Benien E van Aken's research while affiliated with Academisch Medisch Centrum Universiteit van Amsterdam and other places

Hyperhomocysteinemia has been associated with vascular disease in many epidemiological studies. However, the pathophysiology is unclear. It is postulated that increased levels of homocysteine induce an inflammatory response in endothelial cells, mediated by pro-inflammatory cytokines and chemokines. The aim of this study was to investigate whether plasma concentrations of interleukin-6, interleukin-8, C-reactive protein, and monocyte chemoattractant protein-1 are increased with higher plasma homocysteine concentrations and whether decreasing homocysteine by vitamin supplementation decreases the concentration of these markers. Plasma homocysteine, interleukin-6, interleukin-8, C-reactive protein, and monocyte chemoattractant protein-1 concentrations were measured in 230 volunteers before and after 8 weeks of multivitamin supplementation (folic acid, B(6), and B(12)). At baseline, plasma homocysteine concentration was weakly associated with interleukin-8, but not with interleukin-6, C-reactive protein or monocyte chemoattractant protein-1. Vitamin supplementation resulted in a significant decrease in homocysteine concentration, but no effect on interleukin-6, interleukin-8, C-reactive protein or monocyte chemoattractant protein-1 was observed. At baseline homocysteine was only weakly correlated with interleukin-8, but not with interleukin-6, C-reactive protein or monocyte chemoattractant protein-1. Vitamin supplementation affected homocysteine concentration, but not cytokine levels. The hypothesis that hyperhomocysteinemia increases arteriosclerotic or thrombotic risk through vascular inflammation was not supported by this study.

In coronary in-stent restenosis (ISR), a substantial contribution of inflammation is assumed. We evaluated the association between polymorphisms in the Toll-like receptor 4 (TLR4) gene and cytokine response after lipopolysaccharide (LPS) challenge and the development of ISR. Patients were included after successful elective stent placement in a native coronary artery and were scheduled for follow-up angiography after 6 months. Quantitative coronary analysis was performed off-line. Patient whole blood was challenged with LPS for 24 h. Baseline and stimulated concentrations of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, and IL-10 were assessed by ELISA. Two cosegregating single-nucleotide polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) were analyzed by allele-specific PCR amplification of genomic DNA. A total of 236 consecutive patients were included, and 40 (17%) developed ISR. Median baseline and stimulated cytokine concentrations did not differ between patients with and without ISR. In multivariate analysis, male sex, unstable angina, hypertension, and chronic total occlusion were predictors of ISR. TLR4 genotypes were not associated with baseline or stimulated cytokine concentrations or with angiographic variables at follow-up. In vitro cytokine response to LPS challenge is not increased in patients with ISR. Functionality of the TLR4 Asp299Gly polymorphism could not be demonstrated in this setting, and this polymorphism was not associated with angiographic outcome, calling into question its role in the progression of neointimal tissue growth.

One of the possible pathological mechanisms behind the increased vascular injury in diabetes mellitus type 2 (DM2) is the formation of advanced glycation end products (AGEs). The aim of this study was to investigate whether the presence of AGEs and specific markers for coagulation and inflammation in symptomatic atherosclerotic plaques from DM2 patients differs from plaques from nondiabetics. Carotid atherectomies were obtained from DM2 patients (n=11) and controls without DM2 matched for age and other cardiovascular risk factors (n=12) who were treated for symptomatic carotid artery stenosis. Plaques were graded according to the American Heart Association classification of lesions. More fibrosis and more thrombotic complications (p=0.007) were observed in carotid atherectomies from DM2 patients. Percentages of immunostained smooth muscle cells and macrophages in the lesions, quantified planimetrically, did not differ between the two groups. No differences were found in the immunostaining for T cells, tissue factor (TF), endothelial protein C receptor (EPCR), nuclear factor kappaB, and the AGE carboxymethyllysine. These findings demonstrate that DM2 is associated with increased plaque complications; however, a local changed presence of AGEs, TF, and EPCR seems not to be involved in this end stage of atherosclerosis.

We investigated whether the anticoagulant effect of idraparinux, a selective long-acting factor Xa inhibitor, could be neutralized by recombinant factor VIIa (rFVIIa) in healthy male volunteers. We performed a randomized, placebo-controlled trial, comparing idraparinux [7.5 mg subcutaneous (s.c.)] followed at 3 h by rFVIIa [90 microg/kg intravenous (i.v.)] (n = 6), or idraparinux (7.5 mg s.c) followed after 1 week by rFVIIa (90 microg/kg i.v.)(n = 6). rFVIIa, given 3 h after idraparinux, significantly reversed the increased thrombin generation time (TGT), the increased activated partial thromboplastin time (aPTT) and prothrombin time (PT), and the reduced prothrombin fragment 1+2 (F1+2) levels caused by idraparinux, although no clear effect of rFVIIa on the endogenous thrombin potential (ETP) was observed. One week after idraparinux, injection of rFVIIa resulted in a similar relative reduction of the remaining increased aPTT, PT and TGT, with correction to pre-idraparinux values. A clear increase of F1+2 was observed, together with a small increase in ETP. We conclude that rFVIIa has significant effects on the idraparinux-inhibited thrombin generation and clotting parameters. These results suggest that rFVIIa may be useful in serious bleeding complications in idraparinux treated patients.

