research article epicatechin and catechin modulate...
TRANSCRIPT
Research ArticleEpicatechin and Catechin Modulate Endothelial ActivationInduced by Platelets of Patients with Peripheral Artery Disease
R Carnevale1 L Loffredo1 C Nocella1 S Bartimoccia1 T Bucci1 E De Falco2
M Peruzzi2 I Chimenti2 G Biondi-Zoccai2 P Pignatelli1 F Violi1 and G Frati23
1 Department of Internal Medicine and Medical Specialties Sapienza University of Rome 00161 Rome Italy2 Department of Medical-Surgical Sciences and Biotechnologies Sapienza University of Rome Latina Italy3 Department of AngioCardioNeurology IRCCS NeuroMed 86077 Pozzilli Italy
Correspondence should be addressed to R Carnevale robertocarnevaleuniroma1it
Received 7 July 2014 Accepted 20 July 2014 Published 7 August 2014
Academic Editor Liang-Jun Yan
Copyright copy 2014 R Carnevale et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Platelet activation contributes to the alteration of endothelial function a critical initial step in atherogenesis through the productionand release of prooxidant mediatorsThere is uncertainty about the precise role of polyphenols in interaction between platelets andendothelial cells (ECs) We aimed to investigate whether polyphenols are able to reduce endothelial activation induced by activatedplatelets First we compared platelet activation and flow-mediated dilation (FMD) in 10 healthy subjects (HS) and 10 patientswith peripheral artery disease (PAD)Then we evaluated the effect of epicatechin plus catechin on platelet-HUVEC interaction bymeasuring soluble cell adhesionmolecules (CAMs)NOxproduction and eNOSphosphorylation (p-eNOS) inHUVECComparedto HS PAD patients had enhanced platelet activation Conversely PAD patients had lower FMD than HS Supernatant of activatedplatelets from PAD patients induced an increase of sCAMs release and a decrease of p-eNOS and nitric oxide (NO) bioavailabilitycompared to unstimulated HUVEC Coincubation of HUVEC with supernatant of PAD platelets patients pretreated with a scalardose of the polyphenols resulted in a decrease of sCAMs release and in an increase of p-eNOS and NO bioavailability This studydemonstrates that epicatechin plus catechin reduces endothelial activation induced by activated platelets
1 Introduction
Atherosclerosis is a chronic vascular disease Such a conditionis not to be considered a disease in its own right but rathera process that contributes to the pathogenesis of severalserious diseases There is no selective agent responsible forthe entire atherosclerotic process which is considered theproduct of several concomitant interplays among differentrisk factors such as genetic predisposition hyperlipidemiadiabetes mellitus smoking and hypertension [1] Atheroscle-rosis develops progressively through continuous evolution ofarterial wall lesions due to the accumulation of cholesterol-rich lipids and the associated inflammatory response [1]
It is clear that functional or morphological alterationsin endothelial cells (ECs) platelets and leukocytes appearto be critical in the evolution progression and clinicalmanifestation of the atherosclerosis [2] Their interactionplays an important role and represents a key event triggering
and sustaining the inflammatory process in the arterial wall[2] Platelets have a major role in vascular inflammationthrough the production and release of proinflammatory aswell as prooxidant mediators and by interacting with ECsSuch interplay between platelets and endothelium affects theentire course of development of atherosclerosis
Platelets sustain the inflammatory process at all stagesof atherosclerosis by expressing membrane molecules suchas intercellular adhesion molecule-2 (ICAM-2) P-selectinCD95L and CD40L that regulate several biological func-tions in the vessel wall including cellular adhesion andaggregation chemotaxis survival and differentiation andangiogenesis [3 4]
The most important damage to the endothelium is in-duced by reactive oxygen species (ROS) and prooxidant mol-ecules which are associated to various risk factors of athero-sclerosis and produced by different cells including platelets
Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 691015 9 pageshttpdxdoiorg1011552014691015
2 Oxidative Medicine and Cellular Longevity
Vascular endothelium responds to mechanical and hor-monal stimuli by releasing signaling molecules involved inthe modulation of haemostasis such as nitric oxide (NO)[5 6] which acts by enabling mechanisms of relaxation andvasodilatation in vascular smooth muscle cells (VSMCs) andby exerting profound functional and morphological effectson the vascular wall Nitric oxide also exerts importantanti-inflammatory and antithrombotic effects [7] In par-ticular ROS production rapidly inactivates the vasorelax-ation molecule NO contributing to endothelial dysfunctiondescribed as impaired vasodilatation [8]
In addition to the reduction in NO bioavailabilityplatelet-induced endothelium activation leads to increasedexpression of cell adhesion molecules (CAMs) such as inter-cellular adhesion molecule-1 (ICAM-1) vascular adhesionmolecule-1 (VCAM-1) and E-selectin [9]
Among different clinical settings associated with athero-sclerosis peripheral artery disease (PAD) is considered perse a sign of systemic atherosclerosis Moreover PAD patientsdisplay endothelial dysfunction as demonstrated by lowflow-mediated dilatation (FMD) [10] which has become aconventional measure of endothelial dysfunction in humanatherosclerosis [11] Consistently with reduced FMD PADpatients have enhanced NOX2 activation [12] and the cat-alytic subunit ofNADPHoxidase aswell as themajor enzymegenerating ROS involved in the modulation of arterial tone[13 14]
Epidemiological studies have shown that a diet rich inpolyphenols is able to reduce cardiovascular events [15 16]Polyphenols a heterogeneous group of molecules foundprimarily in fruits and vegetables along with their effects onplatelets function have been investigated by several authorsAmong other activities an increase in NO production asso-ciated with a decrease in ROS formation has been described[17 18] This finding is in accordance with our previousreport indicating that polyphenols synergize in exertingantioxidant and antiplatelet effects [19] In fact we observedthat epicatechin plus catechin is able to exert an antioxidanteffect greater than individual polyphenols [19] Neverthelessfew data concerning the effects of polyphenols on platelet-endothelium interaction are available in PAD patients Theaim of this study was to investigate whether polyphenolsare able to modulate endothelial function via inhibition ofplatelet-endothelial interaction Furthermore we tested theeffect of epicatechin plus catechin on the interaction betweenECs and platelets
2 Materials and Methods
21 Cross-Sectional Study The study has been carried out inthe outpatient clinic of our division between January 2013 andSeptember 2013 on 10 patients aged between 40 and 80 yearspresenting with symptoms of intermittent claudication (for atleast 6 months) and corresponding to Fontaine stage IIb Tenconsecutive subjects matched for age gender cardiovascularrisk factors and pharmacological treatments were enrolled inthe same period as controls Each PAD patient to be enrolledin the study had
(1) claudication (defined as leg pain occurring after lessthan 200 meters of walking relieved within 10 min-utes after cessation of exercise and being of presumedatherosclerotic origin)
(2) anklebrachial index (ABI) which was assessed asanklearm systolic blood pressure ratio by Dopplerultrasonography lower than 090 on the worst leg atrest
All subjects underwent a full medical anamnestic collec-tion physical examination 12-lead ECG laboratory tests andmeasurement of ABI Patients had to be in stable conditionswithout abrupt changes of ABI (gt20) in the last monthbefore the enrolment Exclusion criteria were represented byliver insufficiency serious renal disorders (serum creatininegt 28mgdL) acute cerebrovascular disease acutemyocardialinfarction deep venous thrombosis tabagism and ongoingtherapy with antioxidants Written informed consent wasobtained from all subjects the study was conformed to theethical guidelines of the 1975 Declaration of Helsinki and wasapproved by the Ethical Committee of Policlinico UmbertoI Sapienza University of Rome (ClinicalTrialsgov identifierNCT01947712)
22 Platelet Preparation Venous blood was drawn intrisodium citrate (38 110 (v v)) from fasting HS andPAD patients who had not taken any drugs affecting plateletfunction for at least 14 days To obtain platelet rich plasma(PRP) blood was centrifuged at 15min at 180 g at roomtemperature and the supernatant PRPwas separated To avoidleukocyte contamination only the top 75 of the PRP wascollected To obtain washed platelets the PRP was washedwith ACD (101 vv) and suspended in fatty acid-free Tyrodersquosbuffer (2 times 108 plateletsmL unless otherwise noted)
Washed platelets were treated with a scalar dose of epi-catechin (01ndash10 120583M) plus catechin (01ndash10 120583M) and activatedwith arachidonic acid (AA) (05mM) for 10min at 37∘C andsupernatant was stored at minus80∘C unless otherwise indicated
23 Human Umbilical Vein Endothelial Cells (HUVEC)Human umbilical vein endothelial cells (HUVEC) werecultured as described [20] Briefly cells were expanded (2000cellscm2) in complete medium (EndoGRO-LS CompleteMedia Kit Millipore) Cell morphology and growth weremonitored by light microscopy and assessed by Trypan Blue(Sigma St Louis USA) The culture was expanded untilpassage 5
24 Platelet ROS Production Cell suspension was incubatedwith 2101584071015840-dichlorofluorescein diacetate (5 120583molL) for 15minutes at 37∘C After incubation platelets were activatedwith AA Platelet ROS production was expressed as stim-ulation index (mean level of fluorescence in stimulatedcellsmean level of fluorescence in unstimulated cells) (SI)Fluorescence intensity was analyzed on an Epics XL-MCLcytometer (Coulter Electronics) equipped with an argonlaser at 510ndash550 nm (green) For every histogram 50000plateletswere counted to determine the proportion of positive
Oxidative Medicine and Cellular Longevity 3
platelets The fluorescent signal generated by the probe wasexpressed as mean fluorescence intensity (SI) Intra-assaycoefficient of variation was 5
25 Platelet 8-iso-PGF2120572 Assays To measure isoprostanesformation platelets were activated with AAThe supernatantwas stored at minus80∘C until measurement Quantification ofisoprostanes was performed measuring 8-iso-PGF2120572 by apreviously described and validatedEIA assaymethod [21] andwas evaluated in vivo and in vitro Intra-assay and interassaycoefficients of variation were 58 and 50 respectively
26 Platelet NOx A colorimetric assay kit (Tema RicercaItaly) was used to determine the NO metabolites nitrite andnitrate (NOx) in 100 120583L of platelet or HUVEC suspensionmaintained under stirring condition for 10min at 37∘C Intra-assay and interassay coefficients of variation were 29 and17 respectively
27 Platelet H2O2Concentration A colorimetric assay kit
(Tema Ricerca Italy) was used to determine the H2O2
concentrationin 100120583L of platelet suspension maintainedunder stirring condition for 10min at 37∘C Intra-assayand interassay coefficients of variation were 21 and 37respectively
28 ELISA Detection of Platelet sNOX2-dp NOX2-derivedpeptide (NOX2-dp) a marker of NADPH oxidase activationwas detected by ELISA method as previously described byPignatelli et al [22] The peptide was recognized by a specificmonoclonal antibody against the amino acidic sequence(224ndash268 residues) of the extra membrane portion of NOX2To measure sNOX2-dp platelet suspension was activatedwith AA and 100 120583L of the supernatant stored at minus80∘C untilmeasurement Values were expressed as pgmL intra-assayand interassay coefficients of variation were 52 and 6respectively
29 Detection of HUVEC sICAM1 sVCAM1 and sE-SelectinA Custom Human 3-Plex Array (Tema Ricerca Italy) wasused to determine the sICAM1 sVCAM1 and sE-selectinconcentration Values were expressed as ngmL intra-assayand interassay coefficients of variation were 52 and 6respectively
210 Western Blot Analysis of p-eNOS and Total eNOS Equalamounts of protein (30 120583glane) estimated by Bradford assaywere solubilized in a 2X Laemmli sample buffer containing2-mercaptoethanol and loaded in a denaturing SDS10polyacrylamide gel Western blot analysis was performedwith monoclonal anti-mouse eNOS-PSer1177 (1 1000 BDTransduction Laboratory) (2 120583gmL) and anti-mouse eNOS(1 1000 BD Transduction Laboratory) (2120583gmL) incubatedovernight at 4∘C After incubation the pure nitrocellulosemembranes (045 120583m) were washed and incubated withgoat anti-mouse IgG1-horseradish peroxidase for 2 hoursImmune complexes were detected by enhanced chemilumi-nescence The developed spots were quantified by densito-metric analysis on a NIH Image 162f analyzer and the value
was expressed as arbitrary units Each sample was analyzed intriplicate The results were expressed as P-eNOStotal eNOSratio
211 FMD and Carotid Intima-Media Thickness Ultrasoundassessment of FMDwas investigated according to the recentlyreported guidelines [23] as previously described [24] Thecoefficient of variation for FMDmeasurements obtained on 3separate occasions was 125 Longitudinal ultrasonographicscans of the carotid artery were obtained on the same day asthe studies of the brachial artery reactivity and included theevaluation of the right and left common carotid arteries 1 cmproximal to the carotid bulb FMD was performed with a 75MHz linear-array transducer ultrasound system (SonomedLake Success NY)
212 In Vitro Study In vitro study was performed in bloodtaken from 5 PAD patients and 5 HS (3 males and 2 femalesmean age 56 plusmn 6 years) We analysed the effect of scalardoses of epicatechin (01ndash10120583M)plus catechin (01ndash10 120583M) onplatelet-HUVEC interaction
Only PAD patients platelets (2 times 108mL) were incu-bated for 30min at 37∘C with epicatechin (01ndash10 120583M) pluscatechin (01ndash10 120583M) (mix) and stimulated for 10min withAA (05mM) After incubation samples were immediatelypelleted at 4∘C and the supernatants were added to semicon-fluent endothelial monolayers with and without endothelialgrowth factor (EGF 10 ngmL)
After 60 minutes of coincubation supernatants wereremoved by gentle washing which was confirmed by lightmicroscopy observation Afterwards cultures were left for afurther period of 2 hours in incubator with basalmedium andharvested by trypsin Then both HUVEC and HUVEC cul-ture supernatants were collected for the analysis of sICAM1sVCAM1 sE-selectin NOx concentration eNOS phosphory-lation and total eNOS
213 Sample Size Determination We computed the mini-mum sample size with respect to a two-tailed one-sampleStudentrsquos 119905-test considering (i) a difference for NOX to bedetected between the healthy subjects and PAD patients as|120575| 15 120583M (ii) homogeneous SDs between groups as 4 and(iii) type I error probability as 120572 = 005 and power 1 minus 120573 =090 This resulted in a minimum sample size of 119899 = 4for group Additional patients and cases were included fora subset of experiments in order to provide more externallyvalid findings
214 Statistical Analyses Continuous variables are reportedas mean plusmn standard deviation and were compared withStudentrsquos 119905-test Categorical variables are reported as andcompared with the 1205942 test Statistical significance was set atthe 2-tailed 005 level Computations were performed withSPSS 18 (IBM Armonk NY USA)
3 Results
31 Cross-Sectional Study Clinical characteristics of PADpatients and HS are reported in Table 1
4 Oxidative Medicine and Cellular Longevity
Table 1 Clinical characteristics of patients with peripheral artery disease (PAD) and healthy subjects (HS)
Variables PAD (119899 = 10) HS (119899 = 10) 119875 valueMean age (year)a 63 plusmn 5 65 plusmn 6 0854Malesfemales 64 64 10Hypertension (119899)b 98 (9) 0 (0) lt0001Diabetes mellitus (119899) 30 (3) 0 (0) lt0001Dyslipidemia (119899) 80 (8) 0 (0) lt0001Former smokers (119899) 40 (4) 50 (5) 0621CHD (119899) 20 (2) 0 (0) lt0001Glycemia (mgdL) 108 plusmn 6 104 plusmn 4 0589Pharmacological treatments (119899)
ACE-inhibitors 80 (8) 0 (0) lt0001Oral antidiabetic drugs 30 (3) 0 (0) lt0001Insulin 0 (0) 0 (0) 10Statin 70 (7) 0 (0) lt0001Antiaggregants 60 (6) 0 (0) lt0001Oral anticoagulants 20 (2) 0 (0) lt0001
aData are expressed as mean plusmn SDbgt139ndash89mmHg
PAD patients showed higher platelet ROS production(106 plusmn 14 SI versus 34 plusmn 09 SI lowast119875 lt 0001) (Figure 1(a))H2O2concentration (184plusmn07 120583Mversus 54plusmn05 120583M lowast119875 lt
0001) (Figure 1(b)) sNOX2-dp release (298 plusmn 134 pgmLversus 79 plusmn 23 pgmL lowast119875 lt 0001) (Figure 1(c)) and 8-iso-PGF2120572 production (1626 plusmn 428 pmolL versus 670 plusmn64 pmolL lowast119875 lt 0001) (Figure 1(d)) compared to HS PADpatients displayed lower NOx production (287 plusmn 63 120583Mversus 103 plusmn 26 120583M 119875 lt 0001) (Figure 1(e)) and FMDvalues (20plusmn25 versus 77plusmn10 lowast119875 lt 0001) (Figure 1(f))compared with HS
32 In Vitro Study HUVEC stimulated with EGF releaseda higher amount of sICAM1 (Figure 2(a) lowast119875 lt 0001)sVCAM1 (Figure 2(b) lowast119875 lt 0001) and E-selectin(Figure 2(c) lowast119875 lt 0001) versus unstimulated HUVEC
Supernatant of activated platelets from PAD patients(SAPAD) was able to increase sICAM1 sVCAM1 and sE-selectin versus nonstimulated HUVEC (Figure 2) Con-versely supernatant of activated platelets from HS (SAHS)was not able to change markers of endothelial dysfunction(Figure 2)
When SAPAD pretreated with mix of epicatechin (01ndash10 120583M) and catechin (01ndash10 120583M) was added to HUVEC asignificant reduction of sICAM1 sVCAM1 and sE-selectinlevels was observed compared to sCAMs levels induced bySAPAD (Figures 2(a) 2(b) and 2(c))
Both the P-eNOStotal eNOS ratio (Figure 3(a) lowast119875 lt0001) andNOx concentration (Figure 3(b) lowast119875 lt 0001) weredecreased in HUVEC when stimulated with EGF Moreoverwe found that SAPAD contributed to the reduction of P-eNOStotal eNOS ratio compared to untreated HUVEC(Figure 3(a) lowast119875 lt 0001) with consequent reduction of NObioavailability (Figure 3(b) lowast119875 lt 00001) This phenomenonwas not observed in HUVEC treated with SAHS (Figure 3)
When SAPAD pretreated with mix of epicatechin (01ndash10 120583M) and catechin (01ndash10 120583M) was added to HUVECboth the P-eNOStotal eNOS ratio and NO bioavailabilityincreased compared to P-eNOStotal eNOS ratio and NObioavailability induced by SAPAD (Figures 3(a) and 3(b))
4 DiscussionThe major findings of this paper are as follows (a) PADpatients show higher platelet activation than HS (b) super-natant of activated platelets derived from PAD patients(SAPAD) is able to activate ECs resulting in an upregula-tion of soluble molecules derived from ECs (ie sICAM-1sVCAM-1 and E-selectin) (c) PAD platelets induced ECsactivation by reduction of p-eNOS and bioavailability of NO(d) polyphenols are able to reduce endothelial activationinduced by activated platelets
These novel findings support the hypothesis that platelets-induced endothelial activation and thus the progression ofatherosclerosis are a predisposing factor to more complexdiseases such as PAD [25]
Previous findings showed that in PAD patients there isan imbalance between NOX2-mediated oxidative stress andFMD [11] In the present study we expanded such evidencedemonstrating the key oxidative role of platelets in PADpatients by measuring their ROS production and sNOX2-dp release as markers of NADPH oxidase activation [22]as well as their 8-iso-PGF2120572-III production We observeda significant increase of these oxidative stress markers inplatelets of PAD patients compared to HS
