at 37°C, HUVEC were grown in normal glucose (5.5 mmol), 1.5% gelatin‐coated tissue culture plates in endothelial growth medium composed by DMEM/M199 (1:1) supplemented with 1% L‐glutamine, 1% penicillin‐streptomycin, 20% FBS, 10 μg/mL heparin, and 50 μg/mL endothelial cell growth factor. Primary C‐HUVEC and GD‐HUVEC cells were characterized as Von Willebrand factor positive and alpha‐ smooth muscle cell actin negative; for all experiments, cells were used between the third and fifth passage in vitro.
HUVEC were grown to sub‐confluence in complete growth medium, after which cells were serum‐starved and incubated for 16 hours with TNF‐α (10 ng/mL) following 24 hours' pre‐incubation with Liraglutide 100 nM/L (Novo Nordisk A/S Novo Allé, 2880 Bagsvaerd Denmark), in the presence or absence of the GLP‐1 receptor antagonist Exendin 9‐39 (100 nM/L). Exendin 9‐39 was added 30 minutes before Liraglutide stimulation. PKA and CREB phos-phorylation levels were evaluated after 30‐minute Liraglutide treat-ment in the presence or absence of the GLP‐1 receptor antagonist Exendin 9‐39.
2.3
|Preparation of U937 cells and adhesion assays
We evaluated U937 monocyte adhesion to C‐HUVEC and GD‐HUVEC using a cell adhesion assay, in the basal state, after 16 hours' TNF‐α (10 ng/mL) exposure and in the presence of Liraglutide. Cells were grown to confluence in 6‐well tissue culture plates, and U937 cell adhesion was evaluated as previously described.17 One millilitre ofU937 cell suspensions with 1 × 106cells was added to each HUVEC monolayer under rotating conditions (63 rev/min) at RT. After 20 minutes, non‐adhering cells were removed and monolayers fixed with 1% paraformaldehyde. Some monolayers were treated for 1 hour before the assay with mouse anti‐human monoclonal antibody against VCAM‐1 and ICAM‐1 (1 μg/1 × 106cells). The number of adherent
cells was assessed by counting 8 different high‐power field (3.5 mm2). Photographs were randomly chosen high
‐power fields taken at half‐radius distance from the center of the well in 1 of 3 com-parative experiments of similar design, showing U937 monocytoid cell adhesion to endothelial cells.
2.4
|Flow cytometry analysis
At the basal state and after stimulations, total protein levels (perme-abilized cells) and surface exposure (not perme(perme-abilized cell) on plasma membrane were evaluated by fluorescent‐activated cell sorting (FACS) Calibur or FACSCanto II (BD Biosciences, California, USA).
Non‐permeabilized cells were detached by EDTA 5 mM, washed, and resuspended in bovine serum albumin (BSA) 0.5%. Cells were pelleted by centrifugation at 800 rpm for 15 minutes and then incu-bated at the same time with anti‐VCAM‐1 PE‐conjugate (1:100) and with anti‐ICAM‐1 FITC‐conjugate (1:100), both for 30 minutes at room temperature. Permeabilized cells (using FACS Lysing and Permeabilizing Solution, BD, CAT. 349202 and 340973, respectively) were processed and incubated with p44/42 MAPK (1:30), Phospho‐ p44/42 MAPK (Thr202/Tyr204) (1:200), NF‐kB p65 (1:100), PKA C (1:100), Phospho‐PKA C (1:50), CREB (1:400), and Phospho‐ CREB (1:800) primary antibodies, and the secondary antibody FITC‐ conjugated (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used.
In addition, the intracellular levels of peroxynitrite (ONOO−−) were detected in C‐and GD‐HUVEC by using the HKGreen‐4A probe (10μM, 30 minutes at 37°C) synthesized by Prof. Dan Yang's lab.18 This probe is highly sensitive and selective toward peroxynitrite detec-tion in biological samples. As convenient positive control for endoge-nous peroxynitrite induction in C‐HUVEC and GD‐HUVEC were used PMA (200 ng/mL) and Ionomycin (50 nM) 30 minutes before the assay. All data were analysed using FACS Diva (BD Biosciences) and FlowJo v.8.8.6 software (TreeStar, Ashland, OR) and expressed as mean fluorescence intensity (MFI) ratio. The MFI ratio was calculated by dividing the MFI of positive events by the MFI of negative events (MFI of secondary antibody).
