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Vascular and Interventional Radiology |
1 From the Departments of Medical and Chemical Laboratory Diagnostics (M.E., C.M., O.W.) and Angiology (M.S., E.M., W.M., S.S., G.E., M.R.), University of Vienna Medical School, Waehringer Guertel 18–20, A-1090 Vienna, Austria. Received May 3, 2003; revision requested July 11; final revision received September 11; accepted October 21. M.E. and M.S. supported by grant P15231 from the Fonds zur Förderung der Wissenschaftlichen Forschung. Address correspondence to O.W. (e-mail: oswald.wagner@univie.ac.at).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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MATERIALS AND METHODS: A total of 281 patients underwent PTA of the femoropopliteal artery during the study period; 23 (8%) patients had to be excluded due to missing genetic data. We studied 258 patients with intermittent claudication (n = 174) or critical limb ischemia (n = 84). The IL-6 promoter genotype was determined from venous blood samples before intervention by using a mutagenically separated polymerase chain reaction, and patients were followed up for 6 months with duplex ultrasonography for the occurrence of restenosis (
50%) after angioplasty. Multivariate Cox proportional hazards analysis was performed to assess the association between the IL-6 promoter genotype and restenosis, with adjustment for possible confounders such as atherosclerotic risk factors and angiographic covariates.
RESULTS: The 6-month restenosis rate was 26% (23 of 90) in patients with the (–174)GG genotype, 28% (33 of 117) with the (–174)GC genotype, and 43% (22 of 51) with the (–174)CC genotype (P = .044). Homozygous carriers of the (–174)C allele ([–174]CC) exhibited a 2.42-fold increased adjusted risk for restenosis (95% CI: 1.28, 4.58; P = .007) compared with homozygous (–174)G allele carriers ([–174]GG). Heterozygous carriers ([–174]GC) had no significantly increased restenosis risk (hazard ratio, 1.37; 95% CI: 0.84, 2.22; P = .21).
CONCLUSION: The IL-6 promoter polymorphism (–174)G/C seems to influence the occurrence of restenosis after PTA. Homozygous carriers of the (–174)C allele have an increased rate of intermediate-term restenosis.
© RSNA, 2004
Index terms: Arteries, femoral • Arteries, popliteal • Arteries, restenosis, 92.458, 92.721 • Arteries, transluminal angioplasty, 92.1282, 92.454 • Genes and genetics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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A functional polymorphism has been described in the interleukin (IL)-6 promoter region at nucleotide position –174, which is located 174 base pairs upstream (against thedirection of transcription) from the point in the DNA molecule at which the messenger RNA synthesis begins. This polymorphism involves an exchange of guanine (G) for cytosine (C) and is either heterozygous (–174)GC or homozygous (–174)CC. The (–174)G/C polymorphism modulates the cytokine expression as follows: Homozygous carriers of the (–174)C allele exhibit higher IL-6 levels in response to vascular injury compared with heterozygous (–174)G/C carriers and homozygous (–174)G carriers (17). IL-6 represents a key factor in the vascular inflammatory cascade after vascular injury from balloon dilation and is directly involved in the regulation of the acute-phase response (18). Results of several studies have demonstrated a close relationship between the extent of the acute-phase response and the occurrence of restenosis (1–4,8,9,18). Therefore, we hypothesized that carriers of the (–174)C allele in the IL-6 gene promoter would show an increased rate of restenosis after PTA, presumably through enhanced vascular inflammation in response to balloon injury. Thus, the aim of our study was to investigate whether there is an association between a functional polymorphism in the IL-6 gene promoter ([–174]G/C) and restenosis after PTA of the femoropopliteal artery.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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During the study period, 281 patients underwent PTA of the femoropopliteal artery and fulfilled the inclusion criteria. The median age was 72 years (interquartile range [IQR], 65–78 years), and 127 (45%) of the patients were men. In 23 (8%) of 281 patients, genetic data were unavailable because of missing blood samples (n = 21) or problems with genetic testing (n = 2), and these patients had to be excluded.
Definitions
The primary end point in the study was the occurrence of restenosis within a follow-up period of 6 months. Restenosis was defined as a 50% or greater reduction in the diameter of the dilated vessel segment, in comparison with the diameter of the proximal adjacent nondiseased vessel segment, as indicated at duplex ultrasonography (US) and confirmed at angiography. The diagnosis of peripheral artery disease was assessed with clinical evaluation, ankle-brachial index measurement, and duplex US, and was confirmed at angiography of the lower limbs in all patients. Diabetes mellitus, hyperlipidemia, and arterial hypertension were diagnosed according to previously published criteria (4). We defined primary technical success as a remaining stenosis of no more than 30% in the treated segment, as seen on the final angiogram obtained after PTA. Cases of primary successful intervention were grouped as "primary technical success without residual stenosis" or "primary technical success with residual stenosis." Residual stenosis, which indicated a suboptimal result after PTA, was categorized as a remaining diameter reduction of 10%–30% in the treated segment as measured on the final angiogram. Poor runoff was defined as either occlusion or marked stenosis of the femoral or popliteal artery distal to the treated segment and/or occlusion or marked stenosis of at least two crural arteries.
