Induction of SPARC by VEGF in human vascular endothelial cells.

SPARC/osteonectin/BM-40 is a matricellular protein that is thought to be involved in angiogenesis and endothelial barrier function. Previously, we have detected high levels of SPARC expression in endothelial cells (ECs) adjacent to carcinomas of kidney and tongue. Although SPARC-derived peptide showed an angiogenic effect, intact SPARC itself inhibited the mitogenic activity of vascular endothelial growth factor (VEGF) for ECs by the inhibiting phosphorylation of flt-1 (VEGF receptor 1) and subsequent ERK activation. Thus, the role of SPARC in tumor angiogenesis, stimulation or inhibition, is still unclear. To clarify the role of SPARC in tumor growth and progression, we determined the effect of VEGF on the expression of SPARC in human microvascular EC line, HMEC-1, and human umbilical vein ECs. VEGF increased the levels of SPARC protein and steady-state levels of SPARC mRNA in serum-starved HMEC-1 cells. Inhibitors (SB202190 and SB203580) of p38, a mitogen-activated protein (MAP) kinase, attenuated VEGF-stimulated SPARC production in ECs. Since intact SPARC inhibits phosphorylation ERK MAP kinase in VEGF signaling, it was suggested that SPARC plays a dual role in the VEGF functions, tumor angiogenesis, and extravasation of tumors mediated by the increased permeability of endothelial barrier function.

Down-regulation of vascular endothelial growth factor by tissue inhibitor of metalloproteinase-2: effect on in vivo mammary tumor growth and angiogenesis.

The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis.

Induction of endothelial cell apoptosis by solid tumor cells.

The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performed in vivo and in vitro demonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two- to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation.

Inhibition of stromal matrix metalloproteases: effects on breast-tumor promotion by fibroblasts.

Co-injection of fibroblasts with human epithelial breast-tumor MCF7 cells in the presence of Matrigel enhances tumor growth in nude mice. While most of the matrix metalloproteinases (MMPs) have been shown to be produced by stromal cells, tumor cells such as MCF7 cells are unable to produce MMPs. We therefore, hypothesized that the tumor-promoting effect of fibroblasts could be related to their production of MMPs. In order to inhibit stromal proteases, over-production of TIMP-2 was induced in MCF7 cells by in vitro retroviral-mediated gene transfer. TIMP-2-producing MCF7 cells were then co-injected with fibroblasts into nude mice. Alternatively, we evaluated the effect of Batimastat, a synthetic inhibitor of MMPs, on the tumorigenicity of MCF7 cells co-inoculated with fibroblasts into nude mice. Both physiological (TIMP-2) and synthetic (Batimastat) inhibitors of MMPs were able to abolish the tumor-promoting effect of fibroblasts. On the contrary, they failed to modulate the tumorigenicity of MCF7 cells injected alone. Interestingly, Matrigel from which low-molecular-weight proteins or growth factors had been removed failed to favor the tumorigenicity of MCF7 cells inoculated with fibroblasts. These findings emphasize the importance of fibroblasts in cancer progression, and suggest that their role could be related at least in part to production of proteases which can induce the release of factors from the extracellular matrix.

Endothelial cell intracellular Ca2+ concentration is increased upon breast tumor cell contact and mediates tumor cell transendothelial migration.

Tumor cell extravasation is a determinant step in the process of hematogenous metastasis. The signal transduction pathways involved in the interactions between tumor cells and the vascular endothelium during transendothelial migration are still undefined. In the present study, we have investigated the influence of human breast adenocarcinoma cells (MCF7) on human umbilical vein endothelial cell (HUVEC) intracellular Ca2+ concentration ([Ca2+]i). We show that the contact between MCF7 cells and a confluent HUVEC monolayer induces an immediate and transient increase in HUVEC [Ca2+]i. This [Ca2+]i rise could not be elicited by tumor cell-conditioned medium, isolated tumor cell membranes, inert beads or normal breast epithelial cells, demonstrating the involvement of specific recognition mechanisms between MCF7 cells and HUVEC. Depletion of HUVEC intracellular Ca2+ stores by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin as well as the selective depletion of inositol 1,4,5-triphosphate (IP3)-sensitive Ca2+ stores by prior activation of HUVEC using histamine resulted in a complete inhibition of tumor cell-induced [Ca2+]i elevation. Similar results were obtained when HUVEC monolayers were treated with the tyrosine kinase inhibitor herbimycin A, suggesting a role for tyrosine kinase-associated cell surface receptors in tumor cell-endothelial cell interactions. The depletion of HUVEC intracellular Ca2+ stores by thapsigargin was also shown to delay MCF7-induced endothelial cell disjunction, to prevent their spreading on the subendothelial extracellular matrix and transendothelial migration in vitro. These results suggest that transient changes in endothelial [Ca2+]i may govern multiple steps of tumor cell extravasation.

