Roy Asaf

Haptoglobin: Basic and Clinical Aspects

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.

Haptoglobin Genotype Is a Determinant of Iron, Lipid Peroxidation, and Macrophage Accumulation in the Atherosclerotic Plaque

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−/− Hp 2-2 mice were associated with increased iron (P=0.008), lipid peroxidation (4-hydroxynonenal and ceroid) and macrophage accumulation (P=0.03) as compared with plaques from C57Bl/6 ApoE−/− Hp 1-1 mice. Conclusions—Increased iron, lipid peroxidation and macrophage accumulation in ApoE−/− Hp 2-2 plaques suggests that the Hp genotype plays a critical role in the oxidative and inflammatory response to intraplaque hemorrhage.

Correction of HDL Dysfunction in Individuals With Diabetes and the Haptoglobin 2-2 Genotype

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.

Vitamin E therapy results in a reduction in HDL function in individuals with diabetes and the haptoglobin 2-1 genotype

OBJECTIVE Vitamin E provides cardiovascular protection to individuals with diabetes and the haptoglobin 2-2 genotype but appears to increase cardiovascular risk in individuals with diabetes and the haptoglobin 2-1 genotype. We have previously demonstrated that the haptoglobin protein is associated with HDL and that HDL function and its oxidative modification are haptoglobin genotype dependent. We set out to test the hypothesis that the pharmacogenetic interaction between the haptoglobin genotype on cardiovascular risk might be secondary to a parallel interaction between the haptoglobin genotype and vitamin E on HDL function. RESEARCH DESIGN AND METHODS Fifty-nine individuals with diabetes and the haptoglobin 2-1 or 2-2 genotypes were studied in a double-blind placebo controlled crossover design. Participants were treated with either vitamin E (400IU) or placebo for 3 months and crossed over for an equivalent duration. Serum was collected at baseline and after the completion of each treatment. HDL functionality as well as HDL associated markers of oxidation and inflammation were measured after each interval in HDL purified from the cohort. RESULTS Compared to placebo, vitamin E significantly increased HDL function in haptoglobin 2-2 but significantly decreased HDL function in haptoglobin 2-1. This pharmacogenetic interaction was paralleled by similar non-significant trends in HDL associated lipid peroxides, glutathione peroxidase, and inflammatory cargo. CONCLUSION There exists a pharmacogenetic interaction between the haptoglobin genotype and vitamin E on HDL function (clinicaltrials.gov NCT01113671).

Pharmacogenomic effect of vitamin E on kidney structure and function in transgenic mice with the haptoglobin 2-2 genotype and diabetes mellitus

Polymorphic loci regulating oxidative stress are potential susceptibility genes for diabetic nephropathy (DN). Haptoglobin (Hp) is an antioxidant protein which serves to protect against oxidative stress induced by extracorpuscular hemoglobin. There are two alleles at the Hp locus, 1 and 2. The Hp 1 protein is a superior antioxidant to the Hp 2 protein. The Hp 2 allele has been associated with increased prevalence of DN and appears to be associated with a more rapid progression to end-stage renal disease. We sought to recapitulate this association between Hp genotype and DN in mice genetically modified at the Hp locus. We assessed morphometric, histologic, and functional parameters involved in the development and progression of DN in mice with diabetes mellitus (DM) with either the Hp 2-2 or Hp 1-1 genotype. Morphometric analysis demonstrated that glomerular and proximal tubular hypertrophy were significantly increased in Hp 2-2 DM mice. Histological analysis demonstrated that Hp 2-2 DM mice had significantly more collagen type IV, smooth muscle actin, and increased renal iron deposition. Studies of renal function demonstrated creatinine clearance time and albuminuria were increased in Hp 2-2 DM mice. Vitamin E provided significant protection against the development of functional and histological features characteristic of DN to Hp 2-2 DM but not to Hp 1-1 DM mice. These studies serve to strengthen the association between the Hp 2-2 genotype and diabetic renal disease and suggest a pharmacogenomic interaction may exist between the Hp genotype and vitamin E.

Retinal capillary basement membrane thickness in diabetic mice genetically modified at the haptoglobin locus

Individuals with diabetes mellitus (DM) homozygous for the haptoglobin (Hp) 1 allele are at decreased risk of retinopathy as compared to DM individuals with the Hp 2 allele. We sought to recapitulate these findings in DM mice genetically modified at the Hp locus.

Increased renal hypertrophy in diabetic mice genetically modified at the haptoglobin locus

The human haptoglobin (Hp) gene is polymorphic with two functional classes of alleles, denoted 1 and 2. We have demonstrated in three longitudinal studies and several cross‐sectional studies that the Hp genotype is an independent risk factor for diabetic vascular disease. These studies have presented a compelling argument that diabetic individuals homozygous for the Hp 1 allele are at decreased risk of vascular complications as compared to diabetic individuals with the Hp 2 allele.

Haptoglobin genotype is a determinant of survival and cardiac remodeling after myocardial infarction in diabetic mice.

BackgroundWe 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.MethodsThe Hp 2 allele exists only in man. Wild type C57Bl/6 mice carry the Hp 1 allele with high homology to the human Hp 1 allele. We genetically engineered a murine Hp 2 allele and targeted its insertion by homologous recombination to the murine Hp locus to create Hp 2 mice. Diabetes Mellitus (DM) was induced with streptozotocin. MI was produced by occlusion of the left anterior descending artery in DM C57Bl/6 mice carrying the Hp 1 or Hp 2 allele. MI size was determined with TTC staining. Left ventricular (LV) function and dimensions were assessed by 2-dimensional echocardiography.ResultsIn the absence of DM, Hp 1-1 and Hp 2-2 mice had similar LV dimensions and LV function. MI size was similar in DM Hp 1-1 and 2-2 mice 24 hours after MI (50.2 ± 2.1%and 46.9 ± 5.5%, respectively, p = 0.6). However, DM Hp 1-1 mice had a significantly lower mortality rate than DM Hp 2-2 mice 30 days after MI (HR 0.41, 95% CI (0.19–0.95), p = 0.037 by log rank). LV chamber dimensions were significantly increased in DM Hp 2-2 mice compared to DM Hp 1-1 mice 30 days after MI (0.196 ± 0.01 cm2 vs. 0.163 ± 0.01 cm2, respectively; p = 0.029).ConclusionIn DM mice the Hp 2-2 genotype is associated with increased mortality and more severe cardiac remodeling 30 days after MI.

BXT-51072 and the Prevention of Myocardial Ischemia-Reperfusion Injury

Oxidative stress is responsible for myocardial injury occurring after ischemia and reperfusion (IR) and has been shown to be modulated by the Haptoglobin (Hp) genotype. In this manuscript we demonstrate that the antioxidant BXT - 51072, a glutathione peroxidase synthetic mimic, provides protection against IR injury in a Hp genotype dependent fashion.