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.

Down-regulation of histone deacetylase 7 reduces biological activities of retinal microvascular endothelial cells under high glucose condition and related mechanism

AIM:To investigate the expression and effect of histone deacetylase 7(HDAC7)in human retinal microvascular endothelial cells(HRMECs)under high glucose condition and related mechanism,and the expression of HDAC7 in the retinal tissue in diabetic rats.METHODS:The expression of HDAC7 in HRMECs under high glucose and the retinal tissue from normal or diabetic rats were detected with immunohistochemistry and Western blot.LV-shHDAC7 HRMECs were used to study the effect of HDAC7 on cell activities.Cell count kit-8(CCK-8),5-ethynyl2’-deoxyuridine(EdU),flow cytometry,scratch test,Transwell test and tube formation assay were used to examine the ability of cell proliferation,migration,and angiogenesis.Finally,a preliminary exploration of its mechanism was performed by Western blot.RESULTS:The expression of HDAC7 was both upregulated in retinal tissues of diabetic rats and high glucosetreated HRMECs.Down-regulation of HDAC7 expression significantly reduced the ability of proliferation,migration,and tube formation,and reversed the high glucose-induced high expression of CDK1/Cyclin B1 and vascular endothelial growth factor in high glucose-treated HRMECs.CONCLUSION:High glucose can up-regulate the expression of HDAC7 in HRMECs.Down-regulation of HDAC7 can inhibit HRMECs activities.HDAC7 is proposed to be involved in pathogenesis of diabetic retinopathy and a therapeutic target.

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.

Effect of luteolin on apoptosis and vascular endothelial growth factor in human choroidal melanoma cells

AIM:To investigate the effects of luteolin on apoptosis,the cell cycle,and the expression and secretion of vascular endothelial growth factor(VEGF)in human choroidal melanoma cells(C918 and OCM-1).METHODS:C918 and OCM-1 cells cultured in vitro were treated with various concentrations of luteolin(0,5,10,15μmol/L).Cell growth was observed with an inverted microscope,and cell cycle arrest was detected by propidium iodide(PI)staining using flow cytometry.Apoptosis was detected by Hoechst33342 staining,and apoptosis rate was determined by Annexin V-FITC/PI experiments using flow cytometry.The expression of apoptosis-related proteins Bcl-2,Bax and VEGF was analyzed using Western blots.The levels of VEGF secreted by the cells into the supernatant was analyzed using ELISA.RESULTS:After treating with 5 to 15μmol/L luteolin for 48 h,the fusion degree of C918 and OCM-1 cells decreased,and more floating apoptotic cells appeared.Luteolin treatment increased the G0-G1 phase ratio of the C918 and OCM-1 cells,blocked cell cycle progression,and increased the apoptosis rate of the C918 and OCM-1 cells.Western blot showed that luteolin decreased the expression of Bcl-2 and VEGF in the C918 and OCM-1 cells and increased the expression of Bax protein.The ELISA results showed that 10 to 15μmol/L luteolin decreased the cell secretion of VEGF.CONCLUSION:Luteolin may induce apoptosis by regulating the levels of apoptosis-related proteins in C918 and OCM-1 cells.Luteolin can induce cell cycle arrest,decrease the expression of VEGF.

New categorization of human vascular endothelial cells by pro-vs anti-proliferative phenotypes

AIM: To integrally understand the effects of human vascular endothelial cells(VECs) on the proliferation of vascular smooth muscle cells(VSMCs).METHODS: Various kinds of human VECs of different origins were co-cultured with human aortic smooth muscle cells, a representative of human VSMCs. To exclude the irrelevant effects due to growth competition between VECs and VSMCs, the proliferation of VECs had previously been arrested via a low-dose gamma rayirradiation. To discriminately analyze the proliferation of VSMCs from that of VECs, the former cells were labeled with red fluorescent dye while the latter cells were labeled with green fluorescent dye before performing coculture experiments. After 4 d, total cells were harvested and subjected to flow cytometric analyses. Decrements in red fluorescence intensities due to proliferationmediated dilutions were measured and mathematically processed using a specific software to quantitatively evaluate the proliferation of VSMCs. The findings obtained from the flow cytometry-based analyses were further validated by microscopic observations. RESULTS: Commercially available primary cultured human VECs exclusively promoted VSMC proliferation regardless of their tissue origins and we termed these pro-proliferative VECs as "typeⅠ". By contrast, VECs freshly generated from human bone marrow-derived endothelial progenitors cells or human pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells suppressed VSMC proliferation and we termed these anti-proliferative VECs as "typeⅡ". Repetitive subcultures as well as oxidative stress induced "type Ⅱ VECs to typeⅠ" conversion along with an induction of Regulator of G-protein signaling 5(RGS5)Compatibly, anti-oxidant treatments suppressed both the subculture-dependent "typeⅡ to typeⅠ" conversion and an induction of RGS5 gene. Immunostaining studies of clinical specimens indicated that RGS5 protein expressions in endothelial layers were low in norma arteries but they were up-regulated in pathologica arteries including hypertension, atherosclerosis and autoimmune vasculitis in a dose-dependent manner Overexpression and knockdown of RGS5 caused that"typeⅡ to typeⅠ" and "typeⅠ to type Ⅱ" phenotype conversions of VECs, respectively. CONCLUSION: Human VECs are categorized into two types: pro-proliferative RGS5^(high) VECs(typeⅠ) and antiproliferative RGS5 ^(low) VECs(typeⅡ).

