Low pH-induced changes in Pseudomonas exotoxin and its domains: increased binding of Triton X-114.

Pseudomonas exotoxin (PE), which is composed of three structural domains, is a 66-kilodalton protein secreted by P. aeruginosa that is cytotoxic for mammalian cells. After binding to cell surface receptors and internalization into low-pH endocytic vesicles, PE or an active fragment kills mammalian cells by translocating across an intracellular membrane to the cytoplasm and shutting down protein synthesis. To investigate possible conformational changes associated with the translocation process, full-length PE or recombinant proteins containing the PE cell recognition domain, translocation domain, enzymatic domain, or translocation plus enzymatic domains were incubated with Triton X-114 at pH values ranging from 3.0 to 7.0. The truncated forms used were intact domains that had been expressed in Escherichia coli and subsequently purified. Previous studies (K. Sandvig and J. O. Moskaug, Biochem. J. 245:899-901, 1987) had shown that full-length PE bound more Triton X-114 at a low pH than at a physiologic pH. Therefore, we investigated whether this increased binding was due to a global change in PE or a change within a particular domain. Results showed that all the truncated toxin proteins displayed a similar pH-dependent entry into the detergent phase as native PE, with a transition point of 4.2 for PE and 4.4 to 4.5 for the truncated toxins. The isoelectric points of the recombinant proteins were measured and indicate that, at a low pH (5.0), the cell recognition domain bears a net positive charge, the translocation domain bears a net negative charge, and the enzymatic domain bears no charge. The results suggest that upon acidification in the endosome, PE becomes globally hydrophobic and is converted into a translocation-competent form.     

肝素结合生长因子的纯化及其刺激人血管内皮细胞增生作用的研究

用40—80%饱和度硫酸铵沉淀、CM-葡聚糖C-50离子交换层析及肝素琼脂糖亲和层析从牛脑中纯化了肝素结合生长因子,SDS-PAGE显示单一带型,分子量约为16kD;以人脐带静脉内皮细胞为靶细胞,对肝素结合生长因子刺激内皮细胞增生作用进行了研究,结果表明,细胞培养时加入肝素结合生长因子,可使内皮细胞长期传代,冷冻后可成功复苏,培养所需血清浓度可降至10—15%。     

Platelet activating factor modulates peptidoleukotriene effects on the permeability of bovine aortic endothelial cell to albumin

The purpose of the present experiments was to study the potency of various inflammatory mediators to modulate the clearance rate of¹²⁵I-albumin through bovine aortic endothelial cell (BAEC) monolayer on polycarbonate micropore membrane. The incubation of BAEC with LTD₄ (10⁻⁸–10⁻⁶ M) increased dose-dependently the permeability to radioactive albumin. The effect was potentiated by addition of PAF (10⁻⁵ M). LTC₄ had a less potent action on the clearance rate of¹²⁵I-albumin, since only the highest dose of LTC₄ used (10⁻⁶ M) had a significant effect, and it was not potentiated by PAF (10⁻⁵ M). The treatment with PAF alone at concentration up to 10⁻⁵ M did not increase the¹²⁵I-albumin clearance rate. Our results suggest that BAEC may bear specific receptors for LTC₄ and LTD₄ or that LTC₄ is not converted to LTD₄ by BAEC. Our results also suggest that LTD₄ could possibly stimulate the expression of PAF receptors on BAEC.

     

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.     

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.     

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.     

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.     

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.     

Cationic lipid-mediated transfection of bovine aortic endothelial cells inhibits their attachment

The need for a small-diameter vascular graft for coronary artery and peripheral vascular replacement is great and is projected to increase as the population ages. Synthetic small-diameter vascular grafts fail because of acute thrombosis or chronic intimal hyperplasia leading to restenosis. Endothelial cell seeding has been attempted with limited success in the femoral artery by Zilla and others. However, patency rates have not increased sufficiently to justify large clinical trials. Genetic engineering of endothelial cells before seeding has been proposed to encourage endothelial cell phenotypes that would predispose the graft to patency. In this study, we investigate the effect cationic lipid-mediated transfection of endothelial cells with respect to their attachment to a potential graft material, Fluoropassiv (Vascutek). Liposomal transfection was optimized for maximum gene expression. We report that transfection decreases the ability of bovine aortic endothelial cells to attach by approximately 100% as compared with nontransfected control over 18 h. Further, when placed under physiologic shear conditions, this difference is sustained. The effects of gene transfer on endothelial cell adhesion must be included as an important optimization criterion along with gene expression for engineered endothelial cell-seeding applications.     