Activated platelets can interact with the endothelium trig-gering an inflammatory response that may contribute to theearly stages of atherosclerosis [26] Upon activation plateletsrelease granule contents able to activate ECs It has beenshown that activated platelets interact with the endotheliuminducing accumulation and transmigration of circulating
Oxidative Medicine and Cellular Longevity 5
0
2
4
6
8
10
12
14
HS PAD
lowast
Plat
elet
RO
S pr
oduc
tion
(SI)
(a)
0
5
10
15
20
HS PAD
lowast
Plat
elet
H2O2
conc
entr
atio
n (120583
M)
(b)
0
5
10
15
20
25
30
35
40
HS PAD
lowast
Plat
elet
sNO
X2-d
p (p
gm
L)
(c)
0
50
100
150
200
HS PAD
lowastPl
atel
et8
-iso-
PGF2120572
(pm
olL
)
(d)
0
5
10
15
20
25
30
35
HS PAD
lowast
Plat
elet
NO
x pr
oduc
tion
(120583M
)
(e)
0
1
2
3
4
5
6
7
8
9
10
11
HS PAD
lowast
FMD
()
(f)
Figure 1 Platelet-induced oxidative stress in PAD patients Platelet ROS production (a) platelet H2O2concentration (b) platelet sNOX2-dp
levels (c) platelet 8-iso-PGF2120572 production (d) platelet NOx production (e) and FMD (f) in PAD patients (119899 = 10) and healthy subjects(119899 = 10) (lowast119875 lt 0001)
6 Oxidative Medicine and Cellular Longevity
0
50
100
150
200
250
300
350
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
lowastlowast
lowast
NS
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sICA
M1
leve
ls (n
gm
L)
(a)
0
5
10
15
20
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sVCA
M1
leve
ls (n
gm
L)(b)
0
50
100
150
200
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sE-s
elec
tion
leve
ls (n
gm
L)
(c)
Figure 2 Biomarkers of HUVEC activation sICAM1 (a) sVCAM1 (b) and E-selectin (c) levels released by HUVEC after incubation withEGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activated PAD patients (SAPAD) (blackbar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalar doses of epicatechin (01ndash10120583M) plus catechin(01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001)
monocytes finally contributing to the atherosclerotic processat early stages [27] Moreover activated platelets can releasein their microenvironment prooxidant molecules such asisoprostanes and ROS In fact we found that supernatant ofPAD platelets is able to stimulate HUVEC with consequent
release of cellular adhesion molecules in their soluble formnamely CAMs
The soluble forms of adhesion molecules reflect endothe-lial CAM surface expression [28] and therefore can be usedas biomarkers of endothelial activation in different diseases
Oxidative Medicine and Cellular Longevity 7
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
P-eNOS
120573-Actin
0
5
10
15
20
25
30
35
40
45
lowast
lowastlowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
Den
sitom
etric
anal
ysis
ofP-
eNO
Sto
tal e
-NO
S (r
atio
)
Total eNOS
(a)
50
0
5
10
15
20
25
30
35
40
45 lowastlowast
lowast lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
HU
VEC
s NO
x pr
oduc
tion
(120583M
)
(b)
Figure 3 HUVEC eNOS activation and NOx production Densitometric analysis of P-eNOStotal eNOS ratio (a) and NOx production (b)in HUVEC incubated with EGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activatedplatelets from PAD patients (SAPAD) (black bar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalardoses of epicatechin (01ndash10120583M) plus catechin (01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001) A representative western blot analysis ofeNOS phosphorylation (a)
8 Oxidative Medicine and Cellular Longevity
Other studies have shown that PAD patients have lowFMD [24] strictly dependent on NO release [29] In accor-dance with these findings our PAD population had reducedFMD and lower nitrite and nitrate (markers of NO gener-ation) compared to controls NO is synthesized from theamino acid L-arginine by a family of enzymes known asnitric oxide synthase (NOS) [30] Among three isoformseNOS is the major responsible for NO production in thecardiovascular system [31] Enhanced eNOS inactivationcaused by excessive ROS production may play an importantrole in the impairment of endothelium-dependent vasodi-latation When HUVEC were coincubated with SAPAD asignificant reduction in p-eNOSwith consequent drop inNObioavailability was observed
In a previous study we showed that the combination ofepicatechin with catechin was able to boost the antioxidantand antiplatelet effects achieved by a single polyphenol[19] Moreover in a recent study we observed that PADpatients treated with dark chocolate rich in epicatechin andcatechin showed a decrease of oxidative stress 2 hours afterits assumption [32]
In our study we explored the hypothesis that polyphe-nols can downregulate platelets activation and subsequentlyreduce ECs activation Therefore we incubated HUVECwith supernatants of activated platelets pretreated with scalardoses of epicatechin plus catechinThese experiments showedthat this mutual combination leads to a decrease in sCAMlevels and an increase in eNOS phosphorylationwith a higherbioavailability of NO
The present study has implications and limitations thatshould be acknowledged This paper suggests a potential roleof polyphenols in the modulation of endothelial function butlacks a specific in vivo proof Given that supernatants havebeen used in the study it is possible to hypothesize by ourresults that platelets can exert their action by an indirectfashion A deeper investigation on supernatant derived fromPAD patients could highlight which specific molecules areresponsible for these effects and their biochemical mecha-nism Last since ROS have a short half-life thus representinga potential limitation of the study a further step forwardwill be the evaluation of other mechanisms independentfrom oxidative stress or more stable markers of oxidativestress able to induce endothelial dysfunction Besides theseresults require further investigation in order to assesswhethersimilar effects can be obtained in vivo by the administrationof aliments or surrogates rich in polyphenols to PAD patientsas in the hierarchy of events that underlie atheroscleroticremodeling of the adult human vessels and the modulationof endothelial activation in PAD patients seems to play a keyrole
In conclusion this study demonstrates that polyphenolsare able to modulate HUVEC activation induced by plateletsderived from PAD patients
Conflict of Interests
The authors confirm that there is no conflict of interests
Acknowledgment
This work was supported by the Grant University of RomeldquoSapienzardquo year 2011 Project no C26A11J528 to GiacomoFrati
References
[1] W Insull Jr ldquoThe pathology of atherosclerosis plaque develop-ment and plaque responses tomedical treatmentrdquoTheAmericanJournal ofMedicine vol 122 no 1 supplement pp S3ndashS14 2009
[2] J I Borissoff H M H Spronk and H T Cate ldquoThe hemostaticsystem as a modulator of atherosclerosisrdquo The New EnglandJournal of Medicine vol 364 no 18 pp 1746ndash1760 2011
[3] S M Schoenwaelder Y Yuan E C Josefsson et al ldquoTwodistinct pathways regulate platelet phosphatidylserine exposureand procoagulant functionrdquo Blood vol 114 no 3 pp 663ndash6662009
[4] J E Italiano Jr J L Richardson S Patel-Hett et al ldquoAngiogen-esis is regulated by a novel mechanism pro- and antiangiogenicproteins are organized into separate platelet 120572 granules anddifferentially releasedrdquo Blood vol 111 no 3 pp 1227ndash1233 2008
[5] J D Pearson ldquoNormal endothelial cell functionrdquo Lupus vol 9no 3 pp 183ndash188 2000
[6] M T Gentile C Vecchione G Marino et al ldquoResistin impairsinsulin-evoked vasodilationrdquo Diabetes vol 57 no 3 pp 577ndash583 2008
[7] G R Wang Y Zhu P V Halushka T M Lincoln and M EMendelsohn ldquoMechanism of platelet inhibition by nitric oxidein vivo phosphorylation of thromboxane receptor by cyclicGMP-dependent protein kinaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no9 pp 4888ndash4893 1998
[8] Y Ohara T E Peterson and D G Harrison ldquoHypercholes-terolemia increases endothelial superoxide anion productionrdquoJournal of Clinical Investigation vol 91 no 6 pp 2546ndash25511993
[9] D D Wagner and P S Frenette ldquoThe vessel wall and itsinteractionsrdquo Blood vol 111 no 11 pp 5271ndash5281 2008
[10] G Brevetti A Silvestro V Schiano and M ChiarielloldquoEndothelial dysfunction and cardiovascular risk predictionin peripheral arterial desease additive value of flow-mediatedilatation to ankle-brachial pressure indexrdquo Circulation vol108 no 17 pp 2093ndash2098 2003
[11] C Heiss T Lauer A Dejam et al ldquoPlasma nitroso compoundsare decreased in patients with endothelial dysfunctionrdquo Journalof the American College of Cardiology vol 47 no 3 pp 573ndash5792006
[12] L Loffredo R Carnevale R Cangemi et al ldquoNOX2 up-regulation is associated with artery dysfunction in patients withperipheral artery diseaserdquo International Journal of Cardiologyvol 165 no 1 pp 184ndash192 2013
[13] J K Bendall R Rinze D Adlam A L Tatham J de Bonoand K M Channon ldquoEndothelial Nox2 overexpression poten-tiates vascular oxidative stress and hemodynamic response toangiotensin II Studies in endothelial-targeted Nox2 transgenicmicerdquo Circulation Research vol 100 no 7 pp 1016ndash1025 2007
[14] O Jung J G Schreiber H Geiger T Pedrazzini R Busse andR P Brandes ldquogp91phox-containing NADPH oxidase mediatesendothelial dysfunction in renovascular hypertensionrdquoCircula-tion vol 109 no 14 pp 1795ndash1801 2004
Oxidative Medicine and Cellular Longevity 9
[15] M de Lorgeril P Salen J L Martin I Monjaud J Delaye andN Mamelle ldquoMediterranean diet traditional risk factors andthe rate of cardiovascular complications aftermyocardial infarc-tion final report of the Lyon Diet Heart Studyrdquo Circulation vol99 no 6 pp 779ndash785 1999
[16] T L Zern and M L Fernandez ldquoCardioprotective effects ofdietary polyphenolsrdquo Journal of Nutrition vol 135 no 10 pp2291ndash2294 2005
[17] O Vitseva S Varghese S Chakrabarti J D Folts and JE Freedman ldquoGrape seed and skin extracts inhibit plateletfunction and release of reactive oxygen intermediatesrdquo Journalof Cardiovascular Pharmacology vol 46 no 4 pp 445ndash4512005
[18] P Pignatelli A Ghiselli B Buchetti et al ldquoPolyphenols syner-gistically inhibit oxidative stress in subjects given red and whitewinerdquo Atherosclerosis vol 188 no 1 pp 77ndash83 2006
[19] R Carnevale L Loffredo P Pignatelli et al ldquoDark chocolateinhibits platelet isoprostanes via NOX2 down-regulation insmokersrdquo Journal of Thrombosis and Haemostasis vol 10 no1 pp 125ndash132 2012
[20] C Menna E de Falco L Pacini G Scafetta P Ruggieri and RPuca ldquoAxitinib affects cell viability and migration of a primaryfoetal lung adenocarcinoma culturerdquo Cancer Investigation vol32 no 1 pp 13ndash21 2014
[21] S W Hoffman R L Roof and D G Stein ldquoA reliableand sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F(2120572) a marker for lipid peroxidation afterexperimental brain injuryrdquo Journal of Neuroscience Methodsvol 68 no 2 pp 133ndash136 1996
[22] P Pignatelli R Carnevale R Cangemi et al ldquoAtorvastatininhibits gp91119901ℎ119900119909 circulating levels in patients with hypercholes-terolemiardquo Arteriosclerosis Thrombosis and Vascular Biologyvol 30 no 2 pp 360ndash367 2010
[23] M C Corretti T J Anderson E J Benjamin et al ldquoGuidelinesfor the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery a report of theinternational brachial artery reactivity task forcerdquo Journal of theAmerican College of Cardiology vol 39 no 2 pp 257ndash265 2002
[24] L Loffredo A Marcoccia P Pignatelli et al ldquoOxidative-stress-mediated arterial dysfunction in patients with peripheralarterial diseaserdquo European Heart Journal vol 28 no 5 pp 608ndash612 2007
[25] A T Hirsch Z J Haskal N R Hertzer et al ldquoACCAHA2005 Practice Guidelines for the management of patients withperipheral arterial disease (lower extremity renal mesentericand abdominal aortic) a collaborative report from the Amer-ican Association for Vascular SurgerySociety for VascularSurgery Society for Cardiovascular Angiography and Interven-tions Society for Vascular Medicine and Biology Society ofInterventional Radiology and the ACCAHA Task Force onPractice Guidelines (Writing Committee to Develop Guidelinesfor the Management of Patients With Peripheral Arterial Dis-ease) endorsed by the American Association of Cardiovascularand Pulmonary Rehabilitation National Heart Lung andBlood Institute Society for Vascular Nursing TransAtlanticInter-Society Consensus and Vascular Disease FoundationrdquoCirculation vol 113 no 11 pp e463ndashe654 2005
[26] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999
[27] T Dickfeld E Lengyel A E May et al ldquoTransient interactionof activated platelets with endothelial cells induces expression
of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway implications foratherogenesisrdquo Cardiovascular Research vol 49 no 1 pp 189ndash199 2001
[28] A G Kjaeligrgaard A Dige J Krog E Toslashnnesen and LWogensen ldquoSoluble adhesion molecules correlate with surfaceexpression in an in vitro model of endothelial activationrdquo Basicand Clinical Pharmacology and Toxicology vol 113 no 49 pp273ndash279 2013
[29] R Joannides W E Haefeli L Linder et al ldquoNitric oxide isresponsible for flow-dependent dilatation of human peripheralconduit arteries in vivordquoCirculation vol 91 no 5 pp 1314ndash13191995
[30] R Bruckdorfer ldquoThe basics about nitric oxiderdquo MolecularAspects of Medicine vol 26 no 1-2 pp 3ndash31 2005
[31] D M Dudzinski and T Michel ldquoLife history of eNOS partnersand pathwaysrdquo Cardiovascular Research vol 75 no 2 pp 247ndash260 2007
[32] L Loffredo L Perri E Catasca et al ldquoDark chocolate acutelyimproves walking autonomy in patients with peripheral arterydiseaserdquo Journal of the American Heart Association vol 3Article ID e001072 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Oxidative Medicine and Cellular Longevity
Vascular endothelium responds to mechanical and hor-monal stimuli by releasing signaling molecules involved inthe modulation of haemostasis such as nitric oxide (NO)[5 6] which acts by enabling mechanisms of relaxation andvasodilatation in vascular smooth muscle cells (VSMCs) andby exerting profound functional and morphological effectson the vascular wall Nitric oxide also exerts importantanti-inflammatory and antithrombotic effects [7] In par-ticular ROS production rapidly inactivates the vasorelax-ation molecule NO contributing to endothelial dysfunctiondescribed as impaired vasodilatation [8]
In addition to the reduction in NO bioavailabilityplatelet-induced endothelium activation leads to increasedexpression of cell adhesion molecules (CAMs) such as inter-cellular adhesion molecule-1 (ICAM-1) vascular adhesionmolecule-1 (VCAM-1) and E-selectin [9]
Among different clinical settings associated with athero-sclerosis peripheral artery disease (PAD) is considered perse a sign of systemic atherosclerosis Moreover PAD patientsdisplay endothelial dysfunction as demonstrated by lowflow-mediated dilatation (FMD) [10] which has become aconventional measure of endothelial dysfunction in humanatherosclerosis [11] Consistently with reduced FMD PADpatients have enhanced NOX2 activation [12] and the cat-alytic subunit ofNADPHoxidase aswell as themajor enzymegenerating ROS involved in the modulation of arterial tone[13 14]
Epidemiological studies have shown that a diet rich inpolyphenols is able to reduce cardiovascular events [15 16]Polyphenols a heterogeneous group of molecules foundprimarily in fruits and vegetables along with their effects onplatelets function have been investigated by several authorsAmong other activities an increase in NO production asso-ciated with a decrease in ROS formation has been described[17 18] This finding is in accordance with our previousreport indicating that polyphenols synergize in exertingantioxidant and antiplatelet effects [19] In fact we observedthat epicatechin plus catechin is able to exert an antioxidanteffect greater than individual polyphenols [19] Neverthelessfew data concerning the effects of polyphenols on platelet-endothelium interaction are available in PAD patients Theaim of this study was to investigate whether polyphenolsare able to modulate endothelial function via inhibition ofplatelet-endothelial interaction Furthermore we tested theeffect of epicatechin plus catechin on the interaction betweenECs and platelets
2 Materials and Methods
21 Cross-Sectional Study The study has been carried out inthe outpatient clinic of our division between January 2013 andSeptember 2013 on 10 patients aged between 40 and 80 yearspresenting with symptoms of intermittent claudication (for atleast 6 months) and corresponding to Fontaine stage IIb Tenconsecutive subjects matched for age gender cardiovascularrisk factors and pharmacological treatments were enrolled inthe same period as controls Each PAD patient to be enrolledin the study had
(1) claudication (defined as leg pain occurring after lessthan 200 meters of walking relieved within 10 min-utes after cessation of exercise and being of presumedatherosclerotic origin)
(2) anklebrachial index (ABI) which was assessed asanklearm systolic blood pressure ratio by Dopplerultrasonography lower than 090 on the worst leg atrest
All subjects underwent a full medical anamnestic collec-tion physical examination 12-lead ECG laboratory tests andmeasurement of ABI Patients had to be in stable conditionswithout abrupt changes of ABI (gt20) in the last monthbefore the enrolment Exclusion criteria were represented byliver insufficiency serious renal disorders (serum creatininegt 28mgdL) acute cerebrovascular disease acutemyocardialinfarction deep venous thrombosis tabagism and ongoingtherapy with antioxidants Written informed consent wasobtained from all subjects the study was conformed to theethical guidelines of the 1975 Declaration of Helsinki and wasapproved by the Ethical Committee of Policlinico UmbertoI Sapienza University of Rome (ClinicalTrialsgov identifierNCT01947712)
22 Platelet Preparation Venous blood was drawn intrisodium citrate (38 110 (v v)) from fasting HS andPAD patients who had not taken any drugs affecting plateletfunction for at least 14 days To obtain platelet rich plasma(PRP) blood was centrifuged at 15min at 180 g at roomtemperature and the supernatant PRPwas separated To avoidleukocyte contamination only the top 75 of the PRP wascollected To obtain washed platelets the PRP was washedwith ACD (101 vv) and suspended in fatty acid-free Tyrodersquosbuffer (2 times 108 plateletsmL unless otherwise noted)
Washed platelets were treated with a scalar dose of epi-catechin (01ndash10 120583M) plus catechin (01ndash10 120583M) and activatedwith arachidonic acid (AA) (05mM) for 10min at 37∘C andsupernatant was stored at minus80∘C unless otherwise indicated
23 Human Umbilical Vein Endothelial Cells (HUVEC)Human umbilical vein endothelial cells (HUVEC) werecultured as described [20] Briefly cells were expanded (2000cellscm2) in complete medium (EndoGRO-LS CompleteMedia Kit Millipore) Cell morphology and growth weremonitored by light microscopy and assessed by Trypan Blue(Sigma St Louis USA) The culture was expanded untilpassage 5
24 Platelet ROS Production Cell suspension was incubatedwith 2101584071015840-dichlorofluorescein diacetate (5 120583molL) for 15minutes at 37∘C After incubation platelets were activatedwith AA Platelet ROS production was expressed as stim-ulation index (mean level of fluorescence in stimulatedcellsmean level of fluorescence in unstimulated cells) (SI)Fluorescence intensity was analyzed on an Epics XL-MCLcytometer (Coulter Electronics) equipped with an argonlaser at 510ndash550 nm (green) For every histogram 50000plateletswere counted to determine the proportion of positive
