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OBJECTIVE: To investigate the effect of stromal interaction molecule 1 (STIM1) silencing on EPCs cell cycle. METHODS: Rat bone marrow derived endothelial progenitor cells (EPCs) were isolated and cultured in L-DMEM with 20% FBS. Ad-si/rSTIM1 and Ad-hSTIM1 were then transfected into EPCs and the expression of STIM1 mRNA was detected by RT-PCR. The cell cycle was determined using flow cytometry analysis and intracellular free Ca2+ was measured using LSCM. Co-immunoprecipitation was performed to examine the interaction between STIM1 and TRPC1. Protein levels of inositol 1, 4, 5-trisphosphate were analyzed with ELISA assay. RESULTS: Forty-eight hours after transfection, the expression of STIM1 mRNA was significantly downregulated (0.37 +/- 0.02 vs. 1.00 +/- 0.02, P < 0.05) and intracellular free Ca2+ level was significantly reduced (34.07 +/- 4.10 vs. 86.51 +/- 14.12, P < 0.05) in Ad-si/rSTIM1 group compared with control group. The cell cycle was arrested at G1 phase [(90.91 +/- 1.10)% vs. (77.10 +/- 0.56)%, P < 0.05] and the store-operated channel entry was strikingly inhibited in EPCs after treatment with Ad-si/rSTIM1. However, cotransfection of Ad-hSTIM1 with Ad-si/rSTIM1 significantly reversed these responses. Interestingly, co-immunoprecipitation study showed that STIM1 co-precipitated with TRPC1, and IP3 levels measured by ELISA were similar among three groups (P > 0.05). CONCLUSION: siRNA-mediated knockdown of STIM1 inhibited EPCs proliferation by reducing intracellular free Ca2+ through TRPC1-SOC signaling pathway.
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Células Endoteliales/citología , Silenciador del Gen , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , ARN Interferente Pequeño , Células Madre/citología , Adenoviridae/genética , Animales , Ciclo Celular , Proliferación Celular , Células Cultivadas , Vectores Genéticos , Ratas , Molécula de Interacción Estromal 1 , Transfección , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Knockdown of stromal interaction molecule 1 (STIM1) significantly suppresses neointima hyperplasia after vascular injury. Endothelial progenitor cells (EPCs) are the major source of cells that respond to endothelium repair and contribute to re-endothelialization by reducing neointima formation after vascular injury. We hypothesized that the effect of STIM1 on neointima hyperplasia inhibition is mediated through its effect on the biological properties of EPCs. In this study, we investigated the effects of STIM1 on the proliferation and migration of EPCs and examined the effect of STIM1 knockdown using cultured rat bone marrow-derived EPCs. STIM1 was expressed in EPCs, and knockdown of STIM1 by adenoviral delivery of small interfering RNA (siRNA) significantly suppressed the proliferation and migration of EPCs. Furthermore, STIM1 knockdown decreased store-operated channel entry 48h after transfection. Replenishment with recombinant human STIM1 reversed the effects of STIM1 knockdown. Our data suggest that the store-operated transient receptor potential canonical 1 channel is involved in regulating the biological properties of EPCs through STIM1. STIM1 is a potent regulator of cell proliferation and migration in rat EPCs and may play an important role in the biological properties of EPCs.
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Movimiento Celular , Proliferación Celular , Células Endoteliales/fisiología , Glicoproteínas de Membrana/fisiología , Células Madre/fisiología , Animales , Técnicas de Silenciamiento del Gen , Humanos , Glicoproteínas de Membrana/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Ratas , Molécula de Interacción Estromal 1RESUMEN
Excessive proliferation of vascular smooth cells (VSMCs) plays a critical role in the pathogenesis of diverse vascular disorders, and inhibition of VSMCs proliferation has been proved to be beneficial to these diseases. In this study, we investigated the antiproliferative effect of B-type natriuretic peptide (BNP), a natriuretic peptide with potent antioxidant capacity, on rat aortic VSMCs, and the possible mechanisms involved. The results indicate that BNP potently inhibited AngiotensinII (AngII)-induced VSMCs proliferation, as evaluated by [(3)H]-thymidine incorporation assay. Consistently, BNP significantly decreased AngII-induced intracellular reactive oxygen species (ROS) and NAD(P)H oxidase activity. 8-Br-cGMP, a cGMP analog, mimicked these effects. To confirm its mechanism, siRNA of natriuretic peptide receptor-A(NRPA) strategy technology was used to block cGMP production in VSMCs, and siNPRA attenuated the inhibitory effects of BNP in VSMCs. Taken together, these results indicate that BNP was capable of inhibiting VSMCs proliferation by NPRA/cGMP pathway, which might be associated with the suppression of ROS production. These results might be related, at least partly, to the anti-oxidant property of BNP.
