Weightlessness acts on human breast cancer cell line MCF-7.

Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. Human breast cancer cells MCF-7, flown in space in a Photon capsule, were fixed after 1.5, 22 and 48 h in orbit. Cells subjected to weightlessness were compared to 1 g in-flight and ground controls. Post-flight, fluorescent labeling was performed to visualize cell proliferation (Ki-67), three cytoskeleton components and chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network and chromatin structure. Several main phenomena were observed in weightlessness: The perinuclear cytokeratin network and chromatin structure were looser; More cells were cycling and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade; Microtubules were altered in many cells. The results reported in the first point are in agreement with basic predictions of cellular tensegrity. The prolongation of mitosis can be explained by an alteration of microtubules. We discuss here the different mechanisms involved in weightlessness alteration of microtubules: i) alteration of their self-organization by reaction-diffusion processes, and a mathematical model is proposed, ii) activation or deactivation of microtubules stabilizing proteins, acting on both microtubule and microfilament networks in cell cortex.

Shear stress modulates tumour cell adhesion to the endothelium.

The adhesion of breast adenocarcinoma cells (MDA-MB-231) to human umbilical vein endothelial cells (HUVEC) was studied in whole blood and under varying flow conditions. This study was done on HUVEC either kept under static conditions or pre-conditioned in flow for 2 hours at a shear stress of 5 or 13 dyn/cm(2). Coverslips coated by HUVEC were placed in a parallel plate perfusion chamber and perfused at a shear rate of 300 or 1500 sec(-1) with heparin-anticoagulated blood containing 111In labelled MDA-MB-231 cells. We report here the optimal conditions for studying the adhesion of MDA-MB-231 to endothelial cells under shear constraints corresponding to those observed into small and medium sized arteries.

A monoclonal antibody to platelet type III collagen-binding protein (TIIICBP) binds to blood and vascular cells, and inhibits platelet vessel-wall interactions.

TIIICBP is a new platelet receptor involved in platelet-type III collagen and platelet-subendothelium interactions. This receptor is composed of a doublet of 72-68 kDa proteins. In this study, the major protein (68 kDa) was purified and used to produce monoclonal antibodies. One of these antibodies, 7F4, binds to platelets as confirmed by flow cytometry. 7F4 inhibited platelet contact, spreading and aggregation induced by type III collagen. Under flow conditions, 7F4 prevented platelet interactions with type III collagen, endothelial cell matrix and the KOGEOGPK type II collagen octapeptide: the specific sequence recognized by TIIICBP. On the other hand, 7F4 had no effect on platelet-type I collagen interactions. TIIICBP was also detected on lymphocytes, granulocytes and monocytes. TIIICBP was expressed on endothelial cells and fibroblasts but not on smooth-muscle cells. These results show that TIIICBP is expressed on several cell types and participates in cell adhesion to the subendothelium.

The subendothelium of the HMEC-1 cell line supports thrombus formation in the absence of von Willebrand factor and collagen types I, III and VI.

The macromolecular composition of the extracellular matrix (ECM) produced by the human microvascular endothelial cell line (HMEC-1) was determined by ELISA and its thrombogenicity was studied in blood perfusion assays. Results were compared with those obtained with the ECM produced by human umbilical vein endothelial cells (HUVEC). The HMEC-1's ECM contains collagen type IV, fibronectin, laminin and thrombospondin, but no detectable levels of collagen types I, III and VI, or von Willebrand factor (vWF), whereas all these components were found in the ECM synthesized by HUVEC. HMEC-1's ECM was perfused with low-molecular-weight heparin-anticoagulated blood at two wall shear rates (650/s and 2,600/s), representative of moderate and high arterial wall shear rates, in parallel plate flow chambers for 5 min. This resulted in the formation of large platelet aggregates, compared to essentially a monolayer of adherent platelets on HUVEC's ECM. Interestingly, large thrombi were formed at 2,600/s when HMEC-1's ECM was perfused with the blood of a patient with severe type III von Willebrand disease lacking both plasma and platelet vWF, indicating that vWF was not absolutely required for thrombus formation on this matrix. Thrombin generated on the HMEC-1's ECM contributed importantly to the large platelet thrombi formed, shown by performing blood perfusion experiments in the presence of thrombin inhibitors. Our results indicate that 1) platelet adhesion and aggregate formation on a subendothelium may occur at a high shear rate (2600/s) without the participation of collagen types I, III and VI, and vWF; and 2) the HMEC-1 cell line may prove useful for in vitro studies of the thrombogenic properties of microvascular subendothelium which in most cases does not contain fibrillar collagens and vWF.

