Periprostatic adipose inflammation is associated with high-grade prostate cancer.

BACKGROUND: Obesity, a cause of subclinical inflammation, is associated with increased risk of high-grade prostate cancer (PC) and poor outcomes. Whether inflammation occurs in periprostatic white adipose tissue (WAT), and contributes to the negative impact of obesity on PC aggressiveness, is unknown. METHODS: In a single-center, cross-sectional design, men with newly diagnosed PC undergoing radical prostatectomy were eligible for study participation. The primary objective was to examine the prevalence of periprostatic WAT inflammation defined by the presence of crown-like structures (CLS-P) as detected by CD68 immunohistochemistry. Secondary objectives were to explore the clinical and systemic correlates of periprostatic WAT inflammation. Tumor characteristics and host factors including BMI, adipocyte diameter, and circulating levels of lipids, adipokines, and other metabolic factors were measured. Wilcoxon rank-sum, Chi-square, or Fisher's exact tests, and generalized linear regression were used to examine the association between WAT inflammation and tumor and host characteristics. RESULTS: Periprostatic fat was collected from 169 men (median age 62 years; median BMI 28.3). Periprostatic WAT inflammation was identified in 49.7% of patients and associated with higher BMI (P=0.02), larger adipocyte size (P=0.004) and Gleason grade groups IV/V tumors (P=0.02). The relationship between WAT inflammation and high Gleason grade remained significant after adjusting for BMI (P=0.04). WAT inflammation correlated with higher circulating levels of insulin, triglycerides, and leptin/adiponectin ratio, and lower high density lipoprotein cholesterol, compared to those without WAT inflammation (P's <0.05). CONCLUSION: Periprostatic WAT inflammation is common in this cohort of men with PC and is associated with high-grade PC.

Plasminogen-mediated matrix invasion and degradation by macrophages is dependent on surface expression of annexin II.

Genetic evidence demonstrates the importance of plasminogen activation in the migration of macrophages to sites of injury and inflammation, their removal of necrotic debris, and their clearance of fibrin. These studies identified the plasminogen binding protein annexin II on the surface of macrophages and determined its role in their ability to degrade and migrate through extracellular matrices. Calcium-dependent binding of annexin II to RAW264.7 macrophages was shown using flow cytometry and Western blot analysis of EGTA eluates. Ligand blots demonstrated that annexin II comigrates with one of several proteins in lysates and membranes derived from RAW264.7 macrophages that bind plasminogen. Preincubation of RAW264.7 macrophages with monoclonal anti-annexin II IgG inhibited (35%) their binding of 125I-Lys-plasminogen. Likewise, plasmin binding to human monocyte-derived macrophages and THP-1 monocytes was inhibited (50% and 35%, respectively) when cells were preincubated with anti-annexin II IgG. Inhibition of plasminogen binding to annexin II on RAW264.7 macrophages significantly impaired their ability to activate plasminogen and degrade [3H]-glucosamine-labeled extracellular matrices. The migration of THP-1 monocytes through a porous membrane, in response to monocyte chemotactic protein-1, was blocked when the membranes were coated with extracellular matrix. The addition of plasminogen to the monocytes restored their ability to migrate through the matrix-coated membrane. Preincubation of THP-1 monocytes with anti-annexin II IgG inhibited (60%) their plasminogen-dependent chemotaxis through the extracellular matrix. These studies identify annexin II as a plasminogen binding site on macrophages and indicate an important role for annexin II in their invasive and degradative phenotype.

A mediator of cell surface-specific plasmin generation.

It has become increasingly evident that the generation of cell surface proteases including plasmin is fundamental to a wide variety of in vivo biological processes. Cell surface receptors allow for specific controlled proteolysis, provide protection from inhibitors, and enhance catalytic efficiency. Here we describe one such receptor, annexin II, which serves as a coreceptor for tissue plasminogen activator and plasminogen and is found on a wide variety of cell types including endothelial cells, some tumor cells, monocytes and macrophages, and neuronal cells. Evidence indicates that annexin II may be crucial to the efficient generation of cell surface plasmin, endothelial cell formation of new blood vessels, and maintenance of vascular patency. Additionally, it has been shown that annexin II expression in acute promyelocytic leukemia contributes to the bleeding diathesis seen in this disease and that inhibition of annexin II may be an important mechanism in the formation of atherosclerotic plaque. Furthermore, emerging evidence reveals the importance of annexin II on the surface of monocytes and macrophages, where it may contribute to the cells' ability to degrade extracellular matrix proteins and migrate to sites of injury or inflammation.

