Preliminary study on HUVEC permeability changes in FNAIT induced by CD36 antibody / 中国输血杂志

【Objective】 To explore the pathogenesis of fetal edema caused by CD36 antibody in fetal/neonatal alloimmune thrombocytopenia (FNAIT), and to provide reference for clinical prevention and treatment. 【Methods】 The established CD36 monoclonal antibody was incubated with human peripheral blood mononuclear cells (PBMC), and the concentrations of cytokines (TNF-α and IL-1β) in the supernatant of cell culture were detected by ELISA. The permeability of endothelial cells were investigated by detecting the fluorescence intensity of FITC-albumin by incubating cytokine-rich cell supernatant with human umbilical vein endothelial cells (HUVEC). 【Results】 Flow cytometry showed that CD36 monoclonal antibody could bind to human monocytes. Compared with isotype IgG control, increased cytokine TNF-α (pg/mL) (407.73±20.40 vs 29.38 ±4.72, P<0.05) and IL-1β (pg/mL) (247.14±83.59 vs 53.68±26.96, P<0.05) were detected in the supernatant of cell culture after incubation of CD36 monoclonal antibody with human PBMC. Detection of fluorescence intensity of FITC-albumin in transwell cultured HUVEC showed that cytokine-rich cell supernatant derived from CD36 monoclonal antibody incubated with human PBMC can increase the permeability of endothelial cells significantly (CD36 antibody vs isotype IgG, MFI value: 492±16 vs 320±11, P<0.05). 【Conclusion】 The effect of CD36 monoclonal antibody on PBMC can increase HUVEC permeability, which may be one of the pathogenesis of fetal edema with FNAIT.

Fucoxanthin suppresses OxLDL-induced inflammation via activation of Nrf2 and inhibition of NF-κB signaling

Objective: To explore the impact of fucoxanthin on oxidized low-density lipoprotein (OxLDL)-induced stress and inflammation in human endothelial cells and its underlying mechanisms. Methods: HUVECs were treated with OxLDL and/or fucoxanthin for a range of time points and concentrations. We evaluated the effects of fucoxanthin on OxLDL-induced HUVECs using the MTT assay, reactive oxygen species accumulation assay, ELISA, RT-PCR, immunofluorescence, and Western blotting. Results: Fucoxanthin enhanced the cell viability in a dose dependent manner after OxLDL exposure. Furthermore, fucoxanthin pretreatment significantly decreased OxLDL-induced reactive oxygen species production and prevented the activation of the nuclear factor kappa-B pathway, which led to substantial suppression of pro-inflammatory gene expressions. OxLDL-induced upregulation of interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, interleukin-1β, monocyte chemotactic protein-1, cyclooxygenase-1, and tumor necrosis factor-α was significantly reduced by fucoxanthin. Conclusions: Fucoxanthin can inhibit OxLDL-induced vascular inflammation and oxidative stress in HUVECs by targeting Nrf2 signaling pathways.

Protective effect of recombinant / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

This study probed the protective effect of recombinant

Changes of connexin 43 in irradiated human vascular endothelial cells and its influence on cell stiffness / 中华放射医学与防护杂志

Objective:To investigate the changes of connexin 43 (Cx43) in human umbilical vein endothelial cells (HUVEC) after X-ray irradiation and its influence on the stiffness of irradiated cells.Methods:Western blot was used to detect the expression of Cx43 in HUVEC cells at different time points (0, 6, 12, 24 and 48 h) after different doses of X-ray irradiation (0, 2.5, 5, 10 and 20 Gy), and the phosphorylation levels of three phosphorylation sites (Ser279/282, Ser368 and Tyr265) of Cx43 at different time points (3, 6, 24 and 48 h) after 0, 5 and 10 Gy irradiation. The distribution of Cx43 protein in the irradiated HUVEC cells was detected by immunofluorescence. The stiffness changes of cells were detected by atomic force microscopy (AFM) at the depths of 50, 100 and 200 nm.Results:The expression of Cx43 in HUVEC cells was reduced at 6, 12, 24 and 48 h after 10 Gy X-ray irradiation( t=3.262, 3.708, 3.686, 6.825, P<0.05)and this decrease had a dose dependent manner at 24 h after 2.5, 5, 10 and 20 Gy irradiation ( t=3.034, 10.720, 13.130, 13.650, P<0.05). At 24 h after 5, 10 and 20 Gy X-ray irradiation, the distribution of Cx43 in HUVEC cells was transported from intercellular gap junctions to nucleus and perinuclear region. At 24-48 h after irradiation, the phosphorylation level of Ser368 at Cx43 increased and in a dose dependent manner. At 24 h after irradiation, the stiffness of the irradiated cells decreased significantly under the conditions of 100 and 200 nm ( t=3.362, 5.122, P<0.05), and recovered with overexpression of Cx43 ( t=2.674, 4.398, P<0.05). Conclusions:X-ray irradiation leads to the phosphorylation of Ser368 at Cx43, which promotes the degradation and nucleus/perinuclear translocation of Cx43 and reduces the stiffness of HUVEC. Increasing the expression level of Cx43 is helpful to the stiffness recovery of irradiated vascular endothelial cells, suggesting that Cx43 may be a potential target for regulating radiation injury of vascular endothelial cells.

