Binding of the basic fibroblast growth factor (bFGF) by soluble components of human umbilical cord

Pre-eclampsia, the most common pregnancy associated syndrome, is connected with remodelling of extracellular matrix of the umbilical cord tissues. Since the fibroblast growth factor (FGF) is known to be a stimulator of collagen and glycosaminoglycan biosynthesis, one may expect that it plays an important role in such a remodelling. Studies performed on the umbilical cords of 10 control and 10 pre-eclamptic newborns demonstrated that both the umbilical cord arterial wall and Wharton's jelly contain FGF mainly in complexes with the components of different molecular mass. Pre-eclampsia is associated with a decrease of endogenous FGF-binding by soluble high molecular mass components of the umbilical cord. It is suggested that FGF released from these complexes may be actively bound by fibroblasts of the umbilical cord, stimulating them to produce collagen and sulphated glycosaminoglycans.     

Effects of basic fibroblast growth factor on angiogenin expression and cell proliferation in H7402 human hepatoma cells

Hepatocellular carcinoma （HCC） is one of the most common cancers worldwide. Basic fibroblast growth factor （bFGF）, which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Its expression is essential for the progression and metastasis of HCC. This study aims to investigate the effects of bFGF on the expression of angiogenin, another growth factor, which plays an important role in tumor angiogenesis, and on cell proliferation in H7402 human hepatoma cells. The bFGF sense cDNA or antisense cDNA was stably transfected into H7402 cells. Genomic DNA PCR analysis demonstrated that human bFGF sense cDNA or antisense cDNA was inserted into the genome. Furthermore, the expression of bFGF and angiogenin was examined by RT-PCR and Western blot assays. MTT and colony formation assays were employed to determine cell proliferation. Stable bFGF over-expressing and under-expressing transfectants were successfully established. Expression of angiogenin was decreased in the over-expressing bFGF cells （sense transfectants） and was increased in the under-expressing bFGF cells （antisense transfectants）. Cell proliferation increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results demonstrated that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. This study may be helpful in finding a potential therapeutic approach to HCC.     

Cloning and expression of cDNA encoding human basic fibroblast growth factor

A cDNA encoding human basic fibroblast growth factor (bFGF) was isolated from a human foreskin fibroblast cDNA library. The cDNA, 4 kilobases in size, had a coding sequence, 5' and 3' untranslated regions, and a poly(A) chain. Isolation of additional cDNA clones that had a short 3' untranslated region suggested the presence of multiple mRNA forms. By Northern blot analysis, at least five bFGF mRNA species were detected in cultured fibroblast cells. Transfection of the cDNA to COS cells resulted in the detection of mitogenic activity in the culture medium of the transfected cells, suggesting that a part of the synthesized protein might be secreted from cells despite its unusual short signal sequence.     

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.     

Regulation of retinal capillary cells by basic fibroblast growth factor, vascular endothelial growth factor, and hypoxia

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) feature prominently in retinal neovascular diseases. Although the role of VEGF in retinal angiogenesis is well established, the importance of bFGF in this process requires further clarification. This study was undertaken to investigate the responses of retinal capillary cells (endothelial cells and pericytes) to bFGF under hypoxic conditions, as well as the potentially synergistic effects of bFGF and VEGF on the proliferation and cord formation of retinal endothelial cells. Cell proliferation was determined by cell number and by 3H-thymidine incorporation. Cord formation was assessed in three-dimensional gels of collagen type I. VEGF and bFGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced in a hypoxic environment. Moreover, the proliferation of pericytes was stimulated to a greater extent by bFGF relative to VEGF. Endothelial migration in collagen gels, however, was induced more effectively by VEGF than by bFGF. A synergistic effect of VEGF and bFGF on cell invasion was observed in the collagen gel assay. VEGF and bFGF each augment proliferation of these cells, especially under hypoxia. We thus propose that these two cytokines have a synergistic effect at several stages of angiogenesis in the retina.     

