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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1172-1180

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1172-1180.)
© 1995 American Heart Association, Inc.

Articles

Iron Overload Augments the Development of Atherosclerotic Lesions in Rabbits

Jesús A. Araujo; Egidio L. Romano; Beatriz E. Brito; Valentín Parthé; Mirtha Romano; María Bracho; Ramón F. Montaño; José Cardier

From the Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.

Correspondence to Egidio Romano, MD, PhD, Fisiopatología, Medicina Experimental, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020-A, Venezuela.


*    Abstract
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 *Abstract
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Abstract Iron, a major oxidant in vivo, could be involved in atherosclerosis through the induction of the formation of oxidized LDL, a major atherogenic factor. This study was designed to test this hypothesis experimentally. Four groups of New Zealand White rabbits were included: iron-overloaded/hypercholesterolemic (group A, n=8), iron-overloaded (group B, n=6), hypercholesterolemic (group C, n=6), and untreated (group D, n=6). Iron overload was achieved by the intramuscular administration of 1.5 g of iron dextran divided in 30 doses. Hypercholesterolemia was produced by feeding rabbit chow enriched with 0.5% (wt/wt) cholesterol. Serum iron, ferritin, cholesterol, triglycerides, and lipoperoxides in serum were measured throughout the study. Lipoperoxides were measured at the end of the study in liver, aorta, and spleen homogenates. Aortas of groups A and C had multiple lesions; however, group A had greater lesional involvement than group C (P<.05). Lesions were not observed in rabbits fed normal chow (group D). As expected, serum iron and ferritin were above normal levels in groups A and B. Serum cholesterol increased in groups A and C. Lipoperoxides in liver and spleen homogenates of iron-overloaded rabbits were increased. Interestingly, iron deposits were seen by ultrastructural studies in the arterial walls of rabbits in groups A and B. Our study suggests that iron overload augments the formation of atherosclerotic lesions in hypercholesterolemic rabbits.


Key Words: oxidized LDL • atherosclerosis • ferritin • iron • cholesterol fed-rabbits


*    Introduction
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 *Introduction
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The hypothesis that iron deficiency would be protective against coronary heart disease (CHD) was stated by Sullivan1 in 1981 as a potential explanation for the well-known differences in CHD risk for men and women. This hypothesis suggests that the development of CHD may be related to the acquisition of iron stores.1 2 3 4 Likewise, the role of high iron stores as a risk factor for CHD has been emphasized by Salonen.5 Interest in iron as a potential risk factor for CHD has been stimulated by experiments in animals showing that iron overload increases myocardial damage caused by anoxia and reperfusion and that the use of the iron chelator deferoxamine results in a decrease in myocardial damage in several animal models.6 7 8 9 10 11 This effect may result from the ability of free iron to catalyze the formation of highly reactive radicals such as the hydroxyl radical from superoxide and hydrogen peroxide.12 Both, produced during ischemia and reperfusion,13 have a low cellular reactivity without a transition metal.14 Only hydroxyl radical has sufficient reactivity15 and would be generated by a sequence of reactions catalyzed by ferric iron.16

Substantial evidence has accumulated to support the idea that generation of free radicals might be involved in the pathogenesis of atherosclerosis.17 Consequently, iron could be involved in the pathogenesis of CHD through the promotion of oxidative modifications of LDL, increasing its atherogenic potential. The oxidation of LDL is thought to probably occur in the subendothelial layer of the arteries prone to atherosclerosis.18 In studies in vitro, oxidative modification of LDL depends on the concentrations of iron and copper and can be inhibited by metal chelators.19 20 21 Atherosclerotic lesions have recently been shown to be rich in both iron and copper, and the crater from these lesions has been found to promote lipid peroxidation inhibited by deferoxamine.22 Because of these findings, it would be feasible to postulate an association between iron and atherosclerosis and so between iron and CHD. Salonen et al23 found an elevated serum ferritin, considered to be the best estimator of body iron stores,24 to be a strong risk factor for acute myocardial infarction (AMI); they also detected a synergistic association of serum ferritin and LDL cholesterol concentration with the risk of AMI. Moreover, Salonen et al25 confirmed these findings in a longer follow-up study. Similarly, Morrison et al26 found a positive correlation between serum iron and AMI; Ascherio et al27 found that the incidence of AMI was higher among men with a higher heme iron intake than among men with a lower intake. However, Stampfer et al28 did not detect any correlation between ferritin and the risk of AMI in a preliminary analysis among US physicians. Similarly, Magnusson et al29 also did not find ferritin to be a risk factor but found an inverse relation between serum ferritin and the risk of AMI, and determined the total iron-binding capacity (TIBC) to be a strong independent negative risk factor in men. Other groups, on the basis of data from the First National Health and Nutrition Examination Survey Epidemiologic Follow-up Study,30 31 32 have not found an association between iron status, assessed by serum iron, TIBC, and transferrin saturation, and risk of AMI. An inverse relation between transferrin saturation and risk of death from cardiovascular causes was also found in another Finnish population study.33