Study on Inhibitory Effects of Total Alkaloids of Gelsemium elegans on the Proliferation and Angiogenesis of Human Colon Cancer Cells / 中国药房

OBJECTIVE：To investigate the inhibitory effects of total alkaloids of Gelsemium elegans （TAG） on the proliferation and angiogenesis of human colon cancer cells. METHODS ：Human colon cancer cell line HT- 29 and HUVEC were cultured in vitro . After the intervention of low- ，medium-，high-dose TAG （40，80，120 μg/mL），the morphology of the two cells was observed by fluorescence inversion microscope. The survival rate of HT- 29 cells and HUVEC was detected by CCK- 8 assay. Flow cytometry was used to detect HT- 29 cell cycle. The migration rate ，invasion rate and tube number of HUVEC were observed by scratching test ，Transwell invasion experiment and tube formation experiment. RESULTS ：Compared with blank group ，HT-29 cells and HUVEC were decreased to different extents in TAG groups ；dead cells were observed ，and the survival rate of both decreased significantly （P＜0.05 or P＜0.01）. The proportion of HT- 29 cells at G 2/M phase in TAG groups as well as those at G 0/G1 phase in medium-dose group were increased significantly ；the proportion of HT- 29 cells at S phase in TAG groups as well as those at G 0/G1 phase in high-dose group were decreased significantly （P＜0.05 or P＜0.01）. Survival rate ，migration rate and invasion rate of HUVEC were decreased significantly in TAG groups ，and tube number was also decreased significantly at each time point during 4-24 h（P＜0.01）. CONCLUSIONS ：TAG have inhibitory effect on the proliferation of human colon cancer HT- 29 cells and HUVEC，can change HT- 29 cell cycle ，inhibit the migration ，invasion and tube formation of HUVEC.

Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17

BACKGROUND Streptococcus agalactiae capsular type III strains are a leading cause of invasive neonatal infections. Many pathogens have developed mechanisms to escape from host defense response using the host membrane microdomain machinery. Lipid rafts play an important role in a variety of cellular functions and the benefit provided by interaction with lipid rafts can vary from one pathogen to another. OBJECTIVES This study aims to evaluate the involvement of membrane microdomains during infection of human endothelial cell by S. agalactiae. METHODS The effects of cholesterol depletion and PI3K/AKT signaling pathway activation during S. agalactiae-human umbilical vein endothelial cells (HUVEC) interaction were analysed by pre-treatment with methyl-β-cyclodextrin (MβCD) or LY294002 inhibitors, immunofluorescence and immunoblot analysis. The involvement of lipid rafts was analysed by colocalisation of bacteria with flotillin-1 and caveolin-1 using fluorescence confocal microscopy. FINDINGS In this work, we demonstrated the importance of the integrity of lipid rafts microdomains and activation of PI3K/Akt pathway during invasion of S. agalactiae strain to HUVEC cells. Our results suggest the involvement of flotillin-1 and caveolin-1 during the invasion of S. agalactiae strain in HUVEC cells. CONCLUSIONS The collection of our results suggests that lipid microdomain affects the interaction of S. agalactiae type III belonging to the hypervirulent ST-17 with HUVEC cells through PI3K/Akt signaling pathway.

Preparation and preliminary evaluation of quercetin and microRNA-150 co-loaded cationic solid lipid nanoparticles for ocular administration / 中草药

Objective: To prepare the cationic solid lipid nanoparticles (Que/mR150 SLNs) co-loaded with quercetin (Que) and microRNA-150 (mR150) and investigate the preparation process, then assess its in vitro release, cell uptake capacity and safety of ocular administration. Method: First, thin-film dispersion method was used to prepare quercetin-encapsulated cationic solid lipid nanoparticles (Que-SLNs), and the preparation process was optimized based on the particle size, PDI and encapsulation rate; Using electrostatic adsorption method to co-load mR150 in nanoparticles (Que/mR150 SLNs), and the adsorption efficiency of the miRNA by the nanoparticles was examined by agarose gel electrophoresis experiment; The in vitro release performance of quercetin in Que/mR150 SLNs was investigated; The effect of Que/mR150 SLNs on the proliferation of HUVEC of human umbilical vein endothelial cells was measured by MTT method, and fluorescence labeling was used to observe their uptake in HUVEC; And the irritancy of Que/mR150 SLNs to rabbit eyes was examined by pathological tissue sections of rabbit eyes. Result: After process optimization, the cationic nano Que-SLNs had good drug-loading, particle size distribution and stability. The appearance of the cationic nano-Que-SLNs was spherical, and it could be kept stable for two months. The quercetin encapsulation rate was (85.25 ± 1.29)%, the drug load was (1.67 ± 0.02)%, the average particle size was (110.00 ± 2.10) nm, and the Zeta potential is (53.2 ± 5.12) mV; The in vitro drug release results showed that the release of quercetin in the nanoparticles was slow, and the cumulative release amount within 48 h was about (80.69 ± 1.29)%; When the mass ratio of dioctadecyl dimethyl ammonium bromide to mR150 (DDAB/RNA) of different cationic materials was 6:1, the cationic solid lipid nanoparticles basically encapsulated mR150 completely with little effect on its particle size and potential. MTT experiments showed that blank nanometer mass concentration of 50-150 mg/L had no significant proliferation toxicity on HUVEC cells; Cell uptake experiments showed that Cy5 and coumarin-6 dual fluorescently labeled and co-loaded nanometers could effectively enter HUVEC cells; Pathological tissues of rabbit eyes showed that Que/mR150 SLNs had no obvious damage to the eyes. Conclusion: The preparation process of Que/mR150 SLNs solid lipid nanoparticles is stable and reliable, with good reproducibility, storage stability and good biological safety, which is conducive to the efficient delivery of quercetin and mR150 into HUVEC cells, which provides the ideas for the treatment of diseases related to angiogenesis

Effects of Dendrobium officinale Polysaccharides on Gene Expression Profile of HUVEC / 中国药房

OBJECTIVE： To investigate the effects of Dendrobium officinale polysaccharides on gene expression profile of HUVEC. METHODS： HUVEC was selected as objects. MTS method was used to detect the effects of different doses of D. officinale polysaccharides （50， 100， 200， 400， 800 μg/mL） on the proliferation activity of HUVEC. The growth inhibitory concentration of 30％ cells （IC30） was calculated to screen the dose of follow-up tests. cDNA microarray assay was used to detect the changes of gene expression profile for HUVEC after treated with D. officinale polysaccharides for 24 h， so as to screen differentially expressed genes. GO enrichment analysis and KEGG pathway enrichment analysis were performed for top 5 differentially expressed genes by using DAVID bioinformatics resource database. Real-time fluorescence quantitative polymerase chain reaction （qRT-PCR） was used to validate the results of microarray detection with immunity-related differentially expressed genes as objects. RESULTS： After treated with 100， 200， 400， 800 μg/mL D. officinale polysaccharides， survival rate of HUVEC  were decreased significantly （P＜0.05 or P＜0.01）. IC30 value was 408 μg/mL. After treated with 400 μg/mL （by IC30） D. officinale polysaccharides， there were 91 differentially expressed genes in HUVEC cells， of which 84 were up-regulated and 7 were down-regulated. Top 5 genes of up-regulated and down- regulated expression were SELE， CCL2， CXCL6， IL8， ICAM1 as well as VWCE， CPT1A， CLU， CCL14， CINS4， which may be mainly associated with immune conditions and inflammatory responses. The differentially expressed genes mainly distributed in extracellular domain， and were enriched in biological processes such as production and response of cytokines and stimulus response， and played molecular functions such as chemokine and its receptor activity. The up-regulated genes as SELE， ICAM1 and CXCL2 were mainly enriched in TNF signaling pathway， influenza A （H1N1）， herpes simplex virus infection and other pathways. The down-regulated gene CCL14 was mainly enriched in chemokine signaling pathway. Results of qRT-PCR validation tests showed that relative expression of ICAM1 was increased significantly， while that of CCL14 was decreased significantly （P＜0.05）， which was in agreement with microarray detection results. CONCLUSIONS： After treated with D. officinale polysaccharides， the expression of 91 genes in HUVEC cells are different significantly， mainly being up-regulated. The differentially expressed genes may participate in immune regulation through TNF signaling pathway， influenza A （H1N1） and herpes simplex virus infection.

Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines / 中华微生物学和免疫学杂志

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells ( HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate periph-eral blood mononuclear cells ( PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor ( M-CSF) to prepare macro-phages. The purity of CD14+CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-in-fected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pre-treating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2. 66±0. 53, P<0. 05) and then de-creased. The purity of macrophages detected by flow cytometry was (89. 16±2. 07) ％. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16. 10±0. 17 and 29. 76±0. 58, while the expression levels at 24 h in the un-infected group were 1. 46±0. 67 and 1. 60±0. 54, respectively. Expression of IL-6, IL-8, TNF-αand IL-1βat mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-αand IL-1β expression at 24 h were 45. 82±3. 72, 52. 34±1. 69 (12 h), 8. 94±1. 75 and 30. 96±1. 44 in the infected macrophages, and 1. 16±0. 22, 1. 15±0. 21, 1. 11±0. 09 and 1. 47±0. 31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infec-ted HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-cul-ture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0. 01) and that of IL-6, IL-8, TNF-αand IL-1βin macrophages (P<0. 01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-αand IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent.

TAZ promoting angiogenesis and its mechanism in gastric cancer / 中国肿瘤临床

Objective: To determine the expression of TAZ and its role in angiogenesis in gastric carcinoma. Methods: Immunohistochemical staining was performed to investigate the expression of TAZ and to determine whether a direct relationship exists between TAZ and β-catenin. Transfection with TAZ overexpression plasmid in MKN28 cells was conducted to induce exogenous expression of TAZ and a TAZ knockdown plasmid was transfected into MGC803 cells to reduce TAZ levels. The effects on endothelial cell formation, proliferation, and migration were determined by Matrigel three-dimensional culture, MTT proliferation assay and Transwell migration assay. In addition, the expression of TAZ and β-catenin in transfected gastric cancer cells was detected by Western blot. Results: Immunohistochemistry showed that TAZ protein was expressed in 64 of 150 gastric cancer sample tissues (43%), TAZ was localized in the nucleus, and its expression was associated with tumor grade, TNM stage, metastasis, and microvessel density (MVD) (P<0.05). In addition, the expression frequency of β-catenin in the TAZ positive group was 67.2%, which was significantly higher than that in the TAZ negative group, and the expression of TAZ was positively correlated with β-catenin. After transfection, TAZ overexpression increased the expression of β-catenin and enhanced HUVECs tube formation, proliferation, and migration. In the MGC803 cells transfected with the knockdown plasmid, β-catenin levels were decreased and HUVECs motility was inhibited. Conclusions: TAZ may promote angiogenesis in gastric cancer by promoting β-catenin expression.

Effect of Tripterygium Glycosides Tablets on synovial angiogenesis in rats with type Ⅱ collagen induced arthritis / 中国中药杂志

To observe the effect of Tripterygium Glycosides Tablets on angiogenesis of rats with type Ⅱ collagen-induced arthritis( CIA) and on the tube formation of human umbilical vein endothelial cells( HUVEC) in vitro. The HUVEC were induced by 20 μg·L-1 vascular endothelial growth factor( VEGF) in vitro,and were treated with 0. 1,1,10 mg·L-1 Tripterygium Glycosides Tablets continuously for 7 hours. The numbers of branches of tube formation were measured. SD rats were immunized to establish CIA. CIA rats were treated with 9,18,36 mg·kg-1·d-1 Tripterygium Glycosides Tablets for 42 days. Histopathological examination( HE) was performed to observe the vascular morphology and vascular density in the synovial membrane of the inflamed joints. Immunohistochemistry and immunofluorescence were performed to observe the expression of platelets-endothelial cell adhesion molecule( CD31) and αsmooth muscle actin( αSMA) in synovial membrane. Immunohistochemistry and Western blot were performed to observe the expression of hypoxia-inducible factors 1α( HIF1α) and angiotensin 1( Ang1) in the synovial tissue. The results showed that the numbers of branches of tube formation of HUVEC induced by VEGF were improved,and declined significantly after treated by Tripterygium Glycosides Tablets. Compared with the normal group,the vascular density,CD31 positive expression,CD31 +/αSMA-immature and total vascular positive expression in the synovial membrane of the model group were significantly increased,and so as HIF1α and Ang1 in the synovium. Tripterygium Glycosides Tablets reduced the synovial vascular density and inhibited the positive expression of CD31,CD31+/αSMA-immature blood vessels and total vascular,but has no effect on CD31+/αSMA+mature blood vessels. Tripterygium Glycosides Tablets also inhibited the expression of HIF1α and Ang1 in synovial membrane of inflammatory joints. Our results demonstrated that Tripterygium Glycosides Tablets could inhibit the angiogenesis of synovial tissue in CIA rats and the tube formation of HUVEC,which is related to the down-regulation of HIF1α/Ang1 signal axis.

Effect of Fengshi Qutong Capsule on Human Umbilical Vein Endothelial Cells Induced by VEGF / 中国实验方剂学杂志

Objective: To study the effect of Fengshi Qutong capsule on the migration, adhesion, invasion and tube formation of human synovial cells and the phosphorylation and protein expression of vascular endothelial growth factor receptor 2 (VEGFR2). Method: With human umbilical vein endothelial cells (HUVEC) as the research object, low, middle and high-dose Fengshi Qutong capsule(0.02,0.1,0.5 μg·L-1) on HUVEC was determined by methye thiazolye telrazlium (MTT) colorimetric assay for the follow-up experiment. The transwell migration, adhesion and transwell invasion test were used to detect the migration and adhesion of the different concentrations of Fengshi Qutong capsule in HUVEC. The expression of VEGFR2 in HUVEC was detected by Western blot, and Real-time PCR was used to detect the content of VEGFR2 mRNA in cells. Result: Compared with normal group, the proliferation of HUVEC was significantly increased after 24 h and 48 h of VEGF induction (PPP-1 Fengshi Qutong capsule were administered in vitro for 48 h to inhibit HUVEC proliferation activity in a dose-dependent manner (PPPPConclusion: Fengshi Qutong capsule can inhibit the migration, adhesion, invasion and tube formation of HUVEC. This effect may be related to the inhibition of phosphorylation, and protein and mRNA expression level of VEGFR2.

Effects of TET2-targeting miR-27b on oxidized low-density lipoprotein-induced inflammatory responses and apoptosis in endothelial cells / 中华微生物学和免疫学杂志

Objective@#To investigate the effects of miR-27b targeting ten-eleven translocation methylcytosine dioxygenase 2 (TET2) on oxidized low-density lipoprotein (ox-LDL) induced inflammatory responses and apoptosis in endothelial cells.@*Methods@#Double luciferase reporter gene analysis verified the targeting effect of miR-27b on TET2. Human umbilical vein endothelial cells (HUVECs) were induced by ox-LDL in vitro. Eight groups were set up including control group, ox-LDL group, ox-LDL+ anti-miR-con group, ox-LDL+ anti-miR-27b group, ox-LDL+ pcDNA group, ox-LDL+ pcDNA-TET2 group, anti-miR-27b+ si-con group and anti-miR-27b+ si-TET2 group. qRT-PCR was used to detect the expression of miR-27b and TET2 at mRNA level. Cell viability was detected by MTT assay. Cell apoptosis rate was detected by flow cytometry. Western blot was used to detect the expression of TET2, Cyclin D1 and caspase-3 at protein level.@*Results@#TET2 was the target gene of miR-27b. TET2 expression could be negatively regulated by miR-27b. ox-LDL increased the expression of miR-27b and reduced the expression of TET2 in HUVECs. The secretion of inflammatory factors and apoptosis rates of HUVECs in the control, ox-LDL+ anti-miR-27b, ox-LDL+ pcDNA-TET2 and anti-miR-27b+ si-con groups were significantly lower than those in the ox-LDL, ox-LDL+ anti-miR-con, ox-LDL+ pcDNA and anti-miR-27b+ si-TET2 groups, respectively (P<0.05).@*Conclusions@#miR-27b promoted ox-LDL-induced inflammatory responses and apoptosis in endothelial cells through down-regulating the expression of TET2.

Oxidação da proteína dissulfeto isomerase pelo hidroperóxido de urato e as implicações sobre o endotélio vascular / Oxidation of protein disulfide isomerase by urate hydroperoxide and implications in vascular endothelium

