MicroRNAs (miRNAs) are endogenous -22 nt RNAs that play important regulatory roles in targeting mRNAs for cleavage or translational repression. Despite the discovery of increasing numbers of human and mouse miRNAs, ...MicroRNAs (miRNAs) are endogenous -22 nt RNAs that play important regulatory roles in targeting mRNAs for cleavage or translational repression. Despite the discovery of increasing numbers of human and mouse miRNAs, little is known about miRNAs from pig. In this study, we sought to extend the repertoire of porcine small regulatory RNAs using Solexa sequencing. We sequenced a library of small RNAs prepared from immortalized swine umbilical vein endothelial cells (SUVECs). We produced over 13.6 million short sequence reads, of which 8 547 658 perfectly mapped to the pig genome. A bioinformatics pipeline was used to identify authentic mature miRNA sequences. We identified 154 porcine miRNA genes, among which 146 were conserved across species, and 8 were pig-specific miRNA genes. The 146 miRNA genes encoded 116 conserved mature miRNAs and 66 miRNA^*. The 8 pig-specific miRNA genes encoded 4 mature miRNAs. Four potential novel miRNAs were identified. The secondary structures of the 154 miRNA genes were predicted; 13 miRNAs have 2 structures, and miR-9 and miR-199 have 4 and 3 structures, respectively. 36 miRNAs were organized into 19 compact clusters, miR-206, miR-21 and miR-378 were the relatively highly expressed miRNAs. In conclusion, Solexa sequencing allowed the successful discovery of known and novel porcine miRNAs with high accuracy and efficiency. Furthermore, our results supply new data to the somewhat insufficient pig miRBase, and are useful for investigating features of the blood-brain barrier, vascular diseases and inflammation.展开更多
Martentoxin, a 4,046 Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch, has been demonstrated to block large-conductance Ca2+-activated K+ (BKca) channels; however, its biologica...Martentoxin, a 4,046 Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch, has been demonstrated to block large-conductance Ca2+-activated K+ (BKca) channels; however, its biological roles are still largely unknown. In the present study, we investigated the pharmacological effects of martentoxin on regulating the production of nitric oxide induced by TNF-a in human umbilical vein endothelial cells (HU- VECs). We found that, 1, 10 and 100 ~tmol/L martentoxin decreased nitric oxide production by HUVECs ex- posed to 10 ng/mL TNF for 6, 12 and 24 hours. We further demonstrated that martentoxin inhibited the activity of iNOS and retarded the down-regulation of eNOS mRNA induced by TNF-a. Therefore, martentoxin could be a potential therapeutic agent for vascular diseases.展开更多
By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascula...By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdMNOR, CdM-CdMHYP and HUVEC-CdMHYP were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdMNOR and HUVEC-CdMHYP groups, the survival rate, migra-tion and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdMHYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.展开更多
Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells(ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radix...Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells(ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin(ERM) family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells(HUVECs) in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.展开更多
AIM:To compare conventional slow equilibrium cooling and directional freezing(DF) by gauze package for cryopreservation of human umbilical vein endothelial cells(HUVECs).METHODS:HUVECs were randomly assigned to conven...AIM:To compare conventional slow equilibrium cooling and directional freezing(DF) by gauze package for cryopreservation of human umbilical vein endothelial cells(HUVECs).METHODS:HUVECs were randomly assigned to conventional freezing(CF) and DF by gauze package group. The two groups of HUVECs were incubated with a freezing liquid consisting of 10% dimethylsulfoxide(DMSO), 60% fetal bovine serum(FBS) and 30%Dulbecco’s modified Eagle’s medium(DMEM) and then put into cryopreserved tubes. CF group, slow equilibrium cooling was performed with the following program:precool in 4℃ for 30 min,-20℃ for 1h, and then immersion in-80℃ refrigerator. DF group, the tubes were packaged with gauze and then directional freezing in-80℃ refrigerator straightly. One month later, the vitality of HUVECs were calculated between two groups.RESULTS:There was no significant difference in the survival rate and growth curve between CF and DF groups. The DF group was significantly better than CFgroup in adherent rates, morphological changes and proliferative ability.CONCLUSION:In the conventional cryopreserved method, cells are slow equilibrium cooling by steps(4℃,-20℃ and finally-80℃), which is a complicated and time-consuming process. But the improved DF by gauze package method is better than conventional method, for which is convenient and easy to operate.展开更多
This study investigated the changes in human umbilical vein endothelial cells (HUVECs) induced by overexpression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) and its role in cellular injury. Reco...This study investigated the changes in human umbilical vein endothelial cells (HUVECs) induced by overexpression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor Ⅷ-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the prolifera- tive ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS (eNOS) activity was assayed by using spectrophotometry, and NO2-/NO3- levels were measured usin~ nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma mem- brane and cytoplasm, and Western blot analysis confirmed that NOSTR1N levels were significantly higher in cells transfected with the NOSTR1N plasmid (P〈0.01). The activity of eNOS and the levels of NO2-/NO3 were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells (P〈0.01 and P〈0.01, respectively). Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOS- TRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overex- pression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.展开更多
Objective:To investigate the effects of microRNA-21 antisense nucleotide(AS-miR-21)on the proliferation,migration and autophagy of human umbilical vein endothelial cells(HUVECs).Methods:HUVECs were treated with1,000 n...Objective:To investigate the effects of microRNA-21 antisense nucleotide(AS-miR-21)on the proliferation,migration and autophagy of human umbilical vein endothelial cells(HUVECs).Methods:HUVECs were treated with1,000 nmol/L rapamycin for 6 h(rapamycin group)or ASmiR-21 transfection followed by 1,000 nmol/L rapamycin for6 h(AS-miR-21+rapamycin group).HUVECs without any treatment were defined as control group.The proliferation and migration abilities of HUVECs were detected by methyl thiazolyl tetrazolium(MTT)assay,scratch wound healing assay and transwell test,respectively.The expressions of microtubule-associated protein light chain 3 Ⅱ/Ⅰ(LC3 Ⅱ/Ⅰ)and Becline-1 were determined by western blotting.Results:The rapamycin group showed decreased OD value and migration rate,an increased ratio of LC3 Ⅱ/Ⅰ and up-regulated expression of Beclin-1 compared with the control group(P<0.05).The AS-miR-21+rapamycin group demonstrated lower OD value,migration rate,the number of migrated cells,and significantly higher ratio of LC3 Ⅱ/Ⅰ and Beclin-1 protein expression level than the control group and the rapamycin group(P<0.05).Conclusion:AS-miR-21 suppressed the autophagy,proliferation and migration in the HUVECs model of autophagy induced by rapamycin.展开更多