In view of these discrepancies, the current study was designed to test experimentally the potential association between iron and atherosclerosis. We found that iron-overloaded cholesterol-fed rabbits developed more atherosclerotic lesions in their aortas than cholesterol-fed rabbits that were not iron overloaded. We think that this may be due to LDL oxidation promoted by iron dextran sequestered in the aorta intima and/or to synthesis in the liver of LDL that has a low antioxidant reserve and is thus easily oxidized in the arterial wall.


*    Methods
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Animals and Treatments
Twenty-six male New Zealand White rabbits were randomly sorted into four groups: group A, hyperferremic and hypercholesterolemic; group B, hyperferremic; group C, hypercholesterolemic; and group D, control untreated rabbits. Commercial rabbit chow having no detectable cholesterol (Alimentos Protinal) was used to prepare the diets. We prepared the cholesterol-enriched diet by dissolving cholesterol (Sigma Chemical Co) in dehydrated ethanol at 60°C and then impregnating the feed pellets with it. The ethanol was eliminated by evaporation. Ethanol determinations done by an enzymatic method (Sigma Diagnostics 333-B) showed no detectable residual alcohol in the diets. Ethanol with no cholesterol was added to the control diets and eliminated by evaporation. Either the cholesterol-enriched chow, fed to groups A and C, or the normal chow, fed to groups B and D, was given ad libitum to the rabbits over 17 weeks. Iron overload was reached in groups A and B by intramuscular injection of a total dose of 1.5 g of iron dextran (Fisons Pharmaceuticals), divided in doses of 50 mg every 3 days. Doses totaling 0.5 g were administered before the beginning of the experimental diets and the remaining 1 g was given simultaneously with the diets. Blood specimens were collected from rabbits fasted for 14 to 16 hours, at the start of iron overload (-4 weeks), at the start of the experimental diets (0 weeks), and at 4, 9, 13, and 17 weeks after the start of the experimental diets. Estimations were made of serum cholesterol, iron, ferritin, and lipoperoxides, and determinations of hematocrit and anti-dextran antibodies were done. Animals were killed after 17 weeks on diets.

Atherosclerotic Lesion Measurement
Thoracic and abdominal aortas were cut open longitudinally for macroscopic documentation of intimal lesions. Samples were taken for histological and ultrastructural studies. Abdominal aortas were used for tissue homogenates and posterior determination of lipoperoxides. Thoracic aortas were fixed in a 4% formaldehyde solution (Poly/Lem fixative, Polysciences, Inc), stained with 0.2% vol/vol Sudan Black B (Sigma Chemical Co) in 60% ethanol (Merck) for 40 minutes, and washed with 60% ethanol (Merck) three times, at 1, 2, and 12 hours. Stained lesional areas appeared black or dark gray, whereas nonlesional areas were light gray. Aortas were placed on plastic templates and luminal surfaces were photographed with black and white films. Slides were made and magnified 9 to 13 times the original size on homogeneous light plain paper. Lesional and nonlesional areas were drawn, cut, and weighed. Lesional area was estimated by calculation of the percentage of the intimal surface area affected by atherosclerotic lesions. Three parameters were estimated to make analysis more accurate: aortic arch (from the aorta root to 3 mm before the first intercostal hole), descending aorta (from 3 mm before the first intercostal hole to 3 mm after the sixth intercostal hole), and thoracic aorta (from the aorta root to 3 mm after the sixth intercostal hole).

