Dapagliflozin attenuates endothelial cell pyroptosis and dysfunction induced by oxidized low-density lipoprotein / 中国组织工程研究

BACKGROUND:Dapagliflozin,an inhibitor of sodium-glucose cotransporter 2,can delay the progression of atherosclerosis by regulating glucose metabolism,inhibiting inflammation and improving endothelial cell function. OBJECTIVE:To study the effect of dapagliflozin on cell pyroptosis and endothelial dysfunction induced by oxidized low-density lipoprotein. METHODS:Human umbilical vein endothelial cells were divided into a control group(no intervention),a model group(treated with oxidized low-density lipoprotein for 24 hours),and a dapagliflozin group(treated with oxidized low-density lipoprotein + dapagliflozin for 24 hours).Endothelial cell proliferation activity was measured by cell counting kit-8 assay.The levels of intercellular adhesion molecule 1,vascular cell adhesion molecule 1,and monocyte chemotactic protein-1 in cell supernatant were detected using ELISA.Nitric oxide level in the cells was detected by nitrate reductase assay.The pyroptosis rate and characteristics of endothelial cells were detected by Hoechst 33342/PI fluorescence co-staining and lactate dehydrogenase release assay.The protein expression levels of NLRP3,caspase-1,GSDMD,interleukin-1β,and interleukin-18 were detected by western blot assay. RESULTS AND CONCLUSION:(1)Oxidized low-density lipoprotein could cause pyroptosis and dysfunction of endothelial cells.(2)Compared with the control group,the level of nitric oxide and cell activity were decreased(P<0.05),while lactate dehydrogenase,intercellular adhesion molecule 1,vascular cell adhesion molecule 1,and monocyte chemotactic protein-1 levels were significantly increased in the model group(P<0.05).Compared with the model group,cell activity and nitric oxide levels significantly increased(P<0.05),but lactate dehydrogenase,intercellular adhesion molecule 1,vascular cell adhesion molecule 1,and monocyte chemotactic protein-1 levels were significantly diminished in the dapagliflozin group(P<0.05).(3)Compared with the model group,cell pyroptosis rate and the protein expression of pyroptosis factor NLRP3,caspase-1,GSDMD,interleukin-18 and interleukin-1β significantly reduced in the dapagliflozin group(P<0.05).(4)The results indicate that dapagliflozin inhibits oxidized low-density lipoprotein-induced endothelial pyroptosis and ameliorates endothelial cell dysfunction.

3D-printed methacrylated gelatin scaffold loaded with fibroblasts promoting angiogenesis in vitro / 中国组织工程研究

BACKGROUND:Combining seed cells with 3D bioprinting technology enables the specific construction of various tissues and organs to meet the demands of tissue repair.However,further research is needed on the promotion of angiogenesis in damaged tissues. OBJECTIVE:By cultivating a 3D scaffold structure of methacrylated gelatin loaded with fibroblasts,obtaining the supernatant,and mixing it in different proportions with a complete culture medium to simulate the cellular microenvironment during tissue repair,this study aimed to explore the role of various cellular microenvironments in promoting angiogenesis in endothelial cells. METHODS:A methacrylated gelatin scaffold structure loaded with fibroblasts was prepared using an extrusion-based 3D bioprinting process.Hydrogel scaffold extract was prepared and mixed with a complete culture medium in ratios of 1:1,1:2,and 1:4 to obtain conditioned medium.Mouse embryonic fibroblasts BALB3T3 and human umbilical vein endothelial cells were co-cultured with complete medium(control group)and hydrogel scaffold extract,respectively.Cell proliferation was assessed using the CCK-8 assay and cell viability was analyzed using live/dead staining.Three kinds of conditioned medium and complete medium(control group)were used to co-culture with human umbilical vein endothelial cells for tube formulation assay,vascular genetic testing,and immunofluorescence staining of CD31. RESULTS AND CONCLUSION:(1)Scanning electron microscopy revealed that the methacrylated gelatin scaffold exhibited a porous structure,and rheological results demonstrated excellent mechanical properties of the hydrogel.CCK-8 assay and live/dead cell staining showed that the hydrogel scaffold extract had no obvious cytotoxicity.(2)Tube formulation assay indicated that the hydrogel showed the total length of cell tubules in 1:1 conditioned medium group was smaller than that in the control group(P<0.05).There were no statistical differences among the four groups in the number of vascular branches formed by endothelial cells(P>0.05).(3)qRT-PCR results showed that for vascular endothelial growth factor mRNA expression,the 1:2 conditioned medium group was lower than the 1:1 conditioned medium group on day 1(P<0.01).On day 3,the expression level of vascular endothelial growth factor in the 1:2 conditioned medium group was higher than that in the control group(P<0.01).On day 5,the cytokine expression level in the 1:2 conditioned medium group was significantly higher than that in the other three groups(P<0.01 or P<0.000 1).The expression in the 1:1 conditioned medium group was significantly lower than that in the other three groups(P<0.05 or P<0.01).On day 1,the expression level of basic fibroblast growth factor in the 1:1 conditioned medium group was significantly higher than that in the control group and 1:4 conditioned medium group(P<0.01,P<0.05).The expression was higher in the 1:2 conditioned medium group than that in the control group(P<0.05).On day 3,the expression levels of cytokines in the 1:4 conditioned medium group was higher than that in the control group(P<0.05).(4)On day 3,the expression of CD31 in the 1:2 conditioned medium group was higher than that in the control group and the 1:4 conditioned medium group(P<0.05).(5)The results indicate that the resulting conditioned media can simulate the microenvironment of vascular regeneration after tissue damage,promoting the vascularization process of endothelial cells.The best promotion of vascularization in endothelial cells was observed when the ratio of supernatant to complete culture medium was 1:2.

