Role of leptin in atherothrombosis: focus on leukocyte derived microparticles

(1)

Università di Pisa

Programma di dottorato di ricerca in Fisiopatologia e Clinica

dell’Apparato Cardiovascolare e Respiratorio

Presidente prof. Alfredo Mussi

Tesi di dottorato

Role of leptin in atherothrombosis:

focus on leukocyte derived microparticles

SSD:MED/10

Tutor: Dr. Alessandro Celi Candidata: Dr.ssa Silvia Petrini

Revisori: Nigel Mackman PhD, FAHA John C. Parker Distinguished Professor of Medicine Director of the UNC McAllister Heart Institute, Co‐Director of the Thrombosis and Hemostasis Program Division of Hematology/Oncology, Department of Medicine University of North Carolina at Chapel Hill, USA Gregory YH Lip MD FRCP [Lond Edin Glas] FESC FACC Consultant Cardiologist and Professor of Cardiovascular Medicine Director, Haemostasis Thrombosis & Vascular Biology Unit, Birmingham, UK. Anno accademico: 2013/2014

(2)

Un ricercatore è qualcuno che cerca, non necessariamente qualcuno che trova. E non è necessariamente qualcuno che sa cosa stia cercando, è semplicemente qualcuno per cui la vita è una ricerca. Jorge Bucay

(3)

SUMMARY

Background: Microparticles are phospholipid vesicles shed by cells upon activation or during apoptosis. Microparticles are involved in numerous

physiological processes, including coagulation and inflammation. Leptin,

synthesized by adipose tissue, has been implicated in the regulation of

inflammation and the pathogenesis of thrombosis, and its concentration is

linked to the risk of cardiovascular events.

Aim: The aim of our study is to test the hypothesis that one of the mechanisms whereby leptin increases the risk of cardiovascular events is

linked to the induction of procoagulant microparticles by human

peripheral blood mononuclear cells and to investigate the intracellular

mechanisms leading to microparticles release upon incubation with leptin.

Methods: Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy donors. Cells were incubated with leptin.

Leptin‐stimulated cells were also pre‐incubated (30 minutes) with a

phospholipase C inhibitor, U73122; with verapamil, L‐type calcium channel

current blocker; with W‐7, a calmodulin inhibitor or with three different

inhibitors of mitogen activated protein kinases. Microparticle generation

was assessed as phosphatidylserine concentration by a prothrombinase

(4)

by cytofluorimentric analysis. Peripheral blood mononuclear cell‐derived

microparticles were discriminated first by size, as events conforming to a

light scatter distribution within the 0.5‐0.9μm bead range in a side scatter

channel vs. forward scatter channel window and further identified as

CD14 and annexin V positive events after incubation with

fluoresceinisothyocianate‐annexin V and allophycocyanin‐anti CD14

antibody, in anallophycocyanin vs. fluoresceinisothyocianate window.

Tissue factor expression on microparticles was measured with a one‐stage

clotting assay. Intracellular calcium concentration was assessed by a

fluorescent probe.

Results: Leptin significantly stimulates the generation of tissue factor‐ bearing MP by peripheral blood PBMC, as assessed by phosphatidylserine

quantification and clotting tests. These results are confirmed by

cytofluorimetric analysis. U73122, PD98059 (an extracellular signal‐

regulated kinase1/2 inhibitor), and verapamil, significantly inhibit leptin‐

induced MP release. SP600125 (a p38 inhibitor), SB203580 (a c‐JunN‐

terminal kinase inhibitor), and W‐7, have no effect.

Conclusions: Leptin induces the release of tissue factor bearing microparticles with a procoagulant potential by peripheral blood

(5)

type calcium channels and extracellular signal‐regulated kinase1/2

activation. These data are consistent with a role of leptin‐induced

procoagulant microparticles shed by peripheral blood mononuclear cells

in vascular diseases linked to obesity.

Key words: leptin, microparticles, tissue factor, bl

ood coagulation,

cardiovascular

risk factors, obesity

Abbreviations: tumor necrosis factor (TNF), interleukin (IL), tissue factor (TF), microparticles (MP), phosphatidylserine (PS), peripheral blood

mononuclear cells (PBMC), fetal bovine serum (FBS),phosphate buffered

saline (PBS), calcium ionophore (A23187), lypopolysaccharide (LPS),

fluoresceinisothyocianate (FITC), allophycocyanin (APC), side scatter

channel (SSC) and forward scatter channel (FSC), immunoglobulin (Ig),

relative fluorescence units (RFU), arbitrary units (AU), phpspholipase C

(PLC), verapamil (Ver), mitogen activated protein kinases (MAPK),

extracellular signal‐regulated kinase (ERK), c‐JunN‐terminal kinase (JNK),

peroxisome proliferator‐activated receptor (PPAR), phosphatidylinositol

(6)

INTRODUCTION

Obesity is an established risk factor for vascular disorders such as

hypertension and coronary artery disease1,2. Obesity is characterized by

excessive body fat due to an abnormal accumulation of adipose tissue.

