Protective effect of Shenmajing formula on ischemic brain injury in mice / 药学实践杂志

Objective To observe the protective effect of Shenmajing formula on brain tissue of mice with cerebral ischemic injury and explore the possible mechanism. Methods Thirty SPF-grade C57 BL/6 male mice were randomly divided into model control group, Shenmajing group and nimodipine group, and the animal models of cerebral ischemic injury in mice were prepared by electrocoagulation. The protein expression level in endothelial progenitor cells were detected by Western blot. Results Compared with the model control group, the infarct volume of mice in the Shenmajing group was significantly reduced, and the migration, adhesion and tubule formation ability of endothelial progenitor cells were significantly improved, and the expression level of BDNF protein in endothelial progenitor cells was significantly increased. Conclusion The protective effect of Shenmajing granules on brain tissue of mice with cerebral ischemic injury could be closely related to the regulation of BDNF expression in endothelial progenitor cells and improvement of endothelial progenitor cell function of bone marrow origin.

Enrichment of miR-17-5p enhances the protective effects of EPC-EXs on vascular and skeletal muscle injury in a diabetic hind limb ischemia model

BACKGROUND/AIMS: Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXsmiR-17-5p) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo. METHODS: EPCs transfected with scrambled control or miR-17-5p mimics were used to generate EPC-EXs and EPC-EXsmiR-17-5p. Db/db mice were subjected to hind limb ischemia. After the surgery, EPC-EXs and EPC-EXsmiR-17-5p were injected into the gastrocnemius muscle of the hind limb once every 7 days for 3 weeks. Blood flow, microvessel density, capillary angiogenesis, gastrocnemius muscle weight, structure integrity, and apoptosis in the hind limb were assessed. Vascular endothelial cells (ECs) and myoblast cells (C2C12 cells) were subjected to hypoxia plus high glucose (HG) and cocultured with EPC-EXs and EPC-EXsmiR-17-5p. A bioinformatics assay was used to analyze the potential target gene of miR-17-5p, the levels of SPRED1, PI3K, phosphorylated Akt, cleaved caspase-9 and cleaved caspase-3 were measured, and a PI3K inhibitor (LY294002) was used for pathway analysis. RESULTS: In the DHI mouse model, miR-17-5p was markedly decreased in hind limb vessels and muscle tissues, and infusion of EPC-EXsmiR-17-5p was more effective than EPC-EXs in increasing miR-17-5p levels, blood flow, microvessel density, and capillary angiogenesis, as well as in promoting muscle weight, force production and structural integrity while reducing apoptosis in gastrocnemius muscle. In Hypoxia plus HG-injured ECs and C2C12 cells, we found that EPC-EXsmiR-17-5p could deliver their carried miR-17-5p into target ECs and C2C12 cells and subsequently downregulate the target protein SPRED1 while increasing the levels of PI3K and phosphorylated Akt. EPC-EXsmiR-17-5p were more effective than EPC-EXs in decreasing apoptosis and necrosis while increasing viability, migration, and tube formation in Hypoxia plus HG-injured ECs and in decreasing apoptosis while increasing viability and myotube formation in C2C12 cells. These effects of EPC-EXsmiR-17-5p could be abolished by a PI3K inhibitor (LY294002). CONCLUSION: Our results suggest that miR-17-5p promotes the beneficial effects of EPC-EXs on DHI by protecting vascular ECs and muscle cell functions.

EPCs-exos combined with tanshinone Ⅱ_A protect vascular endothelium cells from oxidative damage via PI3K/Akt pathway / 中国中药杂志

This study aims to investigate the molecular mechanism of tanshinone Ⅱ_(A )(TaⅡ_A) combined with endothelial progenitor cells-derived exosomes(EPCs-exos) in protecting the aortic vascular endothelial cells(AVECs) from oxidative damage via the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt) pathway. The AVECs induced by 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine(POVPC) were randomly divided into model, TaⅡ_A, EPCs-exos, and TaⅡ_A+EPCs-exos groups, and the normal cells were taken as the control group. The cell counting kit-8(CCK-8) was used to examine the cell proliferation. The lactate dehydrogenase(LDH) cytotoxicity assay kit, Matrigel assay, DCFH-DA fluorescent probe, and laser confocal microscopy were employed to examine the LDH release, tube-forming ability, cellular reactive oxygen species(ROS) level, and endothelial cell skeleton morphology, respectively. The enzyme-linked immunosorbent assay was employed to measure the expression of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were employed to determine the mRNA and protein levels, respectively, of PI3K and Akt. Compared with the control group, the model group showed decreased cell proliferation and tube-forming ability, increased LDH release, elevated ROS level, obvious cytoskeletal disruption, increased expression of IL-1β, IL-6, and TNF-α, and down-regulated mRNA and protein levels of PI3K and Akt. Compared with the model group, TaⅡ_A or EPCs-exos alone increased the cell proliferation and tube-forming ability, reduced LDH release, lowered the ROS level, repaired the damaged skeleton, decreased the expression of IL-1β, IL-6, and TNF-α, and up-regulated the mRNA and protein levels of PI3K and Akt. TaⅡ_A+EPCs-exos outperformed TaⅡ_A or EPCs-exos alone in regulating the above indexes. The results demonstrated that TaⅡ_A and EPCs-exos exerted a protective effect on POVPC-induced AVECs by activating the PI3K/Akt pathway, and the combination of the two had stronger therapeutic effect.