Effect of cyclic RNA hsa-circ-0001360 on homocysteine-induced apoptosis of human umbilical vein endothelial cells / 中国组织工程研究

BACKGROUND:Increased homocysteine level induces apoptosis of human umbilical vein endothelial cells,but the mechanism remains unclear. OBJECTIVE:To investigate the role of hsa-circ-0001360 in human umbilical vein endothelial cell apoptosis induced by homocysteine. METHODS:In vitro cultured human umbilical vein endothelial cells were divided into control group,homocysteine group,interference control group,interference control + homocysteine group,hsa-circ-0001360 interference group,hsa-circ-0001360 + homocysteine interference group,overexpression control group,overexpression control + homocysteine group,hsa-circ-0001360 overexpression group and hsa-circ-0001360 + homocysteine overexpression group.All groups were treated with 100 μmol/L homocysteine.After 72 hours of intervention,the expressions of apoptosis-related proteins Bax,Bcl-2,and Caspase-3 were detected by western blot assay.The apoptotic rate was detected by flow cytometry.Quantitative real-time PCR was used to detect the expression of hsa-circ-0001360. RESULTS AND CONCLUSION:(1)Compared with the control group,the expression of Caspase-3 and Bax was significantly increased(P<0.01),and the expression of Bcl-2 was significantly decreased(P<0.01),and the apoptotic rate was significantly increased(P<0.01)in the homocysteine group.(2)Compared with control group,the expression of hsa-circ-0001360 was significantly increased in the homocysteine group(P<0.01).(3)The expression of hsa-circ-0001360 was significantly higher in the cytoplasm than that in the nucleus(P<0.01).(4)Compared with the interference control C group and interference control + homocysteine group,the expressions of Caspase-3 and Bax were significantly decreased(P<0.01),while the expression of Bcl-2 was significantly increased(P<0.01);the apoptotic rate was significantly decreased(P<0.01)in sh-hsa-circ-0001360 interference group and sh-hsa-circ-0001360 + homocysteine interference group.(5)Compared with overexpression control group and overexpression control + homocysteine group,the expressions of Caspase-3 and Bax were significantly increased(P<0.01),while the expression of Bcl-2 was significantly decreased(P<0.01);the apoptotic rate was significantly increased(P<0.01)in the hsa-circ-0001360 overexpression group and the hsa-circ-0001360 + homocysteine overexpression group.(6)In conclusion,hsa-circ-0001360 can promote the apoptosis of human umbilical vein endothelial cells induced by homocysteine.

Effect of procalcitonin on lipopolysaccharide-induced expression of nucleotide-binding oligomerization domain-like receptor protein 3 and caspase-1 in human umbilical vein endothelial cells / 中国当代儿科杂志

OBJECTIVES@#To study the effect of procalcitonin (PCT) on lipopolysaccharide (LPS)-induced expression of the pyroptosis-related proteins nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and caspase-1 in human umbilical vein endothelial cells (HUVECs).@*METHODS@#HUVECs were induced by LPS to establish a model of sepsis-induced inflammatory endothelial cell injury. The experiment was divided into two parts. In the first part, HUVECs were randomly divided into four groups: normal control, LPS (1 μg/mL), PCT (10 ng/mL), and LPS+PCT (n=3 each). In the second part, HUVECs were randomly grouped: normal control, LPS, and LPS+PCT of different concentrations (0.1, 1, 10, and 100 ng/mL) (n=3 each). Quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of NLRP3 and caspase-1 in each group.@*RESULTS@#In the first experiment: compared with the normal control group, the PCT, LPS, and LPS+PCT groups had significantly upregulated mRNA and protein expression levels of NLRP3 and caspase-1 (P<0.05); compared with the LPS group, the LPS+PCT group had significantly downregulated mRNA and protein expression levels of NLRP3 and caspase-1 (P<0.05). In the second experiment: compared with those in the LPS group, the mRNA and protein expression levels of NLRP3 and caspase-1 in the LPS+PCT of different concentrations groups were significantly downregulated in a concentration-dependent manner (P<0.05).@*CONCLUSIONS@#LPS can promote the expression of the pyroptosis-related proteins NLRP3 and caspase-1 in HUVECs, while PCT can inhibit the LPS-induced expression of the pyroptosis-related proteins NLRP3 and caspase-1 in HUVECs in a concentration-dependent manner.

