Lactoferrin inhibits the inflammatory and angiogenic activation of bovine aortic endothelial cells

Objective  

Lactoferrin (Lf) is known to have anti-cancer and anti-inflammatory activities; however, its therapeutic mechanism has not been defined. In this study, to explain the therapeutic mechanism of Lf, we examined the effect of Lf on endothelial cell activation, leukocyte integration, and angiogenesis in vitro.     

骨膜蛋白siRNA转染抑制ox-LDL诱导的人主动脉内皮细胞系损伤

目的探讨骨膜蛋白(Postn)对氧化型低密度脂蛋白(ox-LDL)诱导的人主动脉内皮细胞系(HAECs)损伤的影响及其作用机制。方法将HAECs随机分为4组:对照组、ox-LDL组、Postn siRNA组和negative siRNA组。RT-q PCR检测mRNA水平;Western blot检测蛋白表达;MTT检测细胞增殖;流式细胞仪检测细胞凋亡;EMSA检测NF-κB的DNA结合能力。结果与对照组相比,ox-LDL组Postn的mRNA和蛋白水平显著提高(P0.05);细胞增殖能力降低(P0.05);细胞凋亡率升高(P0.05);VCAM1、ICAM1、E-selectin、IL-1β、IL-6、TNF-α、p65和p-IκB-α的蛋白表达水平显著上调(P0.05),且NF-κB的DNA结合能力提高(P0.05),Postn siRNA转染能够逆转以上结果。结论 Postn siRNA转染能够抑制ox-LDL诱导的内皮细胞损伤,这可能与抑制NF-κB信号通路有关。     

Fetal bovine serum inhibits hepatitis C virus attachment to host cells

Fetal bovine serum (FBS), used normally as a basic cell culture supplement, inhibits influenza virus growth. However, the role of FBS in the regulation of hepatitis C virus (HCV) infection has not been studied extensively and remains largely unclear. We adopted the established cell-cultured HCV (HCVcc) isolated from the JFH-1 strain and two sets of solutions (cDMEM7.4 and cDMEM6.8; RHMNB6.8 and RHMN6.8) to investigate the effect of FBS on HCV infection. Our data indicate that FBS blocks HCV infection in a dose-dependent manner. The infectivity of HCV diluted in the RHMNB solution was more susceptible to the addition of FBS than that diluted in the cDMEM solution. In addition, FBS-mediated blocking of HCV infection occurred at the step of virus attachment to the target cells, suggesting that FBS contains factors that interfere with the early steps in HCV infection.     

非诺贝特上调血管内皮细胞一氧化氮合酶的表达

目的:观察PPARα激动剂非诺贝特对牛主动脉(BAECs)内皮细胞一氧化氮合酶(eNOS)活性和表达的影响。方法:制备5-9代BAECs,加入不同浓度的非诺贝特(0, 5, 10, 50, 100 μmol/L)后,用NOS Assay Kit测定eNOS活性,RT-PCR法检测eNOS mRNA表达,Western blot分析检测eNOS蛋白质表达。结果: 非诺贝特以浓度和时间依赖的方式增加eNOS活性,非诺贝特浓度10 μmol/L以上时,明显增加eNOS活性。50μmol/L非诺贝特处理48 h时eNOS活性最大(为对照组的2.32±0.47倍,P<0.01)。非诺贝特处理1 h和12 h不增加eNOS活性。RT-PCR分析表明,非诺贝特浓度大于5 μmol/L以上时,明显增加eNOS mRNA水平,在非诺贝特浓度为50 μmol/L时作用最大,为对照组的2.08±0.33倍(P<0.01)。此作用在6 h时出现,持续到48 h。Western blot显示,非诺贝特处理48 h,eNOS蛋白表达明显增加,在浓度为10,50 和100 μmol/L时,eNOS蛋白表达分别为对照组的1.80±0.45, 2.70±0.42 和 2.20±0.32 倍,均P<0.01。在非诺贝特处理12 h后出现,持续到48 h。结论:PPARα激动剂非诺贝特增加BAECs eNOS基因表达,提高eNOS活性及增加蛋白表达。     

Susceptibility of irradiated bovine aortic endothelial cells to injury.

