Cholesterol induce oligomerization of Vibrio vulnificus cytolysin specifically

Vibrio vulnificus cytolysin (VVC) has been implicated as one of the important virulence determinants of V. vulnificus that causes serious septicemia and wound infection. An attempt was made to investigate that VVC could act as a ligand which stimulates intracellular signaling systems. Cholesterol dose-dependently blocked VVC hemolytic activity through oli-gomerization of cytolysin. Among cholesterol derivatives including 7-dehydrocholesterol, cholesteryl esters, deoxycholate, and cholestane tested, only 7-dehydrocholesterol induced oligomerization as well as inactivation of VVC. These results show that oligomerization of VVC is completely dependent on three-dimensional structure of cholesterol where specific interaction of cholesterol at oligomerization sites of VVC is very selective. These findings support the idea that cholesterol which constitute many of cellular plasma membrane could be a receptor of VVC on plasma membrane of target cells.     

Enterococcal cytolysin: a novel two component peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells

The cytolysin is a novel, two-peptide lytic toxin produced by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The cytolysin exerts activity against a broad spectrum of cell types including a wide range of gram positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes, and human intestinal epithelial cells. The cytolysin likely originated as a bacteriocin involved with niche control in the complex microbial ecologies associated with eukaryotic hosts. However, additional anti-eukaryotic activities may have been selected for as enterococci adapted to eukaryotic cell predation in water or soil ecologies. Cytolytic activity requires two unique peptides that possess modifications characteristic of the lantibiotic bacteriocins, and these peptides are broadly similar in size to most cationic eukaryotic defensins. Expression of the cytolysin is tightly controlled by a novel mode of gene regulation in which the smaller peptide signals high-level expression of the cytolysin gene cluster. This complex regulation of cytolysin expression may have evolved to balance defense against eukaryotic predators with stealth.     

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.     

Covalent immobilization of p-selectin enhances cell rolling

Cell rolling is an important physiological and pathological process that is used to recruit specific cells in the bloodstream to a target tissue. This process may be exploited for biomedical applications to capture and separate specific cell types. One of the most commonly studied proteins that regulate cell rolling is P-selectin. By coating surfaces with this protein, biofunctional surfaces that induce cell rolling can be prepared. Although most immobilization methods have relied on physisorption, chemical immobilization has obvious advantages, including longer functional stability and better control over ligand density and orientation. Here we describe chemical methods to immobilize P-selectin covalently on glass substrates. The chemistry was categorized on the basis of the functional groups on modified glass substrates: amine, aldehyde, and epoxy. The prepared surfaces were first tested in a flow chamber by flowing microspheres functionalized with a cell surface carbohydrate (sialyl Lewis(x)) that binds to P-selectin. Adhesion bonds between P-selectin and sialyl Lewis(x) dissociate readily under shear forces, leading to cell rolling. P-selectin immobilized on the epoxy glass surfaces exhibited enhanced long-term stability of the function and better homogeneity as compared to that for surfaces prepared by other methods and physisorbed controls. The microsphere rolling results were confirmed in vitro with isolated human neutrophils. This work is essential for the future development of devices for isolating specific cell types based on cell rolling, which may be useful for hematologic cancers and certain metastatic cancer cells that are responsive to immobilized selectins.     

Antithrombogenicity of cultured endothelial cell-detached surface

To examine the antithrombogenicity of cultured endothelial cell-detached surface, a simple hybrid vascular model tube consisting of a glass tube and endothelial cells was constructed. To detach the endothelial cells from the inner surface of the model tube, a steady shear stress of 2 or 8 N m(-2) was imposed onto the surface of endothelial cell monolayer by means of a coaxial double cylinder rotational-type apparatus. Coagulation of blood in contact with the endothelial cell-detached surface was examined using a damped oscillation rheometer. Coagulation of whole blood in the cell-detached tube occurred at about 40 min, which was almost the same as that in the endothelial cell-coated tube. A few platelets without shape change adhered to the endothelial cell-detached surface. These data suggest that the endothelial cell-detached surface may exhibit antithrombogenic and anticoagulant surfaces. Biochemical analyses showed that the glass surface, where endothelial cell was detached, was covered with components such as collagen type IV that is considered to be produced from the endothelial cells on the glass surface.     

