Introduction
Atherosclerosis is the major cause of myocardial infarction, stroke and gangrene of lower extremities. This disease is responsible for over 50% of deaths in the USA, Europe and Japan [1]. In the pathomechanism of atherosclerosis, the major role refers to the damage of the arterial wall by pathogenic factors, e.g. oxidized lipids, hypertension, components of tobacco smoke, various infections or mechanical and postoperative injuries. Damage to the arterial wall results in excessive proliferation of endothelial and smooth muscle cells in the vessels and the accumulation of inflammatory and immune cells in the injured site. The activation of these cells triggers many inflammatory markers, cytokines, growth factors and vasoregulatory molecules which participate in this process. Atherosclerosis, including peripheral arterial disease (PAD), has been classified as an inflammatory disease. Inflammation is associated with the pathophysiology and clinical progress of atherosclerosis in various vascular regions [2–4]. Currently, a number of experimental and clinical studies are being carried out in different countries on inflammation mediators and pro-inflammatory cytokines in various human diseases [5]. Previous studies also demonstrated that patients with PAD have increased blood coagulation, thrombotic and embolic complications [6–8]. Currently, researchers point out the close correlation between blood coagulation, fibrinolysis and vascular inflammation [9, 10]. Certain pro-inflammatory cytokines, e.g. interleukin-6 (IL-6), can activate blood coagulation and fibrinolysis, while interleukin-10 (IL-10) can inhibit tissue factor and, indirectly, atherosclerosis development [5, 10, 11].
Fibrinogen, C-reactive protein (CRP) and cytokines: IL-6, tumour necrosis factor (TNF) and IL-10 are the most frequently described inflammatory markers in atherosclerosis, particularly in patients with coronary artery disease, but also in PAD [5, 12–15]. Fibrinogen, synthesized in the liver, is therefore an important blood clotting factor, but also, like CRP is a general inflammatory marker. C-reactive protein is also synthesized in the liver. Increased CRP activity is a response to general inflammatory stimuli mediated by the system of interleukins [16, 17]. CRP is a marker of the grade of the inflammatory process, but also indirectly indicates a cytokine-dependent inflammatory process in the arterial wall, macrophage activation and proliferation of endothelial and smooth muscle cells in the vessels. The inflammatory process is associated with the formation of atherosclerotic plaques, but is also intensified in patients who are subject to revascularization, both surgical (e.g. endarterectomy or bypass surgery) and endovascular (e.g. angioplasty, with or without stent implantation), to improve revascularization in the lower extremities [18]. The inflammatory process is also associated with the formation of in-stent restenosis or postoperative arterial thrombosis [14, 19, 20]. The pathophysiology of restenosis has not been fully explained. It is claimed that the proliferation of smooth muscle cells at the injury site, and neointimal hyperplasia, induces the formation of thrombus and narrows the arterial lumen. The results of Tschöpl and al. indicate that high levels of pro-coagulant factors and persistent thrombin generation by haemostatic system might promote restenosis [21]. Increased levels of CRP also indicate an increased risk of thrombosis or restenosis after revascularization in patients with lower limb ischaemia [2, 14, 22–25]. Most cytokines synthesized by various blood cells and arterial walls have a pleiotropic effect. In many cases, one type of cytokine may functionally, in part or completely, replace the other. Interleukin 6 (IL-6) with 26 kD molecular weight is a cytokine synthesized by monocytes, macrophages, B and T lymphocytes and endothelial cells in blood vessels. It is one of the factors responsible for haemostasis, the synthesis of blood clotting factors in the liver, and its level is increased in various disseminated intravascular coagulation diseases (DIC) [9].
Interleukin 10 (IL-10) with 35 kD molecular weight is also synthesized by monocytes, macrophages and T lymphocytes, but has an anti-inflammatory effect. It inhibits the synthesis of tissue factor (TF) in arterial wall, suppressing the activation of the extrinsic pathway of coagulation and, indirectly, the atherosclerotic process [5, 10, 26].
Transforming growth factor beta-1 (TGF-β1) is synthesized by most of the nuclear cells, including fibroblasts, macrophages, lymphocytes and endothelial cells. Its molecular weight is 25 kD; it has a homodimeric structure and is activated by plasmin [9]. TGF-β1 promotes the in vitro transformation of fibroblasts into tumour-like cells. Studies by Taniguchi et al. demonstrated that this cytokine is involved in the thickening of the arterial wall and reducing the expression of thrombomodulin (TM) [2, 27].