Objectives To investigate the effects and mechanism of glycated serum albumin(GSA) on expression of Monocyte chemoattratant protein-1(MCP-1) in Endothelial Cells. Methods Human Umbilical Vein Endothelial Cells (HUVEC)...Objectives To investigate the effects and mechanism of glycated serum albumin(GSA) on expression of Monocyte chemoattratant protein-1(MCP-1) in Endothelial Cells. Methods Human Umbilical Vein Endothelial Cells (HUVEC)are cultured with GSA of different concentrations and interfered by glycosylation products inhibitor Aminoguanidine (AG) and anti-oxidant N-acetylcy-steine (NAC), The expression of MCP-1 are evaluated by Immunocytochemistry and Sandwich ELISA. MDA content and SOD activity are determined by the technique of TBA and XOD respectively. Results GSA can stimulate MCP-1 production and secretion. Immunocytochemistry showed that after HUVECs were cultured with 50 mg/L GSA, expression of MCP-1 in group 4hrs, 8hrs and 12hrs was 1.3, 1.9 and 2.8 fold as much as that in control group (P < 0.01), and there was significant difference among the experiment groups(P < 0.01). Sandwich ELISA showed that expression of MCP-1 in three different groups was 1.6, 2.4 and 3.0 fold as much as that in control group(P < 0.01), and there was significant difference among the experiment groups(P < 0.01); GSA can cause the decrease of SOD activity(P < 0.05) and increase of MDA content(P < 0.01); AG and NAC can restrain obviously the expression of MCP-1 of HUVECs stimulated by GSA(P < 0.01); NAC can restrain the effect of GSA on SOD activity and MDA content in HUVECs (P < 0.05). Conclusions GSA can stimulate the expression of MCP-1 of endothelial cells by inducing endothelial cells oxidative stress.展开更多
Vascular endothelial growth factors(VEGFs) respectively bind to each of three receptor tyrosine kinases(RTKs),known as Flt-1,KDR and Flt-4.Since VEGFs and their respective families of receptor tyrosine kinases(VE...Vascular endothelial growth factors(VEGFs) respectively bind to each of three receptor tyrosine kinases(RTKs),known as Flt-1,KDR and Flt-4.Since VEGFs and their respective families of receptor tyrosine kinases(VEGFRs) are critical proteins which can regulate vascular development during angiogenesis,we decided to explore the inhibitory effects of soluble kinase insert domain-containing receptor(sKDR) on endothelial cells and angiogenesis.Total RNA was extracted from human umbilical vein endothelial cells(HUVEC),and cDNA of extracellular domains 1―4 was amplified and recombined with pQE40 vector.After being expressed,affinity purified,renatured and analyzed by Western blot,the sKDR was assayed for its effects on endothelial cells by [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide](MTT),and on angiogenesis by chick chorioallantoic membrane(CAM) experiment.sKDR cDNA of 1150 bp was obtained via real-time polymerase chain reaction(RT-PCR),and sKDR was expressed by pQE40 procaryotic expression system,purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis with only one band and proved by Western blot.MTT assay demonstrateds that sKDR could inhibit the VEGF-stimulated HUVEC from proliferation,and CAM experiment showed sKDR could block the VEGF-induced angiogenesis.sKDR has the biological activity to bind with VEGF ligands and is a potential target for tumor anti-angiogenesis therapy.展开更多
Salidroside is extensively used as a herbal medicine worldwide, and it has been shown to protect against disruption of endothelial homeostasis and act as an anti-aging agent. The present study aimed to investigate the...Salidroside is extensively used as a herbal medicine worldwide, and it has been shown to protect against disruption of endothelial homeostasis and act as an anti-aging agent. The present study aimed to investigate the ameliorative effects of salidroside on homocysteine (Hcy)-induced cell senescence in human umbilical vein endothelial cells (HUVECs) that were mediated via inhibition of Krüppel-like factor 4 (KLF4). An endothelial cell senescence model was induced by Hcy. The cell viability, activities of telomerase and lactate dehydrogenase (LDH), and the level of reactive oxygen species were determined using commercial kits. The expression levels of KLF4, p53 and p21 were determined via western blot analysis, whereas the mRNA expression levels of KLF4 were detected by reverse transcription-quantitative PCR. Small interfering RNA-mediated knockdown of KLF4 was found to reverse Hcy-induced cell senescence. Hcy treatment led to an accelerated cell senescence, as evidenced by decreases in both cell viability and telomerase activity, whereas increases were noted in the leakage of LDH and the level of reactive oxygen species, in addition to an up-regulation of the protein levels of p53 and p21, and up-regulation of KLF4 at both the mRNA and protein level. Treatment with salidroside ameliorated Hcy-induced cell senescence in a dose-dependent manner. Taken together, these results suggested that Hcy may induce cell senescence through upregulation of KLF4, and this may be reversed by treatment with salidroside. Therefore, salidroside was shown to inhibit Hcy-induced cell senescence through KLF4 inhibition.展开更多
AIM: To explore the possibility of human umbilical cord mesenchymal stem cells(h UCMSCs), human umbilical vein endothelial cells(h UVECs), human dental pulp stem cells(h DPSCs) and human periodontal ligament st...AIM: To explore the possibility of human umbilical cord mesenchymal stem cells(h UCMSCs), human umbilical vein endothelial cells(h UVECs), human dental pulp stem cells(h DPSCs) and human periodontal ligament stem cells(h PDLSCs) serving as feeder cells in co-culture systems for the cultivation of limbal stem cells.METHODS: Different feeder layers were cultured in Dulbecco's modified Eagle's medium(DMEM)/F12 and were treated with mitomycin C. Rabbits limbal stem cells(LSCs) were co-cultured on h UCMSCs, h UVECs, h DPSCs, h PDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency(CFE) assay and immunofluorescence(IPO13,CK3/12).RESULTS: The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But h UCMSCs, h DPSCs and h PDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to h UVECs and feedercell-free culture.CONCLUSION: hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.展开更多
Background Parthenolide has been tested for anti-tumor activities, such as anti-proliferation and pro-apoptosis in recent studies. However, little is known about its role in the process of tumor angiogenesis. This stu...Background Parthenolide has been tested for anti-tumor activities, such as anti-proliferation and pro-apoptosis in recent studies. However, little is known about its role in the process of tumor angiogenesis. This study aims to investigate the effects and potential mechanisms of parthenolide on the proliferation, migration and lumen formation capacity of human umbilical vein endothelial cells. Methods Different concentrations of parthenolide were applied to the human breast cancer cell line MDA-MB-231 cells. After 24-hour incubation, the culture supernatants were harvested and used to treat human umbilical vein endothelial cells for 24 hours. Then an inverted fluorescence phase contrast microscope was used to evaluate the human umbilical vein endothelial cells. The secretion of vascular endothelial growth factor (VEGF), interleukin (IL)-8 and matrix metalloproteinases (MMP)-9 in the culture supernatant of the MDA-MB-231 cells was then measured with enzyme-linked immunosorbent assay (ELISA) assays. Results Suppression of proliferation, migration, and the lumen formation capacity of human umbilical vein endothelial cells was observed in the presence of the culture supernatants from the breast cancer cell line treated with different concentrations of parthenolide. Parthenolide decreased the levels of the angiogenic factors MMP-9, VEGF, and IL-8 secreted by the MDA-MB-231 cells. Conclusions Parthenolide may suppress angiogenesis through decreasing angiogenic factors secreted by breast cancer cells to interfere with the proliferation, migration and lumen-like structure formation of endothelial cells, thereby inhibiting tumor growth. It is a promising potential anti-angiogenic drug.展开更多
