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Biomedical subjects

I Vlodavsky

Publications and source records attributed to I Vlodavsky.

At least 109 records · Page 6Linked to original sources
PubMed1991

Production of heparanase by normal and neoplastic murine B-lymphocytes.

The production of heparanase, an endoglycosidase capable of degrading heparan sulfate from the subendothelial extracellular matrix (ECM), was investigated in various murine B-lymphoid tumors representing distinct maturation stages of the B-cell lineage. We found that heparanase is produced and released by 3 out of 4 pre-B lymphomas and by 4 B lymphomas examined. In contrast, 5 plasmacytomas and resting normal B lymphocytes, expressed little, if any, heparanase activity. Treatment with LPS resulted in high expression of the enzyme by normal B-lymphocytes, but there was no effect on the constitutive production of heparanase by myeloma or B-lymphoma cells. Our results indicate that heparanase is produced by B cells during discrete stages of their maturation. We suggest that heparanase may play a role in B-cell migration by enabling pre-B and B lymphocytes to leave the bone-marrow compartment and recirculate among peripheral lymphoid organs.

Animals↗
PubMed1991

Coordinate secretion and functional synergism of T cell-associated serine proteinase-1 (MTSP-1) and endoglycosidase(s) of activated T cells.

Cell lysates and exocytosed soluble mediator(s) (ESM) released from CD8+ T cell lines (TCL) by receptor-triggered secretory exocytosis were tested for degradation of proteoglycans associated with in vitro produced subendothelial extracellular matrix (ECM). ESM was found to release low-molecular weight (kav 0.5-0.6) fragments from the sulfated proteoglycans in ECM. In the presence of heparin, an inhibitor for endoglycosidase activity, only high-molecular-weight products (kav 0.2) were formed. Preincubation of ESM with HD-prolylphenylalanyl-arginyl-chloromethylketone (PFR-CK) an inhibitor for the T cell-associated serine proteinase-1 (MTSP-1) totally prevented release of high- and low-molecular weight proteoglycan fragments. Furthermore, it was shown that purified MTSP-1 is able to release from ECM high-molecular weight proteoglycans and that this process is inhibitable by PFR-CK but not by heparin. Further treatment of these soluble high-molecular weight sulfated proteoglycans with ESM from TCL 1.D9 led to appearance of low-molecular weight split products (kav 0.5-0.6). This conversion was inhibitable by heparin but not by PFR-CK. These findings indicate that activated T cells contain two enzymatic activities, i.e. MTSP-1 and at least one endoglycosidase, which after receptor-triggered secretion can synergize in the degradation of sulfated proteoglycans in subendothelial ECM.

Animals↗
PubMed1991

Extracellular matrix-resident basic fibroblast growth factor: implication for the control of angiogenesis.

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.

Animals↗
PubMed1991

Heparanase activity in cultured endothelial cells.

This study was undertaken to identify a heparan sulfate (HS) degradation endoglycosidase (heparanase) in cultured endothelial cells (EC) and to characterize the requirements for its release and subsequent degradation of HS side chains in the subendothelial extracellular matrix (ECM). Intact EC, EC lysates, or EC conditioned media from different sources were incubated with metabolically Na2(35)SO4-labeled ECM produced by bovine EC. The released sulfated products were analyzed by gel filtration on Sepharose 6B. Human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) lysates expressed heparanase activity as indicated by release of most of the radioactivity from ECM as HS fragments that are one-fifth to one-sixth the size of the intact HS side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin. Rabbit coronary microvascular EC and bovine brain capillary EC lysates showed less heparanase activity (30-35%), whereas bovine aortic and corneal EC showed no activity. Intact HUVEC, plated directly on the labeled ECM, expressed low enzyme activity that was not changed when cells were exposed to various agents. Exposure of HUVEC to interleukin-1, phorbol myristate acetate, tumor necrosis factor, endotoxin, thrombin, calcium ionophore A23187, fibroblast growth factor, or radiation did not induce release of the enzyme to the medium or degradation of HS in the ECM, as long as the cells remained viable. EC differ from various normal and malignant cells that degrade HS by virtue of their inability to release the enzyme. We suggest that heparanase release during vessel wall injury may regulate the growth of EC and smooth muscle by release of HS degradation products in processes such as wound healing, neovascularization, and atherosclerosis.

