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Research ArticleElastase and Cathepsin G from Primed Leukocytes CleaveVascular Endothelial Cadherin in Hemodialysis Patients

Meital Cohen-Mazor12 Rafi Mazor12 Batya Kristal34 and Shifra Sela24

1 Bruce Rappaport School of Medicine Technion 31096 Haifa Israel2 Eliachar Research Laboratory Western Galilee Hospital 22100 Naharyia Israel3 Nephrology Unit Western Galilee Hospital 22100 Nahariya Israel4 Faculty of Medicine in the Galilee Bar Ilan University 13100 Safed Israel

Correspondence should be addressed to Meital Cohen-Mazor mazmeitalgmailcom

Received 3 February 2014 Accepted 31 March 2014 Published 4 May 2014

Academic Editor Beatrice Charreau

Copyright copy 2014 Meital Cohen-Mazor et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Aims To test the hypothesis that primed PMNLs in blood of chronic kidney disease patients release the active form of elastase andcathepsin G causing degradation of vital proteins and promote tissue damage Methods RT-PCR immunocytochemical stainingimmunoblotting and FACS analyses were used to study these enzymes in hemodialysis patients (HD) versus healthy normalcontrols (NC) Using PMNLs and endothelial cells cocultivation system we measure the effect of HD PMNLs on the endothelialVE-cadherin an essential protein for maintaining endothelial integrity Results Levels of elastase and cathepsin G were reduced inPMNLs of HD patients while mRNA enzymes levels were not different Elevated levels of the active form of these enzymes werefound in blood of HD patients compared to NCHD plasma had higher levels of soluble VE-cadherin present in three molecularforms whole 140 kDa molecule and two fragments of 100 and 40 kDa Cocultivation studies showed that primed PMNLs cleavethe endothelial cadherin resulting in a 100 kDa fragment Conclusions Elastase and cathepsin G are elevated in the plasma of HDpatients originating from primed PMNLs In these patients chronic elevation of these enzymes contributes to cleavage of VE-cadherin and possible disruption of endothelial integrity

1 Introduction

Elastase and cathepsin G are serine proteinases containedin the azurophilic granules of peripheral polymorphonuclearleukocytes (PMNLs) and degrade proteins of phagocytosedmicroorganisms [1 2] Stored after synthesis in their activeform these proteolytic enzymes have potential deleteriouseffects When released they act on substrates comprisingthe connective tissue of the lung blood vessels joints andother organs causing tissue destruction [3ndash9] Circulatingproteinase inhibitors such as serum proteins 1205721-antitrypsin(1205721-AT) and 1205721-antichymotrypsin (1205721-ACT) neutralize theactivity of these proteinases in plasma [10 11] The clinicalsignificance of elastase and cathepsin G ismost evident underconditions in which an imbalance develops between theseenzymes and their inhibitors enabling the circulating activeenzymes to induce vascular injury

PMNL serine proteinases have been implicated in thepathogenesis of diseases associated with PMNL primingsuch as rheumatoid arthritis [12 13] PMNL priming isalso associated with pathological disorders associated withatherosclerosis and cardiovascular diseases such as hyper-tension diabetes and end stage renal disease treated withchronic hemodialysis (HD) [14ndash16] Studies have shown thatpurified elastase and cathepsin G can cleave the extracellularpart of vascular endothelial (VE) cadherin an essentialprotein formaintaining vascular endothelial integrity [17 18]and that elastase and cathepsin G bound to their inhibitor arereleased during hemodialysis sessions [19]

In this study we sought out to test the hypothesis thateven before dialysis HD PMNLs are primed thus contribut-ing to elevated levels of elastase and cathepsin G in theplasma We have also evaluated cleavage of VE-cadherinmediated by these proteinases found in HD plasma

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 459640 10 pageshttpdxdoiorg1011552014459640

2 BioMed Research International

Our findings consist of elevated levels of these proteins inHD plasma Plasma of HD had higher levels of VE-cadherinfragments the result of this molecule cleavage by active elas-tase and cathepsin G HDPMNLs as well as purified enzymescleaved VE-cadherin from cultured endothelial cells Takentogether this study provides a novel mechanism by whichactive elastase and cathepsin G originating from primedPMNLs of HD patients can initiate endothelial dysfunction

2 Materials and Methods

21 Patients and Blood Samples Ten hemodialysis patientsand healthy control subjects were enrolled in this study(Table 1) Blood for the determination of biochemical andhematological parameters and for the isolation of PMNLswas drawn into citrate tubes and in HD patients fromthe arterial line immediately before a dialysis session Allpatients underwent hemodialysis three times a week eachdialysis treatment lasted 4 hours and was carried out withlowflux polysulfonemembranes (F8 FreseniusMedical CareBad Homburg Germany) The water for dialysis met thestandards of the Association for the Advancement of MedicalInstrumentation (AAMI) Patients with evidence of acute orchronic infection malignancy or who had received a bloodtransfusion within three months prior to blood samplingwere excluded All participants signed an informed consentfor blood sampling and the study was approved by theInstitutional Committee in accordance with the Helsinkideclaration

22 PMNL Isolation and Analysis PMNLs were isolatedas described previously [15] Isolated PMNLs (gt98 pureapproximately 107 cells per isolation) were resuspended andcounted in phosphate buffered saline (PBS Beit HaemekIsrael) containing 01 glucose PMNL priming was assessedby the rate of superoxide release [14] and by the surfacelevels of CD11b as described previously [20] The rate ofsuperoxide release was determined after cell stimulation with032 times 10minus7M phorbol 12-myristate 13-acetate (PMA SigmaSt LouisMO)The assay is based upon superoxide dismutase(SOD) inhibitable reduction of 80 120583M cytochrome C (SigmaSt Louis MO) to its ferrous form The change in opticaldensity was monitored at 549 nm as described previously[14] Expression of CD11b on PMNLs in whole blood wasdetermined using the FC500 flow cytometer (Beckman-Coulter) Fifty 120583L of blood was incubated for 10min with antiCD11b-PE conjugated monoclonal antibody (IQ ProductsThe Netherlands) followed by RBC lysis (Q-prep methodCoulter Corporation Hialeah FL USA) To enable gatingon the PMNL population anti CD16 conjugated to PC5monoclonal antibody (Immunotech Marseille France) wasused Surface levels of CD11b on PMNLs are expressedas mean fluorescence intensity (MFI) after subtracting thenonspecific background

23 RNA Isolation and RT-PCR of Elastase and CathepsinG RNA was isolated using Tri Reagent according to themanufacturerrsquos (Sigma) instructions Complementary DNA

Table 1 Demographics of HD patients and NC subjects used in thisstudy

Hemodialysispatients (119899 = 10)

Normal controls(119899 = 10)

