Bovine fibroblast growth factor: comparison of brain and pituitary preparations

Bovine brain and pituitary fibroblast growth factors (FGF) have been compared with regard to their chemical and biological properties. Pituitary and one preparation of brain FGF (Prep A) contain a basic mitogenic activity, which migrates to the same position on electrophoresis in acid pH gels as detected by incorporation of [methyl- 3H]-thymidine into BALB/c 3T3 cells. In contrast, another preparation of brain FGF (Prep B) contains two mitogens, one (20-30%) indistinguishable from the basic components in pituitary and brain (Prep A) FGF preparations and an acidic activity (70-80%), pl 5-6, that migrates more slowly on acid gels, corresponding to the acidic component of brain FGF described previously (Thomas, K. A., M. C. Riley, S. K. Lemmon, N. C. Baglan, and R. A. Bradshaw. 1980. J. Biol. Chem. 255:5517-5520.) In agreement with that report, none of the mitogens comigrates with fragments of myelin basic protein. Pituitary FGF was virtually inactive, brain (Prep A) FGF had a small amount of activity, and brain (Prep B) FGF was highly potent (50% maximal stimulation at 15-30 ng/ml) in stimulating the growth of human umbilical vein endothelial (HUVE) cells. The acidic component of brain FGF, which is much more unstable at pH 8.5 than the basic one, can be protected by reducing agents, whereas the basic constituent of brain FGF as well as pituitary FGF is unaffected by reducing conditions. Thus, brain FGF preparations may contain two distinct mitogenic activities, one that is acidic and contains HUVE cell activity, and a basic mitogen that is similar to and may be identical with pituitary FGF.     

Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.     

Identification of the fibroblast growth factor receptor in human vascular endothelial cells

The fibroblast growth factor (FGF) receptor of human umbilical vein-derived endothelial (HUE) cells has been identified by affinity labeling. It has an apparent molecular weight of 130,000. It binds both basic and acidic FGF, but not with epidermal growth factor, insulin, or transferrin. The lectin concanavalin-A does not inhibit the binding of ¹²⁵l-bFGF to HUE cell-surface receptors, whereas it inhibits bFGF binding to BHK-21 cell-surface FGF receptor. This suggests that both types of receptors may differ in their degree of glycosylation. In contrast to other cell types, heparin only slightly inhibits the binding of basic FGF to its receptor. Protamine sulfate, which is anti-angiogenic in vivo, and suramin, a drug used in the therapy of trypanosomiasis and onchocerciasis, also inhibit the binding of basic FGF to the receptor.     

Control of proliferation of bovine vascular endothelial cells.

The effects of Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF) on the proliferation of bovine vascular endothelial cells has been examined. FGF induces the initiation of DNA synthesis and cell proliferation in cloned endothelial cells of fetal and adult origin at concentrations as low as 1 ng/ml and is saturating at 50 ng/ml. EGF had no effect over the same range of concentrations. The mitogenic effect of FGF is blocked by a crude extract of cartilage. Platelet extract is also mitogenic for vascular endothelial cells although to a lesser extent than the purified FGF. In contrast to vascular endothelial cells, both EGF and FGF are mitogenic for vascular smooth muscle cells although EGF is less mitogenic than FGF at 100 ng/ml. The mitogenic effect of EGF and FGF on vascular smooth muscle is not blocked by the addition of a crude extract of cartilage, thus demonstrating the specificity of the chalone like effect of cartilage crude extract for endothelial cells.     

Effect of substrata and fibroblast growth factor on the proliferation in vitro of bovine aortic endothelial cells