Oxidative Medicine and Cellular Longevity 3
platelets The fluorescent signal generated by the probe wasexpressed as mean fluorescence intensity (SI) Intra-assaycoefficient of variation was 5
25 Platelet 8-iso-PGF2120572 Assays To measure isoprostanesformation platelets were activated with AAThe supernatantwas stored at minus80∘C until measurement Quantification ofisoprostanes was performed measuring 8-iso-PGF2120572 by apreviously described and validatedEIA assaymethod [21] andwas evaluated in vivo and in vitro Intra-assay and interassaycoefficients of variation were 58 and 50 respectively
26 Platelet NOx A colorimetric assay kit (Tema RicercaItaly) was used to determine the NO metabolites nitrite andnitrate (NOx) in 100 120583L of platelet or HUVEC suspensionmaintained under stirring condition for 10min at 37∘C Intra-assay and interassay coefficients of variation were 29 and17 respectively
27 Platelet H2O2Concentration A colorimetric assay kit
(Tema Ricerca Italy) was used to determine the H2O2
concentrationin 100120583L of platelet suspension maintainedunder stirring condition for 10min at 37∘C Intra-assayand interassay coefficients of variation were 21 and 37respectively
28 ELISA Detection of Platelet sNOX2-dp NOX2-derivedpeptide (NOX2-dp) a marker of NADPH oxidase activationwas detected by ELISA method as previously described byPignatelli et al [22] The peptide was recognized by a specificmonoclonal antibody against the amino acidic sequence(224ndash268 residues) of the extra membrane portion of NOX2To measure sNOX2-dp platelet suspension was activatedwith AA and 100 120583L of the supernatant stored at minus80∘C untilmeasurement Values were expressed as pgmL intra-assayand interassay coefficients of variation were 52 and 6respectively
29 Detection of HUVEC sICAM1 sVCAM1 and sE-SelectinA Custom Human 3-Plex Array (Tema Ricerca Italy) wasused to determine the sICAM1 sVCAM1 and sE-selectinconcentration Values were expressed as ngmL intra-assayand interassay coefficients of variation were 52 and 6respectively
210 Western Blot Analysis of p-eNOS and Total eNOS Equalamounts of protein (30 120583glane) estimated by Bradford assaywere solubilized in a 2X Laemmli sample buffer containing2-mercaptoethanol and loaded in a denaturing SDS10polyacrylamide gel Western blot analysis was performedwith monoclonal anti-mouse eNOS-PSer1177 (1 1000 BDTransduction Laboratory) (2 120583gmL) and anti-mouse eNOS(1 1000 BD Transduction Laboratory) (2120583gmL) incubatedovernight at 4∘C After incubation the pure nitrocellulosemembranes (045 120583m) were washed and incubated withgoat anti-mouse IgG1-horseradish peroxidase for 2 hoursImmune complexes were detected by enhanced chemilumi-nescence The developed spots were quantified by densito-metric analysis on a NIH Image 162f analyzer and the value
was expressed as arbitrary units Each sample was analyzed intriplicate The results were expressed as P-eNOStotal eNOSratio
211 FMD and Carotid Intima-Media Thickness Ultrasoundassessment of FMDwas investigated according to the recentlyreported guidelines [23] as previously described [24] Thecoefficient of variation for FMDmeasurements obtained on 3separate occasions was 125 Longitudinal ultrasonographicscans of the carotid artery were obtained on the same day asthe studies of the brachial artery reactivity and included theevaluation of the right and left common carotid arteries 1 cmproximal to the carotid bulb FMD was performed with a 75MHz linear-array transducer ultrasound system (SonomedLake Success NY)
212 In Vitro Study In vitro study was performed in bloodtaken from 5 PAD patients and 5 HS (3 males and 2 femalesmean age 56 plusmn 6 years) We analysed the effect of scalardoses of epicatechin (01ndash10120583M)plus catechin (01ndash10 120583M) onplatelet-HUVEC interaction
Only PAD patients platelets (2 times 108mL) were incu-bated for 30min at 37∘C with epicatechin (01ndash10 120583M) pluscatechin (01ndash10 120583M) (mix) and stimulated for 10min withAA (05mM) After incubation samples were immediatelypelleted at 4∘C and the supernatants were added to semicon-fluent endothelial monolayers with and without endothelialgrowth factor (EGF 10 ngmL)
After 60 minutes of coincubation supernatants wereremoved by gentle washing which was confirmed by lightmicroscopy observation Afterwards cultures were left for afurther period of 2 hours in incubator with basalmedium andharvested by trypsin Then both HUVEC and HUVEC cul-ture supernatants were collected for the analysis of sICAM1sVCAM1 sE-selectin NOx concentration eNOS phosphory-lation and total eNOS
213 Sample Size Determination We computed the mini-mum sample size with respect to a two-tailed one-sampleStudentrsquos 119905-test considering (i) a difference for NOX to bedetected between the healthy subjects and PAD patients as|120575| 15 120583M (ii) homogeneous SDs between groups as 4 and(iii) type I error probability as 120572 = 005 and power 1 minus 120573 =090 This resulted in a minimum sample size of 119899 = 4for group Additional patients and cases were included fora subset of experiments in order to provide more externallyvalid findings
214 Statistical Analyses Continuous variables are reportedas mean plusmn standard deviation and were compared withStudentrsquos 119905-test Categorical variables are reported as andcompared with the 1205942 test Statistical significance was set atthe 2-tailed 005 level Computations were performed withSPSS 18 (IBM Armonk NY USA)
3 Results
31 Cross-Sectional Study Clinical characteristics of PADpatients and HS are reported in Table 1
4 Oxidative Medicine and Cellular Longevity
Table 1 Clinical characteristics of patients with peripheral artery disease (PAD) and healthy subjects (HS)
Variables PAD (119899 = 10) HS (119899 = 10) 119875 valueMean age (year)a 63 plusmn 5 65 plusmn 6 0854Malesfemales 64 64 10Hypertension (119899)b 98 (9) 0 (0) lt0001Diabetes mellitus (119899) 30 (3) 0 (0) lt0001Dyslipidemia (119899) 80 (8) 0 (0) lt0001Former smokers (119899) 40 (4) 50 (5) 0621CHD (119899) 20 (2) 0 (0) lt0001Glycemia (mgdL) 108 plusmn 6 104 plusmn 4 0589Pharmacological treatments (119899)
ACE-inhibitors 80 (8) 0 (0) lt0001Oral antidiabetic drugs 30 (3) 0 (0) lt0001Insulin 0 (0) 0 (0) 10Statin 70 (7) 0 (0) lt0001Antiaggregants 60 (6) 0 (0) lt0001Oral anticoagulants 20 (2) 0 (0) lt0001
aData are expressed as mean plusmn SDbgt139ndash89mmHg
PAD patients showed higher platelet ROS production(106 plusmn 14 SI versus 34 plusmn 09 SI lowast119875 lt 0001) (Figure 1(a))H2O2concentration (184plusmn07 120583Mversus 54plusmn05 120583M lowast119875 lt
0001) (Figure 1(b)) sNOX2-dp release (298 plusmn 134 pgmLversus 79 plusmn 23 pgmL lowast119875 lt 0001) (Figure 1(c)) and 8-iso-PGF2120572 production (1626 plusmn 428 pmolL versus 670 plusmn64 pmolL lowast119875 lt 0001) (Figure 1(d)) compared to HS PADpatients displayed lower NOx production (287 plusmn 63 120583Mversus 103 plusmn 26 120583M 119875 lt 0001) (Figure 1(e)) and FMDvalues (20plusmn25 versus 77plusmn10 lowast119875 lt 0001) (Figure 1(f))compared with HS
32 In Vitro Study HUVEC stimulated with EGF releaseda higher amount of sICAM1 (Figure 2(a) lowast119875 lt 0001)sVCAM1 (Figure 2(b) lowast119875 lt 0001) and E-selectin(Figure 2(c) lowast119875 lt 0001) versus unstimulated HUVEC
Supernatant of activated platelets from PAD patients(SAPAD) was able to increase sICAM1 sVCAM1 and sE-selectin versus nonstimulated HUVEC (Figure 2) Con-versely supernatant of activated platelets from HS (SAHS)was not able to change markers of endothelial dysfunction(Figure 2)
When SAPAD pretreated with mix of epicatechin (01ndash10 120583M) and catechin (01ndash10 120583M) was added to HUVEC asignificant reduction of sICAM1 sVCAM1 and sE-selectinlevels was observed compared to sCAMs levels induced bySAPAD (Figures 2(a) 2(b) and 2(c))
Both the P-eNOStotal eNOS ratio (Figure 3(a) lowast119875 lt0001) andNOx concentration (Figure 3(b) lowast119875 lt 0001) weredecreased in HUVEC when stimulated with EGF Moreoverwe found that SAPAD contributed to the reduction of P-eNOStotal eNOS ratio compared to untreated HUVEC(Figure 3(a) lowast119875 lt 0001) with consequent reduction of NObioavailability (Figure 3(b) lowast119875 lt 00001) This phenomenonwas not observed in HUVEC treated with SAHS (Figure 3)
When SAPAD pretreated with mix of epicatechin (01ndash10 120583M) and catechin (01ndash10 120583M) was added to HUVECboth the P-eNOStotal eNOS ratio and NO bioavailabilityincreased compared to P-eNOStotal eNOS ratio and NObioavailability induced by SAPAD (Figures 3(a) and 3(b))
4 DiscussionThe major findings of this paper are as follows (a) PADpatients show higher platelet activation than HS (b) super-natant of activated platelets derived from PAD patients(SAPAD) is able to activate ECs resulting in an upregula-tion of soluble molecules derived from ECs (ie sICAM-1sVCAM-1 and E-selectin) (c) PAD platelets induced ECsactivation by reduction of p-eNOS and bioavailability of NO(d) polyphenols are able to reduce endothelial activationinduced by activated platelets
These novel findings support the hypothesis that platelets-induced endothelial activation and thus the progression ofatherosclerosis are a predisposing factor to more complexdiseases such as PAD [25]
Previous findings showed that in PAD patients there isan imbalance between NOX2-mediated oxidative stress andFMD [11] In the present study we expanded such evidencedemonstrating the key oxidative role of platelets in PADpatients by measuring their ROS production and sNOX2-dp release as markers of NADPH oxidase activation [22]as well as their 8-iso-PGF2120572-III production We observeda significant increase of these oxidative stress markers inplatelets of PAD patients compared to HS
Activated platelets can interact with the endothelium trig-gering an inflammatory response that may contribute to theearly stages of atherosclerosis [26] Upon activation plateletsrelease granule contents able to activate ECs It has beenshown that activated platelets interact with the endotheliuminducing accumulation and transmigration of circulating
Oxidative Medicine and Cellular Longevity 5
0
2
4
6
8
10
12
14
HS PAD
lowast
Plat
elet
RO
S pr
oduc
tion
(SI)
(a)
0
5
10
15
20
HS PAD
lowast
Plat
elet
H2O2
conc
entr
atio
n (120583
M)
(b)
0
5
10
15
20
25
30
35
40
HS PAD
lowast
Plat
elet
sNO
X2-d
p (p
gm
L)
(c)
0
50
100
150
200
HS PAD
lowastPl
atel
et8
-iso-
PGF2120572
(pm
olL
)
(d)
0
5
10
15
20
25
30
35
HS PAD
lowast
Plat
elet
NO
x pr
oduc
tion
(120583M
)
(e)
0
1
2
3
4
5
6
7
8
9
10
11
HS PAD
lowast
FMD
()
(f)
Figure 1 Platelet-induced oxidative stress in PAD patients Platelet ROS production (a) platelet H2O2concentration (b) platelet sNOX2-dp
levels (c) platelet 8-iso-PGF2120572 production (d) platelet NOx production (e) and FMD (f) in PAD patients (119899 = 10) and healthy subjects(119899 = 10) (lowast119875 lt 0001)
6 Oxidative Medicine and Cellular Longevity
0
50
100
150
200
250
300
350
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
lowastlowast
lowast
NS
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sICA
M1
leve
ls (n
gm
L)
(a)
0
5
10
15
20
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sVCA
M1
leve
ls (n
gm
L)(b)
0
50
100
150
200
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sE-s
elec
tion
leve
ls (n
gm
L)
(c)
Figure 2 Biomarkers of HUVEC activation sICAM1 (a) sVCAM1 (b) and E-selectin (c) levels released by HUVEC after incubation withEGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activated PAD patients (SAPAD) (blackbar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalar doses of epicatechin (01ndash10120583M) plus catechin(01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001)
monocytes finally contributing to the atherosclerotic processat early stages [27] Moreover activated platelets can releasein their microenvironment prooxidant molecules such asisoprostanes and ROS In fact we found that supernatant ofPAD platelets is able to stimulate HUVEC with consequent
release of cellular adhesion molecules in their soluble formnamely CAMs
The soluble forms of adhesion molecules reflect endothe-lial CAM surface expression [28] and therefore can be usedas biomarkers of endothelial activation in different diseases
Oxidative Medicine and Cellular Longevity 7
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
P-eNOS
120573-Actin
0
5
10
15
20
25
30
35
40
45
lowast
lowastlowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
Den
sitom
etric
anal
ysis
ofP-
eNO
Sto
tal e
-NO
S (r
atio
)
Total eNOS
(a)
50
0
5
10
15
20
25
30
35
40
45 lowastlowast
lowast lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
HU
VEC
s NO
x pr
oduc
tion
(120583M
)
(b)
Figure 3 HUVEC eNOS activation and NOx production Densitometric analysis of P-eNOStotal eNOS ratio (a) and NOx production (b)in HUVEC incubated with EGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activatedplatelets from PAD patients (SAPAD) (black bar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalardoses of epicatechin (01ndash10120583M) plus catechin (01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001) A representative western blot analysis ofeNOS phosphorylation (a)
8 Oxidative Medicine and Cellular Longevity
Other studies have shown that PAD patients have lowFMD [24] strictly dependent on NO release [29] In accor-dance with these findings our PAD population had reducedFMD and lower nitrite and nitrate (markers of NO gener-ation) compared to controls NO is synthesized from theamino acid L-arginine by a family of enzymes known asnitric oxide synthase (NOS) [30] Among three isoformseNOS is the major responsible for NO production in thecardiovascular system [31] Enhanced eNOS inactivationcaused by excessive ROS production may play an importantrole in the impairment of endothelium-dependent vasodi-latation When HUVEC were coincubated with SAPAD asignificant reduction in p-eNOSwith consequent drop inNObioavailability was observed
In a previous study we showed that the combination ofepicatechin with catechin was able to boost the antioxidantand antiplatelet effects achieved by a single polyphenol[19] Moreover in a recent study we observed that PADpatients treated with dark chocolate rich in epicatechin andcatechin showed a decrease of oxidative stress 2 hours afterits assumption [32]
In our study we explored the hypothesis that polyphe-nols can downregulate platelets activation and subsequentlyreduce ECs activation Therefore we incubated HUVECwith supernatants of activated platelets pretreated with scalardoses of epicatechin plus catechinThese experiments showedthat this mutual combination leads to a decrease in sCAMlevels and an increase in eNOS phosphorylationwith a higherbioavailability of NO
The present study has implications and limitations thatshould be acknowledged This paper suggests a potential roleof polyphenols in the modulation of endothelial function butlacks a specific in vivo proof Given that supernatants havebeen used in the study it is possible to hypothesize by ourresults that platelets can exert their action by an indirectfashion A deeper investigation on supernatant derived fromPAD patients could highlight which specific molecules areresponsible for these effects and their biochemical mecha-nism Last since ROS have a short half-life thus representinga potential limitation of the study a further step forwardwill be the evaluation of other mechanisms independentfrom oxidative stress or more stable markers of oxidativestress able to induce endothelial dysfunction Besides theseresults require further investigation in order to assesswhethersimilar effects can be obtained in vivo by the administrationof aliments or surrogates rich in polyphenols to PAD patientsas in the hierarchy of events that underlie atheroscleroticremodeling of the adult human vessels and the modulationof endothelial activation in PAD patients seems to play a keyrole
In conclusion this study demonstrates that polyphenolsare able to modulate HUVEC activation induced by plateletsderived from PAD patients
Conflict of Interests
The authors confirm that there is no conflict of interests
Acknowledgment
This work was supported by the Grant University of RomeldquoSapienzardquo year 2011 Project no C26A11J528 to GiacomoFrati
References
[1] W Insull Jr ldquoThe pathology of atherosclerosis plaque develop-ment and plaque responses tomedical treatmentrdquoTheAmericanJournal ofMedicine vol 122 no 1 supplement pp S3ndashS14 2009
[2] J I Borissoff H M H Spronk and H T Cate ldquoThe hemostaticsystem as a modulator of atherosclerosisrdquo The New EnglandJournal of Medicine vol 364 no 18 pp 1746ndash1760 2011
[3] S M Schoenwaelder Y Yuan E C Josefsson et al ldquoTwodistinct pathways regulate platelet phosphatidylserine exposureand procoagulant functionrdquo Blood vol 114 no 3 pp 663ndash6662009
[4] J E Italiano Jr J L Richardson S Patel-Hett et al ldquoAngiogen-esis is regulated by a novel mechanism pro- and antiangiogenicproteins are organized into separate platelet 120572 granules anddifferentially releasedrdquo Blood vol 111 no 3 pp 1227ndash1233 2008
[5] J D Pearson ldquoNormal endothelial cell functionrdquo Lupus vol 9no 3 pp 183ndash188 2000
[6] M T Gentile C Vecchione G Marino et al ldquoResistin impairsinsulin-evoked vasodilationrdquo Diabetes vol 57 no 3 pp 577ndash583 2008
[7] G R Wang Y Zhu P V Halushka T M Lincoln and M EMendelsohn ldquoMechanism of platelet inhibition by nitric oxidein vivo phosphorylation of thromboxane receptor by cyclicGMP-dependent protein kinaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no9 pp 4888ndash4893 1998
[8] Y Ohara T E Peterson and D G Harrison ldquoHypercholes-terolemia increases endothelial superoxide anion productionrdquoJournal of Clinical Investigation vol 91 no 6 pp 2546ndash25511993
[9] D D Wagner and P S Frenette ldquoThe vessel wall and itsinteractionsrdquo Blood vol 111 no 11 pp 5271ndash5281 2008
[10] G Brevetti A Silvestro V Schiano and M ChiarielloldquoEndothelial dysfunction and cardiovascular risk predictionin peripheral arterial desease additive value of flow-mediatedilatation to ankle-brachial pressure indexrdquo Circulation vol108 no 17 pp 2093ndash2098 2003
[11] C Heiss T Lauer A Dejam et al ldquoPlasma nitroso compoundsare decreased in patients with endothelial dysfunctionrdquo Journalof the American College of Cardiology vol 47 no 3 pp 573ndash5792006
[12] L Loffredo R Carnevale R Cangemi et al ldquoNOX2 up-regulation is associated with artery dysfunction in patients withperipheral artery diseaserdquo International Journal of Cardiologyvol 165 no 1 pp 184ndash192 2013
[13] J K Bendall R Rinze D Adlam A L Tatham J de Bonoand K M Channon ldquoEndothelial Nox2 overexpression poten-tiates vascular oxidative stress and hemodynamic response toangiotensin II Studies in endothelial-targeted Nox2 transgenicmicerdquo Circulation Research vol 100 no 7 pp 1016ndash1025 2007
[14] O Jung J G Schreiber H Geiger T Pedrazzini R Busse andR P Brandes ldquogp91phox-containing NADPH oxidase mediatesendothelial dysfunction in renovascular hypertensionrdquoCircula-tion vol 109 no 14 pp 1795ndash1801 2004
Oxidative Medicine and Cellular Longevity 9
[15] M de Lorgeril P Salen J L Martin I Monjaud J Delaye andN Mamelle ldquoMediterranean diet traditional risk factors andthe rate of cardiovascular complications aftermyocardial infarc-tion final report of the Lyon Diet Heart Studyrdquo Circulation vol99 no 6 pp 779ndash785 1999