Age (years) 54 plusmn 7 47 plusmn 5

MaleFemale 55 55

Diabetes 3 mdashTime on hemodialysis(months) 38 plusmn 6 mdash

was synthesized according to Krug and Berger [21] usingan OmniGene thermal cycler (Hybaid UK) The resultingcDNAswere amplified by PCRusing 2120583L aliquots of RT reac-tion incubated with 02mM dNTPs 2120583L of Taq DNA poly-merase buffer 06U of TaqDNA polymerase (BioLine UK)and 10 pmoles of the specific primers The oligonucleotidesequence for elastase according to Hirata et al [22] is sense(51015840-AGTGCCTGGCCATGGGCTGG-31015840) and antisense (51015840-CACCGGGGCAAAGGCATCGG-31015840) with a final PCR prod-uct of 257 bp The PCR conditions were initial denaturationat 94∘C for 5 minutes 30 PCR cycles of denaturation at94∘C for 1 minute annealing at 55∘C for 2 minutes andextension at 72∘C for 3minutesTheoligonucleotide sequencefor cathepsin G according to Hirata et al [22] is sense(51015840-TGAGAGTGCAGAGGGATAGG-31015840) and antisense (51015840-CAGGAAACTTGAGACCCTGC-31015840) with a final PCR prod-uct of 216 bp The PCR conditions were identical to thosedescribed above for elastase Aliquots (15 120583L) of the amplifiedcDNA of elastase and cathepsin G were separated on 1agarose gel electrophoresis visualized by ethidium bromidestaining and compared to a housekeeping gene 120573-actin The200 bp product of actin was amplified using the followingprimers sense (51015840-CCTTCCTGGGCATGGAGTCCTG-31015840)and antisense (51015840-GAGCAATGATCTTGATCTTC-31015840) Thedensities of the enzyme PCRproducts were calculated in eachgel relative to the actin product

24 Detection and Quantificationof Elastase and Cathepsin Gin PMNLs

(1) Intracellular Levels of Elastase and Cathepsin G in PMNLsThe levels of intracellular elastase and cathepsin G weremeasured in whole blood by flow cytometry using polyclonalrabbit anti-human elastase and anti-human cathepsin Gantibodies (United States Biological Massachusetts USA)followed by a secondary FITC-conjugated fluorescent anti-rabbit antibody (Chemicon International CA) PMNLs werepermeabilized with the FIX amp PERM cell permeabilizationkit Anti CD16- PC5 (Immunotech Marseille France) wasused for gating on PMNLs Total rabbit IgG antibodies(United States Biological Massachusetts USA) followed by asecondary FITC-conjugated fluorescent anti-rabbit antibodyserved as nonspecific controls of the fluorochrome Theresults are presented as MFI per cell after subtracting thenonspecific background

BioMed Research International 3

(2) Localization of Elastase and Cathepsin G in PMNLsHuman PMNLs were stained by indirect immunohisto-chemical staining Whole blood slides were prepared byfixation and permeabilization with methanol (minus20∘C 3minutes) The slides were stained with specific anti-elastaseand anti-cathepsin G antibodies (United States BiologicalMassachusetts USA)TheHistofine Simple Stain kit (NichireiCorporation Japan) was used for immunohistochemicalstaining of PMNLs according to the manufacturerrsquos instruc-tions

25 Detection and Quantification of Elastase and CathepsinG in Plasma Plasma of NC subjects and HD patientswas depleted of albumin and immunoglobulins by Proteo-Prep Blue Albumin Depletion Kit (PROT-BA KIT) (Sigma-Aldrich USA) Following the addition of Laemli loadingbuffer containing 3 120573-mercaptoethanol the plasma wasboiled at 100∘C for 5 minutes Samples were loaded onto15 polyacrylamide SDS gels for separation of proteins andthen transferred to nitrocellulose filters by semidry transfer(Biometra Germany)The filters were blocked by 1 fat milkfor 1 hour at room temperature and then incubated at roomtemperature first with rabbit anti-human elastaserabbitanti-human cathepsin G antibodies (Fitzgerald IndustriesInternational USA) (diluted 1 1000) for 2 hours followedby incubation with goat anti rabbit-HRP conjugate (diluted1 25000) for an additional hour The amount of the enzymein each sample was calculated according to a positive controlloaded on the same gel active elastase (Sigma-Aldrich-catnumber E-8140)active cathepsin G (Fitzgerald IndustriesInternational USA)

The enzyme signal was detected on X-ray films using thechemiluminescence reagents of the EZ-ECL kitThe densitiesof the enzyme bands were determined by the BioCapt andBio-Profile (Bio-1D) software

26 Endothelial Cell Culture Human umbilical veinendothelial cells (HUVEC) were cultured as described byJaffe et al [23] with minor modifications according to Laniret al [24] Endothelial cell specificity (gt95) was confirmedby flow cytometry of cells stained with anti-human CD-34PE-conjugated antibody (Becton Dickinson USA) after celldetachment by trypsinEDTA (Biological Industries BeitHaemek Israel)

27 Cocultivation of HUVEC with PMNLs HUVEC wereseeded in growth medium at a density of 1 times 105 cells insix-well plates (Nalge Nunc International USA) for 2 daysunder subconfluent conditions Experiments were performedalways between passages 2 to 4 In all experiments prior tothe addition of PMNLs HUVEC were deprived of culturemedium and kept in phosphate buffered saline (PBS) for90min at 37∘C [25] To prevent the direct contact of PMNLswith HUVEC 045120583m pore size cell culture inserts (25mmtissue culture inserts Nalge Nunc International) were placedinto each well on top of HUVEC in direct contact with themedium 1mL of PBS [26] PMNLs (106) from NC or HD

were seeded on these inserts and coincubated for 15 minutesat 37∘C with HUVEC as previously described [25]

28 Assessment of VE-Cadherin on HUVEC

(1) By Flow Cytometry HUVEC following 15 minute ofcocultivation with PMNLs were rinsed with PBS scrapedcentrifuged resuspended and incubated for 10 minutes withmouse anti VE-cadherinmonoclonal antibody (United StatesBiological MA USA) followed by incubationwith PE conju-gated anti-mouse antibody (IQ products The Netherlands)Irrelevant IgG-PE served as a nonspecific control of thefluorochrome (IQ products The Netherlands)

(2) By Immunohistochemical Staining HUVEC were grownon a chamber a procedure that does not require cell transferprior to visualizationstaining and cocultivated with PMNLsas previously described [25] After cocultivation the growthchamber was removed and cells were fixed on the slidesin 95 ethanol and stained with mouse anti VE-cadherinmonoclonal antibody (United States Biological MA USA)

29 Evaluation of Soluble VE-Cadherin Fragments

(1) In Plasma Soluble VE-cadherin was measured usingwestern blot analysis The plasma of NC subjects and HDpatients was depleted of albumin and immunoglobulinsFollowing the addition of Laemli loading buffer containing3 120573-mercaptoethanol the plasma was boiled at 100∘C for 5minutes Samples were loaded onto 6 polyacrylamide SDSgels as described above The whole 140 kDa molecule andthe 100 kDa fragment were detected usingmouse anti-humanVE-cadherin (Cell Signaling Technology MA USA) whilethe 40 kDa fragment was detected using mouse anti-humanVE-cadherin (United States Biological Massachusetts USA)which is not able to recognize the 140 kDa and the 100 kDamolecules Thus due to the different antibodies used plasmasampled was loaded onto two gels one stained for theevaluation of 100 + 140 kDa forms and one for the 40 kDaform The levels of both whole and cleaved forms of VE-cadherin were assessed in HD plasma and compared withthose of NC plasma To calculate the levels of the enzymesthe bands were all compared to the same plasma sample runon all gels serving as a normalizing control

