Electro-osmotic studies of endothelial cell surface

The surface properties of human umbilical vein endothelial cells (HUVEC), which form the inner walls of blood vessels, have been studied by electro-osmosis measurements. The data are analyzed on the basis of an electrophoresis theory of “soft” particles (that is, particles covered by polyelectrolyte layers). This theory utilizes two parameters, i.e. the density of fixed charges in the cell surface layer and a “softness” parameter (1/λ) which corresponds to the reciprocal of the coefficient of the frictional forces exerted by the surface layer on the liquid flow in the surface layer. It is found that the “softness” parameter for HUVEC is very large, i.e. the surface of HUVEC is very soft compared with those of other biological cells such as erythrocytes or human promyelocytic leukemia cell lines (HL-60RG cells) which we have studied before. The obtained value of 1/λ is comparable to that observed for a poly (NIPAAm) hydrogel layer in a swollen state. The charge density in the surface layer of HUVEC, however, is found to be similar to those for other biological cells.     

Stimulants from gardeniae fructus for cultured endothelial cell proliferation.

Bioassay-guided fractionation of Gardeniae Fructus extract (GFE), which stimulates the proliferation of cultured endothelial cells, led to the isolation of glycerol and D-mannitol. Both compounds significantly increased the incorporation of [3H]thymidine and [14C]leucine into the acid-insoluble fraction of bovine aortic endothelial cell layers in culture. This clearly indicated that glycerol and D-mannitol are active components of GFE on endothelial cell proliferation. On the other hand, they did not change the number of cultured vascular smooth muscle cells from bovine aorta. Glycerol and D-mannitol may be beneficial drugs for vascular disorders.     

Effect of laser modified surface microtopochemistry on endothelial cell growth

The introduction of microelectronics technology in the area of biological sciences has brought forth previously unforeseeable applications such as DNA or protein biochips, miniaturized, multiparametric biosensors for high performance multianalyte assays, DNA sequencing, biocomputers, and substrates for controlled cell growth (i.e. tissue engineering). We developed and investigated a new method using “cold” excimer laser beam technology combined with microlithographical techniques to create surfaces with well defined 3D microdomains in order to delineate critical microscopic surface features governing cell–material interactions. Microfabricated surfaces with microgrooves 30–3 μm deep, 10–1 μm wide spaced 30 μm apart were obtained with micron resolution, by “microsculpturing” polymer model surfaces using a computer controlled laser KrF excimer beam coupled with a microlithographic projection technique. The laser beam after exiting a mask was focused onto the polymer target surface via an optical setup allowing for a 10-fold reduction of the mask pattern. Various 3D micropatterned features were obtained at the micron level. Reproducible submicron features could also be obtained using this method. Subsequently, model human umbilical endothelial cells (HUVEC) were cultured on the laser microfabricated surfaces in order to study the effects of specific microscopic surface features on cell deposition and orientation. Cell deposition patterns were found to be microstructure dependant, and showed cell orientation dependency for features in the cell range dimension, a behaviour significantly different from that of a previously studied cell model (osteoprogenitor cell). This model may be a promising in so far as it is very rapid (a time frame less than a second per square centimeter of micropatterned surface) and provides further insights into the effects of surface microtopography on cell response with possible applications in the field of biosensors, biomedical and/or pharmaceutical engineering sciences.     

A stimulatory effect of Artemisia leaf extract on the proliferation of cultured endothelial cells

To investigate the effect of the hot water extract from Artemisia leaf (Artemisia princeps Panpanini) (AFE) on the proliferation of endothelial cells, the cells from bovine aorta were cultured for up to 96 h in the presence of 1, 5, 10 or 50 micrograms/ml AFE in RPMI1640 medium supplemented with 10% fetal bovine serum. After a 72 h culture, the cell number was significantly increased by AFE at 1, 5 and 10 micrograms/ml. An increase in the cell number by 5 micrograms/ml AFE observed after a 72 or 96 h treatment. The incorporations of both [3H]thymidine and [14C]leucine by the growing cells were significantly increased by 5 micrograms/ml AFE after a 72 h treatment. In addition, the incorporation of [3H]thymidine by either growing or confluent cells was significantly increased by 50 micrograms/ml AFE after a 72 h treatment. The stimulatory activity of AFE was recognized in the low-molecular-weight fraction (molecular weight less than or equal to 10000 dalton). These results clearly indicated that AFE contained some low-molecular-weight component(s) which stimulates the proliferation of vascular endothelial cells in vitro.     

Low molecular weight heparin enhances prostacyclin production by cultured human endothelial cells.

Confluent cultures of vascular endothelial cells derived from the human umbilical vein were incubated in a serum-free medium in the presence of low molecular weight heparin (LMWH) with molecular weights of 4000-6000 dalton (Da), or of unfractionated heparin (UFH) with average molecular weight 12,000 Da, and prostacyclin production was determined by radioimmunoassay for 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. LMWH at 1 U/ml as anti-factor Xa activity significantly increased prostacyclin production after 6h or longer; however, UFH at 1 USP U/ml did not induce such a significant change. The LMWH-induced increase in prostacyclin production occurred at 0.1 U/ml and above after 6 h of treatment. Since prostacyclin is both a potent inhibitor of platelet aggregation and a vasodilator, it was suggested that the increased endothelial cell prostacyclin production induced by LMWH may be a component of the anticoagulant activity of the drug.     

Control of endothelial cell adhesion to polymer surface by ion implantation

The bio‐compatibility of ion implanted polymers has been studied by means of in vitro attachment measurements of bovine aorta endothelial cells. The specimens used were polystyrene (PS), polyethylene (PE), polypropylene (PP) and expanded polytetrafluoroethylene (ePTFE). He⁺ and Ne⁺ ion implantation were performed at an energy of 150 keV with fluences between 1 × 10 ¹³ to 1 × 10 ¹⁷ ions/cm ² at room temperature. Wettability was estimated by means of a sessile drop method. The chemical and physical structures of ion implanted polymers were investigated by contact angle measurements, atomic force microscopy and X‐ray photoelectron spectroscopic analysis in relation to cell attachment behavior. The strength of cell attachment on ion implanted specimens at static and under flow conditions was also measured. Ion implanted PP and ePTFE were found to exhibit remarkably higher adhesion and spreading of endothelial cells than non‐implanted specimens. In contrast to these findings, ion implanted PS and PE only demonstrated a little improvement of cell adhesion in this assay. Copyright © 2001 John Wiley & Sons, Ltd.     

聚氯乙烯表面共价键合肝素及抗凝血性的研究

采用Ar等离子体引发聚乙二醇(PEG)在聚氯乙烯(PVC)表面固定化,进一步对固定PEG后的PVC进行肝素化处理,以改善PVC材料的抗凝血性能。探讨了PEG浓度对Ar等离子体固定化反应效果的影响。通过X射线光电子能谱(XPS)、衰减全反射红外光谱(ATR-FTIR)、扫描电镜(SEM)和接触角测定研究了固定PEG前后PVC的表面性能和表面形貌的变化。XPS分析证实肝素已成功地共价键合于PVC表面。采用体外凝血时间测定和血小板粘附实验对材料的抗凝血性能进行评价,结果表明,被修饰PVC材料的抗凝血性能显著提高。     

选择性接聚乙二醇枝聚醚聚酯的合成及其血液相容性

水解α－甲基－ω（２，３－环氧）丙基聚乙二醇（Ⅱ）得到接有聚乙二醇（ＰＥＧ）枝的丙二醇（Ⅲ），用其与对苯二甲酰氯及聚丁二醇反应制得在硬链段接有ＰＥＧ枝的聚醚聚酯（Ｈ－ＰＥＥｓ），以四氢呋喃与Ⅱ进行正离子开环共聚合制得每他链接有１．２５个ＰＥＧ枝的聚丁二醇（ＩＶ），用Ⅳ合成了在软链上接ＰＥＧ枝的聚醚聚酯（Ｓ－ＰＥＥｓ），不同位置接权的ＰＥＥｓ亲水性均较母体明显改进，抗凝血性则以Ｈ－ＰＥＥｓ改进显著。     

AFM study of hippocampal cells cultured on silicon wafers with nano-scale surface topograph

The rat hippocampal cells were selected as model to study the interaction between the neural cells and silicon substrates using atomic force microscopy (AFM). The hippocampal cells show tight adherence on silicon wafers with nano-scale surface topograph. The lateral friction force investigated by AFM shows significant increase on the boundary around the cellular body. It is considered to relate to the cytoskeleton and cellular secretions. After ultrasonic wash in ethanol and acetone step by step, the surface of silicon wafers was observed by AFM sequentially. We have found that the culture leftovers form tight porous networks and a monolayer on the silicon wafers. It is concluded that the leftovers overspreading on the silicon substrates are the base of cell adherence on such smooth inert surfaces.     