Endothelial cell death due to increased reactive oxygen species(ROS) may contribute to the initial endothelial injury,which promotes atherosclerotic lesion formation.Piper sarmentosum(PS),a natural product,has been sh...Endothelial cell death due to increased reactive oxygen species(ROS) may contribute to the initial endothelial injury,which promotes atherosclerotic lesion formation.Piper sarmentosum(PS),a natural product,has been shown to have an antioxidant property,which is hypothesized to inhibit production of ROS and prevent cell injury.Thus,the present study was designed to determine the effects of PS on the hydrogen peroxide(H2O2)-induced oxidative cell damage in cultured human umbilical vein endothelial cells(HUVECs).In this experiment,HUVECs were obtained by collagenase perfusion of the large vein in the umbilical cord and cultured in medium M200 supplemented with low serum growth supplementation(LSGS).HUVECs were treated with various concentrations of H2O2(0-1000 μmol/L) and it was observed that 180 μmol/L H2O2 reduced cell viability by 50% as denoted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay.Using the above concentration as the positive control,the H2O2-induced HUVECs were concomitantly treated with various concentrations(100,150,250 and 300 μg/ml) of three different extracts(aqueous,methanol and hexane) of PS.Malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT) and glutathione peroxidase(GPX) levels showed a significant increase(P<0.05) in HUVECs compared to the negative control.However,PS extracts showed a protective effect on HUVECs from H2O2-induced cell apoptosis with a significant reduction in MDA,SOD,CAT and GPX levels(P<0.05).Furthermore,PS had exhibited ferric reducing antioxidant power with its high phenolic content.Hence,it was concluded that PS plays a beneficial role in reducing oxidative stress in H2O2-induced HUVECs.展开更多
Objective:To investigate whether ginsenoside Rb1(Rb1)can protect human umbilical vein endothelial cells(HUVECs)against high glucose-induced apoptosis and examine the underlying mechanism.Methods:HUVECs were divided in...Objective:To investigate whether ginsenoside Rb1(Rb1)can protect human umbilical vein endothelial cells(HUVECs)against high glucose-induced apoptosis and examine the underlying mechanism.Methods:HUVECs were divided into 5 groups:control group(5.5 mmol/L glucose),high glucose(HG,40 mmol/L)treatment group,Rb1(50μmol/L)treatment group,Rb1 plus HG treatment group,and Rb1 and 3-(1 H-1,2,3-triazol-4-yl)pyridine(3-TYP,16μmol/L)plus HG treatment group.Cell viability was evaluated by cell counting kit-8 assay.Mitochondrial and intracellular reactive oxygen species were detected by Mito Sox Red mitochondrial superoxide indicator and dichloro-dihydro-fluorescein diacetate assay,respectively.Annexin V/propidium iodide staining and fluorescent dye staining were used to measure the apoptosis and the mitochondrial membrane potential of HUVECs,respectively.The protein expressions of apoptosis-related proteins[Bcl-2,Bax,cleaved caspase-3 and cytochrome c(Cyt-c)],mitochondrial biogenesis-related proteins[proliferator-activated receptor gamma coactivator 1-alpha,nuclear respiratory factor-1 and mitochondrial transcription factor A],acetylation levels of forkhead box O3 a and SOD2,and sirtuin-3(SIRT3)signalling pathway were measured by immunoblotting and immunoprecipitation.Results:Rb1 ameliorated survival in cells in which apoptosis was induced by high glucose(P<0.05 or P<0.01).Upon the addition of Rb1,mitochondrial and intracellular reactive oxygen species generation and malondialdehyde levels were decreased(P<0.01),while the activities of antioxidant enzymes were increased(P<0.05 or P<0.01).Rb1 preserved the mitochondrial membrane potential and reduced the release of Cyt-c from the mitochondria into the cytosol(P<0.01).In addition,Rb1 upregulated mitochondrial biogenesis-associated proteins(P<0.01).Notably,the cytoprotective effects of Rb1 were correlated with SIRT3 signalling pathway activation(P<0.01).The effect of Rb1 against high glucose-induced mitochondria-related apoptosis was restrained by 3-TYP(P<0.05 or P<0.01).Conclusion:Rb1 could protect HUVECs from high glucose-induced apoptosis by promoting mitochondrial function and suppressing oxidative stress through the SIRT3 signalling pathway.展开更多
Objective:To investigate the anti-angiogenic effect of cryptotanshinone(CPT) on human umbilical vein endothelial cells(HUVECs) and the effect of CPT on Wnt/β-catenin signaling pathway.Methods:HUVECs were incuba...Objective:To investigate the anti-angiogenic effect of cryptotanshinone(CPT) on human umbilical vein endothelial cells(HUVECs) and the effect of CPT on Wnt/β-catenin signaling pathway.Methods:HUVECs were incubated with 0,2.5,5,10,and 20 μmol/L CPT for detecting cell viability with dimethyl thiazolyl-2,5-diphenyltetrazolium bromide(MTT) assay.Then,HUVECs were incubated with 0,2.5,5,and 10 μmol/L CPT for detecting endothelial cell migration,invasion,and tubular-like structure formation with wound healing,transwell invasion and matrigel tube formation assays,respectively.To gain insight into CPT-mediated signaling,the effects of CPT on T-cell factor/lymphocyte enhancer factor(TCF/LEF) transcription factors were detected by the Dual-luciferase reporter assay.Next,the nuclear expression of p-catenin was evaluated using Western blot and immunochemistry.Finally,vascular endothelial growth factor(VEGF) and cyclin D1,downstream proteins of the Wnt pathway were examined with Western blot.Results:CPT dose-dependently suppressed endothelial cell viability,migration,invasion,and tubular-like structure formation.In particular,CPT blocked β-catenindependent transcription in HUVECs in a dose-dependent manner.In Western bolt,10 μ mol/L CPT decreased expression of β-catenin in nucleus of HUVECs(P〈0.01).In immunohistochemistry,β-catenin was more potent in response to LiCI(an activator of the pathway) treatment.However,the signals were weaker in the nucleus of the CPT(10 μmol/L) group,compared to the positive control.Also,VEGF and cyclin D1 were both eliminated by CPT in 5 and 10 μ mol/L doses(P〈0.05).Conclosion:Our study supported the role of CPT as an angiogenic inhibitor,which may impact on the Wnt/β-catenin signaling pathway.展开更多
Objective: This study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and e...Objective: This study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish. Methods: The in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rgl and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit lI (XTI') assay. R1, Rgl and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigel- coated wells was performed to evaluate the pro-angiogenic actions of RI. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-N (o -nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor ]1 (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels. Results: R1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/FIk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemically- induced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs. Conclusion: R1, similar to Rgl and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/FIk-1 and PI3K- Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.展开更多
Objective: To investigate the pro-angiogenic effects of paeoniflorin(PF) in a vascular insufficiency model of zebrafish and in human umbilical vein endothelial cells(HUVECs). Methods: In vivo, the pro-angiogenic...Objective: To investigate the pro-angiogenic effects of paeoniflorin(PF) in a vascular insufficiency model of zebrafish and in human umbilical vein endothelial cells(HUVECs). Methods: In vivo, the pro-angiogenic effects of PF were tested in a vascular insufficiency model in the Tg(fli-1:EGFP)y1 transgenic zebrafish. The 24 h post fertilization(hpf) embryos were pretreated with vascular endothelial growth factor(VEGF) receptor tyrosine kinase inhibitor Ⅱ(VRI) for 3 h to establish the vascular insufficiency model and then post-treated with PF for 24 h. The formation of intersegmental vessels(ISVs) was observed with a fluorescence microscope. The m RNA expression of fms-like tyrosine kinase-1(flt-1), kinase insert domain receptor(kdr), kinase insert domain receptor like(kdrl) and von Willebrand factor(v WF) were analyzed by real-time polymerase chain reaction(PCR). In vitro, the pro-angiogenic effects of PF were observed in HUVECs in which cell proliferation, migration and tube formation were assessed. Results: PF(6.25–100 μmol/L) could rescue VRI-induced blood vessel loss in zebrafish and PF(25–100 μmol/L), thereby restoring the m RNA expressions of flt-1, kdr, kdrl and v WF, which were down-regulated by VRI treatment. In addition, PF(0.001–0.03 μmol/L) could promote the proliferation of HUVECs while PF stimulated HUVECs migration at 1.0–10 μmol/L and tube formation at 0.3 μmol/L. Conclusion: PF could promote angiogenesis in a vascular insufficiency model of zebrafish in vivo and in HUVECs in vitro.展开更多
