title: Effects of everolimus on macrophage-derived foam cell behavior.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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).
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors: Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054, USA. Electronic address: steven.hsu@av.abbott.com.
authors: Steven
authors: S
authors: Hsu
authors: Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054, USA.
authors: Eugen
authors: E
authors: Koren
authors: Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054, USA.
authors: Yen
authors: Y
authors: Chan
authors: Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054, USA.
authors: Mirna
authors: M
authors: Koscec
authors: Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054, USA.
authors: Alexander
authors: A
authors: Sheehy
authors: CVPath Institute, Inc., 19 Firstfield Road, Gaithersburg, MD 20878, USA.
authors: Frank
authors: F
authors: Kolodgie
authors: CVPath Institute, Inc., 19 Firstfield Road, Gaithersburg, MD 20878, USA.
authors: Renu
authors: R
authors: Virmani
authors: Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054, USA.
authors: Debra
authors: D
authors: Feder
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002317:Cardiovascular Agents / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D054855:Drug-Eluting Stents; D000068338:Everolimus; D005487:Foam Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D008264:Macrophages / Q000166:cytology*; D009336:Necrosis / Q000139:chemically induced; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D002317:Cardiovascular Agents; D000068338:Everolimus; D020123:Sirolimus
keywords: Atherosclerosis; Drug-eluting stents; Everolimus; Foam cells