Absence Of Fibroblast Growth Factor Research Articles

IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.

Activation of unliganded FGF receptor by extracellular phosphate potentiates proteolytic protection of FGF23 by its O-glycosylation

Fibroblast growth factor (FGF) 23 produced by bone is a hormone that decreases serum phosphate (Pi). Reflecting its central role in Pi control, serum FGF23 is tightly regulated by serum Pi alterations. FGF23 levels are regulated by the transcriptional event and posttranslational cleavage into inactive fragments before its secretion. For the latter, O-glycosylation of FGF23 by GALNT3 gene product prevents the cleavage, leading to an increase in serum FGF23. However, the molecular basis of Pi sensing in the regulation of serum FGF23 remains elusive. In this study, we showed that high Pi diet enhanced the skeletal expression of Galnt3, but not Fgf23, with expected increases in serum FGF23 and Pi in mice. Galnt3 induction by high Pi was further observed in osteoblastic UMR 106 cells, and this was mediated by activation of the extracellular signal-regulated kinase (ERK) pathway. Through proteomic searches for the upstream sensor for high Pi, we identified one subtype of the FGF receptor (FGFR1c), which was phosphorylated by high Pi in the absence of FGFs. The mode of unliganded FGFR activation by high Pi appeared different from that of FGFR bound to a canonical FGFR ligand (FGF2) when phosphorylation of the FGFR substrate 2α and ERK was monitored. Finally, we showed that an FGFR inhibitor and conditional deletion of Fgfr1 in osteoblasts/osteocytes abrogated high Pi diet-induced increases in serum FGF23 and femoral Galnt3 expression in mice. Thus, these findings uncover an unrecognized facet of unliganded FGFR function and illustrate a Pi-sensing pathway involved in regulation of FGF23 production.

Esrrb plays important roles in maintaining self-renewal of trophoblast stem cells (TSCs) and reprogramming somatic cells to induced TSCs

Trophoblast stem cells (TSCs), which can be derived from the trophoectoderm of a blastocyst, have the ability to sustain self-renewal and differentiate into various placental trophoblast cell types. Meanwhile, essential insights into the molecular mechanisms controlling the placental development can be gained by using TSCs as the cell model. Esrrb is a transcription factor that has been shown to play pivotal roles in both embryonic stem cell (ESC) and TSC, but the precise mechanism whereby Esrrb regulates TSC-specific transcriptome during differentiation and reprogramming is still largely unknown. In the present study, we elucidate the function of Esrrb in self-renewal and differentiation of TSCs, as well as during the induced TSC (iTSC) reprogramming. We demonstrate that the precise level of Esrrb is critical for stem state maintenance and further trophoblast differentiation of TSCs, as ectopically expressed Esrrb can partially block the rapid differentiation of TSCs in the absence of fibroblast growth factor 4. However, Esrrb depletion results in downregulation of certain key TSC-specific transcription factors, consequently causing a rapid differentiation of TSCs and these Esrrb-deficient TSCs lose the ability of hemorrhagic lesion formation in vivo. This function of Esrrb is exerted by directly binding and activating a core set of TSC-specific target genes including Cdx2, Eomes, Sox2, Fgfr4, and Bmp4. Furthermore, we show that Esrrb overexpression can facilitate the MEF-to-iTSC conversion. Moreover, Esrrb can substitute for Eomes to generate GEsTM-iTSCs. Thus, our findings provide a better understanding of the molecular mechanism of Esrrb in maintaining TSC self-renewal and during iTSC reprogramming.