Adipose tissue is an active endocrine and paracrine organ that releases a

large number of peptides. In mammals, two different adipose tissues with

different functions are present: white adipose tissue and brown adipose

tissue. Brown adipose tissue is responsible for generating heat, is mainly

found in hibernating animals and in infants and only rarely detected in

adult humans; in contrast white adipose tissue, which was originally

considered to be a mere large but inert energy store, is metabolically

active and is the source of molecules such as hormones, cytokines and

chemokines3. These molecules influence not only body weight

homeostasis but also inflammation, coagulation, fibrinolysis, insulin

resistance, diabetes, and some forms of cancer4–6. The secretory products

of adipose tissue contribute to the elevated risks of cardiovascular disease

with mechanisms that are complex and only partially understood7. Obesity

is intimately linked to insulin resistance, type 2 diabetes, vascular

inflammation and atherothrombosis8.Dysregulation of the secretion of

(7)

plasminogen activator inhibitor‐1 by the adipocyte has been suggested to

have a pivotal role in enhanced inflammation, vascular damage and

subsequent thrombogenicity9. Leptin is a 16 kDa non‐glycosylated

polypeptide product of the ob gene and is mainly produced and secreted

by fat cells in proportion to fat mass to signal the repletion of body energy

stores to the hypothalamus10–14. Circulating leptin concentrations have

been reported to correlate closely with both the body mass index and the

total amount of body fat10–12. Initially, the effects of leptin were thought to

be only centrally mediated. However, leptin shares with other members of

the long‐chain helical cytokine family an extreme functional pleiotropy.

Although originally isolated in relation to a particular biological action,

many cytokines have subsequently been shown to be capable of

stimulating a variety of biological responses in a wide spectrum of cell

types. Based on an almost ubiquitous distribution of receptors, leptin has

been reported to play a role in a quite diverse range of physiological

functions12,15–17. Therefore, since its discovery, leptin has caused

upheavals not only in the fields of appetite and body mass control, but

also in the broader spheres of general endocrinology, metabolism,

reproduction, immunology, cardiovascular pathophysiology, respiratory

(8)

development18.Increased circulating levels of leptin are directly associated

with myocardial infarction and stroke and, more in general, with

cardiovascular events in humans19. The effect has been observed

independent of obesity status and traditional cardiovascular risk factors.

Also, leptin upregulates the production of pro‐inflammatory cytokines

such as TNF‐α, interleukin (IL)‐6 and IL‐12 and the tissue factor (TF) gene

expression in monocytes and macrophages3,20.

Microparticles (MP) are small membrane vesicles (0.1 – 1 μm) shed from

cells in response to activation, injury and/or apoptosis21. MP were first

described in 1967 when Wolf reported platelet membrane fragments in

human plasma22. For many years, MP have been considered inert cell

debris; however, increasing evidence has more recently suggested MP as

active biological mediators in diverse responses, including cell‐cell

communication23, immune modulation24, inflammation25,26and blood

coagulation27,28. MP circulate in the bloodstream of healthy individuals

and patients, and their number, cellular origin and composition can

change accordingly to the different diseases and different states of the

disease. The impact of these changes on their in vivo effects is still

unknown29. MP circulating in the bloodstream may originate from

(9)

and endothelial cells30. The mechanisms leading to the generation of MP

have not been fully elucidated; it is known that there are two different

cellular processes that can lead to the formation of MP: chemical and

physical cell activation induced by agonists or shear stress respectively,

through an increase in intracellular calcium concentration, and apoptosis

induced both by deprivation of growth factors or by apoptotic

inducers21,29,31. MP are composed of a phospholipid bilayer that exposes

transmembrane proteins and receptors, and encloses cytosolic

components such as enzymes, transcription factors, and mRNA derived

from their parent cells21. The loss of the asymmetrical distribution of

phospholipids on the cell membrane after stimulation, that leads to the

externalization of phosphatidylserine (PS), normally segregated in the

inner leaflet of resting cells, is a crucial step in the generation of MP32. PS

represents a catalytic phospholipid surface for the assembly of the

multimolecular complexes of the coagulation cascade (such as the

prothrombinase and the tenase complexes) that lead to thrombin

generation33. Thus, MP represent an ideal surface for the assembly of such

complexes. MP released from mononuclear cells can also harbor active TF,

the main initiator of the blood coagulation cascade in vivo34,35. The

(10)

been suggested that the so called blood‐borne TF is in fact TF bound to

MP27,36. Several studies have shown that circulating MP are increased in

most cardiovascular diseases and in patients with cardiovascular risk

factors37. In obese and overweight subjects, two pilot studies reported an

increased number of circulating MP38,39. In the latter study, the cellular

origin of MP was not identified. Finally, Morel and coworkers observed

that during weight loss leptin plasma levels decrease in parallel with

procoagulant platelet‐ and leukocyte‐derived MP40.

Although TF‐bearing MP can in principle be generated by several cell

types, monocytes are currently considered the most important cell

source27. Our working hypothesis is that the prothrombotic effect of leptin is linked to the induction of procoagulant MP by human peripheral blood mononuclear cells (PBMC).

