Human coronary endothelial cell activation by endotoxin is characterized by NF-kappa B activation and TNF-alpha synthesis

Tumor necrosis factor-alpha (TNF-alpha), an early inflammatory mediator typ ically regulated by nuclear factor kappa B (NF-kappaB), plays a critical ro le in the development of cardiovascular dysfunction in sepsis. While severa l myocardial cell types synthesize TNF-alpha, the importance of the myocard ial endothelium in sepsis-related cardiac cytokine production is unclear. T o determine the role of the human coronary artery endothelial cell (HCA-EC) in the cytokine response to endotoxin we measured in vitro TNF-alpha synth esis, TNF-alpha mRNA, and the associated NF-kappaB response to LPS. To dete rmine the magnitude of the HCA-EC response we assessed the TNF-alpha and NF -kappaB response to LIPS in a human monocytic cell line (THP-1) as well. We observed an increase in supernatant TNF-alpha from LPS-stimulated HCA-EC ( 12 h) that was ablated by the proteosome Inhibitor, ALLN (N-acetyl-Leu-Leu- norleucinal). Similarly, ALLN-sensitive TNF-alpha was produced by monocytes following LPS, although at concentrations 100-fold higher than HCA-EC. TNF -alpha mRNA from HCA-EC was detected at 60 min in LPS-stimulated cells, but not in unstimulated cells or cells pretreated with ALLN. NF-kappaB p50/p65 subunits were detectable In endothelial nuclear protein 60 min following L PS. In contrast, NF-kappaB subunits from monocytes were detected at 15 min. Also, while ALLN only attenuated endothelial NF-kappaB translocation, mono cyte NF-kappaB translocation was completely inhibited, These data suggest e ndotoxin-stimulated human coronary endothelial cells express TNF-alpha, whi ch is regulated in part by NF-kappaB activation, in a manner and degree dis tinct from human monocytes.