Exogenous FGF-2 Research Articles

Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1

The combined treatment of peripheral nerve (PN) graft and fibroblast growth factor (FGF)-1 for spinal cord injury produces functional recovery, but how it affects injury events is still unknown. This project studied the effect of PN graft and FGF-1 on white matter degeneration following spinal cord injury. Rats were divided into four groups: (i) complete spinal cord transection and T8 segment removed; the remaining three groups underwent transection followed by (ii) PN grafting; (iii) supply of exogenous FGF-1; and (iv) PN grafting plus FGF-1 treatment. Chondroitin sulphate proteoglycan (CSPG) deposition, astrocytes and macrophage activation, cavity size, and calcitonin gene-related peptide and synaptophysin immunoreactivity were compared. Peripheral nerve grafting increased CSPG levels compared to transection surgery alone. This CSPG was associated with the proximity to the PN graft. FGF-1 reduced CSPG deposition in grafted animals regardless of the proximity to the graft. The CSPG reduction was accompanied by reduced GFAP expression and macrophage activation. The amount of CSPG with dissociated glycosaminoglycan did not differ between groups. FGF-1 in Schwann cell-astrocyte coculture did not reduce CSPG deposition. Furthermore, the PN graft increased the calcitonin gene-related peptide immunoreactivity and altered the distribution of synaptophysin-positive axons. Peripheral nerve graft supported sensory re-innervation and partial protection of the grey matter, but up-regulated CSPG in the graft-stump junction compared to non-grafted rats. The reduction of CSPG was caused by FGF-1-PN synergy, and did not involve dissociation of CSPG or the suppression of a general immune response.

Maintenance of human pluripotent stem cells using 4SP-hFGF2-secreting STO cells

Human embryonic stem cells (hESCs) are typically cultured on fibroblast feeder cells or in fibroblast conditioned medium supplemented with fibroblast growth factor 2 (FGF2, also known as bFGF). FGF signaling appears to be important for hESC self-renewal and is required to enable the culture of hESCs in an undifferentiated state. In this study, we generated a transgenic fibroblast feeder line stably expressing a secretable FGF4 signal peptide tagged hFGF2 (4SP-hFGF2). The expression of this transgene functionally replaced the requirement for exogenous FGF2 when using these cells as feeders for the maintenance of hESCs. Under these conditions, hESCs maintained the typical marker of pluripotency assessed after long term culture, while still retaining the capacity for differentiation to all three germ layers. This transgene could be applied to mass produce 4SP-hFGF2 protein, serving to be an economical and effective strategy for culturing pluripotent stem cells as feeder cells.

Differential effects of TGF‐β and FGF‐2 on in vitro proliferation and migration of primate retinal endothelial and Müller cells

During retinal development, the pattern of blood vessel formation depends upon the combined effects of proliferation and migration of endothelial cells, astrocytes and Müller cells. In this study, we investigated the potential for transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF-2) to influence this process by regulating proliferation and migration of retinal endothelial and macroglial cells. We assessed the effects of exogenous TGF-β and FGF-2 on the proliferation and migration of cultured endothelial (RF/6A) and Müller cell (MIO-M1) lines. Cell proliferation was measured using a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay over 72 hr. Cell migration was measured using a scratch-wound assay over 72 hr. Transforming growth factor-β inhibited the proliferation of endothelial and Müller cells and inhibited the migration of Müller cells, but not endothelial cells, compared to untreated controls. Conversely, FGF-2 increased endothelial cell proliferation but inhibited endothelial cell migration. Fibroblast growth factor-2 increased migration of Müller cells but had little effect on proliferation except at higher concentrations (20 ng/ml). Taken together, these observations indicate that TGF-β and FGF could work in concert to inhibit endothelial cell proliferation and migration, respectively; this may have implications for establishing and maintaining the avascular zone of primate fovea.

