1 University Division of Medicine and Departments of 2 Cardiology and 3 Renal Medicine, Medical School Unit, Southmead Hospital, Bristol, and 4 Department of Medicine, Manchester Royal Infirmary, Manchester, UK
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Abstract |
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Methods. We carried out cross-sectional analysis of 23 dialysed and 16 non-dialysed CRF patients, 28 healthy controls, and 20 patients with stable angina and normal renal function. The following were determined: (i) LDL oxidation by Cu2+ and ultraviolet light, serum autoantibodies to oxidized LDL (oxLDL); (ii) forearm flow-mediated vasodilatation, plasma concentrations of adhesion molecules, and von Willebrand factor (vWF); and (iii) circulating levels of TNF- and IL-6, C-reactive protein (CRP), and fibrinogen.
Results. Endothelium-dependent vasodilatation (EDV) was lower in angina, pre-dialysis, and dialysis CRF patients than in controls (all P<0.005). Compared with controls, vWf (P<0.005) and adhesion molecules (vCAM-1, P<0.005; iCAM-1, P=0.01; E-selectin, P=0.05) were raised in dialysis, and vCAM-1 (P=0.01) in pre-dialysis CRF patients. Dialysed patients had lower HDL cholesterol (P=0.01) and higher triglyceride (P=0.05) than controls, but LDL-oxidation was similar in all groups. Autoantibodies to oxLDL were raised in angina (P<0.005) and pre-dialysis (P=0.006), but were absent in most dialysed patients. Concentrations of IL-6, TNF-, CRP and fibrinogen were elevated in CRF compared with control and angina patients (P<0.005). In the whole population, IL-6 and TNF-
correlated negatively with EDV, HDL cholesterol, and positively with triglyceride, blood pressure, vWf, iCAM-1, vCAM-1 and E-selectin (r=-0.43 to +0.70, all P<0.05).
Conclusions. Endothelial dysfunction is unrelated to LDL oxidation, suggesting that LDL oxidation might not be a major cause of VED in CRF. In contrast VED was more severe in CRF than in angina patients and is associated with increased acute-phase proteins and plasma cytokines, demonstrating a chronic inflammatory state. These observations may explain the VED and increased IHD risk of patients with CRF.
Keywords: cytokines; endothelial function; ischaemic heart disease; lipid oxidation; renal failure; vasodilatation
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Introduction |
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The assessment of endothelial damage in vivo is complex in view of the multifunctional nature of endothelial cells. Endothelial activation is an injury response mechanism, with impaired endothelium-dependent vasodilatation (EDV), increased adhesion of platelets and leukocytes, and is a putative first step in atherogenesis. Exposure of endothelial cells to oxLDL in vitro releases soluble cell adhesion molecules (CAMs) (vascular cell adhesion molecule-1, vCAM-1; intercellular adhesion molecule-1, iCAM-1; E-selectin, E-sel), and vWF [4], which therefore have been interpreted as markers of endothelial activation. Vasomotor function of endothelium can be assessed as EDV measured by ultrasound. Endothelial dysfunction may be responsible for accelerated atherogenesis in CRF patients and therefore be evident prior to clinical manifestation of IHD.
Recent studies observed elevated levels of C-reactive protein (CRP) in subjects at risk of IHD [5] and in patients with CRF [6]. Thus it has been proposed that a chronic inflammatory state could account for the high risk of IHD in patients with CRF. The inflammatory response is orchestrated by cytokines, especially tumour necrosis factor- (TNF-
) and interleukin-6 (IL-6). Both regulate the production of acute-phase proteins, have potent effects on lipid and carbohydrate metabolism, and are linked with an increased risk of IHD in subjects with normal renal function [7,8]. Proinflammatory cytokines also regulate vascular adhesion. TNF-
in particular promotes the expression of CAMs by endothelial cells [9]. Furthermore, increased plasma levels of soluble CAMs have been reported in CRF patients [10]. Thus it is possible that cytokines could drive the increased levels of acute-phase proteins and CAMs observed in patients at risk of IHD.
In order to compare the relationships of LDL oxidation and the inflammatory state with vascular function in patients with CRF we undertook a cross-sectional investigation of the relationship of endothelial dysfunction, as determined both by plasma CAMs and EDV, to indices of LDL oxidation and to circulating inflammatory markers, in groups of subjects with CRF and angina, and in healthy control subjects.
