Biomedical subjects

I Vlodavsky

Publications and source records attributed to I Vlodavsky.

At least 55 records · Page 3Linked to original sources

PubMed1997

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.

3T3 Cells↗

PubMed1997

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

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

3T3 Cells↗

PubMed1997

VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix.

A vascular endothelial growth factor (VEGF) mRNA species containing exons 1-6 and 8 of the VEGF gene was found to be expressed as a major VEGF mRNA form in several cell lines derived from carcinomas of the female reproductive system. This mRNA is predicted to encode a VEGF form of 145 amino acids (VEGF145). Recombinant VEGF145 induced the proliferation of vascular endothelial cells and promoted angiogenesis in vivo. VEGF145 was compared with previously characterized VEGF species with respect to interaction with heparin-like molecules, cellular distribution, VEGF receptor recognition, and extracellular matrix (ECM) binding ability. VEGF145 shares with VEGF165 the ability to bind to the KDR/flk-1 receptor of endothelial cells. It also binds to heparin with an affinity similar to that of VEGF165. However, VEGF145 does not bind to two additional endothelial cell surface receptors that are recognized by VEGF165 but not by VEGF121. VEGF145 is secreted from producing cells as are VEGF121 and VEGF165. However, VEGF121 and VEGF165 do not bind to the ECM produced by corneal endothelial cells, whereas VEGF145 binds efficiently to this ECM. Basic fibroblast growth factor (bFGF)-depleted ECM containing bound VEGF145 induces proliferation of endothelial cells, indicating that the bound VEGF145 is active. The mechanism by which VEGF145 binds to the ECM differs from that of bFGF. Digestion of the ECM by heparinase inhibited the binding of bFGF to the ECM and released prebound bFGF, whereas the binding of VEGF145 was not affected by heparinase digestion. It therefore seems that VEGF145 possesses a unique combination of biological properties distinct from those of previously characterized VEGF species.

Endothelial Growth Factors↗

PubMed1997

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

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

Actins↗

PubMed1997

Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone.

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.

Animals↗

PubMed1997

Inhibition of collagen synthesis, smooth muscle cell proliferation, and injury-induced intimal hyperplasia by halofuginone.

Proliferation of vascular smooth muscle cells (SMCs) and accumulation of extracellular matrix (ECM) components within the arterial wall in response to local injury are important etiologic factors in vascular proliferative disorders such as arteriosclerosis and restenosis after angioplasty. Fibrillar and nonfibrillar collagens are major constituents of the ECM that modulate cell shape and proliferative responses and thereby contribute to the pathogenesis of intimal hyperplasia. Halofuginone, an anticoccidial quinoazolinone derivative, inhibits collagen type I gene expression. We investigated the effect of halofuginone on (1) proliferation of bovine aortic endothelial cells and SMCs derived from the same specimen and maintained in vitro, (2) ECM deposition and collagen type I synthesis and gene expression, and (3) injury-induced intimal hyperplasia in vivo. DNA synthesis and proliferation of vascular SMCs in response to serum or basic fibroblast growth factor were abrogated in the presence of as little as 0.1 microgram/mL halofuginone; this inhibition was reversible upon removal of the compound. Under the same conditions, halofuginone exerted a relatively small antiproliferative effect on the respective vascular endothelial cells. Halofuginone also inhibited the synthesis and deposition of ECM components by vascular SMCs as indicated both by a substantial reduction in the amount of sulfated proteoglycans and collagen type I synthesis and gene expression. Local administration of halofuginone in the rabbit ear model of crush injury-induced arterial intimal hyperplasia resulted in a 50% reduction in intimal thickening as measured by a morphometric analysis of the neointima/media ratio. The differential inhibitory effect of halofuginone on vascular SMCs versus endothelial cells, its inhibition of ECM deposition and collagen type I synthesis, and its ability to attenuate injury-induced intimal hyperplasia may place halofuginone alone or in combination with other antiproliferative compounds as a potential candidate for prevention of arterial stenosis and accelerated atherosclerosis.

Animals↗

PubMed1997

Antiproliferative activity to glomerular mesangial cells and receptor binding of a heparin-mimicking polyaromatic anionic compound.

