)JOEBXJ1VCMJTIJOH$PSQPSBUJPO … · membrane antigens were detected on un xed cells, using...

Research ArticleThe Combination of N-Acetyl Cysteine Alpha-Lipoic Acid andBromelain Shows High Anti-Inflammatory Properties in NovelIn Vivo and In Vitro Models of Endometriosis

C Agostinis1 S Zorzet2 R De Leo1 G Zauli1 F De Seta13 and R Bulla2

1 Institute for Maternal and Child Health IRCCS Burlo Garofolo 34137 Trieste Italy2Department of Life Sciences University of Trieste Via Valerio 28 34127 Trieste Italy3Department of Medical Surgical and Health Sciences University of Trieste Ospedale di Cattinara Strada di Fiume 44734149 Trieste Italy

Correspondence should be addressed to R Bulla rbullaunitsit

Received 4 August 2014 Accepted 3 October 2014

Academic Editor Lifei Hou

Copyright copy 2015 C Agostinis 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

To evaluate the efficacy of an association of N-acetyl cystein alpha-lipoic acid and bromelain (NACLABr) in the treatmentof endometriosis we set up a new in vivo murine model We explored the anti-inflammatory and proapoptotic effect of thiscombination on human endometriotic endothelial cells (EECs) and on endothelial cells isolated from normal uterus (UtMECs)We implanted fragments of human endometriotic cysts intraperitoneally into SCID mice to evaluate the efficacy of NACLABrtreatment UtMECs and EECs untreated or treatedwithNACLABr were activatedwith the proinflammatory stimulus TNF-120572 andtheir response in terms of VCAM1 expressionwas evaluatedThe proapoptotic effect of higher doses ofNACLABr onUtMECs andEECs was measured with a fluorogenic substrate for activated caspases 3 and 7 The preincubation of EECs with NACLABr priorto cell stimulation with TNF-120572 prevents the upregulation of the expression of the inflammatory ldquomarkerrdquo VCAM1 FurthermoreNACLABr were able to induce EEC but not UtMEC apoptosis Finally the novel mouse model allowed us to demonstrate thatmice treated with NACLABr presented a lower number of cysts smaller in size compared to untreated mice Our findingssuggest that these dietary supplements may have potential therapeutic uses in the treatment of chronic inflammatory diseaseslike endometriosis

1 Introduction

Endometriosis (EM) is a chronic estrogen-dependent dis-order characterized by the presence of endometrium-liketissue outside the uterine cavity It is associated with dys-menorrhea dyspareunia noncyclic pelvic pain subfertil-ity and infertility [1] This frequent gynaecological diseaseaffects 10ndash15 of women in reproductive age [2] It is wellaccepted that a blood supply is essential for the survivalof endometriotic implants and the development of EM asblood is crucial for providing nutrients and growth factorsand for promoting recruitment of inflammatory cells tothe endometriotic lesions as described by Groothuis [3]Endometriotic lesions are highly vascularized and it is nowwidely accepted that the formation of new blood vessels

at implantation sites plays a key role in the growth ofendometriotic cells [4] Furthermore eutopic endometriumfrom women with EM has greater angiogenic potential thaneutopic endometrium from healthy subjects [5] Since thegrowth of newly formed blood vessels is of pivotal importancein the development of EM the inhibition of angiogenesismay offer an opportunity for treatment [6ndash8] In this respectit is noteworthy that vascular endothelium is known toplay a critical role in regulation of inflammatory processes[9] and that cell adhesion molecules such as vascular celladhesion molecule-1 (VCAM1) as well as proinflamma-tory cytokines play key roles in the pathogenesis of EM[10]

EM can be treated by excising peritoneal implants deepnodules and ovarian cysts Although lesion eradication is

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 918089 9 pageshttpdxdoiorg1011552015918089

2 Mediators of Inflammation

considered a fertility-enhancing procedure the benefit onreproductive performance is moderate [11] Surgical removalof ectopic lesions represents the first line of intervention forthe treatment of EM but is characterized by a relevant per-centage of recurrences [12] In addition a variety of medicalhormonal therapies that all aimed to reduce the levels ofcirculating estrogens are currently available [13] Howeverthese treatments are often unsatisfactory and cannot be usedover long periods of time due to the occurrence of severeadverse effects [14]Therefore new and improved therapeuticsolutions that can efficiently reduce lesions with limitedside effects and no interference with the patientrsquos fertilityare definitely desirable In this respect it has been recentlyshown that N-acetyl cysteine (NAC) effectively treats ovarianendometriosis In terms of reduction in cysts size the datareported by Porpora et al are even more favorable than thosegranted by the currently adopted hormonal treatments withthe further advantages of fertility preservation and of thevirtual absence of undesired side effects [15]

On these bases the aim of this study was to investigatethe effects of an association of NAC alpha-lipoic acid (LA)and bromelain (Br) in vivo after establishing a novel modelof EM based on the injection of human endometrial tissue inthe peritoneumof SCIDmice In addition the of NACLABrcombinationwas analyzed in vitro onmicrovascular endothe-lial cells isolated from human endometriotic tissues (EECs)as well as in microvascular endothelial cells isolated fromhuman endometrium (UtMECs)

2 Materials and Methods

21 Preparation of the Compound Mixture All componentsof the mixture were purchased from Sigma-Aldrich (MilanItaly) and solutionswere sterilized by 022120583mfiltration Stockconcentrations were 10mgmL in H

2O for NAC 5mgmL

in absolute ethanol for LA and Br 1mgmL in PBS forBr NAC and LA solutions were stored at 4∘C while Br atminus20∘C until use All reagents were tested for sterility andLPS For cell culture studies the three drugs were combinedin complete cell culture medium at a final concentration of1000 120583gmL NAC + 500120583gmL LA + 50120583gmL Br In theseconditions the solution did not form visible precipitates andpH was stable (measured with pH-Meter BASIC20+ CRI-SON INSTRUMENT) To choose the optimal concentrationsfor the in vitro studies we have referred to the concentrationsof NAC LA and Br proportionally to those present in thenew dietary supplement Naxend (Pizeta Pharma PerugiaItaly 7272 NAC 2424 LA and 303 Br) consideringalso their bioavailability the absorption and the peak plasmaof each compound

