TRPC1 Deficiency Impairs the Endothelial Progenitor Cell Function via Inhibition of Calmodulin/eNOS Pathway

Endothelial progenitor cells (EPCs) promote angiogenesis and play a pivotal role in endothelial repair and re-endothelialization after vascular injury. Transient receptor potential-canonical1 (TRPC1) has been recently implied to play important roles on EPC function. Here, we studied the role of TRPC1 in regulating EPC function in vivo and in vitro. EPCs were cultured from TRPC1-knockout mice and their controls. In vitro, TRPC1 knockout reduced EPC functional activities, including migration and tube formation. Additionally, calmodulin (CaM)/endothelial nitric oxide synthase (eNOS) signaling activity were downregulated after TRPC1 knockout. Administration of CaM or eNOS inhibitor ameliorated TRPC1 knockout-reduced EPC migration and tube formation. In vivo Matrigel plug assay confirmed that TRPC1 knockout decreased formation of functional blood vessels of EPCs compared with wild-type EPCs. Taken together, these data suggest that TRPC1 is a critical regulator of angiogenesis.     

Globular adiponectin improves high glucose-suppressed endothelial progenitor cell function through endothelial nitric oxide synthase dependent mechanisms

Plasma levels of adiponectin, an adipose-specific protein with putative anti-atherogenic properties, could be down-regulated in obese and diabetic subjects. Recent insights suggest that the injured endothelial monolayer is regenerated by circulating endothelial progenitor cells (EPCs), but high glucose reduces number and functions of EPCs. Here, we tested the hypothesis that globular adiponectin can improve high glucose-suppressed EPC functions by restoration of endothelial nitric oxide synthase (eNOS) activity. Late EPCs isolated from healthy subjects appeared with cobblestone shape at 2-4 weeks. EPCs were incubated with high glucose (25 mM) and treatment with globular adiponectin for functional study. Migration and tube formation assays were used to evaluate the vasculogenetic capacity of EPCs. The activities of eNOS, Akt and concentrations of nitric oxide (NO) were also determined. Administration of globular adiponectin at physiological concentrations promoted EPC migration and tube formation, and dose-dependently upregulated phosphorylation of eNOS, Akt and augmented NO production. Chronic incubation of EPCs in high-glucose medium significantly impaired EPC function and induced cellular senescence, but these suppression effects were reversed by treatment with globular adiponectin. Globular adiponectin reversed high glucose-impaired EPC functions through NO- and p38 MAPK-related mechanisms. In addition, nude mice that received EPCs treated with adiponectin in high glucose medium showed a significant improvement in blood flow than those received normal saline and EPCs incubated in high glucose conditions. The administration of globular adiponectin improved high glucose-impaired EPC functions in vasculogenesis by restoration of eNOS activity. These beneficial effects may provide some novel rational to the vascular protective properties of adiponectin.     

Shear stress-induced activation of Tie2-dependent signaling pathway enhances reendothelialization capacity of early endothelial progenitor cells

Although endothelial progenitor cells (EPCs) play a pivotal role in the endothelial repair following arterial injury and shear stress has a beneficial effect on EPCs, however, the molecular mechanism underlying the influence of EPCs on the endothelial integrity and the regulation of shear stress on the EPC signaling remained to be studied. Here, we investigated the effects of laminar shear stress on the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2)-dependent signaling and its relation to in vivo reendothelialization capacity of human early EPCs. The human early EPCs were treated with shear stress. Shear stress in a dose-dependent manner increased angiopoietin-2 (Ang2)-induced migratory, adhesive and proliferatory activities of EPCs. Transplantation of EPCs treated by shear stress facilitated in vivo reendothelialization in nude mouse model of carotid artery injury. In parallel, the phosphorylation of Tie2 and Akt of EPCs in response to shear stress was significantly enhanced. With treatment of Tie2 knockdown or Akt inhibition, shear stress-induced phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) of EPCs was markedly suppressed. After Tie2/PI3K/Akt/eNOS signaling was blocked, the effects of shear stress on in vitro function and in vivo reendothelialization capacity of EPCs were significantly inhibited. The present findings demonstrate for the first time that Tie2/PI3k/Akt/eNOS signaling pathway is, at least in part, involved in the EPCs-mediated reendothelialization after arterial injury. The upregulation of shear stress-induced Tie2-dependent signaling contributes to enhanced in vivo reendothelialization capacity of human EPCs.     

