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The T29C (rs1800470) polymorphism of the transforming growth factor-β1 (TGF-β1) gene is associated with restenosis after coronary stenting in Mexican patients
Experimental and Molecular Pathology 98 (2015) 13–17
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The T29C (rs1800470) polymorphism of the transforming growth factor-β1 (TGF-β1) gene is associated with restenosis after coronary stenting 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, Mexico Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico c Laboratory of Genomics, Instituto Nacional de Medicina Genomica, Mexico City, Mexico d Interventional Cardiology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico e Interventional Genetic Study Group in Cardiovascular Disease's, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico b
a r t i c l e
i n f o
Article history: Received 3 November 2014 Accepted 12 November 2014 Available online 13 November 2014 Keywords: Coronary artery disease Genetic susceptibility Polymorphisms Restenosis Transforming growth factor-β1
a b s t r a c t The aim of the present study was to establish the role of IL-6 and TGF-β1 gene polymorphisms in the risk of developing in-stent restenosis. Two IL-6 [rs1800796 (− 572 G NC), rs2069827 (− 1426 T N G)] and two TGF-β1 [rs1800469 (−509 TN C), rs1800470 (T29C)] gene polymorphisms were analyzed by 5′ exonuclease TaqMan genotyping assays in a group of 244 patients, who underwent coronary artery stenting. Basal and procedure coronary angiography were analyzed, looking for angiographic predictors of restenosis and follow-up angiography was performed to screen for binary restenosis. Under the dominant and additive models adjusted for hypertension, stable angina, stent used, and diameter of stent, the TGF-β1 T29C (rs1800470) polymorphism was significantly 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 polymorphisms were in linkage disequilibrium and one haplotype (TT) was significantly increased in patients with restenosis when compared to patients without restenosis (OR = 2.03, P = 0.041). In summary, our results suggest that the TGF-β1 T29C gene polymorphism could be involved in the risk of developing restenosis after coronary stent placement. © 2014 Published by Elsevier Inc.
1. Introduction The coronary artery disease (CAD) is the major cause of morbidity and mortality in the world, according with the World Health Organization statistics. The treatment strategies are coronary artery bypass grafting, percutaneous transluminal coronary angioplasty (PTCA), and intracoronary stent. However, after PTCA, restenosis occurs in about 30 to 32% of patients and after intracoronary stent placement in 12 to 32% of patients (Kuchulakanti et al., 2006; Latib et al., 2011; Lee et al., 2011). The restenosis is the arterial wall's healing response to mechanical injury and comprises two main processes—neointimal hyperplasia (i.e., smooth muscle migration/proliferation, extracellular matrix deposition) and vessel remodeling (Costa and Simon, 2005). Immediately after coronary stenting, thrombus formation and acute inflammation
⁎ Corresponding author at: Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Juan Badiano No. 1, Tlalpan 14080, Mexico City, Mexico. E-mail address: [email protected] (G. Vargas-Alarcón).
http://dx.doi.org/10.1016/j.yexmp.2014.11.007 0014-4800/© 2014 Published by Elsevier Inc.
occur, followed by neointimal hyperplasia (Jian-Jun, 2008; Mitra and Agrawal, 2006). Several studies establish that cytokines such as the interleukin-6 (IL6) and transforming growth factor beta (TGF-β1) have an important role in the instability of the atherosclerotic plaque (Abeywardena et al., 2009; Aukrust et al., 2008; Border and Noble, 1994; Fonseca et al., 2009). IL-6 is a pleiotropic proinflammatory cytokine capable of regulating proliferation, differentiation, and activity of a variety of cell types, and it plays a pivotal role in the acute phase response (Dienz and Rincon, 2009; Zhang et al., 2008). The IL-6 has an important role in atherosclerotic plaque development and plaque destabilization. In addition, the IL-6 exert several detrimental effects that augment atherogenesis, and promotes endothelial dysfunction, smooth muscle cell proliferation and migration as well as recruitment and activation of inflammatory cells, thereby perpetuating vascular inflammation (Schuett et al., 2009). On the other hand, TGF-β1 is pleiotropic cytokine, which has been demonstrated to regulate a wide array of biological processes. It plays a major role in the regulation of vascular function and homeostasis. In general, TGF-β1 is considered as an anti-inflammatory