Isolation,characterisation and comparative analysis of human umbilical cord vein perivascular cells and cord blood mesenchymal stem cells

Perivascular cells are known to be ancestors of mesenchymal stem cells (MSCs) and can be obtained from heart, skin, bone marrow, eye, placenta and umbilical cord (UC). However detailed characterization of perivascular cells around the human UC vein and comparative analysis of them with MSCs haven’t been done yet. In this study, our aim is to isolate perivascular cells from human UC vein and characterize them versus UC blood MSCs (UCB-MSCs). For this purpose, perivascular cells around the UC vein were isolated enzymatically and then purified with magnetic activated cell sorting (MACS) method using CD146 Microbead Kit respectively. MSCs were isolated from UCB by Ficoll density gradient solution. Perivascular cells and UCB-MSCs were characterized by osteogenic and adipogenic differentiation procedures, flow cytometric analysis [CD146, CD105, CD31, CD34, CD45 and alpha-smooth muscle actin (α-SMA)], and immunofluorescent staining (MAP1B and Tenascin C). Alizarin red and Oil red O staining results showed that perivascular cells and MSCs had osteogenic and adipogenic differentiation capacity. However, osteogenic differentiation capacity of perivascular cells were found to be less than UCB-MSCs. According to flow cytometric analysis, CD146 expression of perivascular cells were appeared to be 4.8-fold higher than UCB-MSCs. Expression of α-SMA, MAP1B and Tenascin-C from perivascular cells was determined by flow cytometry analysis and immunfluorescent staining. The results appear to support the fact that perivascular cells are the ancestors of MSCs in vascular area. They may be used as alternative cells to MSCs in the field of vascular tissue engineering.     

Effect of forced expression of basic fibroblast growth factor in human bone marrow-derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) derived from human bone marrow are expected to be utilized for the purpose of tissue engineering, because of their extensive self-renewal or proliferation capability. The capability decreases after several passages, however. Basic fibroblast growth factor (bFGF) is commonly used for culture of various cells including bone marrow-derived MSCs. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the bFGF gene into the passaged cells by retroviral system. The bFGF-expressing MSCs, even at 7 to 8 passages after the infection, showed consistent proliferation capability. The capability was not detected in control cells even in culture media containing the bFGF protein. Thus, we could not mimic the effect of forced expression of bFGF by exogenously adding the bFGF protein in culture media. Although we expressed the shortest isoform of bFGF, which was considered to be mostly cytosolic, we found the protein mostly in the nucleus. Our observations demonstrate not only an effective way to maintain proliferation potentials of MSCs, but also a possibility that there may be mechanistic and functional differences in the signal transduction events between endogenously expressed and exogenously added bFGF protein in MSCs.     

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

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

Senescence of aortic endothelial cells in culture: Effects of basic fibroblast growth factor expression on cell phenotype,migration, and proliferation

Bovine aortic endothelial cells (BAEC) can be isolated in large numbers without major contamination by other cells and maintained in culture with a limited life span for about 100 population doublings. In order to study phenotypic changes of BAEC during long-term culture, stocks of different passages of BAEC were established and their morphological, migratory, and proliferative properties analyzed. Early-passage BAEC (passages 5–15) rapidly produce dense, cobblestone-like monolayers. Their growth beyond the monolayer configuration is characterized by the formation of an irregular network of spindle-shaped, crisscrossing BAEC growing either on top or beneath the monolayer, and by the assembly of elongated BAEC into well-differentiated capillary-like tubes. In contrast, senescent BAEC (passages 35–45) form perfect cobblestone monolayers that contain several, often multinucleated giant cells and a few capillary-like tubes but not the crisscrossing networks of their early-passage counterparts. The rates of BAEC migration and proliferation gradually decline during in vitro senescence. This decline is neutralized by exogenous basic fibroblast growth factor (bFGF) which elevates the migratory and proliferative capacities of early-passage and senescent BAEC to uniformly high levels. Northern blot analysis shows a gradual decline in bFGF message and an increase in laminin message during in vitro BAEC senescence. The present study supports the concept of autocrine growth regulation of BAEC and associates a decreased bFGF message with decreased rates of migration and proliferation as well as loss of the crisscrossing BAEC morphotype in senescent cultures. © 1993 Wiley-Liss, Inc.     

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child’s birth; however, its importance as a “store house” of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton’s jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.     

Suppression of basic fibroblast growth factor expression by antisense oligodeoxynucleotides inhibits the growth of transformed human astrocytes.

Basic fibroblast growth factor (bFGF) is a heparin-binding protein expressing potent mitogenic and angiogenic properties. Elevated levels of bFGF have recently been described in human glioma cell lines. The high degree of vascularity and invasiveness which characterize human gliomas suggest that activated expression of bFGF or similar proteins may be related to the aberrant growth patterns of these tumors. The influence of endogenous bFGF on glioma cell growth in vitro was evaluated in the present study by down-regulating bFGF expression using antisense oligonucleotide primers. The addition of 50 microM bFGF-specific antisense primer to the human glioma cell line SNB-19 resulted in an 80% inhibition in glioma growth. This effect was saturable and specific. Antisense primers directed to two different sites of bFGF mRNA were effective in suppressing SNB-19 growth, whereas sense strand primer was ineffective. Furthermore, only the antisense primer significantly reduced the specific activity of bFGF protein in SNB-19 cell extracts. Neither antisense or sense primers inhibited the growth of non-transformed human glia. bFGF mRNA was detected in both transformed and non-transformed human glia by polymerase chain reaction analysis suggesting that alterations in bFGF isoform content or activity may be specifically related to abnormal growth control in human gliomas.     