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.

β-Estradiol 17-acetate enhances the in vitro vitality of endothelial cells isolated from the brain of patients subjected to neurosurgery

In the current landscape of endothelial cell isolation for building in vitro models of the blood-brain barrier,our work moves towards reproducing the features of the neurovascular unit to achieve glial compliance through an innovative biomimetic coating technology for brain chronic implants.We hypothesized that the autologous origin of human brain mic rovascular endothelial cells(hBMECs)is the first requirement for the suitable coating to prevent the glial inflammato ry response trigge red by foreign neuroprosthetics.Therefo re,this study established a new procedure to preserve the in vitro viability of hBMECs isolated from gray and white matter specimens taken from neurosurge ry patients.Culturing adult hBMECs is generally considered a challenging task due to the difficult survival ex vivo and progressive reduction in proliferation of these cells.The addition of 10 nMβ-estradiol 17-acetate to the hBMEC culture medium was found to be an essential and discriminating factor promoting adhesion and proliferation both after isolation and thawing,suppo rting the well-known protective role played by estrogens on microvessels.In particular,β-estradiol 17-acetate was critical for both freshly isolated and thawed female-derived hBMECs,while it was not necessary for freshly isolated male-derived hBMECs;however,it did countera ct the decay in the viability of the latter after thawing.The tumo r-free hBMECs were thus cultured for up to 2 months and their growth efficiency was assessed befo re and after two periods of cryopreservation.Des pite the thermal stress,the hBMECs remained viable and suitable for re-freezing and storage for several months.This approach increasing in vitro viability of hBMECs opens new perspectives for the use of cryopreserved autologous hBMECs as biomimetic therapeutic tools,offering the potential to avoid additional surgical sampling for each patient.

通过调控VEGF信号通路探讨补肾通络方对子宫内膜损伤后HUVECs促增殖、血管生成和迁移作用

目的探析补肾通络方对子宫内膜损伤后人脐静脉内皮细胞模型(human umbilical vein endothelial cells,HUVECs)促增殖、血管生成和迁移作用,及对调控血管内皮生长因子(vascular endothelial growth factor,VEGF)信号通路的影响。方法选择2020年1月—2021年12月,购自武汉大学典型培养物保藏中心的12份HUVECs,给予50 ng/mL VEGF处理后,分别实施20%、10%、5%补肾通络方含药血清和无补肾通络方含药血清干预处理。比较补肾通络方对子宫内膜损伤后,VEGF信号通路的影响及对HUVECs增殖、迁移、血管生成的影响。结果比较20%、10%、5%补肾通络方含药血清和无补肾通络方含药血清干预后24 h的HUVECs活性和24 h细胞生长检测所得OD值,差异无统计学意义(P>0.05);20%补肾通络方含药血清组48 h、72 h的HUVECs活性、细胞生长检测所得OD值及VEGF表达量、HUVECs细胞迁移数均高于10%、5%补肾通络方含药血清组和无补肾通络方血清组,差异有统计学意义(P<0.05)。20%补肾通络方含药血清干预组的血管网状结构完整度优于10%、5%补肾通络方含药血清和无补肾通络方血清,无补肾通络方血清干预组的血管网状结构完整度最差,补肾通络方可逆转VEGF抑制细胞活性的趋势,促进HUVECs的血管形成。结论将补肾通络方应用到子宫内膜损伤中,可逆转VEGF抑制细胞活性的趋势,增强HUVECs的细胞活性,促进细胞增殖、迁移,促进血管新生,促进内皮细胞网络结构的重建,利于促进内皮细胞损伤的修复,利于子宫内膜功能的恢复,且与补肾通络方含药血清浓度呈一定量效关系。

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

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.