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cytokine in the vessel wall. In normal vessel, TGF-β1 inhibits endothelial and vascular smooth muscle cell proliferation (Frangogiannis, 2014; Lan et al., 2013). In the atherosclerosis, TGF-β1 is considered to be antiatherogenic factor, specifically in the early stages of the disease. Thus, TGF-β1 inhibits excessive vascular smooth muscle cell accumulation in the neointima (Redondo et al., 2012). IL-6 and TGF-β1 polymorphisms have been associated with the risk of developing atherosclerosis, myocardial infarction, blood pressure, acute coronary syndrome, and restenosis (Cambien et al., 1996; Funayama et al., 2006; Gao et al., 2013; Hojo et al., 2000; Kurihara et al., 1989; Li et al., 1999; Qi et al., 2005; Tanaka et al., 2005). Considering the prominent role of these cytokines, the objective of this study was to establish the role of the IL-6 and TGF-β1 gene polymorphisms in the risk of developing restenosis after coronary stent placement in a group of Mexican patients. 2. Materials and methods 2.1. Subjects The study included 244 Mexican Mestizo patients with symptomatic coronary artery disease who underwent coronary stent implantation at our institutions and went to follow-up coronary angiography because of symptoms of ischemia documented in a myocardial perfusion imaging test. Basal and procedure coronary angiographies were analyzed for angiographic predictors of restenosis, and follow-up angiography was performed to screen for binary restenosis. Using a N50% stenosis at followup (50% reduction in the luminal diameter of the stenosis compared with the coronary angiography findings immediately following angioplasty) as the criterion to define restenosis, there were 78 patients with restenosis and 166 without restenosis. All subjects were ethnically matched, and we considered as Mexican Mestizos only those individuals who for three generations, including their own, had been born in Mexico. The Institutional Ethics and Research Committee approved the study, and all subjects signed informed consent. 2.2. Genetic analysis The DNA extraction method proposed by Lahiri and Nurnberger was used for DNA isolation (Lahiri and Nurnberger, 1991). The IL-6 − 572 GNC (rs1800796), IL-6 − 1426 TNG (rs2069827), TGF-β1 − 509 TNC (rs1800469), and TGF-β1 T29C (rs1800470) single nucleotide polymorphisms were genotyped using 5′ exonuclease TaqMan genotyping assays on an ABI Prism 7900HT Fast Real-Time PCR system, according to the 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-Identifiler Kit. PCR amplification was using 1 ng of DNA according to the manufacturer's protocol. PCR products were analyzed on the ABI Prism 3100 Genetic Analyzer using GeneMapper 4.0 software (Life Technologies).
2.5. Statistical analysis Gene frequencies of IL-6 and TGF-β1 polymorphisms were obtained by direct counting. Hardy–Weinberg equilibrium was evaluated by χ2 test. All calculations were performed using SPSS version 18.0 (SPSS, Chicago, IL) statistical package. Chi-square tests were used to compare frequencies and ANOVA and Student's t-test were used to compare means. Logistic regression analysis was performed to look for associations of polymorphisms with restenosis using the following models: codominant (major allele homozygotes vs. heterozygotes and major allele homozygotes vs. minor allele homozygotes), dominant (major allele homozygotes vs. heterozygotes + minor allele homozygotes), recessive (major allele homozygotes + heterozygotes vs. minor allele homozygotes), heterozygous (homozygote for the minor allele + homozygote for the major allele vs heterozygote) and log-additive (major allele homozygotes vs. heterozygotes vs. minor allele homozygotes). The inheritance models were adjusted for clinical and angiographic variables. SNPstats (http://bioinfo.iconcologia.net/custom.php) software for Windows® were used to analyze the genetic frequencies and to evaluate the linkage disequilibrium (LD, D′) among polymorphisms, as well 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 T29C polymorphism. FastSNP analyzes the location of the SNP (5′ upstream, 3′ untranslated region, intronic) and possible functional effects such as amino acid changes in protein structures, transcription factor binding sites in promoter or intronic enhancer regions, and alternative splicing regulation by disrupting exonic splicing enhancers or silencers (Yuan et al., 2006). TFSEARCH is made up of three databases: transcription factor binding sites (TRANSFAC), transcription regulatory database (TRRD), and compound regulatory elements (COMPEL). They provide possible transcription factor effects and their binding sites, splicing regulation, and structural and functional properties of composite elements to 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 and without restenosis included in the study are shown in Table 1. As expected, patients who underwent coronary BMS implantation develop more restenosis (72.0%) than those patients who underwent DES implantation (28.0%) (P b 0.001). Also, those patients treated with stents with a diameter b2.5 mm presented more restenosis (P = 0.026). As expected, admixture estimates obtained by the maximum likelihood method revealed a greater Amerindian (49.1%) and European contribution (43.7%) in our Mexican Admixed patients. The African component was lower than 1% in our groups. These admixture estimations confirm that our study group has the genetic characteristics of Mexican Admixed individuals. 3.2. Allele and genotype frequencies
2.4. Admixture estimations using STR markers Individual and group ancestry estimations were calculated in the groups of patients and controls. The individual and group admixture estimations were calculated using the STR frequencies in a trihybrid model with the software Structure version 2.3.4. The STR data from three parental populations (Amerindians, Spaniards and Africans) were obtained from previous published databases (Barrot et al., 2005; Calzada et al., 2005; Sanz et al., 2001).