Another cytokine studied by us, the basic fibroblast growth factor (bFGF), is involved in many normal processes, such as angiogenesis, wound healing, differentiation and tissue repair, and its levels is correlated with the tissue vascularization level [9, 10]. Despite many experimental and clinical studies, the activity of these cytokines in humans with different diseases, particularly PAD, has not been fully explained.
Aim
1. To determine the levels of fibrinogen, C-reactive protein (CRP), interleukin 6 (IL-6), interleukin 10 (IL-10) basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-β1) in the blood of patients with PAD after endovascular revascularization of lower limbs.
2. To observe these patients for 12 months for appearance of restenosis.
3. To compare the inflammatory factors in both groups of PAD patients after endovascular revascularization — before and after restenosis.
Material and methods
The study included 150 patients with PAD who underwent endovascular revascularization at the Angiology and Vascular Surgery Centres in Wroclaw and Krakow. These patients were referred to the Angiology Unit of the Regional Specialist Hospital in Wroclaw. There were 90 men and 60 women, aged 44 to 88 (mean 65.5 years). One man withdrew his consent to participate in the study, and two patients died during the observation period due to malignant cancer. These patients were excluded from the examined group. Patients were recruited between 18.08.2010 and 28.06.2012, and a revascularization procedure had been performed from 1 to 18 months before recruitment. The time from revascularization procedure (PTA and/or stenting of iliac, femoral, popliteal and crural arteries) was one to 3 months in 77 patients, over 3 to 6 months — in 30 patients and over 6 months — in 43 patients. In these patients detailed medical history was obtained and physical examination, basic blood and biochemical laboratory tests and clinical observations were carried out every 3 months with blood collection (5 times over one year). Eighteen patients had small ischaemic ulcers on the extremities with stable inflammation (CRP = 5.2 ± 3.7 mg/l). Intermittent claudication and ankle brachial pressure index (ABPI) were also measured. Ankle brachial pressure index (ABPI) below 0.9 on the right extremity was found in 63 (42%) patients and on the left extremity in 76 (50.7%) patients. In 11 patients, ABPI was below 0.5. Patients had previously been treated with angioplasty (PTA) alone — 21, angioplasty plus stent implantation — 80 or stent implantation alone — 49 patients. The ischaemia of the lower limbs was diagnosed based on an ultrasonography scan (USG duplex Doppler), computed tomography and arteriography. Of the patients included in the study, 101 (67.0%) had various forms of ischaemic heart disease (in some patients with myocardial infarction), 126 (84.0%) arterial hypertension, 120 (80.0%) hyperlipidaemia, 90 (60.0%) were ex-smokers, 41 (27.3%) current smokers, and 19 (12.7%) never-smokers. Ninety one (60.6%) PAD patients were diagnosed with type 2 diabetes and 115 (76.6%) were overweight or obese. All revascularization procedures were successful. No amputations were performed. In 38 PAOD patients new restenosis occurred within 12 months of observation. Control group for cytokines consisted of 15 clinically healthy subjects aged 60–83 years without cardiovascular symptoms, but who reported a number of risk factors.
Blood samples (4.5 ml) were drawn from the cubital vein in the morning in a fasting state and put into a test tube with 0.5 ml of 3.2% sodium citrate. Plasma with sodium citrate was analyzed for levels of fibrinogen and CRP. Cytokines were determined in 2.6 ml blood samples mixed with EDTA. Plasma was obtained through centrifuging at 2500 g for 15 minutes. 0.2 ml plasma samples were then distributed to Eppendorf test tubes and frozen at −80oC until they were used for testing. The plasma levels of tested cytokines were determined using commercial kits for enzyme immunoassays; fibrinogen and CRP levels were measured with a coagulometer.
The following commercial kits were used for the determination of individual parameters:
1. Fibrinogen – Multibren U, Siemens;
2. CRP — CRP VARIO, Abbott;
3. IL 6 — Human IL 6 Quantikine ELISA, R&D Systems;
4. IL 10 — Human IL 10 Quantikine ELISA, R&D Systems;
5. bFGF — Human bFGF Quantikine ELISA, R&D Systems;
6. TGF-β1 — Human TGF-β1 Quantikine ELISA, R&D Systems.
The protocol of the study was approved by the Bioethical Committee at the Regional Specialist Hospital in Wroclaw.