The hypothesis that, in the case of clonal or low-density cultures, cells which do not readily proliferate are those that do not produce an extracellular matrix (ECM), while those that proliferate actively are cells that have retained their ability to produce it, has been tested using low-density vascular endothelial cell cultures maintained on either plastic or ECM-coated dishes and exposed to various combinations of media and sera. Proliferation of low-density vascular endothelial cell cultures seeded on plastic and exposed to DMEM, RPMI-1640, or medium 199 plus thymidine is a function of the batch of calf serum used to supplement the various media. In all three cases, such cultures proliferated at a slow rate and fibroblast growth factor (FGF) greatly accelerated their proliferation. In contrast, when similar cultures were seeded on ECM-coated dishes, they actively proliferated regardless of the batch of calf serum to which they were exposed. FGF was no longer required in order for cultures to become confluent. In the case of cultures exposed to RPMI-1640 or medium 199 plus thymidine, it was even toxic. When cultures were exposed to either medium 199 or Waymouth medium, cells did not proliferate, regardless of the substrate (either plastic or ECM) upon which they were maintained and of the batch of serum to which they were exposed. Addition of FGF to such media had no effect. It is therefore likely that nutrient limitations in both of these media restrict the ability of low-density vascular endothelial cells to respond to the mitogenic stimuli provided by either serum or FGF. These restrictions cannot be relieved by maintaining cells on ECM-coated dishes, and modifications of the nutrient composition of both media is required in order to allow cells to respond to either FGF or serum when maintained on plastic or to serum alone when maintained on ECM. These results suggest that, when low-density cell cultures are maintained on plastic and exposed to an adequate medium, their proliferation will be a function of both serum and FGF. When maintained on ECM, their proliferation will depend only on serum. It is therefore possible that the inability of serum to stimulate optimal cell proliferation when cells are maintained on plastic results from an inability of the cells to produce an ECM, and that FGF could induce such production.     

Regulation of skeletal muscle satellite cell proliferation by bovine pituitary fibroblast growth factor

Satellite cells in skeletal muscle have been implicated in muscle growth processes and regeneration. However, very little is known about the regulation of their proliferation and differentiation. The effect of fibroblast growth factor (FGF) on the proliferation of myogenic cells from adult rat skeletal muscle, presumably satellite cells, has been examined, and FGF has been found to be a potent mitogen for these cells. The mitogenic properties of serum were also documented and studied in conjunction with FGF. Even under conditions of maximal stimulation by serum, the addition of FGF caused a substantial increase in proliferation of satellite cells. The additive nature of the FGF and serum-stimulatory activity suggests that FGF-like molecules are not the active agents in serum and that more than one pathway may be involved in stimulating satellite cell proliferation.     

Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation

Survival and proliferation of many types of vascular endothelial cells are influenced by fibroblast growth factor (FGF)1. Removal of FGF from the medium of human umbilical vein endothelial cells (HUVEC) in culture resulted in death of the cells. Here we show that the death caused by deprivation of FGF is active death or apoptosis, and the process of apoptosis can be inhibited by cycloheximide, an inhibitor of protein synthesis. The present study shows apoptosis occurs in endothelial cells in culture. The process of active death of vascular endothelial cells is inhibited by growth factor. This mechanism may be important for the regulation of vascular organization through the degeneration of vessels.     

Lysates of two established human tumor lines contain heparin-binding growth factors related to bovine acidic brain fibroblast growth factor

Cell lysates of two established human tumor lines, a medulloblastoma (TE671), and a rhabdomyosarcoma (RD), contain mitogenic activity which elutes from heparin-Sepharose under conditions typical of class 1 heparin-binding growth factors, such as acidic brain fibroblast growth factor. The presence of this class of mitogen in both cell lines was confirmed by their chromatographic behavior on reversed-phase C3 columns, and by the ability of heparin to enhance their mitogenic activity. Using a specific synthetic DNA probe, RNA's were isolated from both cell lines by hybridization-selection, translated in vitro, and translated proteins affinity fractionated on heparin-Sepharose. The results demonstrate that TE671 and RD cell lysates contain mRNA's for mitogens related to acidic brain fibroblast growth factor, and also suggest that high molecular weight proteins exist that are closely related to, or are precursor forms of, the class 1 mitogens.     

Expression and functions of the vascular endothelial growth factors and their receptors in human basophils

Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of vascular endothelial growth factors (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (121, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 +/- 10.8 pg/10(6) cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10-500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.     

