FGF-2 Protein Research Articles

Expression and synthesis of fibroblast growth factor-9 in human gammadelta T-lymphocytes. Response to isopentenyl pyrophosphate and TGF-beta1/IL-15.

Gammadelta T-lymphocytes are believed to play a role in maintaining the normal configuration of epithelial tissue. As little is known about the factors mediating this function, we addressed the question of whether gammadelta T-lymphocytes produce fibroblast growth factor (FGF)-9 as well as two other growth factors associated with epithelial tissue reconstitution. Blood gammadelta T cells isolated from healthy donors were grown in the presence of isopentenyl pyrophosphate (IPP) or transforming growth factor-beta1 (TGF-beta1)/interleukin-15 (IL-15) for 24 h and were assessed for the expression and synthesis of FGF-9, keratinocyte growth factor (KGF), and epidermal growth factor (EGF). Resting human gammadelta T cells constitutively expressed KGF and FGF-9 mRNA but no EGF mRNA. In the presence of IPP, FGF-9 mRNA expression significantly increased in a dose-dependent manner, expression of KGF remained unaltered, and EGF mRNA could not be detected. In contrast to IPP, stimulation of the cells with TGF-beta1/IL-15 did not alter FGF-9 expression. Moreover, stimulation with anti-CD3 does not induce FGF-9 expression but triggers a high signal of interferon-gamma mRNA. Western blot analysis of gammadelta T cell lysates, prepared 4 days following stimulation with IPP, showed an increase of FGF-9 protein as compared with control cells. In conclusion, the results demonstrate for the first time that human blood and bronchoalveolar lavage gammadelta T-lymphocytes are capable of expressing FGF-9. The data also provide novel evidence that immunoregulatory cells can synthesize FGF-9.

218.FGF9 stimulates ovarian progesterone production

FGF9, a member of the fibroblast growth factor family (FGF), is known to be a male sex-determining factor involved in testicular cord formation (1). FGF9 knockout males are sex-reversed (2). However, nothing is known about FGF9's role in folliculogenesis because these mice die at birth (2). We previously reported the presence of FGF9 mRNA and protein in the immature rat ovary (3). In these studies we investigated: (1) the presence of FGF9 receptors (FGFR3) on granulosa cells (GC); and (2) the impact of FGF9 on GC progesterone production. GC isolated from 21 day old diethylstilboestrol (DES)-treated rats were cultured for either 2 hours (RNA) or 2 days (progesterone) in McCoys 5C with FGF9 (0.1-50ng/ml) � FSH (100ng/ml). Progesterone was measured in conditioned media by radioimmunoassay. RNA was extracted from the granulosa cells and reverse-transcribed for PCR. Specific primers for P450 side chain cleavage (SCC) amplified a 329�bp cDNA fragment. GAPDH was used for data normalisation. The FGF9 receptor FGFR3, was immunolocalised on formalin-fixed, paraffin-embedded sections of immature rat ovary. FGFR3 protein was localised only to GC of the ovary. Progesterone production by cultured GC was significantly elevated by FGF9, consistent with the presence of FGFR3. Relative to a maximally stimulating dose of FSH, FGF9 increased progesterone production 10- fold. In preliminary studies, FGF9 increased the expression of P450 SCC mRNA by cultured GC revealing a mechanism by which FGF9 increases progesterone production. These data suggest a role for FGF9 not just in testicular formation, but in the regulation of ovarian steroidogenesis. Supported by the NH&MRC of Australia (Regkey 241000 & 198705). (1) Cotinot et al. (2002) Semin. Reprod. Med. 20, 157. (2) Colvin et al. (2001) Cell 104, 875. (3) Drummond et al. (2003) SRB Abstract 90.

Adenovirus-Mediated Transfer of a Truncated Fibroblast Growth Factor (FGF) Type I Receptor Blocks FGF-2 Signaling in Multiple Pancreatic Cancer Cell Lines

