Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.

Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury

Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation-induced hypoxia inducible factor-1α expression.In this study,we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression.MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner.Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α.Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression;however,DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA.These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.

Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3p and underlying mechanism

AIM: To investigate the mechanism by which miR-204-3p inhibits the growth of hepatocellular carcinoma (HCC) tumor endothelial cells (TECs).

Epigallocatechin-3-gallate attenuates lipopolysaccharideinduced inflammation in human retinal endothelial cells

AIM:To investigate the mechanism underlying the anti-inflammatory effects of epigallocatechin-3-gallate(EGCG)in lipopolysaccharide(LPS)-stimulated human retinal endothelial cells(HRECs).METHODS:HRECspre-treatedwithEGCG(0-100μmol/L)were stimulated with LPS(250 ng/mL).Levels of tumor necrosis factor alpha(TNF-α),vascular endothelial growth factor(VEGF),monocyte chemotactic protein-1(MCP-1)and nitric oxide(NO)in the supernatants were determined by enzyme-linked immunosorbent assay(ELISA)and Griess assay.The protein expression of phosphorylated extracellular signal-regulated kinase(ERK)1/2 and p38 mitogen-activated protein kinases(p38)were determined by Western blot analysis.RESULTS:EGCG pre-treatment significantly inhibited the secretion of TNF-α,VEGF,MCP-1 and NO in LPSstimulated HRECs.Moreover,EGCG effectively attenuated LPS-induced activation and phosphorylation of ERK1/2 and p38 in HRECs in a dose-dependent manner.CONCLUSION:EGCG exhibited inhibitory effects on LPS-induced pro-inflammatory cytokines production by modulating ERK1/2 and p38 pathways in HRECs,suggesting EGCG as a potential candidate for antiinflammatory intervention.

Angiogenesis in a 3D model containing adipose tissue stem cells and endothelial cells is mediated by canonical Wnt signaling

Adipose-derived stromal cells （ASCs） have gained great attention in regenerative medicine. Progress in our understanding of adult neovascularization further suggests the potential of ASCs in promoting vascular regeneration, although the specific cues that stimulate their angiogenic behavior remain controversial In this study, we established a three-dimensional （3D） angiogenesis model by co-culturing ASCs and endothelial cells （ECs） in collagen gel and found that ASC-EC-instructed angiogenesis was regulated by the canonical Wnt pathway. Furthermore, the angiogenesis that occurred in implants collected after injections of our collagen gel- based 3D angiogenesis model into nude mice was confirmed to be functional and also regulated by the canonical Wnt pathway. Wnt regulation of angiogenesis involving changes in vessel length, vessel density, vessel sprout, and connection numbers occurred in our system. Wnt signaling was then shown to regulate ASC- mediated paracrine signaling during angiogenesis through the nuclear translocation of β-catenin after its cytoplasmic accumulation in both ASCs and ECs. This translocation enhanced the expression of nuclear cofactor Lef-1 and cyclin D1 and activated the angiogenic transcription of vascular endothelial growth factor A （VEGFA）, basic fibroblast growth factor （bFGF）, and insulin-like growth factor 1 （IGF-1）. The angiogenesis process in the 3D collagen model appeared to follow canonical Wnt signaling, and this model can help us understand the importance of the canonical Wnt pathway in the use of ASCs in vascular regeneration.

PI3K-like Kinases Restrain Pim Gene Expression in Endothelial Cells

Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

miR-146a-3p targeted RELB to regulate cytokine secretion in endothelial cells of atherosclerotic mice