Imaging flow cytometry (ImageStream AMNIS by using IDEAS software, BD) was used to determine the levels of VCAM‐1 and ICAM‐1 membrane exposure and cytoplasm‐nucleus translocation of NF‐kB p65.
2.5
|MV staining for flow cytometry
At the basal state and after stimulations, the culture media of C‐HUVEC and GD‐HUVEC was collected. For each sample, 100 μL of supernatant was added to 200μL of PBS and processed by a com-mon flow cytometry no‐lyse and no‐wash method.19Briefly, samples were stained using a cocktail of reagents (only direct immunolabelling was performed, in order to avoid immune complex formation), as described in Table 1. After 30 minutes of staining (4°C in the dark), 500μL of PBS was added to each tube, and 1 × 106events/sample were acquired by flow cytometry (FACSVerse, BD—3 laser, 8 colour
TABLE 1 Polychromatic flow cytometry panel
Reagent Vendor Clone Catalogue number Dilution/amount per test
MitoTracker Green FM Life Technologies – M7514 1 nM
CD31 PECy7 BD Biosciences WM59 563651 5μL
R‐phycoerythrin‐cyanine 7 (PE‐Cy7). Cocktail of reagents used for EMV staining.
configuration, or FACSCanto II, BD—3 laser, 8 colour configuration). The threshold was placed on the fluorescein isothiocyanate (FITC) channel, while, in order to avoid MV loss, no threshold on morpholog-ical parameters was applied, as previously suggested.20Amplifier set-tings for forward scatter (FSC) and side scatter (SSC) as well as for any fluorescence channel were set in logarithmic mode. MV scatter properties were confirmed by running Megamix Plus—Side Scatter (SSC) beads (Biocytex, Marseille, France, CAT 7803) at the same photomultiplier (PMT) voltages used for MV detection. Each anti-body/reagent was titrated (8 point titration) under assay conditions; dilutions were established based on achieving the highest signal (mean fluorescence intensity, MFI) for the positive population and the lowest signal for the negative population, representing the optimal signal to noise ratio,21and stain indexes were calculated.22Because rare events were analysed, extensive rinsing was performed, to reduce “contami-nation” between samples.
Instrument performances, data reproducibility, and fluorescence cal-ibrations were sustained and checked by the Cytometer Setup & Track-ing Module and further validated by the acquisition of Spherotech 8 peak Rainbow Beads (BD, CAT. RCP‐30‐20A). To evaluate non‐specific fluorescence, fluorescence minus one (FMO) controls were used.23
Com-pensation was assessed using CompBeads (BD, CAT. 552843) and single
stained fluorescent MV. Data were analysed using FACSDiva v 6.1.3 (BD), FACSuite v 1.0.5 (BD) and FlowJo v 8.8.6 (TreeStar, Ashland, OR, USA) software. MV numbers were obtained by a single platform counting method (using Troucount tubes, BD, CAT. 340334).
2.6
|Statistical analysis
Results are presented as means ± standard deviation (SD) of more than 3 different experiments on 3 different C‐HUVEC and GD‐HUVEC explants.
A statistical analysis was performed by applying Student's t‐test and 1‐way analysis of variance (ANOVA) followed by Bonferroni mul-tiple comparison test for post hoc comparisons. P values lesser or equal to 0.05 were considered statistically significant.