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Angiotensina II/farmacología , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Péptido Natriurético Encefálico/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Receptores del Factor Natriurético Atrial/fisiología , Animales , Aorta/citología , Células Cultivadas , GMP Cíclico/metabolismo , Miocitos del Músculo Liso/citología , NADPH Oxidasas/antagonistas & inhibidores , ARN Interferente Pequeño/farmacología , Ratas , Receptores del Factor Natriurético Atrial/genética , Receptores del Factor Natriurético Atrial/metabolismoRESUMEN
OBJECTIVE: To investigate the correlation between the number and activity of circulating endothelial progenitor cells (EPCs), insulin resistance and severity of coronary lesions in patients with coronary artery disease (CAD). METHODS: Patients with coronary angiography evidenced CAD were divided in insulin resistance group (IR, n = 25) and insulin sensitive group (IS, n = 44) according to insulin level, 25 health volunteers served as control. Circulating EPCs were marked as KDR/CD133+ cells via fluorescence-activated cell sorter analysis. EPCs were also isolated from peripheral blood and cultured in vitro for 7 days, identified by DiI-acLDL uptake and lectin staining methods. EPCs migration activities were determined by modified Boyden chamber assay, EPCs proliferation activities were determined by MTT assay. RESULT: Circulating EPCs number was significantly lower in IR group compared with IS group [(0.34 +/- 0.08) per thousand vs. (0.47 +/- 0.09) per thousand, P < 0.01] and control group (P < 0.05). Both insulin resistance index (r = -0.291, P = 0.01)and Gensini score (r = -0.3984, P = 0.006)were negatively correlated with number of circulating EPCs. Proliferation and migration capacities of EPCs were also significantly lower in IR group compared to those in IS group (all P < 0.05) and control group (all P < 0.05). CONCLUSIONS: Insulin resistance/hyperinsulinemia could aggravate severity of coronary artery lesions via reducing the number and activities of circulating EPCs in patients with CAD.
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Enfermedad de la Arteria Coronaria/sangre , Células Endoteliales/citología , Resistencia a la Insulina , Células Madre/citología , Anciano , Recuento de Células Sanguíneas , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Angiografía Coronaria , Enfermedad de la Arteria Coronaria/patología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Previous animal studies and clinical trials report inconsistent findings regarding the role of statins in pulmonary hypertension (PH). Systematic reviews have shown no use of statins on pulmonary arterial hypertension (PAH). This is the first meta-analysis of randomized controlled trials (RCTs) determining the clinical impacts of statin therapy on patients with PH secondary to lung diseases. METHODS: Electronic databases and manual bibliographical searches were conducted. Eligible studies included RCTs of at least 3 months that evaluated statin therapy as compared with control in adult patients with PH due to pulmonary diseases. Statistical analyses were performed to calculate mean difference, relative risks (RRs), and 95% confidence intervals (CIs) using random-effect model. RESULTS: A total of 6 RCTs were identified and included in this study. Five trials reported the effects of statins in patients with both chronic obstructive pulmonary disease (COPD) and PH, and the remaining 1 was based on PH due to pneumoconiosis. We found that statin therapy was associated with increased 6-minute walk distance and reduced pulmonary artery systolic pressure. There was no observed difference in the incidence of death, drug withdrawal, and adverse event between statin and control group. CONCLUSIONS: Our findings suggest that statins might be safe and beneficial for patients with PH due to chronic lung diseases. However, larger RCTs with more patients and longer observational duration are needed.
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Synthetic grafts are of limited use in small-diameter vessels (Φ<6mm) due to the poor patency rate. The inability of such grafts to achieve early endothelialization together with the compliance mismatch between the grafts and the native vessels promote thrombosis, which eventually leads to graft occlusion. In the current study, stromal cell-derived factor (SDF)-1α/vascular endothelial growth factor (VEGF)-loaded polyurethane (PU) conduits were simply prepared via electrospinning. The mechanical property, drug release behavior and cytocompatibility of the conduits were investigated. The effects of the conduits on endothelial progenitor cell (EPC) mobilization and differentiation were examined in vitro. Then, the conduits were implanted as canine femoral artery interposition grafts. The results revealed that SDF-1α and VEGF were quickly released shortly after implantation, and the conduits exhibited slow and sustained release thereafter. The cytokines had definite effects on EPC mobilization and differentiation in vitro and promoted conduit endothelialization in vivo. The conduits had good tissue compatibility and favorable compliance. All of these features inhibited the conduits from being occluded, thereby improving their long-term patency rate. At 6th month postoperatively, 5 of the 8 grafts were patent while all the 8 grafts without the cytokines were occluded. These findings provide a simple and effective method for the construction of small-diameter artificial blood vessels with the aim of facilitating early endothelialization and improving long-term patency. STATEMENT OF SIGNIFICANCE: (1) SDF-1α/VEGF loaded PU conduits were simply prepared by electrospinning. The cytokines with definite and potent effects on angiogenesis were used to avoid complicated mechanism researches. Compared with most of the current vascular grafts which are of poor strength or elasticity, the conduits have favorable mechanical property. All of these inhibit the conduits from occlusion, and thus improve their long-term patency rate. (2) For the in vivo tests, the dogs did not receive any anticoagulant medication in the follow-up period to expose the grafts to the strictest conditions. In vivo endothelialization of the conduits was thoroughly investigated by Sonography, HE staining, SEM and LSCM. The study will be helpful for the construction of small-diameter artificial blood vessels.
