research article role of transforming growth factor- 1 and...

Research ArticleRole of Transforming Growth Factor-1205731 and Smads SignalingPathway in Intrauterine Adhesion

Umme Salma1 Min Xue1 Md Sayed Ali Sheikh2 Xiaoming Guan3 Bin Xu1 Aiqian Zhang1

Lihua Huang4 and Dabao Xu1

1Department of GynecologyThird Xiangya Hospital of Central South University 138 Tongzipo Road Changsha Hunan 410013 China2Department of Cardiology Xiangya Hospital Central South University Changsha Hunan China3Department of OBGYN Baylor College of Medicine 6651 Main Street 10th Floor Houston TX 77030 USA4Center for Medical Experiments Third Xiangya Hospital Central South University Changsha Hunan China

Correspondence should be addressed to Dabao Xu dabaoxuyahoocom

Received 8 November 2015 Revised 19 January 2016 Accepted 26 January 2016

Academic Editor Marc Pouliot

Copyright copy 2016 Umme Salma 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

The aimof the studywas to evaluate the role of Smad3 Smad7 andTGF-1205731 in intrauterine adhesion (IUA) patients and experimentalrabbit models 60 IUA patients 30 control participants and 18 female rabbits were enrolled in this study We found that the plasmaconcentrations and protein expressions of TGF-1205731 were significantly increased in patients and experimental rabbits compared tothose in controls (119875 lt 005) Furthermore the mRNA and protein expression levels of Smad3 were significantly elevated whileSmad7 level was markedly decreased in the patients and experimental rabbits compared with controls (119875 lt 005)This altered ratiorecommended that IUA was positively correlated to the mRNA and protein expression levels of Smad3 Smad7 and TGF-1205731 inblood and uterine tissue Moreover we used the specific inhibitor of Smad3 (SIS3) in experimental rabbit SIS3 obviously reducedthemRNA and protein expression of smad3 and TGF-1205731 while it increased Smad7 expression in the treatment groups as comparedwith IUA rabbits (119875 lt 005) Our study suggested that TGF-1205731Smad3smad7 is a major pathway which plays an important role inthe regulation of the IUA and specific inhibitor of Smad3 (SIS3) may provide a new therapeutic strategy for IUA

1 Introduction

Intrauterine adhesion (IUA) is one of the important causes forinfertility or recurrent pregnancy loss In the past few yearsIUAs represented the significant clinical problem of womenwho mostly oppose infertility and there is no ideal therapyand thus it represents amajor unmetmedical need Intrauter-ine adhesion results from trauma to increase the endometrialfibrosis On the other hand IUAs are bands of scar-link tissuethat form between two surfaces inside the uterine cavity [1]It has been more than a century since Heinrich Fritsch [2]first described a case of posttraumatic intrauterine adhesionIn general adhesions are composed of fibrotic tissue whichmay result in the adherence of opposing surfaces

It has been identified that the tissue injury is the specificrisk factor and it is possible that after an injury to theendometrium fibrosis may follow with the potential for

adhesion formation [3] Conversely in the tissue the fibrinis rapidly deposited after trauma then it either reverts orturns into fibrous adhesions [4] It varies still questionablyin patients with experience of resorption or adhesion for-mation after trauma In addition the characterizations oftissue fibrosis are considered to arise due to a failure ofthe normal wound healing response [3 5] During normalwound healing a network of negative feedback mechanismsactivated after successful healing is responsible for the propertermination of the proliferative and fibrotic processes thusrestoring tissue integrity Deregulation of this healing processresults in subsequent continuous secretion and depositionof ECM that may lead to the development of tissue fibrosisIn contrast the repetitive trauma to the tissue may leadto continuous activation of fibrin proliferative responseHowever the exact cause of deregulation healing process andcontinuous deposition of ECM components is still enigmatic

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 4158287 13 pageshttpdxdoiorg10115520164158287

2 Mediators of Inflammation

Conversely the facts of clinical experience of fibrosis mayconsider as a somewhat permanent scar tissue during whichresolution of the healing process does not occur Fibrosisis still a major clinical problem the region of insufficientpathological understanding TGF-1205731 stimulates synthesis ofECM proteins and inhibits matrix degradation resulting inthe promotion of fibrosis and tissue repair TGF-1205731 belongsto a family of growth factors of critical importance in woundhealing and fibrogenesis Also platelets are an abundantsource of TGF-1205731 this isoform is released by degranulatingplatelets at the site of an injury hence TGF-1205731 isoformconsideration to play the essential role in wound healing andpossible subsequent fibrosis [6]

It has been studied extensively in models of liver kidneyand lung fibrosis [7ndash9] The TGF-1205731 is secreted as part ofan inactive tripartite complex consisting of a homodimerof the TGF-1205731 with latency associated peptide (LAP) anda molecule of latent TGF binding protein (LTBP) [10] Thecomplex is transported to the ECM and is the major formof TGF1205731 found in vivo [10 11] LTBPs interact with variousmatrix components including collagen and fibronectin [12]Nevertheless TGF-1205731 is an important growth regulator ofhuman endometrium the various cellular responses elicitedby TGF-1205731 are mediated through the activation of ser-inethreonine kinase receptors from the cell surface to thenucleus Once activated TGF-1205731 binds its receptors and thenactivates its downstream signaling mediators of Smad3 toexert its biological activities [13ndash16] Altered expression ofTGF-1205731 in human endometrium has also been associatedwith various abnormalities including extracellular matrixdeposition and adhesion formation [17ndash19] Deregulation ofthe TGF-1205731Smads signaling pathway is responsible for tissuefibrosis in several organs On the other hand the involvementof Smad3 is observed in fibrosis in many animal models ofscleroderma cystic fibrosis [20 21] Verrecchia et al [22]identified a number of collagen gene promoters in humandermal fibroblasts which were induced by TGF-1205731 and itdepends upon Smad3 It has been recognized that the severalabnormalities of endometrium occur with the action of TGF-1205731 which Smads mediate [23 24]

It is well accepted that Smad3 is a critical downstreammediator responsible for the biological effects of TGF-1205731Alternatively Smad7 is an inhibitory Smad and negativelyregulates Smad3 activation by its negative feedback mech-anism Expression of Smad7 is induced by TGF-1205731 whichin turn exerts its negative feedback mechanism by causingdegradation of T120573RI and Smads [25] however the overtexpression of TGF-1205731 and Smad3 and decreased Smad7 aremostly observed in renal [26] cardiac [27] and bleomycin(BMI) induced lungfibrosis [28] this above evidence suggeststhat TGF-1205731 acts by stimulating Smad3 to mediate fibrosis

Conversely given the importance of TGF-1205731 in regu-lating several endometrial cellular activities the expressionand regulation of Smads are necessary to mediate theseactions Besides specific inhibitor of Smad3 (SIS3) to inhibitendothelial-myofibroblast transition and renal fibrosis in atype 1 diabetic kidney disease demonstrates a therapeuticpotential for kidney disease by targeting Smad3 signaling[29] On the other hand SIS3 is a specific inhibitor of

Smad3 phosphorylation via inhibiting the effects of TGF-1205731Furthermore it has been reported that SIS3 abolishes theECM overexpression in the normal dermal fibroblasts andscleroderma fibroblasts in vitro [30]

However the relationship between IUA and Smad3Smad7 with TGF-1205731 remains unknown

Based on the knowledge of the basic properties of TGF-1205731 we aimed to investigate TGF-1205731Smad3 signaling pathwayand regulatory mechanisms of inhibitory Smad7 may beessential in IUA remodeling Therefore we demonstratedthe expression of TGF-1205731 Smad3 and Smad7 in both IUApatients and experimental rabbit model Furthermore wedesigned a study to evaluate the possible therapeutic effectsof SIS3 in experimental rabbit model

2 Materials and Methods

The overview of study design is represented in Figure 1

21 Human Experimental Protocol

211 Study Groups We carried out our study on 60 patientsof IUA mean age 2806 years (range 19ndash34) taking asample of peripheral venous blood and eleven samples ofendometrial fibrosis tissue from March 2014 to December2014 that were enrolled in this study and that were selectedto be of moderate type of the IUA The score and the gradeof the IUA were evaluated during hysteroscopy examinationaccording to the revised criteria (Table 1) of the AmericanFertility Society (AFS) finding where tissue samples frompatients undergoing hysteroscopy adhesiolysis for infertilityare Exclusion criteria were infection any disease in theuterus chronic inflammatory andmalignant disease and anymajor operation within the previous 3 months All patientshad signed a written informed consent before surgery andhad agreed to the collection of tissues for research

212 Control Groups We carried out our study on 30 fertilewomen with mean age 2843 years (range 19ndash34) takingsamples of peripheral venous blood and endometrial tissuebiopsy of nonpathological site of uterine cavity to observe theexpression of TGF-1205731 Smad3 and Smad7 inclusion criteriaof control women included normal menstrual cycles nohistory of previous reproductive surgery or chronic systemicdisease and no hormonal or immune modulator therapyfor at least 6 months before the initial examination andbeing matched for age having hormone reports withinnormal limit and not being hospitalized for at least 1 monthprior to participation The study was approved by the LocalMedical Ethics Committee of the Third Xiangya HospitalCentral South University Hunan China All the participantsprovided written informed consent at the time of enrollment

22 Rabbit Experimental Protocol Experimental protocolswere approved by the Institutional Animal Ethics Committeeof the Third Xiangya Hospital Central South UniversityHunan China From July 2 2014 to July 23 2014 18 maturefemale fertile New Zealand white rabbits weighing 2500ndash3000 g age 5ndash8months were enrolled in this studyThey had