Statistical analysis
Results are presented in tables as mean values (M), standard deviation (SD), medians (Me) and interquartile range (lower Q1 and upper Q3). Normality of distribution was assessed using D’Agostino-Pearson test. The statistical significance of differences between groups was analyzed with the T-student test or nonparametric Mann-Whitney test (in case of non-normal data). The correlations between the measured parameters characterized by non-normal data distribution were calculated using the Spearman rank correlation coefficient (rs) with significance level p. The level of statistical significance was adopted at p < 0.05. Statistical analysis was performed with R for Windows (The R Foundation for Statistical Computing, Vienna, Austria) and MedCalc for Windows (MedCalc Software, Mariakerke, Belgium).
Results
Table 1 presents parameters most frequently determined in inflammatory diseases, i.e. levels of fibrinogen and CRP, in patients with PAD after endovascular revascularization.
In comparison with the most commonly used laboratory normal values for fibrinogen (1.8–3.5 g/l) and for CRP (below 5 mg/l), 71 (47.3%) patients with PAD had elevated fibrinogen concentration, and 50 (33.3%) patients had elevated CRP levels. However, when the results were compared with the normal value for CRP proposed by Nijm et al. in 2005 (1.3 ± 1.1 mg/l), as many as 97 (64.7%) patients presented with increased CRP levels. Unfortunately, we did not perform these studies in our own control group; more detailed data are presented in Figure 1.
We determined the levels of fibrinogen and CRP in all patients from the study group (150 subjects) every 3 months (5 times over a year). The lowest (yet elevated) mean levels of fibrinogen and CRP were found shortly after revascularization. The levels of these markers increased over 3-months intervals, and the increase for CRP was more pronounced than that for fibrinogen.
Table 2 presents the levels of 4 cytokines (IL-6, IL-10, bFGF and TGF-β1) in 150 patients with PAD after peripheral endovascular revascularization and in 10 control subjects.. Laboratory normal values specified by the producers of reagents are also presented. In the group of patients, a statistically significant increase in comparison with the controls was only found for IL-10 and lower for bFGF levels, while the concentrations of IL-6 and TGF-β1 increased insignificantly. All cytokine levels were within the specified normal limits, apart from TGF-β1 which levels were several times higher.
We calculated also the correlations between the tested inflammatory markers and cytokines in patients with PAD after endovascular revascularization. Results of significant correlations are demonstrated in table 3.
Table 4 presents all tested inflammatory parameters in 38 patients with PAD, who, at various time points of observation, developed restenosis. In table 4 are comprised the results 3 months before and after the formation of restenosis. Only the fibrinogen (p < 0.02) and TGF-β1 (p < 0.025) levels were significantly higher in patients with restenosis. The levels of CRP, IL-6 and IL-10 were also higher, but the differences were not statistically significant.
There was an interesting correlation with high significance level between pro-inflammatory CRP and anti-inflammatory IL-10. This indicates on a common action in the inflammation process (Fig. 2).
Discussion
The studied 150 patients with PAD had ischaemia of the lower extremities and were treated with PTA, with or without stent implantation, with a good outcome and without amputation. The patients were presented with almost all risk factors for atherosclerosis and had many other serious diseases, such as ischaemic heart disease, often with a history of myocardial infarction, arterial hypertension and type 2 diabetes. For these reasons, the patients received intensive treatment with cardiovascular and antihypertensive drugs, anticoagulants, statins and diabetic drugs. Therefore, it can be questioned as to whether the obtained results may refer solely to ischaemia and procedures on lower extremities or not. Vascular inflammation is associated with the pathogenesis, but also the progression, of atherosclerosis [3, 4, 20]. Both surgical (bypass, mechanical restoration of patency) and endovascular arterial revascularization (balloon angioplasty, with or without stenting) may intensify the inflammatory reaction, similar to restenosis in the implanted stents, or thrombus in surgically or endovascular treated arteries [16, 23]. Inflammatory markers circulating in the blood stream, e.g. CRP, fibrinogen and amyloid A, reflect disease activity and indirectly indicate the activation of macrophages and proliferation of endothelial and smooth muscle cells in the vessels [19, 22, 24]. However, Brevetti et al. (2008) claimed myeloperoxidase to be a more important mediator of inflammation than CRP [2]. The levels of fibrinogen and CRP found in our study are much higher than the values reported by Schillinger et al., who studied PAD patients before endovascular revascularization [17].
