Molecular mediators involved in the persistent effect of advanced glycation products on the inflammatory response and cholesterol efflux disruption in macrophages

• Grant number: 21/04989-3
• Support Opportunities: Scholarships in Brazil - Master
• Effective date (Start): December 01, 2021
• Effective date (End): June 30, 2023
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Marisa Passarelli
• Grantee: Sayonara Ivana Santos de Assis
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Advanced glycation products (AGE) are important contributors to metabolic memory by mediating cellular modifications during the decompensation metabolic period, which are sustained after adequate metabolic control in diabetes mellitus (DM). We have recently demonstrated a persistent effect of albumin modified by advanced glycation (albumin-AGE) on impaired cholesterol removal from macrophages by high-density lipoprotein (HDL) and sensitivity of these cells to inflammation. The aim of this study is to evaluate the molecular mechanisms that mediate the persistent cellular response to AGEs. The persistence of NFKB pathway activation and expression of inflammatory genes involved in cholesterol export will be evaluated in macrophages treated with AGE and control (C) -albumin. Albumin will be glycated in vitro by incubation with 10mM glycolaldehyde for 4 days at 37ºC, in the dark. C-albumin will be incubated with phosphate-buffered solution only. Macrophages differentiated from isolated mouse bone-marrow cells (BMDM) will be incubated for 48 h with C or AGE albumin, then maintained in a medium free of these albumins for different intervals of time, and later incubated for 24 h with lipopolysaccharide. Total cellular content of ABCA-1, ABCG-1 (HDL receptors), IKBA, IKBB, total and phosphorylated IKKA and IKKB, toll-like receptor 4 (TLR4), and AGE receptor (RAGE), and nuclear contente of p65 and p50 will be determined by immunoblot. RT-qPCR will be used for gene expression analysis of RelA, Nfkb1, Ager, Tlr4, IL6, TNF, Abca1, and Abcg1. The secretion of inflammatory cytokines will be determined by ELISA. The results will help in better understanding the molecular mechanisms involved in the sustained cellular actions of AGE compromising lipid homeostasis and favoring atherosclerosis.