There is no clear consensus regarding how cells respond to hydrostatic pressure. This is largely attributable to the high heterogeneity among cell types and the diverse custom-made devices used in previous studies. Th...There is no clear consensus regarding how cells respond to hydrostatic pressure. This is largely attributable to the high heterogeneity among cell types and the diverse custom-made devices used in previous studies. The aim of this work was to develop a facile device that could mimic various pressure environments and then delineate the cellular response to pressure stimulus. The device described here achieved both stable and periodic pressurization without oxygen deprivation. The biological utility of the device was assessed using human umbilical vein endothelial cells. We found more stereoscopic nuclear morphology and re-distribution of lamin A/C under high hydrostatic pressure compared to control cells. Mass spectrometry-based proteomics analysis showed significant changes in mitochondria-related pathways. Western blot analysis confirmed that high hydrostatic pressure induced a tendency toward mitochondrial fusion. Increased mitochondrial activity was observed as well. In conclusion, this device can be readily applied in biological research and extend our understanding of cellular mechano-sensation and the associated changes in mitochondrial behaviors.展开更多
Objective To determine whether advanced glycosylation end products modified bovine serum albumin (AGEs-BSA) affects endothelial cell lateral junction protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1) in...Objective To determine whether advanced glycosylation end products modified bovine serum albumin (AGEs-BSA) affects endothelial cell lateral junction protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the presence or absence of inflammatory mediators.Methods Cultured human umbilical vein endothelial cells (HUVECs) were exposed to AGEs-BSA for 6, 12, 24, and 36 hours, and exposed to AGEs-BSA glycosylated with different concentrations of glucose, tumor necrosis factord-α (TNF-α), interferon (IFN-γ), TNF-α + IFN-y and AGEs-BSA + TNF-α for 24 hours, respectively. Expression of PECAM-1 mRNA was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) with β-actin as an internal standard, and sequencing of RT-PCR products was performed to confirm the specificity of amplification for PECAM-1 gene. The endothelial cell surface expression of PECAM-1 was determined by flow cytometry (FCM).Results There were no significant changes in the expression of PECAM-1 mRNA and protein when the cells were exposed to AGEs-BSA with different concentrations or periods ( P>0. 05). However, PECAM-1 expression was reduced in the cells treated with TNF-α, IFN-y, TNF-α + IFN-γ and AGEs-BSA + TNF-α. The level of PECAM-1 treated with AGEs-BSA + TNF-α was lower than that of TNF-α treated alone (P<0. 01).Conclusions AGEs-BSA had no effect on the expression of PECAM-1 mRNA and protein in cultured HUVEC. With the presence of inflammatory mediator TNF-α, AGEs-BSA decreased the level of PECAM-1, which might reduce the adhesion interaction between adjacent endothelial cells, enhance the permeability of endothelial cells, and might be implicated in the endothelial dysfunction and pathogenesis of atherosclerosis in patients with diabetes mellitus. The significance of this phenomenon in intracellular signal transduction remains to be determined.展开更多
Objective:Gestational diabetes mellitus(GDM)is the most common metabolic disorder during pregnancy.LncRNA HLA complex group 27(HCG27)plays a crucial role in various metabolic diseases.However,the relationship between ...Objective:Gestational diabetes mellitus(GDM)is the most common metabolic disorder during pregnancy.LncRNA HLA complex group 27(HCG27)plays a crucial role in various metabolic diseases.However,the relationship between lncRNA HCG27 and GDM is not clear.This study aimed to verify a competing endogenous RNA(ceRNA)interaction regulation axis of miR-378a-3p/mitogen-activated protein kinase 1(MAPK1)regulated by HCG27 in GDM.Methods:LncRNA HCG27 and miR-378a-3p were detected by RT-qPCR.The expression of MAPK1 in umbilical vein endothelial cells(HUVECs)was detected by RT-qPCR and that in the placenta by Western blotting.To explore the relationship among lncRNA HCG27,miR-378a-3p,MAPK1 and the glucose uptake ability of HUVECs,vector HCG27,si-HCG27,miR-378a-3p mimic and inhibitor were transfected to achieve overexpression and inhibition of HCG27 or miR-378a-3p.The interaction between miR-378a-3p and lncRNA HCG27 or MAPK1 was confirmed by the dual-luciferase reporter assay.Besides,glucose consumption by HUVECs was detected by the glucose assay kit.Results:HCG27 expression was significantly decreased in both the placenta and primary umbilical vein endothelial cells,while the expression of miR-378a-3p was significantly increased in GDM tissues,and the expression of MAPK1 was decreased in GDM tissues.This ceRNA interaction regulation axiswas proved to affect the glucose uptake function of HUVECs.The transfection of si-HCG27 could significantly reduce the expression of the MAPK1 protein.If the MAPK1 overexpression plasmid was transfected simultaneously with si-HCG27 transfection,the reduced glucose uptake in HUVECs resulting from the decrease in lncRNA HCG27 was reversed.MiR-378a-3p mimic can significantly reduce the mRNA expression of MAPK1 in HUVECs,whereas miR-378a-3p inhibitor can significantly increase the mRNA expression of MAPK1.The inhibition of miR-378a-3p could restore the decreased glucose uptake of HUVECs treated with si-HCG27.Besides,overexpression of lncRNA HCG27 could restore the glucose uptake ability of the palmitic acid-induced insulin resistance model of HUVECs to normal.Conclusion:LncRNA HCG27 promotes glucose uptake of HUVECs by miR-378a-3p/MAPK1 pathway,which may provide potential therapeutic targets for GDM.Besides,the fetal umbilical cord blood and umbilical vein endothelial cells collected from pregnant women with GDM after delivery could be used to detect the presence of adverse molecular markers of metabolic memory,so as to provide guidance for predicting the risk of cardiovascular diseases and health screening of offspring.展开更多
基金supported by grants from the National Natural Science Foundation of China (30771607)
文摘MicroRNAs (miRNAs) are endogenous -22 nt RNAs that play important regulatory roles in targeting mRNAs for cleavage or translational repression. Despite the discovery of increasing numbers of human and mouse miRNAs, little is known about miRNAs from pig. In this study, we sought to extend the repertoire of porcine small regulatory RNAs using Solexa sequencing. We sequenced a library of small RNAs prepared from immortalized swine umbilical vein endothelial cells (SUVECs). We produced over 13.6 million short sequence reads, of which 8 547 658 perfectly mapped to the pig genome. A bioinformatics pipeline was used to identify authentic mature miRNA sequences. We identified 154 porcine miRNA genes, among which 146 were conserved across species, and 8 were pig-specific miRNA genes. The 146 miRNA genes encoded 116 conserved mature miRNAs and 66 miRNA^*. The 8 pig-specific miRNA genes encoded 4 mature miRNAs. Four potential novel miRNAs were identified. The secondary structures of the 154 miRNA genes were predicted; 13 miRNAs have 2 structures, and miR-9 and miR-199 have 4 and 3 structures, respectively. 36 miRNAs were organized into 19 compact clusters, miR-206, miR-21 and miR-378 were the relatively highly expressed miRNAs. In conclusion, Solexa sequencing allowed the successful discovery of known and novel porcine miRNAs with high accuracy and efficiency. Furthermore, our results supply new data to the somewhat insufficient pig miRBase, and are useful for investigating features of the blood-brain barrier, vascular diseases and inflammation.