Expansion and Phenotypic Changes of Mouse Bone Marrow Mesenchymal Cells Cultured with FGF-2 and Facial Nerve-Conditioned Medium

Mesenchymal cells (MCs) exhibit great regenerative potential due to their intrinsic properties and ability to restore tissue function, either directly through transdifferentiation or indirectly through paracrine effects. This study aimed to evaluate morphometric and phenotypic changes in MCs grown with facial nerve-conditioned medium in the presence or absence of fibroblast growth factor 2 (FGF-2). For quantitative phenotypic analysis, the expression of GFAP, OX-42, MAP-2, β-tubulin III, NeuN, and NF-200 was analyzed by immunocytochemistry. Cells cultured with facial nerve-conditioned medium in the presence of FGF-2 expressed GFAP, OX-42, MAP-2, β-tubulin III, NeuN, and NF-200. On average, the area and perimeter of GFAP-positive cells were higher in the group cultured with facial nerve-conditioned medium compared to the group cultured with conditioned medium and FGF-2 (p=0.0001). This study demonstrated the plasticity of MCs for neuronal and glial lineages and opens up new research perspectives in cell therapy and trans.differentiation

Reducing the risk of impaired bone apposition to titanium screws with the use of fibroblast growth factor-2\u2212apatite composite layer coating

BackgroundLoosening of screws is a common problem in orthopedic and maxillofacial surgery. Modifying the implant surface to improve the mechanical strength of screws has been tried and reported. We developed screws coated with fibroblast growth factor-2 (FGF-2)−apatite composite layers. We then showed, in a percutaneous external fixation model, that this composite layer had the ability to hold and release FGF-2 slowly, thereby reducing the risk of pin tract infection of the percutaneous external fixation. The objective of the current study was to clarify the effect of FGF-2−apatite composite layers on titanium screws on bone formation around the screw.MethodsWe analyzed samples of previously performed animal experiments. The screws were coated with FGF-2−apatite composite layers by immersing them in supersaturated calcium phosphate solutions containing FGF-2. Then, the uncoated, apatite-coated, and FGF-2−apatite composite layer-coated screws were implanted percutaneously in rabbits. Finally, using inflammation-free histological sections, we histomorphometrically assessed them for the presence of bone formation. Weibull plot analysis was then applied to the data.ResultsOn average, screws coated with FGF-2−apatite composite layers showed a significantly higher bone apposition rate than the uncoated or apatite-coated screws. Although the difference in the average bone apposition rate was small, the FGF-2−apatite composite layers produced a significant, marked reduction in the incidence of impaired bone formation around the screw compared with the incidence in the absence of FGF-2 (uncoated and apatite-coated screws). The probability of resulting in a bone apposition rate equal to or less than 63.75%, for example, is 3.5 × 10-4 for screws coated with the FGF-2−apatite composite layers versus 0.05 for screws in the absence of FGF-2.ConclusionsFGF-2-apatite composite layer coating significantly reduced the risk of impaired bone apposition to the screw. Thus, it is feasible to use titanium screws coated with FGF-2−apatite composite layers as internal fixation screws to decrease the risk of loosening.

β-Catenin inactivation is a pre-requisite for chick retina regeneration.

In the present study we explored the role of β-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear β-catenin in the CM and RPE after injury (retinectomy). β-catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear β-catenin was maintained as long as FGF2 was present. However, nuclear β-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear β-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear β-catenin. Moreover, retinectomy followed by disruption of active β-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that β-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of β-catenin is a pre-requisite for chick retina regeneration.

Engineering de novo assembly of fetal pulmonary organoids.

Induction of morphogenesis by competent lung progenitor cells in a 3D environment is a central goal of pulmonary tissue engineering, yet little is known about the microenvironmental signals required to induce de novo assembly of alveolar-like tissue in vitro. In extending our previous reports of alveolar-like tissue formation by fetal pulmonary cells stimulated by exogenous fibroblast growth factors (FGFs), we identified some of the key endogenous mediators of FGF-driven morphogenesis (organoid assembly), for example, epithelial sacculation, endothelial network assembly, and epithelial-endothelial interfacing. Sequestration of endogenously secreted vascular endothelial growth factor-A (VEGF-A) potently inhibited endothelial network formation, with little or no effect on epithelial morphogenesis. Inhibition of endogenous sonic hedgehog (SHH) partially attenuated FGF-driven endothelial network formation, while the addition of exogenous SHH in the absence of FGFs was able to induce epithelial and endothelial morphogenesis, although with distinct morphological characteristics. Notably, SHH-induced endothelial networks exhibited fewer branch points, reduced sprouting behavior, and a periendothelial extracellular matrix (ECM) virtually devoid of tenascin-C (TN-C). By contrast, focal deposition of endogenous TN-C was observed in the ECM-surrounding endothelial networks of FGF-induced organoids, especially around sprouting tips. In the FGF-induced organoids, TN-C was also observed in the clefts of sacculated epithelium and at the epithelial-endothelial interface. In support of a critical role in the formation of alveolar-like tissue in vitro, TN-C blocking inhibited endothelial network formation and epithelial sacculation. Upon engraftment of in-vitro-generated pulmonary organoids beneath the renal capsule of syngeneic mice, robust neovascularization occurred in 5 days with a large contribution of patent vessels from engrafted organoids, providing proof of principle for exploring intrapulmonary engraftment of prevascularized hydrogel constructs. Expression of proSpC, VEGF-A, and TN-C following 1 week in vivo mirrored the patterns observed in vitro. Taken together, these findings advance our understanding of endogenous growth factor and ECM signals important for de novo formation of pulmonary tissue structures in vitro and demonstrate the potential of an organoid-based approach to lung tissue augmentation.

