Livrables
Ethylenediamine-tetraacetic acid (EDTA) chelation therapy is practiced as an alternative treatment for coronary artery disease, believing that removal of metal ions like copper and iron by EDTA is able to prevent atherogenic oxidative stress. Little is known, however, on its biological effects on vascular endothelium. In this study, we examined the effects of metal chelation with EDTA on the expression of endothelial ICAM-1, VCAM-1 and E-selectin as well as the adhesion of monocytes to the endothelium, being a crucial event in atherogenesis.
In a serum free condition, addition of EDTA enhanced, rather than reversed, iron-induced endothelial adhesion molecule expression. In the presence of copper, EDTA induced ICAM-1 expression. In 50% human pooled serum, EDTA alone, without added iron or copper, enhanced the expression of adhesian molecules. EDTA appeared to promote accumulation of intracellular labile iron, inducing oxygen radical mediated endothelial cell activation.
This activation could be reversed by deferoxamine or deferiprone, two iron chelators widely used for the treatment of secondary iron overload. High doses vitamin C, routinely added to EDTA therapy, augmented rather than reversed EDTA effects, and moreover inducing monocyte adherence to vascular endothelium. Endothelial activation by EDTA-vitamin-C treatment, following interaction with transition metal iron, therefore, may cause harmful effects when being used for the treatment of coronary artery disease.
Conclusion: In particular subjects with iron overload could be at risk after EDTA chelation therapy.
Infiltration of monocytes across the endothelial barrier is not only considered to contribute to the initiation but also to the progression of atherosclerosis. Given the plausible roles of iron in inflammatory processes, we have investigated the effects of iron on monocyte-endothelial cell interactions.
Monocyte rolling and firm adhesion to TNF-a-activated endothelial cells were performed in a physiological flow model, while their diapedesis was assessed by an in vitro transendothelial migration assay. The expression of surface proteins and the formation of intracellular radicals were measured by flow cytometry. Iron enhanced TNF-a-induced endothelial activation, resulting in a rise on monocyte adhesion. Moreover, an increase in adhesion was observed using iron-loaded monocytes compared to controls. Blockade of the integrins, VLA-4 and LFA-1, restored more than 60% of monocyte rolling.
However, in this condition, the number of firmly adhered cells was higher for iron-loaded monocytes than controls, while no difference in the rolling velocity was observed, suggesting that iron could have affected receptors other than the blocked integrins. Iron loading indeed upregulated CCR-2 and CXCR-2 but not PECAM-1 expression on monocytes, and resulted in increased MCP-1-dependent transendothelial migration. Furthermore, both iron-induced adhesion and transmigration could be mitigated by iron chelation. Additionally, accumulation of oxygen-derived radicals due to iron loading was evidenced.
Conclusion: We conclude that intracellular iron is an important enhancer of cytokine-mediated consecutive processes of monocyte-endothelial interactions. This finding reveals an immunomodulatory function of iron in inflammation.
The toxicity of iron is believed to originate from the reaction of a Fe(II) complex with hydrogen peroxide that produces HO". NO" slows the rate of the oxidation (as studied by stopped?flow spectrophotometry) of Fe(II) bound to citrate, atp and various aminopolycarboxylates by H2O2 by a factor between 10 and 100. The reaction of tert?butyl hydroperoxide with Fe(II) complexes is similarly inhibited by nitrogen monoxide. Generally, NO" replaces a water bound to iron(II), and the rate of oxidation is influenced by the rate of dissociation of the nitrogen monoxide from iron(II).
Iron(II) dithiocarbamate complexes are used to trap NO" in biological samples, and the resulting nitrosyliron(II)dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox-cycle and produce O2"? and HO" radicals. The electrode potentials of iron complexes of N?(dithiocarboxy)sarcosine and N?methyl?D?glucamine dithiocarbamate as measured by cyclic voltammetry alow thermodynamically redox cycling, and the rates of oxidation by dioxygen and hydrogen peroxide and reduction by ascorbate, cysteine and glutathione are such that redox cycling is an efficient process. Again, NO" inhibits the oxidation of iron(II) dtcs and iron(II) mgd by H2O2.
Hereditary hemochromatosis (HC) is the most common hereditary disease in European populations. Moreover, iron excess is a major cause of organ disease in many other pathologic conditions. The pathogenesis of HC and iron-driven tissue damage and fibrosis is still unclear.