Human placental cell surface antigens:expression by cultured cells of diverse phenotypic origin

The present work examined the expression of cell surface glycoprotein antigens in cultured human cell lines. The set of glycoproteins studied was defined by their immunoreactivity with antiserum developed to Triton-solubilized extracts of placental brush border membranes. Studies were performed using cell lines of trophoblastic (BeWo, JEG-3) and nontrophoblastic (Chang liver cells) origin, as well as diploid fibroblast cell lines (WI-38, GM-38). Antiplacental brush border antiserum reacts with at least 19 distinct antigens present in placental membrane preparations, each of which can be resolved and identified in two-dimensional electrophoresis. The subunit molecular weight and isoelectric point for all components were defined by their positions in the two-dimensional matrix. Thirteen of these could be detected among the five cell lines examined by lactoperoxidase-catalyzed cell surface iodination. One of these 13 antigens has been identified as the placental isoenzyme of alkaline phosphatase (PAP). The expression of this component is limited to choriocarcinoma cells and Chang liver cells and it is not present in diploid fibroblasts. Under normal circumstances expression of PAP is unique to the differentiated placenta but has been frequently demonstrated in both trophoblastic and nontrophoblastic neoplasms. Two other antigens are variably expressed among the different cell types examined in the present study and their presence or absence was independent of the trophoblastic, epithelial nontrophoblastic, or fibroblastic origin of the cells. Ten surface antigens were expressed in all five cell lines. Six of these had previously been found common to membranes from three adult differentiated tissues, including liver and kidney, as well as placenta (Wada et al, J Supramol Struc 10(3): 287-305, 1979). The presence of this set of antigens in cultured cells as well extends the possibility that these are ubiquitously expressed on human cell surfaces. Two other antigens observed in all cultured cells had been found in both placental and either kidney or liver membranes and may represent common functions shared by many tissues which are also necessary for growth in vitro. The two remaining placental antigens seen in all cultured cells have previously been shown to be absent in adult tissues. Their presence in cultured cells but not in the membranes of resting differentiated tissues may signify the expression of glycoproteins characteristic of trophoblasts in all cells adapted to growth in culture.     

Evaluation of surface charge density and surface potential by electrophoretic mobility for solid lipid nanoparticles and human brain-microvascular endothelial cells

Electrophoretic mobility, zeta potential, surface charge density, and surface potential of cacao butter-based solid lipid nanoparticles (SLN) and human brain-microvascular endothelial cells (HBMEC) were analyzed in this study. Electrophoretic mobility and zeta potential were determined experimentally. Surface charge density and surface potential were evaluated theoretically via incorporation of ion condensation theory with the relationship between surface charge density and surface potential. The results revealed that the lower the pH value, the weaker the electrostatic properties of the negatively charged SLN and HBMEC. A higher content of cacao butter or a slower stirring rate yielded a larger SLN and stronger surface electricity. On the contrary, storage led to instability of SLN suspension and weaker electrical behavior because of hydrolysis of ionogenic groups on the particle surfaces. Also, high H+ concentration resulted in excess adsorption of H+ onto HBMEC, rendering charge reversal and cell death. The largest normalized discrepancy between surface potential and zeta potential occurred at pH = 7. For a fixed biocolloidal species, the discrepancy was nearly invariant at high pH value. However, the discrepancy followed the order of electrical intensity for HBMEC system at low pH value because mammalian cells were sensitive to H+. The present study provided a practical method to obtain surface charge properties by capillary electrophoresis.     

Effects of calphobindin II (annexin VI) on procoagulant and anticoagulant activities of cultured endothelial cells.

Effects of human placental calphobindin II (CPB-II) on the protein C activation and prothrombin activation on the cell surface of cultured calf pulmonary arterial endothelial cells have been investigated. CPB-II inhibited thrombin generation by factor Xa bound to the surface of the cultured endothelial cells in a dose-dependent manner. The amount (IC50) of CPB-II causing the inhibition at 50% was estimated to be approximately 10 nM. CPB-II was found to be ineffective, however, in the protein C activation by thrombin-thrombomodulin (TM) complex on the cell surface. Assay using purified TM revealed that CPB-II was able to exhibit the inhibitory potency for the protein C activation exclusively in the reconstituted system with negatively charged phospholipids. These results suggest that the neutral phospholipids participate in the protein C activation through the thrombin-TM system on the endothelial cell surface. The ability of CPB-II to inhibit procoagulant activity without affecting anticoagulant activity on the cultured endothelial cells is probably related to its potential physiological function, while it is able to exert various degrees of influence upon these activities in blood coagulation by interacting with negatively charged phospholipids in vitro.     

