Extracellular Matrix Heparan Sulfate Research Articles

Brain metastases in melanoma: roles of neurotrophins.

Brain metastasis, which occurs in 20% to 40% of all cancer patients, is an important cause of neoplastic morbidity and mortality. Successful invasion into the brain by tumor cells must include attachment to microvessel endothelial cells, penetration through the blood-brain barrier, and, of relevance, a response to brain survival and growth factors. Neurotrophins (NTs) are important in brain-invasive steps. Human melanoma cell lines express low-affinity NT receptor p75NTR in relation to their brain-metastatic propensity with their invasive properties being regulated by NGF, or nerve growth factor, the prototypic NT. They also express functional TrkC, the putative receptor for the invasion-promoting NT-3. In brain-metastatic melanoma cells, NTs promote invasion by enhancing the production of extracellular matrix (ECM)-degradative enzymes such as heparanase, an enzyme capable of locally destroying both ECM and the basement membrane of the blood-brain barrier. Heparanase is an endo-beta-d-glucuronidase that cleaves heparan sulfate (HS) chains of ECM HS proteoglycans, and it is a unique metastatic determinant because it is the dominant mammalian HS degradative enzyme. Brain-metastatic melanoma cells also produce autocrine/paracrine factors that influence their growth, invasion, and survival in the brain. Synthesis of these factors may serve to regulate NT production by brain cells adjacent to the neoplastic invasion front, such as astrocytes. Increased NT levels have been observed in tumor-adjacent tissues at the invasion front of human brain melanoma. Additionally, astrocytes may contribute to the brain-metastatic specificity of melanoma cells by producing NT-regulated heparanase. Trophic, autocrine, and paracrine growth factors may therefore determine whether metastatic cells can successfully invade, colonize, and grow in the CNS.

Cell surface localization of heparanase on macrophages regulates degradation of extracellular matrix heparan sulfate.

Extravasation of peripheral blood monocytes through vascular basement membranes requires degradation of extracellular matrix components including heparan sulfate proteoglycans (HSPGs). Heparanase, the heparan sulfate-specific endo-beta-glucuronidase, has previously been shown to be a key enzyme in melanoma invasion, yet its involvement in monocyte extravasation has not been elucidated. We examined a potential regulatory mechanism of heparanase in HSPG degradation and transmigration through basement membranes in leukocyte trafficking using human promonocytic leukemia U937 and THP-1 cells. PMA-treated cells were shown to degrade 35S-sulfated HSPG in endothelial extracellular matrix into fragments of an approximate molecular mass of 5 kDa. This was not found with untreated cells. The gene expression levels of heparanase or the enzyme activity of the amount of cell lysates were no different between untreated and treated cells. Immunocytochemical staining with anti-heparanase mAb revealed pericellular distribution of heparanase in PMA-treated cells but not in untreated cells. Cell surface heparanase capped into a restricted area on PMA-treated cells when they were allowed to adhere. Addition of a chemoattractant fMLP induced polarization of the PMA-treated cells and heparanase redistribution at the leading edge of migration. Therefore a major regulatory process of heparanase activity in the cells seems to be surface expression and capping of the enzyme. Addition of the anti-heparanase Ab significantly inhibited enzymatic activity and transmigration of the PMA-treated cells, suggesting that the cell surface redistribution of heparanase is involved in monocyte extravasation through basement membranes.

Heparanase Degrades Syndecan-1 and Perlecan Heparan Sulfate: FUNCTIONAL IMPLICATIONS FOR TUMOR CELL INVASION

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.

Inhibition of heparanase activity and heparanase-induced angiogenesis by suramin analogues.

