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.

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.

Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes.

Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ.

Subcellular localization of heparanase in human neutrophils.

The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel filtration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECM-derived, heparan sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.

The extracellular matrix produced by bovine corneal endothelial cells contains progelatinase A.

Progelatinase A is a matrix metalloproteinase involved in the turnover of extracellular matrix (ECM). We report that the ECM produced by bovine corneal endothelial (BCE) cells contains progelatinase A free of tissue inhibitor of metalloproteinase (TIMP2). The matrix-bound progelatinase A can be activated by APMA to generate a 62 kDa and a 45 kDa species with enzymatic activity and is inhibited by TIMP2. The bound progelatinase can be released after treatment of the ECM with gelatinase B. These studies suggest that the ECM can function as a reservoir for progelatinase A which may be readily available for cells in processes such as metastasis, angiogenesis, inflammation and wound healing.

Poly(N-acryl amino acids): a new class of biologically active polyanions.

Poly(N-acryl amino acids) bearing side groups with a lipophilic character or having charged functional groups (i.e. -NH(2), -COOH, -SH, -OH, and phenols) were synthesized from the radical polymerization of N-acryl amino acid monomers. Monomers were prepared from the reaction of acryloyl chloride and amino acid esters in dry solvents. Polymers of a broad molecular weight ranging from 3 000 to 60 000 Da were obtained. The polymers were optically active, and their structures were confirmed by (1)H NMR and IR spectra and elemental analysis. Hydroxyl-containing polymers were sulfated in high conversion yields by SO(3)/pyridine complex. The newly synthesized linear homopolyanions were tested for heparin-like activities: (i) inhibition of heparanase enzyme, (ii) release of basic fibroblast growth factor (bFGF) from the extracellular matrix (ECM), and (iii) inhibition of smooth muscle cell (SMC) proliferation. Polymers based on tyrosine and leucine were highly active in all three tests (microgram level). Polymers based on phenylalanine, tert-leucine, and proline were active as heparanase inhibitors and FGF release, and polymers of trans-hydroxyproline, glycine, and serine were active only as heparanase inhibitors. The polymer of cis-hydroxyproline was inactive. It was found that a net anionic charge (i.e. carboxylic acid) is essential for biological activity. Thus, methyl ester derivatives of the active polymers, zwitterionic amino acid dependent groups (lysine, histidine), and decarboxylated amino acids (tyramine, ethanolamine) were inactive. The above active polymers did not exhibit anticoagulation activity which is considered the main limitation of heparin and heparinomimetics for clinical use. These synthetic poly(N-acryl amino acids) may have potential use in the inhibition of heparanase-mediated degradation of basement membranes associated with tumor metastasis, inflammation, and autoimmunity.

Mammalian heparanase as mediator of tumor metastasis and angiogenesis.

Expression of heparan sulfate-degrading endoglycosidases, commonly referred to as heparanases, correlates with the metastatic potential of tumor cell lines, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. We purified a 50 kDa heparanase from human hepatoma and placenta and cloned a cDNA and gene encoding a protein of 543 amino acids. Only one heparanase sequence was identified, suggesting that this enzyme is the dominant endoglucuronidase in mammalian tissues. Expression of the cloned cDNA in insect and mammalian cells yielded 65 kDa and 50 kDa recombinant proteins. The 50 kDa enzyme represents an N-terminal processed enzyme that is at least 200-fold more active than the full-length 65 kDa form. Processing was demonstrated following incubation of the full-length recombinant enzyme with intact tumor cells. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and in specimens of human melanomas and carcinomas. In the colon, both the heparanase mRNA and protein are expressed already at the stage of tubulovillous adenoma, but not in the adjacent 'normal-looking' colon epithelium. Non-metastatic murine T lymphoma and melanoma cells transfected with the heparanase gene acquired a highly metastatic phenotype in vivo. Apart from its involvement in the egress of cells from the vasculature, heparanase is tightly involved in angiogenesis, both directly--by promoting invasion of endothelial cells (vascular sprouting), and indirectly--by releasing heparan sulfate-bound basic fibroblast growth factor, and generating HS degradation fragments that promote bFGF activity. The angiogenic potential of heparanase was demonstrated in vivo (Matrigel plug assay) by showing a three to fourfold increase in neovascularization induced by Eb T lymphoma cells following their transfection with the heparanase gene. The ability of heparanase to promote both tumor angiogenesis and metastasis makes it a promising target for cancer therapy.

