Differential inhibition of transforming growth factor beta 1 and beta 2 activity by alpha 2-macroglobulin.

Affinity labeling and immunoprecipitation studies demonstrate that alpha 2-macroglobulin (alpha 2M) is the major serum-binding protein for transforming growth factors beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2). Purified alpha 2M inhibits the binding of both 125I-TGF-beta 1 and 125I-TGF-beta 2 to cell surface receptors at I50 values of 200 and 10 micrograms/ml, respectively. alpha 2M (200 micrograms/ml) does not block TGF-beta 1 inhibition of CCL-64 mink lung cell growth but reduces this activity of TGF-beta 2 10-fold. The electrophoretic migration of 125I-TGF-beta.alpha 2M complexes on polyacrylamide gels under nondenaturing conditions demonstrates that alpha 2M has 10-fold greater affinity for TGF-beta 2 than for TGF-beta 1. Each of these complexes comigrates as a single band with the fast form of alpha 2M. We suggest that alpha 2M is an important differential regulator of the biological activities of TGF-beta 1 and TGF-beta 2 in vivo.     

Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor

Heparin and related glycosaminoglycans are potent inhibitors of both in vivo and in vitro smooth muscle cell (SMC) proliferation. We have found that epidermal growth factor (EGF) reverses the antiproliferative effects of heparin. Other known SMC mitogens, including platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), and thrombin, were unable to prevent heparin action. The EGF specificity was further demonstrated by developing a biological growth assay in which EGF or PDGF, at concentrations as low as 1 ng/ml, stimulated SMC growth in the absence of other serum components. Under these conditions, EGF, but not PDGF, suppressed heparin inhibition as well. The ability of EGF to reverse heparin inhibition was only observed when mitogen and glycosaminoglycan were added to SMC at similar times. If SMC were pretreated with heparin for 48 hours prior to EGF addition, the protective effects of EGF were lost. Heparin did not directly prevent 125I-EGF or platelet-derived EGF-like peptides from binding to the EGF receptor on SMC. However, cultures that were pretreated with heparin for 48 hours bound 49% less 125I-EGF than cultures that had been pretreated with the mucopolysaccharide for only 2 hours or that had not been preexposed to heparin. In previous studies, we have established that heparin exerts its maximal inhibitory activity after a 48-hour treatment of SMC (Reilly et al. 1986). Taken together, these data suggest that heparin may exert its antiproliferative potential by slowly and specifically altering SMC response to EGF-like mitogens of platelet origin.     

Evidence for an age-related dysfunction in the antiproliferative response to transforming growth factor-beta in vascular smooth muscle cells.

Previous studies have indicated that aged animals show an increased intimal hyperplasia after arterial injury. The present studies examined the hypothesis that the increased serum-free proliferation of aged smooth muscle cells (SMC), in vitro, was due to a loss of an antiproliferative signal, such as transforming growth factor-beta 1 (TGF-beta 1). Northern blot analysis of the mRNA derived from old (> 19 mo) or young (3-4 mo) rat aortic SMC indicated that both groups had an equivalent level of the 2.5 kB TGF-beta 1 message. Metabolic labeling with 35S-methionine and immunoprecipitation for TGF-beta 1 confirmed the de novo synthesis of TGF-beta 1 in rat SMC. Old and young SMC supernatants showed equal levels of active or latent (acid-activated) TGF-beta activity. Despite the similarities in the production of TGF-beta 1, old SMC were refractory to inhibition by TGF-beta 1, whereas young SMC were markedly inhibited (80%) by low levels of TGF-beta 1 (IC50 < 5 pg/ml). Binding studies at 4 degrees C indicated that old SMC exhibited reduced binding capacity for 125I-TGF-beta 1. Cross-linking studies confirmed that old SMC showed reduced binding of 125I-TGF-beta 1 to membrane sites corresponding to the high molecular weight type III receptor, as well as the 85-kDa type II and 65-kDa type I receptor. However, at 37 degrees C, old SMC degraded 125I-TGF-beta 1 more rapidly than young SMC. Combined, this data suggests that SMC derived from older animals are capable of normal production of TGF-beta 1 but fail to respond to the autocrine growth inhibitory effects of this agent, thereby leading to enhanced proliferation.     

