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Induction of Tissue Factor Expression in Endothelial Cells by Basic Fibroblast Growth Factor and its Modulation by Fenofibric acid
Takeaki Kaneko, Satoshi Fujii, Akio Matsumoto, Daisuke Goto, Naomasa Makita, Junichi Hamada, Tetsuya Moriuchi, Akira Kitabatake
Thrombosis Journal , 2003, DOI: 10.1186/1477-9560-1-6
Abstract: bFGF increased TF mRNA and protein expression in HUVECs. Increased TF mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. Transient transfection assays of the human TF promoter-luciferase construct (-786/+121 bp) demonstrated that bFGF induced transcription was dependent on the elements within the -197 to -176 bp relative to the transcription start site of the human TF gene. This region contains NF-κB like binding site. Electro mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of NF-κB transcription factor to TF promoter. Nucleotide substitution to disrupt NF-κB like site reduced bFGF stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator activated receptor-α, reduced basal and bFGF stimulated TF expression.These results indicate that bFGF may increase TF production in ECs through activation of transcription at NF-κB binding site, and control coagulation in vessel walls. Fibrate can inhibit TF expression and therefore reduce the thrombogenecity of human atherosclerotic lesions.Tissue factor (TF) is an integral membrane protein, which binds to coagulation factor VII/VIIa and initiates the coagulation cascade [1]. TF expression can be inducible with inflammatory cytokines, lipopolysaccharide (LPS) and oxidized low-density lipoprotein in fibroblast, vascular smooth muscle cells and monocytes in human atherosclerotic lesions [2]. TF expressed on the surface of vascular wall acts as the major procoagulant for thrombus formation [3].Basic fibroblast growth factor (bFGF) elaborated by vascular cells can induce proliferation, morphological changes and migration of smooth muscle cells and endothelial cells (ECs) and modulate angiogenesis [4]. bFGF promotes re-endothelialization with functional endothelium after balloon injury or induced atherosclerosis and modulates TF expression in monocytes and smooth muscle cells in vivo [5]. In ECs TF expressi
Chemically-Induced RAT Mesenchymal Stem Cells Adopt Molecular Properties of Neuronal-Like Cells but Do Not Have Basic Neuronal Functional Properties  [PDF]
Gabriela F. Barnabé, Telma T. Schwindt, Maria E. Calcagnotto, Fabiana L. Motta, Gilberto Martinez, Allan C. de Oliveira, Leda M. N. Keim, Vania D'Almeida, Rosália Mendez-Otero, Luiz E. Mello
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005222
Abstract: Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, β-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na+ or K+ currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro.
Induction-dependent neural marker expression and electrophysiological characteristics of bone marrow mesenchymal stem cells that naturally express high levels of nestin
Yu Liu,ZhiZhong Ye,YaZhu Wang,YuBin Deng,GuangQian Zhou
Chinese Science Bulletin , 2011, DOI: 10.1007/s11434-010-4310-1
Abstract: Under certain experimental conditions, bone marrow mesenchymal stem cells (MSCs) express neuronal phenotypes and neuronal markers, which suggests that they could be used to treat various neurological diseases. In the present study, MSCs were isolated from adult rat bone marrow, cultivated, and evaluated for neurotrophin expression profiles, as well as the potential to differentiate into functional neuronal-like cells in vitro. MSCs from passage 5 were pre-induced with DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 10 ng/mL bFGF (fibroblast growth factor-2). Subsequently, a chemical inductor containing Dimethyl Sulphoxide (DMSO), Butylated Hydroxyanisole (BHA) and forskolin were used to induce neural expression of MSCs. Expression patterns of nestin, NF-200, and GFAP at time points before and after induction were detected by immunofluorescence. Nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF) expressions in MSCs were evaluated by RT-PCR. The whole-cell patch clamp technique was utilized to elucidate the electrical behavior of MSC before and after 24-h differentiation induction. Immunofluorescence analysis revealed that MSCs expressed nestin (57.1% ± 6.9%), but not NF-200 or GFAP. Following neural induction, the cells exhibited a neuronal-like appearance. Nestin and NF-200 expression was positive in the neuronal-like cells, but GFAP expression was negative. After 6-, 12- and 24-h induction, the ratio of nestin-positive cells was 96.5% ± 1.9%, 88.1% ± 5.4%, and 33.5% ± 5.4%. NF-200 positive cells were 90.1% ± 2.9%, 97.5% ± 1.3%, and 98.1% ± 1.6%, respectively. However, prior to induction, MSCs already expressed NGF and BDNF. With a stimulus impulse of 40 mV, the density of the transient outward K current was (9.95 ± 4.85) pA/pF (n = 9) and (328.50 ± 30.62) pA/pF (n = 9) before and after induction, and the density of transient calcium ion currents was ( 0.059 ± 0.027) pA/pF (n = 7) and ( 6.66 ± 0.50) pA/pF (n = 7), respectively. Transient outward potassium currents and calcium ions currents gradually increased following induction. In addition, MSCs isolated from bone marrow exhibited characteristics of neuronal progenitor cells and expressed neurotrophins. These cells exhibited the capacity to differentiate into functional neuronal-like cells in vitro. These results suggested that MSCs express high levels of nestin and could be utilized for therapeutic strategies to treat nervous system diseases.
Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli  [PDF]
Mona Alibolandi,Hasan Mirzahoseini
Biotechnology Research International , 2011, DOI: 10.4061/2011/973741
Abstract: This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8?M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution. 1. Introduction Production of therapeutic proteins in inclusion bodies is useful due to the efficacy of insoluble expression such as high product yield and protection against degradation by proteases [1]. The recovery of biologically active protein from such inclusion bodies requires refolding protocols. In general, the methods used for inclusion body solubilization result in a soluble protein that is biologically inactive. The solublized proteins do not have native conformation and must be transferred into conditions that allow the formation of the native structure. During this period, the correct folding pathway competes, often in disadvantage with misfolding and aggregation of the target protein. Protein refolding involves intramolecular interactions and follows first-order kinetics [2]. Refolding yields commonly decrease with increasing initial concentrations of the unfolded protein independent of the refolding method applied [3]. Aggregates are formed by nonnative intramolecular hydrophobic interactions between protein folding intermediates, which have not yet buried their hydrophobic amino acid stretches. Therefore, prevention of hydrophobic intermolecular interaction during the first steps of refolding is crucial to allow successful renaturation at high protein concentrations [4]. A very efficient strategy to prevent aggregation is to minimize the risk of
The Anticonvulsant Ethosuximide Disrupts Sensory Function to Extend C. elegans Lifespan  [PDF]
James J. Collins,Kimberley Evason,Christopher L. Pickett,Daniel L. Schneider,Kerry Kornfeld
PLOS Genetics , 2008, DOI: 10.1371/journal.pgen.1000230
Abstract: Ethosuximide is a medication used to treat seizure disorders in humans, and we previously demonstrated that ethosuximide can delay age-related changes and extend the lifespan of the nematode Caenorhabditis elegans. The mechanism of action of ethosuximide in lifespan extension is unknown, and elucidating how ethosuximide functions is important for defining endogenous processes that influence lifespan and for exploring the potential of ethosuximide as a therapeutic for age-related diseases. To identify genes that mediate the activity of ethosuximide, we conducted a genetic screen and identified mutations in two genes, che-3 and osm-3, that cause resistance to ethosuximide-mediated toxicity. Mutations in che-3 and osm-3 cause defects in overlapping sets of chemosensory neurons, resulting in defective chemosensation and an extended lifespan. These findings suggest that ethosuximide extends lifespan by inhibiting the function of specific chemosensory neurons. This model is supported by the observation that ethosuximide-treated animals displayed numerous phenotypic similarities with mutants that have chemosensory defects, indicating that ethosuximide inhibits chemosensory function. Furthermore, ethosuximide extends lifespan by inhibiting chemosensation, since the long-lived osm-3 mutants were resistant to the lifespan extension caused by ethosuximide. These studies demonstrate a novel mechanism of action for a lifespan-extending drug and indicate that sensory perception has a critical role in controlling lifespan. Sensory perception also influences the lifespan of Drosophila, suggesting that sensory perception has an evolutionarily conserved role in lifespan control. These studies highlight the potential of ethosuximide and related drugs that modulate sensory perception to extend lifespan in diverse animals.
