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Brain tissue was obtained from human cadavers, with approval of the Ethical Committee of Health and Research Council. Microvessel endothelial cells were isolated by enzymatic digestion, followed by gradient centrifugation. ET-1 was measured from the supernatant by RIA developed at our laboratory, using an antibody purchased from Peninsula (sensitivity 0.8 pg/tube, 50% intercept 18.2 pg/tube). Basal level of ET-1 in the supernatant increased with time (3 hrs: 18.3 6 4.3 pg/ml; 6 hrs: 31.3 6 1.1 pg/ml; 24 hrs: 88.0 6 5.7 pg/ml; 48 hrs: 86.3 6 11.2 pg/ml; MEAN 6 STDEV). TNF-a (270 U/ml) increased ET-1 concentration (3 hrs: 190 6 70%; 24 hrs: 217 6 39%; 48 hrs: 207 6 5%; values are relative changes to control, run parallel to the stimulated wells). A lower dose of TNF-a (210 U/ml) revealed dose dependence of the effect (3 hrs: 137%; 24 hrs: 170%; 48 hrs: 212%). IL-1-a also increased ET-1 dose dependently (IL-1-a 38,8 U/ml: 3 hrs: 129%; 24 hrs: 161%; 48 hrs: 212%; IL-1-a 1,4 U/ml: 3 hrs: 116%; 24 hrs: 122%; 48 hrs: 180%). Lipoprotein(a) (Lp(a)) was prepared from human plasma by gradient centrifugation. Lp(a) had a dual effect on ET-1, increasing ET-1 in the first three hours, but reducing it by the end of the 48 hour observation period. This effect was not dose dependent in the concentration range tested (Lp(a) 450 mg/ml: 3 hrs: 188%; 24 hrs: 91%; 48 hrs: 85%; Lp(a) 360 mg/ml: 3 hrs: 180%; 24 hrs: 94%; 48 hrs: 52%). Lp(a) reduced the stimulatory effect of cytokines on ET-1. Maximal values at 48 hrs were: TNF-a: 207%, TNF-a1Lp(a): 91%; IL-1-a: 212%, IL-1a1Lp(a): 64%. Characterization of ET-1-like immunoreactivity by HPLC revealed that the total ET-1 like immunoreactivity coeluted with the synthetic human ET-1 standard. A cell culture of human brain microvessels was established for studying the regulation of ET-1 release. The cells secrete intact ET-1 to the supernatant. TNF-a and IL-1-a increased ET-1 concentration of the supernatant, whereas Lp(a) alone had a dual effect on it. When given together Lp(a) reduces the effect of cytokines on ET-1.

Characterisation of ET-1 production of cultured endothelial cells isolated from human brain microvessel: effect of cytokines, and lipoprotein(a)