Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells

AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript Ⅱ KDR-TK plasmid by enzymatic digestion with Xho I and Sal I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble, pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP) expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to theprodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively. CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs. Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.

Protective effects of icariin on human umbilical vein endothelial cell injured by angiotensin Ⅱ

To investigate the effects of icariin （ICA） on angiotensin Ⅱ（Ang Ⅱ）-induced injury in human umbilical vein endothelial cells line （ECV-304）. The ECV-304 cells were cultured in vitro. After 24 h incubating with icariin, the model of AngⅡ-induced injury in ECV-304 was established. The cell viability （MTT method）, Lactate dehydrogenase （LDH） release and Nitric oxide （NO） production in the medium, the capacity of scavenging superoxide anion radicals （O2^-） and hydroxyl radicals （.OH） were measured. The activities of superoxide dismutase （SOD）, total nitric oxide synthase （T-NOS）, inducible nitric oxide synthase （iNOS） and constitutive nitric oxide synthase （cNOS） in the cells were determined. Compared with the Ang Ⅱ-treated group, ICA can significantly raise the viability of EC, increase the activities of SOD, T-NOS and cNOS, increase the production of NO, enhance the capacity of scavenging superoxide anion radicals （ O2^- ） and hydroxyl radicals（.OH）, and lower LDH leakage and iNOS activity. The results suggest that ICA can protect endothelial cells （ECV-304） from Ang II-induced injury.

Rhizoma Chuanxiong regulates vascular endothelial growth factor production in hypoxic human umbilical vein endothelial cells in vitro and in peri-infarct rat brain tissue

BACKGROUND： Vascular endothelial growth factor （VEGF） acts as ＂molecular bridge＂ following ischemic stroke to improve and restore blood supply and reduce infarction volume. Clinical studies have demonstrated the efficacy of Rhizoma Chuanxiong （Chuanxiong） in the treatment of ischemic cerebrovascular diseases. However, whether it promotes endogenous VEGF expression in ischemic stroke remains unknown. OBJECTIVE： To investigate the influence of Rhizoma Chuanxiong on VEGF production in vitro cultured human umbilical vein endothelial cells and on VEGF expression in ischemic cerebral tissues to explore its role in angiogenesis. DESIGN, TIME AND SETTING： In vitro basic comparison of traditional Chinese drug-containing serum pharmacology; in vivo randomized, controlled, animal experiment. This study was performed at the Medical Laboratory of West China Hospital, Sichuan University between December 2002 and April 2004. MATERIALS： Two Chinese rabbits were selected. One was intragastrically perfused with 5.8 g/kg Rhizoma Chuanxiong extract twice per day for three consecutive days to prepare Rhizoma Chuanxiong extract-containing serum. The remaining rabbit was intragastrically perfused with the same volume of normal saline twice per day for three consecutive days. Rhizoma Chuanxiong extract was provided by Beijing Traditional Chinese Medicine Research Institute, predominantly composed of ligustrazine, ligustilide, and ferulic acid. ChemiKineTM human VEGF Kit was purchased from Chemicon, USA; mouse anti-VEGF monoclonal antibody and biotin-goat anti-mouse IgG were purchased from Santa Cruz Biotechnology. Inc., USA. METHODS： （1） In vitro experiment： in vitro cultured human umbilical vein endothelial cells were separately incubated in rabbit serum with 10% Rhizoma Chuanxiong extract, normal medium without rabbit serum, and rabbit serum without Rhizoma Chuanxiong extract （blank control）. In addition, cells from the three groups were incubated under normoxia （5% CO2, 95% air） and hypoxia （1% 02, 5% CO2, 94% N2）, respectively, for 24 hours. （2） In vivo experiment： a total of 4/44 Sprague Dawley rats were selected for the sham-operated group （no occlusion）, and the remaining rats were used to establish a cerebral ischemiaJreperfusion model by suture occlusion. 32 animals with ischemia/reperfusion injury were randomly divided into treatment and model groups, with 16 rats in each group. Both groups were intraperitoneally infused with 0.58 g/kg Rhizoma Chuanxiong extract and normal saline two hours following reperfusion. The sham-operated group was administrated normal saline. Animals were treated with saline or Chuanxiong extracts （0.58 g/kg） twice per day for three consecutive days. MAIN OUTCOME MEASURES： In vitro experiment： VEGF concentration was detected in each group by enzyme-linked immunosorbent assay. In vivo experiment： behavioral alterations of rats were evaluated by neurological function scale; infarct volume was assessed by hematoxylin-eosin staining; VEGF protein expression in the infarct regions was determined by immunohistochemistry. RESULTS： （1） VEGF levels were similar between the three groups under normexic condition （P 〉 0.05）; while hypoxia induced VEGF production （P 〈 0.01 ）. VEGF levels in the drug-containing serum group were particularly higher compared with the other groups （P 〈 0.01）. （2） Compared with normal saline treatment, Rhizoma Chuanxiong extract significantly improved the neurological scale and reduced cerebral infarct volumes （P〈 0.05）. The percent of VEGF-positive cells was significantly greater than the model group （P 〈 0.05）. The sham-operated group exhibited normal neurological function, with no infarct focus. CONCLUSION： Rhizoma Chuanxiong extract-containing rabbit serum effectively promoted cultured VEGF production under hypoxia. Rhizoma Chuanxiong extract upregulated VEGF expression in the infarct region, improved neurological function, and reduced infarct size.

