| Research background and objective:Precious studies show that endothelial dysfunction is the initiating process for atherosclerosis (AS) formation. Endothelium repair plays a vital part in postponing the progression of AS. Therefore, how to repair effectively endothelial dysfunction is being the focus on the study to prevent and treat AS. More and more researchers are throwing their attention on endothelial progenitor cells (EPCs), as endothelial precursors, for their ability to differentiate into endothelial cells and to promote angiogenesis. Although recent studies found that injecting EPCs into animal body could effectively put off progression of AS, there exist problems such as low survival ability. Progression of AS is affected by variety of factors,among which oxidative stress directly and/or indirectly impairs endothelial cells and endothelium. And oxidative stress induces the occurrence of atherosclerosis and throughout the entire process of AS. Moreover,numerous studies reported that oxidative stress could promote the reduction in cell proliferation, differentiation, migration and adhesion, increase in cell apoptosis. Thus, improvement in the survival ability and reduction in apoptosis under oxidative stress may provide a guarantee to the treatment of atherosclerosis.Store-operated calcium entry (SOCE) is a universally calcium influx manner in cells of mammals. SOCE mediated by store-operated calcium channel (SOCC) plays an essential part in many cell activities. Our precious studies found that SOCC locates in EPCs. Alterations in the main components of SOCC complex influences in cell functions such as proliferation,differentiation, migration and adhesion. However, there is no relative reports about whether SOCE is involved in oxidative stress-induced injuries in EPCs. In addition, more and more evidence shows that calcium concentration and calcium signaling pathway play a vital part in oxidative stress.Therefore, our studies were designed to utilize hydrogen peroxide to induce occurrence of oxidative stress, and to investigate what a potential role of SOCE may play in oxidative stress-induced injuries in EPCs and relative mechanism. Moreover, we also try our best to find a target and strategy for attenuating or even reversing endothelial injuries.Methods:1. Influences of hydrogen peroxide on cell viability and apoptosis in EPCs.1.1 Isolation, culture and characterization of EPCsMale Sprague-Dawley rats (150g-180g) were sacrificed by injecting sodium pentobarbital (100 mg/kg) into the peritoneal cavity,according to the accepted instructions.SD rats were disinfected by povidone iodine solution. Then spleen was taken out from the peritoneal cavity. Mononuclear cells were obtained from spleen through density-gradient centrifugation according to established methods, and then were seeded into medium with low sugar. When EPCs were cultured for 7days by using the condition for culturing endothelial cells. EPCs were observed under the microscope about whether cells were adherent to wall of culture bottle and changes in cell morphology. Cell morphology of adherent cells was consistent to EPCs, and then these cells were stained with DiI-Ac-LDL and FITC-UEA-I to detect whether they have characteristics of ECs.1.2 Influence of hydrogen peroxide on cell viabilityEPCs were cultured for 5 days, digested by trypsin, and then seeded and cultured in 96 well plates for 2 days. EPCs were treated with hydrogen peroxide, and then cell viability was detected cck-8 after cells being adherent cells.1.3 Influence of hydrogen peroxide on cell apoptosisAfter being cultured for 7 days, EPCs were treated with hydrogen peroxide. Finally, cell apoptosis was detected by FITC-Annexin V Apoptosis Detection Kit.2. Influences of hydrogen peroxide on intracellular calcium, SOCE and SOCC complex2.1 Influence of hydrogen peroxide on intracellular calcium concentration2.1.1 EPCs (cultured for 7 days) were incubated with Fluo-3AM avoiding light, and detected changes in calcium concentration after treatment with hydrogen peroxide.2.1.2 EPCs (cultured for 7 days) were pretreated with a calcium chelator (BAPTA)before expourse to hydrogen peroxide, and then respectively observed cell viability and cell apoptosis through using cck-8 and apoptosis kit.2.2 Influence of hydrogen peroxide on SOCETo investigate change in intracellular calcium concentration due to hydrogen peroxide whether is associated with SOCE, we incubated EPCs (cultured for 7 days) with Fluo-3AM without light, utilized hydrogen peroxide and thapsigargin (TG) separately to treat EPCs in free calcium medium, and then observed changes in intracellular calcium concentration under laser confocal microscope.2.3 Effects of hydrogen peroxide on SOCC complex2.3.1 Total RNA was extracted from EPCs (cultured for 7 days). Alteration in transcription level of main components of SOCC complex (Orail, Stiml and Trpcl) was tested through semi-quantitative PCR.2.3.2 Total proteins were extracted from EPCs (cultured for 7 days). Alteration in translation level of main components of SOCC complex (Orail, Stiml and Trpcl) was tested by western blot.2.4 Influence of SOCE inhibition on hydrogen peroxide-induced