Heparanase, a heparan sulfate-specific endo-beta-D-glucuronidase, plays an important role in tumor cell metastasis through the degradation of extracellular matrix heparan sulfate proteoglycans (ECM HSPG). Heparanase activity correlates with the metastatic propensity of tumor cells. Suramin, a polysulfonated naphthylurea, is an inhibitor of heparanase with suramin analogues shown to possess antiangiogenic and antiproliferative properties. We investigated the effects of selected suramin analogues (NF 127, NF 145 and NF 171) on heparanase activity and heparanase-driven angiogenesis. Studies of the ability of cellular extracts and purified heparanase from human, highly invasive and brain-metastatic melanoma (70W) cells revealed that heparanase expressed by these cells was effectively inhibited by suramin analogues in a dose-dependent manner. These analogues possessed more potent heparanase inhibitory activities than suramin: The concentrations required for 50% heparanase inhibition (IC(50)) were 20-30 microM, or at least 2 times lower than that for suramin. One hundred percent inhibition was observed at concentrations of 100 microM and higher. Of relevance, these compounds significantly decreased (i) the invasive capacity of human 70W cells by chemoinvasion assays performed with filters coated with purified HSPG or Matrigel trade mark, and (ii) blood vessel formation by in vivo angiogenic assays, thus linking their antiangiogenic properties with impedance of heparanase-induced angiogenesis. Specifically, inhibition of invasion by NF 127, NF 145 and NF 171 was found at 10 microM concentrations of compounds with a significant decrease of invasive values at concentrations as low as 1.5 microM. In addition, NF 127, NF 145 and NF 171 promoted nearly complete inhibition of heparanase-induced angiogenesis at values ranging from 236 microM (for NF 145) to 362 microM (for NF 127). These results further emphasize the importance of heparanase in invasive and angiogenic mechanisms and the potential clinical application of heparanase inhibitors such as suramin analogues in cancers and angiogenesis-dependent diseases.

The role of extracellular matrix heparan sulfate glycosaminoglycan in the activation of growth factor signaling pathways.

The role of extracellular matrix heparan sulfate glycosaminoglycan in the activation of growth factor signaling pathways.

Agrin Binds to β-Amyloid (Aβ), Accelerates Aβ Fibril Formation, and Is Localized to Aβ Deposits in Alzheimer's Disease Brain

Agrin is an extracellular matrix heparan sulfate proteoglycan (HSPG) well known for its role in modulation of the neuromuscular junction during development. Although agrin is one of the major HSPGs of the brain, its function there remains elusive. Here we provide evidence suggesting a possible function for agrin in Alzheimer's disease brain. Agrin protein binds the amyloidogenic peptide Aβ (1–40) in its fibrillar state via a mechanism that involves the heparan sulfate glycosaminoglycan chains of agrin. Furthermore, agrin is able to accelerate Aβ fibril formation and protect Aβ (1–40) from proteolysis, in vitro. Supporting a biological significance for these in vitro data, immunocytochemical studies demonstrate agrin's presence within senile plaques and cerebrovascular amyloid deposits, and agrin immunostained capillaries exhibit pathological alterations in AD brain. These data therefore suggest that agrin may be an important factor in the progression of Aβ peptide aggregation and/or its persistence in Alzheimer's disease brain.

Inhibition of heparanase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate oligodeoxynucleotides.

Heparanase activity correlates with the metastatic potential of tumor cells. Moreover, the anti-metastatic effect of non-anti-coagulant species of heparin and certain sulfated polysaccharides was attributed to their heparanase-inhibiting activity. We investigated the effect of a chemically sulfated polysaccharide (laminarin), consisting primarily of beta-1,3 glucan (sodium laminarin), and of synthetic phosphorothioate oligodeoxynucleotides, primarily phosphorothioate homopolymer of cytidine (SdC28), on heparanase activity and tumor metastasis. Investigation of the ability of tumor cells to degrade heparan sulfate in intact extracellular matrix revealed that heparanase activity expressed by B16-BL6 mouse melanoma cells and 13762 MAT rat mammary adenocarcinoma cells was effectively inhibited by LS (50% inhibition at 0.2-1 microgram/ml), but there was no inhibition by sodium laminarin up to a concentration of 50 microgram/ml. Complete inhibition of the melanoma heparanase was obtained in the presence of 0.1 microM SdC28. A single i.p. injection of laminarin sulfate, but not of sodium laminarin, before i.v. inoculation of the melanoma or breast-carcinoma cells inhibited the extent of lung colonization by the tumor cells by 80 to 90%. Similar inhibition was exerted by 0.1 microM SdC28. At the effective concentrations, both compounds had a small effect on proliferation of the tumor cells and on growth of the primary tumors in vivo. These results further emphasize the involvement of heparanase in tumor metastasis and the potential clinical application of diverse heparanase-inhibiting molecules such as sulfated polysaccharides and synthetic polyanionic molecules.