(2) In the Cocultivation Media The media of four coculti-vation experiments were pooled in order to get detectableamount of these proteins These media samples were treatedwith Vivaspin concentrators (Sartorius AG Germany) forimproved recovery of the low-concentration protein samplesFollowing the addition of Laemli loading buffer containing3 120573-mercaptoethanol the plasma was boiled at 100∘C for5 minutes Samples were loaded onto 6 polyacrylamideSDS gels as described above using mouse anti-human VE-cadherin (Cell Signaling Technology MA USA)

210 Statistical Analysis Data is expressed as mean plusmn SDIn the boxes and whiskers presentations the horizontal line


in the middle shows the median (50th percentile) the topand bottom of the box show the 75th and 25th percentilesrespectively and the whiskers show the maximum and theminimum values The nonparametric Mann-Whitney testwas used for comparing two independent groups The two-paired Wilcoxon Signed Ranks test was used for comparingtwo dependent groups Statistical significancewas consideredat 119875 lt 005

3 Results

31 PMNLs Priming in HD Patients We reported previouslythat PMNLs from HD patients are primed [16] To confirmthese results we isolated PMNLs and measured the rate ofsuperoxide release and the levels of surface CD11b indices ofPMNL priming [20]The rate of superoxide release followingPMA stimulation was higher in PMNLs isolated from HDpatients than in those from NC (385 plusmn 39 versus 247 plusmn 5nmoles106 cells10min resp 119875 lt 005) The membranelevels of CD11b were higher in PMNLs isolated from HDpatients than in PMNLs from NC (499 plusmn 78 versus 32 plusmn 4MFI resp 119875 lt 005)

32 Elastase and Cathepsin GmRNA in PMNLs Elastase andcathepsin G mRNA levels were not significantly different inNC versus HD PMNLs 039 plusmn 014 versus 033 plusmn 022 relativedensity units respectively for elastase 119875 = ns and 074 plusmn01 versus 086 plusmn 047 relative density units respectively forcathepsin G 119875 = ns

33 PMNL Intracellular Protein Levels of Elastase and Cathep-sin G Flow cytometry measurements of elastase and cathep-sin G in PMNLsmeasured in whole blood (Figures 1(a)ndash1(e))showed higher levels of these enzymes in NC PMNLs than inHDPMNLs (average of 229plusmn27 versus 123plusmn17MFI respfor elastase 119875 lt 005 and 35plusmn52 versus 128plusmn14MFI respfor cathepsin G 119875 lt 005)

A significant negative correlation was found between thefluorescent intensity of intracellular elastase and cathepsin Gand membrane CD11b expression on PMNLs (119903 = minus043and 119903 = minus051 resp 119875 lt 005) This negative correlationindicates that the higher the priming the lower the amount ofthe intracellular enzymes

34 Immunohistochemical Staining of Elastase and CathepsinG in PMNLs The intracellular levels and locations of elas-tase and cathepsin G were also evaluated by immunohisto-chemical staining of PMNLs in smears of whole blood asrepresented in Figure 2 Examination of the cells under alight microscope revealed that elastase and cathepsin G wereabundant and distributed in PMNLs of NC while sparse inHD PMNLs and even missing in some patients

35 Plasma Levels of Elastase and Cathepsin G Plasma ofNC and HD was depleted of albumin and immunoglobulinsin order to enrich it with elastase and cathepsin G Afterdepletion plasma proteins were separated on SDS-PAGEfollowed by western blot analysis (Figure 3) HD plasma

contained higher levels of free elastase and cathepsin G(30KDa) than NC plasma (average of 13 plusmn 014 versus076 plusmn 008 relative density units resp for plasma elastase119875 lt 005 and 052 plusmn 004 versus 034 plusmn 005 relative densityunits resp for plasma cathepsin G 119875 lt 005) (Figures 3(b)and 3(c)) However no significant differences in the levels ofthe higher molecular weight complexes (40 50 and 70Da)were found between NC and HD A significant negativecorrelation was found between the amounts of the plasmalevels of elastase and cathepsin G (30KDa) and their levelsin PMNLs (Figures 3(d) and 3(e)) (119903 = minus05 and 119903 = minus06resp 119875 lt 005)

36 Plasma Levels of Soluble VE-Cadherin Fragments Asignificant difference in the levels of soluble VE-cadherinbetween NC and HD plasma was found HD plasma con-tains higher levels of soluble VE-cadherin both the wholemolecule 140KDa (146 plusmn 007 versus 125 plusmn 006 relativedensity units 119875 lt 005) and the cleaved form 100KDa(047 plusmn 006 versus 03 plusmn 004 relative density units 119875 lt 005)(Figures 4(a) and 4(b)) Using anti VE-cadherin monoclonalantibody we were able to detect the 40 kDa fragment of VE-cadherin in plasma Again HD plasma contains higher levelsof 40 kDa VE-cadherin (05 plusmn 007 versus 027 plusmn 006 relativedensity units 119875 lt 005) (Figures 4(a) and 4(b))

In addition significant positive correlations were foundbetween plasma active elastase and cathepsin G and the100KDa soluble VE-cadherin (Figures 5(a) and 5(b)) (119903 =051 and 119903 = 053 for elastase and cathepsin G resp119875 lt 005) suggesting that these proteins are involved in thedegradation of VE-cadherin No significant correlation wasfound between plasma active elastase and cathepsin G andthe 140 and 40KDa VE-cadherin forms

37 HUVEC Membrane 40 kDa form of VE-Cadherin afterExposure to PMNLs Control HUVEC and HUVEC exposedto NC PMNLs show light staining which indicates that theanti VE-cadherin did not bind to the VE-cadherin (Figure 6)However when HUVEC were exposed to HD PMNLs thestaining was much intense indicating strong binding of theantibody to VE-cadherin (Figure 6) When HUVEC wereexposed only to proteases (002UmL elastase and 002UmLcathepsin G [12]) VE-cadherin staining was comparableto that after exposure to HD PMNLs HUVEC membraneVE-cadherin was also determined by flow cytometry afterexposure to HD and NC PMNLs The level of VE-cadherinon unexposed HUVECwas 703plusmn384MFI Cocultivation ofHUVECwith NC PMNLs resulted in a significant increase inVE-cadherin staining compared to untreatedHUVEC (985plusmn577 119875 lt 005) Cocultivation of HUVEC with HD PMNLsresulted in a greater increase in VE-cadherin staining (134 plusmn757 119875 lt 005)

38 Cocultivation Media Levels of Soluble 100 kDa Cleavedform of VE-Cadherin We could not detect any soluble VE-cadherin in the control HUVEC which was not exposed toPMNLs On the other hand there was a detectable amountof soluble 100 kDa VE-cadherin in the cocultivation media


629

SS L

in

1020

120

100 103101

CD16 CyQ (a)

Elastase

Fluorescence intensity-FITC Ev

ents

HD PMNLs

NC PMNLs

HDPMNLs

NCPMNLs

(b)

Even

ts

Cathepsin G

Fluorescence intensity-FITC

NC PMNLsHD

PMNLsNC

PMNLsHDPMNLs

(c)