Puerarin reduces oxidative damage to vascular endothelial cells by improving mitochondrial respiratory function / 药学学报

This study investigated the effect of puerarin on human umbilical vein endothelial cells (HUVEC) injured with hydrogen peroxide (H2O2). HUVEC were divided into three groups: a control group, a model group (H2O2 400 μmol·L-1) and a puerarin-treated group (3, 10, 30 and 100 μmol·L-1). HUVEC were cultured with varied concentration of puerarin for 2 h and treated with H2O2 for another 24 h. Cell proliferation was detected by a CCK-8 assay. The mitochondrial membrane potential was measured by a JC-1 fluorescent probe. A transwell chamber assay was adopted to observe cell migration ability. Mitochondrial respiratory function was measured in a two-chamber titration injection respirometer (Oxygraph-2k). The expression of interleukin-1β (IL-1β), interleukin-18 (IL-18) and tumor necrosis factor-α (TNF-α) was detected by quantitative real-time PCR. The expression of pyroptosis-mediated proteins, including cleaved-cysteinyl aspartate-specific proteinase-1 (caspase-1), N-gasdermin D (N-GSDMD), NOD-like receptor protein 3 (NLRP3) and purinergic ligand-gated ion channel 7 receptor (P2X7R) was detected by Western blot. The results show that 400 μmol·L-1 H2O2 treatment for 24 h causes obvious damage to HUVEC. Compared with the model group, puerarin protected against cellular injury in a dose-dependent manner, with the greatest effect at a dose of 30 and 100 μmol·L-1. Puerarin significantly decreased the mitochondrial membrane potential and improved mitochondrial function. Puerarin inhibited cell migration induced by H2O2, suppressed the expression of IL-1β, IL-18 and TNF-α, and down-regulated the pyroptosis-mediated protein. These changes are statistically significant (P < 0.05). These findings demonstrate that puerarin has a protective effect against H2O2-induced oxidative damage of HUVEC by inhibiting the migration of HUVEC cells. The mechanism may be related to improved mitochondrial respiratory function and inhibition of pyroptosis.

Effect of endogenous nitric oxide on superoxide dismutase-1 activity and apoptosis of vascular endothelial cells / 中华实用儿科临床杂志

Objective:To investigate the regulatory effects of endogenous nitric oxide (NO) on the activity of superoxide dismutase-1 (SOD1) and apoptosis of human umbilical vein endothelial cells (HUVECs).Methods:HUVECs were taken as the research object.The endothelial NO synthase (eNOS) short hairpin RNA(shRNA) lentivirus was employed to transfect HUVECs to knock down eNOS.HUVECs were divided in 4 groups: the scramble group, the eNOS shRNA group, the eNOS shRNA + sodium nitroprusside(SNP) group and the eNOS shRNA+ SNP+ tris (2-carboxyethyl) phosphine hydrochloride (TCEP) group.The protein expressions of eNOS and SOD1 dimer/monomer in cells were detected by western blot.The activity of SOD was detected by the enzyme-linked immunosorbent assay.The NO content in cells was detected with NO fluorescence probe.The level of superoxide anion in HUVECs was detected with dihydropyridine (DHE). The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was adopted to detect the apoptosis of HUVECs in situ.Results:Compared with the scramble group, the endogenous NO content (2.690±0.420 vs.15.029±2.193, P<0.01), eNOS protein expression (1.000±0.778 vs.3.141±0.199, P<0.01), SOD1 dimer/monomer ratio (4.6±1.0 vs.7.6±2.0, P<0.05) and SOD activity [(0.432±0.254) Carmen′s unit/10 4 cell vs.(1.000±0.116) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of intracellular superoxide anion (11.180±1.560 vs.6.146±1.007, P<0.01) and HUVECs apoptosis [75.0 (55.0, 100.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA group.Compared with the eNOS shRNA group, the content of endogenous NO (16.705±0.116 vs.2.690±0.420, P<0.01), the ratio of SOD1 dimer/monomer (7.3±2.0 vs.4.6±1.0, P<0.05) and the activity of SOD [(0.737±0.060) Carmen′s unit/10 4 cell vs.(0.432±0.254) Carmen′s unit/10 4 cell, P<0.05] were significantly increased, while the level of superoxide anion (6.897±1.648 vs.11.180±1.560, P<0.01) and the HUVECs apoptosis [0 (0, 0)% vs.75.0 (55.0, 100.0)%, P<0.01] were significantly decreased in the eNOS shRNA+ SNP group.Compared with the eNOS shRNA + SNP group, the ratio of SOD1 dimer/monomer (4.4±0.9 vs.7.3±2.0, P<0.05) and the activity of SOD [(0.214±0.084) Carmen′s unit/10 4 cell vs.(0.737±0.060) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of superoxide anion (10.917±1.552 vs.6.897±1.640, P<0.01) and the apoptosis level of HUVECs[63.6 (55.0, 90.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA+ SNP+ TCEP group.However, there was no significant difference in the NO content (16.112±0.926 vs.16.705±0.116, P>0.05). Conclusions:Endogenous NO could effectively antagonize the apoptosis of endothelial cells by increasing the cysteine-dependent SOD1 dimer/monomer ratio, enhancing SOD activity and inhibiting the accumulation of reactive oxygen species.

