Expression of Acidic and Basic Fibroblast Growth Factors in Human and Bovine Vascular Smooth Muscle Cells

Abstract

The expression and synthesis of acidic and basic fibroblast growth factors (aFGF and bFGF) in cultures of bovine and human vascular smooth muscle cells (BSMC and HSMC) was studied. BSMC express and synthesize only bFGF, whereas HSMC express and synthesize both bFGF and aFGF. The presence of bFGF in BSMC is shown by the following criteria: (1) the growth factor activity in BSMC lysates binds to a heparin-affinity column and elutes as a single peak at 1.5-1.7 m NaCl, characteristic for bFGF; (2) this extract is mitogenic for smooth muscle cells; (3) Northern blot analysis demonstrates three distinct bFGF mRNAs of 7.0, 4.0, and 1.9 kb; no aFGF mRNA species were detected. Analysis of human umbilical vein endothelial cells yielded similar results: Heparin-affinity chromatography and Northern blot analysis failed to demonstrate the presence of aFGF despite the detection of bFGF by these techniques. In contrast, HSMC synthesize two growth factor activities: First, they bind to an immobilized heparin column and elute as two separate peaks at 1.2 and 1.5-1.7 m NaCl, characteristic for aFGF and bFGF; and second, Northern blot analysis demonstrates the expression of aFGF mRNA of 4.6 kb and bFGF mRNAs of 7.0, 4.0 and 1.9 kb. Furthermore, it is shown that aFGF and bFGF are potent mitogens for smooth muscle cells in vitro.     

Immunohistochemical localization of epidermal growth factor and acidic and basic fibroblast growth factors in postnatal developing and adult rat lungs.

Histologic preparations of lungs form 1-, 5-, 10-, 18-, and 25-day-old rats and adult rats were probed immunohistochemically with specific antibodies for the distribution of epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF). Immunoperoxidase staining of sections of adult rat lungs with a rabbit polyclonal antibody to bovine EGF was strong in ciliated cells of airways and mast cells, nonciliated cells of bronchioles, smooth muscle, type II cells of alveoli, and interstitial and epithelial cells of alveolar septal regions. Developing postnatal lungs had moderate and somewhat diffuse immunoreactivity in all epithelial cells, and more intense staining in vascular smooth muscle. Immunoperoxidase staining of adult rat lungs with a rabbit polyclonal antibody against bovine aFGF was distributed in identical fashion to EGF, with the exception of mast cells which were not reactive. These same sights were localized using an immunoperoxidase sequence with a rabbit polyclonal antibody to the leu 60-leu 98 fragment of aFGF (aFGFfr) with less background. In postnatal developing lungs, immunoreactivity with aFGF and aFGFfr preparations was diffuse and moderately intense in epithelial cells and vascular smooth muscle. Immunoperoxidase staining of adult rat lung sections with a monoclonal antibody to bovine bFGF was strong in hyaluronidase-digested preparations. Reactivity was principally confined to alveolar and vascular basement membrane regions and external laminae of smooth muscle. In early postnatal development, immunoreactivity for bFGF was found in basement membranes beneath developing epithelium and the endothelium of vessel wall intima and in the adventitia, with reactivity increasing with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia-induced expression of vascular endothelial growth factor by retinal glial cells promotes in vitro angiogenesis

