Experimental and Molecular Pathology 98 (2015) 13–17

Contents lists available at ScienceDirect

Experimental and Molecular Pathology

j ourna l homepage: www.e lsev ie r .com/ locate /yexmp

The T29C (rs1800470) polymorphism of the transforming growthfactor-β1 (TGF-β1) gene is associated with restenosis after coronarystenting in Mexican patients

José Manuel Fragoso a,e, Joaquín Zuñiga-Ramos b, Marva Arellano-González a, Edith Alvarez-León a,Beatriz E. Villegas-Torres c, Alfredo Cruz-Lagunas b, Hilda Delgadillo-Rodriguez d,e,Marco Antonio Peña-Duque d,e, Marco Antonio Martínez-Ríos d,e, Gilberto Vargas-Alarcón a,e,⁎a Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexicob Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexicoc Laboratory of Genomics, Instituto Nacional de Medicina Genomica, Mexico City, Mexicod Interventional Cardiology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexicoe Interventional Genetic Study Group in Cardiovascular Disease's, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico

⁎ Corresponding author at: Department of MolecularCardiología Ignacio Chávez, Juan Badiano No. 1, Tlalpan 14

E-mail address: gvargas63@yahoo.com (G. Vargas-Ala

http://dx.doi.org/10.1016/j.yexmp.2014.11.0070014-4800/© 2014 Published by Elsevier Inc.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 November 2014Accepted 12 November 2014Available online 13 November 2014

Keywords:Coronary artery diseaseGenetic susceptibilityPolymorphismsRestenosisTransforming growth factor-β1

The aim of the present study was to establish the role of IL-6 and TGF-β1 gene polymorphisms in the risk of de-veloping in-stent restenosis. Two IL-6 [rs1800796 (−572 GNC), rs2069827 (−1426 TNG)] and two TGF-β1[rs1800469 (−509 TNC), rs1800470 (T29C)] gene polymorphisms were analyzed by 5′ exonuclease TaqMangenotyping assays in a group of 244 patients, who underwent coronary artery stenting. Basal and procedure cor-onary angiography were analyzed, looking for angiographic predictors of restenosis and follow-up angiographywas performed to screen for binary restenosis. Under the dominant and additive models adjusted for hyperten-sion, stable angina, stent used, and diameter of stent, the TGF-β1 T29C (rs1800470) polymorphism was signifi-cantly associated with an increase risk of restenosis when compared to patients without restenosis (OR =2.06, 95% CI: 1.03–4.11, PDom = 0.034 and OR= 1.64, 95% CI: 1.09–2.45, PAdd = 0.016). TGF-β1 polymorphismswere in linkage disequilibrium and one haplotype (TT) was significantly increased in patients with restenosiswhen compared to patients without restenosis (OR = 2.03, P = 0.041). In summary, our results suggest thatthe TGF-β1 T29C gene polymorphism could be involved in the risk of developing restenosis after coronarystent placement.

© 2014 Published by Elsevier Inc.

1. Introduction

The coronary artery disease (CAD) is the major cause of morbidityand mortality in the world, according with the World Health Organiza-tion statistics. The treatment strategies are coronary artery bypassgrafting, percutaneous transluminal coronary angioplasty (PTCA), andintracoronary stent. However, after PTCA, restenosis occurs in about30 to 32% of patients and after intracoronary stent placement in 12 to32% of patients (Kuchulakanti et al., 2006; Latib et al., 2011; Lee et al.,2011). The restenosis is the arterial wall's healing response to mechan-ical injury and comprises two main processes—neointimal hyperplasia(i.e., smooth muscle migration/proliferation, extracellular matrix depo-sition) and vessel remodeling (Costa and Simon, 2005). Immediatelyafter coronary stenting, thrombus formation and acute inflammation

Biology, Instituto Nacional de080, Mexico City, Mexico.rcón).

occur, followed by neointimal hyperplasia (Jian-Jun, 2008; Mitra andAgrawal, 2006).