基金supported by the National Science Foundation of China(No.30271137No.30771831+1 种基金No.81072329)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)
文摘Martentoxin, a 4,046 Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch, has been demonstrated to block large-conductance Ca2+-activated K+ (BKca) channels; however, its biological roles are still largely unknown. In the present study, we investigated the pharmacological effects of martentoxin on regulating the production of nitric oxide induced by TNF-a in human umbilical vein endothelial cells (HU- VECs). We found that, 1, 10 and 100 ~tmol/L martentoxin decreased nitric oxide production by HUVECs ex- posed to 10 ng/mL TNF for 6, 12 and 24 hours. We further demonstrated that martentoxin inhibited the activity of iNOS and retarded the down-regulation of eNOS mRNA induced by TNF-a. Therefore, martentoxin could be a potential therapeutic agent for vascular diseases.
基金supported by agrant from the National Natural Sciences Foundation of China(No.30750010)
文摘By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdMNOR, CdM-CdMHYP and HUVEC-CdMHYP were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdMNOR and HUVEC-CdMHYP groups, the survival rate, migra-tion and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdMHYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.
基金supported by grants from the National Natural Science Foundation of China(No.81101950)Research Project Foundation of Health and Family Planning Commission of Wuhan City(No.WX15C37)
文摘Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells(ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin(ERM) family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells(HUVECs) in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.
基金Supported by Science and Technology Foundation of Zhuhai(No.PB200510142013D0401990017)
文摘AIM:To compare conventional slow equilibrium cooling and directional freezing(DF) by gauze package for cryopreservation of human umbilical vein endothelial cells(HUVECs).METHODS:HUVECs were randomly assigned to conventional freezing(CF) and DF by gauze package group. The two groups of HUVECs were incubated with a freezing liquid consisting of 10% dimethylsulfoxide(DMSO), 60% fetal bovine serum(FBS) and 30%Dulbecco’s modified Eagle’s medium(DMEM) and then put into cryopreserved tubes. CF group, slow equilibrium cooling was performed with the following program:precool in 4℃ for 30 min,-20℃ for 1h, and then immersion in-80℃ refrigerator. DF group, the tubes were packaged with gauze and then directional freezing in-80℃ refrigerator straightly. One month later, the vitality of HUVECs were calculated between two groups.RESULTS:There was no significant difference in the survival rate and growth curve between CF and DF groups. The DF group was significantly better than CFgroup in adherent rates, morphological changes and proliferative ability.CONCLUSION:In the conventional cryopreserved method, cells are slow equilibrium cooling by steps(4℃,-20℃ and finally-80℃), which is a complicated and time-consuming process. But the improved DF by gauze package method is better than conventional method, for which is convenient and easy to operate.
文摘This study investigated the changes in human umbilical vein endothelial cells (HUVECs) induced by overexpression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor Ⅷ-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the prolifera- tive ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS (eNOS) activity was assayed by using spectrophotometry, and NO2-/NO3- levels were measured usin~ nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma mem- brane and cytoplasm, and Western blot analysis confirmed that NOSTR1N levels were significantly higher in cells transfected with the NOSTR1N plasmid (P〈0.01). The activity of eNOS and the levels of NO2-/NO3 were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells (P〈0.01 and P〈0.01, respectively). Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOS- TRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overex- pression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.
基金supported by the National Natural Science Foundation of China(No.81373403)
文摘Objective:To investigate the effects of microRNA-21 antisense nucleotide(AS-miR-21)on the proliferation,migration and autophagy of human umbilical vein endothelial cells(HUVECs).Methods:HUVECs were treated with1,000 nmol/L rapamycin for 6 h(rapamycin group)or ASmiR-21 transfection followed by 1,000 nmol/L rapamycin for6 h(AS-miR-21+rapamycin group).HUVECs without any treatment were defined as control group.The proliferation and migration abilities of HUVECs were detected by methyl thiazolyl tetrazolium(MTT)assay,scratch wound healing assay and transwell test,respectively.The expressions of microtubule-associated protein light chain 3 Ⅱ/Ⅰ(LC3 Ⅱ/Ⅰ)and Becline-1 were determined by western blotting.Results:The rapamycin group showed decreased OD value and migration rate,an increased ratio of LC3 Ⅱ/Ⅰ and up-regulated expression of Beclin-1 compared with the control group(P<0.05).The AS-miR-21+rapamycin group demonstrated lower OD value,migration rate,the number of migrated cells,and significantly higher ratio of LC3 Ⅱ/Ⅰ and Beclin-1 protein expression level than the control group and the rapamycin group(P<0.05).Conclusion:AS-miR-21 suppressed the autophagy,proliferation and migration in the HUVECs model of autophagy induced by rapamycin.
