Morphology Of Human Umbilical Vein Endothelial Cells Research Articles

Senescence affects endothelial cells susceptibility to dengue virus infection.

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.

ENOS Phosphorylation Under Flow-Mediated Shear Stress: Effects of Flow Pattern and Ambient Oxygen Concentration

Vascular endothelial cells (EC) are continuously exposed to flow-mediated shear stress, a friction force generated by flowing blood, which is increased during aerobic exercise. The flow-mediated shear stress is the most important physiological stimulus for the formation of nitric oxide (NO) via activation of endothelial nitric oxide synthase (eNOS). It has been demonstrated that flow-mediated shear stress modulates vascular structure and function, and is a major determinant for vascular remodeling and arterial tone, which depends primarily on NO produced by EC in response to shear stress. NO plays many essential roles including vessel relaxation, inhibition of apoptosis, and anti-inflammation. However, effects of flow patterns and ambient oxygen concentration on the eNOS activation and NO production are largely unknown. PURPOSE: To determine the effects of flow patterns (low and high) and ambient oxygen concentration (5%, 10% and 20% O2) on eNOS activation and NO production in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were grown in M199 supplemented with 20% FBS, Endothelial Cell Growth Supplement, Heparin, P/S and Fungizone. At 100% confluency, HUVECs were subjected to physiological level of low (l-flow) or high (h-flow) shear stress (5 or 20 dyne/cm2) using a cone-and-plate shear apparatus under 21% O2 (atmospheric oxygen), 10% O2 (arterial blood level), or 5% O2 (venous blood level). Protein samples were analyzed by western blotting. RESULTS: The morphology of HUVECs was not different among the ECs grown under different oxygen concentrations. However, the growth rate was slightly higher at 10% O2 at low confluency (< 30%) while it was similar at high confluency (60-100%). During 5-60 mins of h-flow, eNOS phosphorylation at the Ser633 residue was similar. However, under 24 hrs of h-flow, the levels of eNOS phosphorylation at both Ser1177 and Ser633 were 2-3 fold higher in 10% O2 compared to that of 21% O2. NO production level was also significantly higher in 10% O2 than in 21% O2 under 24 hrs of h-flow. CONCLUSION: Physiological level of h-flow under 10% O2 increases eNOS phosphorylation and NO production greater than that of under 21% O2 in human endothelial cells. The present study may provide a better in vitro model for the area of vascular mechanotransduction research.

Effect of different mitogens and serum concentration on HUVEC morphology and characteristics: Implication on use of higher passage cells

Human umbilical vein endothelial cells (HUVEC) were cultured in two different media, viz. the commonly used M199 containing 20% fetal bovine serum (FBS) and endothelial cell growth factor and a defined media EGM-2 containing 2% FBS along with growth supplements in known concentrations. The purpose of this study was to determine the effect of different media on the growth potential and cell morphology in subsequent passages. We have established that a dual coating of gelatin and human fibronectin extracellular matrix provides optimal cell attachment. Growth rate for primary culture was almost double in defined media. For secondary culture a two fold higher proliferation rate was observed in defined EGM-2 media. Histological studies were done using phase contrast, confocal and scanning electron microscopy which showed that cells cultured in M199 started losing their morphological characteristic from 3rd passage and after 6th passage appeared to come in senescent stage, while in case of defined media there was no change observed in the cells up to 10th passage. A significant difference was found in the expression of soluble intracellular adhesion molecule-1 (sICAM-1) which is an endothelial cell marker on cells cultured in different media. Additionally it was observed that exposure duration to trypsin–EDTA during cell detachment also plays an important role in maintaining cell morphological characteristics. These results show that significant morphological changes appear in higher order passages if cells are grown in routine medium for a long time and therefore may not be suitable for cell signaling experiments.

