Abstract-A major function of haptoglobin (Hp) is to bind hemoglobin (Hb) to form a stable Hp-Hb complex and thereby prevent Hb-induced oxidative tissue damage. Clearance of the Hp-Hb complex can be mediated by the monocyte/macrophage scavenger receptor CD163. We recently demonstrated that diabetic individuals homozygous for the Hp 2 allele (Hp 2-2) were at 500% greater risk of cardiovascular disease (CVD) compared with diabetic individuals homozygous for the Hp 1 allele (Hp 1-1). No differences in risk by Hp type were seen in individuals without diabetes. To understand the relationship between the Hp polymorphism and diabetic CVD, we sought to identify differences in antioxidant and scavenging functions between the Hp types and to determine how these functions were modified in diabetes. The scavenging function of Hp was assessed using rhodamine-tagged and 125 I-Hp in cell lines stably transfected with CD163 and in macrophages expressing endogenous CD163. We found that the rate of clearance of Hp 1-1-Hb by CD163 is markedly greater than that of Hp 2-2-Hb. Diabetes is associated with an increase in the nonenzymatic glycosylation of serum proteins, including Hb. The antioxidant function of Hp was assessed with glycosylated and nonglycosylated Hb. We identified a severe impairment in the ability of Hp to prevent oxidation mediated by glycosylated Hb. We propose that the specific interaction between diabetes, CVD, and Hp genotype is the result of the heightened urgency of rapidly clearing glycosylated Hb-Hp complexes from the subendothelial space before they can oxidatively modify low-density lipoprotein to atherogenic oxidized low-density lipoprotein.

Abstract-We have recently demonstrated in multiple independent population-based longitudinal and cross sectional analyses that the haptoglobin 2-2 genotype is associated with an increased risk for diabetic cardiovascular disease. The chief function of haptoglobin (Hp) is to bind to hemoglobin and thereby prevent hemoglobin-induced oxidative tissue damage. This antioxidant function of haptoglobin is mediated in part by the ability of haptoglobin to prevent the release of iron from hemoglobin on its binding. We hypothesized that there may be diabetes-and haptoglobin genotypedependent differences in the amount of catalytically active redox active iron derived from hemoglobin. We tested this hypothesis using several complementary approaches both in vitro and in vivo. First, measuring redox active iron associated with haptoglobin-hemoglobin complexes in vitro, we demonstrate a marked increase in redox active iron associated with Hp 2-2-glycohemoglobin complexes. Second, we demonstrate increased oxidative stress in tissue culture cells exposed to haptoglobin 2-2-hemoglobin complexes as opposed to haptoglobin 1-1-hemoglobin complexes, which is inhibitable by desferrioxamine by either a chelation or reduction mechanism. Third, we demonstrate marked diabetes-dependent differences in the amount of redox active iron present in the plasma of mice genetically modified expressing the Hp 2 allele as compared with the Hp 1 allele. Taken together these data implicate redox active iron in the increased susceptibility of individuals with the Hp 2 allele to diabetic vascular disease. Key Words: diabetes Ⅲ oxidative stress Ⅲ iron Ⅲ haptoglobin Ⅲ hemoglobin T he haptoglobin (Hp) gene locus on chromosome 16q22 is polymorphic with two common alleles existing in man, denoted 1 and 2. 1 We have recently established in multiple independent population-based longitudinal and cross sectional studies that the haptoglobin genotype is an independent risk factor for diabetic cardiovascular disease. 2-6 Specifically, we have demonstrated that diabetic individuals homozygous for the haptoglobin 2 allele (Hp 2-2) are at significantly greater risk of developing cardiovascular disease as compared with diabetic individuals homozygous for the haptoglobin 1 allele (Hp 1-1) with an intermediate risk being found in the heterozygote. 3 Haptoglobin is an antioxidant as a direct result of its ability to prevent hemoglobin-driven oxidation. 7 The stoichiometric binding of haptoglobin to hemoglobin not only stabilizes the heme iron moiety in hemoglobin (Hb) 8 but also promotes its scavenging by the CD163 macrophage receptor by receptormediated endocytosis. 9 We have recently demonstrated that the ability of haptoglobin to protect against hemoglobindriven oxidative injury is abrogated when hemoglobin becomes glycated, a process that is markedly accelerated in the diabetic state. 10 Glycohemoglobin-haptoglobin complexes are catalytically redox active and therefore the rate at which haptoglobin-hemoglobin complexes are cleared from the serum and extravascular ...

Objective-Intraplaque hemorrhage increases the risk of plaque rupture and thrombosis. The release of hemoglobin (Hb) from extravasated erythrocytes at the site of hemorrhage leads to iron deposition, which may increase oxidation and inflammation in the atherosclerotic plaque. The haptoglobin (Hp) protein is critical for protection against Hb-induced injury. Two common alleles exist at the Hp locus and the Hp 2 allele has been associated with increased risk of myocardial infarction. We have demonstrated decreased anti-oxidative and anti-inflammatory activity for the Hp 2 protein. We tested the hypothesis that the Hp 2-2 genotype is associated with increased oxidative and macrophage accumulation in atherosclerotic plaques. Methods and Results-The murine Hp gene is a type 1 Hp allele. We created a murine type 2 Hp allele and targeted its insertion to the Hp locus by homologous recombination. Atherosclerotic plaques from C57Bl/6 ApoE Ϫ/Ϫ Hp 2-2 mice were associated with increased iron (Pϭ0.008), lipid peroxidation (4-hydroxynonenal and ceroid) and macrophage accumulation (Pϭ0.03) as compared with plaques from C57Bl/6 ApoE Ϫ/Ϫ Hp 1-1 mice. Conclusions-Increased iron, lipid peroxidation and macrophage accumulation in ApoEϪ/Ϫ Hp 2-2 plaques suggests that the Hp genotype plays a critical role in the oxidative and inflammatory response to intraplaque hemorrhage. Key Words: atherosclerotic plaque Ⅲ hemoglobin Ⅲ inflammation Ⅲ iron Ⅲ macrophages T he major cause of acute coronary thrombosis is atherosclerotic plaque rupture and the precursor lesion has been termed the high-risk plaque. [1][2][3][4][5][6] Pathological features of highrisk plaques include a large lipid necrotic core, thin fibrous cap, inflammatory infiltrate, and intraplaque hemorrhage. [1][2][3][4][5][6] Extracorpuscular hemoglobin (Hb) released from red blood cells after intra-plaque hemorrhage represents a potent stimulus for inflammation within the plaque. It is becoming apparent that the frequency of microvascular hemorrhages has been severely underestimated and may occur in up to 40% of all advanced atherosclerotic plaques. 7 An important defense mechanism to counteract the effects of intra-plaque hemorrhage is mediated by haptoglobin (Hp), an abundant serum protein whose primary function is to bind to extracorpuscular Hb, thereby attenuating its oxidative and inflammatory potential. 8 Hp also promotes the clearance of extracorpuscular Hb via the CD163 scavenger receptor present on macrophages. 9 This scavenging pathway is the only mechanism that exists for removing free Hb released at extravascular sites, ie, at sites of hemorrhage within the atherosclerotic plaque.In humans there exist 2 classes of alleles for Hp, designated 1 and 2. The Hp polymorphism is a common polymorphism. In the western world, 16% of the population is Hp 1-1 (homozygous for the Hp 1 allele), 36% is Hp 2-2 (homozygous for the Hp 2 allele), and 48% is Hp 2-1 (heterozygote). 8 The Hp 2 allele is found only in humans. All other mammals, including higher primates have only th...

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