3
|R E S U L T S
3.1
|Effect of Liraglutide on TNF
‐α stimulated U937
monocyte adhesion to C and GD
‐HUVEC
In order to study the potential vascular protective effects of Liraglutide treatment on endothelial alterations related to hyperglycemia and to a
FIGURE 1 Effect of Liraglutide on monocyte adhesion in C‐HUVEC and GD‐HUVECC‐HUVEC and GD‐HUVEC at basal state or after TNF‐α (10 ng/ mL) exposure in the absence or presence of Liraglutide 100 nM. Quantitative data (upper side) express the number of U937 cells adhering within a high‐power field (3.5 mm2). Each measurement is the mean ± SD of adhering cells from 3 experiments, each consisting of 8 counts per condition. Anti
‐ VCAM‐1 and anti‐ICAM‐1 antibodies at saturating concentrations (1 μg/1 × 106cells) were used as control. Representative images of U937 cell adhesion to C‐HUVEC and GD‐HUVECs (lower side). ANOVA test: P < 0.0001 in C‐HUVEC and GD‐HUVEC. Bonferroni multiple comparison test: *P < 0.05 vs Basal and **P < 0.05 vs TNF‐α in C‐HUVEC, ‡P < 0.05 vs Basal and #P < 0.05 vs TNF‐α in GD‐HUVEC. T‐Test: ΨP < 0.05 Liraglutide vs Basal GD‐HUVEC, †P < 0.0001 Basal GD‐HUVEC vs Basal C‐HUVEC, §P < 0.00002 TNF‐α GD‐HUVEC vs TNF‐α C‐HUVEC.
pro‐inflammatory environment, we examined monocyte (U937) adhe-sion to C‐HUVEC and GD‐HUVEC, in the basal state and after TNF‐α stimulation with or without Liraglutide pre‐incubation.
As shown in Figure 1 and as previously observed (3), already in the unstimulated state GD‐HUVEC presented a significant increase in monocyte adhesion as compared with control cells; after TNF‐α stim-ulation, the number of U937 cells adherent to the HUVEC monolayer dramatically increased in both cellular models; this effect was however significantly more prominent in GD‐HUVEC than in C‐HUVEC. Inter-estingly, treatment with Liraglutide significantly reduced monocyte adhesion to GD‐HUVEC in the basal state. Pre‐treatment with Liraglutide also significantly reduced monocyte adhesion after TNF‐α stimulation in both C‐HUVEC and GD‐HUVEC.
Treating cells with anti‐VCAM‐1 or anti‐ICAM‐1 antibodies at sat-urating concentrations suppressed U937 adhesion to both C‐HUVEC and GD‐HUVEC, thus suggesting that adhesion molecule hyper‐ expression on the cell surface was crucial for increased U937 adhesion to HUVEC.
3.2
|Effect of Liraglutide on TNF
‐α increased
adhesion molecule protein expression and membrane
exposure in C
‐HUVEC and GD‐HUVEC
To confirm the role of adhesion molecules in driving the results of our previous experiment, we investigated whether expression and plasma
FIGURE 2 Effect of Liraglutide on TNF‐α induced VCAM‐1 and ICAM‐1 in C‐HUVEC and GD‐HUVECC‐HUVEC and GD‐HUVEC at basal state or after TNF‐α (10 ng/mL) exposure in the absence or presence of Liraglutide 100 nM. (A) and (B): VCAM‐1 and ICAM‐1 total cell protein expression. (C) and (D) upper side: VCAM‐1 and ICAM‐1 membrane exposure. (C) and (D) lower side: representative single cell images of VCAM‐1 and ICAM‐1 membrane exposure. Quantitative data are results from 4 different experiments expressed as mean fluorescence intensity (MFI) ratio (signal to noise ratio). ANOVA test: in (A) P = 0.0053 in C‐HUVEC and P < 0.0001 in GD‐HUVEC, in (B) P = 0.0047 in C‐HUVEC and P < 0.0001 in GD‐HUVEC in (C) P = 0.0124 in C‐HUVEC and P < 0.0001 in GD‐HUVEC, in (D) P = 0.017 in C‐HUVEC and P = 0.0012 in GD‐HUVEC.Bonferroni multiple comparison test: *P < 0.05 vs Basal C‐HUVEC, **P < 0.05 vs Basal and #P < 0.05 vs TNF‐α in GD‐HUVEC. T‐Test: ‡P < 0.001 and §P < 0.01 TNF‐α GD‐HUVEC vs TNF‐α C‐HUVEC.
membrane exposure of VCAM‐1 and ICAM‐1 were affected by Liraglutide on cells exposed to inflammatory stimuli.