Pancreatic ductal adenocarcinomas (PDACs) overexpress several members of the fibroblast growth factor (FGF) family of ligands and the type I FGF receptor (FGFR-1), and enhanced FGF-2 protein levels correlate with shorter postoperative survival of patients with PDAC. In this study, we investigated the effects of FGF-2 on cell proliferation and mitogen-activated protein kinase (MAPK) activation before and after abrogation of FGFR-1-dependent signaling in 4 pancreatic cancer cell lines (ASPC-1, COLO-357, MIA-PaCa-2, and PANC-1). Signaling was blocked by infecting the cells with an adenoviral vector encoding for a truncated FGFR-1 (AdtrFGFR-1). FGF-2 enhanced the growth of all 4 cell lines and activated MAPK in 3 of these cell lines. Infection with the AdtrFGFR-1 virus resulted in abundant expression of the truncated FGFR-1 at the RNA and protein level, markedly attenuated FGF-2-induced proliferation in all 4 tested cell lines, and decreased FGF-2-dependent MAPK activation in the 3 cell lines in which FGF-2 activated this pathway. These findings suggest that FGFR-1-mediated mitogenesis in multiple pancreatic cancer cells can be efficiently blocked with an adenoviral vector encoding a truncated FGFR-1, raising the possibility that AdtrFGFR-1 may ultimately have a therapeutic role in PDAC.

Effects of heavy ions on visual function and electrophysiology of rodents: the ALTEA-MICE project

ALTEA-MICE will supplement the ALTEA project on astronauts and provide information on the functional visual impairment possibly induced by heavy ions during prolonged operations in microgravity. Goals of ALTEA-MICE are: (1) to investigate the effects of heavy ions on the visual system of normal and mutant mice with retinal defects; (2) to define reliable experimental conditions for space research; and (3) to develop animal models to study the physiological consequences of space travels on humans. Remotely controlled mouse setup, applied electrophysiological recording methods, remote particle monitoring, and experimental procedures were developed and tested. The project has proved feasible under laboratory-controlled conditions comparable in important aspects to those of astronauts' exposure to particle in space. Experiments are performed at the Brookhaven National Laboratories [BNL] (Upton, NY, USA) and the Gesellschaft für Schwerionenforschung mbH [GSI]/Biophysik (Darmstadt, FRG) to identify possible electrophysiological changes and/or activation of protective mechanisms in response to pulsed radiation. Offline data analyses are in progress and observations are still anecdotal. Electrophysiological changes after pulsed radiation are within the limits of spontaneous variability under anesthesia, with only indirect evidence of possible retinal/cortical responses. Immunostaining showed changes (e.g increased expression of FGF2 protein in the outer nuclear layer) suggesting a retinal stress reaction to high-energy particles of potential relevance in space.

Fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo.

Formation and patterning of the vertebrate embryo occur in a head-to-tail sequence. This progressive mode of body formation from the posterior end of the embryo requires a strict temporal coordination of tissue differentiation--a process involving fibroblast growth factor (FGF) signalling. Here we show that transcription of fgf8 messenger RNA is restricted to the growing posterior tip of the embryo. fgf8 mRNA is progressively degraded in the newly formed tissues, resulting in the formation of an mRNA gradient in the posterior part of the embryo. This fgf8 mRNA gradient is translated into a gradient of FGF8 protein, which correlates with graded phosphorylation of the kinase Akt, a downstream effector of FGF signalling. Such a mechanism provides an efficient means to monitor the timing of FGF signalling, coupling the differentiation of embryonic tissues to the posterior elongation of the embryo. In addition, this mechanism provides a novel model for morphogen gradient formation.

Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development.

Fibroblast growth factor 8 (Fgf8) is expressed in many domains of the developing embryo. Globally decreased FGF8 signaling during murine embryogenesis results in a hypomorphic phenotype with a constellation of heart, outflow tract, great vessel and pharyngeal gland defects that phenocopies human deletion 22q11 syndromes, such as DiGeorge. We postulate that these Fgf8 hypomorphic phenotypes result from disruption of local FGF8 signaling from pharyngeal arch epithelia to mesenchymal cells populating and migrating through the third and fourth pharyngeal arches. To test our hypothesis, and to determine whether the pharyngeal ectoderm and endoderm Fgf8 expression domains have discrete functional roles, we performed conditional mutagenesis of Fgf8 using novel Crerecombinase drivers to achieve domain-specific ablation of Fgf8 gene function in the pharyngeal arch ectoderm and endoderm. Remarkably, ablating FGF8 protein in the pharyngeal arch ectoderm causes failure of formation of the fourth pharyngeal arch artery that results in aortic arch and subclavian artery anomalies in 95% of mutants; these defects recapitulate the spectrum and frequency of vascular defects reported in Fgf8 hypomorphs. Surprisingly, no cardiac, outflow tract or glandular defects were found in ectodermal-domain mutants, indicating that ectodermally derived FGF8 has essential roles during pharyngeal arch vascular development distinct from those in cardiac, outflow tract and pharyngeal gland morphogenesis. By contrast, ablation of FGF8 in the third and fourth pharyngeal endoderm and ectoderm caused glandular defects and bicuspid aortic valve, which indicates that the FGF8 endodermal domain has discrete roles in pharyngeal and valvar development. These results support our hypotheses that local FGF8 signaling from the pharyngeal epithelia is required for pharyngeal vascular and glandular development, and that the pharyngeal ectodermal and endodermal domains of FGF8 have separate functions.