Akt/eNOS信号途径调节内皮祖细胞存活和功能的实验研究

目的研究Akt/eNOS信号途径是否调节内皮祖细胞（EPC）的存活和功能。方法分离、培养EPC,然后与不同浓度氧化型低密度脂蛋白（oxLDL）、硝基精氨酸甲酯（L—NAME）或triciribine孵育48h,一部分EPC与左旋精氨酸预处理后,再与oxLDL孵育。然后,检测EPC凋亡率及迁移、黏附和管状结构形成能力,同时检测磷酸化Akt的蛋白表达、内皮型一氧化氮合酶（eNOS）的蛋白及mRNA表达、一氧化氮的产生。结果oxLDL剂量依赖性地诱导EPC凋亡,抑制EPC迁移、黏附及管状结构形成能力,L—NAME和triciribine具有与oxLDL相似的作用。oxLDL的作用能被左旋精氨酸抑制。oxLDL降低磷酸化Akt及eNOS蛋白表达,oxLDL剂量为50μg／ml时下降率分别为（664±4）％和（684±9）％。而且,oxLDL降低EPCeNOSmRNA的表达及一氧化氮的产生,oxLDL剂量为50μg／ml时eNOSmRNA表达的下降率为（594±14）％。结论oxLDL通过调节Akt／eNOS信号途径调节EPC的存活和功能。     

SDF-1alpha/CXCR4 decreases endothelial progenitor cells apoptosis under serum deprivation by PI3K/Akt/eNOS pathway

Recent studies have demonstrated that stromal cell-derived factor-1alpha (SDF-1alpha)/CXCR4 interaction regulates multiple cell signal pathways and a variety of cellular functions such as cell migration, proliferation, survival and angiogenesis. In present study, we aimed to determine the effect of SDF-1alpha on endothelial progenitor cells (EPCs) apoptosis induced by serum deprivation and the implication of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) signaling in this effect. EPCs were isolated and characterized. SDF-1alpha decreased EPCs apoptosis induced by serum deprivation in a dose-dependent manner and the inhibitory effect was CXCR4 dependent as confirmed by the total abolishment by AMD3100, a CXCR4-specific peptide antagonist. SDF-1alpha treatment also significant decreased caspase-3 expression and activity. The inhibitory effect of SDF-1alpha on EPCs apoptosis was nearly completely abolished by PI3K inhibitors (either Wortmannin or LY294002) and partially abolished by NOS inhibitor, N(G)-nitro-arginine methyl ester, whereas inhibitors of MAPKs had no significant effect on this inhibitory effect. The treatment of EPCs with SDF-1alpha resulted in time-dependent Akt, eNOS, extracellular-regulated kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase (JNK) phosphorylations. These findings suggest that PI3K/Akt/eNOS activation, but not MAPKs activation, is required for the inhibitory effect of SDF-1alpha on EPCs apoptosis.     

阿托伐他汀对同型半胱氨酸所诱导内皮祖细胞功能损伤的保护作用

目的:探讨同型半胱氨酸(homocysteine,Hcy)诱导小鼠骨髓内皮祖细胞(endothelial progenitor cells,EPCs)功能损伤的氧化应激机制及阿托伐他汀的拮抗作用。方法:密度梯度离心法获取小鼠骨髓单个核细胞,培养7 d后收集贴壁细胞,FITC-UEA-1荧光染色鉴定EPCs。EPCs与不同浓度Hcy(0μmol/L,50μmol/L,500μmol/L)共孵育24 h或分别经不同浓度的阿托伐他汀(0.1μmol/L,1μmol/L,10μmol/L)预孵育0.5 h后,再加入500μmol/LHcy共孵育24 h。用MTT比色法,改良Boyden小室、黏附能力测定和体外血管生成试剂盒,分别观察EPCs的增殖能力、迁移能力、黏附能力和体外血管生成能力。荧光探针H2DCF-DA法检测细胞内活性氧水平,光泽精化学发光法检测NADPH氧化酶活性,硝酸还原酶法测定细胞培养液中一氧化氮(NO)含量,RT-PCR法测定eNOS基因表达。结果:Hcy诱导EPCs增殖、迁移、黏附和体外血管生成功能下降,活性氧的产生及NADPH氧化酶的活性增加,eNOS基因表达及细胞培养液中NO含量减少,与无Hcy处理组相比有明显差异(P0.05或P0.01)。与500μmol/L Hcy组相比,阿托伐他汀预处理可呈剂量依赖性地拮抗Hcy诱导的上述改变(P0.05或P0.01)。结论:Hcy可能通过激活NADPH氧化酶,诱导EPCs活性氧的产生及降低eNOS基因表达和NO水平,导致EPCs增殖、迁移、黏附和体外血管生成功能下降。阿托伐他汀部分拮抗Hcy的作用。     

