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*

The expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) was examined in the umbilical vessels of the patients with pre-eclampsia (PE) to explore its possible role in the pathogenesis of PE. The NOSTRIN mRNA in umbilical tissues was determined by RT-PCR. The eNOS activity in umbilical vessels was spectrophotometrically detected. NO2-/NO3-, the stable metabolic end products of NO, was measured by using nitrate reductase. RT-PCR showed that the expression level of NOSTRIN was significantly higher in women with PE than in the normal group (P<0.01). The activity of eNOS was significantly decreased in PE group [(12.83+/-3.61) U/mg] than in normal group [(21.72+/-3.83) U/mg] (P<0.01). The level of NO2-/NO3- in PE patients (27.53+/-7.48) micromol/mg was significantly lower than that of normal group (54.27+/-9.53) micromol/mg (P<0.01). The significant negative correlation existed between the expression of NOSTRIN and the activity of eNOS in umbilical vessels of women with PE (r=-0.58, P<0.01). It was concluded that the level of NOSTRIN expression was increased in umbilical vessel of women with PE, indicating that it may be involved in the pathogenesis of PE.
Intracellular Signaling Peptides and Proteins/genetics ; Intracellular Signaling Peptides and Proteins/*metabolism ; Pre-Eclampsia/*enzymology ; Pre-Eclampsia/etiology ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Umbilical Arteries/cytology ; Umbilical Arteries/*enzymology ; Umbilical Veins/cytology ; Umbilical Veins/*enzymology

OBJECTIVETo observe the distribution and expression changes of caveolin-1 under shear stress in a turbulence flow channel model mimicking susceptible sites of atherosclerosis in vitro.

METHODSHuman umbilical vein endothelial cells (HUVECs) were inoculated to flow channel and then exposed to disturbed shear stress for 6, 12, 24 and 48 hours, respectively. HUVECs inoculated to flow channel without shear stress served as controls. Immunofluorescence staining was performed with SABC-FITC to detect the caveolin-1 distribution and RT-PCR was used to determine the mRNA expression of caveolin-1 gene in these cells.

RESULTSConfocal microcopy revealed ellipse-like shaped HUVECs under disturbed shear stress and caveolin-1 didn't move toward to the upstream edge of HUVECs but moved toward to the edge of HUVECs, and expression of caveolin-1 gene in HUVECs was significantly downregulated after exposure to shear stress for 48 hours compared to HUVECs without shear stress stimulation.

CONCLUSIONDisturbed shear stress affected disposition and expression of caveolin-1 which might be responsible for shear stress induced endothelial cell pathological atherosclerotic changes.

Caveolin 1 ; Cells, Cultured ; Endothelium, Vascular ; metabolism ; Humans ; Interleukin-8 ; Stress, Mechanical ; Umbilical Veins

OBJECTIVETo observe the effect of Porphyromonas gingivalis (Pg) on the production of nitric oxide (NO) in cultured human umbilical vein endothelial cells (HUVEC), and to investigate the pathway of damaging endothelial function by Pg.

METHODSPg ATCC33277 was cultured in anaerobic jar, and HUVEC was treated with various concentrations of Pg ATCC33277 at multiplicity of infection (MOI) of 1:10, 1:100 and 1:1000 for 4, 8, 12, 24 h respectively. The cells supernatants were collected and stored at -70 degrees C and NO concentration in the cells supernatants was measured by nitrate reductase assay.

RESULTSWithin 24 h, Pg at MOI of 1:10 and 1:100 stimulated the release of nitric oxide in cultured HUVEC. Within 12 h, Pg at an MOI of 1:1000 group increased NO production, and NO decreased at 24 h.

CONCLUSIONSPg has an effect on the production of NO. Low concentrations of Pg stimulated release of nitric oxide in endothelial cells but high concentrations can decrease the release of NO.