[16] T L Zern and M L Fernandez ldquoCardioprotective effects ofdietary polyphenolsrdquo Journal of Nutrition vol 135 no 10 pp2291ndash2294 2005
[17] O Vitseva S Varghese S Chakrabarti J D Folts and JE Freedman ldquoGrape seed and skin extracts inhibit plateletfunction and release of reactive oxygen intermediatesrdquo Journalof Cardiovascular Pharmacology vol 46 no 4 pp 445ndash4512005
[18] P Pignatelli A Ghiselli B Buchetti et al ldquoPolyphenols syner-gistically inhibit oxidative stress in subjects given red and whitewinerdquo Atherosclerosis vol 188 no 1 pp 77ndash83 2006
[19] R Carnevale L Loffredo P Pignatelli et al ldquoDark chocolateinhibits platelet isoprostanes via NOX2 down-regulation insmokersrdquo Journal of Thrombosis and Haemostasis vol 10 no1 pp 125ndash132 2012
[20] C Menna E de Falco L Pacini G Scafetta P Ruggieri and RPuca ldquoAxitinib affects cell viability and migration of a primaryfoetal lung adenocarcinoma culturerdquo Cancer Investigation vol32 no 1 pp 13ndash21 2014
[21] S W Hoffman R L Roof and D G Stein ldquoA reliableand sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F(2120572) a marker for lipid peroxidation afterexperimental brain injuryrdquo Journal of Neuroscience Methodsvol 68 no 2 pp 133ndash136 1996
[22] P Pignatelli R Carnevale R Cangemi et al ldquoAtorvastatininhibits gp91119901ℎ119900119909 circulating levels in patients with hypercholes-terolemiardquo Arteriosclerosis Thrombosis and Vascular Biologyvol 30 no 2 pp 360ndash367 2010
[23] M C Corretti T J Anderson E J Benjamin et al ldquoGuidelinesfor the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery a report of theinternational brachial artery reactivity task forcerdquo Journal of theAmerican College of Cardiology vol 39 no 2 pp 257ndash265 2002
[24] L Loffredo A Marcoccia P Pignatelli et al ldquoOxidative-stress-mediated arterial dysfunction in patients with peripheralarterial diseaserdquo European Heart Journal vol 28 no 5 pp 608ndash612 2007
[25] A T Hirsch Z J Haskal N R Hertzer et al ldquoACCAHA2005 Practice Guidelines for the management of patients withperipheral arterial disease (lower extremity renal mesentericand abdominal aortic) a collaborative report from the Amer-ican Association for Vascular SurgerySociety for VascularSurgery Society for Cardiovascular Angiography and Interven-tions Society for Vascular Medicine and Biology Society ofInterventional Radiology and the ACCAHA Task Force onPractice Guidelines (Writing Committee to Develop Guidelinesfor the Management of Patients With Peripheral Arterial Dis-ease) endorsed by the American Association of Cardiovascularand Pulmonary Rehabilitation National Heart Lung andBlood Institute Society for Vascular Nursing TransAtlanticInter-Society Consensus and Vascular Disease FoundationrdquoCirculation vol 113 no 11 pp e463ndashe654 2005
[26] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999
[27] T Dickfeld E Lengyel A E May et al ldquoTransient interactionof activated platelets with endothelial cells induces expression
of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway implications foratherogenesisrdquo Cardiovascular Research vol 49 no 1 pp 189ndash199 2001
[28] A G Kjaeligrgaard A Dige J Krog E Toslashnnesen and LWogensen ldquoSoluble adhesion molecules correlate with surfaceexpression in an in vitro model of endothelial activationrdquo Basicand Clinical Pharmacology and Toxicology vol 113 no 49 pp273ndash279 2013
[29] R Joannides W E Haefeli L Linder et al ldquoNitric oxide isresponsible for flow-dependent dilatation of human peripheralconduit arteries in vivordquoCirculation vol 91 no 5 pp 1314ndash13191995
[30] R Bruckdorfer ldquoThe basics about nitric oxiderdquo MolecularAspects of Medicine vol 26 no 1-2 pp 3ndash31 2005
[31] D M Dudzinski and T Michel ldquoLife history of eNOS partnersand pathwaysrdquo Cardiovascular Research vol 75 no 2 pp 247ndash260 2007
[32] L Loffredo L Perri E Catasca et al ldquoDark chocolate acutelyimproves walking autonomy in patients with peripheral arterydiseaserdquo Journal of the American Heart Association vol 3Article ID e001072 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 3
platelets The fluorescent signal generated by the probe wasexpressed as mean fluorescence intensity (SI) Intra-assaycoefficient of variation was 5
25 Platelet 8-iso-PGF2120572 Assays To measure isoprostanesformation platelets were activated with AAThe supernatantwas stored at minus80∘C until measurement Quantification ofisoprostanes was performed measuring 8-iso-PGF2120572 by apreviously described and validatedEIA assaymethod [21] andwas evaluated in vivo and in vitro Intra-assay and interassaycoefficients of variation were 58 and 50 respectively
26 Platelet NOx A colorimetric assay kit (Tema RicercaItaly) was used to determine the NO metabolites nitrite andnitrate (NOx) in 100 120583L of platelet or HUVEC suspensionmaintained under stirring condition for 10min at 37∘C Intra-assay and interassay coefficients of variation were 29 and17 respectively
27 Platelet H2O2Concentration A colorimetric assay kit
(Tema Ricerca Italy) was used to determine the H2O2
concentrationin 100120583L of platelet suspension maintainedunder stirring condition for 10min at 37∘C Intra-assayand interassay coefficients of variation were 21 and 37respectively
28 ELISA Detection of Platelet sNOX2-dp NOX2-derivedpeptide (NOX2-dp) a marker of NADPH oxidase activationwas detected by ELISA method as previously described byPignatelli et al [22] The peptide was recognized by a specificmonoclonal antibody against the amino acidic sequence(224ndash268 residues) of the extra membrane portion of NOX2To measure sNOX2-dp platelet suspension was activatedwith AA and 100 120583L of the supernatant stored at minus80∘C untilmeasurement Values were expressed as pgmL intra-assayand interassay coefficients of variation were 52 and 6respectively
29 Detection of HUVEC sICAM1 sVCAM1 and sE-SelectinA Custom Human 3-Plex Array (Tema Ricerca Italy) wasused to determine the sICAM1 sVCAM1 and sE-selectinconcentration Values were expressed as ngmL intra-assayand interassay coefficients of variation were 52 and 6respectively
210 Western Blot Analysis of p-eNOS and Total eNOS Equalamounts of protein (30 120583glane) estimated by Bradford assaywere solubilized in a 2X Laemmli sample buffer containing2-mercaptoethanol and loaded in a denaturing SDS10polyacrylamide gel Western blot analysis was performedwith monoclonal anti-mouse eNOS-PSer1177 (1 1000 BDTransduction Laboratory) (2 120583gmL) and anti-mouse eNOS(1 1000 BD Transduction Laboratory) (2120583gmL) incubatedovernight at 4∘C After incubation the pure nitrocellulosemembranes (045 120583m) were washed and incubated withgoat anti-mouse IgG1-horseradish peroxidase for 2 hoursImmune complexes were detected by enhanced chemilumi-nescence The developed spots were quantified by densito-metric analysis on a NIH Image 162f analyzer and the value
was expressed as arbitrary units Each sample was analyzed intriplicate The results were expressed as P-eNOStotal eNOSratio
211 FMD and Carotid Intima-Media Thickness Ultrasoundassessment of FMDwas investigated according to the recentlyreported guidelines [23] as previously described [24] Thecoefficient of variation for FMDmeasurements obtained on 3separate occasions was 125 Longitudinal ultrasonographicscans of the carotid artery were obtained on the same day asthe studies of the brachial artery reactivity and included theevaluation of the right and left common carotid arteries 1 cmproximal to the carotid bulb FMD was performed with a 75MHz linear-array transducer ultrasound system (SonomedLake Success NY)
212 In Vitro Study In vitro study was performed in bloodtaken from 5 PAD patients and 5 HS (3 males and 2 femalesmean age 56 plusmn 6 years) We analysed the effect of scalardoses of epicatechin (01ndash10120583M)plus catechin (01ndash10 120583M) onplatelet-HUVEC interaction
Only PAD patients platelets (2 times 108mL) were incu-bated for 30min at 37∘C with epicatechin (01ndash10 120583M) pluscatechin (01ndash10 120583M) (mix) and stimulated for 10min withAA (05mM) After incubation samples were immediatelypelleted at 4∘C and the supernatants were added to semicon-fluent endothelial monolayers with and without endothelialgrowth factor (EGF 10 ngmL)
After 60 minutes of coincubation supernatants wereremoved by gentle washing which was confirmed by lightmicroscopy observation Afterwards cultures were left for afurther period of 2 hours in incubator with basalmedium andharvested by trypsin Then both HUVEC and HUVEC cul-ture supernatants were collected for the analysis of sICAM1sVCAM1 sE-selectin NOx concentration eNOS phosphory-lation and total eNOS
213 Sample Size Determination We computed the mini-mum sample size with respect to a two-tailed one-sampleStudentrsquos 119905-test considering (i) a difference for NOX to bedetected between the healthy subjects and PAD patients as|120575| 15 120583M (ii) homogeneous SDs between groups as 4 and(iii) type I error probability as 120572 = 005 and power 1 minus 120573 =090 This resulted in a minimum sample size of 119899 = 4for group Additional patients and cases were included fora subset of experiments in order to provide more externallyvalid findings
214 Statistical Analyses Continuous variables are reportedas mean plusmn standard deviation and were compared withStudentrsquos 119905-test Categorical variables are reported as andcompared with the 1205942 test Statistical significance was set atthe 2-tailed 005 level Computations were performed withSPSS 18 (IBM Armonk NY USA)
3 Results
31 Cross-Sectional Study Clinical characteristics of PADpatients and HS are reported in Table 1
4 Oxidative Medicine and Cellular Longevity
Table 1 Clinical characteristics of patients with peripheral artery disease (PAD) and healthy subjects (HS)
Variables PAD (119899 = 10) HS (119899 = 10) 119875 valueMean age (year)a 63 plusmn 5 65 plusmn 6 0854Malesfemales 64 64 10Hypertension (119899)b 98 (9) 0 (0) lt0001Diabetes mellitus (119899) 30 (3) 0 (0) lt0001Dyslipidemia (119899) 80 (8) 0 (0) lt0001Former smokers (119899) 40 (4) 50 (5) 0621CHD (119899) 20 (2) 0 (0) lt0001Glycemia (mgdL) 108 plusmn 6 104 plusmn 4 0589Pharmacological treatments (119899)
ACE-inhibitors 80 (8) 0 (0) lt0001Oral antidiabetic drugs 30 (3) 0 (0) lt0001Insulin 0 (0) 0 (0) 10Statin 70 (7) 0 (0) lt0001Antiaggregants 60 (6) 0 (0) lt0001Oral anticoagulants 20 (2) 0 (0) lt0001
aData are expressed as mean plusmn SDbgt139ndash89mmHg
PAD patients showed higher platelet ROS production(106 plusmn 14 SI versus 34 plusmn 09 SI lowast119875 lt 0001) (Figure 1(a))H2O2concentration (184plusmn07 120583Mversus 54plusmn05 120583M lowast119875 lt
0001) (Figure 1(b)) sNOX2-dp release (298 plusmn 134 pgmLversus 79 plusmn 23 pgmL lowast119875 lt 0001) (Figure 1(c)) and 8-iso-PGF2120572 production (1626 plusmn 428 pmolL versus 670 plusmn64 pmolL lowast119875 lt 0001) (Figure 1(d)) compared to HS PADpatients displayed lower NOx production (287 plusmn 63 120583Mversus 103 plusmn 26 120583M 119875 lt 0001) (Figure 1(e)) and FMDvalues (20plusmn25 versus 77plusmn10 lowast119875 lt 0001) (Figure 1(f))compared with HS
32 In Vitro Study HUVEC stimulated with EGF releaseda higher amount of sICAM1 (Figure 2(a) lowast119875 lt 0001)sVCAM1 (Figure 2(b) lowast119875 lt 0001) and E-selectin(Figure 2(c) lowast119875 lt 0001) versus unstimulated HUVEC
Supernatant of activated platelets from PAD patients(SAPAD) was able to increase sICAM1 sVCAM1 and sE-selectin versus nonstimulated HUVEC (Figure 2) Con-versely supernatant of activated platelets from HS (SAHS)was not able to change markers of endothelial dysfunction(Figure 2)
When SAPAD pretreated with mix of epicatechin (01ndash10 120583M) and catechin (01ndash10 120583M) was added to HUVEC asignificant reduction of sICAM1 sVCAM1 and sE-selectinlevels was observed compared to sCAMs levels induced bySAPAD (Figures 2(a) 2(b) and 2(c))
Both the P-eNOStotal eNOS ratio (Figure 3(a) lowast119875 lt0001) andNOx concentration (Figure 3(b) lowast119875 lt 0001) weredecreased in HUVEC when stimulated with EGF Moreoverwe found that SAPAD contributed to the reduction of P-eNOStotal eNOS ratio compared to untreated HUVEC(Figure 3(a) lowast119875 lt 0001) with consequent reduction of NObioavailability (Figure 3(b) lowast119875 lt 00001) This phenomenonwas not observed in HUVEC treated with SAHS (Figure 3)
When SAPAD pretreated with mix of epicatechin (01ndash10 120583M) and catechin (01ndash10 120583M) was added to HUVECboth the P-eNOStotal eNOS ratio and NO bioavailabilityincreased compared to P-eNOStotal eNOS ratio and NObioavailability induced by SAPAD (Figures 3(a) and 3(b))
4 DiscussionThe major findings of this paper are as follows (a) PADpatients show higher platelet activation than HS (b) super-natant of activated platelets derived from PAD patients(SAPAD) is able to activate ECs resulting in an upregula-tion of soluble molecules derived from ECs (ie sICAM-1sVCAM-1 and E-selectin) (c) PAD platelets induced ECsactivation by reduction of p-eNOS and bioavailability of NO(d) polyphenols are able to reduce endothelial activationinduced by activated platelets
These novel findings support the hypothesis that platelets-induced endothelial activation and thus the progression ofatherosclerosis are a predisposing factor to more complexdiseases such as PAD [25]
Previous findings showed that in PAD patients there isan imbalance between NOX2-mediated oxidative stress andFMD [11] In the present study we expanded such evidencedemonstrating the key oxidative role of platelets in PADpatients by measuring their ROS production and sNOX2-dp release as markers of NADPH oxidase activation [22]as well as their 8-iso-PGF2120572-III production We observeda significant increase of these oxidative stress markers inplatelets of PAD patients compared to HS
Activated platelets can interact with the endothelium trig-gering an inflammatory response that may contribute to theearly stages of atherosclerosis [26] Upon activation plateletsrelease granule contents able to activate ECs It has beenshown that activated platelets interact with the endotheliuminducing accumulation and transmigration of circulating
Oxidative Medicine and Cellular Longevity 5
0
2
4
6
8
10
12
14
HS PAD
lowast
Plat
elet
RO
S pr
oduc
tion
(SI)
(a)
0
5
10
15
20
HS PAD
lowast
Plat
elet
H2O2
conc
entr
atio
n (120583
M)
(b)
0
5
10
15
20
25
30
35
40
HS PAD
lowast
Plat
elet
sNO
X2-d
p (p
gm
L)
(c)
0
50
100
150
200
HS PAD
lowastPl
atel
et8
-iso-
PGF2120572
(pm
olL
)
(d)
0
5
10
15
20
25
30
35
HS PAD
lowast
Plat
elet
NO
x pr
oduc
tion
(120583M
)
(e)
0
1
2
3
4
5
6
7
8
9
10
11
HS PAD
lowast
FMD
()
(f)
Figure 1 Platelet-induced oxidative stress in PAD patients Platelet ROS production (a) platelet H2O2concentration (b) platelet sNOX2-dp
levels (c) platelet 8-iso-PGF2120572 production (d) platelet NOx production (e) and FMD (f) in PAD patients (119899 = 10) and healthy subjects(119899 = 10) (lowast119875 lt 0001)
6 Oxidative Medicine and Cellular Longevity
0
50
100
150
200
250
300
350
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
lowastlowast
lowast
NS
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sICA
M1
leve
ls (n
gm
L)
(a)
0
5
10
15
20
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sVCA
M1
leve
ls (n
gm
L)(b)
0
50
100
150
200
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sE-s
elec
tion
leve
ls (n
gm
L)
(c)
Figure 2 Biomarkers of HUVEC activation sICAM1 (a) sVCAM1 (b) and E-selectin (c) levels released by HUVEC after incubation withEGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activated PAD patients (SAPAD) (blackbar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalar doses of epicatechin (01ndash10120583M) plus catechin(01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001)
monocytes finally contributing to the atherosclerotic processat early stages [27] Moreover activated platelets can releasein their microenvironment prooxidant molecules such asisoprostanes and ROS In fact we found that supernatant ofPAD platelets is able to stimulate HUVEC with consequent
release of cellular adhesion molecules in their soluble formnamely CAMs
The soluble forms of adhesion molecules reflect endothe-lial CAM surface expression [28] and therefore can be usedas biomarkers of endothelial activation in different diseases
Oxidative Medicine and Cellular Longevity 7
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
P-eNOS
120573-Actin
0
5
10
15
20
25
30
35
40
45
lowast
lowastlowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
Den
sitom
etric
anal
ysis
ofP-
eNO
Sto
tal e
-NO
S (r
atio
)
Total eNOS
(a)
50
0
5
10
15
20
25
30
35
40
45 lowastlowast
lowast lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
HU
VEC
s NO
x pr
oduc
tion
(120583M
)
(b)
Figure 3 HUVEC eNOS activation and NOx production Densitometric analysis of P-eNOStotal eNOS ratio (a) and NOx production (b)in HUVEC incubated with EGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activatedplatelets from PAD patients (SAPAD) (black bar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalardoses of epicatechin (01ndash10120583M) plus catechin (01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001) A representative western blot analysis ofeNOS phosphorylation (a)
8 Oxidative Medicine and Cellular Longevity
Other studies have shown that PAD patients have lowFMD [24] strictly dependent on NO release [29] In accor-dance with these findings our PAD population had reducedFMD and lower nitrite and nitrate (markers of NO gener-ation) compared to controls NO is synthesized from theamino acid L-arginine by a family of enzymes known asnitric oxide synthase (NOS) [30] Among three isoformseNOS is the major responsible for NO production in thecardiovascular system [31] Enhanced eNOS inactivationcaused by excessive ROS production may play an importantrole in the impairment of endothelium-dependent vasodi-latation When HUVEC were coincubated with SAPAD asignificant reduction in p-eNOSwith consequent drop inNObioavailability was observed
In a previous study we showed that the combination ofepicatechin with catechin was able to boost the antioxidantand antiplatelet effects achieved by a single polyphenol[19] Moreover in a recent study we observed that PADpatients treated with dark chocolate rich in epicatechin andcatechin showed a decrease of oxidative stress 2 hours afterits assumption [32]
In our study we explored the hypothesis that polyphe-nols can downregulate platelets activation and subsequentlyreduce ECs activation Therefore we incubated HUVECwith supernatants of activated platelets pretreated with scalardoses of epicatechin plus catechinThese experiments showedthat this mutual combination leads to a decrease in sCAMlevels and an increase in eNOS phosphorylationwith a higherbioavailability of NO
The present study has implications and limitations thatshould be acknowledged This paper suggests a potential roleof polyphenols in the modulation of endothelial function butlacks a specific in vivo proof Given that supernatants havebeen used in the study it is possible to hypothesize by ourresults that platelets can exert their action by an indirectfashion A deeper investigation on supernatant derived fromPAD patients could highlight which specific molecules areresponsible for these effects and their biochemical mecha-nism Last since ROS have a short half-life thus representinga potential limitation of the study a further step forwardwill be the evaluation of other mechanisms independentfrom oxidative stress or more stable markers of oxidativestress able to induce endothelial dysfunction Besides theseresults require further investigation in order to assesswhethersimilar effects can be obtained in vivo by the administrationof aliments or surrogates rich in polyphenols to PAD patientsas in the hierarchy of events that underlie atheroscleroticremodeling of the adult human vessels and the modulationof endothelial activation in PAD patients seems to play a keyrole
