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Introduction
In the past decade, the prevalence of restenosis worldwide has increased considerably, due to that intracoronary stenting as a strategy for treating coronary artery disease (CAD), promotes thrombosis and restenosis in a percentage of patients (12-32%) often leading to device failure1-4. Clinically, the in-stent restenosis is characterized by delayed recurrence of signs and symptoms of cardiac ischemic after angioplasty. In-stent restenosis is a result of stent-induced mechanical injury, which involves two main processes; the first process is the vascular remodeling, and neointimal hyperplasia, which involves smooth muscle migration/proliferation and extracellular matrix deposition, a phenomenon that may provoke coronary artery re-occlusion through an inflammatory and proliferative response against the foreign body1-4.
Recent studies have shown that the dipeptidyl peptidase 4 protein (DPP4) plays an important role in glucose, lipids metabolism, vascular remodeling, and with inflammatory process regulation5-9. In this context, experimental studies have shown that DPP4 overexpression has an important role in vascular remodeling by the linkage with collagen I, collagen III, and fibronectin through the DPP4 C-terminal noncatalytic segment10,11. In addition, DPP4 co-stimulates T cells by binding to adenosine deaminase (ADA) through its extracellular domain, pro-inflammatory interleukins regulating such as IL-6, IL-12, and TNF-alpha12-15. DPP4 is encoded for the DPP4 gene located in chromosome 2 (2q24.3)8 and is preceded by a promoter region controlled by transcription factors such as the nuclear-factor kappa-light-chain enhancer of activated B cells (NF-kB), and the signal transducer and activator of transcription-alpha (STAT1α), both involved in cell growth, proliferation, and immune response16,17. Recent studies have shown that the same polymorphisms such as rs12617336 C/G, rs12617656 C/T, rs1558957 C/T, rs3788979 C/T, and rs17574 G/A are associated with the presence of the hypoalphalipoproteinemia, coronary artery stenosis, CAD, T2DM, myocardial infarction, obesity, and premature CAD18-25.
Based on this data, we propose that DPP4 gene polymorphisms may be associated with the prevalence of in-stent restenosis. To explore this possibility, the present study aimed to establish whether the DPP4 rs12617336 C/G, rs12617656 C/T, rs1558957 C/T, rs3788979 C/T, and rs17574 G/A polymorphisms are associated with prevalence of in-stent restenosis.
Materials and methods
Study subjects
Our study included 190 patients with CAD (60 patients restenosis, and 130 patients without restenosis). The sample size was calculated to one cohort study matched, using as parameters a power of 80% and an alpha level of 0.05. According to this calculation 154 individuals are required to carry out this study (http://www.openepi.com/SampleSize/SSCohort.html [accessed on 17 April 2023]). This study included 190 patients who underwent coronary stent implantation, previously diagnosed with CAD, in accordance with the European Society of Cardiology and American College of Cardiology guidelines26,27. After 6 months, 190 patients went to a follow-up coronary angiography, to evaluate whether they presented angiographic predictors of restenosis such as a significant narrowing of the coronary lumen in the area of previously stented lesion, as well as signs and symptoms of cardiac ischemia after angioplasty4. The follow-up coronary angiography revealed that 60 patients developed restenosis, and 130 patients did not develop restenosis of the 190 patients. Restenosis is commonly defined as a binary event with a renarrowing of ≥ 50% of the vessel diameter. The term "binary" indicates that patients are allocated to one of two groups: either the group with narrowing ≥ 50% or the group with narrowing < 50%4. All the included subjects were ethnically matched and considered Mexican mestizos only if they and their ancestors (at last three generations) had been born in the country. The study was approved by the Ethics and Research Committee of our institution, under project number: 23-1361, complying with the Declaration of Helsinki. In addition, written informed consent was obtained from all individuals enrolled in the study.
Laboratory analysis
Plasma concentrations of cholesterol and triglycerides were performed using enzymatic/colorimetric assays (Randox Laboratories, Crumlin, UK). High-density lipoprotein-cholesterol (HDL-C) concentrations were determined by the phosphotungstic acid-Mg2+ method. LDL-C concentrations were calculated using Friedewald’s formula in samples with triglyceride concentrations lower than 400 mg/dL28. Dyslipidemia was defined as increased plasma levels of one or more of the lipid profile parameters (total cholesterol > 200 mg/dL, LDL-C > 130 mg/dL, HDL-C < 40 mg/dl, or triglycerides > 150 mg/dL), according to the National Cholesterol Education Project Adult Treatment Panel III guidelines (NCEP-ATP III) [https://www.nhlbi.nih.gov/resources/third-report-expert-panel-detection-evaluation-and-treatment-high-blood-cholesterol-0 (accessed on May 2, 2023)]. Systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg values, or the use of oral antihypertensive therapy were considered to establish the hypertension presence.