Heparanase activity expressed by platelets, neutrophils, and lymphoma cells releases active fibroblast growth factor from extracellular matrix.

Incubation of platelets, neutrophils, and lymphoma cells with Descemet's membranes of bovine corneas and with the extracellular matrix (ECM) produced by cultured corneal endothelial cells resulted in release of basic fibroblast growth factor (bFGF), which stimulated the proliferation of 3T3 fibroblasts and vascular endothelial cells. Similar requirements were observed for release of endogenous bFGF stored in Descemet's membrane and of exogenous bFGF sequestered by the subendothelial ECM. Release of ECM-resident bFGF by platelets, neutrophils, and lymphoma cells was inhibited by carrageenan lambda, but not by protease inhibitors, in correlation with the inhibition of heparanase activity expressed by these cells. Degradation of the ECM-heparan sulfate side chains by this endo-beta-D-glucuronidase is thought to play an important role in cell invasion, particularly in the extravasation of blood-borne tumor cells and activated cells of the immune system. We propose that both heparanase and ECM-resident bFGF may modulate the cell response to contact with its local environment. Heparanase-mediated release of active bFGF from storage in basement membranes provides a novel mechanism for a localized induction of neovascularization in various normal and pathological processes, such as wound healing, inflammation, and tumor development.

Laminarin sulfate mimics the effects of heparin on smooth muscle cell proliferation and basic fibroblast growth factor-receptor binding and mitogenic activity.

Heparin and heparin-like molecules may function, apart from their effect on hemostasis, as regulators of cell growth and neovascularization. We investigated whether similar effects are exerted by laminarin sulfate, an unrelated polysulfated saccharide isolated from the cell wall of seaweed and composed of chemically O-sulfated beta-(1,3)-linked glucose residues. Laminarin sulfate exhibits about 30% of the anticoagulant activity of heparin and is effective therapeutically in the prevention and treatment of cerebrovascular diseases. We characterized the effect of laminarin sulfate on interaction of the heparin-binding angiogenic factor, basic fibroblast growth factor (bFGF), with a naturally produced subendothelial extra-cellular matrix (ECM) and with cell surface receptor sites. Laminarin sulfate (1-2 micrograms/ml) inhibited the binding of bFGF to ECM and to the surface of vascular smooth muscle cells (SMC) in a manner similar to that observed with heparin. Likewise, laminarin sulfate efficiently displaced both ECM- and cell-bound bFGF at concentrations as low as 1 microgram/ml. Both laminarin sulfate and heparin efficiently induced restoration of bFGF receptor binding in xylosyltransferase-deficient CHO cell mutants defective in initiation of glycosaminoglycan synthesis. Moreover, laminarin sulfate elicited bFGF receptor activation and mitogenic response in heparan sulfate (HS)-deficient, cytokine-dependent lymphoid cells. These results indicate that laminarin sulfate effectively replaced the need for heparin and HS in the induction of bFGF receptor binding and signaling. In other experiments, laminarin sulfate was found to inhibit the proliferation of vascular SMC in a manner similar to that observed with heparin. These effects of laminarin sulfate may have potential clinical applications in diverse situations such as wound healing, angiogenesis, and atherosclerosis.

Sulfate moieties in the subendothelial extracellular matrix are involved in basic fibroblast growth factor sequestration, dimerization, and stimulation of cell proliferation.

