Rachel Miller-Lotan

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.

Objective—Clinical trials of vitamin E have failed to demonstrate a decrease in cardiovascular events. However, these studies did not address possible benefit to subgroups with increased oxidative stress. Haptoglobin (Hp), a major antioxidant protein, is a determinant of cardiovascular events in patients with Type 2 diabetes mellitus (DM). The Hp gene is polymorphic with 2 common alleles, 1 and 2. The Hp 2 allelic protein product provides inferior antioxidant protection compared with the Hp 1 allelic product. We sought to test the hypothesis that vitamin E could reduce cardiovascular events in DM individuals with the Hp 2-2 genotype, a subgroup that comprises 2% to 3% of the general population. Methods and Results—1434 DM individuals ≥55 years of age with the Hp 2-2 genotype were randomized to vitamin E (400 U/d) or placebo. The primary composite outcome was myocardial infarction, stroke, and cardiovascular death. At the first evaluation of events, 18 months after initiating the study, the primary outcome was significantly reduced in individuals receiving vitamin E (2.2%) compared with placebo (4.7%; P=0.01) and led to early termination of the study. Conclusions—Vitamin E supplementation appears to reduce cardiovascular events in individuals with DM and the Hp 2-2 genotype (ClinicalTrials.gov NCT00220831).

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- and diabetes-dependent differences in iron-mediated oxidative stress in vitro and in vivo.

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 genotype–dependent 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.

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.

Consumption of wonderful variety pomegranate juice and extract by diabetic patients increases paraoxonase 1 association with high-density lipoprotein and stimulates its catalytic activities.

Association of paraoxonase 1 (PON1) with high-density lipoprotein (HDL) stabilizes the enzyme. In diabetic patients, PON1 dissociates from HDL and, as a consequence, is less biologically active. Our aim was to investigate the effects of Wonderful variety pomegranate juice (WPJ) and pomegranate polyphenol extract (WPOMxl) consumption on PON1 association with HDL in diabetic patients. Thirty patients with type 2 diabetes mellitus participated in the study. Ten male patients and 10 female patients received concentrated WPJ (50 mL/day for 4 weeks), while another group of 10 male patients received WPOMxl (5 mL/day for 6 weeks). There were no significant effects of WPJ or WPOMxl consumption on fasting blood glucose or hemoglobin A1c levels. After 4 weeks of WPJ consumption by male patients, basal serum oxidative stress was significantly decreased by 35%, whereas serum concentrations of thiol groups significantly increased by 25%. Moreover, HDL-associated PON1 arylesterase, paraoxonase, and lactonase activities increased significantly after WPJ consumption by 34-45%, as compared to the baseline levels. PON1 protein binding to HDL was significantly increased by 30% following WPJ consumption, and the enzyme became more stable. In male patients that consumed WPOMxl and in female patients that consumed PJ, a similar pattern was observed, although to a lesser extent. We conclude that WPJ as well as WPOMxl consumption by diabetic patients does not worsen their diabetic parameters. Furthermore, WPJ as well as WPOMxl consumption contribute to PON1 stabilization, increased association with HDL, and enhanced catalytic activities. These beneficial effects of pomegranate consumption on serum PON1 stability and activity could lead to retardation of atherosclerosis development in diabetic patients.

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 reduces cardiovascular disease in individuals with diabetes mellitus and the haptoglobin 2-2 genotype

AIMS Individuals with both diabetes mellitus (DM) and the Haptoglobin (Hp) 2-2 genotype are at increased risk of cardiovascular disease. As the antioxidant function of the Hp 2-2 protein is impaired, we sought to test the pharmacogenomic hypothesis that antioxidant vitamin E supplementation would provide cardiovascular protection to Hp 2-2 DM individuals. MATERIALS & METHODS We determined the Hp genotype on DM participants from two trials (HOPE and ICARE) and assessed the effect of vitamin E by Hp genotype on their common prespecified outcome, the composite of stroke, myocardial infarction and cardiovascular death. Data was analyzed with a fixed-effect model. These results were input into a simulation model, the Evidence Based Medicine Integrator, in order to estimate their long-term implications in a real-world population from Kaiser Permanente (CA, USA). RESULTS Meta-analysis of the two trials demonstrated a significant overall reduction in the composite end point in Hp 2-2 DM individuals with vitamin E (odds ratio: 0.58; 95% CI: 0.40-0.86; p = 0.006). There was a statistically significant interaction between the Hp genotype and vitamin E on the composite end point. In these trials, Hp typing of 69 DM individuals and treating those with the Hp 2-2 with vitamin E prevented one myocardial infarct, stroke or cardiovascular death. Lifelong administration of vitamin E to Hp 2-2 DM individuals in the Kaiser population would increase their life expectancy by 3 years. CONCLUSION A pharmacogenomic strategy of screening DM individuals for the Hp genotype and treating those with Hp 2-2 with vitamin E appears to be highly clinically effective.

