Heparanase is expressed in adult human osteoarthritic cartilage and drives catabolic responses in primary chondrocytes.

OBJECTIVES: The chondrocytes' pericellular matrix acts as a mechanosensor by sequestering growth factors that are bound to heparan sulfate (HS) proteoglycans. Heparanase is the sole mammalian enzyme with HS degrading endoglycosidase activity. Here, we aimed to ascertain whether heparanase plays a role in modulating the anabolic or catabolic responses of human articular chondrocytes. METHODS: Primary chondrocytes were incubated with pro-heparanase and catabolic and anabolic gene expression was analyzed by quantitative polymerase chain reaction (PCR). MMP13 enzymatic activity in the culture medium was measured with a specific fluorescent assay. Extracellular regulated kinase (ERK) phosphorylation was evaluated by Western blot. Human osteoarthritis (OA) cartilage was assessed for heparanase expression by reverse-transcriptase PCR, by Western blot and by a heparanase enzymatic activity assay. RESULTS: Cultured chondrocytes rapidly associated with and activated pro-heparanase. Heparanase induced the catabolic genes MMP13 and ADAMTS4 and the secretion of active MMP13, and down-regulated the anabolic genes ACAN and COL2A1. PG545, a HS-mimetic, inhibited the effects of heparanase. Heparanase expression and enzymatic activity were demonstrated in adult human osteoarthritic cartilage. Heparanase induced ERK phosphorylation in cultured chondrocytes and this could be inhibited by PG545, by fibroblast growth factor 2 (FGF2) neutralizing antibodies and by a FGF-receptor inhibitor. CONCLUSIONS: Heparanase is active in osteoarthritic cartilage and induces catabolic responses in primary human chondrocytes. This response is due, at least in part, to the release of soluble growth factors such as FGF2.

Transgenic over-expression of mammalian heparanase delays prion disease onset and progression.

Cellular heparan sulfate (HS) has a dual role in scrapie pathogenesis; it is required for PrP(Sc) (scrapie prion protein) formation and facilitates infection of cells, mediating cellular uptake of prions. We examined the involvement of heparanase, a mammalian endoglycosidase degrading HS, in scrapie infection. In cultured cells, heparanase treatment or over-expression resulted in a profound decrease in PrP(Sc). Moreover, disease onset and progression were dramatically delayed in scrapie infected transgenic mice over-expressing heparanase. Together, our results provide direct in vivo evidence for the involvement of intact HS in the pathogenesis of prion disease and the protective role of heparanase both in terms of susceptibility to infection and disease progression.

Heparanase expression in periapical granulomas and radicular cysts.

Heparanase is an endo-ß-D-glucuronidase enzyme which degrades heparan sulfate glycosaminoglycan side chains of proteoglycans in the extracellular matrix and in basement membranes. The aim of this study was to evaluate the expression of heparanase in periapical granulomas (PGs) and radicular cysts (RCs). Immunohistochemistry was used to assess heparanase expression in PGs and RCs. Parameters including stain intensity, location and cell type were used to characterize heparanase expression in the periapical lesions. Ordered categories (from weak to strong) were used to compare the level of heparanase staining in the PG and RC groups. Both epithelial cells and inflammatory cells were positive for heparanase. The relative staining of the epithelial cells was strong, whereas the relative staining of the inflammatory cells was weak. Significant differences in immunohistochemical staining of epithelial cells were observed between RCs and PGs (p = 0.002). The relative expression of heparanase in epithelial cells in RCs was strong. In PGs, lesions with few or no epithelial cells, heparanase was predominantly expressed weakly by inflammatory cells. PGs and RCs have the same infectious origin. Therefore, the different cellular sources of heparanase in these periapical lesions may imply that this enzyme has specific pathogenetic functions in RCs and PGs.

Thrombin receptor overexpression in malignant and physiological invasion processes.

Although the involvement of soluble and matrix-immobilized proteases in tumor cell invasion and metastasis is well recognized, the role of proteolytically activated cell surface receptors has not been elucidated. We report here that thrombin receptor, a member of the protease-activated receptor family, is preferentially expressed in highly metastatic human breast carcinoma cell lines and breast carcinoma biopsy specimens. Introduction of thrombin receptor antisense cDNA considerably inhibited the invasion of metastatic breast carcinoma cells in culture through a reconstituted basement membrane. During placental implantation of the human embryo, thrombin receptor is transiently expressed in the invading cytotrophoblasts. These results emphasize the involvement of thrombin receptor in cell invasion associated with tumor progression and normal embryonic development.

Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis.

Heparan sulfate proteoglycans interact with many extracellular matrix constituents, growth factors and enzymes. Degradation of heparan sulfate by endoglycosidic heparanase cleavage affects a variety of biological processes. We have purified a 50-kDa heparanase from human hepatoma and placenta, and now report cloning of the cDNA and gene encoding this enzyme. Expression of the cloned cDNA in insect and mammalian cells yielded 65-kDa and 50-kDa recombinant heparanase proteins. The 50-kDa enzyme represents an N-terminally processed enzyme, at least 100-fold more active than the 65-kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and specimens of human breast, colon and liver carcinomas. Low metastatic murine T-lymphoma and melanoma cells transfected with the heparanase cDNA acquired a highly metastatic phenotype in vivo, reflected by a massive liver and lung colonization. This represents the first cloned mammalian heparanase, to our knowledge, and provides direct evidence for its role in tumor metastasis. Cloning of the heparanase gene enables the development of specific molecular probes for early detection and treatment of cancer metastasis and autoimmune disorders.

Molecular behavior adapts to context: heparanase functions as an extracellular matrix-degrading enzyme or as a T cell adhesion molecule, depending on the local pH.

Migration of lymphocytes into inflammatory sites requires their adhesion to the vascular endothelium and subendothelial extracellular matrix (ECM). The ensuing penetration of the ECM is associated with the expression of ECM-degrading enzymes, such as endo-beta-D glucuronidase (heparanase), which cleaves heparan sulfate (HS) proteoglycans. We now report that, depending on the local pH, a mammalian heparanase can function either as an enzyme or as an adhesion molecule. At relatively acidified pH conditions, heparanase performs as an enzyme, degrading HS. In contrast, at the hydrogen ion concentration of a quiescent tissue, heparanase binds specifically to HS molecules without degrading them, and thereby anchors CD4+ human T lymphocytes. Thus, the local state of a tissue can regulate the activities of heparanase and can determine whether the molecule will function as an enzyme or as a proadhesive molecule.

Vascular endothelial cells maintained in the absence of fibroblast growth factor undergo structural and functional alterations that are incompatible with their in vivo differentiated properties.

Vascular endothelial cells cultured in the presence of fibroblast growth factor (FGF) adopt at confluence a morphological appearance similar to that of the vascular endothelium in vivo. Similarly, their apical cell surface is, as in vivo, nonthrombogenic. In contrast, when the cultures are maintained in the absence of FGF, the cells undergo within two to three passages structural and functional alterations that are incompatible with their in vivo morphological appearance and physiological function. Cultures maintained in the absence of FGF no longer adopt, upon reaching confluence, the configuration of a monolayer composed of small closely apposed and nonoverlapping, cuboidal cells. Instead, confluent cultures deprived of FGF consist of large, overlapping cells which have lost the polarity of cell surface characteristic of the vascular endothelium. The apical cell surface becomes thrombogenic, as reflected by its ability to bind platelets, whereas fibronectin, which at confluence is normally associated only with the basal cell surface, can be found both on top of and underneath the cell layer. Among other changes, both sparse and confluent cultures maintained in the absence of FGF showed a greatly increased production of fibronectin. CSP-60, a cell surface protein whose appearance is correlative with the adoption of a cell monolayer configuration, can no longer be detected in cultures maintained in the absence of FGF. Overlapping endothelial cells maintained in the absence of FGF can also no longer function as a protective barrier against the uptake of ligands such as low density lipoprotein. Exposure of the culture to FGF induces a restoration of the normal endothelial characteristics concomitant with the adoption of a flattened cell monolayer morphology. These results demonstrate that, in addition to being a mitogen. FGF is involved in controlling the differentiation and phenotypic expression of the vascular endothelium. This is reflected by its effect on the morphological appearance, polarity of cell surfaces, platelet binding capacity, and barrier function of the vascular endothelium.

Thrombin adhesive properties: induction by plasmin and heparan sulfate.

We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild-type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and ECM.

Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells.