(11)

MATERIALS AND METHODS

Reagents and kits

Leptin, U73122, SP600125, SB203580, W‐7, RPMI 1640 medium, penicillin,

streptomycin, L glutamine, fetal bovine serum (FBS), trypan blue,

phosphate buffered saline (PBS), Ficoll‐Hystopaque, dextran, calcium

ionophore (A23187), were obtained from Sigma (Milan, Italy). The

absence of lypopolysaccharide (LPS) in leptin powder was attested by

Sigma (Milan, Italy). Thromboplastin standard was obtained from

Beckman Coulter (Milano, Italy). Human anti‐TF antibody was obtained

from America Diagnostica (Instrumentation Laboratory, Milano, Italy).

PD98059 was obtained from Cayman Chemical (Ann Arbor, MI, USA). The

Zymuphen MP‐Activity kit was obtained from Hyphen BioMed (Neuville‐

sur‐Oise, France). The Fluo‐4 NW Calcium Assay kit was obtained from

Molecular Probes (Invitrogen, Milan, Italy). Annexin V‐

fluoresceinisothyocianate (FITC) was obtained from Alexis (Vinci

Biochemicals, Firenze, Italy). Allophycocyanin (APC)‐labeled mouse anti

human CD14 antibody was purchased from BD Pharmingen (San Jose, CA,

USA). Megamix™, a blend of monodisperse fluorescent beads of three

(12)

France). All other chemicals were obtained from the hospital pharmacy

and were of the best grade available.

PBMC isolation and culture

PBMC were isolated either from fresh buffy coats obtained from the local

blood bank or from the peripheral blood of normal volunteers as

described27. Briefly, a fresh buffy coat was mixed gently with an equal

volume of 2,5% Dextran T500, and left for 40 minutes for erythrocyte

sedimentation. 10 mL ok leukocyte‐rich supernatant was recovered and

layered over 5 mL of Ficoll‐Hystopaque and centrifuged for 30 minutes at

350 x g at 4°C. The PBMC‐rich ring was recovered and washed twice in

PBS. PBMC were then resuspended in RPMI/1% penicillin and

streptomycin/1% L‐glutamine and allowed to adhere for 30 minutes at

37°C on 96‐well plates (0.33x106 cells/well). Then the cells were washed

two times with pre‐warmed PBS and resuspended in RPMI/1% penicillin

and streptomycin/1% L‐glutamine/5%FBS and allowed o.n. at 37°C.

MP generation and purification

PBMC were washed two times with pre‐warmed PBS. For MP generation,

leptin or A23187 (12 µM) were added; after the time of incubation at 37°C

the supernatants were recovered, cleared by centrifugation at 14,000 x g

(13)

fragments that might have detached during the stimulation, and

immediately used for further experiments. In selected experiments, MP

(12 mL) were further purified by ultracentrifugation (100,000 x g for 2

hours, 4°C); the pellet was resuspended in 250 μL of normal saline and

used in a one‐stage clotting assay to measure TF‐dependent coagulation.

Measurement of MP

PS‐positive MP in each sample were detected using the Zymuphen MP‐

activity kit (Hyphen BioMed, Neuville‐sur‐Oise, France) according to the

manufacturer’s instructions and expressed as PS equivalents (nM PS).

Flow cytometry detection of MP

PBMC were treated as described above and the supernatant submitted to flow cytometry using a FACScanto™II flow cytometer (BD Biosciences, San Jose, CA, USA). A mixture of 30 μL of supernatant, 3 μL of APC conjugated anti CD14 antibody, 3 μL of FITC labeled annexin V and 30 μL of annexin V

2x binding buffer was incubated for 15 minutes at room temperature in

the dark. Immediately prior to flow cytometric acquisition, 400 μL of PBS

were added to each mixture. Events acquisition was obtained at high flow

rate and was stopped after 210 seconds. The side scatter channel (SSC)

and forward scatter channel (FSC) parameters were set at log scale.

(14)

conjugated mouse immunoglobulin (Ig)G. Monocyte derived MP were

discriminated first by size, as events conforming to a light scatter

distribution within the 0.5‐0.9 μm bead range in a SSC vs. FSC window,

according to Lacroix and coworkers41 and further identified as CD14 and

annexin V positive events in a APC vs. FITC window.

Measurement of intracellular calcium concentration

Molecular Probes Fluo‐4 NW Calcium Assay kit was used to measure the

changes in the intracellular calcium concentration ([Ca2+]i) of PBMC. Pre‐

washed PBMC on 96‐multiwell plate (0.33 x 106 cells/well) were loaded

with 100 μL of the dye loading solution containing Fluo‐4 NW dye and

probenecid, according to the manufacturer’s instructions. The 96‐well

plate was incubated at 37 °C for 45‐60 min in the dark and leptin

(10µg/mL) and A23187 (12 µM) as a positive control were added to the

cells. The changes in Fluo‐4 NW fluorescence were measured by the

Wallac 1420 Victor 2 (PerkinElmer, Milan, Italy) at λex 494 nm and λem 516

nm. Calcium mobilization was observed over time (up to 120 sec) and

analyzed by the Wallac 1420 Software version 3 (PerkinElmer Life and

Analytical Sciences, Wallac, Milan, Italy). The increase in [Ca2+]i

(15)

Assessment of MP‐bound TF activity

TF activity was measured in MP generated in vitro from PBMC by a one‐

stage clotting time assay as described27, except that the normal human

plasma was made MP‐poor by ultracentrifugation (100,000xg for 2 h, 4

°C). Briefly, disrupted MP (100 µl) were mixed with 100 ul of MP‐poor

normal human plasma at 37°C; 100 µI of 25 mM CaCl2 at 37°C was added

to the mixture and the time to clot formation was recorded. The results

were expressed in arbitrary units (AU) of procoagulant activity by

comparison with a standard curve obtained using a human brain

thromboplastin standard. This preparation was assigned a value of 1,000

AU for a clotting time of 30 s. An anti‐human TF antibody (30 µg/mL) was

used to assess the specificity of the test (data not shown).