Mediators of Inflammation 3

Table 1TheAmerican Fertility Society classification of intrauterineadhesions 1988

Extent of cavityinvolved

lt13 13ndash23 gt231 2 4

Type of adhesions Filmy Filmy amp dense Dense1 2 4

Menstrual pattern Normal Hypomenorrhea Amenorrhea0 2 4

Stage l (mild) 1ndash4 Stage ll (moderate) 5ndash8 and Stage lll (severe) 9ndash12

A total of 90

IUA Control

Collection of blood and tissue samples

IUAnontreated

IUA amp SIS3treated

Experimentalcontrol groupwith DMSO

Normalgroup

Scarified at 2weeks

ELISA RT-qPCR Western blot

(n = 2) (n = 2)group (n = 30)(n = 60) (n = 7)

group (n = 7)

Total rabbits (n = 18)women

Figure 1 Overview of study design

free access to food and water under standardized laboratoryconditions in a temperature and light controlled environmentfor 2 weeks

23 IUA Rabbit Models Surgeries in rabbits were performedby the same operator Female rabbits weighing 2500 to3000 g (age 5-6 months) were used in all experiments andeach underwent operation under aseptic conditions afterinduction of general anesthesia with pentobarbital sodium(30mgkg) Our aimwas to propose amodel of the pathogen-esis condition in the rabbit similar to intrauterine adhesionobserved in the human Rabbits were killed with an overdoseof pentothal 2 weeks after the operation Tissues were cutout and collection of inferior vena blood of rabbits 2 weeksafter operation was performed Fourteen rabbits submittedto experimental curettage Totally eighteen female rabbits(Table 2) were randomly divided into four groups

231 Intrauterine Adhesion Nontreated Group Seven rabbitswere operated for a 05ndash10 cm longitudinal incision in themiddle of the uterus and the upper or lower half of theendometrium was randomly scraped using the sharp side ofa uterine curette All rabbits were sacrificed after operationeach at 2 weeks after curettage and the abdominal incisionsof the rabbits were sutured after flushing the uterine andperitoneal cavities This method resulted in the developmentof adhesions within 2 to 3 weeks Tissues were cut out andcollection of inferior vena blood of rabbits 2 weeks afteroperation was performed

232 Intrauterine Adhesion and SIS3 Treated Group Afteruterine curettage performed in an identical manner to thatof the endometrial curettage group then 3 120583MSIS3 (SIS3 wasused after diluting with solution in DMSO) was intrauterineapplied the abdominal incisions of the rabbits were suturedin 2 layers with continuous 2-0 chromic catgut after flushingthe uterine and peritoneal cavities Rabbits were killed for thecollection of uterine tissue and inferior vena blood 2 weeksafter operation

233 Experimental Control Group Two rabbits were oper-ated and DMSO solution in the uterus was injected for 2weeks as an experimental control group Tissues were cut outand collection of inferior vena blood 2 weeks after operationwas performed

234 Normal Group In the normal group (119899 = 2) wereeuthanized only for collection of endometrial tissue andblood samples which were obtained from the inferior venacava The macroscopic appearance of the rabbit uterus isrepresented in Figure 2

24 Collection of Blood and Tissue Samples from Patients andRabbits Peripheral 5mL blood samples (human and rabbit)were obtained in K2-EDTA coated tubes and processedwithin 30min of collection using two-step centrifugationSamples were first centrifuged at 1500timesg for 151015840 at 4∘CThe supernatant was collected and then centrifuged againat 14000timesg for 151015840 at 4∘C to obtain pure plasma Finallyplasma was transferred to RNase-free tubes and stored atminus80∘C until RNA extraction Furthermore tissues of bothIUAs and controls (human and animal) were snap frozen inliquid nitrogen and then stored at minus80∘C until use

25 Measurement of TGF-1205731 We measured transforminggrowth factor-1205731 (TGF-1205731) in plasma obtained from humanand rabbit groups by an enzyme-linked immunosorbent assay(ELISA) according to manufacturerrsquos instructions

26 Analysis of mRNA of Smad3 and Smad7 by RT-qPCR Weused real-time quantitative reverse-transcription polymerasechain reaction (RT-qPCR) to detect the mRNA expressionof Smad3 and smad7 in human and rabbit tissue Total RNAfrom different samples was isolated by using TRIzol reagent(TaKaRa Dalian China) and the concentration and purity ofRNA were determined spectrophotometrically Firstly 4 120583Lof pure RNA (OD 18ndash22) was reverse-transcribed (RT) tocDNA at 42∘C for 30 minutes using reverse-transcriptionkits (TaKaRa Dalian China) according to the instructionsof the manufacturer through an RT-PCR System (Bio-RadUSA) Secondly 2 120583L of cDNA was used as the templatein qRT-PCR Then Smad3 and smad7 mRNA expressionwere detected by using SYBR Premix Ex Taq (TaKaRaDalian China) according to the manufacturerrsquos protocolusing a 7300 Real-Time PCR System (Applied BiosystemsCA USA) Briefly a 10 120583L reaction mixture containing 2 120583LcDNA template 5 120583L SYBRMaster Mix 020120583L ROX 24 120583LH2O and 020120583L of each primer was amplified by the

following thermal parameters initial incubation at 95∘C for


Table 2 Rabbit groups with protocol

Group Number Protocol ScarifiedIntrauterine adhesionnontreated group 7 IUA established by endometrial curettage At 2 weeks

Intrauterine adhesion ampSIS3 treated group 7 IUA established by endometrial curettage with

applied SIS3 (3120583M) as previously mentioned [30] At 2 weeks

Experimental control group 2 Rabbits were operated and DMSO solution wasinjected At 2 weeks

Normal group 2 Euthanized only for taking samples At 2 weeks

(a) (b)

(c)

Figure 2 Macroscopic appearance of rabbit uterus (a) Cut section shown normal uterine endometrium (b) The IUA rabbit established byendometrial curettage after 2 weeks and shown adherence of uterine wall (c) Following SIS3 intrauterine administration only slight damageendometrial

15 s followed by 40 cycles of denaturation at 95∘C for 5 sannealing and extension at 60∘C for 31 s The real-time PCRprimers were shown in Table 3 Data analysis was performedby comparative Ct method using the ABI software For themeasurement of Smad3 and Smad7 mRNA expression 120573-actin was served endogenous control and the results wereexpressed as the ratio of Smad3 Smad7 mRNA to 120573-actinmRNA

27 Western Blot Analysis Human and rabbit tissues wereharvested and lysed with ice-cold protein lysis solutionand protein lysates were centrifuged at 12000 g for 15minat 4∘C Total protein in the supernatant was determinedby a BCA Protein Assay Kit (Beyotime Shanghai China)Samples containing 30ndash60120583g of proteins were separated by10SDS-PAGEgel and transferred to polyvinylidene fluoride(PVDF) membranes The membranes were blocked with 5nonfat milk in TBST buffer (100mM NaCl 10mM tris-HCl pH 74 and 01 Tween-20) for 1 hr at room temper-ature The membranes were then incubated with primary

antibodies against Smad3 P-Smad3 (1 200 Santa Cruz CAUSA) Smad7 (1 500 Santa Cruz CA USA) TGF1205731 (1 200Santa Cruz CA USA) or 120573-actin (1 4000 Santa CruzCA USA) with gentle agitation at 4∘C for 18 h followedby horseradish peroxidase-conjugated secondary antibodies(goat anti-rabbit IgG 1 1000 Santa Cruz anti-mouse IgG1 6000 Santa Cruz) for 60min at room temperature

The protein bands were determined by LuminataCrescendo Western HRP Substrate (Millipore BillericaMA USA) through Molecular Imager ChemiDoc XRSSystem (Bio-Rad Philadelphia PA USA) Quantity One1-D Analysis Software (National Institutes of Health USA)was utilized for densitometry analysis Finally the proteinexpression results were measured as the ratio of target(Smad3 P-Smad3 Smad7 or TGF1205731) density to endogenouscontrol (120573-actin) density

28 Statistical Analyses Data were analyzed with SPSS soft-ware (version 200 SPSS Chicago IL) and reported asmean plusmn standard deviation (SD) Differences among groups


0

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IUA + drugIUAVehicleControlExpr

essio

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in ra

bbit

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ma

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F-1205731

in h

uman

pla

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Figure 3 Plasma concentration of TGF-1205731 level was increased in IUA patients and rabbit compared to that in controls After administrationof SIS3 a significantly decreased plasma TGF-1205731 level in the treated groups compared with nontreated groups of IUA rabbit All data wereexpressed as the means plusmn SEM lowast119875 lt 005 versus controls lowastlowast119875 lt 005 versus IUA

Table 3 Primers used for PCR analysis

Target mRNA Primer sequenceSmad3-forward 51015840 CGACCACCAGATGAACCACA-31015840

Smad3-reverse 51015840 GCTGTGAAGCGTGGAATGTCT-31015840

Smad7-forward 51015840 AACTGCAGACTGTCCAGACG-31015840

Smad7-reverse 51015840 GAGGTTGGGAATCTGAAAGC-31015840

120573-actin-forward 51015840 CATCCTGCGTCTGGACCTGG-31015840

120573-actin-reverse 51015840 TAATGTCACGCACGATTTCC

were compared using Studentrsquos 119905-test one-way ANOVA forcategorical variables Fischerrsquos exact test or the chi-square(1205942) test and for non-Gaussian distribution rank transfor-mation and Wilcoxon test were used The Smads and TGF-1205731 expression data were used and graphs were made withGraphPad Prism version 6 forWindows (GraphPad SoftwareSanDiego CAUSA) andpresented asmeanplusmn standard errorAll tests were 2-sided and a significant leveled 119875 lt 005 wasconsidered to be statically significant