Light and Electron Microscopy
Several portions of apparently healthy and lesioned aortic intimas from each group were processed for histological and ultrastructural examinations. For conventional histology, the tissue fragments were fixed in 4% vol/vol buffered formaldehyde (pH 7.2), embedded in paraffin, and sectioned (4 µm) for staining. Prussian blue reaction was used to detect the presence of iron complexes. Color contrast was obtained with yellow methanile and nuclear red staining. For transmission electron microscopy, fragments were fixed with 2.5% vol/vol glutaraldehyde (Polysciences, Inc) in 0.1 mol/L cacodylate buffer, pH 6.8 (Sigma Chemical Co), and postfixed in 2% wt/vol OsO4 (Polysciences, Inc). Specimens were dehydrated in a graded series of ethanol and embedded in a 1:1 mixture of Medcast and propylene oxide (Pelco, Ted Pella Inc). Polymerization was completed in a Pelco 110 flat embedding mold (Ted Pella Inc) at 60°C for no less than 48 hours. Sections were cut with an ultramicrotome (Porter Blum), placed on 300-mesh grids, counterstained with lead citrate and uranyl acetate, and examined with a transmission electron microscope (Phillips 100B).

Serum Iron
Serum iron was measured according to recommendations made by the International Committee for Standardization in Hematology (ICSH) in 1971,34 with the volumes of the reagents scaled down by a factor of three.

Serum Ferritin
We determined serum ferritin by adapting a standard enzyme-linked sandwich-type immunoassay described for human ferritin determination35 with homemade reagents. Rabbit ferritin was obtained from livers and spleens of two male New Zealand White rabbits iron-overloaded with 2 g of iron dextran, by use of a method described by the ICSH.36 Anti-rabbit ferritin antibodies were generated by immunization of a goat according to a standard protocol. An IgG-enriched fraction of antiserum was isolated by sodium sulfate precipitation.37 Anti-ferritin antibody was conjugated with HRP Type XII (Sigma Chemical Co) by use of a periodate method.38 The assay was standardized with optimum concentration of ferritin antiserum as capture antibody and anti-ferritin conjugated with HRP as indicator antibody. Microplates (Immulon, Dynatech Laboratories, Inc) were coated by incubation of ferritin antiserum diluted in 0.05 mol/L carbonate buffer, pH 9.6, for 30 minutes and were then washed with 0.15 mol/L PBS-Tween (PBST) three times. Samples of sera diluted 1:10 with 0.5% wt/vol BSA in PBST were added to the wells and incubated for 2 hours at room temperature. Plates were washed three times with PBST and incubated with the IgG anti-ferritin HRP-conjugate for 2 hours at room temperature. Color reagent was then added (OPD, Sigma Chemical Co) in 0.15 mol/L citrate phosphate buffer, pH 5.0, and 0.03% vol/vol hydrogen peroxide and incubated for 30 minutes at room temperature. Reaction was stopped by using 25% vol/vol sulfuric acid (Merck), and after 30 minutes the absorbance at 490 nm was measured with a Microplate reader EL307C (Biotek Instruments Inc). Ferritin concentration was then derived from the standard curve.

Hematocrit
Hematocrit was determined by use of the capillary microhematocrit technique in blood obtained from the marginal artery of the ear.

Serum Cholesterol
Serum cholesterol was determined by use of the method of Bowman and Wolf39 with proportions reduced by 2.5 times the values described for the original method.

Determination of Serum Lipid Peroxide Content
We determined the content of serum lipid peroxides by analyzing thiobarbituric acid–reactive substances (TBARS) and expressing them as malondialdehyde equivalents using the method of Yagi40 with reduced proportions. In brief, 20 µL serum was added to 2 mL 40 mmol/L H2SO4 (Merck), then 0.25 mL 10% wt/vol phosphotungstic acid (Sigma Chemical Co) was added and mixed. The mixture was centrifuged at 3000 rpm for 10 minutes, the supernatant was discarded, and the sediment was mixed with 1 mL 40 mmol/L H2SO4 and 0.15 mL 10% wt/vol phosphotungstic acid. The mixture was centrifuged at 3000 rpm for 10 minutes. The sediment was suspended in 2 mL distilled water, and 0.5 mL 0.33% wt/vol thiobarbituric acid reagent (Sigma Chemical Co) was added. The mixture was heated for 60 minutes at 95°C in a water bath, then 2.5 mL n-butanol (Sigma Chemical Co) was added and the mixture was vigorously shaken. The butanol layer was taken after centrifugation at 3000 rpm for 15 minutes and absorbance was taken for fluorometric measurement at 553 nm with 515-nm excitation by an Aminco-Bowman spectrofluorometer (American Instrument Company Inc).