Vascular endothelial cells (ECs) seeded sparsely on extracellular matrix (ECM) will proliferate in the absence of exogenous basic fibroblast growth factor (bFGF). This ECM will also stimulate neurite outgrowth in PC12 cells in the absence of exogenous growth factors. We have previously shown that bFGF is found in subendothelial ECM (Vlodavsky, I., J. Folkman, R. Sullivan, R. Fridman, R. Ishai-Michaeli, J. Sasse, and M. Klagsburn. 1987. Proc. Natl. Acad. Sci. USA. 84:2292-2296) and in basement membranes (Folkman, J., M. Klagsburn, J. Sasse, M. Wadzinski, D. Ingber, and I. Vlodavsky. 1988. Am. J. Pathol. 130:393-400). The actual requirement of ECM-associated bFGF for the growth of ECs and differentiation of PC12 cells was shown in two ways. First, polyclonal anti-bFGF antibodies added to subendothelial ECM inhibited both EC proliferation and PC12 neurite outgrowth. Secondly, PF-HR-9 cells, which do not synthesize bFGF and which produce an ECM not permissive for EC proliferation and PC12 neurite outgrowth, were transfected with bFGF cDNA. PF-HR-9 cells transfected with bFGF, but not with the dominant selectable marker SV2-neomycin, were found to express bFGF and to produce an ECM which did support both EC proliferation and PC12 differentiation. The ECM-mediated stimulatory effects were inhibited by anti-bFGF antibodies but not by anti-nerve growth factor antibodies or nonimmune rabbit IgG. These results indicate that bFGF associated with ECM is a required ECM component for ECM-mediated cell proliferation and differentiation.

An Arg-Gly-Asp sequence within thrombin promotes endothelial cell adhesion.

Thrombin, in addition to its central role in hemostasis, possesses diverse cellular bioregulatory functions implicated in wound healing, inflammation, and atherosclerosis. In the present study we demonstrate that thrombin molecules modified either at the procoagulant or catalytic sites induce endothelial cell (EC) adhesion, spreading, and cytoskeletal reorganization. The most potent adhesive thrombin analogue (NO2-alpha-thrombin) was obtained by nitration of tyrosine residues. The cell adhesion promoting activity of NO2-alpha-thrombin was blocked upon the formation of thrombin-antithrombin III (ATIII) complexes and by antiprothrombin antibodies, but was unaffected by hirudin. Arg-Gly-Asp-containing peptides, fully inhibited EC adhesion to NO2-alpha-thrombin, while synthetic peptides corresponding to thrombin "Loop B" mitogenic site and the thrombin-derived chemotactic fragment "CB67-129", were uneffective. Immunofluorescence studies indicated that EC adhesion to NO2-alpha-thrombin was followed by cell spreading, actin microfilament assembly, and formation of focal contacts. By the use of specific antibodies, the vitronectin (vn) receptor (alpha v beta 3) was found to be localized in clusters upon cell adhesion to NO2-alpha-thrombin. An anti alpha v beta 3 antibody blocked EC adhesion and spreading while antifibronectin (fn) receptor (alpha 5 beta 1) antibodies were uneffective. While native thrombin exhibited a very low cell attachment activity, thrombin that was incubated at 37 degrees C before coating of plastic surfaces induced EC attachment and spreading. We propose that under certain conditions the naturally hindered RGD domain within thrombin is exposed for interaction with alpha v beta 3 on EC. This in turn promotes cell adhesion, spreading, and reorganization of cytoskeletal elements, which may altogether contribute to repair mechanisms in the disturbed vessel wall. This study defines a new biological role of thrombin and characterizes a new recognition mechanism on EC for this molecule.

Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism?

Basic fibroblast growth factor, (bFGF), promotes the formation of new blood capillaries and is sequestered and protected by binding to heparan sulfate (HS), both on the cell surface and in the extracellular matrix. Release of HS-bound bFGF by heparin-like molecules and HS-degrading enzymes (i.e., heparanase) provides a novel mechanism for regulation of the growth of capillary blood vessels in normal and pathological situations. The extracellular matrix also serves as a storage depot for other growth factors and enzymes.

Association of heparanase gene (HPSE) single nucleotide polymorphisms with hematological malignancies.

