Olivier Gallet

Transmigration of human ovarian adenocarcinoma cells through endothelial extracellular matrix involves αv integrins and the participation of MMP2

The growth of ovarian carcinoma is dependent upon their vascularistion, but the interaction of ovarian cancer cells with the endothelium and their invasion through an endothelial environment remain poorly understood at the molecular level. To investigate adhesive events underlying this process with focusing on the role of αv integrins and MT1MMP‐MMP2 proteinases, we used in vitro models of cocultures of human ovarian adenocarcinoma cell lines (IGROV1 and SKOV3) with human umbilical vein endothelial cells (HUVECs). Immunostaining of HUVECs revealed the network organisation of fibrillar fibronectin (Fn) and pericellular vitronectin (Vn). During coculture, IGROV1 and SKOV3 cells gain access to subendothelial basement membrane of HUVECs and dislocated endothelial Fn without affecting endothelial Vn. Transmigration assays revealed that tumour cells invade Vn and, with an higher efficiency, Fn. Our data also highlighted that ovarian carcinoma cells migrated through the Fn‐rich HUVEC‐ECM. The expression of MMP2 and MT1‐MMP was revealed in tumour cells within an endothelial environment. Furthermore, we found that cell migration through the endothelial ECM was almost totally dependent on αv integrin function, whereas β1 integrins were not solicited. In addition, inhibitors of MMP2 activity (alone or combined with anti‐αv integrin MAb) or TSRI265 (which blocks MMP2‐αvβ3 association) were found to impede this process. Finally, αv integrins, MT1‐MMP and MMP2 were found in ovarian carcinoma cells within the 3‐dimensional architecture of intraperitoneal tumour nodes collected from nude mice xenografted with IGROV1 or SKOV3 cell lines or within human tumour tissues. αv integrins therefore appear as essential to the migration properties of human ovarian carcinoma cells, especially in an endothelial environment, with MMP2 participating to this process.

Fibronectin layers by matrix-assisted pulsed laser evaporation from saline buffer-based cryogenic targets

The deposition of fibronectin (FN) from saline buffer-based cryogenic targets by matrix-assisted pulsed laser evaporation (MAPLE) onto silicon substrates is reported. A uniform distribution of FN was revealed by Ponceau staining after control experiments on nitrocellulose paper. Well-organized particulates with heights from hundreds of nanometers up to more than 1 μm packed in homogeneous layers were evidenced by optical microscopy and profilometry on Si substrates. Atomic force microscopy images showed regions composed of buffer and FN aggregates forming a compact film. Comparison of infrared spectra of drop-cast and MAPLE-deposited FN confirmed the preservation of composition and showed no degradation of the protein. The protein deposition on Si was confirmed by antibody staining. Small aggregates and fluorescent fibrils were visualized by fluorescence microscopy. Superior attachment of human osteoprogenitor cells cultivated for 3 h proved the presence of stable and intact FN molecules after transfer.

The sucrose carrier of the plant plasma membrane. II. Immunological characterization

Polyclonal antibodies were raised against either whole plasma membrane of sugar beet (Beta vulgaris L.) leaves or against the 42 kDa region polypeptides of the plasma membrane. The effect of these antibodies on the uptake of sucrose, 3-O-methyl glucose and α-aminoisobutyric acid by broad bean (Vicia faba L.) leaf protoplasts was tested. The anti-plasma membrane serum did not affect the uptake of these solutes, while the anti-42 kDa serum markedly and selectively inhibited the uptake of sucrose without effect on the absorption of hexoses or of amino acids. Sucrose is very poorly metabolized before or after its penetration into the cells, and the inhibition of uptake cannot be explained in terms of side-effects on invertase or glucan synthase activities. The data provide functional evidence confirming the previous hypothesis (Gallet, O., Lemoine, R., Larsson, C. and Delrot, S. (1989) Biochim. Biophys. Acta 978, 56–64) that the 42 kDa polypeptide is a component of the sucrose uptake system of plant plasma membrane. The immunoglobulins purified from the serum were unable to inhibit sucrose uptake, suggesting that the activity of the antibody subclass responsible for inhibition was lost during purification or that an additional component of the serum is necessary to observe this inhibition.