We have used an integrated approach to investigate in animal models, hepatic stellate cells (HSCs), the key players in liver fibrosis, and human cells, the basis for deranged iron homeostasis in HC, the dynamic of iron-driven fibrogenesis as well as its prevention using iron chelators. We found that the key pathogenic event in HFE-HC is a failure of hepatocytes to produce hepcidin, an iron hormone that controls iron egress from macrophages and intestine and that Kupffer cells are dispensable for this activity. This does not apply to other forms of HC such as ferroportin-HC in which the primary event is a loss of iron export.
The resulting iron excess and oxidative stress in HC leads to activation of HSC and liver fibrosis. Active fibrogenesis in HSC is associated with loss of heme oxygenase, decreased carbon monoxide production and increased sinusoidal tone in the liver. In this settings, we discovered that iron chelators che prevent fibrogenesis and activation of HSC.
Non-transferin-bound iron (NTBI) represents forms of loosely bound iron that appear in the plasma of iron overloaded patients. A major component of NTBI, LPI (labile plasma iron), is chelatable and redox active, namely potentially toxic as it can engage in the formation of oxidizing species that can compromise the components of body fluids and cells. LPI can permeate into cells in an uncontrolled manner and lead to iron overload of critical tissues such as heart and endocrine glands and be fatal if not treated (with chelators). We recognized NTBI-LPI as a clinical parameter with both diagnostic and therapeutic value as the target of iron chelation or phlebotomy in the therapy of iron overload diseases such as thalassemia and hemochromatosis. For that purpose we developed 2 high throughput methods for detecting fluorimetrically NTBI and LPI in clinical specimens using original concepts and approaches. FeRiskTM and FeRosTM have been implemented for surveillance of NTBI and LPI in sera, respectively. The patented methods have been adapted as kits which are in the final validation stages by Aferrix, Ltd., an Israeli based company which is presently providing world-wide services to clinics and pharmaceutical companies carrying out clinical studies with novel iron chelators.
Chronic inflammation has been implemented in the pathogenesis of inflammatory diseases like atherosclerosis. Several pathogens like Chlamydia pneumoniae (Cp) and cytomegalovirus (CMV) result in inflammation and thereby are potentially artherogenic. Those infections could trigger endothelial activation, the starting point of the atherogenic inflammatory cascade. Considering the role of iron in a wide-range of infection processes, the presence of iron may complicate infection-mediated endothelial activation. In this study, we measure endothelial ICAM-1, VCAM-1 and Eselection expression using flow cytometry, as an indication of endothelial activation.
An increased number of infected endothelial cells in a monolayer population lead to a raised expression of adhesion molecules of the whole cell population, suggesting paracrine interactions. Iron additively upregulated Cp-induced VCAM-1 expression, while synergistically potentiated Cp-induced ICAM-1 expression. Together with CMV, iron also stimulated ICAM-1 and VCAM-1 expression.
Moreover, the effects of iron could be reversed by intracellular iron chelation or radical scavenging, conforming modulating effects of iron on endothelial activation after infections through an increased cellular oxidative stress. We conclude that endothelial response towards chronic infections is alterable by intracellular iron levels.
Conclusion: This finding implies that iron status in populations positive for Cp or CMV infections could be an important determinant in having increased risk of developing atherosclerosis.
Haemochromatosis leads to the hyperabsorption of iron from the diet. It is a relatively common disease in North Europe. Over a long period of time, typically 30 years, body iron accumulates in such patients to a toxic level. Toxicity can be expressed by adverse cardiac, endocrine or liver symptoms.
One possible strategy to avoid the accumulation of body iron is to introduce a biocompatible polymer in the diet, which is capable of selectively binding iron and thereby preventing its absorption. A range of such polymers have been synthesised and tested in this project and several show potential for clinical development. We have designed an extremely active iron binding polymer which inhibits gastrointestinal absorption of iron. A Swiss pharmaceutical company is currently in negotiation with King's College London concerning IP.
Not all of the non-transferrin-bound iron is redox-active. We attempted to measure the redox-active fraction of NTBI in serum samples provided by Prof. J. Marx by reducing iron(III) by ascorbate, a physiological reductant, and trapping the iron(II) with ferrozine. We found that about 1/3 of NTBI is redox-active, in general agreement with the literature. More studies are needed for a publication.