Observed and expected frequencies of the studied polymorphisms were in Hardy–Weinberg equilibrium. The estimated risk analysis of these polymorphisms in patients with and without restenosis was tested according to five models: co-dominant, dominant, recessive, heterozygous and additive. Under dominant and additive models, the TGF-β1 T29C (rs1800470) polymorphism was significantly associated with an increased 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,
J.M. Fragoso et al. / Experimental and Molecular Pathology 98 (2015) 13–17
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Table 1 Clinical and angiographic characteristics of the studied individuals.
Mena Hypertensiona Type II diabetes mellitusa Hypercholesterolemiaa Smokinga Unstable anginaa Stable anginaa Statin therapya DESa BSMa Diameter smaller b 2.5 mm Stent lengtha (b 22 mm) Bifurcationa Age (years)
With restenosis (n (%))
Without restenosis (n (%))
OR
CI (95%)
P
61 (78) 49 (63) 32 (41) 42 (54) 49 (63) 27 (35) 9 (12) 65 (83) 22 (28) 56 (72) 24 (31) 32 (41) 22 (28) Median (percentile 25–75) 59.9 (54–67)
131 (79) 89 (54) 60 (36) 99 (60) 104 (63) 45 (27) 37 (22) 142 (86) 100 (60) 66 (40) 32 (19) 75 (45) 41 (25) Median (percentile 25–75) 58.6 (53–65)
– – – – – – 0.45 – 0.25 3.85 1.86 – –
– – – – – – 0.20–0.99 – 0.14–0.46 2.15–6.90 0.99–3.44 – –
NS NS NS NS NS NS 0.045 NS b00001 b00001 0.026 NS NS
–
–
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.
95% CI: 1.09–2.45, PAdd = 0.016). Models were adjusted for the variables that were significant in the univariate analysis (hypertension, stable angina, stent used and diameter of stent) (Table 2). 3.3. Linkage disequilibrium analysis
allele of the TGF-β1 T29C (rs1800470) polymorphism is associated with an exonic splicing enhancer that produces a binding site for SF2/ ASF proteins.
4. Discussion
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 of developing restenosis after adjusting for hypertension, presence of stable 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 and TFSEARCH bioinformatics tools showed that the presence of the “T”
In our study, two IL-6 [rs1800796 (−572 GNC), rs2069827 (−1426 T NG)] and two TGF-β1 [rs1800469 (− 509 TNC), rs1800470 (T29C)] polymorphisms were determined in a group of patients who underwent coronary stent implantation in order to establish its role in the genetic susceptibility to developing restenosis. Similar distribution of IL-6 polymorphisms was observed in patients with and without restenosis, whereas, the TGF-β1 T29C (rs1800470) polymorphism was associated with 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 role in mediating the acute inflammatory process (Aukrust et al., 2008) and are involved in the restenosis phenomenon in patients with stent implantation. Sardella et al. reported that IL-6 levels were significantly
Table 2 Association of the IL-6 and TGF-B1 polymorphisms with restenosis. Genotype frequency n (%) IL-6 −572 CNG rs1800796 Without restenosis (n = 166)
With restenosis (n = 78) TGF-B1 −509 TNC rs1800469 Without restenosis (n = 166)
With restenosis (n = 78)
TGF-B T29C rs1800470 Without restenosis (n = 166)
With restenosis (n = 78)
MAF
Model
OR (95% CI)
P
Co-dominant Dominant Recessive Heterozygous Log-additive
1.57 (0.53–4.66) 1.24 (0.69–2.21) 1.44 (0.51–4.07) 1.11 (0.62–1.97) 1.22 (0.77–1.94)
0.68 0.48 0.50 0.73 0.39
Co-dominant Dominant Recessive Heterozygous Log-additive
0.92 (0.43–1.98) 0.95 (0.52–1.74) 0.95 (0.50–1.78) 1.00 (0.58–1.71) 0.96 (0.66–1.41)
0.98 0.87 0.86 0.99 0.83
Co-dominant Dominant Recessive Heterozygous Log-additive
2.69 (1.19–6.08) 2.06 (1.03–4.11) 1.87 (0.98–3.58) 1.06 (0.59–1.90) 1.64 (1.09–2.45)
0.053 0.034 0.06 0.84 0.016
GG 78 (0.469)
GC 77 (0.463)
CC 11 (0.066)
0.300
32 (0.410)
39 (0.500)
7 (0.089)
0.340
CC 43 (0.259)
CT 83 (0.500)
TT 40 (0.240)
0.491
21 (0.269)
39 (0.500)
18 (0.230)
0.475
CC 58 (0.349)
CT 74 (0.446)
TT 34 (0.204)
0.428
15 (0.192)
36 (0.461)
27 (0.346)
0.576
MAF = minor allele frequency, OR = odds ratio, CI = confidence intervals, P = P value. The P values were calculated from logistic regression analysis and the ORs were adjusted for blood pressure, stable angina, stent used and diameter of stent. Bold numbers indicate significant associations. The IL-6 −1426 TNG polymorphism is not shown in the table because the TT genotypes were not detected.