Selective activation of MAPK(erk1/2) by laminin-1 peptide alpha1:Ser(2091)-Arg(2108) regulates macrophage degradative phenotype.

Components of the extracellular matrix contain cryptic domains, which are exposed by proteolysis and elicit biological responses distinct from intact molecules. The disparate cellular response to extracellular matrix fragments and parent intact molecules suggests differential recognition and signaling pathways. In experiments reported here, we demonstrate that urokinase and matrix metalloproteinase-9 expression by RAW264.7 macrophages is stimulated by a synthetic laminin peptide derived from the alpha1-chain (SRARKQAASIKVAVSADR), whereas intact laminin-1 has no effect on proteinase expression by macrophages. Incubation of macrophages with alpha1:SRARKQAASIKVAVSADR stimulates tyrosine phosphorylation of several proteins including mitogen-activated protein kinase (MAPK)(erk1/2). In contrast, neither intact laminin-1 nor the beta1-chain peptide CDPGYIGSR stimulated protein tyrosine phosphorylation in these cells. Inhibition of tyrosine kinases or protein kinase C blocked alpha1-chain peptide-induced phosphorylation of MAPK(erk1/2) and the up-regulation of steady state levels of urokinase mRNA and matrix metalloproteinase-9 activity. A MAPK kinase inhibitor blocked alpha1-chain-induced phosphorylation of MAPK(erk1/2) and the induction of proteinase expression. Intact laminin-1, which was unable to induce macrophage proteinase expression, failed to stimulate the phosphorylation of MAPK(erk1/2). These data demonstrate that incubation of macrophages with alpha1:SRARKQAASIKVAVSADR, but not intact laminin-1, triggers protein kinase C-dependent activation of MAPK(erk1/2), leading to the up-regulation of proteinase expression.

Macrophage formation of angiostatin during inflammation. A byproduct of the activation of plasminogen.

Angiostatin is a potent inhibitor of tumor angiogenesis and the growth of metastatic foci. Recent studies have indicated that neoplastic cells can generate angiostatin directly or in cooperation with tumor-associated macrophages. In studies reported here, we determined whether angiostatin is generated in mice under non-neoplastic settings. Utilizing murine RAW264.7 macrophages and thioglycollate-elicited peritoneal macrophages, we demonstrate that angiostatin-like fragments are generated as a byproduct of the proteolytic regulation of membrane-bound plasmin. Plasmin proteolysis and subsequent loss in membrane-bound plasmin activity requires active plasmin but was unaffected by inhibitors of metalloproteinases. Lysine binding fragments of plasmin, isolated from macrophage-conditioned media utilizing affinity chromatography, appeared as a major (48 kDa) and two minor bands (42 and 50 kDa) in SDS-polyacrylamide gel electrophoresis and were immunoreactive with anti-kringle 1-3 IgG. Each peptide begins with Lys77 and contains the entire sequence of angiostatin. The affinity isolated plasmin fragments inhibited bFGF-induced endothelial cell proliferation. Lavage fluid recovered from the peritoneal cavities of mice previously injected with thioglycollate contained angiostatin-like plasmin fragments similar to those generated in vitro. This is the first demonstration that angiostatin-like plasmin fragments are generated in a non-neoplastic inflammatory setting. Thus, in addition to regulating pericellular plasmin activity, proteolysis of plasmin generates inactive kringle-containing fragments expressing angiostatic properties.

Ligand binding to macrophage scavenger receptor-A induces urokinase-type plasminogen activator expression by a protein kinase-dependent signaling pathway.

