Lipopolysaccharide inhibits activation of latent transforming growth factor-β in bovine endothelial cells

The activation of latent transforming growth factor-β (TGF-β) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF-β levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor-1 (PAI-1), we have tested whether LPS might suppress latent TGF-β activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 μM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF-β secreted into the culture modium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF-β expression as well as the suppression of surface activation of latent TGF-β. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF-β did not occur in cells maintained in LPS-contaminated culture medium. © 1995 Wiley-Liss, Inc.     

Mechanism of retinoid-induced activation of latent transforming growth factor-β in bovine endothelial cells

Cell-associated plasmin is a putative physiological activator of latent transforming growth factor-β (LTGF-β). Since retinoids enhance the production of plasminogen activator (PA) and thereby increase cell-associated plasmin activity, we tested the possibility that retinoids might induce the activation of LTGF-β using bovine endothelial cells (ECs) as a model system. ECs treated with physiological concentrations of retinol or retinoic acid formed active TGF-β in the culture media in a dose- and time-dependent fashion. Cells were treated with 2 μM retinol for 24 h, and the amount of TGF-β produced during a subsequent 12-h incubation period was measured. Out of a total of 14 pM LTGF-β secreted, 0.7 pM was converted to active TGF-β. Northern blot analyses showed that mRNA levels for TGF-β2 but not for TGF-β1 increased in cells treated with retinol. Inclusion of either inhibitors of PA or of plasmin or antibody against PA in the culture medium as well as depletion of plasminogen from the serum blocked the formation of TGF-β, suggesting that PA, plasminogen, and the resulting plasmin are essential for activation of LTGF-β in retinoid-stimulated cells. Antibody against the LTGF-β binding protein blocked activation implying that localization of LTGF-β through its binding protein may be important. However, inhibition of binding of LTGF-β to the cell surface mannose 6-phosphate receptor did not prevent activation. These data indicate that retinoids up-regulate the production of LTGF-β in ECs and induce activation of LTGF-β, perhaps, by increasing PA and plasmin levels. Thus, TGF-β might be a local mediator of some of the biological activities of retinoids both in vivo and in vitro. © 1993 Wiley-Liss, Inc.     

Protection of transforming growth factor β activity by heparin and fucoidan

The transforming growth factor-β (TGF-β) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF-β activity in vitro or in vivo. Our previous work indicated that (1) TGF-β1 has strong heparin-binding properties that were not previously recognized because of neutralization by iodination, and (2) heparin, and certain other polyanions, could block the binding of TGF-β1 to α₂-macroglobulin (α₂-M). The present studies investigated the influence of heparin-like molecules on the stability of the TGF-β1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L-fucose polymer, suppress the formation of an initial non-covalent interaction between ₁₂₅I-TGF-β1 and activated α₂-M. Electrophoresis of ₁₂₅I-TGF-β1 showed that fucoidan protects TGF-β1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF-β derived from vascular smooth muscle cells (SMC), plasmin degraded acid-activated TGF-β, and purified TGF-β1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half-life of ₁₂₅I-TGF-β1 and doubled the amount of cell-associated ₁₂₅I-TGF-β1. Consistent with this protective effect, heparin- and fucoidan-treated SMC demonstrated elevated levels of active, but not latent, TGF-β activity. © 1994 wiley-Liss, Inc.     

Retinoids potentiate transforming growth factor-β activity in bovine endothelial cells through up-regulating the expression of transforming growth factor-β receptors

Retinoic acid (RA) induces the activation of latent transforming growth factor-β (TGF-β) in bovine aortic endothelial cells (BAECs) via enhancement of cellular plasminogen activator (PA)/plasmin levels. The resultant TGF-β suppresses the excessive fibrinolytic activity by decreasing PA expression and stimulating expression of the PA inhibitor, PA inhibitor-1 (PAI-1), and inhibits cell proliferation. Here, we report that, in this regulatory system, RA simultaneously up-regulates the expression of TGF-β receptor types I and II, resulting in enhancement of TGF-β activity in the cells. RA increased the numbers of high- and low-affinity binding sites for ¹²⁵I-TGF-β1 2.1-fold and 1.5-fold, respectively, without alteration of their Kd values. Affinity labeling and Western and Northern blotting studies showed that, following RA treatment, surface levels of both type I and type II receptors increased due to augmentation in their mRNA levels. The effect was dose- and time-dependent. Treatment with 1 μM RA for 15 hr increased mRNA levels of type I and II receptor threefold and eightfold, respectively. Pretreatment of BAECs with either RA or retinol lowered the concentration of TGF-β1 required to suppress PA levels, to enhance PAI-1 levels, and to inhibit cell proliferation. Thus, retinoids may regulate cellular functions of BAECs not only by inducing the formation of active TGF-β but also by stimulating TGF-β receptor expression. This regulatory mechanism may sustain TGF-β-mediated regulation of EC function at a focal site where RA is acting. J. Cell. Physiol. 176:565–573, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of a 60-kDa cell surface-associated transforming growth factor-β binding protein that can interfere with transforming growth factor-β receptor binding

