Interactions of Bartonella henselae with vascular endothelial cells

The emerging human pathogen Bartonella henselae has the remarkable capacity to colonise vascular tissues and to stimulate vasoproliferative tumour growth. Although the molecular principle of bacterium-induced neovascularisation (angiogenesis) is still unclear, recent studies have indicated a novel mechanism of endothelial colonisation that involves the formation, engulfment and uptake of a large bacterial aggregate.     

Staining of Bartonella henselae with carboxyfluorescein diacetate succinimidyl ester for tracking infection in erythrocytes and epithelial cells

Bartonella infection (Bartonella henselae in particular) is responsible for a widening spectrum of human diseases. The persistent colonization of erythrocytes is a feature of Bartonella infection. Endothelial and epithelial cells are also widely used to study the pathogenesis of bartonellosis in vitro. Exploring a convenient method for visualizing the bacillus without affecting infectivity would be very interesting. Carboxyfluorescein diacetate succinimidyl ester (CFSE) has been previously used for staining several bacterial species to study their adhesion to host cells. The present study demonstrated the efficiency and safety of using CFSE in staining B. henselae. The staining of bacillus-invaded erythrocytes and epithelial cells in vitro successfully allowed for flow cytometry and confocol microscopy analyses. Parallel tests using untreated bacteria confirmed that CFSE staining did not result in side effects on the infectivity of B. henselae. Labeling Bartonella with CFSE is a valuable method for studying the bacteria-host interaction.     

Interaction of Bartonella henselae with endothelial cells results in rapid bacterial rRNA synthesis and replication

Bartonella henselae is a slow-growing microorganism and the causative pathogen of bacillary angiomatosis in man. Here, we analysed how interaction of B. henselae with endothelial cells might affect bacterial growth. For this purpose, bacterial rRNA production and ribosome content was determined by fluorescence in situ hybridization (FISH) using rRNA-targeted fluorescence-labelled oligonucleotide probes. B. henselae grown on agar plates showed no detectable rRNA content by means of FISH, whereas B. henselae co-cultured with endothelial cells showed a rapid increase of rRNA production within the first 18 h after inoculation. The increased rRNA synthesis was paralleled by a ∼1000-fold intracellular bacterial replication, whereas bacteria grown on agar base showed only a ∼10-fold replication within the first 48 h of culture. Pretreatment of host cells with paraformaldehyde prevented adhesion, invasion, intracellular replication and bacterial rRNA synthesis of B. henselae . In contrast, inhibition of host cell protein synthesis by cycloheximide did not affect bacterial adhesion and invasion, but prevented intracellular replication although bacterial rRNA content was increased. Inhibition of actin polymerization by cytochalasin D did not affect adhesion, invasion, increased rRNA content or intracellular replication of B. henselae. These results demonstrate that rRNA synthesis and replication of B. henselae is promoted by viable host cells with intact de novo protein synthesis.     

Unusual trafficking pattern of Bartonella henselae -containing vacuoles in macrophages and endothelial cells

Bartonella henselae, the agent of cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen, whose interaction with macrophages and endothelial cells (ECs) is crucial in the pathogenesis of these diseases. However, little is known about the subcellular compartment in which B. henselae resides. Two hours after infection of murine macrophages and human ECs, the majority of B. henselae-containing vacuoles (BCVs) lack typical endocytic marker proteins, fail to acidify, and do not fuse with lysosomes, suggesting that B. henselae resides in a non-endocytic compartment. In contrast to human umbilical vein endothelial cells, bacterial death and lysosomal fusion with BCVs is apparent in J774A.1 macrophages at 24 h. This phenomenon of delayed lysosomal fusion requires bacterial viability, and is confined to the BCV itself. Using magnetic selection, we enriched for transposon-mutagenized B. henselae trapped in lysosomes of macrophages 2 h after infection. Genes affected appear to be relevant to the intracellular lifestyle in macrophages and ECs and include some previously implicated in Bartonella pathogenicity. We conclude that B. henselae has a specific capacity to actively avoid the host endocytic pathway after entry of macrophages and ECs, from within a specialized non-endocytic membrane-bound vacuole.     