Relationship between promoting effect of Zuogui Pills on angiogenesis of reproductive organs and mobilization factors GM-CSF, SDF-1, and their receptors of EPCs in early-aging rats / 中国中药杂志

This study aimed to investigate the relationship between the promoting effect of Zuogui Pills on ovarian and vaginal angiogenesis in early-aging rats and mobilization factors granulocyte-macrophage colony-stimulating factor(GM-CSF), stromal cell-derived factor-1(SDF-1), and their receptors of endothelial progenitor cells(EPCs) and explore the mechanism of Zuogui Pills in improving reproductive hypofunction in early-aging rats. Ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS) was used to analyze the chemical components of the extract of Zuogui Pills. Forty 14-month-old female early-aging rats with estrous cycle disorder were randomly divided into a blank group, a conjugated estrogen group(conjugated estrogen suspension, 65 μg·kg~(-1)), and low-(11 g·kg~(-1)) and high-dose(33 g·kg~(-1)) Zuogui Pills groups, with 10 rats in each group. In addition, 10 4-month-old female rats were assigned to the youth control group. The rats in the blank group and the youth control group were treated with 20 g·kg~(-1) distilled water by gavage, while those in the groups with drug intervention were treated with corresponding drugs by gavage, once a day for 15 days. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of SDF-1 and GM-CSF in the mobilization of EPCs in serum. Hematoxylin-eosin(HE) staining was used to observe the changes in the number of ovarian follicles at all levels and corpus luteum, the number of vaginal epithelial layers, the number of vaginal folds, and the blood vessels of ovarian and vaginal tissues in the groups with drug intervention. Western blot was used to detect the expression of ER, GM-CSFR, CXCR4, and CXCR7 proteins in ovarian and vaginal tissues. As revealed by the results, the blank group showed decreased number of corpus luteum, gro-wing follicles at all levels, and blood vessels(P<0.05), decreased thickness of vaginal mucosa, the number of epithelial layers, the number of vaginal folds, and the number of vessels in the lamina propria(P<0.05), reduced content of SDF-1 and GM-CSF in the peripheral blood(P<0.05), and down-regulated levels of ER, CXCR4, CXCR7, and GM-CSFR proteins in ovarian and vaginal tissues(P<0.05). The groups with drug intervention showed increased number of growing follicles at all levels, corpus luteum, and blood vessels(P<0.05), decreased number of atresia follicles(P<0.05), increased thickness of vaginal mucosa, the number of epithelial layers, the number of vaginal mucosal folds, and the number of blood vessels in the lamina propria(P<0.05), increased content of SDF-1 and GM-CSF in the peripheral blood(P<0.05), and up-regulated levels of ER, CXCR4, CXCR7, and GM-CSFR proteins in ovarian and vaginal tissues(P<0.05). This experiment suggests that Zuogui Pills may promote ovarian and vaginal angiogenesis and improve the reproductive function of early-aging rats by up-regulating the levels of mobilization factors SDF-1, GM-CSF, and their receptors of EPCs.

Endothelial Progenitor Cells and NADPH Oxidase Enzyme Activity in the Development of an Aortic Aneurysm

Abstract Introduction: Endothelial progenitor cells (EPCs) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme activity may affect the vessel wall and have a role in development of aortic aneurysms. EPCs originate from hematopoietic stem cells and can be enumerated from peripheral blood samples by flow cytometry. In this study, we aimed to evaluate the relation of EPC number and NADPH oxidase enzyme activity in the development of thoracic aortic aneurysm (TAA). Methods: Patients with TAA (n=30) and healthy individuals without TAA (control, n=10) were included in our study. Characterization and enumeration of EPC from peripheral blood samples were performed by flow cytometry with panels including markers of EPCs (CD34/CD133/CD309/CD146/CD144). Additionally, NADPH oxidase enzyme activity (capacity) was also measured by the dihydrorhodamine 123 (DHR 123) test. Results: The enumeration of EPC with CD34+/CD146+ marker showed that the number of mean EPC/106 cells was increased in the patient group (41.5/106 cells), but not in the control group (20.50/105 cells) (P<0.01). Additionally, patients with TAA presented significantly lower NADPH oxidase activity by DHR assay than healthy controls (mean stimulation index: 60.40± 7.86 and 75.10±5.21, respectively) (P<0.01). Conclusion: Our results showed that the number of EPCs is significantly higher in aortic aneurysm patients and may have a role in disease progression. The crosstalk between NADPH oxidase enzyme capacity and EPC number may be useful as a parameter to explain the clinical progression of TAA.