Genistein activates endothelial nitric oxide synthase in broiler pulmonary arterial endothelial cells by an Akt-dependent mechanism

Deregulation of endothelial nitric oxide synthase (eNOS) plays an important role in the development of multiple cardiovascular diseases. Our recent study demonstrated that genistein supplementation attenuates pulmonary arterial hypertension in broilers by restoration of endothelial function. In this study, we investigated the molecular mechanism by using broiler pulmonary arterial endothelial cells (PAECs). Our results showed that genistein stimulated a rapid phosphorylation of eNOS at Ser(1179) which was associated with activation of eNOS/NO axis. Further study indicated that the activation of eNOS was not mediated through estrogen receptors or tyrosine kinase inhibition, but via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent signaling pathway, as the eNOS activity and related NO release were largely abolished by pharmacological inhibitors of PI3K or Akt. Thus, our findings revealed a critical function of Akt in mediating genistein-stimulated eNOS activity in PAECs, partially accounting for the beneficial effects of genistein on the development of cardiovascular diseases observed in animal models.     

Modification of polyetherurethane for endothelial cell seeding

Until now no synthetic polymer surface was available suitable for cardiovascular implantation which effectively prevents microthrombosis for a long time. The presented concept of our work is to develop a polymer surface to promote the growth of a durable endothelial cell monolayer which would be formed on the prothesis prior to implantation. A microporous polyetherurethane foil is being used as carrier polymer for the cell monolayer. Suitable functional groups are grafted on the polymer surface by plasma polymerization. The success of the first modification steps is followed by ESCA-analysis and scanning electron microscopy. The modified carrier polymer is the starting material for covalent binding of biopolymers which react as adhesion supporter between the synthetic polymer surface and the endothelial cells.     

Investigation of cytolysin variants by peptide mapping: enhanced protein characterization using complementary ionization and mass spectrometric techniques

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have been used in conjunction with time-of-flight (TOF) and quadrupole ion trap (IT) mass spectrometry, respectively, to analyze various cytolysin proteins isolated from the sea anemone Stichodactyla helianthus and digested by the protease trypsin. By employing different ionization methods, the subsequent changes in ionization selectivity for the peptides in the digested protein samples resulted in ion abundance variation reflected in the mass spectra. Upon investigation of this variation generated by the two ionization processes, it has been shown in this study that enhanced protein coverage (e.g., >95% for cytolysin III) can be achieved. Additionally, capillary and microbore reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ESI mass spectrometry (MS) as well as flow injection analysis by nanoflow ESI-MS afforded the necessary limit of detection (LOD) for detailed structural information of the cytolysin proteins by tandem mass spectrometry (MS/MS) methods. It can be concluded that cytolysins II and III correspond to sticholysins I and II, that "cytolysin I" is a mixture of modified forms of cytolysins II and III, and that "cytolysin IV" is an incompletely processed precursor of cytolysin III.     

Chemiluminescence response and endothelial cell damage following lipopolysaccharide priming of polymorphonuclear leukocytes

Conclusions Neutrophils can be directly stimulated by endothelial cell suspensions, an observation that has also been made by experiments with nylon fiber, as an artificial model for PMNL/EC interactions [5]. As shown by experiments with endothelial cell suspensions a further amplification of this process following LPS-priming up to 10 ng/ml blood has been demonstrated.Unstimulated neutrophils do not lead to an endothelial cell damage, as also could be demonstrated by electron micrographs. An increased ¹¹¹In release occurred following neutrophil stimulation between 10 and 20 ng LPS/ml blood, but cannot be explained by an increased oxygen radical production of neutrophils. Therefore, these data suggest, that other neutrophil derived products rather than oxygen radicals contribute to an endothelial cell damage in this system.

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Chemiluminescenz-Antwort und Endothelzellschaden nach Lipopolysaccharid-Stimulation polymorphkerniger Leukocyten

     

Flow goes forward and cells step backward: endothelial migration

Systemic and pulmonary circulations constitute a complex organ that serves multiple important biological functions. Consequently, any pathological processing affecting the vasculature can have profound systemic ramifications. Endothelial and smooth muscle are the two principal cell types composing blood vessels. Critically, endothelial proliferation and migration are central to the formation and expansion of the vasculature both during embryonic development and in adult tissues. Endothelial populations are quite heterogeneous and are both vasculature type- and organ-specific. There are profound molecular, functional, and phenotypic differences between arterial, venular and capillary endothelial cells and endothelial cells in different organs. Given this endothelial cell population diversity, it has been challenging to determine the origin of endothelial cells responsible for the angiogenic expansion of the vasculature. Recent technical advances, such as precise cell fate mapping, time-lapse imaging, genome editing, and single-cell RNA sequencing, have shed new light on the role of venous endothelial cells in angiogenesis under both normal and pathological conditions. Emerging data indicate that venous endothelial cells are unique in their ability to serve as the primary source of endothelial cellular mass during both developmental and pathological angiogenesis. Here, we review recent studies that have improved our understanding of angiogenesis and suggest an updated model of this process.Subject terms: Cell lineage, Chemotaxis     