Objective:To explore how miR-146a-3p can regulate the cytokine secretion of endothelial cells in atherosclerotic mice through the nuclear factorκB subunit(RELB).Methods:Apolipoprotein knockout(ApoE-/-)mouse atherosclerosis model was established by feeding program of high-fat diet formula and vascular endothelial cells were performed on primary culture.The difference in expression levels of miR-146a-3p in endothelial tissues of atherosclerotic mice and control mice was detected by real-time fluorescence quantitative PCR.The match between mir-146a-3p and RelB was analyzed by TargetScanHuman.Then whether miR-146a-3p targeted RELB was checked by luciferase reporter system.In the case of miR-146a-3p mimics overexpression or miR-146a-3p inhibitor knockdown,the granulocyte colony stimulating factor(GCSF),the levels of granulocyte macrophage colony stimulating factor(GM-CSF),interleukin 2(IL-2),interleukin 3(IL-3),interleukin 4(IL-4),interleukin 5(IL-5),interleukin 6(IL-6),interleukin 9(IL-9),interleukin 10(IL-10),interleukin 12 p40(IL-12 p40),interleukin 12 p70(IL-12 p70),interleukin 13(IL-13),interleukin 17(IL-17),interferon gamma(IFN-γ),monocyte chemotactic protein 1(MCP-1),monocyte chemoattractant protein 5(MCP-5),small inducible cytokine A5(RANTES),and tumor necrosis factor-α(TNFα)were detected by enzyme-linked immunosorbent assay.Results:Compared with the control group,the expression of miR-146a-3p in vascular endothelial tissue of atherosclerotic mice was reduced(P<0.05);MiR-146a-3p targeted the 3'UTR of RELB;After overexpression of miR-520a-3p,the expression level of RELB was decreased,and the levels of GCSF,GM-CSF,IL-2,IL-3,IL-4,IL-5,IL-6,IL-9,IL-10,IL-12 p40,IL-12 p70,IL-13,IL-17,IFN-γ,MCP-1,MCP-5,RANTES,and TNFαcytokines secreted by endothelial cells in atherosclerotic mice were decreased(P<0.05);After knocking down miR-520a-3p,the expression level of RELB was increased,and the levels of GCSF,GM-CSF,IL-2,IL-3,IL-4,IL-5,IL-6,IL-9,IL-10,IL-12 p40,IL-12 p70,IL-13,IL-17,IFN-γ,MCP-1,MCP-5,RANTES,and TNFαcytokines secreted by endothelial cells in atherosclerotic mice were increased(P<0.05).Conclusion:MiR-146a-3p inhibited the cytokine secretion of vascular endothelial cells in atherosclerotic mice by targeting the key molecule of nuclear factorκB(NF-kB)signaling pathway RELB.

Small interfering RNA targeting PGC-1α inhibits VEGF expression and tube formation in human retinal vascular endothelial cells

AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O2) or hypoxic (1%, O2) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O2) or hypoxic (1%, O2) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.

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.

Integrin binding peptides facilitate growth and interconnected vascular-like network formation of rat primary cortical vascular endothelial cells in vitro

Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2′-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional(3 D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3 D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3 D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3 D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3 D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.

Comparison of three fluorescence labeling and tracking methods of endothelial progenitor cells in laser-injured retina

AIM： To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells（EPCs） in a mouse model of laser-induced retinal injury.METHODS： EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes： 5-（and-6）-carboxyfluorescein diacetate succinimidyl ester（CFSE）, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein（Di I-Ac LDL）, and green fluorescent protein（GFP）. The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo.RESULTS： EPCs labeled with CFSE and Di I-Ac LDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28 d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and Di I-Ac LDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina.CONCLUSION： The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and Di I-Ac LDL are suitable for short-term EPClabeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.

Bilayer nicorandil-loaded small-diameter vascular grafts improve endothelial cell function via PI3K/AKT/eNOS pathway

For the surgical treatment of cardiovascular disease(CVD),there is a clear and unmet need in developing small-diameter(diameter<6 mm)vascular grafts.In our previous work,sulfated silk fibroin(SF)was successfully fabricated as a potential candidate for preparing vascular grafts due to the great cytocompatibility and hemocompatibility.However,vascular graft with single layer is difficult to adapt to the complex internal environment.In this work,polycaprolactone(PCL)and sulfated SF were used to fabricate bilayer vascular graft(BLVG)to mimic the structure of natural blood vessels.To enhance the biological activity of BLVG,nicorandil(NIC),an FDA-approved drug with multi-bioactivity,was loaded in the BLVG to fabricate NIC-loaded BLVG.The morphology,chemical composition and mechanical properties of NIC-loaded BLVG were assessed.The results showed that the bilayer structure of NIC-loaded BLVG endowed the graft with a biphasic drug release behavior.The in vitro studies indicated that NIC-loaded BLVG could significantly increase the proliferation,migration and antioxidation capability of endothelial cells(ECs).Moreover,we found that the potential biological mechanism was the activation of PI3K/AKT/eNOS signaling pathway.Overall,the results effectively demonstrated that NIC-loaded BLVG had a promising in vitro performance as a functional small-diameter vascular graft.