0

10

20

30

40

50

Intr

acel

lula

r ela

stas

e MFI

lowast

NC PMNLs HD PMNLs

(d)

lowast

0

20

40

60

80

NC PMNLs HD PMNLs

Intr

acel

lula

r cat

heps

in G

MFI

(e)

Figure 1 Intracellular levels of elastase and cathepsin G in PMNLsmeasured in whole blood (a) Representative histogram of flow cytometryshowing gating on the PMNL population which is CD16 positive cells (b c) Representative histogram of flow cytometry showing intracellularelastase and cathepsin G intensity in HD and NC PMNLs respectively (d e) PMNL intracellular elastase and cathepsin G from NC subjectsand HD patients detected by flow cytometry (119899 = 10) lowast119875 lt 005HD versus NC

when HUVEC were exposed to PMNLs (Figure 7) WhenHUVEC were exposed to HD PMNLs we detected 3 timesmore soluble VE-cadherin versus when exposed to NCPMNLs (Figure 7) We could not detect the 140 and 40 kDaforms of VE-cadherin in the media

4 Discussion

The results of the present study demonstrate that the intracel-lular levels of elastase and cathepsin G in primed peripheralPMNLs of HD patients before starting hemodialysis sessionare significantly lower compared to healthy controls althoughtheir mRNA levels are similar Lower intracellular levels ofthese proteases are associated with a significant increase intheir plasma concentration especially of the active formThis suggests that PMNL priming common to HD patientscauses an increased release of these enzymes resulting intheir higher plasma levels in HD Plasma from HD patients

also contains higher levels of soluble VE-cadherin in threemolecular forms 140 kDa the whole molecule and twomolecular fragments of this protein 100 and 40 kDa

We investigated the degree of PMNL priming of HDpatients and the possible correlation between PMNL primingand the release of elastase and cathepsinG First we supportedour previous studies showing that PMNLs in HD patientsare primed by using two different markers elevated levelsof CD11b and higher rate of superoxide release Next usingimmunocytochemical staining immunoblotting and FACSanalyses we demonstrated that the staining intensities of theintracellular elastase and cathepsin G are reduced in theprimed PMNLs of HD patients versus NC A significantnegative correlation was found between the intensity ofintracellular elastase and cathepsin G and the degree ofPMNL priming as assessed by membrane CD11b expressionsuggesting that the release of these proteolytic enzymes isgreater when PMNLs are primed Moreover our findings


HDNC

Elastase Cathepsin G

HD

(a) (c) (e) (g)

NC

(b) (d) (f) (h)

Figure 2 Localization of elastase and cathepsin G in PMNLs in whole blood smears Indirect immunostaining of elastase (andashd) and cathepsinG (endashh) in blood smears of two NC subjects and two HD patients (light microscopy magnification times100)

refute the possibility that lower synthesis of these enzymesin the HD PMNLs caused their reduced intracellular levelssince the transcription levels of these enzymes were com-parable in HD and healthy controls This is in accordancewith previous studies demonstrating that azurophilic granuleproteins such as these serine proteinases are synthesizedonly at the promyelocyte and metamyelocyte stages andremain stored in granules throughout terminal granulocyticdifferentiation [27 28]

Since the intracellular protein levels of these enzymeswere lower in HD one would expect to find these enzymes inthe surrounding milieu the blood As expected significantlyelevated levels of both proteolytic enzymes were found inblood of HD patients compared to NC

The continuous release of these enzymes from PMNLswas already demonstrated during hemodialysis sessions [19]but to the best of our knowledge a comparison with healthysubjects especially as to the amounts of the active non-inhibitor bound enzymes was not reported Although theplasma levels of the higher molecular weights complexesof the enzymes (40 50 and 70 kDa) were similar betweenplasma of HD and NC the levels of the 30 kDa inhibitorunbound enzymes were significantly higher in HD plasmaEnzyme-inhibitor complexes are formed when elastase orcathepsin G binds to inhibitory proteins of the acute phasesuch as serum proteins 1205721-antitrypsin (1205721-AT) and 1205721-antichymotrypsin (1205721-ACT) [10 11] The significant negativecorrelation between intracellular elastase and cathepsin Gand the degree of PMNL priming and plasma levels ofthese enzymes further supports the enhanced degranulationof these primed PMNLs resulting in the release of the

two proteolytic enzymes This uncontrolled degranulation ofprimed PMNLs to the blood is potentially destructive in twoways (1) reduced intracellular levels of these enzymes withinthe PMNLs decrease their capacity to neutralize and killpathogens (2) the continuous interactions of circulating HDPMNL with the blood vessel endothelial monolayer exposethe vascular wall to chronic injury by these active circulatinggranular proteinases

In the ldquoresponse-to-injuryrdquo hypothesis suggested by Rossatherosclerosis begins as a response to chronic minimalinjury to the endothelium [29] This injury leads to anarray of endothelial cell responses such as disruption ofendothelial integrity which will in the long run result inatherosclerosis [30] Adherence junction (AJs) proteins suchas VE-cadherin maintain endothelial integrity Hermant atal have shown that cleavage of VE-cadherin occurs followingadhesion of fMLP-primed neutrophils to endothelial cellmonolayer resulting in a soluble fragment of molecularmass of 38KDa Using specific inhibitors of neutrophilproteases these researchers were able to identify elastaseand cathepsin G as the major proteases involved in thecleavage of VE-cadherin In addition they demonstratedthat purified elastase and cathepsin G are able to increaseendothelial monolayer permeability in vivo [17] Soeki et alhave shown that enhanced secretion of VE-cadherin fromthe arteries is associated with coronary atherosclerosis [31]We imply that the elevated plasma levels of elastase andcathepsin G in HD patients especially the active formscleave AJs proteins such as VE-cadherin in the endotheliuman action associated with early steps in the atheroscleroticprocess


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HDNCHDNC

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(kD

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a)

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Rela

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ensit

ypl

asm

a act

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last

ase

posit

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ontro

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(b)

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04

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08

1

lowast

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive c

athe

psin

Gp

ositi

ve co

ntro

l

(c)

0

05

1

15

2

25

3

0 5 10 15 20 25 30 35 40 45PMNL intracellular elastase MFI

Plas

ma a

ctiv

e ela

stas

epo

sitiv

e con

trol

(rel

ativ

e den

sity)

r = minus05

P lt 005

(d)

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0 10 20 30 40 50 60 70PMNL intracellular cathepsin G MFI Pl

asm

a ac

tive c

athe

psin

Gp

ositi

ve c

ontr

ol

(rel

ativ

e den

sity)

r = minus06

P lt 005

(e)

Figure 3 Levels of elastase and cathepsin G inHD andNC plasma Proteins of plasma samples depleted from albumin and immunoglobulinsof NC subjects and HD patient were separated on SDS-PAGE followed by western blot analysis (a) A representative western blot of elastaseand cathepsin G Bands were visible at 30 kDa (inhibitor unbound) and higher MW complexes of 40 50 and 70 kDa in NC and HD plasma((b) (c)) Densities of unbound elastase and cathepsin G (30 kDa) fromNC subjects and HD patientsrsquo plasma relative to commercial enzymesbands ( lowast119875 gt 005HD versus NC 119899 = 10) (d) A negative correlation between plasma elastase levels of NC (e) andHD (998771) and the expressionof their PMNLmembrane CD11bmeasured in whole blood (119903 = minus05119875 lt 005) (e) A negative correlation between plasma cathepsin G levelsof NC (e) and HD (998771) and the expression of their PMNL membrane CD11b measured in whole blood (119903 = minus06 119875 lt 005)