3 Results

31 Clinical Characteristics of the Study Groups A total60 IUA patients and 30 control women matched for agehistory of normal menses hypomenorrhea cyclical lowerabdominal pain and abortion were enrolled in this studyThe laboratory findings including RBC WBC Hb FSH LHprolactin estrogen progesterone and testosterone hormonesrecords were collected respectively There were no statisticaldifferences between study group and control group (119875 gt005) The details clinical characteristics of the study groupwere shown in Table 4

32 Plasma Concentration of TGF-1205731 in IUA Patient andExperimental Rabbit Model We measured plasma concen-tration of TGF-1205731 in IUA patients and intrauterine adhesionnontreated group of rabbit to determine whether TGF-1205731 wascorrelated with IUA or not (Figure 3) We found TGF-1205731 wassignificantly increased 27- and 71-fold in IUA patients andintrauterine adhesion nontreated group of rabbit compared

to that in controls (119875 lt 005) Thus increased concentrationof TGF-1205731 is established as a key factor in IUA and Smad3 isproposed as amajor player in TGF-1205731 signaling pathways thatlead to IUA In this conception further study to demonstratethe therapeutic effect of SIS3 in the expression of TGF-1205731 inintrauterine adhesion and SIS3 treated group of rabbit wasperformed and it was examined by using ELISA We found amarked 25-fold decrease of TGF-1205731 in intrauterine adhesionand SIS3 treated group compared to intrauterine adhesionnontreated group of rabbit (119875 lt 005)

Our results indicate SIS3 exhibited a strong inhibitoryeffect of TGF-1205731 signaling in IUA via the inhibition ofsmad3 Also Smad3 is considered to be the primary signalingmolecule involved in TGF-1205731 signal transduction in IUAbecause TGF-1205731 elicits potent profibrotic responses in IUAand Smad3 which are activated by TGF-1205731 and are indis-pensable for most of its profibrotic actions Therefore theinhibition of Smad3 is a necessary component for repressingIUA which is inhibited TGF-1205731 that enhances reduction ofthe occurrence of the IUA Furthermore we confirmed thisresult within tissue fibrosis in IUA patients and experimentalrabbit by RT-qPCR and Western blot analysis

33 Tissue Expression of TGF-1205731 Smad3 and Smad7 in IUAPatient and Experimental Rabbit Model It has been evidentthat TGF-1205731 plays an essential role in fibrosis which isregulated by Smad3 It is well known that the TGF-1205731Smad3signaling system for an autoinhibitory loop involves Smad7as a result the expression of Smad7 influences the activity ofTGF-1205731 As an initial experiment we measured the fibroustissue expression of mRNA and protein Smad3 Smad7and TGF-1205731 in patients and experimental induced rabbitsto determine whether TGF-1205731Smads differentially regulatetheir expression in IUA or not Consistent with the RT-qPCR(cDNA marker) revealed that mRNA expression of Smad3was significantly increased 28-fold while mRNA expressionof Smad7 was significantly decreased 21-fold in patientscompared with controls (119875 lt 005) (Figure 4)

Further our study demonstrated protein expression ofTGF-1205731 Smad3 and Smad7 by Western blot Significant38- and 25-fold increases of protein expression of TGF-1205731


Table 4 Clinical and laboratory findings of the study groups

Variable IUA (119899 = 60) Control (119899 = 30) 119875

Clinical findingsAge 2806 plusmn 363 2843 plusmn 344 0319Normal menses mdash 30 0Hypomenorrhea 60 mdash 0Cyclical lower abdominal pain 24 mdash 0Abortion 60 mdash 0Laboratory findingsRBC (lowast10minus12L) 441 plusmn 028 442 plusmn 021 0948WBC (lowast10minus9L) 538 plusmn 133 547 plusmn 083 0748Hb (gL) 1315 plusmn 779 13313 plusmn 614 0089FSH (mlUmL) 758 plusmn 176 818 plusmn 153 0117LH (mlUmL) 764 plusmn 244 768 plusmn 167 0209PRL (ngmL) 1759 plusmn 295 1657 plusmn 386 0168Estrogen (pmolL) 27725 plusmn 1554 28265 plusmn 12191 0868Progesterone (0ngmL) 0651 plusmn 102 0822 plusmn 007 0366Testosterone (ngdL) 2876 plusmn 761 2842 plusmn 582 0831The data are represented as mean plusmn SD FSH = follicle stimulating hormone LH = luteinizing hormone and PRL = prolactin All 119875 values are representedcomparisons between IUA patient and control groups

0

2

4

6

8

Smad

3 ex

pres

sion

in IU

A p

atie

nts

IUAControl IUAControl00

05

10

15

20

Smad

7 ex

pres

sion

in IU

A p

atie

nts

lowast

lowast

Figure 4 Tissue mRNA expression of Smad3 was increased while Smad7 was decreased in IUA patients compared with controls All datawere expressed as the means plusmn SEM lowast119875 lt 005 versus control

and Smad3 were noticed while protein expression of Smad7significantly decreased 501-fold in patients compared tocontrols (119875 lt 005) (Figure 5)

The mRNA expression of Smad3 was significantlyincreased 39-fold while mRNA expression of Smad7 wassignificantly decreased 27-fold in experimental IUA rabbitcompared with controls (119875 lt 005) (Figure 6) In thisconception we therefore examined the protein expressionof p-Smad3 Smad3 Smad7 and TGF-1205731 in IUA rabbit(Figure 7) Total protein expressions of Smad3 P-Smad3and TGF-1205731 were markedly increased 26- 32- and 201-fold while protein expression of Smad7 was significantlydecreased 404-fold compared to that in controls (119875 lt 005)

Similar results were obtained using fibrous tissue ofexperimental rabbit in contrast to IUA patient Howeverthese findings suggest that increased Smad3 and decreasedSmad7 are an important mechanism underlying the action ofTGF-1205731 activity in IUA

34 Effect of SIS3 on Smad3 P-Smad3 Smad7 and TGF1205731in Experimental Rabbit Model Next we investigated thepossible effects of specific inhibitor of Smad3 (SIS3 withdose of 3 120583M) in IUA which was associated with increasedSmad3 P-Smad3 and TGF1205731 and decreased Smad7 It hasbeen reported that SIS3 with a dose of 3 120583M is able to reducethe Smad3 and inhibited Smad3phosphorylation andTGF-1205731induced fibrotic response in fibroblasts [30] Few studies havedemonstrated that pretreatment of cells with SIS3 (3120583M)in human dermal fibroblasts was completely abrogated toTGF-1205731 signaling that contributes to the upregulation oftype I procollagen via Smad3 [30] Consistent with thesefindings the present study demonstrates that the expressionof fibrous tissuemRNA Smad3 Smad7 and protein of Smad3P-Smad3 Smad7 and TGF-1205731 were determined by RT-qPCRand Western blot analysis respectively (Figures 6 and 7)We found the mRNA and protein expression of Smad3were markedly decreased 16- and 26-fold while Smad7 was


lowast lowast

lowast

TGF-1205731

120573-actin

120573-actin

120573-actin

Smad3 Smad7

0

10

20

30

40

Smad

7 (fo

ld ch

ange

) in

IUA

pat

ient

IUAControlControl IUA

0

10

20

30Sm

ad3

(fold

chan

ge) i

n IU

A p

atie

nt

0

10

20

30

40

TGF-1205731

(fold

chan

ge) i

n IU

A p

atie

nt

IUAControl

Figure 5 Tissue protein expressions of Smad3 and TGF-1205731 were increased while Smad7 was decreased in IUA patients compared withcontrols 120573 actin for loading controls All data were expressed as the means plusmn SEM lowast119875 lt 005 versus control

0

5

10

15

20

25

Smad

3 ex

pres

sion

in IU

A ra

bbit

Vehicle IUA IUA + drugControl Vehicle IUA IUA + drugControl00

05

10

15

20

Smad

7 ex

pres

sion

in IU

A ra

bbit

lowastlowast

lowastlowast

lowast

lowast

Figure 6 Tissue mRNA expression of Smad3 was increased while Smad7 was decreased in IUA rabbit compared with controls After use ofSIS3 the mRNA expression of Smad3 was decreased whereas Smad7 was increased in the treatment groups compared with the nontreatedgroup of IUA rabbits All data were expressed as the means plusmn SEM lowast119875 lt 005 versus control lowastlowast119875 lt 005 versus IUA

significantly increased 24- and 31-fold in the intrauterineadhesion and SIS3 treated group compared to intrauterineadhesion nontreated group (119875 lt 005) We also found theP-Smad3 was decreased 31-fold in treated group comparedto nontreated group of IUA rabbit (119875 lt 005) Furtherwe found fibrous tissue protein expression of TGF-1205731 was

significantly decreased 201-fold in the intrauterine adhesionand SIS3 treated group compared to the intrauterine adhesionnontreated group (119875 lt 005)

Together these results indicated that SIS3 inhibits theupregulation of Smad3 by the suppression of the Smad3phosphorylation and increased Smad7 was associated with


