Activation of Intrinsic and Extrinsic Proapoptotic Signaling Pathways in Interleukin-18-mediated Human Cardiac Endothelial Cell Death

Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-κB DNA binding activity; 2) induces κB-driven luciferase activity; 3) induces IL-1β and TNF-α expression via NF-κB activation; 4) inhibits anti-apoptotic Bcl-2 and Bcl-X_L; 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X _s expression; 6) induces fas and Fas-L promoter activities via NF-κB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X_L chimeric phosphorothioated 2′-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-α, and NF-κB p65 knockdown or dominant negative IκB-α and dominant negative IκB-β or kinase dead IKK-β significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1β, tumor necrosis factor-α, or interferon-γ. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.