Hereditary hemochromatosis (HH) is a disorder characterized by inappropriately high intestinal iron absorption. In populations of Northern European descent, HH is most commonly caused by mutations (C282Y/H63D) in the HFE gene. We investigated the effects of dietary heme iron intake and HFE mutations on serum ferritin concentrations in a population-based random sample of 1611 women aged O50 years using analysis of covariance (ANCOVA). Higher heme iron intake was associated with significantly higher serum ferritin concentrations (Ptrend !0.001).
Also, women with the compound or C282Y homozygous genotype had significantly higher serum ferritin concentrations (geometric mean 115.2 mg/L (95% CI 81.4e162.9 mg/L) than women carrying normal alleles (geometric mean 76.6 mg/L (95% CI 72.5e80.9 mg/L).
We observed the highest serum ferritin concentrations among postmenopausal women who are compound heterozygous or C282Y homozygous, and who consume relatively high amounts of heme iron (geometric mean 183.9 mg/L (95% CI 97.2e347.8 mg/L).
Conclusion: No evidence of strong gene-nutrient interaction on serum ferritin concentrations in middle-aged women.
The reaction of iron(II) with hydrogen peroxide is normally studied by stopped-flow spectrophotometry. Given a mixing time of at best 2 ms, it is possible that the reaction proceeds via an ironoxo(IV) intermediate that decays within the mixing time. With the pulse radiolyis technique we reduced Fe(III) with hydrated electrons in the presence of hydrogen peroxide. No intermediates were observed: iron(III) was reduced within microseconds, after which the oxidation by hydrogen peroxide proceeded with a rate constant as determined by the stopped-flow technique.
Lipid peroxidation is a major contributor in the pathogenesis of atherosclerosis. Oxidized low density lipoprotein (oxLDL) is recognized by scavenger receptors on monocyte derived macrophages which attach and migrate to the intima under various chemotactic gradients. The uncontrollable uptake of oxLDL results in the formation of foam cells, which will eventually contribute to atherogenic plaque formation. Among the various pathways whereby low-density lipoprotein is oxidized is by products from the reaction between neutrophil myeloperoxidase (MPO) and H2O2. Iron and copper have also been implicated in oxLDL formation.
The aim of this study was to investigate the involvement of iron and iron chelators in the in vitro oxidation of human, native LDL. The iron chelators deferoxamine, deferiprone and the experimental iron chelator CP502 were used. Their contribution in the MPO/H2O2 and Copper(II)-catalyzed oxidation of LDL was examined. The activity of the heme containing MPO enzyme was also monitored in the presence of these compounds. 10 µmol/L deferoxamine and 30 µmol/L of each deferiprone and CP502 inhibited the rate of diene formation in both catalyzed reactions.
An increase in lag times of oxidation indicate that all three chelators act as antioxidants in the process of LDL oxidation. The enzymatic activity of MPO remained unchanged. Iron chelation can significantly inhibit LDL oxidation without affecting MPO enzymatic activity.
Conclusion: LDL oxidation is a pathway whereby iron could participate in the pathogenesis of atherosclerosis.
Redox cycling of iron is considered important for its toxicity. Reduction of a low molecular weight Fe(III)-complex followed by oxidation of the Fe(II)-complex by H2O2 may yield the reactive OH" radical (Fenton reaction). Complexation of iron that shifts the one-electron reduction potential to either far below that of reductants, such as monohydroascorbate or to far above that of peroxides, is essential to limit Fenton reactivity.
The bidentate ligand Deferiprone, or CP20, and its derivatives, CP502, CP509, supplied by Prof. R. C. Hider, and the tridentate ligand ICL670 are effective oral chelating agents for the removal of iron. The standard electrode potentials of the iron complexes, measured by cyclic voltammetry are -620 mV, -535 mV, -535 mV and -600 mV, respectively. However, at micromolar concentrations of iron and excess ligand, these values are significantly higher, and under these conditions Fe(II) should not be fully complexed.
By pulse radiolysis, we showed that after diffusion-controlled (6.4 × 10'10 M-1s-1) reduction of Fe(III)(CP20)3 by e-aq, ligand dissociation occurs with a rate constant of (8 +/- 1) x 10'3 s-1. This finding illustrates that, indeed, at dilute conditions iron(II) is not fully complexed by CP20 and could therefore be oxidised by dioxygen to produce superoxide.