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Subjects and methods |
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Brachial artery studies
Measurements were made by a single observer using an ATL 9 ultrasound system (Advanced Technology Laboratories Inc., Bothell, WA., USA) with a 7.5-Mhz probe, according to Celermajer et al. [13] and as previously reported by our group [14]. The non-fistula arm in the dialysis group was used. Subjects were recumbent and relaxed for 15 min before measurements. The brachial artery was imaged between 0 and 10 cm above the antecubital fossa, and the transducer secured with a retort clamp to reduce probe movement. Three adjacent measurements of end-diastolic brachial artery diameter were made from single 2-D frames. Blood flow was determined with the Doppler sampling box positioned mid-artery with an ultrasound beam at 60° angle to flow, in the same frame as used for 2-D measurements. Data were recorded on videotape and digitized using frame-grabbing software (Snap Magic, Quantum Leap Software, West Lothian, UK). A pneumatic tourniquet was inflated to 300 mmHg with obliteration of the radial pulse. After 4 min the cuff was deflated. Flow measurements were made 60 s post-deflation. After a further 15 min measurements were repeated, and again 3 min after administration of sublingual glyceryl trinitrate (GTN) (400 µg). Endothelium-dependent (flow-mediated) vasodilatation (EDV) was measured as percentage increase in arterial diameter induced by hyperaemia. Endothelium-independent vasodilatation (EIV) was measured as percentage increase in diameter induced by GTN. The within-subject reproducibility of the technique based on 10 studies was 10% for brachial artery flow-mediated dilatation.
Lipids
Total and HDL cholesterol, and total triglyceride concentrations were measured using standard enzymatic methods on a DAX (Baer) Auto-Analyser. LDL cholesterol concentration was calculated (unless triglycerides were 4.5 mmol/l) using the Friedewald equation [15].
LDL oxidation studies
LDL was isolated from EDTA plasma as described by Kleinfeld et al. [16]. After dialysis it was stored under nitrogen at 4°C for no more than 24 h. Protein content was measured using the Coomassie protein assay (Pierce Chemical Co., Cheshire, UK) and LDL solutions diluted in phosphate-buffered saline (pH 7.4) to 100 µg/ml protein before oxidation using copper sulphate (5 µmol/l) and UV irradiation. Extent of oxidation with copper was followed by measuring the generation of conjugated dienes (CD) at 234 nm over a 6-h period in a PerkinElmer Lambda 5 recording spectrophotometer [16]. The curve was characterized by a lag-phase (Cu lag time, min) before oxidation commenced, and total LDL oxidation, defined as increase in absorption (arbitrary units) from baseline to the plateau of oxidation (Cu Ox max). UV oxidation was carried out over a 4-h period using a 254-nm UV lamp (Camag, UK), placed 10 cm above LDL samples (100 µl) contained in silica tubes [17], and followed by measuring generation of malondialdehyde (MDA) to its maximum (MDAmax) by HPLC with fluorimetric detection (Shimadzu RF535) of its complex with thiobarbituric acid (TBA) [18]. Peaks were quantitated by a CR6A computing integrator. The inter-assay coefficient of variation (CV) was 5.9%.
Plasma conjugated dienes and antioxidant vitamins
Both of these were analysed by HPLC using programmed UV detection. The CD analysis was performed on a 3-µm MOS (150 mm) column with internal standard (eicosadienoic acid) detection at 210 nm and simultaneous analysis at 234 nm giving the CD content [19]. Inter-assay CV was 6.9%. Vitamins (retinol, -tocopherol, and ß-carotene) were analysed on a 5-µm ODS (250 mm) column [20]. Both HPLC analyses were carried out on Shimadzu equipment (pump LC9A, detector SPD-10AV) fitted with a CR4A computing integrator. Inter-assay CVs for all vitamins were <8%.
Endothelial cell activation
Plasma levels of iCAM-1, vCAM-1, and E-sel were measured by ELISA (R&D Systems Europe, Abingdon, Oxon, UK) and analysed using BioLynx software (Dynex Technologies, Middlesex, UK). Inter-assay CVs were <10% for iCAM-1 and vCAM-1, and 6% for E-sel. vWF was measured using an in-house ELISA with antibodies supplied by Dako Ltd., Cambridge, UK. The inter-assay CV was 12%.
