Size exclusion HPLC method for the determination of acidic fibroblast growth factor in viscous formulations

A size exclusion HPLC method has been developed to determine the protein concentration of pharmaceutical formulations of recombinant acidic fibroblast growth factor (aFGF). These topical aFGF formulations not only contain low levels of protein mass (50 micrograms ml-1), but also include buffer ions, polysaccharide polyanions to conformationally stabilize aFGF and 1% hydroxyethylcellulose to increase the solution's viscosity. A cesium chloride mobile phase is utilized during SEC-HPLC to dissociate aFGF from the pharmaceutical excipients and to minimize nonspecific interaction of the protein with the column matrix. The protein content of a viscous aFGF formulation is determined by comparison of aFGF peak areas to standards of known concentration. Fluorescence spectroscopy was utilized to directly demonstrate that the protein remains in its native conformation during sample preparation and analysis.     

Effects of acidic fibroblast growth factor on neuronal activity of the parvocellular part in rat paraventricular nucleus

The effects of acidic fibroblast growth factor (aFGF) and its amino-terminal and carboxyl-terminal fragments (aFGF(1-15) and aFGF(114-140), respectively were examined on the neuronal activity in the parvocellular part of the paraventricular nucleus. As well known, this part contains a lot of corticotropin-releasing factor (CRF)-immunoreactive neurons. Application of 1 pg/ml and 2 pg/ml aFGF produced responses in 29.7% and 46.7% of neurons tested, respectively. Half or more than half of the responding neurons increased their discharge rate. Application of 0.2 ng/ml and 0.4 ng/ml aFGF(1-15) (1-15) also elicited response in 46.2% and 68.8% of neurons tested, respectively. Of these responding neurons, more than two third increased their firing rate. However, most of neurons tested for 0.67 ng/ml and 1.33 ng/ml aFGF(114-140) did not respond. Results suggest that aFGF and aFGF(1-15) promote the release of CRF through the activation of CRF-containing neurons.     

Brain-derived neurotrophic factor, acidic and basic fibroblast growth factors, insulin-like growth factor-I, and various antioxidants do not prevent the apoptotic death of developing septal cholinergic neurons following nerve growth factor withdrawal in vitro

The degree to which growth factors act alone or in combination to influence neuronal survival during the development of the central nervous system is not well understood. In this study, we investigated whether multiple growth factors might interact to regulate the survival of developing basal forebrain cholinergic neurons in vitro, in the rat. We have previously shown that most embryonic septal cholinergic neurons grown in sandwich cultures in serum-free, completely defined medium are dependent on nerve growth factor during a critical period of their development, such that nerve growth factor withdrawal during this period results in the protein synthesis-dependent, apoptotic death of most, but not all, of these neurons. Here we report that brain-derived neurotrophic factor, acidic and basic fibroblast growth factors, and insulin-like growth factor-I applied individually in serum-free, completely defined medium, were not able either to support the development of septal cholinergic neurons from plating at embryonic day 16, or to prevent the cell death of these neurons induced by nerve growth factor withdrawal during days 14-18 after plating. We also found that the apoptotic death of developing septal cholinergic neurons induced by nerve growth factor withdrawal was not prevented by a number of antioxidants, with the exception of a high concentration (50 mM) of ascorbic acid. However, this effect of ascorbic acid was prevented when pH was buffered, and is likely to have been mediated via a proton-induced sustained neuronal depolarization. These findings suggest that in the absence of serum and other additives, brain-derived neurotrophic factor, acidic and basic fibroblast growth factors, and insulin-like growth factor-I do not interact with nerve growth factor to regulate the survival of septal cholinergic neurons during the developmental period spanned by this in vitro model. In addition, the findings suggest that the apoptotic death of septal cholinergic neurons induced by nerve growth factor withdrawal is not mediated by oxidative stress or free radical generation.     