Color-coded Duplex US
The entire vessel segment from the iliac bifurcation to the origin of the anterior tibial artery was visualized at all baseline and follow-up duplex US investigations (XP128; Acuson, Mountain View, Calif). The exact extent and location of the treated lesions were documented during the baseline evaluation as follows: The distance from the femoral bifurcation to the proximal beginning of luminal diameter reduction and the length of the target lesion were determined and plotted on a graph. The peak systolic velocity in the target lesion was then determined and compared with the peak systolic velocity in the preceding normal segment. A focal increase in peak systolic velocity of at least 140% (corresponding to at least 2.4 times the normal peak systolic velocity) was considered indicative of a stenosis of 50% or more at that site (19). Duplex US investigations were performed by a medical technician; supervision of the investigations and interpretation of the images were performed by one of the authors (E.M.) (4).
Patient Data
At admission, two independent observers (M.S., W.M.) recorded patient medical history, data from physical examination, routine laboratory findings, and current medication regimen by means of a standardized questionnaire (4). Data were checked for interobserver agreement on the day of patient discharge; in case of discrepancy, the patient was reevaluated by both investigators in consensus.
Interventions
Peripheral angiography and PTA were performed by using a previously published protocol that featured transfemoral puncture and administration of 5,000 IU of heparin intraarterially (4). After the location, grade, and length of the stenosis or occlusion were documented, PTA was performed with balloon dilation to a diameter that corresponded to the diameter of the proximal nondiseased vessel segment. We used a nonionic low-osmolality contrast agent (Optiray 320; Mallinckrodt Medical, St Louis, Mo). Angiograms were interpreted by one of the authors (E.M.) immediately after PTA. Color-coded duplex US was performed and ankle-brachial index measurements were obtained 24 hours after PTA to document primary technical success or early reocclusion. Periprocedural and postprocedural complications were recorded by one of the authors other than the interventionist (W.M.). All patients received antithrombotic medication of 100 mg of acetylsalicylic acid daily.
IL-6 Genotype Assessment
Genomic DNA was isolated from whole blood collected 1 day before PTA by using a DNA isolation kit (Puregene; Gentra Systems, Minneapolis, Minn). To identify the IL-6 promoter polymorphism, a mutagenically separated polymerase chain reaction was used as described by Endler et al (20). However, blood samples from the treated vascular segment before and after PTA were not available for assessment of local IL-6 levels.
Follow-up for Restenosis
Patients were scheduled to undergo routine follow-up investigations at the outpatient clinic at 3 and 6 months after PTA. However, patients were advised to visit the outpatient clinic at any new onset of claudication or increase in symptoms. The occurrence of restenosis was analyzed by means of ankle-brachial index measurement, evaluation of patient complaint, physical reexamination, and duplex US as described previously (16,21). Routine duplex US was performed at 6-month follow-up in 243 (94%) of 258 patients. In the remaining 15 (6%) patients, who remained asymptomatic during the follow-up period and had no deterioration in ankle-brachial index, patency of the vessel segment (freedom from restenosis) was assumed without follow-up morphologic imaging. All patients who were suspected at US of having restenosis underwent follow-up angiography; overall, 168 (65%) of 258 patients underwent follow-up angiography. Two independent observers who were blinded with regard to patients’ genotypes evaluated the follow-up data (M.S., S.S.). Data were then checked for interobserver agreement, and, if there were discrepancies, data were reevaluated by both investigators in consensus.
Statistical Analysis
A power calculation (
= .05; 1 – ß = .80) was performed to estimate the number of patients needed to detect a statistically significant difference in restenosis rates between different IL-6 genotypes. We estimated 6-month restenosis rates of 25%–30% and 40%–45% for genotypes with dominant and recessive effects, respectively, and calculated a sample size of 200–250 patients plus 10% patients with missing or incomplete data.
Continuous data are presented as the median and the IQR (range from the 25th to the 75th percentile). Discrete data are given as numbers and percentages. The 
2 test or, as appropriate, Fisher exact test was used to compare groups of categorical data. The Kruskal-Wallis test was used to compare continuous data. Cumulative patency rates (freedom from restenosis) were analyzed with the Kaplan-Meier method and compared by means of the log-rank test. We applied the Cox proportional hazards model to assess the effect of the IL-6 genotype on patency rates while adjusting for the potentially confounding effects of other variables. Variables were selected for the model if they either (a) had an established relationship with the outcome, according to the TransAtlantic InterSociety Consensus recommendations, or (b) appeared to be unequally distributed between the IL-6 genotypes, as indicated by P < .20.