Transforming growth factor-beta complexes with thrombospondin.

Thrombospondin (TSP) was demonstrated to inhibit the growth of bovine aortic endothelial cells, an activity that was not neutralized by antibodies to TSP or by other agents that block TSP-cell interactions but that partially was reversed by a neutralizing antibody to transforming growth factor-beta (TGF-beta). Similar to TGF-beta, TSP supported the growth of NRK-49F colonies in soft agar in a dose-dependent manner, which required epidermal growth factor and was neutralized by anti-TGF-beta antibody. Chromatography of a TSP preparation did not separate the TGF-beta-like NRK colony-forming activity from high molecular weight protein. However, when chromatography was performed at pH 11, this activity was dissociated from TSP. These results suggest that at least some growth modulating activities of TSP are due to TGF-beta associated with TSP by strong non-covalent forces. Most of the active TGF-beta released from platelets after degranulation was associated with TSP, as demonstrated by anti-TSP immunoaffinity and gel permeation chromatography. 125I-TGF-beta binds to purified TSP in an interaction that is specific in the sense that bound TGF-beta could be displaced by TGF-depleted TSP but not significantly by native TSP, heparin, decorin, alpha 2-macroglobulin, fibronectin, or albumin. Hence, TGF-beta can bind to TSP, and the complex forms under physiological conditions. Furthermore, TSP-associated TGF-beta is biologically active, and the binding of TGF-beta to TSP may protect TGF-beta from extracellular inactivators.     

Heparin-like molecules regulate the number of epidermal growth factor receptors on vascular smooth muscle cells

The effect of heparin on the binding of epidermal growth factor (EGF) to vascular smooth muscle cells (SMC) was examined. Heparin pretreatment of SMC obtained from bovine aortic explant tissue resulted in significant reductions in the amount of EGF bound. Decreases in mitogen binding were observed with both growth arrested as well as exponentially growing cultures. The heparin concentrations (10-100 micrograms/ml) and pretreatment times (48-72 h) necessary for suppression of EGF binding correlated with the concentrations and temporal requirements necessary for growth inhibition. Chondroitin sulfate, which has negligible antiproliferative activity, had no effect on EGF binding. However, a highly inhibitory heparan sulfate species obtained from postconfluent SMC suppressed EGF binding by 45%. Platelet-derived growth factor and insulin-like growth factor-1 binding were unaffected by heparin. Scatchard analysis revealed that heparin induced 50 to 60% reductions in the numbers of high and low affinity EGF receptors without detectable changes in the binding affinity or ratio of high to low receptors. Experiments were also performed with enzymatically dispersed SMC. These cultures were inhibited by heparin in a time dependent manner which was partially reversible in the presence of EGF. Subsequent studies revealed that heparin suppressed EGF binding in these cultures by 20 to 40%. In summary, heparin reduces the number of EGF receptors on both explant and enzyme dispersed SMC by a mechanism which closely parallels the antiproliferative effects of this glycosaminoglycan.     

Latent transforming growth factor-beta in serum. A specific complex with alpha 2-macroglobulin

The biological latency of serum transforming growth factor-beta (TGF-beta) was shown to be due to the interaction of TGF-beta with a specific serum binding protein. This binding protein was affinity labeled with 125I-TGF-beta, and its Mr and subunit structure were determined using sodium dodecyl sulfate-gel electrophoresis and gel filtration chromatography. Its Mr is reminiscent of that of the serum protease inhibitor, alpha 2-macroglobulin (alpha 2M). Immunoprecipitation of the 125I-TGF-beta-binding protein complex by a specific anti-alpha 2M antibody, and the formation of identical complexes between 125I-TGF-beta and purified alpha 2M, confirmed that alpha 2M is the TGF-beta-binding protein in serum. Immunoblot analysis showed that endogenous serum TGF-beta is also bound to alpha 2M. However, in contrast to added 125I-TGF-beta, the majority of the endogenous TGF-beta is linked to alpha 2M covalently. Alpha 2M and acid-activated TGF-beta co-eluted from a Superose 6 fast protein liquid chromatography column, confirming that the interaction of TGF-beta with alpha 2M accounts for the latency of serum TGF-beta. It is proposed that alpha 2M may serve an important multifunctional role at sites of inflammation by scavenging both active peptides and proteases that are released by platelets at the site of injury.     