Roles of exogenous and endogenous FGF-2 in animal models of depression

Several members of the fibroblast growth factor (FGF) family have been shown to be dysregulated in individuals with major depression, and treatment with antidepressants has been reported to increase FGF-2 mRNA levels in the forebrain. We have used the tail suspension test (TST), and olfactory bulbectomy (OBX), and FGF-2 deficient mice to investigate putative roles of FGF-2 as an antidepressant and mediator of antidepressive drug actions. FGF-2 applied intraventricularly generated antidepressant-like effects in the TST. FGF-2, similar to the antidepressant amitriptyline, attenuated neuron demise in the piriform cortex and posterolateral cortical nucleus of the amygdala following OBX. Moreover, OBX induced reduction in hippocampal neurogenesis could be ameliorated by subsequent treatment with either amitriptyline or FGF-2. Furthermore, FGF-2 was effective in reversing depressive-like behavior induced by OBX, monitored in the locomotor activity and the passive avoidance test. In bulbectomized FGF-2 deficient mice, treatment with amitriptyline protected neurons, but failed to reverse behavioral alterations. Together, these results suggest that FGF-2 constitutes both a potential target for antidepressive treatments and an important growth factor in the cytokine network underlying the actions of antidepressive drugs. The results further suggest a requirement of endogenous FGF-2 for mediating behavioral, but not neuroprotective actions of amitriptyline.

New Vandetanib analogs: fused tricyclic quinazolines with antiangiogenic potential

The antiangiogenic effects of three novel anilinoquinazoline derivatives were studied with the aim to find new multi-kinase inhibitors as anticancer agents. The compounds are characterized by dioxolane, dioxane and dioxepine rings and bear the same aniline substituent in 4 position as vandetanib, known antiangiogenic agent. The in vitro assays were carried out on human umbilical vein endothelial cells (HUVECs), whereas in vivo angiogenesis was evaluated by means of Matrigel plug assay. The results showed that these compounds exert, even though to different extents, antiangiogenic activity affecting the various step of the process that leads to the formation of new blood vessels. At high concentrations they induced antiproliferative effects, whereas at non-cytotoxic concentrations they inhibited cell migration and the formation of tubular structures in Matrigel. In in vitro assays the dioxolane derivative 1 was more effective than vandetanib. Indeed, it inhibited the effects induced by exogenous VEGF and FGF-2 on both cell proliferation and morphogenesis, whereas vandetanib was completely ineffective. Moreover, all the compounds, as vandetanib, counteracted the FGF-2-induced increase in the hemoglobin content in the Matrigel plugs. Our results showed that all the three novel derivatives possess both in vitro and in vivo antiangiogenic activity, with compound 1 more effective than vandetanib to inhibit in vitro angiogenesis induced by exogenous cytokines.

Cell fate mediators Notch and Twist in mouse mandibular condylar cartilage

ObjectiveThe objectives of this study were to examine if Twist and Notch 1 are present in the mandibular condylar cartilage (MCC) and whether their gene expression can be altered by exogenous FGF-2 and TGF-β2. DesignHalf-heads from CD-1 mice pups harvested at embryonic day 17 (E17) were fixed, decalcified, and sectioned in the sagittal plane for immunohistochemical detection of Notch and Twist using confocal microscopy. Other mandibular condyles and adjacent ramus from E17 mice were cultured in serum-free DMEM containing 0, 3, or 30ng/mL of FGF-2 (10–12 condyles per treatment group). This experimental design was repeated with medium containing 0, 3, or 30ng/mL of TGF-β2. After 3 days of culture, the pooled RNA from each group was extracted for examination of Notch and Twist gene expression using quantitative real-time RT-PCR. ResultsImmunohistochemical examination revealed that Notch and Twist were localized to the prechondroblastic and upper chondroblastic layers of the cartilage. Exogenous FGF-2 up-regulated Notch 1, Twist 1 and Twist 2 gene expression in MCC explants from E17 mice, whilst TGF-β2 had the opposite effect. ConclusionsThe gene expression data demonstrate that MCC explants are sensitive to growth factors known to affect Notch and Twist in other tissues. The subset of cells in which Twist and Notch immunoreactivity was found is suggestive of a role for FGF-2 and TGF-β2 as regulators of cell differentiation of the bipotent MCC cell population, consistent with the role of Notch and Twist as downstream mediators of these growth factors in other tissues.