Animals↗
PubMed1991

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.

Capillaries↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

The effect of flow on the interaction of isolated megakaryocytes with subendothelial extracellular matrix.

We have previously shown that human, guinea pig, or rat megakaryocytes, incubated under static conditions on an extracellular matrix (ECM) produced by endothelial cells, readily adhered to the matrix and underwent platelet-like shape change and thromboxane A2 secretion. We have now exposed megakaryocytes to ECM in a perfusion system similar to that used to study platelets circulated over aortic subendothelium. We used a continuous flow circuit incorporating a parallel plate perfusion chamber. Megakaryocytes were isolated to high purity from guinea pig marrow by centrifugal elutriation and velocity sedimentation. The cells were introduced into the flowing medium while the surface of an ECM-coated coverslip mounted in the chamber was observed continuously by phase-contrast video microscopy for up to 18 hours. Megakaryocytes from the flowing suspension started to adhere to the ECM within seconds. Significant adhesion occurred over a range of shear rates, from 10 to 190 seconds-1, did not appear above 300 seconds-1 and was greatest at a shear rate of 60 seconds-1. Adhesion to the ECM was specific, since there was no adherence to glass coverslips, glutaraldehyde-fixed ECM-coated coverslips, or to endothelial cells cultured on ECM-coated coverslips. At low shear rates large aggregates of megakaryocytes formed on the ECM surface; these could be detached and washed away by higher shear forces. Megakaryocytes thus acquire, even before platelet formation, an adhesive capacity similar to that of platelets. In addition, a significant fraction of the adherent megakaryocytes underwent elongation and pseudopod formation similar to that seen in marrow sinusoids.

Animals↗
PubMed1990

Maintenance on extracellular matrix and expression of heparanase activity by human ovarian carcinoma cells from biopsy specimens.

A routine procedure has been developed for the isolation and maintenance in culture of human ovarian carcinoma cells derived from biopsy specimens. Cell attachment, plating efficiency and initial outgrowth were greatly improved by seeding the cells on a basement-membrane-like extracellular matrix (ECM) deposited by cultured corneal endothelial cells. These effects were most significant in serum-free conditions which markedly reduced the rate of cell attachment and growth on regular tissue culture plastic. In 60-80% of the cases and regardless of the patient's age, cells cultured on ECM in the absence of serum divided actively and formed a tightly packed epithelial cell monolayer. Fibroblast overgrowth and cell detachment often occurred on ECM in the presence of serum. Incubation of the human ovarian carcinoma cells with sulfate-labelled ECM, resulted in the release of heparan sulfate degradation fragments, 4- to 7-fold smaller than intact heparan sulfate side chains. This degradation was brought about by endoglycosidase (heparanase) activity expressed to a higher extent by cells that were first maintained in primary cultures as compared with cell aggregates taken directly from the biopsy specimen. In most cases, cells derived from metastatic tumors expressed a higher heparanase activity than cells from the primary ovarian tumor. This result corroborates previous studies, performed with cell lines, on the possible involvement of heparanase in tumor cell invasion and metastasis.

Autoradiography↗
PubMed1990

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.

Animals↗
PubMed1990

Interaction of lipoprotein lipase with subendothelial extracellular matrix.