Several studies establish that cytokines such as the interleukin-6 (IL-6) and transforming growth factor beta (TGF-β1) have an importantrole in the instability of the atherosclerotic plaque (Abeywardenaet al., 2009; Aukrust et al., 2008; Border and Noble, 1994; Fonsecaet al., 2009). IL-6 is a pleiotropic proinflammatory cytokine capable ofregulating proliferation, differentiation, and activity of a variety of celltypes, and it plays a pivotal role in the acute phase response (Dienzand Rincon, 2009; Zhang et al., 2008). The IL-6 has an important rolein atherosclerotic plaque development and plaque destabilization. Inaddition, the IL-6 exert several detrimental effects that augment athero-genesis, and promotes endothelial dysfunction, smoothmuscle cell pro-liferation and migration as well as recruitment and activation ofinflammatory cells, thereby perpetuating vascular inflammation(Schuett et al., 2009). On the other hand, TGF-β1 is pleiotropic cytokine,which has been demonstrated to regulate awide array of biological pro-cesses. It plays amajor role in the regulation of vascular function andho-meostasis. In general, TGF-β1 is considered as an anti-inflammatory

14 J.M. Fragoso et al. / Experimental and Molecular Pathology 98 (2015) 13–17

cytokine in the vesselwall. In normal vessel, TGF-β1 inhibits endothelialand vascular smoothmuscle cell proliferation (Frangogiannis, 2014; Lanet al., 2013). In the atherosclerosis, TGF-β1 is considered to beantiatherogenic factor, specifically in the early stages of the disease.Thus, TGF-β1 inhibits excessive vascular smooth muscle cell accumula-tion in the neointima (Redondo et al., 2012). IL-6 and TGF-β1polymorphisms have been associated with the risk of developing ath-erosclerosis, myocardial infarction, blood pressure, acute coronary syn-drome, and restenosis (Cambien et al., 1996; Funayama et al., 2006; Gaoet al., 2013; Hojo et al., 2000; Kurihara et al., 1989; Li et al., 1999; Qiet al., 2005; Tanaka et al., 2005). Considering the prominent role ofthese cytokines, the objective of this study was to establish the role ofthe IL-6 and TGF-β1 gene polymorphisms in the risk of developing reste-nosis after coronary stent placement in a group of Mexican patients.

2. Materials and methods

2.1. Subjects

The study included 244MexicanMestizo patients with symptomaticcoronary artery disease who underwent coronary stent implantation atour institutions andwent to follow-up coronary angiography because ofsymptoms of ischemia documented in a myocardial perfusion imagingtest. Basal and procedure coronary angiographies were analyzed for an-giographic predictors of restenosis, and follow-up angiographywas per-formed to screen for binary restenosis. Using a N50% stenosis at follow-up (50% reduction in the luminal diameter of the stenosis comparedwith the coronary angiography findings immediately following angio-plasty) as the criterion to define restenosis, there were 78 patientswith restenosis and 166 without restenosis. All subjects were ethnicallymatched, and we considered as Mexican Mestizos only those individ-uals who for three generations, including their own, had been born inMexico. The Institutional Ethics and Research Committee approvedthe study, and all subjects signed informed consent.

2.2. Genetic analysis

The DNA extractionmethod proposed by Lahiri and Nurnberger wasused for DNA isolation (Lahiri and Nurnberger, 1991). The IL-6 −572GNC (rs1800796), IL-6 −1426 TNG (rs2069827), TGF-β1 −509 TNC(rs1800469), and TGF-β1 T29C (rs1800470) single nucleotide polymor-phisms were genotyped using 5′ exonuclease TaqMan genotyping as-says on an ABI Prism 7900HT Fast Real-Time PCR system, according tothe manufacturer's instructions (Applied Biosystems, Foster City, CA,USA).