New culture system for human embryonic stem cells: Autologous mesenchymal stem cell feeder without exogenous fibroblast growth factor 2

Human embryonic stem (hES) cells have been successfully maintained using human-cell feeder systems or feeder-free systems. However, despite advances in culture techniques, hES cells require supplementation with fibroblast growth factor 2 (FGF-2), an exogenous stemness factor, which is needed to sustain the authentic undifferentiated status. We developed a new culture system for hES cells; this system does not require supplementation with FGF-2 to obtain hES cells that are suitable for tissue engineering and regenerative medicine. This culture system employed mesenchymal stem cells derived from hES cells (hESC-MSCs) as autologous human feeder cells in the absence of FGF-2. The hES cell line SNUhES3 cultured in this new autologous feeder culture system maintained the typical morphology of hES cells and expression of pluripotency-related proteins, SSEA-4, TRA-1-60, OCT4, and alkaline phosphatase, without development of abnormal karyotypes after more than 30 passages. RNA expression of the pluripotency-related genes OCT4 and NANOG was similar to the expression in SNUhES3 cells maintained on xenofeeder STO cells. To identify the mechanism that enables the cells to be maintained without exogenous FGF-2, we checked the secretion of FGF-2 from the mitomycin-C treated autofeeder hESC-MSCs versus xenofeeder STO cells, and confirmed that hESC-MSCs secreted FGF-2 whereas STO cells did not. The level of FGF-2 in the media from the autofeeder system without exogenous FGF-2 was comparable to that from the xenofeeder system with addition of FGF-2. In conclusion, our new culture system for hES cells, which employs a feeder layer of autologous hESC-MSCs, supplies sufficient amounts of secreted FGF-2 to eliminate the requirement for exogenous FGF-2.

Protective effects of basic fibroblast growth factor in the development of emphysema induced by interferon-γ

Recent clinical evidence indicates that the non-eosinophilic subtype of severe asthma is characterized by fixed airway obstruction, which may be related to emphysema. Transgenic studies have demonstrated that high levels of IFN-γ in the airways induce emphysema. Fibroblast growth factor 2 (FGF2), which is the downstream mediator of TGF-β, is important in wound healing. We investigated the role of FGF2 in IFN-γ-induced emphysema and the therapeutic effects of recombinant FGF2 in the prevention of emphysema in a severe non-eosinophilic asthma model. To evaluate the role of FGF2 in IFN-γ-induced emphysema, lung targeted IFN-γ transgenic mice were cross-bred with FGF2-deficient mice. A severe non-eosinophilic asthma model was generated by airway application of LPS-containing allergens twice a week for 4 weeks. To evaluate protective effects of FGF2, recombinant FGF2 (10 μg) was injected subcutaneously during allergen challenge in the severe asthma model. We found that non-eosinophilic inflammation and emphysema induced by transgenic overexpression of IFN-γ in the airways were aggravated by the absence of FGF2. Airway challenge with LPS-containing allergens induced more inflammation in mice sensitized with LPS-containing allergens compared to challenge with allergens alone. In addition, LPS-induced lung inflammation and emphysema depended on IFN-γ but not on IL-13. Interestingly, emphysema in the severe asthma model was significantly inhibited by treatment with recombinant FGF2 during allergen challenge, whereas lung inflammation was unaffected. Therefore, our present data suggest that FGF2 may help protect against IFN-γ-induced emphysema, and that recombinant FGF2 may help lessen the severity of emphysema.