Autocrine Fibroblast Growth Factor-2 Signaling Contributes to Altered Endothelial Phenotype in Pulmonary Hypertension

Pulmonary vascular remodeling is key to the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH). We recently reported that fibroblast growth factor (FGF)2 is markedly overproduced by pulmonary endothelial cells (P-ECs) in IPAH and contributes significantly to smooth muscle hyperplasia and disease progression. Excessive FGF2 expression in malignancy exerts pathologic effects on tumor cells by paracrine and autocrine mechanisms.We hypothesized that FGF2 overproduction contributes in an autocrine manner to the abnormal phenotype of P-ECs, characteristic of IPAH. In distal pulmonary arteries (PAs) of patients with IPAH, we found increased numbers of proliferating ECs and decreased numbers of apoptotic ECs, accompanied with stronger immunoreactivity for the antiapoptotic molecules, B-cell lymphoma (BCL)2, and BCL extra long (BCL-xL) compared with PAs from control patients. These in situ observations were replicated in vitro, with cultured P-ECs from patients IPAH exhibiting increased proliferation and diminished sensitivity to apoptotic induction with marked increases in the antiapoptotic factors BCL2 and BCL-xL and levels of phosphorylated extracellular signal-regulated (ERK)1/2 compared with control P-ECs. IPAH P-ECs also exhibited increased FGF2 expression and an accentuated proliferative and survival response to conditioned P-EC media or exogenous FGF2 treatment. Decreasing FGF2 signaling by RNA interference normalized sensitivity to apoptosis and proliferative potential in the IPAH P-ECs. Our findings suggest that excessive autocrine release of endothelial-derived FGF2 in IPAH contributes to the acquisition and maintenance of an abnormal EC phenotype, enhancing proliferation through constitutive activation of ERK1/2 and decreasing apoptosis by increasing BCL2 and BCL-xL.

Antagonistic effects of hi-FGF-2 on myocardial ischemia/reperfusion injury in rats

Objective To investigate the effect of CUG-initiated, high molecular weight fibroblast growth factor-2 (hi-FGF-2) on myocardial ischemia/reperfusion injury in rats. Methods Ex vivo rat hearts were subjected to 30 rain ischemia and 60 min reperfusion. Normal saline, hi-FGF-2 or low molecular weight FGF-2 (lo-FGF-2) (10 μg in 10 ml normal saline) was perfused with perfusate into the heart during the first 10 min of reperfusion. Distribution of exogenous FGF-2, changes of vinculin as well as cardiac mechanic functions were examined. Results Cardiac functions including systolic pressure, dp/dtmax and developed pressure were significantly improved in hi- and lo-FGF-2 groups compared to saline controls (P <0.05 or 0. 01 ). The exogenous hi- or lo-FGF-2 was broadly distributed into the myocardium during 1 h reperfusion. Intensive and intact vinculin staining was seen in both lo- and hi-FGF-2, but not in normal saline, administered hearts. Conclusion hi-FGF-2, like its low molecular weight isoform, is cardioprotective against myocardial ischemia/reperfasion injury. Key words: FGF-2; Myocardial ischemia; Reperfusion injury

Abstract 3132: Proliferative effects of FGF1 on ovarian cancer cells correlate with FGFR4 over expression and are mediated by MAPK/ERK signaling

