Erythropoietin improves the efficiency of endothelial progenitor cell therapy after myocardial infarction in mice: effects on transplanted cell survival and autologous endothelial progenitor cell mobilization

BackgroundEndothelial progenitor cells (EPCs) participate in vascular repair and angiogenesis. Thus, EPC transplantation into ischemic myocardium might improve cardiac function; however, the vast majority of cells die within a short period. The present study was designed to investigate whether exogenous erythropoietin (EPO) delivery could improve the survival of transplanted EPCs and enhance the efficiency of EPC-based cell therapy.MethodsMyocardial infarction was induced in wild-type mice by ligating the left anterior descending coronary artery. Enhanced green fluorescent protein-EPCs with or without EPO were transplanted into peri-infarct myocardium. Enhanced green fluorescent protein-EPCs were detected 7 and 28 d after surgery. The amount of circulating EPCs was analyzed 3 and 28 d after surgery. The stromal cell-derived factor-1α and vascular endothelial growth factor concentrations, microvessel density, apoptosis, fibrosis in the peri-infarct myocardium, and cardiac function were compared among the groups.ResultsMore enhanced green fluorescent protein-EPCs were found in the hearts treated with EPC + EPO than in those treated with EPC alone. The circulating EPC level was markedly elevated after EPC + EPO treatment compared with EPC application alone. Stromal cell-derived factor-1α and vascular endothelial growth factor were increased accordingly, along with increased microvessel density, decreased apoptosis, and reduced fibrosis in the peri-infarct myocardium. Left ventricular fractional shortening was greater and the interventricular septum was thicker after EPC + EPO treatment compared with EPC treatment alone.ConclusionsEPO improved the efficiency of EPC therapy in mice with myocardial infarction. This effect was associated with enhanced transplanted EPC survival and autologous EPC mobilization.     

Pterostilbene protects against uraemia serum-induced endothelial cell damage via activation of Keap1/Nrf2/HO-1 signaling

International Urology and Nephrology - Chronic kidney disease causes uremia-related endothelial cell dysfunction associated with high risk for cardiovascular diseases. The vascular endothelium is...     

Endothelial cell activation of the smooth muscle cell phosphoinositide 3-kinase/Akt pathway promotes differentiation

OBJECTIVE: Interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) are fundamental in diverse cardiovascular processes such as arteriogenesis, atherosclerosis, and restenosis. We aimed to determine the intracellular signaling mechanisms by which ECs promote a differentiated SMC phenotype. METHODS: Bovine thoracic aorta ECs and SMCs were isolated and cultured. For co-culture studies, ECs were grown to confluence on one side of a semi-permeable Cyclopore membrane. SMCs were then plated on the opposite side of the membrane and cultured for 24 to 48 hours. For adenovirus experiments, SMCs were infected prior to plating opposite ECs. For conditioned media studies, SMCs cultured alone on plastic were treated with media harvested from EC/SMC in co-culture. SMC phenotype was assayed by microscopy and measurement of two-dimensional area, or by western blotting for contractile protein markers of differentiation. Akt activation was measured by western blotting for phospho-Serine 473. RESULTS: Although SMCs cultured alone exhibit a dedifferentiated synthetic phenotype, we report that bilayer co-culture with ECs induced a differentiated SMC phenotype as measured by morphology and cell area and expression of protein markers of differentiation, including contractile proteins and the cyclin-dependent kinase inhibitor p27 kip . The EC/SMC bilayer co-culture resulted in activation of the SMC protein kinase Akt, with no effect on total Akt expression. Similarly, conditioned media from co-cultured EC/SMC promoted rapid Akt phosphorylation and subsequent expression of differentiation protein markers in SMCs cultured alone. Adenoviral overexpression of constitutively active Akt in SMCs cultured alone mimicked the ability of ECs to induce SMC differentiation. Notably, inhibition of phosphoinositide 3 (PI 3)-kinase activity with wortmannin or adenoviral overexpression of a dominant-negative Akt prevented the EC-mediated effect on SMC morphology and differentiation protein marker expression. CONCLUSIONS: ECs direct SMCs towards a differentiated phenotype through activation of the SMC PI 3-kinase/Akt pathway. CLINICAL RELEVANCE: Interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) are fundamental in diverse cardiovascular processes such as arteriogenesis, collateral blood vessel development, atherosclerosis, and restenosis. Alterations in SMC phenotype occur in each of these processes. Endothelial denudation has been suggested to contribute to the SMC proliferative response to vessel injury by angioplasty or other catheterization procedures. We have employed a co-culture approach to dissect the molecular signals that are dependent on the spatial relationship between ECs and SMCs, and have identified the importance of the PI3K/Akt pathway in EC-induced SMC differentiation. This pathway may suggest targets for therapeutic interventions for intimal hyperplasia and restenosis.     

血管内皮细胞生长因子体外转染血管内皮祖细胞的研究

目的 探讨脂质体介导血管内皮细胞生长因子(VEGF)基因转染血管内皮祖细胞(EPC)应用于基因治疗的可行性、安全性。方法 体外分离、培养、免疫组织化学及流式细胞仪鉴定EPC,转染VEGF后用免疫组织化学和ELISA检测EPC表达VEGF蛋白,噻唑蓝(MTT)检测EPC对VEGF质粒转染的敏感性。结果 EPC表达CD_(34)、CD_(31)、KDR及CD_(133)细胞表面标志,转染后EPC胞内表达VEGF。脂质体介导PcDNA3.1(-)/VEGF_(165)质粒转染、PcDNA3.1(-)/VEGF_(165)空质粒转染、不转染任何质粒的3组EPC培养基上清中表达的VEGF分别为(352±35)ng/L、(45±5)ng/L、0 ng/L(P<0.05),VEGF质粒转染对EPC增殖无影响。结论 EPC可作为VEGF转染的靶细胞,用于基因治疗。     

内皮祖细胞移植对大鼠肝硬化作用的研究

目的 探讨内皮祖细胞(endothelial progenitor cells,EPCs)移植对四氯化碳(carbon tetrachloride,CCl4)诱导的大鼠肝硬化的作用.方法 本组38只SD大鼠中8只大鼠为正常对照,其余30只采用25%的CCl4/橄榄油灌胃制备肝硬化模型.再将肝硬化大鼠分为3组,每组10只.12周后直接处死的为肝硬化模型组,门静脉输入大鼠EPCs为EPCs移植组,经门脉输入生理盐水为移植对照组.移植4周后检测移植组和移植对照组大鼠肝组织胶原Ⅲ(collagen Ⅲ,COL Ⅲ)、平滑肌动蛋白(smooth muscle actin α,α-SMA)和Ki67的表达,检测外周血肝功能和血凝分析.结果 肝硬化模型组大鼠肝脏体积增至正常时的2倍.EPCs移植组大鼠与肝硬化模型组比较,肝组织学活动指数(histological activity index,HAI)(F=75.062,P<0.01),丙氨酸氨基转移酶(alanine aminotransferase,ALT)(F=29.942,P<0.05),门冬氨酸氨基转移酶(aspartate aminotransferase,AST)(F=16.618,P<0.05)和总胆红素(total bilirubin,TBIL)(F=9.911,P<0.05)水平降低,白蛋白(albumin,Alb)(F=4.944,P<0.05)和Ki67(F=45.966,P<0.01)水平升高,纤维化面积(F=25.025,P<0.05)减少,α-SMA(F=7.86,P<0.05)和COL Ⅲ(F=135.787,P<0.01)表达降低;与正常肝脏相比,移植对照组HAI,ALT,AST和TBIL水平升高,Alb和Ki67水平降低,纤维化面积增加,α-SMA和COL Ⅲ表达升高(P<0.05).移植对照组大鼠凝血酶原时间延长,差异有统计学意义(P<0.05). 结论移植EPCs可以促进大鼠肝硬化的肝细胞增生,减轻肝纤维化程度.     

肿瘤相关巨噬细胞通过PI3K/Akt途径促进胰腺癌浸润迁移的研究

目的研究PI3K/Akt信号通路对胰腺癌细胞PANC-1浸润迁移能力的影响,进一步探讨肿瘤相关巨噬细胞促进胰腺癌发生发展的分子机制。 方法通过密度梯度离心法从健康成人外周血中分离单个核细胞,用IL-4体外诱导选择性激活的巨噬细胞(M2)。采用实时荧光定量PCR和Western blotting法检测胰腺癌PANC-1细胞PI3K、Akt mRNA和蛋白表达水平的变化,利用Transwell侵袭实验与划痕实验观察细胞浸润迁移能力的变化。 结果体外模拟胰腺癌微环境,将胰腺癌PANC-1细胞与不同激活状态的巨噬细胞共培养,证明M2可显著上调胰腺癌PANC-1细胞PI3K、Akt的mRNA和蛋白水平,共培养20 h后可明显促进胰腺癌PANC-1细胞的浸润与迁移能力。 结论肿瘤相关巨噬细胞可通过PI3K/Akt信号通路促进胰腺癌细胞的浸润与迁移。     

Laparoscopic surgery protects against the oncologic adverse effects of open surgery by attenuating endothelial progenitor cell mobilization

Background Endothelial progenitor cells (EPCs) derived from bone marrow incorporate into foci of neovascularization to propogate tumor growth. These cells are mobilized in response to surgical injury. Laparoscopic surgery may protect against the oncologic adverse effects of open surgical tumor excision, and this may be related to attenuated mobilization of EPCs. Methods For this study, 132 C57BL/6 mice were randomized to standardized laparotomy, laparoscopy, or control groups. The animals were killed at 6, 24, 48, and 72 h. Femur bone marrow and peripheral blood were harvested. Bone marrow EPCs were detected by flow cytometric dual staining for the stem cell antigen-1/cKit phenotype. Circulating EPCs were characterized in blood by vascular endothelial growth factor receptor 2 positive/macrophage activating complement-1 negative staining. Separately, 12 C57/bl6 mice bearing 3LL Lewis lung tumors 12 days after laparotomy or laparoscopy had their tumors excised and examined for endothelial cell expression (marker P1H12). Results Laparoscopy decreased circulating EPCs and bone-marrow EPC levels, as compared with laparotomy, at all time points. Bone marrow EPC levels were 2.95% ± 0.32% after laparotomy, as compared with 0.65 ± 0.21 in the laparoscopy group (p < 0.05). The circulating EPC level in the laparotomy group was 35.2% ± 6% of cells, as compared with 3.1% ± 0.2% in the laparoscopy group (p < 0.05). In homogenized tumors, the percentage of P1H12 expression among laparoscopy-treated animals was 22.1% ± 4.2%, as compared with 39% ± 8% in the laparotomy group (p < 0.05). Conclusion Laparoscopy decreased EPC levels in both bone marrow and circulation, resulting in decreased tumor endothelial cell burden. This may represent a novel mechanism by which laparoscopy protects against the oncologic adverse effects of open surgical tumor excision.     

miR-135a modulates tendon stem/progenitor cell senescence via suppressing ROCK1

Tendon stem/progenitor cell (TSPC) senescence may lead to age-related tendon disorders and impair tendon regeneration and replacement capacity in humans. However, the mechanisms governing TSPC aging and degeneration remain obscure. Recently, it has been reported that Rho-associated coiled-coil protein kinase 1 (ROCK1) might be a key player in TSPC aging process. miRNAs are also involved in cellular senescence. In this study, whether miRNAs modulate senescence of TSPCs through targeting ROCK1 was evaluated. We found that miR-135a, which directly binds to the 3′-untranslated region of ROCK1, is significantly downregulated in aged compared with young TSPCs. Overexpression of miR-135a in young TSPCs suppresses senescence, promotes proliferation, and induces migration and tenogenic differentiation, whereas suppression of miR-135a in aged TSPCs has the opposite effects. By gain-of-function and loss-of-function studies, we confirmed that ROCK1 mediates the effects of miR-135a in TSPCs. Taken together, our data suggest that miR-135a plays an important role in TSPC senescence via targeting ROCK1.     

High uric acid induces phenotypic transition of renal tubular cells via PI3K/Akt signaling pathway

Objective To investigate the effect and the mechanism of epithelial-mesenchymal transition (EMT) in renal tubular cells induced by uric acid. Methods Normal rat kidney tubular cell line (NRK-52E) were exposed to different concentrations of uric acid (100, 200, 400, 600, 800 μmol/L UA) for 48 hours to induce EMT. Morphological changes of the NRK-52E cells were examined under an inverted phase contrast microscope. The protein expression of E-cadherin, α-SMA, p-Akt and Akt were detected by Western blotting. The distribution of E-cadherin and α-SMA were detected by immunofluorescence. NRK-52E cells were pretreated by different concentrations of LY294002(0, 2.5, 5, 10, 15 μmol/L), the inhibitor of PI3K/p-Akt signaling pathway, and then processed by uric acid (400 μmol/L) for 48 hours. Western blotting was used to detect the protein expression of p-Akt and Akt. NRK-52E cells were then divided into four groups: normal group (N), uric acid group (UA), LY294002 group (LY), uric acid with LY294002 group (UA+LY). The protein expression of E-cadherin and α-SMA were detected by Western blotting, the distribution of E-cadherin, α-SMA and p-Akt were detected by immunofluorescence. Results There was abundant cellular expression of E-cadherin in unstimulated renal tubular cells whereas its expression was significantly decreased in uric acid-stimulated cells (P＜0.05). In addition, uric acid induced de novo expression of α-SMA in contrast to almost negative staining in untreated cells (P＜0.05). p-Akt were obviously increased in high uric acid group (P＜0.05) and Akt changed not significantly (P＞0.05). NRK-52E cells transformed into elongated fibroblast-like cells from cuboidal clustered epithelial cells. These indicated that uric acid has induced EMT and activated PI3K/p-Akt signaling pathway in NRK-52E cells. However, the above effects of uric acid were abolished when p-Akt was blocked by the PI3K inhibitor (10, 15 μmol/L LY294002), indicated that LY294002 has reversed the trend of EMT. Conclusions High uric acid induces phenotypic transition of renal tubular cells probably via activating PI3K/Akt signaling pathway.     

血管紧张素II通过PI3K/Akt/FOXO1信号通路诱导骨骼肌细胞萎缩

目的探讨血管紧张素II(Ang II)对骨骼肌细胞的调控作用及其作用机制。方法使用Ang II及其1型受体(AT1R)拮抗剂(ARB)奥美沙坦干预C2C12成肌细胞,分为Control、Ang II和Ang II+ARB三组。使用2%马血清诱导成肌细胞分化为肌管细胞。免疫荧光染色检测肌管细胞的平均面积改变,Western blot检测肌管细胞泛素连接酶、生肌调节因子(MRFs)和PI3K/Akt/FOXO1信号通路蛋白表达的变化。结果免疫荧光染色显示,Ang II干预后,肌管细胞的平均直径和面积减小(P0.001);使用奥美沙坦干预后,肌管细胞平均直径和面积明显增大(P 0.01)。Western blot显示,Ang II干预后,肌管细胞pPI3K/PI3K(P0.01),p-Akt/Akt (P 0.001)和p-FOXO1/FOXO1 (P 0.01)的比率降低; MURF1 (P 0.05)和MAFbx (P 0.001)蛋白表达明显升高,MHC(P0.01)和MyoD(P 0.05)蛋白表达明显降低;使用奥美沙坦处理后,能够减轻Ang II对肌管细胞的干预作用。结论 Ang II能够通过抑制PI3K/Akt/FOXO1信号通路的磷酸化,促进蛋白的降解,抑制蛋白的合成,诱导骨骼肌细胞萎缩。     

人脐血间充质干/祖细胞诱导为心肌样细胞的实验研究

目的　了解脐血间充质干 /祖细胞用于心肌细胞再生的可行性 ,并探讨其最佳的诱导培养条件。方法　将脐血单个核细胞置于低血清 (2 % )DMEM培养基中生成贴壁细胞层 ,依传代方法 ,用相同培养条件进行扩增 ,扩增后的贴壁细胞置入心肌诱导培养液中 ,并添加 5 氮杂胞苷进行诱导分化、采用心肌特异性收缩蛋白 肌钙蛋白T染色鉴定被诱生的心肌样细胞。结果　脐血间充质干 /祖细胞克隆在脐血单个核细胞中出现频率为 0 .5× 10 -6,在传 2 0代时 ,可有效扩增 1.3× 10 7倍 ,诱导后 ,70 %的脐血间充质干 /祖细胞分化为心肌样细胞。结论　采用上述扩增与诱导条件 ,脐血间充质干 /祖细胞可得到有效扩增 ,并可高效向心肌样细胞分化。     

Protective effect of emodin on renal interstitial fibrosis in mice of unilateral ureteral obstruction via PI3K/Akt/mTOR signaling pathway

Objective To investigate the effect and mechanism of emodin (EM) in renal interstitial fibrosis of unilateral ureteral obstruction (UUO) mice. Methods Male C57BL/6J mice were randomly divided into 4 groups, including sham operation group (n=8), UUO operation group (n=8), UUO operation+losartan (LST) group (n=8) and UUO operation+EM group (n=8). The mice in each group were ingested the suspensions by gavage for 14 days after surgery. Mice in UUO+LST and UUO+EM groups were given 10 mg?kg-1?d-1 LST and 20 mg?kg-1?d-1 EM, respectively. LST and EM were mixed with 0.5% sodium carboxymethyl cellulose. Mice in sham group and UUO group were given 0.5% sodium carboxymethyl cellulose. The mice were sacrificed at the 14th day. Interstitial fibrosis was observed by HE, Masson and PAS stain. Real-time PCR was used to detect LC3, Beclin-1 and mTOR mRNA. Protein expressions of TGF-β1, α-SMA, E-cadherin, LC3, Beclin-1, PI3K, p-Akt and mTOR were detected by Western blotting. The autophagy was observed with transmission electron microscopy in the renal tissue. Results Compared with sham mice, UUO mice at the 14th day displayed obvious renal fibrosis. Meanwhile, UUO mice had increased expressions of TGF-β1 and α-SMA (all P＜0.01), and decreased expressions of E-cadherin (P＜0.01). Their renal expressions of PI3K, p-Akt and mTOR were also raised (all P＜0.01). Compared with those in UUO group, in UUO+LST group and UUO+EM group, expressions of autophagy protein LC3 and Beclin-1 were increased (all P＜0.01), and the number of autophagic was increased. Additionally, expressions of TGF-β1 and α-SMA were reduced in UUO+LST group and UUO+EM group (all P＜0.01), while the expression of E-cadherin was increased by emodin treatment (P＜0.05). And expressions of PI3K, p-Akt and mTOR were decreased in UUO+LST group and UUO+EM group (all P＜0.05), meanwhile renal tissue fibrosis significantly reduced. Conclusions Emodin can promote autophagy, ameliorate renal interstitial fibrosis and protect renal function through PI3K/Akt/mTOR signaling pathway.     

内皮型一氧化氮合酶基因体外转染对犬内皮祖细胞功能的影响

目的探讨人内皮型一氧化氮合酶基因(heNOS)转染对犬骨髓源内皮祖细胞(EPC)功能的影响。方法用携带heNOS基因的5型腺病毒作为载体(Ad5-heNOS),对体外定向培养扩增的骨髓源犬EPC进行转染,以未转染的犬EPC作为对照。用酶联免疫吸附试验(ELISA)和硝酸还原酶法检测转染后的EPC中heNOS蛋白的表达和NO的产量,并检测转染后EPC的增殖、黏附、迁移、抗衰老和成血管能力等功能。结果 Ad5-heNOS转染48 h后,转染组EPC的eNOS蛋白表达量和NO产量均明显高于未转染组[(2091.67±172.489)pg/mL vs.(158.00±30.914)pg/mL;(49.5±5.16)μmol/Lvs.(39.7±7.24)μmol/L](均P<0.01);转染组EPC的细胞增殖数、黏附数、迁移数均明显高于未转染组(0.52±0.03 vs.0.31±0.02;28.00±1.41 vs.11.83±1.45;109.67±6.95 vs.72.67±6.29)(均P<0.01),而衰老细胞百分数明显低于未转染组(0.22±0.02 vs.0.32±0.01)(P<0.01);转染heN...     

Calcineurin inhibitor effects on glucose metabolism and endothelial function following renal transplantation

Abstract: Background:  Calcineurin inhibitors (CNI) are involved in the development of post-transplant diabetes mellitus (PTDM). Changes in insulin secretion and sensitivity contribute to the development of PTDM and are associated with endothelial function.
Methods:  In a pre-defined substudy of a previously published randomized trial in renal transplant recipients we compared the effect of CNI treatment (n = 23) with complete CNI-avoidance (n = 21) on insulin secretion and sensitivity (oral glucose tolerance test) as well as endothelial function (laser Doppler flowmetry), 10 wk and 12 months following transplantation.
Results:  Insulin sensitivity differed 10 wk post-transplant and was significantly better after 12 months in patients never treated with CNI drugs [0.091 (0.050) vs. 0.083 (0.036) μmol/kg/min/pmol/L, p = 0.043]. Insulin secretion tended to be higher in CNI treated patients at both time points (p = 0.068). Endothelial function was not significantly different at week 10 [540 (205) vs. 227 (565) arbitary units × minutes, p = 0.35] or month 12 [510 (620) vs. 243 (242), p = 0.33].
Conclusions:  Findings in the present study indicate that long-term CNI treatment negatively affects glucose metabolism and this may contribute to the increased risk for premature cardiovascular disease in CNI treated renal transplant recipients. Further studies to elucidate this hypothesis are, however, needed.     

Mikania micrantha extract enhances cutaneous wound healing activity through the activation of FAK/Akt/mTOR cell signaling pathway

Mikania micrantha (MM) has been traditionally used for various health benefits, including mental health, anti-inflammatory, wound dressing, and healing of sores. However, the molecular mechanisms and dose required for the wound healing activity of MM have yet to be reported. Therefore, a study was conducted to evaluate the wound healing potential of a cold methanolic extract of MM through in vitro and in vivo studies. Human dermal fibroblast adult (HDFa) cells were treated with 0 (control), 75 ng/ml, 125 ng/ml, 250 ng/ml, and 500 ng/ml of MMmethanolic extract (MME) for 24 h. MME at 75 ng/ml has significantly (p˂0.05) promoted HDFa cell proliferation and migration. Further, MME has also been shown to enhance the invasiveness of human umbilical vascular endothelial cells (HUVECs), indicating the neovasculature for wound healing. The tube formation assay demonstrated a significant (p<0.05) increase in the angiogenic effect of the MME starting at a concentration of 75 ng/ml as compared to the control. Treatment of excision wounds in Wistar rats with 5% and 10% MME ointment significantly enhanced wound contraction compared to control animals. Incision wounds in rats treated with 5% and 10% MME showed a significant (p<0.01) increase in tensile strength compared to control. HDFa cells, and granulation tissue collected on day 14 post-wounding, revealed the modulation of the FAK/Akt/mTOR cell signaling pathway during the enhancement of wound healing. The results of gel zymography showed increased activity of MMP-2 and MMP-9 in the HDFa cells after treatment with the extract.  It is concluded that MMEcan potentially accelerate cutaneous wound healing.