Reaction of alpha 2-macroglobulin with plasmin increases binding of transforming growth factors-beta 1 and beta 2.

The binding of 125I-transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) to alpha 2-macroglobulin (alpha 2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-beta and native or reacted forms of alpha 2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native alpha 2M with plasmin or methylamine markedly increased the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to alpha 2M. The alpha 2M-plasmin/TGF-beta complexes were minimally dissociated by heparin. Reaction of alpha 2M with thrombin or trypsin reduced the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-beta 2 and native or reacted forms of alpha 2M were less dissociable by heparin than the equivalent complexes with TGF-beta 1. These studies demonstrate that the TGF-beta-binding activity of alpha 2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that alpha 2M-plasmin preferentially binds TGFs-beta suggest a mechanism by which alpha 2M may regulate availability of TGFs-beta to target cells in vivo.     

Transforming growth factor-beta 1 is a heparin-binding protein: identification of putative heparin-binding regions and isolation of heparins with varying affinity for TGF-beta 1.

Previous studies indicated that a major factor in heparin's ability to suppress the proliferation of vascular smooth muscle cells is an interaction with transforming growth factor-beta 1 (TGF-beta 1). Heparin appeared to bind directly to TGF-beta 1 and to prevent the association of TGF-beta 1 with alpha 2-macroglobulin (alpha 2-M). The present studies indicate that 20-70% of iodinated TGF-beta 1 binds to heparin-Sepharose and the retained fraction is eluted with approximately 0.37 M NaCl. Native, unlabelled platelet TGF-beta 1, however, is completely retained by heparin-Sepharose and eluted with 0.9-1.2 M NaCl. Using synthetic peptides, the regions of TGF-beta 1 that might be involved in the binding of heparin and other polyanions were examined. Sequence analysis of TGF-beta 1 indicated three regions with a high concentration of basic residues. Two of these regions had the basic residues arranged in a pattern homologous to reported consensus heparin-binding regions of other proteins. The third constituted a structurally novel pattern of basic residues. Synthetic peptides homologous to these three regions, but not to other regions of TGF-beta 1, were found to bind to heparin-Sepharose and were eluted with 0.15 M-0.30 M NaCl. Only two of these regions were capable of blocking the binding of heparin to 125I-TGF-beta. Immobilization of these peptides, followed by affinity purification of heparin, indicated that one peptide was capable of isolating subspecies of heparin with high and low affinity for authentic TGF-beta 1. The ability of TGF-beta 1 to bind to heparin or related proteoglycans under physiological conditions may be useful in understanding the biology of this pluripotent growth and metabolic signal. Conversely, a subspecies of heparin molecules with high affinity for TGF-beta 1 may be a factor in some of the diverse biological actions of heparin.     

Interaction of transforming growth factor-beta 1 with alpha 2-macroglobulin. Role in transforming growth factor-beta 1 clearance.

It has been widely assumed that the interaction of transforming growth factor-beta 1 (TGF-beta 1) with its serum-binding protein, alpha 2-macroglobulin (alpha 2M), mediates the rapid clearance of TGF-beta 1 from the circulation. To test this, we have analyzed the effect of TGF-beta 1 binding on the conformational state of alpha 2M. Our results demonstrate that the binding of TGF-beta 1 to alpha 2M does not lead to the conformational change in the alpha 2M molecule that is required for the clearance of the alpha 2M.TGF-beta 1 complex via the alpha 2M receptor. Furthermore, endogenous TGF-beta 1 is associated with the conformationally unaltered slow clearance form of alpha 2M. Clearance studies in mice show that the half-life of 125I-TGF-beta 1 in the circulation (1.6 +/- 0.71 min) is not affected by blocking the alpha 2M receptor with excess conformationally altered alpha 2M. These results suggest that TGF-beta 1 is rapidly cleared from the circulation after injection by a pathway not involving alpha 2M.     