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Table 3 Frequencies (%) of TGF-B1 haplotypes [rs1800469 (−509 TNC) and rs1800470 (T29C)] in patients with and without restenosis.
Haplotype H1 (TC) H2 (CT) H3 (CC) H4 (TT)
With restenosis (n = 78)
Without restenosis (n = 166)
Hf 0.332 0.428 0.092 0.149
Hf 0.413 0.349 0.160 0.078
P
OR
95% CIa
NS NS NS 0.041
– – – 2.03
– – – 1.03–3.98
Hf = haplotype frequency, ACS = acute coronary syndrome, P = P value, OR = odds ratio, CI = confidence intervals. The order of the polymorphisms in the haplotypes is according to the positions in the chromosome (rs1800469 and rs1800470). a The ORs were adjusted for hypertension, presence of stable angina, stent used, and diameter of stent.
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 peripheral blood of patients with chronic stable angina, measured 4 weeks after coronary angioplasty with stent implantation (Kazmierczak et al., 2014). According to Exner et al. (2004), the − 174 G/C polymorphism of the IL-6 gene is associated with the occurrence of restenosis after percutaneous transluminal angioplasty. Recently, another IL-6 polymorphism was associated with increased levels of IL-6 in patients with bare metal stent implantation (Gao et al., 2013). In this case, the polymorphism was not associated with the risk of developing restenosis in these patients, a result that agrees with our study. On the other hand, the TGF-β1 has been shown to be involved in restenosis, recruiting mesenchymal stem cells to the injured tissue, affecting lesion repair (Suwanabol et al., 2011). Polymorphisms in this gene have been associated with the risk of developing cardiovascular diseases (Cruz et al., 2013; Fragoso et al., 2012), but no studies in restenosis have been reported. In our study the TGF-β1 T29C T allele was associated with the risk of developing restenosis after coronary stent implantation. This allele was previously associated with the risk of developing silent myocardial ischemia (Cruz et al., 2013), and acute coronary syndrome (Fragoso et al., 2012) in Mexican individuals. In addition, in agreement with our data, Yokota et al. reported an association of the T allele of the T29C polymorphism with the risk of myocardial infarction in a Japanese population (Yokota et al., 2000). The functional software used here predicted that TGF-β1 T29C (rs1800470) polymorphism is functional. This analysis showed that the 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 proteins that regulate alternative splicing (Sureau et al., 2001) suggesting that the TGF-β1 T29C polymorphism could have a functional effect. The functional effect of the polymorphism detected here should be taken with care because it is only an informatics approach and need to be confirmed with experimental testing. In the linkage disequilibrium analysis and after adjusting for hypertension, presence of stable angina, stent used and diameter of stent, one TGF-β1 risk (TT) haplotype for developing restenosis was detected. Crobu et al., studied three TGF-β1 polymorphisms and reported the association of a haplotype named “GTC” with the risk of developing myocardial infarction (Crobu et al., 2008). This haplotype includes the two polymorphisms analyzed in our study (−509 TNC and T29C). Recent experimental and clinical results support the concept of a protective role of TGF-β1 in the circulatory system, even though some articles indicate its proinflammatory features. Thus, individuals with the risk haplotype (TT) could produce less TGF-β1 with consequent decreasing of its anti-inflammatory effects and increasing the susceptibility to restenosis in these individuals. In summary, our data suggest that the TGF-β1 T29C (rs1800470) polymorphism plays an important role in the risk of developing restenosis. In addition, in our study, it was possible to distinguish one TGF-
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The T29C (rs1800470) polymorphism of the transforming growth factor-β1 (TGF-β1) gene is associated with restenosis after coronary stenting 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, Mexico Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico c Laboratory of Genomics, Instituto Nacional de Medicina Genomica, Mexico City, Mexico d Interventional Cardiology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico e Interventional Genetic Study Group in Cardiovascular Disease's, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico b
a r t i c l e
i n f o
Article history: Received 3 November 2014 Accepted 12 November 2014 Available online 13 November 2014 Keywords: Coronary artery disease Genetic susceptibility Polymorphisms Restenosis Transforming growth factor-β1