Cells, Cultured ; Endothelial Cells ; metabolism ; Humans ; Nitric Oxide ; biosynthesis ; Porphyromonas gingivalis ; isolation & purification ; Umbilical Veins ; cytology

High concentrations of oxygen, indispensable for the treatment of severe hypoxemia from neonatal as well as adult respiratory distress syndrome, increase the risk of oxygen toxicity. Biochemical mechanisms are lipid peroxidation, protein sulfhydryl oxidation, enzyme inactivation, and DNA damage. Recent reports suggest that cytokines might be involved in free radical injury as well as in adaptive response to hyperoxic injury. However, actual signal transduction pathways involving cytokines have not yet been clarified. In this study we exposed cultured human umbilical vein endothelial cells (HUVECs) to either ambient air or 100% oxygen, and compared for the rate of DNA synthesis ([3H]thymidine uptake) at different time points up to 72 h. After exposing the cells to each treatment condition, we extracted RNA, constructed complementary DNA using reverse transcriptase, amplified the specific DNA segments of cytokines by polymerase chain reaction (PCR), and used the PCR products for gel electrophoresis to examine the bands which signified mRNA levels of corresponding cytokines. There was a significant decrease in the rate of DNA synthesis as early as 24 h. The mRNA expression of IL-1 beta and TNFa seemed less influenced by hyperoxia, while IL-8 and TGF beta showed marked increase in mRNA levels at 6 h of 100% oxygen exposure.
Cells, Cultured ; Cytokines/genetics* ; DNA/biosynthesis ; Endothelium, Vascular/metabolism* ; Human ; Hyperoxia/metabolism* ; RNA, Messenger/analysis* ; Umbilical Veins

OBJECTIVETo investigate the effect of different patterns of intermittent hypoxia on the levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) of vascular endothelial cell.

METHODSThe human umbilical vein endothelial cells of the line ECV304 were used to set up the cells model. The experiment cell strains contained one control group and eight experimental groups, 1 constance normoxia group (control group), 3 different intermittent hypoxia (IH) degree groups (10% O₂ group, 1.5% O₂ group, 0.5% O₂ group with 12 times/h), 4 different IH frequency groups (40 times/h, 20 times/h, 12 times/h, 9 times/h and 6 times/h with 0.5%O2). The specimen were put into a program-controlled gas delivery system with different level and frequency of IH respectively. Then enzyme linked immunosorbent assay (ELISA) was used to examine the levels of IL-6 and IL-8.

RESULTSHigher level of IL-6 and IL-8 were found in 3 different IH degree groups than in control group (F were 1961.100 and 103.855 respectively, P all < 0.001). The level of IL-6 and IL-8 was gradually increased with the decreasing of IH degree. The difference of IL-6 and IL-8 levels between each two different groups in IH degree was significant (P all < 0.05). The level of IL-6 and IL-8 groups was significantly different in severe IH exposure (F were 544.396 and 149.328 respectively, P all < 0.001 in both groups). There was a trend as the IH frequency decreased, the level of IL-6 and IL-8 was tenderly increased in 20/hour group (P < 0.05), highest in 12/hour group (P < 0.05), decreased in 9/hour group (P < 0.05), and decreased further in 6/hour group (P < 0.05) in severe IH expose. There was statistical significance between each two different frequency IH groups (P < 0.05).

CONCLUSIONSIntermittent hypoxia caused significant inflammatory reaction in vascular endothelial cells. The secreted level of IL-6 and IL-8 depended on the degree of hypoxia. There was a trend as the level of IH frequency decreased, the level of IL-6 and IL-8 gradually increased at first, and then decreased in the same way.

Cell Hypoxia ; Cell Line ; Endothelium, Vascular ; metabolism ; Humans ; Interleukin-6 ; secretion ; Interleukin-8 ; secretion ; Umbilical Veins ; cytology ; metabolism

OBJECTIVETo investigate the effects of tumor necrosis factor-alpha(TNF-alpha) on syndecan-4 protein expression and proliferation of cultured human umbilical vein endothelial-like cells (HUVECs) in vitro.

