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Recent studies have suggested that inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (statins) can play a role in protection against vascular risk, which is independent of cholesterol reduction. It could act by inhibiting the synthesis of isoprenoids (farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP)), which are respectively essential for membrane attachment and biological activity of GTPases Ras and RhoA. This study demonstrates that a statin (cerivastatin) inhibits angiogenesis. This effect was due to a decrease in endothelial cell locomotion which was reversed by GGPP. It was mainly related to delocalization of RhoA from cell membrane to cytoplasm, responsible for the disorganization of actin stress fibers. Furthermore, a decrease in MMP-2 secretion, involved in cell invasion, was also observed. This effect is rather due to Ras inhibition as it was reversed by FPP. This anti-angiogenic activity could explain the beneficial effect of statins on atherosclerosis and on cancer prevention as shown by clinical studies.

Macrophages play a major role in angiogenesis. We recently reported that oncostatin M (OSM), a cytokine of the interleukin (IL)-6 family secreted by macrophages, has a potent angiogenic activity on human microvascular endothelial cells (HMEC-1), but has no effect on macrovascular cells (human umbilical vein endothelial cells (HUVECs)). In this work, we show that in HMEC-1, OSM (0.5-2.5 ng/ml), leukemia inhibitory factor (LIF) (25 ng/ml), bFGF (25 ng/ml) and IL-1beta (5 ng/ml) induced production of cyclooxygenase (COX)-2. In contrast, in HUVECs, neither OSM nor LIF induced COX-2 mRNA, suggesting that COX-2 might be implicated in the angiogenic activity of OSM. This was confirmed by the inhibiting effect on OSM-induced HMEC-1 proliferation of specific COX-2 inhibitors. In vivo studies confirmed this findings. We conclude that induction of COX-2 by OSM is necessary for its angiogenic activity, but is not sufficient since IL-1beta, which also induces COX-2 in HMEC-1, has only a poor proliferative effect.

Hyaluronic acid (HA) is a glycosaminoglycan of the extracellular matrix. Its fragmentation by the hyaluronidase, secreted by tumor cells, facilitates tumor invasion and the HA degradation products generated stimulate angiogenesis. We report here that the HA-binding protein hyaluronectin (HN) inhibits the stimulatory effect of HA-derived fragments on the proliferation and migration of endothelial cells in vitro, and hampers the organization of endothelial cells into capillary-like structures. Since HN strongly inhibits endothelial cell adhesion to immobilized HA, it is postulated that HN acts by impairing the binding to endothelial cells of HA fragments generated by hyaluronidase, thereby neutralizing the effect of HA degradation products on angiogenesis. Our results reveal a new mechanism by which the angiogenesis induced by HA fragments is modulated by HN.

The urokinase receptor (u-PAR), a protein anchored to cell membrane by a glycosyl phosphatidylinositol, plays a central role in cancer cell invasion and metastasis by binding urokinase plasminogen activator (u-PA), thereby facilitating plasminogen activation. Plasmin can promote cell migration either directly or by activating metalloproteinases that degrade some of the components of the extra cellular matrix. However, the IGR-OV1-Adria cell line contains the u-PAR but does not migrate even in the presence of exogenous u-PA, although the parental IGR-OV1 cell line migrates normally in the presence of u-PA. We therefore investigated the role of cell signalling for u-PA induced cell locomotion. We show that cell migration induced by u-PA-u-PAR complex is always associated with tyrosine kinase activation for the following reasons: (1) the blockade of the u-PAR by a chimeric molecule (albumin-ATF) inhibits not only the u-PA-induced cell migration, but also the signalling in IGR-OV1 line; (2) the binding of u-PA to u-PAR on non-migrating IGR-OV1-Adria cells was not associated with tyrosine kinase activation; (3) the inhibition of tyrosine kinase also blocked cell migration of IGR-OV1. Therefore tyrosine kinase activation seems to be essential for the u-PA-induced cell locomotion possibly by the formation of a complex u-PAR-u-PA with a protein whose transmembrane domain can ensure cell signalling. Thus, IGR-OV1 and IGR-OV1-Adria cell lines represent a good model for the analysis of the mechanism of u-PA-u-PAR-induced cell locomotion.