Proliferation of mesangial cells (MC) is a key feature in the pathogenesis of numerous renal diseases involving the glomerulus. Heparin, one of several compounds capable of suppressing MC proliferation, did not prove beneficial in the treatment of human glomerular diseases. In a search for a superior antiproliferative agent, a synthetic polyaromatic "heparin mimicking" compound (RG-13577, polymer of 4-hydroxyphenoxy acetic acid, M(r) approximately 5800), previously reported to inhibit the proliferation of vascular smooth muscle cells, was applied. RG-13577 exhibits approximately 1% of the anticoagulant activity of heparin and is nontoxic in animal experiments. Proliferation of primary rat MC was almost completely inhibited in the presence of 10 to 25 micrograms/ml RG-13577, and 50% inhibition was obtained at 1 to 5 micrograms/ml RG-13577. The cells resumed their normal growth rate after removal of RG-13577 from the culture medium. Under the same conditions, heparin exerted only a small inhibitors effect. RG-13577 inhibited signaling (i.e., tyrosine phosphorylation) and MC proliferation induced by both basic fibroblast growth factor and platelet-derived growth factor. RG-13577 binds to a naturally produced extracellular matrix, and the bound molecule retained its antiproliferative effect toward MC. 14C-Labeled RG-13577 also binds to cultured MC in a specific and saturable manner. Binding of 14C-RG-13577 was reduced by 80 to 90% in the presence of excess unlabeled RG-13577, apolipoprotein E, or lactoferrin, but there was no effect with heparin. Furthermore, the antiproliferative effect of RG-13577 was abolished in the presence of lactoferrin. It is proposed that compound RG-13577 inhibits MC proliferation through neutralization of growth-promoting factors, primarily heparin-binding growth factors, and possibly through binding to specific cell surface receptors, most likely the LDL receptor-related protein. RG-13577 and related polyanionic compounds may be applied to inhibit MC proliferation in glomerular diseases.

Animals↗

PubMed1997

Naturally produced extracellular matrix is an excellent substrate for canine endothelial cell proliferation and resistance to shear stress on PTFE vascular grafts.

PURPOSE: Successful development of a vascular prosthesis lined with endothelial cells (EC) may depend on the ability of the attached cells to resist shear forces after implantation. The present study was designed to investigate EC detachment from extracellular matrix (ECM) precoated vascular prostheses, caused by shear stress in vitro and to test the performance of these grafts in vivo. METHODS: Bovine aortic endothelial cells were seeded inside untreated polytetrafluoro-ethylene (PTFE) vascular graft (10 x 0.6 cm), PTFE graft precoated with fibronectin (FN), or PTFE precoated with FN and a naturally produced ECM (10(6) cells/graft). Sixteen hours after seeding the medium was replaced and unattached cells counted. The strength of endothelial cell attachment was evaluated by subjecting the grafts to a physiologic shear stress of 15 dynes/cm2 for 1 h. The detached cells were collected and quantitated. PTFE or EC preseeded ECM coated grafts were implanted in the common carotid arteries of dogs. RESULTS: While little or no differences were found in the extent of endothelial cell attachment to the various grafts (79%, 87% and 94% of the cells attached to PTFE, FN precoated PTFE, or FN+ECM precoated PTFE, respectively), the number of cells retained after a shear stress was significantly increased on ECM coated PTFE (20%, 54% and 85% on PTFE, FN coated PTFE, and FN+ECM coated PTFE, respectively, p <0.01). Implantation experiments in dogs revealed a significant increase in EC coverage and a reduced incidence of thrombus formation on ECM coated grafts that were seeded with autologous saphenous vein endothelial cells prior to implantation. CONCLUSION: ECM coating significantly increased the strength of endothelial cell attachment to vascular prostheses subjected to shear stress. The presence of adhesive macromolecules and potent endothelial cell growth promoting factors may render the ECM a promising substrate for vascular prostheses.

Animals↗

PubMed1996

Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1.