Identification of the high affinity binding site in transforming growth factor-beta involved in complex formation with alpha 2-macroglobulin. Implications regarding the molecular mechanisms of complex formation between alpha 2-macroglobulin and growth factors, cytokines, and hormones

The biological activities of transforming growth factor-beta isoforms (TGF-beta(1,2)) are known to be modulated by alpha(2)-macroglobulin (alpha(2)M). alpha(2)M forms complexes with numerous growth factors, cytokines, and hormones, including TGF-beta. Identification of the binding sites in TGF-beta isoforms responsible for high affinity interaction with alpha(2)M many unravel the molecular basis of the complex formation. Here we demonstrate that among nine synthetic pentacosapeptides with overlapping amino acid sequences spanning the entire TGF-beta(1) molecule, the peptide (residues 41-65) containing Trp-52 exhibited the most potent activity in inhibiting the formation of complexes between (125)I-TGF-beta(1) and activated alpha(2)M (alpha(2)M*) as determined by nondenaturing polyacrylamide gel electrophoresis and by plasma clearance in mice. TGF-beta(2) peptide containing the homologous sequence and Trp-52 was as active as the TGF-beta(1) peptide, whereas the corresponding TGF-beta(3) peptide lacking Trp-52, was inactive. The replacement of the Trp-52 with alanine abolished the inhibitory activities of these peptides. (125)I-TGF-beta(3), which lacks Trp-52, bound to alpha(2)M* with an affinity lower than that of (125)I-TGF-beta(1). Furthermore, unlabeled TGF-beta(3) and the mutant TGF-beta(1)W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-beta(1) in blocking I(125)-TGF-beta(1) binding to alpha(2)M*. TGF-beta(1) and TGF-beta(2) peptides containing Trp-52 were also effective in inhibiting I(125)-nerve growth factor binding to alpha(2)M*. Tauhese results suggest that Trp-52 is involved in high affinity binding of TGF-beta to alpha(2)M*. They also imply that TGF-beta and other growth factors/cytokines/hormones may form complexes with alpha(2)M* via a common mechanism involving the interactions between topologically exposed Trp and/or other hydrophobic residues and a hydrophobic region in alpha(2)M*.     

Binding of transforming growth factor-beta 1 to immobilized human alpha 2-macroglobulin.

Native alpha 2-macroglobulin (alpha 2M) and alpha 2M-methylamine were immobilized in 96-well microtiter plates. 125I-labeled transforming growth factor-beta 1 (TGF-beta 1) bound to both alpha 2M variants; however, greater binding was observed with alpha 2M-methylamine. Binding of 125I-TGF-beta 1 (0.2 nM) to immobilized alpha 2M-methylamine was inhibited by nonradiolabeled TGF-beta 1 (up to 74% with 0.4 microM TGF-beta 1). Approximately 10% of the TGF-beta 1-alpha 2M-methylamine complex was covalent. Treatment of alpha 2M-methylamine with iodoacetamide prior to immobilization completely eliminated covalent TGF-beta 1 binding; the total amount of 125I-TGF-beta 1-alpha 2M-methylamine complex detected was unchanged. The binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was not significantly inhibited by increasing the ionic strength to 1.0 M. Binding and complex dissociation were also unaffected by changes in pH within the range 6.9-8.9. Acidic pH dramatically decreased binding and promoted complex dissociation; no binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was detected at pH 3.5. The interaction of TGF-beta 1 with immobilized alpha 2M-methylamine was not significantly changed by 1.0 mM EDTA or 1.0 mM CaCl2. ZnCl2 (1.0 mM) completely eliminated binding. This result was not due to TGF-beta 1 precipitation or aggregation. Inhibition of 125I-TGF-beta 1 binding to alpha 2M-methylamine was 50% complete (IC50) with 30 microM ZnCl2. Native alpha 2M, thrombospondin, and alpha 2M-methylamine (in solution) decreased binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine. The IC50 values for these three proteins were 520, 160, and 79 nM, respectively. The TGF-beta 1-binding activity of native alpha 2M may have reflected, at least in part, trace-contamination with alpha 2M-proteinase complex.     