a b s t r a c t The aim of the present study was to establish the role of IL-6 and TGF-β1 gene polymorphisms in the risk of developing in-stent restenosis. Two IL-6 [rs1800796 (− 572 G NC), rs2069827 (− 1426 T N G)] and two TGF-β1 [rs1800469 (−509 TN C), rs1800470 (T29C)] gene polymorphisms were analyzed by 5′ exonuclease TaqMan genotyping assays in a group of 244 patients, who underwent coronary artery stenting. Basal and procedure coronary angiography were analyzed, looking for angiographic predictors of restenosis and follow-up angiography was performed to screen for binary restenosis. Under the dominant and additive models adjusted for hypertension, stable angina, stent used, and diameter of stent, the TGF-β1 T29C (rs1800470) polymorphism was significantly 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 polymorphisms were in linkage disequilibrium and one haplotype (TT) was significantly increased in patients with restenosis when compared to patients without restenosis (OR = 2.03, P = 0.041). In summary, our results suggest that the TGF-β1 T29C gene polymorphism could be involved in the risk of developing restenosis after coronary stent placement. © 2014 Published by Elsevier Inc.
1. Introduction The coronary artery disease (CAD) is the major cause of morbidity and mortality in the world, according with the World Health Organization statistics. The treatment strategies are coronary artery bypass grafting, percutaneous transluminal coronary angioplasty (PTCA), and intracoronary stent. However, after PTCA, restenosis occurs in about 30 to 32% of patients and after intracoronary stent placement in 12 to 32% of patients (Kuchulakanti et al., 2006; Latib et al., 2011; Lee et al., 2011). The restenosis is the arterial wall's healing response to mechanical injury and comprises two main processes—neointimal hyperplasia (i.e., smooth muscle migration/proliferation, extracellular matrix deposition) and vessel remodeling (Costa and Simon, 2005). Immediately after coronary stenting, thrombus formation and acute inflammation
⁎ Corresponding author at: Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Juan Badiano No. 1, Tlalpan 14080, Mexico City, Mexico. E-mail address: [email protected] (G. Vargas-Alarcón).
http://dx.doi.org/10.1016/j.yexmp.2014.11.007 0014-4800/© 2014 Published by Elsevier Inc.
occur, followed by neointimal hyperplasia (Jian-Jun, 2008; Mitra and Agrawal, 2006). Several studies establish that cytokines such as the interleukin-6 (IL6) and transforming growth factor beta (TGF-β1) have an important role in the instability of the atherosclerotic plaque (Abeywardena et al., 2009; Aukrust et al., 2008; Border and Noble, 1994; Fonseca et al., 2009). IL-6 is a pleiotropic proinflammatory cytokine capable of regulating proliferation, differentiation, and activity of a variety of cell types, and it plays a pivotal role in the acute phase response (Dienz and Rincon, 2009; Zhang et al., 2008). The IL-6 has an important role in atherosclerotic plaque development and plaque destabilization. In addition, the IL-6 exert several detrimental effects that augment atherogenesis, and promotes endothelial dysfunction, smooth muscle cell proliferation and migration as well as recruitment and activation of inflammatory cells, thereby perpetuating vascular inflammation (Schuett et al., 2009). On the other hand, TGF-β1 is pleiotropic cytokine, which has been demonstrated to regulate a wide array of biological processes. It plays a major role in the regulation of vascular function and homeostasis. In general, TGF-β1 is considered as an anti-inflammatory
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cytokine in the vessel wall. In normal vessel, TGF-β1 inhibits endothelial and vascular smooth muscle cell proliferation (Frangogiannis, 2014; Lan et al., 2013). In the atherosclerosis, TGF-β1 is considered to be antiatherogenic factor, specifically in the early stages of the disease. Thus, TGF-β1 inhibits excessive vascular smooth muscle cell accumulation in the neointima (Redondo et al., 2012). IL-6 and TGF-β1 polymorphisms have been associated with the risk of developing atherosclerosis, myocardial infarction, blood pressure, acute coronary syndrome, and restenosis (Cambien et al., 1996; Funayama et al., 2006; Gao et al., 2013; Hojo et al., 2000; Kurihara et al., 1989; Li et al., 1999; Qi et al., 2005; Tanaka et al., 2005). Considering the prominent role of these cytokines, the objective of this study was to establish the role of the IL-6 and TGF-β1 gene polymorphisms in the risk of developing restenosis after coronary stent placement in a group of Mexican patients. 