22 Human Tissues The Maternal-Childrenrsquos Hospital (RC0813 IRCCS ldquoBurlo Garofolordquo Trieste Italy) approved thisstudy and following informed consent endometriosis speci-mens were obtained from women undergoing laparoscopy toremove endometrial cysts and endometrial tissue was fertilewomen undergoing hysterectomy for leiomyomatosis in themidproliferative and midsecretory phase defined accordingto Noyes criteria [16]

23 Animals Female SCID mice (4ndash6 weeks of age) werepurchased from Charles River (Milan Italy) and maintainedunder pathogen-free conditions All the experimental proce-dures involving animals were done in compliance with theguidelines of the European (86609EEC) and the Italian(DL11692) laws and were approved by both the ItalianMinistry of Health and the Administration of the UniversityAnimal House

24 Animal Model and Ex Vivo Analysis of Cysts Endometri-otic tissue from three peritoneal cysts was collected in sterilePBS and then suspended as coarse fragments loaded in3mL syringes and standardized with respect to volume andweight A volume of 05mL of cyst suspension approxi-mately equal to 04 g of wet tissue was injected by 16 ganeedle intraperitoneally into SCID mice (Charles River)Hormonal therapy with 17-120573-estradiol-3-benzoate (Sigma-Aldrich 30 120583gkg im) was initiated at the time of cyst tissueinjection and at 3-day intervals thereafter The day afterinjection mice were divided randomly in two groups and weadministered only to the first one NAC 250mgkgdie LA125mgkgdie and Br 125mgkgdie per os Twenty-one daysfollowing injection the animals were killed and implantedendometriotic lesions in treated (119899 = 7) or untreated mice(119899 = 9) were identified counted resected and collected informalin 10

Endometriotic lesions excised fromSCIDmicewere fixedin 10 buffered formalin and paraffin embedded Four-micrometers-thick sections were stained with Diff-Quick(Biomap Milan Italy) staining (following the manufacturerinstructions) and examined for the presence and distributionof vessels and glands For immunohistochemical analysisthe slides were microwaved three times in Tris-HClEDTA(ethylenediamine tetraacetic acid) pH 90 buffer (Dako) for5min brought to RT andwashed in PBS After neutralizationof the endogenous peroxidase with H

2O2for 10min the

sections were first incubated with protein block (Dako)for 10min and then with the primary antibodies for 1 hat RT (polyclonal rabbit anti-human vWF from Dako)The bound antibodies were revealed using the horseradishperoxidase- (HRP-) conjugated anti-rabbit IgG antibodies(Sigma-Aldrich) and diaminobenzidine (DAB) as substrate(Dako) Slides were evaluated under Leica DM3000 micro-scope (Leica Wetzlar Germany) and the pictures werecollected using a Leica DFC320 digital camera (Leica)

25 Immunofluorescent Staining Endometriotic tissue frag-ments approximately 1 cm3 were embedded in OCT (BioOp-tica Milan Italy) snap-frozen in liquid nitrogen and keptat minus80∘C until use Cryostat sections of about 6 120583m wereair dried fixed in acetone and either used immediately orkept atminus80∘C Binding ofmouse anti-human cytokeratin 818(CK818) ormouse anti-human vWF (DakoMilan Italy) wasdetected by incubating the sections with goat anti-mouse IgGCy3-conjugated secondary antibodies 30min at RT and thenthe nuclei were stained blue with DAPI (410158406-diamidino-2-phenylindole Sigma-Aldrich) 1120583gmL

Mediators of Inflammation 3

26 Cell Isolation and Culture UtMECs and EECs wereisolated and characterized as previously described by Bullaet al [17] Both ECs were positively selected with DynabeadsM-450 (Life Technologies Milan Italy) coated with Ulexeuropaeus 1 lectin (Sigma-Aldrich) seeded on 125 cm2 flaskprecoated with 2 120583gcm2 fibronectin (Roche Milan Italy)andmaintained in serum-free endothelial basalmedium (LifeTechnologies Monza Italy) supplemented with 20 ngmLbFGF (basic Fibroblast Growth Factor) 10 ngmL EGF (Epi-dermal Growth Factor) 10 FCS (all from Life Technolo-gies) and 10 human serum and incubated at 37∘C 5 CO

2

The purity of the resulting EC populations was more than98 as verified by staining with antibodies to VWF CD105VE cadherin (Dako Milano Italy) and CD31PECAM-1kindly provided by M R Zocchi (San Raffaele HospitalMilan Italy)

27 Immunofluorescence on Endothelial Cells ECs wereplated in 8-chamber culture slides (BDBiosciencesDiscoveryLabware Milan Italy) coated with 2 120583gcm2 fibronectin(Roche) and incubated at 37∘C in CO

2enriched atmosphere

When cells grew to confluence they were fixed and perme-abilized with FIX amp PERM cell permeabilization kit (SocietaItalianaChimici Rome Italy)Then cells were incubatedwithprimarymAb (cloneF886)mouse anti-human vWF (Dako)or mouse anti-human CD31 (Immunotools Germany) for 1 hat room temperature (RT) followed by FITC-conjugated goatanti-mouse IgG for 1 h at RT Images were acquiredwith LeicaDM3000 microscope (Leica) and the pictures were collectedusing a Leica DFC320 digital camera (Leica)

28 Cytofluorimetric Analysis ECs were detached from cul-ture flasks with 5mM EDTA at 37∘C and a total number of 5times 105 were fixed with FIX amp PERM cell permeabilization kit(Societa Italiana Chimici) and incubated in permeabilizationsolution in ice for 301015840 with mAb (clone 9) mouse anti-human vimentin (Sigma-Aldrich) mAb (cloneF886) mouseanti-human vWF or mAb (cloneV9) mouse anti-humanCK818 The binding of primary antibodies was detected byincubation with FITC-conjugated goat anti-mouse IgG Themembrane antigens were detected on unfixed cells usingmonoclonal anti-human CD31 CD45 CD34 and CD105directly FITC-conjugated all purchased from Immunotools(Germany) The cells were fixed with 1 paraformalde-hyde (Sigma-Aldrich) and analyzed for fluorescence witha FACScalibur instrument (BD Falcon Milan Italy) usingCellQuest software