Abstract Objectives: To identify the effect of FGF1 on cell proliferation in ovarian cancer and endothelial cells, its relation to FGFR4 over expression and signaling pathways involved Methods: Amplification of FGFR4 on 5q31-35, which was previously shown to be associated with poor survival in patients with advanced stage high-grade serous ovarian cancer (HGSC), was validated by quantitative PCR in 52 HGSC. Association between DNA copy numbers of FGFR4 and FGF1 located on the same segment was performed by Spearman correlation analysis. Over expression of FGFR4 protein was confirmed in ovarian cancer cell lines and tissue by western blot analysis and immunohistochemistry, respectively. Endogenous FGF1 production by several ovarian cancer cell lines in conditioned medium was quantified using ELISA. Effect of exogenous FGF1 on cell proliferation of high and low-FGFR4 expressing ovarian cancer cell lines and an immortalized microvascular endothelial cell line was determined by WST-1 assay. Signaling pathways activation was screened by treating ovarian cancer cell lines and endothelial cells transduced with six different transcription response elements using the Cignal-Lenti reporter system with exogenous FGF1. Results: FGFR4 was amplified and over-expressed in a majority of ovarian cancer cell lines and tumor tissue and FGFR4 DNA copy number was significantly correlated with that of FGF1 (r=0.4, p=0.04). Endogenous FGF1 production was detected by ELISA in 2 of 5 screened ovarian cell lines. FGF1 significantly induced cell proliferation in high FGFR4 expressing ovarian cancer cells (20%, p&amp;lt;0.001) and in endothelial cells (15%, p&amp;lt;0.001); but not in low FGFR4 expressers. Significant activation of MAPK/ERK signal transduction pathways was noted after treatment of ovarian(1.43 fold, P &amp;lt; 0.001) and endothelial(1.33 fold, P &amp;lt; 0.001) cell lines transduced with the Cignal-Lenti reporter system. Conclusions: Our report provides preliminary evidence that poor survival associated with FGF1 amplification and over expression appears to be mediated through FGFR4, which is also amplified and over-expressed in ovarian cancer cell. In addition, FGF1 produced by ovarian cancer cells may exert both autocrine and paracrine effect on cancer cells and endothelial cells through the activation of MAPK/ERK pathway. These data suggests that new regimens targeting FGF1, FGFR4 and downstream signaling pathways may improve ovarian cancer patient survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3132.

Synthesis of novel celluloses derivatives and investigation of their mitogenic activity in the presence and absence of FGF2

Novel cellulose sulfates (CS) with a controlled degree of sulfation (DS S) were synthesized through acetosulfation as well as direct sulfation. CS containing carboxyl (CO) or carboxymethyl (CM) groups were prepared by TEMPO oxidation or by carboxymethylation with chloroacetic acid. The derivatization was characterized by nuclear magnetic resonance and Raman spectroscopy. The derivatives were investigated regarding their cytotoxicity and mitogenic activity by modulation of 3T3 fibroblast proliferation with or without exogenous FGF2. All derivatives were non-toxic for 3T3 cells. CS strongly promoted FGF2-induced proliferation, which was positively related to overall DS S. In the absence of FGF2, minute quantities of CS with intermediate degrees of sulfation exerted stronger mitogenic effects than heparin. No significant promoting effects of CO and CM on cell proliferation were found, though the structure of CO shows similarities to heparin.

VEGF-C regulates lymphangiogenesis and capillary stability by regulation of PDGF-B

Emerging evidence indicates that the tight communication between vascular endothelial cells and mural cells using platelet-derived growth factor (PDGF)-BB is essential for capillary stabilization during the angiogenic process. However, little is known about the related regulator that determines PDGF-BB expression. Using murine models of therapeutic neovascularization, we here show that a typical lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, is an essential regulator determining PDGF-BB expression for vascular stabilization via a paracrine mode of action. The blockade of VEGF type 3 receptor (VEGFR3) using neutralizing antibody AFL-4 abrogated FGF-2-mediated limb salvage and blood flow recovery in severely ischemic hindlimb. Interestingly, inhibition of VEGFR3 activity not only diminished lymphangiogenesis, but induced marked dilatation of capillary vessels, showing mural cell dissociation. In these mice, VEGF-C and PDGF-B were upregulated in the later phase after induced ischemia, on day 7, when exogenous FGF-2 expression had already declined, and blockade of VEGFR3 or PDGF-BB activities diminished PDGF-B or VEGF-C expression, respectively. These results clearly indicate that VEGF-C is a critical mediator, not only for lymphangiogenesis, but also for capillary stabilization, the essential molecular mechanism of communication between endothelial cells and mural cells during neovascularization.