Effects of an extract of human brain containing growth factor activity on the proliferation of human vascular endothelial cells in primary culture

Lesions of vascular human EC play an important role in the development of thrombi and atherosclerosis. The factors which control the repair of vascular lesions are not well known. In addition, they are difficult to study because vascular EC from large vessels are fastidious cells to grow in tissue culture. We have investigated some of the factors that may be important in human umbilical vein EC growth in primary culture. Because of reported species differences in EC culture, we have decided to culture human EC only in the presence of biological culture reagents of human origin. Human umbilical vein EC, at low seed density, can be grown to confluency on a human FN matrix or on human ECM providing the medium is supplemented with a high concentration (30%) of human serum. The optimal proliferation of EC (even when seeded at clonal density) is obtained if HBE is added. HBE cannot completely replace serum, but EC proliferate to a similar extent whether they are grown on FN or on ECM in the presence of 30% human serum of 10% human serum plus HBE. Thus, HBE contains a growth factor activity for human EC which stimulates cell growth and DNA replication. Further work is needed to purify HBE and to compare it to other endothelial cell growth factors isolated from bovine brain and bovine eye.     

Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex, and corpus luteum capillaries

Bovine adrenal and brain cortex and corpus luteum-derived capillary endothelial cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM) coated dishes in the presence of serum supplemented medium. All three cell types formed at confluency a monolayer of small, tightly packed, contact inhibited cells that express factor VIII related antigen. Their proliferative response to basic and acidic FGF when cells were maintained on plastic and exposed to serum supplemented medium was similar to that previously reported for endothelial cells derived from large vessels, with acidic FGF being 30-fold less potent than basic FGF. Their requirement for high density lipoproteins and transferrin in order to proliferate actively when maintained on ECM-coated dishes and exposed to serum-free conditions was also similar to that previously reported for endothelial cells derived from large vessels. Heparin strongly reduced the proliferative response of capillary endothelial cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on plastic or on ECM-coated dishes. The present data indicate that bovine endothelial cells derived from large or small vessels are indistinguishable in so far as their response to growth factors, plasma factors, and substrata are concerned.     

Protein phosphatase 3 differentially modulates vascular endothelial growth factor- and fibroblast growth factor 2-stimulated cell proliferation and signaling in ovine fetoplacental artery endothelial cells

A critical process for vascular endothelial growth factor (VEGF)- and fibroblast growth factor 2 (FGF2)-regulated cellular function is reversible protein phosphorylation, which is tightly controlled by a balance of protein kinases and phosphatases. We have reported that in ovine fetoplacental artery endothelial (OFPAE) cells, VEGF and FGF2 stimulate cell proliferation in part via activation of mitogen-activated protein kinase kinase 1/2 (MAP2K1/2)/mitogen-activated protein kinase 3/1 (MAPK3/1) and phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog 1 (AKT1) pathways. In the present study, we examined if protein phosphatase 3 (PPP3) mediated VEGF- and FGF2-stimulated OFPAE cell proliferation via modulating activation of MAPK3/1 and AKT1. Small interfering RNA (siRNA) targeting human PPP3 catalytic subunit alpha (PPP3CA) was used to suppress PPP3CA protein expression in OFPAE cells. Compared with the scrambled siRNA, PPP3CA siRNA decreased PPP3CA protein levels by approximately 97% without altering protein levels of protein phosphatase 2 catalytic subunit alpha, total MAPK3/1, total AKT1, or glyceraldehyde-3-phosphate dehydrogenase. Knockdown of PPP3CA protein expression enhanced VEGF-stimulated, but not FGF2-stimulated, cell proliferation. Knockdown of PPP3CA protein expression did not significantly affect VEGF-induced MAPK3/1 and AKT1 phosphorylation but attenuated FGF2-induced MAPK3/1 and AKT1 phosphorylation. Thus, to our knowledge, the present study is the first to demonstrate successful knockdown of PPP3CA protein expression in any cell model using a single pair of double-strained siRNA. Moreover, specific knockdown of PPP3CA protein expression enhances VEGF-stimulated, but not FGF2-stimulated, OFPAE cell proliferation and attenuates FGF2-induced, but not VEGF-induced, MAPK3/1 and AKT1 activation. Thus, PPP3CA differentially modulates the VEGF- and FGF2-stimulated cell proliferation and signaling cascades in OFPAE cells. These data also suggest that signaling molecules other than MAPK3/1 and AKT1 play an important role in VEGF- and FGF2-stimulated cell proliferation after knockdown of PPP3CA in OFPAE cells.     