基于NOD样受体3炎性小体通路对利拉鲁肽在氧化低密度脂蛋白诱导内皮细胞损伤的作用机制研究

背景动脉粥样硬化是世界范围内引起心脑血管疾病最主要的原因,炎症是目前研究热点,其中NOD样受体3(NLRP3)是研究最为深入的炎症小体。胰高糖素样肽1(GLP-1)受体激动剂有抗动脉粥样硬化作用,具体机制尚不明确。目的研究利拉鲁肽通过拮抗氧化低密度脂蛋白(ox-LDL)诱导的内皮细胞损伤的作用机制。方法2022-03-25—05-19培养人脐静脉内皮细胞(HUVEC),取HUVEC加空白血清作为对照组,100μg/mL的ox-LDL干预HUVEC 48 h作为模型组,100μg/mL的ox-LDL干预HUVEC 24 h后分别加入100、200、400 nmol/L利拉鲁肽处理24 h作为利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组。CCK-8法计算细胞增殖率。通过扫描电镜观察焦亡细胞形态。检测乳酸脱氢酶(LDH)活力。酶联免疫吸附试验(ELISA)检测白介素(IL)-1β、IL-18表达水平。蛋白质免疫印迹试验(Western blot)检测NLRP3、接头蛋白凋亡相关斑点样蛋白(ASC)、天冬氨酸蛋白水解酶1(Caspase-1)、焦亡执行蛋白(GSDMD)、N端结构域的焦亡执行蛋白(N-GSDMD)表达水平。结果模型组、利拉鲁肽低浓度组和利拉鲁肽中浓度组细胞增殖率低于对照组,利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组细胞增殖率高于模型组(P<0.05)。细胞扫描电镜结果示模型组细胞焦亡明显,利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组细胞焦亡情况明显改善。模型组、利拉鲁肽低浓度组LDH活力高于对照组,利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组低于模型组(P<0.05)。模型组、利拉鲁肽低浓度组IL-1β表达水平高于对照组,利拉鲁肽中浓度组、利拉鲁肽高浓度组IL-1β表达水平低于模型组(P<0.05);模型组IL-18表达水平高于对照组,利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组IL-18表达水平低于模型组(P<0.05)。模型组NLRP3、ASC、Caspase-1、GSDMD、N-GSDMD表达水平高于对照组,利拉鲁肽低浓度组ASC、Caspase-1表达水平高于对照组,利拉鲁肽中浓度组NLRP3、ASC表达水平低于模型组,利拉鲁肽高浓度组NLRP3、ASC、Caspase-1表达水平低于模型组(P<0.05)。结论利拉鲁肽显著抑制ox-LDL诱导的内皮细胞NLRP3炎性小体活化,并且能够抑制内皮细胞的焦亡,具有抗动脉粥样硬化作用。

基于p38MAPK-PPARγ/NF-κB信号通路探讨消肿止痛合剂对血管内皮细胞bEND-3凋亡的影响

目的从细胞及信号通路层面探讨消肿止痛合剂对皮瓣缺血再灌注损伤中血管内皮细胞凋亡的影响。方法血管内皮细胞bEND-3培养。TNF-α体外细胞凋亡诱导模型建立并将细胞分为:p38MAPK抑制剂组、PPARγ抑制剂组、NF-κB抑制剂组、模型对照组、空白组。微丝绿色荧光探针观察细胞骨架微丝。RT-PCR法检测血管内皮细胞bEND-3细胞中p38MAPK、PPARγ、IκBα、NF-κBmRNA的表达。结果空白组中p38MAPK、PPARγ、IκBα、NF-κB的表达量无明显差异,模型对照组中p38MAPK与NF-κB的表达量最高,与模型对照组相比,p38MAPK抑制剂组bEnd.3细胞中p38MAPK与NF-κB的表达被抑制,PPARγ、IκBα表达量升高(P<0.05);PPARγ抑制剂组中PPARγ、IκBα、NF-κB的表达被抑制,p38MAPK表达量升高(P<0.05),NF-κB抑制剂组中p38MAPK与NF-κB的表达被抑制,IκBα与PPARγ的表达量升高(P<0.05);与空白组相比,p38MAPK抑制剂组与NF-κB抑制剂组中p38MAPK、PPARγ、IκBα、NF-κB的表达量升高(P<0.05),PPARγ抑制剂组中p38MAPK与NF-κB表达量明显升高,PPARγ、IκBα表达被抑制(P<0.05)。结论消肿止痛合剂可抑制bEND-3细胞凋亡,其机制与影响可能与p38MAPK-PPARγ/NF-κB信号通路有关。

Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment

BACKGROUND To solve the problem of liver transplantation donor insufficiency,an alternative cell transplantation therapy was investigated.We focused on amniotic epithelial cells(AECs)as a cell source because,unlike induced pluripotent stem cells,they are cost-effective and non-tumorigenic.The utilization of AECs in regenerative medicine,however,is in its infancy.A general profile for AECs has not been comprehensively analyzed.Moreover,no hepatic differentiation protocol for AECs has yet been established.To this end,we independently compiled human AEC libraries,purified amniotic stem cells(ASCs),and co-cultured them with mesenchymal stem cells(MSCs)and human umbilical vein endothelial cell(HUVECs)in a 3D system which induces functional hepatic organoids.AIM To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells METHODS AECs,MSCs,and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients.Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry,Alkaline phosphatase(AP)staining,and flow cytometry.An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay.AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics.The 2D and 3D culture were compared by relative gene expression using several differentiation protocols.ASCs,MSCs,and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging,periodic acid Schiff,and an indocyanine green(ICG)test.RESULTS AECs have certain stemness markers such as EPCAM,SSEA4,and E-cadherin.One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers.Moreover,it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage.Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells.TJP1,associated with epidermal growth factor receptor,and MET,associated with hepatocyte growth factor receptor,were upregulated and may be important for hepatic differentiation.In conventional flat culture,the cells turned unviable and did not readily differentiate into hepatocytes.In 3D culture,however,hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol.Furthermore,the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity,hepatic-like glycogen storage,and ICG absorption/elimination.CONCLUSION Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness.Under a 3D co-culture system,functional hepatic organoids were generated in a multicellular microenvironment.

Knockdown of STAT3 by iRNA Inhibiting Migration and Invasion of Epithelial Ovarian Cancer Cells

Signal transducer and activator of transcription 3（STAT3） is a dual functional transcription factor with the functions of signal transduction and transcription regulation. It is reported that the expression of STAT3 in ovarian cancer is significantly higher and STAT3 can facilitate ovarian cancer growth and metastasis. To clarify the definite effect and molecular mechanism of STAT3 involved in ovarian cancer growth and metastasis, STAT3 expression was significantly downregulated by transfecting ovarian cancer model SK-OV-3 cells with the plasmid vector which express specific RNAi that targets human STAT3. The downregulated STAT3 not only decreased the invasion and migration but also inhibited the proliferation of SK-OV-3 cells. Western blot assay shows that the expression of vascular endothelial growth factor（VEGF） and that of Survivin were reduced in the cells with the plasma vector expressing specific RNAi that targets human STAT3. These results demonstrate that STAT3 involved in the invasion and migration of SK-OV-3 regulates the expression of VEGF and Survivin. In addition, VEGF and Survivin could play an important role in ovarian cancer growth and metastasis.

Inhibition Mechanism of Novel Pyrazolo[1,5-a]pyrazin-4(5H)-one Derivatives Against Proliferation of A549 and H322 Cancer Cells

Objective To explore the inhibition mechanism and safety of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives against proliferation of human lung cancer A549 cells, H322 cells, and human umbilical vein endothelial cell(HUVEC). Methods Cells were treated with 40 μmol/L of the ppo3 a, ppo3 b, ppo3 i, and 0.1% DMSO(control) for 48 hours, respectively. Apoptosis was determined by Hoechst 33258 staining assay in H322 and A549 cells. Cell cycle distribution was determined by flow cytometry analysis in A549 cell. LC3-II, p53, and heat shock protein(HSP) 70 protein levels were detected by Western blotting in A549 cells treated with ppo3 b for 48 hours. The morphology and viability of HUVEC were observed by inverted microscope and sulforhodamine B(SRB) assay. Results Ppo3 a, ppo3 b, and ppo3 i significantly induced apoptosis in H322 and A549 cells. A strong G1-phase arrest was concomitant with the growth inhibitory effect on A549 cells. Ppo3 b effectively elevated the p53 protein level, but significantly reduced the HSP70 protein level. There were no significantly inhibitory effect on the morphology and viability of HUVEC when treated with ppo3 a, ppo3 b, and ppo3 i. Conclusions ppo3 a, ppo3 b, and ppo3 i could inhibit H322 proliferation through apoptosis and inhibit A549 through apoptosis and G1-phase arrest. The protein p53 and HSP70 might involve in the inhibition effects. These derivatives might be a clue to find effective and safe drug for lung cancers.