The role of iron in polymorphonuclear cell (PMN, neutrophil) endothelial cell interactions has scarcely been studied. In innate immunity this is one of the key interactions in fighting and clearing out an invading pathogen. As non-transferrin bound iron (NTBI) has been detected in various patient groups, including end-stage renal disease patients, and even in healthy humans, we questioned whether NTBI could be contributing to neutrophil recruitment to the endothelium. 10µmol/L Fe(III)citrate incubation on endothelial cells for 18 hours, increased the adherence of both treated and non-treated PMN. The increased neutrophil recruitment did not proceed via the classic CD11b/CD18 and ICAM-1 interactions, instead we noted enhancement of the pro-adhesive P-selectin on HUVECs after iron incubation.
Furthermore, increased amount of lactoferrin on the phagocyte membrane was noted on iron-treated PMN. Iron dextran incubation on PMN significantly enhanced transendothelial migration at concentrations of as low as 25µg/mL, without significant enhancement of PECAM-1 expression. However, there is no different in the level of transmigration of untreated PMN through endothelial cells which had been incubated with concentrations of up to 100µg/mL iron dextran, compared to controls. In addition, iron sacharate did not enhance neutrophil transmigration, in the same way as iron dextran, although from both iron preparations, NTBI concentrations of up to 6 µmol/L were measured. In conclusion, this study reports the enhanced adhesion and transmigration of PMN trough an endothelial monolayer. Pharmacological forms of iron given in end-stage renal disease patients on dialysis could modulate the degree of PMN recruitment in inflammation.
Conclusion: This finding implicates that iron is a modulator of innate immune activation.
Cellular labile iron pools (LIPs) represent important physiological and pathophysiological parameters whole levels are indicative of cell iron status in health and disease. In iron overload conditions (thalassemia and hemochromatosis), elevated LIPs can compromise organ function, such as heart and endocrine glands. We developed novel fluorescent probes and microscopic methods for identifying labile iron in cells and organelles, tracing their levels in iron overload and assessing their involvement in iron toxicity.
Most importantly, we introduced the means for evaluating chelator efficacy in living cells by continuous inspection of LIP levels and have adapted those methods for high throughput screening of novel chelators in living cells and their compartments. The information gathered indicates that endocytosis represent a major route of labile plasma iron (LPI) (but also of chelator) entrance into cells such as hepatocytes, cardiomyocytes and macrophages. The discovery of such routes provides a new methodological platform for designing novel chelators as potential therapeutic agents targeted to particular cells, namely those that have high endocytic activity and accumulate relative large amount of LPI , such macrophages. The adaptation of the principle for high throughput screening of novel chelators is highly feasible and of potential commercial value.
Epidemiologic studies aimed at correlating coronary heart disease (CHD) with serum ferritin levels have thus far yielded inconsistent results. We hypothesized that, non-transferrin-bound iron (NTBI) that appears in individuals with overt or cryptic iron overload might be more suitable for establishing correlations with CHD. We therefore investigated the relation of NTBI, serum iron, transferrin saturation and serum ferritin with risk of CHD and acute myocardial infarction (AMI).
The cohort used comprised a population-based sample of 11,471 postmenopausal women aged 49-70 years at enrolment in 1993-1997. During a median follow-up of 4.3 years (quartile limits Q1-Q3, 3.3-5.4), 185 CHD events were identified, including 66 AMI events. We conducted a case-cohort study using all CHD cases and a random sample from the baseline cohort (n=1,134). A weighted Cox proportional hazards model was used to estimate hazard ratios for tertiles of iron parameters in relation to CHD and AMI. Adjusted hazard ratios of women in the highest NTBI tertile (range: 0.38; 3.51) as compared to the lowest (range: -2.06; -0.32) were 0.84 (95% CI: 0.61-1.16) for CHD and 0.47 (95% CI: 0.31-0.71) for AMI. The results were similar for serum iron, transferrin saturation and serum ferritin.
Conclusions: Our results show no excess risk of CHD or AMI within the highest NTBI tertile as compared to the lowest, but rather seem to demonstrate a decreased risk. Additional studies are warranted to confirm our findings.