Transforming growth factor-beta,basic fibroblast growth factor,and platelet-derived growth factor-BB interact to affect proliferation of clonally derived porcine satellite cells

Transforming growth factor beta-1 (1GF-β) stimulated porcine satellite cell proliferation in basal serum-free medium by 25%, but inhibited growth in serumcontaining medium by 58%. The effect of TGF-β on cell proliferation in serumfree medium was examined in combination with the following human recombinant growth factors: platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). TGF-β inhibited PDGF-stimulated proliferation, enhanced FGF-stimulated proliferation, and had no effect on proliferation stimulated by IGF-I. The response of satellite cells to EGF and TGF-β in serum-free medium was not different than TGF-β alone. TGF-β depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF-I, PDGF and EGF, PDGF and FGF, and IGF-I and EGF. In combination with IGF-I and FGF, TGF-β did not affect proliferation. TGF-β inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF-I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM-10% PS) by 13% above control. When the combination of TGF-β and FGF was added to MEM-10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF-AB (this form neutralizes PDGF-AA, AB, and BB) reduced proliferation in MEM-10% PS by 44%. The combination of TGF-β and anti-PDGF-AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF-β interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF-β on proliferation of clonally derived porcine satelite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF. © 1993 Wiley-Liss, Inc.     

Specific blockade of basic fibroblast growth factor gene expression in endothelial cells by antisense oligonucleotide.

The migration and proliferation of endothelial cells play a pivotal role in various vascular diseases. To elucidate the role of endogenous basic fibroblast growth factor (bFGF) produced within endothelial cells on cell growth, we introduced the antisense oligonucleotide complementary to bFGF mRNA into cultured bovine aortic endothelial cells by cationic liposome to block the production of autocrine bFGF. The treatment of the endothelial cells with the specific antisense oligomer efficiently inhibited the synthesis of bFGF with the concomitant suppression of endothelial proliferation, indicating the significant role of bFGF as an endothelial growth promotor. The neutralizing antibody against bFGF had no inhibition on basal DNA synthesis of the endothelial cells, in contrast to marked suppressive action of bFGF antisense oligomer. The results provide the new analytic and therapeutic implications in the use of the antisense methodology for the study of vascular biology.     

High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus

A hybrid of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which is infectious to both Spodoptera frugiperda and Bombyx mori, was prepared in our previous study. Two recombinant hybrid baculoviruses, carrying cDNAs of human acidic and basic fibroblast growth factors, respectively, were successfully constructed in this study, for the large-scale production of human aFGF and bFGF using silkworm as host. These recombinant viruses were used to inoculate silkworm larvae. After the infection, the recombinant proteins were not found in the hemolymph. Such nonsecretion from cells has also been observed in the established insect cell lines, Sf21 and Tn-5. Tissue distribution analysis indicated that the expressed products were mainly located in fat body and the production of the recombinant aFGF and bFGF was maximal at around 80 h postinfection. Therefore, silkworm larvae infected with recombinant viruses were dissected and fat bodies were collected for the purification of recombinant aFGF and bFGF. The expression levels in both cases were estimated to be as high as approximately 600-700 microg per larva. Furthermore, the recombinant proteins were characterized and their biological activities were evaluated by in vitro bioassay using cell culture.     

Inhibition of cell growth and tumorigenesis of human glioblastoma cells by a neutralizing antibody against human basic fibroblast growth factor.

We report here that a neutralizing mouse monoclonal antibody against basic FGF inhibited both anchorage-dependent and anchorage-independent growth of U-87MG and T98G human glioblastoma cells and HeLa cells, all of which express both the basic FGF and the FGF receptor genes. In addition, the subcutaneous administration of this antibody significantly suppressed the tumor development of these tumor cells in nude mice. Therefore, basic FGF plays an important role in neoplastic growth of these cells. The neutralization of basic FGF will be effective in controlling the growth of tumors, such as glioblastoma and other cancer cells which bear basic FGF and FGF receptors.