2.3. STR genotyping

Fifteen STR markers (CSF1PO, FGA, THO1, TPOX, VWA, D3S11358,D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11,D19S433 and D2S1338) were typed using the AmpFl STR-IdentifilerKit. PCR amplification was using 1 ng of DNA according to themanufacturer's protocol. PCR products were analyzed on the ABIPrism 3100 Genetic Analyzer using GeneMapper 4.0 software (LifeTechnologies).

2.4. Admixture estimations using STR markers

Individual and group ancestry estimations were calculated in thegroups of patients and controls. The individual and group admixture es-timations were calculated using the STR frequencies in a trihybridmodel with the software Structure version 2.3.4. The STR data fromthree parental populations (Amerindians, Spaniards and Africans)were obtained from previous published databases (Barrot et al., 2005;Calzada et al., 2005; Sanz et al., 2001).

2.5. Statistical analysis

Gene frequencies of IL-6 and TGF-β1 polymorphisms were obtainedby direct counting. Hardy–Weinberg equilibrium was evaluated by χ2

test. All calculationswere performed using SPSS version 18.0 (SPSS, Chi-cago, IL) statistical package. Chi-square tests were used to compare fre-quencies and ANOVA and Student's t-test were used to comparemeans.Logistic regression analysis was performed to look for associations ofpolymorphisms with restenosis using the following models: co-dominant (major allele homozygotes vs. heterozygotes andmajor allelehomozygotes vs. minor allele homozygotes), dominant (major allelehomozygotes vs. heterozygotes+minor allele homozygotes), recessive(major allele homozygotes + heterozygotes vs. minor allele homozy-gotes), heterozygous (homozygote for the minor allele + homozygotefor the major allele vs heterozygote) and log-additive (major allelehomozygotes vs. heterozygotes vs. minor allele homozygotes). Theinheritance models were adjusted for clinical and angiographic vari-ables. SNPstats (http://bioinfo.iconcologia.net/custom.php) softwarefor Windows® were used to analyze the genetic frequencies and toevaluate the linkage disequilibrium (LD, D′) among polymorphisms, aswell as to construct haplotypes.

2.6. Functional prediction analysis

Two in silico programs were used [FastSNP (http://fastsnp.ibms.sinica.edu.tw) and TFSEARCH database (http://mbs.cbrc.jp/research/db/TFSEARCH.html)] to predict the potential effect of the TGF-β1 T29Cpolymorphism. FastSNP analyzes the location of the SNP (5′ upstream,3′ untranslated region, intronic) and possible functional effects such asamino acid changes in protein structures, transcription factor bindingsites in promoter or intronic enhancer regions, and alternative splicingregulation by disrupting exonic splicing enhancers or silencers (Yuanet al., 2006). TFSEARCH is made up of three databases: transcriptionfactor binding sites (TRANSFAC), transcription regulatory database(TRRD), and compound regulatory elements (COMPEL). They providepossible transcription factor effects and their binding sites, splicing reg-ulation, and structural and functional properties of composite elementsto predict the functional effects of SNPs (Heinemeyer et al., 1998).

3. Results

3.1. Characteristics of the study sample

Clinical and angiographic characteristics of the patients with andwithout restenosis included in the study are shown in Table 1. As ex-pected, patients who underwent coronary BMS implantation developmore restenosis (72.0%) than those patients who underwent DES im-plantation (28.0%) (P b 0.001). Also, those patients treated with stentswith a diameter b2.5mmpresentedmore restenosis (P=0.026). As ex-pected, admixture estimates obtained by the maximum likelihoodmethod revealed a greater Amerindian (49.1%) and European contribu-tion (43.7%) in our Mexican Admixed patients. The African componentwas lower than 1% in our groups. These admixture estimations confirmthat our study grouphas the genetic characteristics ofMexicanAdmixedindividuals.