29 Whole Cell VCAM1 ELISA Both types of cells weregrown to the confluence in 96-well plates and incubated withdrugs (concentration of NAC 10 120583gmL LA 9120583gmL andBr 2120583gmL) alone or in association for 48 h 37∘C 5 CO

2

Successively the cells were stimulated overnight with TNF-120572 (100 ngmL) washed with Dulbeccorsquos PBS added with 2BSA (Bovine Serum Albumine fraction V Sigma-Aldrich)and CaCl

2-MgCl

207mM (Sigma-Aldrich) and then incu-

batedwithmousemAb anti-humanVCAM1 (Sigma-Aldrich)5 120583gmL for 90min at RT The binding of primary anti-body was reveled incubating the cells with a polyclonal

anti-mouse IgG conjugated with alkaline phosphatase Theenzymatic reactionwas developedwith PNPP (p-nitrophenylphosphate) (Sigma-Aldrich 1mgmL) as substrate and readkinetically at 405 nmusing aTitertekMultiskanELISA reader(Flow Labs Milano Italy)

210 Apoptosis Assay Both types of cells were grown to80 of confluence in 96-well plates and incubated with thecompounds alone or in association for 72 h 37∘C After thencells were incubated with 5120583M of CellEvent Caspase-37Green Detection Reagent (Life Technologies) a fluorogenicsubstrate for activated caspases 3 and 7 The reagent consistsof a four amino acid peptide (DEVD) conjugated to a nucleicacid binding dye This cell-permeant substrate is intrinsicallynonfluorescent because the DEVD peptide inhibits the abil-ity of the dye to bind to DNA After activation of caspase-3or caspase-7 in apoptotic cells the DEVD peptide is cleavedenabling the dye to bind to DNA and produce a brightfluorogenic response with an absorptionemission maximaof sim502530 nm The fluorescence data were acquired withTECAN Infinite200 and normalized for total protein presentin each well For the protein quantitation the cells were thenlysed with NaOH 1M and evaluated by Bradford assay aspreviously reported [18]

3 Statistical Analysis

For each set of experiments values are reported as means plusmnSE The results were evaluated by using the Mann-Whitneytest Statistical significance was defined as 119875 lt 005

4 Results

41 Establishment of a Relevant In Vivo Model for EM andEfficacy of the NACLABr Combination in Decreasing theNumber of Cysts Formation In Vivo The primary aim of ourstudy was the evaluation of the effect of NACLABr in a rel-evant model of EM For this purpose we set up a new mousemodel modifying the animal model described by Awwadand colleagues [19] and by Grummer and colleagues [20]Specifically we used human endometriotic tissue obtainedfrom ovarian cysts instead of normal human endometrium tocreate the endometriotic lesions into the peritoneal cavity ofSCID mice Endometriotic tissue from three peritoneal cystswas injected in the peritoneal cavity of SCIDmice Hormonaltherapy with 17-120573-estradiol-3-benzoate was initiated at thetime of injection and at intervals of 3 days thereafter Twenty-one days following injection the animals were killed andimplanted endometriotic lesions were identified analyzedand characterized This protocol provided an implantationrate of 100 and the dimension and the histology of thesecysts were evaluated Excised explants revealed the presenceof EM-like features upon histologic examination that isstroma and endometrial glands All the implants showeda well restructured columnar andor cuboidal glandularepithelium with cytogenetic stroma A nascent capillarynetwork was present at the interface between the implantand the underlying murine tissue The presence of new


vessels indicated with black arrows in Figure 1(a) was con-firmed by immunohistochemistry staining the sections withanti-vWF polyclonal antibodies (Figure 1(b)) For these invivo experiments we treated the animals daily with NAC(250mgkg) LA (125mgkg) andBr (125mgkg) provided inthe animalsrsquo water bottles a per os administration thatmimicshuman dosing [21] To choose the optimal concentrations ofNAC LA and Br we have referred to the concentrations ofNAC LA and Br proportionally to those present in the newdietary supplement Naxend (Pizeta Pharma Perugia Italy7272 NAC 2424 LA and 303 Br)

As shown in Figure 1(c) all control group animals devel-oped at least 1 cyst with up to 4 in some control animals In 4mice treated with the NACLABr combination no cyst wasvisible and in 3 mice only 1 cyst was present The number ofcysts developing in treated compared to untreated animalswas significantly (119875 lt 005) lower (Figure 1(d)) It is alsonoteworthy that the cysts present in the untreated animalwere larger than those in the treated mice

42 Isolation and Characterization of Endometriotic Endothe-lial Cells (EECs) and Evaluation of Their In Vitro Responseto the NACLABr Combination Since endothelial cells playa significant role in the development of endometrioticlesions [4] as also confirmed in our animal model we nextdeveloped a new protocol for the isolation and culture ofendothelial cells from human endometriotic ovarian cystsFor this purpose the presence and the density of the vesselsinside human endometriotic cysts were initially analyzed byimmunofluorescence on sections of human endometriotictissues The sections were stained with mAb anti-humanvWF a classical endothelial cell marker and with CK818in order to evidence the presence of endometriotic glandsFigure 2 clearly shows the presence of several vessels insidethe cysts and the presence of one representative glandThese samples were then used to isolate the endothelialcells The isolated endothelial cells cultured on fibronectineasily reach confluence within few days The morphology ofcultured EECs stained with mAb anti-CD31 another typicalmarker of endothelial cells is shown in Figure 3 We thencharacterized EECs by cytofluorimetric analysis in order toevaluate the purity of these cells and to exclude the presenceof contaminating cells 100 of these cells were positive forclassical endothelial cell markers (CD31 CD105 vWF andvimentin) and 80 were positive for CD34 a marker fornewly formed vessels (Figure 3) Cultured EECs were devoidof expression of the epithelial marker CK818 and leukocytemarker CD45 Consistent results were obtained for EECpopulations derived from five different patients