2. Materials and methods 2.1. Subjects The study included 244 Mexican Mestizo patients with symptomatic coronary artery disease who underwent coronary stent implantation at our institutions and went to follow-up coronary angiography because of symptoms of ischemia documented in a myocardial perfusion imaging test. Basal and procedure coronary angiographies were analyzed for angiographic predictors of restenosis, and follow-up angiography was performed to screen for binary restenosis. Using a N50% stenosis at followup (50% reduction in the luminal diameter of the stenosis compared with the coronary angiography findings immediately following angioplasty) as the criterion to define restenosis, there were 78 patients with restenosis and 166 without restenosis. All subjects were ethnically matched, and we considered as Mexican Mestizos only those individuals who for three generations, including their own, had been born in Mexico. The Institutional Ethics and Research Committee approved the study, and all subjects signed informed consent. 2.2. Genetic analysis The DNA extraction method proposed by Lahiri and Nurnberger was used for DNA isolation (Lahiri and Nurnberger, 1991). The IL-6 − 572 GNC (rs1800796), IL-6 − 1426 TNG (rs2069827), TGF-β1 − 509 TNC (rs1800469), and TGF-β1 T29C (rs1800470) single nucleotide polymorphisms were genotyped using 5′ exonuclease TaqMan genotyping assays on an ABI Prism 7900HT Fast Real-Time PCR system, according to the 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-Identifiler Kit. PCR amplification was using 1 ng of DNA according to the manufacturer's protocol. PCR products were analyzed on the ABI Prism 3100 Genetic Analyzer using GeneMapper 4.0 software (Life Technologies).
2.5. Statistical analysis Gene frequencies of IL-6 and TGF-β1 polymorphisms were obtained by direct counting. Hardy–Weinberg equilibrium was evaluated by χ2 test. All calculations were performed using SPSS version 18.0 (SPSS, Chicago, IL) statistical package. Chi-square tests were used to compare frequencies and ANOVA and Student's t-test were used to compare means. Logistic regression analysis was performed to look for associations of polymorphisms with restenosis using the following models: codominant (major allele homozygotes vs. heterozygotes and major allele homozygotes vs. minor allele homozygotes), dominant (major allele homozygotes vs. heterozygotes + minor allele homozygotes), recessive (major allele homozygotes + heterozygotes vs. minor allele homozygotes), heterozygous (homozygote for the minor allele + homozygote for the major allele vs heterozygote) and log-additive (major allele homozygotes vs. heterozygotes vs. minor allele homozygotes). The inheritance models were adjusted for clinical and angiographic variables. SNPstats (http://bioinfo.iconcologia.net/custom.php) software for Windows® were used to analyze the genetic frequencies and to evaluate the linkage disequilibrium (LD, D′) among polymorphisms, as well 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 T29C polymorphism. FastSNP analyzes the location of the SNP (5′ upstream, 3′ untranslated region, intronic) and possible functional effects such as amino acid changes in protein structures, transcription factor binding sites in promoter or intronic enhancer regions, and alternative splicing regulation by disrupting exonic splicing enhancers or silencers (Yuan et al., 2006). TFSEARCH is made up of three databases: transcription factor binding sites (TRANSFAC), transcription regulatory database (TRRD), and compound regulatory elements (COMPEL). They provide possible transcription factor effects and their binding sites, splicing regulation, and structural and functional properties of composite elements to 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 and without restenosis included in the study are shown in Table 1. As expected, patients who underwent coronary BMS implantation develop more restenosis (72.0%) than those patients who underwent DES implantation (28.0%) (P b 0.001). Also, those patients treated with stents with a diameter b2.5 mm presented more restenosis (P = 0.026). As expected, admixture estimates obtained by the maximum likelihood method revealed a greater Amerindian (49.1%) and European contribution (43.7%) in our Mexican Admixed patients. The African component was lower than 1% in our groups. These admixture estimations confirm that our study group has the genetic characteristics of Mexican Admixed individuals. 3.2. Allele and genotype frequencies
2.4. Admixture estimations using STR markers Individual and group ancestry estimations were calculated in the groups of patients and controls. The individual and group admixture estimations were calculated using the STR frequencies in a trihybrid model with the software Structure version 2.3.4. The STR data from three parental populations (Amerindians, Spaniards and Africans) were obtained from previous published databases (Barrot et al., 2005; Calzada et al., 2005; Sanz et al., 2001).