Different role of platelet glycoprotein GP Ia/IIa in platelet contact and activation induced by type I and type III collagens.

The role of glycoprotein Ia/IIa was studied during platelet contact and aggregation induced by type I and type III collagen. The anti-glycoprotein Ia/IIa (6F1) antibody inhibited type I collagen-induced aggregation but did not inhibit the first contact between platelets and collagen. In contrast, it was without effect either on type III collagen-induced contact or platelet interaction with the subendothelium in a static assay. Platelet aggregation induced by type III collagen was only slightly slowed down by 6F1 but pp72 spleen tyrosine kinase phosphorylation was not modified even at concentrations of 6F1 that completely blocked platelet activation induced by type I collagen. Our results indicate that glycoprotein Ia/IIa is not a primary binding site for type I or type III collagen on the platelet membrane. This receptor is more specifically involved in type I collagen-induced platelet spreading and aggregation.

A new platelet receptor specific to type III collagen. Type III collagen-binding protein.

Platelet interaction with type III collagen is mediated by several platelet receptors that recognize specific sequences in collagen. We previously described an octapeptide KP*GEP*GPK within the alpha(1)III-CB4 fragment that binds to platelets and specifically inhibits platelet aggregation induced by type III collagen. In this study, we demonstrated that the octapeptide prevented platelet contact and spreading on type III collagen and subendothelium under static and flow conditions. Platelets adhered to the immobilized octapeptide, and anti-bodies directed against other platelet collagen receptors (glycoprotein (GP) Ia/IIa, GP IV, p65, p47) did not impair this adhesion. The platelet octapeptide receptor was identified by ligand blotting as a protein doublet with molecular masses of 68 and 72 kDa and does not correspond to any other already known platelet collagen receptors (GP Ia, GP IV GP VI, and p65). Our results indicate that a specific type III collagen receptor, expressed on the platelet surface, is involved in the first stages of platelet type III collagen interaction.

Adhesion-induced intracellular signalling in endothelial cells depends on the nature of the matrix.

The adhesion of a human microvascular endothelial cell line to its own matrix was studied in comparison with adhesion of the same cells to fibronectin or thrombospondin-1. These endothelial cells adhered preferentially to their matrix whereas an equal cell number was attached to fibronectin or thrombospondin-1. The adhesion of cells to thrombospondin-1 was mediated by the N-terminal heparin binding domain of thrombospondin-1 as shown by the use of a recombinant fragment, N18. Cells adhering to their matrix displayed a morphology and a cytoskeleton organization very similar to that observed in vivo with an apical immunostaining for actin stress fibers and a fine basal labeling for vinculin. Cells on fibronectin were extensively spread and rapidly assembled stress fibers and focal contacts. Cells adherent to thrombospondin-1 presented large lamellae rich in actin but devoid of vinculin and only few actin fibers were observed. Depending on the substratum used, adhering endothelial cells displayed also different tyrosine phosphorylation patterns on electrophoresis. Our observations indicate that endothelial cells adhering to their matrix present an activation state intermediate between that induced by a "hyperadhesive" protein like fibronectin and that generated by a moderate, indeed anti-adhesive, protein like thrombospondin-1.

Transforming growth factor-beta 1 increases the adhesion of MDA-MB-231 mammary adenocarcinoma cells to the microvascular subendothelium.

The increase of tumor cell adhesion to the subendothelium in the presence of TGF-beta 1 is thought to be mediated by two major events: an enrichment of extracellular matrix proteins secreted by endothelial cells and an increase of the integrins on the surface of tumor cells. In this study, we analyzed the effect of TGF-beta 1 on the adhesion of a mammary adenocarcinoma cell line (MDA-MB-231) to the matrix of human microvascular endothelial cells (HMEC-1). The adhesion of TGF-beta 1-treated tumor cells to a non-treated matrix or to purified matrix proteins was enhanced, while no increase was observed when non-treated tumor cells were let to adhere to a matrix secreted by HMEC-1 in the presence of the cytokine. Thus, the increase of cell adhesion was due to the effect of TGF-beta 1 on tumor cells and not to the matrix enrichment induced by this cytokine. The hyper-adhesion was inhibited by the RGD peptide and EDTA indicating that integrins were involved. Integrin subunits concentrations (alpha 5, alpha v and beta 1) on the surface of TGF-beta 1-treated tumor cells were not modified, while confocal microscopy showed a reorganization of beta 1 integrin subunits on the cell surface and in the cytoplasm resulting in actin fibers reorganization in the cytoskeleton. This indicates that the enhanced adhesion of TGF-beta 1-treated MDA-MB-231 cells to the subendothelium is due to a qualitative change of integrins.