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Prótesis Vascular , Quimiocina CXCL12 , Células Progenitoras Endoteliales/metabolismo , Poliuretanos , Factor A de Crecimiento Endotelial Vascular , Animales , Quimiocina CXCL12/química , Quimiocina CXCL12/farmacocinética , Quimiocina CXCL12/farmacología , Perros , Células Progenitoras Endoteliales/citología , Masculino , Poliuretanos/química , Poliuretanos/farmacología , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/farmacocinética , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Synthetic small-diameter vascular grafts are of limited use mainly due to the lack of endothelial cells (ECs), which inhibit intraluminal thrombosis and intimal hyperplasia. Grafts loaded with homing factors for circulating endothelial progenitor cells (EPCs) have the potential to achieve in situ endothelialization. In view of the important role that EPCs play in the construction of small-diameter artificial blood vessels, antioxidant therapy aiming to inhibit oxidative stress-induced EPC apoptosis should be the focus of clinical interest. In this study, polyethylene glycol coated cerium oxide nanoparticles (CNPs-PEG) with antioxidant properties were successfully synthesized and characterized. The effects of CNPs-PEG on EPC viability and EPC apoptosis induced by oxidative stress were examined. Then, CNPs-PEG together with a potent angiogenesis cytokine vascular endothelial growth factor (VEGF) were encapsulated into polyurethane (PU) scaffolds via electrospinning. The morphology, mechanical properties and CNPs-PEG/VEGF release profiles of the scaffolds were investigated. The growth of EPCs on the scaffolds, and the effects of the released CNPs-PEG and VEGF on anti-EPC apoptosis and endothelialization in vitro were studied. The results showed that CNPs-PEG had favorable stability and cytocompatibility. They could effectively inhibit H2O2-induced EPC apoptosis. The scaffolds showed sustained release behavior of CNPs-PEG/VEGF and favorable cytocompatibility. The released CNPs-PEG retained the anti-apoptosis properties and, moreover, enhanced the effects of VEGF on the mobilization and differentiation of EPCs. It is concluded that the combined application of CNPs-PEG and VEGF in electrospun PU scaffolds facilitated endothelialization in vitro, and thus should be promising for the construction of small-diameter artificial blood vessels.
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AIM: To explore mir-542-3p mediated inhibition of vascular smooth muscle cell (VSMC) proliferation through the inhibition of Syk activation. METHODS AND RESULTS: MicroRNA (mir)-542-3p was selected for analysis based on miRNA microarray and qRT-PCR results. In vitro mir-542-3p expression was significantly downregulated in old (o)VSMCs compared with young (y)VSMCs under serum stimulation conditions. Upregulation of mir-542-3p in oVSMCs significantly inhibited VSMC proliferation, whereas downregulation of mir-542-3p in yVSMCs increased VSMC proliferation. We identified spleen tyrosine kinase (Syk) as a direct target of mir-542-3p by database search, and showed that its expression and phosphorylation were higher in oVSMCs than in yVSMCs after serum stimulation. Luciferase assays confirmed that Syk is a direct target of miR-3542-3p. Knock-down of mir-542-3p in yVSMCs inhibited the activation of the Syk downstream effectors STAT3 and STAT5, whereas mir-542-3p overexpression enhanced STAT3 and STAT5 activities. In a rat balloon injury model, mir-542-3p inhibited neointima formation and proliferating cell nuclear antigen (PCNA) protein expression. CONCLUSION: Mir-542-3p modulates VSMC proliferation via the Syk/STAT3-STAT5 axis. Downregulation of mir-542-3p may explain age-related neointimal hyperplasia in rats.