Observed and expected frequencies of the studied polymorphisms were in Hardy–Weinberg equilibrium. The estimated risk analysis of these polymorphisms in patients with and without restenosis was tested according to five models: co-dominant, dominant, recessive, heterozygous and additive. Under dominant and additive models, the TGF-β1 T29C (rs1800470) polymorphism was significantly associated with an increased 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,
J.M. Fragoso et al. / Experimental and Molecular Pathology 98 (2015) 13–17
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Table 1 Clinical and angiographic characteristics of the studied individuals.
Mena Hypertensiona Type II diabetes mellitusa Hypercholesterolemiaa Smokinga Unstable anginaa Stable anginaa Statin therapya DESa BSMa Diameter smaller b 2.5 mm Stent lengtha (b 22 mm) Bifurcationa Age (years)
With restenosis (n (%))
Without restenosis (n (%))
OR
CI (95%)
P
61 (78) 49 (63) 32 (41) 42 (54) 49 (63) 27 (35) 9 (12) 65 (83) 22 (28) 56 (72) 24 (31) 32 (41) 22 (28) Median (percentile 25–75) 59.9 (54–67)
131 (79) 89 (54) 60 (36) 99 (60) 104 (63) 45 (27) 37 (22) 142 (86) 100 (60) 66 (40) 32 (19) 75 (45) 41 (25) Median (percentile 25–75) 58.6 (53–65)
– – – – – – 0.45 – 0.25 3.85 1.86 – –
– – – – – – 0.20–0.99 – 0.14–0.46 2.15–6.90 0.99–3.44 – –
NS NS NS NS NS NS 0.045 NS b00001 b00001 0.026 NS NS
–
–
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.
95% CI: 1.09–2.45, PAdd = 0.016). Models were adjusted for the variables that were significant in the univariate analysis (hypertension, stable angina, stent used and diameter of stent) (Table 2). 3.3. Linkage disequilibrium analysis
allele of the TGF-β1 T29C (rs1800470) polymorphism is associated with an exonic splicing enhancer that produces a binding site for SF2/ ASF proteins.
4. Discussion
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 of developing restenosis after adjusting for hypertension, presence of stable 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 and TFSEARCH bioinformatics tools showed that the presence of the “T”
In our study, two IL-6 [rs1800796 (−572 GNC), rs2069827 (−1426 T NG)] and two TGF-β1 [rs1800469 (− 509 TNC), rs1800470 (T29C)] polymorphisms were determined in a group of patients who underwent coronary stent implantation in order to establish its role in the genetic susceptibility to developing restenosis. Similar distribution of IL-6 polymorphisms was observed in patients with and without restenosis, whereas, the TGF-β1 T29C (rs1800470) polymorphism was associated with 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 role in mediating the acute inflammatory process (Aukrust et al., 2008) and are involved in the restenosis phenomenon in patients with stent implantation. Sardella et al. reported that IL-6 levels were significantly
Table 2 Association of the IL-6 and TGF-B1 polymorphisms with restenosis. Genotype frequency n (%) IL-6 −572 CNG rs1800796 Without restenosis (n = 166)
With restenosis (n = 78) TGF-B1 −509 TNC rs1800469 Without restenosis (n = 166)
With restenosis (n = 78)
TGF-B T29C rs1800470 Without restenosis (n = 166)
With restenosis (n = 78)
MAF
Model
OR (95% CI)
P
Co-dominant Dominant Recessive Heterozygous Log-additive
1.57 (0.53–4.66) 1.24 (0.69–2.21) 1.44 (0.51–4.07) 1.11 (0.62–1.97) 1.22 (0.77–1.94)
0.68 0.48 0.50 0.73 0.39
Co-dominant Dominant Recessive Heterozygous Log-additive
0.92 (0.43–1.98) 0.95 (0.52–1.74) 0.95 (0.50–1.78) 1.00 (0.58–1.71) 0.96 (0.66–1.41)
0.98 0.87 0.86 0.99 0.83
Co-dominant Dominant Recessive Heterozygous Log-additive
2.69 (1.19–6.08) 2.06 (1.03–4.11) 1.87 (0.98–3.58) 1.06 (0.59–1.90) 1.64 (1.09–2.45)
0.053 0.034 0.06 0.84 0.016
GG 78 (0.469)
GC 77 (0.463)
CC 11 (0.066)
0.300
32 (0.410)
39 (0.500)
7 (0.089)
0.340
CC 43 (0.259)
CT 83 (0.500)
TT 40 (0.240)
0.491
21 (0.269)
39 (0.500)
18 (0.230)
0.475
CC 58 (0.349)
CT 74 (0.446)
TT 34 (0.204)
0.428
15 (0.192)
36 (0.461)
27 (0.346)
0.576
MAF = minor allele frequency, OR = odds ratio, CI = confidence intervals, P = P value. The P values were calculated from logistic regression analysis and the ORs were adjusted for blood pressure, stable angina, stent used and diameter of stent. Bold numbers indicate significant associations. The IL-6 −1426 TNG polymorphism is not shown in the table because the TT genotypes were not detected.