Determination of Lipid Peroxide Content in Tissue Homogenates
Abdominal aorta and samples of spleen and liver were promptly excised after the animals were killed and were weighed and then chilled in ice-cold PBS. After being washed with PBS, tissue homogenates were prepared in a ratio of 1 g of wet tissue to 9 mL of PBS by use of a glass homogenizer. Homogenates were stored at -40°C until analysis. When needed, specimens were thawed and TBARS were determined as described above.

Anti-Dextran Antibodies
The presence of anti-dextran antibodies was investigated by the immunoenzymatic method described by Kao and Sharon.41 Iron dextran or dextran with a molecular weight of 40 000 was used as coating antigen.

Anti-Dextran Cellular Proliferative Assays
At the 17th week of the diet, blood mononuclear cells (MNC) of rabbits from groups A and B were isolated following the technique suggested by Walstra et al.42 Spleen MNC were isolated by use of the same method on a cellular suspension prepared by mechanical disruption of spleen in RPMI-1640 culture media. Rabbit blood or spleen MNC were then tested in standard proliferative assays with concanavalin A, iron dextran, and dextran used as mitogens. The proliferative response was estimated by measurement of the [3H]thymidine uptake.

Statistics
All data were expressed as mean±SD. Data were evaluated statistically by unpaired Mann-Whitney test. A probability value less than .05 was taken as the level of statistical significance.


*    Results
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 *Results
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Evaluation of Atherosclerotic Lesions
Cholesterol-fed rabbits (groups A and C) had gross intimal lesions in the whole extension of the aorta that were more pronounced in the ascending and arch portions and in the branching points of large and intercostal arteries. Involvement of other vessels such as the primitive carotid, subclavian, iliac, and even pulmonary arteries was also seen. One rabbit in group B, and none in group D, also had a small single lesion in the aortic arch. A comparison of the percentage of areas with lesions between groups A and C for three portions of the aortas is presented in Fig 1Down. It can be seen that group A rabbits, on the average, had greater lesional areas than group C rabbits. Group A had an estimated lesion area of 18.47±15.33% that was statistically significant (P=.0256) compared with 5.67±4.70% in group C in the descending aorta. Similarly, in the aortic arch, group A had an estimated lesion area of 73.80±18.37%, compared with 60.88±16.00% in group C. In the whole thoracic aorta, group A had an estimated lesion area of 46.93±16.13% compared with 37.08±13.05% in group C. Even though group A had greater estimated lesion areas than group C in the aortic arch and thoracic aorta, the differences were not statistically significant (P=.0903 and .1474, respectively). In view of the global tendency observed and the statistically significant difference detected between groups A and C in the descending aorta, it can be concluded that among cholesterol-fed rabbits, those that were iron overloaded developed more atherosclerotic lesions than those that were not.



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  		Figure 1. Comparison of the percentages of the areas affected by lesions in rabbits from the iron-loaded hypercholesterolemic group (A) and the hypercholesterolemic group (C). *P<.05 for the descending aorta portion.

Light and Electron Microscopy
An interesting finding was the detection of iron deposits, both intracellular and extracellular, in sections of aortas from animals that were iron overloaded (groups A and B). These deposits were not found in aortas of nontreated rabbits. Fig 2Down represents histology features of atherosclerotic lesions in groups A and C. Fig 2Down depicts the histological appearance of the aorta wall of a group C rabbit showing an atheroma and surrounding normal tissue (top left) and an advanced stage of lesion with cell proliferation, mononuclear cell infiltration, and iron complexes stained by Prussian blue, typically found in group A (top right). Similar iron complexes were found in group B rabbits, especially in hyperpigmented regions typically located around branching of intercostal and large arteries. However, no atherosclerotic lesions were found in these points. Fig 2Down (bottom) shows the presence of iron complexes stained by Prussian blue inside a macrophage and also in the extracellular space in a group A rabbit.




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  		Figure 2. Photomicrographs show paraffin-embedded sections of lesions of iron-loaded hypercholesterolemic rabbits stained with yellow methanile and nuclear fast red. Iron deposits were stained with Prussian blue. Top left, typical atherosclerotic lesion observed in hypercholesterolemic rabbits. Photomicrographs at top right and at bottom left show lesions observed in iron-loaded hypercholesterolemic rabbits. Note the blue-stained material corresponding to iron deposits associated with foam cells and with extracellular matrix (top right). At higher magnification (bottom left) iron deposits are seen inside macrophages.