Study on the serum level of CoQ10B in patients with Moyamoya disease and its mechanism of affecting disease progression / Estudo sobre o nível sérico de CoQ10B em pacientes com doença de Moyamoya e seu mecanismo de afetar a progressão da doença

ABSTRACT Background: At present, the etiology and pathogenesis of Moyamoya disease (MMD) are not completely clear. Patients are usually diagnosed after cerebrovascular events. Therefore, it is of great clinical significance to explore the predictive factors of MMD. Objective: This study aimed to investigate the serum level of CoQ10B, the amount of endothelial progenitor cells (EPCs), and mitochondrial function of EPCs in MMD patients. Methods: Forty-one MMD patients and 20 healthy controls were recruited in this study. Patients with MMD were divided into two groups: Ischemic type (n=23) and hemorrhagic type (n=18). Blood samples were collected from the antecubital vein and analyzed by CoQ10B ELISA and flow cytometry. Measures of mitochondrial function of EPCs include oxygen consumption rate (OCR), mitochondrial membrane potential, Ca2+ concentration, adenosine triphosphatases activity and ROS level. Results: The serum CoQ10B level in MMD patients was significantly lower than that in healthy controls (p<0.001). The relative number of EPCs in MMD patients was significantly higher than that in healthy controls (p<0.001). Moreover, the OCR, mitochondrial membrane potential and ATPase activity were decreased and the Ca2+ and reactive oxygen species levels were increased in MMD patients (p<0.001). Conclusions: Our results showed obviously decreased serum CoQ10B level and increased EPCs number in patients with MMD compared with healthy patients, and the mitochondria function of EPCs in MMD patients was abnormal.

RESUMO Antecedentes: No momento, a etiologia e a patogênese da doença de Moyamoya (DMM) não são completamente claras. Os pacientes geralmente são diagnosticados após eventos cerebrovasculares. Sendo assim, é de grande importância clínica explorar os fatores preditivos de DMM. Objetivo: Este estudo teve como objetivo investigar o nível sérico de CoQ10B, a quantidade de células progenitoras endoteliais (CPE) e a função mitocondrial de CPE em pacientes com DMM. Métodos: Quarenta e um pacientes com DMM e 20 controles saudáveis foram recrutados neste estudo. Aqueles com DMM foram divididos em dois grupos: tipo isquêmico (n=23) e tipo hemorrágico (n=18). Amostras de sangue foram coletadas da veia antecubital e analisadas por CoQ10B Ensaio de Imunoadsorção Enzimática (ELISA) e citometria de fluxo. As medidas da função mitocondrial de CPE incluem taxa de consumo de oxigênio (TCO), potencial de membrana mitocondrial, concentração de Ca2+, atividade de adenosina trifosfatases (ATPase) e nível de espécies reativas de oxigênio (ROS). Resultados: O nível sérico de CoQ10B em pacientes com DMM foi significativamente menor do que em controles saudáveis (p<0,001). O número relativo de CPE em pacientes com MMD foi significativamente maior do que em controles saudáveis (p<0,001). Além disso, a TCO, o potencial de membrana mitocondrial e a atividade ATPase diminuíram e os níveis de Ca2+e ROS aumentaram em pacientes com MMD (p<0,001). Conclusões: Nossos resultados mostraram obviamente diminuição do nível sérico de CoQ10B e aumento do número de CPE em pacientes com DMM em comparação com pacientes saudáveis, e a função mitocondrial de CPE em pacientes com DMM estava anormal.

Effect and mechanism of estrogen on EPCs function in diabetic rats / 药学实践杂志

Objective To explore the effect and mechanism of estrogen on endothelial progenitor cells（EPCs）function in diabetic rats. Methods EPCs were isolated from bone marrow of rats and characterized by fluorescence microscopy and flow cytometry. Rat diabetic model was established via streptozotocin induction. The bone marrow was taken to culture EPCs. EPCs of diabetes were incubated with Estrogen 10 nmol/L for 24h. The functions and proliferation of EPCs in vitro were detected. The levels of MnSOD and NO in EPCs and TSP-1 in supernatant were assayed. Results Compared with control group, EPCs proliferation, adhesion and angiogenesis functions were impaired in diabetic rats. The level of MnSOD and NO in diabetic EPCs were significantly decreased, while TSP-1 protein level in the supernatant increased. The above changes can be reversed with estrogen incubation. Conclusion Estrogen improved the EPCs migration and tubule formation in diabetic rats. The mechanism may be related to the reduction of oxidative stress and downregulation of TSP-1 expression in diabetic EPCs.