Differential effects of Bartonella henselae on human and feline macro- and micro-vascular endothelial cells

Bartonella henselae, a zoonotic agent, induces tumors of endothelial cells (ECs), namely bacillary angiomatosis and peliosis in immunosuppressed humans but not in cats. In vitro studies on ECs represent to date the only way to explore the interactions between Bartonella henselae and vascular endothelium. However, no comparative study of the interactions between Bartonella henselae and human (incidental host) ECs vs feline (reservoir host) ECs has been carried out because of the absence of any available feline endothelial cell lines.To this purpose, we have developed nine feline EC lines which allowed comparing the effects of Bartonella strains on human and feline micro-vascular ECs representative of the infection development sites such as skin, versus macro-vascular ECs, such as umbilical vein.Our model revealed intrinsic differences between human (Human Skin Microvascular ECs -HSkMEC and Human Umbilical Vein ECs - iHUVEC) and feline ECs susceptibility to Bartonella henselae infection.While no effect was observed on the feline ECs upon Bartonella henselae infection, the human ones displayed accelerated angiogenesis and wound healing.Noticeable differences were demonstrated between human micro- and macro-vasculature derived ECs both in terms of pseudo-tube formation and healing. Interestingly, Bartonella henselae effects on human ECs were also elicited by soluble factors.Neither Bartonella henselae-infected Human Skin Microvascular ECs clinically involved in bacillary angiomatosis, nor feline ECs increased cAMP production, as opposed to HUVEC.Bartonella henselae could stimulate the activation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) in homologous cellular systems and trigger VEGF production by HSkMECs only, but not iHUVEC or any feline ECs tested.These results may explain the decreased pathogenic potential of Bartonella henselae infection for cats as compared to humans and strongly suggest that an autocrine secretion of VEGF by human skin endothelial cells might induce their growth and ultimately lead to bacillary angiomatosis formation.     

Granulocyte interactions with GM-CSF and G-CSF secretion by endothelial cells and monocytes

We have, in previous studies, characterized the cytokine and cellular regulation of GM-CSF and G-CSF production by monocytes and endothelial cells. In this study, we investigated the regulatory role of granulocytes. The addition of granulocytes to endotoxin-stimulated monocytes dose-dependently decreased both GM-CSF and G-CSF concentrations, presumably by absorbing the cytokines. A similar dose-dependent decrease in GM-CSF concentration was found when granulocytes were added to IL-1-stimulated endothelial cells. In contrast, G-CSF secretion by endothelial cells responded to granulocytes in a biphasic fashion. At low granulocyte concentrations, endothelial cells responded with an increased G-CSF secretion, but at high concentrations of granulocytes G-CSF secretion was down modulated. Our results suggest that there exist two loops between granulocytes and endothelial cells for regulating G-CSF activity. Granulocytes can stimulate G-CSF secretion by activated endothelial cells but can also decrease the biological activity by absorbing the cytokine. These mechanisms might be involved in the regulation of the local and systemic levels of granulocytes.     

The Bartonella henselae VirB/Bep system interferes with vascular endothelial growth factor (VEGF) signalling in human vascular endothelial cells

The vasculotropic pathogen Bartonella henselae (Bh) intimately interacts with human endothelial cells (ECs) and subverts multiple cellular functions. Here we report that Bh specifically interferes with vascular endothelial growth factor (VEGF) signalling in ECs. Bh infection abrogated VEGF-induced proliferation and wound closure of EC monolayers as well as the capillary-like sprouting of EC spheroids. On the molecular level, Bh infection did not alter VEGF receptor 2 (VEGFR2) expression or cell surface localization, but impeded VEGF-stimulated phosphorylation of VEGFR2 at tyrosine(1175) . Consistently, we observed that Bh infection diminished downstream events of the tyrosine(1175) -dependent VEGFR2-signalling pathway leading to EC proliferation, i.e. phospholipase-Cγ activation, cytosolic calcium fluxes and mitogen-activated protein kinase ERK1/2 phosphorylation. Pervanadate treatment neutralized the inhibitory activity of Bh on VEGF signalling, suggesting that Bh infection may activate a phosphatase that alleviates VEGFR2 phosphorylation. Inhibition of VEGFR2 signalling by Bh infection was strictly dependent on a functional VirB type IV secretion system and thereby translocated Bep effector proteins. The data presented in this study underscore the role of the VirB/Bep system as important factor controlling EC proliferation in response to Bh infection; not only as previously reported by counter-acting an intrinsic bacterial mitogenic stimulus, but also by restricting the exogenous angiogenic stimulation by Bh-induced VEGF.     