Electron microscopy revealed typical foam cells in sections of aortas from rabbits in groups A and C (Fig 3ADown), as well as hyperdense material inside foam cells in aortas of group A rabbits (Fig 3BDown) and in both the cytosol and the interior of well-defined vesicles (Fig 3CDown). Sometimes such hyperdense material was found in apparent association with lipids in vacuoles in the interior of macrophages (Fig 3CDown). Hyperdense material was also found extracellularly in aortas of rabbits from groups A and B (Fig 3DDown), and also as myelin-like organized structures (not shown). These aggregates of hyperdense material were not found in aortas of rabbits from groups C and D. Because of the exclusive finding of these aggregates in iron-overloaded rabbits and in view of the correspondence with anatomic areas shown to have iron complexes by Prussian blue reaction, it suggests that this hyperdense material corresponds to iron-complex aggregates.



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  		Figure 3. Representative electron photomicrographs from rabbit aortas show typical foam cell seen in sections of hypercholesterolemic rabbits from groups A (iron-loaded) and C (A); foam cell with lipid vacuole and with vacuole containing myelin-like hyperdense material from a group A rabbit (B); higher magnification of a macrophage showing lipid vacuole, one also containing hyperdense material (arrow) (C); and hyperdense material associated with extracellular matrix (arrow) (D).

Serum Iron
Basal serum iron was an average of 149.6±33.46 µg/dL, ranging from 98 to 237 µg/dL. In iron-overloaded animals (groups A and B), blood samples were taken 72 hours after the last iron dextran dose. In both groups iron levels increased progressively to approximately 1200 µg/dL (group A, 1263.71±110.15 µg/dL; group B, 1163±291.47 µg/dL), measured at the end of the iron-loading protocol corresponding to week 9 of the diets, as shown in Fig 4Down. Serum iron returned to basal levels 4 weeks after the iron overload was finished (week 13 of the diets). Serum iron levels of nontreated animals (groups C and D) remained unchanged during the study. As expected, serum iron levels were not affected by the type of diet administered; there were no differences between groups A and B or between groups C and D.



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  		Figure 4. Average serum iron concentration during the study in the four groups of rabbits: A, iron-loaded hypercholesterolemic; B, iron-loaded; C, hypercholesterolemic; and D, normal. Week 0 indicates the time in which the high-cholesterol diet was started. Iron overload protocol started at week -4 and ended at week 9.

Serum Ferritin
The immunoenzymatic assay developed to determine rabbit serum ferritin levels had a lower limit of detection of 8 µg/L. Basal serum ferritin levels ranged from 8 to 95 µg/L, with an average of 31.19±25.78 µg/L. Serum ferritin levels for the control rabbits (group D) fell below detection limits after a few weeks of blood sampling. In iron-overloaded animals, serum ferritin levels were progressively raised to 17.42±14.16 times (group A) and 6.77±6.48 times (group B) their basal levels by week 9 of the diets (72 hours after the iron-overload protocol was finished). Afterwards the levels decreased, as shown in Fig 5Down. In fact, levels in group A reached 302.12±131.20 µg/L and in group B they reached 123.33±40.70 µg/L by the week 9 of the diets. A striking feature was the difference between groups A and B in the pattern of serum ferritin. Although serum ferritin levels were gradually augmented in both groups while iron dextran was being administered and then decreased when iron was stopped, the increase was much more pronounced in group A than in group B, and whereas group B levels returned to almost basal levels by the end of the study, group A levels remained significantly high. Similarly, group C (cholesterol-fed rabbits) had higher levels than group D (normal control). Because there were no differences between groups A and B or between groups C and D except for the type of diet administered, it may be deduced that in some way the cholesterol-enriched diet influenced serum ferritin levels.



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  		Figure 5. Bar graphs show absolute serum ferritin levels (left y axis) and line plot showing values relative to basal levels (right y axis) in group A (hypercholesterolemic and hyperferremic) rabbits and group B (hyperferremic) rabbits at -4 weeks (beginning of iron overload), 0 weeks (beginning of diets), and 4, 9, 13, and 17 weeks on diets. Values shown are the means.