VCAM‐1 and ICAM‐1 protein expression was relatively low in the basal state and significantly increased after TNF‐α exposure. As shown in Figure 2, in TNF‐α stimulated GD‐HUVEC Liraglutide pre‐incubation significantly decreased VCAM‐1 (panel A) and ICAM‐1 (panel B) pro-tein expression, while in control cells, this effect was less pronounced. Consistent with our previously published data (3), when TNF‐α was added to culture media VCAM‐1 and ICAM‐1 exposure on plasma membrane increased. This increase was significantly greater in GD‐ HUVEC as compared with C‐HUVEC as shown in Figure 2, panel C and D.
Pre‐incubation of C‐HUVEC with Liraglutide induced a trend toward reduction in TNF‐α stimulated VCAM‐1 and ICAM‐1 mem-brane exposure. On the other hand, Liraglutide significantly reduced both VCAM‐1 (panel C) and ICAM‐1 (panel D) membrane exposure in GD‐HUVEC.
3.3
|Effect of Liraglutide on TNF
‐α induced
inflammation signalling molecules
Because adhesion molecule expression is at least partially controlled by MAPK and NF‐kB signalling pathways, we then evaluated expres-sion and phosphorylation of MAPK42/44 and NF‐kB p65 nuclear translocation in C‐HUVEC and GD‐HUVEC.
Both in the basal state and after TNF‐α stimulation, MAPK42/44 as well as phospo‐MAPK42/44 were significantly higher in GD‐ HUVEC as compared with C‐HUVEC, as shown in Figure 3, panel A and B. In TNF‐α stimulated cells, Liraglutide treatment reduced phospo‐MAPK42/44 protein level, although this reduction reached statistical significance only in GD‐HUVEC (Figure 3B). In addition, NF‐kB p65 cytoplasm‐nucleus translocation was inhibited by Liraglutide in TNF‐α stimulated in both C and GD‐HUVEC (panel C and D).
We then evaluated the intracellular nitro oxidative stress levels in our cellular models. Peroxynitrite levels were significantly higher in GD‐HUVEC as compared with C‐HUVEC both in the basal state and after TNF‐α incubation. Pretreatment with Liraglutide significantly decreased peroxynitrite levels in TNF‐α stimulated GD‐HUVEC; a trend toward reduction was also observed in control cells (Figure 4).
3.4
|Role of the GLP
‐1 receptor on Liraglutide
endothelial effects
We then investigated the involvement of the GLP‐1‐Rx intracellular signalling cascade on the Liraglutide vascular protective effects by evaluating PKA and its downstream target CREB activation.
In both C‐HUVEC and GD‐HUVEC, Liraglutide significantly increased PKA and CREB phosphorylation (indicating protein activa-tion), and this effect was reversed by the GLP‐1 Rx antagonist Exendine 9‐39 (Figure 5, panel A and B).
In the presence of the GLP‐1‐Rx antagonist Exendin 9‐39, the effect of Liraglutide in reducing VCAM‐1 and ICAM‐1 exposure on cell membrane persisted (Figure 5, panel C and D), suggesting that its
protective effect on adhesion molecules exposure and on leucocytes adhesion might involve pathways independent of the GLP‐1‐Rx.
3.5
|Effect of Liraglutide on endothelial
microvesicles release in culture media
To characterize Liraglutide protective effect against inflammatory stimuli in our cellular model, we determined Liraglutide effects on endothelial microvesicles (EMV) release in the culture medium.
MV were identified as small particles characterized by mitochon-drial activity, as we have previously proposed.20 As shown in
Figure 6 panel A1, MV were gated on a SSC‐Area (FSC)/MitoTracker GreenFMdot plot, as events staining positive for MitotrackerGreen.
Such MV population falls within the 0.3 to 0.9‐μm size range (panel A2), as confirmed by running Megamix beads (Biocytex) at the same PMT voltages (not shown). Finally, the surface expression of CD31 of such MV confirmed their endothelial origin (panel A3).
As shown in Figure 6 panel B, no difference in EMV release was observed between C‐HUVEC and GD‐HUVEC in the basal state. After TNF‐α exposure EMV release significantly increases in both cells lines; however, EMV level in culture media was significantly higher in GD‐ HUVEC. Liraglutide pre‐incubation induced a reduction in EMV release in both cells lines; however, this effect was statistically significant only in GD‐HUVEC. Addition of the GLP‐1 Rx antagonist Exendine9‐39 to the culture medium reversed the Liraglutide effect, indicating that acti-vation of the GLP‐1‐Rx might be involved in the molecular mechanisms determining EMV release.