Immunolocalization of acidic fibroblast growth factor and receptors in the tubulointerstitial compartment of chronically rejected human renal allografts

Tubular damage and loss associated with interstitial inflammation and fibrosis may be the most important determinants in chronic renal allograft rejection. To elucidate potential pathophysiologic mechanisms associated with tubulointerstitial lesions, we examined the expression of a fibrogenic cytokine, acidic fibroblast growth factor (FGF-1) and its high-affinity receptors, in both relevant renal transplant controls (n=5) and tissue from patients (n=19) who underwent nephrectomy after graft loss, secondary to chronic rejection. In situ hybridization and immunohistochemical analyses demonstrated minimal expression of FGF-1 mRNA and protein in the tubulointerstitial compartment of the normal human kidney. In contrast, tubulointerstitial lesions in kidney allografts experiencing chronic rejection demonstrated the exaggerated appearance of both FGF-1 protein and mRNA in resident inflammatory and tubular epithelial cells. Patterns of staining were consistent throughout tubular compartments and did not appear to be localized to any particular region. The tubulointerstitium in kidneys with findings of chronic rejection also exhibited increased immunodetection of proliferating cell nuclear antigen in the tubular epithelium, inflammatory cell infiltrate, and neovascular structures. The enhanced appearance of FGF-1 and readily detectable fibroblast growth factor receptors suggests that this polypeptide mitogen may serve as an important mediator of growth and repair responses, associated with development of angiogenesis and tubulointerstitial lesions during chronic rejection of human renal allografts.     

The location of acidic fibroblast growth factor in the breast is dependent on the activity of proteases present in breast cancer tissue

Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 37 degrees C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast.     

Solution structure of acidic fibroblast growth factor bound to 1,3, 6-naphthalenetrisulfonate: a minimal model for the anti-tumoral action of suramins and suradistas

Recent data show that anti-angiogenesis may provide a promising route to treat cancer. Fibroblast growth factors (FGFs) are powerful angiogenic polypeptides, whose mitogenic activity requires the presence of heparin-like compounds. It has been shown that angiogenesis promoted by FGFs on inhibition by monoclonal antibodies and antisense targeting can also inhibit tumour growth. Derivatives of suramin, a polysulfonated binaphthyl urea and binaphthylsulfonated derivatives of distamycin, suradistas, constitute an important group of potential anti-cancer agents. These compounds compete with heparin in forming tight complexes with FGFs. This inhibits the recognition of these growth factors by their tyrosine kinase membrane receptors thereby suppressing their angiogenic activity. Here we show that 1,3,6-naphthalenetrisulfonate, a common chemical function of the suramins and suradistas with the highest anti-angiogenic activity inhibits the mitogenic activity of acidic fibroblast growth factor, and that this inhibition is relieved by increasing concentrations of heparin in the assay. We have also solved the three-dimensional structure in solution of the protein complexed to this compound. The structural data provide clues that may help in understanding the inhibitory effect of suramins and suradistas, and could contribute to the development of new anti-tumoral drugs.     

Peroxynitrite induces covalent dimerization of epidermal growth factor receptors in A431 epidermoid carcinoma cells

Irreversible tyrosine modifications by inflammatory oxidants such as peroxynitrite (ONOO-) can affect signal transduction pathways involving tyrosine phosphorylation. The epidermal growth factor receptor (EGFR), a member of the c-ErbB receptor tyrosine kinase family, is involved in regulation of epithelial cell growth and differentiation, and possible modulation of EGFR-dependent signaling by ONOO- was studied. Exposure of epidermoid carcinoma A431 cells to 0.1-1.0 mM ONOO- resulted in tyrosine nitration on EGFR and other proteins but did not significantly affect EGFR tyrosine autophosphorylation. A high molecular mass tyrosine-phosphorylated protein (approximately 340 kDa) was detected in A431 cell lysates after exposure to ONOO-, most likely representing a covalently dimerized form of EGFR, based on immunoprecipitation and/or immunoblotting with alpha-EGFR antibodies and co-migration with ligand-induced EGFR dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Covalent EGFR dimerization by ONOO- probably involved intermolecular dityrosine cross-linking and was enhanced after receptor activation with epidermal growth factor. Furthermore, irreversibly cross-linked EGFR was more extensively tyrosine-phosphorylated compared with the monomeric form, indicating that ONOO- preferentially cross-links activated EGFR. Exposure of A431 cells to ONOO- markedly reduced the kinetics of tyrosine phosphorylation of a downstream EGFR substrate, phospholipase C-gamma1, which may be related to covalent alterations in EGFR. Alteration of EGFR signaling by covalent EGFR dimerization by inflammatory oxidants such as ONOO- may affect conditions of increased EGFR activation such as epithelial repair or tumorigenesis.     