Serum Cholesterol
Basal serum cholesterol was on average 1.83±0.43 mmol/L, ranging from 0.83 to 2.64 mmol/L. Iron-overloaded and standard diet–fed rabbits (group B) had serum cholesterol levels that remained below 2.72 mmol/L, as in the untreated control group (group D). Cholesterol-fed rabbits, whether iron-overloaded or not, had serum cholesterol levels around 15.52 mmol/L as soon as 4 weeks after the start of the diet, levels that remained unchanged until the end of the study, oscillating between 14.97 and 16.63 mmol/L. Iron overload did not influence blood cholesterol levels, because there were no differences between groups A and C or between groups B and D.

Lipid Peroxides
Levels of lipid peroxides in liver, spleen, and aorta homogenates were determined by measuring TBARS levels and expressed in nmol malondialdehyde/100 mg wet weight. Results are shown in Fig 6Down. Liver homogenate TBARS levels in cholesterol-fed rabbits (group A, 27.21±19.36 nmol/100 mg; group C, 4.36±5.14 nmol/100 mg) were significantly higher (P=.0013 for group A versus group D and P=.0040 for group C versus group D) than control group levels (group D, 0.74±0.22 nmol/100 mg); however, iron-overloaded rabbits (group A) had much higher levels than rabbits that were not treated (P=.0013 for group A versus group C). Standard diet–fed and iron-overloaded rabbits (group B, 10.41±3.07 nmol/100 mg) had higher levels (P=.0040 for group B versus group D) than the control group as well. Although group A had higher levels than group B, they were not statistically significant (P=.0745). It may be concluded that iron overload stimulated generation of lipid peroxides in liver.



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  		Figure 6. Bar graphs show lipid peroxides in tissue homogenates of liver, spleen, and aorta of group A (hypercholesterolemic and hyperferremic), group B (hyperferremic), group C (hypercholesterolemic), and group D (control) rabbits. Thiobarbituric acid–reactive substances (TBARS) were determined as described in "Methods" and values shown are the means. Tissue homogenates were prepared at week 17 on diets immediately after the animals were killed. *P<.05.

Spleen homogenate TBARS levels in control group D were 2.68±1.13 nmol/100 mg. Group A rabbits (11.98±5.86 nm/100mg) had significantly higher levels than group D rabbits (P=.0051). Although TBARS levels in group B (6.18±6.20 nmol/100 mg) and group C (5.90±5.91 nmol/100 mg) were greater than in group D, they were not statistically significant (P=.4206 and P=.2143, respectively). Interestingly, group A TBARS levels were significantly higher than group C levels (P=.0367) but not significantly higher than those of group B rabbits (P=.1010). Thus, it is concluded that iron overload also stimulated generation of lipoperoxides in the spleen parenchyma.

In aorta homogenates there were no statistically significant differences in TBARS levels among the four groups. Here, although levels in group A were considerably higher (5.65±5.67 nmol/100 mg) than in the control group (2.25±0.13 nmol/100 mg), this difference did not reach statistical significance (P=.5364) because of the great individual variability observed.

In serum TBARS levels, although there were important intragroup and intergroup variations during the study, there were no consistent and significant differences because these variations were not related to either the diet or the iron overload.

Hematocrit
Basal hematocrit levels were, on average, .39±.03, ranging from .33 to .43. Hematocrits of standard diet–fed rabbits (groups B and D) remained unchanged during the study. However, in cholesterol-fed rabbits hematocrit decreased progressively and significantly (data not shown). Thus, the development of hypercholesterolemia was accompanied by a decrease in hematocrit levels.

Anti-Dextran Immune Response
Because iron overload was accomplished by administration of iron dextran complexes, it was important to rule out the potential immune response that might have been elicited against iron dextran and particularly against immunogenic epitopes present in the dextran portion. It is well known that the pathogenesis of atherosclerosis has a core of inflammatory and immune elements43 ; if an immune response against dextran epitopes had been elicited, it could be argued that immune mechanisms may have been involved. However, no anti-dextran antibodies in the sera of iron-overloaded rabbits (groups A and B) were detected. Similarly, none of the animals had significant cellular proliferative responses upon evaluation of either blood MNC or spleen MNC at the week 17 of the diet (data not shown). Therefore, it seems that in our study no cellular or humoral anti-dextran immune response was elicited by the iron dextran administration.