4
|D I S C U S S I O N
Because vascular complications are the main cause of morbidity and mortality in diabetic patients,24in treating diabetes, a pharmacological intervention capable not only to improve metabolic control but also to target mechanisms underlying endothelial dysfunction would repre-sent a remarkable advantage.
Epidemiological and clinical studies have suggested that incretin‐ based therapies might exert a protective action on the vessels.25 In
particular, GLP‐1 Rx agonists exhibited anti‐oxidant, anti‐inflamma-tory, and anti‐atherogenic activities in several clinical, animal, and in vitro models.25GLP‐1 and GLP‐1 Rx agonists have been reported to induce reduction of several inflammatory mediators in parallel with suppression of NF‐κB signalling and MMP‐9 activity, in both ex vivo and in vivo studies in type 2 diabetes.26,27
Recently, large clinical trials have demonstrated that exposure to GLP‐1 receptor agonists such as Liraglutide4or Semaglutide5is
associ-ated with a reduced risk of occurrence of cardiovascular events in pop-ulations of T2D subjects at very high cardiovascular risk.
The results of the present study unveil the effects of Liraglutide on cultured endothelial cells which might be related to a potential direct protective cardiovascular effect of this molecule. We investigated, in a cellular model of diabetes, the effect of the GLP‐1Rx agonist Liraglutide on the early events characterizing the pathogenesis of dia-betes cardiovascular complications. To investigate such effects, we
choose as a cellular model HUVEC obtained from umbilical cords of women affected by gestational diabetes (GDM).12
GDM and T2DM share common pathophysiological mechanisms, such as increased insulin resistance, hyperglycemia, chronic
inflammation, and oxidative stress. All of these mechanisms impact negatively on endothelial function and lead to atherosclerotic plaque formation and CVDs.28,29The model we used thus represents a useful cellular model of early vascular damage: indeed, HUVEC obtained from
FIGURE 3 Effect of Liraglutide on TNF‐α induced inflammation pathways in C‐HUVEC and GD HUVECC‐HUVEC and GD‐HUVEC at basal state or after TNF‐α (10 ng/mL) exposure in the absence or presence of Liraglutide 100 nM. (A) Total p42/44 MAPK expression and (B) p42/44 MAPK phosphorylation levels. (C) Representative single cell images of NF‐kB p65 cytoplasm‐nucleus translocation. Nuclei stained in red and NF‐kB p65 stained in green. (D) Nuclear NF‐kB levels. Data are expressed as mean fluorescence intensity (MFI) ratio (signal to noise ratio) of 3 or 4 different experiments.ANOVA test: in (A) P = 0.29 in C‐HUVEC and P = 0.397 in GD‐HUVEC, in (B) P = 0.86 in C‐HUVEC and P = 0.007 in GD‐HUVEC, in (D) P = 0.01 in C‐HUVEC and P = 0.012 in GD‐HUVEC.Bonferroni multiple comparison test: §P < 0.05 vs TNF‐α C‐HUVEC, ΨP < 0.05 vs Basal and #P < 0.05 vs TNF‐α in GD‐HUVEC. T‐Test: *P < 0.002 and **P < 0.005 Basal GD‐HUVEC vs Basal C‐HUVEC, †P < 0.002 and ‡P < 0.005 TNF‐α GD‐ HUVEC vs TNF‐α C‐HUVEC.