We have analyzed the binding of lipoprotein lipase (LPL) to the subendothelial extracellular matrix produced by cultured endothelial cells. Binding was linear up to a concentration of 0.5 microgram/ml (10 nM) enzyme used in this study, and equilibrium was achieved after 2 h of incubation with bovine 125I-LPL at 4 degrees C. Heparin and heparan sulfate effectively inhibited the binding of LPL to extracellular-matrix-coated plates; chondroitin sulfate had no effect, while high concentrations of dermatan sulfate or keratan sulfate inhibited binding of LPL to extracellular matrix by only 40%. Basic fibroblast growth factor (bFGF) did not affect LPL binding, while antithrombin-III (AT-III) caused up to a 50% inhibition of enzyme binding to extracellular matrix. alpha-Thrombin. 5.10(-6) M, and its esterolytically inactive derivative, DIP-alpha-thrombin, effectively inhibited binding of LPL to extracellular-matrix-coated plates. alpha-Thrombin was also able to release the extracellular-matrix-bound LPL in an active form. Extracellular-matrix-bound LPL detached into medium containing triolein emulsion and/or serum, and was catalytically active after being released. Extracellular-matrix-bound LPL lost 30% of its activity following incubation at 37 degrees C for 4 h. in contrast to soluble LPL which lost 75% of its activity. It is plausible to conclude from these data that in vivo the subendothelial basement membrane, similarly to extracellular matrix, sequesters and stabilizers LPL secreted into the subendothelial space by non-endothelial cells, and thus may play an important role in determining the route of LPL from its site of synthesis to its site of action.

Animals↗
PubMed1990

Inhibition of T lymphocyte heparanase by heparin prevents T cell migration and T cell-mediated immunity.

Previously we reported that activated T lymphocytes express a heparanase enzyme that degrades the heparan sulfate moiety of the proteoglycan of the extracellular matrix (ECM). Expression of the heparanase enzyme was found to be associated with the ability of activated T lymphocytes to penetrate blood vessel walls and accumulate in target organs. We recently found that relatively low doses of heparin administered to mice or rats inhibited T cell-mediated immune reactions. In the present study we investigated the effects in vitro and in vivo of the heparanase inhibitor, heparin, on the expression of T lymphocyte heparanase and on the ability of T lymphocytes to mediate a delayed-type hypersensitivity (DTH) reaction. We found that heparanase was induced by immunizing mice with antigen in vivo or by activating T lymphocytes with concanavalin A in vitro. Relatively low doses of heparin administered once daily in vivo (5 micrograms) or present in vitro (0.1 microgram/ml) inhibited the expression of heparanase induced by immunization or by concanavalin A incubation. Higher or lower doses of heparin did not have these effects. The same doses of heparin that inhibited expression of heparanase also inhibited the ability of the lymph node cells to migrate to a site of antigen and adoptively produce a DTH reaction. These findings suggest that modulation of cell-mediated immune reactions may be achieved by relatively low doses of heparin which inhibit expression of T lymphocyte heparanase.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

Selective inhibition of neutrophil activation by the subendothelial extracellular matrix: possible role in protection of the vessel wall during diapedesis.

Mobilization of circulating neutrophils toward an inflamed area involves adherence of the cells to the vascular endothelium and subsequent penetration through the endothelial cell layer without causing significant damage. To investigate the nature of a possible protective mechanism, granulocytes were incubated with the extracellular matrix (ECM) produced by cultured endothelial cells and tested for release of enzymes, chemoattractants, and free oxygen radicals. In the absence of exogenously added stimuli, the neutrophils adhered to the ECM but there was no detectable release of lysozyme, chemotactic activity, or production of O2-. In contrast, the cells readily released a heparan sulfate-degrading endoglycosidase (heparanase) to an extent comparable with that released in contact with polystyrene surfaces. Neutrophils treated with the calcium ionophore A23187 or with the peptide FMLP produced O2- to a much lesser degree when incubated in contact with ECM-coated surfaces than did those incubated in contact with uncoated polystyrene culture dishes. The ECM itself was devoid of superoxide dismutase activity. Stimulation with opsonized zymosan was not inhibited by the ECM. Experiments with isolated constituents of the ECM revealed that fibronectin but not collagen type IV or laminin could partially inhibit O2- production by Ca2+ ionophore-stimulated neutrophils. Treatment of the ECM with proteolytic enzymes, but not with heparanase, abolished its inhibitory effect on neutrophil activation. These results indicate that the subendothelial basement membrane has the capacity to inhibit release of potentially noxious agents excluding heparanase, suggesting a preferential involvement of this enzyme in neutrophil diapedesis.