3.2. Allele and genotype frequencies

Observed and expected frequencies of the studied polymorphismswere in Hardy–Weinberg equilibrium. The estimated risk analysis ofthese polymorphisms in patients with and without restenosis was test-ed according to five models: co-dominant, dominant, recessive, hetero-zygous and additive. Under dominant and additive models, the TGF-β1T29C (rs1800470) polymorphism was significantly associated with anincreased risk of restenosis when compared to patients without reste-nosis (OR = 2.06, 95% CI: 1.03–4.11, PDom = 0.034 and OR = 1.64,

Table 1Clinical and angiographic characteristics of the studied individuals.

With restenosis (n (%)) Without restenosis (n (%)) OR CI (95%) P

Mena 61 (78) 131 (79) – – NSHypertensiona 49 (63) 89 (54) – – NSType II diabetes mellitusa 32 (41) 60 (36) – – NSHypercholesterolemiaa 42 (54) 99 (60) – – NSSmokinga 49 (63) 104 (63) – – NSUnstable anginaa 27 (35) 45 (27) – – NSStable anginaa 9 (12) 37 (22) 0.45 0.20–0.99 0.045Statin therapya 65 (83) 142 (86) – – NSDESa 22 (28) 100 (60) 0.25 0.14–0.46 b00001BSMa 56 (72) 66 (40) 3.85 2.15–6.90 b00001Diameter smaller b 2.5 mm 24 (31) 32 (19) 1.86 0.99–3.44 0.026Stent lengtha (b 22 mm) 32 (41) 75 (45) – – NSBifurcationa 22 (28) 41 (25) – – NS

Median (percentile 25–75) Median (percentile 25–75)Age (years) 59.9 (54–67) 58.6 (53–65) – – NS

Abbreviations: BMS = bare-metal stent; DES = drug-eluting stent; NS = not significant; OR = odds ratio; CI = confidence intervals.a (n (%)) number and proportion of subjects with the clinical and angiographic characteristic in both groups.

15J.M. Fragoso et al. / Experimental and Molecular Pathology 98 (2015) 13–17

95%CI: 1.09–2.45, PAdd=0.016).Modelswere adjusted for the variablesthat were significant in the univariate analysis (hypertension, stable an-gina, stent used and diameter of stent) (Table 2).

3.3. Linkage disequilibrium analysis

The TGF-β1 −509 TNC (rs1800469) and TGF-β1 T29C (rs1800470)polymorphisms showed a moderate linkage disequilibrium (D′ =0.56) and were used to construct four haplotypes, H1 (TC), H2 (CT),H3 (CC) and H4 (TT). The H4 haplotype was associated with the risk ofdeveloping restenosis after adjusting for hypertension, presence of sta-ble angina, stent used and diameter of stent (OR = 2.03, 95% CI: 1.03–3.98, P = 0.041) (Table 3).

3.4. Functional analysis

The functional prediction analysis obtained by FastSNP andTFSEARCH bioinformatics tools showed that the presence of the “T”

Table 2Association of the IL-6 and TGF-B1 polymorphisms with restenosis.

Genotype frequency n (%)

IL-6 −572 CNGrs1800796

Without restenosis (n = 166) GG GC CC78 (0.469) 77 (0.463) 11 (0.0

With restenosis (n = 78) 32 (0.410) 39 (0.500) 7 (0.08

TGF-B1 −509 TNCrs1800469

Without restenosis (n = 166) CC CT TT43 (0.259) 83 (0.500) 40 (0.2

With restenosis (n = 78) 21 (0.269) 39 (0.500) 18 (0.2

TGF-B T29Crs1800470

Without restenosis (n = 166) CC CT TT58 (0.349) 74 (0.446) 34 (0.2

With restenosis (n = 78) 15 (0.192) 36 (0.461) 27 (0.3

MAF = minor allele frequency, OR = odds ratio, CI = confidence intervals, P = P value. Theblood pressure, stable angina, stent used and diameter of stent. Bold numbers indicate significThe IL-6−1426 TNG polymorphism is not shown in the table because the TT genotypes were n

allele of the TGF-β1 T29C (rs1800470) polymorphism is associatedwith an exonic splicing enhancer that produces a binding site for SF2/ASF proteins.