As shown in Figure 1, CRP is a much better inflammatory marker than fibrinogen. In the course of one year of observation, fibrinogen levels increased by only about 10%, while the level of CRP more than doubled. As found by Buffon et al., increased CRP levels can predict early complications and late restenosis, although this refers to coronary angioplasty [29]. In the analysis of increased fibrinogen levels in patients with PAD, the fact that these levels are higher in older people than in the young (p < 0.02) should be considered. We faced difficulties in the assessment of CRP levels in the studied patients because of the wide limit of the laboratory normal values (up to 5 mg/l). However, we used also normal values proposed by other researchers, e.g. Nijm et al. (1.3 ± 1.1mg/l), and found elevated CRP levels in 64.7% of patients, which was in line with the findings of most published studies [20, 24, 25]. In addition, we tested the levels of 4 other cytokines: IL-6, IL-10, bFGF and TGF-β1 in PAD patients after endovascular revascularization. Only IL-10 was higher and bFGF lower than in the control group (p < 0.027 and p < 0.046). Levels of IL-6 and TGF-β1 in patients compared were also higher, but not significant. All cytokines except for TGF-β1 were still within the range of normal values. It appears that these parameters do not play an important role in the laboratory diagnostics of PAD. However, if more or less significant correlations between CRP and fibrinogen and all 4 cytokines are considered, the results could be important for the pathogenesis of thrombosis and PAD. Seo et al. (2007), reported high levels of IL-10 in patients with different disseminated intravascular coagulation diseases (DIC) [26]. Also Pinderski et al. reported in 1999 that IL-10 blocks atherosclerotic events in vitro and in vivo and inhibits tissue factor (TF) in mice. IL-10 also plays an active modulatory role in endothelial cell damage and accelerates platelet adhesion in vitro [10].
In table 4, we present the characteristics of 38 patients with PAD after endovascular revascularization, who over one year of observation developed restenosis. For comparison, the parameters of the same patients three months before restenosis are presented. Patients with restenosis had significantly higher levels of fibrinogen and TGF-β1 and also higher, but not statistically significant, were levels of CRP, IL-6 and IL-10. Despite many experimental and clinical studies, the pathomechanism of arterial thrombosis and in-stent restenosis after revascularization in patients with PAD has not been fully explained. It has been claimed that the inflammatory process stimulates endothelial cells and smooth muscle cells proliferation, as well as late neointimal hyperplasia in the surgically and endovascularly treated part of the artery, leading to restenosis [24]. The frequency of restenosis in our patients (about 25.3%) corresponds with the statistics reported by other authors [16, 17]. Vascular stents, particularly those made of polytetrafluoroethylene (ePTFE), are regarded as neutral. However, after implantation in patients, they are covered with an ultra-thin biofilm containing mainly fibrinogen and blood cells, including platelets, which may be involved in the pathomechanism of restenosis [30, 31].
The highest values of CRP were found at 24 to 72 hours after revascularization [17].
Although CRP is a marker of systemic inflammation in the human body, it also plays a role in the assessment of abnormal blood supply to the lower extremities and early and late complications after revascularization procedures [2]. Other cytokines, i.e. bFGF and TGF-β1, despite their low pro-inflammatory activity, are less useful in the clinical assessment of patients with PAD. Our examination connected with CRP level confirms earlier observations of other authors, who recommend the determination of CRP in PAD patients after revascularization to predict early and late thrombotic complications, e.g. as occurrence of restenosis.
Conclusions
1. In PAD patients after endovascular revascularization the level of CRP and fibrinogen during one-year observation significantly increased.
2. In these patients significant correlation between pro-inflammatory CRP and anti-inflammatory IL-10 was observed.
3. The concentrations of CRP, fibrinogen and cytokines (IL-6, bFGF, TGF-β1 and IL-10) were also higher in PAD-patients with restenosis indicating the involvement of low-grade inflammation in this process.
This publication is part of project „Wrovasc — Integrated Cardiovascular Centre”, co-financed by the European Regional Development Fund, within Innovative Economy Operational Programme, 2007–2013 realized in Regional Specialist Hospital, Research and Development Centre in Wroclaw.
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Adres do korespondencji:
prof. dr hab. n. med. Maria Kotschy
Wojewódzki Szpital Specjalistyczny,
Ośrodek Badawczo-Rozwojowy we Wrocławiu
ul. H.M. Kamieńskiego 73a, 51–124 Wrocław
tel.: (+48 71) 327 01 35
Acta Angiol Vol. 20, No. 2 pp. 47–59
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