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.     

Fibronectin expression correlates with U937 cell adhesion to migrating bovine aortic endothelial cells in vitro.

A blood vessel's response to denudation injury will determine its final luminal diameter as well as its function. The synthesis, deposition, and remodeling of extracellular matrix components and migration by vascular endothelial cells are major factors in determining luminal diameter, cellular proliferative and migratory responses, and mononuclear cell adhesion at sites of injury. Previously, we have shown that after in vivo and in vitro denudation injury, endothelial cell migration is dramatically influenced by the amount of fibronectin synthesized and deposited by the responding endothelial cell population. The aim of this study was to elucidate the roles of fibronectin in modulating mononuclear cell adhesion to the endothelial cell population during in vitro migration. In this report we demonstrate that U937 cell binding to the migrating fronts of endothelial cell monolayers is modulated by the amount of fibronectin synthesized and deposited by the endothelial cells. Agents which increase fibronectin deposition, such as transforming growth factor-beta 1, elicit greater U937 cell adhesion. Manipulations that decrease fibronectin deposition, such as transfection and overexpression of pp60c-src proto-oncogene in endothelial cells, reduce U937 cell adhesion. These results suggest that changes in endothelial cell extracellular matrix synthesis and deposition modulate, in part, the adhesive properties of the vessel wall after injury. In turn, the intensity and duration of mononuclear cell adhesion at sites of vessel wall injury determines, in part, the vessel wall response.     

FIZZ1 could enhance the angiogenic ability of rat aortic endothelial cells

Found in inflammatory zone (FIZZ1), also known as hypoxia-induced mitogenic factor (HIMF), is a secreted protein formed by 111 amino acid residues. FIZZ1 is mainly located in alveolar epithelial cells, white adipose tissue and the heart. This study aimed to explore the effects of FIZZ1 on the angiogenic ability of cultured rat aortic endothelial cells (RAECs) and the potential mechanism. The RAECs were cultured in the extracellular matrix (ECM) supplemented with 10% fetal bovine serum (FBS). Matrigel assay was used to detect the angiogenic ability of the RAECs and Agilent Rat Microarray containing 41,000 genes/ESTs was used to screen the differentially expressed genes of the RAECs after they were treated with FIZZ1 (5 x 10^-9~2 x 10^-8 mol/L). The results were verified using RT-PCR method. We found that FIZZ1 markedly enhanced the angiogenic ability of RAECs (22.6 ± 2.94 vs. 19.7 ± 2.57, P < 0.01; 28.5 ± 3.32 vs. 19.7 ± 2.57, P < 0.01; 36.9 ± 5.01 vs. 19.7 ± 2.57, P < 0.01) in a dose-dependent manner (5 x 10^-9~2 x 10^-8 mol/L). 440 genes (Gng8, Atg9a, Gdf6, etc.) were found to be up-regulated and 497 genes (Hbb-b1, Camk1g, etc.) down-regulated in the experimental group. Changes in Gng8 and Atg9a were revealed by RT-PCR. FIZZ1 could enhance angiogenesis of RAECs by up-regulating Gng8 and Atg9a.     

Cationization of bovine serum albumin alters its conformation as well as its charge

The molecular charge on bovine serum albumin (BSA) was modified by substituting carboxyl groups on the protein with ethylenediamine, thereby producing a highly cationic derivative with a pI of 9.3 to 9.5. Gel-filtration studies showed that the molecular weight of BSA was not significantly altered after cationization. When the cationized BSA was administered to rabbits using a chronic serum sickness schedule of injections, the animals developed a membranous glomerulopathy similar to the human disease, except that approximately one-third of the animals also showed focal and segmental endocapillary proliferation. Comparison of the circular dichroism spectra of native and cationized BSA showed that the substitution of the carboxyl groups resulted in a 50% reduction in the alpha-helical content of the native molecule. This conformational change should be considered as a possible determinant of the different immune response and immunopathology associated with the cationized molecule compared with native BSA.     

Bovine caveolin-2 cloning and effects of shear stress on its localization in bovine aortic endothelial cells.