Genetic analysis
DNA was obtained from total blood samples29. The location of the rs12617336 C/G, rs12617656 C/T, rs1558957 C/T, rs3788979 C/T, and rs17574 G/A polymorphisms included in this study are shown in table 1. The determination of the DPP4 polymorphisms were performed using 5’ exonuclease TaqMan genotyping assays on a QuantStudio 12K Flex Real-Time PCR system using a replaceable 384-well block in accordance with manufacturer’s instructions (Applied Biosystems, Foster City, USA). As a quality control, 10% of the samples were genotyped twice; results were concordant for all cases.
Table 1 Information of the studied polymorphism tested
| Genea symbol | dbSNPa | Chromosome (NCBI Build 156)* | Position (NCBI Build 156)* | Change base | Location in gene* |
|---|---|---|---|---|---|
| DPP4 | rs12617336 | 2q24.3 | 161992802 | C>G | 3’- UTR |
| DPP4 | rs12617656 | 2q24.3 | 161994637 | C>T | Intron 1 |
| DPP4 | rs1558957 | 2q24.3 | 162033665 | T>C | Intron 14 |
| DPP4 | rs3788979 | 2q24.3 | 162044379 | T>C | Intron 22 |
| DPP4 | rs17574 | 2q24.3 | 162929979 | G>A | Exon 26 |
*The chromosomal location for each SNP table was obtained by querying each SNP "rs" number in the NCBI Single Nucleotide Polymorphism database (dbSNP), build 156 on the 21st of September 2022 (http://www.ncbi.nlm.nih.gov/projects/SNP/).
Statistical analysis
Allelic and genetic distributions of DPP4 polymorphisms in patients with or without restenosis were obtained by direct counting. The HardyWeinberg equilibrium was evaluated in both groups by chi-squared test. Data analysis was performed with SPSS program version 18.0 (IBM, Chicago, Il). The MannWhitney U or Student’s t-test was used to compare the continuous variables. In addition, categorical variables were analyzed with Chi-squared or Fisher’s exact test. The association of the rs12617336 C/G, rs12617656 C/T, rs1558957 C/T, rs3788979 C/T, and rs17574 G/A polymorphisms with prevalence of in-stent restenosis was determined by the following inheritance models: additive, codominant, dominant, heterozygous, and recessive30,31. These analyses were adjusted for cardiovascular risk factors using logistic regression to determine whether the presence of the genetic variant was associated with the prevalence of the disease. The p values were corrected by the Bonferroni test based on the number of SNPs. The values of p < 0.01 were considered statistically significant, and all odds ratios (OR) were presented as 95% confidence intervals. The statistical power of the association of the DPP4 gene polymorphisms in patients with restenosis was 0.80 calculated by the OpenEpi software (http://www.openepi.com/SampleSize/SSCC.htm [accessed on 17 June 2022]).
Haplotypes and linkage disequilibrium analyses (LD, D’) were performed by Haploview version 4.1 software (Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA). This software uses the Human Haplotype Map project data to determine the combination of alleles in a single gene, or alleles in multiple genes along a chromosome that tend to be inherited together due to their chromosomal proximity, providing statistical values of LD, D’, logarithm of the odds (LOD) and r squared32.
Results
Characteristics of the study population
The clinical and angiographic analysis of the patients with and without restenosis is presented in table 2. This analysis showed that the patients with restenosis present significant differences in concentrations of total cholesterol and HDL-C (p < 0.05) when compared with patients without restenosis, despite statin therapy. In addition, restenosis was more frequent in patients who underwent bare coronary stenting (BSM) with diameter less than or equal to 2.5 mm than in those who underwent drug-eluting stenting (DES) (75%, and 25%, respectively) (p ≤ 0.001). On the other hand, the stable angina in the patients with restenosis was less (9%) when compared to patients without restenosis (23%). Finally, in the patients with restenosis the unstable angina, as well as the stent diameter less than or equal to 2.5 mm were more frequent (41%, and 32%, respectively), than in patients without restenosis (27%, and 18%, respectively).
