Effects of synthetic polycations on leucine incorporation, lactate dehydrogenase release, and morphology of human umbilical vein endothelial cells

Naturally occurring cationic proteins secreted by human granulocytes have pro-inflammatory effects including induction of increased vascular permeability and oedema, which are likely to be mediated by damage to vascular endothelium. Synthetic cationic polyamino acids have been shown to exert similar inflammatory effects in vivo. We have therefore used a range of synthetic polycationic amino acids to investigate the characteristics required to cause endothelial cell damage, assessed by in vitro inhibition of leucine incorporation into macromolecules by human umbilical vein endothelial cells (HUVEC) in culture. Exposure of HUVEC to 20 nM–2 μM cationic polypeptides of similar M_r(av) (≈40000) in the presence of 20% serum produced a dose-dependent inhibition of [³H] leucine incorporation by polymers of ornithine, arginine or lysine. Similar results were obtained using [³H]thymidine. Neutral or anionic polypeptides of similar M_r were without effect. The molar potency of polylysines increased over the range M_r 40000–120000, while polylysines of M_r(av; <25 000 had no effect. In the absence of serum, inhibition occurred more rapidly and at lower doses. Inhibition of leucine and thymidine incorporation was time-dependent, e.g. exposure to 800 nM-polylysine, M_{r (av)} 90 000, led to progressively increasing inhibition that was complete after 24 h exposure, and was irreversible. The effects of polycations could not be blocked by pretreatment of the cells with polyanions. Precoating of the culture surface with polylysines had no effect on leucine incorporation by HUVEC or their subsequent response to polylysines in solution. Exposure to the peptide Arg-Gly-Asp-Ser inhibited incorporation by 30% but did not increase susceptibility to polylysine. The extent of inhibition of radiolabel incorporation was correlated with changes in cell morphology and release of a cytoplasmic enzyme (1actate dehydrogenase). We conclude that cationic proteins can exert significant, essentially irreversible, cytotoxic effects on endothelium, the magnitude of which depends on the density of cationic residues interacting with the cell and the time of exposure to the polycation. The use of synthetic amino acid polymers in such a model system provides a means of investigating the characteristics of cationic polypeptides that affect endothelial cells and the nature of the responses induced.