文摘Objectives To investigate the effects and mechanism of glycated serum albumin(GSA) on expression of Monocyte chemoattratant protein-1(MCP-1) in Endothelial Cells. Methods Human Umbilical Vein Endothelial Cells (HUVEC)are cultured with GSA of different concentrations and interfered by glycosylation products inhibitor Aminoguanidine (AG) and anti-oxidant N-acetylcy-steine (NAC), The expression of MCP-1 are evaluated by Immunocytochemistry and Sandwich ELISA. MDA content and SOD activity are determined by the technique of TBA and XOD respectively. Results GSA can stimulate MCP-1 production and secretion. Immunocytochemistry showed that after HUVECs were cultured with 50 mg/L GSA, expression of MCP-1 in group 4hrs, 8hrs and 12hrs was 1.3, 1.9 and 2.8 fold as much as that in control group (P < 0.01), and there was significant difference among the experiment groups(P < 0.01). Sandwich ELISA showed that expression of MCP-1 in three different groups was 1.6, 2.4 and 3.0 fold as much as that in control group(P < 0.01), and there was significant difference among the experiment groups(P < 0.01); GSA can cause the decrease of SOD activity(P < 0.05) and increase of MDA content(P < 0.01); AG and NAC can restrain obviously the expression of MCP-1 of HUVECs stimulated by GSA(P < 0.01); NAC can restrain the effect of GSA on SOD activity and MDA content in HUVECs (P < 0.05). Conclusions GSA can stimulate the expression of MCP-1 of endothelial cells by inducing endothelial cells oxidative stress.
文摘Vascular endothelial growth factors(VEGFs) respectively bind to each of three receptor tyrosine kinases(RTKs),known as Flt-1,KDR and Flt-4.Since VEGFs and their respective families of receptor tyrosine kinases(VEGFRs) are critical proteins which can regulate vascular development during angiogenesis,we decided to explore the inhibitory effects of soluble kinase insert domain-containing receptor(sKDR) on endothelial cells and angiogenesis.Total RNA was extracted from human umbilical vein endothelial cells(HUVEC),and cDNA of extracellular domains 1―4 was amplified and recombined with pQE40 vector.After being expressed,affinity purified,renatured and analyzed by Western blot,the sKDR was assayed for its effects on endothelial cells by [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide](MTT),and on angiogenesis by chick chorioallantoic membrane(CAM) experiment.sKDR cDNA of 1150 bp was obtained via real-time polymerase chain reaction(RT-PCR),and sKDR was expressed by pQE40 procaryotic expression system,purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis with only one band and proved by Western blot.MTT assay demonstrateds that sKDR could inhibit the VEGF-stimulated HUVEC from proliferation,and CAM experiment showed sKDR could block the VEGF-induced angiogenesis.sKDR has the biological activity to bind with VEGF ligands and is a potential target for tumor anti-angiogenesis therapy.
文摘Salidroside is extensively used as a herbal medicine worldwide, and it has been shown to protect against disruption of endothelial homeostasis and act as an anti-aging agent. The present study aimed to investigate the ameliorative effects of salidroside on homocysteine (Hcy)-induced cell senescence in human umbilical vein endothelial cells (HUVECs) that were mediated via inhibition of Krüppel-like factor 4 (KLF4). An endothelial cell senescence model was induced by Hcy. The cell viability, activities of telomerase and lactate dehydrogenase (LDH), and the level of reactive oxygen species were determined using commercial kits. The expression levels of KLF4, p53 and p21 were determined via western blot analysis, whereas the mRNA expression levels of KLF4 were detected by reverse transcription-quantitative PCR. Small interfering RNA-mediated knockdown of KLF4 was found to reverse Hcy-induced cell senescence. Hcy treatment led to an accelerated cell senescence, as evidenced by decreases in both cell viability and telomerase activity, whereas increases were noted in the leakage of LDH and the level of reactive oxygen species, in addition to an up-regulation of the protein levels of p53 and p21, and up-regulation of KLF4 at both the mRNA and protein level. Treatment with salidroside ameliorated Hcy-induced cell senescence in a dose-dependent manner. Taken together, these results suggested that Hcy may induce cell senescence through upregulation of KLF4, and this may be reversed by treatment with salidroside. Therefore, salidroside was shown to inhibit Hcy-induced cell senescence through KLF4 inhibition.
基金Supported by the Project Plan of Science and Technology Assistance in Xinjiang Autonomous Region(No.201491171)
文摘AIM: To explore the possibility of human umbilical cord mesenchymal stem cells(h UCMSCs), human umbilical vein endothelial cells(h UVECs), human dental pulp stem cells(h DPSCs) and human periodontal ligament stem cells(h PDLSCs) serving as feeder cells in co-culture systems for the cultivation of limbal stem cells.METHODS: Different feeder layers were cultured in Dulbecco's modified Eagle's medium(DMEM)/F12 and were treated with mitomycin C. Rabbits limbal stem cells(LSCs) were co-cultured on h UCMSCs, h UVECs, h DPSCs, h PDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency(CFE) assay and immunofluorescence(IPO13,CK3/12).RESULTS: The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But h UCMSCs, h DPSCs and h PDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to h UVECs and feedercell-free culture.CONCLUSION: hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.
文摘Background Parthenolide has been tested for anti-tumor activities, such as anti-proliferation and pro-apoptosis in recent studies. However, little is known about its role in the process of tumor angiogenesis. This study aims to investigate the effects and potential mechanisms of parthenolide on the proliferation, migration and lumen formation capacity of human umbilical vein endothelial cells. Methods Different concentrations of parthenolide were applied to the human breast cancer cell line MDA-MB-231 cells. After 24-hour incubation, the culture supernatants were harvested and used to treat human umbilical vein endothelial cells for 24 hours. Then an inverted fluorescence phase contrast microscope was used to evaluate the human umbilical vein endothelial cells. The secretion of vascular endothelial growth factor (VEGF), interleukin (IL)-8 and matrix metalloproteinases (MMP)-9 in the culture supernatant of the MDA-MB-231 cells was then measured with enzyme-linked immunosorbent assay (ELISA) assays. Results Suppression of proliferation, migration, and the lumen formation capacity of human umbilical vein endothelial cells was observed in the presence of the culture supernatants from the breast cancer cell line treated with different concentrations of parthenolide. Parthenolide decreased the levels of the angiogenic factors MMP-9, VEGF, and IL-8 secreted by the MDA-MB-231 cells. Conclusions Parthenolide may suppress angiogenesis through decreasing angiogenic factors secreted by breast cancer cells to interfere with the proliferation, migration and lumen-like structure formation of endothelial cells, thereby inhibiting tumor growth. It is a promising potential anti-angiogenic drug.