The sucrose carrier of the plant plasmalemma. III. Partial purification and reconstitution of active sucrose transport in liposomes

The proteins from plasma membranes from sugar beet leaves were solubilized by 1% CHAPS and separated by size exclusion chromatography and by ion-exchange chromatography. The fractions enriched in sucrose transporter were monitored in three ways: differential labeling, ELISA, and reconstitution in proteoliposomes. When the plasma membranes were differentially labeled by N-ethylamaleimide in the presence of sucrose, a major peak of differential labeling was found at 120 kDa upon gel filtration. When this peak was recovered, denaturated by sodium dodecyl sulfate and reinjected on the gel filtration column, it yielded a peak of differential labeling at 42 kDa. When unlabeled membranes were used, the fractions eluted from the column were monitored by ELISA for their ability to recognize a serum directed against a 42 kDa previously identified as a putative sucrose carrier. The results paralleled those obtained by differential labeling, i.e. a major ELISA-reactive peak was found at 120 kDa upon gel filtration, and this peak yielded a peak most reactive at 40 kDa after denaturation. The 120 kDa peak prepared from unlabeled membranes was further separated on a Mono-Q column. The fractions were monitored by ELISA as described above, and reconstituted into proteoliposomes using asolectin. Active transport of sucrose, but not of valine could be observed with the reconstituted 120 kDa fraction. When the eluates from the Mono-Q column were reconstituted, the fractions exhibiting highest transport activity were enriched with a 42 kDa band. The data provide the first report concerning reconstitution of sucrose transport activity and confirm the involvement of a 42 kDa polypeptide in sucrose transport.

Pre-osteoblasts on poly(l-lactic acid) and silicon oxide: Influence of fibronectin and albumin adsorption

Cell adhesion and subsequent viability are critical initial steps in biomaterial-tissue integration and are strongly dependent on the material properties and the presence of matrix proteins. In the present study MC3T3-E1 osteoblast-like cell behavior on silicon oxide (SO) and poly(L-lactic acid) (PLLA) substrates has been examined, with a focus on the influence of the adhesive protein fibronectin and the non-adhesive protein albumin adsorbed on the substrates. Quartz crystal microgravimetry showed adsorption of fibronectin and albumin to be nearly identical on SO and PLLA. Subsequent exposure a previously adsorbed fibronectin layer to albumin decreased the rigidity of the adsorbed layer without any measurable increase in adsorbed mass. Cell adhesion and spreading were significantly enhanced on both SO and PLLA substrates coated with fibronectin or with fibronectin and albumin, compared with uncoated or albumin-coated substrates. The only statistically significant difference between the two substrates in these assays was increased spreading on PLLA compared with SO in the presence of fibronectin and albumin. Cell proliferation was significantly higher on SO compared with PLLA after 7 days culture, but depended on the presence of fibronectin only in the PLLA system. In contrast, mitochondrial activity was higher on PLLA than on SO, and was enhanced by fibronectin on both substrates. PLLA substrates coated with fibronectin and subsequently exposed to albumin exhibited the highest level of cell differentiation, as assayed via alkaline phosphatase activity. These results demonstrate the importance of adsorbed proteins on osteoblast-like cell-surface interactions.

Sulfur dioxide inhibits the sucrose carrier of the plant plasma membrane

Plasma membrane vesicles were prepared by phase partition from a microsomal fraction of broad bean (Vicia faba L.) leaf. In order to study the effects of sodium sulfite on active uptake of sucrose, the vesicles were artificially energized by a transmembrane pH gradient (delta pH) and/or a transmembrane electrical gradient (delta psi). At 1 mM, sulfite strongly inhibited sucrose uptake but did not affect the two components of the proton motive force, delta pH (measured by dimethyloxazolidine dione) and delta psi (measured by tetraphenylphosphonium). Moreover, sulfite did not inhibit the proton-pumping ATPase of the plasma membrane vesicles. These data demonstrate that sulfite may inhibit transport of photoassimilates in plant by a direct inhibition of the sucrose carrier of the plasma membrane.