The growth promoting activity of the subendothelial extracellular matrix (ECM) is attributed to sequestration of basic fibroblast growth factor (bFGF) by heparan sulfate proteoglycans and its regulated release by heparin-like molecules and heparan sulfate (HS) degrading enzymes. HS is also involved in bFGF receptor binding and activation. The present study focuses on the growth promoting activity and bFGF binding capacity of sulfate-depleted ECM. Corneal endothelial cells (EC) maintained in the presence of chlorate, an inhibitor of phosphoadenosine phosphosulfate synthesis, produced ECM containing 10-15% of the sulfate normally present in ECM. Incorporation of sulfate into HS was reduced by more than 90%. Binding of 125I-bFGF to sulfate-depleted ECM was reduced by 50-60% and only about 10% of the ECM-bound bFGF was accessible to release by heparin. Incubation of 125I-bFGF on top of native ECM resulted in dimerization of the ECM-bound bFGF, but there was a markedly reduced binding and dimerization of bFGF on sulfate-depleted ECM. ECM produced in the presence of chlorate contained a nearly 10-fold less endogenous bFGF as compared to native ECM and exerted little or no mitogenic activity toward vascular EC and 3T3 fibroblasts. In other studies, we investigated the interaction between chlorate-treated vascular EC and either native or sulfate-depleted ECM. Exogenous heparin stimulated the proliferation of chlorate-treated EC seeded on native ECM, suggesting its interaction with ECM-bound bFGF and subsequent presentation to high affinity cell surface receptors. On the other hand, heparin had no effect on chlorate-treated cells seeded in contact with sulfate-depleted ECM or regular tissue culture plastic. Altogether, the present experiments indicate that heparan sulfate proteoglycans associated with the cell surface and ECM act in concert to regulate the bioavailability and growth promoting activity of bFGF. While HS in the subendothelial ECM functions primarily in sequestration of bFGF in the vicinity of responsive cells, HS on cell surfaces is playing a more active role in displacing the ECM-bound bFGF and its subsequent presentation to high affinity signal transducing receptors.

Lung colonization by and heparanase activity in in vitro transformed 3T3 cells rendered highly tumorigenic by an in vivo passage.

Cloned BALB/c 3T3 cells transformed in vitro with polyoma virus and maintained in culture (C cells) were compared with respect to their ability to form experimental metastases, with cells from the same clones that were passaged subcutaneously in vivo (CTC cells), thereby gaining a high tumorigenicity phenotype. No correlation was found between a high subcutaneous tumorigenicity potential or the progression state of these cells, and their capacity to form experimental metastases. Both cell types were also tested for their ability to release heparan sulfate degradation products from a naturally produced, sulfate-labeled extracellular matrix (ECM). Whereas the in vitro maintained C cells did not express an endo-beta-D-glucuronidase (heparanase) activity, some of the in vivo passaged CTC cells expressed such an activity. No strict correlation was found, however, between heparanase activity and the ability of CTC cells from individual tumors to form experimental metastases. However, A9 CTC 220 cells which produced a large number of lung metastases also expressed a high heparanase activity. Both heparanase and lung colonization by A9 CTC 220 cells were inhibited to a large extent by heparin, suggesting the involvement of heparanase in the extravasation of these highly metastatic cells. A9 CTC 220 cells were found to release basic fibroblast growth factor (bFGF) from ECM. This release was partially inhibited by carrageenan lambda which also completely inhibited the heparanase activity expressed by these cells. The in vivo acquisition of heparanase activity was not a result of cell fusion between heparanase expressing host-derived cells and heparanase-negative transformed cells. This conclusion was based on the fact that both the in vitro maintained, heparanase-negative as well as the in vitro passaged, heparanase-positive cells exhibited a similar membrane and molecular market profile.

Thrombin-induced release of active basic fibroblast growth factor-heparan sulfate complexes from subendothelial extracellular matrix.

The angiogenic factor, basic fibroblast growth factor (bFGF), is sequestered and protected by binding to heparan sulfate proteoglycans (HSPG) in the subendothelial extracellular matrix (ECM). Release of ECM-bound bFGF provides a novel mechanism for regulation of cell proliferation and neovascularization in normal and pathologic situations. Exposure of ECM to thrombin, the final activation product of the clotting cascade, resulted in release of high molecular weight HSPG-bFGF complex, as indicated by its immunoprecipitation with anti-bFGF antibodies, susceptibility to degradation by bacterial heparinase, and inhibition of its mitogenic activity in the presence of neutralizing anti-bFGF antibodies. The ECM-resident bFGF-HSPG complex was not released by thrombin in the presence of hirudin or antithrombin III, or by catalytically blocked thrombin preparations. A threefold to fivefold higher mitogenic activity was released by thrombin from ECM that was preheated (1 hour, 80 degrees C), as compared with native ECM. This difference is attributed to heat stable bFGF-HSPG complexes that are more readily released after heat treatment of the ECM and to activation and release of ECM-resident transforming growth factor-beta (TGF-beta) activity. Our results indicate that the large reservoir of proteolytic activity present in plasma in the form of prothrombin may participate in release from the subendothelial ECM of biologically active bFGF and TGF-beta, depending on the accessibility of thrombin. Thrombin may gain access to the subendothelium on clot formation after tissue injury and as a result of the conversion of prothrombin to thrombin induced by the ECM itself.

Extracellular matrix-resident growth factors and enzymes: possible involvement in tumor metastasis and angiogenesis.

Neoplastic cells require an appropriate pericellular environment and new formation of stroma and blood vessels in order to constitute a solid tumor. Tumor progression also involves degradation of various extracellular matrix (ECM) constituents. In this review we have focused on the possible involvement of ECM-resident growth factors and enzymes in neovascularization and cell invasion. We demonstrate that the pluripotent angiogenic factor, basic fibroblast growth factor (bFGF) is an ECM component required for supporting cell proliferation and differentiation. Basic FGF has been identified in the subendothelial ECM produced in vitro and in basement membranes of the cornea and blood vessels in vivo. Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell (EC) proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Our results indicate that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by cellular heparanase. We propose that restriction of bFGF bioavailability by binding to ECM and local regulation of its release, provides a novel mechanism for regulation of capillary blood vessel growth in normal and pathological situations. Heparanase activity correlates with the metastatic potential of various tumor cells and heparanase inhibiting molecules markedly reduce the incidence of lung metastasis in experimental animals. Heparanase may therefore participate in both tumor cell invasion and angiogenesis through degradation of the ECM-HS and mobilization of ECM-resident EC growth factors. The subendothelial ECM contains also tissue type- and urokinase type- plasminogen activators (PA), as well as PA inhibitor which may regulate cell invasion and tissue remodeling. Heparanase and the ECM-resident PA participate synergistically in sequential degradation of HS-proteoglycans in the ECM. These results together with similar observations on the properties of other ECM-immobilized enzymes and growth factors, suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized, regulated and persistent mode of action, as compared to the same molecules in a fluid phase.

Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix.

Bovine aortic and corneal endothelial cells synthesize a growth factor that remains mostly cell-associated but can also be extracted from the subendothelial extracellular matrix (ECM) deposited by these cells. The endothelial cell-derived growth factors extracted from cell lysates and from the extracellular matrix appear to be structurally related to basic fibroblast growth factor by the criteria that they bind to heparin-Sepharose and are eluted at 1.4-1.6 M NaCl, have a molecular weight of about 18,400, cross-react with anti-basic fibroblast growth factor antibodies when analyzed by electrophoretic blotting and immunoprecipitation, and are potent mitogens for bovine aortic and capillary endothelial cells. It is suggested that endothelium can store growth factors capable of autocrine growth promotion in two ways: by sequestering growth factor within the cell and by incorporating it into the underlying extracellular matrix.

Endogenous basic fibroblast growth factor displaced by heparin from the lumenal surface of human blood vessels is preferentially sequestered by injured regions of the vessel wall.

BACKGROUND: Proliferation of smooth muscle cells (SMCs) of the arterial wall in response to local injury is an important factor in vascular proliferative disorders. Among the growth factors that promote SMC proliferation is basic fibroblast growth factor (bFGF), which is characterized by a high affinity for heparin and is associated with heparan sulfate on cell surfaces and extracellular matrices. We investigated whether heparin can displace endogenous active bFGF from the lumenal surface of blood vessels, whether bFGF is preferentially bound to injured blood vessels, and whether a synthetic, polyanionic, heparin-mimicking compound (RG-13577) can prevent sequestration of bFGF by the vessel wall. METHODS AND RESULTS: Injured and noninjured saphenous vein segments were perfused with or without heparin, in the absence or presence of 125I-bFGF and/or RG-13577 (a polymer of 4-hydroxyphenoxy acetic acid). Heparin displaced bFGF from the lumenal surface of the vein, and the released bFGF stimulated proliferation of SMCs. Likewise, systemic administration of heparin during open heart surgery resulted in a marked increase in plasma bFGF levels. Injured veins sequestered 125I-bFGF to a much higher extent than noninjured vein segments, both in the absence and presence of heparin. This sequestration was inhibited by compound RG-13577. CONCLUSIONS: Despite its beneficial effects, heparin may displace active bFGF, which subsequently may be preferentially deposited on injured vessel walls, thus contributing to the pathogenesis of restenosis. This effect may be prevented by a synthetic heparin-mimicking compound.

Importance of size and sulfation of heparin in release of basic fibroblast growth factor from the vascular endothelium and extracellular matrix.

We have characterized the importance of size, sulfation, and anticoagulant activity of heparin in release of basic fibroblast growth factor (bFGF) from the subendothelial extracellular matrix (ECM) and the luminal surface of the vascular endothelium. For this purpose, 125I-bFGF was first incubated with ECM and confluent endothelial cell cultures, or administered as a bolus into the blood of rats, the immobilized 125I-bFGF was then subjected to release by various chemically modified species of heparin and size-homogeneous oligosaccharides derived from depolymerized heparin. Both totally desulfated and N-desulfated heparin failed to release the ECM-bound bFGF. Likewise, substitution of N-sulfate groups of heparin and low molecular weight heparin (fragmin) by acetyl or hexanoyl residues resulted in an almost complete inhibition of bFGF release by these polysaccharides. The presence of O-sulfate groups in heparin increased but was not critical for release of ECM-bound bFGF. Similar structural requirements were identified for release of 125I-bFGF bound to low-affinity sites on the surface of vascular endothelial cells. Oligosaccharides derived from depolymerized heparin and containing as little as 8-10 sugar units were, on a weight basis, equivalent to whole heparin in their ability to release bFGF from ECM. Low-sulfate oligosaccharides were less effective releasers of bFGF as compared to medium- and high-sulfate fractions of the same size oligosaccharides. Heparin fractions with high and low affinity to antithrombin III exhibited a similar high bFGF-releasing activity despite a 200-fold difference in their anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequential degradation of heparan sulfate in the subendothelial extracellular matrix by highly metastatic lymphoma cells.

A highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma elaborates a heparan sulfate (HS) degrading endoglycosidase (heparanase) to a much higher extent than its non-metastatic parental subline (Eb). Whereas a serum-free medium conditioned by either subline contained a trypsin-like serine protease, heparanase activity was detected only in the ESb-conditioned medium (CM). ESb CM was incubated with a naturally produced, sulfate-labelled subendothelial extracellular matrix (ECM) or with a soluble, high-MW labelled proteoglycan first released from the ECM by incubation with Eb CM or with the partially purified ESb protease. Sulfate labelled degradation products were analyzed by gel filtration on Sephrose 6B. The optimal pH for degradation of ECM-bound HS was 6.2 as compared to pH 5.2 for degradation of the soluble proteoglycan. Heparanase-mediated degradation of both ECM-bound and soluble HS was inhibited by heparin. Addition of either trypsin, plasmin or to a lower extent, the purified ESb protease, stimulated between 5- and 20-fold the ESb CM-mediated degradation of ECM-bound HS but had no effect on heparanase-mediated degradation of the soluble proteoglycan. This stimulation was inhibited in the presence of heparin or protease inhibitors. These results indicate that both a protease and heparanase are involved in the ESb-mediated degradation of ECM-bound HS and that one enzyme produces a more accessible substrate for the next enzyme. This sequential cleavage is characteristic of degradation of a multimolecular structure such as the subendothelial ECM and hence cannot be detected in studies with its isolated constituents.