Microfibrils from the arterial subendothelium.

Microfibrillar structures of the subendothelium are represented by either type VI collagen or elastin-associated microfibrils which are also referred to as fibrillin-containing microfibrils. These structures are present throughout the subendothelium irrespective of the presence of elastin. The localization, structure, and protein composition of microfibrils are reviewed. The arterial subendothelium is thrombogenic despite its very low content in fibrillar collagens. This thrombogenicity is linked to the microfibrillar structures, essentially to type VI collagen and to thrombospondin-containing microfibrils. Their respective ability to bind the von Willebrand factor and to activate blood platelets is discussed.

Endothelial cell proliferation regulated by cytokines modulates thrombospondin-1 secretion into the subendothelium.

Endothelial cells activation and proliferation are induced by cytokines and modulated by adhesive molecules of the extracellular matrix. In a previous study, we showed that IL-1beta and TNF-alpha inhibited thrombospondin-1 (TSP) secretion by human umbilical vein endothelial cells. This work was carried on by studying the effects of other cytokines [interleukin 6 (IL-6), IL-8, basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta)] known to modulate endothelial cell proliferation. We show here that TSP incorporation into the subendothelial matrix is inversely proportional to cell density. Proliferative cytokines such as IL-6 and bFGF inhibit TSP secretion, whereas the anti-proliferative TGF-beta enhances it. IL-8 has no effect either on cell proliferation or TSP secretion. Thus, the modulation of TSP secretion by these cytokines is apparently due to changes in cell proliferation. Therefore, when studying the effects of cytokines on TSP secretion, it is important to correlate the concentration of subendothelial matrix TSP to the cell density.

Induction of thrombospondin-1 by all-trans retinoic acid modulates growth and differentiation of HL-60 myeloid leukemia cells.

Thrombospondin-1 (TSP), a multifunctional extracellular matrix protein, modulates human hematopoietic stem cell adherence and thus may play a role in blood cell proliferation and/or differentiation. The expression of TSP was studied in the human myeloid leukemia cell line, HL-60, upon differentiation into monocytes by phorbol-13-monoacetate (PMA) or into granulocytes by all-trans retinoic acid (RA). HL-60 cells cultured under serum-free conditions constitutively secreted low amounts of TSP into the cultured medium, approximately 13 ng/10(6) cells/24 h. PMA used at 4 x 10(-8) M did not significantly modulate TSP secretion over a 24 h period. In contrast, RA at 10(-7) M induced a 5- to 10-fold increase in TSP secreted by HL-60 cells during their differentiation into granulocytes over a 5 day period. The role of secreted TSP in RA-dependent cessation of growth and differentiation was examined using blocking anti-TSP antibodies. In the presence of the polyclonal anti-TSP antibody R5 (25 microg/ml), growth of RA-treated HL-60 cells was maintained at control levels for up to 3 days and a concomitant delay in granulocytic differentiation was observed. Moreover, the addition of soluble TSP (0.5-5 microg/ml) to untreated HL-60 cells decreased their growth and promoted their differentiation in a dose-dependent manner. Using a neutralizing antibody to transforming growth factor beta (TGF-beta) or purified TGF-beta1 we further demonstrated that the effects of TSP were not mediated through activation of latent TGF-beta. These studies indicate that TSP decreases the proliferation and promotes the differentiation of HL-60 cells.

Stromal cells from human benign prostate hyperplasia produce a growth-inhibitory factor for LNCaP prostate cancer cells, identified as interleukin-6.

To understand specific interactions between stromal cells and epithelial cells in benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma, we developed stromal-cell cultures from normal human prostate (PNX) and BPH (BH101), composed of fibroblasts and myofibroblasts. Their role in epithelial-cell growth was studied using the established cancer cell lines LNCaP, PC3 and DU145 and an SV40 large T-immortalized normal epithelial-cell line, PNT1A, in double-diffusion co-culture chambers. PNT1A was stimulated by PNX (x1.6) and more strongly by BH101 stromal cells (x2.7). Conversely, LNCaP growth decreased by 50% in the presence of BH101 stromal cells (stromal/epithelial ratio: 10). A BH101-conditioned medium (CM), obtained in serum-free conditions, induced 90% inhibition of [3H]thymidine incorporation of the LNCaP androgen-sensitive cell line. Two other androgen-independent prostate cancer cell lines were either insensitive to BH101 CM (PC3) or slightly inhibited (40% for DU145). BH101 produced large amounts of IL-1beta, IL-6 and IL-8. HPLC gel filtration enabled separation of an inhibitory fraction which contained IL-6. IL-6 was demonstrated to be responsible for the strong inhibitory effect since an IL-6-neutralizing antibody abolished this inhibition, which was reproduced by human recombinant IL-6. Recombinant IL-6 growth inhibition was observed only on LNCaP prostate cancer androgen-sensitive cells.