43 NACLABr Exerts Anti-Inflammatory Effect Reducing theExpression of VCAM1 in TNF-120572 Stimulated EECs In nextgroup of experiments EECs from 5 distinct patients weregrown to confluence and then stimulated with 100 ngmL ofthe proinflammatory cytokine TNF-120572 in order to upregulatethe expression of the adhesion molecule VCAM1 [22] which

represent a proinflammatory marker We compared the totalamount of VCAM1 by ELISA on EECs untreated EECs stim-ulated for 12 hours with TNF-120572 and cells treated with TNF-120572previously preincubated for 72 hours with NAC LA and Br(NAC 10 120583gmL AL 9 120583gmL and Br 2 120583gmL) used aloneor in association No reduction in VCAM1 expression wasobserved in cells treated with individual drugs (Figure 4(a))Only the drug combination exerted a statistically (119875 lt005) significant although incomplete decrease of VCAM1levels as compared to TNF-120572-treated cultures For compar-ison we have used endothelial cells isolated from normalhuman endometrium fromwomen undergoing hysterectomy(UtMECs) [17] As shown in Figure 4(b) the downregulationof VCAM1 in TNF-120572-treated UtMECs was complete in thepresence of the combination of NAC + Br + LA (MIX) Theaddition of NACLABr to the endothelial cell culture mediadid not alter the medium pH and no effect to the cell viabilitywas observed for ECs treated with the same concentrationsof compounds as assessed by trypan blue staining (data notshown)

44 NACLABr Combination Selectively Exerts ProapoptoticActivity on EECs In order to evaluate whether besides theanti-inflammatory activity the NAC LA and Br mixturemight also affect endothelial cell viability EECs were platedon 8-chamber culture slides grown to 80 confluence andincubated for 72 h with NAC LA and Br (NAC 20120583gmLAL 18 120583gmL and Br 4 120583gmL) used alone or in combinationAs shown in Figures 5(a)-5(b) EECs incubated with themixture of compounds showed a decreased number of viablecells as compared to untreated cultures Based on theseresults we then investigated the ability of NACLABr toinduce apoptosis Both EECs and UtMECs were incubatedwith a fluorogenic substrate for activated caspases 3 and 7and the fluorescence values obtained were normalized fortotal protein present in each well As shown in Figure 5(c)treatment of the cells with the NACLABr mixture atthese higher concentrations was able to induce a statisticallysignificant (119875 lt 005) increase of apoptosis of EECs In factthe induction of caspase activity induced by the drug com-bination was comparable to that of positive control (H

2O2)

Of note the NACLABr mixture was totally ineffective inUtMECs (Figure 5(d))

5 Discussion

Most recent guidelines for the treatment of endometriosis-associated symptoms recommend to surgically treat endo-metriosis as this is effective for reducing endometriosis-associated pain for those in whom medical treatment hasfailed [23] Medical treatments for EM are usually aimed atreducing the production of endogenous estrogens or induc-ing endometrial differentiation with progestins The painassociated with endometriosis is usually treated initially withoral contraceptive agents or non-steroidal anti-inflammatorydrugs because these agents have fewer side effects and are lessexpensive than other treatment options GnRH agonists aswell as other agents such as danazol or progestational agents


(a) (b)

Untreated Treated0

1

2

3

4

Num

ber o

f cys

ts

P lt 0001

(c)

Treated with Naxend Untreated

(d)

Figure 1 NACLABr reduces the number and the dimension of cysts in a new in vivo model of EM (a) Histochemical analysis of anendometriotic lesion in mice with Diff-Quick staining Original Magnification 100x (b) Immunohistochemical analysis of a cyst excisedfrom the peritoneum of mice Sections were stained with rabbit anti-vWF followed by a secondary antibody to rabbit IgG HRP conjugatedand revealed with DAB The inset showed the staining obtained with only secondary antibody Original Magnification 100x (c) Number ofcysts counted in untreatedmice (119899 = 9) or treated with NAC 250mgkgdie AL 125mgkgdie and Br 125mgkgdie (119899 = 7) Mann-Whitneytest 119875 lt 0001 (d) Representative image of the different morphological appearance of cysts in treated or untreated SCID mice

Figure 2 Immunofluorescence analysis of human endometriotic cysts The frozen sections were stained with mAb anti-human vWF tohighlight the vessels or with mAb anti-human CK 818 to show the glands The binding of mouse monoclonal antibodies was revealed bythe incubation with goat anti-mouse IgG Cy3-conjugated secondary antibodies Nuclei were stained in blue by DAPI original magnification100x

and recently aromatase inhibitors are usually reserved foruse if the first-line agents fail to provide an acceptabledegree of relief These agents represent standard therapiesfor EM but are associated with long-term side effects [14]Although currently available medical therapies are not cura-tive per se they are important for pain suppression andlesion regression Thus efforts are still being focused on theimprovement and promotion of new treatments with higher

efficacy and fewer side effects A high number of medicationshave been tested in preclinical models of endometriosisdue to their theoretical capacity of disrupting importantpathophysiologic pathways of the disease such as inflamma-tory response angiogenesis and cell survival proliferationmigration adhesion and invasion TNF-120572 blockers nuclearfactor kB inhibitors antiangiogenic agents statins antioxi-dants immune-modulators flavonoids histone deacetylase


100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0C

ount

s10

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

010

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts

CD45 VimentinvWFCD105CD31CD34

Figure 3 Characterization of the purity of endothelial cells isolated from endometriotic tissue (A) EECs were characterized bycytofluorimetric analysis for the expression of CD45 CD34 CD31 CD105 vWF and vimentin and the expression of these markers (greenlines)was comparedwith correlated control antibodies (black lines)The expression of vWFandCD31was confirmedby immunofluorescenceon EECs grown to confluence in 8-chamber culture slides After fixation and permeabilization the cells were stained with mAb anti-humanCD31 or anti-vWFThe binding of mouse monoclonal antibodies was revealed by the incubation with goat anti-mouse IgG FITC-conjugatedsecondary antibodies Nuclei were stained in blue by DAPI original magnification 200x

NAC Br LA MIX Resting0

20

40

60

80

100 EEC

lowast

VCA

M-1

expr

essio

n (

)

(a)


20

40

60

80

100 UtMEC

lowast

lowast

VCA

M-1

expr

essio

n (

)

(b)

