CLINICAL RESEARCH: ATHEROSCLEROSIS
Haptoglobin Genotype Is a Major Determinant of the Amount of Iron in the Human Atherosclerotic Plaque
Pedro R. Moreno, MD, FACC*,
K. Raman Purushothaman, MD*,
Meera Purushothaman, PhD*,
Paul Muntner, PhD*,
Nina S. Levy, PhD ,
Valentin Fuster, MD, PhD, FACC*,
John T. Fallon, MD, PhD ,
Patrick A. Lento, MD,
Aaron Winterstern, BS and
Andrew P. Levy, MD, PhD, FACC,*
* Department of Medicine (Cardiology), Mount Sinai Medical Center, New York, New York
Department of Pathology, Mount Sinai Medical Center, New York, New York
Rappaport Faculty of Medicine, Technion Israel Institute of Technology, Haifa, Israel
Manuscript received April 7, 2008;
revised manuscript received June 5, 2008,
accepted June 6, 2008.
* Reprint requests and correspondence: Dr. Andrew P. Levy, Technion Faculty of Medicine, POB 9649, Haifa, Israel 31096 (Email: alevy{at}tx.technion.ac.il).
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Abstract
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Objectives: We sought to test the hypothesis that haptoglobin (Hp) genotype is a determinant of the amount of iron in the atherosclerotic plaque.
Background: In atherosclerotic lesions, intraplaque hemorrhage releases free hemoglobin (Hb), whose incorporated iron can act as an oxidant and inflammatory stimulus. These effects are antagonized by Hp, which binds free Hb and facilitates its clearance from the plaque. The Hp gene has 2 alleles (1 and 2), giving rise to 3 genotypes: Hp 1-1, Hp 2-1, and Hp 2-2. We previously hypothesized that Hp 2-2 individuals with diabetes mellitus (DM) have impaired clearance of Hb and its iron cargo from the plaque.
Methods: We identified the presence or absence of Perl's iron stain in 189 plaques obtained from 37 decedents at autopsy.
Results: Among DM, the prevalence of Perl's iron stain was increased in Hp 2-2 compared with that seen in Hp 1-1 or 2-1 (46.2% vs. 11.8%). After accounting for the within-decedent correlation of plaques, the prevalence ratio of Perl's iron stain associated with Hp 2-2 was 3.97 (95% confidence interval: 1.38 to 11.5; p = 0.025). In non-DM plaques, there was a nonsignificant trend towards a higher prevalence of iron staining in Hp 2-2 compared with that in Hp 1-1 or 2-1 (26.8% vs. 11.1%; prevalence ratio =2.40 [95% confidence interval: 0.81 to 7.09]; p = 0.114).
Conclusions: These data support an impaired clearance of Hb from plaques in Hp 2-2 individuals, particularly in DM. The higher prevalence of plaque iron in Hp 2-2 DM individuals may contribute to the increased incidence of atherothrombotic events in these patients.
Key Words: atherosclerotic plaque • iron • intraplaque hemorrhage • diabetes mellitus
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Abbreviations and Acronyms
| | DM = diabetes mellitus | | Hb = hemoglobin | | Hp = haptoglobin | | TCFA = thin-cap fibroatheromas |
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Intraplaque hemorrhage from neovessels is evolving as a major factor in the progression of atherothrombosis, especially in patients with diabetes mellitus (DM) (1). Extravasation of red blood cells into the atherosclerotic plaque results in the release of free hemoglobin (Hb), whose incorporated iron can act as an oxidant (2). These effects are antagonized by haptoglobin (Hp) (3). Haptoglobin acts as an antioxidant by binding Hb, resulting not only in the stabilization of Hb-derived iron in the heme pocket of Hb (3), but also in the rapid clearance of Hb by the CD163 Hp-Hb receptor (4).
The Hp gene is polymorphic, with 2 common alleles, 1 and 2 (3). This polymorphism is predictive of the risk of major adverse cardiovascular events in DM, as demonstrated in 5 independent longitudinal studies (5–9). Specifically, Hp 2-2 DM individuals have been found to have a 2- to 5-fold increase in major adverse cardiovascular events compared with that seen in Hp 2-1 or 1-1 DM individuals (5–9).
