Effects of everolimus on macrophage-derived foam cell behavior.

Abstract

The purpose of this study was to investigate the effects of everolimus on foam cell (FC) viability, mRNA levels, and inflammatory cytokine production to better understand its potential inhibitory effects on atheroma progression. Human THP1 macrophage-derived FC were formed using acetylated LDL (acLDL, 100 μg/mL) for 72 hours, followed by everolimus treatment (10(-5)-10(-11) M) for 24 hours. FC viability was quantified using fluorescent calcein AM/DAPI staining. FC lysates and media supernatants were analyzed for apoptosis and necrosis using a Cell Death ELISA(PLUS) assay. FC lysates and media supernatants were also analyzed for inflammatory cytokine (IL1β, IL8, MCP1, TNFα) mRNA levels and protein expression using quantitative reverse transcription real-time polymerase chain reaction (QPCR) and a Procarta® immunoassay, respectively. mRNA levels of autophagy (MAP1LC3), apoptosis (survivin, clusterin), and matrix degradation (MMP1, MMP9) markers were evaluated by Quantigene® Plex assay and verified with QPCR. Additionally, hypercholesterolemic rabbits received everolimus-eluting stents (EES) for 28 or 60 days. RAM-11 immunohistochemical staining was performed to compare %RAM-11 positive area between stented sections and unstented proximal sections. Statistical significance was calculated using one-way ANOVA (p≤0.05). Calcein AM/DAPI staining showed that FC exposed to everolimus (10(-5) M) had significantly decreased viability compared to control. FC apoptosis was significantly increased at a high dose of everolimus (10(-5)M), with no necrotic effects at any dose tested. Everolimus did not affect endothelial (HUVEC) and smooth muscle (HCASMC) cell apoptosis or necrosis. Everolimus (10(-5)M) significantly increased MAP1LC3, caused an increased trend in clusterin (p=0.10), and significantly decreased survivin and MMP1 mRNA levels in FC. MCP1 cytokine mRNA levels and secreted protein expression was significantly decreased by everolimus (10(-5) M) in FC. Percentage of RAM-11 positive area exhibited a reduction trend within sections stented with EES compared to unstented proximal sections at 60 days (p=0.09). Everolimus, a potent anti-proliferative agent used in drug-eluting stents and bioresorbable vascular scaffolds, may inhibit atheroma progression and/or promote atheroma stabilization through diminished viability of FC, decreased matrix degradation, and reduced pro-inflammatory cytokine secretion. We explored the effects of everolimus on the behavior of human THP1 macrophage-derived foam cells in culture, including cell viability, mRNA levels, and pro-inflammatory cytokine production. We conclude that everolimus, a potent anti-proliferative agent used in drug-eluting stents/bioresorbable vascular scaffolds, may potentially inhibit atheroma progression and/or promote atheroma stabilization through diminished viability of foam cells, decreased matrix degradation, and reduced pro-inflammatory cytokine secretion.