Basic Fibroblast Growth Factor for Treatment of Onychomadesis with Delayed Regrowth of the Nail  [PDF]
Tomito Oji,Masaki Yazawa,Kazuo Kishi
Case Reports in Dermatological Medicine , 2013, DOI: 10.1155/2013/214810
Abstract: Onychomadesis usually arises from an inflammation of the paronychium or as a result of blisters and hemorrhaging under a nail that has been struck or compressed. No documented interactions between basic fibroblast growth factor (bFGF) and onychomadesis have hitherto been reported. This case report describes a 25-year-old woman with onychomadesis following infection of the ingrown nail of her left thumb. After ten months of observation with no treatment showed no regrowth of her left thumbnail, the external use of bFGF and antibiotic ointment was started. One month later, nail regrowth was observed up to the halfway point of the nail bed, and after treatment for three months, the regrown nail reached the top of the nail bed. Both thumbnails now looked identical. This case suggests that external use of bFGF can promote nail regrowth in cases of onychomadesis with delayed regrowth of the nail. 1. Introduction Onychomadesis usually arises from an inflammation of the paronychium or blisters and hemorrhage under the nail caused by a blow or compression. Systemic diseases, such as hand-foot-and-mouth disease, pemphigus vulgaris, and Stevens-Johnson syndrome, occasionally result in onychomadesis. Normally, simple observation or supportive treatment of inflammation will promote the regrowth of the fallen nail. However, in cases where nail regrowth does not occur, the condition tends to be refractory. Hitherto, there have been no effective treatments that promote the regrowth of fallen nails. In this paper, we report a notable case of onychomadesis after infection of an ingrown nail, in which the external use of basic fibroblast growth factor (bFGF; Trafermin; Fiblast Spray) and antibiotic ointment (Bacitracin-fradiomycin sulfate; Baramycin) led to normal nail regrowth. 2. Case Presentation The case was a 25-year-old woman. She was a nursery teacher and routinely washed her hands many times daily. She had no previous medical history and was taking no medications or dietary supplements. Her left thumb had an ingrown nail, but she had not had it examined in a clinic, since it showed no troublesome symptoms. However, after her ingrown nail became infected, it presented with pain, swelling, and discharge of pus. Those symptoms continued for a month, after which the nail fell off the nail bed. She went to a clinic to have her left thumbnail examined at that time, but underwent observation rather than receiving treatment due to the expectation that it would spontaneously regenerate. No further infections flared up, but no regrowth of her left thumbnail was seen in four
Differential Fibroblast Growth Factor 8 (FGF8)-Mediated Autoregulation of Its Cognate Receptors, Fgfr1 and Fgfr3, in Neuronal Cell Lines  [PDF]
Natasha N. Mott,Wilson C. J. Chung,Pei-San Tsai,Toni R. Pak
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0010143
Abstract: Fibroblast growth factors (FGFs) mediate a vast range of CNS developmental processes including neural induction, proliferation, migration, and cell survival. Despite the critical role of FGF signaling for normal CNS development, few reports describe the mechanisms that regulate FGF receptor gene expression in the brain. We tested whether FGF8 could autoregulate two of its cognate receptors, Fgfr1 and Fgfr3, in three murine cell lines with different lineages: fibroblast-derived cells (3T3 cells), neuronal cells derived from hippocampus (HT-22 cells), and neuroendocrine cells derived from hypothalamic gonadotropin-releasing hormone (GnRH) neurons (GT1-7 cells). GnRH is produced by neurons in the hypothalamus and is absolutely required for reproductive competence in vertebrate animals. Several lines of evidence strongly suggest that Fgf8 is critical for normal development of the GnRH system, therefore, the GT1-7 cells provided us with an additional endpoint, Gnrh gene expression and promoter activity, to assess potential downstream consequences of FGF8-induced modulation of FGF receptor levels. Results from this study suggest that the autoregulation of its cognate receptor represents a common downstream effect of FGF8. Further, we show that Fgfr1 and Fgfr3 are differentially regulated within the same cell type, implicating these two receptors in different biological roles. Moreover, Fgfr1 and Fgfr3 are differentially regulated among different cell types, suggesting such autoregulation occurs in a cell type-specific fashion. Lastly, we demonstrate that FGF8b decreases Gnrh promoter activity and gene expression, possibly reflecting a downstream consequence of altered FGF receptor populations. Together, our data bring forth the possibility that, in addition to the FGF synexpression group, autoregulation of FGFR expression by FGF8 represents a mechanism by which FGF8 could fine-tune its regulatory actions.
