טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRozenberg Orit
SubjectThe Effect of Paraoxonase (PON1) on Macrophage Foam Cells
Formation and on the Development of
Atherosclerosis
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Michael Aviram
Full Thesis textFull thesis text - English Version


Abstract

Paraoxonase1 (PON1) is a high density lipoprotein (HDL) - associated lactonase, which possesses anti-oxidative and anti-atherogenic properties. Increased susceptibility to atherosclerosis was observed in PON1 knockout (PON10) mice, which was associated with increased serum and macrophage oxidative stress and atherogenicity, two phenomena which are found to be enhanced in diabetes, together with PON1 inactivation. As PON1 undergoes inactivation under oxidative stress its activity is preserved by dietary antioxidants, and is affected by cysteine residues, which are redox sensitive.

The aims of the present study were to characterize the direct effect of PON1 on macrophage oxidative status, on foam cell formation, and on atherosclerotic lesion development. We also questioned possible regulation of PON1 activity under oxidative stress by S-glutathionylation.

In vitro expression of PON1 in macrophages was studied in Mouse Peritoneal Macrophages (MPM) harvested from PON10 mice transfected with human PON1, while in vivo expression was achieved by MPM harvested from human PON1 transgenic (PON1Tg) mice. Both in vitro and in vivo macrophage expression of PON1 resulted in decrement in cellular oxidative status and atherogenicity compared to control cells. Moreover, PON1Tg mice on the genetic background of the atherosclerotic apolipoproteinE knockout mice (PON1Tg/E0) were characterized by 2.7-fold decreased atherosclerotic lesion size, in comparison to E0 mice.

Incubation of J774A.1 macrophages under diabetic conditions, represented by elevated glucose levels (30mM), increased macrophage oxidative stress and atherogenicity, whereas a further incubation of the cells with PON1 or with HDL, decreased cellular oxidation. Moreover, PON1 expression in MPM harvested from PON1Tg mice protected the cells from elevated peroxide levels caused by 30mm glucose, compared to control MPM harvested from C57BL/6 mice.

We next studied PON1 regulation, following incubation of PON1 or of HDL with oxidized glutathione (GSSG). A dose- and a time-dependent inactivation of PON1 activities were noted. PON1 inactivation was associated with the formation of a mixed disulfide bond between GSSG and PON1's cysteine residue(s), as detected by immunoblotting with anti-glutathione IgG. PON1 activity was recovered following the addition of the reducing agent DL-Dithiothreitol, to the PON1-SSG complex.

We conclude that PON1 directly reduced macrophage oxidative status and protected macrophages from oxidative stress even under sever diabetic conditions. These phenomena were associated with decreased cellular atherogenicity. Consequently, PON1 attenuated atherosclerosis development, as shown in atherosclerotic PON1Tg mice. Furthermore, PON1 could undergo S-glutathionylation with a consequent reversible inactivation under oxidative stress, a protective mechanism which prevents an irreversible inactivation of the enzyme under oxidative stress.