基金Project (Nos UKM-FF-03-FRGS0005-2007 and FF-138-2007) supported by the Ministry of Higher Education and Universiti Kebangsaan Malaysia
文摘Endothelial cell death due to increased reactive oxygen species(ROS) may contribute to the initial endothelial injury,which promotes atherosclerotic lesion formation.Piper sarmentosum(PS),a natural product,has been shown to have an antioxidant property,which is hypothesized to inhibit production of ROS and prevent cell injury.Thus,the present study was designed to determine the effects of PS on the hydrogen peroxide(H2O2)-induced oxidative cell damage in cultured human umbilical vein endothelial cells(HUVECs).In this experiment,HUVECs were obtained by collagenase perfusion of the large vein in the umbilical cord and cultured in medium M200 supplemented with low serum growth supplementation(LSGS).HUVECs were treated with various concentrations of H2O2(0-1000 μmol/L) and it was observed that 180 μmol/L H2O2 reduced cell viability by 50% as denoted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay.Using the above concentration as the positive control,the H2O2-induced HUVECs were concomitantly treated with various concentrations(100,150,250 and 300 μg/ml) of three different extracts(aqueous,methanol and hexane) of PS.Malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT) and glutathione peroxidase(GPX) levels showed a significant increase(P<0.05) in HUVECs compared to the negative control.However,PS extracts showed a protective effect on HUVECs from H2O2-induced cell apoptosis with a significant reduction in MDA,SOD,CAT and GPX levels(P<0.05).Furthermore,PS had exhibited ferric reducing antioxidant power with its high phenolic content.Hence,it was concluded that PS plays a beneficial role in reducing oxidative stress in H2O2-induced HUVECs.
基金Supported by the National Natural Science Foundation of China(No.81370447)Science and Technology Planning Project of Guangdong Province,China(No.2016A050502014)the Ph.D.Start-up Fund of Natural Science Foundation of Guangdong Province,China(No.2015A030310048,and 2016A030310203)。
文摘Objective:To investigate whether ginsenoside Rb1(Rb1)can protect human umbilical vein endothelial cells(HUVECs)against high glucose-induced apoptosis and examine the underlying mechanism.Methods:HUVECs were divided into 5 groups:control group(5.5 mmol/L glucose),high glucose(HG,40 mmol/L)treatment group,Rb1(50μmol/L)treatment group,Rb1 plus HG treatment group,and Rb1 and 3-(1 H-1,2,3-triazol-4-yl)pyridine(3-TYP,16μmol/L)plus HG treatment group.Cell viability was evaluated by cell counting kit-8 assay.Mitochondrial and intracellular reactive oxygen species were detected by Mito Sox Red mitochondrial superoxide indicator and dichloro-dihydro-fluorescein diacetate assay,respectively.Annexin V/propidium iodide staining and fluorescent dye staining were used to measure the apoptosis and the mitochondrial membrane potential of HUVECs,respectively.The protein expressions of apoptosis-related proteins[Bcl-2,Bax,cleaved caspase-3 and cytochrome c(Cyt-c)],mitochondrial biogenesis-related proteins[proliferator-activated receptor gamma coactivator 1-alpha,nuclear respiratory factor-1 and mitochondrial transcription factor A],acetylation levels of forkhead box O3 a and SOD2,and sirtuin-3(SIRT3)signalling pathway were measured by immunoblotting and immunoprecipitation.Results:Rb1 ameliorated survival in cells in which apoptosis was induced by high glucose(P<0.05 or P<0.01).Upon the addition of Rb1,mitochondrial and intracellular reactive oxygen species generation and malondialdehyde levels were decreased(P<0.01),while the activities of antioxidant enzymes were increased(P<0.05 or P<0.01).Rb1 preserved the mitochondrial membrane potential and reduced the release of Cyt-c from the mitochondria into the cytosol(P<0.01).In addition,Rb1 upregulated mitochondrial biogenesis-associated proteins(P<0.01).Notably,the cytoprotective effects of Rb1 were correlated with SIRT3 signalling pathway activation(P<0.01).The effect of Rb1 against high glucose-induced mitochondria-related apoptosis was restrained by 3-TYP(P<0.05 or P<0.01).Conclusion:Rb1 could protect HUVECs from high glucose-induced apoptosis by promoting mitochondrial function and suppressing oxidative stress through the SIRT3 signalling pathway.
基金Supported by National Natural Science Foundation of China(No.81170270)Medicine and Technology Program of Zhejiang Province(No.2013KYB188)
文摘Objective:To investigate the anti-angiogenic effect of cryptotanshinone(CPT) on human umbilical vein endothelial cells(HUVECs) and the effect of CPT on Wnt/β-catenin signaling pathway.Methods:HUVECs were incubated with 0,2.5,5,10,and 20 μmol/L CPT for detecting cell viability with dimethyl thiazolyl-2,5-diphenyltetrazolium bromide(MTT) assay.Then,HUVECs were incubated with 0,2.5,5,and 10 μmol/L CPT for detecting endothelial cell migration,invasion,and tubular-like structure formation with wound healing,transwell invasion and matrigel tube formation assays,respectively.To gain insight into CPT-mediated signaling,the effects of CPT on T-cell factor/lymphocyte enhancer factor(TCF/LEF) transcription factors were detected by the Dual-luciferase reporter assay.Next,the nuclear expression of p-catenin was evaluated using Western blot and immunochemistry.Finally,vascular endothelial growth factor(VEGF) and cyclin D1,downstream proteins of the Wnt pathway were examined with Western blot.Results:CPT dose-dependently suppressed endothelial cell viability,migration,invasion,and tubular-like structure formation.In particular,CPT blocked β-catenindependent transcription in HUVECs in a dose-dependent manner.In Western bolt,10 μ mol/L CPT decreased expression of β-catenin in nucleus of HUVECs(P〈0.01).In immunohistochemistry,β-catenin was more potent in response to LiCI(an activator of the pathway) treatment.However,the signals were weaker in the nucleus of the CPT(10 μmol/L) group,compared to the positive control.Also,VEGF and cyclin D1 were both eliminated by CPT in 5 and 10 μ mol/L doses(P〈0.05).Conclosion:Our study supported the role of CPT as an angiogenic inhibitor,which may impact on the Wnt/β-catenin signaling pathway.
基金Supported by grants from the Overseas and Hong Kong,Macao Young Scholars Collaborative Research Fund by the National Natural Science Foundation of China(No.81328025)the Science and Technology Development Fund of Macao SAR(Ref.No.014/2011/A1),Research Committee,University of Macao
文摘Objective: This study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish. Methods: The in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rgl and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit lI (XTI') assay. R1, Rgl and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigel- coated wells was performed to evaluate the pro-angiogenic actions of RI. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-N (o -nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor ]1 (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels. Results: R1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/FIk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemically- induced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs. Conclusion: R1, similar to Rgl and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/FIk-1 and PI3K- Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.