We have characterized a 60-kDa transforming growth factor-β (TGF-β) binding protein that was originally identified on LNCaP adenocarcinoma prostate cells by affinity cross-linking of cell surface proteins by using ¹²⁵I-TGF-β1. Binding of ¹²⁵I-TGF-β1 to the 60-kDa protein was competed by an excess of unlabeled TGF-β1 but not by TGF-β2, TGF-β3, activin, or osteogenic protein-1 (OP-1), also termed bone morphogenetic protein-7 (BMP-7). In addition, no binding of ¹²⁵I-TGF-β2 and ¹²⁵I-TGF-β3 to the 60-kDa binding protein on LNCaP cells could be demonstrated by using affinity labeling techniques. The 60-kDa TGF-β binding protein showed no immunoreactivity with antibodies against the known type I and type II receptors for members of the TGF-β superfamily. Treatment of LNCaP cells with 0.25 M NaCl, 1 μg/ml heparin, or 10% glycerol caused a release of the 60-kDa protein from the cell surface. In addition, we found that the previously described TGF-β type IV receptor on GH3 cells, which does not form a heteromeric complex with TGF-β receptors, could be released from the cell surface by these same treatments. This suggests that the 60-kDa protein and the similarly sized TGF-β type IV receptor are related proteins. The eluted 60-kDa LNCaP protein was shown to interfere with the binding of TGF-β to the TGF-β receptors. Thus, the cell surface-associated 60-kDa TGF-β binding protein may play a role in regulating TGF-β binding to TGF-β receptors. J. Cell. Physiol. 173:447–459, 1997. © 1997 Wiley-Liss, Inc.     

THP-1 macrophage membrane-bound plasmin activity is up-regulated by transforming growth factor-β1 via increased expression of urokinase and the urokinase receptor

Receptors for urokinase (uPA) and plasminogen provide a mechanism to direct the cellular activation of plasminogen. The regulation of these receptors is important for several macrophage functions. In these studies, the effect of transforming growth factor-b?1 (TGF-b?1) on uPA, uPA receptor, and plasminogen receptor expression by human THP-1 macrophage was examined. TGF-b?1 induction of uPA expression by THP-1 cells was differentiation dependent. Suspension and adherent cultures expressed similar constitutive levels of uPA. Exposure of adherent cells to TGF-b?1 led to a dose- and time-dependent increase in uPA activity which was paralleled by an increase in uPA antigen and uPA mRNA. In contrast, uPA expression by suspension cultures was unresponsive to TGF-b?1. The differential response exhibited by suspension and adherent THP-1 cells may reflect differences in their expression of TGF-b?1 receptors, since when assayed by crosslinking techniques, suspension cells primarily expressed a 65 kDa receptor; whereas, the adherent cells expressed 65 and 100 kDa receptors. TGF-b?1-induced alterations in uPA receptor expression by adherent THP-1 cells were examined by quantitating membrane-bound uPA activity. Membrane-bound uPA activity increased three-fold when cells were incubated with TGF-b?1. The increase in membrane-uPA activity expressed by TGF-b?1-treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF-b?1 primed cells with exogenous uPA did not lead to an increase in membrane-bound uPA activity. Furthermore, immunoreactive uPA receptor was increased in TGF-b?1-treated cells. Following incubation with plasminogen, membrane-bound plasmin activity increased three-fold in TGF-b?1-treated cells. However, no change in immunoreactive membrane-bound plasmin(ogen) was observed. In addition, binding of ¹²⁵I-Lys-plasminogen to THP-1 cells was not affected by TGF-b?1 treatment. We conclude that TGF-b?1 stimulates membrane-bound plasmin activity, without affecting plasminogen receptor expression, through the up-regulation of uPA and the uPA receptor expression. © 1995 Wiley-Liss, Inc.     