Haptoglobin 2-2 Genotype Determines Chronic Vasospasm After Experimental Subarachnoid Hemorrhage

Background and Purpose— Chronic cerebral arterial vasospasm is the leading cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). Not all cases of SAH, however, develop chronic vasospasm. Inflammation, specifically leukocyte–endothelial cell interactions, appears to be critical in vasospasm development. Haptoglobin (Hp) is a serum protein that limits the extent of inflammation after a hemorrhagic event. An individual’s Hp genotype may predict the severity of the inflammatory response during a hemorrhagic event, and consequently modulate the risk for vasospasm. Methods— Sixty mice (Hp 1-1, n=30; Hp 2-2, n=30) underwent injection of either autologous blood or normal saline solution into the cisterna magna. An additional 30 mice (15 per genotype) served as controls. The extent and manifestations of vasospasm were assessed by measuring lumen patency, quantifying activity levels, and counting the number of vessel-infiltrated macrophages/neutrophils at 24 hours after injection, which corresponds to the time of peak vasospasm in mice. Results— Genetically modified Hp 2-2 mice with SAH had significantly lower basilar artery lumen patencies (mean±SEM; 52.9±1.9% vs 82.3±1.3%; P<0.01), reduced activity levels (0.8±0.3 vs 2.4±0.2; P<0.01), and increased macrophage/neutrophil counts in the subarachnoid space (31.2±6.3 vs 8.8±1.7, P<0.01) as compared with wild-type Hp 1-1 mice. Conclusions— These findings suggest that the Hp 2-2 genotype is critical for the development of severe vasospasm, which typically occurs 24 hours after SAH in mice.

Hypothesis--haptoglobin genotype and diabetic nephropathy.

Vascular complications cause serious morbidity in patients with diabetes mellitus. Three such complications are nephropathy, retinopathy and accelerated atherosclerotic cardiovascular disease. There is currently scant evidence of a genetic marker that predicts which patients will have vascular complications. Oxidative stress has an important role in the development of diabetic vascular complications. Haptoglobin (Hp) is a hemoglobin-binding protein that has a major role in protecting against heme-driven oxidative stress. There are two common alleles for Hp (1 and 2) and, therefore, three common Hp genotypes: Hp 1-1, Hp 2-1, and Hp 2-2. The antioxidant protection provided by Hp is genotype-dependent; the protein encoded by Hp 1-1 provides superior antioxidant protection compared with that encoded by Hp 2-2. We have shown that diabetic individuals with Hp 2-2 are more likely to develop nephropathy, retinopathy, and cardiovascular disease than those with the Hp 2-1 or Hp 1-1 genotypes.

Haptoglobin genotype predicts development of coronary artery calcification in a prospective cohort of patients with type 1 diabetes

BackgroundCoronary artery disease has been linked with genotypes for haptoglobin (Hp) which modulates extracorpuscular hemoglobin. We hypothesized that the Hp genotype would predict progression of coronary artery calcification (CAC), a marker of subclinical atherosclerosis.MethodsCAC was measured three times in six years among 436 subjects with type 1 diabetes and 526 control subjects participating in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. Hp typing was performed on plasma samples by polyacrylamide gel electrophoresis.ResultsThe Hp 2-2 genotype predicted development of significant CAC only in subjects with diabetes who were free of CAC at baseline (OR: 1.95, 95% CI: 1.07-3.56, p = 0.03), compared to those without the Hp 2-2 genotype, controlling for age, sex, blood pressure and HDL-cholesterol. Hp 2 appeared to have an allele-dose effect on development of CAC. Hp genotype did not predict CAC progression in individuals without diabetes.ConclusionsHp genotype may aid prediction of accelerated coronary atherosclerosis in subjects with type 1 diabetes.