Macrophage scavenger receptor-type A (MSR-A) has been implicated in the transmission of cell signals and the regulation of diverse cellular functions (Falcone, D. J., and Ferenc, M. J. (1988) J. Cell. Physiol. 135, 387-396; Falcone, D. J., McCaffrey, T. A., and Vergilio, J. A. (1991) J. Biol. Chem. 266, 22726-22732; Palkama, T. (1991) Immunology 74, 432-438; Krieger, M., and Herz, J. (1994) Annu. Rev. Biochem. 63, 601-637); however, the signaling mechanisms are unknown. In studies reported here, we demonstrate that binding of both lipoprotein and non-lipoprotein ligands to MSR-A induced protein tyrosine phosphorylation and increased protein kinase C (PKC) activity leading to up-regulated urokinase-type plasminogen activator (uPA) expression. Specifically, the binding of acetylated low density lipoprotein and fucoidan to MSR-A in human THP-1 macrophages triggered tyrosine phosphorylation of many proteins including phospholipase C-gamma1 and phosphatidylinositol-3-OH kinase. Inhibitors of tyrosine kinase dramatically reduced MSR-induced protein tyrosine phosphorylation and PKC activity. Moreover, inhibitors of tyrosine kinase and PKC reduced uPA activity expressed by THP-1 macrophages exposed to MSR-A ligands. The intracellular signaling response for tyrosine phosphorylation following ligand binding was further demonstrated by using the stable MSR-transfected Bowes cells that express surface MSR-A. These findings establish for the first time a signaling pathway induced by ligand binding to MSR-A and suggest a molecular model for the regulation of macrophage uPA expression by specific ligands of the MSR-A.

Role of laminin in matrix induction of macrophage urokinase-type plasminogen activator and 92-kDa metalloproteinase expression.

Urokinase-type plasminogen activator (uPA) and 92-kDa matrix metalloproteinase (MMP-9) expression by RAW264.7 macrophages were up-regulated when plated on extracellular matrices. Collagen IV, fibronectin, and tenascin stimulated macrophages' MMP-9 expression. In contrast, laminin stimulated both uPA and MMP-9 expression in a dose- and time-dependent manner. The increase in macrophage uPA activity was preceded by an increase in their steady state levels of uPA mRNA. Laminin-induced uPA expression was most pronounced in RAW264.7 macrophages followed by THP-1 monocytes, J774A.1 macrophages, and bone marrow-derived macrophages. Neither laminin nor matrix induced alterations in THP-1 monocyte expression of plasminogen activator inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-2. Synthetic laminin peptides were utilized to identify the laminin domain(s) responsible for induction of uPA expression. Peptides derived from the beta1 chain of laminin had no effect on macrophage uPA expression, whereas SIKVAV, derived from alpha1 chain, stimulated uPA expression 20-fold. Preincubation of THP-1 monocytes with a monoclonal antibody directed against the alpha6 subunit of the alpha6beta1 laminin receptor blocked matrix induction of uPA without affecting the induction of MMP-9. These results demonstrate that macrophage binding to laminin plays an important role in the regulation of their degradative phenotype via the up-regulation of uPA and MMP-9.

Health care reform? An American obsession with prescriptive incrementalism.

A rounded evaluation of the national health insurance proposals that now seem to be taken seriously by political elites requires conceptual organization. This article adopts a typology that describes each major proposal as a social, mixed or a private insurance scheme depending on the source(s) of funding, method of compensating hospitals and physicians, the unit of payment, and mechanism for financing capital. Not surprisingly, the analysis suggests that the social insurance model, closely resembling the Canadian system, is more likely to control inflation and redress distributional inequities than are other approaches. Why, then, has this approach not been adopted? The answer may be found in the widespread acceptance of disjointed incrementalism as a valid description of the policy process which yields an ideological orientation that can be termed "prescriptive incrementalism." This orientation is closely related to a belief in an "American exceptionalism," a belief that is not warranted by a cross-sectional examination of the political culture infusing issues about the proper role of government in health care financing and delivery. Unfortunately for advocates, the truly exceptional factor restricting the United States' ability to effect national health reform is a quite delberately obstruction-oriented political structure.