Figure 4 Anti-inflammatory effect of NACLABr on ECs analysis of VCAM1 expression Five different populations of endometrioticendothelial cells (EECs) and 5 different populations of uterine microvascular endothelial cell (UtMECs) were isolated as described by Bullaet al [17] with some modifications ECs were grown to confluence in a 96-well plate and then incubated with NAC 10120583gmL AL 9 120583gmLand Br 2120583gmL alone or in association (MIX) Successively the cells were stimulated overnight with TNF-120572 (100 ngmL) and incubated withanti-human VCAM1The binding of primary antibody was revealed incubating the cells with a goat anti-mouse IgG conjugated with alkalinephosphatase The 100 of VCAM1 expression is referred to the TNF-120572-treated cells Data are expressed as mean plusmn SE of results from fiveexperiments each performed in triplicate lowast119875 lt 005 with respect to the untreated (Mann-Whitney test)

inhibitors matrix metalloproteinase inhibitors metforminnovel modulators of sex steroids expression and apoptoticagents were all effective in vitro andor in animal modelsMost of these agents did not reach the clinical setting mainlybecause of the high risk of adverse effects [24]

An alternative approach for treatment of EM is repre-sented by anti-inflammatory compounds [11] In particularPittaluga and colleagues [25] and Onalan et al [26] recentlydemonstrated the efficacy of NAC in two different in vivomodels of EM No data are available on the use of LA


(a) (b)

Untr NAC LA Br MIX

EEC

100

200

300

400

500

600

700

800lowast

ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein

H2O2

(c)

UtMEC

100

200

300

400

500

600

700

800

ns ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein

Untr NAC LA Br MIX H2O2

(d)

Figure 5 Evaluation of the proapoptotic effect of NACLABr on ECs (a) (b) Morphologic appearance of EECs untreated (a) or incubatedwith NAC 20 120583gmL AL 18 120583gmL and Br 4120583gmL in association (MIX) (b) (c) (d) Both types of cells were grown to 80 of confluencein 96-well plates and incubated with NAC 20120583gmL AL 18 120583gmL and Br 4 120583gmL alone or in association (MIX) for 72 h 37∘C The cellswere then incubated with 5120583M of CellEvent Caspase-37 Green Detection Reagent (Life Technologies) a fluorogenic substrate for activatedcaspases 3 and 7 The fluorescence data were normalized for the total protein present in each well Data are expressed as mean plusmn SE of resultsfrom three experiments each performed in triplicate lowast119875 lt 005 with respect to the untreated (Mann-Whitney test)

and Br for the treatment of EM but they are currently inclinical use for the treatment of inflammatory diseases [2728] Endometriotic lesions are highly vascularized and itis now widely accepted that the formation of new bloodvessels in implanted places plays a key role in the growth ofendometriotic cells [4] The growth of newly formed bloodvessels is of pivotal importance in the development of EMso inhibition of angiogenesis may offer a new opportunityfor treatment [29] Therefore in this study we have proposedan innovative in vitro model to study the efficacy of a newtreatment for EM based on the analysis of endometrioticendothelial cell response in consideration of the key roleof endothelial cells in controlling inflammation and angio-genesis A first important achievement of our study wasthe establishment of a novel mouse model based on theintraperitoneal injection of endometriotic human tissue inSCID mice In analogous models available in the literaturefragments of human endometriotic cysts were surgically fixedto the peritoneal wall [20] The benefit of our model is thatour procedures reduce animal suffering and animal lossesThe fact that all treated animals developed at least one cyst is

a profoundly beneficial aspect of this model Immunohisto-chemical observations indicated that the tissue excised frommurine peritoneumdeveloped several new vessels indicatingthat the cysts had a morphological organization similar tohuman cysts with new blood vessel formations Thanks tothe development of this mouse model we have been able todemonstrate the effectiveness of NACLABr in vivo with theresult that treated mice presented a lower number of cystswhich were also smaller in size than those in untreated mice

A second important finding of our study was thesuccessful isolation of pure endothelial cells from humanendometriotic lesions Using these cells to set up an in vitromodel that exploits the endothelial cells is preferable sincemany differences exist between endothelial cells isolated fromdifferent sites [30] Our results indicated that when usedalone the three studied compounds are able to induce onlya modest or null inhibition of TNF-120572 activation of VCAM1and a strong inhibitionwhen used in combination suggestingthe presence of an additive effect of the three compoundsIn line with our current data previous findings obtained onhypertensive patients (with type 2 diabetes) treatedwithNAC


experienced reduction of C-reactive protein intracellularadhesion molecule and vascular cell adhesion molecule[31] In addition Tisato et al documented that LA signifi-cantly decreased the baseline levels of PDGF RANTES andCXCL10 expression and counteracted TNF-120572-induced NF-Band p38MAPK activation in endothelial cells from chronicvenous disease patients [27] A second important finding ofour study was the ability of NACLABr to promote apoptosisin EEC In this respect it should be underlined that the invitro behavior of UtMECs was different as these cells weretotally unaffected by the NACLABr combination in termsof apoptosis induction These findings underline the impor-tance not only of tissue-specificity but also of pathologicalspecificity of endothelial cells This might explain the partialdiscrepancies of our current data with those of Cai et al[32] and Mohr and Desser [33] which respectively indicatedthat Br inhibits endothelial cell invasion and angiogenesiswhile Larghero et al [34] demonstrated that LA inducedapoptosis through the production of the proapoptotic TNF-alpha-related apoptosis-inducing ligand (TRAIL) cytokine inendothelial cells [34]

6 Conclusions

In conclusion we have adopted an improved in vivomodel ofEMwhich reduces animal suffering by nonsurgical implanta-tion of tissue fromhuman cystsMoreover EECs are a uniquehuman-derived easily available in vitro model of EM thatmay advance study of the inflammatory process and the roleof angiogenesis in endometriosisThanks to thesemodels wecould demonstrate that NACLABr is an effective treatmentfor EM that may have potential therapeutic uses in theprevention and treatment of patients It would be interestingin a further study to compare the effect of NACLABr withstandard therapies and to evaluate if the use of NACLABrin combination with standard therapies may lead to theimprovement of the standard medical treatment for EM

