Proteomic Analysis Of Endothelial Progenitor Cells Exposed To Oxidative Stress

Endothelial progenitor cells (EPCs) can differentiate into endothelial cells. When thetissue is damaged or ischemic, they can be recruited to the damaged area and participate inthe neovascularization to repair damaged tissue. The cumulative evidence indicates that inpatients with cerebrovascular risk factors, including hyperlipidemia, hypertension, diabetesmellitus, cardiovascular diseases, and also in smokers, there are reduced numbers andimpaired functionality of EPCs. They not only are the indicator of stroke risk and prognosis,but also have therapeutic potential to acute ischemic stroke patients because of the ability torepair vascular endothelium, form new vessels, delay the form of atherosclerosis plaque, andpromote neurogenesis.Reactive oxygen species (ROS) which includeΟ2, H2O2, ΗΟ2, ΟΗand so on areproduced in the process of metabolization by aerobic cells. Previous studies havedemonstrated that multiple pathological conditions can increase the production of ROS in thevascular wall, including hyperlipidemia, hypertension, and diabetes mellitus. In addition, theproduction of ROS is also increased in the environment of acute ischemia. It has been shownthat higher expression of intracellular antioxidative enzymes catalase, manganese superoxidedismutase (MnSOD), glutathione peroxidase-1(GPx-1), and haemoxygensae1(HO-1) arecritical mechanisms protecting EPCs against oxidative stress. But under the same condition,after inhibiting the first three antioxidative enzymes, the level of ROS in EPCs is still lowerthan that in human umbilical vein endothelial cells (HUVECs), indicating that there mustalso be some other antioxidative enzymes exist in EPCs to eliminate ROS. About themechanism of oxidative damage, one study has shown that enhanced apoptosissignal-regulating kinase1(ASK1) promoted apoptosis and leaded to the diminishedvessel-forming ability of EPCs after oxidative stress. Another study revealed that theincreased ROS impaired the telomerase reverse transcriptase (TERT) activity and inducedthe translocating of TERT protein from nucleus into the cytosol, finally reduced the prolongability of telomere which is followed by the onset of cell senescence. Other investigationsabout mechanisms of oxidative damage were indirect involving in different conditionsincluding high glucose condition, ox-LDL and angiotensin II, in which ROS production wasincreased. But the knowlege about oxidative stress and EPCs is limited. The aim of this study is to find new clues about antioxidative defensive mechanism and oxidative damagemechanism of EPCs with the help of proteomic analysis.To investigate the effect of oxidative stress on EPCs, cells were treated with H2O2atdifferent final concentrations (100,200,300and400μM) for3hours. MTT assay,scratch-wound assay and matrigel assay showed that cell number reduced, and migration andtubule formation function were impaired under H2O2stress in a concentration-dependentmanner. To identify proteins in response to H2O2stress, EPCs were treated in200μM H2O2for3hours and then two-dimensional differential in-gel electrophoresis (2D-DIGE) combinedwith matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF/TOF MS) analysis were performed. Eight proteins were identifiedsuccessfully, six of which were up-regulated and the rest were down-regulated. To furtherconfirm the results from the MS analysis, the expression pattern of peroxiredoxin-3inresponse to H2O2stress was examined by2D-Western blot and the result showed that MS wasreliable. The results suggested that in order to protect themselves from oxidative damage,EPCs upregulated antioxidative enzymes peroxiredoxin-2, thioredoxin-dependent peroxidereductase, peroxiredoxin-6and cytoskeleton proteins EGF-containing fibulin-like extracelluarmatrix protein1and vimentin. About the oxidative damaged mechanism, we found that RabGDP dissociation inhibitor alpha which inhibited signal transduction was upregulated. BothADP-sugar pyrophosphatase which eliminated oxidative nucleotide and reduced errors duringDNA replication and triosephosphate isomerase which catalyzed "aerobic glycolysis" toproduce energy were downregulated. In summary, though the defensive responses existed,including the up-regulation of antioxidative enzymes and cytoskeleton proteins, EPCs stilldysfunctioned due to the altered proteins associated with signal transduction, oxidativenucleotide eliminating, and energy production. These results indicated that we should focuson the proteins which were susceptible to oxidative stress, as well as protective proteins, suchas Rab GDI α, NUDT5and TIM. Because these negative pathways can't be avoided by theprotection of defensive responses and may be the key pathways of oxidative damage to EPCsfunctions.This study performed2D-DIGE combined with MALDI-TOF/TOF MS analysis to studythe proteomic alters of EPCs oxidative stress model. This hasn't been reported in the literature.All of the eight altered proteins discovered in this study also haven't been reported in theprevious studies of EPCs exposed to oxidative stress. The discovery of new antioxidativeenzymes and cytoskeleton proteins provide novel insights into antioxidative defensive mechanim of EPCs and the alteration of proteins associated with signal transduction,oxidative nucleotide eliminating, and energy production offers new clues to the study ofoxidative damage mechanism of EPCs.