In conclusion this study demonstrates that polyphenolsare able to modulate HUVEC activation induced by plateletsderived from PAD patients
Conflict of Interests
The authors confirm that there is no conflict of interests
Acknowledgment
This work was supported by the Grant University of RomeldquoSapienzardquo year 2011 Project no C26A11J528 to GiacomoFrati
References
[1] W Insull Jr ldquoThe pathology of atherosclerosis plaque develop-ment and plaque responses tomedical treatmentrdquoTheAmericanJournal ofMedicine vol 122 no 1 supplement pp S3ndashS14 2009
[2] J I Borissoff H M H Spronk and H T Cate ldquoThe hemostaticsystem as a modulator of atherosclerosisrdquo The New EnglandJournal of Medicine vol 364 no 18 pp 1746ndash1760 2011
[3] S M Schoenwaelder Y Yuan E C Josefsson et al ldquoTwodistinct pathways regulate platelet phosphatidylserine exposureand procoagulant functionrdquo Blood vol 114 no 3 pp 663ndash6662009
[4] J E Italiano Jr J L Richardson S Patel-Hett et al ldquoAngiogen-esis is regulated by a novel mechanism pro- and antiangiogenicproteins are organized into separate platelet 120572 granules anddifferentially releasedrdquo Blood vol 111 no 3 pp 1227ndash1233 2008
[5] J D Pearson ldquoNormal endothelial cell functionrdquo Lupus vol 9no 3 pp 183ndash188 2000
[6] M T Gentile C Vecchione G Marino et al ldquoResistin impairsinsulin-evoked vasodilationrdquo Diabetes vol 57 no 3 pp 577ndash583 2008
[7] G R Wang Y Zhu P V Halushka T M Lincoln and M EMendelsohn ldquoMechanism of platelet inhibition by nitric oxidein vivo phosphorylation of thromboxane receptor by cyclicGMP-dependent protein kinaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no9 pp 4888ndash4893 1998
[8] Y Ohara T E Peterson and D G Harrison ldquoHypercholes-terolemia increases endothelial superoxide anion productionrdquoJournal of Clinical Investigation vol 91 no 6 pp 2546ndash25511993
[9] D D Wagner and P S Frenette ldquoThe vessel wall and itsinteractionsrdquo Blood vol 111 no 11 pp 5271ndash5281 2008
[10] G Brevetti A Silvestro V Schiano and M ChiarielloldquoEndothelial dysfunction and cardiovascular risk predictionin peripheral arterial desease additive value of flow-mediatedilatation to ankle-brachial pressure indexrdquo Circulation vol108 no 17 pp 2093ndash2098 2003
[11] C Heiss T Lauer A Dejam et al ldquoPlasma nitroso compoundsare decreased in patients with endothelial dysfunctionrdquo Journalof the American College of Cardiology vol 47 no 3 pp 573ndash5792006
[12] L Loffredo R Carnevale R Cangemi et al ldquoNOX2 up-regulation is associated with artery dysfunction in patients withperipheral artery diseaserdquo International Journal of Cardiologyvol 165 no 1 pp 184ndash192 2013
[13] J K Bendall R Rinze D Adlam A L Tatham J de Bonoand K M Channon ldquoEndothelial Nox2 overexpression poten-tiates vascular oxidative stress and hemodynamic response toangiotensin II Studies in endothelial-targeted Nox2 transgenicmicerdquo Circulation Research vol 100 no 7 pp 1016ndash1025 2007
[14] O Jung J G Schreiber H Geiger T Pedrazzini R Busse andR P Brandes ldquogp91phox-containing NADPH oxidase mediatesendothelial dysfunction in renovascular hypertensionrdquoCircula-tion vol 109 no 14 pp 1795ndash1801 2004
Oxidative Medicine and Cellular Longevity 9
[15] M de Lorgeril P Salen J L Martin I Monjaud J Delaye andN Mamelle ldquoMediterranean diet traditional risk factors andthe rate of cardiovascular complications aftermyocardial infarc-tion final report of the Lyon Diet Heart Studyrdquo Circulation vol99 no 6 pp 779ndash785 1999
[16] T L Zern and M L Fernandez ldquoCardioprotective effects ofdietary polyphenolsrdquo Journal of Nutrition vol 135 no 10 pp2291ndash2294 2005
[17] O Vitseva S Varghese S Chakrabarti J D Folts and JE Freedman ldquoGrape seed and skin extracts inhibit plateletfunction and release of reactive oxygen intermediatesrdquo Journalof Cardiovascular Pharmacology vol 46 no 4 pp 445ndash4512005
[18] P Pignatelli A Ghiselli B Buchetti et al ldquoPolyphenols syner-gistically inhibit oxidative stress in subjects given red and whitewinerdquo Atherosclerosis vol 188 no 1 pp 77ndash83 2006
[19] R Carnevale L Loffredo P Pignatelli et al ldquoDark chocolateinhibits platelet isoprostanes via NOX2 down-regulation insmokersrdquo Journal of Thrombosis and Haemostasis vol 10 no1 pp 125ndash132 2012
[20] C Menna E de Falco L Pacini G Scafetta P Ruggieri and RPuca ldquoAxitinib affects cell viability and migration of a primaryfoetal lung adenocarcinoma culturerdquo Cancer Investigation vol32 no 1 pp 13ndash21 2014
[21] S W Hoffman R L Roof and D G Stein ldquoA reliableand sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F(2120572) a marker for lipid peroxidation afterexperimental brain injuryrdquo Journal of Neuroscience Methodsvol 68 no 2 pp 133ndash136 1996
[22] P Pignatelli R Carnevale R Cangemi et al ldquoAtorvastatininhibits gp91119901ℎ119900119909 circulating levels in patients with hypercholes-terolemiardquo Arteriosclerosis Thrombosis and Vascular Biologyvol 30 no 2 pp 360ndash367 2010
[23] M C Corretti T J Anderson E J Benjamin et al ldquoGuidelinesfor the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery a report of theinternational brachial artery reactivity task forcerdquo Journal of theAmerican College of Cardiology vol 39 no 2 pp 257ndash265 2002
[24] L Loffredo A Marcoccia P Pignatelli et al ldquoOxidative-stress-mediated arterial dysfunction in patients with peripheralarterial diseaserdquo European Heart Journal vol 28 no 5 pp 608ndash612 2007
[25] A T Hirsch Z J Haskal N R Hertzer et al ldquoACCAHA2005 Practice Guidelines for the management of patients withperipheral arterial disease (lower extremity renal mesentericand abdominal aortic) a collaborative report from the Amer-ican Association for Vascular SurgerySociety for VascularSurgery Society for Cardiovascular Angiography and Interven-tions Society for Vascular Medicine and Biology Society ofInterventional Radiology and the ACCAHA Task Force onPractice Guidelines (Writing Committee to Develop Guidelinesfor the Management of Patients With Peripheral Arterial Dis-ease) endorsed by the American Association of Cardiovascularand Pulmonary Rehabilitation National Heart Lung andBlood Institute Society for Vascular Nursing TransAtlanticInter-Society Consensus and Vascular Disease FoundationrdquoCirculation vol 113 no 11 pp e463ndashe654 2005
[26] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999
[27] T Dickfeld E Lengyel A E May et al ldquoTransient interactionof activated platelets with endothelial cells induces expression
of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway implications foratherogenesisrdquo Cardiovascular Research vol 49 no 1 pp 189ndash199 2001
[28] A G Kjaeligrgaard A Dige J Krog E Toslashnnesen and LWogensen ldquoSoluble adhesion molecules correlate with surfaceexpression in an in vitro model of endothelial activationrdquo Basicand Clinical Pharmacology and Toxicology vol 113 no 49 pp273ndash279 2013
[29] R Joannides W E Haefeli L Linder et al ldquoNitric oxide isresponsible for flow-dependent dilatation of human peripheralconduit arteries in vivordquoCirculation vol 91 no 5 pp 1314ndash13191995
[30] R Bruckdorfer ldquoThe basics about nitric oxiderdquo MolecularAspects of Medicine vol 26 no 1-2 pp 3ndash31 2005
[31] D M Dudzinski and T Michel ldquoLife history of eNOS partnersand pathwaysrdquo Cardiovascular Research vol 75 no 2 pp 247ndash260 2007
[32] L Loffredo L Perri E Catasca et al ldquoDark chocolate acutelyimproves walking autonomy in patients with peripheral arterydiseaserdquo Journal of the American Heart Association vol 3Article ID e001072 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Oxidative Medicine and Cellular Longevity
Table 1 Clinical characteristics of patients with peripheral artery disease (PAD) and healthy subjects (HS)
Variables PAD (119899 = 10) HS (119899 = 10) 119875 valueMean age (year)a 63 plusmn 5 65 plusmn 6 0854Malesfemales 64 64 10Hypertension (119899)b 98 (9) 0 (0) lt0001Diabetes mellitus (119899) 30 (3) 0 (0) lt0001Dyslipidemia (119899) 80 (8) 0 (0) lt0001Former smokers (119899) 40 (4) 50 (5) 0621CHD (119899) 20 (2) 0 (0) lt0001Glycemia (mgdL) 108 plusmn 6 104 plusmn 4 0589Pharmacological treatments (119899)
ACE-inhibitors 80 (8) 0 (0) lt0001Oral antidiabetic drugs 30 (3) 0 (0) lt0001Insulin 0 (0) 0 (0) 10Statin 70 (7) 0 (0) lt0001Antiaggregants 60 (6) 0 (0) lt0001Oral anticoagulants 20 (2) 0 (0) lt0001
aData are expressed as mean plusmn SDbgt139ndash89mmHg
PAD patients showed higher platelet ROS production(106 plusmn 14 SI versus 34 plusmn 09 SI lowast119875 lt 0001) (Figure 1(a))H2O2concentration (184plusmn07 120583Mversus 54plusmn05 120583M lowast119875 lt
0001) (Figure 1(b)) sNOX2-dp release (298 plusmn 134 pgmLversus 79 plusmn 23 pgmL lowast119875 lt 0001) (Figure 1(c)) and 8-iso-PGF2120572 production (1626 plusmn 428 pmolL versus 670 plusmn64 pmolL lowast119875 lt 0001) (Figure 1(d)) compared to HS PADpatients displayed lower NOx production (287 plusmn 63 120583Mversus 103 plusmn 26 120583M 119875 lt 0001) (Figure 1(e)) and FMDvalues (20plusmn25 versus 77plusmn10 lowast119875 lt 0001) (Figure 1(f))compared with HS
32 In Vitro Study HUVEC stimulated with EGF releaseda higher amount of sICAM1 (Figure 2(a) lowast119875 lt 0001)sVCAM1 (Figure 2(b) lowast119875 lt 0001) and E-selectin(Figure 2(c) lowast119875 lt 0001) versus unstimulated HUVEC
Supernatant of activated platelets from PAD patients(SAPAD) was able to increase sICAM1 sVCAM1 and sE-selectin versus nonstimulated HUVEC (Figure 2) Con-versely supernatant of activated platelets from HS (SAHS)was not able to change markers of endothelial dysfunction(Figure 2)
When SAPAD pretreated with mix of epicatechin (01ndash10 120583M) and catechin (01ndash10 120583M) was added to HUVEC asignificant reduction of sICAM1 sVCAM1 and sE-selectinlevels was observed compared to sCAMs levels induced bySAPAD (Figures 2(a) 2(b) and 2(c))
Both the P-eNOStotal eNOS ratio (Figure 3(a) lowast119875 lt0001) andNOx concentration (Figure 3(b) lowast119875 lt 0001) weredecreased in HUVEC when stimulated with EGF Moreoverwe found that SAPAD contributed to the reduction of P-eNOStotal eNOS ratio compared to untreated HUVEC(Figure 3(a) lowast119875 lt 0001) with consequent reduction of NObioavailability (Figure 3(b) lowast119875 lt 00001) This phenomenonwas not observed in HUVEC treated with SAHS (Figure 3)
When SAPAD pretreated with mix of epicatechin (01ndash10 120583M) and catechin (01ndash10 120583M) was added to HUVECboth the P-eNOStotal eNOS ratio and NO bioavailabilityincreased compared to P-eNOStotal eNOS ratio and NObioavailability induced by SAPAD (Figures 3(a) and 3(b))
4 DiscussionThe major findings of this paper are as follows (a) PADpatients show higher platelet activation than HS (b) super-natant of activated platelets derived from PAD patients(SAPAD) is able to activate ECs resulting in an upregula-tion of soluble molecules derived from ECs (ie sICAM-1sVCAM-1 and E-selectin) (c) PAD platelets induced ECsactivation by reduction of p-eNOS and bioavailability of NO(d) polyphenols are able to reduce endothelial activationinduced by activated platelets
These novel findings support the hypothesis that platelets-induced endothelial activation and thus the progression ofatherosclerosis are a predisposing factor to more complexdiseases such as PAD [25]
Previous findings showed that in PAD patients there isan imbalance between NOX2-mediated oxidative stress andFMD [11] In the present study we expanded such evidencedemonstrating the key oxidative role of platelets in PADpatients by measuring their ROS production and sNOX2-dp release as markers of NADPH oxidase activation [22]as well as their 8-iso-PGF2120572-III production We observeda significant increase of these oxidative stress markers inplatelets of PAD patients compared to HS
Activated platelets can interact with the endothelium trig-gering an inflammatory response that may contribute to theearly stages of atherosclerosis [26] Upon activation plateletsrelease granule contents able to activate ECs It has beenshown that activated platelets interact with the endotheliuminducing accumulation and transmigration of circulating
Oxidative Medicine and Cellular Longevity 5
0
2
4
6
8
10
12
14
HS PAD
lowast
Plat
elet
RO
S pr
oduc
tion
(SI)
(a)
0
5
10
15
20
HS PAD
lowast
Plat
elet
H2O2
conc
entr
atio
n (120583
M)
(b)
0
5
10
15
20
25
30
35
40
HS PAD
lowast
Plat
elet
sNO
X2-d
p (p
gm
L)
(c)
0
50
100
150
200
HS PAD
lowastPl
atel
et8
-iso-
PGF2120572
(pm
olL
)
(d)
0
5
10
15
20
25
30
35
HS PAD
lowast
Plat
elet
NO
x pr
oduc
tion
(120583M
)
(e)
0
1
2
3
4
5
6
7
8
9
10
11
HS PAD
lowast
FMD
()
(f)
Figure 1 Platelet-induced oxidative stress in PAD patients Platelet ROS production (a) platelet H2O2concentration (b) platelet sNOX2-dp
levels (c) platelet 8-iso-PGF2120572 production (d) platelet NOx production (e) and FMD (f) in PAD patients (119899 = 10) and healthy subjects(119899 = 10) (lowast119875 lt 0001)
6 Oxidative Medicine and Cellular Longevity
0
50
100
150
200
250
300
350
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
lowastlowast
lowast
NS
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sICA
M1
leve
ls (n
gm
L)
(a)
0
5
10
15
20
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sVCA
M1
leve
ls (n
gm
L)(b)
0
50
100
150
200
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sE-s
elec
tion
leve
ls (n
gm
L)
(c)
Figure 2 Biomarkers of HUVEC activation sICAM1 (a) sVCAM1 (b) and E-selectin (c) levels released by HUVEC after incubation withEGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activated PAD patients (SAPAD) (blackbar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalar doses of epicatechin (01ndash10120583M) plus catechin(01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001)
monocytes finally contributing to the atherosclerotic processat early stages [27] Moreover activated platelets can releasein their microenvironment prooxidant molecules such asisoprostanes and ROS In fact we found that supernatant ofPAD platelets is able to stimulate HUVEC with consequent
release of cellular adhesion molecules in their soluble formnamely CAMs
The soluble forms of adhesion molecules reflect endothe-lial CAM surface expression [28] and therefore can be usedas biomarkers of endothelial activation in different diseases
Oxidative Medicine and Cellular Longevity 7
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
P-eNOS
120573-Actin
0
5
10
15
20
25
30
35
40
45
lowast
lowastlowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
Den
sitom
etric
anal
ysis
ofP-
eNO
Sto
tal e
-NO
S (r
atio
)
Total eNOS
(a)
50
0
5
10
15
20
25
30
35
40
45 lowastlowast
lowast lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
HU
VEC
s NO
x pr
oduc
tion
(120583M
)
(b)
Figure 3 HUVEC eNOS activation and NOx production Densitometric analysis of P-eNOStotal eNOS ratio (a) and NOx production (b)in HUVEC incubated with EGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activatedplatelets from PAD patients (SAPAD) (black bar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalardoses of epicatechin (01ndash10120583M) plus catechin (01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001) A representative western blot analysis ofeNOS phosphorylation (a)
8 Oxidative Medicine and Cellular Longevity
Other studies have shown that PAD patients have lowFMD [24] strictly dependent on NO release [29] In accor-dance with these findings our PAD population had reducedFMD and lower nitrite and nitrate (markers of NO gener-ation) compared to controls NO is synthesized from theamino acid L-arginine by a family of enzymes known asnitric oxide synthase (NOS) [30] Among three isoformseNOS is the major responsible for NO production in thecardiovascular system [31] Enhanced eNOS inactivationcaused by excessive ROS production may play an importantrole in the impairment of endothelium-dependent vasodi-latation When HUVEC were coincubated with SAPAD asignificant reduction in p-eNOSwith consequent drop inNObioavailability was observed
In a previous study we showed that the combination ofepicatechin with catechin was able to boost the antioxidantand antiplatelet effects achieved by a single polyphenol[19] Moreover in a recent study we observed that PADpatients treated with dark chocolate rich in epicatechin andcatechin showed a decrease of oxidative stress 2 hours afterits assumption [32]
In our study we explored the hypothesis that polyphe-nols can downregulate platelets activation and subsequentlyreduce ECs activation Therefore we incubated HUVECwith supernatants of activated platelets pretreated with scalardoses of epicatechin plus catechinThese experiments showedthat this mutual combination leads to a decrease in sCAMlevels and an increase in eNOS phosphorylationwith a higherbioavailability of NO
The present study has implications and limitations thatshould be acknowledged This paper suggests a potential roleof polyphenols in the modulation of endothelial function butlacks a specific in vivo proof Given that supernatants havebeen used in the study it is possible to hypothesize by ourresults that platelets can exert their action by an indirectfashion A deeper investigation on supernatant derived fromPAD patients could highlight which specific molecules areresponsible for these effects and their biochemical mecha-nism Last since ROS have a short half-life thus representinga potential limitation of the study a further step forwardwill be the evaluation of other mechanisms independentfrom oxidative stress or more stable markers of oxidativestress able to induce endothelial dysfunction Besides theseresults require further investigation in order to assesswhethersimilar effects can be obtained in vivo by the administrationof aliments or surrogates rich in polyphenols to PAD patientsas in the hierarchy of events that underlie atheroscleroticremodeling of the adult human vessels and the modulationof endothelial activation in PAD patients seems to play a keyrole
In conclusion this study demonstrates that polyphenolsare able to modulate HUVEC activation induced by plateletsderived from PAD patients
Conflict of Interests
The authors confirm that there is no conflict of interests
Acknowledgment
This work was supported by the Grant University of RomeldquoSapienzardquo year 2011 Project no C26A11J528 to GiacomoFrati
References
[1] W Insull Jr ldquoThe pathology of atherosclerosis plaque develop-ment and plaque responses tomedical treatmentrdquoTheAmericanJournal ofMedicine vol 122 no 1 supplement pp S3ndashS14 2009
[2] J I Borissoff H M H Spronk and H T Cate ldquoThe hemostaticsystem as a modulator of atherosclerosisrdquo The New EnglandJournal of Medicine vol 364 no 18 pp 1746ndash1760 2011
[3] S M Schoenwaelder Y Yuan E C Josefsson et al ldquoTwodistinct pathways regulate platelet phosphatidylserine exposureand procoagulant functionrdquo Blood vol 114 no 3 pp 663ndash6662009
[4] J E Italiano Jr J L Richardson S Patel-Hett et al ldquoAngiogen-esis is regulated by a novel mechanism pro- and antiangiogenicproteins are organized into separate platelet 120572 granules anddifferentially releasedrdquo Blood vol 111 no 3 pp 1227ndash1233 2008
[5] J D Pearson ldquoNormal endothelial cell functionrdquo Lupus vol 9no 3 pp 183ndash188 2000
[6] M T Gentile C Vecchione G Marino et al ldquoResistin impairsinsulin-evoked vasodilationrdquo Diabetes vol 57 no 3 pp 577ndash583 2008
[7] G R Wang Y Zhu P V Halushka T M Lincoln and M EMendelsohn ldquoMechanism of platelet inhibition by nitric oxidein vivo phosphorylation of thromboxane receptor by cyclicGMP-dependent protein kinaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no9 pp 4888ndash4893 1998
[8] Y Ohara T E Peterson and D G Harrison ldquoHypercholes-terolemia increases endothelial superoxide anion productionrdquoJournal of Clinical Investigation vol 91 no 6 pp 2546ndash25511993
[9] D D Wagner and P S Frenette ldquoThe vessel wall and itsinteractionsrdquo Blood vol 111 no 11 pp 5271ndash5281 2008
[10] G Brevetti A Silvestro V Schiano and M ChiarielloldquoEndothelial dysfunction and cardiovascular risk predictionin peripheral arterial desease additive value of flow-mediatedilatation to ankle-brachial pressure indexrdquo Circulation vol108 no 17 pp 2093ndash2098 2003
[11] C Heiss T Lauer A Dejam et al ldquoPlasma nitroso compoundsare decreased in patients with endothelial dysfunctionrdquo Journalof the American College of Cardiology vol 47 no 3 pp 573ndash5792006
[12] L Loffredo R Carnevale R Cangemi et al ldquoNOX2 up-regulation is associated with artery dysfunction in patients withperipheral artery diseaserdquo International Journal of Cardiologyvol 165 no 1 pp 184ndash192 2013
[13] J K Bendall R Rinze D Adlam A L Tatham J de Bonoand K M Channon ldquoEndothelial Nox2 overexpression poten-tiates vascular oxidative stress and hemodynamic response toangiotensin II Studies in endothelial-targeted Nox2 transgenicmicerdquo Circulation Research vol 100 no 7 pp 1016ndash1025 2007
[14] O Jung J G Schreiber H Geiger T Pedrazzini R Busse andR P Brandes ldquogp91phox-containing NADPH oxidase mediatesendothelial dysfunction in renovascular hypertensionrdquoCircula-tion vol 109 no 14 pp 1795ndash1801 2004
Oxidative Medicine and Cellular Longevity 9
[15] M de Lorgeril P Salen J L Martin I Monjaud J Delaye andN Mamelle ldquoMediterranean diet traditional risk factors andthe rate of cardiovascular complications aftermyocardial infarc-tion final report of the Lyon Diet Heart Studyrdquo Circulation vol99 no 6 pp 779ndash785 1999
[16] T L Zern and M L Fernandez ldquoCardioprotective effects ofdietary polyphenolsrdquo Journal of Nutrition vol 135 no 10 pp2291ndash2294 2005
[17] O Vitseva S Varghese S Chakrabarti J D Folts and JE Freedman ldquoGrape seed and skin extracts inhibit plateletfunction and release of reactive oxygen intermediatesrdquo Journalof Cardiovascular Pharmacology vol 46 no 4 pp 445ndash4512005
[18] P Pignatelli A Ghiselli B Buchetti et al ldquoPolyphenols syner-gistically inhibit oxidative stress in subjects given red and whitewinerdquo Atherosclerosis vol 188 no 1 pp 77ndash83 2006
[19] R Carnevale L Loffredo P Pignatelli et al ldquoDark chocolateinhibits platelet isoprostanes via NOX2 down-regulation insmokersrdquo Journal of Thrombosis and Haemostasis vol 10 no1 pp 125ndash132 2012
[20] C Menna E de Falco L Pacini G Scafetta P Ruggieri and RPuca ldquoAxitinib affects cell viability and migration of a primaryfoetal lung adenocarcinoma culturerdquo Cancer Investigation vol32 no 1 pp 13ndash21 2014
[21] S W Hoffman R L Roof and D G Stein ldquoA reliableand sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F(2120572) a marker for lipid peroxidation afterexperimental brain injuryrdquo Journal of Neuroscience Methodsvol 68 no 2 pp 133ndash136 1996
[22] P Pignatelli R Carnevale R Cangemi et al ldquoAtorvastatininhibits gp91119901ℎ119900119909 circulating levels in patients with hypercholes-terolemiardquo Arteriosclerosis Thrombosis and Vascular Biologyvol 30 no 2 pp 360ndash367 2010
[23] M C Corretti T J Anderson E J Benjamin et al ldquoGuidelinesfor the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery a report of theinternational brachial artery reactivity task forcerdquo Journal of theAmerican College of Cardiology vol 39 no 2 pp 257ndash265 2002
[24] L Loffredo A Marcoccia P Pignatelli et al ldquoOxidative-stress-mediated arterial dysfunction in patients with peripheralarterial diseaserdquo European Heart Journal vol 28 no 5 pp 608ndash612 2007
[25] A T Hirsch Z J Haskal N R Hertzer et al ldquoACCAHA2005 Practice Guidelines for the management of patients withperipheral arterial disease (lower extremity renal mesentericand abdominal aortic) a collaborative report from the Amer-ican Association for Vascular SurgerySociety for VascularSurgery Society for Cardiovascular Angiography and Interven-tions Society for Vascular Medicine and Biology Society ofInterventional Radiology and the ACCAHA Task Force onPractice Guidelines (Writing Committee to Develop Guidelinesfor the Management of Patients With Peripheral Arterial Dis-ease) endorsed by the American Association of Cardiovascularand Pulmonary Rehabilitation National Heart Lung andBlood Institute Society for Vascular Nursing TransAtlanticInter-Society Consensus and Vascular Disease FoundationrdquoCirculation vol 113 no 11 pp e463ndashe654 2005
[26] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999
[27] T Dickfeld E Lengyel A E May et al ldquoTransient interactionof activated platelets with endothelial cells induces expression
of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway implications foratherogenesisrdquo Cardiovascular Research vol 49 no 1 pp 189ndash199 2001
[28] A G Kjaeligrgaard A Dige J Krog E Toslashnnesen and LWogensen ldquoSoluble adhesion molecules correlate with surfaceexpression in an in vitro model of endothelial activationrdquo Basicand Clinical Pharmacology and Toxicology vol 113 no 49 pp273ndash279 2013
[29] R Joannides W E Haefeli L Linder et al ldquoNitric oxide isresponsible for flow-dependent dilatation of human peripheralconduit arteries in vivordquoCirculation vol 91 no 5 pp 1314ndash13191995
[30] R Bruckdorfer ldquoThe basics about nitric oxiderdquo MolecularAspects of Medicine vol 26 no 1-2 pp 3ndash31 2005
[31] D M Dudzinski and T Michel ldquoLife history of eNOS partnersand pathwaysrdquo Cardiovascular Research vol 75 no 2 pp 247ndash260 2007
[32] L Loffredo L Perri E Catasca et al ldquoDark chocolate acutelyimproves walking autonomy in patients with peripheral arterydiseaserdquo Journal of the American Heart Association vol 3Article ID e001072 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 5
0
2
4
6
8
10
12
14
HS PAD
lowast
Plat
elet
RO
S pr
oduc
tion
(SI)
(a)
0
5
10
15
20
HS PAD
lowast
Plat
elet
H2O2
conc
entr
atio
n (120583
M)
(b)
0
5
10
15
20
25
30
35
40
HS PAD
lowast
Plat
elet
sNO
X2-d
p (p
gm
L)
(c)
0
50
100
150
200
HS PAD
lowastPl
atel
et8
-iso-
PGF2120572
(pm
olL
)
(d)
0
5
10
15
20
25
30
35
HS PAD
lowast
Plat
elet
NO
x pr
oduc
tion
(120583M
)
(e)
0
1
2
3
4
5
6
7
8
9
10
11
HS PAD
lowast
FMD
()
(f)
Figure 1 Platelet-induced oxidative stress in PAD patients Platelet ROS production (a) platelet H2O2concentration (b) platelet sNOX2-dp
levels (c) platelet 8-iso-PGF2120572 production (d) platelet NOx production (e) and FMD (f) in PAD patients (119899 = 10) and healthy subjects(119899 = 10) (lowast119875 lt 0001)
6 Oxidative Medicine and Cellular Longevity
0
50
100
150
200
250
300
350
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
lowastlowast
lowast
NS
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sICA
M1
leve
ls (n
gm
L)
(a)
0
5
10
15
20
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sVCA
M1
leve
ls (n
gm
L)(b)
0
50
100
150
200
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sE-s
elec
tion
leve
ls (n
gm
L)
(c)
Figure 2 Biomarkers of HUVEC activation sICAM1 (a) sVCAM1 (b) and E-selectin (c) levels released by HUVEC after incubation withEGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activated PAD patients (SAPAD) (blackbar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalar doses of epicatechin (01ndash10120583M) plus catechin(01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001)
monocytes finally contributing to the atherosclerotic processat early stages [27] Moreover activated platelets can releasein their microenvironment prooxidant molecules such asisoprostanes and ROS In fact we found that supernatant ofPAD platelets is able to stimulate HUVEC with consequent
release of cellular adhesion molecules in their soluble formnamely CAMs
The soluble forms of adhesion molecules reflect endothe-lial CAM surface expression [28] and therefore can be usedas biomarkers of endothelial activation in different diseases
Oxidative Medicine and Cellular Longevity 7
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
P-eNOS
120573-Actin
0
5
10
15
20
25
30
35
40
45
lowast
lowastlowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
Den
sitom
etric
anal
ysis
ofP-
eNO
Sto
tal e
-NO
S (r
atio
)
Total eNOS
(a)
50
0
5
10
15
20
25
30
35
40
45 lowastlowast
lowast lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
HU
VEC
s NO
x pr
oduc
tion
(120583M
)
(b)
Figure 3 HUVEC eNOS activation and NOx production Densitometric analysis of P-eNOStotal eNOS ratio (a) and NOx production (b)in HUVEC incubated with EGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activatedplatelets from PAD patients (SAPAD) (black bar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalardoses of epicatechin (01ndash10120583M) plus catechin (01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001) A representative western blot analysis ofeNOS phosphorylation (a)
8 Oxidative Medicine and Cellular Longevity
Other studies have shown that PAD patients have lowFMD [24] strictly dependent on NO release [29] In accor-dance with these findings our PAD population had reducedFMD and lower nitrite and nitrate (markers of NO gener-ation) compared to controls NO is synthesized from theamino acid L-arginine by a family of enzymes known asnitric oxide synthase (NOS) [30] Among three isoformseNOS is the major responsible for NO production in thecardiovascular system [31] Enhanced eNOS inactivationcaused by excessive ROS production may play an importantrole in the impairment of endothelium-dependent vasodi-latation When HUVEC were coincubated with SAPAD asignificant reduction in p-eNOSwith consequent drop inNObioavailability was observed
In a previous study we showed that the combination ofepicatechin with catechin was able to boost the antioxidantand antiplatelet effects achieved by a single polyphenol[19] Moreover in a recent study we observed that PADpatients treated with dark chocolate rich in epicatechin andcatechin showed a decrease of oxidative stress 2 hours afterits assumption [32]
In our study we explored the hypothesis that polyphe-nols can downregulate platelets activation and subsequentlyreduce ECs activation Therefore we incubated HUVECwith supernatants of activated platelets pretreated with scalardoses of epicatechin plus catechinThese experiments showedthat this mutual combination leads to a decrease in sCAMlevels and an increase in eNOS phosphorylationwith a higherbioavailability of NO
The present study has implications and limitations thatshould be acknowledged This paper suggests a potential roleof polyphenols in the modulation of endothelial function butlacks a specific in vivo proof Given that supernatants havebeen used in the study it is possible to hypothesize by ourresults that platelets can exert their action by an indirectfashion A deeper investigation on supernatant derived fromPAD patients could highlight which specific molecules areresponsible for these effects and their biochemical mecha-nism Last since ROS have a short half-life thus representinga potential limitation of the study a further step forwardwill be the evaluation of other mechanisms independentfrom oxidative stress or more stable markers of oxidativestress able to induce endothelial dysfunction Besides theseresults require further investigation in order to assesswhethersimilar effects can be obtained in vivo by the administrationof aliments or surrogates rich in polyphenols to PAD patientsas in the hierarchy of events that underlie atheroscleroticremodeling of the adult human vessels and the modulationof endothelial activation in PAD patients seems to play a keyrole
In conclusion this study demonstrates that polyphenolsare able to modulate HUVEC activation induced by plateletsderived from PAD patients
Conflict of Interests
The authors confirm that there is no conflict of interests
Acknowledgment
This work was supported by the Grant University of RomeldquoSapienzardquo year 2011 Project no C26A11J528 to GiacomoFrati
References
[1] W Insull Jr ldquoThe pathology of atherosclerosis plaque develop-ment and plaque responses tomedical treatmentrdquoTheAmericanJournal ofMedicine vol 122 no 1 supplement pp S3ndashS14 2009
[2] J I Borissoff H M H Spronk and H T Cate ldquoThe hemostaticsystem as a modulator of atherosclerosisrdquo The New EnglandJournal of Medicine vol 364 no 18 pp 1746ndash1760 2011
[3] S M Schoenwaelder Y Yuan E C Josefsson et al ldquoTwodistinct pathways regulate platelet phosphatidylserine exposureand procoagulant functionrdquo Blood vol 114 no 3 pp 663ndash6662009
[4] J E Italiano Jr J L Richardson S Patel-Hett et al ldquoAngiogen-esis is regulated by a novel mechanism pro- and antiangiogenicproteins are organized into separate platelet 120572 granules anddifferentially releasedrdquo Blood vol 111 no 3 pp 1227ndash1233 2008
[5] J D Pearson ldquoNormal endothelial cell functionrdquo Lupus vol 9no 3 pp 183ndash188 2000
[6] M T Gentile C Vecchione G Marino et al ldquoResistin impairsinsulin-evoked vasodilationrdquo Diabetes vol 57 no 3 pp 577ndash583 2008
[7] G R Wang Y Zhu P V Halushka T M Lincoln and M EMendelsohn ldquoMechanism of platelet inhibition by nitric oxidein vivo phosphorylation of thromboxane receptor by cyclicGMP-dependent protein kinaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no9 pp 4888ndash4893 1998
[8] Y Ohara T E Peterson and D G Harrison ldquoHypercholes-terolemia increases endothelial superoxide anion productionrdquoJournal of Clinical Investigation vol 91 no 6 pp 2546ndash25511993
[9] D D Wagner and P S Frenette ldquoThe vessel wall and itsinteractionsrdquo Blood vol 111 no 11 pp 5271ndash5281 2008
[10] G Brevetti A Silvestro V Schiano and M ChiarielloldquoEndothelial dysfunction and cardiovascular risk predictionin peripheral arterial desease additive value of flow-mediatedilatation to ankle-brachial pressure indexrdquo Circulation vol108 no 17 pp 2093ndash2098 2003
[11] C Heiss T Lauer A Dejam et al ldquoPlasma nitroso compoundsare decreased in patients with endothelial dysfunctionrdquo Journalof the American College of Cardiology vol 47 no 3 pp 573ndash5792006
[12] L Loffredo R Carnevale R Cangemi et al ldquoNOX2 up-regulation is associated with artery dysfunction in patients withperipheral artery diseaserdquo International Journal of Cardiologyvol 165 no 1 pp 184ndash192 2013
[13] J K Bendall R Rinze D Adlam A L Tatham J de Bonoand K M Channon ldquoEndothelial Nox2 overexpression poten-tiates vascular oxidative stress and hemodynamic response toangiotensin II Studies in endothelial-targeted Nox2 transgenicmicerdquo Circulation Research vol 100 no 7 pp 1016ndash1025 2007
[14] O Jung J G Schreiber H Geiger T Pedrazzini R Busse andR P Brandes ldquogp91phox-containing NADPH oxidase mediatesendothelial dysfunction in renovascular hypertensionrdquoCircula-tion vol 109 no 14 pp 1795ndash1801 2004
Oxidative Medicine and Cellular Longevity 9
[15] M de Lorgeril P Salen J L Martin I Monjaud J Delaye andN Mamelle ldquoMediterranean diet traditional risk factors andthe rate of cardiovascular complications aftermyocardial infarc-tion final report of the Lyon Diet Heart Studyrdquo Circulation vol99 no 6 pp 779ndash785 1999
[16] T L Zern and M L Fernandez ldquoCardioprotective effects ofdietary polyphenolsrdquo Journal of Nutrition vol 135 no 10 pp2291ndash2294 2005
[17] O Vitseva S Varghese S Chakrabarti J D Folts and JE Freedman ldquoGrape seed and skin extracts inhibit plateletfunction and release of reactive oxygen intermediatesrdquo Journalof Cardiovascular Pharmacology vol 46 no 4 pp 445ndash4512005
[18] P Pignatelli A Ghiselli B Buchetti et al ldquoPolyphenols syner-gistically inhibit oxidative stress in subjects given red and whitewinerdquo Atherosclerosis vol 188 no 1 pp 77ndash83 2006
[19] R Carnevale L Loffredo P Pignatelli et al ldquoDark chocolateinhibits platelet isoprostanes via NOX2 down-regulation insmokersrdquo Journal of Thrombosis and Haemostasis vol 10 no1 pp 125ndash132 2012
[20] C Menna E de Falco L Pacini G Scafetta P Ruggieri and RPuca ldquoAxitinib affects cell viability and migration of a primaryfoetal lung adenocarcinoma culturerdquo Cancer Investigation vol32 no 1 pp 13ndash21 2014
[21] S W Hoffman R L Roof and D G Stein ldquoA reliableand sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F(2120572) a marker for lipid peroxidation afterexperimental brain injuryrdquo Journal of Neuroscience Methodsvol 68 no 2 pp 133ndash136 1996
[22] P Pignatelli R Carnevale R Cangemi et al ldquoAtorvastatininhibits gp91119901ℎ119900119909 circulating levels in patients with hypercholes-terolemiardquo Arteriosclerosis Thrombosis and Vascular Biologyvol 30 no 2 pp 360ndash367 2010
[23] M C Corretti T J Anderson E J Benjamin et al ldquoGuidelinesfor the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery a report of theinternational brachial artery reactivity task forcerdquo Journal of theAmerican College of Cardiology vol 39 no 2 pp 257ndash265 2002
[24] L Loffredo A Marcoccia P Pignatelli et al ldquoOxidative-stress-mediated arterial dysfunction in patients with peripheralarterial diseaserdquo European Heart Journal vol 28 no 5 pp 608ndash612 2007
[25] A T Hirsch Z J Haskal N R Hertzer et al ldquoACCAHA2005 Practice Guidelines for the management of patients withperipheral arterial disease (lower extremity renal mesentericand abdominal aortic) a collaborative report from the Amer-ican Association for Vascular SurgerySociety for VascularSurgery Society for Cardiovascular Angiography and Interven-tions Society for Vascular Medicine and Biology Society ofInterventional Radiology and the ACCAHA Task Force onPractice Guidelines (Writing Committee to Develop Guidelinesfor the Management of Patients With Peripheral Arterial Dis-ease) endorsed by the American Association of Cardiovascularand Pulmonary Rehabilitation National Heart Lung andBlood Institute Society for Vascular Nursing TransAtlanticInter-Society Consensus and Vascular Disease FoundationrdquoCirculation vol 113 no 11 pp e463ndashe654 2005
[26] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999
[27] T Dickfeld E Lengyel A E May et al ldquoTransient interactionof activated platelets with endothelial cells induces expression
of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway implications foratherogenesisrdquo Cardiovascular Research vol 49 no 1 pp 189ndash199 2001
[28] A G Kjaeligrgaard A Dige J Krog E Toslashnnesen and LWogensen ldquoSoluble adhesion molecules correlate with surfaceexpression in an in vitro model of endothelial activationrdquo Basicand Clinical Pharmacology and Toxicology vol 113 no 49 pp273ndash279 2013
[29] R Joannides W E Haefeli L Linder et al ldquoNitric oxide isresponsible for flow-dependent dilatation of human peripheralconduit arteries in vivordquoCirculation vol 91 no 5 pp 1314ndash13191995
[30] R Bruckdorfer ldquoThe basics about nitric oxiderdquo MolecularAspects of Medicine vol 26 no 1-2 pp 3ndash31 2005
[31] D M Dudzinski and T Michel ldquoLife history of eNOS partnersand pathwaysrdquo Cardiovascular Research vol 75 no 2 pp 247ndash260 2007