Emerging roles for proteinases in cancer.

Metalloproteinases and serine proteinases have been associated with tumor invasion and formation of metastasis which represent the major obstacles to cancer cure. The contribution of proteinases in these processes was initially thought to be the destruction of extracellular matrices. However, recent evidence suggests that they mainly affect tumor growth rather than invasion. Proteinases can indeed generate active matrix protein fragments, influence the release, the activation and the bioavailability of growth factors, and consequently modulate tumor cell growth, apoptosis and angiogenesis. Additionally, proteinases, their receptors and/or inhibitors can be directly involved in cell migration and in the processing or shedding of cell surface proteins. Further elucidation of the functions of proteinases is essential for the development of novel anticancer strategies.

Alteration of interendothelial adherens junctions following tumor cell-endothelial cell interaction in vitro.

The integrity of the vascular endothelium is mainly dependent upon the organization of interendothelial adherens junctions (AJ). These junctions are formed by the homotypic interaction of a transmembrane protein, vascular endothelial cadherin (VE-cadherin), which is complexed to an intracellular protein network including alpha-, beta-, and gamma-catenin. Additional proteins such as vinculin and alpha-actinin have been suggested to link the VE-cadherin/catenin complex to the actin-based cytoskeleton. During the process of hematogenous metastasis, circulating tumor cells must disrupt these intercellular junctions in order to extravasate. In the present study, we have investigated the influence of tumor cell-endothelial cell interaction upon interendothelial AJ. We show that human breast adenocarcinoma cells (MCF-7), but not normal human mammary epithelial cells, induce a rapid endothelial cell (EC) dissociation which correlates with the loss of VE-cadherin expression at the site of tumor cell-EC contact and with profound changes in vinculin distribution and organization. This process could not be inhibited by metalloproteinase nor serine protease inhibitors. Immunoprecipitations and Western blot analysis demonstrate that the overall expression of VE-cadherin and vinculin as well as the composition of the VE-cadherin/catenins complex are not affected by tumor cells while the tyrosine phosphorylation status of proteins within the complex is significantly altered. Our data suggest that tumor cells modulate AJ protein distribution and phosphorylation in EC and may, thereby, facilitate EC dissociation.

Wavelet analysis of olfactory nerve response to stimulus.

Multiunit electrophysiological activity recorded by gross electrodes from the olfactory nerve was analyzed by wavelet decomposition, a relatively new method of signal processing. The analysis was run on data from the unstimulated olfactory system as well as on data evoked in response to six different odorant stimuli. Like Fourier analysis, wavelet analysis provides a spectral decomposition of the signal. Unlike Fourier, wavelet analysis also locates the dominant spectral features in time. The output of a wavelet analysis can be further processed to enhance selected features. The increased amplitude of the nerve response evoked by stimulation was the most obvious feature, but efforts to learn from it were unproductive. The temporal pattern of receptor cell activity was much more yielding. The analysis resolved the nerve activity into three classes of events based on duration. On wavelet maps these classes of events separate out into three shifting and overlapping but distinct bands, one of which was interpreted as being associated with individual receptor cell firings and the other two as short and somewhat longer duration bursts of activity that was attributed to the synchronized firing of a group of receptor cells. This interpretation is supported by experiments in which waveforms simulating action potentials and bursts of action potentials are added to recorded data. Stimulation of the olfactory system with odorant molecules evokes a significant increase in the number of short duration bursts, and an amplitude increase that can be related to the number of receptor cells responding. Changes in the patterns of wavelet events can be associated with synchrony of cell firing, reset times for bursts of firing, and possibly other physiological dynamics. A number of differences in activity patterns with different odorants were observed, but without sufficient repeatability to allow reliable discrimination among them. While this study is clearly preliminary in that regard, it shows the potential of the wavelet method for contributing to the understanding of olfaction.

Plasma membrane-dependent activation of gelatinase A in human vascular endothelial cells.