Role of microRNA-23b in flow-regulation of Rb phosphorylation and endothelial cell growth

MicroRNAs (miRs) can regulate many cellular functions, but their roles in regulating responses of vascular endothelial cells (ECs) to mechanical stimuli remain unexplored. We hypothesize that the physiological responses of ECs are regulated by not only mRNA and protein signaling networks, but also expression of the corresponding miRs. EC growth arrest induced by pulsatile shear (PS) flow is an important feature for flow regulation of ECs. miR profiling showed that 21 miRs are differentially expressed (8 up- and 13 downregulated) in response to 24-h PS as compared to static condition (ST). The mRNA expression profile indicates EC growth arrest under 24-h PS. Analysis of differentially expressed miRs yielded 68 predicted mRNA targets that overlapped with results of microarray mRNA profiling. Functional analysis of miR profile indicates that the cell cycle network is highly regulated. The upregulation of miR-23b and miR-27b was found to correlate with the PS-induced EC growth arrest. Inhibition of miR-23b using antagomir-23b oligonucleotide (AM23b) reversed the PS-induced E2F1 reduction and retinoblastoma (Rb) hypophosphorylation and attenuated the PS-induced G1/G0 arrest. Antagomir AM27b regulated E2F1 expression, but did not affect Rb and growth arrest. Our findings indicate that PS suppresses EC proliferation through the regulation of miR-23b and provide insights into the role of miRs in mechanotransduction.     

2型糖尿病外周血内皮祖细胞增殖、分化及细胞周期变化

目的观察2型糖尿病（T2DM）患者外周血内皮祖细胞（EPC）增殖、分化能力及细胞周期分布。方法T2DM患者（DM组）和非T2DM患者（Con组）各20例，离心法获取外周血单个核细胞，培养7天后，鉴定EPC，检测EPC增殖能力、EPC分化及细胞周期分布。结果DM组外周血EPC数量及增殖能力明显降低；EPC数量、增殖能力与HbA1c水平和DM病程呈负相关；EPC表达CD14^＋、CD64^＋明显高于Con组，而表达vWF^＋明显低于Con组；EPC在S期的比例明显减少，在G0／G1期的比例增高。结论T2DM患者外周血EPC数量减少、增殖能力受损、向内皮细胞系分化减少。     

Estrogen-mediated endothelial progenitor cell biology and kinetics for physiological postnatal vasculogenesis

Estrogen has been demonstrated to promote therapeutic reendothelialization after vascular injury by bone marrow (BM)-derived endothelial progenitor cell (EPC) mobilization and phenotypic modulation. We investigated the primary hypothesis that estrogen regulates physiological postnatal vasculogenesis by modulating bioactivity of BM-derived EPCs through the estrogen receptor (ER), in cyclic hormonally regulated endometrial neovascularization. Cultured human EPCs from peripheral blood mononuclear cells (PB-MNCs) disclosed consistent gene expression of ER alpha as well as downregulated gene expressions of ER beta. Under the physiological concentrations of estrogen (17beta-estradiol, E2), proliferation and migration were stimulated, whereas apoptosis was inhibited on day 7 cultured EPCs. These estrogen-induced activities were blocked by the receptor antagonist, ICI182,780 (ICI). In BM transplanted (BMT) mice with ovariectomy (OVX) from transgenic mice overexpressing beta-galactosidase (lacZ) regulated by an endothelial specific Tie-2 promoter (Tie-2/lacZ/BM), the uterus demonstrated a significant increase in BM-derived EPCs (lacZ expressing cells) incorporated into neovasculatures detected by CD31 immunohistochemistry after E2 administration. The BM-derived EPCs that were incorporated into the uterus dominantly expressed ER alpha, rather than ER beta in BMT mice from BM of transgenic mice overexpressing EGFP regulated by Tie-2 promoter with OVX (Tie-2/EGFP/BMT/OVX) by ERs fluorescence immunohistochemistry. An in vitro assay for colony forming activity as well as flow cytometry for CD133, CD34, KDR, and VE-cadherin, using human PB-MNCs at 5 stages of the female menstrual-cycle (early-proliferative, pre-ovulatory, post-ovulatory, mid-luteal, late-luteal), revealed cycle-specific regulation of EPC kinetics. These findings demonstrate that physiological postnatal vasculogenesis involves cyclic, E2-regulated bioactivity of BM-derived EPCs, predominantly through the ER alpha.     