These epidemiological findings may reflect functional differences between the Hp allelic products. The Hp 2 protein is inferior to the Hp 1 protein in blocking oxidative damage induced by Hb (10). Furthermore, the Hp 1-1-Hb complex is cleared more rapidly than the Hp 2-2-Hb complex, particularly in DM (11,12). As a result of the impaired clearance of Hb and its iron cargo, we hypothesized that atherosclerotic plaques from Hp 2-2 DM individuals would have increased iron. In order to test this hypothesis, we assessed Perl's iron in human atherosclerotic plaques stratified by Hp genotype and DM.
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Methods
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Ethical approval.
The study was approved by the investigational review board at the Mount Sinai Medical Center in New York.
Characteristics of decedents used in this study.
Thirty-seven male cadavers were used in these studies (21 with and 16 without DM). All DM decedents in this analysis had type II DM. A fasting glucose of >125 mg/dl on 2 different occasions was used as the definition of DM. All DM decedents were known to have DM (mean duration 8 ± 4 years) and were being treated with sulfonylureas. None of the decedents were insulin dependent. The prevalence of cardiac death was similar in DM and non-DM decedents (33% vs. 31%). Furthermore, the prevalence of cardiac death was similar by Hp genotype within the DM group (31% for Hp 1-1/2-1 vs. 30% for Hp 2-2). Noncardiac death was due to respiratory, gastrointestinal, or renal causes.
Determination of Hp genotype.
When fresh frozen tissue was available (18 cadavers), Hp genotype was determined by polymerase chain reaction (13). When only formalin-fixed paraffin embedded tissue was available (19 cadavers), Hp genotype was determined by examining the Hp protein (liver tissue) by Western blot. Paraffin was first removed with dewaxing solution (Dexmor Scientific, Tel Aviv, Israel) and then incubated in a Tris buffered 2% sodium dodecyl sulfate solution at 65°C for 2 h. The supernatant was then removed and electrophoresed on a 15% sodium dodecyl sulfate polyacrylamide gel. The proteins were then electro-transferred to Immobilon (Tamara Ltd., Mevaseret Zion, Israel) PVDF and blotted against a rabbit polyclonal serum to Hp. The band pattern on the Western blot determined the Hp type (9 kD for the Hp 1 alpha chain and 16 kD for the Hp 2 alpha chain). The appearance of both a 9 and 16 kD band was defined as Hp 2-1 while the appearance of only the 9 or 16 kD bands defined Hp 1-1 or 2-2 genotypes, respectively.
Selection and characterization of plaques.
Full-thickness aortic wall histological sections from 189 lesions were taken sequentially at autopsy from 37 decedents. The aorta was slit open longitudinally, and the intima was washed with saline and then examined visually. On gross examination, aortas had diffuse atherosclerotic lesions with variably distanced spaces between plaques. No aneurysms were observed. A 20-cm aortic segment from the lower thoracic aorta extending into the abdominal aorta above the renal arteries was selected. Individual atherosclerotic plaques raised above the surface with a long axis >0.75 cm were studied (14). A 1.0-cm long and 0.5-cm wide sample with an edge of normal tissue was obtained for each plaque. The minimum distance between plaques was 0.5 cm. Based on the Virmani classification scheme (15), only fibroatheromas were included in this analysis. Cap thickness was assessed using ocular micrometry (16). Of the 189 fibroatheromas, 29 (15.6%) had a fibrous cap thickness of <65 µm and were classified as thin-cap fibroatheromas (TCFA). The distribution of TCFA was similar in DM and non-DM plaques (15% vs. 14%). Furthermore, within the DM group, the prevalence of TCFA was similar by Hp genotype (TCFA prevalence of 12% in Hp 1-1/2-1 vs. 15% in Hp 2-2). Plaques with gross intraplaque hemorrhage were excluded from analysis. There were no plaque dissections or ruptures in any of the plaques analyzed.
Perl's stain.
Perl's iron stain (17) was performed by a technician who did not have knowledge of the Hp type. Perl's stain was dichotomized as absent or present. The location of iron staining was predominately located in the extracellular matrix and less frequently in the necrotic core.
Statistical analysis.
