Protective effect of recombinant Lactobacillus plantarum against H_(2)O_(2)-induced oxidative stress in HUVEC cells

This study probed the protective effect of recombinant Lactobacillus plantarum against hydrogen peroxide(H_(2)O_(2))-induced oxidative stress in human umbilical vein endothelial cells(HUVECs).We constructed a new functional L.plantarum(NC8-pSIP409-alr-angiotensin-converting enzyme inhibitory peptide(ACEIP))with a double-gene-labeled non-resistant screen as an expression vector.A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)colorimetric assay was carried out to determine the cell viability of HUVEC cells following pretreatment with NC8-pSIP409-alr-ACEIP.Flow cytometry(FCM)was used to determine the apoptosis rate of HUVEC cells.Cysteinyl aspartate specific proteinase(caspase)-3/8/9 activity was also assayed and western blotting was used to determine protein expression of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),inducible nitric oxide synthase(iNOS),nicotinamide adenine dinucleotide phosphate oxidase 2(gp91phox),angiotensin II(AngII),and angiotensin-converting enzyme 2(ACE2),as well as corresponding indicators of oxidative stress,such as reactive oxygen species(ROS),mitochondrial membrane potential(MMP),malondialdehyde(MDA),and superoxide dismutase(SOD).NC8-pSIP409-alr-ACEIP attenuated H_(2)O_(2)-induced cell death,as determined by the MTT assay.NC8-pSIP409-alr-ACEIP reduced apoptosis of HUVEC cells by FCM.In addition,compared to the positive control,the oxidative stress index of the H_(2)O_(2)-induced HUVEC(Hy-HUVEC),which was pretreated by NC8-pSIP409-alr-ACEIP,iNOS,gp91phox,MDA,and ROS,was decreased obviously;SOD expression level was increased;caspase-3 or-9 was decreased,but caspase-8 did not change;Bcl-2/Bax ratio was increased;permeability changes of mitochondria were inhibited;and loss of transmembrane potential was prevented.Expression of the hypertension-related protein(AngII protein)in HUVEC cells protected by NC8-pSIP409-alr-ACEIP decreased and expression of ACE2 protein increased.These plantarum results suggested that NC8-pSIP409-alr-ACEIP protects against H_(2)O_(2)-induced injury in HUVEC cells.The mechanism for this effect is related to enhancement of antioxidant capacity and apoptosis.

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.

Saikosaponins-b suppresses tumor growth and angiogenesis of hepatocellular carcinoma by regulating VEGF/ERK/HIF-1α signal pathway

OBJECTIVE Angiogenesis therapy has attracted interest as a potential treatment for hepatocellular carcinoma(HCC).In this study,we investigated the anti-proliferative activities and antiangiogenesis effects of saikosaponins(SS)-b on hepatocellular carcinoma(HCC)and its regulation on VEGF/ERK/HIF-1 αsignal pathway.METHODS H22 hepatoma-bearing mice model and HepG-2 cells were used to study the anti-tumor and anti-angiogenesis effects of SS-b in vivo and in vitro.Pathological change of tumor tissue was observed by HE staining,the microvascular changes were detected by immunohistochemical method.The effects of SS-b on angiogenesis were examined by using the chick embryo chorioallantoic membrane(CAM)model.The effects of SS-b on proliferation,migration and invasion were investigated by MTT assay,scratch wound healing assay and transwell assay inhuman umbilical vein endothelial cell(HUVEC)and HepG2 cells in vitro.Vascular endothelial growth factor(VEGF),matrix metalloproteinase-2/9(MMP-2/9),hypoxia-inducible factor-1α(HIF-1α)expression and the phosphorylation of extracellular regulated kinase(ERK)were analyzed using RT-PCR and Westernblot.RESULTS SS-b effectively inhibited the tumor growth of H22 mice in vivo.The inhibitory rate of tumor was 49.1%,50.7%,66.1%in SS-b 5,10 and 20 mg·kg-1group respectively.HE staining results showed that SS-b induced tumor necrosis and nuclear dissolution in H22 mice.Moreover,SS-b also reduced the number of microvessels of tumor tissue in H22 mice significantly and suppressed the angiogenesis of CAM induced by b-FGF.SS-b had an obvious inhibitory effect on cell proliferation,migration and invasion of HUVEC cells and HepG-2 cells.These effects were associated with downregulation of the expression of MMP2/9 and suppression of VEGF/ERK/HIF-1αsignaling in H22 mice and Hep-G2 cells.CONCLUSION Our findings showed that SS-b exerts anti-tumor effects by inhibiting tumor angiogenesis via regulating VEGF/ERK/HIF-1α signal pathway in vivo and in vitro.