Using cultured bovine aortic endothelial cells (BAEC), the authors attempted to determine whether prior irradiation would alter the susceptibility of these cells to three known injurious stimuli and, if so, whether the alteration would be related to radiation dose. BAEC were irradiated with 0, 5, or 10 Gy of gamma rays and, on the third postirradiation day, exposed to fibrin, nicotine, or bacterial endotoxin (lipopolysaccharide, LPS). Release of prelabeled 51Cr, representing cell lysis, cell detachment, or a combination of the two, was determined. Significant differences between irradiated and control cells were determined by using paired Student's t-tests. Irradiation did not appear to have altered the sensitivity of BAEC to fibrin-induced injury. Cells irradiated with 10 Gy of gamma rays, but generally not those irradiated with half this dose, showed a heightened susceptibility to nicotine. Contrary to the nicotine results, irradiated cells showed less cell detachment and lysis after exposure to LPS. These results suggest that the susceptibility of irradiated BAEC to harmful stimuli depends largely on the nature of the stimulus as well as the radiation dose.     

Chloride-selective channels of large conductance in bovine aortic endothelial cells

Single-channel currents of an anionic channel in the plasma membrane of cultured bovine aortic endothelial cells have been recorded with the patch-clamp technique. The channel is selective for chloride over cations, and has an average single channel conductance of 382 picosiemens in symmetric 140 millimoles of chloride. In addition to the main conductance state it shows well-defined subconductance states of about 50, 100, 150 and 200 picosiemens. The channel is very active at membrane potentials close to 0 mV, but steps to either positive or negative membrane potentials above ± 20 millivolt lead to a rapid inactivation of the channel. Changes in the concentrations of free calcium or andenosine tri-phosphate on the cytosolic surface do not influence channel activity. The chloride channel rarely opens at resting membrane potential, but it may help repolarize endothelial cells following depolarizing stimuli.     

Junctional transfer in wounded cultures of bovine aortic endothelial cells

Both in vivo and in vitro, endothelial cells form continuous monolayers displaying growth control by cell density as well as cell contact. Several functions of endothelium are dependent upon maintenance of an intact monolayer. When such a monolayer is injured, endothelial cells migrate out from the wound edges and proliferate to cover the area of denudation. The response of endothelium to wounding is therefore a matter of both growth control and cellular motility. Since intercellular gap junctions have been postulated to have a role in growth control, we set out to determine whether junctional communication was altered in the activated, migrating cells. We found extensive transfer of microinjected fluorescent dye molecules (Lucifer yellow CH) in cells at or near the wound edge at various times after wounding (1 minute to 48 hours). However, cells near a wound edge transferred dye at a frequency significantly diminished from that observed in undisturbed monolayers (81.1 +/- 1.5 (SE) versus 92.7 +/- 0.8% of adjacent cells received dye). There was a significant positive correlation between transfer frequency and distance from the wound (expressed as intervening cells) but not between transfer frequency and time after wounding. These results suggest that endothelial cells in a wounded monolayer retain junctional communication at a slightly but significantly reduced level and that increased motility and the wound healing response do not necessarily correlate with total loss of communication.     

Hydrogen peroxide stimulates endocytosis in cultured bovine aortic endothelial cells

Fluid-phase uptake of macromolecules by cultured bovine aortic endothelial cells was measured using FITC-dextran (70000 Da). Low doses of hydrogen peroxide added extracellularly stimulated the uptake of macromolecules by the endothelial cells. There was no general increase in the passive permeation or the transport across the cell layer. Moreover, when endothelial cells were stimulated with phorbol myristate acetate (PMA), macromolecules uptake was also enhanced. The PMA effect was blocked with superoxide dismutase (SOD) and catalase, suggesting a pivotal role of oxygen metabolites in fluid phase uptake of macromolecules by endothelial cells.     

Leukotriene induction of TxB2 in cultured bovine aortic endothelial cells

The leukotrienes (LT) are potent mediators of inflammation, capable of inducing plasma leakage from postcapillary venules and vasoconstriction of terminal arterioles. Some microvascular effects may be attributable to LT stimulation of thromboxane (Tx) synthesis. Incubation of primary cultures of bovine aortic endothelial cells with buffer, LTB₄ (10^–8 M) or LTD₄ (10^–8 M), resulted in TxA₂ production in pg/10⁵ cells to the extent of: 67 ± 20, 571 ± 180, and 333 ± 60 respectively, as measured by radioimmunoassay of the stable metabolite TxB₂. Endothelial pretreatment with the LTD₄ receptor antagonist FPL55712 (10^–5 M) significantly blocked any LTB₄- or LTD₄-stimulated TxA₂ synthesis. Pretreatment with the TxA₂ synthetase inhibitor ketoconazole (10^–6 M) also prevented LTB₄ of LTD₄ stimulation of TxA₂. Preincubation with DMTU (10^–5 M), an hydroxyl radical scavenger, decreased LTB₄-induced release of TxA₂ (405 ± 40 and 366 ± 20, respectively). These findings suggest that LT may mediate their inflammatory actions in vascular beds by stimulation of Tx release from endothelial cells.     