Data presentation and statistical analysis

Unless otherwise indicated, data are shown as means+SEM from n

independent, consecutive experiments; comparisons among groups were

made by either ANOVA for repeated measures followed by Bonferroni’s

analysis or Student’s paired t‐test, as appropriate, using Prism Software

(GraphPad, San Diego, CA, USA). Values of P <0.05 were considered

(16)

R

Le

To st in PS st 10

RESULTS

eptin ind

o investig timulated ncubation; S concen timulation 0µg/mL

uced MP

gate whet with 3 dif ; the supe ntration. A n with le

P generati

ther lepti fferent co ernatants As shown eptin. The for

ion by PB

n induces ncentratio were col n in figu e effect r

BMC

s the rele ons of lep lected, an ure 1, PB reached ease of M tin for 3 d nd MP we BMC gene statistical 4 MP, PBMC different ti ere quanti erate MP significa C were imes of ified as P upon nce at hours.

(17)

C -Lep tin 0.0 0.1 0.2 0.3 0.4 0.5

***

Micr o p ar ti cles ( n M P S )

Figure 1. MP generation, expressed as PS concentration, by PBMC incubated with leptin. (A) Dose–response curve for leptin (4 h) (B) and time‐response curve for leptin (10µg/mL). Data are from one experiment representative of 3. (C) MP generation by PBMC for 4 h with leptin (10µg/mL). ***p<0.001 for leptin treated cells compared with baseline (Student’s paired t‐test); n=8.

To confirm the results and to investigate the cellular origin of these MP,

flow cytometry was used. MP generation was expressed as events

conforming to light scatter distribution within the 0.5‐0.9 µm bead range

in a SSC vs FSC window (SSC+) and further identified as annexin V positive

(18)

leptin increases the number of annV+ MP and the number of CD14+. MP, confirming their monocytic origin (figure 2). A B C D -Lept in 0 500 1000 1500 2000 ** M icro p a rt ic les ( a n n V + eve n ts) -Lepti n 0 200 400 600 800 ** M icr opar ti cl es ( a nnV+ /CD14+ e v e n ts )

(19)

E

F

Figure 2. (A) MP generation, expressed as events SSC+ and annV+. Dose‐response curve for leptin. Data are from one experiment representative of 3. (B) MP generation, expressed as events SSC+ and annV+ and CD14+. Dose‐response curve for leptin. Data are from one experiment representative of 3. (C) MP generation by PBMC for 4 h with leptin (10µg/mL) SSC+/annV+, (D) SSC+/CD14+/annV+. **P , 0.01 for leptin treated cells compared with baseline (Student’s paired t‐test); n=11. (E) FACS analysis of MP generation express as events SSC+/annV+ and (F) SSC+/CD14+/ann V+.

(20)

Leptin induces the expression of MP‐bound TF by PBMC

The procoagulant activity of MP is due to the exposure of PS on their

surface and is also enhanced by the presence of functional TF27. To

evaluate whether leptin induces the generation of TF‐bearing MP by

PBMC, we analysed the procoagulant activity of purified MP released by

treated and untreated cells through a one‐stage clotting test. As shown in

figure 3, leptin induces an increase in procoagulant activity of MP. A

monoclonal antibody to TF (30 mg/mL) inhibited most of the procoagulant activity (not shown), confirming the identity of this activity with TF.

(21)

-Lept in 0.0 0.2 0.4 0.6 0.8 1.0

***

T

F

A

c

ti

vi

ty

(A

U

)

Figure 3. Effect of leptin on the generation of TF‐bearing MP by PBMC expressed as TF activity in AU. The cells were incubated with leptin 10µg/mL for 4 h; the supernatant was then tested for TF activity with a one‐stage clotting assay. ***p<0.001 for leptin treated cells compared with baseline (Student’s paired t‐test); n=3.

Leptin induces the mobilization of [Ca

2+

]

I

in PBMC

Leptin has been shown to induce a mobilization of intracellular calcium

concentration42,43. Because calcium mobilization is involved in MP

generation21,44–47, we investigated whether leptin increases [Ca2+]i in our

experimental conditions. Figure 4 shows that leptin (10µg/mL) induces a

(22)

0 50 100 150 0.0 0.2 1.6 1.8 2.0 2.2 2.4 Baseline Leptin Time (sec) In tr acel lu la r ca lc iu m concen tr at io n ( R FU ) Figure 4. Evaluation of intracellular calcium mobilization in PBMC treated with leptin (10μg/mL) ( )compared with untreated cells ( )as assessed by Fluo4‐NW incorporation; n= 3.