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Normally, cells that express caveolin-1 also express caveolin-2, but this has not been demonstrated in bovine aortic endothelial cells (BAECs). Here, we show that BAECs express caveolin-2, which localizes in caveolae with caveolin-1. We have cloned the bovine caveolin-2 gene and after comparison with known protein sequences (human, murine, rat, and canine) have found divergent immunogenic regions (amino acid [aa] 21 to aa 50 and aa 79 to 88), which may explain the inability to detect caveolin-2 in different cell types. We developed a bovine caveolin-2-specific antibody to examine this protein's expression and localization in BAECs. We used differential gradient centrifugations and immunoprecipitation to show that bovine caveolin-2 and caveolin-1 form a hetero-oligomer in plasma membrane caveolae. Using immunocytochemistry we show that a pool of caveolin-2 also colocalizes with the cis-Golgi in static culture, but unlike caveolin-1, this Golgi associated pool is maintained after 1 day of shear exposure. Therefore, the interaction of caveolin-2 with caveolin-1 could play an important role in caveolae biogenesis and shear stimulated mechano-signal transduction.     

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.     

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).     

激活的固醇调节元件结合蛋白在内皮细胞移行中的作用

目的 探讨固醇调节元件结合蛋白(SREBP)在内皮细胞移行中的激活及其作用.方法 培养牛主动脉内皮细胞,应用Western blot分析、即时定量PCR、细胞移行测定、Cdc42活性测定等方法 ,研究了SREBP的激活在内皮细胞移行中的作用,以及这个过程中涉及SREBP的潜在的信号转达.结果 (1)内皮细胞移行组与非移行组相比,SREBP被显著激活(P<0.05);(2)内皮细胞移行组与非移行组相比,SREBP下游基因,低密度脂蛋白受体、羟甲基戊二酰辅酶A还原酶及脂肪酸合成酶的mRNA的表达显著增加(P<0.05);(3)SREBP裂解激活蛋白RNAi和SREBP抑制剂(25-羟化胆固醇)能够明显抑制内皮细胞的移行;(4)在各种模型中SREBP的抑制导致内皮细胞移行的减少;(5)SREBP裂解激活蛋白缺陷型、S2P缺陷型及S1P缺陷型细胞株,表现为内皮细胞移行损伤;(6)抑制内皮细胞SREBP活性后,Rho、Cdc42活性也被抑制.结论 SREBP是内皮细胞移行中的关键调控分子,靶向干预SREBP将可能成为治疗异常内皮细胞移行相关疾病的一条新途径.     

Transient disruptions of aortic endothelial cell plasma membranes.

Cells of gut, skin, and muscle frequently suffer transient survivable plasma membrane disruptions ("wounds") under physiological conditions, but it is not known whether endothelial cells of the aorta, which are constantly exposed to hemodynamically generated mechanical forces, similarly are injured in vivo. We have used serum albumin as a molecular probe for identifying endothelial cells of the rat aorta that incurred and survived transient plasma membrane wounds in vivo. Such wounded endothelial cells were in fact observed in the aortas of all rats examined. However, the percentage of wounded cells in the total aortic endothelial population varied remarkably between individuals ranging from 1.4% to 17.9% with a mean of 6.5% (+/- 4.6% SD). Wounded endothelial cells were heterogeneously distributed, being found in distinct clusters often in the shape of streaks aligned with the long axis of the vessel, or in the shape of partial or complete rims surrounding bifurcation openings, such as the ostia of the intercostal arteries. Physical exercise (running) did not increase the frequency of aortic endothelial cell membrane wounding, nor did spontaneous hypertension. Surprisingly, 80% of mitotic endothelial cell figures were identified as wounded. This article identified a previously unrecognized form of endothelial cell injury, survivable disruptions of the plasma membrane, and shows that injury to the endothelial cells of the normal aorta is far more commonplace than previously suspected. Plasma membrane wounding of endothelial cells could be linked to the initiation of atherosclerosis.     

Cytoskeletal alterations in lipopolysaccharide-induced bovine vascular endothelial cell injury and its prevention by sodium arsenite

Morphological changes, especially cytoskeletal alterations, in lipopolysaccharide (LPS)-induced vascular endothelial cell injury were studied by using LPS-susceptible bovine aortic endothelial cells (BAEC). BAEC in cultures with LPS showed cell rounding, shrinking, and intercellular gap formation. In those cells, LPS caused the disorganization of actin, tubulin, and vimentin. LPS also induced a reduction in the F-actin pool and an elevation in the G-actin pool. Cytoskeletal disorganization affected transendothelial permeability across the endothelial monolayer. Pretreatment of BAEC with sodium arsenite (SA) prevented alterations in LPS-induced BAEC injury. However, posttreatment with SA had no protective effect on them. SA upregulated the expression of heat shock protein in the presence of LPS. The role of SA in prevention of LPS-induced BAEC injury is discussed.     

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.