Acurhagin‐C, an ECD disintegrin, inhibits integrin αvβ3‐mediated human endothelial cell functions by inducing apoptosis via caspase‐3 activation

Acurhagin, a member of versatile metalloproteinase disintegrins from Agkistrodon acutus venom, has been identified as a platelet aggregation inhibitor, previously. Here, acurhagin-C, the C-terminal Glu-Cys-Asp (ECD)-containing fragment of acurhagin, was evaluated for its biological activities and potential applications in anti-angiogenic therapy. Human umbilical vein endothelial cells (HUVECs) were treated with acurhagin-C to assay effects on viability, apoptosis, adhesion, migration, invasion, proliferation and angiogenesis. The recognition site and signalling involved for the interactions of acurhagin-C with HUVEC were determined using flow cytometric, electrophoresis and immunoblotting analyses. Acurhagin-C decreased viability and induced apoptosis in HUVEC. It also dose-dependently inhibited HUVEC adhesion to immobilized extracellular matrices fibronectin, collagen I and vitronectin with respective IC(50) values of approximately 0.6, 0.3 and 0.1 microM. Acurhagin-C prevented migration and invasion of HUVEC through vitronectin- and Matrigel-coated barriers respectively. Furthermore, acurhagin-C attenuated fibroblast growth factor-2-primed angiogenesis both in vitro and in vivo, and specifically blocked the binding of anti-alphavbeta3 monoclonal antibody 23C6 to HUVEC in an ECD-dependent manner. However, purified alphavbeta3 also dose-dependently bound to immobilized acurhagin and acurhagin-C with a saturable pattern. Interference with integrin alphavbeta3-mediated functions and promotion of caspase-3 activation by acurhagin-C affected morphology of HUVEC and induced apoptosis. Acurhagin-C elicited endothelial anoikis via disruption of alphavbeta3/focal adhesion kinase/phosphatidylinositol 3-kinase/Akt survival cascade and subsequent initiation of the procaspase-3 apoptotic signalling pathway.

Biocompatibility of pure titanium modified by human endothelial cell-derived extracellular matrix

Extracellular matrix (ECM) used to modify biomaterial surface is a promising method for improving cardiovascular material hemocompatibility. In the present work, human umbilical vein endothelial cells (HUVECs) are cultured and native ECM is obtained on pure titanium surface. Fourier infrared spectrum (FTIR) test proves the existence of amide I and amide II band on the modified titanium surface. X-ray photoelectron spectroscopy (XPS) further confirms the chemical composition and binding types of the ECM proteins on the titanium substrate. The results of light microscopy and atomic force microscopy (AFM) exhibit the morphology of HUVEC derived ECM. There are higher water contact angles on the ECM modified samples. Furthermore, some ECM components, including fibronectin (FN), laminin (LN) and type IV collagen (IV-COL) are presented on ECM-covered titanium surface by immunofluorescence staining. The biological behavior of cultured HUVECs and adherent platelets on different samples are investigated by in vitro HUVECs culture and platelet adhesion. Cells exhibit better morphology and their proliferation ability greatly improve on the ECM-covered titanium. At the same time, the platelet adhesion and spreading are inhibited on ECM-covered titanium surface. These investigations demonstrate that ECM produced by HUVECs cannot only improve adhesion and proliferation ability of endothelial cell but also inhibit adhesion and activation of platelets. Thus, the approach described here may provide a basis for preparation of modified surface in cardiovascular implants application.

T0201-3-2 衝撃圧力が培養血管内皮細胞のVE-カドヘリンに与える影響([T0201-3]細胞の構造と流れにおけるマイクロ・ナノスケール解析(3))

Human umbilical veinendothelial cells (HUVECs) were exposed to an impact pressure of -100 kPa and changes in morphology of HUVECs and expression of vascular entodhelial (VE)-cadherin were examined in order to investigate effect of exposure to impact pressure on endothelial permeability. In the results, HUVECs exposed to impact pressure were absent locally. VE-cadherin in control were continuously expressed along peripheral region of cells. However, VE-cadherin in HUVECs exposed to impact pressure were sparsely expressed along peripheral region of cells and partly distributed in cells. These findings suggest that the exposure to impact pressure may change the expression and the distribution of VE-cadherin, influencing endothelial permeability.