The initiation of the angiogenic process requires a locally confined and time-limited proteolysis of the basement membrane (BM) components at the site of new vessel sprout. Gelatinase A, a member of the matrix metalloproteinase family, degrades BM type IV collagen and is involved in the BM breakdown by migrating tumor cells and endothelial cells (EC). Gelatinase A is synthesized as latent proenzyme and must be activated in order to express its proteolytic activity. A plasma membrane-dependent mechanism of activation has been described for several tumor and transformed cells lines. In the present study, we show that latent (72 kD) and mature (62-59 kD) forms of gelatinase A are present in EC membrane fraction from Triton X-114 extract while only latent form is found in the cytosolic fraction. The incubation of EC membrane fraction with exogenous latent gelatinase A resulted in a significant activation giving rise to 62-59 kD mature forms. 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong potentiator of angiogenesis in vitro and in vivo, increases the amount of both latent and activated forms of gelatinase A in EC membrane fraction as well as the ability of this latter fraction to activate exogenous latent gelatinase A. We show that the mRNA transcript coding for the membrane-integrated MMP, the MT-MMP, previously described as a potential gelatinase A activator in invasive tumor cells is also expressed in vascular EC and is regulated through a TPA sensitive process. This enzyme may be responsible for membrane-dependent gelatinase A activation in normal vascular EC and may therefore be a determinant in the control of BM proteolysis during angiogenesis.

Thrombospondin modulates human breast adenocarcinoma cell adhesion to human vascular endothelial cells.

Thrombospondin (TSP), a M(r) 450,000 cytoadhesive glycoprotein, has been shown to potentiate tumor cell metastasis in mice by a mechanism that involves the hemostatic system of the host. In this study, the potential involvement of TSP in the interaction of human mammary adenocarcinoma MCF-7 cells with human umbilical vein endothelial cells (HUVECs) in culture was investigated. Using an ELISA, preconfluent HUVECs synthesized 100-fold more TSP than did MCF-7 cells during 24 h of culture (20 versus 0.2 microgram/10(6) cells). Confocal microscopy localized TSP within intercellular junctions between aggregated MCF-7 cells in suspension. On adherent cells, TSP exhibited a patchy distribution both on the cell surface and in the cytosol. In HUVECs, TSP strongly stained the perinuclear space and was also found in association with cytoskeletal microfibrils. Flow cytometric analysis indicated the presence of a large number of unoccupied receptors for TSP on MCF-7 cells. Binding studies using [125I]TSP demonstrated the presence of 1.6 x 10(6) sites/cell with an apparent Kd of 28 nM. Attachment of radiolabeled MCF-7 cells to a TSP-coated substrate and to HUVEC monolayers was inhibited in the presence of a polyclonal antibody to TSP (10 micrograms/ml) or increasing concentrations (1-10 micrograms/ml) of soluble TSP. Neither nonimmune IgG nor the cell adhesion peptide Gly-Arg-Gly-Asp-Ser (100 micrograms/ml) inhibited these interactions. Inhibition was also observed with heparin (10 micrograms/ml), suggesting the participation of TSP heparin-binding domain(s) and heparin-like molecules. In the presence of an excess of soluble TSP or anti-TSP antibody, MCF-7 cells did not form aggregates in suspension and preformed aggregates were readily dissociated by the addition of soluble TSP. These results indicate that mammary adenocarcinoma cells use TSP to form aggregates and to attach to human endothelial cells. These interactions may have physiological implications during the hematogenous spread of tumor cells.

Coordinate enhancement of gelatinase A mRNA and activity levels in human fibroblasts in response to breast-adenocarcinoma cells.

Gelatinases/type-IV collagenases are metalloproteinases involved in some carcinoma invasion and metastatic processes. The exact cellular source of the 72-kDa gelatinase A is controversial. We have analyzed the expression of mRNA coding for gelatinase A in vivo by in situ hybridization on breast-cancer tissues. The mRNA for gelatinase A was present in fibroblasts. We have therefore evaluated the gelatinase-A activity in vitro, in co-cultures of different breast adenocarcinoma cell lines and human fibroblasts. In monoculture, none of the tumor cells tested produced detectable amounts of gelatinase A. The gelatinase-A activity was enhanced in cultures of fibroblasts maintained in the presence of MDA-MB 231 or SKBR3 cells, or their conditioned medium. This increased enzymatic activity was evidenced both in the culture medium and in the membrane fraction and was paralleled by enhancement of the steady-state levels of mRNA. These results are an in vitro demonstration of a regulation of fibroblasts gelatinase-A production by soluble factors secreted by breast-tumor cells.

[Coracoid process abutment according to Latarjet versus the Bankart operation. A comparative study of the results in 50 cases]. / Butée coracoïdienne selon Latarjet versus opération de Bankart. Etude comparative des résultats à propos de 50 cas.