Nontransferrin-bound iron (NTBI) has been detected in iron overload diseases. This form of iron may exert pro-oxidant effects and modulate cellular function and inflammatory response. The present study has aimed to investigate the effects of serum NTBI on monocyte adherence to endothelium.
Measured by a recently developed high-throughput fluorescence-based assay, serum NTBI was found to be higher in both homozygotes of HFE C282Y mutation of hereditary hemochromatosis (7.9 +/- 0.6 iM, n=9, P<0.001) and heterozygotes (4.0±0.5 ìM, n=8, P<0.001), compared with controls (1.6 +/-0.2 iM, n=21). The effects of these sera on monocyte adhesion and endothelial activation were examined. Adhesion of normal human monocytes to C282Y homozygote- and heterozygote-serum-treated human umbilical vein endothelial cells was higher (25.0±0.9 and 22.1 +/-0.7%, respectively) compared with controls (17.6 +/- 0.5%, both P<0.001). For the three groups combined, the expression of adhesion molecules, ICAM-1, VCAM-1, and E-selectin, was positively correlated to NTBI levels but not to the inflammatory marker C-reactive protein. Furthermore, accumulation of intracellular labile iron and oxidative radicals within the cells due to NTBI was evidenced. Finally, counteraction of NTBI-induced endothelial activation was observed using iron chelators.
Conclusion: These findings therefore identify a physiological function of NTBI in monocyte-endothelial interactions that may also contribute to the development of atherosclerosis and neurodegenerative diseases.
Four main thematics have been developed:
- Characterization of hepcidin. The discovery of the link between hepcidin and iron metabolism, achieved by our Group just before the starting of the contract, represented a major breakthrough for the understanding of iron metabolism. Indeed, numerous other studies have since led to conclude that hepcidin is the key hormonal regulator of iron status in the body. During the contract period, we showed that hepcidin expression, at the transcriptional level : i) was modulated in mice by the genetic background including gender and sex, and in humans by hepatic function, hepatic iron concentration and haemoglobin levels ; ii) was well correlated in humans with the quantification of hepcidin in urine, therefore validating this urinary quantitative approach. However, our efforts for producing a specific anti-hepcidin antibody have not yet been successful.
- Regulation of the expression of hepatic genes, including iron metabolism genes, by body iron status. Using a home-made hepatic c-DNA chips (dedicated c-DNA microarray), we were able, in the original human HepaRG cell line, to associate iron storage capacity and hepatocyte differentiation. Through a macroarray approach, we have identified Cyclin D1 overexpression in the liver of iron overloaded mice ; in view of the parallel increase in liver mass, hepatocyte ploidy and mitotic index, this Cyclin D1 overexpression could induce or reflect an abnormal progression of hepatocytes in the cell cycle.
- Determination of abnormal iron species in various pathological groups of patients. Two main types of complementary data were obtained : i) the demonstration of the presence of non-transferrin bound iron (NTBI) in the plasma of untreated HFE hemochromatosis (once transferrin saturation is above 45%) and of alcoholic cirrhotic patients ; ii) the detection of labile plasma iron (LPI), a redox active component of non-transferrin bound iron, in untreated HFE hemochromatosis and in alcoholic cirrhosis, when transferrin saturation is over 75%.
- Use of new-iron trapping agents. We showed, in liver cell cultures, that the bidentate hydroxypyridinone CP411 and the hexadentate O-trensox were more efficient than CP20 for protecting iron-related hepatocyte toxicity and for inhibiting tumor cell proliferation. Studying the link between polyamines and iron, we demonstrated that :
-- Polyamine-iron complexes exhibit an active cellular uptake through the polyamine transport system, and
-- The high antiproliferative effect of both O-trensox and the new oral iron tridentate chelator ICL670 may be the consequence of both iron and polyamine depletion.
We think that these data fulfil our main objectives which were :
- To improve our understanding of the mechanisms accounting for liver iron excess and liver cell iron toxicity at the organ, cellular and molecular levels ;
- To define (or to pave the road for defining) new genetic and biochemical markers having a diagnostic or prognostic value in iron overloaded situations ;
- To define (or to pave the road for defining) a therapeutic approach based on the use of iron-trapping agents not only for counteracting the toxicity related to cellular iron excess but also for contributing to prevent the development of hepatocellular carcinoma.
Absorption of nutrient iron is of key importance for iron metabolism and developmen of both iron deficiency and iron overload. We have identified some iron transport proteins of intestinal mucosal cells. In this project we investigated molecular and functional roles of duodenal cytochrome B (dcytb) in iron metabolism. Most important, however, was the identification of the molecule that transports heme.
Dietary heme iron is an important nutritional source of iron in carnivores and omnivores that is more readily absorbed than non-heme iron derived from vegetables and grain. Most heme is absorbed in the proximal intestine with absorptive capacity decreasing distally. We utilised a subtractive hybridization approach to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. Here we show a membrane protein named HCP1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature dependent and saturable manner. HCP1 mRNA was highly expressed in duodenum and regulated by hypoxia. HCP1 protein was iron-regulated and localised to the brush-border membrane of duodenal enterocytes in iron deficiency. Our data indicate that HCP1 is the long sought intestinal heme transporter.
A role for iron in the risk of ischaemic heart disease has been supported by in vitro and in vivo studies. We investigated whether dietary haem iron intake is associated with coronary heart disease (CHD) risk in a large population-based cohort of middle-aged women. We used data of 16 136 women aged 49 70 years at recruitment between 1993 and 1997. Follow-up was complete until 1 January 2000 and 252 newly diagnosed CHD cases were documented. Cox proportional hazards analysis was used to estimate hazard ratios of CHD for quartiles of haem iron intake, adjusted for cardiovascular and nutritional risk factors.
We strati.ed by the presence of additional cardiovascular risk factors, menstrual periods, and antioxidant intake to investigate the possibility of effect modi.cation. High dietary haem iron intake was associated with a 65% increase in CHD risk [hazard ratio (HR) 1/4 1.65; 95% con.dence interval (CI): 1.07 2.53], after adjustment for cardiovascular and nutritional risk factors. This risk was not modi.ed by additional risk factors, menstruation, or antioxidant intake.
Conclusion: The results indicate that middle-aged women with a relatively high haem iron intake have an increased risk of CHD.
Aside from the reaction of iron(II) complexes with peroxides that yield reactive radicals, the Fenton reaction, iron also catalyzes the decompositions of nitrosothiols that play a role in the homostasis of vascular tension.
The mechanisms of S-nitrosothiol transformation into paramagnetic dinitrosyl iron complexes with thiol- or non-thiol ligands, or into mononitrosyl iron complex with N-methyl-D-glucamine dithiocarbamate, catalyzed by Fe2+ ions under anaerobic conditions, were studied by monitoring EPR and optical features of the complexes and S-nitrosothiols. Kinetic investigations demonstrated the appearance of a short-living paramagnetic mononitrosyl iron complex with L-cysteine prior to formation of a stable dinitrosyl iron complex with cysteine in a solution, micromolar in Fe2+-citrate and S-nitrosocysteine and millimolar in L-cysteine at pH 7.4.
The addition of deoxyhemoglobin did not influence the process, which points to a direct interaction between S-nitrosocysteine and Fe2+ ions to yield dinitrosyl iron compounds. The reaction to form dinitrosyliron cysteine is first- and second-order in iron and S-nitrosocysteine, respectively. The third-order rate constant is (1.0+/-0.2) x 10'5 M-2s-1 (estimated from EPR results) or (2.0+/-0.1) x 10'4 M-2s-1, as estimated by optical methods. The formation of di- and mononitrosyl iron complexes from S-nitrosothiols and Fe2+ ions takes place in a concerted mechanism.
Iron is an essential mineral for the human body. It has, however, been suggested that excessive iron stores may increase the risk of cardiovascular diseases. So far, results of epidemiological studies have been inconclusive. We use data of a cohort of 11,471 Dutch postmenopausal women to investigate whether high serum ferritin concentrations increase the risk of coronary heart disease. Women were included in the study between 1993 and 1997 and were followed until January 1st 2000 for cardiovascular events.
Casecohort sampling was used to reduce costs and save valuable biologic material. Serum ferritin levels were assessed with an immuno-metric assay on all 185 coronary heart disease cases and a random sample of 1,134 of the total cohort. We used a weighted Cox proportional hazards model to estimate crude, age-adjusted and multivariate adjusted hazard ratios for serum ferritin levels in relation to coronary heart disease. The multivariate adjusted hazard ratio for the highest serum ferritin tertile compared to the lowest tertile was 0.80 (95% CI: 0.46-1.38) for coronary heart disease (CHD) and 0.62 (95% CI: 0.26-1.50) for acute myocardial infarction (AMI).
