A study on oxidative modification of low-density lipoprotein and the response of vascular endothelial cells

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Radical species cause oxidative modification of low-density lipoprotein(LDL), which plays a key role in the very early stage of atherogenesis. However, the exact mechanism how oxidized LDL acts as an initiator of atherosclerosis and what causes abonormal signals in the endothelial cells are not still clear. In this study, we conducted experiments as how LDL is oxidized, what kind of modification plays an important role for the endothelial cells.First we examined the structure of sugar chain on the LDL particles and identified eight types of sugar chains. There was no significant difference between native LDL and oxidized LDL in the structure. When we released mannose by adding an enzyme, a significant prolongation of the lag phase was observed Second we analyzed the amino acid sequence of each fragment that was generated by oxidation of LDL, and found that various serum proteins such as apolipoprotein A-I, fibrin, α2-macrqglobulin, haptoglobin, etc. were non-specifically bound to LDL. … More Finally we established a method to purify thus contaminated LDL by using gel permeation chromatography, and obtained purified fragments of oxidized LDL.We already found that vascular endothelial adhesion molecule-1(VCAM-1) expressed on the endothelial cells by the stimulation of oxidized LDL. VCAM-1 plays a key role to adhere macophages in the every early stage of atherogenesis. So we conducted experiments to examine whether the level of messenger RNA(mRNA) of VCAM-1 would rise or not by adding purified oxidized LDL. First we added IL-1 to the endothelial cells by changing its concentration and incubation time. It was found that two hours incubation was the most effective time to induce mRNA of VCAM-1. Finally we succeeded in finding the most appropriate condition and observing the induction of mRNA of VCAM-1 by adding fragments of oxidized LDL.Another result of the study was to find a method to estimate three-dimensional structure of apolipoprotein B-100(apoB-100). Because of its extraordinary size and insoluble nature, the three dimensional structure has been difficult to deduce. The method was developed based on the consideration that sites of α-helices with higher hydrophilicity are more likely to combine with each other and form bundles in the non-polar environment. Two cylinder models were established and the interactive force (E) between them was calculated according to hydrophobicity and the charge on the amino acids. Consequently, a series of values of E ranging from 383 to 7315 was obtained. Extremely high E values were considered to represent regions of α-helices. If we combined the regions in close proximity to each other, 5 candidates for α-helix bundles were identified. These regions matched well with the lipid-binding motifs of apoB-100 as reported by others. Thus, our algorithm was useful in predicting the higher-order structure of apoB-100, and proper use of this method may enable identification of potential lipid-associating domains of membrane proteins. Less