Table 2 Baseline clinical and angiographic characteristics of the patients with and without restenosis
| Clinical characteristics | With restenosis (n = 60) | Without restenosis (n = 130) | p |
|---|---|---|---|
| Gender, n (%) | |||
| Male | 46 (77) | 105 (80) | 0.647 |
| Female | 14 (23) | 25 (20) | |
| Age (years) | 61.9 ± 10.62 | 63.3 ± 12.49 | 0.453 |
| BMI (kg/m2) | 27.1 ± 4.26 | 28.4 ± 3.9 | 0.039 |
| Blood pressure (mmHg) | |||
| Systolic | 127.1 ± 19.3 | 122.8 ± 16.6 | 0.117 |
| Diastolic | 78.5 ± 11.5 | 75.7 ± 10.6 | 0.101 |
| Total cholesterol (mg/dl) | 150 (114-181.2) | 167 (136-200) | 0.003 |
| HDL-C (mg/dl) | 41 (36-49) | 38 (33-49.7) | 0.029 |
| LDL-C (mg/dl) | 103 (66-127.7) | 103 (70-139.5) | 0.559 |
| Triglycerides (mg/dl) | 156 (110-207.7) | 169 (128.5-210.7) | 0.253 |
| Hypertension, n (%) | |||
| Yes | 30 (50) | 55 (42) | 0.321 |
| Type II diabetes mellitus, n (%) | |||
| Yes | 39 (65) | 90 (69) | 0.895 |
| Smoking, n (%) | |||
| Yes | 37 (61) | 83 (64) | 0.463 |
| Unstable angina, n (%) | |||
| Yes | 25 (41) | 35 (27) | 0.042 |
| Stable angina, n (%) | |||
| Yes | 5 (9) | 30 (23) | 0.014 |
| Statin therapy, n (%) | |||
| Yes | 48 (80) | 110 (85) | 0.820 |
| DES, n (%) | |||
| Yes | 15 (25) | 79 (61) | < 0.0001 |
| BSM, n (%) | |||
| Yes | 45 (75) | 51 (39) | < 0.0001 |
| Stent diameter ≤ 2.5 mm, n (%) | |||
| Yes | 19 (32) | 24 (18) | 0.043 |
| Stent length < 20 mm, n (%) | |||
| Yes | 25 (42) | 56 (43) | 0.855 |
Data such as gender, hypertension, type 2 diabetes mellitus, smoking, unstable angina, stable angina, statin therapy, bare metal stent, drug-eluting stent, stent diameter, and stent length are expressed as n (frequency), and p values were calculated to Chi-square. Other variables are expressed as mean ± SD or median [25th-75th interquartile interval] for normally or non-normally distributed variables and groups were compared by t-test or Mann-Whitney U-test, respectively. BMS: bare metal stent; DES: drug-eluting stent.
Association of DPP4 polymorphisms with restenosis
Patients with or without restenosis were in Hardy-Weinberg equilibrium. In the first analysis, the rs12617336 C/G, rs1558957 C/T, rs3788979 C/T, and rs17574 G/A polymorphisms showed a genetic distribution similar between patients with and without restenosis. Nonetheless, the rs12617656 CC genotype frequency of the rs12617656 C/T SNP showed significant differences between patients with or without restenosis (Supplementary Table 1). In addition, when we analyzed the rs12617656 C/T SNP under the different inheritance models, adjusted by age, gender, BMI, total cholesterol, HDL-C, as well as with the angiographic characteristics, such as unstable angina, drug-eluting stent (DES), bare metal stent (BMS), and stent diameter. We observed that the CC genotype maintains the associated risk of development restenosis after coronary stent, under the co-dominant and recessive models (OR = 3.32, 95% CI: 1.15-9.62, PCo-Dominant = 0.0009, and OR = 4.96, 95%CI: 1.89-13.01, PRecessive = 0.0008, respectively) (Table 3). On the other hand, we observed that CT genotype was associated with protection against development restenosis after coronary stent under heterozygous model (OR = 0.32, 95%CI: 0.15-0.69, PHeterozygous = 0.0027) (Table 3).