0

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0

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IUA + drugIUAVehicleControl


lowastlowast

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7 (fo

ld ch

ange

) in

IUA

rabb

itTG

F-1205731

(fold

chan

ge) i

n IU

A ra

bbit

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10

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lowastlowast

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ld ch

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) in

IUA

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Smad3

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lowastlowast

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P-Sm

ad3

(fold

chan

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n IU

A ra

bbit

120573-actin

P-Smad3

120573-actin

Smad7

TGF-1205731

120573-actin

Figure 7 Tissue protein expressions of Smad3 phosphorylation (P) Smad3 and TGF-1205731 were increased while Smad7 was decreased in IUArabbit compared with controls DMSOwas used as a vehicle control After use of SIS3 the protein expressions of Smad3 P-Smad3 and TGF-1205731were decreased whereas Smad7 was increased in the treated group compared with the nontreated group of IUA rabbit 120573 actin for loadingcontrols All data were expressed as the means plusmn SEM lowast119875 lt 005 versus control lowastlowast119875 lt 005 versus IUA

inhibitory effect of TGF-1205731 in IUA However SIS3 whichinhibited increased TGF-1205731 signaling could be used as atherapeutic intervention for IUA via the inhibition of Smad3

4 Discussion

This study described for the first time the role of individualexpression of TGF-1205731 Smad3 and Smad7 signal pathway inIUA patients and experimental rabbit modelWe investigatedin patients and created a rabbit model of postoperativeIUA using the endometrial curettage to establish a similarcondition of IUA in human which will be help in our under-standing of molecular mechanisms of intrauterine adhesionand also help to evaluate appropriate therapeutic alternativefor prevention and treatment of the IUA In the past decadesextensive studies have been recognized in the role of variouscytokines and growth factors that can lead to the developmentof tissue fibrosis in different organs Among them TGF-1205731is a multifunctional regulatory cytokine with crucial roles in

fibrotic processes Therefore it is important that only a fewstudies about the potential role of TGF-1205731 in fibrosis havebeen published so far [31 32] It is well accepted that activeTGF-1205731 binds its receptors and then activates its downstreammediators Smad2 and Smad3 to exert its biological effectsincluding fibrosis [33 34]The basis for TGF-1205731 regulation ofcollagen synthesis has developed rapidly as previous studiesidentified by Smad family [35] However Smads componentis amajor specific signal transduction pathway of TGF1205731 [35]In the present study we determine the expression of TGF-1205731Smad3 and Smad7 levels in IUA patients and experimentalrabbit model to identify the molecular defects underlying theTGF-1205731 action We divided three experimental approachesin the first approach we demonstrated patient plasma forTGF-1205731 and fibrous tissue for mRNA and protein that hasexpressed the regulatory Smad3 and inhibitory Smad7 inaddition to TGF-1205731 respectively

In the second approach we establish a rabbit modelof IUA using the endometrial curettage The delay in the


regeneration of endometrial epithelial cells may be importantto the formation of IUA after endometrial damage Because ofthe insufficient epithelial cells covering the uterine cavity thestroma is bared allowing fibroblast activity and connectivetissue formation leading to endometrial fibrosis and possiblyto the development of the IUAWith our experimental designthe whole process of injury or trauma caused by sharp deeplyendometrial curettage could be evaluated and the partialclearance of endometriumwas preceded that may contact thebare endometrium or myometrium and then caused moreinjury effect Due to epithelial loss or epithelialization delaythe presence of fibrosis or the varying degrees of scarsadhesions began to appear Usually the epithelialization didnot start until 2 weeks after injury considerable granulationtissue hyperplasia and fibrosis appeared When a sufficientpercentage of fibrosis appears it will hinder the regenerationof the endometrium and formation of the adhesion [36 37]However trauma to endometrium goes through pathologicalchanges and limited repair consistent with the clinical obser-vation in IUA patient

Our purpose was a model of the pathogenesis conditionin the rabbit similar to IUA observed in the patient Indeedour initial experiment of rabbits the time points of 2 weeksafter endometrial curettage has been characterized as theearly stage of IUA with healing scar may produce fibrosisovert an IUA stage respectively despite the fact that TGF-1205731has powerful stimulation in tissue adhesions and continuousTGF-1205731 activation is associated with pathological cause of theIUA

In the present study confirms to establish IUA a rabbitmodel by visible to the naked eye plasma concentration ofTGF-1205731 and tissue mRNA and protein expression of Smad3Smad7 and TGF-1205731

In the third approach the study was to assess the possibletherapeutic effect as a treatment of intrauterine applicationof SIS3 after endometrial curettage of IUA rabbit that wascompared with the intrauterine adhesion nontreated groupof rabbit

ELISA analysis was used to determine the expressions ofplasma TGF-1205731 RT-qPCR were used to determine Smad3and Smad7 mRNA and Western analysis was used to deter-mine total Smad3 P-Smad3 Smad7 and TGF-1205731 proteinexpression in both patient and rabbit model In the presentstudy we demonstrated that the plasma concentration andprotein expression of TGF-1205731 levels significantly increasein IUA patients and experimental rabbit model comparedto those in controls respectively (119875 lt 005) Previouslydata showed that increased TGF-1205731 induces fibroblasts tosynthesize and contract ECM this cytokine has long beenbelieved to be a central mediator of the fibrotic response[10 27]

On the basis of this conception our results indicateincreased expression of TGF-1205731 levels have preceded the for-mation of fibrosis that enhances the development of the IUAConsistent with our observation increased TGF-1205731 has beenshown in atrial fibrosis as well as transient overexpression ofporcineTGF-1205731 in rat lungs using the adenovirus transfectionsystem induced prolonged lung fibrosis Moreover TGF-1205731 is expressed at greater levels in keloid fibroblasts when

compared with normal dermal fibroblasts There was analso growing evidence support that TGF-1205731 stimulates theprogression of cardiac fibrosis during cardiac hypertrophyand heart failure [38]

Indeed in the present study we found the marked dif-ferent expression of tissue mRNA and protein smad3 Smad7in patients and experimental rabbit model we addressed thisissue and found in other fibrotic disease therefore our studybelieves these different expressions of Smad may alter thedevelopment of the IUA

However the expression of Smads in IUA as well astheir isolated adhesions tissue indicates that these adhesionspossess the necessary components of the TGF-1205731 signalingpathway that can be recruited and activated by Smadscomponent A significantly increased TGF-1205731 level had aneffect on the expression of Smad3 and Smad7 in the IUA Ithas been evident that Smad3 plays a role in the developmentof pathological fibrosis in different organs for consistence inthis regard we further demonstrate the tissue mRNA andprotein expression of Smad3 levels in IUA

We found the tissue mRNA and protein expression ofSmad3 levels are significantly increased in patients andexperimental rabbit model compared to those in controls(119875 lt 005)We alsomeasured phosphorylation of Smad3 thatsignificantly increased in the experimental IUA rabbit modelcompared to controls (119875 lt 005)

Our result suggests the altered TGF-1205731 expression haschanged the Smad3 expression and activation that may bemore important in inducing the fibrosis in the IUA Howeverincreased expression of Smad3 is equally important as anincreased expression of TGF-1205731 in the development of theIUA Also TGF-1205731 is acting as a key regulator to change theSmad3 expression in IUA Thus previously published datataken together with our present study propose the existenceof species specificity in the profibrotic effects of TGF-1205731 onendometrial stroma [17 25ndash28] Consistent with our observa-tion few studies have shown increased Smad3 expression inscleroderma skin scleroderma fibroblasts and postinfarctionmyocardial fibrosis [27 28 39] Takagawa et al [40] alsodemonstrated the expression of Smad3 increased in a murinemodel of BM-induced scleroderma these observations haveinitiated and facilitate our study to establish that Smad3 is animportant step in signal transduction or the activation of theIUA

In the present study we believed the IUAs occur dueto the abnormal responses or functional activities exhibitedby tissue fibroblasts towards the mediators of TGF-1205731 whichSmadsmediate On the other hand few studies demonstratedthe evidence that found an underlying correlation betweendecreased Smad7 and skin lesions in a murine model ofscleroderma andmyocardial fibrosis in infracted heart [20 2128] It was previously demonstrated that the Smad7 presentwithin the cytoplasm of endometrium that regulates TGF-1205731 ligand initiated signaling by competing with the receptorregulated Smads for receptor based phosphorylation andactivation by an autocrineparacrine action of TGF-120573 [7]Generally Smad7 not only is constitutively expressed butalso appears to be rapidly induced by TGF-1205731 in differentfibroblast including dermal primary fibroblasts [20 21]


On the basis of our data increased levels of Smad7 areopposed to the fibrotic actions of TGF-1205731 in IUA whereasdecreased levels of Smad7 are more responsible for fibroticeffect in IUA Consistent with these findings our resultindicates the IUA associated with marked decrease of tissuemRNA and protein expression of Smad7 and it will probablychallenge the homeostatic role of Smad7 in modulating ofTGF-1205731 responses

As an inhibitory regulator in the TGF-120573Smad signalingpathway Smad7 can be induced by a Smad3-dependentmechanism which in turn blocks the signal transduction ofTGF-1205731 via its negative feedback loop [25 41ndash43] ElevatedTGF-1205731 level and decreased Smad7 level are often present intissues where an uncontrolled fibrotic response occurs Thisfinding is consistentwith our present study inwhichwe foundthe tissue mRNA and protein expression of Smad7 levels aresignificantly decreased in patients and experimental rabbitmodel compared to those in controls (119875 lt 005) DecreasedSmad7 is induced activation of Smad3 via TGF-1205731 dependentpathways as a result Smad3-mediated fibrosis occurs inIUA Previous studies also reported reducing Smad7 byactivating the Smad3-dependent Smurf2 degradation path-way [41 43] Reduction of Smad7 is caused by ubiquitin-dependent protein degradation that could be mediated atleast in part by Smurf2 proteins and that plays an importantrole in the fibrosis [44] Consistent with this observationTGF-1205731 induces Smurf2 expression via a Smad3-dependentmechanism [44] However collectively evidence indicatedthat dysregulation of Smad3 and Smad7 was established tobe one of the major mechanisms in mediating the fibroticresponse in IUA In this regard dysregulation of Smad3and Smad7 in IUA may regulate through decreased Smad3and increased Smad7 simultaneously which seems to be aneffective strategy for treatment of the IUA