Purification and partial characterization of bovine pituitary fibroblast growth factor

A purification procedure and partial characterization of bovine pituitary fibroblast growth factor (FGF) are described. The steps of the published methods [3,4] which yield inhomogeneous material, were retained, with modifications. The final isolation, with an additional purification of ～20-fold, was achieved by electro-phoresis in polyacrylamide gels at acid pH. The mitogenic peptide has a molecular weight of 14,500–15,00 as determined on SDS gels, chromatographs as a monomer in nondenaturing conditions, and is active at the picomolar level in effecting the incorporation of ³H-thymidine in Balb/c 3T3 cells. A preliminary amino acid composition is presented.     

Biological characterization of an acid-sensitive growth factor from human platelets: role in the proliferation of human melanoma and bovine endothelial cells

An acid-sensitive growth factor for human melanoma has been partially purified from human platelets. TSK gel filtration HPLC provides a molecular weight estimation of 60,000 daltons. This factor is not only mitogenic for human melanoma, but also stimulates 3H-thymidine uptake and increases the number of bovine aortic endothelial cells, while fibroblasts are nonresponsive. Radioiodination of active HPLC fraction has been accomplished. The human melanoma cell line, M1RW5 demonstrates specific, time-dependent binding of the labeled protein. These studies suggest that the growth of human melanoma may in part be regulated by a newly described growth factor present in human platelets.     

Comparison of human and bovine brain derived heparin-binding growth factors

Two growth factors have been purified to homogeneity from human brain using heparin affinity chromatography. They have apparent molecular weights of 17 Kd and 18 Kd. Their amino acid compositions differ, but are similar to those of the two heparin-binding growth factors present in bovine neural tissue. These results suggest that the heparin-binding growth factors in neural tissue can be grouped into two distinct classes.     

Eicosapentaenoic acid attenuates vascular endothelial growth factor-induced proliferation via inhibiting Flk-1 receptor expression in bovine carotid artery endothelial cells

Eicosapentaenoic acid (EPA; 20:5, n-3) can restrain tumor growth and metastasis in vivo; however, the mechanism of its antitumor effect is still not fully understood. Angiogenesis is a crucial process for tumor growth and metastasis and inhibition of tumor angiogenesis can suppress tumor growth and metastasis in vivo. Vascular endothelial growth factor (VEGF) is an important angiogenic factor. In this study, we investigated the mechanisms of the inhibitory effect of EPA on VEGF-induced proliferation of bovine carotid artery endothelial (BAE) cells. BAE cells, treated with 0–5 μg/ml EPA for 48 h, displayed a dose-dependent suppression to VEGF (0.2 nM)-induced proliferation. Similar inhibitory effect was not found in BAE cells treated with arachidonic acid (AA; 20:4, n-6), or docasahexaenoic acid (DHA; 22:5, n-3). In contrast to its effect on VEGF-induced proliferation, EPA had no inhibition to basic fibroblast growth factor (bFGF, 0.2 nM)-induced proliferation in BAE cells. Both VEGF and bFGF activated mitogen-activated protein (MAP) kinase in BAE cells; however, EPA selectively inhibited VEGF-induced, but not bFGF-induced activation of MAP kinase. Flk-1 expression was inhibited dose-dependently in EPA-treated cells, whereas Flt-1 expression was increased in EPA treated cells. This in vitro inhibitory effect by EPA on Flk-1 receptor expression provides indirect evidence that one of the mechanisms of EPA for antitumor action in vivo maybe related to its antiangiogenic action. J. Cell. Physiol. 176:342–349, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Inhibition of endothelial cell proliferation by type beta-transforming growth factor: interactions with acidic and basic fibroblast growth factors

TGF beta is a potent (ED50 approximately 10(-11) M) inhibitor of the proliferative activities of both acidic and basic FGF on vascular and capillary endothelial cells in vitro. The inhibition of cell growth is dose-dependent and characteristic of a non-competitive interaction. The results demonstrate that TGF beta and FGF can interact at the cellular level to modulate growth and suggest that many of the biological activities of FGF observed in vitro and in vivo (ie angiogenesis, cell growth, cell differentiation) may be regulated by the presence of TGF beta and related proteins (ie inhibin) in the local cellular milieu. The possible identity of TGF beta with the inhibitors of endothelial cell growth detected in in vitro assays of crude extracts is discussed.