טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentViener Hilla Lee
SubjectHaptoglobin Polymorphism and Diabetes Mellitus: The Effects
of Vitamin E treatment on CVD
DepartmentDepartment of Medicine
Supervisor Professor Andrew Peter Levy
Full Thesis textFull thesis text - English Version


Abstract

Hatoglobin (Hp), polymorphic plasma protein whose primary function is to bind free hemoglobin (Hb) released into the blood stream following red blood cell lysis. Binding of Hp to Hb neutralizes the toxic effects of heme iron, which acts as a pro-oxidative agent. In the absence of Hp, heme iron can participate in various oxidative reactions resulting in the production of highly reactive oxygen species and causing damage to surrounding plasma proteins and tissues.

Hp gene has two alleles in humans, designated Hp1 and Hp2. Consequently there are three Hp phenotypes which exist in humans: Hp 1-1, Hp 2-1, and Hp 2-2. More than ten independent clinical studies comprised of over 30,000 diabetic individuals from ethnically and geographically diverse backgrounds showed direct association between Hp polymorphism and CVD susceptibility. Specifically, diabetic patients who have Hp 2-2 genotype exhibit up to five-fold increased risk of CVD compared to Hp 1-1 or Hp 2-1 diabetic patients.

A likely mechanism for this association involves the inferior anti-oxidant activity of the Hp 2-2 protein. Specifically, heme iron in the Hp 2-2 -Hb complex thought to be more redox active and result in more oxidative damage to vascular tissues. In support of this hypothesis, vitamin E, a well-studied anti-oxidant, was shown to reduce myocardial infarction by as much as 50% in diabetic Hp 2-2 individuals but not Hp 1-1 or Hp 2-1 diabetics in a prospective, double blind randomized trial.

 In this study we wanted to develop a mouse model of diabetic vascular disease to understand the interaction between Hp polymorphism and vitamin E treatment.

 Specifically, we chose to study atherosclerotic plaque development in apoE KO mice transgenic for the Hp allele using microultrasound technology which allows for serial in vivo imaging of the same mouse over time. Hence Fewer animals are needed for analysis and the results are more accurate since the same plaque is being followed over time rather than many different plaques being averaged at any given time point in addition to intact tissue viewed in vivo. 

This study demonstrated by in vivo repeated measurements of the brachiocephalic plaque that plaques in Hp 2-2 ApoE-/- mice grow more rapidly and exhibit more unstable characteristics (thinner cap, more intraplaque iron) as compared to plaques from Hp 1-1 ApoE -/- mice. Vitamin E  shown to arrest plaque progression and increase plaque stability in Hp 2-2 mice while providing no benefit to these parameters in Hp 1-1 mice.

These studies confirm the interaction between vitamin E and Hp genotype on the development of atherosclerosis in diabetic mice. The ability of vitamin E to prevent plaque growth strengthens the hypothesis that redox active iron underlies the cause of vascular disease progression in Hp 2-2 diabetic individuals. These results prompted further studies in these mice aimed at trying to measure tissue iron using MRI technology. Preliminary results show that increased iron is measurable in the kidneys of mice with experimentally induced hemolysis. Further studies are underway to assess iron levels in diabetic mice transgenic for Hp.