水飞蓟素对雷帕霉素诱导的内皮祖细胞凋亡和生长抑制的拮抗作用

目的:水飞蓟素对雷帕霉素诱导的内皮祖细胞（EPCs）凋亡和生长抑制的拮抗作用。方法:通过密度梯度离心法分离、培养、鉴定得到EPCs;在EPCs培养物中加入不同浓度（0、01、1、10、100 ng/ml）的雷帕霉素干预24 h和加入（1 ng/ml）雷帕霉素干预不同时间（0 、6、12、24、48 h）。收集细胞检测其增殖和迁移能力。在EPCs培养物中加入不同浓度（0、25、50、100 μg/ml）的水飞蓟素干预24 h后,收集细胞检测其增殖、迁移和凋亡的水平。在EPCs培养物中同时加入（1 ng/ml）雷帕霉素和不同浓度（25、50、100 μg/ml）水飞蓟素干预24 h后,收集细胞检测其增殖、迁移、凋亡和体外血管生成的能力。结果:与未药物干预的EPCs培养物相比较,雷帕霉素（1、10、100 ng/ml）能抑制EPCs增殖和迁移,并随干预浓度的增加和干预时间的延长而增加（P<0.05）。水飞蓟素干预24 h后,50 μg/ml和100 μg/ml的水飞蓟素可明显增加EPCs增殖和迁移的能力（P<0.05）,25~100 μg/ml的水飞蓟素可明显抑制EPCs凋亡（P<005）,并呈浓度依赖性。EPCs在加入雷帕霉素（1 ng/ml）和不同浓度水飞蓟素（25、50、100 μg/ml）共同干预24 h后,50 μg/ml和100 μg/ml的水飞蓟素都可明显改善雷帕霉素对EPCs的促凋亡作用,并能逆转雷帕霉素对EPCs增殖、迁移和血管形成能力的抑制作用（P<0.05）。结论:雷帕霉素能抑制EPCs增殖和迁移,并呈浓度和时间依赖性。水飞蓟素能增强EPCs增殖和迁移,抑制其凋亡,并呈浓度依赖性。水飞蓟素能抑制雷帕霉素对EPCs的促凋亡作用,并能逆转雷帕霉素对EPCs增殖、迁移和血管形成能力的抑制作用。     

MicroRNA changes in human arterial endothelial cells with senescence: relation to apoptosis, eNOS and inflammation

A senescent phenotype in endothelial cells is associated with increased apoptosis, reduced endothelial nitric oxide synthase (eNOS) and inflammation, which are implicated in arterial dysfunction and disease in humans. We tested the hypothesis that changes in microRNAs are associated with a senescent phenotype in human aortic endothelial cells (HAEC). Compared with early-passage HAEC, late-passage HAEC had a reduced proliferation rate and increased staining for senescence-associated beta-galactosidase and the tumor suppressor p16^INK4a. Late-passage senescent HAEC had reduced expression of proliferation-stimulating/apoptosis-suppressing miR-21, miR-214 and miR-92 and increased expression of tumor suppressors and apoptotic markers. eNOS-suppressing miR-221 and miR-222 were increased and eNOS protein and eNOS activation (phosphorylation at serine1177) were lower in senescent HAEC. Caveolin-1 inhibiting miR-133a was reduced and caveolin-1, a negative regulator of eNOS activity, was elevated in senescent HAEC. Inflammation-repressing miR-126 was reduced and inflammation-stimulating miR-125b was increased, whereas inflammatory proteins were greater in senescent HAEC. Development of a senescent arterial endothelial cell phenotype featuring reduced cell proliferation, enhanced apoptosis and inflammation and reduced eNOS is associated with changes in miRNAs linked to the regulation of these processes. Our results support the hypothesis that miRNAs could play a critical role in arterial endothelial cell senescence.     