Leptin‐induced MP generation is mediated by mobilization on

intracellular calcium

Because leptin acts through calcium mobilization we investigate which

proteins of leptin intracellular pathway were involved in MP generation.

We used a phpspholipase C (PLC) inhibitor, U73122, to investigate the role

of calcium ions stored in the endoplasmic reticulum. Cells were pre‐

treated with U73122 (1µM) for 30 minutes. To investigate the role of

calcium ions entrance through membrane channels we used verapamil

(23)

treated with Ver (0,650µM) for 30 minutes. Finally, to investigate the role

of downstream proteins in the leptin pathway we pre‐treated the PBMC

with W‐7, a calmodulin inhibitor, for 30 minutes. Cell treatment with

U73122 inhibited MP production induced by leptin (figure 5). Further,

treatment with Ver inhibited the effect of leptin (figure 6). In contrast,

pre‐treatment with W‐7 had no effect on leptin MP generation (figure 7). -Lept in Leptin + U7 3122 0.0 0.2 0.4 0.6

*

M

icr

o

p

a

rt

icles (

n

M P

S

)

Figure 5. MP generation, expressed as PS concentration, by PBMC incubated for 4 h with leptin (10µg/mL) and pre‐treated with U73122 (1µM) for 30 minutes. *P , 0.05 for leptin treated cells compared with baseline and for leptin treated cells compared with U73122 and leptin treated cells (ANOVA analysis with Bonferroni post test);n=7.

(24)

-Lept in Lept in + Ver 0.0 0.1 0.2 0.3 0.4 0.5

**

*

M

icro

p

a

rt

icles (

n

M

P

S

)

Figure 6. MP generation, expressed as PS concentration, by PBMC incubated for 4 h with leptin (10µg/mL) and pre‐treated with Ver (0.650µM) for 30 minutes. **P , 0.01 for leptin treated cells compared with baseline (ANOVA analysis with Bonferroni post test); n=5. *P , 0.05 for leptin treated cells compared with leptin and Ver treated cells (ANOVA analysis with Bonferroni post test); n=6.

(25)

-Lept in Lept in + W-7 0.0 0.2 0.4 0.6 NS

**

Mi

cro

p

a

rt

ic

le

s

(

n

M PS)

Figure 7. MP generation, expressed as PS concentration, by PBMC incubated for 4 hours with leptin (10µg/mL) and pre‐treated with W‐7 (0.650µM) for 30 minutes. **P , 0.01 for leptin treated cells compared with baseline (ANOVA analysis with Bonferroni post test); n=6. Leptin‐induced MP generation is mediated by activation of ERK1/2 but not by activation JNK and p38. To investigate the implication of various proteins that compose the leptin

intracellular pathway we focused the attention on mitogen activated

protein kinases (MAPK). To this end, we used 3 different inhibitors of

(26)

SP600125, c‐JunN‐terminal kinase (JNK) inhibitor, and SB203580, p38

inhibitor. The cells were pre‐treated with PD98159 (1µM),SP600125

(0.650µM) and SB203580 (1µM) for 30 minutes. As shown in figure 8,only

PD98159 inhibited MP generation by PBMC.

(27)

(28)

C -Lept in Lept in + SB 20358 0 0.0 0.1 0.2 0.3 0.4 NS ** M icro p art icles ( n M PS)

Figure 8. MP generation, expressed as PS concentration, by PBMC incubated for 4 hours with leptin (10µg/mL) and pre‐treated for 30 minutes (A) with PD98059 (1µM), (B) with SP600125 (0.650µM), (C) with SB203580 (1µM). *P , 0.05,**P , 0.01 for leptin treated cells compared with baseline (ANOVA analysis with Bonferroni post test); n=6.

(29)

DISCUSSION

Adipose tissue is a source of inflammatory molecules that can change

vascular integrity. Leptin, besides playing an important role in the central

control of food intake and energy expenditure, has recently been shown

to exert systemic effects in a variety of physiologic and pathologic

processes48. It has been demonstrated that plasma leptin is higher in

patients who subsequently develop first ever myocardial infarction than in

control subjects in a population‐based case controlled study49. In addition,

leptin levels predicted myocardial infarction independently of traditional

risk factors. This finding was later conformed in a larger study50. Leptin is

also an independent predictor of myocardial infarction in men and women

with arterial hypertension51. Plasma leptin is higher in the offspring with

paternal history of premature myocardial infarction than in those without

family history of cardiovascular events52. In addition it was observed that

leptin is able to activate platelet53and human peripheral blood

mononuclear cells20 suggesting its leading role in atherothrombotic

disease in obesity. MP, shed from the surface of many cells upon

stimulation, considered for a long time to be artifacts, are now recognized

(30)

exocytosis of multivesicular bodies. MP have important physiological and

pathological roles: in coagulation, by mediating the coordinate

contribution of platelets, macrophages and neutrophils; in inflammatory

diseases, via the release of cytokines; and in tumor progression,

facilitating the spreading and release of cancer cells to generate

metastases54. Because plasma leptin in obese subjects is more elevated

than normal weight subjects55 and since MP are more elevated in plasma

of obese individuals than controls38,39, our aim was to test the hypothesis

that the prothrombotic effect of leptin is potentially linked, at least in

part, to the induction of procoagulant MP by PBMC. Indeed, leptin causes

procoagulant MP release by PBMC in a dose‐ and time dependant fashion;

flow cytometric analysis identified a portion of these MP as of monocytic

origin. Napoleone et al. observed that leptin induced TF expression in

PBMC20. Based on this observation we investigate on the generation of TF‐

bearing MP by PBMC. We demonstrate that leptin induces a significant

increase of TF activity in MP release by PBMC. It has been demonstrated

that plasma levels of leptin, even in obese patients (1‐5 nM), is much

lower than the concentration used in our in vivo experiments (650 nM).

