Effects Of STIM1/TRPC1 On The EPCs Involved In The Repair Process After Vascular Injury

1.Background and Objective:Endothelial cell (EC) damage is an important pathophysiological step of atherosclerosis,hypertension and restenosis following percutaneous coronary intervention (PCI) such as angioplasty and stenting. Accelerated reendothelialization effectively inhibits smooth muscle cell (SMC) migration, proliferation, and resulting neointimal formation, and is therefore of special interest with regard to prevention of the early stages of atherosclerosis and restenosis. The traditional paradigm of vascular repair is based on the proliferation and migration of pre-existing mature endothelial cells from the adjacent vasculature. Recently, there is a growing understanding that endothelial progenitor cells also contribute to this process. EPCs, which can be mobilized and recruited into injured vessels, differentionate EC, replace dysfunctional endothelium,are increasingly recognized to play a key role in the maintenance of vascular integrity and to act as“repair”cells in response to vascular injury. During re-endothelialization, the key steps regulated by various mechanisms are migration and proliferation of EPCs.However; the mechanisms by which the biological properties of EPCs are regulated remain unclear, especially with respect to those ion channels.Calcium (Ca²⁺) signaling is essential for a variety of cell functions, such as the regulation of proliferation, differentiation and so on. Previous study suggested that Ca²⁺ was involved to regulate the proliferation?differentiation and homing of EPCs. The effects of Ca²⁺ on cell function depend on Ca²⁺ entry induced by Ca²⁺channels on the plasma membrane. A major Ca²⁺ entry pathway component in non-excitable cells, including EPCs,are store-operated Ca²⁺ channels (SOCs). Major components of SOCs are stromal interaction molecule 1 (STIM1), Orai and transient receptor potential canonical (TRPC) protein families. STIM1 is a sensor of SOCs that aggregates and relocates into clusters at the ER-plasma membrane junctions, where it functionally interacts with and activates plasma membrane TRPC and Orai channels, and then mediate store-operated Ca²⁺ entery (SOCE) under store depletion.Recently, our study demonstrated that knockdown of STIM1 significantly suppressed neointimal hyperplasia by inhibiting vascular smooth cell (SMC) proliferation after vascular injury. In addition, It has been reported that an antibody to TRPC1 decreased neointimal hyperplasia after vascular injury by inhibiting SMC proliferation. Moreover, EPCs also play a crucial role on the process of re-endothelialization by reducing neointima formation after vascular injury. However, it remains unclear whether STIM1 and TRPC1 effect on EPCs function. Our previous study found that STIM1 and TRPC1 were expressed in EPCs. Therefore, we hypothesized that STIM1 and TRPC1 affect the biological properties and re-endothelialization of EPCs.2. Methods2.1 Effect of STIM1 on EPCs proliferation, migration and involved in the repair process after vascular injury2.1.1 Effect of STIM1 on EPCs proliferation, migration2.1.1.1 Isolation and characterization of rat bone marrow derived EPCsRat bone marrow derived (BM-derived) EPCs were isolated using density-gradient centrifugation at 1500×g for 20 min. Following purification with three washing steps, cells were resuspended in low-glucose Dulbecco's Modified Eagle's Medium (L-DMEM) supplemented with 10 ng/mL vascular endothelial growth factor (VEGF),20% fetal calf serum. To confirm the phenotype of EPCs, first, cells were incubated with Dil–Ac-LDL for 4 h, in addition, cells were fixed with 4% paraformaldehyde, last, and cells were incubated with FITC-labeled lectin (UEA-1) for 1 h and examined under a laser confocal scanning microscope (LCSM). Cells that stained positive for both acLDL-DiI and UEA-1 were identified as EPCs, nearly all adherent cells (90%) were positive for both markers. Additionally, antibodies against rat CD34, CD45, VEGFR-2, CD133 and the corresponding isotype control antibodies were examined by flow cytometry analysis.2.1.1.2 Effect of STIM1 on EPCs proliferation