Effects of endothelial progenitor cell-derived exosomes on neuronal injury induced by oxygen-glucose deprivation and restoration / 中华麻醉学杂志

Objective:To evaluate the effects of endothelial progenitor cell (EPC)-derived exosomes on neuronal injury induced by oxygen-glucose deprivation and restoration (OGD/R).Methods:HT22 neurons of mice were cultured and divided into 3 groups ( n=30 each) using a random number table method: control group (C group), OGD/R group and OGD/R plus EPC-derived exosome group (OGD/R+ EXO group). Cells in group C were cultured in normal atmosphere.In group OGD/R, the cells were exposed to 94%N 2-1%O 2-5%CO 2 for 6 h in glucose- and serum-free DMEM medium, followed by 24 h restoration of O 2 and glucose in the normal medium.In group OGD/R+ EXO, 20 μg/ml EPC-derived exosomes were added to the culture medium at 24 h before developing the model.EPCs were identified by immunofluorescence staining.Exosomes were identified by Western blot, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Cell viability was measured by CCK-8 assay, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by enzyme-linked immunosorbent assay.The neuronal apoptosis was detected by TUNEL staining, and the apoptosis rate was calculated.The expression of Bax, Bcl-2, caspase-3, and cleaved caspase-3 was determined by Western blot, and cleaved caspase-3/caspase-3 ratio was calculated. Results:The cultured cells were EPCs, and EPC-derived exosomes were successfully extracted.Compared with group C, the cell viability was significantly decreased, the content of MDA was increased, the activity of SOD was decreased, the apoptosis rate was increased, the expression of Bax was up-regulated, the expression of Bcl-2 was down-regulated, and the ratio of cleaved-caspase-3/caspase-3 was increased in group OGD/R and group OGD/R+ EXO ( P<0.05). Compared with group OGD/R, the cell viability was significantly increased, the content of MDA was decreased, the activity of SOD was increased, the apoptosis rate was decreased, the expression of Bax was down-regulated, the expression of Bcl-2 was up-regulated, and the ratio of cleaved-caspase-3/caspase-3 was decreased in group OGD/R+ EXO ( P<0.05). Conclusions:EPC-derived exosomes can reduce OGD/R-induced neuronal injury, which is related to inhibition of oxidative stress and neuronal apoptosis.

Application studies and challenges of endothelial progenitor cells transplantation for diabetes retinopathy / 中华实验眼科杂志

Endothelial progenitor cells (EPCs) are progenitor cells possessing vasculogenic potential.The main function of EPCs is to play a role by paracrine angiogenic factors and neuroprotective factors.EPCs also have the ability to differentiate into endothelial cells and integrate themselves into newly formed capillaries.Therefore, EPCs play an important role in vascular repair and neuroprotection.The research on surface markers and functions of EPCs is the basis of EPCs research.A series of clinical trials, animal and cell experiments show that EPCs transplantation and joint transplantation of EPCs and other cells can promote vascular repair and improve retinal function with good safety.EPCs are expected to be an effective treatment for diabetic retinopathy (DR). DR is now defined as a refractory eye disease with retinal neurovascular unit (NVU) injury associated with systemic metabolism anbormality.Change in EPCs count and damage of EPCs function are involved in the occurrence and development of DR.Ophthalmologists should pay attention to the early managing approach of EPCs.Current treatment strategies include transplantation of EPCs, joint transplantation of EPCs with other cells, and regulation of endogenous EPCs.The unique biological characteristics of EPCs provide many possibilities in repairing retinal NVU injury and DR prevention and treatment.This article introduces the latest research progress of EPCs for DR from five aspects including the origin of EPCs, physiological and pathological state, function, treatment strategy and clinical application.At the same time, the existing problems and technical bottlenecks will also be discussed.

Endothelial Progenitor Cells Derived from mESCs Were Directed to Differentiate into Vascular Endothelial Cells and Promote Wound Healing / 中国生物化学与分子生物学报

Ischemic dysfunction is an important global health problem. Vascular endothelial cells(VECs) play a key role in angiogenesis, and insufficient vascular remodeling may lead to chronic nonhealing wounds. Therefore, effective VEC generation strategies of exploration help improve angiogenesisin damaged tissues. Embryonic stem cells (ESCs) are widely used in the study of tissueendothelialization, and endothelial progenitor cells (EPCs) are indispensable parts of the development ofVECs. The aims of this study were to find a rapid, easily screened and reproducible method for thederivation of EPCs from mouse embryonic stem cells (mESCs), and obtain VECs with high survival ratesand strong functions from the directed differentiation of the EPCs. The results showed that mESCs weredifferentiated into " stepping stone" -like progenitor cells with active proliferative ability by 10 ng / mLVEGF and 5 ng / mL bFGF. At the same time, the method of differential adherence was helpful for theselection of EPCs, and EPCs induced high expression of CD133 and CD34 (The relative expressionlevels were 0. 88 ± 0. 04 and 2. 12 ± 0. 02, respectively) for 3 days. Then EPCs were digested withacctuse enzymes, and induced to differentiate into vascular endothelial-like cells by 50 ng / mL VEGF and25 ng / mL bFGF for 7 days. The endothelial cells not only expressed endothelial marker genes (CD31, CD144, LAMA5, Tek, KDR and vWF),and marker proteins CD31, CD144 and LAMA5 (The relativeexpression levels were 1. 07 ± 0. 03, 0. 60 ± 0. 02 and 0. 70 ± 0. 02, respectively), but also had thegood ability of migration, tubulogenesis and formation of W-P bodies. Moreover, PBS, EPC and VECwere used to treat wounds of the same size. Both EPC and VEC could accelerate the degree of tissuehealing (The relative healing rates were 78. 93 ± 75. 35%, 95. 57 ± 83. 73% and 100. 00 ± 0. 00%, respectively), and VEC significantly enhanced the ability of wound angiogenesis and inflammatoryresponses. In consequence, this study preliminarily confirmed that mESC-derived EPCs coulddifferentiate into VECs after directional induction for 7 days, which had good function of tissue repair. The physiological pathway on stem cells by stimulating angiogenesis is expected to become a new target fortissue remodeling.