Heparanase, endo-beta-D-glucuronidase, degrades heparan sulfate glycosaminoglycans - the principal polysaccharide of the basement membrane and extracellular matrix. Heparanase activity plays a decisive role in biological processes associated with remodeling of the extracellular matrix, such as cancer metastasis, angiogenesis and inflammation. In the hematopoietic system, heparanase is thought to be associated with normal differentiation and function of myeloid cells and platelets. We investigated heparanase polymorphisms in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), Hodgkin's disease (HD) and multiple myeloma (MM). Significant correlation was found between rs11099592 and rs6535455 heparanase gene (HPSE) single nucleotide polymorphisms (SNPs) and ALL (chi2(1d.f.)=4.96, P=0.026). Genotype frequency comparisons revealed a significant association with rs4693602 (chi2(2d.f.)=7.276, P=0.026) in MM patients and rs4364254 (chi2(2d.f.)=6.226, P=0.044) in AML patients. Examination of HPSE gene mRNA expression by real-time RT-PCR indicated a significant low HPSE gene expression level in ALL patients and a high expression level in MM and AML patients, compared to healthy controls. Moreover, statistically significant correlation was found between heparanase mRNA expression level and three HPSE gene SNPs (rs4693608, rs11099592 and rs4364254) among healthy individuals. These data suggest that certain HPSE gene SNPs may contribute to basal heparanase gene expression and that alterations in this gene are an important determinant in the pathogenesis of ALL, AML and MM.

Tumor suppressor p53 regulates heparanase gene expression.

Mammalian heparanase degrades heparan sulfate, the most prominent polysaccharide of the extracellular matrix. Causal involvement of heparanase in tumor progression is well documented. Little is known, however, about mechanisms that regulate heparanase gene expression. Mutational inactivation of tumor suppressor p53 is the most frequent genetic alteration in human tumors. p53 is a transcription factor that regulates a wide variety of cellular promoters. In this study, we demonstrate that wild-type (wt) p53 binds to heparanase promoter and inhibits its activity, whereas mutant p53 variants failed to exert an inhibitory effect. Moreover, p53-H175R mutant even activated heparanase promoter activity. Elimination or inhibition of p53 in several cell types resulted in a significant increase in heparanase gene expression and enzymatic activity. Trichostatin A abolished the inhibitory effect of wt p53, suggesting the involvement of histone deacetylation in negative regulation of the heparanase promoter. Altogether, our results indicate that the heparanase gene is regulated by p53 under normal conditions, while mutational inactivation of p53 during cancer development leads to induction of heparanase expression, providing a possible explanation for the frequent increase of heparanase levels observed in the course of tumorigenesis.

Differential effect of components of the extracellular matrix on differentiation and apoptosis.

BACKGROUND: Epithelial cells are closely associated with a basement membrane, but the intimate relationships that affect growth, differentiation and survival remain enigmatic. We have previously reported that granulosa cells adjacent to the basement membrane of the ovarian follicle have a higher degree of differentiation compared with cells located distal to the basement membrane. By contrast, granulosa cells distal to the basement membrane are the first to undergo apoptosis during follicular atresia. Moreover, growth of granulosa cells in vitro on a naturally produced basement-membrane-like extracellular matrix (ECM) enhances progesterone production and the cellular response to gonadotropic hormones by an undefined mechanism. RESULTS: To investigate the effect of the ECM on granulosa cell differentiation and death, primary granulosa cells were cultured on ECMs that lacked or contained bFGF (basic fibroblast growth factor). These otherwise identical ECMs were deposited by HR9 mouse endodermal cells, which do not synthesize bFGF, or by HR9 cells transfected with the bFGF gene. Both ECMs provided protection against apoptosis in serum-free medium, but only the bFGF-containing ECM maintained expression of the steroidogenic P450scc enzyme system and the production of progesterone. Moreover, culturing the cells on this ECM enhanced the expression of the 30 kDa steroid acute regulatory protein which plays a key role in steroid hormone biosynthesis. Laminin, but not fibronectin, was able to replace the ECM in protecting the cells from apoptosis; but not in maintaining steroidogenesis, whereas bFGF was able to enhance steroidogenesis without protecting the cells against apoptosis. Cells cultured on both ECMs or laminin had a well-developed actin cytoskeleton compared with cells cultured on non-coated dishes, which underwent apoptosis. CONCLUSIONS: Cellular responses to ECM are mediated by the combined action of macromolecular constituents and regulatory molecules, such as bFGF, that are sequestered and stored in the ECM. ECM or laminin protects against cell death by interacting with specific integrin receptors and maintaining a well-developed actin cytoskeleton. ECM-bound bFGF provides differentiation signals for granulosa cells, which are in intimate contact with the ECM. Thus, a clear distinction can be made between the survival activity and the differentiation stimulus exerted by the ECM on epithelial cells.

Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties.