Recent studies have demonstrated the existence of a soluble fibroblast growth factor (FGF) receptor type 1 (FGFR1) extracellular domain in the circulation and in vascular basement membranes. However, the process of FGFR1 ectodomain release from the plasma membrane is not known. Here we report that the 72-kDa gelatinase A (matrix metalloproteinase type 2, MMP2) can hydrolyze the Val368-Met369 peptide bond of the FGFR1 ectodomain, eight amino acids upstream of the transmembrane domain, thus releasing the entire extracellular domain. Similar results were obtained regardless of whether FGF was first bound to the receptor or not. The action of MMP2 abolished binding of FGF to an immobilized recombinant FGFR1 ectodomain fusion protein and to Chinese hamster ovary cells overexpressing FGFR1 The released recombinant FGFR1 ectodomain was able to bind FGF after MMP2 cleavage, suggesting that the cleaved soluble receptor maintained its FGF binding capacity. The activity of MMP2 could not be reproduced by the 92-kDa gelatinase B (MMP9) and was inhibited by tissue inhibitor of metalloproteinase type 2. These studies demonstrate that FGFR1 may be a specific target for MMP2 on the cell surface, yielding a soluble FGF receptor that may modulate the mitogenic and angiogenic activities of FGF.

Amino Acid Sequence↗

PubMed1996

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.

3T3 Cells↗

PubMed1996

A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix.

Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N-terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel 'MGGQSES' motif. RD2 binds to the N-terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface-exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.

Adhesins, Bacterial↗

PubMed1996

Specific involvement of glypican in thrombin adhesive properties.

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.

Animals↗

PubMed1996

Involvement of heparan sulfate and related molecules in sequestration and growth promoting activity of fibroblast growth factor.

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues [1, 2]. The basic HSPG structure consists of a protein core to which several linear heparan sulfate (HS) chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups [1, 2]. Beside serving as a scaffold for the attachment of various ECM components (e.g., collagen, laminin, fibronectin), the binding of HS to certain proteins has been suggested to induce a conformational change which may lead to the exposure of novel reactive determinants or conversely stabilize an inert protein configuration [1-4]. Of particular significance is the interaction of HS with fibroblast growth factors (FGFs), mediating their sequestration, stabilization and high affinity receptor binding and signaling [3-7]. Cellular responses to FGFs may hence be modulated by metabolic inhibitors of HS synthesis and sulfation, HS-degrading enzymes, and synthetic mimetics of heparin/HS. In the present review we focus on the involvement of HS in basic FGF (bFGF) receptor binding and mitogenic activity and its modulation by species of heparin, HS, and synthetic polyanionic 'heparin-mimicking' compounds. The results are discussed in relation to the current thoughts on the dual involvement of low and high affinity receptor sites in the growth promoting and angiogenic activities of bFGF and other heparin-binding growth factors.

Animals↗

PubMed1996

Purification and characterization of placental heparanase and its expression by cultured cytotrophoblasts.

The role of different extracellular matrix (ECM)-degrading enzymes in the normal functioning of the placenta is well documented. Heparan sulphate proteoglycan (HSPG) is an integral constituent of the placental and decidual ECM. Because this proteoglycan specifically interacts with various macromolecules in the ECM, its degradation may disassemble the matrix. Hence, in the case of the placenta, this may facilitate normal placentation and trophoblast invasion. Crude placental specimens were collected from first and third trimester placentas. Heparanase (endo-beta-glucuronidase) was isolated and purified by ammonium sulphate precipitation followed by sequential chromatographies on carboxymethyl-, heparin- and ConA-Sepharose columns. The placental enzyme was further characterized for its molecular weight and specific inhibition by heparin, and was shown to resemble heparanase expressed by highly metastatic tumor cells and activated cells of the immune system. In order to locate the source of heparanase activity in the placenta, primary cytotrophoblast cultures were established. Intact cells, as well as conditioned medium and cell lysates, were analysed for heparanase activity using metabolically sulphate-labelled ECM as a natural substrate. Heparanase was highly active in lysates of cytotrophoblasts. This activity was also expressed by intact cytotrophoblasts seeded on ECM, but no activity could be detected in the culture medium. Incubation of the cytotrophoblasts in contact with ECM resulted in release of ECM-bound basic fibroblast growth factor (bFGF). We propose that the cytotrophoblastic heparanase facilitates placentation, through cytotrophoblast extravasation and localized neovascularization.

Cells, Cultured↗

PubMed1996

Hyaluronan, CD44 and its variant exons in human trophoblast invasion and placental angiogenesis.