Oxidized LDL autoantibodies: Autoantibodies to MDA-LDL in plasma were measured using an in-house ELISA. Briefly, plates were coated with normal LDL and LDL modified with MDA prepared by acid hydrolysis of tetra-methoxypropane (Sigma Chemical Co., Poole, UK) (based on [21]). Bound autoantibodies present in plasma samples were located by goat anti-human IgG (fab specific) phosphatase-conjugated antibody and detected with 1 mg/ml p-nitrophenylphosphate at 405 nm. The inter-assay CV was 9.7%.
Cytokines and acute phase proteins
TNF and IL-6 were measured by ELISA (R&D Systems Europe, Abingdon, Oxon, UK). Inter-assay CVs were <7.5% for both. CRP was measured by nephelometry (Behring, Sussex, UK; lower limit of detection 1.5 mg/l) and fibrinogen level was derived from prothrombin time. Interassay CVs were <10% for both.
Study power and statistical methods
Based on data from healthy controls in our laboratory, power calculations for EDV predicted a requirement for 36 CRF and 36 control subjects to show a 10% difference in means with 80% power at the 5% level of significance. During the course of the study it became apparent that there was a greater than 50% impairment in EDV in the CRF group compared with control and angina patients, and so the sizes of these groups were correspondingly reduced. It was also estimated that approximately 40 subjects per group (renal vs non-renal) would give at least 90% power to detect a 10% difference in IL-6 and iCAM-1, at the 5% level of significance (two-tailed). The CRF and the control groups were then divided into pre-dialysis/dialysis and IHD/non-IHD respectively. Comparisons between groups were made primarily using KruskalWallis analysis of variance and MannWhitney U-test, and correlations tested using the Spearman R correlation coefficient. For cytokine data, which were normally distributed after log transformation, parametric methods were used. Post-hoc adjustments for multiple comparisons were made using Bonferroni's test. Statistical significance was defined as P<0.05. Interactions between variables were determined by partial correlation coefficients and multiple regression. All data were analysed with SPSS for Windows (v 9; SPSS UK Ltd., Chertsey, UK).
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Results |
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Endothelial function
Table 2 shows results of brachial artery studies. EDV was reduced in IHD and both CRF groups compared with controls, but there were no differences in EIV. Baseline arterial diameters and blood flow were similar in all groups (not shown). Soluble CAMs and vWF were elevated in dialysed patients (Figure 2
). vCAM-1 was higher in both CRF groups compared with controls and angina patients. There was a trend to increased levels of vWF and E-sel in the pre-dialysis group. ANOVA demonstrated increased levels of vCAM-1, iCAM-1, and vWF in dialysed patients vs controls. When all subjects were grouped together there were significant correlations between EDV and inflammatory markers (IL-6 r=-0.43, TNF
r=-0.28, fibrinogen r=-0.31, (all P<0.01), CRP r=-0.26, P<0.05) and soluble CAMs vCAM-1 (r=-0.35, P=0.001), iCAM-1 (r=-0.29, P=0.006) and E-sel (r=-0.27, P=0.011), but not vWF. EDV did not correlate significantly with markers of lipid oxidation but did correlate with HDL cholesterol (r=0.25, P=0.017). There were no significant correlations within groups between EDV and lipids, oxidation markers and CAMs. Correlations across groups were heavily weighted by the differences between the renal and other groups. However within the control group there were correlations between HDL-cholesterol and iCAM-1 (r=-0.39, P=0.04), and triglycerides and E-sel (r=0.45, P=0.02). Within the dialysis group vCAM-1 and iCAM-1 correlated with systolic blood pressure (r=0.58, P=0.003; r=0.53, P=0.009).
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Lipid oxidation and antioxidants
Compared with control subjects there were no differences in copper oxidation characteristics or in plasma conjugated diene levels between groups, but the extent of oxidation by UV irradiation measured by maximum MDA concentration was higher in both angina and pre-dialysis groups (Table 3). Plasma levels of MDA were similar in all groups but dialysis subjects had a lower LDL-MDA concentration. Oxidized LDL autoantibodies were higher than control in both angina and pre-dialysis patients but were absent in most dialysed subjects (Figure 1c
). Table 3
shows levels of antioxidant vitamins and markers of lipid peroxidation. There were marked elevations in plasma retinol concentrations in both pre-dialysis and dialysed subjects. ß-Carotene was lower in angina than in control subjects. Vitamin E concentrations were within the reference range (1039 µmol/l), although levels in pre-dialysis subjects were higher than controls (0.05>P>0.01). Although one dialysed patient was taking a vitamin C supplement, his exclusion from the analyses did not affect the results.