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

Experimental study of enhanced anti-pancreatic cancer effects by combination treatment with HUVEC vaccine and gemcitabine / 中国药理学通报

Aim To investigate whether low dose gem- citabine ( GEM) could enhance the anti-pancreatic cancer effects of human umbilical vein endothelial cell (HUVEC) vaccine.Methods C57BL/6 mice were randomly divided into four groups; PBS control group, GEM group, HUVEC vaccine group and HUVEC vaccine combined GEM group ( HUVEC-GEM group).Mice were inoculated with Pan02 pancreatic cancer cells to establish a subcutaneous xenograft model to observe tumor growth and adverse reactions in tumor bearing mice.Whether GEM could enhance the immune responses induced by HUVEC vaccine was determined by ELISA analysis of the immune serum, splenic lymphocyte proliferation assay, cytotoxic T lymphocyte killing assay and IFN-7 content assay.Results The results of subcutaneous transplantation tumor model showed that the introduction of GEM into the HU-VEC vaccine treatment could enhance the therapeutic anti-pancreatic cancer effects of HUVEC vaccine.Enzyme-linked immunoassay results confirmed that GEM acted as an immune adjuvant could effectively increase the HUVEC antibodies and I FN-7 level in the immune serum of mice.The results of lymphocyte proliferation experiment and CTL killing activity assay indicated that GEM could effectively enhance the spleen lymphocyte transformation activity and CTL killing ability of HUVEC vaccine immunized mice.Conclusions Low dose GEM could enhance the immune responses induced by HUVEC vaccine and thus enhance the anti- Pan02 pancreatic cancer effects of HUVEC vaccine.

The effect and mechanism of YAP1 expression on placental angiogenesis in preeclampsia exposed to hypoxia environment / 西安交通大学学报(医学版)

【Objective】 To investigate the effect and mechanism of inhibiting Yes-associated protein1 (YAP1) expression by verteporfin on proliferation, migration and invasion of human umbilical vein endothelial cells (HUVECs) exposed to hypoxia environment and the possible mechanisms that further affect placental angiogenesis in preeclampsia. 【Methods】 MTT method was used to detect the cell viability of HUVECs at different concentrations (0, 4, 8, 12 and 16 μg/mL) after 12 h and 24 h treatment with verteporfin under hypoxia and calculate the IC50 value to select the subsequent experimental drug concentration. Flow cytometry was made to analyze verteporfin’s effect on HUVEC apoptosis in hypoxic environment. The wound healing assay and Transwell invasion assay were used to determine the effect of verteporfin on HUVEC cell migration and invasion abilities under hypoxic environment. Angiogenesis test was used to detect the effect of verteporfin on the angiogenesis of HUVECs under hypoxic environment. The effects of verteporfin on the expression levels of YAP1 and TEAD1 in Hippo signaling pathway under normoxia and hypoxia were determined by Western blotting. 【Results】 Under hypoxic environment, verteporfin could inhibit the proliferation of HUVECs by calculating the IC50 value, the subsequent experimental group selected 16 μg/mL verteporfin to treat cells. Flow cytometry showed that verteporfin induced the apoptosis rate of HUVECs under hypoxia (P<0.01). The results of wound healing, Transwell invasion and the angiogenesis experiments confirmed that compared with the control group, verteporfin could inhibit the migration, invasion and angiogenesis of HUVECs in hypoxic environment (P<0.05). Western blotting assay indicated that under normoxia and hypoxia, the expressions of YAP1 and TEAD1 were reduced (P<0.01). 【Conclusion】 In hypoxic environment, verteporfin inhibits the proliferation of HUVECs by inhibiting the expressions of YAP1 and TEAD1, and reduces the migration, invasion and angiogenesis of HUVECs. It is confirmed that the Hippo-YAP1 signaling pathway may affect the placental angiogenesis of preeclampsia and participate in the occurrence of preeclampsia by regulating the proliferation and invasion of vascular endothelial cells.