VE-cadherin

1 2 3 4

40

100

140

(kD

a)

(a)

0

02

04

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08

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12

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18

Rela

tive d

ensit

ypl

asm

a VE-

cadh

erin

sam

ple c

ontro

lNCHD

lowast

lowast lowast

140 100 40

(kDa)

(b)

Figure 4 Soluble VE-cadherin fragments (140 and 100 kDa forms) in HD plasma versus NC plasmaProteins of plasma samples depletedfrom albumin and immunoglobulins of NC subjects and HD patient were separated on SDS-PAGE followed by western blot analysis (a)Representative gels showing three sizes of VE-cadherin the whole molecule 140 kDa and the cleaved forms 100 and 40 kDa in NC (13) andHD plasma (24) (b) Relative densities of VE-cadherin from NC subjects and HD patient plasma ( lowast119875 gt 005HD versus NC 119899 = 10)

0

01

02

03

04

05

06

07

08

0 02 04 06 08 1

Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)

Plasma cathepsin G (relative density)

(a)

0

01

02

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04

05

06

07

08

0 05 1 15 2 25 3Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

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Plasma elastase (relative density)

(b)

Figure 5 (a) Positive correlations between plasma active cathepsin G in plasma of NC (e) and HD patients (998771) and soluble VE-cadherin100 kDa fragment (119903 = 051 119875 lt 005 continuous line) (b) Positive correlations between plasma active elastase in plasma of NC and HDpatients and VE-cadherin 100 kDa fragment (119903 = 053 119875 lt 005 continuous line)

This study demonstrates that HD plasma contains higherlevels of soluble VE-cadherin present in three molecularforms whole 140 kDa molecule and two fragments the 100and the 40 kDa forms The 100 kDa protein is probablya product of the proteolysis of VE-cadherin by elastaseand cathepsin G [17] This assumption is supported by thesignificant positive correlation found between the amountof plasma active elastase and cathepsin G and the plasmalevels of the 100 kDa fragment the product of VE-cadherindegradation

Our in vitro experiments showed that HD PMNLs canmediate cleavage of VE-cadherin from endothelial cells Thiscleavage resulted in intense staining of the 40 kDa fragment

on endothelial cells concomitantly with the appearance ofthe 100 kDa in the culture media These results apparentlyconflict with our findings regarding low levels of elastaseand cathepsin G found in HD PMNLs compared to controlsYet we propose that while in NC PMNLs these enzymesare stored within specific granules HD PMNLs secretetheir enzyme content to the surrounding milieu Thereforeeven trace amounts could elicit VE-cadherin cleavage asdescribe hereinThe potential role of elastase and cathepsinGreleased from primedHD PMNLs inmediating VE-cadherincleavage is further supported by our in vitro experimentswhere applying purified elastase and cathepsin G to HUVECresulted in an increase staining of the 40 kDa fragment


HUVEC

(a)

Cocultivation ofHUVEC with HD PMNLs

(b)

HUVEC with NC PMNLsCocultivation of

(c)

HUVEC + elastase and cathepsin G

(d)

Figure 6 Indirect immunohistochemical staining of VE-cadherin (40 kDa form) in HUVEC after cocultivation with PMNLs and afterexposure to elastase and cathepsin G (light microscopy magnification times20)

HUVEC

VE-cadherin

HUVEC + NC PMNLs

HUVEC +HD PMNLs

100 kDa

Figure 7 A representative gel showing soluble VE-cadherin(100 kDa form) in PMNLs and HUVEC cocultivation media sep-arated by SDS-PAGE followed by western blot analysis

Our in vitro studies utilizing these proteinases onendothelial cells are in agreement with previous studydemonstrating that after treatment with cathepsin Gendothelial cells became contracted and did not interactwith neighbor cellsThese effects depended on concentrationand length of exposure to cathepsin G and ultimately cellsbecame star-shaped until totally detached [26]

For conclusion this study shows decreased elastase andcathepsin G expression inHDPMNLs while increased inHDplasma most importantly in their active form Furthermorehigher levels of soluble VE-cadherin were found in HDpatient plasma Thus this study can provide a mechanismby which active elastase and cathepsin G are released from

primed PMNLs mediate VE-cadherin cleavage and initiateendothelial dysfunction in these patients Moreover theimplications of this study are beyond HD patients and canbe implicated in all clinical states associated with primedPMNLs and accelerated atherosclerosis such as hypertensionand diabetes

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Meital Cohen-Mazor and Rafi Mazor equally contributed tothe preparation of this paper

Acknowledgments

This research was supported in part by a Grant of the publiccommittee for the designation of estate funds the Ministryof Justice Israel (6014 3-1726) The authors are grateful toMSc G Shapiro for her excellent technical assistance in thisproject


References

[1] G Feinstein and A Janoff ldquoA rapid method for purificationof human granulocyte cationic neutral proteases purificationand characterization of human granulocyte chymotrypsin likeenzymerdquo Biochimica et Biophysica Acta vol 403 no 2 pp 477ndash492 1975

[2] K Ohlsson and I Olsson ldquoThe neutral proteases of humangranulocytes Isolation and partial characterization of granulo-cyte elastasesrdquo European Journal of Biochemistry vol 42 no 2pp 519ndash527 1974

[3] A Janoff ldquoHuman granulocyte elastase Further delineationof its role in connective tissue damagerdquo American Journal ofPathology vol 68 no 3 pp 579ndash592 1972

[4] H Keiser R A Greenwald G Feinstein and A Janoff ldquoDegra-dation of cartilage proteoglycan by human leukocyte granuleneutral proteases a model of joint injury II Degradation ofisolated bovine nasal cartilage proteoglycanrdquo Journal of ClinicalInvestigation vol 57 no 3 pp 625ndash632 1976

[5] M Ziff H J Gribetz and J Lospalluto ldquoEffect of leukocyte andsynovial membrane extracts on cartilage mucoproteinrdquo Journalof Clinical Investigation vol 39 no 2 pp 405ndash412 1960

[6] A Janoff and J D Zeligs ldquoVascular injury and lysis of basementmembrane in vitro by neutral protease of human leukocytesrdquoScience vol 161 no 3842 pp 702ndash704 1968

[7] A Heidland W H Horl and N Heller ldquoProteolytic enzymesand catabolism enhanced release of granulocyte proteinases inuremic intoxication and during hemodialysisrdquo Kidney Interna-tional Supplement vol 24 no 16 pp S27ndashS36 1983

[8] D Farley G Salvesen and J Travis ldquoMolecular cloning ofhuman neutrophil elastaserdquo Biological Chemistry Hoppe-Seylervol 369 no 5 pp 3ndash7 1988

[9] G Doring ldquoThe role of neutrophil elastase in chronic inflam-mationrdquo American Journal of Respiratory and Critical CareMedicine vol 150 no 6 pp S114ndashS117 1994

