The cytoprotective enzyme heme oxygenase-1 (HO-1) influences endothelial cell survival, proliferation, inflammatory response, and angiogenesis in response to various angiogenic stimuli. In this study, we investigate the involvement of HO-1 in the angiogenic activity of orexin-A. We showed that orexin-A stimulates expression and activity of HO-1 in human umbilical vein endothelial cells (HUVECs). Furthermore, we showed that inhibition of HO-1 by tin (Sn) protoporphryin-IX (SnPP) reduced orexin-A-induced angiogenesis in vivo and ex vivo. Orexin-A-stimulated endothelial tube formation and chemotactic activity were also blocked in SnPP-treated vascular endothelial cells. Orexin-A treatment increased the expression of nuclear factor erythroid-derived 2 related factor 2 (Nrf2), and antioxidant response element (ARE) luciferase activity, leading to induction of HO-1. Collectively, these findings indicate that HO-1 plays a role as an important mediator of orexin-A-induced angiogenesis, and provide new possibilities for therapeutic approaches in pathophysiological conditions associated with angiogenesis.
Antioxidant Response Elements ; Endothelial Cells* ; Heme Oxygenase-1* ; Human Umbilical Vein Endothelial Cells ; Luciferases ; Tin ; Orexins

Angiogenesis has an essential role in many pathophysiologies. Here, we show that phospholipase C-β3 (PLC-β3) isoform regulates endothelial cell function and retinal angiogenesis. Silencing of PLC-β3 in human umbilical vein endothelial cells (HUVECs) significantly delayed proliferation, migration and capillary-like tube formation. In addition, mice lacking PLC-β3 showed impaired retinal angiogenesis with delayed endothelial proliferation, reduced endothelial cell activation, abnormal vessel formation and hemorrhage. Finally, tumor formation was significantly reduced in mice lacking PLC-β3 and showed irregular size and shape of blood vessels. These results suggest that regulation of endothelial function by PLC-β3 may contribute to angiogenesis.
Animals ; Blood Vessels ; Endothelial Cells ; Hemorrhage ; Human Umbilical Vein Endothelial Cells ; Mice ; Phospholipases* ; Retinaldehyde*

OBJECTIVETo study the cultural method and identification of human umbilical vein endothelial cells (HUVECs), and investigate the expression of tyrosine kinase-2 with immunoglobulin-like and epidermal growth factor homology domains(Tie-2) in HUVECs.

METHODSHUVECs were isolated from umbilical veins by the technique of irrigative digestion, and were cultivated in plates. The cells were identified by VIII monoclonal antibody. Tie-2 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and SABC immunocytochemistry.

RESULTSHUVECs could adhere to the plates completely after 24 hours, and confluence a monolayer 4-5 days later. The band of Tie-2 mRNA was obviously and the expression of Tie-2 protein was strongly positive by immunocytochemistry in HUVECs. The positive rate was over 85%.

CONCLUSIONHighly purified endothelial cells were isolated. And there were overexpression of Tie-2 in HUVECs.

OBJECTIVETo study the effects of Saccharomyces albicans (S. albicans) on the cell cycle distribution and proliferation of human umbilical vein endothelial cell ECV304 cells in vitro.

METHODSThe line of ECV304 cultured in vitro were divided into four groups which were treated by S. albicans supernatant, S. albicans inactivated bacilli, supernatant and inactivated bacilli mixture, normal culture medium. The proliferous effect of ECV304 induced by supernatant, inactivated bacilli, supernatant and inactivated bacilli mixture using the methods of MTT, cell count, microscope and flow cytometry were conducted.

RESULTSIn the condition of different times and different culture concentrations, ECV304 cells incubated with 4-fold diluted S. albicans supernatant for 48 h increased the proliferation rate. The S and G2/M population of ECV304 cells increased after incubated with S. albicans supernatant for 40 h, which showed significant increasing cell proliferation index (PI) (P < 0.05). The PI of the cells treated by inactivated bacilli showed no significant change (P > 0.05).

CONCLUSIONS. albicans could induce ECV304 cell proliferation which depends on the release of metabolic products of S. albicans.

OBJECTIVETo evaluate the effects of non-Saccharomyces albicans metabolic products on the cell cycle distribution and proliferation of human umbilical vein endothelial cell ECV304 cells in vitro.

METHODSThe parallel dilution supernatant of Saccharomyces tropicalis, Saccharomyces krusei and Saccharomyces glabrata were prepared, and 1, 4, 16-fold(s) diluted concentration and control group were set up. The line of human umbilical vein endothelial cell ECV304 was cultured in vitro and treated by non-Saccharomyces albicans supernatant. The proliferous effect of ECV304 induced by non-Saccharomyces albicans supernatant after 24, 48, 72 h was detected by the methods of MTT, and the changes of cell density and cycle after 48 h were investigated by inverted microscope and flow cytometry.