Ascites-induced shift along epithelial-mesenchymal spectrum in ovarian cancer cells: enhancement of their invasive behavior partly dependant on αv integrins

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence. The presence of ascites, which acts as a dynamic reservoir of active molecules and cellular components, correlates with the OC peritoneal metastasis and is associated with poor prognosis. Since epithelial-mesenchymal transition (EMT) is involved in different phases of OC progression, we have investigated the effect of the unique ascitic tumor microenvironment on the EMT status and the behavior of OC cells. The exposure of three OC cell lines to ascites leads to changes in cellular morphologies. Within ascites, OC cells harboring an initial intermediate epithelial phenotype are characterized by marked dislocation of epithelial markers (E-cadherin, ZO-1 staining) while OC cells initially harboring an intermediate mesenchymal phenotype strengthen their mesenchymal markers (N-cadherin, vimentin). Ascites differentially triggers a dissemination phenotype related to the initial cell features by either allowing the proliferation and the formation of spheroids and the extension of colonies for cells that present an initial epithelial intermediate phenotype, or favoring the migration of cells with a mesenchymal intermediate phenotype. In an ascitic microenvironment, a redeployment of αv integrins into cells was observed and the ascites-induced accentuation of the two different invasive phenotypes (i.e. spheroids formation or migration) was shown to involve αv integrins. Thus, ascites induces a shift toward an unstable intermediate state of the epithelial-mesenchymal spectrum and confers a more aggressive cell behavior that takes on a different pathway based on the initial epithelial-mesenchymal cell features.

Interactions de protéines de membrane externe de Pseudomonas aeruginosa avec la fibronectine plasmatique. Recherche d'adhésines bactériennes

Abstract Pseudomonas aeruginosa adherence is a complex phenomenon largely mediated by pili involving specific receptor–ligand interactions. Anti-fibronectin antibodies as well as plasmatic fibronectin are able to inhibit P. aeruginosa adherence onto A549 cells showing that matricial fibronectin is an actual receptor for this bacterium. Experiments performed in vitro with human plasmatic fibronectin used as receptor and outer membrane proteins of P. aeruginosa as ligands show the presence of four fibronectin-binding proteins. These proteins with molecular mass of 70 ± 2, 60 ± 2, 48 ± 2 and 36 ± 1 kDa should be adhesins of P. aeruginosa on epithelial cell matrix in a non-pilus mediated adherence.

Inorganic–Organic Thin Implant Coatings Deposited by Lasers

The lifetime of bone implants inside the human body is directly related to their osseointegration. Ideally, future materials should be inspired by human tissues and provide the material structure-function relationship from which synthetic advanced biomimetic materials capable of replacing, repairing, or regenerating human tissues can be produced. This work describes the development of biomimetic thin coatings on titanium implants to improve implant osseointegration. The assembly of an inorganic-organic biomimetic structure by UV laser pulses is reported. The structure consists of a hydroxyapatite (HA) film grown onto a titanium substrate by pulsed-laser deposition (PLD) and activated by a top fibronectin (FN) coating deposited by matrix-assisted pulsed laser evaporation (MAPLE). A pulsed KrF* laser source (λ = 248 nm, τ = 25 ns) was employed at fluences of 7 and 0.7J/cm(2) for HA and FN transfer, respectively. Films approximately 1500 and 450 nm thick were obtained for HA and FN, respectively. A new cryogenic temperature-programmed desorption mass spectrometry analysis method was employed to accurately measure the quantity of immobilized protein. We determined that less than 7 μg FN per cm(2) HA surface is adequate to improve adhesion, spreading, and differentiation of osteoprogenitor cells. We believe that the proposed fabrication method opens the door to combining and immobilizing two or more inorganic and organic materials on a solid substrate in a well-defined manner. The flexibility of this method enables the synthesis of new hybrid materials by simply tailoring the irradiation conditions according to the thermo-physical properties of the starting materials.

Reconstitution of active sucrose transport in plant proteoliposomes.

The proteins of purified plasma membranes from sugar beet (Beta vulgaris L.) leaf were solubilized and separated on a size exclusion column. The fractions eluted from the column were monitored by ELISA with antibodies directed to a putative sucrose carrier protein. The peak most reactive in ELISA was approximately 120 kDa, and yielded a 40 kDa peak after denaturation by SDS. The 120‐kDa peak was recovered and used for reconstitution experiments with asolectin. Upon imposition of an artificial pH gradient and electrical gradient, the obtained proteoliposomes exhibited active transport of sucrose, but not of valine. The active transport of sucrose was inhibited by N‐ethylmaleimide and HgCl2.