Hsa_circ_0074491 regulates the malignance of cholesteatoma keratinocytes by modulating the PI3K/Akt pathway by binding to miR-22-3p and miR-125a-5p: An observational study

Cholesteatoma is a benign cystic lesion that can continue to grow like a tumor. Circular ribonucleic acid (RNA) hsa_circ_0074491 (circ_0074491) has been reported to be down-regulated in cholesteatoma tissues. However, the role and regulatory mechanism of circ_0074491 in the growth of cholesteatoma are unclear.The expression of circ_0074491, microRNA (miR)-22-3p, and miR-125a-5p in cholesteatoma tissues was detected by quantitative real-time polymerase chain reaction. The proliferation, cell cycle, apoptosis, migration, and invasion of cholesteatoma keratinocytes were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, plate clone, flow cytometry, or transwell assays. Several protein levels were examined by western blotting. The targeting relationship between miR-22-3p or miR-125a-5p and circ_0074491 was verified via dual-luciferase reporter and RNA pull-down assays.We observed the downregulation of circ_0074491 in cholesteatoma tissues. Furthermore, circ_0074491 knockdown facilitated cell proliferation, migration, invasion, and repressed cell apoptosis in cholesteatoma keratinocytes. Circ_0074491 was verified as a decoy for miR-22-3p and miR-125a-5p in cholesteatoma keratinocytes. Both miR-22-3p and miR-125a-5p silencing reversed the impacts of circ_0074491 silencing on proliferation, apoptosis, migration, and invasion of cholesteatoma keratinocytes. Also, circ_0074491 knockdown activated the PI3K/Akt pathway in cholesteatoma keratinocytes via miR-22-3p and miR-125a-5p.Circ_0074491 played a suppressive role in cholesteatoma through inactivating the PI3K/Akt pathway via binding to miR-22-3p and miR-125a-5p, which provided a novel evidence for the involvement of circRNA in the development of cholesteatoma.     

High-density lipoprotein cholesterol regulates endothelial progenitor cells by increasing eNOS and preventing apoptosis

OBJECTIVE: Endothelial progenitor cells (EPCs) are implicated as an important marker of endothelial function and cardiovascular risk. In the present study, we examined whether high-density lipoprotein (HDL) cholesterol plays a role in the peripheral EPC levels and its underlying mechanisms in the HDL cholesterol-induced elevation of EPCs. METHODS: For the clinical study, vascular risk factors and blood markers were measured and EPC colony forming units were counted after 7 days of culture. For the in vitro study, after 7 days of culture, EPCs were incubated in the presence or absence of HDL for 24h followed by measurements of eNOS and pro-MMP-9 expression and caspase-3 activity. RESULTS: EPC colony levels significantly correlated with HDL levels (P=0.017). HDL treatment significantly increased eNOS protein expression in EPCs (P<0.001) while it significantly decreased pro-MMP-9 levels at the concentration of 50 microg/mL (P=0.002). Homocysteine treatment significantly increased caspase-3 activity whereas HDL significantly decreased it as compared to the homocysteine-only treated group. INTERPRETATION: The data demonstrate that EPC colony levels are significantly lower in individuals with low HDL and that HDL increases eNOS and decreases pro-MMP-9 in EPCs. HDL also prevents EPC apoptosis through inhibition of caspase-3 activity suggesting a possible mechanism for its positive effects on circulating EPC numbers.     

miR-21/PTEN/Akt参与白藜芦醇促EPC的体外成血管能力

目的 探讨miR-21及其下游PTEN/Akt信号通路在白藜芦醇促进大鼠骨髓源性内皮祖细胞(EPC)体外成血管能力中的作用.方法 采用密度梯度离心法分离大鼠四肢骨髓的单个核细胞,培养于含10％胎牛血清的EGM-2MV完全培养基中诱导分化成EPC,实验用3～5代的EPC.白藜芦醇(20 μmol/L)干预12 h后,采用...     