[10] T Chandra R Stackhouse V J Kidd K J H Robsonand S L C Woo ldquoSequence homology between human 1205721-antichymotrypsin 1205721-antitrypsin and antithrombin IIIrdquo Bio-chemistry vol 22 no 22 pp 5055ndash5061 1983

[11] R A Stockley ldquoProteolytic enzymes their inhibitors and lungdiseasesrdquo Clinical Science vol 64 no 2 pp 119ndash126 1983

[12] A Janoff L Raju and R Dearing ldquoLevels of elastase activity inbronchoalveolar lavage fluids of healthy smokers and nonsmok-ersrdquo American Review of Respiratory Disease vol 127 no 5 pp540ndash544 1983

[13] S D Swain T T Rohn and M T Quinn ldquoNeutrophil primingin host defense role of oxidants as priming agentsrdquoAntioxidantsand Redox Signaling vol 4 no 1 pp 69ndash83 2002

[14] B Kristal R Shurtz-Swirski J Chezar et al ldquoParticipationof peripheral polymorphonuclear leukocytes in the oxidativestress and inflammation in patients with essential hyperten-sionrdquo American Journal of Hypertension vol 11 no 8 pp 921ndash928 1998

[15] R Shurtz-Swirski S Sela A T Herskovits et al ldquoInvolvementof peripheral polymorphonuclear leukocytes in oxidative stressand inflammation in type 2 diabetic patientsrdquo Diabetes Carevol 24 no 1 pp 104ndash110 2001

[16] S Sela R Shurtz-Swirski M Cohen-Mazor et al ldquoPrimedperipheral polymorphonuclear leukocyte a culprit underlyingchronic low-grade inflammation and systemic oxidative stress

in chronic kidney diseaserdquo Journal of the American Society ofNephrology vol 16 no 8 pp 2431ndash2438 2005

[17] B Herman S Bibert E Concord et al ldquoIdentification ofproteases involved in the proteolysis of vascular endotheliumcadherin during neutrophil transmigrationrdquo Journal of Biologi-cal Chemistry vol 278 no 16 pp 14002ndash14012 2003

[18] D Gulino E Delachanal E Concord et al ldquoAlteration ofendothelial cell monolayer integrity triggers resynthesis of vas-cular endothelium cadherinrdquo Journal of Biological Chemistryvol 273 no 45 pp 29786ndash29793 1998

[19] R M Schaefer N Herfs W Ormanns W H Horl and AHeidland ldquoChange of elastase and cathepsin G content inpolymorphonuclear leukocytes during hemodialysisrdquo ClinicalNephrology vol 29 no 6 pp 307ndash311 1988

[20] JW YoonM V Pahl andND Vaziri ldquoSpontaneous leukocyteactivation and oxygen-free radical generation in end-stage renaldiseaserdquo Kidney International vol 71 no 2 pp 167ndash172 2007

[21] M S Krug and S L Berger ldquoFirst-strand cDNA synthesisprimed with oligo(dT)rdquo Methods in Enzymology vol 152 pp316ndash325 1987

[22] R K Hirata S-T Chen and S C Weil ldquoExpression of granuleprotein mRNAs in acute promyelocytic leukemiardquoHematologicPathology vol 7 no 4 pp 225ndash238 1993

[23] E A Jaffe R L Nachman C G Becker and C R MinickldquoCulture of human endothelial cells derived from umbilicalveins Identification bymorphologic and immunologic criteriardquoJournal of Clinical Investigation vol 52 no 11 pp 2745ndash27561973

[24] N Lanir M Zilberman I Yron G Tennenbaum Y Shechterand B Brenner ldquoReactivity patterns of antiphospholipid anti-bodies and endothelial cells effect of antiendothelial antibodieson cell migrationrdquo Journal of Laboratory and Clinical Medicinevol 131 no 6 pp 548ndash556 1998

[25] J Jacobi S Sela H I Cohen J Chezar and B Kristal ldquoPrimingof polymorphonuclear leukocytes a culprit in the initiation ofendothelial cell injuryrdquo American Journal of Physiology Heartand Circulatory Physiology vol 290 no 5 pp H2051ndashH20582006

[26] L Iacoviello V Kolpakov L Salvatore et al ldquoHuman endothe-lial cell damage by neutrophil-derived cathepsin G role ofcytoskeleton rearrangement and matrix-bound plasminogenactivator inhibitor-1rdquo Arteriosclerosis Thrombosis and VascularBiology vol 15 no 11 pp 2037ndash2046 1995

[27] N Borregaard K Theilgaard-Monch O E Soslashrensen and JB Cowland ldquoRegulation of human neutrophil granule proteinexpressionrdquo Current Opinion in Hematology vol 8 no 1 pp23ndash27 2001

[28] J B Cowland and N Borregaard ldquoThe individual regulationof granule protein mRNA levels during neutrophil maturationexplains the heterogeneity of neutrophil granulesrdquo Journal ofLeukocyte Biology vol 66 no 6 pp 989ndash995 1999

[29] R Ross ldquoThe pathogenesis of atherosclerosis a perspective forthe 1990srdquo Nature vol 362 no 6423 pp 801ndash809 1993

[30] T G DeLoughery and S H Goodnight ldquoThe hypercoagula-ble states diagnosis and managementrdquo Seminars in VascularSurgery vol 6 no 1 pp 66ndash74 1993

[31] T Soeki Y Tamura H Shinohara K Sakabe Y Onose and NFukuda ldquoElevated concentration of soluble vascular endothelialcadherin is associated with coronary atherosclerosisrdquo Circula-tion Journal vol 68 no 1 pp 1ndash5 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Our findings consist of elevated levels of these proteins inHD plasma Plasma of HD had higher levels of VE-cadherinfragments the result of this molecule cleavage by active elas-tase and cathepsin G HDPMNLs as well as purified enzymescleaved VE-cadherin from cultured endothelial cells Takentogether this study provides a novel mechanism by whichactive elastase and cathepsin G originating from primedPMNLs of HD patients can initiate endothelial dysfunction

2 Materials and Methods

21 Patients and Blood Samples Ten hemodialysis patientsand healthy control subjects were enrolled in this study(Table 1) Blood for the determination of biochemical andhematological parameters and for the isolation of PMNLswas drawn into citrate tubes and in HD patients fromthe arterial line immediately before a dialysis session Allpatients underwent hemodialysis three times a week eachdialysis treatment lasted 4 hours and was carried out withlowflux polysulfonemembranes (F8 FreseniusMedical CareBad Homburg Germany) The water for dialysis met thestandards of the Association for the Advancement of MedicalInstrumentation (AAMI) Patients with evidence of acute orchronic infection malignancy or who had received a bloodtransfusion within three months prior to blood samplingwere excluded All participants signed an informed consentfor blood sampling and the study was approved by theInstitutional Committee in accordance with the Helsinkideclaration