4. Discussion

In our study, two IL-6 [rs1800796 (−572 GNC), rs2069827 (−1426TNG)] and two TGF-β1 [rs1800469 (−509 TNC), rs1800470 (T29C)]polymorphismswere determined in a group of patientswhounderwentcoronary stent implantation in order to establish its role in the geneticsusceptibility to developing restenosis. Similar distribution of IL-6 poly-morphisms was observed in patients with and without restenosis,whereas, the TGF-β1 T29C (rs1800470) polymorphism was associatedwith the risk of developing restenosis after coronary stent implantation.It is well known that cytokines such as IL-6 and TGF-β1 have a key rolein mediating the acute inflammatory process (Aukrust et al., 2008) andare involved in the restenosis phenomenon in patients with stent im-plantation. Sardella et al. reported that IL-6 levels were significantly

MAF Model OR (95% CI) P

66) 0.300 Co-dominant 1.57 (0.53–4.66) 0.68Dominant 1.24 (0.69–2.21) 0.48Recessive 1.44 (0.51–4.07) 0.50

9) 0.340 Heterozygous 1.11 (0.62–1.97) 0.73Log-additive 1.22 (0.77–1.94) 0.39

40) 0.491 Co-dominant 0.92 (0.43–1.98) 0.98Dominant 0.95 (0.52–1.74) 0.87Recessive 0.95 (0.50–1.78) 0.86

30) 0.475 Heterozygous 1.00 (0.58–1.71) 0.99Log-additive 0.96 (0.66–1.41) 0.83

04) 0.428 Co-dominant 2.69 (1.19–6.08) 0.053Dominant 2.06 (1.03–4.11) 0.034Recessive 1.87 (0.98–3.58) 0.06

46) 0.576 Heterozygous 1.06 (0.59–1.90) 0.84Log-additive 1.64 (1.09–2.45) 0.016

P values were calculated from logistic regression analysis and the ORs were adjusted forant associations.ot detected.

Table 3Frequencies (%) of TGF-B1 haplotypes [rs1800469 (−509 TNC) and rs1800470 (T29C)] inpatients with and without restenosis.

With restenosis(n = 78)

Without restenosis(n = 166)

P OR 95% CIa

Haplotype Hf HfH1 (TC) 0.332 0.413 NS – –

H2 (CT) 0.428 0.349 NS – –

H3 (CC) 0.092 0.160 NS – –

H4 (TT) 0.149 0.078 0.041 2.03 1.03–3.98

Hf = haplotype frequency, ACS = acute coronary syndrome, P = P value, OR = oddsratio, CI = confidence intervals.The order of thepolymorphisms in thehaplotypes is according to thepositions in the chro-mosome (rs1800469 and rs1800470).

a The ORs were adjusted for hypertension, presence of stable angina, stent used, anddiameter of stent.

16 J.M. Fragoso et al. / Experimental and Molecular Pathology 98 (2015) 13–17

increased in the coronary sinus of patients receiving either bare,paclitaxel- or sirolimus-eluting stents (Sardella et al., 2006). In addition,Kazmierczak et al. reported increased levels of IL-6 in the peripheralblood of patients with chronic stable angina, measured 4 weeks aftercoronary angioplasty with stent implantation (Kazmierczak et al.,2014). According to Exner et al. (2004), the −174 G/C polymorphismof the IL-6 gene is associatedwith the occurrence of restenosis after per-cutaneous transluminal angioplasty. Recently, another IL-6 polymor-phism was associated with increased levels of IL-6 in patients withbare metal stent implantation (Gao et al., 2013). In this case, the poly-morphism was not associated with the risk of developing restenosis inthese patients, a result that agrees with our study. On the other hand,the TGF-β1 has been shown to be involved in restenosis, recruitingmes-enchymal stem cells to the injured tissue, affecting lesion repair(Suwanabol et al., 2011). Polymorphisms in this gene have been associ-ated with the risk of developing cardiovascular diseases (Cruz et al.,2013; Fragoso et al., 2012), but no studies in restenosis have been re-ported. In our study the TGF-β1 T29C T allele was associated with therisk of developing restenosis after coronary stent implantation. This al-lelewaspreviously associatedwith the risk of developing silentmyocar-dial ischemia (Cruz et al., 2013), and acute coronary syndrome (Fragosoet al., 2012) in Mexican individuals. In addition, in agreement with ourdata, Yokota et al. reported an association of the T allele of the T29Cpoly-morphism with the risk of myocardial infarction in a Japanese popula-tion (Yokota et al., 2000).