and migrationRat BM-derived EPCs were isolated and cultured, and EPCs between days 5 and 7 were used in vitro experiments.EPCs were transduced with Ad-si/rSTIM1, Ad-hSTIM1, non silence control Adenovirus (NSC) for 48 h and used in experiments.STIM1 mRNA and protein levels were examined by PCR and Western blot. The proliferation of EPCs was measured by [3^H]-Thymidine incorporation and cell counting. EPCs migration was determined using a modified Boyden chamber assay. Cell-cycle distribution was analyzed using fluorescence-activated cell sorting (FACS).2.1.2 Effect of STIM1 on EPCs reendothelialization: to determin the effect of EPCs induced by STIM1 during vascular repair in vivo, angioplasty of the rat left carotid artery was constructed by using a balloon embolectomy catheter. Firstly, animals were subjected to anesthesia and surgical procedure. Secondly, we transduced Ad-si/rSTIM1, Ad-hSTIM1 and NSC into EPCs, and then EPCs were labelled with acLDL-DiI. Last, these transfected EPCs were transplanted by intravenous tail vein after induction of carotid arterial injury. Evans Blue dye was performed to measured reendothelialization at 7 and 14 day after carotid arterial injury. In addition, the neointimal formation was evaluated by staining with hematoxylin and eosin at 14 day after injury.2.2 The interaction between STIM1 and TRPC1 regulate SOCE of EPCsChanges in intracellular Ca²⁺ in individual cells were examined by an Anquacosmos system.The interaction between STIM1 and TRPC1 were examined by Co-immunoprecipitation. Protein levels of inositol 1, 4, 5-trisphosphate (IP3) in the cell supernatants were determined with an ELISA kit.2.3 The role and mechanism of TRPC1 on EPCs proliferation and migrationRat BM-derived EPCs were isolated and cultured, and EPCs between days 5 and 7 was used in vitro experiments. EPCs were transduced with siRNA-TRPC1,shRNA-TRPC1, siRNA-control and shRNA-control respectively for 48?72 h and used in experiments. TRPC1 mRNA and protein levels were examined by realtime PCR and Western blott. The proliferation of EPCs was measured by [3^H]-Thymidine incorporation and cell counting. EPCs migration was determined using a modified Boyden chamber assay. Cell-cycle distribution was analyzed using fluorescence- activated cell sorting. Changes in intracellular Ca²⁺ in individual cells were examined by an Anquacosmos system. Cell cycle gene was measured by a Cell Cycle PCR Array.3.Result3.1.Effect of STIM1 on EPCs proliferation?migration and reendothelialization during vascular repair process after vascular injury3.1.1 Effect of STIM1 on EPCs proliferation?migration. 3.1.1.1 Rat BM-derived EPCs isolation and characterizationRepresentative BM-derived EPCs exhibited a cord-like, cluster-like, or tubular-like appearance. After 5–7 days in culture, attached EPCs were examined by flow cytometry analysis and immunofluorescence. LSCM showed that the majority of cells were dual-stained cells (N>90%), positive for both acLDL-DiI and UEA-1.Flow cytometry analysis demonstrated that the cells expressed endothelial /stem cell markers, including VEGFR-2, CD34, and CD133, but not CD45.3.1.1.2 Effect of STIM1 on EPCs proliferation and migrationSTIM1 was expressed in EPCs by semi-quantitative RT-PCR and Western blotting. Immunocytochemistry was used to further investigate the cellular location of STIM1 in EPCs, and STIM1 was found to be localized in the cytoplasm of EPCs.Rat bone-marrow-derived EPCs were cultured for in vitro experiments. Adenovirus constructs expressing NSC, Ad-hSTIM1, and Ad-si/rSTIM1 were transfected into those EPCs. Transduction of EPCs with Ad-si/rSTIM1 at multiplicities of infection (MOI) of 10 and 20 plaque forming units (pfu)/cell effectively decreased STIM1 mRNA and protein expression at 48 h post-transduction. However, the co-transfection of Ad-hSTIM1 (MOI 10 pfu/cell) with Ad-si/rSTIM1 (MOI 10 pfu/cell) restored the expression of STIM1 both at mRNA level and protein level. Transfection of EPCs with Ad-si/rSTIM1 decreased the uptake of [3^H]-thymidine at 48 h after infection when compared with NSC. The co-transfection of Ad-hSTIM1 (MOI 10 pfu/cell) reversed the effects of STIM1 knockdown