umbilical cords of women affected by GD exhibit and maintain in cul-ture a pro‐atherogenic phenotype.12
We observed that exposure to Liraglutide reduces monocyte adhesion to GD‐HUVEC both in the basal state and after TNF‐α stim-ulation. This finding is consistent with the observations by Krasner et al, who demonstrated suppressed THP‐1 monocyte adhesion in human aortic endothelial cells exposed to an inflammatory insult, such as TNF‐α, after Liraglutide treatment.30The mitigation of the pro ‐ath-erosclerotic changes we observed in vitro is in keeping with the obser-vations in vivo by Nagashima et al, who demonstrated reduced foam cell formation and atherosclerotic lesion development following GLP‐ 1 infusion in apolipoprotein E‐deficient mice.31Our observations are also consistent with data by Gaspari et al who observed inhibited pro-gression of early onset, low burden atherosclerotic disease following Liraglutide treatment in apolipoprotein E‐deficient mice.32
The protective effect that Liraglutide appears to exert might be related to downregulation of several pro‐inflammatory factors and cell adhesion molecules; this effects appears to be more evident in GD‐ HUVEC where the molecular mechanism determining inflammation and oxidative stress is more pronounced. Indeed, we show that Liraglutide reduces the TNF‐α triggered vascular adhesion molecule expression, and it does so more in GD‐HUVEC than in C‐HUVEC. Hattori and Liu obtained similar results in a different experimental model: according to their observations, Liraglutide exposure inhibited VCAM‐1 and ICAM‐1 expression as well as mRNA levels in control endothelial cells exposed to high glucose and/or TNF‐α.11
We also demonstrated that Liraglutide treatment significantly reduces VCAM‐1 and ICAM‐1 exposure on plasma membrane. This find-ing confers to our results a functional meanfind-ing, further reinforced by the results of the monocyte adhesion assay, showing decreased monocyte adhesion to the endothelial layer following Liraglutide exposure.
We hypothesized that Liraglutide modulates cellular adhesion molecules probably by reducing inflammation.33Thus, we investigated MAPK and NF‐kB activation in our cellular model, and we observed that Liraglutide significantly downregulates MAPK activation, confirming, as related to the latter, previous observations in different models.33,34
Sixteen hours after TNF‐α stimulation, no change in phosphory-lated MAPK levels relative to baseline was detectable neither in the control nor in the GD‐HUVEC. It must be considered, however, that whether, in endothelial cell, TNF‐α acutely stimulates MAPK phos-phorylation, this effect is hardly detectable with chronic stimula-tion.35,36The effects of TNF‐α stimulation on the activated MAPK dependent pathways, instead, do persist for hours. Indeed, as a conse-quence, an increase in endothelial leukocytes adhesion molecules expression occurs, which favours monocytes diapedesis within the vessel and which can be observed from 6 up to 16 hours after stimulation.12,37
It is true that, as compared with controls, HUVECs obtained from cords of women affected by GDM present increased levels of total and phosphorylated MAPK in the basal state. It is however entirely possi-ble (and indeed very likely) that right after TNF‐α stimulation an increase in MAPK activation occurred which is not anymore detectable at the time of our observation. This could still explain the more pro-nounced effect of TNF‐α on VCAM‐1 and ICAM‐1 total protein levels and membrane exposure as observed in GD‐HUVEC 16 hours after stimulation.
Similarly to MAPK, NF‐kB activation shows a peak 30 to 60 minutes after TNF‐α exposure; then, it progressively declines while the effects of its activation on adhesion molecules expression can be observed a few hours later.38
It should however be considered that MAPK and NF‐kB activation might be just a part of the story. Diabetes‐induced endothelial dys-function is characterized by increased oxidative stress and reduced NO bioavailability. In GD‐HUVEC, NO bioavailability is reduced; thus, reduced NO bioavailability together with increased MAPK and inflam-matory pathway activation (eg, NF‐kB) might contribute to the increased adhesion molecule expression and exposure and increased leucocyte adhesion observed in diabetes vessels.
It has been observed that Liraglutide exerts favourable effects on NO production and bioavailability34 as well as on oxidative stress reduction as shown by Shiraki A.10,33and in our study (Figure 4). Thus,
it is plausible that the modulation of the adhesion molecule expression and exposure we observed in Liraglutide‐treated GD‐HUVEC may be determined by a synergic actions of Liraglutide on most of the hall-marks of endothelial dysfunction, such as modulation of MAPK and NF‐kB signalling pathways, impaired NO production and/or bioavail-ability, and oxidative stress.
In our study, GD‐HUVEC exposed to Liraglutide exhibited less peroxynitrite levels as compared with untreated GD‐HUVEC indicating that vascular protective effects of GLP1Rx agonists might be partially mediated by their anti‐oxidant properties.