Em condições inflamatórias do sistema vascular, altas concentrações de mieloperoxidase somada à presença do ácido úrico, sugerem a formação local do oxidante hidroperóxido de urato. A ação desse peróxido já foi demonstrada sobre glutationa e peroxirredoxinas, tornando plausível a possibilidade de que outras proteínas tiólicas também pudessem ser alvo de oxidação. A proteína dissulfeto isomerase é uma ditiol-dissulfeto oxidoredutase e chaperona, localizada principalmente no retículo endoplasmático, onde participa do enovelamento de proteínas nascentes. Além disso, um pool dessas enzimas foi identificado na superfície da célula e no meio extracelular (secretada) e parece ser especialmente importante em eventos vasculares como ativação e agregação de plaquetas, trombose e remodelamento vascular. Primeiramente, foi investigado se o hidroperóxido de urato era capaz de oxidar a PDI. Pelo ensaio do DTNB foi verificado que os tióis livres da proteína eram consumidos após reação com o peróxido e, em seguida, por nLC-MS/MS os resíduos de cisteínas dos sítios catalíticos foram identificados como os principais alvos de oxidação. Embora não tenham sido verificadas outras modificações além de dissulfetos, foi observado que o tratamento com hidroperóxido promoveu agregação e inativação da proteína. Os estudos subsequentes envolveram uma linhagem de células endoteliais (HUVECs). Análises preliminares de citotoxicidade (detecção da atividade da enzima lactato desidrogenase no sobrenadante e incorporação de sondas fluorescentes ao DNA) mostraram que tratamentos com concentrações de até 400 µM de hidroperóxido de urato não são letais às células em cultura. Usando alquilantes impermeáveis à membrana celular foi mostrado que o hidroperóxido de urato oxida não só a proteína dissulfeto isomerase, mas também proteínas tiólicas totais expressas na superfície das HUVECs. Experimentos de wound healing foram feitos para avaliação da capacidade de migração das células mediante o tratamento com hidroperóxido de urato, mas nenhuma diferença foi observada. Contudo, a incubação das células com os agentes oxidantes hidroperóxido de urato e diamida, inibidores de PDI e integrina e um alquilante de tiol, resultaram, pelo menos nos trinta primeiros minutos, em menor capacidade de adesão das células à fibronectina. Além disso, as células tratadas com hidroperóxido de urato se tornaram mais sensíveis ao destacamento da placa de cultura e apresentaram alteração na morfologia. O tratamento com o peróxido também afetou a homeostase redox das HUVECs, observado pela diminuição da razão GSH/GSSG. Finalmente foram apresentadas evidênciasindiretas de que o ácido úrico é substrato da peroxidasina, uma heme peroxidase abundantemente expressa no sistema vascular. Primeiro, pelo ensaio do Amplex Red foi observado que a presença de ácido úrico na mistura reacional resultou em menor taxa de oxidação do reagente. Depois, por LC-MS/MS, também em amostra na qual o ácido úrico estava presente, foi identificado o hidroxiisourato, álcool resultante da decomposição do hidroperóxido de urato. Todo o conjunto de dados deverá contribuir para o maior entendimento da participação do hidroperóxido de urato em processos oxidativos vasculares − especialmente a oxidação de proteínas − que pode ser um dos mecanismos responsáveis pela alteração da função endotelial e da homeostase vascular

During vascular inflammatory conditions, high amounts of myeloperoxidase added to the presence of uric acid, suggest the local formation of urate hydroperoxide. Its oxidative action has already been demonstrated on glutathione and peroxiredoxins, making plausible the possibility that other thiol proteins could also be a target for oxidation. The protein disulfide isomerase is a dithiol-disulfide oxidoreductase and chaperone, located mainly in the endoplasmic reticulum, where it is involved in the correct folding of nascent proteins. Also, a pool of these enzymes has been identified in cell surface and the extracellular (secreted) milieu and appears to be important in vascular events, such as platelet activation and aggregation, thrombosis and vascular remodeling. First, it was investigated whether urate hydroperoxide was capable of oxidizing PDI. By the DTNB assay, it was found that the free thiols of the protein were consumed after reaction with the peroxide and then, by nLC-MS / MS, the active redox cysteine residues were identified as the main oxidation targets. Although no modifications other than disulfides have been found, hydroperoxide treatment has been shown to promote protein aggregation and inactivation. Subsequent studies involved an endothelial cell line (HUVECs). Preliminary cytotoxicity analyzes (detection of lactate dehydrogenase enzyme activity in the supernatant and incorporation of fluorescent probes into DNA) have shown that treatments with concentrations up to 400 µM are not lethal to cells in culture. Then, using alkylating agents impermeable to the cell membrane, urate hydroperoxide was shown to oxidize not only PDI but also total thiol proteins expressed on HUVECs surface. Wound healing experiments were performed to evaluate cell migration after treatment with urate hydroperoxide, but no difference was observed. However, incubation of the cells with the oxidizing agents urate hydroperoxide and diamide, inhibitors of both PDI and integrin and a thiol alkylator, resulted, at least for the first thirty minutes, in reduced cell adhesion to fibronectin. In addition, cells treated with urate hydroperoxide became more sensitive to detachment from the culture dish and exhibited alterations in morphology. Treatment with the peroxide also affected the redox homeostasis of the HUVECs, observed by a decrease in the GSH / GSSG ratio. Finally, indirect evidence was presented that uric acid is a substrate of peroxidasin, a heme peroxidase abundantly expressed in the vascular system. First, with the Amplex Red assay it was observed that the presence of uric acid in the reaction mixture resulted in lower oxidation rates of the reagent. Then, by LC-MS / MS, hydroxyisourate, which is the alcohol derived from urate hydroperoxide decomposition, was also identified in samples containing uric acid. Taken together, the data presented should contribute to a better understanding of the involvement of urate hydroperoxide in vascular oxidative processes − especially protein oxidation − that may be one mechanism associated to disturbances in endothelial function and vascular homeostasis