However, Napoleone et al. have demonstrated an effect of leptin in

(31)

this work, at the same time confirming that weight loss causes a

significant reduction in both TF activity and antigen in the blood stream20.

These considerations lend strength to our claim that in vitro data can

be extrapolated to the in vivo situation. Endothelial cells and PBMC can

generate MP upon stimulation with various stimuli, including bacterial

LPS, cytokines such as TNF‐α or IL‐1 or other molecules of different nature

such as A23187, angiotensin II, peroxisome proliferator‐activated

receptor (PPAR)‐γ agonists, cigarette smoke extract46,47,56–61. Because the

kinetics of MP generation differ according to the different agonists, the

involvement of different intracellular pathways can be postulated. We

therefore investigated the specific mechanism(s) whereby leptin induced

MP generation in our experimental conditions. Because cells can generate

MB by activation, a mechanism that involved an increase of intracellular

calcium concentration, or following apoptosis21, we investigated the effect

of leptin on intracellular calcium concentration. The observation that

leptin induced calcium mobilization led us to conclude that release of MP

by PBMC take place through cell activation. To better understand this

pathway of action we used three different molecules involved on calcium

mobilization: U73122, a PLC inhibitor, Ver, an L‐type calcium channel

(32)

leptin‐induced MP release, this observation is consistent with the

hypothesis that MP shedding after calcium mobilization from intracellular

storage pools takes place via phosphatidylinositol (3,4,5) trisphosphate

(PI3)18 and calcium entry via L‐type channels. According to this model, we

postulated that calmodulin, a protein that gets activated upon binding

Ca2+62,63, was implicated in this pathway, however, our experimental

results did not confirm the hypothesis. Because the leptin intracellular

pathway includes MAPK cascade activation and PLC is implicate in

activation of Ras, the initial protein involved in MAPK cascade18,64, we

investigated the role of ERK1/2, JNK and p38 on leptin‐induced MP release

by PBMC. We found that only ERK1/2 inhibitor, PD98059, decreases MP

generation. This observation led us to conclude that leptin‐induced MP

generation is mediated by the activation of ERK1/2. This conclusion is

consistent with the previous observation that MAPK in phosphorylated by

leptin stimulation in human PBMC65. All inhibitors were used at a

concentration higher or equal than leptin concentration; all were tested

for toxicity (data not shown).

In conclusion, our data demonstrate that leptin induces the generation of

procoagulant, TF bearing MP by PBMC through a mechanism involving

(33)

These procoagulant structures likely contribute to the increased risk of

thrombotic events in obese patients. Pharmacological modulation of MP

generation, for example through the inhibition of ERK1/2 and/or of

calcium mobilization, might prove helpful in preventing such events.

(34)

REFERENCES

1. Haynes, W. G., Morgan, D. A., Walsh, S. A., Sivitz, W. I. & Mark, A. L. Cardiovascular consequences of obesity: role of leptin. Clin. Exp. Pharmacol. Physiol.25, 65–9 (1998).

2. Williams, I. L., Wheatcroft, S. B., Shah, A. M. & Kearney, M. T. Obesity, atherosclerosis and the vascular endothelium: mechanisms of reduced nitric oxide bioavailability in obese humans. Int. J. Obes. Relat. Metab. Disord.26, 754– 64 (2002).

3. Lorenzet, R., Napoleone, E., Cutrone, A. & Donati, M. B. Thrombosis and obesity : Cellular bases. Thromb. Res.129, 285–289 (2015).

4. Kopelman, P. G. Obesity as a medical problem. Nature404, 635–43 (2000). 5. Lau, D. C. et al. Paracrine interactions in adipose tissue development and

growth. Int. J. Obes. Relat. Metab. Disord.20 Suppl 3, S16–25 (1996).

6. Mohamed‐Ali, V., Pinkney, J. H. & Coppack, S. W. Adipose tissue as an endocrine and paracrine organ. Int. J. Obes. Relat. Metab. Disord.22, 1145–58 (1998). 7. Lau, D. C. W., Dhillon, B., Yan, H., Szmitko, P. E. & Verma, S. Adipokines:

molecular links between obesity and atheroslcerosis. Am. J. Physiol. Heart Circ. Physiol.288, H2031–41 (2005).

8. Belza, A., Toubro, S., Stender, S. & Astrup, A. Effect of diet‐induced energy deficit and body fat reduction on high‐sensitive CRP and other inflammatory markers in obese subjects. Int. J. Obes. (Lond).33, 456–64 (2009).

9. Reaven, G. M. et al. Hemostatic abnormalities associated with obesity and the metabolic syndrome. J. Thromb. Haemost.3, 1074–85 (2005).