RESULTSAt the 24th hour, all of the higher concentration (1-fold) of non-Saccharomyces albicans supernatant and the 4-folds diluted Saccharomyces krusei could promote ECV304 proliferation(P < 0.05). After adding various non-Saccharomyces albicans supernatant at 48h and 72th hour, Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant significantly increased proliferation rate of ECV304, while Saccharomyces tropicalis supernatant group showed no significant change no matter which concentration was tested. At 48th hour after adding the non-Saccharomyces albicans supernatant, the ECV304 cells density treated by Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant were significantly higher under the inverted microscope. The G0/G1 population of ECV304 cells decreased while cell proliferation index (PI) increased after incubated with Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant for 48 hours (P < 0.05). Saccharomyces tropicalis group showed no significant change (P > 0.05).

CONCLUSIONThe metabolic products of Sacharoymces krusei and Saccharomyces glabrata could induce proliferation of ECV304 cell, which suggests non-Saccharomyces albicans should be undergone more attention clinically in detection and treatment.

The endothelial-mesenchymal transition (EndMT) is known to be involved in the transformation of vascular endothelial cells to mesenchymal cells. EndMT has been confirmed that occur in various pathologic conditions. Transforming growth factor β1 (TGF-β1) is a potent stimulator of the vascular endothelial to mesenchymal transition (EMT). Aspirin-triggered resolvin D1 (ATRvD1) has been known to be involved in the resolution of inflammation, but whether it has effects on TGF-β1-induced EndMT is not yet clear. Therefore, we investigated the effects of AT-RvD1 on the EndMT of human umbilical vein vascular endothelial cells line (HUVECs). Treatment with TGF-β1 reduced the expression of Nrf2 and enhanced the level of F-actin, which is associated with paracellular permeability. The expression of endothelial marker VE-cadherin in HUVEC cells was reduced, and the expression of mesenchymal marker vimentin was enhanced. AT-RvD1 restored the expression of Nrf2 and vimentin and enhanced the expression of VE-cadherin. AT-RvD1 did also affect the migration of HUVEC cells. Inhibitory κB kinase 16 (IKK 16), which is known to inhibit the NF-κB pathway, had an ability to increase the expression of Nrf2 and was associated with the inhibition effect of AT-RvD1 on TGF-β1-induced EndMT, but it had no effect on TGF-β1-induced EndMT alone. Smad7, which is a key regulator of TGF-β/Smads signaling by negative feedback loops, was significantly increased with the treatment of AT-RvD1. These results suggest the possibility that AT-RvD1 suppresses the TGF-β1-induced EndMT through increasing the expression of Smad7 and is closely related to oxidative stress.
Actins ; Endothelial Cells ; Human Umbilical Vein Endothelial Cells ; Humans ; Inflammation ; Oxidative Stress* ; Permeability ; Phosphotransferases ; Transforming Growth Factors ; Umbilical Veins ; Vimentin

The article aims to study the effect and mechanism of shear stress on eicosanoids produced by the metabolism of polyunsaturated fatty acids in endothelial cells. First, human umbilical vein endothelial cells were treated by control (Static), laminar shear stress (LSS) and oscillatory shear stress (OSS) for 6 h. Then the endothelial cells were incubated with fresh M199 medium for 3 h, and the cell culture medium was collected. Ultra-performance liquid chromatography-mass spectrometer was used to detect the level of eicosanoid metabolites secreted by endothelial cells. The results showed that under different shear stress, the level of eicosanoid metabolites were changed significantly. We found 10 metabolites were significantly up-regulated by OSS compared with those in LSS group, including PGD2, PGE2, PGF2α and PGJ2 produced by cyclooxygenase; 11-HETE, 15-HETE, 13-HDoHE produced by lipoxygenase or spontaneous oxidation; 12,13-EpOME, 9,10-EpOME, 9,10-DiHOME produced by cytochrome P450 oxidase and soluble epoxide hydrolase. The transcription levels of these up-regulated eicosanoids metabolic enzyme-related genes were also increased in vitro and in vivo. These results indicate that OSS may promote the increase of metabolites by up-regulating the transcription level of metabolic enzyme-related genes, which playing a key role in the development of atherosclerosis. This study reveals the effect of shear stress on eicosanoid metabolism in endothelial cells, which provides a novel supplement to the systems biology approach to study systemic hemodynamics.
Cells, Cultured ; Eicosanoids ; Human Umbilical Vein Endothelial Cells ; Humans ; Metabolomics ; Stress, Mechanical