Heterogeneity of microfibrils: role of thrombospondin-microfibrils in the thrombogenicity of the subendothelium.

We report the results of an immunogold electron microscopical analysis on microfibrils from the arterial subendothelium showing that thrombospondin (TSP) is present on 40 nm-diameter structures joining 8-10 nm-diameter microfibrils containing fibrillin. They differ from type VI collagen which forms 3-5 nm-diameter microfibrils. TSP containing microfibrils (TSP-MF) extracted from human umbilical arteries did not contain fibrillin or type VI collagen. Blood platelet interactions with TSP-MF were not modified by anti-fibrillin or anti-type VI collagen antibodies. In situ, vWF was bound to cross-linked microfibrils, at the level of their 40 nm junction, and a double-labeling with the anti-thrombospondin and anti-vWF antibodies was observed. In vitro, vWF binding to TSP-MF was not inhibited by anti-fibrillin or anti-type VI collagen antibodies. These results suggest a structural and functional heterogeneity of microfibrils and emphasize the role of TSP-MF in the thrombogenicity of the subendothelium.

Proinflammatory cytokines (interleukin-1 beta and tumor necrosis factor-alpha) down regulate synthesis and secretion of thrombospondin by human endothelial cells.

We examined the effects of proinflammatory cytokines on the expression of two extracellular matrix proteins, e.g., thrombospondin (TSP) and fibronectin (FN) b cultured human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with human recombinant interleukin-1 beta (IL-1 beta) or human tumor necrosis factor-alpha (TNF-alpha) caused a time-and dose-dependent decline in TSP production whereas FN production was not modified. At low concentrations, IL-1 beta and TNF-alpha in combination ha a greater effect than either agent alone. Interferon-gamma (IFN-gamma) was without effect. The decline in TSP synthesis resulted in a decreased secretion of this glycoprotein into the extracellular matrix. Endothelial cell monolayers cultured on porous filters were used to study the polarity of TSP secretion. Approximately two thirds of the synthesized protein was secreted to the apical side medium and one third to the basal side medium and both types of secretion were inhibited to a similar extent by cytokine treatment. Immunoprecipitation experiments revealed no apparent degradation of secreted TSP, either in the apical or in the basal compartment. Treatment of HUVECs with lL-1 beta, either alone or in combination with TNF-alpha, had no significant effect on the steady-state TSP mRNA levels, suggesting a posttranscriptional regulation. Our results indicate that IL-1 beta decreasing TSP deposition and suggest different regulatory mechanisms for the expression of various secreted proteins by endothelial cells.

[Abnormal fibrinolysis in Degos disease. A study of 3 cases]. / Anomalies de la fibrinolyse dans la maladie de Degos. Etude de trois cas.

INTRODUCTION: Degos' disease is a rare dermatosis characterized by papular lesions with a porcelain-white central atrophy and histopathological aspect of wedge-shaped infarction necrosis and an endovasculitis in the dermis. Its pathogenesis is unknown but many abnormalities of haemostasis have been reported. PATIENTS AND METHODS: Platelets functions, coagulation and fibrinolysis were estimated in three patients with Degos' disease. For one patient, direct immunoelectron microscopy using an antibody to von Willebrand factor was performed on lesional skin. RESULTS: In all the patients, prolonged euglobulin lysis time, increased plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activities before and after a venous occlusion test were detected and indicated an inhibition of fibrinolysis. Electron microscopy demonstrated in one case an increased number of Weibel-Palade bodies and a raised staining of von Willebrand factor in endothelial cells. Tests for coagulation and circulating anticoagulant were normal. Results of platelets adhesion showed decrease of adhesion in one case and increased adhesion in another. Platelets aggregation studies were normal in two cases and showed hyperactive spontaneous and induced aggregation in one case. CONCLUSION: We showed an inhibition of fibrinolysis in three patients with Degos' disease. These abnormalities could induce a prethrombotic state. The release of PA and PAI from the endothelial cells into the blood stream and the modifications observed with electron microscopy may signify a primary lesion of endothelial cell of still unknown origin.

Defective adhesion of blood platelets to vascular microfibrils in the Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) platelets, which lack the membrane glycoprotein complex Ib-IX, do not adhere to subendothelium. The adhesion of platelets from two patients with BSS to subendothelial microfibrils (MFs) and type I collagen was compared in an in vitro assay adapted to patients with low platelet count. With both patients, platelet adhesion to MFs was strongly defective, whereas the adhesion to collagen was normal. The involvement of GPIb in the MFs-induced platelet adhesion was confirmed by the inhibitory effect of a MoAb (AN51) to the von Willebrand (vWF) factor binding domain of GPIb. The adhesion of platelets to MFs thus requires GPIb-IX and an axis MFs-vWF-GPIb-IX seems therefore to be prevalent in the reactivity of platelets with subendothelium.

Involvement of thrombospondin in the adherence of human breast-adenocarcinoma cells: a possible role in the metastatic process.

The attachment of cancer cells to adhesive molecules, such as laminin (LN) and fibronectin (FN) in the extracellular matrix is a critical step in tumor invasion and metastasis. Recent data have suggested a potential role for thrombospondin (TSP), a 420-kDa cyto-adhesive glycoprotein, in the growth and spread of breast cancer. In this study, we have measured the ability of the human breast adenocarcinoma cell line, MDA-MB-231, to synthesize TSP and to use this molecule as an adhesion factor. The level of TSP in cells and secreted into the culture medium were determined by an enzyme-linked immunosorbent assay (ELISA). At pre-confluence, MDA-MB-231 cells were shown to produce a high level of TSP, most of which was retained within the cells. In comparison, FN was almost entirely secreted into the culture medium. An increased secretion of TSP was however measured at low cell density, suggesting that TSP might be required for cell/substratum or cell/cell interactions. As shown by flow cytometry, the cells expressed membrane-bound TSP as well as unoccupied TSP receptors. 125I-TSP bound saturably to 1.2 x 10(6) sites per cell with an apparent dissociation constant of 23 nM. The binding was inhibited by an excess of unlabeled TSP and by heparin, suggesting that the receptor could be a heparan-sulfate proteoglycan or a sulfatide. TSP promoted attachment but not spreading of MDA-MB-231 cells which attached and spread on FN and LN substrates. These results suggest that endogenously synthesized TSP may have a role in the cyto-adherence of tumor cells during the spread of breast cancer.

Role of thrombospondin in the adhesion of human endothelial cells in primary culture.

The role of thrombospondin on the adhesion of endothelial cells in primary culture was studied using a serum-free defined medium or thrombospondin-depleted fetal bovine serum. Under these conditions, only 6% of the cells adhered to gelatin-coated dishes, whereas cells adhering to gelatin in the presence of normal fetal bovine serum were considered as 100% adhesion. The percentage of cells attached to fibronectin or thrombospondin-coated dishes in thrombospondin-depleted serum was 66 and 32%, respectively. The addition of purified platelet thrombospondin to thrombospondin-depleted serum increased the adhesion of endothelial cells to gelatin and to thrombospondin, up to 32 and 59%, respectively, and restored the attachment to fibronectin to the same extent as that observed in the presence of normal serum. In contrast to the attachment, the spreading of the adhering cells was not further influenced by the addition of soluble thrombospondin. Subcultured cells did not require any protein for adhering to gelatin substrata. These observations indicate that thrombospondin plays a major role in the adhesion of endothelial cells in primary culture.

Molecular mechanism of the interaction of subendothelial microfibrils with blood platelets.

This short review summarizes the present state of our knowledge concerning the mechanisms of the interaction between subendothelial microfibrils and blood platelets. Microfibrils associated with elastin and with basement membranes have been shown to be able to promote platelet adhesion and subsequent activation and aggregation. A 128 kDa thrombospondin (TSP)-like glycoprotein of microfibrils (GP 128) is involved in their reactivity towards platelets, as established from specific inhibition by antibodies against GP 128 and against TSP of the microfibril-platelet interaction. This interaction only occurs in the presence of plasma von Willebrand factor (vWF). VWF effectively binds to microfibrils; this binding does not implicate GP 128, but a 97 kDa protein of microfibrils not yet characterized. In addition, from data based on inhibition of platelet adhesion and aggregation by monoclonal antibodies against platelet membrane glycoprotein Ib (GP Ib), it appears that GP Ib, a platelet membrane receptor for vWF, acts in the vWF dependent microfibril-platelet interaction as a receptor for VWF bound to protein 97 kDa in microfibrils. A tentative model is presented to illustrate the complex mechanism of the microfibril-platelet interactions: recognition of vWF bound to microfibrils by GP Ib would be the first step, which would then allow subsequent binding of GP 128 to its receptor on the platelet membrane.