Conflict of Interests

The authors declared that they have no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

C Agostinis and S Zorzet contributed equally to this workF De Seta and R Bulla share senior authorship

Acknowledgments

The contribution of Stefania Consenti e Francesca Gelleniis gratefully acknowledged This work was supported byGrants from the Ministry of Health (Ricerca FinalizzataRC4108 e RC 0109) Fondazione Casali to R Bulla andby a generousliberal donation from PiZeta Pharma SpAPerugia Italy

References

[1] N Berlanda P Vercellini E Somigliana M P FrattaruoloL Buggio and U Gattei ldquoRole of surgery in endometriosis-associated subfertilityrdquo Seminars in Reproductive Medicine vol31 no 2 pp 133ndash143 2013

[2] P Vercellini L Fedele G Aimi G Pietropaolo D ConsonniandPGCrosignani ldquoAssociation between endometriosis stagelesion type patient characteristics and severity of pelvic painsymptoms amultivariate analysis of over 1000 patientsrdquoHumanReproduction vol 22 no 1 pp 266ndash271 2007

[3] P G Groothuis ldquoAngiogenesis and vascular remodelling infemale reproductive organsrdquo Angiogenesis vol 8 no 2 pp 87ndash88 2005

[4] Q-Y Jiang and R-J Wu ldquoGrowth mechanisms of endometri-otic cells in implanted places a reviewrdquoGynecological Endocrin-ology vol 28 no 7 pp 562ndash567 2012

[5] S E Hur J Y Lee H-S Moon and H W Chung ldquoAngi-opoietin-1 angiopoietin-2 and Tie-2 expression in eutopicendometrium in advanced endometriosisrdquo Molecular HumanReproduction vol 12 no 7 pp 421ndash426 2006

[6] K Schweppe ldquoHistological and electron-microscopy studies onendometriosisrdquo Hormone Research vol 32 no 1 pp 106ndash1091989

[7] G Hudelist J Keckstein K Czerwenka et al ldquoEstrogenreceptor 120573 and matrix metalloproteinase 1 are coexpressed inuterine endometrium and endometriotic lesions of patientswith endometriosisrdquo Fertility and Sterility vol 84 no 2 pp1249ndash1256 2005

[8] J Meola J C Rosa e Silva D B Dentillo et al ldquoDifferentiallyexpressed genes in eutopic and ectopic endometrium of womenwith endometriosisrdquo Fertility and Sterility vol 93 no 6 pp1750ndash1773 2010

[9] AMantovani F Bussolino and EDejana ldquoCytokine regulationof endothelial cell functionrdquoTheFASEB Journal vol 6 no 8 pp2591ndash2599 1992

[10] R O Burney and L C Giudice ldquoPathogenesis and pathophysi-ology of endometriosisrdquo Fertility and Sterility vol 98 no 3 pp511ndash519 2012

[11] P Crosignani D Olive A Bergqvist andA Luciano ldquoAdvancesin the management of endometriosis an update for cliniciansrdquoHuman Reproduction Update vol 12 no 2 pp 179ndash189 2006

[12] M G Porpora D Pallante A Ferro B Crisafi F Bellati and PBenedetti Panici ldquoPain and ovarian endometrioma recurrenceafter laparoscopic treatment of endometriosis a long-termprospective studyrdquo Fertility and Sterility vol 93 no 3 pp 716ndash721 2010

[13] R W Kistner ldquoConservative management of endometriosisrdquoThe Lancet vol 79 no 5 pp 179ndash183 1959

[14] V M Rice ldquoConventionalmedical therapies for endometriosisrdquoin Annals of the New York Academy of Sciences pp 343ndash3522002

[15] M G Porpora R Brunelli G Costa et al ldquoA promise inthe treatment of endometriosis an observational cohort studyon ovarian endometrioma reduction by N-acetylcysteinerdquoEvidence-based Complementary and Alternative Medicine vol2013 Article ID 240702 7 pages 2013

[16] R W Noyes A T Hertig and J Rock ldquoDating the endometrialbiopsyrdquoAmerican Journal of Obstetrics and Gynecology vol 122no 2 pp 262ndash263 1975

[17] R Bulla C Agostinis F Bossi et al ldquoDecidual endothelialcells express surface-bound C1q as a molecular bridge between


endovascular trophoblast and decidual endotheliumrdquo Molecu-lar Immunology vol 45 no 9 pp 2629ndash2640 2008

[18] P Spessotto R Bulla C Danussi et al ldquoEMILIN1 represents amajor stromal element determining human trophoblast inva-sion of the uterine wallrdquo Journal of Cell Science vol 119 part 21pp 4574ndash4584 2006

[19] J T Awwad R A Sayegh X J Tao T Hassan S T Awwadand K Isaacson ldquoThe SCID mouse an experimental model forendometriosisrdquo Human Reproduction vol 14 no 12 pp 3107ndash3111 1999

[20] R Grummer F Schwarzer K Bainczyk et al ldquoPeritonealendometriosis validation of an in-vivo modelrdquo Human Repro-duction vol 16 no 8 pp 1736ndash1743 2001

[21] A A Bachmanov D R Reed G K Beauchamp and M GTordoff ldquoFood intake water intake and drinking spout sidepreference of 28 mouse strainsrdquo Behavior Genetics vol 32 no6 pp 435ndash443 2002

[22] L Osborn C Hession R Tizard et al ldquoDirect expressioncloning of vascular cell adhesionmolecule 1 a cytokine-inducedendothelial protein that binds to lymphocytesrdquo Cell vol 59 no6 pp 1203ndash1211 1989

[23] G A J Dunselman N Vermeulen C Becker et al ldquoESHREguideline management of women with endometriosisrdquoHumanReproduction vol 29 no 3 pp 400ndash412 2014

[24] S R Soares A Martınez-Varea J J Hidalgo-Mora and A Pel-licer ldquoPharmacologic therapies in endometriosis a systematicreviewrdquo Fertility and Sterility vol 98 no 3 pp 529ndash555 2012

[25] E Pittaluga G Costa E Krasnowska et al ldquoMore than antioxi-dant N-acetyl-L-cysteine in a murine model of endometriosisrdquoFertility and Sterility vol 94 no 7 pp 2905ndash2908 2010