The functional software used here predicted that TGF-β1 T29C(rs1800470) polymorphism is functional. This analysis showed thatthe presence of the T allele produces a binding site for SF2/ASF proteins(Syrris et al., 1998). These proteins belong to the family of SR proteinsthat regulate alternative splicing (Sureau et al., 2001) suggesting thatthe TGF-β1 T29C polymorphism could have a functional effect. The func-tional effect of the polymorphism detected here should be taken withcare because it is only an informatics approach and need to be con-firmed with experimental testing.

In the linkage disequilibrium analysis and after adjusting for hyper-tension, presence of stable angina, stent used and diameter of stent, oneTGF-β1 risk (TT) haplotype for developing restenosis was detected.Crobu et al., studied three TGF-β1 polymorphisms and reported the as-sociation of a haplotype named “GTC”with the risk of developing myo-cardial infarction (Crobu et al., 2008). This haplotype includes the twopolymorphisms analyzed in our study (−509 TNC and T29C). Recent ex-perimental and clinical results support the concept of a protective roleof TGF-β1 in the circulatory system, even though some articles indicateits proinflammatory features. Thus, individuals with the risk haplotype(TT) could produce less TGF-β1 with consequent decreasing of itsanti-inflammatory effects and increasing the susceptibility to restenosisin these individuals.

In summary, our data suggest that the TGF-β1 T29C (rs1800470)polymorphism plays an important role in the risk of developing reste-nosis. In addition, in our study, it was possible to distinguish one TGF-

β1 risk haplotype for the development of restenosis. Our data are pre-liminary due to the study sample size and additional studies in a largernumber of individuals and in other populations could help define thetrue role of these polymorphisms as risk factors for developingrestenosis.

Conflict of interest statement

No competing financial interests exist.

Acknowledgments

This work was supported in part by grants from the ConsejoNacional de Ciencia y Tecnología (project number 182962). The authorsare grateful to the study participants. Institutional Review Board ap-proval was obtained for all sample collections.

References

Abeywardena, M.Y., Leifert, W.R., Warnes, K.E., et al., 2009. Cardiovascular biology ofinterleukin-6. Curr. Pharm. Des. 15, 1809–1821.

Aukrust, P., Halvorsen, B., Yndestad, A., et al., 2008. Chemokines and cardiovascular risk.Arterioscler. Thromb. Vasc. Biol. 28, 1909–1919.

Barrot, C., Sanchez, C., Ortega, M., et al., 2005. Characterization of three Amerindian pop-ulations from Hidalgo State (Mexico) by 15 STR-PCR polymorphisms. Int. J. LegalMed. 119, 111–115.

Border, W.A., Noble, N.A., 1994. Transforming growth factor β in tissue fibrosis. N. Engl. J.Med. 331, 1286–1292.

Calzada, P., Suarez, I., Garcia, S., et al., 2005. The Fang population of Equatorial Guineacharacterized by 15 STR-PCR polymorphisms. Int. J. Legal Med. 119, 107–110.

Cambien, F., Ricard, S., Troesch, A., et al., 1996. Polymorphisms of the transforming growthfactor-B1 gene in relation to myocardial infarction and blood pressure. The EtudeCas-Temoin de I'Infarctus du Myocarde (ECTIM) Study. Hypertension 28, 881–887.