Most decedents had multiple plaques; the mean number of plaques per decedent was 5.4 (range 1 to 10). Analyses were conducted separately for DM and non-DM plaques,. Generalized estimating equations were used to determine the association between Hp 2-2 and the presence of Perl's iron stain accounting for the presence of multiple plaques within each decedent. Specifically, the outcome was the presence of Perl's iron stain, the exposure was having Hp 2-2 (Hp 1-1 or 2-1 was considered unexposed), and each decedent was modeled as the clustering variable. Due to the high prevalence of Perl's iron stain, the generalized estimating equation used a log-binomial link function in order to calculate prevalence ratios (18), in lieu of odds ratios, as the measure of association. Analyses were conducted using STATA 10 (Stata Corp., College Station, Texas).
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Results
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Perl's iron stain was performed on 189 plaques from 37 decedents and dichotomized as absent (i.e., no stain) or present for all analyses. In patients with DM, we analyzed 52 plaques from 9 aortas with the Hp 2-2 genotype, and 51 plaques from 12 aortas with the Hp 1-1 or 2-1 genotype. In patients without DM, we analyzed 41 plaques from 7 aortas with the Hp 2-2 genotype, and 45 plaques from 9 aortas with the Hp 1-1 or 2-1 genotype. In DM plaques, the prevalence of Perl's iron stain was 4-fold greater in Hp 2-2 compared with that in Hp 1-1 or 2-1 (Fig. 1). In non-DM plaques, the prevalence of iron staining was 2-fold greater in Hp 2-2 compared with that in Hp 2-1 or 1-1. After taking into account the within-decedent correlation of plaque characteristics, the prevalence ratio for Perl's iron staining comparing Hp 2-2 with Hp 1-1 and 2-1 was 3.97 (95% confidence interval: 1.38 to 11.5; p = 0.025). The analogous prevalence ratio for non-DM plaques was 2.40 (95% confidence interval: 0.81 to 7.09; p = 0.114).
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Figure 1 Percentage of Plaques With Perl's Iron Stain by Hp Genotype and DM Status
In patients with diabetes mellitus (DM), 24 of 52 plaques from 9 haptoglobin (Hp) 2-2 decedents compared with 6 of 51 plaques from 12 Hp 1-1 or 2-1 decedents demonstrated Perl's iron stain. In patients without DM, 11 of 41 plaques from 7 Hp 2-2 decedents compared with 5 of 45 plaques from 9 Hp 1-1 or 2-1 decedents demonstrated Perl's iron stain.
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Discussion
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In this study, we document a higher prevalence of Perl's iron stain in atherosclerotic plaques from humans with the Hp 2-2 genotype, particularly in the setting of DM. These data are consistent with our previously formulated hypothesis that there is an impaired clearance of Hb-derived iron in Hp 2-2 DM plaques due both to a decreased ability of CD163 to mediate uptake of Hp 2-2-Hb and to marked down-regulation of CD163 in Hp 2-2 DM (11,19).
The relatively small number of total cadavers from whom plaques were sampled is a major limitation of this study. Nonetheless, the extensive mechanistic data that can account for the observed results as well as the fact that the same results were obtained in Hp 2-2 transgenic mice (17) provides reassurance that the results we report here are not due to chance.
We have recently demonstrated that Hb-derived iron in the atherosclerotic plaque is redox active (20) and that free Hb or Hp-Hb can scavenge nitric oxide (21). Therefore, the increased iron demonstrated in these studies would be expected to result in increased oxidative stress and reduced nitric oxide bioavailability within the human atherosclerotic plaque that may contribute to the increased incidence of atherothrombotic events in DM individuals with the Hp 2-2 genotype (5–9).
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Footnotes
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This work was supported by grants from the Israel Science Foundation, U.S.-Israel Binational Science Foundation, and the Kennedy Leigh Trust (to Dr. Levy) and by the Mount Sinai Hospital Norman Levy Foundation. Dr. Levy is a consultant for Synvista Therapeutics.