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.

Expression of Soluble Vascular Endothelial Growth Factor Receptor-2 and Its Effect on Proliferation of Vascular Endothelial Cells

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.

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.

Pro-vs anti-stenotic capacities of type-Ⅰ vs type-Ⅱ human induced pluripotent-derived endothelial cells

AIM:To verify in vivo relevance of the categorization of human vascular endothelial cells(VECs)into type-I(proproliferative)and type-II(anti-proliferative).METHODS:Endothelial layers of murine femoral arteries were removed by wire injury(WI)operation,a common technique to induce arteriostenosis.Type-I and type-II VECs produced from human induced pluripotent stem cells(iPSCs),whose characters were previously determined by their effects on the proliferation of vascular smooth muscle cells in in vitro co-culture experiments,were mixed with Matrigel?Matrix.The mixtures were injected into subcutaneous spaces around WI-operated femoral arteries for the transplanted human iPSC-derived VECs(iPSdECs)to take a route to the luminal surface via vasa vasorum,a nutrient microvessel for larger arteries.Histologies of the femoral arteries were examined over time.The presence of human iPSdECs was checked by immunostaining studies using an antibody that specifically recognizes human VECs.Degrees of stenosis of the femoral arteries were calculated after three weeks.To determine the optimal experimental condition,xenotransplantation experiments were performed under various conditions using immunocompromised mice as well as immunocompetent mice with or without administration of immunosuppressants.RESULTS:Because immunocompromised mice showed unexpected resistance to WI-induced arteriostenosis,we performed xenotransplantation experiments using immunocompetent mice along with immunosuppressant administrations.After one week,luminal surfaces of the WI-operated arteries were completely covered by human iPSdECs,showing the efficacy of our novel transplantation technique.After three weeks,type-IiPSdECs-transplanted arteries underwent total stenosis,while type-II-iPSdECs-transplanted arteries remained intact.However,untransplanted arteries of immunosuppressant-treated mice also remained intact by unknown reasons.We found that transplanted human VECs had already been replaced by murine endothelial cells by this time,indicating that a transient existence of human type-II-iPSdECs on arterial luminal surfaces can sufficiently prevent the development of stenosis.Thus,we re-performed xenotransplantation experiments using immunocompetent mice without administrating immunosuppressants and found that arteriostenosis was accelerated or prevented by transplantation of type-I or type-II iPSdECs,respectively.Similar results were obtained from the experiments using human embryonic stem cell-derived VECs at early passages(i.e.,type-II)and late passages(i.e.,type-I).CONCLUSION:Pro-and anti-stenosis capacities of type-I and type-II human iPSdECs were verified,respectively,promising a therapeutic application of allogenic iPSdECs.

INTERACTIONS BETWEEN THE HUMAN GASTRIC CARCINOMA CELL AND THE HUMAN VASCULAR ENDOTHELIAL CELL

Objective To definite the interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the establishment and maintenance of the tumor vascular system and the tumor hematogenous metastasis.Methods We prepared the conditioned mediums of each cell so as to study the effect of the conditioned medium on itself or others by MTT colorimetry. The comprehensive effect of interactions between two cells was determined by stratified transfilter co culture or direct contact co culture.Results The conditioned medium of human gastric carcinoma cell can stimulate the proliferation of the human vascular endothelial cell, but the CM of HVEC can inhibit the growth of HGCC. Both kinds of cells can inhibit the growth of itself. The ultimate comprehensive effect of the interactions between two kinds of cells was increase of total cell numbers.Conclusion There exist the complicated interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the tumor angiogenesis and the tumor hematogenous metastasis. The ultimate comprehensive effect of the interactions is increase of total cells numbers and tumor volume.