on [3^H]-thymidine uptake. In addition, EPCs were first serum starved for 24 h to obtain synchronization in G₀ phase, and then transduced with Ad-si/rSTIM1, Ad-hSTIM1 and NSC for 48 h. FACS was used to measure the cell cycle distribution. Approximately 12.8% of EPCs infected with the NSC progressed into S phase. EPCs infected with Ad-si/rSTIM1 were distributed mainly in G1 phase, and only 0.967% of cells progressed into S phase. EPCs co-infected with Ad-si/rSTIM1 and Ad-hSTIM1 approximately 10.7% of cells progressed into S phase.Last, we used the modified Boyden's chambers to assess the effects of STIM1 on EPC migration. transfection of EPCs with Ad-si/rSTIM1 decreased the number of migrating cells significantly 48 h after infection compared with NSC-transfected cells In the si/rSTIM1 (MOI 10 pfu/cell) knockdown background, Ad-hSTIM1 (MOI 10 pfu/cell) re-expression reversed the effects of STIM1 knockdown in a number of migrating cells.3.1.2. Effect of STIM1 on EPCs function during vascular repair processEvans Blue dye was performed to measured reendothelialization at 7 and 14 day after vascular injury. Blue represented nonendothelialized lesions at injured vessels, white represented the reendothelialized area at uninjured vessels. At 7 and 14 day, the reendothelialized area in the Ad-si/rSTIM1-EPCs infected arteries was obviously less than that in NSC-infected groups (P<0.05). Interestingly, the reendothelialized area in the Ad-si/rSTIM1+ Ad-hSTIM1-EPCs infected arteries restored the levels of NSC-transduced groups (P> 0.05).The neointimal formation was evaluated by staining with hematoxylin and eosin at 14 day after injury. A marked increase in the neointimal area and I/M ratio(0.50±0.01 vs 0.21±0.02, P<0.05)was shown in Ad-si/rSTIM1-EPCs group compared with NSC-transduced groups at 14 day. I/M ratio in the Ad-si/rSTIM1+ Ad-hSTIM1-EPCs transduced arteries restored the levels of NSC-transduced groups (P >0.05).3.2 Effect of STIM1 and TRPC1 on EPCs SOCE3.2.1 Effect of STIM1 on EPCs SOCEWe investigated the effect of STIM1 on EPCs SOCE, to activate SOCE, we depleted intracellular Ca²⁺ stores by treating cells using 1 mMol/L thapsigargin (TG) without extracellular Ca²⁺ and then adding extracellular Ca²⁺ to 2 mM. The TG-mediated SOCE may be attributed to the release of Ca²⁺ from the sarcoplasmic reticulum. The transfection of Ad-si/rSTIM1 at 48 h resulted in a marked decrease in SOCE compared to NSC group (P<0.05). However, in the Ad-si/rSTIM1 knockdown background, the co-transfection of cells with Ad-hSTIM1 reversed the effects of STIM1 knockdown on intracellular Ca²⁺ in EPCs.3.2.2 TRPC1-SOCs participate with STIM1 in mediating SOCE of EPCsOur results demonstrated that TRPC1 was expressed in EPCs by semi-quantitative PCR and Western blotting. At the same time, TRPC1 was localized to the plasma membrane and at intracellular sites in EPCs as determined by immunocytochemistry. Additionally, semi-quantitative RT-PCR and Western blotting were used to address whether the knockdown or re-expression of STIM1 affected the expression of TRPC1. The results demonstrated that TRPC1 mRNA and protein levels were greatly decreased 48 h after si/rSTIM1 transfection. However, Ad-hSTIM1 re-expression reversed the effects of STIM1 knockdown on TRPC1. To determine if STIM1 was associated with the TRPC1 channel in rat EPCs, a co-immunopreci -pitation study was performed. The results demonstrated that STIM1 co-precipitates with TRPC1, indicating a molecular complex had formed between STIM1 proteins and TRPC1 channels in rat EPCs. To further investigate how TRPC1 was regulated by STIM1, we tested the levels of IP3 by ELISA 48 h after NSC, Ad-hSTIM1 and Ad-si/rSTIM1 transfection. The results were not significantly different when the NSC and si/r STIM1 treatments were compared, nor were they significantly different when the si/r STIM1 and si/rSTIM1 + hSTIM1 groups were compared (P > 0.05). This indicated that the function of TRPC1-SOCs incorporated the regulation of SOCE in EPCs via STIM1.3.3. The role and mechanism of TRPC1 on EPCs proliferation and migration3.3.1 The role of TRPC1 on EPCs proliferation and