Costa, M.A., Simon, D.I., 2005. Molecular basis of restenosis and drug-eluting stents. Circu-lation 111, 2257–2273.

Crobu, F., Palumbo, L., Franco, E., et al., 2008. Role of TGF-B1 haplotypes in the occurrenceof myocardial infarction in young Italian patients. BMC Med. Genet. 9, 13.

Cruz, M., Fragoso, J.M., Alvarez-Leon, E., et al., 2013. The TGF-B1 and IL-10 gene polymor-phisms are associated with risk of developing silent myocardial ischemia in the dia-betic patients. Immunol. Lett. 156, 18–22.

Dienz, O., Rincon,M., 2009. The effects of IL-6 on CD4 T cell responses. Clin. Immunol. 130,27–33.

Exner, M., Schillinger, M., Minar, E., et al., 2004. Interleukin-6 promoter genotype and re-stenosis after femoropopliteal balloon angioplasty: initial observations. Radiology231, 839–844.

Fonseca, J.E., Santos, M.J., Canhão, H., et al., 2009. Interleukin-6 as a key player in systemicinflammation and joint destruction. Autoimmun. Rev. 8, 538–542.

Fragoso, J.M., Martinez-Rios, M.A., Alvarez-Leon, E., et al., 2012. The T29C polymorphismsof the transforming growth factor-B1 (TGF-B1) gene is associated with genetic sus-ceptibility to acute coronary syndrome in Mexican patients. Cytokine 58, 280–283.

Frangogiannis, N.G., 2014. Targeting the transforming growth factor (TGF)-B cascade inthe remodeling heart: benefits and perils. J. Mol. Cell. Cardiol. 76C, 169–171.

Funayama, H., Ishikawa, S., Kubo, N., Yasu, T., Saito, M., Kawakami, M., 2006. Close associ-ation of regional interleukin-6 levels in the infarct-related culprit coronary arterywith restenosis in acute myocardial infarction. Circ. J. 70, 426–429.

Gao, J., Liu, Y., Cui, R.Z., et al., 2013. Relationship of interleukin-6-572C/g promoter poly-morphism and serum levels to post-percutaneous coronary intervention restenosis.Chin. Med. J. 126, 1019–1025.

Heinemeyer, T., Wingender, E., Reuter, I., et al., 1998. Database on transcriptional regula-tion: TRANSFAC, TRRD, and COMPEL. Nucleic Acids Res. 26, 364–370.

Hojo, Y., Ikeda, U., Katsuki, T., et al., 2000. Interleukin 6 expression in coronary circulationafter coronary angioplasty as a risk factor for restenosis. Heart 84, 83–87.

Jian-Jun, L.I., 2008. Inflammatory response, drug-eluting stent and restenosis. Chin. Med. J.121, 566–572.

Kazmierczak, E., Grajek, S., Kowal, J., et al., 2014. Prognostic usefulness of IL-6 and VEGFfor the occurrence of changes in coronary arteries of patients with stable anginaand implanted stents. Eur. Rev. Med. Pharmacol. Sci. 18, 2169–2175.

Kuchulakanti, P.K., Chu, W.W., Torguson, R., et al., 2006. Correlates and long-term out-comes of angiographically proven stent thrombosis with sirolimus- and paclitaxel-eluting stents. Circulation 113, 1108–1113.

Kurihara, H., Yoshizumi, M., Sugiyama, T., et al., 1989. Transforming growth factor β stim-ulates the expression of endothelium mRNA by vascular endothelial cells. Biochem.Biophys. Res. Commun. 159, 1435–1440.

Lahiri, D.K., Nurnberger Jr., J.I., 1991. A rapid non-enzymatic method for the preparationHMW DNA from blood for RFLP studies. Nucleic Acids Res. 19, 5444.

Lan, T.H., Huang, X.Q., Tan, H.M., 2013. Vascular fibrosis in atherosclerosis. Cardiovasc.Pathol. 22, 401–407.