Oxygen ion implantation at 20 to 2000 keV into polysulfone for improvement of endothelial cell adhesion

The irradiation effects of oxygen on polysulfone have been investigated at energies of 20 keV, 150 keV and 2 MeV. The strong improvement of endothelial cell adhesion and proliferation is found on ion irradiated polysulfone at 20 keV. Such improvement is declined with increasing ion energy. The changes of surface color and free energy are strongly dependent on ion energy and dose. The formation of amorphous carbon phase is demonstrated by Raman spectroscopy and its degree is correspondent to the color changes observed. The formations of hydroxyl and carboxyl groups are confirmed by the attenuated total reflectance (ATR) FTIR spectroscopy. The depletions of heteroatoms are conjectured by detail analysis of X-ray photoelectron spectroscopy (XPS). Since no single one of these changes can be related directly to the improved adhesion and proliferation of endothelial cells on irradiated surface, we argue that the distribution of functional groups is crucial in promoting the adhesion of endothelial cells. Although the distribution cannot directly be detected at present, the irradiation effects were related to the results of TRIM simulation. The surface changes can be controlled by adjusting the size energy and dose of irradiating ion for the optimum morphology to cell adhesion.     

Amperometric determination of nitric oxide derived from pulmonary artery endothelial cells immobilized in a microchip channel

A simple method for immobilizing a confluent layer of bovine pulmonary artery endothelial cells (bPAECs) in microchip-based channels is described. The microchips are prepared from poly(dimethylsiloxane) and have channel dimensions that approximate resistance vessels in vivo. The reversibly sealed channels were coated with fibronectin (100 microg ml(-1)) by aspiration. The bPAECs, which were introduced in the same manner, became attached to the fibronectin coating in about 2 h. The microchip could then be resealed over a micromolded carbon ink electrode (24 microm width x 6 microm height). Coating the carbon microelectrode with a 0.05% Nafion solution selectively blocked nitrite (10 microM) from being transported to the electrode surface while nitric oxide (NO, 10 microM) was amperometrically measured. Upon stimulation with adenosine triphosphate (ATP, 100 microM) the immobilized bPAECs produced and released micromolar amounts of NO. This NO production was effectively inhibited when the immobilized cells were incubated with L-nitro-arginine methyl ester (L-NAME), a competitive inhibitor for nitric oxide synthase. Moreover, once the immobilized bPAECs were no longer able to produce NO, incubation with L-arginine allowed for further ATP-stimulated NO production.     

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.     

A rapid method for detection of cell adhesion molecules (CAMs) on human umbilical vein endothelial cells (HUVECs)

Detection of cell surface proteins is widely used as molecular markers for initiation, progression and severity of many diseases. In particular, detection of cell adhesion molecules (CAMs) on endothelial cells is important as it indicates the extent of inflammation associated with several diseases including arthritis, asthma, tumor metastasis, etc. Here, we report, a rapid method for detection of CAMs on endothelial cells by covalently immobilizing TNF-α induced cells on a photoactivated polystyrene microtiter plate at 50 °C in 45 min followed by performing enzyme-linked immunosorbent assay (ELISA) technique at elevated temperature. Our method reduced the time of cell-ELISA to 3 h with results akin to conventional cell-ELISA carried out in 38 h. The method thus described herein could be potentially useful in clinical and research laboratories for rapid detection of cell surface proteins including CAMs on intact cell samples.     

Surface patterning and modification of polyurethane biomaterials using silsesquioxane-gelatin additives for improved endothelial affinity

Polyurethanes (PUs) are well-known for their biocompatibility but their intrinsic inert property hampers cell-matrix interactions. Surface modifications are thus necessary to widen their use for biomedical applications. In this work, surface modifications of PU were achieved first by incorporating polyhedral oligomeric silsesquioxane (POSS), followed by alteration of the surface topography via the breath figures method. Subsequently, surface chemistry was also modified by immobilization of gelatin molecules through grafting, for the enhancement of the surface cytocompatibility. Scanning electron microscopy (SEM) was used to verify the formation of highly ordered microstructures while static contact angle, FTIR and XPS confirmed the successful grafting of gelatin molecules onto the surfaces. In vitro culture of human umbilical vein endothelial cells (HUVECs) revealed that endothelial cell adhesion and proliferation were significantly enhanced on the gelatin-modified surfaces, as shown by live/dead staining and WST-1 proliferation assay. The results indicated that the combination of the strategies yielded an interface that improves cell attachment and subsequent growth. This enhancement is important for the development of higher quality biomedical implants such as vascular grafts.     

