Role Of SOCE On Endothelial Progenitor Cell Function In Atherosclerotic Mice

Background and Objective:It’s generally recognized that endothelial cell injury is the initial process of atherosclerosis(AS). Risk factors of AS, such as hyperlipemia, smoking, diabetes mellitus, hypertension, interfere with endothelial function and promote AS development. Therefore,promoting endothelial repairing is important to prevent AS development. Endothelial progenitor cell(EPC), dependent on the ability to differentiate to endothelial cell, was once thought to be the seed cell for endothelial repairing. However, circulating EPC number and EPC function were declined in patients with AS or risk factors of AS. These factors reduce the efficiency of EPC in repairing endothelial damage and promote AS development. Thus, the efforts to slow down or revise dysfunction of EPC are critical for repairing the endothelial lining and preventing AS. Unfortunately, ambiguity regarding the regulatory mechanism of EPC functions has hindered the development of prevention and treatment strategies that target EPC dysfunction during the process of AS.Store operated Ca2+ entry(SOCE) is the prevalent pattern of Ca2+ entry in nonexcitable cells, which is mediated by store operated channels(SOC) and play a critical role in regulating majority cell function both in physiological and pathological conditions. Previously studies by other researchers and in our laboratory found that EPCs may express most of the SOC component proteins, inhibiting these proteins by gene silencing technique could reduce SOCE and inhibit proliferation and migration of EPC. In addition,given that SOCE has the extensive effects on cell physiological function,the change of SOCE may results a series impacts on cell function. On the other hand, SOCE may be the same target of different pathogenic factors. These imply that in the progression of AS, a variety of harmful factor may influence SOCE, which following results in EPC dysfunction. However, the role of SOCE in AS correlated EPC dysfunction is unclear.In view of those mentioned above, in the present study, we used an atherosclerotic mouse model and oxide low density lipoprotein(representing as a risk factor of AS) to explore the detrimental effects of AS in EPC and the role of SOCE in these processes.Methods1. Change of EPC proliferation, migration and SOCE in AS mice1.1 Replication of AS mice model, culturing and identification of EPCApo E-/- mice were feeding with normal diet and high fat diet respectively. The high fat diet containing 0.15% cholesterol and 20% fat. Mice feeding with high fat diet were sacrificed after 12 weeks and 16 weeks respectively and oil O staining onthe aorta of mice was used to evaluated atherosclerosis burden.Bone marrow of mice were harvested and bone marrow mononuclear cells were isolated using density-gradient centrifugation. Cells were seeded in cell culture flasks or plates(precoated with fibronectin) and cultured in endothelial basal medium. The growth condition and morphological characteristics of cells were observed under an inverted microscope. To confirm the phenotype of the EPCs, the cells were incubated with Di I-ac-LDL-Dil and FITC-UEA-I and the cells double positive for Di I-ac-LDL and FITC-UEA-I were identified as EPCs. In addition, cells were processed for immunofluorescent staining to evaluate the expression of CD34, CD133 and VEGF receptor 2.1.2 Change of proliferation and migration of EPC in AS miceCells were cultured for 5 days, the number of cells was determined by direct counting under microscope, the proliferation of cells was analyzed by CCK-8 dyeing and the migration of cells was evaluated by Transwell.1.3 Change of SOCE in EPC of AS mice1.3.1 Change of the amplitude and waveform of SOCE in EPC of AS miceCells were cultured for 7 days and incubated with calcium fluorescent probe(Fluo-3). Thapsigargin(TG) induced SOCE was observed under laser scanning confocal microscope. Data were analyzed to observed the change of amplitude and waveform of SOCE.1.3.2 Change of Ca2+ oscillation of EPC in AS miceCells were cultured for 7 days and incubated with calcium fluorescent probe(Fluo-3). The change of spontaneous and VEGF induced Ca2+ oscillation were observed under a laser scanning confocal microscope.1.3.3 Expression of SOC components in EPC of AS miceTotal RNA of EPCs were extracted, RT-PCR was undertaken to identify the transcription of SOC components in EPC and RT-q PCR was used to analyze the transcriptional difference of key SOC components. Total protein of EPCs were extracted, Western blotwas used to evaluated the expression difference between the key SOC components.1.4 Change of