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Plasma cytokines
Plasma IL-6 and TNF- were increased in CRF, particularly in dialysed patients (Figure 3
a). Compared with controls, IL-6 was elevated in dialysis patients, and TNF-
was elevated in angina subjects, pre-dialysis and dialysed CRF patients. Analysis of the whole group (n=91) was then undertaken. No significant relationships were observed between plasma cytokines and age, sex, WHR or body mass index (BMI). Plasma creatinine was strongly related to levels of TNF-
and IL-6. In multiple regression analysis (stepwise procedure) creatinine was the sole determinant of IL-6 levels (P<0.0001), displacing all other variables including age, gender, BMI, LDL cholesterol, blood pressure and study group (R2=0.22, P<0.0001). Plasma creatinine and study groups were interchangeable in this respect. Within groups, plasma TNF-
correlated strongly with creatinine in pre-dialysis (r=0.83, P=0.0004) but not dialysis patients (r=-0.07, P=0.75). There were no significant within-group relationships between levels of IL-6 and creatinine. Similarly, no relationships were observed between albumin or haemoglobin and cytokines. However, in the entire group haemoglobin correlated with TNF-
and IL-6 (respectively r=-0.50 and -0.34, P<0.0001 and P=0.0017) and a weak inverse correlation was seen between albumin and IL-6 (r=-0.21, P=0.044).
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Acute-phase proteins
Plasma CRP was elevated in CRF compared with control and angina patients, and was highest in dialysed patients (Figure 3b). Plasma levels of fibrinogen were highest in dialysed patients (Figure 3c
). In the entire group, CRP and fibrinogen levels correlated after controlling for creatinine (partial r=0.46, P<0.001). CRP levels correlated positively with IL-6 and TNF-
(r=0.48 and 0.42, both P<0.001), negatively with HDL cholesterol and positively with BMI, triglyceride, and SBP (r=-0.30 to +0.32; P=0.0020.014). Fibrinogen correlated negatively with HDL cholesterol and positively with triglyceride and SBP (r=-0.29 to 0.31, P=0.003 to 0.006).
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Discussion |
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Impairment of EDV, suggesting large/resistant vessel dysfunction, was observed in pre-dialysis and dialysis patients, and was similar to that in IHD. These results were not influenced by variations in baseline arterial diameter or blood flow which were similar in all groups. These findings are consistent with previous observations [22,23]. In contrast to EDV, circulating CAMs and vWF have been considered to be markers of VED, and as in this study, increased concentrations have been observed previously in CRF [24,25]. The raised level of vCAM-1 suggests that VED is present at the pre-dialysis stage. Soluble CAMs, in contrast to EDV, reflect non-vasomotor aspects of VED and may be produced by a more widespread endothelial surface, including capillary beds. Thus, impaired EDV and increased plasma levels of such markers may well be influenced by different factors. It is interesting to note, however, that in the whole group EDV was inversely related to levels of CAMs, suggesting that both aspects of VED deteriorated in parallel in the presence of CRF. Although EDV was inversely related to plasma levels of cytokines and acute-phase proteins, it is not clear whether this relationship represents cause and effect or association.
Although cholesterol plays a key role in IHD, total and LDL cholesterol levels were similar in all groups. In contrast, dialysed subjects had lower HDL cholesterol and higher triglyceride levels. However, no relationships were observed between either EDV or EIV and cholesterol, HDL cholesterol or triglyceride. In previous studies, impaired EDV has been associated with cholesterol levels in subjects with normal renal function [26], although recent work questions this [27]. In contrast, we observed inverse relationships of HDL cholesterol with iCAM-1 and E-sel, and a positive correlation between triglycerides and E-sel. We have observed similar relationships for E-sel in patients with diabetes [14]. Thus, low HDL cholesterol and raised triglycerides may be more important markers of an atherogenic lipid phenotype, as in type-2 diabetes.
