FIG. 4.

Hp1-1 and Hp2-2 equally restrict trans-endothelial diffusion of extracellular Hb. Confluent HUVEC monolayers grown on a permeable trans-well cell culture insert were incubated on the “luminal” side with 300 μM Hb, Hb:Hp1-1, or Hb:Hp2-2. After 6 h of incubation, total heme concentrations on the “luminal” (A) and “abluminal” (B) side of the endothelial monolayer were quantified by UV-VIS spectrophotometry and by SEC (C), respectively. The results indicate that the large molecular size of the Hb:Hp complexes fully restrict trans-endothelial diffusion of Hb. (D) The integrity of the endothelial monolayer during Hb and Hb:Hp incubation was confirmed by identical experiments performed in an ECIS instrument in order to measure trans-endothelial resistance over time. In absence or presence of Hp of either phenotype, Hb treatment does not affect monolayer electrical resistance. (E) Adherence junction morphology was examined on the ECIS electrodes at the end of the experiments shown in (D), where green indicates β-catenin, blue shows 4′,6-diamidino-2-phenylindole stained nuclei. (F) Cellular ATP was quantified after 6 h of incubation under the same condition as in the translocation assay with a luminescent assay. There was no significant difference among treatments. Data are shown as mean±SE (n=8). The negative control samples (negative) were complete assay reaction mix without cells. HUVECs, human umbilical vein endothelial cells; SEC, size-exclusion chromatography; ECIS, electric cell impedance substrate measurement. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars