High Levels Of bFGF Research Articles

Up-regulation of a fibroblast growth factor binding protein in children with renal diseases

Basic fibroblast growth factor (bFGF) is an angiogenic growth factor that is involved in renal growth and the pathogenesis of renal diseases. We have detected high levels of bFGF accumulated in the kidney of HIV-transgenic mice and in children with HIV-associated renal diseases and the hemolytic uremic syndrome (HUS). However, the mechanism modulating the activity of bFGF under these circumstances is poorly understood. We carried out experiments to determine whether a secreted binding protein (FGF-BP) that modulates the activity of bFGF during the process of tumor growth was expressed in pediatric kidneys and to define whether the expression of FGF-BP was altered in pediatric renal diseases associated with high levels of bFGF. Immunohistochemistry and in situ hybridization studies were done in 41 renal sections from children with HIV nephropathies, HUS, other pediatric renal diseases, controls, and fetal kidneys. Western blots and reverse transcriptase-polymerase chain reaction studies were done in selected urine samples and cultured renal cells. Recombinant FGF-BP was produced to study the mitogenic activity of FGF-BP in cultured human renal proximal tubular epithelial cells (RPTEcs). The expression of FGF-BP was up-regulated predominately in renal tubular epithelial cells in children with renal tubular injury, HIV-associated nephropathy (HIVAN), and HUS, and FGF-BP was secreted in the urine of these patients. FGF-BP was also abundantly expressed in developing fetal renal tubules. Recombinant FGF-BP enhanced the mitogenic effects of bFGF in cultured human RPTEcs. The localization of FGF-BP in renal tubular epithelial cells could provide a mechanism by which the activity of bFGF is modulated in developing and regenerating renal tubules of children.

Basic fibroblast growth factor in mesothelioma pleural effusions: correlation with patient survival and angiogenesis.

The expression of angiogenic factors may represent useful markers for diagnosis and prediction of disease outcome. Basic fibroblast growth factor (b-FGF) is a potent angiogenic factor which promotes in vitro growth of endothelial cells and in vivo vessel formation. We investigated the expression of b-FGF in patients affected with malignant and non-malignant pleural diseases and presenting clinically with non-specific signs and symptoms. We also studied the relationships between the expression of b-FGF in patients with malignant pleural mesothelioma (MM) and tumour aggressiveness, assessed as tumour vessel density (TVD), or patient survival. Basic-FGF was measured by immunoassay in the serum and pleural effusions (PE) of 37 patients. Of these, MM was diagnosed in 15/37 patients while the remaining patients had either peripheral lung adenocarcinoma (PLA) or benign inflammatory pleural disease (BPD). The mean b-FGF level measured 8.5+/-6.1 pg/ml in the PE of the malignant group (MM + PLA) and 23.9+/-19.8 in the PE of the non-malignant group (BPD) (p=0.001). The mean b-FGF level was significantly lower in the PE of MM patients (6.9+/-5.2 pg/ml) compared to BPD patients (p=0.004). Linear regression analysis showed a significant inverse correlation (r=-0.59; p=0.041) between b-FGF levels found in MM PE and patient survival. A noteworthy relationship between high serum b-FGF levels and reduced survival was also observed (r=-0.57; p=0.052). Interestingly, both serum (r=0.48; p=0.114) and PE (r=0.26; p=0.413) b-FGF levels in MM patients correlated poorly with TVD. Our data indicate that b-FGF is significantly more expressed in non-malignant compared to malignant PE, this difference being particularly evident between MM and BPD. Our results also suggest that high b-FGF levels correlate with poor MM patient survival through mechanisms which may be independent of b-FGF angiogenic potential.

Potential role of HOXD9 in synoviocyte proliferation.

To investigate the role of HOXD9 in the proliferation activity of cultured synoviocytes as well as the mechanisms that regulate HOXD9 transcription. Synoviocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were transfected with HOXD9 complementary DNA to establish stable transformants that overexpressed HOXD9. HOXD9 expression was detected by Western blotting with anti-HOXD9 antibody. The growth properties of the transformants were investigated by proliferation and colony formation assays. The expression of basic fibroblast growth factor (bFGF), tumor necrosis factor alpha (TNFalpha), interleukin-1beta, c-Fos, and c-Myc was examined by Western blotting. Transcriptional regulation of HOXD9 was examined by transient cotransfection. HOXD9 protein was highly expressed in RA synoviocytes, but there was no expression in OA synoviocytes. HOXD9 transfection induced stable HOXD9 protein expression in synoviocytes and showed an increased proliferation rate under both normal and serum-starved conditions, as well as an enhanced capacity to proliferate anchorage independently to form colonies in soft agar cultures, compared with control transfectants. Higher levels of bFGF and c-Fos were detected in HOXD9 transformants than in controls. Transient cotransfection assays of NIH3T3 fibroblasts and synoviocytes showed that HOXD9 activated the luciferase reporter construct containing the highly conserved region (HCR), an autoregulatory element of HOXD9 promoter. This activation was significantly increased by bFGF, suppressed by TNFalpha, and unchanged by transforming growth factor beta in synoviocytes. Human T lymphotropic virus type I tax also activated the luciferase reporter construct containing the HCR and had a synergistic effect with HOXD9 on HCR promoter activation. Our data suggest that HOXD9 plays a potential role in synovial proliferation. In addition, they suggest that the involvement of HOXD9 in the regulation of cellular growth might be mediated, at least in part, by up-regulation of growth-related factors such as bFGF and c-Fos and/or might result from increased transcription activity by its regulators.

EFFECT OF A CHINESE HERBAL MEDICINE MIXTURE ON A RAT MODEL OF HYPERCHOLESTEROLEMIC ERECTILE DYSFUNCTION

We examine the effect of a Chinese herbal medicine mixture on erectile function in a rat model of hypercholesterolemic erectile dysfunction. In this study 32, 3-month-old Sprague-Dawley rats were used. The 8 control animals were fed a normal diet and the remaining 24 were fed 1% cholesterol diet for 4 months. After 2 months herbal medicine was added to the drinking water of the treatment group of 16 rats but not the cholesterol only group of 8. Of the 16 rats 8 received 25 mg./kg. per day (group 1) and 8 received 50 mg./kg. per day (group 2) of Chinese herbal medicine mixture. Serum cholesterol levels were measured at 2 and 4 months. At 4 months erectile function was evaluated with cavernous nerve electrostimulation in all animals. Penile tissues were collected for electron microscopy, and to perform Western blot for endothelial nitric oxide synthase, neuronal nitric oxide synthase, basic fibroblast growth factor (bFGF) and caveolin-1. Serum cholesterol levels were significantly higher in animals fed the 1% cholesterol diet compared to controls at 2 and 4 months. Nevertheless, there was no significant difference among group 1 (145 +/- 30 mg./dl.), group 2 (157 +/- 20) and the cholesterol only group (143 +/- 15). Systemic arterial pressure was not significantly different between the animals that were fed the 1% cholesterol diet and the controls. During electrostimulation of the cavernous nerve peak sustained intracavernous pressure was significantly lower in the cholesterol only group (50 +/- 23 cm. H2O) compared to the control group. Conversely erectile function was not impaired in the herbal medicine treated rats. Electron microscopy showed many caveolae with fingerlike processes in the cavernous smooth muscle and endothelial cell membranes in control and treated rats but not in the cholesterol only group of rats. Western blot did not show a difference among groups in protein expression for endothelial nitric oxide synthase and neuronal nitric oxide synthase in penile tissue but caveolin-1 and bFGF protein expression was significantly higher in groups 1 and 2 than in the cholesterol only and control groups. Rats developed erectile dysfunction after being fed a 1% cholesterol diet for 4 months. Although serum cholesterol levels were similar in the cholesterol only rats and those treated with Chinese herbal medicine mixture, erectile response was significantly better in the treated group. The mechanism of the herbal medicine is unknown. High levels of bFGF and caveolin-1 expression in the treated group may protect the cavernous smooth muscle and endothelial cells from the harmful effect of high serum cholesterol.

Basic Fibroblast Growth Factor Is Expressed by CD19/CD11c-Positive Cells in Hairy Cell Leukemia

Several features are characteristic for hairy cell leukemia (HCL). Among those are pancytopenia, bone marrow fibrosis, and the appearance of a defined tumor cell phenotype in peripheral blood (PB), bone marrow (BM), and spleen. Hairy cells (HC) coexpress antigens specific for B lymphocytes and monocytes/macrophages and thus the malignant cell does not seem to be restricted to a defined lineage. When serum or bone marrow aspirate was screened by enzyme-linked immunosorbent assay (ELISA) for basic fibroblast growth factor (bFGF), specimen derived from HCL (serum: mean value, 29 pg/mL; BM aspirate: mean value, 641 pg/mL) contained significantly higher levels than those from healthy subjects. To study whether peripheral blood mononuclear cells (PBMC) derived from patients suffering from HCL and healthy donors (HD) were capable of producing bFGF, culture supernatant (conditioned medium, [CM]) was tested for the presence of this cytokine. While bFGF was not detectable in cell cultures from HD, HCL-derived CM contained relatively high levels of bFGF. CM was successfully used for stimulation of mesenchymal cell proliferation, which could be inhibited by a neutralizing anti-bFGF antibody. Cellular activation by pokeweed mitogen (PWM) or the combination of 12-o-tetradecanoyl-phorbol-13-acetate (TPA) plus calcium ionophore (Ca-Ip) led to an enhanced mRNA expression. Results of Western blot experiments showed that HC synthesize at least three isoforms (approximately 18, 23, and 25 kD), but only the 23-kD isoform is exported. To assess the nature of the producer cell, double immunofluorescence analysis using a bFGF-specific and an anti-CD11c monoclonal antibody (MoAb) was undertaken. The majority of cells scoring positive for CD11c were also reactive with the anti-bFGF MoAb. Furthermore, enrichment of CD19/CD11c-positive cells correlated with enhanced bFGF levels, thereby supporting the argument for HC being the producer cells of bFGF. A biological function of bFGF in HCL might be mediation of chemoresistance, as 2-chlorodeoxyadenosine (2-CdA)-induced inhibition of cell proliferation can be reversed by bFGF. Endogenous bFGF production by HC is not affected by this purine analogue and 2-CdA-induced apoptosis is diminished in bFGF-producing HC as compared with normal PBMC. Therefore, bFGF expression by HC might be important for resistance to chemotherapy and survival of the malignant cells.

Upregulation of basic fibroblast growth factor in breast carcinoma and its relationship to vascular density, oestrogen receptor, epidermal growth factor receptor and survival

Angiogenesis, the process whereby endothelial cells divide and migrate to form new blood capillaries, has been assessed in tumours by measuring microvessel density. High microvessel density is a significant adverse prognostic factor in breast cancer. The angiogenic factor, basic fibroblast growth factor (bFGF), has been associated with tumourigenesis and metastasis in several human cancers. There are few quantitative studies of bFGF expression in normal tissues compared to cancer. We have measured bFGF levels in 149 human primary breast carcinomas and assessed the findings in relation to microvessel density, oestrogen receptor (ER) and epidermal growth factor receptor (EGFR). Basic FGF levels were measured by ELISA. Western blotting and immunohistochemistry were carried out to confirm the presence of bFGF. Levels of bFGF were more than 10-fold higher in tumour cytosols compared to reduction mammoplasty tissue and 3-fold compared to non neoplastic cytosols from the same breast as the tumour (P < 0.0001). Immunohistochemistry showed bFGF protein was localised exclusively in the stroma whereas no bFGF staining was observed in the epithelial cells. High bFGF levels were significantly related to high ER (P = 0.01). Similarly, high bFGF levels were significantly related to low grade (P = 0.046) and to small tumour size (P = 0.04). No significant relationship was observed between bFGF and microvessel count, EGFR or age. In univariate analysis and in a Cox proportional hazard model bFGF did not reach significance for overall or relapse free survival. Our results show that although bFGF is elevated in breast carcinomas compared to normal breast tissue it is not related to microvessel density and it is not an independent predictor of survival in breast cancer patients. Basic FGF may be one of multiple factors that synergise with other growth factors such as VEGF to enhance angiogenesis.

Circulating basic fibroblast growth factor declines during Cy/TBI bone marrow transplantation.

Basic fibroblast growth factor (bFGF) inhibits radiation-induced apoptosis, and radioprotects haematopoietic, cartilage growth plate, pulmonary and gastrointestinal tissues. Conversely, chronic overexpression of bFGF may promote fibrosis. We measured the endogenous circulating bFGF in blood of patients undergoing conditioning TBI. Twenty-six patients with haematopoietic malignancies were conditioned with cyclophosphamide/TBI for allogeneic BMT. Daily blood samples were collected each morning prior to, during, and for several days after TBI. bFGF levels in plasma of normal volunteers are 0.8-26 pg/ml. bFGF was below detectability in 22%, 30% and 45% of patients pre-TBI, during TBI or post-TBI respectively. Mean circulating plasma levels of bFGF decreased from a median of 52 pg/ml pre-TBI to 26 pg/ml during TBI, and to 5 pg/ml post-TBI. Among the 26 patients, 13 had more than one non-detectable plasma bFGF level, an additional five had at least one non-detectable level, and only eight patients had detectable levels in all daily samples. Naturally high levels of bFGF were observed in some patients undergoing fractionated TBI. In contrast, as many as 79% of patients had low bFGF levels in one or more samples. The impact of endogenous bFGF on the tolerance of normal tissues to irradiation is unknown, and warrants further study.

Progressive growth of infantile cutaneous hemangiomas is directly correlated with hyperplasia and angiogenesis of adjacent epidermis and inversely correlated with expression of the endogenous angiogenesis inhibitor, IFN-beta.

Cutaneous infantile hemangioma progresses through proliferation and involution phases. Since treatment with interferon, a negative regulator of angiogenesis, accelerates the involution phase, we hypothesized that cutaneous infantile hemangioma is associated with an imbalance between endogenous positive and negative regulators of angiogenesis. We examined 30 specimens of cutaneous hemangioma [proliferative phase (n=15), involuting phase (n=8), and involuted phase (n=7)] and control human skin (n=17), fixed in formalin and embedded in paraffin. Routine histology, immunohistochemistry, and an mRNA in situ hybridization technique were used to measure expression of the positive angiogenic molecules basic fibroblast growth factor (bFGF) and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), and an endogenous inhibitor of angiogenesis, interferon-beta (IFN-beta). Proliferative phase hemangiomas expressed high levels of bFGF and VEGF/VPF but not IFN-beta (mRNA and protein). The epidermis directly overlying proliferating hemangiomas was hyperplastic, contained numerous dividing cells, and expressed bFGF and VEGF/VPF but not IFN-beta. Epidermis from normal individuals and epidermis directly overlying involuted tumors or at sites distant to the proliferating hemangioma was not hyperplastic and expressed normal levels of bFGF, VEGF/VPF, and IFN-beta. These data suggest that the proliferation of cutaneous hemangiomas and adjacent epidermis is associated with an imbalance between positive and negative angiogenic factors expressed by the neoplasm and adjacent normal tissue.

Quantitative analysis of basic fibroblast growth factor and vascular endothelial growth factor in human colorectal cancer.

Tumour growth is angiogenesis dependent. Some authors suggest a prognostic role of microvessel count in colorectal cancer. We tested the role of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the switch to the angiogenic phenotype in 35 patients with colorectal cancer at different stages of disease. We evaluated the two angiogenic factors, by enzyme-linked immunosorbent assay (ELISA), in tumour, peritumoral mucosa, pathological mesenteric and peripheral blood. We used ten endoscopic intestinal biopsies and ten peripheral blood samples from healthy subjects as control. bFGF was significantly lower in tumour tissues and in peritumoral mucosas than in healthy mucosas, whereas VEGF was up-regulated in tumours but not in peritumoral mucosa. Both angiogenic factors were greatly increased in mesenteric blood. VEGF tumour and serum levels were significantly correlated with the stage of disease. bFGF tumour and serum concentration were not correlated with the stage of disease. The high levels of bFGF in mesenteric blood suggest that this growth factor might be abnormally released from tumour tissue and peritumoral mucosa and could function as an early effector in the switch to the angiogenic phenotype. In contrast, VEGF, whose levels show a significant correlation with the stage of disease, could act in a following step, supporting tumour progression.

Basic FGF attenuates amyloid β-peptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na +/K +-ATPase activity in hippocampal neurons

Basic fibroblast growth factor (bFGF) exhibits trophic activity for many populations of neurons in the brain, and can protect those neurons against excitotoxic, metabolic and oxidative insults. In Alzheimer's disease (AD), amyloid β-peptide (A β) fibrils accumulate in plaques which are associated with degenerating neurons. A β can be neurotoxic by a mechanism that appears to involve induction of oxidative stress and disruption of calcium homeostasis. Plaques in AD brain contain high levels of bFGF suggesting a possible modulatory role for bFGF in the neurodegenerative process. We now report that bFGF can protect cultured hippocampal neurons against A β25-35 toxicity by a mechanism that involves suppression of reactive oxygen species (ROS) accumulation and maintenance of Na +/K +-ATPase activity. A β25-35 induced lipid peroxidation, accumulation of H 2O 2, mitochondrial ROS accumulation, and a decrease in mitochondrial transmembrane potential; each of these effects of A β25-35 was abrogated in cultures pre-treated with bFGF. Na +/K +-ATPase activity was significantly reduced following exposure to A β25-35 in control cultures, but not in cultures pre-treated with bFGF. bFGF did not protect neurons from death induced by ouabain (a specific inhibitor of the Na +/K +-ATPase) or 4-hydroxynonenal (an aldehydic product of lipid peroxidation) consistent with a site of action of bFGF prior to induction of oxidative stress and impairment of ion-motive ATPases. By suppressing accumulation of oxyradicals, bFGF may slow A β-induced neurodegenerative cascades.

Apoptosis of human glioma cells in vitro and in vivo induced by a neutralizing antibody against human basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is mitogenic to neuroectoderm- and mesoderm-derived cells and is a potent angiogenic factor. Abundant amounts of this factor and its receptor are detected in human glioma tissues and cells, and bFGF in glioma is thought to be involved in autonomous cell growth as an autocrine growth factor. A neutralizing mouse monoclonal antibody (MAb) against bFGF, 3H3 MAb, has been shown to inhibit both in vitro and in vivo growth of human glioma cell lines. This study shows that the human glioma cell lines U-87MG and U-251MG, which express high levels of bFGF and its receptor, can be induced to undergo apoptosis when cultured with 3H3 MAb. It is also demonstrated that 3H3 MAb can cause apoptosis in the same glioma cells that were transplanted into nude mice. Furthermore, enforced overexpression of bcl-2 protein by gene transfection prevented 3H3 MAb-induced apoptosis of glioma cells. It is concluded that induction of apoptosis by the neutralizing antibody is a promising therapeutic strategy for glioma.

Inflammatory cytokines induce AIDS-Kaposi's sarcoma-derived spindle cells to produce and release basic fibroblast growth factor and enhance Kaposi's sarcoma-like lesion formation in nude mice.

Homosexual males often present signs of immune activation and are likely to have increased levels of inflammatory cytokines such as IL-1 beta, TNF-alpha, and IFN-gamma. These individuals develop Kaposi's sarcoma (AIDS-KS) more frequently than other HIV-1-infected groups. Our previous work demonstrated that inflammatory cytokines stimulate the growth of spindle cells derived from AIDS-KS lesions (AIDS-KS cells) and that these cells produce high levels of bFGF that mediate autocrine and paracrine (endothelial) cell growth and angiogenesis. Here we show that AIDS-KS cells constitutively produce and release bioactive bFGF in the absence of cell death, and that extracellular bFGF exist in both a soluble and a bound form; the latter can be released by treatment with trypsin, heparin, or heparinase I. Inflammatory cytokines stimulate both the synthesis and release of biologically active bFGF from KS cells and enhance their ability to induce angiogenic KS-like lesions in nude mice. Because bFGF is highly expressed in primary KS lesions, and is a mediator of KS-like lesion formation, these results suggest that the export of bFGF induced by inflammatory cytokines may play a critical role in the induction and progression of KS in HIV-1-infected homosexual men.

Basic fibroblast growth factor (bFGF) in systemic and tumor venous blood of soft tissue sarcoma patients

Soft tissue sarcomas of the extremities contribute to only 0.9% of all neoplasms. Even in experienced hands radical surgical resection is followed by a recurrency rate of 7 to 35%. The reasons for this high percentage of recurrent disease after radical surgery are not yet clear. Since angiogenesis is a key factor in tumor growth we have investigated concentrations of the angiogenic peptide bFGF in systemic blood and soft tissue sarcomas venous blood of 33 consecutive patients undergoing radical tumor resection: liposarcomas (6), malignant fibrous histiozytoma (6), fibrosarcomas (6), clear cell sarcomas (2), leiomyosarcomas (2), others (6). Systemic venous blood was obtained preoperatively, during surgery, lhour, I day and 1 week after operation. 10 cc of venous blood was collected from tumor veins during surgery. Samples wzre centifuged and stored at minus 20*Celsius. bFGF levels were measured with a monoclonal sandwich EL1SA (RD 586+227 pg/ml). Although no pathological bFGF serum levels could be detected in serum of soft tissue sarcoma patients, high bFGF concentrations were found in tumor blood. The fact that non-tumor soft tissue wounds expressed even higher bFGF levels suggests that a marked angiogenic potential is located within the wound bed which might promote tumor growth and development of recurrencies. The data support the importance of radical local tumor resection and anti-angiogenic treatment modalities.

Tumor growth and angiogenesis induced by a secreted binding protein for fibroblast growth factors.

Basic fibroblast growth factor (bFGF) is a strong inducer of angiogenesis and thus may play an important role in the growth of solid tumors. However, bFGF is usually found immobilized on the extracellular matrix, and it is only partly understood how it is solubilized to reach and activate its extracellular receptors. We studied the potential contribution to this process by a secreted binding protein (BP) with high affinity for FGFs. An expression vector for BP was transfected into a human cell line (SW-13) that contains constitutively high levels of bFGF. The BP-expressing cells began to grow colonies in soft agar due to their autocrine stimulation by bFGF and released biologically active bFGF into their media. Furthermore, they grew into well vascularized tumors in athymic nude mice. In addition, we found the BP mRNA expressed at high levels in squamous cell carcinoma (SCC) tissues from patients and in SCC cell lines of different origin as well as in immortalized keratinocytes. However, we failed to detect BP mRNA in normal adult tissues or in a number of non-SCC tumor cell lines. Expression of the secreted BP appears to be a mechanism through which immobilized FGF can be activated to support tumor growth and angiogenesis.

A New Avian Fibroblast Growth Factor Receptor in Myogenic and Chondrogenic Cell Differentiation

We studied the expression of FREK (fibroblast growth factor receptor-like embryonic kinase), a new receptor recently cloned from quail embryo, during the differentiation of skeletal muscle satellite cells and epiphyseal growth-plate chondrocytes. Although FREK mRNA was expressed in both cell types, satellite cells expressed higher levels of this mRNA than chondrocytes. FREK gene expression was found to be modulated by b-FGF in a biphasic manner: low concentrations increased expression, whereas high concentrations attenuated it. In both cell cultures, the levels of FREK mRNA declined during terminal differentiation. Moreover, retinoic acid (RA), which induces skeletal muscle satellite cells to differentiate, also caused a reduction in FREK gene expression in these cells. Induction of chondrocyte differentiation with ascorbic acid was monitored by a decrease in collagen type II gene expression and an increase in alkaline phosphatase activity. Satellite cell differentiation was marked by morphological changes as well as by increased sarcomeric myogenin content and creatine kinase activity and changes in the expression of the regulatory muscle-specific genes, MyoD and myogenin. DNA synthesis in both cell types was stimulated by b-FGF. However, in satellite cells, the response was bell-shaped, peaking at 1 ng/ml b-FGF, whereas in chondrocytes, higher levels of b-FGF were needed. b-FGF-dependent DNA synthesis in satellite cells was decreased by RA at concentrations over 10 -7 M . The observed correlation between the level of FREK gene expression and various stages of differentiation, its modulation by b-FGF and RA, as well as the correlation between FREK gene expression and the physiological response to b-FGF, suggest that this specific FGF receptor plays an important role in muscle and cartilage cell differentiation.

Basic fibroblast growth factor and transforming growth factor beta-1: synergistic mediators of angiogenesis in vitro.

We investigated the relative roles of basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two-dimensional Petri dish cultures and a three-dimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC50 of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three-dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF modification of the antiproliferative effect of TGF-b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF-b and 10-20 ng/ml bFGF in two-dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose-dependent manner: invasion was maximal at 0.3-0.7 ng/ml TGF-b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a fibroblast-like appearance with slender cell extensions. We concluded that bFGF and TGF-b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two-dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis.

Expression of basic fibroblast growth factor in the nervous system of early avian embryos

Basic fibroblast growth factor (bFGF) promotes the survival of a subpopulation of non-neuronal cells developing from trunk neural crest. It was therefore important to determine whether this factor is present in the nervous system at early developmental stages. Immunocytochemistry using specific polyclonal and monoclonal antibodies was combined with three highly sensitive assays: bFGF-induced proliferation of bovine adrenal cortex-derived capillary endothelial cells (ACE), a radioimmunoassay for bFGF (RIA) and Western blot analysis. bFGF immunoreactivity was localized to the cytoplasm of neuroepithelial cells derived from embryonic day 2 (E2) quail neural tubes and cultured for one day in a chemically defined medium. Specific staining was observed in young sensory neurons in cultures of neural crest clusters as well as in a subpopulation of non-neuronal cells. In cultured E7 dorsal root ganglia, immunostaining was confined to neuronal cell bodies and fibers. In situ, staining of spinal cord and ganglionic neurons appeared on E6 and increased in intensity towards E10. Various mesoderm-derived structures such as the limb buds, the mesenchyme dorsal to the neural tube, the vertebral muscles and cartilage showed specific staining patterns in addition to neural tissue. In agreement with the results of immunocytochemical studies, 1.4ng bFGF per mg protein was detected in spinal cord extracts by RIA as early as E3, its concentration increased to 8.0 ng mg-1 on E5 and then to a maximum of 18.0 ng mg-1 protein on E10, this was followed by a subsequent decrease in concentration in older embryos. On the other hand, high levels of bFGF were present in vertebral tissues from E10 onwards. Extracts of immunopositive tissues were subjected to heparin-Sepharose affinity chromatography and eluted in a stepwise salt gradient. Fractions that eluted from the columns at 2 M NaCl contained a bFGF-like protein as revealed by their ability to stimulate the proliferation of ACE cells and by Western blot analysis. These data demonstrate that bFGF is expressed during early nervous system development in both central and peripheral neurons.

Correlation of cell migration, cell invasion, receptor number, proteinase production, and basic fibroblast growth factor levels in endothelial cells.

The levels of endogenous basic fibroblast growth factor (bFGF) in seven clones of cultured bovine capillary endothelial (BCE) cells were assayed, and their relation to cell morphology, bFGF receptor number, cell migration, amniotic membrane invasivity, and proteinase levels were studied. Immunoblotting experiments with anti-bFGF IgG demonstrated that cells from these clones contained different amounts of bFGF. The cells containing high levels of bFGF had a spindle or elongated appearance at confluence and a low number of high affinity receptors for bFGF. The cells containing low levels of bFGF had a cobblestone-like appearance and a higher number of high affinity receptors. When exposed to 10 ng/ml bFGF, cells containing a low level of bFGF took on an elongated appearance with a crisscross pattern similar to that seen with the high producer bFGF cells. The endogenous bFGF levels of the BCE cell clones correlated with the extent of cell migration after wounding of a monolayer and the degree of invasion of the human amniotic membrane. Cells from the clone with the highest endogenous bFGF level migrated well, invaded the amnion membrane without the addition of exogenous bFGF, and were relatively unaffected by the addition of bFGF. Cells from the clone containing the lowest level of bFGF did not migrate or invade under normal conditions. However, the addition of bFGF to the culture medium strongly enhanced both of these processes. The inclusion of anti-bFGF IgG in the media suppressed cell migration and invasion. The plasminogen activator (PA) activities of cell lysates of the clones, assayed by the 125I-fibrin plate technique, indicated that the PA levels did not correlate with the bFGF levels. Metalloproteinase activities in the conditioned medium, assayed by gelatin zymography, correlated with the endogenous bFGF levels, suggesting that the degree of expression of metalloproteinases might be critical for cell migration and invasion. These data suggest that endogenous bFGF may have an important role for migration and invasion of BCE cells during neovascularization via the induction and/or activation of specific metalloproteinases.

Transformation of NIH 3T3 cells with basic fibroblast growth factor or the hst/K-fgf oncogene causes downregulation of the fibroblast growth factor receptor: reversal of morphological transformation and restoration of receptor number by suramin.

When NIH 3T3 cells were transfected with the cDNA for basic fibroblast growth factor (bFGF), most cells displayed a transformed phenotype. Acquisition of a transformed phenotype was correlated with the expression of high levels of bFGF (Quarto et al., 1989). Cells that had been transformed as a result of transfection with bFGF cDNA had a decreased capacity to bind 125I-bFGF to high affinity receptors. NIH 3T3 cells transfected with bFGF cDNA that expressed lower levels of bFGF were not transformed and had a normal number of bFGF receptors. NIH 3T3 cells transfected with the hst/Kfgf oncogene, which encodes a secreted molecule with 45% homology to bFGF, also displayed a transformed phenotype and decreased numbers of bFGF receptors. However, NIH 3T3 cells transfected with the H-ras oncogene were transformed but had a normal number of bFGF receptors. Thus, transformation by bFGF-like molecules resulted in downregulation of bFGF receptors. Receptor number was not affected by cell density for both parental NIH 3T3 cells and transformed cells. In the cells transfected with bFGF cDNA that were not transformed, the receptors could be downregulated in response to exogenous bFGF. Conditioned medium from transformed transfected cells contained sufficient quantities of bFGF to downregulate bFGF receptors on parental NIH 3T3 cells. Thus, the downregulation of bFGF receptors seemed related to the presence of bFGF in an extracytoplasmic compartment. Treatment of the transformed transfected NIH 3T3 cells with suramin, which blocks the interaction of bFGF with its receptor, reversed the morphological transformation and restored receptors almost to normal numbers. These results demonstrate that in these cells bFGF transforms cells by interacting with its receptor and that bFGF and hst/K-fgf may use the same receptor.