Effects of PINK1 mediated mitophagy on senescence and function of rat bone marrow endothelial progenitor cells / 中国药理学通报

Aim To investigate the effects of PTEN-induced putative kinase1(PINK1)mediated mitophagy on senescence and function of rat bone marrow endothelial progenitor cells(EPCs)by using small interfering RNA(siRNA)technology to knock down the PINK1 gene in rat bone marrow EPCs.Methods EPCs from bone marrow in rats were isolated, cultured and identified.After counting, EPCs were randomlydivided into control group, negative control group(NC siRNA), and Pink1 transfection group(PINK1 siRNA).The expression of PINK1 mRNA and protein in cells in various groups were detected by qRT-PCR and Western blot.At the same time, different time points were chosen to simulate the aging process based on the best knock down time.The senescence of cells was detected by SA-β-galactosidase staining and p16 protein expression.The function of cell proliferation, migration and tubule formation was detected by CCK-8, Transwell chamber and in vitro angiogenesis kit.ROS level was detected by flow cytometry.The expressions of PINK1, Parkin, LC3, and p62 were detected by Western blot.Mitochondria and autophagosomes were observed by transmission electron microscope.Results 48 h after PINK1 siRNA transfected, PINK1 was effectively knocked down.Compared with control group, the positive rate of blue staining and the expression of p16 protein in PINK1 siRNA group increased significantly 48 h and 96 h after transfection.The function of cell proliferation, migration and tubule formation decreased significantly.The level of ROS increased significantly, while the expression of PINK1, Parkin and LC3 protein decreased significantly, and p62 protein expression increased significantly.Under the transmission electron microscope, the mitochondria swelled and denatured, and the number of autophagosomes decreased in the PINK1 siRNA group.Conclusions The down-regulation of PINK1 gene can aggravate the senescence of EPCs, and PINK1 mediated mitophagy may participate in the regulation of senescence and function of EPCs.

Exendin-4 attenuates high glucose-induced endothelial progenitor cell dysfunction by regulating SIRTl/p53 signaling pathway / 中国药理学通报

Aim To investigate the effect of Exendin-4 high glucose and the function of silent information reg- on endothelial progenitor cells ( EPCs) induced by ulator 1 (SIRT1 ).Methods EPCs were isolated and cultured by density gradient centrifugation from peripheral blood of healthy volunteers.Different concentrations of Exendin-4( 12.5, 50, 100, 200 (xmol • L"1) induced EPCs were respectively performed by activity test to select the appropriate concentration.The optimal concentrations of Exendin-4 and high glucose (25 mmol • L 1) were incubated together for 72 h to detect the functional activities of EPCs.The capabilities of migration, adhension and tube formation of EPCs in vitro were detected respectively.The levels of LDH and MDA in EPCs were detected.The mRNA expressions of TNF-a, 1L-1 p and IL-6 in EPCs were measured by real-time fluorescent quantitative PCR ( RT-qPCR ).The protein expression of SIRT1 , P53 and Ac-P53 in EPCs were determined by Western blot.Results Ex- endin-4 could increase the viability of EPCs induced by j J high glucose in a dose-dependent manner, especially for 50 (xmol • L "1 (P <0.05 ).Hie results of Western blot showed that the protein expression of SIRT1 was significantly enhanced by 50 |xmol • L"' Exendin-4 treatment ( P < 0.05 ).Compared with high glucose group, Exenclin-4 significantly increased the migration, adhesion, tube formation of EPCs (P<0.05) and decreased the level of LDH and MDA ( P < 0.05 ).The mRNA expression of TNF-cx, lL-(3 and IL-6 in EPCs also decreased (P < 0.05).However, the protective effects of Exendin-4 could he significantly blocked by SIRT1 inhibitor ( EX-527) (P<0.05).In addition, the S1HT1 agonist ( SHT1720 ) could also improve the dysfunction of EPCs induced by high glucose ( P < 0.05).Conclusions Exendin-4 can improve the viability of human EPCs, restore the EPCs normal function, reduce high glucose-induced oxidative damage, and reduce the releases of inflammatory cvtokines un- j j der high glucose condition, which may be related to the regulation of S1HT1/P53 signaling pathway.