To determine whether retinal glial cells (RGCs) participate in the paracrine regulation of retinal neovascularization, we investigated whether cultured RGCs synthesize and release vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) under normoxic or hypoxic conditions. Northern blot analysis demonstrated that cultured RGCs transcribed both VEGF mRNA with two molecular bands approximately 3.9 and 4.3 kilobases (kb), and bFGF mRNA with approximately 3.7 and 6.0 kb. The expression of VEGF mRNA was greatly enhanced by hypoxic cultivation (2% oxygen) when compared with normoxic cultivation (20% oxygen), while the expression of bFGF mRNA by RGCs was not significantly affected by hypoxia. The effects of RGCs-conditioned media (CM) on tritiated-thymidine incorporation and in vitro angiogenesis by retinal capillary endothelial cells (RECs) in producing the formation of capillary-like tubes in type I collagen gels, were evident in the observation that RGCs-CM harvested after hypoxic cultivation significantly enhanced tritiated-thymidine incorporation (1.9 times, P<0.01) and in vitro angiogenesis (2.4 times, P<0.01) compared with the normoxic RGCs-CM. These enhancing effects of RGCs-CM at hypoxia were suppressed by anti-VEGF neutralizing antibody. Furthermore, RECs were shown to express mRNA encoding the VEGF receptor flt-1 by northern blot analysis. These results suggest that VEGF expressed by RGCs under hypoxic conditions plays an integral role in the initiation and progression of retinal neovascularization in a paracrine manner.     

Increased expression of acidic and basic fibroblast growth factors in chronic pancreatitis.

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) belong to a family of mitogenic polypeptides that are involved in cellular proliferation and differentiation. In this study we investigated the potential role of aFGF and bFGF in chronic pancreatitis (CP), a fibrotic condition associated with acinar cell dedifferentiation and atrophy, and fibroblastic proliferation. By immunohistochemistry, aFGF and bFGF were abundant in pancreatic ductal and acinar cells in pancreatic tissues from CP patients. Immunoblotting with the same highly specific monoclonal antibodies demonstrated a marked increase in aFGF and bFGF in pancreatic homogenates from CP patients by comparison with the normal pancreas. Northern blot analysis indicated that, by comparison with normal controls, 16 of 21 CP tissues exhibited a 14-fold increase in aFGF mRNA levels, and 19 of 21 CP tissues exhibited a 15-fold increase in bFGF mRNA levels. In situ hybridization confirmed that this overexpression occurred in ductal and acinar cells, and indicated that both mRNA moieties colocalized with their respective proteins. These findings suggest that aFGF and bFGF may either be involved in the pathobiological mechanisms that occur in CP, or that their overexpression may be the consequence of other perturbations that occur in this disorder.     

Pleiotypic actions of the seminiferous growth factor on two testicular cell lines: comparisons with acidic and basic fibroblast growth factors

Bovine seminiferous growth factor (SGF) stimulated different pleiotypic responses in TM3 Leydig and TM4 Sertoli cells from those obtained with bovine acidic and basic fibroblast growth factors (aFGF, bFGF). First, the three growth factors had distinct mitogenic effects. SGF increased TM3 and TM4 cell numbers, whereas aFGF was mitogenic only for TM3 cells, and bFGF was inactive on both cell lines. Second, only SGF and bFGF stimulated TM4 cells to produce 3- and 2-fold, respectively, greater levels of extracellular, sulfated glycoprotein-1. By Northern analyses, SGF increased the steady-state levels of sulfated glycoprotein-1 mRNA approximately 1.34-fold relative to those of actin mRNA during a 48-hr period. Third, the cell lines secreted different [35S]methionine-labeled proteins. With TM3 cells, some proteins were secreted specifically, whereas other were up- or down-regulated differentially in response to SGF, aFGF, or bFGF. Likewise, with TM4 cells, the three growth factors induced qualitative and quantitative changes in the secretion of specific proteins. On immunoblots, SGF did not bind antibodies specific for an internal domain of aFGF or FGF-5, nor those directed against N-terminal, internal, and C-terminal regions of bFGF. These data suggest SGF, aFGF, and bFGF acted on TM3 Leydig and TM4 Sertoli cells through different receptors and/or diverging pathways of signal transduction to induce different pleiotypic effects.     

Purification and characterization of fibroblast growth factors from the opossum, Monodelphis domestica, brain.

An extract from the brain of the opossum Monodelphis domestica was fractionated by heparin affinity chromatography. A major peak of mitogenic activity (heparin binding growth factor 2, HBGF-2) eluted from heparin-Sepharose between 1.7 and 2.0 M NaCl. Antisera specific for bovine bFGF detected four polypeptides of 17.5–23 kDa in opossum brain HBGF-2 preparations. Opossum brain heparin binding growth factor 1 (HBGF-1), a minor peak of activity, eluted from heparin-Sepharose at 1.1 NaCl and contained a 16.2 kDa protein that cross-reacted with antiserum against bovine aFGF.     

Purification and characterization of fibroblast growth factors from the opossum, Monodelphis domestica, brain

An extract from the brain of the opossum Monodelphis domestica was fractionated by heparin affinity chromatography. A major peak of mitogenic activity (heparin binding growth factor 2, HBGF-2) eluted from heparin-Sepharose between 1.7 and 2.0 M NaCl. Antisera specific for bovine bFGF detected four polypeptides of 17.5–23 kDa in opossum brain HBGF-2 preparations. Opossum brain heparin binding growth factor 1 (HBGF-1), a minor peak of activity, eluted from heparin-Sepharose at 1.1 NaCl and contained a 16.2 kDa protein that cross-reacted with antiserum against bovine aFGF.     

Endothelial cell growth factor derived from a human glioblastoma cell line and possible association with tumor angiogenesis

The cytosol of a human glioblastoma cell line (KNS-42) stimulated the growth of both bovine aortic endothelial cells and smooth muscle cells in a dose-dependent manner. The endothelial cell growth activity eluted at an apparent molecular weight of about 30,000 on a Sephadex G-100 column and bound to a heparin-Sepharose column with high affinity to elute at 1.3-1.7 M NaCl. The growth activity was destroyed by heating at 56 degrees C for 30 min, but not by exposure to trypsin, deoxyribonuclease or ribonuclease at 37 degrees C for 30 min. As this factor stimulated the growth of vascular endothelial cells and smooth muscle cells, and vasoproliferative responses in chick embryo chorioallantoic membranes were apparent, this factor may possibly be related to tumor angiogenesis.     

Effect of FGFs on adult bovine Muller cells: proliferation, binding and internalization.

A new method for culturing retinal Muller cells from adult bovine tissue is described. The identification of these glial cells was based on immunocytochemical analysis of specific Muller cell markers. Cultured cells from fourth to ninth passage showed positive labelling for S 100 protein, carbonic anydrase (CAA), glutamine synthetase (GS), alpha cristallin (alpha C) and polyclonal glial fibrillary acidic protein (GFAP) antibody, but were negative for both monoclonal GFAP antibody and also for Muller cells in the retina. Investigation of the effect of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and epithelial growth factor (EGF) on the proliferation of the Muller cells revealed that bFGF was the most potent mitogen (EC50 = 14 pM). Binding data revealed the presence of two classes of binding sites for aFGF and bFGF: (1) a high affinity binding site (Kd of 14 pM and 27 pM for aFGF and bFGF respectively); (2) a low affinity binding site (Kd of 3.2 nM and 0.6 nM for aFGF and bFGF respectively with great variability in the number of binding sites). In addition, the cross-linking experiments revealed the presence of high molecular weight FGF receptors (110-140 kDa). After aFGF or bFGF binding to Muller cells, aFGF and bFGF-cell surface receptors were rapidly downregulated with a half-life for disappearance of 35-50 min. Internalization and degradation of 125I-bFGF bound to the Muller cell receptors did not occur at 4 degrees C. At 37 degrees C, however, there was a rapid decrease in receptor-bound 125I-bFGF due to the downregulation of bFGF receptors. Concomitantly 125I-bFGF appeared inside the Muller cells. After 2 h, 125I-bFGF began to be degraded and after 6 h three fragments of 16 kDa, 8 kDa and 5.5 kDa were discernible. Degradation of bFGF appeared to occur in the lysosomal compartment since it was inhibited by chloroquine, an inhibitor of lysosomal proteases; aFGF internalization and degradation followed the same kinetics as bFGF with the appearance of 7 kDa and 5 kDa fragments. These results suggest that Muller cells may be the target for aFGF and bFGF contained in other cells of the retina. The fact that aFGF could be released from rod outer segment by a phosphorylation-dependent mechanism, and that apical prolongation of the Muller cells is connected with the photoreceptor cells suggest that these factors may be the mediators involved in the communication between glial cells and neurons.     