Specific RGTA increases collagen V expression by cultured aortic smooth muscle cells via activation and protection of transforming growth factor-beta1.

Regenerating agents (RGTA) are defined as heparan sulfate mimics, which in vivo stimulate tissue repair. RGTA are obtained by controlled grafting of carboxymethyl and sulfate groups on dextran polymers. RGTA are selected in vitro, on their ability to protect heparin binding growth factors such as TGF-beta1 for example, as well as to alter extracellular matrix biosynthesis. We had reported that RGTA were able to modulate smooth muscle cell (SMC) collagen biosynthesis. Here, we demonstrated that a specific RGTA (RG-1503), altered differentially collagen type expression by post-confluent SMC and that this action involves TGF-beta1. RG-1503 decreased, by 50%, collagen I and III biosynthesis and stimulated specifically, by twofold, collagen V biosynthesis. TGF-beta1 stimulated collagen I and V by 1.5- and threefold, respectively. A synergic action for RGTA in association with TGF-beta1 was observed specifically for collagen V expression (eightfold increase). The stimulation of collagen V biosynthesis by RGTA was abolished by TGF-beta1 neutralizing antibodies. These modulations occurred at protein and mRNA levels. RG-1503 did not alter TGF-beta1 mRNA steady state level or total TGF-beta1 protein content (latent+active forms). However, RG-1503 significantly induced an elevated proportion of active TGF-beta1 form, which could result from the selective protection from proteolytic degradation of TGF-beta1 by RG-1503. These data open a rationale for understanding the stimulation of tissue repair induced by RGTA, and also, a new insight for developing drugs adapted to inhibit excess collagen deposition in smooth muscle cells associated vascular disorder, and in fibrotic diseases.     

Lactate, alanine and glutamine metabolism in isolated canine pup liver cells

125I-Labelled alpha 2-macroglobulin-trypsin complex (125I-labelled alpha 2-macroglobulin X trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37 degrees C. The association of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin X trypsin was inhibited by unlabelled alpha 2-macroglobulin X trypsin with a half-inhibition constant of about 8 micrograms/ml (11 nM). 125I-Labelled alpha 2-macroglobulin became cell-associated to a smaller extent (10-40% of that of alpha 2-macroglobulin X trypsin) and the half-inhibition constant was about 35 micrograms/ml in adipocytes. The cell association of 125I-labelled alpha 2-macroglobulin X trypsin was markedly inhibited by dansylcadaverine, bacitracin, omission of Ca2+ from the medium or pretreatment of the cells with trypsin. After incubation for 180 min more than 60% of the cell-associated 125I-labelled alpha 2-macroglobulin X trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized material. 125I-Labelled alpha 2-macroglobulin X trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin was approx. 40% of that of 125I-labelled alpha 2-macroglobulin X trypsin. Degradation of 125I-labelled alpha 2-macroglobulin X trypsin was abolished by a high concentration (0.5 mg/ml) of alpha 2-macroglobulin X trypsin. It is concluded that alpha 2-macroglobulin X trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.     

Histidine-rich glycoprotein plus zinc reverses growth inhibition of vascular smooth muscle cells by heparin

Vascular smooth muscle cell (SMC) hyperplasia is known to be an important component in the pathogenesis of arteriosclerosis and restenosis. Although heparin has been well recognized as the representative molecule suppressing SMC growth in vitro, attempts to use heparin as a therapeutic anti-restenosis drug have not favorably influenced the angiographic or clinical outcome after angioplasty in some clinical trials. In this study, we have examined the effect of histidine-rich glycoprotein (HRG), a relatively abundant serum glycoprotein (~100 micrograms/ml in human serum), on the growth inhibition of cultured vascular SMC by heparin. Vascular SMC growth was significantly inhibited by heparin, giving nearly 85% inhibition with 100 micrograms/ml heparin. HRG reversed heparin-induced SMC growth inhibition in a dose dependent manner; 75% restoration of cell growth was observed when 100 micrograms/ml of HRG was co-added with 100 micrograms/ml heparin. Interestingly, micromolar concentrations of the zinc ion (0-10 microM), compatible with concentrations released from activated platelets, were found to enhance the restorative action of HRG. Western blot experiment demonstrated no significant amounts of the HRG moiety in fetal bovine serum, eliminating the possible contribution of contaminant HRG from culture media. These findings indicate that HRG, in combination with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explain the lack of success of heparin as a therapeutic anti-restenosis drug in clinical trials.     