The binding of urokinase (u-PA) to its cell surface receptor (u-PAR) is critical for tumor cell invasion. Here, we report that the distribution of this binding by a u-PAR antagonist ATF-HSA inhibits in vitro the motility of endothelial cells in a dose-dependent manner. This inhibition was also observed when the cells were first stimulated with potent angiogenic factors, including bFGF or VEGF. [3H]thymidine incorporation assay demonstrated that ATF-HSA did not affect the cell proliferation. ATF-HSA was more potent than plasmin inhibitors, suggesting that it exerts its effects not solely by inhibiting the remodeling of the extracellular matrix. In fact, analysis of the cell shape change during migration revealed for the first time that its effect is related to a decrease in cell deformability. These results suggest that u-PAR antagonist may be a new approach to control angiogenesis.

Receptor-bound urokinase is likely to be a crucial determinant in both tumor invasion and angiogenesis. We report here that a yeast-derived genetic conjugate between human serum albumin and the 1-135 N-terminal residues of urokinase (u-PA) competitively inhibits the binding of exogenous and endogenous u-PA to its cell-anchored receptor (u-PAR). This hybrid molecule (ATF-HSA) also inhibits in vitro pro-urokinase-dependent plasminogen activation in the presence of u-PAR bearing cells. These effects are probably responsible for the observed in vitro inhibition of tumor cell invasion in a reconstituted basement membrane extract (Matrigel).

A mutant antithrombin was isolated from the plasma of a patient with pulmonary embolism. The new protein, which accounted for 55% of the antithrombin, had decreased heparin affinity and contained two components when analysed on the basis of either charge or molecular mass. Sialidase and endo-beta-N-acetylglucosaminidase F treatment suggested that this heterogeneity was due to a partial glycosylation occurring at a new carbohydrate attachment sequence. Peptide mapping by reverse-phase HPLC showed that the abnormality involved the N-terminal tryptic peptide. Sequence analysis demonstrated that the underlying mutation was 7 Ile----Asn which introduces a new Asn-Cys-Thr glycosylation sequence. This new oligosaccharide attachment site occupies the base of the proposed heparin-binding site, and the finding explains the consequent decrease in heparin affinity.

The monoclonal antibody ALB6 directed against the leukocyte differentiation antigen CD9 (p24) increases the calcium incorporation into isolated platelet membrane vesicles enriched in internal membranes. The similarities of the effects of both the monoclonal antibody and the catalytic subunit of the cAMP-dependent protein kinase (C, subunit), which phosphorylates a protein of an apparent molecular mass of 23 kDa, led us to investigate the relationship between CD9 (p24) and the 23-kDa phosphoprotein (p23). ALB6IgG does not inhibit the C.subunit-induced phosphorylation of p23 and the immunoadsorption by ALB6IgG of p24 associated to membrane vesicles does not alter the phosphorylation pattern. Thus, proteins of similar molecular mass appear to be involved in calcium fluxes: one is recognized by the ALB6 antibody while the other can be phosphorylated by the C-subunit.

The leukemia-associated cell surface antigen p 24 is found on normal platelets as well as on Bernard Soulier syndrome and thrombasthenia type I platelets. ALB6 IgG (a monoclonal antibody against p 24) induces the aggregation of platelets from normal donors but not from thrombasthenia. In contrast, ALB6 Fab inhibits platelet aggregation induced by collagen, ADP, thrombin, ionophore A 23187 and ALB6 IgG. The results suggest that ALB6 interferes with a mechanism common to all aggregation pathways; the possible mechanisms are discussed.