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.

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.

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.

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.

Vein transplantation using human umbilical cord blood stem cells in the treatment of stroke sequela

BACKGROUND: Transplanted mononuclear cell (MNC) of umbilical blood can survive in central nervous system (CNS) of host through blood brain barrier, differentiate into nerve cells, migrate to damaged site and integrate morphological structure and function with nerve cells of host so as to improve deficiencies of sensatory function, motor function and cognitive function and influence on stroke sequela. OBJECTIVE: To observe the vein transplantation of human umbilical cord blood stem cells (HUCBSC) for improving neurological function, limb function and activity of daily living of patients with stroke and evaluate the reliability. DESIGN: Self-controlled study. SETTING: Department of Neurosurgery, the Second People's Hospital of Zhengzhou City; Red-crossed Blood Center of Henan Province; Department of Neurosurgery, the Fist Affiliated Hospital of Zhengzhou University. PARTICIPANTS: A total of 10 patients with stroke sequela were selected from Department of Cerebral Surgery, the Second People's Hospital of Zhengzhou City from April to December 2005. There were 9 males and 1 female aged from 35 to 75 years with the mean age of 56 years. All of them were diagnosed with CT and MRI examination and coincidence with diagnostic criteria of stroke established by the Fourth National Academic Meeting for Cerebrovascular Disease. All patients provided informed consent. METHODS: 80-140 mL umbilical blood of term birth of newborn was selected hermetically and maintained in sterile plastic bag. And then, the blood was centrifugated at the speed of 1 500 r/min for 30 minutes at 22 ℃ in order to separate MNC, i.e., HUCBSC. In addition, after final diagnosis during hospitalization, stroke patients were perfused with HUCBSC through superficial vein of back of the hand. Each patient was averagely perfused with 6 portions of HUCBSC (cellular numbers ≥ 1×108/portion) and the interval between each portion was 1-7 days with the mean interval of 4 days. MAIN OUTCOME MEASURES: ① Neurological function of stroke patients was evaluated with neurological function deficiency (NFD) before treatment and at 3 months after treatment. The scale includes consciousness, level fix function, facial paralysis, language, muscle force of upper limbs, muscle force of lower limb and step function. The total scores ranged from 0 to 45; meanwhile, the lower the scores were, the better the neurological function was. ② Motor function of injured limbs was evaluated with Fugl-Meyer Assessment (FMA), including motor function of upper limbs, motor function of lower limbs, balance ability, sensory function and motion of joint. The total scores ranged from 0 to 226; meanwhile, the higher the scores were, the better the motor function of limbs was. ③ Activities of daily living (ADL) was evaluated with Barthel Index (BI), including having meals, taking a bath, dressing oneself, putting on clothes, walking in balance and stair activity. The total scores ranged from 0 to 100; meanwhile, the higher the scores were, the stronger the ADL was. RESULTS: A total of 10 patients were involved in the final analysis. After treatment, NFD of stroke patients was (10.9±5.09) points, which was lower than that before treatment [(25.4±6.09) points, t =8.213, P < 0.01]. In addition, after treatment, FMA and BI of stroke patients were (80.9±25.00) points and (81.1±15.93) points, respectively, which were higher than those before treatment [(31.9±21.85) points, (36.2±19.41) points, t =13.024, 13.670, P < 0.01]. Immuno-suppressive drugs were not used during the whole therapeutic procedure; moreover, immunological rejection and allergic reaction were not observed during the same period. CONCLUSION: Transplanting HUCBSC through superficial vein of back of the hand is regarded as a simple and safe method for the treatment of stroke sequela.

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.

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.

Gadolinium-promoted angiogenesis involves the activation of PKCα/β_2 and MAPKs in human umbilical vein endothelial cells

Gadolinium has been widely used as a contrast agent for magnetic resonance imaging in clinical practice. Recently, it was reported that gadolinium is involved in nephrogenic systemic fibrosis, although the exact mechanism by which gadolinium triggers nephrogenic systemic fibrosis remains unclear. In this study, we show that gadolinium chloride （GdC13） induced human umbilical vein endothelial cells （HUVECs） to migrate in Matrigel and tubulogenesis during wound healing. Chick chorioallantoic membrane assay confirmed that GdC13 stimulates angiogenesis. Under the optimal angiogenic concentration of GdC13 （1 0 ~tM）, intracellular calcium concentration and reactive oxygen species generation were elevated. Moreover, western blotting results indicate that in cells treated with GdC13, Ca2＋-dependent PKCa/132 was phosphorylated, and MAPKs pathways were also activated. Taken together, GdC13 has a potential effect on angiogenesis in HUVECs, and the possible mechanisms may involve oxidative stress and calcium-related signalin~ pathways.

Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

Culture supernatants of breast cancer cell line MDA-MB-231 treated with parthenolide inhibit the proliferation, migration, and lumen formation capacity of human umbilical vein endothelial cells

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.

A comparison study of the targeting properties of NGR-liposomes and RGD-liposomes towards human umbilical vein endothelial cells

Endothelial cells in the angiogenic vessels of solid tumors over-express several proteins, which could be recognized by some peptide ligands. In this study, the targeting properties of two peptides, RGD （arginine-glycine-aspartic acid） and NGR （asparagine-glycine-arginine）, towards human umbilical vein endothelial cells （HUVEC） were compared in vitro using doxorubicin entrapped liposomes as vehicles. The doxorubicin-loaded sterically stabilized liposomes （SSL-DOX） and RGD or NGR modified liposomes （RGD-SSL-DOX or NGR-SSL-DOX） were prepared and characterized. The studied properties included particle size, zeta potential, encapsulation efficiency and in vitro release rate. Flow cytometry, confocal microscopy and SRB assay were used on HUVEC to assess the targeting effect of the two peptides towards endothelial cells of tumor vasculature. All of the liposomes prepared in this study were obtained with encapsulation efficiencies of above 98%, particle sizes of about 65-75 nm and slight negative surface charges. The in vitro release results demonstrated that the modification of RGD or NGR did not alter the release behaviors of liposomes. It was observed in flow cytometry that the uptake of doxorubicin by HUVEC from SSL-DOX, NGR-SSL-DOX, RGD-SSL-DOX and doxorubicin solution followed the order of doxorubicin solution〉RGD-SSL-DOX 〉NGR-SSL-DOX〉SSL-DOX, and the intemalized doxorubicin distributed in both nuclei and cytoplasm for ligand modified SSL and only in nuclei for non-targeted SSL. The order of cytotoxicity in SRB assay was the same as that of the uptake study. The characterization study indicated that modifications did not significantly change the properties of the sterically stabilized liposomes. HUVEC treated with both modified liposomes showed higher uptake of doxorubicin as compared to those with SSL-DOX as a result of the receptor-mediated endocytosis. Moreover, RGD-SSL-DOX exhibited better targeting effect than NGR-SSL-DOX.

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.

Pro-angiogenic Activity of Notoginsenoside R1 in Human Umbilical Vein Endothelial Cells in vitro and in a Chemical-Induced Blood Vessel Loss Model of Zebrafish in vivo

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.

Inhibitory Effect of Cryptotanshinone on Angiogenesis and Wnt/β-Catenin Signaling Pathway in Human Umbilical Vein Endothelial Cells

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.

Ginsenoside Rb1 Protects Human Umbilical Vein Endothelial Cells against High Glucose-Induced Mitochondria-Related Apoptosis through Activating SIRT3 Signalling Pathway

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.

Paeoniflorin Promotes Angiogenesis in A Vascular Insufficiency Model of Zebrafish in vivo and in Human Umbilical Vein Endothelial Cells in vitro

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.