Eicosanoid modulation of stress fibers in cultured bovine aortic endothelial cells

Leukotrienes (LT) B₄, LTD₄, and thromboxanes (TX) are cytotoxic, and increase microvascular permeability. Because endothelial stress fibers are theorized to be part of the cytoskeletal mechanism by which microvessels maintain their barrier function, the effect of these eicosanoids on stress fibers in bovine aortic endothelial cells was tested. LTB₄, LTD₄, and TXB₂ each decreased stress fiber numbers by 93%, 62%, and 66%, respectively, when compared to controls (P < 0.01). In contrast, endothelial cells treated with prostacyclin (PGI₂) at 10^–7 M, produced a significant increase (P >0.01) in stress fiber numbers, 211% to that of controls. Azaprostanoic acid (13-APA) and FPL55712, receptor antagonists to TX and slowreacting substance of anaphylaxis (SRS) receptors, respectively, the cyclooxygenase inhibitor ibuprofen, and the TX synthase inhibitors imidazole and ketoconazole were used to test for possible endothelial cell receptor mediation and de novo prostanoid synthesis associated with inflammatory eicosanoid-induced disassembly of stress fibers. Each pharmacologic agent inhibited the LTD₄- and TXB₂-induced decreases in stress fibers; LTB₄-stimulated stress fiber decreases were inhibited only by pretreatment with TX synthase blockers. These data suggest that increased permeability associated with inflammatory eicosanoid metabolites may have as a common target stress fiber disassembly, and the mechanism may be receptor-mediated. That cyclooxygenase and TX synthase blockers inhibited the eicosanoid action suggests that endogenous TX synthesis may be a step in the mechanism. PGI₂ enhancement of the microvascular barrier may be related to the effect of PGI₂ on promoting stress fiber assembly. In summary, endothelial cell synthesized autocoids derived from arachidonic acid may help to regulate microvascular permeability by way of their action on stress fiber assembly/dissassembly, and unbalanced prostanoid secretion by way of LT stimulation may result in a loss of the microvascular barrier and increased permeability.Supported by NIH grants HL16714, HL33104, and GM24891.     

Chloride-selective channels of large conductance in bovine aortic endothelial cells.

Single-channel currents of an anionic channel in the plasma membrane of cultured bovine aortic endothelial cells have been recorded with the patch-clamp technique. The channel is selective for chloride over cations, and has an average single channel conductance of 382 picosiemens in symmetric 140 millimoles of chloride. In addition to the main conductance state it shows well-defined subconductance states of about 50, 100, 150 and 200 picosiemens. The channel is very active at membrane potentials close to 0 mV, but steps to either positive or negative membrane potentials above +/- 20 millivolt lead to a rapid inactivation of the channel. Changes in the concentrations of free calcium or adenosine tri-phosphate on the cytosolic surface do not influence channel activity. The chloride channel rarely opens at resting membrane potential, but it may help repolarize endothelial cells following depolarizing stimuli.     

Shear activated channels in cell-attached patches of cultured bovine aortic endothelial cells

We investigated the response of inward rectifier K⁺ (IRK) currents in bovine aortic endothelial cells (BAECs) to shear stress. Shear evoked reversible hyperpolarization in current clamped BAECs. Voltage clamped BAECs exhibited large inward and small outward whole cell K⁺ currents blocked by cesium and increased in amplitude by exposure to shear stress. The open state probability of IRK channels in cell-attached membrane patches was increased within minutes of exposure to shear stress. IRK channels in inside-out patches were activated by increases in [Ca²⁺]_i from 10^–7 to 10^–6 mM. We demonstrate that shear stress induces hyperpolarization and gating of single channel and whole cell IRK currents in BAECs.Supported by NIH grant numbers K08-1924, HL49294 (ERJ), HL 29587-09 (DH), R37 HL 33833-07 (DH), and HL15062 (PFD).     

Is there a calcium paradox in isolated bovine aortic endothelial cells?