Roles of hospital administrators in South Carolina.

Using discriminant analyses of data on 916 returned questionnaires from a mailing to 1,650 administrators in 82 South Carolina hospitals, this study examines the allocation of interpersonal, informational, decisional, and treatment roles among executive, administrative, and clinical directors. Educational attainment, years of experience, and gender were found to influence respondents' positions. Results also indicate that executive directors assume responsibility for the organization and its relation to the environment. As expected, those in clinical and administrative positions assume more responsibility for interpersonal and treatment roles than do executive directors.

Decreased type II/type I TGF-beta receptor ratio in cells derived from human atherosclerotic lesions. Conversion from an antiproliferative to profibrotic response to TGF-beta1.

Atherosclerosis and postangioplasty restenosis may result from abnormal wound healing. The present studies report that normal human smooth muscle cells are growth inhibited by TGF-beta1, a potent wound healing agent, and show little induction of collagen synthesis to TGF-beta1, yet cells grown from human vascular lesions are growth stimulated by TGF-beta1 and markedly increase collagen synthesis. Both cell types increase plasminogen activator inhibitor-1 production, switch actin phenotypes in response to TGF-beta1, and produce similar levels of TGF-beta activity. Membrane cross-linking of 125I-TGF-beta1 indicates that normal human smooth muscle cells express type I, II, and III receptors. The type II receptor is strikingly decreased in lesion cells, with little change in the type I or III receptors. RT-PCR confirmed that the type II TGF-beta1 receptor mRNA is reduced in lesion cells. Transfection of the type II receptor into lesion cells restores the growth inhibitory response to TGF-beta1, implying that signaling remains responsive. Because TGF-beta1 is overexpressed in fibroproliferative vascular lesions, receptor-variant cells would be allowed to grow in a slow, but uncontrolled fashion, while overproducing extracellular matrix components. This TGF-beta1 receptor dysfunction may be relevant for atherosclerosis, restenosis and related fibroproliferative diseases.

THP-1 macrophage membrane-bound plasmin activity is up-regulated by transforming growth factor-beta 1 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-beta 1 (TGF-beta 1) on uPA, uPA receptor, and plasminogen receptor expression by human THP-1 macrophage was examined. TGF-beta 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-beta 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-beta 1. The differential response exhibited by suspension and adherent THP-1 cells may reflect differences in their expression of TGF-beta 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-beta 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-beta 1. The increase in membrane-uPA activity expressed by TGF-beta 1-treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF-beta 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-beta 1-treated cells. Following incubation with plasminogen, membrane-bound plasmin activity increased three-fold in TGF-beta 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-beta 1 treatment. We conclude that TGF-beta 1 stimulates membrane-bound plasmin activity, without affecting plasminogen receptor expression, through the up-regulation of uPA and the uPA receptor expression.

Specific inhibition of eIF-5A and collagen hydroxylation by a single agent. Antiproliferative and fibrosuppressive effects on smooth muscle cells from human coronary arteries.

Restenosis occurs in 35% of patients within months after balloon angioplasty, due to a fibroproliferative response to vascular injury. These studies describe a combined fibrosuppressive/antiproliferative strategy on smooth muscle cells cultured from human primary atherosclerotic and restenotic coronary arteries and from normal rat aortas. L-Mimosine suppressed the posttranslational hydroxylation of the precursors for collagen and for eukaryotic initiation factor-5A (eIF-5A) by directly inhibiting the specific protein hydroxylases involved, prolyl 4-hydroxylase (E.C. 1.14.11.2) and deoxyhypusyl hydroxylase (E.C. 1.14.99.29), respectively. Inhibition of deoxyhypusyl hydroxylation correlated with a dose-dependent inhibition of DNA synthesis. Inhibition of prolyl hydroxylation caused a dose-dependent reduction in the secretion of hydroxyproline-containing protein and decreased the formation of procollagen types I and III. The antifibroproliferative action could not be attributed to nonspecific or toxic effects of mimosine, appeared to be selective for the hydroxylation step in the biosynthesis of the procollagens and of eIF-5A, and was reversible upon removal of the compound. The strategy of targeting these two protein hydroxylases has important implications for the pathophysiology of restenosis and for the development of agents to control fibroproliferative diseases.