[32] L Loffredo L Perri E Catasca et al ldquoDark chocolate acutelyimproves walking autonomy in patients with peripheral arterydiseaserdquo Journal of the American Heart Association vol 3Article ID e001072 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Oxidative Medicine and Cellular Longevity
0
50
100
150
200
250
300
350
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
lowastlowast
lowast
NS
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sICA
M1
leve
ls (n
gm
L)
(a)
0
5
10
15
20
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sVCA
M1
leve
ls (n
gm
L)(b)
0
50
100
150
200
lowastlowast
lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
sE-s
elec
tion
leve
ls (n
gm
L)
(c)
Figure 2 Biomarkers of HUVEC activation sICAM1 (a) sVCAM1 (b) and E-selectin (c) levels released by HUVEC after incubation withEGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activated PAD patients (SAPAD) (blackbar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalar doses of epicatechin (01ndash10120583M) plus catechin(01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001)
monocytes finally contributing to the atherosclerotic processat early stages [27] Moreover activated platelets can releasein their microenvironment prooxidant molecules such asisoprostanes and ROS In fact we found that supernatant ofPAD platelets is able to stimulate HUVEC with consequent
release of cellular adhesion molecules in their soluble formnamely CAMs
The soluble forms of adhesion molecules reflect endothe-lial CAM surface expression [28] and therefore can be usedas biomarkers of endothelial activation in different diseases
Oxidative Medicine and Cellular Longevity 7
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
P-eNOS
120573-Actin
0
5
10
15
20
25
30
35
40
45
lowast
lowastlowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
Den
sitom
etric
anal
ysis
ofP-
eNO
Sto
tal e
-NO
S (r
atio
)
Total eNOS
(a)
50
0
5
10
15
20
25
30
35
40
45 lowastlowast
lowast lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
HU
VEC
s NO
x pr
oduc
tion
(120583M
)
(b)
Figure 3 HUVEC eNOS activation and NOx production Densitometric analysis of P-eNOStotal eNOS ratio (a) and NOx production (b)in HUVEC incubated with EGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activatedplatelets from PAD patients (SAPAD) (black bar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalardoses of epicatechin (01ndash10120583M) plus catechin (01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001) A representative western blot analysis ofeNOS phosphorylation (a)
8 Oxidative Medicine and Cellular Longevity
Other studies have shown that PAD patients have lowFMD [24] strictly dependent on NO release [29] In accor-dance with these findings our PAD population had reducedFMD and lower nitrite and nitrate (markers of NO gener-ation) compared to controls NO is synthesized from theamino acid L-arginine by a family of enzymes known asnitric oxide synthase (NOS) [30] Among three isoformseNOS is the major responsible for NO production in thecardiovascular system [31] Enhanced eNOS inactivationcaused by excessive ROS production may play an importantrole in the impairment of endothelium-dependent vasodi-latation When HUVEC were coincubated with SAPAD asignificant reduction in p-eNOSwith consequent drop inNObioavailability was observed
In a previous study we showed that the combination ofepicatechin with catechin was able to boost the antioxidantand antiplatelet effects achieved by a single polyphenol[19] Moreover in a recent study we observed that PADpatients treated with dark chocolate rich in epicatechin andcatechin showed a decrease of oxidative stress 2 hours afterits assumption [32]
In our study we explored the hypothesis that polyphe-nols can downregulate platelets activation and subsequentlyreduce ECs activation Therefore we incubated HUVECwith supernatants of activated platelets pretreated with scalardoses of epicatechin plus catechinThese experiments showedthat this mutual combination leads to a decrease in sCAMlevels and an increase in eNOS phosphorylationwith a higherbioavailability of NO
The present study has implications and limitations thatshould be acknowledged This paper suggests a potential roleof polyphenols in the modulation of endothelial function butlacks a specific in vivo proof Given that supernatants havebeen used in the study it is possible to hypothesize by ourresults that platelets can exert their action by an indirectfashion A deeper investigation on supernatant derived fromPAD patients could highlight which specific molecules areresponsible for these effects and their biochemical mecha-nism Last since ROS have a short half-life thus representinga potential limitation of the study a further step forwardwill be the evaluation of other mechanisms independentfrom oxidative stress or more stable markers of oxidativestress able to induce endothelial dysfunction Besides theseresults require further investigation in order to assesswhethersimilar effects can be obtained in vivo by the administrationof aliments or surrogates rich in polyphenols to PAD patientsas in the hierarchy of events that underlie atheroscleroticremodeling of the adult human vessels and the modulationof endothelial activation in PAD patients seems to play a keyrole
In conclusion this study demonstrates that polyphenolsare able to modulate HUVEC activation induced by plateletsderived from PAD patients
Conflict of Interests
The authors confirm that there is no conflict of interests
Acknowledgment
This work was supported by the Grant University of RomeldquoSapienzardquo year 2011 Project no C26A11J528 to GiacomoFrati
References
[1] W Insull Jr ldquoThe pathology of atherosclerosis plaque develop-ment and plaque responses tomedical treatmentrdquoTheAmericanJournal ofMedicine vol 122 no 1 supplement pp S3ndashS14 2009
[2] J I Borissoff H M H Spronk and H T Cate ldquoThe hemostaticsystem as a modulator of atherosclerosisrdquo The New EnglandJournal of Medicine vol 364 no 18 pp 1746ndash1760 2011
[3] S M Schoenwaelder Y Yuan E C Josefsson et al ldquoTwodistinct pathways regulate platelet phosphatidylserine exposureand procoagulant functionrdquo Blood vol 114 no 3 pp 663ndash6662009
[4] J E Italiano Jr J L Richardson S Patel-Hett et al ldquoAngiogen-esis is regulated by a novel mechanism pro- and antiangiogenicproteins are organized into separate platelet 120572 granules anddifferentially releasedrdquo Blood vol 111 no 3 pp 1227ndash1233 2008
[5] J D Pearson ldquoNormal endothelial cell functionrdquo Lupus vol 9no 3 pp 183ndash188 2000
[6] M T Gentile C Vecchione G Marino et al ldquoResistin impairsinsulin-evoked vasodilationrdquo Diabetes vol 57 no 3 pp 577ndash583 2008
[7] G R Wang Y Zhu P V Halushka T M Lincoln and M EMendelsohn ldquoMechanism of platelet inhibition by nitric oxidein vivo phosphorylation of thromboxane receptor by cyclicGMP-dependent protein kinaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no9 pp 4888ndash4893 1998
[8] Y Ohara T E Peterson and D G Harrison ldquoHypercholes-terolemia increases endothelial superoxide anion productionrdquoJournal of Clinical Investigation vol 91 no 6 pp 2546ndash25511993
[9] D D Wagner and P S Frenette ldquoThe vessel wall and itsinteractionsrdquo Blood vol 111 no 11 pp 5271ndash5281 2008
[10] G Brevetti A Silvestro V Schiano and M ChiarielloldquoEndothelial dysfunction and cardiovascular risk predictionin peripheral arterial desease additive value of flow-mediatedilatation to ankle-brachial pressure indexrdquo Circulation vol108 no 17 pp 2093ndash2098 2003
[11] C Heiss T Lauer A Dejam et al ldquoPlasma nitroso compoundsare decreased in patients with endothelial dysfunctionrdquo Journalof the American College of Cardiology vol 47 no 3 pp 573ndash5792006
[12] L Loffredo R Carnevale R Cangemi et al ldquoNOX2 up-regulation is associated with artery dysfunction in patients withperipheral artery diseaserdquo International Journal of Cardiologyvol 165 no 1 pp 184ndash192 2013
[13] J K Bendall R Rinze D Adlam A L Tatham J de Bonoand K M Channon ldquoEndothelial Nox2 overexpression poten-tiates vascular oxidative stress and hemodynamic response toangiotensin II Studies in endothelial-targeted Nox2 transgenicmicerdquo Circulation Research vol 100 no 7 pp 1016ndash1025 2007
[14] O Jung J G Schreiber H Geiger T Pedrazzini R Busse andR P Brandes ldquogp91phox-containing NADPH oxidase mediatesendothelial dysfunction in renovascular hypertensionrdquoCircula-tion vol 109 no 14 pp 1795ndash1801 2004
Oxidative Medicine and Cellular Longevity 9
[15] M de Lorgeril P Salen J L Martin I Monjaud J Delaye andN Mamelle ldquoMediterranean diet traditional risk factors andthe rate of cardiovascular complications aftermyocardial infarc-tion final report of the Lyon Diet Heart Studyrdquo Circulation vol99 no 6 pp 779ndash785 1999
[16] T L Zern and M L Fernandez ldquoCardioprotective effects ofdietary polyphenolsrdquo Journal of Nutrition vol 135 no 10 pp2291ndash2294 2005
[17] O Vitseva S Varghese S Chakrabarti J D Folts and JE Freedman ldquoGrape seed and skin extracts inhibit plateletfunction and release of reactive oxygen intermediatesrdquo Journalof Cardiovascular Pharmacology vol 46 no 4 pp 445ndash4512005
[18] P Pignatelli A Ghiselli B Buchetti et al ldquoPolyphenols syner-gistically inhibit oxidative stress in subjects given red and whitewinerdquo Atherosclerosis vol 188 no 1 pp 77ndash83 2006
[19] R Carnevale L Loffredo P Pignatelli et al ldquoDark chocolateinhibits platelet isoprostanes via NOX2 down-regulation insmokersrdquo Journal of Thrombosis and Haemostasis vol 10 no1 pp 125ndash132 2012
[20] C Menna E de Falco L Pacini G Scafetta P Ruggieri and RPuca ldquoAxitinib affects cell viability and migration of a primaryfoetal lung adenocarcinoma culturerdquo Cancer Investigation vol32 no 1 pp 13ndash21 2014
[21] S W Hoffman R L Roof and D G Stein ldquoA reliableand sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F(2120572) a marker for lipid peroxidation afterexperimental brain injuryrdquo Journal of Neuroscience Methodsvol 68 no 2 pp 133ndash136 1996
[22] P Pignatelli R Carnevale R Cangemi et al ldquoAtorvastatininhibits gp91119901ℎ119900119909 circulating levels in patients with hypercholes-terolemiardquo Arteriosclerosis Thrombosis and Vascular Biologyvol 30 no 2 pp 360ndash367 2010
[23] M C Corretti T J Anderson E J Benjamin et al ldquoGuidelinesfor the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery a report of theinternational brachial artery reactivity task forcerdquo Journal of theAmerican College of Cardiology vol 39 no 2 pp 257ndash265 2002
[24] L Loffredo A Marcoccia P Pignatelli et al ldquoOxidative-stress-mediated arterial dysfunction in patients with peripheralarterial diseaserdquo European Heart Journal vol 28 no 5 pp 608ndash612 2007
[25] A T Hirsch Z J Haskal N R Hertzer et al ldquoACCAHA2005 Practice Guidelines for the management of patients withperipheral arterial disease (lower extremity renal mesentericand abdominal aortic) a collaborative report from the Amer-ican Association for Vascular SurgerySociety for VascularSurgery Society for Cardiovascular Angiography and Interven-tions Society for Vascular Medicine and Biology Society ofInterventional Radiology and the ACCAHA Task Force onPractice Guidelines (Writing Committee to Develop Guidelinesfor the Management of Patients With Peripheral Arterial Dis-ease) endorsed by the American Association of Cardiovascularand Pulmonary Rehabilitation National Heart Lung andBlood Institute Society for Vascular Nursing TransAtlanticInter-Society Consensus and Vascular Disease FoundationrdquoCirculation vol 113 no 11 pp e463ndashe654 2005
[26] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999
[27] T Dickfeld E Lengyel A E May et al ldquoTransient interactionof activated platelets with endothelial cells induces expression
of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway implications foratherogenesisrdquo Cardiovascular Research vol 49 no 1 pp 189ndash199 2001
[28] A G Kjaeligrgaard A Dige J Krog E Toslashnnesen and LWogensen ldquoSoluble adhesion molecules correlate with surfaceexpression in an in vitro model of endothelial activationrdquo Basicand Clinical Pharmacology and Toxicology vol 113 no 49 pp273ndash279 2013
[29] R Joannides W E Haefeli L Linder et al ldquoNitric oxide isresponsible for flow-dependent dilatation of human peripheralconduit arteries in vivordquoCirculation vol 91 no 5 pp 1314ndash13191995
[30] R Bruckdorfer ldquoThe basics about nitric oxiderdquo MolecularAspects of Medicine vol 26 no 1-2 pp 3ndash31 2005
[31] D M Dudzinski and T Michel ldquoLife history of eNOS partnersand pathwaysrdquo Cardiovascular Research vol 75 no 2 pp 247ndash260 2007
[32] L Loffredo L Perri E Catasca et al ldquoDark chocolate acutelyimproves walking autonomy in patients with peripheral arterydiseaserdquo Journal of the American Heart Association vol 3Article ID e001072 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 7
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
P-eNOS
120573-Actin
0
5
10
15
20
25
30
35
40
45
lowast
lowastlowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
Den
sitom
etric
anal
ysis
ofP-
eNO
Sto
tal e
-NO
S (r
atio
)
Total eNOS
(a)
50
0
5
10
15
20
25
30
35
40
45 lowastlowast
lowast lowast
NS
1 10
minus
minus
minus
minus
minus
minus
minus
minus minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
minus
+
+
+ + + +
01
EGF (10ngmL)SAPADSAHSMix (120583M)
HU
VEC
s NO
x pr
oduc
tion
(120583M
)
(b)
Figure 3 HUVEC eNOS activation and NOx production Densitometric analysis of P-eNOStotal eNOS ratio (a) and NOx production (b)in HUVEC incubated with EGF (white bar) supernatant of activated platelets from HS (SAHS) (dark gray bars) supernatant of activatedplatelets from PAD patients (SAPAD) (black bar) and supernatant of activated platelets from PAD patients (SAPAD) pretreated with scalardoses of epicatechin (01ndash10120583M) plus catechin (01ndash10120583M) (mix) (light gray bars) (lowast119875 lt 0001) A representative western blot analysis ofeNOS phosphorylation (a)
8 Oxidative Medicine and Cellular Longevity
Other studies have shown that PAD patients have lowFMD [24] strictly dependent on NO release [29] In accor-dance with these findings our PAD population had reducedFMD and lower nitrite and nitrate (markers of NO gener-ation) compared to controls NO is synthesized from theamino acid L-arginine by a family of enzymes known asnitric oxide synthase (NOS) [30] Among three isoformseNOS is the major responsible for NO production in thecardiovascular system [31] Enhanced eNOS inactivationcaused by excessive ROS production may play an importantrole in the impairment of endothelium-dependent vasodi-latation When HUVEC were coincubated with SAPAD asignificant reduction in p-eNOSwith consequent drop inNObioavailability was observed
In a previous study we showed that the combination ofepicatechin with catechin was able to boost the antioxidantand antiplatelet effects achieved by a single polyphenol[19] Moreover in a recent study we observed that PADpatients treated with dark chocolate rich in epicatechin andcatechin showed a decrease of oxidative stress 2 hours afterits assumption [32]
In our study we explored the hypothesis that polyphe-nols can downregulate platelets activation and subsequentlyreduce ECs activation Therefore we incubated HUVECwith supernatants of activated platelets pretreated with scalardoses of epicatechin plus catechinThese experiments showedthat this mutual combination leads to a decrease in sCAMlevels and an increase in eNOS phosphorylationwith a higherbioavailability of NO
The present study has implications and limitations thatshould be acknowledged This paper suggests a potential roleof polyphenols in the modulation of endothelial function butlacks a specific in vivo proof Given that supernatants havebeen used in the study it is possible to hypothesize by ourresults that platelets can exert their action by an indirectfashion A deeper investigation on supernatant derived fromPAD patients could highlight which specific molecules areresponsible for these effects and their biochemical mecha-nism Last since ROS have a short half-life thus representinga potential limitation of the study a further step forwardwill be the evaluation of other mechanisms independentfrom oxidative stress or more stable markers of oxidativestress able to induce endothelial dysfunction Besides theseresults require further investigation in order to assesswhethersimilar effects can be obtained in vivo by the administrationof aliments or surrogates rich in polyphenols to PAD patientsas in the hierarchy of events that underlie atheroscleroticremodeling of the adult human vessels and the modulationof endothelial activation in PAD patients seems to play a keyrole
In conclusion this study demonstrates that polyphenolsare able to modulate HUVEC activation induced by plateletsderived from PAD patients
Conflict of Interests
The authors confirm that there is no conflict of interests
Acknowledgment
This work was supported by the Grant University of RomeldquoSapienzardquo year 2011 Project no C26A11J528 to GiacomoFrati
References
[1] W Insull Jr ldquoThe pathology of atherosclerosis plaque develop-ment and plaque responses tomedical treatmentrdquoTheAmericanJournal ofMedicine vol 122 no 1 supplement pp S3ndashS14 2009
[2] J I Borissoff H M H Spronk and H T Cate ldquoThe hemostaticsystem as a modulator of atherosclerosisrdquo The New EnglandJournal of Medicine vol 364 no 18 pp 1746ndash1760 2011
[3] S M Schoenwaelder Y Yuan E C Josefsson et al ldquoTwodistinct pathways regulate platelet phosphatidylserine exposureand procoagulant functionrdquo Blood vol 114 no 3 pp 663ndash6662009
[4] J E Italiano Jr J L Richardson S Patel-Hett et al ldquoAngiogen-esis is regulated by a novel mechanism pro- and antiangiogenicproteins are organized into separate platelet 120572 granules anddifferentially releasedrdquo Blood vol 111 no 3 pp 1227ndash1233 2008
[5] J D Pearson ldquoNormal endothelial cell functionrdquo Lupus vol 9no 3 pp 183ndash188 2000
[6] M T Gentile C Vecchione G Marino et al ldquoResistin impairsinsulin-evoked vasodilationrdquo Diabetes vol 57 no 3 pp 577ndash583 2008
[7] G R Wang Y Zhu P V Halushka T M Lincoln and M EMendelsohn ldquoMechanism of platelet inhibition by nitric oxidein vivo phosphorylation of thromboxane receptor by cyclicGMP-dependent protein kinaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no9 pp 4888ndash4893 1998
[8] Y Ohara T E Peterson and D G Harrison ldquoHypercholes-terolemia increases endothelial superoxide anion productionrdquoJournal of Clinical Investigation vol 91 no 6 pp 2546ndash25511993
[9] D D Wagner and P S Frenette ldquoThe vessel wall and itsinteractionsrdquo Blood vol 111 no 11 pp 5271ndash5281 2008
[10] G Brevetti A Silvestro V Schiano and M ChiarielloldquoEndothelial dysfunction and cardiovascular risk predictionin peripheral arterial desease additive value of flow-mediatedilatation to ankle-brachial pressure indexrdquo Circulation vol108 no 17 pp 2093ndash2098 2003
[11] C Heiss T Lauer A Dejam et al ldquoPlasma nitroso compoundsare decreased in patients with endothelial dysfunctionrdquo Journalof the American College of Cardiology vol 47 no 3 pp 573ndash5792006
[12] L Loffredo R Carnevale R Cangemi et al ldquoNOX2 up-regulation is associated with artery dysfunction in patients withperipheral artery diseaserdquo International Journal of Cardiologyvol 165 no 1 pp 184ndash192 2013
[13] J K Bendall R Rinze D Adlam A L Tatham J de Bonoand K M Channon ldquoEndothelial Nox2 overexpression poten-tiates vascular oxidative stress and hemodynamic response toangiotensin II Studies in endothelial-targeted Nox2 transgenicmicerdquo Circulation Research vol 100 no 7 pp 1016ndash1025 2007