Vascular endothelial cells as targets for photochemical internalization (PCI)

Cancer treatment can be exerted by targeting both cancer cells and the vasculature supplying solid tumors. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, but recent data on contrast‐enhanced MRI have indicated that the method also reduces blood perfusion in HT1080 fibrosarcoma xenografts. The present report aims to investigate if PCI may exert direct cytotoxic effects on endothelial cells. PCI of saporin was performed on endothelial human umbilical vein endothelial cell (HUVEC) and fibrosarcoma cells (HT1080) using two PCI‐relevant photosensitizers, TPPS_2a and AlPcS_2a. A 22‐ and 13‐fold higher photosensitizer uptake was detected in the endothelial cells compared with the HT1080 cells for AlPcS_2a and TPPS_2a, respectively. PCI of saporin was, however, found more effective in HT1080 cells. For HT1080 cells, PCI with saporin increased cell killing 1.9‐fold over photodynamic therapy alone, but under the same conditions, only increased HUVEC cell killing by 1.6‐ and 1.3‐fold with AlPcS_2a and TPPS_2a, respectively. Saporin uptake was higher in HUVECs than in the HT1080 cells, hence did not reflect the cell line differences in PCI efficacy. This is the first report on PCI‐mediated kill of endothelial cells and lays the foundation for further preclinical evaluation of the PCI technology as an antivascular strategy to ablate tumors.     

On-chip diagnosis of subclinical mastitis in cows by electrochemical measurement of neutrophil activity in milk

Subclinical mastitis is a common infectious disease affecting dairy cows. To develop an early diagnostic device for this disease, we focused on measuring an increase in the number of neutrophils in raw milk of mastitic cows. Superoxide anions (O(2)(-)), secreted by neutrophils, can be a good indicator of neutrophil concentration, and therefore, the seriousness of the mastitis. In this study, neutrophils in raw milk samples were separated from fat globules in a flow channel using differences in specific gravity and specific adhesion of neutrophils to P-selectin. Neutrophils trapped in the flow channel were subsequently concentrated in an array of micropillars of a working electrode modified with P-selectin and superoxide dismutase. The O(2)(-) secreted from the trapped neutrophils was electrochemically detected. A difference in the detection current was observed between normal and mastitic milk samples. A clear linear relationship between the electric current and cell density was observed.     

The identification of proliferation and tumour-induced proteins in human endothelial cells: a possible target for tumour therapy

The continued growth and spread of tumours is dependent on the proliferation of the endothelial cells of their vasculature. The presence of proliferation- or tumour-induced surface proteins on these endothelial cells would offer a suitable epitope for monoclonal antibody therapy of tumours. Using cultured human umbilical and capillary endothelial cells, we have stimulated them with simple mitogens and tumour conditioned media and examined the proteins induced by [35S]methionine incorporation and 125I-surface-labelling. Two-dimensional polyacrylamide gel electrophoresis revealed the induction of proliferation and tumour-related antigens on the surface of the endothelial cells. Subsequent monoclonal antibody studies suggest that tumour specific surface proteins are present on most tumour endothelium.     

Development of an efficient endothelial cell specific vector using promoter and 5' untranslated sequences from the human preproendothelin-1 gene

We report here, that a vector constructed based on ppET-1 gene promoter and 5' untranslated region induced a high level of gene expression in endothelial cells and the specificity is even further enhanced under hypoxia-mimic conditions due to a natural hypoxia responsive element within the promoter region. A naked DNA vector that confers endothelial cell specific gene expression as well as efficient levels of gene expression was constructed with an endothelial cell specific naked DNA vector, pETlong, by using the full length promoter of the preproendothelin-1 gene and the entire 5' untranslated region upstream from the start codon. Inclusion of the entire 5' untranslated region in pETlong increased gene expression 2.96 fold as compared with that from pETshort, which contains only the promoter sequences. Reporter gene expression from pETlong was 7.9 fold higher as compared with that from CMV-driven promoter based vector in calf pulmonary endothelial cells. However, in nonendothelial COS cells, luciferase activity from pETlong was only 0.3 fold as compared with that of CMV-based vector. Similar results were observed in other nonendothelial cells. These results demonstrate that the pETlong drives gene expression in endothelial cells with high efficacy and specificity. We have examined hypoxia responsiveness of pETlong as the promoter region of the preproendothelin-1 gene contains hypoxia responsive elements. The activity of the pETlong vector was increased 1.6 fold under hypoxia-mimic conditions using cobalt chloride. The high levels of hypoxia-inducible expression in endothelial cells relative to the low levels of background expression in other cells shows that pETlong could be a useful tool for vascular targeting of vascular disease and cancer gene therapy.