基金Supported by the Science and Technology Development Fund of Macao SAR(No.078/2011/A3 and 134/2014/A3)Research Committee of University of Macao(No.MYRG2015-00182-ICMS-QRCM)Beijing Municipal Science and Technology Commission(No.Z141100002214011),China
文摘Objective: To investigate the pro-angiogenic effects of paeoniflorin(PF) in a vascular insufficiency model of zebrafish and in human umbilical vein endothelial cells(HUVECs). Methods: In vivo, the pro-angiogenic effects of PF were tested in a vascular insufficiency model in the Tg(fli-1:EGFP)y1 transgenic zebrafish. The 24 h post fertilization(hpf) embryos were pretreated with vascular endothelial growth factor(VEGF) receptor tyrosine kinase inhibitor Ⅱ(VRI) for 3 h to establish the vascular insufficiency model and then post-treated with PF for 24 h. The formation of intersegmental vessels(ISVs) was observed with a fluorescence microscope. The m RNA expression of fms-like tyrosine kinase-1(flt-1), kinase insert domain receptor(kdr), kinase insert domain receptor like(kdrl) and von Willebrand factor(v WF) were analyzed by real-time polymerase chain reaction(PCR). In vitro, the pro-angiogenic effects of PF were observed in HUVECs in which cell proliferation, migration and tube formation were assessed. Results: PF(6.25–100 μmol/L) could rescue VRI-induced blood vessel loss in zebrafish and PF(25–100 μmol/L), thereby restoring the m RNA expressions of flt-1, kdr, kdrl and v WF, which were down-regulated by VRI treatment. In addition, PF(0.001–0.03 μmol/L) could promote the proliferation of HUVECs while PF stimulated HUVECs migration at 1.0–10 μmol/L and tube formation at 0.3 μmol/L. Conclusion: PF could promote angiogenesis in a vascular insufficiency model of zebrafish in vivo and in HUVECs in vitro.
基金supported by grants from the National Key R&D Program of China(No.2018YFC1005002)the National Natural Science Foundation of China(Nos.82070482,81772007,21734003 and 51927805)+1 种基金the Shanghai Municipal Science and Technology Major Project(No.2017SHZDZX01)the Shanghai Municipal Education Commission(Innovation Program No.2017-01-07-00-07E00027)。
文摘There is no clear consensus regarding how cells respond to hydrostatic pressure. This is largely attributable to the high heterogeneity among cell types and the diverse custom-made devices used in previous studies. The aim of this work was to develop a facile device that could mimic various pressure environments and then delineate the cellular response to pressure stimulus. The device described here achieved both stable and periodic pressurization without oxygen deprivation. The biological utility of the device was assessed using human umbilical vein endothelial cells. We found more stereoscopic nuclear morphology and re-distribution of lamin A/C under high hydrostatic pressure compared to control cells. Mass spectrometry-based proteomics analysis showed significant changes in mitochondria-related pathways. Western blot analysis confirmed that high hydrostatic pressure induced a tendency toward mitochondrial fusion. Increased mitochondrial activity was observed as well. In conclusion, this device can be readily applied in biological research and extend our understanding of cellular mechano-sensation and the associated changes in mitochondrial behaviors.
基金This study was supported by the grants from the Jiangsu Technologic Foundation (No. BJ98324).
文摘Objective To determine whether advanced glycosylation end products modified bovine serum albumin (AGEs-BSA) affects endothelial cell lateral junction protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the presence or absence of inflammatory mediators.Methods Cultured human umbilical vein endothelial cells (HUVECs) were exposed to AGEs-BSA for 6, 12, 24, and 36 hours, and exposed to AGEs-BSA glycosylated with different concentrations of glucose, tumor necrosis factord-α (TNF-α), interferon (IFN-γ), TNF-α + IFN-y and AGEs-BSA + TNF-α for 24 hours, respectively. Expression of PECAM-1 mRNA was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) with β-actin as an internal standard, and sequencing of RT-PCR products was performed to confirm the specificity of amplification for PECAM-1 gene. The endothelial cell surface expression of PECAM-1 was determined by flow cytometry (FCM).Results There were no significant changes in the expression of PECAM-1 mRNA and protein when the cells were exposed to AGEs-BSA with different concentrations or periods ( P>0. 05). However, PECAM-1 expression was reduced in the cells treated with TNF-α, IFN-y, TNF-α + IFN-γ and AGEs-BSA + TNF-α. The level of PECAM-1 treated with AGEs-BSA + TNF-α was lower than that of TNF-α treated alone (P<0. 01).Conclusions AGEs-BSA had no effect on the expression of PECAM-1 mRNA and protein in cultured HUVEC. With the presence of inflammatory mediator TNF-α, AGEs-BSA decreased the level of PECAM-1, which might reduce the adhesion interaction between adjacent endothelial cells, enhance the permeability of endothelial cells, and might be implicated in the endothelial dysfunction and pathogenesis of atherosclerosis in patients with diabetes mellitus. The significance of this phenomenon in intracellular signal transduction remains to be determined.
基金supported by the National Key Research and Development Program of China(No.2021YFC2701502)the Health Commission of Scientific Research Project of Hubei Province(No.WJ2021M129).
文摘Objective:Gestational diabetes mellitus(GDM)is the most common metabolic disorder during pregnancy.LncRNA HLA complex group 27(HCG27)plays a crucial role in various metabolic diseases.However,the relationship between lncRNA HCG27 and GDM is not clear.This study aimed to verify a competing endogenous RNA(ceRNA)interaction regulation axis of miR-378a-3p/mitogen-activated protein kinase 1(MAPK1)regulated by HCG27 in GDM.Methods:LncRNA HCG27 and miR-378a-3p were detected by RT-qPCR.The expression of MAPK1 in umbilical vein endothelial cells(HUVECs)was detected by RT-qPCR and that in the placenta by Western blotting.To explore the relationship among lncRNA HCG27,miR-378a-3p,MAPK1 and the glucose uptake ability of HUVECs,vector HCG27,si-HCG27,miR-378a-3p mimic and inhibitor were transfected to achieve overexpression and inhibition of HCG27 or miR-378a-3p.The interaction between miR-378a-3p and lncRNA HCG27 or MAPK1 was confirmed by the dual-luciferase reporter assay.Besides,glucose consumption by HUVECs was detected by the glucose assay kit.Results:HCG27 expression was significantly decreased in both the placenta and primary umbilical vein endothelial cells,while the expression of miR-378a-3p was significantly increased in GDM tissues,and the expression of MAPK1 was decreased in GDM tissues.This ceRNA interaction regulation axiswas proved to affect the glucose uptake function of HUVECs.The transfection of si-HCG27 could significantly reduce the expression of the MAPK1 protein.If the MAPK1 overexpression plasmid was transfected simultaneously with si-HCG27 transfection,the reduced glucose uptake in HUVECs resulting from the decrease in lncRNA HCG27 was reversed.MiR-378a-3p mimic can significantly reduce the mRNA expression of MAPK1 in HUVECs,whereas miR-378a-3p inhibitor can significantly increase the mRNA expression of MAPK1.The inhibition of miR-378a-3p could restore the decreased glucose uptake of HUVECs treated with si-HCG27.Besides,overexpression of lncRNA HCG27 could restore the glucose uptake ability of the palmitic acid-induced insulin resistance model of HUVECs to normal.Conclusion:LncRNA HCG27 promotes glucose uptake of HUVECs by miR-378a-3p/MAPK1 pathway,which may provide potential therapeutic targets for GDM.Besides,the fetal umbilical cord blood and umbilical vein endothelial cells collected from pregnant women with GDM after delivery could be used to detect the presence of adverse molecular markers of metabolic memory,so as to provide guidance for predicting the risk of cardiovascular diseases and health screening of offspring.