When bovine aortic endothelial (BAE) cells are superfused with a solution containing low calcium (approximately 10 nM) and subsequently exposed to a solution containing normal Ca2+ (2 mM) a large transient increase in intracellular calcium is seen. This elevation in [Ca2+]i is similar to that described as the calcium paradox in cardiac cells. If the cells are exposed to the agonist, ATP, during the period in low-Ca2+ solution the paradoxical rise in [Ca2+]i is increased. Removal of external Na+ from the low-Ca2+ solution reduces the rise in [Ca2+]i on returning to 2mM-Ca2+ solution. These data are consistent with the presence of a calcium paradox in these cells and with the hypothesis that the underlying mechanism involves the loading of the cell with Na+ during the period in low Ca2+. This process may occur as a result of the altered selectivity of the ATP-activated Ca2+ influx mechanism.     

人白介素10对异种内皮细胞保护作用的研究

目的：研究人白介素10(HIL-10)对活化的牛主动脉内皮细胞(BAECs)的保护作用，为HIL-10用来减轻异种排斥反应、诱导异种移植免疫耐受提供实验依据。方法：用不同浓度的HIL-10(2、5、10、20、40ng/ml)孵育BAECs2小时后，再与肿瘤坏死因子a(TNF-a，ng/ml)共孵育6小时或18小时；应用细胞-酶联免疫吸附分析方法(Cell-ELISA)检测细胞表面的E-seleetin和ICAM-1的表达。用MTT方法测定药物对细胞活性的影响。结果：用HIL-10在一定浓度内预处理BAECs后，能明显抑制TNF-a诱导的E-seleetin与ICAM-1的表达，并呈现一定的剂量依赖性。用MTT方法测定细胞活性的实验表明，各实验组细胞活性与对照组无明显差异性。结论：HIL-10能明显抑制TNF-a活化的BAECs表达Bseleetin与ICAM-1；HIL-10对BAECs有一定的保护作用。     

Cationic lipid bilayer coated gold nanoparticles-mediated transfection of mammalian cells

Here, we demonstrated dimethyldioctadecylammonium bromide (DODAB), a cationic lipid, bilayer coated Au nanoparticles (AuNPs) could efficiently deliver two types of plasmid DNA into human embryonic kidney cells (HEK 293) in the presence of serum. The transfection efficiency of AuNPs was about five times higher than that of DODAB. The interaction of AuNPs with DNA was characterized with dye intercalation assay and agarose gel electrophoresis. The morphology of the complex of AuNPs with DNA was observed with scanning electron microscope (SEM). The intracellular trafficking of the complex was monitored with transmission electron microscope (TEM). Based on experimental results, the possible mechanism was proposed and the barriers in the process of transfection were discussed. This work demonstrates a simple way to increase the transfection efficiency of cationic lipid through changing the stability of the complex of cationic lipid with DNA. It may provide some insights into understanding and controlling the interaction of cationic lipid with DNA. It also provides a novel way to construct gold nanoparticles-based gene vectors and some insights into learning the process of nanomaterials-mediated transfection.     

Toxic effects of aldehydes released from fixed pericardium on bovine aortic endothelial cells

Glutaraldehyde (GA) and formaldehyde (FA) were shown to be released from 1 cm2 fixed pericard patches into 2-mL storage solutions after three 2-min washings in concentrations of about 1 mg/L. The cytotoxicity of these aldehyde concentrations on bovine aortic endothelial cells (BAECs) was evaluated in vitro, by proliferation capacity, cellular ATP content, PGI2 release and cyclic AMP synthesis. Continuous incubation of BAECs with GA greater than 0.1 mg/L and FA greater than 0.5 mg/L resulted in a significantly inhibited proliferation and in an increase of the intracellular Ca2+ triggered parameters, PGI2 and cyclic AMP, up to three fold. This strongly suggests an aldehyde-induced inhibition of the plasma-membrane bound Ca2+-ATPase, an enzyme which normally maintains low intracellular Ca2+-level. From these findings there is evidence that aldehydes released from bioprosthetic valve tissue may contribute to the lack of endothelial cell coverage in human implants.     

