Does Haptoglobin Phenotype Influence Postnatal Morbidity in Preterm Neonates?

BACKGROUND: Haptoglobin (Hp) is an acute phase protein with antioxidant, bacteriostatic, and anti-inflammatory activities. Hp proteins associated with the three major phenotypes differ in their proinflammatory and anti-inflammatory action. Inflammation and oxidative stress are both involved in most pathophysiological processes in premature infants. The objective of this study was to determine whether Hp phenotype influences clinical manifestations and sepsis incidence in the premature infants. OBJECTIVE: Infants born before 35 weeks gestational age were prospectively evaluated for Hp phenotype and clinical events, including sepsis, necrotizing enterocolitis, bronchopulmonary dysplasia, intraventricular hemorrhage, and retinopathy of prematurity. The participants were observed until discharge. METHODS: A total of 122 preterm infants were enrolled in the study. Clinical events were not affected by the Hp phenotype. The expression of Hp protein was extremely low in the study population. More septic episodes were found in infants with a birth weight greater than 1,500 g, although, the difference was not statistically significant. RESULTS: Extremely low expression of Hp may explain the lack of a correlation between Hp phenotype and sepsis in preterm infants. Further research involving a larger neonatal population is required to better understand the role of the Hp phenotype in morbidity of premature infants.

Pharmacogenomic interaction between the Haptoglobin genotype and vitamin E on atherosclerotic plaque progression and stability.

OBJECTIVE: Homozygosity for a 1.7 kb intragenic duplication of the Haptoglobin (Hp) gene (Hp 2-2 genotype), present in 36% of the population, has been associated with a 2-3 fold increased incidence of atherothrombosis in individuals with Diabetes (DM) in 10 longitudinal studies compared to DM individuals not homozygous for this duplication (Hp 1-1/2-1). The increased CVD risk associated with the Hp 2-2 genotype has been shown to be prevented with vitamin E supplementation in man. We sought to determine if there was an interaction between the Hp genotype and vitamin E on atherosclerotic plaque growth and stability in a transgenic model of the Hp polymorphism. METHODS AND RESULTS: Brachiocephalic artery atherosclerotic plaque volume was serially assessed by high resolution ultrasound in 28 Hp 1-1 and 26 Hp 2-2 mice in a C57Bl/6 ApoE(-/-) background. Hp 2-2 mice had more rapid plaque growth and an increased incidence of plaque hemorrhage and rupture. Vitamin E significantly reduced plaque growth in Hp 2-2 but not in Hp 1-1 mice with a significant pharmacogenomic interaction between the Hp genotype and vitamin E on plaque growth. CONCLUSIONS: These results may help explain why vitamin E supplementation in man can prevent CVD in Hp 2-2 DM but not in non Hp 2-2 DM individuals.

Vitamin E and diabetic nephropathy in mice model and humans.

Diabetes mellitus (DM) is associated with increased oxidative stress due to elevated glucose levels in the plasma. Glucose promotes glycosylation of both plasma and cellular proteins with increased risk for vascular events. Diabetic patients suffer from a higher incidence of cardiovascular complications such as diabetic nephropathy. Haptoglobin (Hp) is an antioxidant plasma protein which binds free hemoglobin, thus preventing heme-iron mediated oxidation. Two alleles exist at the Hp gene locus (1 and 2) encoding three possible Hp genotypes that differ in their antioxidant ability, and may respond differently to vitamin E treatment. Several clinical studies to have shown that Hp 1-1 genotype is a superior antioxidant to the Hp 2-2 genotype and Hp 2-2 genotype is associated with a higher incidence of cardiovascular disease. Vitamin E was found to have beneficial effect in patient and mice with Hp 2-2 genotype. In this review we have summarized the results of our studies in patients with diabetic nephropathy treated with vitamin E and in diabetic mice with different haptoglobin genotypes.

Is the Hp 2-2 diabetic mouse model a good model to study diabetic nephropathy?

Diabetic nephropathy (DN) is the leading cause of end stage renal disease and dialysis worldwide. Despite aggressive treatment, the number of patients on hemodialysis due to type 1 and type 2 diabetes mellitus is increasing annually. The lack of reliable animal models that mimic human disease has delayed the identification of specific factors that cause or predict DN. Different investigators around the world are testing different murine models. Validation criteria for early and advanced DN, phenotypic methods, background strain have recently been developed. Establishment of an authentic mouse model of DN will undoubtedly facilitate the understanding of the underlying genetic mechanisms that contribute to the development of DN and to study new treatments. Here we describe the characteristics of our new mouse model with type 1 diabetes mellitus and different haptoglobin genotypes that can mimic human DN.