In our cell model, the GLP‐1 Rx antagonist Exendin 9‐39 was not able to block Liraglutide induced modulation of adhesion molecules; this suggests, consisting with previous observations, that other path-ways, independent of the classical GLP‐1 Rx, may be involved.32,33
FIGURE 4 Effect of Liraglutide on TNF‐α induced oxidative stress in C‐HUVEC and GD‐HUVECC‐HUVEC and GD‐HUVEC at basal state or after TNF‐α (10 ng/mL) exposure in absence or presence of Liraglutide 100 nM. Total peroxynitrite levels. Data are expressed as mean fluorescence intensity (MFI) ratio (signal to noise ratio) of 4 different experiments. Phorbol myristate acetate (PMA, 200 ng/mL) and Ionomycin (IONO, 50 nM) are used as positive control. ANOVA test: P = 0.05 in C‐HUVEC, P = 0.0008 in GD‐HUVEC. Bonferroni multiple comparison test:‡P < 0.05 vs Basal in C‐HUVEC and GD‐ HUVEC, **P < 0.05 vs TNF‐α GD‐HUVEC. T‐Test: *P < 0.001 Basal GD‐ HUVEC vs Basal C‐HUVEC, #P = 0.0006 TNF‐α GD‐HUVEC vs TNF‐α C‐HUVEC.
Endothelial microvesicles (EMV) have the potential to act as bio-markers of CVD status. Microvesicles (MV) are anucleoid submicron fragments of plasma membranes made up of oxidized phospholipids
and specific proteins representing the cells they originate from. EMV are released into the circulation following endothelial cell activation or apoptosis.39,40
FIGURE 5 Role of the GLP‐1 receptor on Liraglutide vascular effectsC‐HUVEC and GD‐HUVEC after TNF‐α (10 ng/mL) stimulation in absence or presence of Liraglutide (100 nM) with or without Exendin 9‐39 inhibitor (100 nM) preincubation. (A) and (B): PKA and CREB phosphorylation levels. Quantitative data are results from 3 different experiments expressed as Fold Increase (FI) of positive cells, versus TNF‐α condition. (C) and (D) upper side: VCAM‐1 and ICAM‐1 membrane exposure. Quantitative data result from 3 different experiments expressed as fold increase (FI) of mean fluorescence intensity (MFI) ratio (signal to noise ratio), versus TNF‐α condition. (C) and (D) lower side: representative single cell images of VCAM‐1 and ICAM‐1 membrane exposure. ANOVA test: in (A) P = 0.0054 in C‐HUVEC and P = 0.0028 in GD‐HUVEC, in (B) P = 0.0001 in C‐HUVEC and P = 0.0027 in GD‐HUVEC, in (C) P = 0.37 in C‐HUVEC and P = 0.026 in GD‐HUVEC, in (D) P = 0.92 in C‐HUVEC and P = 0.042 in GD‐HUVEC. Bonferroni multiple comparison test: *P < 0.05 vs TNF‐α and #P < 0.05 vs TNF‐α + Liraglutide in C‐HUVEC, **P < 0.05 vs TNF‐α and §P < 0.05 vs TNF‐α + Liraglutide in GD‐HUVEC.T‐Test: †P < 0.005 and ‡P < 0.01 TNF‐α GD‐HUVEC vs TNF‐α C‐HUVEC.
Recent studies demonstrate significantly elevated EMV levels in patients who are symptomatic for carotid artery disease,41coronary artery disease,42,43stroke,44,45or diabetes20as compared with age
‐ matched controls. Moreover, elevated EMV are associated with coro-nary endothelial dysfunction46 and correlate with increased risk of
major cardiovascular events as well.42,43
Our paper provides the first evidence that, after TNF‐α exposure, EMV release is significantly higher in GD‐HUVEC culture media as compared with control cells, thus suggesting that EMV could represent a reliable biomarker of endothelial dysfunction. EMV release in culture media was significantly reduced by Liraglutide pre‐incubation, provid-ing further evidence for the protective Liraglutide effects against endothelial cell stress/damage.
Flow cytometry is used to detect MV, by means of antibody ‐con-jugated fluorophores47; this technique allows an accurate MV
quantification.48No single marker for endothelial MV exists, as many
antigens are also present on a number of different cell sub‐sets; there-fore, a panel of markers needs to be used for accurate identification. Unfortunately, consensus guidelines on EMV identification have not been reached so far. However, we developed an optimized PFC proto-col (without centrifugation, thus preserving MV structure) allowing to specifically distinguishing MV from other non‐MV particles and used it in the present study for EMV count. We believe that our results indi-cate specific and highly reproducible MV enumeration.