LPS-stimulated bovine aortic endothelial cells produce IL-1 and IL-6 like activities

Vascular endothelium is known to closely interact with leukocytes and immunocompetent cells. We report here that cultured bovine aortic endothelial cells (BAEC) synthesize both interleukin 1 (IL-1) and interleukin 6 (IL-6) like activities in response to bacterial lipopolysaccharide stimulation. Our results agree with previous data obtained from human venous endothelia and support the concept that IL-1 and IL-6 synthesis are properties common to endothelial cells from different vascular beds. The IL-1 activity was measured by murine thymocyte proliferation assay and by an indirect bioassay using NOB1 cells, which evidenced higher IL-1 amounts than the former. This discrepancy appeared to be partly due to the simultaneous production of one or more inhibitor(s) of the thymocyte proliferation by BAEC. The IL-6 assay was performed with the murine hydridoma cell line B9. In other respects, the cyclooxygenase inhibitor indomethacin enhanced the IL-1 like production, but was ineffective on IL-6 like production. The present study provides additional evidence that endothelial cells from large arteries may also participate in inflammatory and immunological processes.     

Voltage-activated potassium,but not calcium currents in cultured bovine aortic endothelial cells

The electrophysiological properties of cultured bovine aortic endothelial cells were characterized using the patch clamp technique. Resting potentials were measured on passing to the whole cell recording configuration and were close to –65 mV in healthy cells. In cell-attached recordings with a high potassium pipette solution, inward single channel currents were observed with zero applied pipette potential. A linear slope conductance of 25 pS was found for a wide range of hyperpolarizing patch potentials and also for depolarizing patch potentials of up to 50–60 mV. A pronounced inward rectification was apparent as no reversal of these currents was seen for larger depolarizations. Whole cell recording in physiological solutions revealed the presence of a hyperpolarization-activated inward current with strong inward rectification and no voltage-dependent ionic current was observed upon depolarization in this subset of cells. Substitution of potassium for external sodium resulted in a shift in the zero current potential consistent with potassium being the main permeant ion. Together with the characteristic voltage-dependent blocking actions of external sodium ions and low concentrations of barium and caesium ions, our results indicate that this current is very similar to the classical inward rectifier as originally described in skeletal muscle and in tunicate eggs. In a second population of cells, a depolarization-activated outward current displaying characteristics of the fast transient A-type potassium current as first reported in molluscan neurones was also observed. No evidence for inward voltage dependent sodium or calcium currents was found.     

Effect of flow direction on the morphological responses of cultured bovine aortic endothelial cells

The effect of flow direction on the morphology of cultured bovine aortic endothelial cells is studied. Fully confluent endothelial cells cultured on glass were subjected to a fluid-imposed shear stress of 2 Pa for 20 min and 24 h using a parallel plate flow chamber. Experiments on shear flow exposure were performed for (i) one-way flow, (ii) reciprocating flow with a 30 min interval and (iii) alternating orthogonal flows with a 30 min interval. After flow exposure, the endothelial cells were fixed and F-actin filaments were stained with rhodamine phalloidin. Endothelial cells were observed and photographed by means of a microscope equipped with epifluorescence optics. The shape index (SI) and angle of cell orientation were measured, and F-actin distributions in the cells were statistically studied. Endothelial cells under the one-way flow condition showed marked elongation (SI=0.39±0.16, mean±S.D.) and aligned with the flow direction. In the case of the reciprocating (SI=0.63±0.14) and the alternating orthogonal flows (0.64±0.14), cells did not elongate so strongly as in the case of one-way flow. Although most cells in the reciprocating flow aligned with the flow direction, the cell axes in the alternate orthogonal flow distributed around a mean value of −45.1° with a large S.D. value. Endothelial cells can be expected to recognise the flow direction, and change their shape and F-actin structure.     

Adhesion and proliferation of bovine aortic endothelial cells on monoamine- and diamine-containing polystyrene derivatives.

Adhesion and proliferation of bovine aortic endothelial cells on polystyrene derivatives having monoamine or diamine side chain was investigated focusing on the chemical structure of amino groups. Copolymers, SE8.5, is composed of polystyrene with 8.5 mol% of monoamine side chains, and SED8, which is with 8 mol% of diamine side chains, were estimated to contain almost the same amount of protonated amino groups in bulk composition at physiological pH (pH 7.4). There observed significant difference in cellular spreading of attached endothelial cells between these two types of copolymer surfaces. Spreading-% of attached cells on SED8 surfaces was approximately 1.6 times greater than that on SE8.5 6 h after seeding. This difference in cellular spreading influenced to subsequent cell growth. Cellular growth on each polymer surface was featured by parameter k, which corresponds to the 'rate constant' of cellular proliferation. While the k-value for SE8.5 decreased with decreasing seeding density as well as the case for polystyrene, SED8 maintained a high k-value even at low seeding density as 2 x 10(3) cells/cm2. These results suggest that cells may recognize the difference in the chemical structure of amine side chains of SE and SED copolymers.