Rapamycin inhibits release of tumor necrosis factor-α from human vascular smooth muscle cells

Neointimal proliferation with plaque formation is the principal cause of coronary artery disease. In the neointima, inflammatory cytokines like tumor necrosis factor-α (TNF-α) are expressed by vascular smooth muscle cells (VSMCs). These cytokines stimulate proliferation and migration of VSMCs, events that are crucial to neointima formation. Stents, liberating rapamycin, have been shown to reduce neointima formation in human coronary arteries. The purpose of this study was to determine if rapamycin could inhibit the production of TNF-α by VSMCs. With institutional review board approval, VSMCs were cultured from saphenous vein segments obtained from five patients. Cells were identified as VSMC by immunostaining for smooth muscle α-actin. Cells were exposed to bacterial lipopolysaccharide (LPS), LPS plus rapamycin, or LPS plus isoproterenol for 24 hours. Cells with no treatment served as controls. The culture medium was then removed and analyzed for TNF-α. Additionally, the effect of treatment on viability was determined by assay of mitochondrial activity. TNF-α released into the culture medium is expressed as pg TNF-α/mg cell protein. Statistical analysis was by ANOVA. In control cells, TNF-α was undetectable in the culture medium. The addition of LPS (10 μg/mL) increased TNF-α release to 4312 ± 705 pg/mg at 24 hours. The addition of 1 ng/mL rapamycin with LPS reduced TNF-α production 50 per cent (P < 0.01 vs LPS alone). A similar reduction of TNF-α release was seen with 1 μM isoproterenol. LPS, rapamycin, or isoproterenol did not affect cell viability. These data show that rapamycin effectively inhibits the release of TNF-α from VSMCs stimulated with inflammatory mediators like LPS. Rapamycin is as effective as agents that raise intracellular cyclic AMP (e.g., isoproterenol). Therefore, a potential mechanism for the effectiveness of rapamycin-releasing stents is reduction of inflammatory cytokine expression by VSMCs.