migrationFirst, siRNA-TRPC1, shRNA-TRPC1, siRNA-control, and shRNA- control were transfected into the EPCs. After 48 h of transfection, TRPC1 levels were assessed by real-time RT-PCR and Western blotting 48 h post-transduction. Compared with the controls, transfection with siRNA-TRPC1 or shRNA-TRPC1 attenuated TRPC1 mRNA and protein expression significantly (P<0.05). Next, EPC proliferation was analyzed by a [3^H]-thymidine incorporation assay. The results indicated that transfection of EPCs with siRNA-TRPC1 decreased the uptake of [3^H]-thymidine post-infection when compared with the siRNA-control (877.67±13.74 vs. 1925.67±24.36, n =3, P< 0.05). Transfection of EPCs with shRNA-TRPC1 also decreased the uptake of [3^H]-thymidine compared with the shRNA-control (941.00±4.04 vs. 2058.67±52.42, P < 0.05). At the same time, we performed cell counting to further determine the proliferation of EPCs. Transfection of EPCs with either siRNA-TRPC1 or shRNA-TRPC1 significantly inhibited EPCs proliferation. We also investigated the effects of TRPC1 on EPCs migration using the modified Boyden chamber assay. We observed a notable inhibition of EPCs migration in the siRNA-TRPC1 group and shRNA-TRPC1 at 48 h post-infection compared with the control (P < 0.05). FACS was used to measure the cell cycle distribution. Approximately 16.14% of EPCs infected with the siRNA-control progressed into S phase. EPCs infected with siRNA-TRPC1 or shRNA-TRPC1 was distributed mainly in G1 phase. Approximately 2.15% of the siRNA-TRPC1 group and 2.67% of the shRNA-TRPC1 progressed into S phase.Lastly, we measured whether silencing of TRPC1 inhibits SOCE. both the siRNA-TRPC1 group and the shRNA-TRPC1 group showed a significant downregulation in SOCE at 48 h after infection compared with the siRNA-control or shRNA-control (siRNA-TRPC1 vs. siRNA-control: 33.88±1.81 vs. 97.33±4.10; shRNA-TRPC1 vs. shRNA-control: 27.93±1.60 vs. 101.33±3.28, n=3, P < 0.05).3.3.2 Effects of TRPC1 on EPC cell cycle gene expressionWe analyzed the expression of cell cycle genes using a Cell Cycle PCR gene chips Array. Genes that were either upregulated or downregulated by at least 2-fold in siRNA-TRPC1-infected EPCs compared with the siRNA-control group (P<0.05), of these genes, 9 genes were upregulated in samples from siRNA-TRPC1-infected EPCs as compared with the siRNA-control. The upregulated genes were as follows: Ak1, Brca2, Camk2b, p21, Ddit3, Inha, Slfn1, Mdm2, and Prm1. Four genes were downregulated in siRNA-TRPC1-infected EPCs compared with the siRNA-control group. The downregulated genes were as follows: Bcl2, Mki67, Pmp22, and Ppp2r3a.3.3.3 Impact of Slfn1 on EPC proliferation following TRPC1 silencingTo further confirm the increased expression of Slfn1 in response to TRPC1 silencing. We performed realtime RT-PCR and Western blot analysis. A 9.1-fold increase in Slfn1 mRNA levels and an 8.6-fold increase in Slfn1 protein levels were detected in siRNA-TRPC1 when compared with the siRNA-control. Next, we tested the impact of Slfn1 inhibition combined with TRPC1 silencing on EPC proliferation. To inhibit Slfn1 expression, EPCs were transfected with either siRNA-TRPC1 alone or with siRNA-TRPC1 and a Slfn1-blocking peptide. Interestingly, we found that the G1 arrest induced by TRPC1 silencing was partially reversed in the presence of the Slfn1-blocking peptide. The S-phase population was increased when EPCs is being stimulated in the presence of siRNA-TRPC1 and the Slfn1- blocking peptide,whereas the control antibody did not affect the cell cycle distribution. In addition, the proliferation of EPCs under siRNA-TRPC1 and Slfn1-blocking peptide stimulation was partially restored compared with the siRNA-TRPC1 group (n=3, P < 0.05). Lastly, the reduced uptake of [3^H]-thymidine by EPCs following TRPC1 silencing was also partially reversed by the Slfn1-blocking peptide. 4. Conclusions4.1 knockdown of endogenous STIM1 significantly inhibited EPCs reendothelialization.4.2 STIM1 co-precipitates with TRPC1, indicating a molecular complex, which regulate SOCE, had formed between STIM1 proteins and TRPC1 channels in rat EPCs.4.3 knockdown of endogenous TRPC1 significantly suppressed EPCs reendothelialization.