Increased adherence of oxidant-treated human and bovine erythrocytes to cultured endothelial cells

Bovine erythrocytes, which normally lack phosphatidyl choline in their membranes, when treated with either H2O2 or diamide (1-3 mM), showed a partial appearance of phosphatidyl ethanolamine (PE 40%) and phosphatidyl serine (PS, 30-33%) in the external leaflet of the bilayer and a concomitant increased (four- to five-fold) propensity to adhere to cultured bovine aortic endothelial cells. Similar treatment of normal human erythrocytes caused an alteration in the organization of the phospholipid bilayer and also resulted in their increased adherence to endothelial cells derived either from human umbilical vein or bovine aorta. Treatment of RBCs with H2O2 at low concentration (0.5 mM) resulted in cross-linking of spectrin without significant changes in the orientation of aminophospholipids but the RBCs exhibited 15-20% increase in adherence to endothelial cells. Pretreatment of either human or bovine erythrocytes with antioxidants such as vitamin E (2 mM) prevented both oxidant-induced reorganization of phospholipids in the bilayer and enhancement of adherence to endothelial cells. Introduction of either phosphatidyl serine or phosphatidyl ethanolamine but not phosphatidyl choline into erythrocyte membranes increased their adherence to endothelial cells threefold. Oxidant-treated RBCs exhibited enhanced binding and fluorescence of Merocyanine 540 dye (MC-540), which is sensitive to the packing of lipids in the lipid bilayer. On flow cytometric analysis, 78% of H2O2 (0.5 mM)-treated erythrocytes compared to 30% of untreated RBCs exhibited MC-540 binding and fluorescence, indicating differences in the lipid packing in the outer leaflet of the bilayer. Oxidant-treated erythrocytes adhere preferentially to endothelial cells rather than to bovine aortic smooth muscle cells and skin fibroblasts. It is suggested that the alterations in the erythrocyte membrane surface due to spectrin cross-linking and the organization of the phospholipids concomitant with less ordered packing in the external leaflet of the bilayer, either induced by oxidative manipulation in normal RBC or in pathological erythrocytes, play a role in erythrocyte-endothelial cell interaction.     

Synergistic interactions of saponins and monoterpenes in HeLa cells, Cos7 cells and in erythrocytes

In phytomedicine complex extracts consisting of phenolics, monoterpenes or saponins are traditionally used. It is often impossible to attribute the biological activity of an extract to one or few compounds. As an explanation of the superior activity of extracts, a synergistic effect of combinations of active compounds has been suggested. Since lipophilic monoterpenes or saponins targeting the biomembrane usually accompany polar polyphenols in phytomedical preparations, we decided to investigate their effect as single substances and in combination to gain further insight into potential synergistic effects of herbal medicine. Combinations of the monoterpenes α-pinene, thymol and menthol with the monodesmosidic saponins digitonin, aescin, glycyrrhizic acid and Quillaja saponin demonstrated strong synergistic activity. The IC(50) of haemolysis was lowered by a factor of 10-100 from 316μg/ml to 2μg/ml for aescin, 157μg/ml to 11μg/ml for Quillaja saponins and 20μg/ml to 3μg/ml for digitonin when combined with thymol. A similar significant synergistic cytotoxicity occurred both in HeLa and Cos7 cells by combining the α-pinene, thymol and menthol with the saponins. The IC(50) of glycyrrhizic acid was lowered by a factor 100 from around 300μg/ml to around 1-10μg/ml and the IC(50) of aescin, digitonin and Quillaja saponins about the factor 10. Monoterpenes and monodesmosidic saponins have a common target, the biomembrane, which is present in all animal, fungal and bacterial cells. Disturbance of membrane fluidity and permeability is the mode of action. This activity is non-specific which makes it extremely difficult for bacteria and fungi to develop resistance. This explains the overall success of these molecules as defence chemicals in the plant kingdom. The synergistic effect of combinations of saponins with monoterpenes opens a complete new field of possible applications in medicine to overcome resistance in multidrug resistant microbial and human cell.     