S07-03 Asymmetric division in zebrafish neural tube is a non-stochastic event and the neuron results from the daughter that inherits the apical domain

Emerging evidence suggests that neurogenesis may occur within the adult hypothalamus but the character and location of hypothalamic neural stem cells is unknown. We have identified a stem cell niche, confined to the central/posterior hypothalamus. Niche-derived cells require FGF signalling to maintain their proliferation exvivo, and proliferate, but do not differentiate, in response to exogenous FGF2 invivo. Enhanced proliferation of hypothalamic neural stem cells occurs concommitant with a reduction in proliferating progenitors. We define two distinct FGF2-dependent cell types, whose position correlates with particular tanycyte populations. Cells that map to GFAP+ive tanycytes show hallmarks of classical neural stem cells; cells that map to adjacent GFAPive ventral a2 tanycytes show characteristics intermediate beween stem and progenitor cells. Both cell types appear largely quiescent invivo, but endogenous FGFs may govern their proliferation: FGF ligands are expressed specifically within different tanycyte populations and FGF signalling elements are stochastically detected in tanycyte cells.

Effect of FGF-binding Protein 3 on Vascular Permeability

Fibroblast growth factor-binding protein 1 (FGF-BP1 is BP1) is involved in the regulation of embryonic development, tumor growth, and angiogenesis by mobilizing endogenous FGFs from their extracellular matrix storage. Here we describe a new member of the FGF-BP family, human BP3. We show that the hBP3 protein is secreted from cells, binds to FGF2 in vitro and in intact cells, and inhibits FGF2 binding to heparin. To determine the function of hBP3 in vivo, hBP3 was transiently expressed in chicken embryos and resulted in > 50% lethality within 24 h because of vascular leakage. The onset of vascular permeability was monitored by recording the extravasation kinetics of FITC-labeled 40-kDa dextran microperfused into the vitelline vein of 3-day-old embryos. Vascular permeability increased as early as 8 h after expression of hBP3. The increased vascular permeability caused by hBP3 was prevented by treatment of embryos with PD173074, a selective FGFR kinase inhibitor. Interestingly, a C-terminal 66-amino acid fragment (C66) of hBP3, which contains the predicted FGF binding domain, still inhibited binding of FGF2 to heparin similar to full-length hBP3. However, expression of the C66 fragment did not increase vascular permeability on its own, but required the administration of exogenous FGF2 protein. We conclude that the FGF binding domain and the heparin binding domain are necessary for the hBP3 interaction with endogenous FGF and the activation of FGFR signaling in vivo.

Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium.

A major limitation in developing applications for the use of human embryonic stem cells (HESCs) is our lack of knowledge of their responses to specific cues that control self-renewal, differentiation, and lineage selection. HESCs are most commonly maintained on inactivated mouse embryonic fibroblast feeders in medium supplemented with FCS, or proprietary replacements such as knockout serum-replacement together with FGF-2. These undefined culture conditions hamper analysis of the mechanisms that control HESC behavior. We have now developed a defined serum-free medium, hESF9, for the culture of HESCs on a type I-collagen substrate without feeders. In contrast to other reported media for the culture of HESCs, this medium has a lower osmolarity (292 mosmol/liter), l-ascorbic acid-2-phosphate (0.1 microg/ml), and heparin. Insulin, transferrin, albumin conjugated with oleic acid, and FGF-2 (10 ng/ml) were the only protein components. Further, we found that HESCs would proliferate in the absence of exogenous FGF-2 if heparin was also present. However, their growth was enhanced by the addition of FGF-2 up to 10 ng/ml although higher concentrations were deleterious in the presence of heparin.

Fibroblast Growth Factor 2 Restrains Ras-Driven Proliferation of Malignant Cells by Triggering RhoA-Mediated Senescence

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.