Effect of brain-derived microvesicles on cytoskeleton of human umbilical vein endothelial cells / 中华神经医学杂志

Objective To observe the effect of brain-derived microvesicles (BDMVs) on cytoskeleton in human umbilical vein endothelial cells (HUVECs).Methods BDMVs were prepared in vitro and identified by transmission electron microscopy and particle size identification.HUVECs were co-cultured with PKH26-1abeled BDMVs for 0.5,1,and 2 h;flow cytometry was used to detect the phagocytosis of HUVECs for BDMVs at different time points.HUVECs cultured in vitro were divided into control group,BDMVs treatment group and nimodipine treatment group;cells in the BDMVs treatment group were given 1.5× 107/mL BDMVs;cells in the nimodipine treatment group were pretreated with 2 μg nimodipine (0.2 mg/mL) for 10 min,and then,given 1.5×107/mL BDMVs.After being stained with rhodamine-labeled phalloidin,the fluorescence intensity and number of stress fibers of fibroactin in HUVECs were observed by laser confocal microscopy.Results BDMVs had complete membrane structure with a diameter of 100-1000 nm under transmission electron microscopy.The proportion of cells phagocytizing BDMVs increased significantly with prolonged incubation time,enjoying significant differences (0.5h:22.7％±1.2％;1 h:52.3％±1.3％;2h:71.6％±1.9％,P＜0.05).Laser confocal microscopy showed that,as compared with the control group,the fluorescence intensity ofcytoskeletal protein was obviously increased and the number of stress fibers increased was obviously larger in the BDMVs treatment group.As compared with those in the BDMVs treatment group,the fluorescence intensity of cytoskeletal protein was decreased and the number of stress fibers was obviously smaller in the nimodipine group.Conclusion The role of BDMVs in phagocytosis of HUVECs becomes stronger as time being prolonged,and BDMVs phagocytosis leads to cytoskeletal remodeling,which can be partially blocked by nimodipine.

Docosahexaenoic acid reduces adenosine triphosphate-induced calcium influx via inhibition of store-operated calcium channels and enhances baseline endothelial nitric oxide synthase phosphorylation in human endothelial cells

Docosahexaenoic acid (DHA), an omega-3-fatty acid, modulates multiple cellular functions. In this study, we addressed the effects of DHA on human umbilical vein endothelial cell calcium transient and endothelial nitric oxide synthase (eNOS) phosphorylation under control and adenosine triphosphate (ATP, 100 µM) stimulated conditions. Cells were treated for 48 h with DHA concentrations from 3 to 50 µM. Calcium transient was measured using the fluorescent dye Fura-2-AM and eNOS phosphorylation was addressed by western blot. DHA dose-dependently reduced the ATP stimulated Ca²⁺-transient. This effect was preserved in the presence of BAPTA (10 and 20 µM) which chelated the intracellular calcium, but eliminated after withdrawal of extracellular calcium, application of 2-aminoethoxy-diphenylborane (75 µM) to inhibit store-operated calcium channel or thapsigargin (2 µM) to delete calcium store. In addition, DHA (12 µM) increased ser1177/thr495 phosphorylation of eNOS under baseline conditions but had no significant effect on this ratio under conditions of ATP stimulation. In conclusion, DHA dose-dependently inhibited the ATP-induced calcium transient, probably via store-operated calcium channels. Furthermore, DHA changed eNOS phosphorylation suggesting activation of the enzyme. Hence, DHA may shift the regulation of eNOS away from a Ca²⁺ activated mode to a preferentially controlled phosphorylation mode.

Effects of short-chain acyl-CoA dehydrogenase on human umbilical vein endothelial cell apoptosis / 中华危重病急救医学

Objective To observe the changes of short-chain acyl-CoA dehydrogenase (SCAD) expression on human umbilical vein endothelial cell (HUVEC) apoptosis and investigate its relationship with apoptosis. Methods The HUVEC was cultured normally for 2-3 days. The apoptotic model of HUVEC was established by tert-butyl hydrogen peroxide (tBHP). The HUVEC was treated by different concentrations of tBHP (0, 10, 20, 30, 40, 50 μmol/L) for 12 hours and different time (0, 3, 6, 9, 12 hours) with 50 μmol/L tBHP to establish the apoptotic model of HUVEC. The cell viability was detected by methyl thiazolyl tetrazolium (MTT), the mRNA expression of SCAD was determined by real-time polymerase chain reaction (PCR), the protein expression of SCAD was achieved by Western Blot. The best concentrate and time were determined to interfere the HUVEC to achieve the apoptotic model of HUVEC. The SCAD gene of HUVEC was knocked down by RNA interference sequence (siRNA274, siRNA414, siRNA679). The mRNA expression of SCAD, the protein expression of SCAD and the activity of SCAD enzyme were detected to achieve the best RNA interference sequence. The HUVEC was intervened by the best RNA interference sequence and tBHP. The cell activity and apoptosis rate, the enzyme activity of SCAD, the mRNA and protein expression of SCAD, the contents of reactive oxygen species (ROS), aderosine triphosphate (ATP) and free fatty acid (FFA) were detected to observe the effect of SCAD on apoptosis of HUVEC. Results ① The cell viability, the mRNA expression and the protein expression of SCAD were decreased gradually in a concentration and time dependent manner with the increase of tBHP concentration and the prolongation of intervention time. The decline was most significant in the group of the 50 μmol/L tBHP to interfere HUVEC for 12 hours. ② The siRNA679 transfection was the most significant in reducing SCAD mRNA and protein expressions among the three interference sequences (siRNA274, siRNA414, siRNA679). ③ Compare with blank control group, the cell viability was significantly decreased in the siRNA679 group (A value: 0.48±0.09 vs. 1.00±0.09, P < 0.01), the apoptotic rate of HUVEC was significantly increased [(29.96±2.09)% vs. (2.90±1.90)%, P < 0.01], the expression of SCAD mRNA and SCAD protein, the activity of SCAD enzyme and the content of ATP were significantly decreased [SCAD mRNA (2-ΔΔCt): 0.50±0.16 vs. 1.34±0.12, SCAD/α-Tubulin: 0.67±0.11 vs. 1.00±0.06, the activity of SCAD enzyme (kU/g): 0.38±0.04 vs. 0.53±0.04, the content of ATP (μmol/g): 0.14±0.02 vs. 0.19±0.01, all P < 0.05], the contents of FFA and ROS were significantly increased [FFA (nmol/g): 0.84±0.07 vs. 0.47±0.04, ROS (average fluorescence intensity): 647.5±23.7 vs. 434.2±46.5, both P < 0.01]. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as HUVEC treated with tBHP. Conclusions Down-regulation of SCAD may play an important role in HUVEC apoptosis. Increase in the expression of SCAD may become an important part in intervening HUVEC apoptosis.