10. Banks, W. a. The many lives of leptin. Peptides25, 331–8 (2004).

11. Frühbeck, G., Jebb, S. A. & Prentice, A. M. Leptin: physiology and pathophysiology. Clin. Physiol.18, 399–419 (1998).

12. Frühbeck, G. A heliocentric view of leptin. Proc. Nutr. Soc.60, 301–318 (2007). 13. Ravussin, E. Cellular sensors of feast and famine. J. Clin. Invest.109, 1537–40

(2002).

14. Unger, R. H. The hyperleptinemia of obesity‐regulator of caloric surpluses. Cell117, 145–6 (2004).

(35)

15. Frühbeck, G. Peripheral actions of leptin and its involvement in disease. Nutr. Rev.60, S47–55; discussion S68–84, 85–7 (2002).

16. Baratta, M. Leptin‐‐from a signal of adiposity to a hormonal mediator in peripheral tissues. Med. Sci. Monit.8, RA282–92 (2002).

17. Muoio, D. M. & Lynis Dohm, G. Peripheral metabolic actions of leptin. Best Pract. Res. Clin. Endocrinol. Metab.16, 653–66 (2002).

18. Frühbeck, G. Intracellular signalling pathways activated by leptin. Biochem. J.393, 7–20 (2006).

19. Gualillo, O., González‐juanatey, J. R. & Lago, F. The Emerging Role of Adipokines as Mediators of Cardiovascular Function : Physiologic and Clinical Perspectives. 17, 275–283 (2007).

20. Napoleone, E. et al. Leptin induces tissue factor expression in human peripheral blood mononuclear cells: a possible link between obesity and cardiovascular risk? J. Thromb. Haemost.5, 1462–8 (2007).

21. VanWijk, M. J., VanBavel, E., Sturk, a & Nieuwland, R. Microparticles in cardiovascular diseases. Cardiovasc. Res.59, 277–87 (2003).

22. Wolf, P. The nature and significance of platelet products in human plasma. Br. J. Haematol.13, 269–88 (1967).

23. Distler, J. H. W., Huber, L. C., Gay, S., Distler, O. & Pisetsky, D. S. Microparticles as mediators of cellular cross‐talk in inflammatory disease. Autoimmunity39, 683–90 (2006).

24. Sadallah, S., Eken, C. & Schifferli, J. A. Ectosomes as modulators of inflammation and immunity. Clin. Exp. Immunol.163, 26–32 (2011).

25. Ardoin, S. P., Shanahan, J. C. & Pisetsky, D. S. The role of microparticles in inflammation and thrombosis. Scand. J. Immunol.66, 159–65 (2007).

26. Distler, J. H. W. & Distler, O. Inflammation: Microparticles and their roles in inflammatory arthritides. Nat. Rev. Rheumatol.6, 385–6 (2010).

27. Celi, A., Lorenzet, R., Furie, B. C. & Furie, B. Microparticles and a P‐selectin‐ mediated pathway of blood coagulation. Dis. Markers20, 347–52 (2004).

28. Leroyer, A. S. et al. Endothelial‐derived microparticles: Biological conveyors at the crossroad of inflammation, thrombosis and angiogenesis. Thromb. Haemost.104, 456–63 (2010).

29. Benameur, T., Andriantsitohaina, R. & Martínez, M. C. Therapeutic potential of plasma membrane‐derived microparticles. Pharmacol. Rep.61, 49–57 (2009).

(36)

30. Hugel, B., Martínez, M. C., Kunzelmann, C. & Freyssinet, J.‐M. Membrane microparticles: two sides of the coin. Physiology (Bethesda).20, 22–7 (2005). 31. Morel, O., Jesel, L., Freyssinet, J.‐M. & Toti, F. Cellular mechanisms underlying

the formation of circulating microparticles. Arterioscler. Thromb. Vasc. Biol.31, 15–26 (2011).

32. Montoro‐García, S., Shantsila, E., Marín, F., Blann, A. & Lip, G. Y. H. Circulating microparticles: new insights into the biochemical basis of microparticle release and activity. Basic Res. Cardiol.106, 911–23 (2011).

33. Morel, O. et al. Procoagulant microparticles: disrupting the vascular homeostasis equation? Arterioscler. Thromb. Vasc. Biol.26, 2594–604 (2006). 34. Lechner, D. & Weltermann, A. Circulating tissue factor‐exposing microparticles.

Thromb. Res.122 Suppl, S47–54 (2008).

35. Mackman, N. Alternatively spliced tissue factor ‐ one cut too many? Thromb. Haemost.97, 5–8 (2007).

36. Giesen, P. L. et al. Blood‐borne tissue factor: another view of thrombosis. Proc. Natl. Acad. Sci. U. S. A.96, 2311–5 (1999).

37. Boulanger, C. M., Amabile, N. & Tedgui, A. Circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease. Hypertension48, 180–6 (2006).

38. Murakami, T. et al. Impact of weight reduction on production of platelet‐derived microparticles and fibrinolytic parameters in obesity. Thromb. Res.119, 45–53 (2007).

