Molecular Insights into IQSEC2 Disease.

Recent insights into IQSEC2 disease are summarized in this review as follows: (1) Exome sequencing of IQSEC2 patient DNA has led to the identification of numerous missense mutations that delineate at least six and possibly seven essential functional domains present in the IQSEC2 gene. (2) Experiments using IQSEC2 transgenic and knockout (KO) mouse models have recapitulated the presence of autistic-like behavior and epileptic seizures in affected animals; however, seizure severity and etiology appear to vary considerably between models. (3) Studies in IQSEC2 KO mice reveal that IQSEC2 is involved in inhibitory as well as stimulatory neurotransmission. The overall picture appears to be that mutated or absent IQSEC2 arrests neuronal development, resulting in immature neuronal networks. Subsequent maturation is aberrant, leading to increased inhibition and reduced neuronal transmission. (4) The levels of Arf6-GTP remain constitutively high in IQSEC2 knockout mice despite the absence of IQSEC2 protein, indicating impaired regulation of the Arf6 guanine nucleotide exchange cycle. (5) A new therapy that has been shown to reduce the seizure burden for the IQSEC2 A350V mutation is heat treatment. Induction of the heat shock response may be responsible for this therapeutic effect.

Daily Brief Heat Therapy Reduces Seizures in A350V IQSEC2 Mice and Is Associated with Correction of AMPA Receptor-Mediated Synaptic Dysfunction.

Purposeful induction of fever for healing, including the treatment of epilepsy, was used over 2000 years ago by Hippocrates. More recently, fever has been demonstrated to rescue behavioral abnormalities in children with autism. However, the mechanism of fever benefit has remained elusive due in large part to the lack of appropriate human disease models recapitulating the fever effect. Pathological mutations in the IQSEC2 gene are frequently seen in children presenting with intellectual disability, autism and epilepsy. We recently described a murine A350V IQSEC2 disease model, which recapitulates important aspects of the human A350V IQSEC2 disease phenotype and the favorable response to a prolonged and sustained rise in body core temperature in a child with the mutation. Our goal has been to use this system to understand the mechanism of fever benefit and then develop drugs that can mimic this effect and reduce IQSEC2-associated morbidity. In this study, we first demonstrate a reduction in seizures in the mouse model following brief periods of heat therapy, similar to what was observed in a child with the mutation. We then show that brief heat therapy is associated with the correction of synaptic dysfunction in neuronal cultures of A350V mice, likely mediated by Arf6-GTP.

IQSEC2 mutation associated with epilepsy, intellectual disability, and autism results in hyperexcitability of patient-derived neurons and deficient synaptic transmission.

Mutations in the IQSEC2 gene are associated with drug-resistant, multifocal infantile and childhood epilepsy; autism; and severe intellectual disability (ID). We used induced pluripotent stem cell (iPSC) technology to obtain hippocampal neurons to investigate the neuropathology of IQSEC2-mediated disease. The neurons were characterized at three-time points during differentiation to assess developmental progression. We showed that immature IQSEC2 mutant dentate gyrus (DG) granule neurons were extremely hyperexcitable, exhibiting increased sodium and potassium currents compared to those of CRISPR-Cas9-corrected isogenic controls, and displayed dysregulation of genes involved in differentiation and development. Immature IQSEC2 mutant cultured neurons exhibited a marked reduction in the number of inhibitory neurons, which contributed further to hyperexcitability. As the mutant neurons aged, they became hypoexcitable, exhibiting reduced sodium and potassium currents and a reduction in the rate of synaptic and network activity, and showed dysregulation of genes involved in synaptic transmission and neuronal differentiation. Mature IQSEC2 mutant neurons were less viable than wild-type mature neurons and had reduced expression of surface AMPA receptors. Our studies provide mechanistic insights into severe infantile epilepsy and neurodevelopmental delay associated with this mutation and present a human model for studying IQSEC2 mutations in vitro.

Housing of A350V IQSEC2 pups at 37 °C ambient temperature prevents seizures and permits the development of social vocalizations in adulthood.

OBJECTIVES: Mutations in the human IQSEC2 gene are associated with drug-resistant epilepsy and severe behavioral dysfunction. We have focused on understanding one human IQSEC2 missense mutation (A350V) for which we have created a corresponding A350V IQSEC2 mouse model by CRISPR which demonstrates seizures when the mice are 15-20 days old and impaired social vocalizations in adulthood. We observed that a child with the A350V mutation stops having seizures when experiencing a fever of greater than 38 °C. In this study, we first sought to determine if we could recapitulate this phenomenon in A350V 15-20 day old mice using a previously established protocol to raise body temperature to 39 °C achieved by housing the mice at 37 °C. We then sought to determine if mice in whom seizure activity had been prevented as pups would develop social vocalization activity in adulthood. METHODS: 15-20 day old A350V male mice were housed either at 37 °C or 22 °C. Ultrasonic vocalizations of these mice were assessed at 8-10 weeks in response to a female stimulus. RESULTS: Housing of 15-20 day old A350V mice at 37 °C resulted in a reduction in lethal seizures to 2% (1/41) compared to 45% (48/108) in mice housed at 22 °C, p = 0.0001. Adult A350V mice who had been housed at 37 °C as pups displayed a significant improvement in the production of social vocalizations. CONCLUSION: Raising the body temperature by raising the ambient temperature might provide a means to reduce seizures associated with the A350V IQSEC2 mutation and thereby allow for an improved neurodevelopmental trajectory.

Haptoglobin Phenotype Is Associated With High-Density Lipoprotein-Bound Hemoglobin Content and Coronary Endothelial Dysfunction in Patients With Mild Nonobstructive Coronary Artery Disease.

Objective- Coronary endothelial dysfunction (ED) is an early stage of atherosclerosis and is associated with impaired high-density lipoprotein (HDL) function. A functional polymorphism at the haptoglobin (Hp) gene locus (rs72294371) has been associated with marked differences in HDL structure and function. We sought to determine whether Hp phenotype was associated with coronary ED and whether the amount of hemoglobin (Hb) tethered to HDL via Hp was Hp-type dependent and associated with ED. Approach and Results- Microvascular and epicardial coronary endothelial function was assessed in 338 individuals with nonobstructive coronary artery disease. Microvascular ED was defined as <50% change in coronary blood flow and epicardial ED as ≥20% decrease in coronary artery diameter after intracoronary acetylcholine infusion. The amount of Hb bound to HDL was measured by ELISA after HDL purification from plasma samples using immune-affinity chromatography. One hundred and seventy of the individuals in this study (50.3%) were diagnosed with microvascular ED, 143 (42.3%) with epicardial ED, and 67 (19.7%) had diabetes mellitus (DM). Hp phenotype was significantly associated with microvascular ( P=0.01) and epicardial ED ( P=0.04) among DM individuals. There was a significant and inverse correlation between the amount of HDL-bound Hb and change in coronary blood flow (r=-0.40; P<0.0001) and in coronary artery diameter (r=-0.44; P<0.0001) in response to acetylcholine infusion. Hb content of HDL was significantly increased in individuals with Hp 2-2 and DM. In a logistic regression model, Hp 2-2 phenotype was associated with microvascular ED (odds ratio, 1.9; P=0.03) and the amount of HDL-bound Hb was an independent predictor of both microvascular (odds ratio, 4.6 for each 1-SD increase; P<0.0001) and epicardial (odds ratio, 2.2; P<0.0001) ED. Conclusions- Hp phenotype is significantly associated with coronary ED in DM individuals. This association is likely related to increased Hb tethering to HDL via Hp 2-2 in DM.

IQSEC2-Associated Intellectual Disability and Autism.

Mutations in IQSEC2 cause intellectual disability (ID), which is often accompanied by seizures and autism. A number of studies have shown that IQSEC2 is an abundant protein in excitatory synapses and plays an important role in neuronal development as well as synaptic plasticity. Here, we review neuronal IQSEC2 signaling with emphasis on those aspects likely to be involved in autism. IQSEC2 is normally bound to N-methyl-D-aspartate (NMDA)-type glutamate receptors via post synaptic density protein 95 (PSD-95). Activation of NMDA receptors results in calcium ion influx and binding to calmodulin present on the IQSEC2 IQ domain. Calcium/calmodulin induces a conformational change in IQSEC2 leading to activation of the SEC7 catalytic domain. GTP is exchanged for GDP on ADP ribosylation factor 6 (ARF6). Activated ARF6 promotes downregulation of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors through a c-jun N terminal kinase (JNK)-mediated pathway. NMDA receptors, AMPA receptors, and PSD-95 are all known to be adversely affected in autism. An IQSEC2 transgenic mouse carrying a constitutively active mutation (A350V) shows autistic features and reduced levels of surface AMPA receptor subunit GluA2. Sec7 activity and AMPA receptor recycling are presented as two targets, which may respond to drug treatment in IQSEC2-associated ID and autism.

Haptoglobin genotype-dependent differences in macrophage lysosomal oxidative injury.

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.

Molecular modeling of ARF6 dysregulation caused by mutations in IQSEC2.

IQSEC2 gene mutations are associated with epilepsy, autism, and intellectual disability. The primary function IQSEC2, mediated via its Sec 7 domain, is to act as a guanine nucleotide exchange factor for ARF6. We sought to develop a molecular model, which may explain the aberrant Sec 7 activity on ARF6 of different human IQSEC2 mutations. We integrated experimental data of IQSEC2 mutants with protein structure prediction by the RaptorX server combined with molecular modeling and molecular dynamics simulations. Normally, apocalmodulin (apoCM) binds to IQSEC2 resulting in its N-terminal fragment inhibiting access of its Sec 7 domain to ARF6. An increase in Ca2+ concentration destabilizes the interaction of IQSEC2 with apoCM and removes steric hindrance of Sec 7 binding with ARF6. Mutations at amino acid residue 350 of IQSEC2 result in loss of steric hindrance of Sec 7 binding with ARF6 leading to constitutive activation of ARF6 by Sec 7. On the other hand, a mutation at amino acid residue 359 of IQSEC2 results in constitutive hindrance of Sec 7 binding to ARF6 leading to the loss of the ability of IQSEC2 to activate ARF6. These studies provide a model for dysregulation of IQSEC2 Sec 7 activity by mutant IQSEC2 proteins.Communicated by Ramaswamy H. Sarma.

Vitamin E in the prevention of cardiovascular disease: the importance of proper patient selection.

Vitamin E is a naturally occurring fat-soluble antioxidant which has been proposed as a treatment for both primary and secondary protection against cardiovascular (CV) events. Promising data from observational epidemiological studies associating higher vitamin E dietary intake with lower risk of CV events have not been validated in randomized controlled clinical trials assessing the effect of vitamin E on CV outcomes. While the pendulum of medical opinion has swung to suggest that high dose vitamin E supplements have no place in the treatment and prevention of CV disease, new data is emerging that allows identification of a specific target population for this treatment, namely patients with diabetes mellitus and the haptoglobin genotype 2-2. This review details the scientific basis and clinical evidence related to the effect of vitamin E on CV outcomes, and the importance of proper patient selection in gaining therapeutic benefit from this intervention.

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.

Haptoglobin genotype and its role in determining heme-iron mediated vascular disease.

Haptoglobin (Hp) is a hemoglobin (Hb) binding protein whose major function is to prevent heme-iron mediated oxidation. The polymorphic nature of the Hp gene results in varying levels of antioxidant function associated with the protein products. Multiple clinical studies have now determined that the Hp 2-2 genotype is associated with an increased risk of developing vascular complications in patients suffering from diabetes. The mechanism for this phenomenon is a decrease in antioxidant capability associated with the Hp 2-2 protein. Specifically, heme iron associated with the Hp2-2/Hb complex is more redox active than other Hp type complexes and has been shown in a number of systems to lead to increased levels of oxidative stress in the form of oxidized lipids and decreased lipoprotein function. In addition, Hp 2-2/Hb complexes are cleared less efficiently from the circulation, leading to a buildup of iron in the plasma and in tissues. Recent analyses from clinical studies utilizing vitamin E treatment have shown beneficial results specifically in the diabetic Hp 2-2 genotype population. The use of vitamin E in the treatment of Hp 2-2 diabetics has the potential to greatly reduce medical costs and improve quality of life in the ever-growing diabetic population.

Characterization of spontaneous seizures and EEG abnormalities in a mouse model of the human A350V IQSEC2 mutation and identification of a possible target for precision medicine based therapy.

IQSEC2 is an X-linked gene localized to the post synaptic density encoding a GTP exchange factor that regulates NMDA mediated changes in synaptic function. Mutations in the IQSEC2 gene are associated with drug resistant epilepsy, intellectual disability and autism. Precision medicine based therapeutics to treat IQSEC2 associated epilepsy requires the development and characterization of mutation specific animal models. To date no EEG recordings have been presented for any mouse model of any IQSEC2 mutation showing seizures. In this study we characterize the seizures and EEG brain wave abnormalities present in mice with a A350V IQSEC2 missense mutation that is associated with drug resistant epilepsy in man. We show that seizures are associated with a greater than 40% mortality rate in male mice and occur exclusively from post-natal day 16-20. EEG recordings of mouse pups during this window demonstrate seizures and the presence of spikes with a marked increase in delta waves. EEG recordings in adult male mice have persistent excessive slow frequency activity and spikes, but seizures were not recorded. RNAseq analysis of the hippocampi of mice prior to the development of seizures demonstrated marked abnormalities in canonical pathways involved in synaptogenesis and dendritic maturation with the most prominently dysregulated gene being that for TRH suggesting a potential target for therapy given the previous demonstration of TRH to decrease seizures in several forms of drug resistant epilepsy.

Genetics of redox systems and their relationship with cardiovascular disease.

As atherosclerosis is still one of the major causes of death in Western populations, it is important to identify those individuals who are at increased risk for the disease so that aggressive treatment may be administered as early as possible. Following the understanding that oxidative stress has a pivotal role in the development and progression of atherosclerosis, many polymorphisms in genes that are related to redox systems were examined for their association with increased risk for cardiovascular disease (CVD). Although many polymorphisms were studied, only a handful showed consistent relevance to CVD in different trials. This article focuses on six of these polymorphisms, examining their effect on the risk for CVD as well as their effect on protein expression and function. Reports regarding pharmacogenetic implications of these polymorphisms, where such exist, are discussed as well.

Reduction in seizure burden in a child with a A350V IQSEC2 mutation using heat therapy with a Jacuzzi.

A child with a A350V IQSEC2 missense mutation resulting in drug-resistant epilepsy stops having seizures when he has a fever. We demonstrate that raising the body temperature of the child using a commercial Jacuzzi dramatically reduces his seizures and appears to improve his social behavioral interactions.

Structural and functional brain-wide alterations in A350V Iqsec2 mutant mice displaying autistic-like behavior.

IQSEC2 is an X-linked gene that is associated with autism spectrum disorder (ASD), intellectual disability, and epilepsy. IQSEC2 is a postsynaptic density protein, localized on excitatory synapses as part of the NMDA receptor complex and is suggested to play a role in AMPA receptor trafficking and mediation of long-term depression. Here, we present brain-wide structural volumetric and functional connectivity characterization in a novel mouse model with a missense mutation in the IQ domain of IQSEC2 (A350V). Using high-resolution structural and functional MRI, we show that animals with the A350V mutation display increased whole-brain volume which was further found to be specific to the cerebral cortex and hippocampus. Moreover, using a data-driven approach we identify putative alterations in structure-function relations of the frontal, auditory, and visual networks in A350V mice. Examination of these alterations revealed an increase in functional connectivity between the anterior cingulate cortex and the dorsomedial striatum. We also show that corticostriatal functional connectivity is correlated with individual variability in social behavior only in A350V mice, as assessed using the three-chamber social preference test. Our results at the systems-level bridge the impact of previously reported changes in AMPA receptor trafficking to network-level disruption and impaired social behavior. Further, the A350V mouse model recapitulates similarly reported brain-wide changes in other ASD mouse models, with substantially different cellular-level pathologies that nonetheless result in similar brain-wide alterations, suggesting that novel therapeutic approaches in ASD that result in systems-level rescue will be relevant to IQSEC2 mutations.

Downregulation of the hemoglobin scavenger receptor in individuals with diabetes and the Hp 2-2 genotype: implications for the response to intraplaque hemorrhage and plaque vulnerability.

In individuals with diabetes mellitus (DM), the haptoglobin (Hp) genotype is a major determinant of susceptibility to myocardial infarction. We have proposed that this is because of DM and Hp genotype-dependent differences in the response to intraplaque hemorrhage. The macrophage hemoglobin scavenging receptor CD163 plays an essential role in the clearance of hemoglobin released from lysed red blood cells after intraplaque hemorrhage. We sought to test the hypothesis that expression of CD163 is DM and Hp genotype-dependent. CD163 was quantified in plaques by immunohistochemistry, on peripheral blood monocytes (PBMs) by FACS, and as soluble CD163 (sCD163) in plasma by ELISA. In DM plaques, despite an increase in macrophage infiltration, CD163 immunoreactivity was lower, resulting in a dramatic reduction in the percentage of macrophages expressing CD163 (27+/-2% versus 70+/-2%, P=0.0001). In individuals with DM as compared with individuals without DM, the percentage of PBMs expressing CD163 was reduced (3.7+/-0.6% versus 7.1+/-0.9%, P<0.002) whereas soluble plasma CD163 was increased (2.6+/-1.1 microg/mL versus 1.6+/-0.8 microg/mL, P<0.0005). Among DM individuals, the Hp 2-2 genotype was associated with a decrease in the percentage of PBMs expressing CD163 (2.3+/-0.5% versus 5.6+/-1.3%, P=0.01) and an increase in plasma soluble CD163 (3.0+/-0.2 microg/mL versus 2.3+/-0.2 microg/mL, P=0.04). Taken together, these results demonstrate an impaired hemoglobin clearance capacity in Hp 2-2 DM individuals and may provide the key insight explaining the increased incidence of myocardial infarction in this population.

An IQSEC2 Mutation Associated With Intellectual Disability and Autism Results in Decreased Surface AMPA Receptors.

We have recently described an A350V mutation in IQSEC2 associated with intellectual disability, autism and epilepsy. We sought to understand the molecular pathophysiology of this mutation with the goal of developing targets for drug intervention. We demonstrate here that the A350V mutation results in interference with the binding of apocalmodulin to the IQ domain of IQSEC2. We further demonstrate that this mutation results in constitutive activation of the guanine nucleotide exchange factor (GEF) activity of IQSEC2 resulting in increased production of the active form of Arf6. In a CRISPR generated mouse model of the A350V IQSEC2 mutation, we demonstrate that the surface expression of GluA2 AMPA receptors in mouse hippocampal tissue was significantly reduced in A350V IQSEC2 mutant mice compared to wild type IQSEC2 mice and that there is a significant reduction in basal synaptic transmission in the hippocampus of A350V IQSEC2 mice compared to wild type IQSEC2 mice. Finally, the A350V IQSEC2 mice demonstrated increased activity, abnormal social behavior and learning as compared to wild type IQSEC2 mice. These findings suggest a model of how the A350V mutation in IQSEC2 may mediate disease with implications for targets for drug therapy. These studies provide a paradigm for a personalized approach to precision therapy for a disease that heretofore has no therapy.

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.

Haptoglobin Genotype Is a Determinant of Hemoglobin Adducts and Vitamin E Content in HDL.

Haptoglobin (Hp) is an abundant hemoglobin- (Hb-) binding serum protein and a constituent of the HDL proteome. In man, there exists a common polymorphism at the Hp locus with two common alleles defined by the presence (Hp 2 allele) or absence (Hp 1 allele) of a 1.7 kb in-frame partial duplication of exons 3 and 4 of the Hp gene. Numerous studies have demonstrated that the Hp 2-2 genotype is associated with a 3-5-fold increase in vascular disease among individuals with diabetes mellitus (DM). Increased Hp-Hb complex has been shown to be associated with the HDL of Hp 2-2 DM individuals. Hb-associated HDL has been proposed to result in the oxidation of HDL and the consumption of antioxidants in HDL, such as vitamin E, rendering the HDL further susceptible to oxidation. In this study, we set out to identify proteins which become cross-linked to Hb in HDL and to measure vitamin E in HDL as a function of the Hp genotype. We report on the identification of a novel 72 kd Hb reactive species which is cross-linked to HDL and demonstrate that vitamin E in HDL is decreased in Hp 2-2 DM individuals.

Haptoglobin genotype modulates the balance of Th1/Th2 cytokines produced by macrophages exposed to free hemoglobin.

The haptoglobin genotype has been demonstrated to be an independent risk factor for CVD in multiple epidemiological studies. The primary function of haptoglobin is to mitigate the deleterious effects of extracorpuscular hemoglobin. We sought to determine if the protein products of the two haptoglobin alleles differed in their ability to modulate the cytokine profile produced by macrophages in response to hemoglobin. Peripheral blood mononuclear cells were isolated from normal human volunteers and cultured in the presence of complexes formed by the protein products of the two different haptoglobin alleles with hemoglobin. The release of specific cytokines in the conditioned media of these cells was assessed by ELISA. We found that the haptoglobin 1 allele protein product-hemoglobin complex stimulated the secretion of significantly more Il-6 and Il-10 than the haptoglobin 2 allele protein product-hemoglobin complex. We demonstrate that the release of these cytokines is dependent on the liganding of the haptoglobin-hemoglobin complex to the CD163 receptor and the activity of casein kinase II. Haptoglobin genotype modulates the balance of inflammatory (Th1) and anti-inflammatory (Th2) cytokines produced by macrophages exposed to free hemoglobin. This may have implications in understanding inter-individual differences in the inflammatory response to hemorrhage.