Molecular studies in flexor tendon wound healing: the role of basic fibroblast growth factor gene expression

Basic fibroblast growth factor (bFGF) is a cytokine that plays a fundamental role in angiogenesis. This study examines bFGF messenger RNA (mRNA) expression in a rabbit flexor tendon wound healing model. Thirty-four New Zealand white rabbit forepaws underwent transection and repair of the middle digit flexor digitorum profundus tendon in zone II. Tendons were harvested at increasing time intervals and analyzed by in situ hybridization and immunohistochemistry. Few tenocytes and tendon sheath cells expressed bFGF mRNA in unwounded tendons. In contrast, tendons subjected to transection and repair exhibited an increased signal for bFGF mRNA in both resident tenocytes concentrated along the epitenon and infiltrating fibroblasts and inflammatory cells from the tendon sheath. These data demonstrate that (1) normal tenocytes and tendon sheath cells are capable of bFGF production, (2) bFGF mRNA is upregulated in the tendon wound environment, and (3) the upregulation of this angiogenic cytokine occurs in tenocytes as well as in tendon sheath fibroblasts and inflammatory cells.     

Intracerebroventricular injection of anti-Fas activates the hypothalamus-pituitary-adrenal axis and induces peripheral interleukin-6 and serum amyloid A in mice: comparison with other ligands of the tumor necrosis factor/nerve growth factor receptor superfamily

Fas is a receptor of the tumor necrosis factor (TNF)/ nerve growth factor (NGF) receptor superfamily that mediates apoptosis and some inflammatory changes. As the central administration of TNF is known to activate the hypothalamus-pituitary-adrenal axis (HPAA) and to induce peripheral responses including induction of serum interleukin (IL)-6 and serum amyloid A (SAA), we investigated the effects of intracerebroventricular (i.c.v.) administration of agonist anti-Fas monoclonal antibody Jo2. Centrally administered anti-Fas (1 microg/mouse, i.c.v.) induced elevated levels of corticosterone, IL-6, and SAA comparable to those observed after i.c.v. administration of recombinant murine TNF. On the other hand, administration of murine NGF did not elevate serum corticosterone or IL-6, but induced SAA. Thus, Fas can trigger a centrally mediated anti-inflammatory response (HPAA activation) and induce a peripheral acute-phase response comparable to that induced with TNF, whereas NGF induces only acute-phase proteins.     

Stored and encountered seeds: A comparison of two spatial memory tasks in marsh tits and chickadees.

Tested food-storing birds in 2 spatial memory tasks involving retrieval of stored seeds and retrieval of items encountered while searching. In Exp I, 4 wild-caught marsh tits (Parus palustris) were equally good, after a 2-hr retention interval, at retrieving stored items and items they had encountered while looking for storage sites. Retrieval of encountered seeds did not depend on retracing a route. In Exp II, 3 black-capped chickadees (Parus atricapillus) retrieved seeds 2 hrs after storing them or after "window-shopping." In window-shopping, the Ss encountered seeds behind small plastic windows; during retrieval, the windows were opened. Window-shopped seeds were retrieved above chance level but slightly less well than stored seeds. In this experiment the Ss seemed to rely in part on retracing a route. In Exp III, chickadees (2 from Exp II) were allowed to eat a small piece of seed by each window-shopped item, but this did not improve window-shopping retrieval performance. (17 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Preferences for emotional information in older and younger adults: A meta-analysis of memory and attention tasks.