22 PMNL Isolation and Analysis PMNLs were isolatedas described previously [15] Isolated PMNLs (gt98 pureapproximately 107 cells per isolation) were resuspended andcounted in phosphate buffered saline (PBS Beit HaemekIsrael) containing 01 glucose PMNL priming was assessedby the rate of superoxide release [14] and by the surfacelevels of CD11b as described previously [20] The rate ofsuperoxide release was determined after cell stimulation with032 times 10minus7M phorbol 12-myristate 13-acetate (PMA SigmaSt LouisMO)The assay is based upon superoxide dismutase(SOD) inhibitable reduction of 80 120583M cytochrome C (SigmaSt Louis MO) to its ferrous form The change in opticaldensity was monitored at 549 nm as described previously[14] Expression of CD11b on PMNLs in whole blood wasdetermined using the FC500 flow cytometer (Beckman-Coulter) Fifty 120583L of blood was incubated for 10min with antiCD11b-PE conjugated monoclonal antibody (IQ ProductsThe Netherlands) followed by RBC lysis (Q-prep methodCoulter Corporation Hialeah FL USA) To enable gatingon the PMNL population anti CD16 conjugated to PC5monoclonal antibody (Immunotech Marseille France) wasused Surface levels of CD11b on PMNLs are expressedas mean fluorescence intensity (MFI) after subtracting thenonspecific background

23 RNA Isolation and RT-PCR of Elastase and CathepsinG RNA was isolated using Tri Reagent according to themanufacturerrsquos (Sigma) instructions Complementary DNA

Table 1 Demographics of HD patients and NC subjects used in thisstudy

Hemodialysispatients (119899 = 10)

Normal controls(119899 = 10)

Age (years) 54 plusmn 7 47 plusmn 5

MaleFemale 55 55

Diabetes 3 mdashTime on hemodialysis(months) 38 plusmn 6 mdash

was synthesized according to Krug and Berger [21] usingan OmniGene thermal cycler (Hybaid UK) The resultingcDNAswere amplified by PCRusing 2120583L aliquots of RT reac-tion incubated with 02mM dNTPs 2120583L of Taq DNA poly-merase buffer 06U of TaqDNA polymerase (BioLine UK)and 10 pmoles of the specific primers The oligonucleotidesequence for elastase according to Hirata et al [22] is sense(51015840-AGTGCCTGGCCATGGGCTGG-31015840) and antisense (51015840-CACCGGGGCAAAGGCATCGG-31015840) with a final PCR prod-uct of 257 bp The PCR conditions were initial denaturationat 94∘C for 5 minutes 30 PCR cycles of denaturation at94∘C for 1 minute annealing at 55∘C for 2 minutes andextension at 72∘C for 3minutesTheoligonucleotide sequencefor cathepsin G according to Hirata et al [22] is sense(51015840-TGAGAGTGCAGAGGGATAGG-31015840) and antisense (51015840-CAGGAAACTTGAGACCCTGC-31015840) with a final PCR prod-uct of 216 bp The PCR conditions were identical to thosedescribed above for elastase Aliquots (15 120583L) of the amplifiedcDNA of elastase and cathepsin G were separated on 1agarose gel electrophoresis visualized by ethidium bromidestaining and compared to a housekeeping gene 120573-actin The200 bp product of actin was amplified using the followingprimers sense (51015840-CCTTCCTGGGCATGGAGTCCTG-31015840)and antisense (51015840-GAGCAATGATCTTGATCTTC-31015840) Thedensities of the enzyme PCRproducts were calculated in eachgel relative to the actin product

24 Detection and Quantificationof Elastase and Cathepsin Gin PMNLs

(1) Intracellular Levels of Elastase and Cathepsin G in PMNLsThe levels of intracellular elastase and cathepsin G weremeasured in whole blood by flow cytometry using polyclonalrabbit anti-human elastase and anti-human cathepsin Gantibodies (United States Biological Massachusetts USA)followed by a secondary FITC-conjugated fluorescent anti-rabbit antibody (Chemicon International CA) PMNLs werepermeabilized with the FIX amp PERM cell permeabilizationkit Anti CD16- PC5 (Immunotech Marseille France) wasused for gating on PMNLs Total rabbit IgG antibodies(United States Biological Massachusetts USA) followed by asecondary FITC-conjugated fluorescent anti-rabbit antibodyserved as nonspecific controls of the fluorochrome Theresults are presented as MFI per cell after subtracting thenonspecific background

















3 Results













629

SS L

in

1020

120

100 103101

CD16 CyQ (a)

Elastase


ents

HD PMNLs

NC PMNLs

HDPMNLs

NCPMNLs

(b)

Even

ts

Cathepsin G


NC PMNLsHD

PMNLsNC

PMNLsHDPMNLs

(c)

0

10

20

30

40

50

Intr

acel

lula

r ela

stas

e MFI

lowast

NC PMNLs HD PMNLs

(d)

lowast

0

20

40

60

80

NC PMNLs HD PMNLs

Intr

acel

lula

r cat

heps

in G

MFI

(e)



4 Discussion





HDNC


HD

(a) (c) (e) (g)

NC

(b) (d) (f) (h)








70

50

40

30

Cathepsin G

HDNCHDNC

Elastase

70

50

40

30

(kD

a)

(kD

a)

(a)

lowast

0

1

2

3

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive e

last

ase

posit

ive c

ontro

l

(b)

0

02

04

06

08

1

lowast

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive c

athe

psin

Gp

ositi

ve co

ntro

l

(c)

0

05

1

15

2

25

3


Plas

ma a

ctiv

e ela

stas

epo

sitiv

e con

trol

(rel

ativ

e den

sity)

r = minus05

P lt 005

(d)

0

02

04

06

08


asm

a ac

tive c

athe

psin

Gp

ositi

ve c

ontr

ol

(rel

ativ

e den

sity)

r = minus06

P lt 005

(e)



VE-cadherin

1 2 3 4

40

100

140

(kD

a)

(a)

0

02

04

06

08

1

12

14

16

18

Rela

tive d

ensit

ypl

asm

a VE-

cadh

erin

sam

ple c

ontro

lNCHD

lowast

lowast lowast

140 100 40

(kDa)

(b)


0

01

02

03

04

05

06

07

08

0 02 04 06 08 1

Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(a)

0

01

02

03

04

05

06

07

08

0 05 1 15 2 25 3Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(b)






HUVEC

(a)


(b)


(c)


(d)


HUVEC

VE-cadherin

HUVEC + NC PMNLs

HUVEC +HD PMNLs

100 kDa









Acknowledgments



References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































3 Results













629

SS L

in

1020

120

100 103101

CD16 CyQ (a)

Elastase


ents

HD PMNLs

NC PMNLs

HDPMNLs

NCPMNLs

(b)

Even

ts

Cathepsin G


NC PMNLsHD

PMNLsNC

PMNLsHDPMNLs

(c)

0

10

20

30

40

50

Intr

acel

lula

r ela

stas

e MFI

lowast

NC PMNLs HD PMNLs

(d)

lowast

0

20

40

60

80

NC PMNLs HD PMNLs

Intr

acel

lula

r cat

heps

in G

MFI

(e)



4 Discussion





HDNC


HD

(a) (c) (e) (g)

NC

(b) (d) (f) (h)








70

50

40

30

Cathepsin G

HDNCHDNC

Elastase

70

50

40

30

(kD

a)

(kD

a)

(a)

lowast

0

1

2

3

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive e

last

ase

posit

ive c

ontro

l

(b)

0

02

04

06

08

1

lowast

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive c

athe

psin

Gp

ositi

ve co

ntro

l

(c)