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Envejecimiento/genética , Regulación hacia Abajo/genética , MicroARNs/genética , Neointima/genética , Animales , Proliferación Celular/genética , Masculino , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Fosforilación/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/genética , Regulación hacia ArribaRESUMEN
The purpose of the present study was to determine whether using hydrogen-rich medium (HRM) to increase hydrogen levels in endothelial cells (ECs) protects ECs from apoptosis induced by advanced glycation end products (AGEs). The thoracic aorta was removed from 2-3-year-old Sprague-Dawley rats, and ECs were isolated and cultured. After culturing ECs in the presence of AGEs and/or with HRM for 24 h, Annexin V/7-AAD and TUNEL staining were carried out to detect apoptosis. Intracellular ROS were detected by fluorescent probe and quantified by flow cytometry. The expression of antioxidative enzymes (superoxide dismutase, glutathione peroxidase) was determined by real-time PCR analysis and enzymatic assay. The relative expression levels of Bcl-2 and Bax were analyzed by western blotting. The addition of AGEs increased the apoptosis of ECs in a concentration-dependent manner and HRM reduced the AGE (400 µg/ml)-induced apoptosis from 21.61±2.52 to 11.32±1.75%. HRM also significantly attenuated the AGE-induced intracellular ROS induction and decrease in the expression of antioxidative enzymes. In conclusion, hydrogen exhibits significant protective effects against AGE-induced EC injury possibly through reducing ROS generation, intracellular antioxidant enzyme system protection and elevation of the Bcl-2/Bax ratio.
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Apoptosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Hidrógeno/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Células Cultivadas , Productos Finales de Glicación Avanzada/farmacología , Estrés Oxidativo/efectos de los fármacos , Ratas , Superóxido Dismutasa/metabolismoRESUMEN
Endothelial progenitor cells (EPCs) play an important role in accelerating endothelial repair after vascular injury. The proliferation and migration of EPCs is a critical first step in restoring endothelial. However, mechanisms for modulating EPC proliferation and migration are still being elucidated. Our previous study found that transient receptor potential canonical-1 (TRPC1) is involved in regulating store-operated Ca(2+) entry in EPCs through stromal interaction molecule 1. Therefore, in the present study, we sought to further investigate the regulation of proliferation and migration of EPCs by TRPC1. We found that the silencing of TRPC1 by 2 different RNA interference methods suppressed the proliferation and migration of EPCs. In addition, knockdown of TRPC1 significantly reduced of the amplitude of store-operated Ca(2+) entry and caused arrest of the EPC cell cycle in G1 phase. Analysis of the expression of 84 cell cycle genes by microarray showed that 9 genes were upregulated and 4 were downregulated by >2-fold in EPCs following TRPC1 silencing. The genes with expression changes were Ak1, Brca2, Camk2b, p21, Ddit3, Inha, Slfn1, Mdm2, Prm1, Bcl2, Mki67, Pmp22, and Ppp2r3a. Finally, we found that a Schlafen 1-blocking peptide partially reversed the abnormal cell cycle distribution and proliferation induced by TRPC1 knockdown, suggesting that Schlafen 1 is downstream of TRPC1 silencing in regulating EPC proliferation. In summary, these findings provide a new mechanism for modulating the biological properties of EPCs and suggest that TRPC1 may be a new target for inducing vascular repair by EPCs.
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Movimiento Celular , Proliferación Celular , Células Endoteliales/citología , Silenciador del Gen , Células Madre/citología , Canales Catiónicos TRPC/metabolismo , Animales , Biomarcadores/metabolismo , Calcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Células Endoteliales/metabolismo , Citometría de Flujo , Puntos de Control de la Fase G1 del Ciclo Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratas , Células Madre/metabolismo , Canales Catiónicos TRPC/genética , Vía de Señalización WntRESUMEN
BACKGROUND: Reactive oxygen species (ROS) play a pivotal role in neointima hyperplasia after balloon injury. Molecular hydrogen has emerged as a novel antioxidant and has been proven effective in treating many diseases. OBJECTIVES: We aimed to determine the mechanism by which hydrogen affects neointima formation. METHODS: We assessed the influence of a hydrogen-rich saline solution (HRSS) by daily injection in rats. Rats were euthanized to evaluate the neointima. ROS, malondialdehyde (MDA) and superoxide dismutase (SOD) and reduced glutathione (GSH), were detected in the injured artery. Macrophage infiltration and the production of inflammatory factors (i.e., IL-6, TNF-α and NF-κB) were also observed. The in vitro effects of hydrogen on vascular smooth muscle cell (VSMC) proliferation were also measured. RESULTS: HRSS decreased the neointima area significantly. The neointima/media ratio was also reduced by HRSS. There was a decline in the number of PCNA-positive cells in the intima treated with HRSS. Meanwhile, HRSS ameliorated the ROS and MDA levels and increased SOD, reduced GSH levels in the injured carotid. In addition, the levels of inflammatory factors, such as IL-6, TNF-α and NF-κB p65, were attenuated by HRSS. In vitro studies also confirmed the anti-proliferative capability of the hydrogen solution and ROS generation in VSMCs induced by PDGF-BB. CONCLUSION: HRSS may have a protective role in the prevention of neointima hyperplasia and restenosis after angioplasty. HRSS may partially exert its role by neutralizing the local ROS and suppressing the TNF-α/NF-κB pathway.