Inhibition of viability of human retinal microvascular endothelial cells by vialinin A under high glucose condition

AIM:To investigate the effects of vialinin A on viability of human retinal endothelial cells(HRECs)under high glucose condition and its potential mechanism.METHODS:The HRECs were divided into four groups:normal glucose control group(NG,5 mmol/L D-glucose),high glucose group(HG,30 mmol/L D-glucose),HG+1μmol/L vialinin A group,and HG+5μmol/L vialinin A group.The cell viabilities were measured with cell counting kit-8(CCK-8)assay for proliferation,with scratch assay for migration,and tube formation,for evaluation of the impact of vialinin A on cellular behaviour.Real-time PCR and Western blotting were used to determine the expression level of vascular endothelial growth factor(VEGF).RESULTS:The proliferative capacity and migration of HRECs was reduced by 5μmol/L vialinin A in high glucose environment(both P<0.05).Vialinin A also inhibited highglucose-induced tube formation of HRECs.The expression level of VEGF and PI3K in HRECs was also significantly decreased by vialinin A(P<0.05).CONCLUSION:Vialinin A inhibits the cell viability of HRECs.It may serve as a potential target for anti-angiogenic therapy.

Martentoxin, a large-conductance Ca^(2+)-activated K^+ channel inhibitor, attenuated TNF-α-induced nitric oxide release by human umbilical vein endothelial cells

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.

The effects of microRNA-34a regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide-induced human umbilical vein endothelial cells

BACKGROUND: Notch-1/NF-κB signaling plays a key role in the cecal ligation and puncture(CLP)-induced sepsis. This study aims to investigate the intervention effects of microRNA-34a(miR-34a) lentivirus regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide(LPS)-induced human umbilical vein endothelial cells(HUVEC).METHODS: HUVEC were divided into four groups as the following: they were infected with negative control lentivirus(NC group) or miR-34a lentivirus(OE group); LPS(1 g/mL) was added on the third day on the basis of NC group and OE group for 24 hours(NC+LPS group or OE+LPS group). The levels of TNF-α, IL-1β, IL-6, and IL-10 in the cell supernatants, and the mRNA and protein expression of Notch-1 and NF-κB in the HUVEC were evaluated.RESULTS: After 24 hours, the levels of TNF-α, IL-1β, IL-6 in the cell supernatants and the protein expression of NF-κB from NC+LPS group were significantly higher than those of NC group, but IL-10 level and the protein expression of Notch-1 in NC+LPS group were the opposite. After intervention of miR-34a lentivirus, the cell supernatants TNF-α and the protein expression of NF-κB in OE+LPS group after 24 hours markedly decreased compared to NC+LPS group. While the cell supernatants IL-1β and IL-6 and the mRNA expression of NF-κB slightly decreased in OE+LPS group, IL-10 and the mRNA and protein expression of Notch-1 were the opposite.CONCLUSION: miR-34a regulating Notch-1/NF-κB signaling pathway can reduce the HUVEC damage caused by LPS stimulation.

Damaging Effect of Cigarette Smoke Extract on PrimaryCultured Human Umbilical Vein Endothelial Cells and Its Mechanism

Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10-4 mol/L) on HUVEC viability, proliferation, angiogenesis and apoptosis were observed. Results CSE decreased HUVEC survival rate and angiogenesis after 24 h as well as its proliferation after 48 h in a dose-dependent manner. Moreover, CSE induced apoptosis of HUVEC as indicated in condensation of nuclear chromatin and the presence of hypodiploid DNA. HUVEC incubated with CSE for 24 h gave a significant decrease in the expression of Bcl-2 as well as the decline in the Bcl-2/Bax ratio accompanied with the loss of mitochondrial membrane potential and excess cytosolic calcium. Our study also observed that p53 protein level decreased, rather than increased in cells treated with CSE. Nicotine had no discernible inhibitory effects on the above indices of HUVEC. Conclusion Exposure to CSE other than nicotine causes inhibition of viability, proliferation and differentiation of HUVEC. CSE-induced HUVEC injury is mediated in part through accelerated apoptosis but independent of p53 pathway. It appears that mitochondria have played a key role in the apoptosis of HUVEC induced by CSE.

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

AIM： To study the selective killing of human umbilical vein endothelial cells （HUVECs） by a double suicide gene under the regulation of a kinase domain insert containing receptor （KDR） promoter and mediated by an adenoviral gene vector. METHODS： Human KDR promoter was cloned by polymerase chain reaction （PCR）, and two recombinant adenoviral plasmids pAdKDR-CdgIyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses （AdKDR-CDglyTK and AdCMV-CDglyTK） respectively, and the infection rates were estimated by detection of green fluorescent protein （GFP） expression. Infected cells were cultured in culture media containing different concentrations of 5-fiuoroo/tosine （5-FC） and ganciclovir （GCV）, and the killing effects were measured. RESULTS： The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs： HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVo CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK （P 〈 0.001）. CONCLUSION： Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Knockdown of Ezrin Suppresses the Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells In Vitro

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.

Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells

AIM： To study the expression of endothelial and inducible nitric oxide synthases （eNOS and iNOS） and their role in inflammatory bowel disease （IBD）. METHODS： We examined the effect of sera obtained from patients with active Crohn＇s disease （CD） and ulcerative colitis （UC） on the function and viability of human umbilical vein endothelial cells （HUVEC）. HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10^-4 mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively. RESULTS： In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control （18%±16% vs 23%± 15% P〈0.01）. UC or CD serum caused a significant increase in iNOS compared to control （UC： 300%±21%; CD： 275% ± 27% vs 108% ± 14%, P〈0.01）. Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum. CONCLUSION： IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced antiinflammatory protection of endothelial cells.

Up-regulation interleukin-6 and interleukin-8 by activated protein C in lipopolysaccharide-treated human umbilical vein endothelial cells

Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.

Effects of Fumonisin B1 on Biomechanics and Cytoskeleton of Human Umbilical Vein Endothelial Cells

Objective Fumonisin B1(FB1)is an important mycotoxin in nature worldwide.The biomechanical properties of cells are closely related to their structure and function,and the cytoskeleton is the structural and functional basis of cells motility,and therefore,from a biomechanical point of view,the purpose of this study is to investigate the effects of FB1 on the biomechanical properties,migration capacity and cytoskeletal structure of human umbilical vein endothelial cells(HUVECs),which may lay an experimental foundation for further exploration of the toxicity mechanism of fumonisin.Methods HUVECs were cultured and treated with different concentrations of FB1.Then,CCK-8 kit was used to detect the effect of FB1 on the survival rate.The osmotic fragility of the cells was measured after treatment with different osmotic pressures for30 min.The cell membrane fluidity was measured by fluorescence polarization method.The cell electrophoretic mobility was measured by cell electrophoretic apparatus.The migration capacity of the cells was observed by scratch repair assay.The changes of reactive oxygen species and cytoskeletal structure were observed by confocal laser scanning microscopy.Finally,the mRNA and protein relative expression levels of cytoskeletal binding proteins were detected by real-time PCR,Western blotting and confocal laser scanning.Results The results of CCK-8 showed that FB1 could significantly inhibit the proliferation of HUVECs in a dose-and time-dependent manner.After treatment of HUVECs with FB1,the hypotonic resistance of the cell,cell surface charge,cell membrane fluidity and migration capacity were all weakened,while reactive oxygen species were significantly increased and the cytoskeletal structure was significantly reorganized.Furthermore,RTPCR results showed that the mRNA relative expression levels of cytoskeletal binding proteins,exception of actin,were down-regulated after treated with FB1.Besides,Western blotting and statistical analysis based on fluorescence intensity of laser confocal microscopy confirmed theses changes in protein level.Conclusions FB1 can significantly affect the biomechanical properties and motility of HUVECs,which may be directly correlated to the remodel of F-actin cytoskeleton,as well as the relative expression changes of cytoskeletal binding proteins.It is significant for further exploring the toxicity mechanism of fumonisin.

Comparison of conventional and directional freezing for the cryopreservation of human umbilical vein endothelial cells

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.

Changes in Human Umbilical Vein Endothelial Cells Induced by Endothelial Nitric Oxide Synthase Traffic Inducer

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.

Effects of miR-21 antisense oligonucleotides on proliferation,migration and autophagy of human umbilical vein endothelial cells

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.

The Effects and Mechanism of GSA on Expression of MCP-1 in Cultured Human Umbilical Vein Endothelial Cells

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.

Human umbilical cord mesenchymal stem cells derivedexosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity

AIM:To observe the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)secretions on the relevant factors in mouse retinal astrocytes,and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A(VEGF-A)and to observe the therapeutic effect on the mouse model of retinopathy of prematurity(ROP).METHODS:Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group.MTT assay,flow cytometry,reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to detect related indicators.Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored.At last,the efficacy of exosomes of UCMSCs in a mouse ROP model was explored.Graphpad6 was used to comprehensively process data information.RESULTS:The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation.Reactive oxygen species(ROS)and hypoxia inducible factor-1α(HIF-1α)of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased,and the ROP cell model was established after 6h of hypoxia.The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α,the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent.Compared with the ROP cell model group,the expression of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signal pathway related factors in the hUCMSCs exocrine group is significantly decreased.The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1αin ROP model tissues.HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.CONCLUSION:In a hypoxia induced mouse retinal astrocyte model,hUCMSCs exosomes are found to effectively reduce the expression of HIF-1αand VEGF-A,which are positively correlated with the concentration of hUCMSCs exosomes.HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1αand VEGF-A proteins in ROP mice,and are positively correlated with drug dosage.Besides,they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

A MicroRNA Catalog of Swine Umbilical Vein Endothelial Cells Identified by Deep Sequencing

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.

Co-culture of Mesenchymal Stem Cells with Umbilical Vein Endothelial Cells under Hypoxic Condition

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.