In our experimental rabbit protocol we observe theexpression of Smad3 at 2 weeks overt the control theseobservations suggest that the prolonged activation of Smad3is important for the initiation to activation of TGF-1205731 signalsHowever our experimental rabbit data show that duringthe development of IUA there is an increase in TGF-1205731signaling as shownby increase of Smad3 in fibrous tissue thusthis was attempted to activate TGF-1205731 signaling by fibroustissue as evidenced by the decreased expression of the Smad7Therefore this negative feedback loop appears sufficient forthe development of the IUAThe action of TGF-1205731 is effectivein IUA possibly due to a higher TGF-1205731 receptor expressionin IUA resulting in increased Smad3 and decreased Smad7expression

Further the present study revealed the therapeutic effectsof SIS3 in experimental rabbit model SIS3 is a potent andselective inhibitor of Smad3 function TGF-1205731 is amajor stim-ulus for tissue fibrosis by enhancing the synthesis of collagensand other matrix components [45 46] TGF-1205731 regulationof collagen synthesis has developed rapidly which Smadsmediate [47] Smad3 are necessary for the transcriptionalactivation of collagen genes [48 49] On the other handseveral studies have evaluated the fibrosis on the basis ofthe accumulation of collagen in tissue due to an imbalancebetween production and deposition of ECM components

including collagen type 1 and it promotes increased TGF-1205731 and Smad3 while decreasing Smad7 [26 50ndash52] TGF-1205731 signaling contributes to the upregulation of type I pro-collagen via Smad3 in human dermal fibroblasts [53 54]Consistent with the above findings the present study foundoverexpression of Smad3 due to increased phosphorylatedSmad3 in uterine fibrous tissues Besides decreased Smad7expression resulted in sustained activation of TGF-1205731 andSmad3 signaling and this trend may be due to a loss ofthe inhibitory effect of Smad7 on Smad3 activation whichis closely associated with fibrosis in IUA Furthermore SIS3has been shown to inhibit collagen synthesis by fibroblastsand fibrosis in several different experimental models SIS3may reduce the upregulated expression of type I collagenthrough the inhibition of phosphorylated Smad3 in scle-roderma fibroblasts From the above evidence the presentstudy recommended that SIS3 is responsible for inhibition ofphosphorylated Smad3 and type I collagen expression in IUA

Moreover 3 120583M SIS3 completely diminished the consti-tutive phosphorylation of Smad3 as well as the excessive typeI procollagen expression in scleroderma fibroblasts Thesefindings supported our results that increased phosphory-lation of Smad3 is an important cause of excessive ECMdeposition and SIS3 is a selective inhibitor of collagen typeI that reduced the occurrence of fibrosis in IUA This findinghas showed consistent with previous studies treatment withSIS3 in fibrosis of several different experimental models[30] Therefore in this study we had aimed to examine theefficacy of SIS3 in the treatment of an IUA rabbit modelConversely we demonstrate the intrauterine application of aspecific inhibitor of Smad3 (SIS3) that alters the expressionof Smad3 and Smad7 activity which enhances altering of thetranscriptional response of TGF-1205731 in IUA

In the present study we found the marked decrease ofexpression of TGF-1205731 and Smad3 levels while Smad7 levelsignificantly increased in the intrauterine adhesion and SIS3treated group compared to intrauterine adhesion nontreatedgroup (119875 lt 005) Also SIS3 is a Smad3 inhibitor andblocks Smad3 signaling directly by inhibiting Smad3 phos-phorylation Moreover SIS3 also acts as a Smad7 agonist andinhibits Smad3 signaling by inducing Smad7 which reducethe fibrosis of IUA (Figure 8)This findingwas consistentwithother study in which there was inhibition of Smad3 with acombination of naringenin (NG) and asiatic acid (AA) (NGis a Smad3 inhibitor and blocks Smad3 signaling directly byinhibiting Smad3 phosphorylation whereas AA functions asa Smad7 agonist and inhibits Smad3 signaling by inducingSmad7) [55]

However Smad3 plays such a critical role in mediatingthe pathobiology of fibrotic disease as demonstrated bythe previously cited studies inhibition of Smad3 signalingcould be a prime target for intervention in fibrotic condi-tions Although increased TGF-1205731 was shown to induce thedecreased expression of Smad7 this induction was transientand therefore indicated the existence of a negative feedbackloop within the pathway and SIS3 has the ability to alter thispathway rather than enhance reduction of the occurrence ofthe IUA In this regard we are attempting to elucidate theantifibrotic effect of SIS3 in IUA


Smad3expression Smad7

expression

IUA

SIS3

IUA expression Treatment expression

Reduce theoccurrence

of IUA

TGF-1205731 expression

Figure 8 Amarked increase of TGF1205731 and Smad3 but a decrease ofSmad7 (red) suggesting that the imbalance between TGF1205731 Smad3and Smad7 signaling could be important in the development ofthe IUA Inhibitory Smad7 competes with Smad3 for access to theactivated TGF-1205731 thereby preventing Smad3 and blocking TGF-1205731induced responses in IUA by SIS3 in the experimentally inducedrabbit We consider it unlikely that increased Smad3 expressionplays a major role in altered TGF-1205731 signaling However inhibitionof Smad3 and the induction of Smad7 are therefore essentialfor preventing excessive TGF-1205731 stimulation (blue) that enhancereduction of the occurrence of the IUA

Also SIS3 recognizes the inhibitory receptor of smad3signal transduction pathways that result in interruption ofTGF-1205731 action SIS3 acts as an important negative feedbackinhibitor of TGF-1205731 signaling by blocking the activationof Smad3 and preventing their interaction with activatedTGF-1205731 receptors as a result disruption of Smad3 confersresistance to the development of the IUA

Together all data in the present study establish that sig-nificantly increased the expression of TGF-1205731 Smad3 whilemarked decreased Smad7 levels in intrauterine adhesionlead to alteration of TGF-1205731 intracellular signaling and theunderlying mechanism of TGF-1205731 action As a substitutespecific inhibitor of smad3 (SIS3) that selectively targetsindividual signaling pathways downstream of the TGF-1205731receptor to significantly decreased TGF-1205731 Smad3 whileincreasing Smad7 levels therefore likely to bemore successfulto elicit to reduce the fibrosis occurrence in experimentalIUA rabbitmodelThese results suggest that TGF-1205731mediatesaction through Smads and most likely signaling pathwaysresult in intrauterine adhesion growth and its regression byspecific inhibitors of Smad3 (SIS3)

5 Conclusion

Our findings indicate that alterations in the expression ofTGF-1205731 and Smad3 components occur during the develop-ment of IUA in both patient and experimental rabbit modelThe patients and rabbit blood have shown significantlyincreased plasma concentration of TGF-1205731 is markedly cor-related to the development of the IUA and IUA is promoted

to the increased tissue expression of TGF-1205731 and Smad3and decreased Smad7 Therefore treatments targeting themodification of TGF-1205731 Smad3 and Smad7 signaling viasuppressing Smad3 with SIS3 may provide a new therapeuticstrategy for IUA

Conflict of Interests

The authors have no conflict of interests to declare

References

[1] H Baakdah and T Tulandi ldquoAdhesion in gynecology com-plication cost and prevention a reviewrdquo Surgical technologyinternational vol 14 pp 185ndash190 2005

[2] H Fritsch ldquoEin Fall von volligen Schwund der Gebaumutter-hohle nach Auskratzungrdquo Zentralblatt fur Gynakologie vol 18pp 1337ndash1342 1894

[3] M P Diamond and M L Freeman ldquoClinical implications ofpostsurgical adhesionsrdquoHumanReproductionUpdate vol 7 no6 pp 567ndash576 2001

[4] H Ellis ldquoThe causes and prevention of intestinal adhesionsrdquoBritish Journal of Surgery vol 69 no 5 pp 241ndash243 1982

[5] B Eckes P Zigrino D Kessler et al ldquoFibroblast-matrix inter-actions in wound healing and fibrosisrdquo Matrix Biology vol 19no 4 pp 325ndash332 2000

[6] G Gabbiani ldquoThe myofibroblast in wound healing and fibro-contractive diseasesrdquo The Journal of Pathology vol 200 no 4pp 500ndash503 2003

[7] A B Roberts and M B Sporn ldquoTransforming growth factor-brdquo in The Molecular and Cellular Biology of Wound Repair pp275ndash308 Springer Berlin Germany 1996

[8] T Nakamura R Sakata T Ueno M Sata and H UenoldquoInhibition of transforming growth factor 120573 prevents progres-sion of liver fibrosis and enhances hepatocyte regeneration indimethylnitrosamine- treated ratsrdquo Hepatology vol 32 no 2pp 247ndash255 2000

[9] J B Kopp V M Factor M Mozes et al ldquoTransgenic mice withincreased plasma levels of TGF-1205731 develop progressive renaldiseaserdquo Laboratory Investigation vol 74 no 6 pp 991ndash10031996

[10] S N Giri D M Hyde and M A Hollinger ldquoEffect of antibodyto transforming growth factor 120573 on bleomycin induced accu-mulation of lung collagen in micerdquo Thorax vol 48 no 10 pp959ndash966 1993