Although oxLDL may be the most atherogenic LDL fraction, we found that copper oxidation lag-time of LDL, plasma MDA, and CD concentrations were similar in all groups. This contrasts with a previous report [28] which showed increased copper lag-time (at lower [Cu2+] than in our study) in dialysis patients, although the authors did not report MDA or CD values. Maximum MDA levels generated by UV oxidation of LDL were significantly higher in IHD and pre-dialysis CRF groups, consistent with greater oxidizability of LDL, although this was not seen in dialysis patients. Similar trends were seen in the copper oxidation maximum. Apart from the known hypervitaminosis A of renal disease, antioxidant defences, at least as indicated by levels of fat-soluble vitamins, were little different in the four groups. The interpretation of LDL oxidation and antioxidant data is complex since it is uncertain how currently used in vitro methods reflect processes in vivo. Therefore, we also measured levels of antibody to oxLDL. One group previously reported increased oxLDL antibodies in CRF [29]. We also observed increases in autoantibodies in the IHD group, in agreement with others [30], as well as in the pre-dialysis group. Antibodies were, however, undetectable in the dialysis group. However, there are a range of technical problems with autoantibody detection. For example oxLDL immune complexes, previously observed in diabetes [31], are not detectable by our assay. Also, it is possible that antigen prepared for our assay, MDA-LDL, may not be optimal with CRF serum, in which the dominant modified LDL might be carbamylated [32], or where LDL oxidation leads to the formation of advanced glycation end-products (AGEs) [33] which might mask detection. We have recently observed that autoantibodies to carbamylated proteins are present in plasma from haemodialysis patients (unpublished findings).
In contrast to the lack of differences in LDL oxidation parameters, increased plasma levels of cytokines were observed in both CRF groups. These were accompanied by increased levels of CRP and fibrinogen, more atherogenic lipid profiles, elevated blood pressure, and increases in circulating CAMs. Recent reports have implicated TNF- and IL-6 as risk markers for IHD in CRF [6,34]. However, our data also show that plasma cytokine levels are related to a range of other aspects of vascular adhesion and coagulation. Since both TNF-
and IL-6 are involved in the regulation of acute-phase proteins [35], it is plausible that a cause-and-effect relationship exists between elevated circulating cytokine levels and acute-phase reactants in CRF. Irish [6] observed increased fibrinogen, reduced HDL cholesterol, and increased IL-6 in patients with CRF, and suggested that this could predispose to vascular disease. In previous studies, fibrinogen levels were elevated in subjects at risk of IHD [36]. Although many factors influence the inflammatory response and immune function may be abnormal in CRF, reduced renal clearance of cytokines could also contribute to increased production of acute-phase proteins. Furthermore, macrophage IL-6 secretion may be increased in CRF [37].
Positive correlations were observed between circulating cytokines, CAMs and vWF. vWF was first to be proposed as a marker of VED, increased levels interpreted as indicating endothelial activation and heightened thrombotic risk [25]. Soluble CAMs are also thought to indicate VED and have been used as markers of injury. E-sel is specific for endothelium, whereas iCAM-1 and vCAM-1 are also expressed by other cells. Such products have been shown to circulate at increased levels in patients with normal renal function and atherosclerosis [24]. The mechanisms resulting in elevated CAM levels are less clear, but cytokines are important. TNF- induces all three CAMs in cultured endothelial cells [9]. Cytokines also regulate vWF production in endothelial cells [38]. Thus, the present results could be explained by the effects of cytokines on endothelium, or by reduced clearance of endothelial products.
The principal limitation of this study is its cross-sectional design, such that cause and effect are not discriminated. In the absence of more patients with CRF of single aetiologies, we cannot determine the impact of individual renal diseases on cytokine levels. However, since CRF was the primary abnormality, it is likely that increases in inflammatory markers and CAMs are secondary to CRF. This is supported by the strong relationship between cytokine and creatinine levels, suggesting that renal clearance may affect circulating cytokine levels. In dialysed patients, there was no reduction in levels of cytokines, which are large enough to be excluded from dialysis, and levels of CAMs were further increased. In conclusion, elevated circulating levels of cytokines offer a plausible mechanism for the increased risk of IHD in CRF, and interventions to reduce their production and action, or increase their clearance, might offer a new approach to IHD prevention in patients with CRF.
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Acknowledgments |
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Notes |
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References |
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