Haptoglobin is an abundant hemoglobin-binding protein present in the plasma. The function of haptoglobin is primarily to determine the fate of hemoglobin released from red blood cells after either intravascular or extravascular hemolysis. There are two common alleles at the Hp genetic locus denoted 1 and 2. There are functional differences between the Hp 1 and Hp 2 protein products in protecting against hemoglobin-driven oxidative stress that appear to have important clinical significance. In particular, individuals with the Hp 2-2 genotype and diabetes mellitus appear to be at significantly higher risk of microvascular and macrovascular complications. A pharmacogenomic strategy of administering high dose antioxidants specifically to Hp 2-2 DM individuals may be clinically effective.

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-Pharmacogenomics is a key component of personalized medicine. The Israel Cardiovascular Events Reduction with Vitamin E Study, a prospective placebo-controlled study, recently demonstrated that vitamin E could dramatically reduce CVD in individuals with diabetes and the haptoglobin (Hp) 2-2 genotype (40% of diabetic individuals). However, because of the large number of clinical trials that failed to demonstrate benefit from vitamin E coupled with the lack of a mechanistic explanation for why vitamin E should be beneficial only in diabetic individuals with the Hp 2-2 genotype, enthusiasm for this pharmacogenomic paradigm has been limited. In this study, we sought to provide such a mechanistic explanation based on the hypothesis that the Hp 2-2 genotype and diabetes interact to promote HDL oxidative modification and dysfunction.RESEARCH DESIGN AND METHODS-Hb and lipid peroxides were assessed in HDL isolated from diabetic individuals or mice with the Hp 1-1 or Hp 2-2 genotypes. HDL function was assessed based on its ability to promote cholesterol efflux from macrophages. A crossover placebo-controlled study in Hp 2-2 diabetic humans and in Hp 1-1 and Hp 2-2 diabetic mice assessed the ability of vitamin E to favorably modify these structural and functional parameters.RESULTS-Hb and lipid peroxides associated with HDL were increased and HDL function was impaired in Hp 2-2 diabetic individuals and mice. Vitamin E decreased oxidative modification of HDL and improved HDL function in Hp 2-2 diabetes but had no effect in Hp 1-1 diabetes.CONCLUSIONS-Vitamin E significantly improves the quality of HDL in Hp 2-2 diabetic individuals.

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...

Inducible nitric oxide synthase (iNOS) is expressed by the liver in a number of physiological and pathophysiological conditions. The aim of this study was to investigate the relationship between the diabetic state, iNOS and oxidative stress in the rat liver and isolated hepatocytes. Hepatic iNOS expression and activity was measured in both healthy and streptozotocin-induced diabetic rats and determined in hepatocytes in the presence and absence of insulin. Cu/Zn superoxide dismutase (SOD) and phosphatidylinositol-3-kinase (PI3K) were also measured. In a separate experiment lasting 3 weeks, diabetic rats received either no treatment, two daily injections of insulin or aminoguanidine in the drinking water. Diabetes led to increased activity (45%) and expression (70%) of liver iNOS, an effect that was attenuated by insulin treatment both in vitro and in whole animals. Hepatocyte iNOS expression increased by 56%. Hepatic SOD expression was elevated in the diabetic state, but activity levels were similar to healthy controls. Insulin treatment in vivo led to increased enzyme activity but expression was not modified. Levels of PI3K protein were significantly lower in diabetic rats while insulin treatment markedly increased expression. Aminoguanidine did not inhibit hepatic iNOS in this study. Glycemic control via insulin administration was able to downregulate enhanced hepatic iNOS activity and expression in the liver observed in the diabetic state and improve SOD activity, responses that can potentially reduce the free radical damage associated with diabetes.

In man the haptoglobin (Hp) gene is polymorphic with two common alleles denoted 1 and 2.1 We have recently demonstrated in multiple independent longitudinal studies that there is an up to 500% increase in the incidence of cardiovascular disease in individuals with the Hp 2-2 genotype and diabetes mellitus (DM).2 -6 The normal function of Hp is to bind extracorpuscular hemoglobin (Hb) and promote its clearance after it is released into either the blood secondary to red cell intravascular hemolysis (6 g per day in a normal man) or into the extravascular compartment after hemorrhage (i.e., after hemorrhage into the atherosclerotic plaque).1 We and others have previously demonstrated that the Hp 1 allele protein product is superior to the Hp 2 allele protein product in blocking Hb induced oxidation of multiple substrates including LDL and HDL.7 -9 The major pathway for clearance of the Hp-Hb complex from the blood as well as from the atherosclerotic plaque is via the monocyte/ macrophage CD163 receptor.10 We have demonstrated that the Hp 1-Hb complex is cleared more rapidly by the CD163 receptor than the Hp 2-Hb complex. 11 Furthermore, the amount of the CD163 receptor expressed on monocyte/macrophages is decreased in Hp 2-2 and DM individuals. 12 In this study we set out to test the hypothesis that the Hp genotype regulates the turnover and oxidative activity of Hb in the atherosclerotic plaque.All protocols were approved by the Animal Care and Use Committee of the Technion. The characterization and generation of Hp 1 and Hp 2 mice in a C57Bl/6 Apo E−/− genetic background has been previously described in detail. 13 Briefly, the murine Hp 1 allele is the wild type murine Hp allele. The Hp 2 allele exists only in man and murine Hp 2 allele was genetically engineered in vitro and its insertion targeted at the Hp locus by homologous recombination. Previous characterization of these mice has demonstrated that there are no differences between Hp 1 and Hp 2 mice in Hp concentration, total cholesterol and HDL cholesterol.Plaque iron in frozen 5 mm sections of brachiocephalic arteries from Hp 1 or Hp 2 Apo E−/− mice was detected and quantified using Perl's stain as previously described. Immunofluorescent identification of hemoglobin in the atherosclerotic plaque was performed in frozen 5 mm sections of brachiocephalic arteries from Hp 1 or Hp 2 Apo E−/− mice. Hemoglobin was identified with a rabbit antiserum to mouse hemoglobin (MP Biochemicals) using a Cy3 conjugated secondary antibody (Jackson Immunoresearch) for antigen detection. As a control for specificity of the fluorescent signal from Hb, sections were also incubated in

These results support a direct linkage between diabetic vascular disease and the Hp genotype. These Hp-modified mice may serve as a platform on which to test a variety of pharmacological agents in order to decrease diabetic vascular disease.

Background: We have recently demonstrated in man that a functional allelic polymorphism in the Haptoglobin (Hp) gene plays a major role in determining survival and congestive heart failure after myocardial infarction (MI). We sought to recapitulate the effect of Hp type on outcomes and cardiac remodeling after MI in transgenic mice.