Faculty Bibliography

Culture medium conditioned by human SK-Hep1 hepatoma cells or mouse S180 sarcoma cells rapidly up-regulates endothelial cell expression of basic fibroblast growth factor (bFGF) and induces formation of capillary-like structures by vascular endothelial cells grown on three-dimensional fibrin gels (in vitro angiogenesis). Incubation of endothelial cells with the tumor cell-conditioned media also results in increased expression of urokinase plasminogen activator (uPA), a key component of the proteolytic system required for cell invasion and capillary formation. Although the tumor cell-conditioned media contain no bFGF, addition of anti-recombinant bFGF IgG abolishes the up-regulation of uPA and blocks in vitro angiogenesis. This indicates that both the increase in uPA production and formation of capillary-like structures are mediated by endogenous bFGF expressed by the endothelial cells. Both the bFGF/uPA-inducing activity and the angiogenic activity of SK-Hep1 cell-conditioned medium copurify with a relatively acid-resistant peptide that has moderate affinity for heparin and M(r) < 18 kDa > 3.5 kDa. Known cytokines with similar biochemical features do not possess the same biological activity. These findings indicate that angiogenesis can be mediated by endothelial cell bFGF through an autocrine mechanism and that the bFGF-inducing peptide may represent a novel tumor-derived angiogenic factor that modulates in endothelial cells the concerted expression of cytokines and proteolytic enzymes required for capillary formation

Multiple forms of FGF-2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF-2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF-2 as the AUG codon is 3' to the CUG codons. Although the 18 kDa form FGF-2 is localized primarily in the cytosol, a significant fraction of the HMW FGF-2 has a nuclear location. The nuclear localization of HMW FGF-2 is determined by amino acid residues in the amino-terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino-terminal extension of HMW FGF-2. The nuclear localization of HMW FGF-2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF-2, or all forms of FGF-2, we have found that HMW FGF-2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF-2. These cells are transformed by what appears to be the intracellular action of HMW FGF-2. The interaction of FGF-2 with heparin has also been examined.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND. Basic fibroblast growth factor (bFGF) is a potent local promoter of vascular smooth muscle cell migration and proliferation and may play a major role in the pathogenesis of intimal fibromuscular lesions. Preliminary studies have shown that exogenous bFGF localizes to injured rabbit aorta and suggest that this interaction might be inhibited by anti-bFGF immunoglobulin (Ig) G. This study was designed to evaluate the possible role of polyclonal anti-bFGF IgG in reducing intimal fibromuscular lesion formation in the injured rabbit aorta. METHODS. The abdominal aortic endothelium was subjected to balloon injury in 13 rabbits. Six rabbits received intravenous rabbit anti-bFGF IgG, and seven received irrelevant rabbit IgG (16 micrograms/kg) 30 minutes before injury and daily for 5 days after injury. At 14 days after injury the aorta was fixed and sectioned, and the intimal and medial areas were measured by computerized digital morphometry with the intimal/medial ratio as an index of neointimal lesion formation. RESULTS. In the control group the intimal/medial ratio was 0.538 +/- 0.046 (mean +/- SEM), which was significantly greater than the anti-bFGF-treated group value of 0.148 +/- 0.021 (p < 0.001). CONCLUSIONS. These results show that daily doses of intravenous polyclonal anti-bFGF IgG for 5 days after balloon aortic injury significantly inhibit intimal fibromuscular lesion formation at 14 days. The results suggest that the process of intimal fibromuscular lesion formation may be susceptible to modification by antagonists to bFGF

Neointimal hyperplasia limits the long-term patency of saphenous vein grafts (SVGs), but is notably absent from most internal mammary artery (IMA) grafts. Basic fibroblast growth factor (bFGF) is a local endothelial and vascular smooth muscle mitogen known to be involved in the pathogenesis of neointimal hyperplasia. This study used an animal model to compare the number of available high-affinity (HAR) and low-affinity (LAR) bFGF receptors in SVGs and IMA grafts and to determine whether distention injury causes an increase in receptor availability. The IMA and SVG specimens were harvested from 12 dogs and distended at 25 or 200 mm Hg for 15 minutes, and then the bFGF receptor uptake was measured in them using iodine 125-labeled bFGF. In the IMA conduits distended at low pressure, there were 2.54 +/- 0.10 (mean +/- standard error of the mean) HARs per mm2 of intimal surface area available and 5.19 +/- 0.40 LARs per mm2. High-pressure distention significantly (p < 0.001) increased the number of available HARs to 5.06 +/- 0.27 per mm2 and of LARs to 7.27 +/- 0.042 per mm2. At low pressure, the SVGs had significantly (p < 0.001) more HARs (9.14 +/- 0.84 per mm2) and LARs (18.2 +/- 0.57 per mm2) available than did the IMA conduits, and high pressure significantly (p < 0.001) increased the number of HARs available in SVGs to 24.1 +/- 2.43 per mm2 and the number of LARs to 44.7 +/- 2.34 per mm2.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic fibroblast growth factor (bFGF) is a hematopoietic cytokine that stimulates stromal and stem cell growth. It binds to a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan on human bone marrow (BM) stromal cells. The bFGF-proteoglycan complex is biologically active and is released by addition of exogenous phosphatidylinositol-specific phospholipase C. In this study, we show the presence of an endogenous GPI-specific phospholipase D (GPI-PLD) that releases the bFGF-binding heparan sulfate proteoglycan and the variant surface glycoprotein (a model GPI-anchored protein) from BM cultures. An involvement of proteases in this process is unlikely, because released proteoglycan contained the GPI anchor component, ethanol-amine, and protease inhibitors did not diminish the release. The mechanism of release is likely to involve a GPI-PLD and not a GPI-specific phospholipase C, because the release of variant surface glycoprotein did not reveal an epitope called the cross-reacting determinant that is exposed by phospholipase C-catalyzed GPI anchor cleavage. In addition, phosphatidic acid (which is specifically a product of GPI-PLD-catalyzed anchor cleavage) was generated during the spontaneous release of the GPI-anchored variant surface glycoprotein. We also detected GPI-PLD-specific enzyme activity and mRNA in BM cells. Therefore, we conclude that an endogenous GPI-PLD releases bFGF-heparan sulfate proteoglycan complexes from human BM cultures. This mechanism of GPI anchor cleavage could be relevant for mobilizing biologically active bFGF in BM. An endogenous GPI-PLD could also release other GPI-anchored proteins important for hematopoiesis and other physiologic processes