Progress toward Healthy People 2000: a preliminary look at Oklahoma.

Over 10,000 Oklahomans die each year from coronary artery disease or stroke. This study examined the behavioral risk factors for these illnesses present in Oklahomans. Oklahomans are at considerably higher risk than the desirable national goals for such risk factors. For example, too many Oklahomans smoke and too few exercise. The data to support these findings are included herein, and some steps to reverse these trends are recommended.

Regulation of macrophage receptor-bound plasmin by autoproteolysis.

The activation of plasminogen by macrophage is regulated by their expression of receptors for urokinase and plasmin(ogen). In these studies we have examined plasmin(ogen) binding to adherent human THP-1 macrophage. Plasmin bound to the THP-1 cells in a time- and dose-dependent manner (Kd 15.8 +/- 6.2 nM; Bmax 1.4 +/- 0.3 x 10(6)/cell). The lysine analog epsilon-aminocaproic acid competitively inhibited plasmin binding. The fraction of membrane-bound plasmin, however, became increasingly resistant to displacement with epsilon-aminocaproic acid. Over a 24-h period, membrane-bound plasmin activity fell 80% despite the presence of catalytically active plasmin in the incubation media. The loss of receptor-bound plasmin activity was not due to proteolytic alterations of its receptor since 125I-Lys-plasminogen bound to THP-1 cells pretreated with plasmin with similar affinity as to untreated cells. Following a 24-h incubation of 125I-Lys-plasminogen or 125I-plasmin with THP-1 cells, several degradative fragments were apparent in their conditioned media. The smaller degradative fragments (28 and 36 kDa) lacked cell binding activity and were demonstrated to be active by casein-zymography. A 48-kDa fragment bound to cells in a lysine-dependent manner but was not active. In contrast, phenylmethylsulfonyl fluoride-inactivated 125I-plasmin retained its binding activity over 24 h, and degradative fragments were not present in the conditioned media. The binding of 125I-Lys-plasmin(ogen) to THP-1 cells was also examined in the presence of excess alpha 2 plasmin inhibitor. Despite the absence of fluid-phase plasmin activity, membrane-bound 125I-Lys-plasmin(ogen) decreased over 24 h. At 24 h a radiolabeled 48-kDa fragment was observed in the conditioned media and together with 125I-Lys-plasmin(ogen) was bound to cells. Unlike 125I-Lys-plasmin, the 48-kDa fragment did not form a complex with alpha 2 plasmin inhibitor. Thus, autoproteolysis of receptor-bound plasmin results in fragments with truncated physiologic properties that possess either cell binding or catalytic activities. We propose that autoproteolysis is a mechanism for regulating membrane-bound plasmin activity.

Protection of transforming growth factor-beta 1 activity by heparin and fucoidan.

The transforming growth factor-beta (TGF-beta) 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-beta activity in vitro or in vivo. Our previous work indicated that 1) TGF-beta 1 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-beta 1 to alpha 2-macroglobulin (alpha 2-M). The present studies investigated the influence of heparin-like molecules on the stability of the TGF-beta 1 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-beta 1 and activated alpha 2-M. Electrophoresis of 125I-TGF-beta 1 showed that fucoidan protects TGF-beta 1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF-beta derived from vascular smooth muscle cells (SMC), plasmin degraded acid-activated TGF-beta, and purified TGF-beta 1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half-life of 125I-TGF-beta 1 and doubled the amount of cell-associated 125I-TGF-beta 1. Consistent with this protective effect, heparin- and fucoidan-treated SMC demonstrated elevated levels of active, but not latent, TGF-beta activity.

Local factors affecting the tendency to bypass local hospitals for inpatient mental health care: an exploratory analysis.

