Despite the ubiquitous presence of basic fibroblast growth factor (bFGF) in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues, suggesting that the extracellular matrix (ECM) may serve as a reservoir for bFGF. Moreover, functional studies indicated that bFGF is an ECM component required for supporting endothelial cell proliferation and neuronal differentiation. We have found 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 heparanase expressed by normal and malignant cells (i.e. platelets, neutrophils, lymphoma cells). It is proposed that restriction of bFGF bioavailability by binding to ECM and local regulation of its release provide a novel mechanism for neovascularization in normal and pathological situations. The subendothelial ECM contains also tissue type- and urokinase type-plasminogen activators which participate in cell invasion and tissue remodeling. These results and studies on the properties of other ECM-immobilized enzymes (i.e. thrombin, plasmin, lipoprotein lipase) and growth factors (GM-CSF, IL-3, osteogenin), suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized and persistent mode of action, as compared to the same molecules in a fluid phase.
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.
SnmlTlal'yMigration 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-B-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.ukocyte-mediated inflammatory reactions and immune surveillance require the activation ofleukocytes by specific antigens, cytokines, and chemokines, and the ensuing extravasation of circulating immune cells from blood vessels to sites of inflammation (1-3). The recognition and the subsequent interactions of the infiltrating immune cells, such as CD4 + T lymphocytes, with glycoproteins of the subendothelial basement membrane and extracellular matrix (ECM) is mediated primarily by integrin receptors of the/~1 subfamily (1). In addition, the physiologically programmed migration of T cells is associated with the secretion of various matrix-degrading enzymes, such as endo-fl-D glucuronidase (heparanase) (4-6). Naive CD4 § T lymphocytes respond to activation by synthesizing heparanase de novo, whereas memory T cells release heparanase from preformed stores within minutes of contact with antigen (4). The cell surface-, basement membrane-, or ECM-associated heparan sulfate (HS) proteoglycans, which serve as substrates for the catalytic activity of heparanase, determine the self-assembly and insolubility of the ECM, stabilize the matrix structure, and maintain the matrix integrity by interacting with ECM glycoproteins (4). The HS molecules also affect the proliferation of immune cells (7) and bind growth factors and cytokines (8, 9). Herein, we examined whether the performance of heparanase is affected by physiological alterations in the environment, specifically, whether in addition to displaying an enzymatic activity, heparanase can also regulate the accumulation ofT cells in inflamed tissues. Materials and MethodsIsolation and Purification of Mammalian Heparanase. Heparanase was purified from human placenta by a modification of a previously described method (10). Briefly, human placentas were homogenized and suspended in 10 mM phosphate-citrate buffer (PCB; pH 6.0), sonicated, and centrifuged (3000 g, 4~ 15 min). Proteins were precipitated with 35-55% AmSO4, resuspended in 10 mM PCB containing 0.1 M NaCl, and dialyzed against the same buffer. The dialyzed material was then subjected to cation exchange chromatogr...
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)
Zinc(II) accumulated by platelets has profound effects on platelet activity. This study is focused on the distribution of Zn(II) between human platelet subcellular compartments. After incubation with 86Rb+ and platelet lysis, the organelles were separated by sucrose density gradient centrifugation. Fibrinogen served as a marker for alpha-granules. 86Rb+ and factor XIII served as markers for the cytoplasmic fractions. Zn(II) was found to be distributed between the cytoplasm and the alpha-granules, with variations between different individual units. The total platelet Zn concentration and its relative subcellular distribution were dependent on its extracellular level. Incubation of platelets with 100 microM Zn(II) resulted in a twofold increase of its level in the cytoplasm and by one order of magnitude in the alpha-granules. In addition to the anticipated factor XIII activity in the cytoplasmic pool fraction, we found thrombin-inducible factor XIII activity within the alpha-granules. Immunoblotting confirmed the presence of both the a and b subunits of plasma factor XIII (a2b2 form) in the alpha-granules. As fibrinogen is not synthesized in the platelet, we propose that by virtue of their mutual binding, fibrinogen, Zn(II) and plasma factor XIII-a2b2 are simultaneously taken up into the alpha-granules by endocytosis, presumably through the vehicle of the GPIIb/IIIa fibrinogen receptor. A rationale for co-packaging these components within the alpha-granules is that Zn(II) inhibits factor XIII activity and thereby prevents the premature cross-linking of the concentrated fibrinogen prior to platelet activation and secretion. By contrast, cytoplasmic Zn(II) may increase platelet responsiveness to agonists due to its interaction with cytoplasmic modulators of platelet activity.
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