[11] T J Broekelmann A H Limper T V Colby and J AMcdonald ldquoTransforming growth factor beta 1 is present at sitesof extracellular matrix gene expression in human pulmonaryfibrosisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 15 pp 6642ndash6646 1991

[12] J P Annes J S Munger and D B Rifkin ldquoMaking sense oflatent TGF120573 activationrdquo Journal of Cell Science vol 116 no 2pp 217ndash224 2003

[13] A Heydemann E Ceco J E Lim et al ldquoLatent TGF-120573-bindingprotein 4 modifies muscular dystrophy in micerdquoThe Journal ofClinical Investigation vol 119 no 12 pp 3703ndash3712 2009

[14] J Taipale J Saharinen K Hedman and J Keski-Oja ldquoLatenttransforming growth factor-1205731 and its binding protein arecomponents of extracellular matrix microfibrilsrdquo Journal ofHistochemistry and Cytochemistry vol 44 no 8 pp 875ndash8891996


[15] P K Mehta and K K Griendling ldquoAngiotensin II cell signalingphysiological and pathological effects in the cardiovascularsystemrdquoAmerican Journal of Physiology vol 292 no 1 pp C82ndashC97 2007

[16] N Chegini Y Zhao R SWilliams andK C Flanders ldquoHumanuterine tissue throughout the menstrual cycle expresses trans-forming growth factor-1205731 (TGF-1205731) TGF-1205732 TGF-1205733 andTGF-120573 type II receptor messenger ribonucleic acid and proteinand contains [125I]TGF-1205731-binding sitesrdquo Endocrinology vol135 no 1 pp 439ndash449 1994

[17] D A Lawrence ldquoTransforming growth factor-120573 a generalreviewrdquo European Cytokine Network vol 7 no 3 pp 363ndash3741996

[18] M H Branton and J B Kopp ldquoTGF-120573 and fibrosisrdquo Microbesand Infection vol 1 no 15 pp 1349ndash1365 1999

[19] B Casslen T Sandberg B Gustavsson R Willen andM Nilbert ldquoTransforming growth factor 1205731 in the humanendometrium Cyclic variation increased expression by estra-diol and progesterone and regulation of plasminogen activatorsand plasminogen activator inhibitor-1rdquo Biology of Reproductionvol 58 no 6 pp 1343ndash1350 1998

[20] T Yamamoto S Takagawa I Katayama et al ldquoAnimal model ofsclerotic skin I local injections of bleomycin induce scleroticskin mimicking sclerodermardquo Journal of Investigative Derma-tology vol 112 no 4 pp 456ndash462 1999

[21] Y Zhang L L McCormick and A C Gilliam ldquoLatency-associated peptide prevents skin fibrosis in murine scleroder-matous graft-versus-host disease a model for human sclero-dermardquo Journal of Investigative Dermatology vol 121 no 4 pp713ndash719 2003

[22] F Verrecchia M-L Chu and A Mauviel ldquoIdentificationof novel TGF-120573smad gene targets in dermal fibroblastsusing a combined cDNA microarraypromoter transactivationapproachrdquoThe Journal of Biological Chemistry vol 276 no 20pp 17058ndash17062 2001

[23] J Massague and D Wotton ldquoTranscriptional control by theTGF-120573Smad signaling systemrdquoThe EMBO Journal vol 19 no8 pp 1745ndash1754 2000

[24] A Moustakas S Souchelnytskyi and C-H Heldin ldquoSmad reg-ulation in TGF-120573 signal transductionrdquo Journal of Cell Sciencevol 114 no 24 pp 4359ndash4369 2001

[25] P Kavsak R K Rasmussen C G Causing et al ldquoSmad7 bindsto Smurf2 to form an E3 ubiquitin ligase that targets the TGF120573receptor for degradationrdquoMolecular Cell vol 6 no 6 pp 1365ndash1375 2000

[26] W Wang X R Huang A G Li et al ldquoSignaling mechanismof TGF-1205731 in prevention of renal inflammation role of Smad7rdquoJournal of the American Society of Nephrology vol 16 no 5 pp1371ndash1383 2005

[27] S M Ka X R Huang H Y Lan P Y Tsai et al ldquoSmad7gene therapy ameliorates an autoimmune crescentic glomeru-lonephritis in micerdquo Journal of the American Society of Nephrol-ogy vol 18 no 6 pp 1777ndash1788 2007

[28] B Wang J Hao S C Jones M-S Yee J C Roth and I MC Dixon ldquoDecreased Smad 7 expression contributes to cardiacfibrosis in the infarcted rat heartrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 282 no 5pp H1685ndashH1696 2002

[29] J Li X Qu J Yao et al ldquoBlockade of endothelial-mesenchymaltransition by a Smad3 inhibitor delays the early development ofstreptozotocin-induced diabetic nephropathyrdquoDiabetes vol 59no 10 pp 2612ndash2624 2010

[30] M Jinnin H Ihn and K Tamaki ldquoCharacterization of SIS3 anovel specific inhibitor of Smad3 and its effect on transform-ing growth factor-1205731-induced extracellular matrix expressionrdquoMolecular Pharmacology vol 69 no 2 pp 597ndash607 2006

[31] P D Arkwright V Pravica P J Geraghty et al ldquoEnd-organdysfunction in cystic fibrosis association with angiotensin Iconverting enzyme and cytokine gene polymorphismsrdquo Amer-ican Journal of Respiratory and Critical Care Medicine vol 167no 3 pp 384ndash389 2003

[32] P D Arkwright S Laurie M Super et al ldquoTGF-1205731genotype

and accelerated decline in lung function of patients with cysticfibrosisrdquoThorax vol 55 no 6 pp 459ndash462 2000

[33] E P Bottinger ldquoTGF-beta in renal injury and diseaserdquo Seminarsin Nephrology vol 27 no 3 pp 309ndash320 2007

[34] R Derynck and Y E Zhang ldquoSmad-dependent and Smad-independent pathways in TGF-120573 family signallingrdquoNature vol425 no 6958 pp 577ndash584 2003

[35] C Savage P Das A L Finelli et al ldquoCaenorhabditis elegansgenes sma-2 sma-3 and sma-4 define a conserved family oftransforming growth factor 120573 pathway componentsrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 93 no 2 pp 790ndash794 1996

[36] J G Schenker ldquoEtiology of and therapeutic approach tosynechia uterirdquo European Journal of Obstetrics Gynecology andReproductive Biology vol 65 no 1 pp 109ndash113 1996

[37] WZ Polishuk ldquoEndometrial regeneration and adhesion forma-tionrdquo SouthAfricanMedical Journal vol 49 no 12 pp 440ndash4421975

[38] J Hao H Ju S Zhao A Junaid T Scammell-La Fleur andI M C Dixon ldquoElevation of expression of Smads 2 3 and 4decorin and TGF-120573 in the chronic phase of myocardial infarctscar healingrdquo Journal of Molecular and Cellular Cardiology vol31 no 3 pp 667ndash678 1999

[39] HHayashi S Abdollah YQiu et al ldquoTheMAD-related proteinSmad7 associates with the TGF120573 receptor and functions as anantagonist of TGF120573 signalingrdquo Cell vol 89 no 7 pp 1165ndash11731997

[40] S Takagawa G Lakos Y Mori T Yamamoto K Nishioka andJ Varga ldquoSustained activation of fibroblast transforming growthfactor-120573Smad signaling in a murine model of sclerodermardquoJournal of Investigative Dermatology vol 121 no 1 pp 41ndash502003

[41] M Afrakhte A Moren S Jossan et al ldquoInduction of inhibitorySmad6 and Smad7 mRNA by TGF-120573 family membersrdquo Bio-chemical and Biophysical Research Communications vol 249no 2 pp 505ndash511 1998

[42] H-J Zhu J Iaria and A M Sizeland ldquoSmad7 differentiallyregulates transforming growth factor 120573-mediated signalingpathwaysrdquo Journal of Biological Chemistry vol 274 no 45 pp32258ndash32264 1999

[43] T Ebisawa M Fukuchi G Murakami et al ldquoSmurf1 interactswith transforming growth factor-beta type I receptor throughSmad7 and induces receptor degradationrdquo Journal of BiologicalChemistry vol 276 no 16 pp 12477ndash12480 2001

[44] R TanWHe X Lin L P Kiss andY Liu ldquoSmadubiquitinationregulatory factor-2 in the fibrotic kidney regulation targetspecificity and functional implicationrdquo American Journal ofPhysiologymdashRenal Physiology vol 294 no 5 pp F1076ndashF10832008

[45] M Kawabata and K Miyazono ldquoSignal transduction of theTGF-120573 superfamily by Smad proteinsrdquoThe Journal of Biochem-istry vol 125 no 1 pp 9ndash16 1999


[46] J Massague ldquoTGF-120573 signal transductionrdquo Annual Review ofBiochemistry vol 67 no 1 pp 753ndash791 1998

[47] C Savage P Das A L Finelli et al ldquoCaenorhabditis elegansgenes sma-2 sma-3 and sma-4 define a conserved familyof transforming growth factor beta pathway componentsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 93 no 2 pp 790ndash794 1996

[48] S-J Chen W Yuan S Lo M Trojanowska and J VargaldquoInteraction of smad3 with a proximal smad-binding elementof the human alpha2(I) procollagen gene promoter requiredfor transcriptional activation by TGF-betardquo Journal of CellularPhysiology vol 183 no 3 pp 381ndash392 2000

[49] L Vindevoghel R J Lechleider A Kon et al ldquoSMAD34-dependent transcriptional activation of the human type VIIcollagen gene (COL7A1) promoter by transforming growthfactor betardquo Proceedings of the National Academy of Sciences ofthe United States of America vol 95 no 25 pp 14769ndash147741998