Homocysteine Inhibits Arterial Endothelial Cell Growth Through Transcriptional Downregulation of Fibroblast Growth Factor-2 Involving G Protein and DNA Methylation

Homocysteine (Hcy) contributes to atherogenesis and angiostasis by altering the phenotype of arterial endothelial cells (ECs). The present study was aimed at elucidating potential mechanisms by which Hcy can slow EC proliferation and induce EC apoptosis, thereby disrupting endothelial integrity. Given the strong mitogenic and antiapoptotic properties of fibroblast growth factor (FGF)2, we examined whether Hcy can modulate its expression. In cultured human coronary and bovine aortic ECs, Hcy exerted time- and concentration-dependent (0 to 500 micromol/L) reduction of the mRNA and protein levels of FGF2, whereas vascular endothelial growth factor expression was not affected until Hcy reached a proapoptotic 500 micromol/L. By testing a panel of signal transduction inhibitors, we found that the Hcy-induced downregulation of FGF2 was specifically attenuated by pertussis toxin, an inhibitor of Gi protein signaling. Hcy induced cell cycle arrest at the G(1)/S transition and increased TUNEL-positive apoptotic cells in a graded manner. These effects were effectively counteracted by exogenous FGF2. Reporter gene assays showed that Hcy downregulated FGF2 by transcriptional repression of the gene promoter encompassed in a CpG dinucleotide-rich island. This region was heavily methylated at the cytosine residues by Hcy despite decreased methylation potential (S-adenosylmethionine to S-adenosylhomocysteine ratio). Normal levels of FGF2 transcription were restored to ECs simultaneously exposed to Hcy and 5-aza-deoxycytidine. We conclude that homocysteine disrupts the growth and survival of ECs through a G protein-mediated pathway associated with altered promoter DNA methylation and the transcriptional repression of FGF2.

FGF signaling is a major regulator of coronary tubulogenesis in the murine embryo

Our previous work has documented that FGF‐2 is a potent stimulator of both coronary endothelial cell proliferation and tube formation in quail embryos. The current study addressed the role of FGF signaling and Sca‐1+ bone marrow cells (BMC) in coronary tubulogenesis. Embryonic day 6 mouse hearts were explanted onto collagen gels and incubated for 7 days. Tubulogenesis (aggregate tube length), migration distance and cell number were quantified. Exogenous FGF‐2 enhanced tube formation 3‐fold, while adenovirus FGFR‐1DN, which virtually abolishes all FGF signaling, decreased tubulogenesis by about 50%, and markedly inhibited cell proliferation. Soluble adenovirus FGFR‐3 IIIb (or soluble R3IIIb‐Fc), which can bind and inhibit FGF‐1 and FGF‐9, did not significantly inhibit tubulogenesis, but reduced the number of endothelial cells. Addition of sca‐1+ BMC caused only a small (20%) increase in tubulogenesis. The data support the hypothesis that cell proliferation and tube formation are regulated by different FGF ligands.

Human Fibroblast Growth Factor Receptor 1-IIIb Is a Functional Fibroblast Growth Factor Receptor Expressed in the Pancreas and Involved in Proliferation and Movement of Pancreatic Ductal Cells

The possible functions of the human IIIb-messenger RNA splice variant of fibroblast growth factor (FGF) receptor 1 (FGFR-1 IIIb) are yet to be delineated. In this study, the expression and functionality of the human FGFR-1 IIIb were characterized in the pancreas. In situ hybridization with a specific FGFR-1 IIIb probe in human pancreatic tissues demonstrated that FGFR-1 IIIb localized in normal pancreatic acinar and in ductal-like pancreatic cancer cells. To further assess the potential role of this receptor, a full-length human FGFR-1 IIIb was stably expressed in TAKA-1 pancreatic ductal cells not expressing endogenous FGFR-1. The FGFR-1 IIIb-expressing TAKA-1 cells synthesized a glycosylated 110-kd protein capable of inducing proliferation on incubation with exogenous FGF-1, -2, and -4. These effects were paralleled by tyrosine phosphorylation of FGFR substrate 2 and association of FGFR substrate 2 with FGFR-1 IIIb. The FGF-1, -2, and -10 induced the activation of p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, and c-Jun N-terminal kinase. Pharmacological inhibition revealed that FGF-induced proliferation was dependent on the concomitant activation of p44/42 MAPK and c-Jun N-terminal kinase. The FGFR-1 IIIb expression enhanced single-cell movement and plating efficacy. Our results demonstrate that the human FGFR-1 IIIb variant is a functional FGFR expressed in the pancreas that can alter pancreatic functions that regulate proliferation, adhesion, and movement.

A tissue-engineered model of fetal distal lung tissue

In extending our previous studies toward development of an engineered distal lung tissue construct (M. J. Mondrinos, S. Koutzaki, E. Jiwanmall, M. Li, J. P. Dechadarevian, P. I. Lelkes, and C. M. Finck. Tissue Eng 12: 717-728, 2006), we studied the effects of exogenous fibroblast growth factors FGF10, FGF7, and FGF2 on mixed populations of embryonic day 17.5 murine fetal pulmonary cells cultured in three-dimensional collagen gels. The morphogenic effects of the FGFs alone and in various combinations were assessed by whole mount immunohistochemistry and confocal microscopy. FGF10/7 significantly increased epithelial budding and proliferation; however, only FGF10 alone induced widespread budding. FGF7 alone induced dilation of epithelial structures but not widespread budding. FGF2 alone had a similar dilation, but not budding, effect in epithelial structures, and, in addition, significantly enhanced endothelial tubular morphogenesis and network formation, as well as mesenchymal proliferation. The combination of FGF10/7/2 induced robust budding of epithelial structures and the formation of uniform endothelial networks in parallel. These data suggest that appropriate combinations of exogenous FGFs chosen to target specific FGF receptor isoforms will allow for control of lung epithelial and mesenchymal cell behavior in the context of an engineered system. We propose that tissue-engineered fetal distal lung constructs could provide a potential source of tissue or cells for lung augmentation in pediatric pulmonary pathologies, such as pulmonary hypoplasia and bronchopulmonary dysplasia. In addition, engineered systems will provide alternative in vitro venues for the study of lung developmental biology and pathobiology.

Selective cyclooxygenase‐2 inhibition suppresses basic fibroblast growth factor expression in human esophageal adenocarcinoma

Inhibition of cyclooxygenase (COX)-2 is reported to suppress growth and induce apoptosis in human esophageal adenocarcinoma (EADC) cells, although the precise biologic mechanism is unclear. In this study we tested the hypothesis that the antitumor activity of COX-2 inhibitors may involve modulation of basic fibroblast growth factor (FGF-2), which is overexpressed in EADC. We evaluated the effects of NS-398, a selective COX-2 inhibitor, on FGF-2 expression and proliferation of EADC cell lines that express COX-2 and those that do not. We also correlated COX-2 and FGF-2 expression with clinico-pathologic findings and outcome in a well-characterized series of surgically resected EADC tissues. Seg-1 cells robustly expressed COX-2 and FGF-2, whereas Bic-1 cells expressed neither transcript. FGF-2 was reduced to undetectable levels in Seg-1 cells following NS-398 treatment, but increased within 4 h of drug removal. NS-398 significantly inhibited the growth of Seg-1 cells, and this effect was ameliorated by addition of exogenous FGF-2. In contrast, NS-398 had no effect on Bic-1 cell proliferation and FGF-2 alone had no effect on proliferation of either cell line. NS-398, or a neutralizing anti-FGF-2 antibody, induced apoptosis in Seg-1 cells, and these effects were inhibited by addition of exogenous FGF-2. COX-2 protein was strongly expressed in 46% (10/22) of EADCs, and was associated with a trend towards reduced disease-free survival. These findings indicate that the antitumor effects of COX-2 inhibition in EADC cells may be mediated via suppression of FGF-2, and that COX-2 may be a clinically relevant molecular marker in the management of human EADC.