0

05

1

15

2

25

3


Plas

ma a

ctiv

e ela

stas

epo

sitiv

e con

trol

(rel

ativ

e den

sity)

r = minus05

P lt 005

(d)

0

02

04

06

08


asm

a ac

tive c

athe

psin

Gp

ositi

ve c

ontr

ol

(rel

ativ

e den

sity)

r = minus06

P lt 005

(e)



VE-cadherin

1 2 3 4

40

100

140

(kD

a)

(a)

0

02

04

06

08

1

12

14

16

18

Rela

tive d

ensit

ypl

asm

a VE-

cadh

erin

sam

ple c

ontro

lNCHD

lowast

lowast lowast

140 100 40

(kDa)

(b)


0

01

02

03

04

05

06

07

08

0 02 04 06 08 1

Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(a)

0

01

02

03

04

05

06

07

08

0 05 1 15 2 25 3Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(b)






HUVEC

(a)


(b)


(c)


(d)


HUVEC

VE-cadherin

HUVEC + NC PMNLs

HUVEC +HD PMNLs

100 kDa









Acknowledgments



References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















629

SS L

in

1020

120

100 103101

CD16 CyQ (a)

Elastase


ents

HD PMNLs

NC PMNLs

HDPMNLs

NCPMNLs

(b)

Even

ts

Cathepsin G


NC PMNLsHD

PMNLsNC

PMNLsHDPMNLs

(c)

0

10

20

30

40

50

Intr

acel

lula

r ela

stas

e MFI

lowast

NC PMNLs HD PMNLs

(d)

lowast

0

20

40

60

80

NC PMNLs HD PMNLs

Intr

acel

lula

r cat

heps

in G

MFI

(e)



4 Discussion





HDNC


HD

(a) (c) (e) (g)

NC

(b) (d) (f) (h)








70

50

40

30

Cathepsin G

HDNCHDNC

Elastase

70

50

40

30

(kD

a)

(kD

a)

(a)

lowast

0

1

2

3

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive e

last

ase

posit

ive c

ontro

l

(b)

0

02

04

06

08

1

lowast

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive c

athe

psin

Gp

ositi

ve co

ntro

l

(c)

0

05

1

15

2

25

3


Plas

ma a

ctiv

e ela

stas

epo

sitiv

e con

trol

(rel

ativ

e den

sity)

r = minus05

P lt 005

(d)

0

02

04

06

08


asm

a ac

tive c

athe

psin

Gp

ositi

ve c

ontr

ol

(rel

ativ

e den

sity)

r = minus06

P lt 005

(e)



VE-cadherin

1 2 3 4

40

100

140

(kD

a)

(a)

0

02

04

06

08

1

12

14

16

18

Rela

tive d

ensit

ypl

asm

a VE-

cadh

erin

sam

ple c

ontro

lNCHD

lowast

lowast lowast

140 100 40

(kDa)

(b)


0

01

02

03

04

05

06

07

08

0 02 04 06 08 1

Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(a)

0

01

02

03

04

05

06

07

08

0 05 1 15 2 25 3Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(b)






HUVEC

(a)


(b)


(c)


(d)


HUVEC

VE-cadherin

HUVEC + NC PMNLs

HUVEC +HD PMNLs

100 kDa









Acknowledgments



References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HDNC


HD

(a) (c) (e) (g)

NC

(b) (d) (f) (h)








70

50

40

30

Cathepsin G

HDNCHDNC

Elastase

70

50

40

30

(kD

a)

(kD

a)

(a)

lowast

0

1

2

3

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive e

last

ase

posit

ive c

ontro

l

(b)

0

02

04

06

08

1

lowast

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive c

athe

psin

Gp

ositi

ve co

ntro

l

(c)

0

05

1

15

2

25

3


Plas

ma a

ctiv

e ela

stas

epo

sitiv

e con

trol

(rel

ativ

e den

sity)

r = minus05

P lt 005

(d)

0

02

04

06

08


asm

a ac

tive c

athe

psin

Gp

ositi

ve c

ontr

ol

(rel

ativ

e den

sity)

r = minus06

P lt 005

(e)



VE-cadherin

1 2 3 4

40

100

140

(kD

a)

(a)

0

02

04

06

08

1

12

14

16

18

Rela

tive d

ensit

ypl

asm

a VE-

cadh

erin

sam

ple c

ontro

lNCHD

lowast

lowast lowast

140 100 40

(kDa)

(b)


0

01

02

03

04

05

06

07

08

0 02 04 06 08 1

Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(a)

0

01

02

03

04

05

06

07

08

0 05 1 15 2 25 3Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(b)






HUVEC

(a)


(b)


(c)


(d)


HUVEC

VE-cadherin

HUVEC + NC PMNLs

HUVEC +HD PMNLs

100 kDa









Acknowledgments



References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















70

50

40

30

Cathepsin G

HDNCHDNC

Elastase

70

50

40

30

(kD

a)

(kD

a)

(a)

lowast

0

1

2

3

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive e

last

ase

posit

ive c

ontro

l

(b)

0

02

04

06

08

1

lowast

NC HD

Rela

tive d

ensit

ypl

asm

a act

ive c

athe

psin

Gp

ositi

ve co

ntro

l

(c)

0

05

1

15

2

25

3


Plas

ma a

ctiv

e ela

stas

epo

sitiv

e con

trol

(rel

ativ

e den

sity)

r = minus05

P lt 005

(d)

0

02

04

06

08


asm

a ac

tive c

athe

psin

Gp

ositi

ve c

ontr

ol

(rel

ativ

e den

sity)

r = minus06

P lt 005

(e)



VE-cadherin

1 2 3 4

40

100

140

(kD

a)

(a)

0

02

04

06

08

1

12

14

16

18

Rela

tive d

ensit

ypl

asm

a VE-

cadh

erin

sam

ple c

ontro

lNCHD

lowast

lowast lowast

140 100 40

(kDa)

(b)


0

01

02

03

04

05

06

07

08

0 02 04 06 08 1

Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(a)

0

01

02

03

04

05

06

07

08

0 05 1 15 2 25 3Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(b)






HUVEC

(a)


(b)


(c)


(d)


HUVEC

VE-cadherin

HUVEC + NC PMNLs

HUVEC +HD PMNLs

100 kDa









Acknowledgments



References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VE-cadherin

1 2 3 4

40

100

140

(kD

a)

(a)

0

02

04

06

08

1

12

14

16

18

Rela

tive d

ensit

ypl

asm

a VE-

cadh

erin

sam

ple c

ontro

lNCHD

lowast

lowast lowast

140 100 40

(kDa)

(b)


0

01

02

03

04

05

06

07

08

0 02 04 06 08 1

Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(a)

0

01

02

03

04

05

06

07

08

0 05 1 15 2 25 3Solu

ble V

E-ca

dher

in (r

elat

ive d

ensit

y)


(b)






HUVEC

(a)


(b)


(c)


(d)


HUVEC

VE-cadherin

HUVEC + NC PMNLs

HUVEC +HD PMNLs

100 kDa









Acknowledgments



References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HUVEC

(a)


(b)


(c)


(d)


HUVEC

VE-cadherin

HUVEC + NC PMNLs

HUVEC +HD PMNLs

100 kDa









Acknowledgments



References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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