טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShur Anna
SubjectCRP Expression and Modulation in Different Subtypes of
Macrophages and the Implications in the
Pathogenesis of Atherogenesis
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Michael Aviram
Dr. Marielle Kaplan
Full Thesis textFull thesis text - English Version


Abstract

Atherosclerosis is a chronic cardiovascular disease, characterized by impaired lipid metabolism and inflammation. Accumulation of Oxidized LDL (Ox-LDL) in arterial macrophages, subsequent foam cells formation and inflammatory response are critical factors to the pathogenesis of the disease. Plaque-associated macrophages comprise a highly heterogenic population including pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes that exert diverse effects on the plaque formation.

C-reactive protein (CRP), an inflammation marker and potent predictor of future cardiovascular disease (CVD), appears to be an active mediator of CVD with distinct pro-atherogenic properties. CRP is present in atherosclerotic lesions and is locally produced by smooth muscle cells and macrophages.

We hypothesize that CRP exhibits different patterns of expression in the two subpopulations of macrophages (M1 and M2).  Therefore CRP pro-inflammatory effects in the arterial wall could depend on the triggers affecting the balance between pro-inflammatory (M1) and anti-inflammatory macrophages (M2).

Our aims included the validation of M1 and M2 activation in human THP-1 cell line of macrophages. Furthermore, we analyzed CRP expression in M1 versus M2 macrophages and questioned whether CRP selective expression involves the NFkB signaling pathway.  Finally, we have determined whether oxidative stress can affect the M1/M2 macrophage phenotype balance.

M1 phenotype differentiation was validated by macrophages incubation with 60ng/ml IFN- γ and M2 phenotype differentiation was validated following macrophages incubation with 60ng/ml IL-4. M1 macrophages were characterized by elevated CRP mRNA expression (by 67%) and CRP protein levels (by 108%) as well as induced NFκB activation compared to control cells, but these features were not shared by M2 macrophages. 

Incubation of macrophages with Ox-LDL led to a M1 phenotype as well as to a M2 phenotype, this in correlation with an increased CRP mRNA expression. The antioxidants vitamin E and punicalagin inhibited by up to 86% IL-6 expression (M1 phenotype) cells but did not significantly affect IL-10 secretion (M2 phenotype), this in correlation with a significant inhibition in CRP mRNA expression and CRP protein in M1 macrophages.

Our data support the conclusion that elevated expression of CRP is characteristic of M1 pro-inflammatory macrophages rather than M2 phenotype and it is mediated through NFkB activation. Oxidized LDL could be one of the endogenous triggers for macrophages activation mainly to M1 phenotype, this in association with increased expression of the inflammatory marker CRP. Antioxidants could be potent triggers for inducing anti-inflammatory pathway by inhibiting induction of macrophages to the M1 pathway.