Although many glycosylphosphatidylinositol (GPI)-anchored proteins have been observed as soluble forms, the mechanisms by which they are released from the cell surface have not been demonstrated. We show here that a cell-associated GPI-specific phospholipase D (GPI-PLD) releases the GPI-anchored, complement regulatory protein decay-accelerating factor (DAF) from HeLa cells, as well as the basic fibroblast growth factor-binding heparan sulfate proteoglycan from bone marrow stromal cells. DAF found in the HeLa cell culture supernatants contained both [3H]ethanolamine and [3H]inositol, but not [3H]palmitic acid, whereas the soluble heparan sulfate proteoglycan present in bone marrow stromal cell culture supernatants contained [3H]ethanolamine. 125I-labeled GPI-DAF incorporated into the plasma membranes of these two cell types was released in a soluble form lacking the fatty acid GPI-anchor component. GPI-PLD activity was detected in lysates of both HeLa and bone marrow stromal cells. Treatment of HeLa cells with 1,10-phenanthroline, an inhibitor of GPI-PLD, reduced the release of [3H]ethanolamine-DAF by 70%. The hydrolysis of these GPI-anchored molecules is likely to be mediated by an endogenous GPI-PLD because [3H]ethanolamine DAF is constitutively released from HeLa cells maintained in serum-free medium. Furthermore, using PCR, a GPI-PLD mRNA has been identified in cDNA libraries prepared from both cell types. These studies are the first demonstration of the physiologically relevant release of GPI-anchored proteins from cells by a GPI-PLD

The role of receptor-bound urokinase-type plasminogen activator (uPA) in cellular activation of latent transforming growth factor-beta (LTGF-beta) 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 neomycin-resistance 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-beta 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-beta 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-beta abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF-beta. 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-beta 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-beta IgG indicated that the inhibition was due to TGF-beta. 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-beta as assayed by both the BAE migration and PA assays, presumably because it interfered with cell-surface localization of LTGF-beta. 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-beta. These results support the conclusion that plasmin-dependent activation LTGF-beta by LB6 cells is promoted by the surface localization of uPA by its receptor

The role of heparin or heparan sulfates in the interaction of basic fibroblast growth factor (bFGF) with its high affinity receptor were investigated using purified extracellular ligand-binding region of FGF receptor-1 (FGFR-1) and intact receptors expressed in a myeloid cell line (32D) that does not express detectable levels of heparan sulfate proteoglycans or in Chinese hamster ovary (CHO) cell mutants defective in heparan sulfate synthesis. The purified extracellular domain of FGFR-1 formed complexes with 125I-bFGF both in the presence or absence of heparin. Intact FGFR-1 expressed in 32D cells also bound the same amount of 125I-bFGF in the presence or absence of heparin when saturating concentrations of bFGF were used. Varying the concentration of 125I-bFGF showed that heparin increased the amount of 125I-bFGF bound at low bFGF concentrations and increased the affinity of bFGF for its receptor by about 3-fold. To eliminate the possibility of alteration of bFGF properties through the chemical modification reactions, bFGF was labeled biosynthetically. The binding of biosynthetically labeled bFGF to FGFR-1 also did not require heparin. When FGFR-1 or FGFR-2 were expressed in mutant CHO cells deficient in heparan sulfate synthesis, the cells also bound 125I-bFGF in the absence of heparin, and the addition of heparin increased the affinity of bFGF for its receptors 2-3-fold. Thus, heparin or heparan sulfate is not required for the binding of bFGF to its receptors but increases the binding affinity to a moderate degree. Finally, the requirement for heparin in signal transduction through the receptor was investigated. Expression of c-fos mRNA was induced by bFGF in 32D cells expressing FGFR-1 to the same extent in the presence or absence of heparin

Transforming growth factor-beta (TGF-beta) is a potent regulator of cellular differentiation, proliferation, migration, and protein expression. These properties have been exploited to create a variety of bioassays for detecting the mature growth factor. In this paper, we describe a highly sensitive and specific, nonradioactive quantitative bioassay for TGF-beta based on its ability to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mink lung epithelial cells (MLEC) were stably transfected with an expression construct containing a truncated PAI-1 promoter fused to the firefly luciferase reporter gene. Addition of TGF-beta (0.2 to > 30 pM) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. Although responsive to TGF-beta, this promoter fragment was only minimally influenced by other known inducers of PAI-1 expression. When compared to the widely used MLEC assay, this assay demonstrated greater sensitivity and specificity, allowing quantification of TGF-beta in complex biological solutions