An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype.

BACKGROUND: Haptoglobin (Hp) is an abundant serum protein which binds extracorpuscular hemoglobin (Hb). Two alleles exist in humans for the Hp gene, denoted 1 and 2. Diabetic individuals with the Hp 2-2 genotype are at increased risk of developing vascular complications including heart attack, stroke, and kidney disease. Recent evidence shows that treatment with vitamin E can reduce the risk of diabetic vascular complications by as much as 50% in Hp 2-2 individuals. We sought to develop a rapid and accurate test for Hp phenotype (which is 100% concordant with the three major Hp genotypes) to facilitate widespread diagnostic testing as well as prospective clinical trials. METHODS: A monoclonal antibody raised against human Hp was shown to distinguish between the three Hp phenotypes in an enzyme linked immunosorbent assay (ELISA). Hp phenotypes obtained in over 8000 patient samples using this ELISA method were compared with those obtained by polyacrylamide gel electrophoresis or the TaqMan PCR method. RESULTS: Our analysis showed that the sensitivity and specificity of the ELISA test for Hp 2-2 phenotype is 99.0% and 98.1%, respectively. The positive predictive value and the negative predictive value for Hp 2-2 phenotype is 97.5% and 99.3%, respectively. Similar results were obtained for Hp 2-1 and Hp 1-1 phenotypes. In addition, the ELISA was determined to be more sensitive and specific than the TaqMan method. CONCLUSIONS: The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies.

Poor lysosomal membrane integrity in proximal tubule cells of haptoglobin 2-2 genotype mice with diabetes mellitus.

The haptoglobin (Hp) genotype is a major determinant of progression of nephropathy in individuals with diabetes mellitus (DM). The major function of the Hp protein is to bind and modulate the fate of extracorpuscular hemoglobin and its iron cargo. We have previously demonstrated an interaction between the Hp genotype and the DM on the accumulation of iron in renal proximal tubule cells. The primary objective of this study was to determine the intracellular localization of this iron in the proximal tubule cell and to assess its potential toxicity. Transmission electron microscopy demonstrated a marked accumulation of electron-dense deposits in the lysosomes of proximal tubules cells in Hp 2-2 DM mice. Energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to perform elemental analysis of these deposits and demonstrated that these deposits were iron rich. These deposits were associated with lysosomal membrane lipid peroxidation and loss of lysosomal membrane integrity. Vitamin E administration to Hp 2-2 DM mice resulted in a significant decrease in both intralysosomal iron-induced oxidation and lysosomal destabilization. Iron-induced renal tubular injury may play a major role in the development of diabetic nephropathy and may be a target for slowing the progression of renal disease.

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

BACKGROUND: Coronary 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. METHODS: CAC 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. RESULTS: The 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. CONCLUSIONS: Hp genotype may aid prediction of accelerated coronary atherosclerosis in subjects with type 1 diabetes.

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).

Haptoglobin phenotype in women with preeclampsia.

In pre-eclampsia, poor placentation causes both oxidative and endoplasmic reticulum stress of the placenta. The anti-oxidative protein Haptoglobin has three phenotypes: 1-1, 1-2, and 2-2. Haptoglobin 1-1 is a more potent antioxidant. Our objective was to determine whether haptoglobin 1-1 was less common in women with preeclampsia which is a disease with an oxidatives-stress component, compared to the healthy population. Haptoglobin phenotype was compared in 240 healthy and 120 preeclamptic gravida in a case-control study. Statistical analysis was performed using Chi square test. The prevalence of haptoglobin 1-1 was 13% among healthy women and 6% among preeclamptic women (P=0.049). Secondary analysis was also performed. The prevalence of haptoglobin 1-1 is higher in healthy compared to preeclamptic subjects, a finding compatible with a protective role. Haptoglobin 1-1 might have a protective role in preeclampsia. Further work is needed with more Hp 1-1 subjects before we can conclude on the possible use of Haptoglobin phenotype to assess the risk of preeclampsia.

Vasospasm after subarachnoid hemorrhage in haptoglobin 2-2 mice can be prevented with a glutathione peroxidase mimetic.