Binding of anti-SSA antibodies to apoptotic fetal cardiocytes stimulates urokinase plasminogen activator (uPA)/uPA receptor-dependent activation of TGF-β and potentiates fibrosis

In congenital heart block (CHB), binding of maternal anti-SSA/Ro Abs to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases urokinase plasminogen activator (uPA)/uPA receptor (uPAR)-dependent plasmin activation. Because the uPA/uPAR system plays a role in TGF-β activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF-β, thereby promoting a profibrosing phenotype. Supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apoptotic-CHB-IgG [apo-CHB-IgG]) exhibited significantly increased levels of active TGF-β compared with supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor. Treatment of the culture medium with anti-TGF-β Ab or TGF-β inhibitor (SB431542) abrogated the luciferase response, thereby confirming TGF-β dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared with healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA Abs or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF-β activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation, as evidenced by increased smooth muscle actin and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR Abs. These data suggested that binding of anti-Ro Abs to apoptotic cardiocytes triggers TGF-β activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promotes myofibroblast transdifferentiation and scar.     

Active transforming growth factor-β in human melanoma cell lines: No evidence for plasmin-related activation of latent TGF-β

Cultured human melanoma cells were found to secrete TGF-β mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF-β in the range from 370 to 610 pg per 10⁶ cells per 24 h. A distinct characteristic of these melanoma cell lines is that they form active surface-bound plasmin by the activation of plasminogen with surface-bound tissue-type plasminogen activator. The present study was performed to assess the role of plasmin in the process of latent TGF-β activation in the melanoma cell lines. No direct correlation was found between cell-associated plasmin activity and the amount of active TGF-β present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF-β1; three cell lines were growth-stimulated, two were growth-inhibited, and one had a very low sensitivity to the growth factor. The active TGF-β produced by the melanoma cells was found to inhibit the natural killer cell function of peripheral blood lymphocytes, suggesting that it may have an immunosuppressive effect and a role in the development of melanomas. © 1996 Wiley-Liss, Inc.     

Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity

Cultured bovine capillary endothelial (BCE) cells synthesize heparan sulfate proteoglycans (HSPG), which are both secreted into the culture medium and deposited in the cell layer. The nonsoluble HSPGs can be isolated as two predominant species: a larger 800-kD HSPG, which is recovered from preparations of extracellular matrix, and a 250-kD HSPG, which is solubilized by nonionic detergent extraction of the cells. Both HSPG species bind bFGF. 125I-bFGF bound to BCE cell cultures is readily released by either heparinase or plasmin. When released by plasmin, the growth factor is recovered from the incubation medium as a complex with the partly degraded high molecular mass HSPG. Endogenous bFGF activity is released by a proteolytic treatment of cultured BCE cells. The bFGF-binding HSPGs are also released when cultures are incubated with the inactive proenzyme plasminogen. Under such experimental conditions, the release of the extracellular proteoglycans can be enhanced by treating the cells either with bFGF, which increases the plasminogen activating activity expressed by the cells, or decreased by treating the cells with transforming growth factor beta, which decreases the plasminogen activating activity of the cells. Specific immune antibodies raised against bovine urokinase also block the release of HSPG from BCE cell cultures. We propose that this plasminogen activator-mediated proteolysis provides a mechanism for the release of biologically active bFGF-HSPG complexes from the extracellular matrix and that bFGF release can be regulated by the balance between factors affecting the pericellular proteolytic activity.     

Alpha 2-macroglobulin is a binding protein for basic fibroblast growth factor

After incubation with human serum or plasma, 125I-basic fibroblast growth factor (bFGF) (molecular mass 18.5 kDa) exhibits molecular mass forms greater than 200 kDa as determined by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. These high molecular mass forms of bFGF are immunoprecipitable with antiserum raised against alpha 2-macroglobulin (alpha 2M). Purified alpha 2M and 125I-bFGF form a covalent complex in a specific, saturable manner. Excess unlabeled bFGF competes with 125I-bFGF for complex formation. Complex formation is complete after 4 h and is inhibited by pretreating alpha 2M with dithiothreitol, iodoacetamide, iodoacetic acid, and N-ethylmaleimide. The complex is resistant to acidic conditions and denaturants such as urea. Heparin, which binds bFGF, has no effect on complex formation. Methylamine, which blocks protease binding to alpha 2M, increases the amount of 125I-bFGF that can be bound 2-fold. Plasmin and trypsin treatment of alpha 2M has no effect on 125I-bFGF binding. The ability of growth factors to compete for binding is specific, as aFGF and TGF-beta compete for binding to alpha 2M, whereas platelet-derived growth factor does not. 125I-bFGF.alpha 2M complexes do not bind to low affinity bFGF binding sites and bind poorly to high affinity bFGF binding sites on BHK-21 cells. In addition, 125I-bFGF bound to alpha 2M has decreased ability to stimulate plasminogen activator production in bovine capillary epithelial cells.     