The authors have analyzed the postoperative results in 50 patients of two surgical techniques used for the treatment of anterior recurrent dislocation of the shoulder. The study only concerns "virgin" shoulders having formally presented numerous anterior recurrent dislocations treated by the Bankart procedure or the Bristow-Latarjet operation. This work consists of a clinical evaluation with objective and subjective results on one hand, and a radiological evaluation on the other hand. The Latarjet operation gives 97% excellent and good results, as compared with 76% for the Bankart procedure. The radiological study confirms the "arthrogenetic" nature of the Latarjet operation and reveals a frequent lysis of the coracoid bone block. Nevertheless, these two factors do not affect in any way the objective and subjective results. The Latarjet operation, insofar as it is carefully executed, remains in our opinion, the prime operation, particularly with respect to the quality of the stability achieved.

[Dislocation of the tibialis posterior tendon]. / La luxation du tendon du jambier postérieur (M. tibialis posterior).

The authors report a case of traumatic dislocation of the tibialis posterior tendon on the medial malleolus. They describe the treatment applied and made a review of the literature about this rare pathology.

Rat chromosome 5 (q22-23) contains elements that control cell morphology and interactions with the extracellular matrix: a study of normal fibroblast x malignant hepatoma cell hybrids.

Cell interactions with the extracellular matrix are consistently modified in neoplasia. Malignant transformation has been correlated with modifications in the synthesis and distribution of matrix components and with alterations of cell adhesive properties to these components. A particular class of genes, able to suppress the transformed phenotype in normal cells, may be involved in those phenotypic changes. By studying somatic cell hybrids between mouse hepatoma (BWTG3) cells and normal rat skin fibroblasts (RSF), Islam and co-workers were able to localize a gene or a group of genes controlling anchorage dependence and cell growth in vitro. This (or these) gene(s) was (were) assigned to the q22-23 fragment of rat chromosome 5. In the present study, we compare the morphology and the interactions with the extracellular matrix proteins (laminin, fibronectin, and collagen IV) and the synthesis of these proteins by RSF X BWTG3 hybrid cells that had either retained (BS181p10) or lost (BS181a5) the q22-23 region of rat chromosome 5. Our results suggest that the rat 5q22-23 fragment controls a part of the cell differentiation program including morphology, attachment to extracellular matrix, and synthesis of some matrix proteins, particularly alpha 1 and alpha 2 chains of collagen IV.

Malignant cell attachment to endothelium of ex vivo perfused human umbilical vein. Modulation by platelets, plasma and fibronectin.

The success of blood-born metastatic spread depends upon a key event: the tumor cell arrest and attachment to the host organ vasculature. In the present study, we have investigated interactions between several normal and cancer cell lines and vascular endothelium in a model of ex vivo perfusion of human umbilical vein. In this system, hydrodynamic parameters are monitored and endothelial cells are kept in their original environment known to modulate their phenotype. Metastatic tumor cell adhesion to the perfused endothelium was found to be significantly higher than that of normal cells tested. Platelets and soluble plasma factors including fibronectin promoted tumor cell arrest and adhesion to endothelium. Altogether our results indicate that the ex vivo perfusion of human umbilical vein allows the study of the interactions between malignant tumor cells, circulating plasma or blood cells and the endothelium during blood-born metastatic spread.

[Multicenter Belgian study of the results of total arthroplasty for persistent congenital hip dislocation]. / Etude multicentrique belge des résultats des arthroplasties totales pour luxation congénitale invétérée de hanche.

The SO.B.C.O.T. proposed a multicenter study of the results of total hip replacement performed in Belgium for treatment of severe congenital dislocation. Eleven departments with about 30 surgeons contributed to this retrospective analysis, in which cases treated in different centers with various techniques are reviewed. Difficulties in data collection are discussed. Owing to the variety of data, the article is divided in 2 sections: an inquiry or descriptive analysis of congenital dislocation of the hip treated in Belgium between January 1972 and December 1987; the midterm results achieved by different techniques.

[The Herbert screw in the treatment of fractures and pseudarthrosis of the carpal scaphoid. Review of 25 cases]. / Utilisation de la vis de Herbert dans le traitement des fractures et pseudarthroses du scaphoïde carpien. Revue de 25 cas.

The Authors have reviewed the charts of 25 patients with scaphoid lesions treated with the Herbert screw and this study reports their personal experience. Between September 1983 and December 1986, 4 fresh fractures, 5 delayed unions and 16 pseudarthrosis were treated by means of this osteosynthesis procedure. The mean follow-up was 22 months. Bony union occurred in 21 patients. The series contains 2 failures defined as an absence of bony union after a post-operative delay of more than 6 months. There were 2 other cases considered as doubtful unions on X-rays. Wrist function, nevertheless, was restored in 23 patients. Plaster immobilization did not appear to be essential for every case. The short leave of absence, averaging 2 months only, is also in favour of this technique.