We have analyzed the binding of thrombin, a serine protease with central roles in hemostasis, to the subendothelial extracellular matrix (ECM) produced by cultured endothelial cells. This substrate provides a thrombogenic surface where hemostasis is initiated. Binding was saturable and equilibrium was achieved after 3 h incubation with 125I-alpha-thrombin. Scatchard analysis of thrombin binding revealed the presence of 5.1 X 10(9) binding sites per squared millimeter ECM, with an apparent Kd of 13 nM. The catalytically blocked enzyme, diisofluorophosphate (DIP)-alpha-thrombin competed efficiently with 125I-alpha-thrombin, indicating that the binding was independent of its catalytic site. Moreover, high concentrations of the synthetic tetradecapeptide, representing residues 367-380 of thrombin B chain (the macrophage mitogenic domain of thrombin), competed with thrombin binding to ECM, indicating that the binding site may reside in the vicinity of "loop B" region. Thrombin binds to dermatan sulfate in the ECM, as demonstrated by the inhibition of 125I-alpha-thrombin binding to ECM pretreated with chondroitinase ABC, but not with heparitinase or chondroitinase AC. This stands in contrast to 125I-FGF (fibroblast growth factor) binding to ECM, which was inhibited by heparitinase but not by chondroitinase ABC, ECM-bound thrombin exhibits an exposed proteolytic site as monitored by the Chromozyme TH assay and by its ability to convert fibrinogen to a fibrin clot and to induce platelet activation as indicated by 14C-serotonin release. ECM-bound thrombin failed to form a complex with its major circulating inhibitor-antithrombin III (AT III), compared with rapid complex formation with soluble thrombin. We propose that thrombin binds to subendothelial ECM where it remains functionally active, localized, and protected from inactivation by circulating inhibitors.

Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix.

Lipoprotein lipase enhances binding at 4 degrees C of human plasma lipoproteins (chylomicrons, VLDL, intermediate density lipoprotein, LDL, and HDL3) to cultured fibroblasts and hepG-2 cells and to extracellular matrix. Heparinase treatment of cells and matrix reduces the lipoprotein lipase enhanced binding by 90-95%. Lipoprotein lipase causes only a minimal effect on the binding of lipoproteins to heparan sulfate deficient mutant Chinese hamster ovary cells while it promotes binding to wild type cells that is abolished after heparinase treatment. With 125I-LDL, lipoprotein lipase also enhances uptake and proteolytic degradation at 37 degrees C by normal human skin fibroblasts but has no effect in heparinase-treated normal cells or in LDL receptor-negative fibroblasts. These observations prove that lipoprotein lipase causes, predominantly, binding of lipoproteins to heparan sulfate at cell surfaces and in extracellular matrix rather than to receptors. This interaction brings the lipoproteins into close proximity with cell surfaces and may promote metabolic events that occur at the cell surface, including facilitated transfer to cellular receptors.

Degradation of sulfated proteoglycans in the subendothelial extracellular matrix by human platelet heparitinase.

Cultured vascular and corneal endothelial cells produce an underlying extracellular matrix (ECM) which induces platelet adherence, aggregation, and release reaction. Incubation of a metabolically (35S)O = 4-labeled ECM with platelet-rich plasma or washed platelets, but not with platelet-poor plasma, resulted in degradation of its heparan sulfate-containing proteoglycans into labeled fragments four to five times smaller than intact glycosaminoglycan side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin, indicating that heparan sulfate in the ECM is susceptible to cleavage by the platelet heparitinase. This degradation required adhesion of platelets to the ECM rather than aggregation since it was not inhibited by aspirin, which prevented platelet aggregation but not adherence. The enzyme was not released during aggregation of platelets on the ECM but was readily liberated upon their exposure to thrombin. This liberation was inhibited in the presence of prostacyclin (PGI2). Isolated high molecular weight proteoglycans first released from the ECM by incubation with platelet poor plasma served as a substrate for further degradation by the platelet heparitinase, suggesting a cascade mechanism for degradation of heparan sulfate in the ECM. Heparitinase, although to a lower level, was also active when washed platelets were added on top of a confluent endothelial cell monolayer covering the (35S)O = 4-labeled ECM. It is suggested that the platelet heparitinase may be involved in the impairment of the integrity of the vessel wall and thus facilitate the extravasation of blood-borne cells.

Interferon enhances prostacyclin production by cultured vascular endothelial cells.