Exercício Físico Melhora as Funções das Células Progenitoras Endoteliais em Pacientes com Síndrome Metabólica / Exercise Training Improves Functions of Endothelial Progenitor Cells in Patients with Metabolic Syndrome

Resumo Fundamento As células progenitoras endoteliais (CPEs) desempenham um papel importante na manutenção da função endotelial. A síndrome metabólica (SM) está associada à disfunção das CPEs. Embora o exercício físico tenha um impacto benéfico na atividade das CPEs, seu mecanismo ainda não está completamente esclarecido. Objetivo O objetivo deste estudo é investigar os efeitos do exercício físico nas funções das CPEs e os mecanismos subjacentes em pacientes com SM. Métodos Os voluntários com SM foram divididos em grupo exercício (n=15) e grupo controle (n=15). Antes e após 8 semanas de treinamento físico, as CPEs foram isoladas do sangue periférico. Foram feitos o ensaio de unidades formadoras de colônias (UFC), o ensaio de formação de tubos, a expressão proteica do óxido nítrico sintase endotelial (eNOS), da fosfatidilinositol-3-quinase (PI3-K) e da proteína quinase B (AKT). Considerou-se um valor de probabilidade <0,05 para indicar significância estatística. Resultados Após 8 semanas, o número de UFCs aumentou significativamente no grupo exercício em comparação com o grupo controle (p<0,05). Além disso, observamos uma diminuição significativa do modelo de avaliação da homeostase da resistência à insulina (HOMA-IR), endotelina-1, proteína C reativa de alta sensibilidade e dos níveis de homocisteína no grupo exercício. A intervenção com exercícios também pode aumentar a capacidade de formação de tubos de CPEs e aumentar o nível de fosforilação de eNOS, PI3-K e AKT. Conclusão O exercício físico aprimorou as funções das CPEs. O mecanismo pode estar relacionado ao exercício, ativando a via PI3-K/AKT/eNOS.

Abstract Background Endothelial progenitor cells (EPCs) play an important role in maintaining endothelial function. Metabolic syndrome (MetS) is associated with EPC dysfunction. Although physical exercise has a beneficial impact on EPC activity, its mechanism is not completely clear yet. Objective The purpose of this study is to investigate the effects of physical exercise on the functions of EPCs and the underlying mechanisms in patients with MetS. Methods Volunteers with MetS were divided into exercise group (n=15) and control group (n=15). Before and after 8 weeks exercise training, EPCs were isolated from peripheral blood. Colony forming unit (CFU) assay, tube-formation assay, the protein expression of endothelial nitric oxide synthase (eNOS), phosphatidylinositol-3-kinase (PI3-K) and protein kinase B (AKT) were determined. A probability value <0.05 was considered to indicate statistical significance. Results After 8 weeks, the number of CFUs was significantly increased in the exercise group compared to the control group (p<0.05). In addition, we observed a significant decrease of homeostasis model assessment for insulin resistance (HOMA-IR), endothelin-1, high-sensitive C-reactive protein, and homocysteine levels in the exercise group. Exercise intervention could also enhance tube-formation capacity of EPCs and increase phosphorylation level of eNOS, PI3-K and AKT. Conclusion Physical exercise enhanced the functions of EPCs. The mechanism may be related to exercise, activating the PI3-K/AKT/eNOS pathway.

Effects of Sirt1 on proliferation, migration, and apoptosis of endothelial progenitor cells in peripheral blood of SD rats with chronic obstructive pulmonary disease

Objective: To explore the effect of Sirt1 on the function of endothelial progenitor cells (EPCs) in rats with chronic obstructive pulmonary disease (COPD). Methods: A rat COPD model was established via smoking and endotoxin administration for three months. The peripheral circulating EPCs were isolated by gradient centrifugation, and their functions, cell cycle distribution, apoptosis, and Sirt1 expression were examined. The function changes of EPCs in the presence or absence of Sirt1 agonist and inhibitor were estimated; meanwhile, the expressions of Sirt1, FOXO3a, NF-κB, and p53 were also evaluated. Results: The proliferation, adhesion, and migration of EPCs decreased while the apoptosis rate was increased in the COPD rats. The expression of Sirt1 protein in EPCs of the COPD group was significantly lower than that in the control group (P<0.01). The overexpression of the Sirt1 gene using a gene transfection technique or Sirt1 agonists (SRT1720) improved the proliferation, migration, and adhesion, and decreased the apoptosis of EPC. However, Sirt1 inhibitor (EX527) decreased EPC functions in the COPD group. The effect of Sirt1 expression on EPC function may be related to reduction of FOXO3a and increase of NF-κB and p53 activity. Conclusions: Increased expression of Sirt1 can improve the proliferation and migration of EPCs and reduce their apoptosis in COPD rats. This change may be related to FOXO3a, NF-κB, and p53 signaling pathways.