Using the records of 2,171 rural residents of Illinois who received inpatient treatment for mental illness or substance abuse, this paper examines factors that influence the tendency to seek service from a distant rather than a local hospital. Results indicate that the age and insurance coverage of the individual, the per capita income of the community area, surrogates for the service orientation of the local hospital and the proximity of the patient's residence to an urban center are significant influences. With the exceptions of drug abuse requiring detoxification or other symptomatic treatment, drug abuse accompanied by comorbidity and psychosocial disorders, psychosis, and childhood disorders, the primary diagnosis of the individual failed to have a significant effect on the propensity to bypass local sources of inpatient treatment.

Macrophage and foam cell release of matrix-bound growth factors. Role of plasminogen activation.

We have determined whether macrophage derived-foam cells, a prominent component of the atherosclerotic lesion, express more urokinase-type plasminogen activator (uPA) and whether their ability to generate plasmin stimulates the release of matrix-bound growth factors. Steady state levels of uPA mRNA and both membrane and intracellular uPA activities were significantly increased in foam cells. When cultured on cell-derived matrices containing bound 125I-basic fibroblast growth factor (bFGF), both macrophage and foam cells released intact 125I-bFGF into their media. The release of 125I-bFGF by either cell was significantly enhanced in the presence of plasminogen. However, foam cells, which expressed more membrane uPA, released more 125I-bFGF than control cells. The release of matrix-bound bFGF was independent of heparanase activity, since neither macrophage nor foam cells degraded 35SO4-labeled heparan sulfate proteoglycans. In addition, media derived from foam cells cultured on cell-derived matrices in the presence of plasminogen had increased levels of transforming growth factor (TGF) beta activity as compared to cells grown in the absence of plasminogen. In contrast, plasminogen had no effect on TGF-beta activity recovered in the media of foam cells grown on plastic. Moreover, when macrophage were cultured on matrices containing bound 125I-TGF-beta, the release of labeled TGF-beta was increased in the presence of plasminogen. This is the first demonstration that foam cells can release two important growth regulators, bFGF and TGF-beta, from the extracellular matrix, and provides a mechanism by which macrophage and foam cells can stimulate atherosclerotic lesion development.

Transforming growth factor-beta 1 stimulates macrophage urokinase expression and release of matrix-bound basic fibroblast growth factor.

Macrophage expression of urokinase-type plasminogen activator (uPA) appears to play a role in their release of matrix-bound basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta). In experiments reported here, we have examined the potential regulatory effects of bFGF and TGF-beta 1 on macrophage uPA expression. TGF-beta 1 stimulated in a dose- and time-dependent manner the expression of secreted membrane and intracellular uPA activities by a macrophage cell line (RAW264.7). When examined at similar concentrations, bFGF had little effect, and interleukin-1 alpha, tumor necrosis factor-alpha, and monocyte colony stimulating factor had no effect on macrophage uPA expression. Exposure of macrophages to TGF-beta 1 led to a rapid and sustained increase in the steady-state levels of uPA mRNA that was independent of de novo protein synthesis and was completely inhibited by actinomycin D. However, the TGF-beta 1-induced increase in uPA mRNA was largely unaffected by subsequent incubation of cells with actinomycin D. The protein kinase C inhibitor H7 markedly reduced the ability of TGF-beta 1 to stimulate expression of uPA activity. Likewise, okadaic acid and microcystin, inhibitors of serine/threonine phosphatases, potentiated the ability of TGF-beta 1 to upregulate macrophage uPA expression. TGF-beta 1 primed cells converted nearly all added plasminogen to plasmin and expressed sixfold more membrane-bound plasmin than control cells. Preincubation of TGF-beta 1 with either serum or methylamine-modified alpha 2-macroglobulin did not affect its ability to induce macrophage uPA expression. When control and TGF-beta 1-primed macrophages were cultured on matrices containing bound 125I-bFGF, their release of 125I-bFGF was increased five and tenfold, respectively, in the presence of plasminogen. The ability of TGF-beta to induce macrophage uPA expression and the plasmin-dependent release of matrix-bound bFGF may provide an indirect mechanism by which TGF-beta stimulates angiogenesis.

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.