Glomerular epithelial cells synthesize endothelin peptides.

Glomerular mesangial and endothelial cells have been reported to synthesize and secrete endothelin-1 (ET-1). Whether glomerular epithelial cells (GEC) have the ability to synthesize ET-peptides is not known. Employing immunocytochemistry we report that the GEC in vitro constitutively express ET-1 and ET-3. ET-1 synthesis by the GEC was further confirmed by detection of a specific 2.3kb mRNA that hybridizes with rat prepro ET-1 genomic DNA on Northern blot analysis. ET-1 is secreted into the medium in a time-dependent manner as measured by radioimmunoassay and radiobinding assay. Synthesis of endothelin peptides by the GEC may have important implications in the pathogenesis of glomerular diseases where GEC injury figures prominently.     

Intimal smooth muscle cell proliferation after balloon catheter injury. The role of basic fibroblast growth factor.

The localization and synthesis of basic fibroblast growth factor (bFGF) in the rat carotid artery were investigated at times of chronic smooth muscle cell proliferation. Immunocytochemical staining showed the presence of bFGF in the uninjured arterial wall, and after balloon injury, this cellular staining was decreased. Western and northern blot analyses likewise showed that the amount of bFGF protein and mRNA decreased after injury. A neutralizing antibody to bFGF was administered 4 and 5 days after injury and was found to have no effect on intimal smooth muscle cell proliferation. These data suggest that an increase in the expression of bFGF is not necessary for chronic smooth muscle cell proliferation observed after balloon catheter injury and that bFGF is not the major mitogen responsible for intimal smooth muscle cell proliferation.     

Immunohistochemical localization of acidic fibroblast growth factor in normal human enterochromaffin cells and related gastrointestinal tumours

Acidic fibroblast growth factor (aFGF) is a member of the structurally related heparin-binding growth factor family. The best studied members of this family are aFGF and basic FGF (bFGF), which are potent mitogens and differentiation factors for mesodermderived cells, including fibroblasts. This study was designed to verify the immunohistochemical expression of aFGF in normal human endocrine cells of the gut and in related endocrine tumours. We examined normal gastrointestinal mucosa from seven different subjects and 41 gut endocrine tumours from different sites, including stomach, duodenum, and small and large intestine, using an aFGF polyclonal antibody with no cross-reactivity for bFGF. We localized aFGF in a fraction of serotonin-producing enterochromaffin (EC) cells of the normal gut, while it was absent in gastrin (G), CCK, secretin (S), somatostatin (D) and glicentin (L) cells. aFGF immunoreactivity was also expressed in serotonin producing EC cell tumours, but not in other functional types of gut endocrine neoplasms investigated, including gastric ECL cell, duodenal somatostatin and gastrin cell, and rectal L cell tumours. A positive correlation was found between expression of aFGF and the amount of tumour fibrous stroma, suggesting that aFGF may be involved in proliferation and activity of stromal fibroblasts.     

Differential changes in expression of a and b FGF, IGF-1 and -2, and TGF-alpha during seasonal growth and involution of roe deer testis

Growth factors are involved in the regulation of testicular growth and involution of seasonal breeders. Therefore, we studied the seasonal expression of several growth factors in roe deer: aFGF, bFGF, IGF-1, IGF-2, and TGF-alpha. Total RNA from testis tissue was extracted monthly and analyzed using quantitative RT-PCR. Localization of mRNAs was examined by in situ-hybridization. Levels of expression differed by more than three orders of magnitude. Expression also showed different seasonal patterns. IGF-1, IGF-2 and bFGF were maximally transcribed during testis recrudescence in spring. In contrast, the mRNA amount of aFGF reached its maximum between July (breeding season) and January. TGF-alpha mRNA-levels were very low and showed poor seasonal variation. Each growth factor showed its own typical expression localization in testicular tissues and cell types. The results suggest the specific role of different growth factors in the paracrine control of spermatogenesis and its seasonal regulation.     