Gualou-Xiebai-Banxia decoction protects against type II diabetes with acute myocardial ischemia by attenuating oxidative stress and apoptosis via PI3K/Akt/eNOS signaling / 中国天然药物

Gualou-Xiebai-Banxia decoction has a long history of medical use for treating cardiovascular diseases in China. In this study, we investigated the protective effect and underlying mechanisms GXB in type II diabetes with acute myocardial ischemia (T2DM-AMI) rats. We hypothesized that GXB may display its protective effect on T2DM-AMI by reducing endothelial progenitor cells (EPCs) apoptosisviaactivating PI3K (phosphatidyl inositol 3-kinase)/Akt (serine/threonine protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling. Rats were challenged with a high-fat diet and intraperitoneal injection of streptozotocin to induce a model of type II diabetes mellitus (T2DM) and coronary ligation to induce acute myocardial infarction (AMI). Changes in metabolites were assessed via enzyme-linked immunoassay and biochemical examination. The number and apoptosis rate of EPCs in peripheral blood were detected by flow cytometry. Target mRNAs and proteins in EPCs were analyzed by RT-PCR and Western blot analysis. The results demonstrated that GXB treatment decreased T2DM-AMI-associated changes in plasma fasting blood glucose, muscular enzymes, and blood lipids, and reduced oxidative stress. Furthermore, EPC apoptosis was increased in T2DM-AMI rats and was associated with decreased mRNA and protein levels of PI3K, Akt, and eNOS compared to the controls. Conversely, T2DM-AMI rats treated with GXB exhibited more circulating EPCs and downregulated levels of cell apoptosis, combined with increased mRNA and protein levels of PI3K, Akt, and eNOS compared to those of untreated T2DM-AMI rats. Our study showed that GXB treatment mitigated EPC apoptosis and promoted PI3K/Akt/eNOS signaling in T2DM-AMI rats.

Proliferation and migration of endothelial progenitor cells promoted by bone marrow mesenchymal stem cells conditioned medium with rosiglitazone / 中国组织工程研究

BACKGROUND: Studies have shown that mesenchymal stem cells can participate in the repair of wound injury caused by diabetes, but the high glucose environment obviously inhibits the function of mesenchymal stem cells and the effect of transplantation. OBJECTIVE: To observe the effect of conditioned medium of bone marrow mesenchymal stem cells intervened by rosiglitazone on the proliferation and migration of endothelial progenitor cells in high glucose environment. METHODS: (1) The bone marrow mesenchymal stem cells from the logarithmic growth period were cultured in three groups. The normal group was cultured with alpha-MEM medium containing 10% fetal bovine serum. The high glucose group was cultured with alpha-MEM medium containing 10% fetal bovine serum and 25 mmol/L glucose. The rosiglitazone group was cultured with alpha-MEM medium containing 10% fetal bovine serum, 25 mmol/L glucose and 10 μmol/L rosiglitazone. After 48 hours of culture, the culture supernatant was extracted as conditioned medium. The levels of vascular endothelial growth factor and matrix cell derived factor 1 in conditioned medium were detected by ELISA. (2) The endothelial progenitor cells from the logarithmic growth period were divided into three groups. The control group was cultured with the EGM-2 MV medium containing 10% fetal bovine serum. The model group was cultured with the EGM-2 MV medium containing 10% fetal bovine serum, 30 mmol/L glucose and conditioned medium of the high glucose group. The experimental group was cultured with EGM-2 MV medium containing 10% fetal bovine serum, 30 mmol/L glucose and conditioned medium of the rosiglitazone group. After 24 hours of culture, the ability of cell proliferation and migration was detected. RESULTS AND CONCLUSION: (1) The levels of vascular endothelial growth factor and matrix cell derived factor 1 in the conditioned medium of high glucose group were significantly lower than that of the normal group (P < 0.05). The levels of vascular endothelial growth factor and matrix cell derived factor 1 in the conditioned medium of the rosiglitazone group were significantly higher than in the high glucose group (P < 0.05). (2) The proliferation and migration ability of endothelial progenitor cells in the model group was lower than that in the control group (P < 0.05). The proliferation and migration ability of endothelial progenitor cells in the experimental group was higher than that in the model group (P < 0.05). (3) It is suggested that the conditioned medium of rosiglitazone intervened bone marrow mesenchymal stem cells can promote the proliferation and migration of endothelial progenitor cells.

Effect of Exendin-4 on endothelial progenitor cell function and AKT/eNOS signaling pathway in a mouse model of type 1 diabetes / 中国药理学通报

Aim To investigate the effect of Exendin4 on proliferation, migration, adhesion and senescence of endothelial progenitor cells (EPCs) in type I diabetic mice and its possible mechanism. Methods EPCs from 6-month diabetic mice were isolated and cultured by density gradient centrifugation. Cells were treated with different concentrations of Exendin-4 (1, 5, 10, 25 μmol · L

Endothelial progenitor cells in the peripheral blood of patients with moyamoya disease labeled with superparamagnetic iron oxide in vitro for MRI detection