Fucoidan is a non-anticoagulant inhibitor of intimal hyperplasia.

We previously reported that heparin inhibits the proliferation of fibroblasts and vascular smooth muscle cells (SMC), in part, by binding to and increasing the antiproliferative activity of transforming growth factor-beta 1 (TGF-beta 1). We now report that certain other polyanions which are structurally distinct from heparin, such as fucoidan and polyinosinic acid, are more avid ligands for TGF-beta 1 and more potent antiproliferative agents than heparin. Fucoidan possessed more potent antiproliferative activity than heparin against rat and bovine aortic SMC in vitro, though possessing much lower anticoagulant activity than heparin. Furthermore, fucoidan suppressed in vivo intimal hyperplasia when continuously infused into rats subjected to balloon-catheter injury. Unlike heparin, which also suppressed intimal hyperplasia, fucoidan did not cause systemic anticoagulation. Thus, fucoidan may be useful as a non-anticoagulant inhibitor of post-angioplasty intimal hyperplasia.     

Transforming growth factor-beta s are equipotent growth inhibitors of interleukin-1-induced thymocyte proliferation

The effects of two forms of transforming growth factor-beta, TGF-beta 1 and TGF-beta 2, upon the proliferative response of murine thymocytes were investigated in this study. TGF-beta 1 and TGF-beta 2 were found to be equipotent growth inhibitors of interleukin-1 (IL-1)- and phytohemagglutinin (PHA)-stimulated thymocytes when added at the initiation of the cultures. These factors suppressed the proliferative response in a dose-dependent fashion between 0.4 and 100 pM. The proliferative response was maximally inhibited (90% inhibition) at 100 pM. The half-maximal inhibitory dose (ID50) was 6 and 4 pM for TGF-beta 1 and TGF-beta 2, respectively. These factors were less effective or ineffective at suppressing the proliferation of thymocytes which had been prestimulated for 24 to 48 hr by IL-1 and PHA. Neither factor inhibited interleukin-2 (IL-2)-dependent thymocyte proliferation or the proliferation of an IL-2-dependent cytotoxic T cell line (CTL-L), suggesting that the anti-proliferative actions of these factors was by inhibition of cellular events triggered by IL-1. Furthermore, anti-TGF-beta 1 antibodies did neutralize the biological actions of TGF-beta 1 and these antibodies did block the binding of 125I-labeled TGF-beta 1 to cell surface receptors showing that the inhibitory action is mediated through specific receptors for TGF-beta 1 on thymocytes. These antibodies, however, did not neutralize the anti-proliferative action of TGF-beta 2. Although TGF-beta 1 and TGF-beta 2 exhibit very similar biological activities, these molecules are antigenically different and, therefore, have different tertiary structures.     

Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta

125I-Labeled human platelet-derived transforming growth factor beta (125I-TGF-beta) and human alpha 2-macroglobulin (alpha 2M) formed a complex as demonstrated by 5% native polyacrylamide gel electrophoresis. The 125I-TGF-beta.alpha 2M complex migrated at a position identical to that of the fast migrating form of alpha 2M. Most of the 125I-TGF-beta.alpha 2M complex could be dissociated by acid or urea treatment. When 125I-TGF-beta was incubated with serum, the high molecular weight form of 125I-TGF-beta could be immunoprecipitated by anti-human alpha 2M anti-sera as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 2M purified from platelet-rich plasma also showed the latent transforming growth factor activity and immunoreactivity of TGF-beta. These results suggest that TGF-beta.alpha 2M complex is a latent form of TGF-beta.     