The proteasome inhibitor bortezomib inhibits FGF‐2‐induced reduction of TAZ levels in osteoblast‐like cells

Bortezomib (PS-341; Velcade), a proteasome inhibitor, is used as a therapeutic agent for multiple myeloma. Bortezomib has been shown to strongly induce osteoblast differentiation and elevate the levels of osteoblast-related differentiation markers in the serum of patients with myeloma. Bortezomib also reportedly increases the activity of the transcription factor, Runx2. However, the mechanism of action by which bortezomib-elevated Runx2 activity mediates osteoblast differentiation remains unclear. On the other hand, fibroblast growth factor 2 (FGF-2) is found at high levels in patients with multiple myeloma. We previously reported that FGF-2 reduces the levels of the transcriptional coactivator with PDZ-binding motif (TAZ). We therefore investigated the effects of bortezomib on TAZ protein levels in the presence of FGF-2. Osteoblastic MC3T3-E1 cells were treated with different concentrations of bortezomib in the presence or absence of FGF-2 and various biologic responses were investigated by immunoblotting, RT-PCR, quantitative PCR, and alizarin red staining. We found that bortezomib inhibited FGF-2-induced reduction of TAZ levels through a pathway other than that used for proteasome inhibition, while maintaining TAZ function, which in turn, enhanced the expression of Runx2-transcribed osteogenic differentiation markers. Bortezomib also suppressed the antimineralization effect of FGF-2. These findings suggest that bortezomib inhibited FGF-2-induced reduction of TAZ and consequently stimulated osteogenic differentiation independently of proteasome inhibition. These findings may contribute to elucidate the osteolytic mechanism in multiple myeloma, and to the development of new drugs for multiple myeloma and other osteolytic diseases.

Mineralization behavior with mesenchymal stromal cells in a biomimetic hyaluronic acid-based scaffold

A biomimetic hyaluronic acid (HA)-based polymer scaffold was analysed in vitro for its characteristics and potential to support mineralization as carrier-vehicle. Biomimetic apatite crystal nucleation on the scaffold surface was obtained by a fine control of the pH level that increased ionic solubility thus controlling apatite formation kinetic. Different concentrations of human mesenchymal stromal cells (h-MSCs) were seeded on the scaffold, osteogenesis was induced in the presence or absence of fibroblast growth factor -2 and mineralization was analysed at different time points. We found that only at the highest h-MSCs concentration tested, the cells were uniformly distributed inside and outside the scaffold and proliferation started to decrease from day 7. Electron microscopy analysis evidenced that h-MSCs produced extracellular matrix but did not establish a direct contact with the scaffold. We found mineralized calcium-positive areas mainly present along the backbone of the scaffold starting from day 21 and increasing at day 35. FGF-2 treatment did not accelerate or increase mineralization. Non-biomimetic HA-based control scaffold showed immature mineralized areas only at day 35. Our data demonstrate that the biomimetic treatment of an HA-based scaffold promotes a faster mineralization process suggesting its possible use in clinics as a support for improving bone repair.

The role of FGF-signaling in early neural specification of human embryonic stem cells

The mechanisms that govern human neural specification are not completely characterized. Here we used human embryonic stem cells (hESCs) to study the role of fibroblast growth factor (FGF)-signaling in early human neural specification. Differentiation was obtained by culturing clusters of hESCs in chemically-defined medium. We show that FGF-signaling, which is endogenously active during early differentiation of hESCs, induces early neural specification, while its blockage inhibits neuralization. The early neuralization effect of FGF-signaling is not mediated by promoting the proliferation of existing neural precursors (NPs) or prevention of their apoptosis. The neural instructive effect of FGF-signaling occurs after an initial FGF-independent differentiation into primitive ectoderm-like fate. We further show that FGF-signaling can induce neuralization by a mechanism which is independent of modulating bone morphogenic protein (BMP)-signaling. Still, FGF-signaling is not essential for hESC neuralization which can occur in the absence of FGF and BMP-signaling. Collectively, our data suggest that human neural induction is instructed by FGF-signaling, though neuralization of hESCs can occur in its absence.