RO-heparin Inhibits L-Selectin-mediated Neutrophils Adhesion to Vascular Endothelium Under Flow Conditions

Selectins are carbohydrate-binding cell adhesion molecules that play a major role in the initiation of inflammatory responses. Accumulaed evidence has suggested that heparin＇s anti-inflammatory effects are mainly mediated by blocking L-or P-selectin-initiated cell adhesion. Recently, we have reported that periodate-oxidized, borohydridereduced heparin （RO-heparln） can inhibit P-selectin-mediated acute inflammation. Here we further examined the effect of RO-heparin on the adhesion of L-selectin-mediated leukocytes to vascular endothelium under flow conditions in vivo and in vitro. The results show that RO-heparin with a low anticoagulant activity can effectively reduce leucocyte roiling on thioglycoUate-induced rat mesenterlc venules and L-selectin-metadiated neutrophil roiling on TNF-α-induced human umbilical vein endothelial cells（HUVECs） under flow conditions. Our findings suggest that the effect of RO-heparin on inflammatory responses is mainly a result of its inhibiting the interaction between P- or L-selectin and its ligands. The findings also suggest that RO-heparin may be useful in preventing inflammation diseases.

Long-term stimulation of angiotensin Ⅱ induced endothelial senescence and dysfunction

Objective Vascular endothelial cells senescence is one of major risk factors for atherosclerotic diseases,which can be induced by endogenous peptides,such as angiotensin Ⅱ(Ang Ⅱ).However,the effect of chronic Ang Ⅱ stimulation on endothelial senescence remains unknown.Therefore,this study aims to investigate the changes in morphology and function of human umbilical vein endothelial cells(HUVECs)in response to the chronic stimulation of Ang Ⅱ.

Piper sarmentosum as an antioxidant on oxidative stress in human umbilical vein endothelial cells induced by hydrogen peroxide

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.

In vitro biocompatibility evaluation of silk-fibroin/ polyurethane membrane with cultivation of HUVECs

In order to investigate the in vitro biocompatibility of a novel polyurethane （PU） membrane modified by incorporation of superfine silk-fibroin powder （SFP）, which was prepared for small-diameter vascular grafts, with the cultivation of human umbilical vein endothelial cells （HUVECs）, PU and SFP were mixed with the ratios of 9：1, 7：3, 5：5, 3：7 （PU：SFP） to make four composite materials. Unmodified PU and polytetrafluoroethylene （PTFE） were added as control groups. CCK-8 assay was used to evaluate the cytotoxicity of these biomaterials. Data were processed using SPSS, and P〈 0.05 was considered to be statistically significant. Adherence and spreading of HUVECs on the surface of specimens was observed using direct contact cultivation. The toxicity ratings of the novel composites were grade 0-1, which is in the acceptable range. In all the experimental groups except control, SFP/PU with ratio of 1：9 had the least cytotoxicity property, and more content of SFP in the composite showed no improvement of the biocompatibility. HUVECs strongly attached to and grew on the surface of the biomaterials, and proliferated rapidly. The proliferation ability increased with increased proportion of SFP; however the cell quantity on the surface of the materials decreased when the proportion of SFP was equal to or larger than that of PU in the composite. It is concluded that this novel material has excellent cellular affinity with no cytotoxicity to HUVECs. Adding SFP gives PU better biocompatibility, while further research on optimum blend ratios is still needed.

Protective Effect of Telmisartan on Endothelial Cells Exposed to High Glucose Levels in vitro

Objectives To investigate the effect of telmisartan on human umbilical vein endothelial cells （HUVEC） exposed to high glucose in vitro and the related mechanism. Methods HUVECs were incubated with telmisartan and glucose （5 mmol/L, 30 mmot/L） at 0 h, 12 h, 24 h, 36 h, 48 h, respectively. The level of malondialdehyde （MDA） and superoxide dismutase （SOD） in the supernatant of cultured endothelial cells was measured by thiobarbituric acid test and xanthine oxidase test. The expression of PPAR-γ was determined at 24 hour with Western blot technique. Results When the endothelial cells were cultured in high glucose environment, the MDA level was significantly increased, but the SOD activity and the protein expression of PPAR-γ were markedly decreased. However, the high glucose-induced effects were inhibited by telmisartan intervention. Conclusion Telmisartan can decrease oxidative stress and increase PPAR-γ expression of endothelial cells in high glucose environment. （S Chin J Cardio12009 ; 10 （4） ： 222 -226）