Moyamoya disease (MMD) is currently thought to involve endothelial progenitor cells (EPCs). We investigated whether superparamagnetic iron oxide (SPIO) can be used to label EPCs. Mononuclear cells from 10 moyamoya disease patients were isolated, and cluster of differentiation 133 (CD133) positive cells sorted by magnetic-activated cell sorting were cultured in vitro. The positive rates of CD133, vascular endothelial growth factor receptor (VEGFR)-2, and cluster of differentiation 34 (CD34) were detected by flow cytometry. The cells were co-cultured with fluorescence labeled Dil-acetylated-low-density lipoprotein (Dil-ac-LDL) and Ulex europaeus agglutinin-1 (UEA-1) to observe the endocytosis of Dil-ac-LDL and binding to UEA-1. Prussian blue staining and transmission electron microscopy were used to observe the endocytosis of different SPIO concentrations in EPCs, and CCK-8 was used to detect proliferation of cells transfected with different concentrations of SPIO. T2 weighted imaging (T2WI) signals from magnetic resonance imaging after SPIO endocytosis were compared. Positive rates of CD133, VEGFR-2, and CD34 on sorted mononuclear cells were 68.2±3.8, 57.5±4.2, and 36.8±6.5%, respectively. The double-positive expression rate of CD34 and VEGFR-2 was 19.6±4.7%, and 83.1±10.4% of cells, which showed the uptake of Dil-ac-LDL and binding with UEA-1. The labeling efficiencies of SPIO at concentrations of 25 and 50 μg/mL were higher than for 12.5 μg/mL. The proliferation of cells was not influenced by SPIO concentrations of 12.5 and 25 μg/mL. After labeling, the T2WI of EPCs was reduced. The concentration of 25 μg/mL SPIO had high labeling efficiency detected by magnetic resonance imaging (MRI) without decreased EPCs viability.

Protective Effect of Danggui Buxuetang on Endothelial Progenitor Cells Against Oxidative Stress Injury Induced by H2O2 / 中国实验方剂学杂志

Objective:To observe protective effect of Danggui Buxuetang (DBT) on oxidative stress injury of mouse bone marrow-derived endothelial progenitor cells (EPCs) against induced by hydrogen peroxide (H2O2). Method:Monocytes from bone marrow of mice were obtained by density gradient centrifugation, and EPCs were obtained by specific culture medium. The experiment was divided into blank group，model group，DBT group (100，200，400 mg·L-1). Methyl thiazolyl tetrazolium（MTT） assay was used to determine the survival rate of EPCs and establish the cell injury model induced by H2O2. MTT，transwell chamber，matrigel and superoxide fluorescent anion probe (DHE) were used to detect the proliferation，migration，in vitro angiogenesis and ROS level，detection of autophagy by Western blot. Result:Compared with blank group，the proliferation ability，migration ability，the number of lumens and the length of tubule branches of EPCs in the model group were significantly reduced (P<0.01)，the level of intracellular reactive oxygen species （ROS） was significantly increased (P<0.01)，the expression of p62，the light chain microtubule associated protein 1 protein light chain 3 Ⅱ type （LC3-Ⅱ） protein of microtubule associated protein 1，was significantly increased (P<0.01). Compared with model group，DBT group increased the ability of cell proliferation and migration （P<0.01）. In addition，DBT increased the expression of LC3-Ⅱ protein in a concentration dependent manner (P<0.01)，and decreased the expression of p62 protein (P<0.01). Conclusion:DBT can improve the autophagy level of EPCs under oxidative stress, promote the proliferation, migration and angiogenesis of injured EPCs, and protect the biological function of EPCs under oxidative stress.

Protective effects of endothelial progenitor cells-derived microvesicles on hypoxic-ischemic brain damage in newborn rats / 中国新生儿科杂志

Objective To study the protective effects and preliminary mechanisms of endothelial progenitor cells-derived microvesicles (EPC-MVs) on hypoxic-ischemic brain damage (HIBD) in newborn rats by using the HIBD model.Method Rat endothelial progenitor cells (EPCs) were cultured and microvesicles were extracted from EPCs culture medium by ultracentrifugation.A total of 60 neonatal SD rats were randomly assigned into control group,HIBD group,saline group and EPC-MVs group.The HIBD model was prepared in HIBD group,saline group and EPC-MVs group.After the preparation of the HIBD model,rats in saline group and EPC-MVs group received intraventricular injection with saline and EPC-MVs,respectively.After 72 hours,the rats were sacrificed for brain tissue,cerebral infarction was detected by TTC staining,vascular endothelial growth factor (VEGF) mRNA was tested by real-time PCR,protein western blot was used to detect changes in VEGF protein expression.Result Cells extracted and cultured from the spleen of 12-week-old SD rats were confirmed as EPCs by morphology and flow cytometry.EPC-MVs isolated by high-speed centrifugation from EPCs culture supernatant met the morphological characteristics of microvesicles by transmission electron microscopy.The infarcted brain tissue was not detected in the control group.The cerebral infarction volume ratios of HIBD group,saline group and EPC-MVs group were (80.3 ± 6.3) ％,(77.9 ± 8.9) ％,(35.2 ± 7.7) ％,respectively.The infarct volume of EPC-MVs group was significantly lower than that of HIBD group and saline group (P ＜ 0.001).The expression of VEGF mRNA and protein in HIBD group,saline group and EPC-MVs group were higher than those in control group (P ＜0.001).Among them,EPC-MVs group had the most significant increase,compared with the other three experimental groups,and the difference was statistically significant (P ＜ 0.001).There was no significant difference between saline group and HIBD group in the expression of VEGF mRNA and protein (P ＞ 0.05).Conclusion Intraventricular injection of EPC-MVs can attenuate HIBD in neonatal rats,and the mechanism may be related to up-regulation of VEGF expression.