The novel anticoagulant fondaparinux proved to be effective and safe in the postoperative prevention of venous thrombosis. Current phase III trials with this synthetic selective factor Xa inhibitor focus on its use in the treatment of patients with venous and arterial thrombosis. As with any anticoagulant therapy, there is a risk of bleeding complications; hence, a strategy to reverse the effects of fondaparinux is desirable. The aim of this study was to investigate whether recombinant factor VIIa (rFVIIa) could neutralize the anticoagulant effects of subcutaneously administered fondaparinux. In a randomized, placebo-controlled design, 16 healthy male subjects received either a single subcutaneous dose of fondaparinux (10 mg) and a single intravenous bolus of rFVIIa (90 microg/kg; n=8), fondaparinux and placebo (n=4), or placebo and rFVIIa (n=4). Fondaparinux (or placebo) was administered 2 hours before rFVIIa (or placebo). Injection of rFVIIa after fondaparinux normalized the prolonged activated partial thromboplastin and prothrombin times and reversed the decrease in prothrombin activation fragments 1+2 (F(1+2)), as observed with fondaparinux alone. Thrombin-generation time and endogenous thrombin potential, which were inhibited by fondaparinux, normalized up to 6 hours after rFVIIa injection. rFVIIa is capable of normalizing coagulation times and thrombin generation during fondaparinux treatment. The duration of this effect ranged from 2 to 6 hours after rFVIIa injection. These results suggest that rFVIIa may be useful to reverse the anticoagulant effect of fondaparinux in case of serious bleeding complications or need for acute surgery during treatment with fondaparinux.

Inflammatory mediators are involved in activation of the coagulation system, and elevated plasma concentrations of IL-6 and IL-8 are associated with an increased risk of venous thrombosis. Using serologic and molecular biologic tests, we investigated in a case-control study on patients with recurrent venous thrombosis the association between Chlamydia (C) pneumoniae and venous thrombosis and we evaluated the relation between C. pneumoniae serology and the cytokines IL-6 and IL-8. The presence of C. pneumoniae antibody titers > or = 1:16 was not associated with an increased risk of venous thrombosis (odds ratio 0.8 95% CI, 0.4-1.7). Circulating C. pneumoniae-DNA was detected in only one patient and two control subjects. IgG antibody titers against C. pneumoniae were not correlated with the concentrations of IL-6 and IL-8. These results indicate that the inflammatory process shown in patients with venous thrombosis is not related to C. pneumoniae.

Elevated plasma levels of interleukin 8 (IL-8) were previously shown to be associated with recurrent venous thrombosis. To assess the risk of venous thrombosis, IL-8 plasma concentrations were measured in patients and control subjects of the Leiden Thrombophilia Study (LETS). This population based case-control study included 474 patients with a first deep-vein thrombosis and 474 age- and sex-matched controls. The risk of venous thrombosis for subjects with elevated IL-8 levels (above 90th percentile of controls) compared with subjects with IL-8 levels below the 90th percentile was increased 1.8-fold (95%CI 1.2-2.8). Adjusted for age and sex, the odds ratio was 1.9 (95%CI 1.3-2.8). IL-8 concentrations were weakly correlated with age, male sex, and concentrations of C-reactive protein, factor VIII coagulation activity and homocysteine, but adjustment for these factors did not substantially affect the association between IL-8 and venous thrombosis. Our results suggest that IL-8 is a risk factor for venous thrombosis.

Hyperhomocysteinemia is a risk factor for atherosclerosis and thrombosis. The aim of this study was to analyze if exposure of monocytic cells to increased levels of homocysteine (HCY) induces the accumulation of inflammatory mediators. Interleukin (IL)-6 production by monocytic cell line Mono Mac 6 (MM6) was 1.7-fold increased in the presence of 50 micromol/l HCY and 3.5-fold with 200 micromol/l HCY. Incubation with homocystine resulted in a comparable dose-dependent increase, but neither cysteine nor methionine stimulated IL-6 accumulation. Elevated homocysteine concentrations did not affect the production of IL-8 and monocytic chemotactic protein-1 (MCP-1) in MM6. Furthermore, lipopolysaccharide (LPS) stimulation of MM6, cultured with elevated HCY (200 micromol/l) levels, resulted in a 3.5-fold increased response after 18 h, whereas no effect on LPS-induced IL-8 and MCP-1 response was observed. In conclusion, increased concentrations of homocysteine induce IL-6 accumulation in monocytic cells. After treatment with homocysteine, monocytic cells become more susceptible to endotoxin. This study is in favor of an association between homocysteine and monocytic IL-6 production.

Inflammatory processes may play a key role in venous thrombosis, by inducing a procoagulant state through the action of cytokines and chemokines on monocytes and endothelial cells. Plasma concentrations of three inflammatory mediators, interleukin 6 (IL-6), interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1), that mediate the cross-talk between inflammation and coagulation, were measured in 182 subjects with recurrent venous thrombosis and 350 healthy subjects recruited through a general practice. Elevated levels of IL-6 (>90th percentile of the control group) were detected in 25.8% of the patients with venous thrombosis in comparison with 10% (by definition) of the controls [odds ratio 2.4 (95%CI 1.5-3.8)]. In 21.5% of the patients elevated plasma levels of IL-8 (>90th percentile) were determined [odds ratio 2.0 (95%CI 1.2-3.5)]. Elevated levels of MCP-1 (>90th percentile) were detected in 24.1% of the patients [odds ratio 1.9 (95%CI 1.2-3.2)]. This is the first large clinical study showing that an increase in inflammatory mediators is associated with venous thrombosis. Future prospective studies are necessary to clarify the causal nature of the inflammatory process with respect to venous thrombosis.