e NOS phosphorilation and expression of EPC in AS miceWestern blot analysis was used to evaluated the change of phosphorylation and expression of e NOS in EPC.2. Effects of ox-LDL in proliferation, migration and SOCE of EPC2.1 Effects of ox-LDL in proliferation and migration of EPCCells culturing in plates were incubated with ox-LDL. The number of cells was determined by direct counting under microscope, the proliferation of cells was analyzed by CCK-8 dyeing, the migration of cells was evaluated by Transwell and the angiogenesis in vitro was evaluated in the basement membrane matrix.2.2 Effects of ox-LDL on SOCE of EPC2.2.1 Effects of ox-LDL on the amplitude and waveform of SOCE in EPCCells were incubated with ox-LDL, then the cells were incubated with calcium fluorescent probe(Fluo-3). ATP induced SOCE was observed under laser scanning confocal microscope. Data were analyzed to observed the change of amplitude and waveform of SOCE.2.2.2 Effects of ox-LDL on expression of SOC components in EPCCells were incubated with ox-LDL, then the total protein of EPCs were extracted, Western blot analysis was used to evaluated the expression difference between the key SOC components.2.2.3 Effects of ox-LDL on phosphorylation of STIM1 proteinCells were incubated with ox-LDL, then cells were stimulated with ATP. Total protein were extracted in different time phase after stimulating, STIM1 protein was enriched by immunoprecipitation and Western blot analysis was used to evaluated the phosphorylation of total serine in STIM1.3. The correlation of SOCE and proliferation and migration of EPC3.1 Effects of inhibiting of SOCE on proliferation and migration of EPCThe culturing EPCs were incubated with specific SOCE inhibitors or infected by lentivirus loaded with sh STIM1 gene. Change of SOCE was observed under a laser scanning confocal microscope.The number of cells was determined by direct counting under microscope, the proliferation of cells was analyzed by CCK-8 dyeing and the migration of cells was evaluated by Transwell.3.2 Effects of inhibiting of SOCE on e NOS phosphorilation and expression in EPCThe culturing EPCs were incubated with specific SOCE inhibitors or infected by lentivirus loaded with sh STIM1 gene. Western blot analysis was used to evaluated the phosphorylation and expression of e NOS in EPC. Results1. Change of EPC proliferation, migration and SOCE in AS mice1.1 Replication of AS mice model, culturing and identification of EPCAS mice model was replicated successfully, the lipid plaque in aorta was more significant in high fat diet feeding mice.Mice bone marrow derived EPCs were cultured successfully. After seeding in culture plates, the bone morrow mononuclear cells became attaching the plate gradually and exhibited fusiform, spindly and irregular shape. Some cells were cloned and linear growth. Most of cells were double positive to expression UEA and banding LDL. Immunofluorescent staining showed that most of cells were positive to CD34, CD133 and VEGFR2.1.2 Change of proliferation and migration of EPC in AS miceNumber of EPCs were reduced along with the progression of AS, the number in high fat diet 12 weeks and 16 weeks EPCs were decreased by 21.5% and 45% respectively compare to normal diet EPCs(p<0.05). The proliferative activity were decreased by 28.2% and 56% respectively compare to normal diet EPCs and the migrate activity were decreased by 34.4% and 43.8%respectively(p<0.05).1.3 Change of SOCE in EPC of AS mice1.3.1 Change of the amplitude and waveform of SOCE in EPC of AS miceTG induced SOCE successfully in EPC. The amplitude of SOCE was reduced along with the progression of AS. In high fat diet 12 weeks and 16 weeks EPCs, the amplitude of SOCE decreased by 65.2% and 67.3% respectively(p<0.05). In addition, the amplitude of Ca2+ release was also decreased. The analyzing of waveform of SOCE showed that the ascending and descending velocity of SOCE were decreased along with the progression of AS. In high fat diet 12 weeks and 16 weeks EPCs, the ascending velocity decreased by 65.1% and 72.1% respectively(p<0.05). In high fat diet 16 weeks EPCs, the descending velocity decreased by 70%(p<0.05). However, in high fat diet 16 weeks EPCs, the descending velocity only decreased by 32.6% and had no statistic significant.1.3.2 Change of Ca2+ oscillation of EPC in AS miceGiven the role of SOCE in activating and maintaining of Ca2+ oscillation and the role of Ca2+ oscillation in regulating cellular function, we next explore the Ca2+ oscillation of EPC in AS mice. We found that the amplitude and frequency of spontaneous and as well as VEGF induced Ca2+ oscillation were decreased in EPC of AS mice(p<0.05).1.3.3 Expression