3D生物打印构建HGFs和HUVECs共培养体系促进HUVECs成血管

目的:构建人牙龈成纤维细胞(human gingival fibroblasts,HGFs)和人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)共培养体系,促进HUVECs成血管。方法:取HGFs和HUVECs培养传代至3~5代进行实验。通过慢病毒转染用红色荧光蛋白标记HUVECs。构建HGFs和HUVECs二维共培养体系,将HGFs与HUVECs以不同比例(4∶1、1∶1、1∶4)共培养,荧光显微镜下观察血管网络形成情况。配制5%甲基丙烯酰化明胶(GelMA)30胶,构建HGFs和HUVECs三维共培养体系,激光共聚焦显微镜下观察血管网络的形成情况。对HUVECs形成的血管网络进行定量分析。将HUVECs从三维共培养系统中分离出来,评估单独培养和共培养一定时间后HUVECs的增殖、迁移和小管形成效果以及相关血管生成基因的表达水平。结果:二维共培养时,当HGFs和HUVECs以比例1∶1共培养时血管网络形成的效果最佳。三维共培养时,HGFs可以促进HUVECs血管生成。HGFs和HUVECs共培养组的增殖、迁移和小管形成效果更好,相关血管生成基因的表达水平远远高于HUVECs单独三维培养组。结论:HGFs和HUVECs共培养能够引导和促进HUVECs出芽及迁移。

非接触共培养体系下抑制ARPE-19中CAMKⅡ表达对HUVECs迁移和侵袭及管腔形成的影响

目的:探讨非接触共培养体系下抑制人视网膜色素上皮细胞(ARPE)中Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CAMKⅡ)表达对人脐静脉内皮细胞(HUVECs)迁移、侵袭、管腔形成的影响。方法:将过表达CAMKⅡ-δ的ARPE-19样本进行RNA测序,应用生物信息学分析差异基因参与的功能。使用transwell小室构建ARPE-19和HUVECs非接触共培养体系,根据实验干预措施分为:空白组:仅接种未共培养的HUVECs,无ARPE-19细胞;对照组:ARPE-19和HUVECs细胞均使用完全培养基进行共培养;AIP组(CAMKⅡ抑制组):ARPE-19使用含有AIP(160 nmol/L)的完全培养基,HUVECs使用完全培养基,进行共培养。检测HUVECs迁移、侵袭和管腔形成能力的变化,并通过Western blotting检测CAMKⅡ/AMPK/mTOR/VEGFA蛋白表达水平。结果:生信分析发现差异基因参与细胞生长与死亡和细胞运动等生物学过程。划痕和transwell迁移实验均表明AIP组的HUVECs相对迁移率均明显低于对照组(均P<0.05)。而侵袭和小管形成实验表明,AIP组的相对侵袭率和相对管腔形成率较对照组无明显改变(均P>0.05)。Western blotting结果表明AIP组CAMKⅡ、P-mTOR、VEGFA蛋白表达较对照组均明显下调,而P-AMPK蛋白表达较明显上调(均P<0.05)。结论:在非接触共培养体系下抑制ARPE-19细胞中CAMKⅡ表达可以显著降低HUVECs迁移能力,但不能改变侵袭和管腔形成能力,这可能是通过AMPK/mTOR/VEGFA信号通路实现的。

基于PI3K/Akt/mTOR信号通路的miR-24调控ox-LDL诱导的HUVECs自噬机制研究

目的探讨miR-24对氧化低密度脂蛋白(ox-LDL)诱导下的人脐静脉内皮细胞(HUVECs)自噬的影响及其相关机制,为进一步阐明miR-24在动脉粥样硬化(AS)中的作用提供理论依据。方法采用实时荧光定量PCR(qRT-PCR)检测miR-24的表达;应用蛋白免疫印迹法(western blot)和qRT-PCR法检测Beclin-1、LC3Ⅰ/LC3Ⅱ、p-mTOR、p-PI3K、p-Akt的蛋白和mRNA表达水平;应用透射电子显微镜技术检测细胞的自噬小体生成情况;应用四甲基偶氮唑盐(MTT)法、细胞划痕法、Caspase-3比色法和Hoechst 33258染色法分别检测细胞活性、迁移和凋亡情况。结果应用ox-LDL诱导HUVECs后,发现HUVECs中miR-24的表达显著降低(P<0.05)。miR-24过表达可明显抑制ox-LDL诱导的HUVECs自噬(P<0.05),而miR-24低表达则会增加ox-LDL诱导的HUVECs自噬(P<0.05)。miR-24过表达可显著降低Beclin-1的表达水平,上调LC3Ⅰ/LC3Ⅱ的水平(P<0.05),同时,miR-24过表达可显著促进p-PI3K、p-Akt和p-mTOR的表达(P<0.05)。此外,miR-24过表达显著抑制HUVECs的活力和迁移,增加Caspase-3活性并促进其凋亡(P<0.05)。结论miR-24的过表达可激活PI3K/Akt/mTOR信号通路而降低ox-LDL诱导的HUVECs的自噬水平并促进其凋亡,miR-24可能成为AS的潜在治疗新靶点。

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles

BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.