Expression and localization of fibroblast growth factor (FGF) family members during the final growth of bovine ovarian follicles.

The aim of this study was to investigate the possible participation of fibroblast growth factor (FGF) family members: FGF1, FGF2, and FGF7, and their receptor variants: FGFR, FGFR2IIIb, and FGFR2IIIc in theca interna (TI) and granulosa cell (GC) compartments of bovine follicles during final growth. A classification of follicles into five groups (<0.5; >0.5-5; >5-20; >20-180; >180 ng/ml, respectively) was performed according to the follicular fluid (FF) oestradiol-17beta (E) content. The mRNA expression and protein localization was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. FGF1 mRNA expression was relatively high in TI and lower in GC, and without any regulatory change for both tissue compartments during final follicular growth. The FGF1 protein could be predominantly localized in the cytoplasm of GC, in smooth muscle cells of blood vessels, in the rete ovarii, and at a lesser degree in theca cells. FGF2 mRNA in TI increased significantly in large follicles and was low and without any regulatory change in GC. FGF7 mRNA expression was relatively high in TI and very low in GC. For FGF7 in mature follicles a marked staining of the TI and the basal layers of the GC could be demonstrated. The mRNA signal for the FGFR in TI increased significantly with beginning of E production (E > 0.5-5 ng/ml FF) and was without any regulatory change in GC. The mRNA expression of FGFR2IIIb was relatively high in GC and increased significantly during final growth of follicles in contrast to the TI with very low expression. The FGFR2IIIc mRNA expression in TI and GC was relatively high but without any clear change. Our results suggest that FGF growth factor family members are involved in process of folliculogenesis and especially during final growth of the preovulatory (dominant) follicle by stimulation of angiogenesis and GC survival and proliferation.

Ephrin-A2 regulates position-specific cell affinity and is involved in cartilage morphogenesis in the chick limb bud

In the developing limb bud, mesenchymal cells show position-specific affinity, suggesting that the positional identity of the cells is represented as their surface properties. Since the affinity is regulated by glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, and by EphA4 receptor tyrosine kinase, we hypothesized that the GPI-anchored ligand, the ephrin-A family, also contributes to the affinity. Here, we describe the role of ephrin-A2 in the chick limb bud. Ephrin-A2 protein is uniformly distributed in the limb bud during early limb development. As the limb bud grows, expression of ephrin-A2 is strong in its proximal-to-intermediate regions, but weak distally. The position-dependent expression is maintained in vitro, and is regulated by FGF protein, which is produced in the apical ectodermal ridge. To investigate the role of ephrin-A2 in affinity and in cartilage morphogenesis of limb mesenchyme, we ectopically expressed ephrin-A2 in the limb bud using the retrovirus vector, RCAS. Overexpressed ephrin-A2 modulated the affinity of the mesenchymal cells that differentiate into autopod elements. It also caused malformation of the autopod skeleton and interfered with cartilage nodule formation in vitro without inhibiting chondrogenesis. These results suggest that ephrin-A2 regulates the position-specific affinity of limb mesenchyme and is involved in cartilage pattern formation in the limb.

Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury

Basic fibroblast growth factor (or FGF-2) has been shown to be a potent stimulator of retinal ganglion cell (RGC) axonal growth during development. Here we investigated if FGF-2 upregulation in adult RGCs promoted axon regrowth in vivo after acute optic nerve injury. Recombinant adeno-associated virus (AAV) was used to deliver the FGF-2 gene to adult RGCs providing a sustained source of this neurotrophic factor. FGF-2 gene transfer led to a 10-fold increase in the number of axons that extended past 0.5 mm from the lesion site compared to control nerves. Detection of AAV-mediated FGF-2 protein in injured RGC axons correlated with growth into the distal optic nerve. The response to FGF-2 upregulation was supported by our finding that FGF receptor-1 (FGFR-1) and heparan sulfate (HS), known to be essential for FGF-2 signaling, were expressed by adult rat RGCs. FGF-2 transgene expression led to only transient protection of injured RGCs. Thus the effect of this neurotrophic factor on axon extension could not be solely attributed to an increase in neuronal survival. Our data indicate that selective upregulation of FGF-2 in adult RGCs stimulates axon regrowth within the optic nerve, an environment that is highly inhibitory for regeneration. These results support the hypothesis that key factors involved in axon outgrowth during neural development may promote regeneration of adult injured neurons.

