This study was aimed to investigate the effect of human umbilical vein endothelial cells (HUVEC) on dendritic cell (DC) development. First, HUVEC were isolated from human umbilical cord by collagenase digestion, and then the morphology, immunophenotypes and functions were identified. Furthermore, the HUVEC were cocultured with CD14(+) monocytes under the cytokine condition for detecting the influence of HUVEC on differentiation of CD14(+) cells to DC. The phenotype of dendritic cells derived from CD14(+) cells was analyzed by flow cytometry, the immunoregulatory function of DC was tested by mixed lymphocyte reaction (MLR). The change of IL-6 and VEGF as well as EPK and p38 signal pathway were analyzed by neutral antibody experiment and Western blot. The results showed that HUVEC isolated from human umbilical cord were characterized by spindle-shaped morphology, homogenous immunophenotypes (vWF(+)CD31(+)CD73(+)CD45(-)HLA-DR(-)CD86(-)CD34(low)), Dil-Ac-LDL incorporation ability and forming capillary-like structures. Following stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4), HUVEC cocultures could inhibit the initial differentiation of CD14(+) monocyte to DC. Interestingly, IL-6 and VEGF enhanced the suppression effect of HUVEC on generation of DC via activation of the ERK or p38 mitogen activated protein kinase pathway. It is concluded that HUVEC are involved in DC development and can suppress the differentiation of monocyte to DC.
Cell Differentiation ; Coculture Techniques ; Dendritic Cells ; cytology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Monocytes ; cytology

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

AIMTo investigate the influence of adenosine on three-dimensional HUVEC tube formation.

METHODSA new three-dimensional culture type was established in which collagen type I was the substance and two layers of HUVEC grew upward and downward separately. In control group no adenosine was added in the holes; in experiment group 10(-4) mol/L adenosine was added in each hole. Under inverted phase contrast microscope, defined sights were observed and the number of sprout was recorded. Observation lasted for 96 hours, and experiments were repeated 3 times.

RESULTSHUVEC grew upward and downward into collage gel respectively. Tube structure and three-dimensional network was built up gradually. In experiment group(adenosine 10(-4) mol/L) HUVEC grew fast with quick infiltration and sprouting. The tube formation was thick and might form three-dimensional network throughout the collagen gel. At each time point, the difference between experiment and control group was significant (P < 0.05 or P < 0.01).

CONCLUSIONAdenosine may promote HUVEC sprouting and tube-formation.

Adenosine ; pharmacology ; Cells, Cultured ; Cellular Structures ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans

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 investigate the constituent expression of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) in human umbilical vein endothelial cells (HUVECs) and the effect of PHLPP1 gene transfer on the proliferation of the cells in vitro.

METHODSCultured HUVECs were transfected with pcDNA3-GFP or pcDNA3HA-PHLPP1 via lipofectamine 2000. The cell proliferation ability was determined by cell counting and MTT colorimetric assay, and Western blotting was used to detect the protein expression of PHLPP1 in the cells.

RESULTSNo PHLPP1 protein was detected in the non-transfected cells or pcDNA3-GFP-transfected cells. pcDNA3HA-PHLPP1 gene transfection significantly increased PHLPP1 expression in the HUVECs (P<0.01), but the cell proliferation status remained unchanged (P>0.05). The absorbance of the cells measured by MTT assay was 0.134-/+0.0152, 0133-/+0.014 and 0.137-/+0.016, with cell counts of (8.293-/+0.962)x10(5), (7.937-/+0.101)x10(5) and (8.127-/+0.112)x10(5), respectively, showing no significant differences between the 3 groups (P>0.05).

CONCLUSIONSPhosphatase PHLPP1 may not be the most important signal protein in the regulation of HUVEC proliferation.

Cell Proliferation ; Gene Transfer Techniques ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Nuclear Proteins ; genetics ; Phosphoprotein Phosphatases ; genetics ; Transfection

In this study for exploring the effect of RGD peptide on adhesive stability of endothelial cells biomaterial surface, all materials were divided into three groups, RGD group (PET covalently grafted synthetic RGD peptides), control group (PET precoated with fibronectin) and blank group (Non-coated surface). Cultured human umbilical vein endothelial cells (HUVECs) were seeded on the materials, then adhesive stability of HUVECs on the varied PET surfaces was observed under steady flow condition, and effects of shear stress and shear time on adherent cells were compared. The results showed that the resistance adherent endothelial cells to detachment by flow was shear stress and shear time dependent. Comparison three groups under the same condition revealed that the ECs retention rates of RGD-grafted or fibronectin-coated group were much higher than that of the non-coated group. Under 8.19 dyne/cm2 shear stress after 4h, retention rates were 13.73% (blank group), 43.33% (RGD group) and 40.75% (control group) respectively. These data indicated that RGD peptide can improve the adhesive stability of endothelial cell on biomaterial and the effect of RGD in vivo needs further studies.
Biocompatible Materials ; chemistry ; Cell Adhesion ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Oligopeptides ; chemistry ; Polyethylene Terephthalates ; chemistry ; Stress, Mechanical