Latib, A., Mussardo, M., Ielasi, A., et al., 2011. Long-term outcomes after the percutaneoustreatment of drug-eluting stent restenosis. JACC Cardiovasc. Interv. 4, 155–164.

Lee, S.W., Park, S.W., Kim, Y.H., et al., 2011. A randomized, double-blind, multicenter com-parison study of triple Antiplatelet therapy with dual antiplatelet therapy to reduce

17J.M. Fragoso et al. / Experimental and Molecular Pathology 98 (2015) 13–17

restenosis after drug-eluting stent implantation in long coronary lesions results fromthe DECLARE-LONG II (Drug-Eluting Stenting Followed by Cilostazol Treatment Re-duces Late Restenosis in Patients with Long Coronary Lesions) trial. J. Am. Coll.Cardiol. 57, 1264–1270.

Li, Baogui, Khana, A., Sharma, V., Singh, T., Suthanthiran, M., August, P., 1999. TGF-β11DNA polymorphisms, protein levels and blood pressure. Hypertension 33 (part II),271–275.

Mitra, A.K., Agrawal, D.K., 2006. In stent restenosis: bane of the stent era. J. Clin. Pathol. 59,232–239.

Qi, S., Cao, B., Jiang, M., et al., 2005. Association of the−183 polymorphisms in the INF-γgene promoter with hepatitis B virus infection in the Chinese population. J. Clin. Lab.Anal. 19, 276–281.

Redondo, S., Navarro-Dorado, J., Ramajo, M., Medina, U., Tejerina, T., 2012. The complexregulation of TGF-B in cardiovascular disease. Vasc. Health Risk Manag. 8, 533–539.

Sanz, P., Prieto, V., Flores, I., Torres, Y., Lopez-Soto, M., Farfan, M.J., 2001. Population data of13 STRS in southern Spain (Andalusia). Forensic Sci. Int. 119, 113–115.

Sardella, G., Mariani, P., D'Alessandro, et al., 2006. Early elevation of interleukin-1B andinterleukin-6 levels after bare of drug-eluting stent implantation in patients with sta-ble angina. Thromb. Res. 117, 659–664.

Schuett, H., Luchtefeld, M., Grothusen, C., et al., 2009. How much is too much?Interleukin-6 and its signalling in atherosclerosis. Thromb. Haemost. 102, 215–222.

Sureau, A., Gattoni, R., Dooghe, Y., Stevenin, J., Soret, J., 2001. SC35 autoregulates itsexpression by promoting splicing events that destabilize its mRNAs. EMBO J. 20,1785–1796.

Suwanabol, P.A., Kent, K.C., Liu, B., 2011. TGF-β and restenosis revisited: a Smad link. J.Surg. Res. 167, 287–297.

Syrris, P., Carter, N.D., Metcalfe, J.C., et al., 1998. Trans-forming growth factor-B1 genepolymorphisms and coronary artery disease. Clin. Sci. 95, 659–667.

Tanaka, C., Mannami, T., Kamide, K., et al., 2005. Single nucleotide polymorphisms in theinterleukin-6 gene associated with blood pressure and atherosclerosis in Japanesegeneral population. Hypertens. Res. 28, 35–41.

Yokota, M., Ichihara, S., Lin, T.L., Nakashima, N., Yamada, Y., 2000. Association of a T29Cpolymorphism of the transforming growth factor-beta1 gene with genetic suscepti-bility to myocardial infarction in Japanese. Circulation 101, 2783–2787.

Yuan, H.Y., Chiou, J.J., Tseng,W.H., et al., 2006. FASTSNP: an always up-to date and extend-able service for SNP function analysis and prioritization. Nucleic Acids Res. 34,W635–W641.

Zhang, D., Zhou, Y., Wu, L., et al., 2008. Association of IL-6 gene polymorphisms with ca-chexia susceptibility and survival time of patients with pancreatic cancer. Ann. Clin.Lab. Sci. 38, 113–119.