The authors conducted a meta-analysis to determine the magnitude of older and younger adults' preferences for emotional stimuli in studies of attention and memory. Analyses involved 1,085 older adults from 37 independent samples and 3,150 younger adults from 86 independent samples. Both age groups exhibited small to medium emotion salience effects (i.e., preference for emotionally valenced stimuli over neutral stimuli) as well as positivity preferences (i.e., preference for positively valenced stimuli over neutral stimuli) and negativity preferences (i.e., preference for negatively valenced stimuli to neutral stimuli). There were few age differences overall. Type of measurement appeared to influence the magnitude of effects; recognition studies indicated significant age effects, where older adults showed smaller effects for emotion salience and negativity preferences than younger adults. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The basic fibroblast growth factor and its receptor in pulmonary adenocarcinomas: an investigation of their expression as prognostic markers

The expression of basic fibroblast growth factor (bFGF) and its receptor, the high-affinity type I basic fibroblast growth factor receptor (FGFR-1): were immunohistologically studied in tissues specimens from 167 patients with a pulmonary adenocarcinoma. Of the 167 specimens, 82 (49%) expressed bFGF and 104 (62%) expressed FGFR-1, bFGF and FGFR-1 were simultaneously expressed in 72 (43%). It was also found that many patients who showed intensely positive staining for bFGF were also positive for FGFR-1, and that the expression of bFGF or FGFR-1 or both was associated with p-stage, T and N factors. The overall prognosis was significantly poorer in the bFGF-positive or FGFR-1-positive patients than in negative patients (P < 0.01). The prognosis was also significantly poorer in all patients positive for both bFGF and FGFR-1 than in those negative for both (P < 0.01); this was also true for stage I patients (P < 0.05). Multivariate analysis showed that bFGF had a significant affect on prognosis, whereas FGFR-1 did not. As FGFR-1 is significantly linked with the bFGF expression, it may be that FGFR-1 interferes with the bFGF effect on survival. These findings suggest that bFGF and FGFR-1 play important roles in tumour progression, and that bFGF expression may be a useful prognostic marker for pulmonary adenocarcinomas.     

UVB radiation preferentially induces recruitment of memory CD4+ T cells in normal human skin: long-term effect after a single exposure

Acute, low-doses of ultraviolet (UV)-B radiation affect the immune competent cells of the skin immune system. In this study, we examined the time-dependent changes of the cutaneous T cell population in normal human volunteers following a single local exposure to UV. Solar-simulated UV radiation caused an initial decrease in intraepidermal T cell numbers, even leading to T cell depletion at day 4, whereupon a considerable infiltration of T cells in the epidermis occurred that peaked at day 14. In the dermis the number of T cells was markedly increased at days 2 (peak) and 4 after irradiation, and subsequently declined to the nonirradiated control values at day 10. Double-staining with several T cell markers showed that the T cells, infiltrating the (epi)dermis upon UV exposure, were almost exclusively CD4+ CD45RO+ T cells, expressing an alpha/beta type T cell receptor, but lacking the activation markers HLA-DR, VLA-1, and IL-2R. Application of UVB radiation resulted in similar dynamics of T cells, indicating that the UVB wavelengths within the solar-simulated UV radiation were responsible for the selective influx of CD4+ T cells. In conjunction with UVB-induced alterations in the type and function of antigen-presenting cells (i.e., Langerhans cells and macrophages), the changes of the cutaneous T cell population may also contribute to UVB-induced immunosuppression at skin level in man.     