The effect of Liraglutide on EMV release seems to be, at least par-tially, dependent on GLP‐1 Rx activation. This is not surprising, because EMV represent a biological marker of endothelial damage potentially linked to activation of a host of intracellular path-ways,16,49,50 some of which Liraglutide likely modulates through GLP‐1 Rx activation.
FIGURE 6 Effect of Liraglutide on TNF‐α induced microvesicles release in C‐HUVEC and GD‐HUVEC(A) Gain strategy. (A1) EMV were gated on the basis of their Mitotracker positivity and their typical SSC features in a SSC‐A versus MitoTracker Green FM dot plot. (A2) Megamix Plus beads were run at the same photomultiplier (PMT) voltages of the sample; a gate including 0.2 and 0.5‐μm Megamix Plus beads was drawn on a FSC‐A/ SSC‐A dot plot and applied to the samples. (A3) CD31 fluorescence of EMV (red) and its respective control (blue). (B) C‐HUVEC and GD‐HUVEC at basal state or after TNF‐α (10 ng/mL) exposure in the absence or presence of Liraglutide 100 nM. Data represent EMV number/mL of culture media expressed as fold increase (FI) vs basal condition. ANOVA test: P = 0.0045 in C‐HUVEC, P < 0.0001 in GD‐HUVEC.Bonferroni multiple comparison test: *P < 0.05 vs Basal C‐HUVEC, **P < 0.05 vs Basal GD‐HUVEC, #P < 0.05 vs TNF‐α GD‐HUVEC, ΨP < 0.05 vs TNF‐α + Liraglutide GD‐HUVEC.T‐Test: †P < 0.002 TNF‐α GD‐HUVEC vs TNF‐α C‐HUVEC.
Thus, according to our observation, Liraglutide vascular protective actions appear to be mediated by both GLP‐1Rx dependent (EMV release) and independent pathways (adhesion molecules modulation). The link between EMV and the alterations of the pro‐inflammatory endothelium phenotype is very interesting as well. However, further and more focused studies are needed to more precisely define to which extent GLP‐1 Rx activation is involved in mediating Liraglutide vascular actions. It could also be intriguing to better characterize EMV phenotype released from a damaged/stress endothelium and the possible direct involvement of specific EMV cluster in determining endothelial dysfunction.
In conclusion, TNF‐α induced pro‐atherogenic alterations are amplified in endothelial cells chronically exposed to hyperglycemia in vivo. Liraglutide mitigates TNF‐α effects and reduces cell stress/ damage indicators, such as EMV release. These results reinforce the notion that Liraglutide could exert a protective effect against endothe-lial dysfunction and mitigate vascular damage induced by inflammatory cytokines and hyperglycemia.
A C K N O W L E D G E M E N T S
We gratefully acknowledge Prof. Dan Yang, University of Hong Kong, for kindly providing us the HKGreen‐4A probe for peroxynitrate detection.
S O U R C E A N D F U N D I N G
This study was partially supported by an unconditioned research grant from Novo Nordisk and by a Ministry of University and Research Gov-ernment grant to A.C. (PRIN 2012, n. 20123BJ89E_003).
C O N F L I C T O F I N T E R E S T
G.F. has received speaker fees from Eli Lilly, Merck Sharp and Dohme, and Servier.
A.C. has received speaker fees and/or consulting honoraria from Astra Zeneca, Boehringer Ingelhaim, Eli Lilly, Merck Sharp and Dhom, Novartis, Novo Nordisk, Roche Diagnostics, Sanofi‐Aventis, Takeda and has received research grant support from Eli Lilly and Novo Nordisk.
O R C I D
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How to cite this article: Di Tomo P, Lanuti P, Di Pietro N, et al. Liraglutide mitigates TNF‐α induced pro‐atherogenic changes and microvesicle release in HUVEC from diabetic women. Diabetes Metab Res Rev. 2017;33:e2925. https://doi.org/ 10.1002/dmrr.2925
