Effect of homocysteine-thiolactone on endothelial cell cultures

CHAMORRO MARÍA EUGENIA; MALTANERI ROMINA EUGENIA; SCHIAPPACASSE AGUSTINA; QUINTANA IRENE; GENOUD VALERIA; VITTORI DANIELA; NESSE, ALCIRA

Estambul

Congreso; 24th Biennial International Congress on Thrombosis; 2016

Background: Hyperhomocystinemia (HHcy) has been associated to the endotelial disfunction and injury involved in the development of thrombosis and atherosclerosis. It has recently been proposed that homocysteine-thiolactone (HTL), a highly-reactive species, could be responsible for the harmful action of HHcy. The N-homocysteinilation reaction occurs when HTL acylates protein lysine residues. Given that serum allows proliferation and migration of endothelial EA.hy926 cells in culture, our aim was to study if such effect is altered after serum proteins have been treated with HTL. Methods: Fetal bovine serum (S) was incubated with HTL (final concentrations 2, 4 and 10 mM) for 18 h at 37°C (S-HTL). Structural modification of serum proteins was assessed by capillary electrophoresis, polyacrilamide gel electrophoresis in native conditions (PAGE) and quantification of free sulphydryl groups produced by N-homocysteinilation (Ellman?s reaction). Cell migration was evaluated in scratching assays and cell invasion was assayed in vitro on Matrigel-coated transwells. Results: Modification in the net charge of S-HTL proteins was observed (capillary electrophoresis and native PAGE) due to the masking of lysine residues as well as the increase in sulphydryl groups, thus confirming the N-homocysteinilation reaction. In the presence of S-HTL, cell migration decreased compared to cultures with untreated serum (S), with dependence on HTL concentration (Migration: S 92±7%, S-HTL 2 mM 64±16%, *S-HTL 4 mM 46±14%, **S-HTL 10mM 30±9%; *P