Extracellular matrix heparan sulfate modulates endothelial cell susceptibility to Staphylococcus aureus.

The ability of extracellular matrix heparan sulfate to alter the susceptibility of human endothelial cells to S. aureus was investigated. Endothelial cells grown on extracellular matrix synthesized by S. aureus-infected endothelial cells were more susceptible to subsequent staphylococcal infection than endothelial cells grown on the extracellular matrix synthesized by untreated endothelial cells. Endothelial cells were more susceptible to S. aureus infection when 1) grown on heparitinase-treated extracellular matrix that removed heparan sulfate chains, 2) grown on extracellular matrix produced by chlorate-treated endothelial cells that reduced sulfation in the matrix heparan sulfate proteoglycans, 3) grown on heparan sulfate purified from extracellular matrix elaborated by infected endothelial cells, and 4) endothelial cells were chlorate-treated and therefore expressed desulfated cellular heparan sulfate proteoglycans. Extracellular matrix produced by S. aureus-infected endothelial cells contained heparan sulfate proteoglycans with reduced sulfation. The altered extracellular matrix with reduced sulfated heparan sulfate proteoglycans signalled the uninfected endothelial cells to produce under sulfated cellular heparan sulfate proteoglycans that increased S. aureus adherence to the endothelial cells.

Distribution and substrate properties of agrin, a heparan sulfate proteoglycan of developing axonal pathways

The distribution and substrate properties of agrin, an extracellular matrix heparan sulfate proteoglycan (HSPG), was investigated in the developing chick nervous system by immunocytochemistry, Western blotting, and in neurite outgrowth assays. By comparing the distribution of agrin with that of laminin-1, merosin (laminin-2), neurofilament, and neural cell adhesion molecule (NCAM), it was found that throughout development, agrin is a constituent of all basal laminae. From embryonic day (E) 4 onwards, agrin is also abundant in axonal pathways of the central nervous system, such as the optic nerve, the tectobulbar pathway, the white matter of the spinal cord, and the marginal and the molecular layers of the forebrain and the cerebellum. The abundance of agrin in brain decreases from E13 onwards. In the peripheral nervous system, agrin is present throughout development as a constituent of the Schwann cell basal laminae. Western blots confirmed the immunocytochemical data, showing maximum expression of agrin occurs during the early to medium stages of brain development. Western blots also showed that in mouse and human brain, agrin exists as an HSPG. Purified agrin did not support neurite outgrowth, rather it inhibited retinal neurite extension on mixed agrin/merosin substrates. Despite the fact that agrin, when used as a substrate inhibited neurite outgrowth, its temporal and spatial overlap with growing axons suggests that agrin has a supportive role in the development of axonal pathways, possibly as a binding component for growth factors and cell adhesion proteins.

Heparan sulfate-binding peptide promotes the deposition of proteoglycans in the extracellular matrix.

A synthetic peptide, which was shown to bind extracellular matrix heparan sulfate chains with a high degree of affinity and specificity [Colburn et al. (1996): Arch Biochem Biophys 325:129-138], has now been found to promote the transfer and the deposition of endothelial cell surface proteoglycans in the extracellular matrix. The peptide also induces preferential binding of extracellular matrix heparan sulfate proteoglycans, which have been added to the supernatant growth medium, and the requirement for its presence is stringent in that only a negligible amount of proteoglycans are bound to the cell layer in the absence of the peptide. In addition, antibodies directed against the peptide detect the accumulation of the peptide in the matrix compartment where the peptide is found associated with the proteoglycans transferred from the cell surface. The sequence of events induced by the peptide appears to be an extension of a naturally occurring process since proteoglycans with properties similar to those of the species ordinarily present in the extracellular matrix have been observed to transfer from the cell surface to the matrix during a pulse-chase experiment. We suggest that formation of the complex peptide-proteoglycan with consequent displacement of the proteoglycan from its anchorage on the cell initiates the process of transfer of the heparan sulfate-bound peptide from the cell surface to the extracellular matrix.

Heparanase and a synthetic peptide of heparan sulfate-interacting protein recognize common sites on cell surface and extracellular matrix heparan sulfate.