We used a hierarchic modeling strategy to assess the effect of demographic variables and procedure-related variables separately and jointly. The following factors were considered for the final model to adjust for confounding effects: sex, age (tertiles), diabetes mellitus, arterial hypertension, statin therapy, critical limb ischemia, length of lesion (tertiles), residual stenosis (less than 10% vs 10%–30%), poor runoff, and recurrent stenosis after prior PTA. Furthermore, we tested for multiple interactive effects between baseline variables that were incorporated into the fully adjusted model. Results of survival analysis with the Cox proportional hazards model are given as the hazard ratio and the 95% CI. An analysis was performed to compare results between patients with missing data and patients with complete data, with respect to differences in demographic variables, clinical characteristics, and angiographic data. All P values were calculated as two sided; P < .05 was considered to denote statistical significance. Calculations were performed by using a statistical software package (SPSS for Windows, version 10.0; SPSS, Chicago, Ill).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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We included 258 patients in the final analysis. The median age of the study population was 72 years (IQR, 65–78 years), and 116 (45%) of 258 patients were men (median age of men, 72 years; IQR, 65–77 years; median age of women, 73 years; IQR, 65–78 years; P = .38). Severe lifestyle-limiting intermittent claudication was found in 174 patients; among these patients, the median maximum walking distance was 80 meters (IQR, 50–100 meters). The remaining 84 patients underwent percutaneous revascularization because of critical limb ischemia. A de novo lesion was treated in 190 (74%) of 258 patients, and 68 (26%) of 258 patients had a recurrent lesion after previous PTA. Primary technical success, defined as less than 30% remaining luminal diameter reduction in the treated segment, was achieved in 245 (95%) of 258 patients; however, 150 (61%) of these 245 patients had a 10%–30% residual stenosis. Complications were observed in 32 (12%) of 258 patients, as follows: three vessel segment perforations, 16 peripheral emboli, four arteriovenous fistulas, and nine pseudoaneurysms at the site of arterial puncture.
IL-6 Genotype
Among 258 patients, homozygous (–174)GG was found in 90 (35%) patients, heterozygous (–174)GC was found in 117 (45%) patients, and homozygous (–174)CC was found in 51 (20%) patients. Demographic data and clinical characteristics according to IL-6 genotype are shown in Table 1. Baseline variables were equally distributed between the three genotypes, with the exception of arterial hypertension and hyperlipidemia, which were less frequently found in homozygous carriers of the (–174)C allele.
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During the study period, homozygous carriers of the (–174)C allele ([–174]CC) had the highest risk for restenosis, heterozygous carriers ([–174]GC) had an intermediate risk, and homozygous (–174)G allele carriers ([–174]GG) had the lowest risk (Figure). We then applied a multivariate Cox proportional hazards model to assess the association between IL-6 genotype and restenosis risk, while adjusting for possible confounding factors (Table 2). Homozygous (–174)CC carriers had a 2.42-fold increased adjusted risk for restenosis (95% CI: 1.28, 4.58; P = .007), compared with the risk to homozygous (–174)GG carriers. Heterozygous (–174)GC carriers had no significantly increased restenosis risk (hazard ratio, 1.37; 95% CI: 0.84, 2.22; P = .21). The final model was adjusted for sex, age (tertiles), length of lesion (tertiles), use of statin therapy, and the presence or absence of the following: diabetes mellitus, arterial hypertension, critical limb ischemia, residual stenosis, poor runoff, and recurrent stenosis.
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We tested for interaction between IL-6 genotype, possible confounding variables, and restenosis. No relevant effect modifications were observed for any baseline variable (particularly age or sex); thus, relevant differences in the observed association between IL-6 genotype and restenosis in various subgroups seem unlikely.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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Although PTA is a minimally invasive procedure for revascularization, it induces a vascular injury manifested by an inflammatory response and subsequent proliferation of vascular smooth muscle cells (5,7). The production of acute-phase proteins after PTA reflects the extent of vascular inflammation at the site of the treated segment (1) and correlates with the restenosis risk (1–4). The cytokine IL-6, which is synthesized in response to diverse inflammatory stimuli, acts as a key regulatory protein in the inflammatory cascade (22–25). IL-6 production is upregulated because of angioplasty, and the local cytokine concentration is associated with recurrent luminal narrowing (18). IL-6 has a pivotal role in stimulating the acute-phase response, including the release of fibrinogen and C-reactive protein, both of which are potent predictors of outcome after angioplasty (1–4). IL-6 may also be directly involved in restenosis, as it has been shown to stimulate endothelial activation (23), leukocyte recruitment into the vessel wall (26,27), and vascular smooth muscle cell proliferation (28), factors that are essential to the pathogenesis of hypertrophic neointima formation after PTA and luminal loss.