A potential role for PTTG/securin in the developing human fetal brain.

Human securin, known also as PTTG, has established oncogenic and cell cycle regulatory functions. PTTG/securin transforms cells in vitro, inhibits sister chromatid separation, and regulates secretion of fibroblast growth factor-2. FGF-2 is a key regulator of CNS development and PTTG/securin expression has been reported in murine fetal brain. We examined the expression and function of securin and FGF-2 in the developing human fetal brain and in a fetal neuronal cell line (NT 2). Securin expression was significantly reduced in first and second trimester fetal cerebral cortex compared with adult cerebral cortex, where immunocytochemistry revealed intense securin staining in neuronal cell bodies. FGF-2 protein was concordantly lower in fetal cortex, whereas pretranslational expression of PTTG binding factor (PBF) was not significantly altered in fetal brain compared with adult. PCNA expression demonstrated that high securin levels in adult cortex were associated with absent cell proliferation. In NT-2 cells, securin stimulated FGF-2 expression, which could be abrogated by a carboxyl-terminal mutation. Low transient expression of securin resulted in a significant proliferative effect, whereas high levels of securin expression inhibited cell turnover. We propose a potential role for human PTTG/securin in modulating cell proliferation and FGF-2 expression during human neurogenesis.

Expression pattern of glypican-1 mRNA after brain injury in mice

Glypican-1, a heparan sulfate proteoglycan, is expressed in various tissues including developing and postnatal central nervous system. It serves as a receptor for heparin-binding molecules such as fibroblast growth factors (FGFs). We investigated whether glypican-1 was expressed after brain injury in adult mice. In situ hybridization study showed that glypican-1 mRNA was expressed in the region surrounding necrotic tissue, and that the signal intensity peaked 7 days after the cryo-injury. In addition, both FGF-2 and amyloid precursor protein (APP) were concurrently upregulated and colocalized with glypican-1 mRNA. Since FGF-2 and APP can bind to glypican-1 in vitro, the present study suggested that their autocrine/paracrine interactions with glypican-1 may be involved in neuronal regeneration and/or neurite-outgrowth inhibition after brain injury.

Heparin-binding EGF-like growth factor regulates elastin and FGF-2 expression in pulmonary fibroblasts.

Elastase degradation of elastin within alveolar walls is an important event in the development of pulmonary emphysema. In addition to elastolytic activities, elastases release growth factors from extracellular matrices and interstitial cell surfaces that can regulate elastogenesis and other cellular responses. In the present study, we demonstrate that brief treatment of matrix-laden rat pulmonary fibroblast cultures with pancreatic elastase results in the release of soluble heparin-binding epidermal growth factor-like growth factor (HB-EGF) concomitant with a decrease in HB-EGF binding to both heparan sulfate proteoglycan and receptor sites on the cells. In undigested, matrix-laden fibroblasts, HB-EGF significantly downregulates elastin mRNA via activation of epidermal growth factor receptor. Results from nuclear run-on analyses show that HB-EGF downregulates elastin mRNA via transcriptional suppression. HBEGF treatment stimulates MAP or ERK kinase (MEK)-dependent ERK1/2 phosphorylation and leads to nuclear accumulation of Fra-1. Blocking ERK1/2 activation by MEK1/2 inhibitors (PD-98059 or U-0126) diminishes HB-EGF-induced Fra-1 accumulation and subsequent downregulation of elastin mRNA. Coaddition of two elastase-released growth factors, HB-EGF and FGF-2, results in an additive inhibitory effect on elastin mRNA levels. Furthermore, HB-EGF addition to pulmonary fibroblasts increases FGF-2 mRNA and protein levels. These data suggest that HB-EGF and FGF-2 act in concert to regulate the synthesis of elastin in injury/repair situations.