of SOC components in EPC of AS miceIn order to explain the mechanism of the change of SOCE, the expression of SOC components was evaluated. At first, RT-PCR was used to identify the transcription of SOC components gene in EPC and found that most of those components were transcriptional in EPCs, which include STIM1-2, Orai1-3, TRPC1 and TRPC6. Then, we found that the key components of SOC such as STIM1, Orai1 and TRPC1 were decreased both in m RNA and protein level in EPC along with the progression of AS(p<0.05).1.4 Change of e NOS phosphorilation and expression of EPC in AS miceGiven the critical role of e NOS in regulating proliferation and migration of EPC, the change of e NOS phosphorilation and expression in EPC of AS mice was explored. The VEGF induced phosphorylation of e NOS was decreased along with the progression of AS(p<0.05). In addition, expression of e NOS also decreased in EPC of AS mice(p<0.05).2. Effects of ox-LDL in proliferation, migration and SOCE of EPCGiven that ox-LDL is the most critical pathogenic factor for AS, we next used ox-LDL in vitro to verify the effects of AS progression on function and SOCE in EPC.2.1 Effects of ox-LDL in proliferation and migration of EPCEPCs incubated with ox-LDL for 72 h, the number and proliferative activity of EPCs were reduced in a dose dependent manner and the maximum dose(100μg/ml) had the most effect(p<0.05). The low dose ox-LDL(25μg/ml) exhibited the inhibiting effect significant(p<0.05) on EPC migration and gradually more significant along with the dose increasing. In addition, high dose of ox-LDL also had the effect in inhibiting EPC angiogenesis in vitro.2.2 Effects of ox-LDL on SOCE of EPC2.2.1 Effects of ox-LDL on the amplitude and waveform of SOCE in EPCAfter incubating with ox-LDL for 24 h, the amplitude of SOCE in EPCs exhibited decreasing tendency but had no statistic significant. However, after incubating with ox-LDL for 72 h, the amplitude of SOCE in EPCs was reduced significantly(p<0.05). The analyzing of waveform of SOCE showed that the ascending and descending velocity of SOCE were decreased significantly after incubating with ox-LDL for 24 h(p<0.05). This effect was more remarkable after incubating with ox-LDL for 72 h.2.2.2 Effects of ox-LDL on expression of SOC components in EPCAfter incubating with ox-LDL for 72 h, the expression of key SOC components including STIM1, Orai1 and TRPC exhibited decreasing tendency but had no statistic significant.2.2.3 Effects of ox-LDL on phosphorylation of STIM1 proteinGiven that the change of SOC components was not enough to explain the change of SOCE, we next observed the change of phosphorylation of STIM1 protein and found that the phosphorylation of total serine in STIM1 protein was reduced significantly after incubating with ox-LDL for 24 h(p<0.05). This effect was more remarkable after incubating with ox-LDL for 72 h.3. The correlation of SOCE and proliferation and migration of EPC3.1 Effects of inhibiting of SOCE on proliferation and migration of EPCThe SOCE inhibitor 2-APB and ML-9 reduced the amplitude of SOCE 65.1% and 34.7% respectively(p<0.05). After incubating with 2-APB for 72 h, the number, proliferative activity and migrate of EPC were decreased by 52.0%, 49.7% and 54.5% respectively(p<0.05). After incubating with ML-9 for 72 h, the number, proliferative activity and migrate of EPC were decreased by 48.4%, 57.0% and 49.7% respectively(p<0.05). In addition, sh STIM1 also reduced amplitude of SOCE significantly. Furthermore, sh STIM1 reduced the number, proliferative activity and migrate of EPC for 59.4%, 64.0% and 66.4% respectively(p<0.05).3.2 Effects of inhibiting of SOCE on e NOS phosphorilation and expression of EPCBoth SOCE inhibitor and sh STIM1 reduced the expression of e NOS for 41.3% and 52.7% respectively(p<0.05). In addition, sh STIM1 also reduced phosphorylation of e NOS significantly(p<0.05). Conclusions1. Proliferation and migration of EPCs were reduced along with the progression of AS2. SOCE reduced along with the progression of AS3. Ca2+ oscillation and e NOS phosphorilation and expression of EPCs reduced along with the progression of AS4. ox-LDL also inhibited proliferation, migration and SOCE of EPCs5. SOCE correlated with proliferative, migratory and e NOS phosphorilation and expression of EPCs6. Both AS and its risk factor(ox-LDL) can reduce the proliferation and migration of EPC. The underlying mechanism of these effects is possible that AS and its risk factors reduced SOCE, which further influence the Ca2+ oscillation and e NOS phosphorilation and expression.These results provide theoretical basis which support the SOCE as targets for treatment of EPC dysfunction.