Influences of interaction between DENV-2-infected HUVECs and regulatory T cells on major inflam-matory cytokines / 中华微生物学和免疫学杂志

Objective To investigate the influences on major inflammatory cytokines after co-cul-turing regulatory T cells (Treg) with human umbilical vein endothelial cells ( HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Peripheral blood mononuclear cells (PBMC) were extrac-ted from concentrated human leukocytes by density gradient centrifugation. Treg cells were sorted by immu-nomagnetic beads. Expression of CD4,CD25 and CD127 molecules on the membrane of Treg cells was detec-ted by flow cytometry to identify the purity of Treg cells. HUVECs pretreated with or without sphingosine-1-phosphate S1P type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h were first infected with DENV-2 and then co-cultured with Treg cells. Expression of IL-6,IL-8,TNF-α,IL-10 and TGF-β at mRNA level was detected by real-time RT-PCR. Levels of IL-6,IL-8,IL-10 and TGF-β in the culture supernatants were detec-ted by a double-antibody sandwich ELISA. Results The purity of Treg cells was (84. 3±0. 5)%. Expression of NS1 at mRNA level in DENV-2-infected HUVECs first gradually increased and then decreased after reac-hing the peak at 24 h (3. 03±0. 26, P<0. 01). Enhanced expression of IL-6,IL-8 and TNF-α at mRNA level in HUVECs was observed after DENV-2 infection ( P<0. 01). Expression of these cytokines at every time point was decreased after co-culturing DENV-2-infected HUVECs with Treg cells ( P<0. 05),but was still higher than that before infection. CYM-5442 pretreatment decreased the expression of IL-6,IL-8 and TNF-α at mRNA level in DENV-2-infected HUVECs and inhibited the secretion of IL-10 and TGF-β by Treg cells that were co-cultured with DENV-2-infected HUVECs. Conclusion Primary HUVECs infected by DENV-2 can enhance the secretion of IL-10 and TGF-β by Treg cells,and the suppressive cytokines produced by Treg cells can reduce the production of inflammatory cytokines by DENV-2-infected HUVECs.

Protective effects of puerarin on high glucose-induced oxidative injury in HUVEC-12 cells / 中成药

AIM To explore the action mechanism of puerarin's protective effects against oxidative stress of HUVEC-12 cells induced by high glucose.METHODS HUVEC-12 cells cultured with 100 mmol/L glucose medium and 10,25,50 μmoL/L puerarin for 36 h had the cell proliferation,the levels of lactate dehydrogenase (LDH),intracellular reactive oxygen species (ROS),the activities of caspase-3,superoxide dismutase (SOD)and catalase (CAT),and the contents of malondialdehyde (MDA) and glutathione (GSH) measured.The mRNA expressions of SIRT1 and PGC-1α were detected by real-time flu orescence quantitative PCR,and the protein contents of SIRT1 and PGC-1 α were determined by enzyme-linked immunosorbent assay.RESULTS The puerarin treatment to HUVEC-12 cells resulted in markedly lowered LDH level,caspase-3 activity,intracellular levels of MDA and ROS,and notable improvement of the cell viability,the activities of SOD and CAT,GSH content,the mRNA expressions and the protein contents of SIRT1 and PGC-1α as well.CONCLUSION The protective effect of puerarin on high glucose-induced oxidative damage of HUVEC-12 cells may be attributed to the SIRT1/PGC-1α pathway activation.