*    Discussion
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 *Discussion
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Salonen and coworkers23 25 recently reported that randomly selected Finnish men between the ages of 42 and 60 years who had serum ferritin levels over 200 µg/L and who were followed up from 1984 to 1991 had a 2.2-fold adjusted risk of AMI compared with men with lower serum ferritin levels. These reports sparked a great interest in the possibility that high iron stores may be a risk factor for CHD. In a small cross-population study comparing data from 10 countries, Lauffer44 found that CHD mortality rates correlated strongly with the product of hepatic storage iron and serum cholesterol concentration in men (r=.72) but only weakly in women (r=.38). Indeed, these findings support the hypothesis made by Sullivan1 in 1981 that the lower incidence of CHD in premenopausal women compared with men and postmenopausal women45 46 47 can be explained by the presence of higher levels of stored iron in the latter two groups. The new iron paradigm of CHD was later postulated to explain the effect of cholestyramine on coronary mortality, the primary prevention of myocardial infarction by aspirin, the impact of fish oils on the reduction of the incidence of CHD through inhibition of iron absorption or gastrointestinal blood loss,3 and the beneficial effects of exercise on cardiovascular disease mortality by exercise-induced reductions in iron levels.48 The confirmation or rejection of this hypothesis has remarkable importance because it would influence iron-enrichment policies for the population's food supply, blood donation programs, and even reconsideration about what are considered normal levels for iron stores and several hematologic parameters. More recently, conflicting evidence has been found in relation to iron stores and CHD risk. Walker et al49 did not find this association in South African black and Indian populations. Stampfer et al,28 in a preliminary analysis among US physicians that included 238 cases of myocardial infarction, did not find any correlation between serum ferritin and the risk of AMI. Magnusson et al,29 in a prospective study of 2036 randomly selected Icelandic men and women aged 25 to 74 years who were followed up for 8.5 years, detected a strong negative relation between TIBC and the risk of AMI after considering age and classic cardiovascular risk factors. These results, showing low TIBC rather than ferritin to be the risk factor, would not support the idea that a high iron store per se is an important determinant of CHD as suggested by Salonen and coworkers. However, iron would continue to be considered an important factor because accumulation of free iron in the subendothelial space is likely to be the critical step in the pathogenesis of atherosclerosis, and TIBC may be a more reliable predictor for the accumulation of free iron in the vessel wall than the total iron stores.29 Giles et al30 did not find any association between TIBC and CHD; Liao et al31 found that serum iron was inversely associated with the risk of AMI in women but not in men, and Sempos et al32 found an association between transferrin saturation and increased risk of CHD or AMI. Serum ferritin is the single best indicator of iron stores that is practical to measure in a population,24 50 because transferrin saturation and TIBC are limited by their diurnal variation and the potential influence of other factors such as infection, inflammation, malignancy, and liver disease.51 Therefore, it is not accurate to compare results obtained by use of different parameters to estimate iron stores.

In experimental animal models, iron overload has been found to increase myocardial damage caused by anoxia and reperfusion, and the use of the iron chelator deferoxamine resulted in a decrease in myocardial damage.6 7 8 9 10 11 This effect may result from the ability of free iron to catalyze the formation of highly reactive radicals such as the hydroxyl radical15 16 from superoxide and hydrogen peroxide12 produced during ischemia and reperfusion.13 There is evidence that supports the hypothesis that free radicals could be involved in the pathogenesis of atherosclerosis.17 Iron may play a role in the pathogenesis of atherosclerosis through the induction of oxidative modifications of LDL. Indeed, there are many lines of evidence to show that oxidized LDL is generated in vivo.17 Oxidized LDL has been proven to be atherogenic by several mechanisms.52 53 Oxidative modification of LDL in vitro is absolutely dependent on low concentrations of copper or iron ions; it is completely inhibited by metal chelators such as EDTA.9 10 11 Recently, iron and copper have been found in atherosclerotic lesions. The content of these lesions was shown to stimulate the peroxidation of rat liver microsomes, and this oxidation was inhibited by deferoxamine.22 It has been hypothesized that iron present in atherosclerotic lesions may stimulate LDL oxidation. Therefore, iron stores might be correlated to CHD because higher iron stores would be accompanied by higher arterial wall deposits. Experimental evidence for such a hypothesis is lacking.

