טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentFaour Zamzam
SubjectIdentification of Metabolomic Signatures of Macrophages
and the Implications for Atherogenesis
DepartmentDepartment of Medicine
Supervisors Dr. Marielle Kaplan
Dr. Elena Dumin
Full Thesis textFull thesis text - English Version


Abstract

Atherosclerosis is a multifactorial chronic cardiovascular disease, characterized by aggregation and accumulation of cholesterol and oxidized LDL, and proliferation of smooth muscle cells. Different cells types are involved including macrophages. Macrophages comprise a heterogeneous population, including several co-existing subtypes with diverse and even opposite roles such as M1 pro-inflammatory and M2 anti-inflammatory. These cells influence the lipid metabolism, inflammatory response and plaque stability. However, it has been recently recognized that the two states M1 and M2 are insufficient to describe the much broader complexity of stimuli and responses that characterizes the different subtypes of macrophages.

Diabetes Mellitus, which is characterized by impaired insulin homeostasis and inability to control glucose levels, increases the risk of cardiovascular diseases including atherosclerosis by enhancing the inflammatory response and the formation of macrophage foam cells.

Metabolomics technology has emerged as a powerful tool for addressing biological questions. This tool can be effective for the systemic study of endogenous chemical signature of a functional metabolic phenotype since it detects the whole metabolites, which they are the final downstream product of gene transcription. Metabolomics has the ability to investigate the cellular state and determine different activation states in response to environmental factors . This could help in investigating different diseases pathways by playing a role in screening, diagnosis and prognosis.

The aim of our study was to decipher the macrophage metabolic signature in different subtypes of macrophages: M1 pro-inflammatory macrophages, M2 anti-inflammatory macrophages, and macrophages which are exposed to high level of glucose or oxidized LDL (considered as pro-atherogenic mediators for atherosclerosis).

In this study, four different methods of extraction and analysis have been developed and optimized for our investigations to determine a wide range of metabolites which differs biologically, physically and chemically. These methods were called based on the class of interest and extracted metabolites; Amino acid, organic acids, metabolomics and acylcarnitines methods.

We have used the High-performance Liquid Chromatography and Gas or Liquid Chromatography Mass Spectrometry analytical techniques to separate and detect the metabolites. Using Agilent’s Chemstation for GC-MS analysis and Clarity for HPLC analysis, metabolites peaks were identified and quantified.

From the four employed different methods. 28 metabolites exhibited high precision and their levels were compared between the different types of macrophages. Different metabolic pathways were identified as characteristic for each specific macrophages phenotypes. Aerobic glycolysis is induced upon activation of M1 macrophages which increased the levels of citric acid, while M2 macrophages obtain their energy mostly from fatty acid oxidation which require increased levels of fatty acids as myristic acid and lauric acid. Fatty Acyl-CoA biosynthesis and eNOS activation and regulation were shown to be characteristic of M1 phenotype. Moreover, transport of fatty acids, ABC-family protein mediators and SLC-mediated transmembrane transport were revealed as characteristic of M2 phenotype. Glutamic acid and inositol were seen as characteristic of M2 macrophages. 

Results from this study could help us understand the diversity of metabolic pathways involved in the regulation of macrophage activation during atherogenesis. Besides, these results could lead to identifying metabolic modules which affect macrophages function.