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Table 3 Frequencies (%) of TGF-B1 haplotypes [rs1800469 (−509 TNC) and rs1800470 (T29C)] in patients with and without restenosis.
Haplotype H1 (TC) H2 (CT) H3 (CC) H4 (TT)
With restenosis (n = 78)
Without restenosis (n = 166)
Hf 0.332 0.428 0.092 0.149
Hf 0.413 0.349 0.160 0.078
P
OR
95% CIa
NS NS NS 0.041
– – – 2.03
– – – 1.03–3.98
Hf = haplotype frequency, ACS = acute coronary syndrome, P = P value, OR = odds ratio, CI = confidence intervals. The order of the polymorphisms in the haplotypes is according to the positions in the chromosome (rs1800469 and rs1800470). a The ORs were adjusted for hypertension, presence of stable angina, stent used, and diameter of stent.
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 peripheral blood of patients with chronic stable angina, measured 4 weeks after coronary angioplasty with stent implantation (Kazmierczak et al., 2014). According to Exner et al. (2004), the − 174 G/C polymorphism of the IL-6 gene is associated with the occurrence of restenosis after percutaneous transluminal angioplasty. Recently, another IL-6 polymorphism was associated with increased levels of IL-6 in patients with bare metal stent implantation (Gao et al., 2013). In this case, the polymorphism was not associated with the risk of developing restenosis in these patients, a result that agrees with our study. On the other hand, the TGF-β1 has been shown to be involved in restenosis, recruiting mesenchymal stem cells to the injured tissue, affecting lesion repair (Suwanabol et al., 2011). Polymorphisms in this gene have been associated with the risk of developing cardiovascular diseases (Cruz et al., 2013; Fragoso et al., 2012), but no studies in restenosis have been reported. In our study the TGF-β1 T29C T allele was associated with the risk of developing restenosis after coronary stent implantation. This allele was previously associated with the risk of developing silent myocardial ischemia (Cruz et al., 2013), and acute coronary syndrome (Fragoso et al., 2012) in Mexican individuals. In addition, in agreement with our data, Yokota et al. reported an association of the T allele of the T29C polymorphism with the risk of myocardial infarction in a Japanese population (Yokota et al., 2000). The functional software used here predicted that TGF-β1 T29C (rs1800470) polymorphism is functional. This analysis showed that the 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 proteins that regulate alternative splicing (Sureau et al., 2001) suggesting that the TGF-β1 T29C polymorphism could have a functional effect. The functional effect of the polymorphism detected here should be taken with care because it is only an informatics approach and need to be confirmed with experimental testing. In the linkage disequilibrium analysis and after adjusting for hypertension, presence of stable angina, stent used and diameter of stent, one TGF-β1 risk (TT) haplotype for developing restenosis was detected. Crobu et al., studied three TGF-β1 polymorphisms and reported the association of a haplotype named “GTC” with the risk of developing myocardial infarction (Crobu et al., 2008). This haplotype includes the two polymorphisms analyzed in our study (−509 TNC and T29C). Recent experimental and clinical results support the concept of a protective role of TGF-β1 in the circulatory system, even though some articles indicate its proinflammatory features. Thus, individuals with the risk haplotype (TT) could produce less TGF-β1 with consequent decreasing of its anti-inflammatory effects and increasing the susceptibility to restenosis in these individuals. In summary, our data suggest that the TGF-β1 T29C (rs1800470) polymorphism plays an important role in the risk of developing restenosis. In addition, in our study, it was possible to distinguish one TGF-
β1 risk haplotype for the development of restenosis. Our data are preliminary due to the study sample size and additional studies in a larger number of individuals and in other populations could help define the true role of these polymorphisms as risk factors for developing restenosis. Conflict of interest statement No competing financial interests exist. Acknowledgments This work was supported in part by grants from the Consejo Nacional de Ciencia y Tecnología (project number 182962). The authors are grateful to the study participants. Institutional Review Board approval was obtained for all sample collections. References Abeywardena, M.Y., Leifert, W.R., Warnes, K.E., et al., 2009. Cardiovascular biology of interleukin-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. 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