[26] G Onalan C Gulumser B Mulayim A Dagdeviren and HZeyneloglu ldquoEffects of amifostine on endometriosis compari-son with N-acetyl cysteine and leuprolide as a new treatmentalternative a randomized controlled trialrdquo Archives of Gynecol-ogy and Obstetrics vol 289 no 1 pp 193ndash200 2014

[27] V Tisato G Zauli E Rimondi et al ldquoInhibitory effect ofnatural anti-inflammatory compounds on cytokines releasedby chronic venous disease patient-derived endothelial cellsrdquoMediators of Inflammation vol 2013 Article ID 423407 13pages 2013

[28] S Muller R Marz M Schmolz B Drewelow K Eschmannand P Meiser ldquoPlacebo-controlled randomized clinical trialon the immunomodulating activities of low- and high-dosebromelain after oral administrationmdashnew evidence on theantiinflammatory mode of action of bromelainrdquo PhytotherapyResearch vol 27 no 2 pp 199ndash204 2013

[29] DDjokovic andCCalhaz-Jorge ldquoAngiogenesis as a therapeutictarget in endometriosisrdquo Acta Medica Portuguesa vol 27 no 4pp 489ndash497 2014

[30] W C Aird ldquoPhenotypic heterogeneity of the endothelium IIRepresentative vascular bedsrdquoCirculation Research vol 100 no2 pp 174ndash190 2007

[31] V Cherubini M Cantarini E Ravaglia and E BartolottaldquoIncidence of IDDM in theMarcheRegion ItalyrdquoDiabetes Carevol 17 no 5 pp 432ndash435 1994

[32] T Cai G Fassina M Morini et al ldquoN-acetylcysteine inhibitsendothelial cell invasion and angiogenesisrdquo Laboratory Investi-gation vol 79 no 9 pp 1151ndash1159 1999

[33] T Mohr and L Desser ldquoPlant proteolytic enzyme papain abro-gates angiogenic activation of human umbilical vein endothelialcells (HUVEC) in vitrordquo BMC Complementary and AlternativeMedicine vol 13 article 231 2013

[34] P Larghero R Vene S Minghelli et al ldquoBiological assays andgenomic analysis reveal lipoic acid modulation of endothelialcell behavior and gene expressionrdquo Carcinogenesis vol 28 no5 pp 1008ndash1020 2007

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


considered a fertility-enhancing procedure the benefit onreproductive performance is moderate [11] Surgical removalof ectopic lesions represents the first line of intervention forthe treatment of EM but is characterized by a relevant per-centage of recurrences [12] In addition a variety of medicalhormonal therapies that all aimed to reduce the levels ofcirculating estrogens are currently available [13] Howeverthese treatments are often unsatisfactory and cannot be usedover long periods of time due to the occurrence of severeadverse effects [14]Therefore new and improved therapeuticsolutions that can efficiently reduce lesions with limitedside effects and no interference with the patientrsquos fertilityare definitely desirable In this respect it has been recentlyshown that N-acetyl cysteine (NAC) effectively treats ovarianendometriosis In terms of reduction in cysts size the datareported by Porpora et al are even more favorable than thosegranted by the currently adopted hormonal treatments withthe further advantages of fertility preservation and of thevirtual absence of undesired side effects [15]

On these bases the aim of this study was to investigatethe effects of an association of NAC alpha-lipoic acid (LA)and bromelain (Br) in vivo after establishing a novel modelof EM based on the injection of human endometrial tissue inthe peritoneumof SCIDmice In addition the of NACLABrcombinationwas analyzed in vitro onmicrovascular endothe-lial cells isolated from human endometriotic tissues (EECs)as well as in microvascular endothelial cells isolated fromhuman endometrium (UtMECs)

2 Materials and Methods

21 Preparation of the Compound Mixture All componentsof the mixture were purchased from Sigma-Aldrich (MilanItaly) and solutionswere sterilized by 022120583mfiltration Stockconcentrations were 10mgmL in H

2O for NAC 5mgmL

in absolute ethanol for LA and Br 1mgmL in PBS forBr NAC and LA solutions were stored at 4∘C while Br atminus20∘C until use All reagents were tested for sterility andLPS For cell culture studies the three drugs were combinedin complete cell culture medium at a final concentration of1000 120583gmL NAC + 500120583gmL LA + 50120583gmL Br In theseconditions the solution did not form visible precipitates andpH was stable (measured with pH-Meter BASIC20+ CRI-SON INSTRUMENT) To choose the optimal concentrationsfor the in vitro studies we have referred to the concentrationsof NAC LA and Br proportionally to those present in thenew dietary supplement Naxend (Pizeta Pharma PerugiaItaly 7272 NAC 2424 LA and 303 Br) consideringalso their bioavailability the absorption and the peak plasmaof each compound

22 Human Tissues The Maternal-Childrenrsquos Hospital (RC0813 IRCCS ldquoBurlo Garofolordquo Trieste Italy) approved thisstudy and following informed consent endometriosis speci-mens were obtained from women undergoing laparoscopy toremove endometrial cysts and endometrial tissue was fertilewomen undergoing hysterectomy for leiomyomatosis in themidproliferative and midsecretory phase defined accordingto Noyes criteria [16]

23 Animals Female SCID mice (4ndash6 weeks of age) werepurchased from Charles River (Milan Italy) and maintainedunder pathogen-free conditions All the experimental proce-dures involving animals were done in compliance with theguidelines of the European (86609EEC) and the Italian(DL11692) laws and were approved by both the ItalianMinistry of Health and the Administration of the UniversityAnimal House

24 Animal Model and Ex Vivo Analysis of Cysts Endometri-otic tissue from three peritoneal cysts was collected in sterilePBS and then suspended as coarse fragments loaded in3mL syringes and standardized with respect to volume andweight A volume of 05mL of cyst suspension approxi-mately equal to 04 g of wet tissue was injected by 16 ganeedle intraperitoneally into SCID mice (Charles River)Hormonal therapy with 17-120573-estradiol-3-benzoate (Sigma-Aldrich 30 120583gkg im) was initiated at the time of cyst tissueinjection and at 3-day intervals thereafter The day afterinjection mice were divided randomly in two groups and weadministered only to the first one NAC 250mgkgdie LA125mgkgdie and Br 125mgkgdie per os Twenty-one daysfollowing injection the animals were killed and implantedendometriotic lesions in treated (119899 = 7) or untreated mice(119899 = 9) were identified counted resected and collected informalin 10