[50] K C Flanders ldquoSmad3 as a mediator of the fibrotic responserdquoInternational Journal of Experimental Pathology vol 85 no 2pp 47ndash64 2004

[51] H Y Lan ldquoSmad7 as a therapeutic agent for chronic kidneydiseasesrdquo Frontiers in Bioscience vol 13 no 13 pp 4984ndash49922008

[52] B Wang A Omar T Angelovska et al ldquoRegulation of collagensynthesis by inhibitory Smad7 in cardiac myofibroblastsrdquo TheAmerican Journal of PhysiologymdashHeart and Circulatory Physiol-ogy vol 293 no 2 pp H1282ndashH1290 2007

[53] S-J ChenW Yuan YMori A LevensonM Trojanowska andJ Varga ldquoStimulation of type I collagen transcription in humanskin fibroblasts by TGF-120573 involvement of Smad 3rdquo Journal ofInvestigative Dermatology vol 112 no 1 pp 49ndash57 1999

[54] B Hu Z Wu and S H Phan ldquoSmad3 mediates transforminggrowth factor-120573-induced 120572-smooth muscle actin expressionrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol29 no 3 pp 397ndash404 2003

[55] X-M Meng Y Zhang X-R Huang et al ldquoTreatment ofrenal fibrosis by rebalancing TGF-120573Smad signaling with thecombination of asiatic acid and naringeninrdquo Oncotarget vol 6no 35 pp 36984ndash36997 2015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Conversely the facts of clinical experience of fibrosis mayconsider as a somewhat permanent scar tissue during whichresolution of the healing process does not occur Fibrosisis still a major clinical problem the region of insufficientpathological understanding TGF-1205731 stimulates synthesis ofECM proteins and inhibits matrix degradation resulting inthe promotion of fibrosis and tissue repair TGF-1205731 belongsto a family of growth factors of critical importance in woundhealing and fibrogenesis Also platelets are an abundantsource of TGF-1205731 this isoform is released by degranulatingplatelets at the site of an injury hence TGF-1205731 isoformconsideration to play the essential role in wound healing andpossible subsequent fibrosis [6]

It has been studied extensively in models of liver kidneyand lung fibrosis [7ndash9] The TGF-1205731 is secreted as part ofan inactive tripartite complex consisting of a homodimerof the TGF-1205731 with latency associated peptide (LAP) anda molecule of latent TGF binding protein (LTBP) [10] Thecomplex is transported to the ECM and is the major formof TGF1205731 found in vivo [10 11] LTBPs interact with variousmatrix components including collagen and fibronectin [12]Nevertheless TGF-1205731 is an important growth regulator ofhuman endometrium the various cellular responses elicitedby TGF-1205731 are mediated through the activation of ser-inethreonine kinase receptors from the cell surface to thenucleus Once activated TGF-1205731 binds its receptors and thenactivates its downstream signaling mediators of Smad3 toexert its biological activities [13ndash16] Altered expression ofTGF-1205731 in human endometrium has also been associatedwith various abnormalities including extracellular matrixdeposition and adhesion formation [17ndash19] Deregulation ofthe TGF-1205731Smads signaling pathway is responsible for tissuefibrosis in several organs On the other hand the involvementof Smad3 is observed in fibrosis in many animal models ofscleroderma cystic fibrosis [20 21] Verrecchia et al [22]identified a number of collagen gene promoters in humandermal fibroblasts which were induced by TGF-1205731 and itdepends upon Smad3 It has been recognized that the severalabnormalities of endometrium occur with the action of TGF-1205731 which Smads mediate [23 24]

It is well accepted that Smad3 is a critical downstreammediator responsible for the biological effects of TGF-1205731Alternatively Smad7 is an inhibitory Smad and negativelyregulates Smad3 activation by its negative feedback mech-anism Expression of Smad7 is induced by TGF-1205731 whichin turn exerts its negative feedback mechanism by causingdegradation of T120573RI and Smads [25] however the overtexpression of TGF-1205731 and Smad3 and decreased Smad7 aremostly observed in renal [26] cardiac [27] and bleomycin(BMI) induced lungfibrosis [28] this above evidence suggeststhat TGF-1205731 acts by stimulating Smad3 to mediate fibrosis

Conversely given the importance of TGF-1205731 in regu-lating several endometrial cellular activities the expressionand regulation of Smads are necessary to mediate theseactions Besides specific inhibitor of Smad3 (SIS3) to inhibitendothelial-myofibroblast transition and renal fibrosis in atype 1 diabetic kidney disease demonstrates a therapeuticpotential for kidney disease by targeting Smad3 signaling[29] On the other hand SIS3 is a specific inhibitor of

Smad3 phosphorylation via inhibiting the effects of TGF-1205731Furthermore it has been reported that SIS3 abolishes theECM overexpression in the normal dermal fibroblasts andscleroderma fibroblasts in vitro [30]

However the relationship between IUA and Smad3Smad7 with TGF-1205731 remains unknown

Based on the knowledge of the basic properties of TGF-1205731 we aimed to investigate TGF-1205731Smad3 signaling pathwayand regulatory mechanisms of inhibitory Smad7 may beessential in IUA remodeling Therefore we demonstratedthe expression of TGF-1205731 Smad3 and Smad7 in both IUApatients and experimental rabbit model Furthermore wedesigned a study to evaluate the possible therapeutic effectsof SIS3 in experimental rabbit model

2 Materials and Methods

The overview of study design is represented in Figure 1

21 Human Experimental Protocol

211 Study Groups We carried out our study on 60 patientsof IUA mean age 2806 years (range 19ndash34) taking asample of peripheral venous blood and eleven samples ofendometrial fibrosis tissue from March 2014 to December2014 that were enrolled in this study and that were selectedto be of moderate type of the IUA The score and the gradeof the IUA were evaluated during hysteroscopy examinationaccording to the revised criteria (Table 1) of the AmericanFertility Society (AFS) finding where tissue samples frompatients undergoing hysteroscopy adhesiolysis for infertilityare Exclusion criteria were infection any disease in theuterus chronic inflammatory andmalignant disease and anymajor operation within the previous 3 months All patientshad signed a written informed consent before surgery andhad agreed to the collection of tissues for research

212 Control Groups We carried out our study on 30 fertilewomen with mean age 2843 years (range 19ndash34) takingsamples of peripheral venous blood and endometrial tissuebiopsy of nonpathological site of uterine cavity to observe theexpression of TGF-1205731 Smad3 and Smad7 inclusion criteriaof control women included normal menstrual cycles nohistory of previous reproductive surgery or chronic systemicdisease and no hormonal or immune modulator therapyfor at least 6 months before the initial examination andbeing matched for age having hormone reports withinnormal limit and not being hospitalized for at least 1 monthprior to participation The study was approved by the LocalMedical Ethics Committee of the Third Xiangya HospitalCentral South University Hunan China All the participantsprovided written informed consent at the time of enrollment

22 Rabbit Experimental Protocol Experimental protocolswere approved by the Institutional Animal Ethics Committeeof the Third Xiangya Hospital Central South UniversityHunan China From July 2 2014 to July 23 2014 18 maturefemale fertile New Zealand white rabbits weighing 2500ndash3000 g age 5ndash8months were enrolled in this studyThey had




lt13 13ndash23 gt231 2 4




A total of 90

IUA Control


IUAnontreated

IUA amp SIS3treated


Normalgroup

Scarified at 2weeks


(n = 2) (n = 2)group (n = 30)(n = 60) (n = 7)

group (n = 7)




















(a) (b)

(c)








0

2

4

6

8

10

Control IUA0

10

20

30

40


essio

n of

TG

F-1205731

in ra

bbit

plas

ma

Expr

essio

n of

TG

F-1205731

in h

uman

pla

sma

lowastlowast

lowastlowast










3 Results











0

2

4

6

8

Smad

3 ex

pres

sion

in IU

A p

atie

nts


05

10

15

20

Smad

7 ex

pres

sion

in IU

A p

atie

nts

lowast

lowast







lowast lowast

lowast

TGF-1205731

120573-actin

120573-actin

120573-actin

Smad3 Smad7

0

10

20

30

40

Smad

7 (fo

ld ch

ange

) in

IUA

pat

ient


0

10

20

30Sm

ad3

(fold

chan

ge) i

n IU

A p

atie

nt

0

10

20

30

40

TGF-1205731

(fold

chan

ge) i

n IU

A p

atie

nt

IUAControl


0

5

10

15

20

25

Smad

3 ex

pres

sion

in IU

A ra

bbit


05

10

15

20

Smad

7 ex

pres

sion

in IU

A ra

bbit

lowastlowast

lowastlowast

lowast

lowast






0

10

20

30

40

0

10

20

30

40

50



lowastlowast

lowast

lowastlowast

lowast

Smad

7 (fo

ld ch

ange

) in

IUA

rabb

itTG

F-1205731

(fold

chan

ge) i

n IU

A ra

bbit

0

10

20

30


lowastlowast

lowast

Smad

3 (fo

ld ch

ange

) in

IUA

rabb

it

120573-actin

Smad3

0

10

20

30

40


lowastlowast

lowast

P-Sm

ad3

(fold

chan

ge) i

n IU

A ra

bbit

120573-actin

P-Smad3

120573-actin

Smad7

TGF-1205731

120573-actin



4 Discussion




























expression

IUA

SIS3



of IUA





5 Conclusion





References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















lt13 13ndash23 gt231 2 4




A total of 90

IUA Control


IUAnontreated

IUA amp SIS3treated


Normalgroup

Scarified at 2weeks


(n = 2) (n = 2)group (n = 30)(n = 60) (n = 7)

group (n = 7)




