Fibroblast growth factor 2 and transforming growth factor beta1 synergism in human bronchial smooth muscle cell proliferation

Bronchial smooth muscle cell (BSMC) hyperplasia is a typical feature of airway remodeling and contributes to airway obstruction and hyperresponsiveness in asthma. Fibroblast growth factor 2 (FGF-2) and transforming growth factor beta1 (TGF-beta1) are sequentially upregulated in asthmatic airways after allergic challenge. Whereas FGF-2 induces BSMC proliferation, the mitogenic effect of TGF-beta1 remains controversial, and the effect of sequential FGF-2 and TGF-beta1 co-stimulation on BSMC proliferation is unknown. This study aimed to assess the individual and sequential cooperative effects of FGF-2 and TGF-beta1 on human BSMC proliferation and define the underlying mechanisms. Mitogenic response was measured using crystal violet staining and [3H]-thymidine incorporation. Steady-state mRNA and protein levels were measured by semiquantitative RT-PCR, Western blot, and ELISA, respectively. TGF-beta1 (0.1-20 ng/ml) alone had no effect on BSMC proliferation, but increased the proliferative effect of FGF-2 (2 ng/ml) in a concentration-dependent manner (up to 6-fold). Two distinct platelet-derived growth factor receptor (PDGFR) inhibitors, AG1296 and Inhibitor III, as well as a neutralizing Ab against PDGFRalpha, partially blocked the synergism between these two growth factors. In this regard, TGF-beta1 increased PDGF-A and PDGF-C mRNA expression as well as PDGF-AA protein expression. Moreover, FGF-2 pretreatment increased the mRNA and protein expression of PDGFRalpha and the proliferative effect of exogenous PDGF-AA (140%). Our data suggest that FGF-2 and TGF-beta1 synergize in BSMC proliferation and that this synergism is partially mediated by a PDGF loop, where FGF-2 and TGF-beta1 upregulate the receptor (PDGFRalpha) and the ligands (PDGF-AA and PDGF-CC), respectively. This powerful synergistic effect may thus contribute to the hyperplastic phenotype of BSMC in remodeled asthmatic airways.     

Leukotriene D4-induced, epithelial cell-derived transforming growth factor β1 in human bronchial smooth muscle cell proliferation

Background Cysteinyl‐leukotrienes (cys‐LTs) orchestrate many pathognomonic features of asthma in animal models of allergic airway inflammation, including bronchial smooth muscle cell (BSMC) hyperplasia. However, because cys‐LTs alone do not induce mitogenesis in monocultures of human BSMC, the effect observed in vivo seemingly involves indirect mechanisms, which are still undefined. Objective This study aims to investigate the regulatory role of leukotriene (LT)D₄ on TGF‐β1 expression in airway epithelial cells and the consequence of this interplay on BSMC proliferation. Methods HEK293 cells stably transfected with cys‐LT receptor 1 (CysLT1) (293LT1) were stimulated with LTD₄ and TGF‐β1 mRNA and protein expression was measured using Northern blot and ELISA, respectively. Conditioned medium (CM) harvested from LTD₄‐treated cells was then assayed for its proliferative effect on primary human BSMC. TGF‐β1 mRNA expression was also determined in tumoural type II pneumocytes A549 and in normal human bronchial epithelial cells (NHBE) following LTD₄ stimulation. Results The results demonstrated that LTD₄‐induced TGF‐β1 mRNA production in a time‐ and concentration‐dependent manner in 293LT1. TGF‐β1 secretion was also up‐regulated and CM from LTD₄‐treated 293LT1 was shown to increase BSMC proliferation in a TGF‐β1‐dependent manner. The increased expression of TGF‐β1 mRNA by LTD₄ also occured in A549 and NHBE cells via a CysLT1‐dependent mechanism. Conclusion In conclusion, elevated expression of cys‐LTs in asthmatic airways might contribute to BSMC hyperplasia and concomitant clinical features of asthma such as airway hyperresponsiveness via a paracrine loop involving TGF‐β1 production by airway epithelial cells.     