Cell-associated activation of latent transforming growth factor-β by calpain

Transforming growth factor-β (TGF-β) is normally secreted in a latent form, and plasmin-mediated proteolytic cleavage of latency-associated peptide (LAP), a component of latent TGF-β complex that makes the complex inactive, activates latent TGF-β. In the present study, we investigated the possible involvement of calpain, one of the cysteine proteases, in the activation of latent TGF-β. When recombinant latent TGF-β was incubated with calpain (1–10 u/ml) in a test tube, calpain cleaved LAP and released mature TGF-β from the latent complex. When calpain was applied to cultured bovine capillary endothelial (BCE) cells, a low concentration of calpain (0.05–0.1 u/ml) inhibited the migration and proliferation of the cells, and these inhibitory effects were abrogated by anti-TGF-β antibody as well as by calpain inhibitor peptide, but not by α₂-antiplasmin, a specific inhibitor of plasmin. Active TGF-β was detected in the conditioned medium of BCE cells collected in the presence of calpain. Chemical cross-linking of ¹²⁵I-calpain to BCE cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that calpain bound to the cell surface through chondroitinase ABC-sensitive proteoglycan. In addition, treatment of the BCE cells with chondroitinase ABC abrogated the inhibitory effect of calpain on the migration of these cells. Our data thus suggest that calpain is able to activate latent TGF-β through a mechanism independent of plasmin. This activation is efficient in the presence of cells, and calpain binds to the cell surface via proteoglycan and activates latent TGF-β, which is targeted to the same surface. J. Cell. Physiol. 174:186–193, 1998. © 1998 Wiley-Liss, Inc.     

Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-β to TGF-β

The role of receptor-bound urokinase-type plasminogen activator (uPA) in cellular activation of latent transforming growth factor-beta (LTGF-β) was investigated in a model system of mouse LB6 cells transfected with either a human uPA receptor cDNA (LhuPAR⁺). a human prouPA cDNA (LhuPA), or a control neomycinresistance cDNA (Lneo). When LhuPAR⁺ cells were co-cultured with LhuPA cells, the plasmin-dependent fibrinolytic activity generated was more than that observed in either homotypic cultures with fivefold greater number of LhuPA cells or co-cultures containing LhuPA and Lneo cells instead of the LhuPAR⁺ cells. The preferential activation of TGF-β by co-cultures with the greatest plasmin-generation potential, LhuPAR⁺ and LhuPA cells, was confirmed by three independent bioassays. In the first assay, a 48% decrease in PA activity, a measure of active TGF-β production, was observed with BAE cells treated with conditioned medium (CM) from co-cultures of LhuPA and LhuPAR⁺ cells. Inclusion of neutralizing antibodies to TGF-β abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF-β. Addition of the amino terminal fragment of uPA (ATF) or omission of plasminogen from co-cultures blocked both the fibrinolytic activity and the generation of TGF-β activity in the CM. In the second assay, CM from co-cultures of LhuPA and LhuPAR⁺ cells inhibited the migration of BAE cells in a wound assay. Controls with anti-TGF-β IgG indicated that the inhibition was due to TGF-β. In the third assay, proliferation of mink lung epithelial cells was inhibited by CM generated by co-cultures of LhuPA and LhuPAR⁺ cells as compared to CM from the same cells cultured in the absence of plasminogen or to CM from a co-culture of LhuPA with LhuPAR^? cells. Excess mannose-6-phosphate (M6P) blocked the generation of TGF-β as assayed by both the BAE migration and PA assays, presumably because it interfered with cellsurface localization of LTGF-β. Additionally, small numbers of LhuPA and LhuPAR⁺ cells co-cultured with BAE cells inhibited the BAE cell PA activity via the paracrine action of TGF-β. These results support the conclusion that plasmindependent activation LTGF-β by LB6 cells is promoted by the surface localization of uPA by its receptor. © 1994 Wiley-Liss, Inc.     