Insulin modulates ischemia-induced endothelial progenitor cell mobilization and neovascularization in diabetic mice

Decreased levels of circulating endothelial progenitor cells (EPCs) predict increased risk of cardiovascular events in diabetic patients. Insulin treatment exerts important cardiovascular protection. Whether and how insulin participates in the EPC regulation of postnatal neovascularization are currently unclear. We employed a mouse hindlimb ischemia model to study EPC mobilization in non-diabetic and streptozotocin-induced diabetic mice. Insulin was administered to diabetic animals postoperatively. To determine the role of EPCs contributing to postnatal vasculogenesis, we used bone marrow-transplanted mice whose bone marrow cells selectively expressed enhanced green fluorescent protein (EGFP). Insulin treatment improved EPC mobilization into peripheral blood, accelerated transcutaneous oxygen pressure restoration and increased capillary density in the ischemic limb associated with partial incorporation of EGFP-positive cells into the capillaries. Insulin treatment restored ischemia-induced release of stromal-derived growth factor 1α and vascular endothelial growth factor (VEGF), and consequently enhanced the activity of Akt and endothelial nitric oxide synthase (eNOS) as well as matrix metalloproteinase-9 in bone marrow. Insulin also augmented tissue-level activation of VEGF/Akt/eNOS pathway. However, all such effects of insulin were completely blocked by combined treatment with a NOS inhibitor. Our data suggested that insulin treatment improved ischemia-induced EPC mobilization and contributed to compensatory neovascularization in diabetic mice through a VEGF/eNOS-related pathway.     

血管内皮生长因子调节内皮祖细胞生物学功能

目的研究血管内皮生长因子（VEGF）对体外培养骨髓源性内皮祖细胞（EPCs）数量及增殖、迁移、黏附功能的影响及机制初探。方法密度梯度离心法获取骨髓单个核细胞,FITC-荆豆凝集素I、DiI-乙酰化低密度脂蛋白荧光双染鉴定。单个核细胞培养7d后分为对照组和VEGF干预组。VEGF干预组加入不同浓度VEGF（25,50,75,100μg／L）培养48h,分别采用四氮唑溴盐比色法、改良的Boyden小室和黏附能力测定观察EPCs的增殖、迁移和黏附能力。RT—PCR法半定量检测VEGF对EPCs内皮型一氧化氮合酶（eNOS）mRNA表达的影响。硝酸还原酶法比色测定VEGF对EPCs分泌一氧化氮的影响。结果VEGF可浓度依赖性地增加EPCs数量并明显促进EPCs的黏附、迁移和增殖能力,与对照组比较差异显著。VEGF可上调EPCseNOSmRNA的表达,促进EPCs分泌一氧化氮。结论VEGF可能通过上调EPCseNOSmRNA的表达影响EPCs部分生物学功能。     

Biphasic response of endothelial progenitor cell proliferation induced by high glucose and its relationship with reactive oxygen species

In this study, the effect of high glucose (HG) on endothelial progenitor cell (EPC) proliferation and its relationship with cyclins and reactive oxygen species (ROS) were investigated. Mouse EPCs were isolated from bone marrow using a magnetic activated cell-sorting system and cultured in the presence or absence of HG (30 mmol/l). We found that in the early stage of incubation (3 days), HG promoted cell proliferation, and increased the expressions of cdk2 and cyclin E, while in the late stage of culture (7 days) it inhibited cell proliferation and decreased the expressions of cdk2, cyclin E, and proliferating cell nuclear antigen (PCNA). Moreover, on the third day after incubation, HG significantly inhibited the apoptosis of EPCs, while in the late stage it markedly activated caspase-3 and promoted apoptosis. ROS generation in cells and maleic dialdehyde level in medium were significantly increased in HG group on the seventh day, whereas the expressions of superoxide dismutase and glutathione levels decreased. Tempol, a membrane-permeable radical scavenger, significantly inhibited ROS production in EPCs and partially reversed the HG-mediated inhibition of EPCs proliferation on the seventh day. We hypothesize that in the HG environment, the biphasic response of EPC proliferation may be related to the generation of ROS, which causes modulation of cyclins and cell cycle effect.     