The influence of polyelectrolyte charges of polyurethane membrane surface on the growth of human endothelial cells

A novel technique to introduce free amino groups onto polyester scaffolds via aminolyzing the ester groups with diamine has been developed recently. The introduction of the free amino groups on these polyester surfaces provides us the possibility to modify polymer surface in a simpler manner, e.g. layer-by-layer assembly of charged species. By this technique, many negatively and positively charged biopolymers were deposited alternatively on polyurethane surface. The deposition process was monitored by fluorescence spectroscopy and advancing contact angle measurements. The result of human endothelial cells cultured in vitro showed that cells on negatively charged surface could not spread and flatten well due to the electrostatic repulsion. The lower attachment ratio induced the lower proliferation ratio. However, after the surface charge was inversed by collagen, both attachment and proliferation ratios increased to different extent. Observed under SEM, cells also presented a flat and spreading morphology.     

Effect of honeycomb-patterned surface topography on the adhesion and signal transduction of porcine aortic endothelial cells

Surface topography has vital roles in cellular response. Here, to investigate the mechanism behind cellular response to surface topography, we prepared honeycomb (HC)-patterned films from poly(epsilon-caprolactone) (PCL) with micropatterned surface topography by casting a polymer solution of water-immiscible solvent under high humidity. We characterized the adsorption of fibronectin (Fn) on the film using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). The response of porcine aortic endothelial cells (PAECs) to adsorbed Fn molecules onto HC-patterned films was observed by immunofluorescence labeling of vinculin and the actin fiber of PAECs cultured for 1 and 72 h in serum-free medium. The expression of focal adhesion kinase autophosphorylated at the tyrosine residue (pFAK) at 1 h culture was determined using an immunoprecipitation method. Fn adsorbed selectively around the pore edges to form ring-shaped aggregates. The immunostaining results revealed that PAECs adhered to the HC-patterned films at focal contact points localized around pore peripheries. These points correspond to adsorption sites of Fn. The expression of pFAK after 1 h on the HC-patterned film was 3 times higher than that on a corresponding flat film, indicating that the signaling mediated by the binding between Fn and the integrin receptor was more highly activated on the HC-patterned film. These results suggest that the cellular response to HC-patterned films (e.g., adhesion pattern and phosphorylation of FAK) originates from the regularly aligned adsorption pattern of Fn determined by the pore structure of the film.     

Synthesis of 1,4-diphenylbutadiene derivatives: novel inducer of tissue-type plasminogen activator (t-PA) in cultured bovine endothelial cells

(E,E)-1,4-Diphenylbutadiene derivatives were synthesized by utilizing the Stobbe reaction of dimethyl succinate as a key step. Their stereoisomers were also synthesized stereoselectively by means of the cross-coupling reaction of the vinylstannanes and the vinylbromides, which were obtained from the propiolic acid esters by stereoselective hydrostannation, as a key step. To discover novel stimulators of fibrinolysis in vascular endothelial cells, the synthesized compounds were added to cultured bovine endothelial cells to determine the activity of the plasminogen activator in the conditioned medium. Of the synthesized compounds, three compounds were found to stimulate the activity of the plasminogen activator in endothelial cells. In addtition, these compounds inhibited thrombus formation in a rat model of venous thrombosis.     

Effect of surface charge of magnetite nanoparticles on their internalization into breast cancer and umbilical vein endothelial cells

Internalization of magnetite nanoparticles with diameter of approximately 40 nm into normal and cancer cells was examined by microscopic observation and flow cytometry. Magnetite nanoparticles were synthesized by hydrolysis in an aqueous solution containing ferrous chloride with organic amines as a base. It was demonstrated that the difference in surface charge of magnetite nanoparticles brought about the difference in uptake efficiency. The nanoparticles with positive charge showed higher internalization into human breast cancer cells than the nanoparticles with negative charge, while the degree of internalization of the positively- and negatively-charged nanoparticles into human umbilical vein endothelial cells (HUVEC) was almost the same.