Differential Changes in Expression of a and b FGF,IGF-1 and -2, and TGF-α During Seasonal Growth and Involution of Roe Deer Testis

Growth factors are involved in the regulation of testicular growth and involution of seasonal breeders. Therefore, we studied the seasonal expression of several growth factors in roe deer: aFGF, bFGF, IGF-1, IGF-2, and TGF- &#33. Total RNA from testis tissue was extracted monthly and analyzed using quantitative RT-PCR. Localization of mRNAs was examined by in situ -hybridization. Levels of expression differed by more than three orders of magnitude. Expression also showed different seasonal patterns. IGF-1, IGF-2 and bFGF were maximally transcribed during testis recrudescence in spring. In contrast, the mRNA amount of aFGF reached its maximum between July (breeding season) and January. TGF- &#33 mRNA-levels were very low and showed poor seasonal variation. Each growth factor showed its own typical expression localization in testicular tissues and cell types. The results suggest the specific role of different growth factors in the paracrine control of spermatogenesis and its seasonal regulation.     

cDNA cloning and expression of a human FGF receptor which binds acidic and basic FGF.

We have isolated and characterized a cDNA clone, phFGFR, encoding a human fibroblast growth factor (FGF) receptor. phFGFR contains an open reading frame which encodes an 820 amino acid polypeptide with three immunoglobulin-like domains in the extracellular part and an intracellular split tyrosine kinase domain. Transient expression in COS-1 cells and immunoprecipitation using an antiserum raised against a C-terminal peptide, gave rise to two components, representing mature (130 kDa) and precursor (115 kDa) forms of the phFGFR encoded polypeptide, which was denoted hFGFR-1. Crosslinking of iodinated acidic FGF (aFGF) and basic FGF (bFGF) to transiently expressing COS-1 cells revealed a major band of 95 kDa, which was competed for by both aFGF and bFGF. From Scatchard analyses, the Kd:s for binding of aFGF and bFGF to hFGFR-1 were estimated to 25 pM and 41 pM, respectively. Thus, phFGFR encodes a human FGF receptor with high affinity for both aFGF and bFGF.     

aFGF immunoreactivity in prostate cancer and its co-localization with bFGF and FGF8

Fibroblast growth factors (FGFs) have been implicated in the development of numerous malignancies including prostate cancer. In a pilot study it has been shown that FGF8 mRNA is up-regulated in prostate cancer. The aim of the present study was to determine whether aFGF and bFGF were co-expressed with FGF8 in human prostate cancer. Twenty-nine cases of prostate cancer of different histological grades were examined. Immunohistochemical analysis was employed to study aFGF and bFGF expression. In the light of the results, aFGF immunoreactivity was studied in a further 43 cases. aFGF and bFGF immunoreactivity was identified in the cytoplasm of the malignant prostatic epithelium. aFGF was overexpressed in 62/72 (86.1 per cent) cases and bFGF in 19/29 (65.5 per cent). High levels of aFGF immunoreactivity were noted in areas of high-grade prostatic intraepithelial neoplasia (PIN). In this series, aFGF immunoreactivity was most commonly observed and correlated closely with Gleason score and tumour stage ( p=0.007 and 0.007, respectively). Co-localization of aFGF, bFGF, and FGF8 was detected in 9/29 (31.0 per cent) cases. There was a significant correlation between aFGF and FGF8 expression. In conclusion, aFGF, bFGF, and FGF8 are co-localized in human prostate cancer; they may have a synergistic effect in prostate cancer growth and progression.