Both hyaluronan and one of its receptors, CD44, can be demonstrated in the early human conceptus and in placental stroma. The variants of CD44 resulting from variable exon splicing are found in metastasizing human malignancies and are also involved in hyaluronan uptake and degradation. The resulting hyaluronan fragments are known to be highly angiogenic. We postulated that the self-limited process of trophoblast invasion of the uterine decidua results in part from the strategy of alternative splicing of CD44, similar to that used by invasive cancer cells in the course of metastatic spread and possibly angiogenesis. Monoclonal antibodies specific for CD44s and for an exon expressed during metastatic tumour progression, CD44v7, were used to examine this hypothesis. In this study we found human trophoblasts, for the first time, to express CD44. Intermediate trophoblasts of first and second trimester exhibited the standard form of CD44 while extravillous trophoblasts, which are responsible for the invading characteristics of the placenta, were positive for the alternatively spliced form, the CD44v7-8. Moreover, in the case of placenta accreta there was a prominent membrane staining of the trophoblasts that were embedded in the fibrin layer over the myometrium. The highly metastatic choriocarcinoma cells also expressed CD44v7-8. We propose, therefore, that the invading trophoblasts utilize the alternatively splicing machinery. These cells retain their invasive capabilities through the permissive ECM by carrying the CD44v7-8 isoform, which binds weakly to hyaluronan and thus prevents it from being degraded by intracellular hyaluronidase.

Alternative Splicing↗

PubMed1996

Heparan sulfate-dependent and low density lipoprotein receptor-related protein-dependent catabolic pathways for lipoprotein lipase in mouse embryonic fibroblasts.

Heparan sulfate and low density lipoprotein receptor related protein (LRP) have been shown to participate in the uptake and degradation of the enzyme lipoprotein lipase (LPL). Yet, the contribution of each of these pathways to LPL metabolism and their possible dependence is unknown. In the present study we examined the metabolism of 125I-labeled LPL in untreated and heparinase-treated primary wild-type mouse embryonic fibroblasts (MEF) and in mouse fibroblasts that express single LRP allele (PEA-10) or are lacking the LRP (PEA-13). The degradation of LPL in PEA-13 cells was 30% lower than in MEF and PEA-10 cells. Heparinase treatment decreased the LPL degradation by 58%, 79% and 92%, whereas heparin reduced such degradation by 87%, 90% and 94% in MEF, PEA-10 and PEA-13 cultures, respectively. Assuming that a) heparinase treatment abolished the heparan-sulfate pathway, and that b) the degradation remaining in heparin-treated cultures represents nonspecific values, it appears that heparan sulfate contributes about 61%, 83% and 95% of total LPL degradation, whereas the LRP pathway contributes 39%, 17% and less than 5% of LPL degradation in MEF, PEA-10 and PEA-13 cells, respectively. In addition, the data indicate that LPL interaction with heparan sulfate and the LRP pathways is independent of each other. The study shows that these cells possess both a heparan sulfate-dependent pathway and an LRP-dependent pathway for LPL metabolism and that the two pathways are independent of each other.

Alleles↗

PubMed1995

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

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

CD4-Positive T-Lymphocytes↗

PubMed1995

Binding to heparan sulfate is a major event during catabolism of lipoprotein lipase by HepG2 and other cell cultures.

Lipoprotein lipase (LPL) is rapidly and efficiently cleared from the circulation by the liver through an as yet unclear mechanism. In the present study, we determined the nature of LPL interactions with the liver parenchimal cell line HepG2 as compared to other cells in culture. Binding, cell association and degradation of 125I-labelled bovine milk LPL by HepG2 cells, normal and low density lipoprotein (LDL) receptor-negative human fibroblasts and Chinese hamster ovary (CHO) cells show similar values irrespective of source and origin. LPL metabolism in HepG2 cells was characterized by a high capacity to degrade the enzyme, an extremely high sensitivity to heparin and was inhibited by 60%-70% after treatment of the cells with sodium chlorate and heparinase (but not chondroitinase). These findings suggested an important role for heparan sulfate in the process of cell interaction and metabolism of LPL. To further clarify the role of heparan sulfate in determining the LPL-cell interactions, we compared the metabolism of LPL in wild type and mutant heparan sulfate-deficient CHO cells. Heparan sulfate-deficient CHO cells show a low capacity to bind and degrade LPL, about 10%-20% that of the wild type cells. In another set of experiments, we sought to determine whether LPL interactions with HepG2 cells are affected by triglyceride-rich lipoproteins. The results clearly show that whereas unlabeled LPL dramatically enhanced the metabolism of radioiodinated very low density lipoprotein (VLDL), unlabeled VLDL had no effect on radioiodinated LPL metabolism in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