Study on Improvement Effects of Different Proportions of Total Ginsenoside of Ginseng ，Total Glucosides of Moutan Cortex and Paeonol Containing Serum on HUVEC Injury Induced by H 2O2 / 中国药房

OBJECTIVE： To study improvement effects of different proportions of total glucosides of ginseng （TGG）， total glucosides of moutan cortex （TGM） and paeonol containing serum on the injury of human umbilical vein endothelial cells （HUVEC） injury induced by hydrogen peroxide （H2O2）， screen the optimal proportion and investigate its mechanism. METHODS： The rats were randomly divided into blank group （distilled water）， TGG group （TGG， 2.025 g/kg）， TGM group （TGM， 4.05      g/kg） and paeonol group （paeonol， 1.08 g/kg）， with 12 rats in each group. They were given relevant medicine twice a day for consecutive 7 days. 1 h after last medication， the blood samples were collected via abdominal aorta to prepare drug containing serum. Using survival rate of HUVEC as evaluation indexes， different proportions of TGG， TGM and paeonol containing serum as factors， L9（34） orthogonal test was designed to optimize the optimal proportion of 3 kinds of drug containing serum. HUVEC were divided into blank group， model group， TGG group， TGM group， paeonol group and optimal proportion group. Except that blank group were treated with relevant medium， other group were treated with 1.2 mmol/L H2O2 to induce HUVEC injury， and then TGG group （volume fraction of drug containing serum was 0.000 5％）， TGM group （volume fraction of drug containing serum was 0.000 5％）， paeonol group （volume fraction of drug containing serum was 1％） and optimal proportion group were intervened with drug containing serum. The levels of LDH， NO and ET-1 in cells were detected by microplate method and ELISA. RESULTS： The optimal proportion of drug containing serum were TGG 0.000 5％， TGM 0.000 5％ and paeonol 1％. Compared with blank group， the levels of LDH and ET-1 were higher in model group （P＜0.01）， while NO level was lower （P＜0.05）. Compared with model group， the levels of NO were higher in TGG group， TGM group and optimal proportion group （P＜0.01）， while the levels of LDH and ET-1 were lower （P＜0.05 or P＜0.01）. Compared with TGG group， TGM group and paeonol group， the level of LDH was lower in optimal proportion group （P＜0.05 or P＜0.01）， while the level of NO was higher （P＜0.05 or P＜0.01）. CONCLUSIONS： TGG and TGM combined with paeonol can significantly improve HUVEC injury induced by H2O2， and the mechanism of which may be associated with the decrease of LDH and ET-1 and the increase of NO.

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.

Paeoniflorin Promotes Angiogenesis in A Vascular Insufficiency Model of Zebrafish in vivo and in Human Umbilical Vein Endothelial Cells in vitro / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the pro-angiogenic effects of paeoniflorin (PF) in a vascular insufficiency model of zebrafish and in human umbilical vein endothelial cells (HUVECs).</p><p><b>METHODS</b>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 mRNA expression of fms-like tyrosine kinase-1 (flt-1), kinase insert domain receptor (kdr), kinase insert domain receptor like (kdrl) and von Willebrand factor (vWF) 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.</p><p><b>RESULTS</b>PF (6.25-100 μmol/L) could rescue VRI-induced blood vessel loss in zebrafish and PF (25-100 μmol/L), thereby restoring the mRNA expressions of flt-1, kdr, kdrl and vWF, 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.</p><p><b>CONCLUSION</b>PF could promote angiogenesis in a vascular insufficiency model of zebrafish in vivo and in HUVECs in vitro.</p>

Influence of aspirin on platelet activation during vascular endothelial injury induced by high blood glucose fluctuations / 中国药理学通报

Aim To investigate the influence of aspirin on platelet activation during vascular endothelial injury induced by high blood glucose fluctuations .Methods In this study , "fluctuant high blood glucose cultured human umbilical vein endothelial cell ( HUVEC ) mod-el" and "human platelet-HUVEC supernate experi-mental system" were established in vitro, as well as type 2 diabetes mellitus ( T2DM) rat model with high blood glucose fluctuation in vivo. There were four groups: normal glucose ( N ) , steady high glucose (W), fluctuant high glucose ( B), and aspirin group ( ASA) .At the end of the study , the peripheral blood platelet maximum aggregation rate , levels of sE-selec-tin, von Willebrand factor ( vWF ) and platelet mem-brane protein level of CD62p were determined.Results In comparison with N group, levels of sE-selectin,vWF, the platelet maximum aggregation rate and plate-let membrane protein level of CD 62 p in W group and B group all significantly increased ( P <0.01 ) , mean-while B group significantly increased further compared with W group ( P <0.05 or P <0.01 ) .Pretreatment with ASA significantly decreased the elevated levels of sE-selectin, vWF, the platelet maximum aggregation rate and CD62p induced by high glucose fluctuations (P<0.01).Conclusions High blood glucose fluctu-ations can not only aggravate endothelial injury , but al-so promote platelet aggregation obviously , while aspirin has obvious antagonistic effects on these effects .

Mechanism of gambogenic acid in resisting angiogenesis of lung cancer in vitro / 中国中药杂志

The aim of this paper was to observe the effect of gambogenic acid on angiogenesis of lung cancer and its preliminary mechanism. After culturing lung adenocarcinoma A549 cells, the conditioned medium was treated with gambogenic acid and then used to culture human umbilical vein endothelial cells (HUVECs) to establish the indirect contact cell co-culture system. A two-dimensional culture model of HUVEC was established with matrigel to observe the effect of gambogenic acid on angiogenesis. DAPI staining was used to observe the morphological changes in HUVEC cells after treatment with gambogenic acid under the fluorescence microscope. Annexin V-FITC/PI staining and flow cytometry analysis were used to determine gambogenic acid's effect on HUVEC cell apoptosis rate. The protein expressions of PI3K, p-PI3K, Akt, p-Akt were measured by Western blot. PTEN-siRNA was transfected into cells, and RT-PCR was used to detect the expression levels of PI3K and Akt genes. Gambogenic acid can significantly inhibit angiogenesis, and its inhibitory effect was dose-dependent. DAPI staining showed apoptotic morphological features of HUVEC cells under fluorescence microscope. Annexin V-FITC/PI staining showed that gambogenic acid induced apoptosis in HUVECs. The results of Western blot showed that the expressions of p-PI3K and p-Akt protein were down-regulated with gambogenic acid, while the expressions of PI3K and Akt protein was insignificant. The results of RT-PCR indicated that the expressions of PI3K and Akt protein were up-regulated by PTEN siRNA. Gambogenic acid can inhibit angiogenesis in lung cancer in vitro, and the mechanism of inhibiting angiogenesis may be related to the PI3K/Akt signaling pathway.

Co-culture of human breast adenocarcinoma cells and human umbilical vein endothelial cells to mimic in vivo tumor microenvironment / 药学学报

The development of tumor tissue is a complicated process, which is closely related to tumor microenvironment. In order to simulate the tumor tissue in vivo, non-contact co-culture of human breast adenocarcinoma cells (MCF-7 cells) and human umbilical vein endothelial cells (HUVECs cells) using transwell cell culture plate was developed in this study. The cell viability, morphology, cell resistance, cell cycle and vascular endothelial growth factor (VEGF) protein content of co-cultured MCF-7 and HUVECs cells were investigated, and compared with those of separately cultivated MCF-7 and HUVECs cells during the same period. Different to the separately cultured MCF-7 and HUVECs cells, co-cultured MCF-7 and HUVECs cells exhibited higher cell viability, deformed cell morphology, lower cell resistance, higher proportion of S and G2/M phases and higher VEGF protein content (about 1.4−2 times). The double cell model via non-contact co-culture of MCF-7 and HUVECs cells constructed in this study could simulate the interaction between tumor cells and tumor vascular endothelial cells in vivo, which may provide a more realistic model for subsequent study of drug release system in the control of breast cancer in vitro.