alteration in intracellular calcium concentration2.4.1 Interfering expression in Stiml with shRNAEPCs (cultured for 5 days) were well adherent to the wall, and degree of cellular ffusion was about 30-50%. Then EPCs were transfected with lentivirus carrying shRNA for 2 days,observed transfected condition under fluorescence microscope and detected the efficiency of transfection via western blot.2.4.2 Influence of SOCE inhibition on hydrogen peroxide-induced alteration in intracellular calcium concentrationEPCs (inhibited by antagonists or interfered with shRNA) were incubated with Fluo-3AM without light, and then washed out with free calcium medium. After treated with hydrogen peroxide, EPCs were observed under laser confocal microscope for their alteration of real-time in intracellular Ca2+ concentration.3. Influences of hydrogen peroxide on cell viability and increase in cell apoptosis via augment in SOCEEPCs (pretreated by antagonists or interfered with shStiml) were treated with hydrogen peroxide, and then separately detected the cell viability with cck-8 and the apoptosis via FITC-Annexin V/PI stainning.4. Mechanism of hydrogen peroxide promotes apoptosis via SOCE inhibition4.1 Effect of SOCE inhibition on intracellular ROS levelEPCs (cultured for 5 days) were pretreated with ROS scavenger and SOCE inhibition separately before hydrogen peroxide insult, and then detected by apoptosis kit comparing with hydrogen peroxide insult.4.2 Influence of SOCE inhibition on caspase familyEPCs (cultured for 7 days) were detected about expression of caspase family via western blot. And then the member of caspase family who has obvious change in expression when exposed to hydrogen peroxide would be detected again via pretreatment with SOCE inhibition before hydrogen peroxide insult comparing with hydrogen peroxide insult.4.3 Influence of SOCE inhibition on ER stress level and mitochondrial dysfunction levelUp-regulation in caspase 9 and caspase 12 were detected by hydrogen peroxide insult,and this shows that hydrogen peroxide insult activates the ER stress apoptotic pathway and mitochondrial dysfunction apoptotic pathway. In order to this inference, we utilized hydrogen peroxide to treat EPCs, detected changes in expression of CHOP and BIP, whose up-regulation suggests occurrence of ER stress and mitochondrial dysfunction specific Cytochrome C via western blot, and tested MMP via Rh123 staining. Then EPCs were pretreated with SOCE inhibition and detected the same measurements as hydrogen peroxide insult.Results:1. Influence of hydrogen peroxide on cell viability and apoptosis in EPCs1.1 Isolation, culture and characterization of EPCsMononuclear EPCs were isolated via density-gradient centrifugation according to established methods. While cells were cultured under 37℃ and 5% CO2, their cell morphology would become more and more bigger than the original state, and ultimately fusiform, spindly, dendritic and so on under the inverted microscope. For the characterization of EPCs, we utilized double fluorescence staining with Dil-Ac-LDL and FITC-UEA-I. The results showed approximately 85% of cells were positive for both markers. And these are EPCs.1.2 Influence of hydrogen peroxide on cell viabilityEPCs (cultured for 7 days) were treated with various concentration (0,200,400,600,800 and 1000μM) of hydrogen peroxide for 6h, and then detected by cck-8 for cell viability. The results showed when concentration of hydrogen peroxide more than 200μM, cell viability was remarkably lower than control group (p<0.05). And when concentration of hydrogen peroxide increased to 600μM, cell viability was about 50%. At the same time, we treated EPCs with hydrogen peroxide (600μM) for various time. Then we found that when the time was longer than 1h, cell viability was remarkably lower than control group (p<0.05). And when the time prolonged to 6h, cell viability was about 50%. Therefore, EPCs were treated with hydrogen peroxide (600μM) for 6h as the following experimental condition.1.3 Influence of hydrogen peroxide on cell apoptosisEPCs were treated with hydrogen peroxide (600μM) for 6h, and then detected with kit for cell apoptosis. The data suggested that hydrogen peroxide insult increases apoptosis significantly(p<0.05). And cell apoptosis of control group and hydrogen peroxide group were separately 5.30% and 40.65%.2. Influence of hydrogen peroxide on intracellular calcium concentration, SOCE and SOCC complex2.1 Influence of hydrogen peroxide on intracellular calcium concentration2.1.1 EPCs (cultured for 7 days) were incubated with Fluo-3AM without light, and then observed under laser confocal microscope after treatment with hydrogen peroxide (600μM).We found that hydrogen peroxide insult increased intracellular concentration rapidly.2.1.2 To investigate relationship between hydrogen peroxide, calcium concentration, and cell viability and apoptosis, we applied BAPTA to pretreat EPCs, and then treat them with hydrogen peroxide (600p,M) for 6h. Cell viability and apoptosis were separately observed with cck-8 and apoptosis kit. Data suggested that pretreatment with BAPTA significantly attenuated hydrogen peroxide-induced reduction in cell