Internalization of basic fibroblast growth factor (bFGF) in cultured endothelial cells: role of the low affinity heparin-like bFGF receptors

We have shown (Presta et al., Cell Regul., 2:719-726, 1991) that a long-lasting interaction of basic fibroblast growth factor (bFGF) with endothelial GM 7373 cells is required to induce cell proliferation. In the present work, we have investigated the interaction of 125I-bFGF with GM 7373 cells, its pathway of internalization, and its intracellular fate under the same experimental conditions previously utilized to assess the mitogenic activity of the growth factor. Cell cultures were incubated with 10 ng/ml 125I-bFGF for 2 h at 4 degrees C. Then, cells were shifted to 37 degrees C without changing the medium. A rapid down-regulation of high affinity sites, paralleled by a rapid internalization of 125I-bFGF, was observed during the first 1-2 h at 37 degrees C. After 6-8 h, also low affinity sites down-regulate. This was paralleled by a continuous internalization of 125I-bFGF and by a slow disappearance of the growth factor from the culture medium. This suggests that GM 7373 cells activate, when exposed to bFGF for a long period of time, a late internalization pathway mediated by low affinity sites. This hypothesis was supported by the following experimental evidence: 1) soluble heparin inhibited the prolonged internalization of 125I-bFGF and its binding to low affinity sites with the same potency; 2) treatment of GM 7373 cells with heparinase, which removes most of the low affinity sites, also inhibited the prolonged internalization of 125I-bFGF. 125I-bFGF internalized via low affinity sites was partially protected from lysosomal degradation. This was the case also when 125I-bFGF was internalized in the presence of soluble heparin, suggesting that the complexes bFGF-cell surface glycosaminoglycan and bFGF-soluble heparin are maintained during the internalization of the growth factor. Moreover, the capacity of soluble heparin to inhibit the mitogenic activity of bFGF also when added to cell cultures several hours after the growth factor indicates that the requirement for a prolonged interaction of bFGF with GM 7373 cells in order to induce cell proliferation might be related to the late internalization of the growth factor via low affinity sites.     

Basic fibroblast growth factor in a porcine model of chronic myocardial ischemia: a comparison of angiographic, echocardiographic and coronary flow parameters

Recently, a number of growth factors including basic fibroblast growth factor (bFGF) have been shown to promote angiogenesis in vivo. In this study, we evaluated dose-dependent effect of bFGF administration in the setting of chronic myocardial ischemia. A total of 18 Yorkshire pigs subjected to ameroid occluder placement on the left circumflex artery were randomized to treatment with 10 (n = 6) or 100 microg (n = 5) of bFGF incorporated into heparin-alginate microspheres or inactive control pellets (n = 7). Eight weeks later, all animals underwent angiographic evaluation of collateral development as well as studies of coronary flow and global and regional left ventricular function. Both bFGF groups had significantly higher angiographic collateral index, TIMI flow scores and coronary flow in the ameroid-compromised territory compared with controls. Left ventricular function studies demonstrated improved global and regional function in both fibroblast growth factor groups with significantly better preservation of regional wall motion in high dose (100 microg) bFGF animals. We conclude that local perivascular delivery of bFGF results in significant improvement in myocardial function in the setting of chronic myocardial ischemia.     

A possible role for multidrug resistance-associated protein in the secretion of basic fibroblast growth factor by osteogenic sarcoma cell line (MG-63)

Basic fibroblast growth factor (bFGF) lacks signal sequence and therefore the mechanism of its secretion is unknown. Multidrug resistance-associated protein (MRP) has been shown to transport a variety of molecules. Therefore, in this study we examined the role of MRP in the secretion of bFGF by osteogenic sarcoma MG-63 cells which spontaneously secrete bFGF. We show that MG-63 cells express MRP both at the protein and at the mRNA level. Furthermore, probenecid (a putative inhibitor of transport activity of MRP), in a concentration-dependent manner, inhibited secretion of bFGF from MG-63 cells with concomitant increase in intracellular contents of bFGF. These results suggest that MRP may have a possible role in the secretion of bFGF.