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References
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1. Moreno PR, Fuster V. New aspects in the pathogenesis of diabetic atherothrombosis J Am Coll Cardiol 2004;44:2293-2300.[Abstract/Free Full Text]2. Levy AP, Moreno PR. Intraplaque hemorrhage Curr Mol Med 2006;6:479-488.[CrossRef][Web of Science][Medline] 3. Bowman BH, Kurosky A. Haptoglobin: the evolutionary product of duplication, unequal crossing over, and point mutation Adv Hum Genet 1982;12:189-261.[Web of Science][Medline] 4. Kristiansen M, Graversen JH, Jacobsen C, et al. Identification of the hemoglobin scavenger receptor Nature 2001;409:198-201.[CrossRef][Web of Science][Medline] 5. Levy AP, Hochberg I, Jablonski K, et al. Haptoglobin phenotype and the risk of cardiovascular disease in individuals with diabetes: the Strong Heart study J Am Coll Cardiol 2002;40:1984-1990.[Abstract/Free Full Text] 6. Roguin A, Koch W, Kastrati A, Aronson D, Schomig A, Levy AP. Haptoglobin genotype is predictive of major adverse cardiac events in the one year period after PTCA in individuals with diabetes Diabetes Care 2003;26:2628-2631.[Abstract/Free Full Text] 7. Suleiman M, Aronson D, Asleh R, et al. Haptoglobin polymorphism predicts 30-day mortality and heart failure in patients with diabetes and acute myocardial infarction Diabetes 2005;19:2802-2806. 8. Milman U, Blum S, Shapira C, et al. Vitamin E supplementation reduces cardiovascular events in a subgroup of middle-aged individuals with both type 2 diabetes mellitus and the haptoglobin 2-2 genotype: a prospective, double-blinded clinical trial Art Thromb Vasc Biol 2008;28:341-347.[Abstract/Free Full Text] 9. Costacou T, Ferrell RE, Orchard TJ. Haptoglobin genotype: a determinant of cardiovascular complication risk in type I diabetes Diabetes 2008;57:1702-1706.[Abstract/Free Full Text] 10. Frank M, Lache O, Enav B, et al. Structure/function analysis of the anti-oxidant properties of haptoglobin Blood 2001;98:3693-3698.[Abstract/Free Full Text] 11. Asleh R, Marsh S, Shiltruck M, et al. Genetically determined heterogeneity in hemoglobin scavenging and susceptibility to diabetic cardiovascular disease Circ Res 2003;92:1193-1200.[Abstract/Free Full Text] 12. Asleh R, Blum S, Kalet-Litman S, et al. Correction of HDL dysfunction in individuals with diabetes and the Haptoglobin 2-2 genotype Diabetes 2008Jul 3 [E-pub ahead of print]. 13. Koch W, Latz W, Eichinger M, et al. Genotyping of common haptoglobin polymorphism Hp1/2 based on the polymerase chain reaction Clin Chem 2002;48:1377-1382.[Abstract/Free Full Text] 14. Moreno PR, Purushothaman KR, Fuster V, Echeverri D, Truszczynska H, O'Connor WN. Tunica media and plaque neovascularization is increased in ruptured atherosclerotic lesions of the human aorta: implications for plaque vulnerability Circulation 2004;110:2032-2038.[Abstract/Free Full Text] 15. Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions Arterioscler Thromb Vasc Biol 2000;20:1262-1275.[Free Full Text] 16. Moreno PR, Lodder RA, Purushothaman KR, Charash WE, O'Connor WN, Muller JE. Detection of lipid pool, thin fibrous cap and inflammatory cells in human aortic atherosclerotic plaques by near-infrared spectroscopy Circulation 2002;105:923-927.[Abstract/Free Full Text] 17. Levy AP, Levy JE, Kalet-Litman S, et al. Haptoglobin genotype is a determinant of iron, lipid peroxidation and macrophage accumulation in the atherosclerotic plaque Arterioscler Thromb Vasc Biol 2007;27:134-140.[Abstract/Free Full Text] 18. Behrens T, Taeger D, Wellman J, Keil U. Different methods to calculate effect estimates in cross-sectional studies. A comparison between prevalence odds ratio and prevalence ratio. Methods Inf Med 2004;43:505-509.[Web of Science][Medline] 19. Levy AP, Purosothaman KR, Levy NS, et al. Downregulation of the hemoglobin scavenger receptor in individuals with diabetes and the Hp 2-2 genotype: implications for the response to intraplaque hemorrhage and plaque vulnerability Circ Res 2007;101:106-110.[Abstract/Free Full Text] 20. Kalet-Litman S, Asleh R, Miller-Lotan R, Levy AP. The haptoglobin genotype is associated with increased redox active hemoglobin derived iron in the atherosclerotic plaque (abstr) J Am Coll Cardiol 2008;51(Suppl A):A283. 21. Azarov I, He X, Jeffers A, et al. Rate of nitric oxide scavenging by hemoglobin bound to haptoglobin Nitric Oxide 2008;18:296-302.[CrossRef][Web of Science][Medline]
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