脂联素对高糖刺激下内皮祖细胞的作用及其可能的作用机制

目的:研究脂联素(adiponectin,APN)对高糖刺激下内皮祖细胞(Endothelial Progenitor Cells,EPCs)的作用,并探讨其可能的机制。方法: 密度梯度离心法分离人外周血单个核细胞,经含血管内皮生长因子、碱性成纤维细胞生长因子和100 ml/L胎牛血清的M199培养基培养7 d,贴壁细胞进行形态学、流式细胞仪测细胞分子标志物(CD34、CD133和KDR)和激光共聚焦倒置显微镜下观察培养细胞摄取ac LDL和结合UEA I经何种方法鉴定为EPCs。细胞同步化后,将其随机分为7组:正常糖浓度对照组(55 mmol/L)、高渗对照组（55 mmol/L葡萄糖+245 mmol/L甘露醇及其浓度）、高糖(30mmol/L)组及高糖APN干预组(30 mmol/L葡萄糖合并APN,125、25、5、10 μg/ml)。干预48 h后,分别采用MTT比色法、Transwell小室检测EPCs的增殖、迁移;以Annexin V FITC凋亡检测试剂盒处理细胞,流式细胞仪检测EPCs的凋亡;用荧光探针(DCFH DA)检测法进行细胞活性氧(reactive oxygen species,ROS)检测。结果: ①经密度梯度离心法分离出的外周血单个核细胞培养7 d后,细胞集落增加明显,梭形细胞增多并呈交叉性生长;用激光共聚焦倒置显微镜观察,细胞摄取DiI acLDL呈红色荧光,摄取FITC UEA I呈绿色荧光,摄取DiI acLDL并结合FITC UEA I的细胞呈黄色荧光,流式细胞仪分析结果提示:细胞表达KDR(9232%)、CD133(107%)、CD34(233%),证实培养的细胞是正在分化的EPCs。②随着糖浓度的增高,EPCs的增殖能力下降,凋亡、ROS水平增加(P<001);当糖浓度为30 mmol/L与50 mmol/L时,两者相比对EPCs的影响无统计学差异。③与正常糖浓度对照组相比,高渗对照组EPCs的增殖、迁移、凋亡和ROS水平无统计学差异。在30 mmol/L葡萄糖条件下,EPCs的数量和迁移功能较正常对照组明显下降,细胞凋亡增多,不同浓度APN干预后能明显提高高糖损伤后EPCs的功能(P<005,P<001),并随着浓度的增加,EPCs的增殖与迁移能力增高,凋亡、ROS水平下降(P<001)。结论: ①与对照组相比,高糖干预可导致EPCs增殖能力下降,细胞凋亡增多;②高糖干预后加入APN,EPCs的增殖、迁移能力恢复,细胞凋亡减少,并在一定范围内,其作用随浓度的增加而增强;③高糖可以引起EPCs功能受损,其作用机制可能与ROS水平升高有关;而APN可以通过降低高糖引起的ROS水平,保护EPCs功能;渗透压对EPCs无影响。     

MicroRNA-20a inhibits stress-induced cardiomyocyte apoptosis involving its novel target Egln3/PHD3

Excessive stress, e.g. due to biomechanical overload or ischemia/reperfusion is a potent inductor of cardiomyocyte apoptosis, which contributes to maladaptive remodeling. Despite substantial progress in the understanding of the molecular pathophysiology, many components of the signaling pathways underlying remodeling in general and apoptosis in particular still remain unknown. Recent evidence suggests that microRNAs (miRs) play an important role in the heart's response to increased cardiac stress. To identify novel modulators of stress-dependent remodeling, we conducted a genome-wide miR-screen of mechanically stretched neonatal rat cardiomyocytes (NRCM). Out of 351 miRs, eight were significantly regulated by biomechanical stress, including microRNA-20a, which is part of the miR17-92 cluster. Interestingly, further expression analyses also revealed upregulation of microRNA-20a in an in vitro hypoxia/"reperfusion" model. Given the potential apoptosis-modulating properties of the miR17-92 cluster, we subjected NRCM to hypoxia and subsequent reoxygenation. AdmiR-20a significantly inhibited hypoxia-mediated apoptosis in a dose-dependent fashion, while targeted knockdown of miR-20a in NRCM induced cardiomyocyte apoptosis. Mechanistically, the antiapoptotic effect of miR-20a appears to be mediated through direct targeting and subsequent downregulation of the proapoptotic factor Egln3. Thus, miR-20a is upregulated in acute biomechanical stress as well as hypoxia and inhibits apoptosis in cardiomyocytes. These properties reveal miR-20a as a cardioprotective micro-RNA and a potential target for novel therapeutic strategies to prevent cardiac remodeling.