Table 3 The inheritance models analysis of DPP4 rs12617656 C/T polymorphisms in patients with or without restenosis
| SNP | Model | Genotype | With restenosis n = 60 (n (%)) | Without restenosis n = 130 (n (%)) | OR (95%CI) | p |
|---|---|---|---|---|---|---|
| rs12617656 C/T | Co-dominant | TT | 20 (0.333) | 46 (0.354) | 3.32 (1.15-9.62) | 0.0009 |
| TC | 23 (0.383) | 69 (0.531) | ||||
| CC | 17 (0.283) | 15 (0.115) | ||||
| Dominant | TT | 20 (0.333) | 46 (0.354) | 0.86 (0.41-1.84) | 0.698 | |
| TC+CC | 40 (0.667) | 84 (0.646) | ||||
| Recessive | TT+TC | 43 (0.717) | 115 (0.885) | 4.96 | 0.0008 | |
| CC | 17 (0.283) | 15 (0.115) | (1.89-13.01) | |||
| Heterozygous | TT+CC | 37 (0.617) | 61 (0.469) | 0.32 (0.15-0.69) | 0.0027 | |
| CT | 23 (0.383) | 69 (0.531) | ||||
| Additive | - | - | - | 1.52 (0.91-2.57) | 0.109 |
The p-values were calculated by the logistic regression analysis, and ORs were adjusted for BMI, total cholesterol, HDL-C, as well as with the angiographic characteristics, such as unstable angina. DES: drug-eluting stent; BMS: bare metal stent, and stent diameter; SNP: single nucleotide polymorphism; OR: odds ratio; CI: confidence interval; pC: p-Corrected.
Haplotype analysis
As shown in figure 1, strong linkage disequilibrium was noted among the rs12617656 C/T, rs1558957 C/T, and rs3788979 C/T polymorphisms in restenosis (D’ > 0.90). In this context, haplotype analyses were performed among the aforementioned three SNPs due to the rs12617336 C/G, and rs17574 G/A SNPs were not informative according to data obtained from Haploview version 4.1 software. As shown in table 4, the analysis exhibited one haplotype (CTC) out of five with significant differences between patients with and without restenosis. The CTC haplotype proved to be more common in patients with restenosis (4.3%) than in those without the condition (0.2%), suggesting that this haplotype could be associated with the risk of development restenosis after coronary stent (p = 0.006).
Table 4 Distribution of the haplotypes between the rs12617656 C/T, rs1558957 C/T, and rs3788979 C/T polymorphisms of the DPP4 gene in the study groups
| Haplotype | With restenosis (n = 60) | Without restenosis (n = 129) | p | ||
|---|---|---|---|---|---|
| rs12617656 C/T | rs1558957 C/T | rs3788979 C/T | Hf | Hf | |
| T | T | C | (0.382) | (0.456) | 0.174 |
| C | C | T | (0.291) | (0.284) | 0.887 |
| T | C | C | (0.126) | (0.162) | 0.353 |
| C | C | C | (0.141) | (0.095) | 0.188 |
| C | T | C | (0.043) | (0.002) | 0.006 |
The polymorphism order in the haplotypes is according to the positions in the chromosome (rs12617656rs1558957-rs3788979 chromosome 2q24.3). Hf: haplotype frequency.