Biomechanical interactions of Schistosoma mansoni eggs with vascular endothelial cells facilitate egg extravasation

The eggs of the parasitic blood fluke, Schistosoma, are the main drivers of the chronic pathologies associated with schistosomiasis, a disease of poverty afflicting approximately 220 million people worldwide. Eggs laid by Schistosoma mansoni in the bloodstream of the host are encapsulated by vascular endothelial cells (VECs), the first step in the migration of the egg from the blood stream into the lumen of the gut and eventual exit from the body. The biomechanics associated with encapsulation and extravasation of the egg are poorly understood. We demonstrate that S. mansoni eggs induce VECs to form two types of membrane extensions during encapsulation; filopodia that probe eggshell surfaces and intercellular nanotubes that presumably facilitate VEC communication. Encapsulation efficiency, the number of filopodia and intercellular nanotubes, and the length of these structures depend on the egg’s vitality and, to a lesser degree, its maturation state. During encapsulation, live eggs induce VEC contractility and membranous structures formation in a Rho/ROCK pathway-dependent manner. Using elastic hydrogels embedded with fluorescent microbeads as substrates to culture VECs, live eggs induce VECs to exert significantly greater contractile forces during encapsulation than dead eggs, which leads to 3D deformations on both the VEC monolayer and the flexible substrate underneath. These significant mechanical deformations cause the VEC monolayer tension to fluctuate with the eventual rupture of VEC junctions, thus facilitating egg transit out of the blood vessel. Overall, our data on the mechanical interplay between host VECs and the schistosome egg improve our understanding of how this parasite manipulates its immediate environment to maintain disease transmission.     

Differences in laminin fragment interactions of normal and transformed endothelial cells

Bovine aortic and microvascular endothelial cells showed good adhesion with spreading on fibronectin or collagen IV and to a lower extent on laminin. Recognition of native laminin was due to its long arm fragment E8 and was mediated by alpha 6 integrins as demonstrated by antibody inhibition. A considerably stronger, RGD-dependent interaction was observed with the isolated laminin short arm fragment P1 previously shown to represent a cryptic cell-binding site. No adhesion was observed with the heparin-binding fragment E3. In contrast, murine microvascular endothelial cells transformed by the polyoma middle T oncogene showed preferential adherence and spreading on laminin via its E8 cell-binding site and also showed adhesion to fragment E3. Attachment to laminin fragment P1 and to collagen IV was low or negative and was never followed by spreading. These data show that the transformation of microvascular endothelial cells, which give them the property to form hemangiomas, also leads to changes in cell adhesion to extracellular matrix proteins, particularly to laminin fragments.     

In vitro interactions between rat bone marrow-derived endothelial progenitor cells and hepatic stellate cells

Transplantation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) has been reported to improve liver fibrosis, but there is no direct evidence for the mechanism of improvement. We investigated the mechanism in vitro by coculturing BM-derived EPCs with activated hepatic stellate cells (HSCs) to mimic the hepatic environment. EPCs and HSCs were cultured alone and indirectly cocultured at a 1:1 ratio in a Transwell system. The characteristics of HSCs and EPCs were examined at different time points. An invasion assay showed the time-dependent effect on degradation of the extracellular matrix (ECM) layer in EPCs cultured alone. Real-time PCR and enzyme-linked immunosorbent assay analysis revealed that EPCs served as a source of matrix metalloproteinase-9 (MMP-9), and MMP-9 expression levels significantly increased during the 2 d of coculture. CFSE labeling showed that EPCs inhibited proliferation of HSCs. Annexin-V/PI staining, erminal deoxynucleotidyl transferase X-dUTP nick end labeling analysis, and (cleaved) caspase-3 activity revealed that EPCs promoted HSC apoptosis. However, the proliferation and apoptosis of EPCs were unaffected by cocultured HSCs. Coculturing increased the expression of inducible nitric oxide synthase, vascular endothelial growth factor, and hepatocyte growth factor (HGF) in EPCs, promoted differentiation of EPCs, and reduced the expression of types I and III collagens and transforming growth factor beta 1. Knockdown of HGF expression attenuated EPC-induced activation of HSC apoptosis and profibrotic ability. These findings demonstrated that BM-derived EPCs could degrade ECM, promoting activated HSC apoptosis, suppressing proliferation and profibrotic ability of activated HSCs. HGF secretion by EPCs plays a key role in inducing activated HSC apoptosis and HSC profibrotic ability.     

Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX

Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, which may result from treatment with the Ca2+ ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.     

CXCL12 mediates trophic interactions between endothelial and tumor cells in glioblastoma

Emerging evidence suggests endothelial cells (EC) play a critical role in promoting Glioblastoma multiforme (GBM) cell proliferation and resistance to therapy. The molecular basis for GBM-EC interactions is incompletely understood. We hypothesized that the chemokine CXCL12 and its receptor CXCR4 could mediate direct interactions between GBM cells and tumor-associated endothelial cells and that disruption of this interaction might be the molecular basis for the anti-tumor effects of CXCR4 antagonists. We investigated this possibility in vivo and in an in vitro co-culture model that incorporated extracellular matrix, primary human brain microvascular ECs (HBMECs) and either an established GBM cell line or primary GBM specimens. Depletion of CXCR4 in U87 GBM cells blocked their growth as intracranial xenografts indicating that tumor cell CXCR4 is required for tumor growth in vivo. In vitro, co-culture of either U87 cells or primary GBM cells with HBMECs resulted in their co-localization and enhanced GBM cell growth. Genetic manipulation of CXCL12 expression and pharmacological inhibition of its receptors CXCR4 and CXCR7 revealed that the localizing and trophic effects of endothelial cells on GBM cells were dependent upon CXCL12 and CXCR4. These findings indicate that the CXCL12/CXCR4 pathway directly mediates endothelial cell trophic function in GBMs and that inhibition of CXCL12-CXCR4 signaling may uniquely target this activity. Therapeutic disruption of endothelial cell trophic functions could complement the structural disruption of anti-angiogenic regimens and, in combination, might also improve the efficacy of radiation and chemotherapy in treating GBMs.     

Formation of stress fibres in human endothelial cells infected with Bartonella bacilliformis is associated with altered morphology, impaired migration and defects in cell morphogenesis

Bartonella bacilliformis , a Gram-negative, flagellated bacterium, infects human erythrocytes (haematic phase) and endothelial cells (tissue phase), resulting in a biphasic disease. In the tissue phase of disease (verruga peruana), infection leads to infection of endothelial cells and a pronounced proliferation of these cells, resulting in characteristic skin eruptions of papules and nodules. We have studied the properties of endothelial cells infected in vitro . Extensive cytoskeletal remodelling of endothelial cells occurred after infection in vitro with B. bacilliformis. The cells became spindle shaped and contained arrays of actin stress fibres orientated parallel to the long axis of the cell. Cell–cell contacts were disrupted , along with the distribution of the plasma membrane marker protein, PECAM-1, which participates in cell–cell junctions. The prominent stress fibres terminated in an increased number of focal contacts, which were studied using immunofluorescent staining for paxillin, a cytoplasmic protein that localizes in the focal adhesions. These morphological changes are consistent with activation of intracellular Rho by B. bacilliformis . Formation of stress fibres and the increased number of focal adhesions could be prevented by preincubation of the endothelial cells with C3 exoenzyme, which inactivates intracellular Rho by ADP ribosylation. Endothelial cell motility was greatly diminished in infected cells and the cells did not respond effectively to a stimulus that would evoke motility. In addition, infection of endothelial cells interfered with their ability to form networks of capillary tubes when suspended within three-dimensional collagen matrices. If the properties of infected endothelial cells in vivo are similar, the infected cells will probably not participate effectively in angiogenesis.