Conclusions: The results of this study do not support an association between high serum ferritin levels and coronary heart disease or acute myocardial infarction.
Iron is an essential element for the human body. It has, however, been suggested that excessive iron stores may increase the risk of vascular disease. So far, epidemiologic studies on stroke are sparse. We studied the association between iron status and stroke risk in a population-based cohort of 11 471 Dutch postmenopausal women between 49 and 70 years of age. Women were included between 1993 and 1997 and followed up until January 1, 2000, for cerebrovascular events.
We conducted a case-cohort study by using all stroke cases (n=63) and a random sample of the baseline cohort (n=1134). Serum ferritin, serum iron, and transferrin saturation were measured as markers of iron status. A weighted Cox proportional-hazards model was used to estimate crude and multivariate-adjusted hazard ratios for tertiles of different iron parameters in relation to stroke. In a multivariate model, the highest tertile of serum ferritin concentration was associated with an increased risk of stroke (hazard ratio [HR], 1.45; 95% confidence interval [CI], 0.87 to 2.42) compared with the lowest tertile. For ischemic stroke, the increase was more pronounced (HR, 2.23; 95% CI, 1.05 to 4.73) and reached statistical significance.
Conclusions: Neither serum iron nor transferrin saturation was associated with an increased stroke risk. However, higher serum ferritin concentrations in postmenopausal women are associated with an increased risk of ischemic stroke.
Although heterozygosity for the C282Y mutation in the HFE gene has been associated with an increased risk of cardiovascular events, epidemiologic studies remain inconclusive. The aim of the present study was to obtain further evidence whether or not HFE mutations are associated with risk of coronary heart disease (CHD) in middle-aged women. We used data of a cohort of 15,236 Dutch middle-aged women to investigate whether C282Y carriers and H63D carriers are at increased risk of coronary heart disease compared to non-carriers. Women were included in the study between 1993 and 1997 and were followed until January 1st 2000 for cardiovascular events. HFE genotyping was performed on all 211 coronary heart disease cases and a randomly selected sample from the baseline cohort (n=1,526).
A weighted Cox proportional hazards model was used to estimate crude, age-adjusted and multivariate adjusted hazard ratios for C282Y and H63D carriership in relation to coronary heart disease. Compared with non-carriers, those that carried the C282Y allele were not at increased risk for CHD (HR = 1.25 95% CI: 0.74-2.09). Neither did we find an association between the H63D mutation and CHD risk (HR = 0.73 95% CI: 0.43-1.24).
Conclusions: Our results are in accordance with similar studies to date, for which we present a meta-analysis. HFE mutations appear not to affect the risk of coronary heart disease.
Elevated iron stores and high plasma iron concentration have been linked to an increased risk of atherosclerosis. Iron may thereby affect the interaction of monocytes to endothelium, an initial event in the formation of atherosclerotic plaques. Addition of 10 µmol/L non transferrin-bound iron to the incubation medium caused a 2-fold increase in monocyte adhesion to human umbilical vein endothelial cells (HUVECs). A concordant increase in the expression of the following adhesion molecules was observed: vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and endothelial selectin on HUVECs as well as very late antigen-4, and lymphocyte function associated antigen-1 on monocytes.
The inclusion of either deferiprone or salicylaldehyde isonicotinoylhydrazone counteracted these effects. Intracellular iron chelation by deferoxamine was completed only after 10 hours of incubation, shown by reversal of ironquenched intracellular calcein signal, and concurrently the effects of iron were blunted. The membraneimpermeable chelator, diethylenetriamine pentaaceticacid, failed to negate iron effects, even after 48 hours of treatment. Furthermore, only membrane-permeable superoxide or hydroxyl radical scavengers were capable of preventing HUVEC activation by iron.
Conclusion: Non transferrin-bound iron increases the level of intracellular labile iron, which promotes monocyte recruitment to endothelium and may thereby contribute to the pathogenesis of atherosclerosis. Iron-induced adhesion molecule expression was observed, and this event may involve the production of oxygen radicals.
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