Cell Adhesion↗
PubMed1990

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.

Animals↗
PubMed1990

Thrombin immobilized to extracellular matrix is a potent mitogen for vascular smooth muscle cells: nonenzymatic mode of action.

Esterolytically inactive diisopropyl fluorophosphate-conjugated thrombin (DIP-alpha-thrombin) stimulated 3H-thymidine incorporation and proliferation of growth-arrested vascular smooth muscle cells (SMCs), similar to native alpha-thrombin. Half-maximal mitogenic response of SMCs was obtained at 1 nM thrombin and was specifically blocked by the leech-derived, high-affinity thrombin inhibitor, hirudin. Native thrombin and a variety of thrombin species that were chemically modified to alter thrombin procoagulant or esterolytic functions were found to induce 3H-thymidine incorporation to a similar extent. Exposure of SMCs to DIP-alpha-thrombin caused a rapid and transient expression of the c-fos protooncogene, determined by Northern blot analysis. These results indicate that thrombin is a potent mitogen for SMCs through a distinct non-enzymatic domain. Binding of 125I-alpha-thrombin to SMC cultures revealed an apparent dissociation constant of 6 nM and an estimated 5.4 x 10(5) binding sites per cell. This binding was inhibited to the same extent by native thrombin and by its nonenzymatic form, DIP-alpha-thrombin. Moreover, the chemotactic fragment of thrombin (CB67-129), which failed to elicit a mitogenic response, competed for 125I-alpha-thrombin binding to SMCs. Cross-linking analysis of 125I-alpha-thrombin to SMCs revealed a specific cell-surface binding site 55 kDa in size. Finally, thrombin immobilized to a naturally produced extracellular matrix retained potent mitogenic activity toward SMCs. These observations lend support to the possibility that in vivo, subendothelial basement membranes sequester thrombin (as well as other bioactive molecules), which may stimulate localized and persistent growth of arterial SMCs. Thrombin may thus be involved directly in progression of atherosclerotic plaque formation.

Animals↗
PubMed1990

Envelope glycoprotein of avian hemangioma retrovirus induces a thrombogenic surface on human and bovine endothelial cells.

Vascular endothelial cells are a target for blood-borne pathogens which may affect their integrity and thromboresistant properties. Here, we report that cultured bovine and human endothelial cells lose their thromboresistance following interaction with the avian hemangioma-inducing retrovirus. We show that the envelope (env) gene product, glycoprotein 85, is responsible for this effect, which appears soon after infection without viral replication or cell transformation. Induction of thrombogenicity is associated with a reduction in prostacyclin release and increased expression of tissue factor. These observations may explain the occurrence of thrombosis frequently observed in association with the hemangiosarcomas induced by avian hemangioma-inducing retrovirus. These unique endothelial cell-virus interactions may also be a model for the pathogenesis of various vascular diseases.

Animals↗
PubMed1990

Interferon enhances phospholipase A2 activity in bovine aortic endothelial cells.

The effect of human interferon-alpha (IFN) on the activity of PLA2 in bovine aortic endothelial cells (BAEC) was studied. A novel method for determination of PLA2 activity in intact cell membranes with a fluorescent analogue of phosphatidylcholine, was employed. It was found that pretreatment of BAEC with human leukocyte IFN alpha or recombinant human IFN alpha 2 enhanced PLA2 activity as a function of IFN concentration. Since PLA2 activity is responsive to the lipid composition of its environment, we analyzed the fatty acyl chain composition of the cell lipids after treatment with IFN. It was found that both IFN alpha and IFN alpha 2 markedly increased the content of arachidonic acid. We suggest that exposure of BAEC to IFN enhances the activity of the cell membrane PLA2 concomitantly with enrichment of the cell membrane lipids with arachidonic acid.

Animals↗