Discussion
In this study, five DPP4 gene polymorphisms (rs12617336 C/G, rs12617656 C/T, rs1558957 C/T, rs3788979 C/T, and rs17574 G/A) were analyzed to establish whether they are involved in the risk of developing in-stent restenosis. In this study, the results show that the CC genotype of the rs12617656 C/T polymorphism located in the intron 1, as well as the CTC haplotype conformed by rs12617656 C/T, rs1558957 C/T, and rs3788979 C/T polymorphisms located in the introns 1, 14 and 22, respectively, confer a high risk of developing in-stent restenosis. To the best of our knowledge, our work is one of few studies that describe the association of the rs12617656 C/T polymorphism with in-stent restenosis. In this context, Ahmed et al. reported that rs12617656 C allele is associated with type 2 diabetes mellitus in Malaysian population7. Nonetheless, controversial with these data, prior studies have shown that the T allele of the rs12617656 C/T polymorphism plays an important role in the risk of development of arthritis rheumatoid in the Asian population33,34. On the other hand, recent studies have shown that SNPs such as rs12617336 C/G, rs17574 G/A, and rs3788979 C/T included in our study play an important role in the development of hypoalphalipoproteinemia, premature CAD in diabetic patients, coronary artery stenosis and T2DM in different populations, but not the rs12617656 C/T SNP23-25. As can be seen, the association of this polymorphism and other polymorphisms of the DPP4 gene with cardiovascular diseases and other pathology in different populations is scarce and controversial. Finally, we also determined that the "CTC" haplotype conformed by the rs12617656 C/T, rs1558957 C/T, and rs3788979 C/T polymorphisms, was associated with an increased risk of developing in-stent restenosis, and a high probably of this block may be segregate together (D’ > 0.90). Nonetheless, as far as we know, there are no studies that showed a haplotype similar with reported in our study. On the other hand, we considered that the association of the rs12617656 C/T polymorphism with in-stent restenosis could be due to the allelic distribution of these polymorphisms, which varies according to the ethnic origin of the study populations. In this sense, we reported that the frequency of the rs12617656 C allele in the Mexican mestizos without restenosis was 38% (Supplementary Table 1). According, to data obtained from NCBI (National Center for Biotechnology Information) (https://www.ensembl.org/index.html [accessed on 17 September 2022]), the distribution of the rs12617656 C allele in our population, as well as in the individuals from Los Angeles with Mexican ancestry, Caucasians and Africans was similar (32.8%, 34.6%, and 36.7%, respectively), but low when compared with Asians (65.3%). Considering our results and the different distribution of the rs12617656 C/T polymorphism, we propose that additional studies such as genome-wide association studies, exome sequencing studies, and recently "exome chip" in other populations with different ethnic origins could help define the true role of this polymorphism as markers of risk of developing restenosis.
On the other hand, recent studies have shown the effect of the DPP4 gene polymorphisms with levels of soluble DPP4, as well as with the plasma lipid concentrations7,19,23-25. For example, Vargas-Alarcón et al. reported that the rs17574 GG genotype is associated with the lowest sDPP4 levels in patients with hypoalphalipoproteinemia and premature CAD with T2DM23,24. Aghili et al., reported that rs3788979 CC genotype decreased plasma sDPP4 level in patients with myocardial infarction19. In the same way, Wang et al, reported that the rs3788979 CC genotype is associated with a reduced ApoB level, a protein that is involved in lipid metabolism, being the main protein constituent of very low-density lipoproteins such as VLDL, and LDL-C25. Controversial with Ahmed et al, who report that the rs4664443 AA genotype is associated with higher serum sDPP4 levels in patients with T2DM20. However, as far as we know, experimental studies about rs12617656 C/T polymorphism with sDPP4 levels are inexistent. In addition, the correlation between the DPP4 gene polymorphisms and concentrations of the sDPP4 is controversial with positive and negative results. However, it has been established that high of soluble sDPP4 levels inactivate glucagon-like peptide-1 (GLP-1) a hormone that regulates glucose metabolisms that could be causing insulin resistance, and hyperinsulinemia that leads to developing T2DM5,35. In addition, it has been reported that increased DPP4 mRNA levels in the liver of patients with non-alcoholic fatty liver disease correlate with insulin resistance36. On the other hand, DPP4 exerts function as co-stimulating of T cells through interaction with ADA, modulating the function of antigen-presenting cells37. These data suggest that sDPP4 could have an important role in the inflammatory process present in T2DM, hypoalphalipoproteinemia, premature CAD in T2DM, insulin resistance, myocardial infarction, and dyslipidemias. In this context, we considered that more studies as these could help to understand the contribution of the rs12617656 C/T polymorphism in the sDPP4 levels, and the risk of developing in-stent restenosis, or some other cardiovascular disease.
Limitations
We recognize that our study has limitations: (a) The limited size of the samples in the group study, as well as the lack of replication in other populations. (b) The sDPP4 levels were not measured in the patients with or without restenosis. Nonetheless, despite these limitations, our study suggests that the rs12617656 C/T polymorphism has a role in the development of in-stent restenosis.
Conclusion
In summary, our findings showed that the DPP4 rs12617656 C/T polymorphism, as well as the haplotype (CTC) composed by the DPP4 rs12617656 C/T, DPP4 rs1558957 C/T, and DPP4 rs3788979 T/C polymorphisms, is associated with risk of developing in-stent restenosis. In addition, we considered that these results in our population justify the design of additional studies with a larger number of individuals from other ethnic origins to confirm the true role of this polymorphism as a marker of risk in-stent restenosis.