The effects of interferon (IFN) on the arachidonate metabolism and physiological functions of cultured endothelial cells and blood platelets have been examined. Cultured bovine aortic endothelial cells were found to be sensitive to the antiviral and antiproliferative activities of human leukocyte (alpha) IFN and to increase their capacity to synthesize prostacyclin (PGI2) upon exposure to IFN. Several observations indicate that IFN stimulates PGI2 synthesis at the level of the enzymes phospholipase A2 and cyclooxygenase: (a) PGI2 production was dependent upon the supply of exogenous arachidonic acid or the liberation of endogenous cellular arachidonate by ionophore A23187, but was not observed when IFN-treated cells were exposed to the endoperoxide prostaglandin H2. (b) IFN had no effect on the spontaneous release of PGI2 into the culture medium during the incubation period (24-72 h). (c) The stimulatory effect of IFN on PGI2 production was inhibited by both glucocorticoids and indomethacin. The effect of IFN on platelet prostaglandin metabolism was also investigated. Incubation of platelet-rich plasma with IFN had no effect on platelet aggregation and thromboxane A2 production. The biological significance of the findings presented in this paper may be considered in view of the protective role of PGI2 in the vessel wall and the fact that infection with certain viruses induces endothelial damage both in man and experimental animal models.

Suppression of experimental autoimmune diseases and prolongation of allograft survival by treatment of animals with low doses of heparins.

The ability of activated T lymphocytes to penetrate the extracellular matrix and migrate to target tissues was found to be related to expression of a heparanase enzyme (Naparstek, Y., I. R. Cohen, Z. Fuks, and I. Vlodavsky. 1984. Nature (Lond.). 310:241-243; Savion, N., Z. Fuks, and I. Vlodavsky. 1984. J. Cell. Physiol. 118:169-176; Fridman, R., O. Lider, Y. Naparstek, Z. Fuks, I. Vlodavsky, and I. R. Cohen. 1987. J. Cell. Physiol. 130:85-92; Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We found previously that heparin molecules inhibited expression of T lymphocyte heparanase activity in vitro and in vivo, and administration of a low dose of heparin in mice inhibited lymphocyte traffic and delayed-type hypersensitivity reactions (Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We now report that treatment with commercial or chemically modified heparins at relatively low doses once daily (5 micrograms for mice and 20 micrograms for rats) led to inhibition of allograft rejection and the experimental autoimmune diseases adjuvant arthritis and experimental autoimmune encephalomyelitis. Higher doses of the heparins were less effective. The ability of chemically modified heparins to inhibit these immune reactions was associated with their ability to inhibit expression of T lymphocyte heparanase. There was no relationship to anticoagulant activity. Thus heparins devoid of anticoagulant activity can be effective in regulating immune reactions when used at appropriate doses.

Modulation of fibroblast growth factor-2 receptor binding, dimerization, signaling, and angiogenic activity by a synthetic heparin-mimicking polyanionic compound.

Heparan sulfate (HS) proteoglycans play a key role in cell proliferation induced by basic fibroblast growth factor (FGF-2) and other heparin-binding growth factors. To modulate the involvement of HS, we have used a synthetic, nonsulfated polyanionic aromatic compound (RG-13577) that mimics functional features of heparin/HS. FGF-2-stimulated proliferation of vascular endothelial cells was markedly inhibited in the presence of 5-10 microg/ml compound RG-13577 (poly-4-hydroxyphenoxy acetic acid; Mr approximately 5 kD). Direct interaction between RG-13577 and FGF-2 was demonstrated by the ability of the former to compete with heparin on binding to FGF-2. RG-13577 inhibited FGF-2 binding to soluble- and cell surface-FGF receptor 1 (FGFR1). Unlike heparin, RG-13577 alone failed to mediate dimerization of FGF-2. Moreover, it abrogated heparin-mediated dimerization of FGF-2 and FGFR1, as well as FGF-2 mitogenic activity in HS-deficient F32 lymphoid cells. The antiproliferative effect of compound RG-13577 was associated with abrogation of FGF-2-induced tyrosine phosphorylation of FGFR1 and of cytoplasmic proteins involved in FGF-2 signal transduction, such as p90 and mitogen-activated protein kinase. A more effective inhibition of tyrosine phosphorylation was obtained after removal of the cell surface HS by heparinase. In contrast, tyrosine phosphorylation of an approximately 200-kD protein was stimulated by RG-13577, but not by heparin or FGF-2. RG-13577 prevented microvessel outgrowth from rat aortic rings embedded in a collagen gel. Development of nontoxic polyanionic compounds may provide an effective strategy to inhibit FGF-2-induced cell proliferation associated with angiogenesis, arteriosclerosis, and restenosis.