Study Of The Injury Mechanism Of Vascular Endothelium Cells Induced By Homocysteine

Background and Objective: Homocysteine (Hcy), a kind of sulfhydryl amino acid, is the demethylated derivative of methionine and mainly in protein binding pattern in vivo. Physiological functions of homocysteine are participating in the metabolism of one carbon unit. Abundant epidemiological evidence has demonstrated that the increasing concentration of Hcy in plasma is an independent risk factor for cardiovascular disease, which included artherosclerosis, coronary heart disease, cerebrovascular disease, diabetes associated vasculopathy and Alzheimer's disease, and the endothelial cell(EC) injuries may be the initial representation. Many reports indicated lots of models about the EC injury caused by Hcy, but a systemic and convictive conclusion remain still unelicitable. Main purpose of this study is to analyze the protein profile changes and the key proteins of Hcy associated physiological changes of HUVEC, based on the comparative proteomics study of HUVEC and the cells incubed with Hcy.Material and methods: Apoptosis cells were detected by the Flow cytometry. The apoptosis gene profile of HUVEC treated with Hcy were analyzed by an apoptosis gene microarray. The results of microarray also validated by RT-PCR and Western blot. The comparative proteomics study were performed by 2DE-MS technique, and the bioinformatics were used to analyze the function and interaction of proteins.Results: Apoptosis cells could be detected after Hcy treatment, and the apoptosis cell counts were positive correlated with the concentration of Hcy. 12 down-regulated apoptosis genes and 16 up-regulated genes were detected by microarray and conformed by RT-PCR and Western-bolt. The down-regulated genes are ASC, Bcl-2, BFL1, Blk, caspase-9, FLIP, DAPK2, DFF40, HRK, LTA, LTB, RPA3; The up-regulated genes are Apaf-1, Bak, caspase-6, caspase-7, caspase-8, DcR-1, TNFR1, Fas/CD95, CD27, TRAIL, TNFSF-12, TNFSF-14, TNFSF-9, P53, TRAF-2, TRAF-3. Many difference displayed on the 2DE gels and 27 proteins were identified by MS. In all these 27 proteins, expression of calreticulin precursor, tubulin, hnRNP K, ATP synthase alpha chain(mitochondrial precursor), Fascin, Keratin, Reticulocalbin 1 precursor, poly(rC)-binding protein 2 isoform a, calponin, acidic isoform, hnRNP A/B, splice isoform 2 of p07226 tropomyosin beta 4 chain, putative RNA-binding protein 3, inorganic pyrophosphatase, proteasome subunit beta type 7 precursor, phosphoglycerate mutase 1, superoxide dismutase[Mn], stathmin, prefoldin subunit 5, eIF-5Aand an unknown protein(for MGC:5321) were decreased; the expression of β-Actin, alpha enolase, Myo 5C, peroxiredoxin 4, peroxiredoxin I, hypothetical protein and annexin XI were increased. After bioinformationdatabase searching and interaction analyzing , four proteins are indicated as the key moleculars of interaction networks, they are c-myc, ATP5A1, FSCN1 and hnRNP-K.Conclusion: The results of apoptosis gene microarray and protein profiles indicated that apoptosis is the main route of Hcy injuring the vascular endothelium cells. The apoptosis induced by Hcy may be mainly through the death receptor passway. This conclusion needs more data back-up.