Phosphorylation of connexin-43 at serine 262 promotes a cardiac injury-resistant state

The cardioprotective agent fibroblast growth factor 2 (FGF-2) was found previously to promote phosphorylation of connexin-43 (Cx43) at protein kinase C (PKC) sites such as serine (S) 262 at levels above those of non-stimulated hearts. We asked if other PKC-dependent cardioprotective treatments cause a similar effect, and if Cx43 phosphorylation at S262 mediates resistance to injury. Isolated perfused adult rat hearts were subjected to the following treatments: ischaemic preconditioning (PC); diazoxide perfusion; FGF-2 pre-treatment followed by 30 min global ischaemia; 30 min global ischaemia followed by 60 min reperfusion in the presence or absence of FGF-2. Cx43 phosphorylation was assessed by western blotting with phospho-specific antibodies. Neonatal cardiomyocyte cultures were used to examine the effect of expressing Cx43 incapable of being phosphorylated at S262 due to an S to alanine (A) substitution on simulated ischaemia-induced cell death (TUNEL staining) and injury (lactic dehydrogenase release). Ischaemic PC, diazoxide, and FGF-2 pre-ischaemic or post-ischaemic treatments elicited a P Cx43 state, defined as above-physiological levels of phospho-S262-Cx43 and phospho-S368-Cx43. P Cx43 was sustained during global ischaemia and was accompanied by attenuation of ischaemia-induced Cx43 dephosphorylation and prevention of Cx43 lateralization. Post-ischaemic FGF-2 treatment also diminished dephosphorylated Cx43. Modest overexpression of S262A-Cx43, but not wild-type Cx43, exacerbated cardiomyocyte death and injury caused by simulated ischaemia in vitro. It also prevented the cytoprotective effects of FGF-2 or overexpressed PKCepsilon. P Cx43 marks a state of enhanced resistance to ischaemic injury promoted by PKC-activating treatments such as FGF-2 administration or ischaemic PC. Cx43 phosphorylation at S262 likely mediates PKCepsilon-dependent cardioprotection.

Fibroblast Growth Factor 2 Promotes Endothelial Differentiation of Adipose Tissue-Derived Stem Cells

Adipose tissue-derived stem cells (ADSC) could potentially restore endothelial function in vasculogenic erectile dysfunction (ED). The mechanism for ADSC endothelial differentiation remained unidentified. To test whether ADSC could differentiate into endothelial cells in the penis and to identify the underlying mechanism of ADSC endothelial differentiation. For in vivo endothelial differentiation, ADSC were labeled with bromodeoxyuridine (BrdU), injected into rat corpora cavernosa, and localized by immunofluorescence and phase-contrast microscopy. For in vitro endothelial differentiation, ADSC were grown in endothelial growth medium 2 (EGM2), stained for endothelial markers CD31, von Willebrand Factor (vWF), and endothelial nitric oxide synthase (eNOS), and assessed for the ability to form tube-like structures in Matrigel and to endocytose acetylated low-density lipoprotein (Ac-LDL). To identify factors that promote ADSC endothelial differentiation, ADSC were grown in various media, each of which contained a specific combination of supplemental factors and assessed for LDL-uptake. PD173074, a selective inhibitor of fibroblast growth factor 2 (FGF2) receptor, was used to confirm the importance of FGF2 signaling for ADSC endothelial differentiation. In vivo endothelial differentiation was assessed by immunofluorescence microscopy. In vitro endothelial differentiation was assessed by immunofluorescence, Matrigel tube formation, and Ac-LDL uptake. Injected ADSC were localized to the sinusoid endothelium, some of which stained positive for both BrdU and endothelial antigen rat endothelial cell antigen. ADSC proliferated at a faster rate in EGM2 than in standard DMEM, expressed endothelial markers CD31, vWF, and eNOS, formed tube-like structures in Matrigel, and endocytosed Ac-LDL. These properties were greatly diminished when ADSC were grown in the absence of FGF2 but were unaffected when grown in the absence of vascular endothelial growth factor, insulin-like growth factor, or epidermal growth factor. Furthermore, ADSC displayed similar endothelial properties when grown in FGF2-supplemented basic medium as in EGM2. Finally, blockade of FGF2 signaling with PD173074 abrogated ADSC endothelial differentiation. ADSC could differentiate into endothelial cells in the penis. FGF2 signaling mediates ADSC endothelial differentiation.