Angiogenic protein therapy

Therapeutic angiogenesis, in the form of growth factor protein administration or gene therapy, has emerged as a new method of treatment for patients with severe, inoperable coronary artery disease. Improved myocardial perfusion and function after the administration of angiogenic growth factors has been demonstrated in animal models of chronic myocardial ischemia. A recent clinical study reported beneficial long-term effects of therapeutic angiogenesis using FGF-2 protein in terms of freedom from angina and myocardial perfusion on nuclear imaging and suggested that protein angiogenic therapy has the potential to extend treatment options to patients who are not optimal candidates for conventional methods of myocardial revascularization. The ultimate role that angiogenesis will play in the treatment of ischemic heart disease will, however, be determined from adequately powered, randomized, double-blind, placebo-controlled trials. It is likely that endogenous antiangiogenic influences, intrinsic lack of response of patients with severe endothelial dysfunction, and other limitations will have to be overcome before angiogenesis becomes standard therapy for the treatment of coronary artery disease. © 2003 Elsevier Inc. All rights reserved.

Impaired Osteoclast Formation in Bone Marrow Cultures of Fgf2 Null Mice in Response to Parathyroid Hormone

Fibroblast growth factor (FGF)-2 and parathyroid hormone (PTH) are potent inducers of osteoclast (OCL) formation, and PTH increases FGF-2 mRNA and protein expression in osteoblasts. To elucidate the role of endogenous FGF-2 in PTH responses, we examined PTH-induced OCL formation in bone marrow cultures from wild type and mice with a disruption of the Fgf2 gene. FGF-2-induced OCL formation was similar in marrow culture from both genotypes. In contrast, PTH-stimulated OCL formation in bone marrow cultures or co-cultures of osteoblast-spleen cells from Fgf2-/mice was significantly impaired. PTH increased RANKL mRNA expression in osteoblasts cultures from both genotypes. After 6 days of treatment, osteoprotegerin protein in cell supernatants was 40-fold higher in vehicle-treated and 30-fold higher in PTH-treated co-cultures of osteoblast and spleen cells from Fgf2-/mice compared with Fgf2+/+ mice. However, a neutralizing antibody to osteoprotegerin did not rescue reduced OCL formation in response to PTH. Injection of PTH caused hypercalcemia in Fgf2+/+ but not Fgf2-/mice. We conclude that PTH stimulates OCL formation and bone resorption in mice in part by endogenous FGF-2 synthesis by osteoblasts. Because RANKL- and interleukin-11-induced OCL formation was also reduced in bone marrow cultures from Fgf2-/mice, we further conclude that endogenous FGF-2 is necessary for maximal OCL formation by multiple bone resorbing factors.

Negative Feedback Regulation of FGF Signaling Levels by Pyst1/MKP3 in Chick Embryos

Background: The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known.Results: We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth.Conclusions: Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.

Tissue interactions pattern the mesenchyme of the embryonic mouse lung

The mechanisms that control proliferation and differentiation of embryonic lung mesenchyme are largely unknown. We describe an explant system in which exogenous recombinant N-Sonic Hedgehog (N-Shh) protein sustains the survival and proliferation of lung mesenchyme in a dose-dependent manner. In addition, Shh upregulates several mesenchymal cell markers, including its target gene Patched (Ptc), intercellular signaling genes Bone Morphogenetic Protein-4 (Bmp4) and Noggin (Nog), and smooth muscle actin and myosin. In explants exposed to N-Shh in the medium, these products are upregulated throughout the mesenchyme, but not in the periphery. This exclusion zone correlates with the presence of an overlying mesothelial layer, which, as in vivo, expresses Fibroblast Growth Factor 9 (Fgf9). Recombinant Fgf9 protein inhibits the differentiation response of the mesenchyme to N-Shh, but does not affect proliferation. We propose a model for how factors made by two epithelial cell populations, the inner endoderm and the outer jacket of mesothelium, coordinately regulate the proliferation and differentiation of the lung mesoderm.

Basic FGF and Ki-67 proteins useful for immunohistological diagnostic evaluations in malignant solitary fibrous tumor.