Role of connexin 43 in X-ray induced apoptosis in human umbilical vein endothelial cells and its mechanism / 中华放射医学与防护杂志

Objective To study the role of Cx43 in X-ray induced apoptosis of HUVEC cells and its mechanism.Methods Flow cytometry was used to detect the apoptosis of HUVEC cells at 48-96 h after 10 Gy X-ray irradiation and at 72 h after irradiation of different doses.Western blot was used to detect the protein expressions of Cx43 and cleaved caspase-3 in HUVEC cells at 72 h after 0,5,10 and 20 Gy irradiation.Small interfering RNA was transfected into HUVEC cells to silence Cx43 expression,the Cx43 bearing plasmid was transfected into cells to overexpress Cx43.The effect of Cx43 knockdown or overexpression on apoptosis induction and cleaved caspase-3 protein expression were detected by flow cytometry and Western blot,respectively.Results The apoptosis of HUVEC increased significantly from 48 h to 96 h after X-ray irradiation and in a dose-dependent manner at 72 h after irradiation.The expression of Cx43 protein was negatively correlated with the dose but the expression of cleaved caspase-3 was positively correlated with the dose in the range of 0-20 Gy.After Cx43 silencing,the proportion of early apoptosis and apoptosis combined with dead cells were significantly higher than that of the siRNA control group(t =3.674,6.375,P ＜ 0.05).After Cx43 overexpression,the proportion of early apoptosis and apoptosis combined with dead cells were significantly lower than that of vector control group(t =9.399,11.190,P ＜ 0.05).The expression of cleaved caspase-3 in the Cx43 silencing group was higher than that in the siRNA control group,but this protein in the Cx43 overexpressed group was lower than that in the vector control group.Conclusions Cx43 may protect X-ray irradiated HUVEC cells from apoptosis by down-regulating the activation of caspase-3.

Protective effect of Polygonum orientale flower extract on H₂O₂-induced oxidative damage of HUVEC cells / 中国中药杂志

To investigate the protective effects and mechanism of Polygonum orientale flower extract on H₂O₂-induced oxidative damage of human umbilical vein endothelial cells (HUVEC), H₂O₂ was used to induce the oxidativestress damage on HUVEC cells and efforts were made to screen the low, medium and high drug concentrations of P.orientale flower extract. Cell viability was detected by the MTS assay. The content of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of superoxidedimutase (SOD) and catalase (CAT) were detected by biochemical kits. The mRNA and protein levels of Bax, Bcl-2 were detected respectively by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The protein level of cleaved caspase-3 was detected by Western blot. According to the results, the viability of HUVEC cells was reduced to around 55% after being treated with 120 μmol·L⁻¹ H₂O₂ for 0.5 h. Treatment of H₂O₂ also could increase LDH leakage rate and MDA content and attenuate the activities of SOD and CAT, up-regulate the expression level of Bax and cleaved caspase-3, and down-regulate the expression level of Bcl-2. As compared with H₂O₂ model group, P.orientale flower extract of 50-200 mg·L⁻¹ could increase the viability of HUVEC cells, reduce LDH release and MDA content, enhance the activities of SOD and CAT, down-regulate pro-apoptotic protein cleaved caspase-3 and Bax, and up-regulate apoptosis inhibitory protein Bcl-2. In summary, P.orientale flower extract showed a protective effect on H₂O₂-induced HUVEC cells injury, which may result from enhancing the cell capability of clearing the oxygen free radial, decreasing the production of lipid peroxidation and inhibiting apoptosis.

Xueshuantong capsule alleviates oxygen-glucose deprivation/reoxygenation-induced tight junction damage of HUVEC cells / 中国比较医学杂志

Objective To study the protective effect of Panax notoginseng saponins(PNS)and its components Rg1 and Rb1 on oxygen-glucose deprivation/reoxygenation(OGD/Reox)-induced tight junction damage. Methods Anaerobic box were used to induce OGD in HUVEC cells for 6 h followed by reoxygenation for 24 h. Transepithelial/endothelial electrical resistance(TEER)and cell permeability were detected,immunefluorescence was used to observe the ZO-1 and claudin-5 protein expression. Results PNS 20,40 mg/L and ginsenoside Rb1 significantly inhibited the OGD/Reox-induced decreased tight junction resistance,and the increased cell permeability(P< 0.05). PNS 20,40 mg/L and ginsenoside Rb1 partly restored the inter-cellular tight junctions which were regularly arranged on the cell membrane, and the cells displayed cobble stone-like arrangement. Conclusions PNS ameliorates ischemia-induced vascular endothelial cell tight junction damage via MMP-9 and VEGF/VEGFR2 signaling pathway. Rb1 is one of the effective monomer components.

Importance of the putative furin recognition site 742RNRR745 for antiangiogenic Sema3C activity in vitro

Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity.