Adenosine ; metabolism ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans

OBJECTIVE: To explore the molecular mechanisms of Porphyromonas gingivalis infection-induced umbilical vein endothelial barrier dysfunction in vitro. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and after the formation of the endothelial barrier, the cells were infected with P. gingivals at a multiplicity of infection (MOI). The transepithelial electrical resistance (TEER) of the cell barrier was measured, and FITC-dextran trans-endothelial permeability assay and bacterial translocation assay were performed to assess the endothelial barrier function. The expression levels of cell junction proteins including ZO-1, occludin and VE-cadherin in the cells were examined by qRT-PCR and Western blotting. RESULTS: In freshly seeded HUVECs, TEER increased until reaching the maximum on Day 5 (94 Ωcm²), suggesting the formation of the endothelial barrier. P. gingivals infection caused an increase of the permeability of the endothelial barrier as early as 0.5 h after bacterial inoculation, and the barrier function further exacerbated with time, as shown by significantly lowered TEER, increased permeability of FITC-dextran (40 000/70 000), and increased translocation of SYTO9-E. coli cross the barrier. MTT assay suggested that P. gingivals infection did not significantly affect the proliferation of HUVECs (P>0.05), but in P. gingivalsinfected cells, the expressions of ZO-1, occludin and VE-cadherin increased significantly at 24 and 48 h after bacterial inoculation (P < 0.05). CONCLUSION P. gingivals may disrupt the endothelial barrier function by down-regulating the expressions of the cell junction proteins (ZO-1, occludin, VE-cadherin) and increasing the permeability of the endothelial barrier.
Humans ; Cadherins/metabolism* ; Escherichia coli/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Occludin ; Porphyromonas gingivalis/metabolism* ; Umbilical Veins/metabolism*

OBJECTIVETo establish a stable cell line overexpression heme oxygenase-1 (HO-1) mediated by a modified lentivirus system and identify its function.

METHODSThe HO-1 gene was amplified by polymerase chain reaction and cloned into the modified pLentiLox3.7 expression vectors. The recombinant plasmids were transfected into HEK293T cells and the HO-1 was detected by Western blot. The recombinant plasmids were transfected into HEK293T cells to produce the viruses, with the helping plasmids including plp1, plp2, and VSVG. HEK293T cells were infected by the viruses and the cells that can express HO-1 were identified by Western blot. The reactive oxygen species were detected in the HO-1-overexpression HEK293T cells and the normal cells after the adding of hydrogen peroxide. The same experiment was performed with human umbilical vein endothelial cells.

RESULTSThe stable cell line that can overexpress HO-1 was established, which was verified by Western blot. The reactive oxygen species in the HO-1-overexpression HEK293T cells and human umbilical vein endothelial cells decreased obviously after exposure to hydrogen peroxide.

CONCLUSIONSThe lentivirus-carrying HO-1 was successfully packaged and the stable cell line overexpression HO-1 was established. HO-1 can play a protective role in the course of oxidative damage.

Cell Line ; HEK293 Cells ; Heme Oxygenase-1 ; metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Plasmids ; Transfection

Caveolin 1 ; genetics ; metabolism ; Cells, Cultured ; Homocysteine ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo prepare the polyclonal antibody of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), and to determine the expression of EOLA1 in human umbilical vein endothelial cell (HUVEC).

METHODSThe protein samples (sample 1 and 2) expressing EOLA1 were purified and renatured. The protein concentrations were determined with bicinchoninic acid assay. The protein samples were identified with peptide mass fingerprinting (PMF) analysis. Protein sample with higher coincidence rate of amino acid sequence with theoretic protein was used to inoculate 4 mice; another 4 mice inoculated with adjuvant were used as control. Serum was isolated from collected mice blood. Polyclonal antibody of EOLA1 was purified with saturated ammonium sulfate precipitation, and was determined with ELISA for the titer (data were denoted by absorbance value). The expression of EOLA1 in HUVEC was determined with Western blot.

RESULTSThe concentration of protein sample 1 and 2 was respectively 0.124 16 mg/mL and 0.132 15 mg/mL. According to PMF analysis, the coincidence rate of amino acid sequence between protein samples and theoretic protein were 32% (protein sample 1) and 24% (protein sample 2). The polyclonal antibody of EOLA1 with titer more than 1:10 000 was obtained from mice inoculated with protein sample 1. The expression of EOLA1 protein in HUVEC was determined with polyclonal antibody of EOLA1.

CONCLUSIONSThe polyclonal antibody of EOLA1 can be prepared by inoculating mice with EOLA1 prokaryotic expressing protein, which can be used for determination of EOLA1 protein.

Animals ; Antibodies ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; metabolism ; Membrane Proteins ; immunology ; metabolism ; Mice

OBJECTIVETo study the subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) protein in endothelial cells.

METHODSHuman umbilical vein endothelial cell strain ECV304 were cultured in vitro. The fusion protein of enhanced green fluorescent protein (EGFP)-EOLA1 expressing plasmid was constructed. Empty plasmid with EGFP at N side (pEGFP-N2) and fusion protein expressing plasmid EGFP-EOLA1 was respectively transfected into ECV304 cells with liposome. After being cultured for 48 hours, the expression levels of EGFP and fusion protein EGFP-EOLA1 in cells were detected with Western blot. The subcellular localization of EOLA1 protein was detected by laser scanning confocal microscope and immunoelectron microscopy.

RESULTSThe EGFP-EOLA1 coexpression plasmid was verified to be successfully constructed by enzyme cutting and gene sequencing. The fusion protein of EGFP-EOLA1 was observed to express in transfected cells through Western blot. Green fluorescence scattered all over the ECV304 cells transfected with empty plasmid and cells transfected with fusion protein expressing plasmid, and it gathered obviously in the nuclei in the latter cells. Immune deposits were observed in the matrix of cells transfected with fusion protein expressing plasmid but not in the cells transfected with empty plasmid.

CONCLUSIONSEOLA1 protein is localized in the nucleus and the matrix of ECV304 cell, and it plays its role as a signal transduction factor.

Cell Line ; Cell Nucleus ; metabolism ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; metabolism ; Membrane Proteins ; metabolism ; Signal Transduction

The present study investigated the mechanism of components in stasis-resolving and collateral-dredging Chinese herbal medicines, including scutellarin(Scu), paeonol(Pae), and hydroxy safflower yellow A(HSYA), in the treatment of psoriasis by regulating angiogenesis and inflammation. The human umbilical vein endothelial cells(HUVECs) cultured in vitro were divided into a normal group, a model group, a VEGFR tyrosine kinase inhibitor Ⅱ(VRI) group, and Scu, Pae, and HSYA groups with low, me-dium, and high doses. Cell viability was detected by the CCK-8 assay. Cell migration was detected by wound healing assay. Tube formation assay was used to measure the tube formation ability. Western blot was used to detect the protein expression of the VEGFR2/Akt/ERK1/2 signaling pathway. The secretion levels of inflammatory cytokines IFN-γ, IL-1β, IL-6, and TNF-α were detected by ELISA. The results showed that compared with the model group, all the Scu, Pae, and HSYA groups could reduce cell viability, inhibit cell migration and tube formation(P<0.05, P<0.01), and down-regulated the protein expression of VEGFR2, p-VEGFR2, Akt, p-Akt, ERK1/2, and p-ERK1/2. Scu and Pae could down-regulate VEGFR2 expression(P<0.05, P<0.01), while other groups only showed a downward trend. Scu and Pae significantly reduced IFN-γ and IL-6 levels(P<0.01), and HSYA significantly reduced the levels of IFN-γ, IL-1β, and IL-6(P<0.01). Scu, Pae, and HSYA had no significant effect on TNF-α. The results suggested that Scu, Pae, and HSYA may exert a therapeutic role in psoriasis-related angiogenesis and inflammation by inhibiting VEGFR2/Akt/ERK1/2 signaling pathway and inhibiting the secretion of IFN-γ, IL-1β, and IL-6.
Angiogenesis Inhibitors/pharmacology* ; China ; Human Umbilical Vein Endothelial Cells ; Humans ; Neovascularization, Pathologic/drug therapy* ; Vascular Endothelial Growth Factor A/metabolism*

