Effect of homocysteine-thiolactone on endotelial cell cultures

CHAMORRO M EUGENIA; QUINTANA IRENE L.; NESSE ALCIRA; SCHIAPPACASSE AGUSTINA; VITTORI DANIELA; MALTANERI ROMINA; GENOUD VALERIA

Estambul

Congreso; 24th Biennial International Congress on Thrombosis; 2016

European and Mediterranean League Against Thrombotic Diseases

Hyperhomocystinemia(HHcy) has been associated to the endothelialdysfunctionand injury involved in thrombosis and atherosclerosis. It hasbeenproposed that homocysteine-thiolactone (HTL), a highly-reactivespecies,could be responsible for the harmful action of HHcy. TheN-homocysteinylationprocess occurs when HTL acylates protein lysineresidues.Given that serum allows proliferation and migration ofendothelialEA.hy926 cells in culture, we aimed to study if such effect isalteredafter serum proteins have been treated with HTL.*2 **Methods * Fetal bovineserum (S) was incubated with HTL (final concentrations 2, 4and 10mM)for 18h at 37°C (S-HTL). Structural modification of serumproteins wasassessed by capillary electrophoresis, polyacrilamide gelelectrophoresisin native conditions (PAGE) and quantification of freesulphydrylgroups produced by N-homocysteinylation (Ellman?s reaction).Cellmigration was evaluated in scratching assays and cell invasion wasassayed onMatrigel-coated transwells*.**3 **Results * Modificationin the net charge of S-HTL proteins was observed (capillaryelectrophoresisand native PAGE) due to the masking of lysine residues andthe increasein sulphydryl groups, thus confirming the N-homocysteinylationreaction.S-HTL decreased migration compared to cultures with untreatedserum (S),with dependence on HTL concentration (Migration: S 92±7%, S-HTL2mM 64±16%,*S-HTL 4mM 46±14%, **S-HTL 10mM 30±9%; *P<0.05 and **P<0.01 vs.S; n=7).Similarly, S-HTL 10mM abrogated the cell invasion observed with S(Cellnumber: without S 2000±800, S 9600±2400, *S-HTL 2900±1400; *P<0.05vs. S; n=3).With aims to determine which serum proteins could be involvedin theinhibition observed with S-HTL, we studied the effect of HTL onAlbumin (A)and Fibronectin (FN). The increase in the sulphydryl contentand theelectrophoretic analysis showed structural alteration of bothproteins.Though albumin and fibronectin enhanced migration, theirmodificationby HTL significantly decreased such effect (Migration: withoutS 44±6%, A78±4%, FN 59±5%, A-HTL 60±3%, FN-HTL 42±3%; A vs. without S;**P<0.01;FN vs. without S, A-HTL vs. A y FN-HTL vs. FN; *P<0.05; n=3).*4 **Conclusions * N-homocysteinylationof serum proteins altered their migratory effect onendothelialEA.hy926 cells, suggesting a possible antiangiogenic mechanism.Modifiedalbumin and fibronectin would be involved in such effect.