Solitary fibrous tumor (SFT) is an uncommon soft tissue tumor initially reported in the pleura but recently described in other sites in the body. Morphological distinction between benign and malignant SFT is often difficult. An immunohistochemical study was performed in pleural and extrapleural sites. The aim of this study was to determine if an immunohistochemical method is helpful in distinguishing benign SFT from malignant SFT, and providing valid information to predict the prognosis associated with malignant SFT. Twenty-four cases of benign (14 patients) and malignant (10 patients) SFT in the pleura, pelvic space, prostate and other sites of soft tissue were analyzed. Tumors from 10 patients were diagnosed as malignant on the basis of markedly increased cellularity, mitotic activity (>4/10 high-power fields), nuclear pleomorphism and areas of necrosis. Immunohistochemically, we found a mean basic fibroblast growth factor (bFGF) labeling index of 48.67% (48.67 +/- 8.52%) for benign SFT and 74.5% (74.5 +/- 6.92%) for malignant SFT (P < 0.05). We also found a mean Ki-67 labeling index of 1.9% (1.9 +/- 0.43%) for benign SFT and 6.11% (6.11 +/- 1.05%) for malignant SFT (P < 0.05). Our results suggest that bFGF and Ki-67 are diagnostically relevant to the evaluation of malignant SFT and these proteins are thought to be potentially useful markers for prognosis of SFT.

Fibroblast growth factor (FGF)-4 can induce proliferation of cardiac cushion mesenchymal cells during early valve leaflet formation

While much has been learned about how endothelial cells transform to mesenchyme during cardiac cushion formation, there remain fundamental questions about the developmental fate of cushions. In the present work, we focus on the growth and development of cushion mesenchyme. We hypothesize that proliferative expansion and distal elongation of cushion mesenchyme mediated by growth factors are the basis of early valve leaflet formation. As a first step to test this hypothesis, we have localized fibroblast growth factor (FGF)-4 protein in cushion mesenchymal cells at the onset of prevalve leaflet formation in chick embryos (Hamburger and Hamilton stage 20–25). Ligand distribution was correlated with FGF receptor (FGFR) expression. In situ hybridization data indicated that FGFR3 mRNA was confined to the endocardial rim of the atrioventricular (AV) cushion pads, whereas FGFR2 was expressed exclusively in cushion mesenchymal cells. FGFR1 expression was detected in both endocardium and cushion mesenchyme as well as in myocardium. To determine whether the FGF pathways play regulatory roles in cushion mesenchymal cell proliferation and elongation into prevalvular structure, FGF-4 protein was added to the cushion mesenchymal cells explanted from stage 24–25 chick embryos. A significant increase in proliferative ability was strongly suggested in FGF-4-treated mesenchymal cells as judged by the incorporation of 5′-bromodeoxyuridine (BrdU). To determine whether cushion cells responded similarly in vivo, a replication-defective retrovirus encoding FGF-4 with the reporter, bacterial β-galactosidase was microinjected into stage 18 chick cardiac cushion mesenchyme along the inner curvature where AV and outflow cushions converge. As compared with vector controls, overexpression of FGF-4 clearly induced expansion of cushion mesenchyme toward the lumen. To further test the proliferative effect of FGF-4 in cardiac cushion expansion in vivo (ovo), FGF-4 protein was microinjected into stage 18 chick inner curvature. An assay for BrdU incorporation indicated a significant increase in proliferative ability in FGF-4 microinjected cardiac cushion mesenchyme as compared with BSA-microinjected controls. Together, these results suggest a role of FGF-4 for cardiac valve leaflet formation through proliferative expansion of cushion mesenchyme.

Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation.

Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

Axonal regeneration across long gaps in silicone chambers filled with Schwann cells overexpressing high molecular weight FGF-2.

Basic fibroblast growth factor (FGF-2) has been shown to enhance the survival and neurite extension of various types of neurons including spinal ganglion neurons. In addition, endogenous FGF-2 and FGF receptors are upregulated following peripheral nerve lesion in ganglia and at the lesion site. FGF-2 protein is expressed in different isoforms (18 kDa, 21 kDa, 23 kDa) and differentially regulated after nerve injury. In the rat we analyzed the regenerative capacity of the high molecular weight (HMW) FGF-2 isoforms (21/23 kDa) to support the regeneration of the axotomized adult sciatic nerve across long gaps. The nerve stumps were inserted into the opposite ends of a silicone chamber resulting in an interstump gap of 15 mm. Silicone tubes were filled with Matrigel or a mixture of Schwann cells (SC) and Matrigel. SC were prepared from newborn rats and transfected to overexpress HMW FGF-2. Four weeks after the operation procedure, channels were analyzed with regard to tissue cables bridging both nerve stumps and myelinated axons distal to the original proximal nerve stump. Peripheral nerves interposed with HMW Schwann cells displayed significantly enhanced nerve regeneration, with the greatest number of tissue cables containing myelinated axons and the highest number of myelinated axons. These results suggest that a cellular substrate together with a source of a trophic factor could be a promising tool to promote nerve regeneration and, therefore, become useful also for a clinical approach to repair long gaps.