Proteomics Analysis Of Oxidative Modification In Endothelial Colony-forming Cells Treated By Hydrogen Peroxide

Endothelial progenitor cells (EPCs) are heterogeneous precursor cell population of endothelial cells. EPCs are considered to play an important role in neovascularization and be as biomarkers to assess cardiovascular diseases risk. There was evidence showed that EPCs can promote the repair of damaged endothelium and attenuate the development of atherosclerosis. Impaired function of EPCs is related to endothelial dysfunction. It is known that oxidative stress is one of the most important pathogenetic factors impairing the endothelial function. The endothelial cells injured by oxidative stress can release some cytokine which mobilized the EPCs to repair the endothelial cells. So EPCs are exposed to the oxidative stress during the process of repairing endothelial. Several pathological conditions, including hypertension, hyperglycemia, hyperlipemia and atherosclerosis can enhance the production of reactive oxygen species (ROS) in vessel, which induce the oxidative stress. Many researches have shown that level of EPCs in patients with cardiovascular diseases was not only lower but also negative related to the Framingham risk factor score. This indicates that the oxidative stress can induce the impairment of EPCs. But the exact mechanism is unknown.EPCs may have the common origin with haemopoietic stem cells which can differentiate into macrophages and monocytes. Though some studies have suggested that the EPCs may be resistant to oxidative stress, the resistibility is considered to have the limitation because the cell populations used by researchers were mixed with macrophages and monocytes, which are known to be resistant to oxidative stress.In order to conquer this problem, the endothelial colony-forming cells (ECFCs) were applied to be the research object. ECFCs, one of EPCs subpopulations, do not express the hematopoietic or monocyte/macrophage cell surface antigens. They have high proliferative potential, relatively high levels of telomerase and form perfused vessels in vivo. The potential of ECFCs in tissue repair and regeneration has been generally accepted.In this study, we treated the ECFCs with hydrogen peroxide (H2O2) as oxidative stress model and observed the changes in cytology and morphology of ECFCs using MTT assay, AO/EB double staining and the tube formation assay in vitro. Then we investigated the alterations in oxidative levels of proteins associated with H2O2-induced morphologic and cytological changes in ECFCs by proteomics analysis of oxidative modification. The results showed that H2O2treatment led to a decreased proliferation, increased apoptosis and impaired tube-forming ability of ECFCs in a dose-dependent manner. The result of redox proteomics analysis showed that eight protein spots were differentially expressed in oxidative levels, of which five were upregulated and three were downregulated. MALDI TOF/TOF mass spectrometry analysis showed that five proteins with upregulated oxidative levels were identified successfully. They are T-complex protein lsubunit alpha, isoform A of prelamin-A/C, cofilin-1, peroxiredoxin-4and actin, which play some important roles in cellular locomotion, apoptosis, folding of protein and tube formation in vitro. The upregulated oxidative levels of these five proteins may take responsibility for the dysfunction of ECFCs under the oxidative stress. Our results may provide some novel insights into the molecular mechanisms of oxidative stress on ECFCs. And our work may offer some theoretic foundation for the clinical therapy of ECFCs.