Endometriotic lesions excised fromSCIDmicewere fixedin 10 buffered formalin and paraffin embedded Four-micrometers-thick sections were stained with Diff-Quick(Biomap Milan Italy) staining (following the manufacturerinstructions) and examined for the presence and distributionof vessels and glands For immunohistochemical analysisthe slides were microwaved three times in Tris-HClEDTA(ethylenediamine tetraacetic acid) pH 90 buffer (Dako) for5min brought to RT andwashed in PBS After neutralizationof the endogenous peroxidase with H

2O2for 10min the

sections were first incubated with protein block (Dako)for 10min and then with the primary antibodies for 1 hat RT (polyclonal rabbit anti-human vWF from Dako)The bound antibodies were revealed using the horseradishperoxidase- (HRP-) conjugated anti-rabbit IgG antibodies(Sigma-Aldrich) and diaminobenzidine (DAB) as substrate(Dako) Slides were evaluated under Leica DM3000 micro-scope (Leica Wetzlar Germany) and the pictures werecollected using a Leica DFC320 digital camera (Leica)

25 Immunofluorescent Staining Endometriotic tissue frag-ments approximately 1 cm3 were embedded in OCT (BioOp-tica Milan Italy) snap-frozen in liquid nitrogen and keptat minus80∘C until use Cryostat sections of about 6 120583m wereair dried fixed in acetone and either used immediately orkept atminus80∘C Binding ofmouse anti-human cytokeratin 818(CK818) ormouse anti-human vWF (DakoMilan Italy) wasdetected by incubating the sections with goat anti-mouse IgGCy3-conjugated secondary antibodies 30min at RT and thenthe nuclei were stained blue with DAPI (410158406-diamidino-2-phenylindole Sigma-Aldrich) 1120583gmL



2







2


2-MgCl







4 Results









2O2)


5 Discussion



(a) (b)

Untreated Treated0

1

2

3

4

Num

ber o

f cys

ts

P lt 0001

(c)


(d)






100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0C

ount

s10

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

010

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts




20

40

60

80

100 EEC

lowast

VCA

M-1

expr

essio

n (

)

(a)


20

40

60

80

100 UtMEC

lowast

lowast

VCA

M-1

expr

essio

n (

)

(b)





(a) (b)

Untr NAC LA Br MIX

EEC

100

200

300

400

500

600

700

800lowast

ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein

H2O2

(c)

UtMEC

100

200

300

400

500

600

700

800

ns ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein


(d)







6 Conclusions






Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2







2


2-MgCl







4 Results









2O2)


5 Discussion



(a) (b)

Untreated Treated0

1

2

3

4

Num

ber o

f cys

ts

P lt 0001

(c)


(d)






100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0C

ount

s10

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

010

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts




20

40

60

80

100 EEC

lowast

VCA

M-1

expr

essio

n (

)

(a)


20

40

60

80

100 UtMEC

lowast

lowast

VCA

M-1

expr

essio

n (

)

(b)





(a) (b)

Untr NAC LA Br MIX

EEC

100

200

300

400

500

600

700

800lowast

ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein

H2O2

(c)

UtMEC

100

200

300

400

500

600

700

800

ns ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein


(d)







6 Conclusions






Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























2O2)


5 Discussion



(a) (b)

Untreated Treated0

1

2

3

4

Num

ber o

f cys

ts

P lt 0001

(c)


(d)






100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0C

ount

s10

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

010

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts




20

40

60

80

100 EEC

lowast

VCA

M-1

expr

essio

n (

)

(a)


20

40

60

80

100 UtMEC

lowast

lowast

VCA

M-1

expr

essio

n (

)

(b)





(a) (b)

Untr NAC LA Br MIX

EEC

100

200

300

400

500

600

700

800lowast

ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein

H2O2

(c)

UtMEC

100

200

300

400

500

600

700

800

ns ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein


(d)







6 Conclusions






Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

Untreated Treated0

1

2

3

4

Num

ber o

f cys

ts

P lt 0001

(c)


(d)






100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0C

ount

s10

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

010

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts




20

40

60

80

100 EEC

lowast

VCA

M-1

expr

essio

n (

)

(a)


20

40

60

80

100 UtMEC

lowast

lowast

VCA

M-1

expr

essio

n (

)

(b)





(a) (b)

Untr NAC LA Br MIX

EEC

100

200

300

400

500

600

700

800lowast

ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein

H2O2

(c)

UtMEC

100

200

300

400

500

600

700

800

ns ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein


(d)







6 Conclusions






Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0C

ount

s10

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

0

100

80

60

40

20

0

Cou

nts

101

102

103

104

FL1-H10

010

110

210

310

4

FL1-H10

0

100

80

60

40

20

0

Cou

nts




20

40

60

80

100 EEC

lowast

VCA

M-1

expr

essio

n (

)

(a)


20

40

60

80

100 UtMEC

lowast

lowast

VCA

M-1

expr

essio

n (

)

(b)





(a) (b)

Untr NAC LA Br MIX

EEC

100

200

300

400

500

600

700

800lowast

ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein

H2O2

(c)

UtMEC

100

200

300

400

500

600

700

800

ns ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein


(d)







6 Conclusions






Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

Untr NAC LA Br MIX

EEC

100

200

300

400

500

600

700

800lowast

ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein

H2O2

(c)

UtMEC

100

200

300

400

500

600

700

800

ns ns

Activ

ated

casp

ases

3 an

d 7

tota

l pro

tein


(d)







6 Conclusions






Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















6 Conclusions






Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















)JOEBXJ1VCMJTIJOH$PSQPSBUJPO … · membrane antigens were detected on un xed cells, using...

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Transcript of )JOEBXJ1VCMJTIJOH$PSQPSBUJPO … · membrane antigens were detected on un xed cells, using...