Heparanase is an endo-beta-D-glucuronidase that degrades the glycosaminoglycan chains of heparan sulfate (HS) proteoglycans at specific sites. Elevated levels of heparanase are associated with the metastatic potential of melanoma and other types of tumor cells. We previously reported heparanase degradation of cell surface HS subpopulations of the human adenocarcinoma cell line RL95. In the present study, heparanase activity was examined on RL95 cell surface HS subpopulations in the presence of a synthetic peptide (CRPKAKAKAKAKDQTK) of heparin/heparan sulfate-interacting protein (HIP; Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817-11823). Heparanase digestion generated HS fragments from cell surface- or extracellular matrix-derived HS of approximately 25 and 9 kDa, respectively. In contrast, HS of various size classes isolated from proteoglycans secreted or released by RL95 and endothelial cells in culture were not susceptible to heparanase digestion. Incubation of heparanase-containing melanoma cellular extracts or partially purified heparanase preparations with cell surface- or ECM-derived HS and HIP peptide, but not a scrambled sequence of this peptide or other HS-binding proteins present in ECM, completely inhibited heparanase action. Conversely, predigestion of cell surface HS with either heparanase-containing cellular extracts or with secreted or partially purified heparanase destroyed binding to HIP peptide. Preincubation of HS with HIP peptide prevented subsequent heparanase digestion. Collectively, these data demonstrate that HIP peptide and heparanase recognize specific, common motifs within HS chains at cell surfaces and in ECM and may mutually modulate HS-dependent activities.

Vascular smooth muscle cell phenotype influences glycosaminoglycan composition and growth effects of extracellular matrix.

Rat neonatal and neointimal vascular smooth muscle cells differ dramatically from adult medical vascular smooth muscle cells in their growth properties, with the neonatal and neointimal cells exhibiting growth in the absence of exogenously added growth factors. Since it has been hypothesized that extra-cellular matrix proteoglycans may influence the growth and differentiation of vascular smooth muscle cells, we examined the ability of matrix derived from these cells to influence vascular smooth muscle cell proliferation. To produce test matrices, cells were grown to confluence and removed by brief alkali treatment. Test cells were seeded onto these matrices and the rates of growth in a growth-factor-deficient medium determined. Compared to plastic wells, matrix from neonatal or neointimal cells stimulated the growth of vascular smooth muscle cells. Interestingly, matrix from adult cells was less efficient at promoting growth. Enzymatic digestion of extracellular matrix heparan sulfate, but not of other glycosaminoglycans, further increased the growth-stimulatory effect of extracellular matrix, suggesting that matrix heparan sulfate acts as a growth inhibitor. Consistent with this, biochemical analysis showed that the adult matrix contained a higher percentage of heparan sulfate compared with neonatal or neointimal matrix. These results suggest that autocrine production of heparan sulfate proteoglycans may play an important role in growth regulation of vascular smooth muscle cells during normal vascular development and differentiation as well as in pathological response to injury.

Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis

A survey of defined species of cell surface and extracellular matrix heparan sulfate proteoglycans (HSPG) was performed in a search for cellular proteoglycans that can promote bFGF receptor binding and biological activity. Of the various affinity-purified HSPGs tested, perlecan, the large basement membrane HSPG, is found to induce high affinity binding of bFGF both to cells deficient in HS and to soluble FGF receptors. Heparin-dependent mitogenic activity of bFGF is strongly augmented by perlecan. Monoclonal antibodies to perlecan extract the receptor binding promoting activity from active HSPG preparations. In a rabbit ear model for in vivo angiogenesis, perlecan is a potent inducer of bFGF-mediated neovascularization. These results identify perlecan as a major candidate for a bFGF low affinity, accessory receptor and an angiogenic modulator.

Differential structural requirements of heparin and heparan sulfate proteoglycans that promote binding of basic fibroblast growth factor to its receptor.