Vasospasm after subarachnoid hemorrhage (SAH) is attributable to inflammation and oxidative stress associated with extracellular hemoglobin (Hb). Haptoglobin (Hp) binds free Hb and the Hp-Hb complex is cleared by macrophages, and the Hp-2 isoform of Hp is associated with more oxidative stress and more severe vasospasm. We hypothesized that treatment with an anti-oxidant, the glutathione peroxidase mimetic SYI-2074, would reduce vasospasm after SAH in Hp-2 mice. We found that SAH induced significant vasospasm in Hp-2 mice (lumen patency reduced to 65.9%), but no vasospasm was seen in mice that received SYI-2074 after SAH (lumen patency of 98.7%). We conclude that vasospasm after SAH in Hp-2 mice can be prevented with SYI-2074, suggesting that oxidative stress contributes significantly to vasospasm.

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: 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.

Controlled delivery of nitric oxide inhibits leukocyte migration and prevents vasospasm in haptoglobin 2-2 mice after subarachnoid hemorrhage.

OBJECTIVE: Cerebral vasospasm is the leading cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH) occurs. The haptoglobin 2-2 genotype likely increases the risk for developing posthemorrhagic vasospasm, but potential treatments for vasospasm have never been tested in an animal model of this genotype. We used the nitric oxide (NO) donor diethylenetriamine (DETA)/NO incorporated into ethylene/vinyl acetate (EVAc) polymers to evaluate the efficacy of controlled NO repletion in a haptoglobin 2-2 mouse basilar artery SAH model. METHODS: Mice were randomized to 3 groups: autologous blood injection and empty polymer implantation into the subarachnoid space (n = 16); blood injection and 30% DETA/NO-EVAc implantation (n = 20); and sham operation (n = 19). At 24 hours after surgery, activity level was assessed on a 3-point scale, and basilar arteries were processed for morphometric measurements. Leukocyte extravasation was assessed by immunohistochemistry (n = 12). RESULTS: Treatment with controlled release of NO from DETA/NO-EVAc polymers after SAH resulted in a significant increase in basilar artery lumen patency (73.3% +/- 4.3% versus 96.5% +/- 4.3%, mean +/- standard error of the mean; P = 0.01), a significant improvement in activity after experimental SAH (2.14 +/- 0.14 versus 2.56 +/- 0.10 points; P = 0.025), and a significant decrease in extravasated leukocytes (21 +/- 4.55 versus 6.75 +/- 3.77 leukocytes per high-power field, untreated versus treated mice; P = 0.001). CONCLUSION: Treatment with controlled release of NO prevented posthemorrhagic vasospasm in haptoglobin 2-2 mice, and mitigated neurological deficits, suggesting that DETA/NO-EVAc would be an effective therapy in patients with a genotype that confers higher risk for vasospasm after SAH. In addition to smooth muscle relaxation, inhibition of leukocyte migration may contribute to the therapeutic mechanism of NO.

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

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. METHODS: The 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. RESULTS: In 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). CONCLUSION: In DM mice the Hp 2-2 genotype is associated with increased mortality and more severe cardiac remodeling 30 days after MI.

Haptoglobin genotype and endothelial function in diabetes mellitus: a pilot study.

Endothelial function (EnF) is impaired in patients with diabetes mellitus (DM) due in large part to an increase in oxidative stress. Haptoglobin (Hp) is a potent antioxidant protein which is encoded by two different alleles (1 and 2) with the Hp 1 protein being a superior antioxidant to the Hp 2 protein. We hypothesized that DM individuals with the Hp 2-2 genotype would have greater endothelial dysfunction as compared to DM individuals with the Hp 1-1 genotype. We studied EnF in 16 Hp 2-2, 14 Hp 1-1 DM individuals and 14 healthy subjects. DM patients' groups were matched in terms of age, cardiovascular risk factors and metabolic characteristics. EnF was assessed using post-ischemic reactive hyperemia and strain gauge plethysmography and expressed either as the maximal flow after the ischemic period or as the area under the flow-time curve (AUC). We showed that EnF indices, AUC and maximal flow, were also higher in the healthy and Hp 1-1 groups compared with Hp 2-2 genotype group (615 +/- 60 and 600 +/- 40 vs. 450 +/- 50 ml dl(-1), 29 +/- 2.6 and 25 +/- 3 vs. 14 +/- 1.8 ml min(-1) dl(-1), P < 0.003 and P < 0.05, for AUC and maximal flow, one-way ANOVA, respectively). We concluded that Hp 2-2 diabetic patients had a worse EnF than controls and Hp 1-1 diabetic subjects.

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.

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.