viability (p<0.05). Moreover,pretreatment with BAPTA obviously lowered hydrogen peroxide-induced increase in cell apoptosis (p<0.05).2.2 Influence of hydrogen peroxide on SOCETo investigate whether hydrogen peroxide insult induced changes in intracellular calcium concentration via SOCE, we incubate EPCs (cultured for 7 days) with Fluo-3AM without light, washed out extracellular calcium, and observed changes in intracellular calcium concentration under laser confocal microscope after separated treatment with hydrogen peroxide and TG. We found that treatment with hydrogen peroxide could induce occurrence of typical process of calcium release activated calcium internal flow as TG.2.3 Influence of hydrogen peroxide on SOCC complexStudies shows that hydrogen peroxide insult can induce changes in SOCE. In order to investigate mechanism of hydrogen peroxide-induced alteration in SOCE, we detected separately alteration in transcription level and translation level of main components of SOCC complex (Orail, Stiml and Trpcl). The results showed that hydrogen peroxide insult down-regulated Orail and Trpcl, and up-regulated Stiml in the transcription level. And hydrogen peroxide insult down-regulated Orail (39.77%, p<0.05), and up-regulated Stiml(122.03%, p<0.05) in translation level.2.4 Influence of SOCE inhibition on hydrogen peroxide-induced alteration in intracellular calcium concentration2.4.1 SOCE was inhibited via interfering Stiml who was up-regulated by hydrogen peroxide insult with shRNA. EPCs were observed about 50% cells carrying fluorescent. And degree of interference was obtained via detecting expression of Stiml. The results showed that shRNA interference down-regulated expression (30.15%, p<0.05).2.4.2 EPCs (pretreated with antagonist or interfered Stiml with shRNA) were treated with hydrogen peroxide (600μM) for 6h. And then EPCs were observed under laser confocal microscope for changes in intracellular calcium concentration. The results showed that SOCE inhibition could reduce hydrogen peroxide-induced elevation in intracellular calcium concentration.3. Influences of hydrogen peroxide on cell viability and increase in cell apoptosis via augment in SOCEEPCs (pretreated with antagonist or interfered Stiml with shRNA) were treated with hydrogen peroxide (600μM) for 6h. Cell viability and apoptosis were separately observed with cck-8 and apoptosis kit. The results showed that cell viability of antagonist group and shRNA interference group increased separately 20.04% (p<0.05) and 27.68% (p<0.05)comparing with hydrogen peroxide group. And cell apoptosis of antagonist group and shRNA interference group increased separately 16.45% (p<0.05) and 23.34% (p<0.05) comparing with hydrogen peroxide group.4. Investigation in mechanism of apoptosis resulted from hydrogen peroxide via SOCE4.1 Effect of SOCE inhibition on intracellular ROS levelHydrogen peroxide insult (600μM, 6h) could significantly rose intracellular ROS(p<0.05), while pretreatment with ROS scavenger could reduce apoptosis (p<0.05). This suggests that elevation in intracellular ROS promotes increase in apoptosis. Then what a role of ROS plays in hydrogen peroxide-induced apoptosis via SOCE? Further, we found SOCE inhibition could significantly reduce hydrogen peroxide-induced elevation in intracellular ROS (p<0.05).4.2 Influence of SOCE inhibition on caspase familyIn order to investigate mechanism of apoptosis due to hydrogen peroxide via SOCE, we firstly observed influence of hydrogen peroxide on caspase family. The results showed that hydrogen peroxide insult could obviously up-regulate expression of caspase 9 and caspase 12(p<0.05).4.3 hydrogen peroxide insult (600μM, 6h) could significantly up-regulate expression of caspase 9 and caspase 12 (p<0.05). This suggests hydrogen peroxide insult may activate the ER stress apoptotic pathway and mitochondrial dysfunction apoptotic pathway. Further, to prove our inference, we detected indexes whose alteration will suggest the occurrence of ER stress and mitochondrial dysfunction. The data suggested that hydrogen peroxide could remarkably up-regulate expression CHOP, BIP and Cytochrome C (p<0.05), and reduced MMP (p<0.05). These state that hydrogen peroxide insult can activate ER stress and tigger mitochondrial dysfunction. While SOCE inhibition significantly lowers up-regulation in ER stress and mitochondrial dysfunction due to hydrogen peroxide (p<0.05).Conclusions:1. Hydrogen peroxide insult promotes decrease in cell viability and increase in cell apoptosis.2. Hydrogen peroxide insult promotes augmentation in SOCE.3. Hydrogen peroxide insult augments SOCE partly through up-regulation in expression of Stiml.4. Hydrogen peroxide insult induces decrease in cell viability and increase in cell apoptosis via augmentation in SOCE.5. Hydrogen peroxide insult activates ER stress apoptotic pathway and mitochondrial dysfunction apoptotic pathway via augmentation in SOCE.6. SOCE inhibition could improve the ability of EPCs for survival under oxidative stress condition. This suggests that SOCE may become an effective target for injury endothelium repair, therapy for AS. |
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