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). The capacity of various species of heparin and heparan sulfate (HS) to promote bFGF receptor binding was investigated using both Chinese hamster ovary mutant cells deficient in cell surface HSPG and a soluble bFGF receptor-alkaline phosphatase fusion protein. Highly sulfated oligosaccharides were more effective than medium and low sulfate fractions of the same size oligosaccharide. O-Sulfation in heparin was found to be critical for its capacity to promote binding of bFGF to its receptors. The highest level of bFGF-receptor binding was achieved in the presence of over-sulfated heparin fragments (% sulfur > 14) regardless of whether the N-position was sulfated or acetylated. Unlike receptor binding of bFGF which requires oligosaccharides containing at least 8-10 sugar units, displacement of heparin- or HS-bound bFGF was obtained by oligosaccharides containing as little as four sugar units and by an N-sulfated, O-desulfated heparin fragment (% sulfur = 5.3). A preparation of total cell surface-derived HS induced bFGF receptor binding. A preliminary survey of several defined and affinity purified species of cell surface HSPG, including syndecan, fibroglycan, and glypican failed to identify natural HSPG that promote high affinity receptor binding of bFGF. A similar lack of activity was observed with species of HS isolated from bovine arterial tissue and characterized for their effect on vascular smooth muscle cell proliferation. Moreover, most of these species of HS inhibited in a dose-dependent manner the restoration of bFGF-receptor binding induced by heparin or by total HSPG. These results suggest the involvement of defined heparin-like oligosaccharide sequences and unique species of cell surface and extracellular matrix HS in the regulation of bFGF receptor binding and biological activity.

Thrombin enhances degradation of heparan sulfate in the extracellular matrix by tumor cell heparanase

The ability of normal and malignant blood-borne cells to extravasate correlates with the activity of an endo-β- d-glucuronidase (heparanase) which degrades heparan sulfate (HS) in the subendothelial extracellular matrix (ECM). The association of malignancy with different types of coagulopathies prompted us to study the effect of thrombin (EC 3.4.21.5), a serine protease elaborated during activation of the clotting cascade, on the ability of heparanase to degrade the ECM-HS. The circulating zymogen form of thrombin, prothrombin, was converted to proteolytically active thrombin during incubation with ECM. Thrombin generation by the ECM was time and dose dependent, reaching maximal conversion by 6 h incubation at 3 U/ml of prothrombin. Heparanase-mediated release of low M r HS cleavage products from sulfate-labeled ECM was stimulated four- to sixfold in the presence of α-thrombin, but there was no effect on degradation of soluble HS. Similar results were obtained with heparanase preparations derived from mouse lymphoma and human hepatoma cell lines and from human placenta. Incubation of ECM with α-thrombin alone resulted in release of nearly intact high- M r labeled proteoglycans. Thrombin stimulation of heparanase action was dose and time dependent, reaching a maximal value at 24 h incubation with 1 μ M α-thrombin. The effect of modified thrombin preparations correlated with their proteolytic activity. Catalytically blocked preparations of thrombin (e.g., DIP-α-thrombin, MeSO 2-α-thrombin) failed to facilitate heparanase action, while catalytically modified preparations (e.g., γ-thrombin, NO 2-α-thrombin) exerted only a slight enhancement. Antithrombin III (ATIII) and hirudin both inhibited thrombin-stimulated heparanase degradation of ECM-bound HS. Heparanase action was also facilitated by ECM-immobilized thrombin to an extent which was similar to that induced by soluble thrombin. This result implies that thrombin sequestered by the subendothelial ECM and protected from interaction with its natural inhibitor ATIII (Bar-Shavit et al., 1989, J. Clin. Invest. 84, 1096–1104) may participate locally in cellular invasion during tumor metastasis, inflammation, and autoimmunity.

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells.

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.

Degranulating mast cells secrete an endoglycosidase that degrades heparan sulfate in subendothelial extracellular matrix

Mast cells are widely distributed in perivascular connective tissues, especially in areas of active tumor growth and vascular reactivity. Incubation of metabolically [35S]O4 = -labeled subendothelial extracellular matrix (ECM) with lysates of bone marrow-derived mouse mast cells (BMMC) resulted in extensive degradation of heparan sulfate (HS) into fragments 5 to 6 times smaller than intact HS side chains. A much lower activity (seven- to eightfold) was expressed by intact BMMC incubated in contact with the ECM. These fragments were not produced in the presence of heparin, were sensitive to deamination with nitrous acid, and resistant to further degradation with papain or chondroitinase ABC. These results indicate that an endoglycosidase (heparanase) is involved in BMMC-mediated degradation of HS in the subendothelial ECM. Heparanase activity was not detected in medium conditioned by cultured BMMC, or in lysates of Ableson transformed BMMC and rat basophilic leukemic (RBL) cells. Both heparanase and beta- hexosaminidase, a mast cell granule enzyme, were released on degranulation of BMMC induced by the calcium ionophore A23187, or by exposure to IgE-Ag, suggesting that heparanase is localized in the cell granules. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase were released. Degradation of the ECM-HS by the mast cell heparanase and the associated release of HS-bound endothelial cell growth factors that are stored in ECM (Vlodavsky et al, Proc Natl Acad Sci USA 84:2292, 1987; Bashkin et al, Biochemistry 28:1737, 1989) may play a role in the proposed mast cell-mediated stimulation of neovascularization.

Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor

Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 35S-HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 35S-HSPG deposited into ECM do not bind aFGF. The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCl. aFGF-binding and aFGF-nonbinding 35S-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography. 35S-HSPG which bind aFGF, designated HSPGP, were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, 35S-HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF-potentiating HSPGP and aFGF-inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively. 3H-Core protein derived from HSPGP or HSPG1 contained only minor biological activity. The ability of heparitinase or heparinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.

Differentiating human leukemia cells express heparanase that degrades heparan sulfate in subendothelial extracellular matrix

Human promyelocytic (HL-60) and monoblast-like (U-937) leukemia cell lines were tested for expression of an endoglycosidase (heparanase) capable of degrading heparan sulfate (HS) side chains in the subendothelial extracellular matrix (ECM). Heparanase activity has been previously shown to be expressed by activated lymphocytes and macrophages and by highly metastatic tumor cells, in correlation with their ability to invade blood vessels and extracellular matrices. Incubation of HL-60 and U-937 cells with sulfate-labeled ECM in the presence of 12- O-tetradecanoyl-phorbol-13-acetate (TPA) resulted in heparanase-mediated release of heparan sulfate degradation products. This degradation was inhibited by heparin, stimulated by plasminogen and not expressed by cells treated with retinoic acid or dimethylsulfoxide and undergoing neutrophilic differentiation. Heparanase activity was not detected in media conditioned by HL-60 and U-937 cells but was found in their cell lysates, regardless of whether or not the cells were exposed to TPA. These findings imply that TPA-induced differentiation of human myeloid leukemic cells to macrophage-like cells, but not to neutrophilic granulocytes, is associated with expression on the cell surface of a preformed heparanase activity. The enzyme may serve as a marker for human cell differentiation into macrophages, allowing the differentiating cells to traverse the vascular compartment and reach their target sites.

Subendothelial extracellular-matrix heparan sulfate proteoglycan-degrading activity of human monocyte macrophages.

At the early stage of atherogenesis, circulating monocyte macrophages appear to adhere to the endothelial cell surface and migrate subendothelially to become foam cells. The mechanism of these macrophage-endothelial cell interactions was investigated. Adherent macrophages isolated from human blood were plated on [35S]O4-prelabeled extracellular matrix-coated dishes prepared from cultured porcine aortic endothelial cells. During incubation for 2-3 days at pH 7.4 either in the presence or absence of serum, macrophages solubilized the labeled extracellular matrix to a lower molecular weight component (Kav approximately equal to 0.5) than the materials (Kav = 0) released into the medium containing no cells. The degrading activity was not stored intracellularly but instead was found pericellularly, requiring continuous cell-matrix contact. Heparin (10 micrograms/ml) inhibited this degrading activity of macrophages. Degradation products were precipitated with cetylpyridinium chloride and were resistant to further digestion with alkali, pronase, or chondroitinase ABC, but were converted to further lower molecular weight fragments (Kav = 0.84) after nitrous acid digestion or heparitinase treatment. The intact glycosaminoglycan side chains determined by subjecting the extracellular matrix to cleavage with alkali or pronase were larger (Kav congruent to 0.20) than those of degradation products released by macrophages. These results suggest that the attachment and subsequent invasion of endothelial cells by monocyte macrophages may involve the production of extracellular-matrix heparan sulfate proteoglycan-degrading activity by these cells.