Cholesterol-fed animals (groups A and C) developed atherosclerotic lesions all over their aortas, but most significantly in the aortic arch and in the branching points of intercostal and large arteries. However, animals that were treated with iron (group A) developed more lesions in the descending aorta (P=.0256) than those that were not (group C), as shown in Fig 2Up. The differences seen in the aortic root and in the entire thoracic aorta were not significant. This could be due to the large effect observed in the aortic root because of the strong hemodynamic stress in the area, preventing evidence of statistical significance. In fact, the aortic roots of some rabbits were nearly 100% affected. In any case, the tendencies in the aortic root and the entire thoracic aorta and the significant differences in the descending aorta allow the conclusion that atherosclerotic lesion development was significantly greater in group A than in group C. When iron dextran was administered to normocholesterolemic rabbits (group B), there were no effects on atherosclerotic lesion development because group B and group D (control) rabbits did not develop lesions.

In the present study, cholesterol was dissolved in ethanol to prepare the cholesterol-enriched diet; ethanol was afterward removed by evaporation. This method eliminates the additional factor to be considered in atherogenesis in animal models when cholesterol is dissolved in vegetable oil to prepare the enriched diets.

As expected, iron dextran treatment resulted in an increase in body iron stores, as shown by an increase in serum ferritin levels in group A and group B rabbits. However, serum ferritin levels in group A rabbits were augmented much more significantly than those in group B rabbits. It is known that elevated serum ferritin may result from chronic inflammatory processes54 because of transfer of iron from transferrin to ferritin. It may be hypothesized that higher ferritin levels observed in group A rabbits and, although at much lower levels, in group C with respect to group D reflected not only iron stores but also an acute phase component. Thus, experimental hypercholesterolemia in rabbits may be associated with chronic inflammation.

Light and electron microscopy examination allowed the visualization of iron aggregates in the arterial intima of rabbits treated with iron dextran. The iron dextran that entered the arterial wall could associate with the negatively charged arterial proteoglycans of the extracellular matrix by means of electrostatic interactions. This association would allow the development and retention of iron aggregates. Some of these aggregates could be taken up by macrophages. The presence of iron aggregates in the arterial intima might explain the greater development of atherosclerotic lesions in group A rabbits. Iron aggregates would stimulate oxidative modifications in LDL in transit through the arterial wall. Camejo et al55 and Olson et al56 suggest that positive residues (the amino acids arginine and lysine) of apo B would interact with the negative charges of glycosaminoglycans from arterial proteoglycans, rendering LDL more susceptible to oxidation.57 If iron dextran interacts with arterial proteoglycans, these proteoglycans could work as encounter platforms between iron dextran and LDL, helping interactions between each other. Further studies are necessary to investigate these hypotheses.

Additionally, it may be postulated than iron dextran induces a pro-oxidant environment in hepatic and spleen parenchymas. Linpisarn et al58 found that iron dextran added to normal liver homogenates accelerated malondialdehyde production. In the present study it was found that iron dextran–treated rabbits had higher TBARS levels in liver homogenates. Thus, we suggest that iron treatment results in a pro-oxidative liver environment that would influence lipoprotein synthesis. Resultant LDL would have a decreased antioxidant reserve and would be more susceptible to oxidation in the arterial wall when exposed to oxidative stress. Supporting this theory is the result obtained with probucol. This lipophilic agent with great antioxidant potential, transported within the LDL particle, can dramatically protect LDL from in vitro–mediated oxidative modification59 and can ameliorate atherosclerosis in hypercholesterolemic rabbits.60 61 We did not observe an increase of serum lipid peroxides as a result of iron dextran treatment. This is in agreement with previous observations52 suggesting that it is most likely that oxidative modification of LDL occurs primarily in the intima in microdomains protected from the various antioxidants found in plasma and in the extracellular space.

We could not find either anti-dextran antibodies or a significant anti-dextran cellular proliferative response in blood and spleen mononuclear cells. This rules out the possibility of the participation of dextran-mediated immune mechanisms in the potentiation of atherosclerotic lesions.

In conclusion, our results are in agreement with the hypothesis of a positive association between iron and atherosclerosis. However, we are aware of the limitations of the experimental animal model that we used and of the artificial way iron overload was achieved. Thus, further studies are needed to solve the present controversy in relation to the role of iron stores in atherogenesis in humans.

Received December 8, 1994; accepted May 3, 1995.


*    References
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