OBJECTIVE: To investigate the damaging of human umbilical vein endothelial cells (HUVEC) induced by antiplatelet integrin β3 antibodies in vitro. METHODS: The serum from 36 chronic ITP patients were collected, flow cytometry and monoclonal antibody specific immobilization of platelet antigen (MAIPA) assay were used to collect antiplatelet integrin β3 antibodies from the serum of the patients. After HUVEC were treated by ITP patient serum (PS) containing anti-integrin β3 antibodies, the cell damage was detected by Lactate dehydrogenase (LDH) assay, cell apoptosis was detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by Reverse transcription-Quantitative real-time PCR (RT-qPCR), and expression of Apoptosis-related signaling pathway protein Akt and related protein Bax were detected by Western blot. HUVEC were treated by PS combined with Akt activator SC79, the cells damage were detected by LDH assay, apoptosis of the cells were detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by RT-qPCR. RESULTS: Among 36 cases of serum from the chronic ITP patients, 5 patients' serum containing anti-integrin β3 antibodies were collected. After HUVEC was treated by PS, the viability of LDH was significant increased（P<0.05）, so as for the apoptosis of the cells（P<0.05）, the expression of gene and protein of Bax was increased up-regulated（P<0.05）, the protein expression of pAkt was down-regulated（P<0.05）. Comparing with HUVEC cultured with PS alone, the viability of LDH of HUVEC treated by PS combined with SC79 was significantly reduced（P<0.05）, so as for the apoptosis of the cells（P<0.05）, and gene expression of Bax was significantly decreased（P<0.05）. CONCLUSION Anti-integrin β3 serum can cause the damage and apoptosis of HUVEC through Akt signaling pathway，the apoptotic effects of anti-integrin β3 antibodies to HUVEC was effectively reversed by SC79.
Apoptosis ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; Humans ; Integrin beta3 ; Signal Transduction

OBJECTIVE: To investigate the effects of heparin on the secretion of monocyte chemotactic protein-1 (MCP-1) in human umbilical vein endothelial cells (HUVEC) and the adhesion of monocytes to endothelial cells stimulated by lipopolysaccharide (LPS). METHODS: HUVEC were cultured in vitro, and the cells between generation 4 and 5 were used for the experiments. The cells were divided into phosphate buffer saline (PBS) control group, heparin control group, LPS group, and heparin+LPS group. The LPS group was challenged with LPS 10 mg/L; the PBS control group was added with the same amount of PBS; the heparin group was added with 10 kU/L unfractionated heparin; the heparin+LPS group was treated with 10 kU/L unfractionated heparin 15 minutes before LPS stimulation. The cells were harvested at 6 hours and 12 hours after LPS stimulation in each group, and the MCP-1 mRNA expression was determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (qRT-PCR). After incubation with each group, the fluorescent dyelabeled human monocyte cell line THP-1 was cultured with each group for 1 hour in the dark, and the adhesion density of THP-1 and HUVEC was observed under fluorescence microscope. RESULTS: Compared with the PBS control group, the MCP-1 mRNA expression significantly increased at 6 hours and 12 hours after LPS stimulation and peaked at 6 hours, then decreased gradually, but remained significantly higher than the PBS control group at 12 hours [2^-ΔΔCt: 16.41 (15.03, 18.00) vs. 1.00 (0.80, 1.26) at 6 hours, 9.27 (8.11, 9.85) vs. 1.00 (0.84, 1.20) at 12 hours, both P < 0.05]. Heparin preconditioning significantly reduced LPS-induced MCP-1 mRNA expression [2^-ΔΔCt: 2.06 (1.72, 2.46) vs. 16.41 (15.03, 18.00) at 6 hours, 2.46 (2.19, 4.56) vs. 9.27 (8.11, 9.85) at 12 hours, both P < 0.05]. There was no significant difference in MCP-1 mRNA expression between the heparin control group and the PBS control group [2^-ΔΔCt: 1.47 (1.29, 1.65) vs. 1.00 (0.80, 1.26) at 6 hours, 2.69 (2.58, 2.77) vs. 1.00 (0.84, 1.20) at 12 hours, both P > 0.05]. Fluorescence microscopy observation showed that LPS stimulation could promote the adhesion of THP-1 to HUVEC; heparin preconditioning could inhibit the adhesion of THP-1 to HUVEC stimulated by LPS. CONCLUSIONS Heparin preconditioning could inhibit the MCP-1 mRNA expression , thereby reduce the adhesion of THP-1 to HUVEC, thus play a protective role in sepsis.
Cells, Cultured ; Fibrinolytic Agents ; Heparin ; Human Umbilical Vein Endothelial Cells ; Humans ; Lipopolysaccharides ; Monocytes

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