Rat vascular smooth muscle cells immortalized with SV40 large T antigen possess defined smooth muscle cell characteristics including growth inhibition by heparin

Rat aortic smooth muscle cells (SMC) have been established by retroviral delivery of the complementary DNA (cDNA) for the simian virus 40 large T antigen (SV40LT) and examined for SMC phenotypic markers and growth characteristics, including responsiveness to the antiproliferative effects of heparin. The transfected cells (SV40LT-SMC) maintain defined SMC characteristics for more than 215 population doublings (PD) as judged by muscle-specific actin expression and growth inhibition by heparin. SV40LT-SMC greater than 129 PD become transformed while SV40LT-SMC less than 77 PD resemble nontransfected SMC morphologically and are nontumorigenic. SV40LT-SMC apparently release a growth factor which acts in an autocrine fashion, since (1) suramin inhibits SV40LT-SMC proliferation, (2) SV40LT-SMC-conditioned medium (CM) contains mitogenic activity, and (3) SV40LT-SMC CM suppresses the binding of platelet-derived growth factor to SMC. Heparin (10-100 micrograms/ml) is a potent inhibitor of both early (less than 80 PD) and late-passage (greater than 80 PD) SV40LT-SMC proliferation. The antiproliferative effects of heparin are similar to those previously observed for SMC by several criteria; the dose-response inhibition curves are indistinguishable from those obtained with nontransfected cells, other glycosaminoglycans have little effect on SV40LT-SMC growth, the antiproliferative effects of heparin are reversed in the presence of epidermal growth factor, and heparin displays high-affinity saturable binding to SV40LT-SMC. In conclusion, SV40LT-SMC are a continuous line of SMC-like cells that are sensitive to the growth inhibitor, heparin. SV40LT-SMC should facilitate studies of heparin inhibition and may be applicable for the study of other SMC characteristics as well.     

Distinct transforming growth factor-beta (TGF-beta) receptor subsets as determinants of cellular responsiveness to three TGF-beta isoforms.

Characterization of the three mammalian transforming growth factor-beta (TGF-beta) isoforms, TGF-beta 1, -beta 2, and -beta 3, indicates that TGF-beta 3 is somewhat more potent (ED50 = 0.5 pM versus 2 pM) than TGF-beta 1 and TGF-beta 2 as a growth inhibitor of the Mv1Lu mink lung epithelial cell line. In the fetal bovine heart endothelial (FBHE) cell line, however, TGF-beta 1 and -beta 3 are at least 50-fold more potent than TGF-beta 2 which is a very weak growth inhibitor (ED50 greater than or equal to 0.5 nM). Thus, as growth inhibitors, TGF-beta 1 and -beta 3 resemble each other more than TGF-beta 2. The presence of serum alpha 2-macroglobulin in the FBHE cell assays decreases the biological potency of TGF-beta s, in particular TGF-beta 2. This effect of alpha 2-macroglobulin, however, is not sufficient to explain the low responsiveness of FBHE cells to TGF-beta 2. Evaluation of the role of TGF-beta receptors as determinants of cell-specific responsiveness to TGF-beta isoforms indicates that TGF-beta 1, -beta 2, and -beta 3 have similar affinity for the membrane proteoglycan, betaglycan. They differ, however, in their ability to bind to receptor types I and II which are implicated in TGF-beta signal transduction. TGF-beta 1 is similar, albeit not identical, to TGF-beta 3 and much more potent than TGF-beta 2 as a competitor for binding to the overall population of receptors I and II in all cell lines tested. A subset of receptors I and II has been identified in Mv1Lu cells which has high affinity for TGF-beta 2 (KD approximately 10 pM) and binds this factor at concentrations that are biologically active in Mv1Lu cells. This receptor subset could not be detected in FBHE cells, suggesting that cell-specific differences in the level of high affinity of TGF-beta 2 receptors may lead to cell-specific differences in responsiveness to this isoform. Thus, despite their structural and biological similarities, TGF-beta 1, -beta 2, and -beta 3 diverge in their ability to bind to receptors in a manner that correlates with their potency as growth inhibitors.