Biosensor incorporating cell barrier architectures for detecting Staphylococcus aureus alpha toxin

Alpha toxin is a common virulent factor of Staphylococcus aureus and is believed to play crucial roles in pathogenicity induced by S. aureus. Alpha toxin is also known to induce permeability to endothelial cell monolayers in vitro due to the formation of interendothelial gaps. The present study is directed towards measuring alpha toxin using a whole-cell-based biosensor. The biosensor, consisting of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) on a potassium ion-selective electrode, takes advantage of cell permeability dysfunction to detect the presence of small quantities of alpha toxin. When a confluent monolayer of cells was formed on the membrane surface, the response of the electrode toward the marker ion, potassium, was inhibited. Upon exposing this sensor to varying concentrations of alpha toxin for 20 min, an increase in sensor response to potassium was observed. The response thus obtained was indirectly related to the concentration of alpha toxin. The detection limit of this sensor for alpha toxin was found to be 0.1 ng/ml. Cell monolayers were stained with silver nitrate to quantify the formation of intercellular gaps as well as to study the effect of this toxin on HUVECs morphology. A strong positive correlation was observed between the response obtained from the biosensor and the area of the intercellular gaps. Silver staining also revealed the tendency of cells to round up upon being exposed to alpha toxin.

ZD6126: A novel small molecule vascular targeting agent

Purpose: The aim of these studies was to evaluate factors that contribute to the selectivity of the novel vascular targeting agent ZD6126.Methods: Human umbilical vein endothelial cells (HUVECs) were treated with ZD6126 phenol, and effects on morphology, detachment, and cytotoxicity (sulforhodamine-B dye incorporation) were determined. Hras5-transformed mouse 3T3 fibroblasts were implanted s.c. in athymic nude rats, and effects on the tumor were assessed after either i.v. bolus or 24-h minipump infusion of ZD6126.Results: In vitro, ZD6126 phenol (∼0.1 μm) rapidly (<40 min) destabilized the tubulin cytoskeleton of proliferating endothelial cells, resulting in cell shape change (“rounding up”) and cell detachment at noncytotoxic drug concentrations. In vivo, in rats, an i.v. bolus dose of ZD6126 (20 mg/kg) was rapidly broken down to ZD6126 phenol, which has a short plasma elimination half-life (∼1 h). Peak plasma levels of ZD6126 phenol were well above the level required to induce HUVEC morphology changes in vitro, but cytotoxic concentrations were not maintained. A single i.v. bolus dose (50 and 20 mg/kg) of ZD6126 was well tolerated and resulted in extensive central tumor necrosis in the Hras5 model. Administration of ZD6126 using a 24-h s.c. minipump resulted in decreased (∼30-fold) peak plasma levels, but maintained cytotoxic drug levels over 24 h. Infusion of 50 mg/kg ZD6126 over 24 h was not tolerated. Infusion of 20 mg/kg ZD6126 resulted in increased toxicity compared with the i.v. bolus doses of ZD6126 and did not result in any increased tumor necrosis after 24 h.Conclusion: ZD6126 phenol induces rapid morphologic changes in HUVECs at noncytotoxic drug levels. These rapid morphologic effects combined with the rapid elimination of ZD6126 phenol contribute to the selective effects of ZD6126 on tumor vasculature at well-tolerated doses.

Sera from preeclamptic women specifically activate human umbilical vein endothelial cells in vitro: morphological and biochemical evidence.

Endothelial cell dysfunction could explain many of the pathophysiological changes observed in preeclampsia. Markers of endothelial cell activation including increased circulating Von Willebrand factor (VWF) and cellular fibronectin (cFN) antedate clinically evident disease. We have therefore proposed that alteration of endothelial cell function by circulating agent(s) produced by the placenta initiates the clinical syndrome. This hypothesis predicts that there are a factor(s) in the blood of women with preeclampsia that are capable of altering endothelial cell function. We and others have examined in vitro interactions of maternal serum and endothelial cells to test this hypothesis. Our initial report indicating increased release of [51Cr]chromate from human umbilical vein endothelial cells (HUVE) suggested a lethal, lytic effect of serum from preeclamptic women. However, more specific indicators of endothelial cell structure and function do not support such a nonspecific effect. The morphology of HUVE was minimally altered after exposure to sera of preeclamptic women, and the entry of propidium iodide entry into cells, a sensitive indicator of membrane integrity, also was not increased. These findings, in combination with the increased expression of mRNA for platelet-derived growth factor (PDGF), suggest endothelial cell activation rather than cell death in response to sera from preeclamptic women. Comparison of the effects of endotoxin and sera from preeclamptic women also supports the specificity of this response. Whereas endotoxin strikingly increased VWF on the surface of HUVE and tissue factor activity in conditioned media while minimally increasing cFN, preeclamptic sera increased cFN but had no demonstrable effect on VWF or tissue factor activity.(ABSTRACT TRUNCATED AT 250 WORDS)