Effect of germacrone in alleviating HUVECs damaged by H2O2-induced oxidative stress / 中国中药杂志

This study focuses on the protective effect of germacrone on human umbilical vein endothelial cells(HUVECs) damaged by H2O2-induced oxidative stress and its possible mechanisms. The oxidative damage model was established by using 500 μmol•L⁻¹ H2O2 to treat HUVECs for 3 hours, and then protected with different concentrations of germacrone for 24 hours. The effect of germacrone on cell viability of HUVECs damaged by H2O2 was detected by MTT. The contents of PGI2, TXB2, ET-1, t-PA, PAI-1, TNF-α and IL-6 were detected by ELISA. The content of NO was detected by using nitrate reductase method. Colorimetry was used to detect NOS and GSH-Px. The contents of MDA, SOD and LDH were detected by TBA, WST-1 and microplate respectively. Apoptosis was observed by Hoechst 33258 fluorescent staining. The mRNA expressions of Bax, Bcl-2 and Caspase-3 in cells were detected by RT-PCR. The results showed that the cell damage rate was 52% after treated with 500 μmol•L⁻¹ H2O2 for 3 hours. The cell activity was increasing with the rise of germacrone concentration within the range of 20-200 mol•L⁻¹. Compared with normal group, the contents of PGI2, NO, T-NOS, t-PA, SOD, GSH-Px and Bcl-2 mRNA expressions were lower after damaged with H2O2. The contents of PAI-1, ET-1, IL-6, TNF-α, TXB2, LDH, MDA, Bax mRNA and Caspase-3 mRNA expressions were increased. Compared with model group, the contents of PGI2, NO, T-NOS, t-PA, SOD, GSH-Px and Bcl-2 mRNA expressions were increased after treated with germacrone. The contents of PAI-1, ET-1, IL-6, TNF-α, TXB2, LDH, MDA, Bax mRNA and Caspase-3 mRNA expressions were lower after treated with germacrone. According to Hoechst 33258 fluorescence staining, compared with normal group, the cell membrane and the nucleus showed strong dense blue fluorescence, and the number of cells significantly decreased in model group. Compared with model group, blue fluorescence intensity decreased in drug group. The above findings demonstrate that germacrone may improve the effect on HUVECs damaged by H2O2-induced oxidative stress by resisting oxidation and inhibiting cell apoptosis.

CCM3 down-regulating the lead acetate-induced migration of human umbilical vein endothelial cells / 中国职业医学

OBJECTIVE: To investigate the effects of knockout cerebral cavernous malformation(CCM) virulence gene CCM3 on the migration induced by lead acetate in immortalized human umbilical vein endothelial cells(HUVECs) and to explore the possible mechanism of endoplasmic reticulum stress(ERS). METHODS: CCM3 wildtype(CCM3-WT) and CCM3 knockout(CCM3-KO) HUVECs were used as experimental cells. a) CCM3-WT and CCM3-KO HUVECs were treated with lead acetate at 0,10,50 and 200 μmol/L for 24 hours. The migration of these cells was observed by woundhealing assay. b) CCM3-WT and CCM3-KO HUVECs were treated with lead acetate at 0,10,50 and 200 μmol/L for 24 hours,and at 50 μmol/L for 0,6,12,24 and 48 hours,and the mRNA expression of genes of unfolded protein response pathway were detected by quantitative real-time polymerase chain reaction; the protein expression of glucose-regulated protein 78(GRP78) was detected by Western blotting. c) CCM3-WT and CCM3-KO HUVECs were divided into lead exposure group and tauroursodeoxycholic acid(TUDCA) group. The former was treated with 50 μmol/L lead acetate for 24 hours,and the latter was pre-treated with ERS inhibitor TUDCA,followed by 50 μmol/L lead acetate. The migration of these cells was observed by wound-healing assay. RESULTS: a) The migration of CCM3-WT and CCM3-KO cells decreased and showed a dose-effect relationship with the increase of lead acetate concentration(P < 0. 05). b) The mRNA relative expression of the GRP78,protein kinase-like endoplasmic reticulum kinase(PERK),transcription activator 4(ATF4) and CCAAT enhancer binding homologous protein(CHOP) in CCM3-KO cells treated with 10,50 and 200 μmol/L lead acetate were higher than that in CCM3-WT cells at the same doses,except for the GRP78 in CCM3-KO cells treated with10 μmol/L lead acetate(P < 0. 05). The mRNA expression of PERK and CHOP in CCM3-KO cells increased in a timeeffect relationship with the increase of lead-exposure time(P < 0. 05). The mRNA relative expression of the four genes in CCM3-KO cells were higher than those in CCM3-WT cells at 48 hours(P < 0. 05). When cells were treated with 50μmol/L lead acetate,the protein expression of GRP78 in CCM3-KO cells was higher than that in CCM3-WT cells(P <0. 05),and the protein expression of GRP78 in CCM3-KO cells increased in a time-effect relationship with the increase of lead-exposure time(P < 0. 05). c) The cell migration of TUDCA group was lower than that of lead-exposure group(P <0. 05). CONCLUSION: Lead acetate may activate ERS by activating the PERK-ATF4-CHOP signaling pathway,thereby reducing the migration of HUVECs. CCM3 gene has a protective effect on cell migration.