Effects of LY294002 on the function of retinal endothelial cell in vitro

AIM： To study the effects of LY294002 [phosphatidylinositol 3-kinase（PI3K） inhibitor] on the function and mechanisms of retinal endothelial cells（RECs） in vitro.METHODS： RECs were randomly divided into control group and LY294002 treatment group. RECs in the control group were placed the incubator for hypoxic exposure in vitro. RECs in the LY294002 treatment group were pretreated with LY294002（40 μmol/L） under hypoxic condition. The expression of matrix metalloproteinase（MMP）-2, MMP-9, vascular endothelial growth factor（VEGF）, and apoptosis and proliferation of RECs were evaluated with Western blot, real-time reverse transcription-polymerase chain reaction（RT-PCR）, and flow cytometric analysis, correspondently.RESULTS： Compared with the control group, treating the RECs with LY294002 was able to remarkably inhibit cell proliferation rates（t_（1d）=2.13, t_（2d）=2.65, t_（3d）=2.36, t_（4d）=2.06, all P〈0.05）. Flow cytometric analysis indicated that a moderate increase in apoptosis in the LY294002 treatment group compared to the control group（t=2.51, P〈0.05）. The expression of MMP-2, MMP-9 and VEGF were downregulated in the LY294002 treatment group by Western blot and real-time RT-PCR（all P〈0.05）.CONCLUSION： LY294002 regulates the function of RECs by reducing the expression of MMP-2, MMP-9, and VEGF in vitro. LY294002 may provide an effective method for preventing pathological angiogenesis.

组织蛋白酶B-RNAi-LV载体构建及其对小鼠脑微血管内皮细胞系bEND.3的影响

目的构建组织蛋白酶B(Cathepsin B,CTSB)小RNA(siRNA)慢病毒载体,并探讨其对小鼠脑微血管内皮细胞(bEND.3)的影响。方法设计并合成含有干扰Cathesin B基因的19 nt的双链寡DNA片段,将此片段克隆到携有绿色荧光蛋白(GFP)的慢病毒表达载体质粒pGCSIL-GFP上,经测序正确后,命名为pGCSIL-GFP-CTSB,将慢病毒表达载体pGCSIL-GFP-CTSB、慢病毒包装载体pHelper1.0和pHelper2.0 3质粒共同转染于293T细胞,获得携带Cathepsin B基因的RNAi慢病毒(Cathesin B-RNAi-Lentivirus,即CTSB-RNAi-LV),通过Real time-PCR和Western blotting方法观察CTSB-RNAi-LV对bEND.3的影响。结果 1.pGCSIL-GFP-CTSB中携带有正确的Cathesin B siRNA基因;2.目的基因Cathepsin B siRNA被RNAi慢病毒高效地转导入靶细胞bEND.3内,并达到稳定的表达;3.CTSB-RNAi-LV能有效降低bEND.3中Cathepsin B mRNA和蛋白表达。结论成功构建了RNAi慢病毒载体pGCSIL-GFP-CTSB;并成功包装了RNAi慢病毒CTSB-RNAi-LV;CTSB-RNAi-LV能有效地抑制bEND.3中Cathepsin B mRNA和蛋白表达水平下调。

小鼠脑微血管内皮细胞株bEnd.3的细胞特征及基因表达谱分析

目的 :对一种小鼠脑组织来源的血管内皮细胞株bEnd .3的主要细胞特征进行研究并做血管内皮细胞生物学功能基因芯片分析。方法 :利用倒置显微镜、扫描和透射电子显微镜观察细胞形态特征 ;免疫细胞化学法显示血管内皮细胞特异性表面标志 ;ELISA定量检测PGE2 ;流式细胞术、MTT法研究增殖和凋亡、血管内皮细胞基因芯片研究正常培养bEnd 3基因表达谱。结果 :bEnd 3具备多种典型的微血管内皮细胞 (MVEC)特征 :细胞呈多边形或梭型、呈铺路石样生长 ;存在管腔样结构 (TLS)和毛细血管样网络 ,细胞表面有丰富的微绒毛 ;vW因子、CD34阳性 ;CD31、CD36、CD10 5等也有不同程度的表达 ;细胞高水平分泌前列腺素E2 (PGE2 ) ;多种与正常血管功能密切相关的基因也有不同程度的表达。结论 :bEnd .3保留了MVEC的基本特性 ,可用于心脑血管疾病。