Regulation of Endothelin Release from Human Brain Microvessel Endothelial Cells

Abstract

After approval by the Local Ethical Committee, brain microvessel endothelial cells from human cadavers were isolated by enzymatic digestion and gradient centrifugation. Basal levels of endothelin-1 (ET) in the supernatant increased over time (3 h, 18.3 +/- 4.3 pg/ml; 6 h, 31.3 +/- 1.1 pg/ml; 24 h, 88.0 +/- 5.7 pg/ml; 48 h, 86.3 +/- 11.2 pg/ml, mean +/- SD). Tumor necrosis factor-alpha (TNF-alpha) (270 U/ml) increased ET concentration dose-dependently: 3 h, 190 +/- 70%; 24 h, 217 +/- 39%; 48 h, 207 +/- 5%; TNF-alpha at 210 U/ml: 3 h, 137%; 24 h, 170%; 48 h, 212% (values are relative changes from control, run in parallel to the stimulated wells). Interleukin-1 alpha (IL-1 alpha) (38.8 U/ml) also increased ET dose-dependently: (3 h, 129%; 24 h, 161%; 48 h, 212%; IL-1 alpha 1.4 U/ml: 3 h, 116%; 24 h, 122%; 48 h, 180%). Lipoprotein (a) (Lp(a)) had a dual effect on ET, increasing ET in the first 3 h but reducing it by the end of the 48-h observation period. This effect was not dose-dependent in the concentration range tested: Lp(a) 450 micrograms/ml; 3 h, 188%; 24 h, 91%; 48 h, 85%; Lp(a) 360 micrograms/ml: 3 h, 180%; 24 h, 94%; 48 h, 52%). Lp(a) reduced the stimulatory effect of cytokines on ET release. Maximal values at 48 h were TNF-alpha 207%, TNF-alpha + Lp(a) 91%, IL-1 alpha 212%, IL-1 alpha + Lp(a) 64%. In HPLC analysis, the total ET-like immunoreactivity co-eluted with the synthetic human ET standard. A cell culture of human brain microvessel endothelial cells was established. TNF-alpha and IL-1 alpha increased ET secretion, whereas Lp(a) had a dual effect. When given together, Lp(a) reduced the effect of cytokines on ETs.