[14] O Jung J G Schreiber H Geiger T Pedrazzini R Busse andR P Brandes ldquogp91phox-containing NADPH oxidase mediatesendothelial dysfunction in renovascular hypertensionrdquoCircula-tion vol 109 no 14 pp 1795ndash1801 2004
Oxidative Medicine and Cellular Longevity 9
[15] M de Lorgeril P Salen J L Martin I Monjaud J Delaye andN Mamelle ldquoMediterranean diet traditional risk factors andthe rate of cardiovascular complications aftermyocardial infarc-tion final report of the Lyon Diet Heart Studyrdquo Circulation vol99 no 6 pp 779ndash785 1999
[16] T L Zern and M L Fernandez ldquoCardioprotective effects ofdietary polyphenolsrdquo Journal of Nutrition vol 135 no 10 pp2291ndash2294 2005
[17] O Vitseva S Varghese S Chakrabarti J D Folts and JE Freedman ldquoGrape seed and skin extracts inhibit plateletfunction and release of reactive oxygen intermediatesrdquo Journalof Cardiovascular Pharmacology vol 46 no 4 pp 445ndash4512005
[18] P Pignatelli A Ghiselli B Buchetti et al ldquoPolyphenols syner-gistically inhibit oxidative stress in subjects given red and whitewinerdquo Atherosclerosis vol 188 no 1 pp 77ndash83 2006
[19] R Carnevale L Loffredo P Pignatelli et al ldquoDark chocolateinhibits platelet isoprostanes via NOX2 down-regulation insmokersrdquo Journal of Thrombosis and Haemostasis vol 10 no1 pp 125ndash132 2012
[20] C Menna E de Falco L Pacini G Scafetta P Ruggieri and RPuca ldquoAxitinib affects cell viability and migration of a primaryfoetal lung adenocarcinoma culturerdquo Cancer Investigation vol32 no 1 pp 13ndash21 2014
[21] S W Hoffman R L Roof and D G Stein ldquoA reliableand sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F(2120572) a marker for lipid peroxidation afterexperimental brain injuryrdquo Journal of Neuroscience Methodsvol 68 no 2 pp 133ndash136 1996
[22] P Pignatelli R Carnevale R Cangemi et al ldquoAtorvastatininhibits gp91119901ℎ119900119909 circulating levels in patients with hypercholes-terolemiardquo Arteriosclerosis Thrombosis and Vascular Biologyvol 30 no 2 pp 360ndash367 2010
[23] M C Corretti T J Anderson E J Benjamin et al ldquoGuidelinesfor the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery a report of theinternational brachial artery reactivity task forcerdquo Journal of theAmerican College of Cardiology vol 39 no 2 pp 257ndash265 2002
[24] L Loffredo A Marcoccia P Pignatelli et al ldquoOxidative-stress-mediated arterial dysfunction in patients with peripheralarterial diseaserdquo European Heart Journal vol 28 no 5 pp 608ndash612 2007
[25] A T Hirsch Z J Haskal N R Hertzer et al ldquoACCAHA2005 Practice Guidelines for the management of patients withperipheral arterial disease (lower extremity renal mesentericand abdominal aortic) a collaborative report from the Amer-ican Association for Vascular SurgerySociety for VascularSurgery Society for Cardiovascular Angiography and Interven-tions Society for Vascular Medicine and Biology Society ofInterventional Radiology and the ACCAHA Task Force onPractice Guidelines (Writing Committee to Develop Guidelinesfor the Management of Patients With Peripheral Arterial Dis-ease) endorsed by the American Association of Cardiovascularand Pulmonary Rehabilitation National Heart Lung andBlood Institute Society for Vascular Nursing TransAtlanticInter-Society Consensus and Vascular Disease FoundationrdquoCirculation vol 113 no 11 pp e463ndashe654 2005
[26] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999
[27] T Dickfeld E Lengyel A E May et al ldquoTransient interactionof activated platelets with endothelial cells induces expression
of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway implications foratherogenesisrdquo Cardiovascular Research vol 49 no 1 pp 189ndash199 2001
[28] A G Kjaeligrgaard A Dige J Krog E Toslashnnesen and LWogensen ldquoSoluble adhesion molecules correlate with surfaceexpression in an in vitro model of endothelial activationrdquo Basicand Clinical Pharmacology and Toxicology vol 113 no 49 pp273ndash279 2013
[29] R Joannides W E Haefeli L Linder et al ldquoNitric oxide isresponsible for flow-dependent dilatation of human peripheralconduit arteries in vivordquoCirculation vol 91 no 5 pp 1314ndash13191995
[30] R Bruckdorfer ldquoThe basics about nitric oxiderdquo MolecularAspects of Medicine vol 26 no 1-2 pp 3ndash31 2005
[31] D M Dudzinski and T Michel ldquoLife history of eNOS partnersand pathwaysrdquo Cardiovascular Research vol 75 no 2 pp 247ndash260 2007
[32] L Loffredo L Perri E Catasca et al ldquoDark chocolate acutelyimproves walking autonomy in patients with peripheral arterydiseaserdquo Journal of the American Heart Association vol 3Article ID e001072 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Oxidative Medicine and Cellular Longevity
Other studies have shown that PAD patients have lowFMD [24] strictly dependent on NO release [29] In accor-dance with these findings our PAD population had reducedFMD and lower nitrite and nitrate (markers of NO gener-ation) compared to controls NO is synthesized from theamino acid L-arginine by a family of enzymes known asnitric oxide synthase (NOS) [30] Among three isoformseNOS is the major responsible for NO production in thecardiovascular system [31] Enhanced eNOS inactivationcaused by excessive ROS production may play an importantrole in the impairment of endothelium-dependent vasodi-latation When HUVEC were coincubated with SAPAD asignificant reduction in p-eNOSwith consequent drop inNObioavailability was observed
In a previous study we showed that the combination ofepicatechin with catechin was able to boost the antioxidantand antiplatelet effects achieved by a single polyphenol[19] Moreover in a recent study we observed that PADpatients treated with dark chocolate rich in epicatechin andcatechin showed a decrease of oxidative stress 2 hours afterits assumption [32]
In our study we explored the hypothesis that polyphe-nols can downregulate platelets activation and subsequentlyreduce ECs activation Therefore we incubated HUVECwith supernatants of activated platelets pretreated with scalardoses of epicatechin plus catechinThese experiments showedthat this mutual combination leads to a decrease in sCAMlevels and an increase in eNOS phosphorylationwith a higherbioavailability of NO
The present study has implications and limitations thatshould be acknowledged This paper suggests a potential roleof polyphenols in the modulation of endothelial function butlacks a specific in vivo proof Given that supernatants havebeen used in the study it is possible to hypothesize by ourresults that platelets can exert their action by an indirectfashion A deeper investigation on supernatant derived fromPAD patients could highlight which specific molecules areresponsible for these effects and their biochemical mecha-nism Last since ROS have a short half-life thus representinga potential limitation of the study a further step forwardwill be the evaluation of other mechanisms independentfrom oxidative stress or more stable markers of oxidativestress able to induce endothelial dysfunction Besides theseresults require further investigation in order to assesswhethersimilar effects can be obtained in vivo by the administrationof aliments or surrogates rich in polyphenols to PAD patientsas in the hierarchy of events that underlie atheroscleroticremodeling of the adult human vessels and the modulationof endothelial activation in PAD patients seems to play a keyrole
In conclusion this study demonstrates that polyphenolsare able to modulate HUVEC activation induced by plateletsderived from PAD patients
Conflict of Interests
The authors confirm that there is no conflict of interests
Acknowledgment
This work was supported by the Grant University of RomeldquoSapienzardquo year 2011 Project no C26A11J528 to GiacomoFrati
References
[1] W Insull Jr ldquoThe pathology of atherosclerosis plaque develop-ment and plaque responses tomedical treatmentrdquoTheAmericanJournal ofMedicine vol 122 no 1 supplement pp S3ndashS14 2009
[2] J I Borissoff H M H Spronk and H T Cate ldquoThe hemostaticsystem as a modulator of atherosclerosisrdquo The New EnglandJournal of Medicine vol 364 no 18 pp 1746ndash1760 2011
[3] S M Schoenwaelder Y Yuan E C Josefsson et al ldquoTwodistinct pathways regulate platelet phosphatidylserine exposureand procoagulant functionrdquo Blood vol 114 no 3 pp 663ndash6662009
[4] J E Italiano Jr J L Richardson S Patel-Hett et al ldquoAngiogen-esis is regulated by a novel mechanism pro- and antiangiogenicproteins are organized into separate platelet 120572 granules anddifferentially releasedrdquo Blood vol 111 no 3 pp 1227ndash1233 2008
[5] J D Pearson ldquoNormal endothelial cell functionrdquo Lupus vol 9no 3 pp 183ndash188 2000
[6] M T Gentile C Vecchione G Marino et al ldquoResistin impairsinsulin-evoked vasodilationrdquo Diabetes vol 57 no 3 pp 577ndash583 2008
[7] G R Wang Y Zhu P V Halushka T M Lincoln and M EMendelsohn ldquoMechanism of platelet inhibition by nitric oxidein vivo phosphorylation of thromboxane receptor by cyclicGMP-dependent protein kinaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no9 pp 4888ndash4893 1998
[8] Y Ohara T E Peterson and D G Harrison ldquoHypercholes-terolemia increases endothelial superoxide anion productionrdquoJournal of Clinical Investigation vol 91 no 6 pp 2546ndash25511993
[9] D D Wagner and P S Frenette ldquoThe vessel wall and itsinteractionsrdquo Blood vol 111 no 11 pp 5271ndash5281 2008
[10] G Brevetti A Silvestro V Schiano and M ChiarielloldquoEndothelial dysfunction and cardiovascular risk predictionin peripheral arterial desease additive value of flow-mediatedilatation to ankle-brachial pressure indexrdquo Circulation vol108 no 17 pp 2093ndash2098 2003
[11] C Heiss T Lauer A Dejam et al ldquoPlasma nitroso compoundsare decreased in patients with endothelial dysfunctionrdquo Journalof the American College of Cardiology vol 47 no 3 pp 573ndash5792006
[12] L Loffredo R Carnevale R Cangemi et al ldquoNOX2 up-regulation is associated with artery dysfunction in patients withperipheral artery diseaserdquo International Journal of Cardiologyvol 165 no 1 pp 184ndash192 2013
[13] J K Bendall R Rinze D Adlam A L Tatham J de Bonoand K M Channon ldquoEndothelial Nox2 overexpression poten-tiates vascular oxidative stress and hemodynamic response toangiotensin II Studies in endothelial-targeted Nox2 transgenicmicerdquo Circulation Research vol 100 no 7 pp 1016ndash1025 2007
[14] O Jung J G Schreiber H Geiger T Pedrazzini R Busse andR P Brandes ldquogp91phox-containing NADPH oxidase mediatesendothelial dysfunction in renovascular hypertensionrdquoCircula-tion vol 109 no 14 pp 1795ndash1801 2004
Oxidative Medicine and Cellular Longevity 9
[15] M de Lorgeril P Salen J L Martin I Monjaud J Delaye andN Mamelle ldquoMediterranean diet traditional risk factors andthe rate of cardiovascular complications aftermyocardial infarc-tion final report of the Lyon Diet Heart Studyrdquo Circulation vol99 no 6 pp 779ndash785 1999
[16] T L Zern and M L Fernandez ldquoCardioprotective effects ofdietary polyphenolsrdquo Journal of Nutrition vol 135 no 10 pp2291ndash2294 2005
[17] O Vitseva S Varghese S Chakrabarti J D Folts and JE Freedman ldquoGrape seed and skin extracts inhibit plateletfunction and release of reactive oxygen intermediatesrdquo Journalof Cardiovascular Pharmacology vol 46 no 4 pp 445ndash4512005
[18] P Pignatelli A Ghiselli B Buchetti et al ldquoPolyphenols syner-gistically inhibit oxidative stress in subjects given red and whitewinerdquo Atherosclerosis vol 188 no 1 pp 77ndash83 2006
[19] R Carnevale L Loffredo P Pignatelli et al ldquoDark chocolateinhibits platelet isoprostanes via NOX2 down-regulation insmokersrdquo Journal of Thrombosis and Haemostasis vol 10 no1 pp 125ndash132 2012
[20] C Menna E de Falco L Pacini G Scafetta P Ruggieri and RPuca ldquoAxitinib affects cell viability and migration of a primaryfoetal lung adenocarcinoma culturerdquo Cancer Investigation vol32 no 1 pp 13ndash21 2014
[21] S W Hoffman R L Roof and D G Stein ldquoA reliableand sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F(2120572) a marker for lipid peroxidation afterexperimental brain injuryrdquo Journal of Neuroscience Methodsvol 68 no 2 pp 133ndash136 1996
[22] P Pignatelli R Carnevale R Cangemi et al ldquoAtorvastatininhibits gp91119901ℎ119900119909 circulating levels in patients with hypercholes-terolemiardquo Arteriosclerosis Thrombosis and Vascular Biologyvol 30 no 2 pp 360ndash367 2010
[23] M C Corretti T J Anderson E J Benjamin et al ldquoGuidelinesfor the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery a report of theinternational brachial artery reactivity task forcerdquo Journal of theAmerican College of Cardiology vol 39 no 2 pp 257ndash265 2002
[24] L Loffredo A Marcoccia P Pignatelli et al ldquoOxidative-stress-mediated arterial dysfunction in patients with peripheralarterial diseaserdquo European Heart Journal vol 28 no 5 pp 608ndash612 2007
[25] A T Hirsch Z J Haskal N R Hertzer et al ldquoACCAHA2005 Practice Guidelines for the management of patients withperipheral arterial disease (lower extremity renal mesentericand abdominal aortic) a collaborative report from the Amer-ican Association for Vascular SurgerySociety for VascularSurgery Society for Cardiovascular Angiography and Interven-tions Society for Vascular Medicine and Biology Society ofInterventional Radiology and the ACCAHA Task Force onPractice Guidelines (Writing Committee to Develop Guidelinesfor the Management of Patients With Peripheral Arterial Dis-ease) endorsed by the American Association of Cardiovascularand Pulmonary Rehabilitation National Heart Lung andBlood Institute Society for Vascular Nursing TransAtlanticInter-Society Consensus and Vascular Disease FoundationrdquoCirculation vol 113 no 11 pp e463ndashe654 2005
[26] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999
[27] T Dickfeld E Lengyel A E May et al ldquoTransient interactionof activated platelets with endothelial cells induces expression
of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway implications foratherogenesisrdquo Cardiovascular Research vol 49 no 1 pp 189ndash199 2001
[28] A G Kjaeligrgaard A Dige J Krog E Toslashnnesen and LWogensen ldquoSoluble adhesion molecules correlate with surfaceexpression in an in vitro model of endothelial activationrdquo Basicand Clinical Pharmacology and Toxicology vol 113 no 49 pp273ndash279 2013
[29] R Joannides W E Haefeli L Linder et al ldquoNitric oxide isresponsible for flow-dependent dilatation of human peripheralconduit arteries in vivordquoCirculation vol 91 no 5 pp 1314ndash13191995
[30] R Bruckdorfer ldquoThe basics about nitric oxiderdquo MolecularAspects of Medicine vol 26 no 1-2 pp 3ndash31 2005
[31] D M Dudzinski and T Michel ldquoLife history of eNOS partnersand pathwaysrdquo Cardiovascular Research vol 75 no 2 pp 247ndash260 2007
[32] L Loffredo L Perri E Catasca et al ldquoDark chocolate acutelyimproves walking autonomy in patients with peripheral arterydiseaserdquo Journal of the American Heart Association vol 3Article ID e001072 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 9
[15] M de Lorgeril P Salen J L Martin I Monjaud J Delaye andN Mamelle ldquoMediterranean diet traditional risk factors andthe rate of cardiovascular complications aftermyocardial infarc-tion final report of the Lyon Diet Heart Studyrdquo Circulation vol99 no 6 pp 779ndash785 1999
[16] T L Zern and M L Fernandez ldquoCardioprotective effects ofdietary polyphenolsrdquo Journal of Nutrition vol 135 no 10 pp2291ndash2294 2005
[17] O Vitseva S Varghese S Chakrabarti J D Folts and JE Freedman ldquoGrape seed and skin extracts inhibit plateletfunction and release of reactive oxygen intermediatesrdquo Journalof Cardiovascular Pharmacology vol 46 no 4 pp 445ndash4512005
[18] P Pignatelli A Ghiselli B Buchetti et al ldquoPolyphenols syner-gistically inhibit oxidative stress in subjects given red and whitewinerdquo Atherosclerosis vol 188 no 1 pp 77ndash83 2006
[19] R Carnevale L Loffredo P Pignatelli et al ldquoDark chocolateinhibits platelet isoprostanes via NOX2 down-regulation insmokersrdquo Journal of Thrombosis and Haemostasis vol 10 no1 pp 125ndash132 2012
[20] C Menna E de Falco L Pacini G Scafetta P Ruggieri and RPuca ldquoAxitinib affects cell viability and migration of a primaryfoetal lung adenocarcinoma culturerdquo Cancer Investigation vol32 no 1 pp 13ndash21 2014
[21] S W Hoffman R L Roof and D G Stein ldquoA reliableand sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F(2120572) a marker for lipid peroxidation afterexperimental brain injuryrdquo Journal of Neuroscience Methodsvol 68 no 2 pp 133ndash136 1996
[22] P Pignatelli R Carnevale R Cangemi et al ldquoAtorvastatininhibits gp91119901ℎ119900119909 circulating levels in patients with hypercholes-terolemiardquo Arteriosclerosis Thrombosis and Vascular Biologyvol 30 no 2 pp 360ndash367 2010
[23] M C Corretti T J Anderson E J Benjamin et al ldquoGuidelinesfor the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery a report of theinternational brachial artery reactivity task forcerdquo Journal of theAmerican College of Cardiology vol 39 no 2 pp 257ndash265 2002
[24] L Loffredo A Marcoccia P Pignatelli et al ldquoOxidative-stress-mediated arterial dysfunction in patients with peripheralarterial diseaserdquo European Heart Journal vol 28 no 5 pp 608ndash612 2007
[25] A T Hirsch Z J Haskal N R Hertzer et al ldquoACCAHA2005 Practice Guidelines for the management of patients withperipheral arterial disease (lower extremity renal mesentericand abdominal aortic) a collaborative report from the Amer-ican Association for Vascular SurgerySociety for VascularSurgery Society for Cardiovascular Angiography and Interven-tions Society for Vascular Medicine and Biology Society ofInterventional Radiology and the ACCAHA Task Force onPractice Guidelines (Writing Committee to Develop Guidelinesfor the Management of Patients With Peripheral Arterial Dis-ease) endorsed by the American Association of Cardiovascularand Pulmonary Rehabilitation National Heart Lung andBlood Institute Society for Vascular Nursing TransAtlanticInter-Society Consensus and Vascular Disease FoundationrdquoCirculation vol 113 no 11 pp e463ndashe654 2005
[26] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999
[27] T Dickfeld E Lengyel A E May et al ldquoTransient interactionof activated platelets with endothelial cells induces expression
of monocyte-chemoattractant protein-1 via a p38 mitogen-activated protein kinase mediated pathway implications foratherogenesisrdquo Cardiovascular Research vol 49 no 1 pp 189ndash199 2001
[28] A G Kjaeligrgaard A Dige J Krog E Toslashnnesen and LWogensen ldquoSoluble adhesion molecules correlate with surfaceexpression in an in vitro model of endothelial activationrdquo Basicand Clinical Pharmacology and Toxicology vol 113 no 49 pp273ndash279 2013
[29] R Joannides W E Haefeli L Linder et al ldquoNitric oxide isresponsible for flow-dependent dilatation of human peripheralconduit arteries in vivordquoCirculation vol 91 no 5 pp 1314ndash13191995
[30] R Bruckdorfer ldquoThe basics about nitric oxiderdquo MolecularAspects of Medicine vol 26 no 1-2 pp 3ndash31 2005
[31] D M Dudzinski and T Michel ldquoLife history of eNOS partnersand pathwaysrdquo Cardiovascular Research vol 75 no 2 pp 247ndash260 2007
[32] L Loffredo L Perri E Catasca et al ldquoDark chocolate acutelyimproves walking autonomy in patients with peripheral arterydiseaserdquo Journal of the American Heart Association vol 3Article ID e001072 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom