טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMeilin Edna
Subject"Heparin Cryo-Precipitation (HCP)" of Plasma Reduces
Plasma Levels of Constituents Involved in
Endothelial Dysfunction
DepartmentDepartment of Medicine
Supervisors Ms. Batya Kristal
Assistant Professor Shifra Sela
Full Thesis textFull thesis text - English Version


Abstract

Introduction: Atherosclerosis and cardiovascular diseases are associated with inflammation, oxidative stress and endothelial dysfunction. We hypothesize that structurally modified molecules accumulate in the circulation of atherosclerotic patients, inducing endothelial dysfunction. We suggest that these modified molecules constituting non-traditional risk factors for atherosclerosis, can be removed from circulation by a combined treatment with heparin, cold temperatures and centrifugation of plasma.

Aims: To show that: 1) Oxidized proteins induce endothelial dysfunction 2) Patients with atherosclerosis have high levels of modified proteins and accumulation of non-traditional risk factors for atherosclerosis 3) To develop a method, which will preferentially and simultaneously reduce the abnormally high levels of modified molecules.

Methods: Carbonylated fibrinogen and albumin reflect modifications induced by oxidation and were used as a model for non-traditional risk factors for atherosclerosis. Their effect on umbilical vein endothelial cell (HUVEC) functions was followed; endothelial cell attachment, expression of cell adhesion molecules and intracellular tissue factor production were studied. We developed a new method - “Heparin Cryo-precipitation” (“HCP”), which combines the use of heparin, freezing temperatures and centrifugation in order to reduce the levels of inflammatory mediators and oxidative adducts. Blood was drawn from atherosclerotic hemodialysis patients (HD) and from healthy subjects (HC) and levels of fibrinogen, albumin, CRP, TNF-α, IL-6, advanced oxidation protein products (AOPP), carbonylated fibrinogen and albumin were determined. Contents of the “cryogel”, the pellet formed by “HCP”, was evaluated.

 Results:  Exposure of HUVEC to oxidized albumin/fibrinogen resulted in increased expression of adhesion molecules. HUVEC incubated with oxidized albumin or high levels of untreated fibrinogen increased production of intracellular tissue factor. HD plasma contained abnormally high levels of modified molecules and non-modified risk factors for atherosclerosis, which concentrations were preferentially, simultaneously and significantly reduced by the “HCP” method and precipitated into the cryogel.

Conclusions:  We suggest that oxidized fibrinogen and oxidized albumin should be recognized as non-traditional risk factors for atherosclerosis, due to their delirious effect on endothelial cells. The simultaneous precipitation of modified and abnormally high non-modified circulating risk factors by “HCP”, will probably prevent endothelial dysfunction by repeated use in the long run.

Future implications: Most patients with atherosclerosis have multiple cardiovascular risk factors, traditional and non traditional, which potentiate each other. The “HCP” method may be used in the future as a new treatment modality complementary to the pharmacological approach, designed to reduce concomitantly several non-traditional risk factors for atherosclerosis and cardiovascular diseases and effectively attenuate the atherosclerotic process.