Transfection of adult canine Schwann cells and olfactory ensheathing cells at early and late passage with human TERT differentially affects growth factor responsiveness and in vitro growth

Adult canine Schwann cells and olfactory ensheathing cells (OECs) are closely related cell types that are considered attractive candidates for translational studies of neural repair. To establish a reliable cell source by comparing the in vitro properties of immortalized Schwann cells and OECs for transplantation purposes, we transfected both cell types with human telomerase reverse transcriptase (hTERT). Ectopic hTERT expression has been shown to induce immortalization of various cell types without substantial alterations of their phenotypes. Schwann cells and OECs were isolated from adult dogs, transfected with hTERT at early (P4) and late passage (P26), characterized regarding in vitro proliferation, antigenic expression and senescence-associated genes in the presence and absence of fibroblast growth factor-2 (FGF-2). Ectopic hTERT expression in late passage glia treated with but not without FGF-2 prevented the decline in proliferation observed in non-transfected cells. Immortalization did not alter p75 NTR and GFAP but O4 and A2B5 expression. Contrary to this, early passage hTERT transfection significantly reduced proliferation independent of FGF-2 and lowered expression of O4 and GFAP in both cell types. Transfection did not alter mRNA expression of senescence-associated genes such as p53 and p16. No substantial differences were found between Schwann cells and OECs underscoring the close relationship of both cell types. Taken together, we established a stable source of adult canine Schwann cells and OECs and demonstrated that the effects of hTERT expression on in vitro growth and growth factor responsiveness depend on the replicative age.

Anti-angiogenic activity of sesterterpenes; natural product inhibitors of FGF-2-induced angiogenesis

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is of physiological and pathological importance. We have investigated the anti-angiogenic potential of two naturally occurring sesterterpenes, leucosesterterpenone (compound 1) and leucosterlactone (compound 2) isolated from the Himalayan plant Leucosceptrum canum and identified as having biological activity in preliminary screening. Compound 1 inhibited fibroblast growth factor-2-induced proliferation, migration in a wounding assay, chemotaxis and tube formation with small vessel (human dermal) and large vessel (bovine aortic) endothelial cells while compound 2 was largely inactive. Both compounds were also active in an in vivo angiogenic model using the chick chorioallantoic membrane. Neither compounds showed inhibitory activity in the absence of fibroblast growth factor-2. We were able to demonstrate in a binding assay that compounds 1 and 2 bound to the fibroblast growth factor-2 receptor-1 with IC(50) values of 1.4 +/- 0.956 and 132.47 +/- 7.90 muM, respectively, with a concomitant down regulation of phosphorylated ERK1/2 but did not bind to receptor-2. Compound 1 was less hydrophobic than compound 2 and this may contribute to its increased activity. Compound 1 is a new addition to the small number of inhibitors of fibroblast growth factor-2-induced angiogenesis. The compound was a specific inhibitor in that it had no effect on vascular endothelial growth factor or epithelial growth factor-induced angiogenesis. Since angiogenesis is essential for tumour development we conclude that these compounds may have potential as anti-tumour agents.

Growth factors, stem cells, and stroke

Postischemic neurogenesis has been identified as a compensatory mechanism to repair the damaged brain after stroke. Several factors are released by the ischemic tissue that are responsible for proliferation, differentiation, and migration of neural stem cells. An understanding of their roles may allow future therapies based on treatment with such factors. Although damaged cells release a variety of factors, some of them are stimulatory whereas some are inhibitory for neurogenesis. It is interesting to note that factors like insulin-like growth factor-I can induce proliferation in the presence of fibroblast growth factor-2 (FGF-2), and promote differentiation in the absence of FGF-2. Meanwhile, factors like transforming growth factor-beta can induce the differentiation of neurons while inhibiting the proliferation of neural stem cells. Therefore, understanding the role of each factor in the process of neurogenesis will help physicians to enhance the endogenous response and improve the clinical outcome after stroke. In this article the authors discuss the role of growth factors and stem cells following stroke.

Non-canonical fibroblast growth factor signalling in angiogenesis

Whereas fibroblast growth factors (FGFs) classically transmit their signals via high-affinity tyrosine kinase receptors (FGFR1-4), recent evidence strongly implicates non-tyrosine kinase receptors (NTKR) or cell-surface FGFR-interacting proteins as important players in FGF signalling. Although NTKR have lower affinity for FGFs in comparison with cognate tyrosine kinase receptors, because of their high abundance they can effectively bind FGFs and produce unique biological effects independent of FGFRs. A prime example of such NTKR is the syndecan family of plasma membrane proteoglycans and, in particular, syndecan-4, which transmits FGF signalling via a protein kinase Calpha pathway. Another NTKR, alpha(v)beta(3) integrin, functions as an FGF signalling modulator by binding both FGF2 and FGFR1. Yet another NTKR, neural cell adhesion molecule (NCAM), can serve as an FGFR ligand and assemble an FGFR signalling complex in the absence of FGFs. Furthermore, N-cadherin, which has been reported to associate with FGFR, appears to activate FGFR in both ligand (FGF)-dependent and ligand-independent manners. Finally, gangliosides are implicated as a co-receptor system of FGFs. The biological consequence of non-canonical FGF signalling tends to be less discernable compared to the canonical FGFR activation because of the overlap between these two pathways; nevertheless, non-canonical signalling is important and sometimes essential for cellular functions. Given the diversity of FGF activities through embryonic development to adult physiology, the existence of the non-canonical signalling system may account for the different cellular response to the FGF input in different biological contexts. In this review, we will discuss recent findings related to non-canonical FGF signalling with emphasis on the endothelial biology and angiogenesis.

Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny

This protocol describes methods of isolating skin-derived precursors (SKPs) from rodent and human skin, and for generating and enriching Schwann cells from rodent SKPs. SKPs are isolated as a population of non-adherent cells from the dermis that proliferate and self-renew as floating spheres in response to fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). Their differentiation into Schwann cells and subsequent enrichment of these differentiated progeny involves culturing SKPs as adherent cells in the absence of FGF2 and EGF, but in the presence of neuregulins, and then mechanically isolating the Schwann cell colonies using cloning cylinders. Methods for expanding and characterizing these Schwann cells are provided. Generation of primary SKPs takes approximately 2 weeks, while differentiation of Schwann cells requires an additional 4-6 weeks.

Elucidation of the mechanism of the regulatory function of the Ig1 module of the fibroblast growth factor receptor 1.

The extracellular part of the fibroblast growth factor (FGF) receptor (FGFR) consists of up to three Ig modules (Ig1-Ig3), in which the Ig2 and Ig3 modules determine affinity and specificity for FGF and heparin. The FGFR isoforms lacking the Ig1 module have higher affinity for FGF and heparin than the triple Ig-module isoforms, suggesting that the Ig1 module is involved in the regulation of the FGFR-ligand interaction. We show here by surface plasmon resonance and NMR analyses that the Ig1 module binds to the Ig2 module, and identify by NMR the binding sites involved in the Ig1-Ig2 interaction. The identified binding site in the Ig2 module was found to be in the area of the FGF-Ig2 and Ig2-heparin contact sites, thus providing direct structural evidence that the Ig1 module functions as a competitive autoinhibitor of the FGFR-ligand interaction. Furthermore, the Ig1 binding site of the Ig2 module overlaps the Ig2-Ig2 contact site. This suggests that the function of the Ig1 module is not only regulation of the FGFR-ligand binding affinity but also prevention of spontaneous FGFR dimerization (through a direct Ig2-Ig2 interaction) in the absence of FGF.