39. Wolk, R. et al. Association between plasma adiponectin levels and unstable coronary syndromes. Eur. Heart J.28, 292–8 (2007). 40. Morel, O. et al. Short‐term very low‐calorie diet in obese females improves the haemostatic balance through the reduction of leptin levels, PAI‐1 concentrations and a diminished release of platelet and leukocyte‐derived microparticles. Int. J. Obes. (Lond).35, 1479–86 (2011). 41. Robert, S. et al. Standardization of platelet‐derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies? J. Thromb. Haemost.7, 190–7 (2009).

42. Padra, J. T., Seres, I., Fóris, G., Paragh, G. & Kónya, G. Leptin triggers Ca(2+) imbalance in monocytes of overweight subjects. Neuropeptides46, 203–9 (2012).

(37)

43. Rajapurohitam, V., Izaddoustdar, F., Martinez‐Abundis, E. & Karmazyn, M. Leptin‐induced cardiomyocyte hypertrophy reveals both calcium‐dependent and calcium‐independent/RhoA‐dependent calcineurin activation and NFAT nuclear translocation. Cell. Signal.24, 2283–90 (2012).

44. Neri, T. et al. Role of NF‐kappaB and PPAR‐gamma in lung inflammation induced by monocyte‐derived microparticles. Eur. Respir. J.37, 1494–502 (2011).

45. Pasquet, J. M., Dachary‐Prigent, J. & Nurden, A. T. Calcium influx is a determining factor of calpain activation and microparticle formation in platelets. Eur. J. Biochem.239, 647–54 (1996).

46. Cordazzo, C. et al. Rapid shedding of proinflammatory microparticles by human mononuclear cells exposed to cigarette smoke is dependent on Ca(2+) mobilization. Inflamm. Res. (2014). doi:10.1007/s00011‐014‐0723‐7

47. Cordazzo, C. et al. Angiotensin II induces the generation of procoagulant microparticles by human mononuclear cells via an angiotensin type 2 receptor‐ mediated pathway. Thromb. Res.131, e168–74 (2013).

48. Fantuzzi, G. & Faggioni, R. Leptin in the regulation of immunity, inflammation, and hematopoiesis. J. Leukoc. Biol.68, 437–46 (2000).

49. Söderberg, S. et al. Leptin is associated with increased risk of myocardial infarction. J. Intern. Med.246, 409–18 (1999). 50. Thøgersen, A. M. et al. Interactions between fibrinolysis, lipoproteins and leptin related to a first myocardial infarction. Eur. J. Cardiovasc. Prev. Rehabil.11, 33– 40 (2004). 51. Wallerstedt, S. M., Eriksson, A.‐L., Niklason, A., Ohlsson, C. & Hedner, T. Serum leptin and myocardial infarction in hypertension. Blood Press.13, 243–6 (2004). 52. Makris, T. K. et al. Markers of risk in young offspring with paternal history of

myocardial infarction. Int. J. Cardiol.89, 287–293 (2003).

53. Konstantinides, S., Schäfer, K., Koschnick, S. & Loskutoff, D. J. Leptin‐dependent platelet aggregation and arterial thrombosis suggests a mechanism for atherothrombotic disease in obesity. 108, (2001).

54. Cocucci, E., Racchetti, G. & Meldolesi, J. Shedding microvesicles: artefacts no more. Trends Cell Biol.19, 43–51 (2009).

55. Considine, R. V et al. Serum immunoreactive‐leptin concentrations in normal‐ weight and obese humans. N. Engl. J. Med.334, 292–5 (1996).

(38)

56. Satta, N. et al. Monocyte vesiculation is a possible mechanism for dissemination of membrane‐associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J. Immunol.153, 3245–55 (1994). 57. Patel, K. D., Zimmerman, G. A., Prescott, S. M. & McIntyre, T. M. Novel leukocyte agonists are released by endothelial cells exposed to peroxide. J. Biol. Chem.267, 15168–75 (1992). 58. Mesri, M. & Altieri, D. C. Endothelial cell activation by leukocyte microparticles. J. Immunol.161, 4382–7 (1998). 59. Neri, T. et al. Effects of peroxisome proliferator‐activated receptor‐γ agonists on the generation of microparticles by monocytes/macrophages. Cardiovasc. Res.94, 537–44 (2012).

60. Cerri, C. et al. Monocyte/macrophage‐derived microparticles up‐regulate inflammatory mediator synthesis by human airway epithelial cells. J. Immunol.177, 1975–80 (2006).

61. Li, M., Yu, D., Williams, K. J. & Liu, M.‐L. Tobacco smoke induces the generation of procoagulant microvesicles from human monocytes/macrophages. Arterioscler. Thromb. Vasc. Biol.30, 1818–24 (2010).

62. Clapham, D. E. Calcium signaling. Cell131, 1047–58 (2007).

63. Hoeflich, K. P. & Ikura, M. Calmodulin in action: diversity in target recognition and activation mechanisms. Cell108, 739–42 (2002).

64. Seger, R. & Krebs, E. G. The MAPK signaling cascade. FASEB J.9, 726–35 (1995). 65. Martín‐Romero, C. & Sánchez‐Margalet, V. Human leptin activates PI3K and

MAPK pathways in human peripheral blood mononuclear cells: possible role of Sam68. Cell. Immunol.212, 83–91 (2001).