Angiogenesis inhibitors targeting the VEGF signaling pathway are developed into drugs for the treatment of vaious diseases, such as cancer, rheumatoid arthritis, and age-related macular degeneration. Recent studies have revealed that oleanolic acid (OA), a natural pentacyclic triterpenoid, inhibited the VEGF/VEGFR2 signaling pathway and angiogenesis in HUVECs, which may represent an attractive VEGF inhibitor. In this paper, rational structural modification towards OA was performed in order to improve its inhibitory effects aganist VEGF and anti-angiogenesis potential. As a result, a series of novel OA derivatives, possessing α,β-unsaturated ketone system in ring A and amide functional group at C-28, were prepared and evaluated for cytotoxicity and their ability to inhibit VEGF-induced abnormal proliferation of HUVECs. The results showed that two promising derivatives, OA-1 and OA-16, exhibited no in vitro cytotoxicity against HUVECs but showed more potent inhibitory activity against VEGF-induced proliferation and angiogenesis in HUVECs, compared with OA. The results of Western blot indicated that OA-1 and OA-16 inhibited VEGF-induced VEGFR2 activation. Furthermore, small interfering RNA experiments were performed to confirm that both compounds inhibited VEGF-induced angiogenesis via VEGFR2. Thus, the present study resulted in the discovery of new promising OA-inspired VEGF inhibitors, which can serve as potential lead compounds for the treatment of angiogenesis-related diseases.
Cell Movement ; Cell Proliferation ; Human Umbilical Vein Endothelial Cells ; Humans ; Oleanolic Acid/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism*

Fluid shear stress plays an important role in many physiological and pathophysiological processes of the cardiovascular system. Previous studies have identified the exposure of vascular endothelial cells to fluid mechanical forces can modulate the expression of many genes, including IL-8 gene expression, and IL-8 expression induced by fluid shear stress was time-dependent. To investigate the role of intensity of fluid shear stress on IL-8 expression in human umbilical vein endothelial cells (HUVECs), we had HUVECs exposed to shear stress 2.23, 4.20, 6.08, 8.19, 9.67, 12.15, 14.40, 16.87, and 19.29 dyne/cm2 respectively and employed quantitative reversal transcription-polymerase chain reaction (qRT-PCR) to assay the expression of IL-8 mRNA. The results show that IL-8 mRNA did not express in HUVECs untreated with fluid shear stress. The IL-8 mRNA expression by shear stress was force intensity-dependent. After HUVECs exposed to low fluid shear stress (2.23 dyne/cm2) for 1 h or 2 h, IL-8 mRNA expression increased near 68 or 52 times as that of HUVECs exposed to high fluid shear stress (19.29 dyne/cm2). The linear regression equations between IL-8 mRNA expression (log (copies), y) and shear stress (dyne/cm2, x) are: y = 7.57 - 0.11x, r = 0.97 (for 1 h); y = 7.92 - 0.10x, r = 0.96 (for 2 h). This in vitro study demonstrates the expression of IL-8 gene can be regulated by fluid shear stress. The low shear stress could induces much more expression of IL-8 mRNA, which plays probably an important role in the pathogenesis of inflammation and arteroatherosclerosis.
Cells, Cultured ; Endothelium, Vascular ; cytology ; Gene Expression ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Interleukin-8 ; metabolism ; Stress, Mechanical

OBJECTIVEto assess direct adverse effects of occupational dusts from pottery factories and tungsten mines on vascular endothelial cells in vitro test.

METHODShuman umbilical vein endothelial cell (HUVEC) line HUV-EC-C were used as target cells. HUVEC were then treated with respirable dust particles from workplaces in pottery factories and tungsten mines in concentrations of 25, 50, 100, 200 and 400 microg/ml for 24 h. Standard quartz was used as control. LDH activity, cell viability, the release of NO and TNF-α levels were determined to assess the biological responses of the dust particles.

RESULTSdose-response relationships between the dust concentrations and the enhancement of the LDH activity, the release of NO and TNF-α were found in both dust particles from pottery factories and tungsten mines. The cell viability decreased with the increase of dust concentration from 25 to 400 microg/ml. Compared with the dust particles from workplaces, the quartz dust induced significantly higher LDH activity (P < 0.05) after cultured with HUVEC. No significant difference of releases of NO were observed among the dust particles from workplaces and standard quartz. However, significantly higher levels of TNF-α were induced by standard quartz compared with dust samples from workplaces at concentrations of 200, 400 microg/ml.

CONCLUSIONoccupational dust particles from workplaces and quartz could induce the injury and the releases of TNF-α from HUVEC.

Cells, Cultured ; Ceramics ; Dust ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Occupational Exposure ; Quartz ; Tumor Necrosis Factor-alpha ; metabolism ; Tungsten