Role for autocrine TGF-β1 in regulating differentiation of a human leukemic cell line toward osteoclast-like cells

Increasing evidence suggests that transforming growth factor-β (TGF-β) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF-β1 and that exogenous or autocrine TGF-β1 can induce the same features of osteoclastic-like cells, exerting its effects through the binding to TGF-β specific receptors. Scatchard analysis of ¹²⁵I-labeled TGF-β1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of ～25 pM and a binding capacity of ～900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF-β receptors. Stimulation of FLG 29.1 cells with low TGF-β1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF-β1 caused a significant and dose-dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF-β1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF-β1 mRNA expression and growth factor release. The majority of TGF-β1 secreted by TPA-treated cells was in its latent form. However, anti-TGF-β antibodies inhibited TGF-β1 and TPA-induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF-β1 and indicating that the cells can activate and respond to the TGF-β that they secrete. These findings support a potential autocrine role for TGF-β1 in osteoclast differentiation. © 1994 Wiley-Liss, Inc.     

Blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathways by statins reduces the expression of bFGF, HGF, and TGF-β as angiogenic factors in mouse osteosarcoma

The tumor microenvironment plays a critical role in modulating malignant behavior and can dramatically influence cancer treatment strategies. We investigated whether statins inhibit the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and transforming growth factor-β (TGF-β) mRNA in the mouse osteosarcoma cell line LM8. We found that statins significantly inhibited mRNA expressions of bFGF, HGF, and TGF-β, and bFGF, HGF, and TGF-β secretions at concentrations that did not have antiproliferative effects on LM8 cells, but had no effect on the mRNA expression and secretion of VEGF. The inhibition of bFGF, HGF, and TGF-β mRNA expression, and bFGF, HGF, TGF-β secretions was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination with statins. Furthermore, statins reduced the membrane localization of K-Ras, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated Akt. Our research indicates that statins inhibit GGPP biosynthesis in the mevalonate pathway, and then inhibit signal transduction in the Ras/ERK and Ras/Akt pathways, thereby inhibiting bFGF, HGF, TGF-β expression in LM8 cells. These results suggest that statins are potentially useful as anti-angiogenic agents for the treatment of osteosarcoma.     

Metabolism of receptor-bound and matrix-bound basic fibroblast growth factor by bovine capillary endothelial cells

Bovine capillary endothelial (BCE) cells were incubated at 4 degrees C with 5 ng/ml 125I-basic fibroblast growth factor (bFGF) to equilibrate 125I-bFGF with high affinity cell surface receptors and low affinity matrix binding sites. 67% of the added 125I-bFGF bound to the matrix and 7% bound to receptors. The fate of bound bFGF was followed after cells were incubated in bFGF-free medium and were shifted to 37 degrees C to restore cell metabolism. 125I-bFGF bound to receptors decreased rapidly while the amount of 125I-bFGF bound to matrix was reduced more slowly. The rapid decrease in receptor-bound 125I-bFGF appeared to be due to a down-regulation of bFGF receptors; cells that had been treated for 5 h with bFGF had 60% fewer high affinity receptors than untreated cells. Despite the initial high level of 125I-bFGF binding to matrix, most of this 125I-bFGF was mobilized and metabolized by the cells. 125I-bFGF was internalized by the cells at 37 degrees C, leading to a constant accumulation of 125I-bFGF within the cell. Internalized bFGF was rapidly cleaved from an 18-kD form to a 16-kD form. The 16-kD form was more slowly degraded with a half-life of approximately 8 h. Degradation of internalized 125I-bFGF was inhibited by chloroquine, suggesting that the digestion occurred in a lysosomal compartment. The role of matrix binding sites in the internalization process was investigated. Binding to matrix sites seemed not to be directly involved in the internalization process, since addition of heparin at a concentration that blocked 95% of the binding to matrix had no effect on the initial rate of internalization of bFGF. BCE cells also released a substance that competed for the binding of bFGF to matrix but not to receptors. This substance bound to DEAE-cellulose and was sensitive to heparinase treatment, suggesting that it was a heparinlike molecule. Thus, heparinlike molecules produced by BCE cells can modulate the cellular interaction with bFGF. Matrix-associated heparinlike molecules bind bFGF which can later be metabolized by the cell, and secreted heparinlike molecules release bFGF from matrices.     

Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.     

Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells

The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells (KD of 42.0 +/- 3.8 pM and 70,526 +/- 6121 binding sites/cell for the high-affinity sites, KD of 0.933 +/- 0.27 nM and 630,252 +/- 172,459 sites/cell for low-affinity binding sites). The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely 125I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound 125I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At 37 degrees C, 30% of the cell-associated 125I-bFGF became resistant to the acidic wash after 90 min, suggesting that this fraction of bound 125I-bFGF was internalized. At this temperature, degradation of the internalized ligand was followed after 1 h by the appearance of three major bands of 15,000, 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.