METHODSHUVECs exposed to different concentrations of TNF-alpha(100, 20, 10, and 1 ng/ml) were cultured for 24 h and 36 h to observe their proliferation in comparison with the control group. The cell proliferation rate was determined by non-radioactive MTS/PES assay. The expression of syndecan-4 protein was evaluated by immunoblotting technique using anti-syndecan-4 antibody. Results The proliferation rate of the endothelial-like cells was 1.956-/+0.214 in the control group, and 2.154-/+0.250, 2.260-/+0.151, 2.118-/+0.205 and 2.106-/+0.136 in TNF-alpha-treated groups corresponding to TNF-alpha concentrations of 100, 20, 10 and 1 ng/ml at 24 h, respectively. It was shown that TNF-alpha significantly stimulated cell proliferation at the concentration above 1 ng/ml (P<0.05) as compared with the control group (P<0.05). The proliferation rate of the endothelial-like cell was 1.915-/+0.236 in the control group, and 2.067-/+0.328, 2.207-/+0.150, 2.052-/+0.126 and 2.051-/+0.180 in TNF-alpha-treated groups corresponding to TNF-alphaconcentrations of 100, 20, 10 and 1 ng/ml at 36 h, respectively. The expression of syndecan-4 protein was significantly enhanced by TNF-alpha.

CONCLUSIONSTNF-alpha can stimulate HUVEC proliferation, and expression of syndean-4 may represent an additional component of the pro-inflammatory, growth-stimulating pathways that are activated in response to changes in the vascular wall.

Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; metabolism ; Humans ; Syndecan-4 ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; Umbilical Veins ; cytology

Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Gene Expression Profiling ; Humans ; Ouabain ; pharmacology ; Umbilical Veins ; cytology

Connexin43 has been shown to play a pivotal role in wound healing process. Wound repair is enhanced by acute downregulation of connexin43, by increasing proliferation and migration of keratinocyte and fibroblast. Angiogenesis is also a central feature of wound repair, but little is known about the effects of connexin43 modulation on functions of endothelial cells. We used connexin43 specific small interference RNA (siRNA) to reduce the expression of connexin43 in human umbilical vein endothelial cell (HUVEC), and investigated the effects of connexin43 downregulation on intercellular communication, viability, proliferation, migration and angiogenic activity of HUVEC. Treatment of siRNA markedly reduced the expression of connexin43 by -80% in HUVEC (P < 0.05), and decreased the intercellular communication by -65% (P < 0.05). The viability, proliferation, migration and angiogenic activity of HUVEC decreased significantly (P < 0.05), compared with that of the normal cells. The results suggest that temporally downregulation of connexin43 expression at early stage of wound to inhibit the abnormal angiogenesis characterized with leaky and inflamed blood vessels, maybe a prerequisite for coordinated normal healing process.
Cell Movement ; Cell Proliferation ; Cell Survival ; Connexin 43 ; metabolism ; Down-Regulation ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Neovascularization, Physiologic ; Umbilical Veins ; cytology ; Wound Healing

OBJECTIVETo investigate the penetrating ability of fusion protein PEP-1-EGFP with human umbilical vein endothelial cells.

METHODSTwo prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E. coli BL21 (DE3) to express EGFP and fusion protein PEP-1-EGFP, respectively. The expressed EGFP and PEP-1-EGFP were purified with Ni(2+) -resin affinity chromatography, and their capabilities of transduction into human umbilical vein endothelial cells were evaluated. The time- and dose-dependent transduction of the fusion protein PEP-1-EGFP and its stability in the human umbilical vein endothelial cells were observed. The toxicity of the fusion protein PEP-1-EGFP was detected by MTT method.

RESULTSEGFP failed to be transduced into human umbilical vein endothelial cells, whereas PEP-1-EGFP fusion protein was transduced into cells shortly in 5 minutes. Its transduction was time- and dose-dependent and the fluorescence in the cells were detected even 27 hours later. No cytotoxicity of the fusion protein PEP-1-EGFP to human umbilical vein endothelial cells was detected even when the dose reached up to 200 micromol/L.

CONCLUSIONPEP-1-EGFP fusion protein can efficiently transduce the target protein into human umbilical vein endothelial cells, which provides a basis for future researches on the transduction of antioxidant enzymes mediated by the cell-penetrating peptide, PEP-1, in ischemia-reperfusion injury therapy.

Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Green Fluorescent Proteins ; metabolism ; Humans ; Peptides ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; metabolism ; toxicity ; Umbilical Veins ; cytology