This study was purposed to characterize the effect of carboxylic acid metabolite (SR26334) of clopidogrel bisulfate deprived of antiplatelet efficacy on the spectrum of gene expression in the cultured human umbilical vein endothelial cell (HUVEC) line (EA.hy926), and to explore the potential molecule mechanism of SR26334 impact on HUVEC. By using a Affymetrix HU133 plus 2.0 oligonucleotide microarray, the alteration of gene expression spectrum induced by SR26334 in HUVEC was detected, the real-time PCR was used to confirm the results of selected differentially expressing genes. The results indicated that total 235 including 176 up-regulated and 59 down-regulated genes were obtained with change more than 1.5-fold after SR26334 (10 µmol/L) acted on HUVEC for 48 h. SR26334 affected the expression levels of genes involved regulation of transcription, transcription, positive regulation of transcription from RNA polymerase II promoter, cell cycle, cell division, protein amino acid dephosphorylation in HUVEC. It is concluded that carboxylic acid metabolite SR26334 of clopidogrel bisulfate modulates function of endothelial cells through different pathway at gene level.
Cell Line ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Ticlopidine ; analogs & derivatives ; pharmacology ; Transcriptome ; drug effects

OBJECTIVETo determine the effects of noncoding repressor of NFAT (NRON) overexpression or silencing on human umbilical vein endothelial cells (HUVECs) functions.

METHODSStable HUVECs cell lines with NRON overexpression and short hairpin RNA (shRNA) interference were obtained. HUVECs, the empty vector pBABE-cell line and the empty vector pSuper-cell line served as controls. Cell proliferations of these cell lines were tested using MTS method, tube formation capacity and migration function were also examined.

RESULTSMTS experiments evidenced dose-dependent cells proliferations in all cell lines after 48 h culture with fetal bovine serum (HUVECs, r = 0.91;pBABE empty vectors cell-line, r = 0.88;NRON overexpression cell-line, r = 0.89;pSuper empty vectors cell-line, r = 0.95;shRNA infererence cell-line, r = 0.97). Proliferation capacity was lower in NRON overexpressed HUVECs and was higher in NRON silencing HUVECs compared with pBABE empty vectors treated and normal HUVECs (all P < 0.05). Tube formation and migration functions were also reduced in NRON overexpressed HUVECs [(8.33 ± 0.12) roots, (1857 ± 65) cells] and increased in shRNA infererence of NRON treated HUVECs [(36.00 ± 0.51) roots, (6987 ± 50) cells] compared with pBABE empty vectors treated HUVECs [(19.67 ± 1.42) roots, (4411 ± 117) cells], pSuper empty vectors treated HUVECs [(17.33 ± 2.93) roots, (3883 ± 109) cells] and normal HUVECs [(23.33 ± 3.01) roots, (5145 ± 72) cells, all P < 0.05].

CONCLUSIONNRON overexpression could reduce and NRON silencing could increase proliferation, tube formation and migration capacities of HUVECs.

Cell Line ; Cell Proliferation ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; NFATC Transcription Factors ; genetics ; RNA, Long Noncoding ; genetics

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 study the biological effects of the paracrine from ADSC after being stimulated by insulin on vascular endothelial cells.

METHODS(1) ADSC was isolated from human adipose tissue and cultured in vitro. The third generation cells were collected and divided into insulin group (I, cultured with serum-free DMEM containing 1 x 10(-7) mol/L insulin) and control group (C, cultured with serum-free DMEM) according to the random number table, with 6 slots in each group. Three days later, ADSC culture medium (ADSC-CM) was collected for determination of levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by ELISA. (2) Human umbilical vein endothelial cells (HUVEC) were cultured to the third generation, and they were cultured with special nutrient solution and divided into ADSC-CM with insulin stimulation group (AI), ADSC-CM without insulin stimulation group (AC), insulin group (I, with same concentration as above), blank control group (BC) according to the random number table. Three days later, proliferation of HUVEC was determined with MTT method (with expression of absorbance value). Another two samples of HUVEC were respectively divided into 4 groups as above for determination of apoptosis rate with Annexin V/FITC double-staining 12 hours after culture, and HUVEC migration with scratch adhesion test at post scratch hour (PSH) 12, 24, 36, 48. Data were processed with t test.

RESULTS(1) Compared with those in C group [(287 +/- 47), (577 +/- 84) pg/mL, respectively], the secretion levels of VEGF and HGF in I group [(643 +/- 64), (930 +/- 68) pg/mL, respectively] were significantly increased (with t value respectively 18.869, 18.475, P values all below 0.05). (2) The absorbance value of HUVEC in AI and AC groups was 0.847 +/- 0.042, 0.798 +/- 0.022, respectively, which were higher than that in I and BC groups [0.665 +/- 0.028 (with t value respectively 4.579, 3.732), 0.674 +/- 0.031 (with t value respectively 3.761, 4.073), P values all below 0.01], and that in AI group was higher than that in AC group (t = 2.576, P < 0.05). The apoptosis rates of HUVEC in AI and AC groups [(5.8 +/- 1.9)%, (9.0 +/- 2.0)%, respectively] were obviously lower as compared with that in I and BC groups [(30.4 +/- 6.0)% (with t value respectively 12.891, 10.417), (31.4 +/- 7.4)% (with t value respectively 11.474, 9.783), P values all below 0.05 ], and that in AC group was higher than that in AI group (t = 8.548, P < 0.05). The distance of migration of HUVEC in AI and AC groups were greater than that in I and BC groups at PSH 36, 48, and that in AI group was greater as compared with that in AC group (with t value respectively 4.076, 4.573, P values all below 0.05).

CONCLUSIONSParacrine from ADSC after being stimulated by insulin can promote proliferation and migration of HUVEC, and suppress its apoptosis, and it is beneficial for tissue vascularization.

Adipocytes ; cytology ; secretion ; Adipose Tissue ; cytology ; Apoptosis ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Hepatocyte Growth Factor ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Insulin ; pharmacology ; Stem Cells ; cytology ; secretion ; Vascular Endothelial Growth Factor A ; metabolism