Ixora coccinea Enhances Cutaneous Wound Healing by Upregulating the Expression of Collagen and Basic Fibroblast Growth Factor  [PDF]
Aadesh Upadhyay,Pronobesh Chattopadhyay,Danswrang Goyary,Papiya Mitra Mazumder,Vijay Veer
ISRN Pharmacology , 2014, DOI: 10.1155/2014/751824
Abstract: Background. Ixora coccinea L. (Rubiaceae) has been documented for traditional use in hypertension, menstrual irregularities, sprain, chronic ulcer, and skin diseases. In the present study, I. coccinea was subjected to in vitro and in vivo wound healing investigation. Methods. Petroleum ether, chloroform, methanol, and water sequential I. coccinea leaves extracts were evaluated for in vitro antioxidant, antimicrobial, and fibroblast proliferation activities. The promising I. coccinea methanol extract (IxME) was screened for in vivo wound healing activity in Wistar rat using circular excision model. Wound contraction measurement, hydroxyproline quantification, and western blot for collagen type III (COL3A1), basic fibroblast growth factor (bFGF), and Smad-2, -3, -4, and -7 was performed with 7-day postoperative wound granulation tissue. Gentamicin sulfate (0.01% w/w) hydrogel was used as reference standard. Results. IxME showed the potent antimicrobial, antioxidant activities, with significant fibroblast proliferation inducing activity, as compared to all other extracts. In vivo study confirmed the wound healing accelerating potential of IxME, as evidenced by faster wound contraction, higher hydroxyproline content, and improved histopathology of granulation tissue. Western blot analysis revealed that the topical application of I. coccinea methanol extract stimulates the fibroblast growth factor and Smad mediated collagen production in wound tissue. 1. Background The World Health Organization estimated that 80% of the world’s population still relies on plant-based medicines for their primary health care, and skin related disorders specially wounds, which is the most common reason for medical visits in the developing countries. Wounds and wound management therapy have a long history and in the different Ayurvedic classics of India like Charaka Samhita, Sushruta Samhita, and Ayurveda Siksha approximately 70% of the wound healing medicines are of plant origin. In the last few decades, traditional wound healing plants have received enough attention for scientific investigations [1–3], where pathophysiological process of wound healing and various related activities such as fibroblast growth stimulation and antioxidant and antimicrobial activities has been extensively studied and correlated to the rationale of the traditional plant medicines [1, 4, 5]. In thrust of finding for an effective wound healing herb,? ?Ixora coccinea L. (Rubiaceae) was selected for the in vitro and in vivo wound healing investigations. I. coccinea is a small-medium evergreen shrub,
Analysis of trophic responses in lesioned brain: focus on basic fibroblast growth factor mechanisms
Chadi, G.;Fuxe, K.;
Brazilian Journal of Medical and Biological Research , 1998, DOI: 10.1590/S0100-879X1998000200007
Abstract: the actions of fibroblast growth factors (fgfs), particularly the basic form (bfgf), have been described in a large number of cells and include mitogenicity, angiogenicity and wound repair. the present review discusses the presence of the bfgf protein and messenger rna as well as the presence of the fgf receptor messenger rna in the rodent brain by means of semiquantitative radioactive in situ hybridization in combination with immunohistochemistry. chemical and mechanical injuries to the brain trigger a reduction in neurotransmitter synthesis and neuronal death which are accompanied by astroglial reaction. the altered synthesis of bfgf following brain lesions or stimulation was analyzed. lesions of the central nervous system trigger bfgf gene expression by neurons and/or activated astrocytes, depending on the type of lesion and time post-manipulation. the changes in bfgf messenger rna are frequently accompanied by a subsequent increase of bfgf immunoreactivity in astrocytes in the lesioned pathway. the reactive astrocytes and injured neurons synthesize increased amount of bfgf, which may act as a paracrine/autocrine factor, protecting neurons from death and also stimulating neuronal plasticity and tissue repair
Analysis of trophic responses in lesioned brain: focus on basic fibroblast growth factor mechanisms  [cached]
Chadi G.,Fuxe K.
Brazilian Journal of Medical and Biological Research , 1998,
Abstract: The actions of fibroblast growth factors (FGFs), particularly the basic form (bFGF), have been described in a large number of cells and include mitogenicity, angiogenicity and wound repair. The present review discusses the presence of the bFGF protein and messenger RNA as well as the presence of the FGF receptor messenger RNA in the rodent brain by means of semiquantitative radioactive in situ hybridization in combination with immunohistochemistry. Chemical and mechanical injuries to the brain trigger a reduction in neurotransmitter synthesis and neuronal death which are accompanied by astroglial reaction. The altered synthesis of bFGF following brain lesions or stimulation was analyzed. Lesions of the central nervous system trigger bFGF gene expression by neurons and/or activated astrocytes, depending on the type of lesion and time post-manipulation. The changes in bFGF messenger RNA are frequently accompanied by a subsequent increase of bFGF immunoreactivity in astrocytes in the lesioned pathway. The reactive astrocytes and injured neurons synthesize increased amount of bFGF, which may act as a paracrine/autocrine factor, protecting neurons from death and also stimulating neuronal plasticity and tissue repair
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