Numerous environmental and genetic factors are believed to influence the expression of IL-6 and the local cytokine concentration. A functional (–174)G/C polymorphism in the IL-6 gene promoter has been demonstrated to modulate IL-6 serum levels after vascular injury (17). Carriers of the (–174)C allele exhibited higher IL-6 serum levels within the first hours after coronary artery bypass surgery than did noncarriers of the (–174)C allele (17). If we combine these data with our observations, it seems reasonable to speculate that the IL-6 promoter genotype (–174)CC is associated with a transiently increased local overexpression of the cytokine in the vascular wall after balloon angioplasty and thus promotes a vascular inflammatory response, although we cannot prove this hypothesis because data on IL-6 serum levels in the treated segment after intervention are lacking. A self-perpetuating inflammatory and proliferative process, once initiated in the treated segment, leads to the hypertrophic wound-healing response known as restenosis. However, as Brull and colleagues (17) outlined, IL-6 serum levels in (–174)C carriers peak only transiently, within the first few hours after vascular injury. This observation gives rise to the suspicion that IL-6 may contribute to the initiation of restenosis rather than drive its perpetuation. Measured serum levels of IL-6 may vary, depending on the time point of the measurement, while determination of genotype is unaffected and will allow more accurate risk estimation. If one assumes that the hazardous effect of the (–174)CC genotype is confirmed in an independent patient sample, one may speculate about the clinical consequences of this finding. Adjunctive measures to reduce restenosis, such as the application of endovascular brachytherapy or of drug-eluting stents, might be adequate, particularly in these patients.
Conflicting data have been published on the importance of the IL-6 genotype in other contexts of vascular disease. Nauck and colleagues (29) found no association between the IL-6 (–174)G/C polymorphism, IL-6 plasma concentrations, and coronary artery disease. Other authors have described a positive association between IL-6 genotype and peripheral artery disease (30).
Significant differences in the frequencies of arterial hypertension and hyperlipidemia between the IL-6 genotypes were observed in our patient sample. Although it has been suggested that IL-6 is involved in the pathogenesis of arterial hypertension (31), an observation was made in a recent article that the (–174)G/C polymorphism was not associated with hypertension in elderly patients (32). To the best of our knowledge, there are no published reports with results that demonstrate a relationship between the IL-6 genotype and hyperlipidemia. However, it has been demonstrated that patients with the (–174)C allele have a better response to lipid-lowering statin treatment (33); therefore, interactions between lipid-lowering medication, hyperlipidemia, and IL-6 promoter genotype may account for the observed differences. Analyses from randomized controlled trials will be necessary to address this issue.
In our study, we were unable to obtain arterial blood samples directly from the treated vessel segment before and after PTA to determine the expression of IL-6 at the site of balloon injury and correlate it with the IL-6 promoter genotype. However, the findings of Brull et al (17) and Hojo et al (18) consistently support our hypothesis.
In conclusion, the functional IL-6 promoter polymorphism (–174)G/C, which has previously been demonstrated to modulate IL-6 serum levels, seems to influence the occurrence of restenosis after PTA. Homozygous carriers of the (–174)C allele have an increased rate of intermediate-term restenosis, presumably through enhanced vascular inflammation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: IL = interleukin, IQR = interquartile range, PTA = percutaneous transluminal angioplasty
Author contributions: Guarantors of integrity of entire study, M.E., M.S., E.M., O.W.; study concepts, M.E., M.S., E.M., G.E., C.M., O.W.; study design, M.E., M.S., W.M., S.S., M.R.; literature research, M.E., M.S., G.E.; clinical studies, E.M., M.S., W.M., S.S.; data acquisition, M.E., M.S., W.M., M.R.; data analysis/interpretation, all authors; statistical analysis, M.S.; manuscript preparation, M.E., M.S.; manuscript editing, M.E.; manuscript definition of intellectual content, revision/review, and final version approval, all authors
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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  M Libra, S S Signorelli, Y Bevelacqua, P M Navolanic, V Bevelacqua, J Polesel, R Talamini, F Stivala, M C Mazzarino, and G Malaponte Analysis of G(-174)C IL-6 polymorphism and plasma concentrations of inflammatory markers in patients with type 2 diabetes and peripheral arterial disease J. Clin. Pathol., February 1, 2006; 59(2): 211 - 215. [Abstract] [Full Text] [PDF]
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