(a) (b)

(c)








0

2

4

6

8

10

Control IUA0

10

20

30

40


essio

n of

TG

F-1205731

in ra

bbit

plas

ma

Expr

essio

n of

TG

F-1205731

in h

uman

pla

sma

lowastlowast

lowastlowast










3 Results











0

2

4

6

8

Smad

3 ex

pres

sion

in IU

A p

atie

nts


05

10

15

20

Smad

7 ex

pres

sion

in IU

A p

atie

nts

lowast

lowast







lowast lowast

lowast

TGF-1205731

120573-actin

120573-actin

120573-actin

Smad3 Smad7

0

10

20

30

40

Smad

7 (fo

ld ch

ange

) in

IUA

pat

ient


0

10

20

30Sm

ad3

(fold

chan

ge) i

n IU

A p

atie

nt

0

10

20

30

40

TGF-1205731

(fold

chan

ge) i

n IU

A p

atie

nt

IUAControl


0

5

10

15

20

25

Smad

3 ex

pres

sion

in IU

A ra

bbit


05

10

15

20

Smad

7 ex

pres

sion

in IU

A ra

bbit

lowastlowast

lowastlowast

lowast

lowast






0

10

20

30

40

0

10

20

30

40

50



lowastlowast

lowast

lowastlowast

lowast

Smad

7 (fo

ld ch

ange

) in

IUA

rabb

itTG

F-1205731

(fold

chan

ge) i

n IU

A ra

bbit

0

10

20

30


lowastlowast

lowast

Smad

3 (fo

ld ch

ange

) in

IUA

rabb

it

120573-actin

Smad3

0

10

20

30

40


lowastlowast

lowast

P-Sm

ad3

(fold

chan

ge) i

n IU

A ra

bbit

120573-actin

P-Smad3

120573-actin

Smad7

TGF-1205731

120573-actin



4 Discussion




























expression

IUA

SIS3



of IUA





5 Conclusion





References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























(a) (b)

(c)








0

2

4

6

8

10

Control IUA0

10

20

30

40


essio

n of

TG

F-1205731

in ra

bbit

plas

ma

Expr

essio

n of

TG

F-1205731

in h

uman

pla

sma

lowastlowast

lowastlowast










3 Results











0

2

4

6

8

Smad

3 ex

pres

sion

in IU

A p

atie

nts


05

10

15

20

Smad

7 ex

pres

sion

in IU

A p

atie

nts

lowast

lowast







lowast lowast

lowast

TGF-1205731

120573-actin

120573-actin

120573-actin

Smad3 Smad7

0

10

20

30

40

Smad

7 (fo

ld ch

ange

) in

IUA

pat

ient


0

10

20

30Sm

ad3

(fold

chan

ge) i

n IU

A p

atie

nt

0

10

20

30

40

TGF-1205731

(fold

chan

ge) i

n IU

A p

atie

nt

IUAControl


0

5

10

15

20

25

Smad

3 ex

pres

sion

in IU

A ra

bbit


05

10

15

20

Smad

7 ex

pres

sion

in IU

A ra

bbit

lowastlowast

lowastlowast

lowast

lowast






0

10

20

30

40

0

10

20

30

40

50



lowastlowast

lowast

lowastlowast

lowast

Smad

7 (fo

ld ch

ange

) in

IUA

rabb

itTG

F-1205731

(fold

chan

ge) i

n IU

A ra

bbit

0

10

20

30


lowastlowast

lowast

Smad

3 (fo

ld ch

ange

) in

IUA

rabb

it

120573-actin

Smad3

0

10

20

30

40


lowastlowast

lowast

P-Sm

ad3

(fold

chan

ge) i

n IU

A ra

bbit

120573-actin

P-Smad3

120573-actin

Smad7

TGF-1205731

120573-actin



4 Discussion




























expression

IUA

SIS3



of IUA





5 Conclusion





References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

Control IUA0

10

20

30

40


essio

n of

TG

F-1205731

in ra

bbit

plas

ma

Expr

essio

n of

TG

F-1205731

in h

uman

pla

sma

lowastlowast

lowastlowast










3 Results











0

2

4

6

8

Smad

3 ex

pres

sion

in IU

A p

atie

nts


05

10

15

20

Smad

7 ex

pres

sion

in IU

A p

atie

nts

lowast

lowast







lowast lowast

lowast

TGF-1205731

120573-actin

120573-actin

120573-actin

Smad3 Smad7

0

10

20

30

40

Smad

7 (fo

ld ch

ange

) in

IUA

pat

ient


0

10

20

30Sm

ad3

(fold

chan

ge) i

n IU

A p

atie

nt

0

10

20

30

40

TGF-1205731

(fold

chan

ge) i

n IU

A p

atie

nt

IUAControl


0

5

10

15

20

25

Smad

3 ex

pres

sion

in IU

A ra

bbit


05

10

15

20

Smad

7 ex

pres

sion

in IU

A ra

bbit

lowastlowast

lowastlowast

lowast

lowast






0

10

20

30

40

0

10

20

30

40

50



lowastlowast

lowast

lowastlowast

lowast

Smad

7 (fo

ld ch

ange

) in

IUA

rabb

itTG

F-1205731

(fold

chan

ge) i

n IU

A ra

bbit

0

10

20

30


lowastlowast

lowast

Smad

3 (fo

ld ch

ange

) in

IUA

rabb

it

120573-actin

Smad3

0

10

20

30

40


lowastlowast

lowast

P-Sm

ad3

(fold

chan

ge) i

n IU

A ra

bbit

120573-actin

P-Smad3

120573-actin

Smad7

TGF-1205731

120573-actin



4 Discussion




























expression

IUA

SIS3



of IUA





5 Conclusion





References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















0

2

4

6

8

Smad

3 ex

pres

sion

in IU

A p

atie

nts


05

10

15

20

Smad

7 ex

pres

sion

in IU

A p

atie

nts

lowast

lowast







lowast lowast

lowast

TGF-1205731

120573-actin

120573-actin

120573-actin

Smad3 Smad7

0

10

20

30

40

Smad

7 (fo

ld ch

ange

) in

IUA

pat

ient


0

10

20

30Sm

ad3

(fold

chan

ge) i

n IU

A p

atie

nt

0

10

20

30

40

TGF-1205731

(fold

chan

ge) i

n IU

A p

atie

nt

IUAControl


0

5

10

15

20

25

Smad

3 ex

pres

sion

in IU

A ra

bbit


05

10

15

20

Smad

7 ex

pres

sion

in IU

A ra

bbit

lowastlowast

lowastlowast

lowast

lowast






0

10

20

30

40

0

10

20

30

40

50



lowastlowast

lowast

lowastlowast

lowast

Smad

7 (fo

ld ch

ange

) in

IUA

rabb

itTG

F-1205731

(fold

chan

ge) i

n IU

A ra

bbit

0

10

20

30


lowastlowast

lowast

Smad

3 (fo

ld ch

ange

) in

IUA

rabb

it

120573-actin

Smad3

0

10

20

30

40


lowastlowast

lowast

P-Sm

ad3

(fold

chan

ge) i

n IU

A ra

bbit

120573-actin

P-Smad3

120573-actin

Smad7

TGF-1205731

120573-actin



4 Discussion




























expression

IUA

SIS3



of IUA





5 Conclusion





References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast lowast

lowast

TGF-1205731

120573-actin

120573-actin

120573-actin

Smad3 Smad7

0

10

20

30

40

Smad

7 (fo

ld ch

ange

) in

IUA

pat

ient


0

10

20

30Sm

ad3

(fold

chan

ge) i

n IU

A p

atie

nt

0

10

20

30

40

TGF-1205731

(fold

chan

ge) i

n IU

A p

atie

nt

IUAControl


0

5

10

15

20

25

Smad

3 ex

pres

sion

in IU

A ra

bbit


05

10

15

20

Smad

7 ex

pres

sion

in IU

A ra

bbit

lowastlowast

lowastlowast

lowast

lowast






0

10

20

30

40

0

10

20

30

40

50



lowastlowast

lowast

lowastlowast

lowast

Smad

7 (fo

ld ch

ange

) in

IUA

rabb

itTG

F-1205731

(fold

chan

ge) i

n IU

A ra

bbit

0

10

20

30


lowastlowast

lowast

Smad

3 (fo

ld ch

ange

) in

IUA

rabb

it

120573-actin

Smad3

0

10

20

30

40


lowastlowast

lowast

P-Sm

ad3

(fold

chan

ge) i

n IU

A ra

bbit

120573-actin

P-Smad3

120573-actin

Smad7

TGF-1205731

120573-actin



4 Discussion




























expression

IUA

SIS3



of IUA





5 Conclusion





References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

0

10

20

30

40

50



lowastlowast

lowast

lowastlowast

lowast

Smad

7 (fo

ld ch

ange

) in

IUA

rabb

itTG

F-1205731

(fold

chan

ge) i

n IU

A ra

bbit

0

10

20

30


lowastlowast

lowast

Smad

3 (fo

ld ch

ange

) in

IUA

rabb

it

120573-actin

Smad3

0

10

20

30

40


lowastlowast

lowast

P-Sm

ad3

(fold

chan

ge) i

n IU

A ra

bbit

120573-actin

P-Smad3

120573-actin

Smad7

TGF-1205731

120573-actin



4 Discussion




























expression

IUA

SIS3



of IUA





5 Conclusion





References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































expression

IUA

SIS3



of IUA





5 Conclusion





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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