Effects Of FGF-2 Research Articles

The effects of growth factors on the proliferation and in vitro angiogenesis of human macular inner choroidal endothelial cells

Aim:To investigate the effect of VEGF165, FGF2, IGF-1, PDGF-AA, PDGF-BB and IL-1β on the proliferation and angiogenic tube formation of human macular inner choroidal endothelial cells (ICEC).Methods:The proliferation of human...

Differential Activation of Multiple Signalling Pathways Dictates eNOS Upregulation by FGF2 but not VEGF in Placental Artery Endothelial Cells

Fibroblast growth factor (FGF2), but not vascular endothelial growth factor (VEGF), upregulates endothelial nitric oxide synthase (eNOS) protein expression, at least partially, via activation of extracellular signal-regulated kinase 2/1 (ERK2/1) in ovine fetoplacental artery endothelial (oFPAE) cells. Herein we further investigated the temporal effects of FGF2 and VEGF on other signalling pathways including members (Jun N-terminal kinase JNK1/2 and p38MAPK) of mitogen-activated protein kinases (MAPK), phosphatidylinositol-3 kinase/ v-akt murine thymoma viral oncogene homologue 1 (PI3K/AKT1), and the tyrosine kinase c-SRC, and examined if either one or more of these pathways play a role in the differential regulation of eNOS by FGF2 and VEGF. We first confirmed that in oFPAE cells, FGF2, but not VEGF, increased eNOS protein. FGF2 stimulated eNOS protein in a time- and concentration-dependent manner, which also depended on cell density. FGF2 provoked sustained (5 min to 12 h) whereas VEGF only stimulated transient (5 min) ERK2/1 phosphorylation. FGF2 was 1.7-fold more potent in stimulating ERK2/1 phosphorylation than VEGF. FGF2 and VEGF only transiently activated JNK1/2 and AKT1 within 5 min; however, FGF2 was a stronger stimulus than VEGF. FGF2 and VEGF did not significantly activate p38MAPK at 5 min; however, VEGF stimulated p38MAPK phosphorylation at 60 min. VEGF but not FGF2 significantly stimulated c-SRC phosphorylation. Inhibitors of MEK-ERK2/1 (PD98059), JNK1/2 (SP600125) and PI3K (wortmannin), but not p38MAPK (SB203580) and SRC (PP2), decreased the FGF2-increased eNOS protein expression. Thus, the FGF2-induced eNOS protein expression requires activation of multiple signalling pathways including ERK2/1, JNK1/2 and PI3K/AKT1. Differences in intensity and temporal patterns of activation of these pathways by FGF2 and VEGF may account for their differential effects on eNOS expression in OFPAE cells.

Antidepressant-like effects of intracerebroventricular FGF2 in rats

The fibroblast growth factor (FGF) system is altered in post-mortem brains of individuals with major depressive disorder (MDD), but the functional relevance of this observation remains to be elucidated. To this end, we tested whether administering agents that act on FGF receptors would have antidepressant-like effects in rodents. We microinjected either FGF2 (200 ng, i.c.v.) or the FG loop (FGL) of neural cell adhesion molecule (NCAM) (5 μg, i.c.v.) into the lateral ventricle of rats and tested them on the forced swim test. Activating FGF receptors acutely had an antidepressant-like effect in the forced swim test. Furthermore, chronic FGF2 decreased depression-like behavior as assessed by two independent tests. Finally, the FGF system itself was altered after FGF2 administration. Specifically, there was an increase in FGFR1 mRNA in the dentate gyrus 24 h post-FGF2, suggesting the potential for self-amplification of the initial signal. These results support the potential therapeutic use of FGF2 or related molecules in the treatment of MDD and point to alternate mechanisms of neuronal remodeling that may be critical in this treatment.

Functional effect of FGF2- and FGF8-expanded ventral mesencephalic precursor cells in a rat model of Parkinson's disease

Ventral mesencephalic (VM) precursor cells are of interest in the search for transplantable dopaminergic neurons for cell therapy in Parkinson's disease (PD). In the present study we investigated the survival and functional capacity of in vitro expanded, primary VM precursor cells after intrastriatal grafting to a rat model of PD. Embryonic day 12 rat VM tissue was mechanically dissociated and cultured for 4 or 8 days in vitro (DIV) in the presence of FGF2 (20 ng/ml), FGF8 (20 ng/ml) or without mitogens (control). Cells were thereafter differentiated for 6 DIV by mitogen withdrawal and addition of serum. After differentiation, significantly more tyrosine hydroxylase-immunoreactive (TH-ir), dopamine-producing neurons were found in FGF2- and FGF8-expanded cultures compared to controls. Moreover, expansion for 4 DIV resulted in significantly more TH-ir cells than expansion for 8 DIV both for FGF2 (2.4 fold; P < 0.001) and FGF8 (3.8 fold; P < 0.001) treated cultures. The functional potential of the expanded cells (4 DIV) was examined after grafting into striatum of aged 6-hydroxydopamine-lesioned rats. Amphetamine-induced rotations performed 3, 6 and 9 weeks postgrafting revealed that grafts of FGF2-expanded cells induced a significantly faster and better functional recovery than grafts of FGF8-expanded cells or control cells ( P < 0.05 for both). Grafts of FGF2-expanded cells also contained significantly more TH-ir cells than grafts of FGF8-expanded cells ( P < 0.05) or control cells ( P < 0.01). In conclusion, FGF2-mediated pregrafting expansion of primary VM precursor cells considerably improves dopaminergic cell survival and functional restoration in a rat model of PD.

Cryptic fragment α4 LG4‐5 derived from laminin α4 chain inhibits de novo adipogenesis by modulating the effect of fibroblast growth factor‐2

Cleavage of the extracellular matrix (ECM) by proteolysis unmasks cryptic sites and generates novel fragments with biological activities functionally distinct from those of the intact ECM molecule. The laminin G-like (LG)4-5 fragment has been shown to be excised from the laminin alpha4 chain in various tissues. However, the functional role of this fragment has remained unknown to date. To investigate this, we prepared alpha4 LG1-3 and alpha4 LG4-5 fragments by elastase digestion of recombinant alpha4 LG1-5, and examined their effects on de novo adipogenesis in mice at the site of injection of basement membrane extract (Matrigel) and fibroblast growth factor (FGF)-2. Although the addition of whole alpha4 LG1-5 suppressed adipogenesis to some extent, the alpha4 LG4-5 fragment could strongly suppress adipogenesis at a concentration of less than 20 nm. Addition of the alpha4 LG4 module, which contains a heparin-binding region, had a suppressive effect, but this was lost in mutants with reduced heparin-binding activity. In addition, antibodies against the extracellular domain of syndecan-2 and -4, which are known receptors for the alpha4 LG4 module, suppressed adipogenesis. Thus, these results suggest that the cryptic alpha4 LG4-5 fragment derived from the laminin alpha4 chain inhibits de novo adipogenesis by modulating the effect of FGF-2 through syndecans.

FGF-2 signaling induces downregulation of TAZ protein in osteoblastic MC3T3-E1 cells

Transcriptional coactivator with PDZ-binding motif (TAZ) protein is a coactivator of Runx2 and corepressor of PPARγ. It also induces differentiation of mesenchymal cells into osteoblasts. In this study, we found that FGF-2, which inhibits bone mineralization and stimulates cell proliferation, reduced the TAZ protein expression level in osteoblast-like cells, MC3T3-E1. This reduction was recovered by removing FGF-2 from the culture medium, which also restored the osteoblastic features of MC3T3-E1 cells. Furthermore, FGF-2-induced reduction of TAZ is blocked by a SAPK/JNK-specific inhibitor. These findings suggest that the expression of TAZ protein is involved in osteoblast proliferation and differentiation. This may help elucidate the discrepancies in the effect of FGF-2 and contribute to the understanding of FGF/FGFR-associated craniosynostosis syndrome etiology and treatment.

The Increase in Retinal Cells Proliferation Induced by FGF2 is Mediated by Tyrosine and PI3 Kinases

Since 1973, multiple effects of basic fibroblast growth factor have been described in a large number of cells. These effects include proliferation, survival and differentiation. The aim of this work was to study the intracellular pathways involved in the basic fibroblast growth factor (FGF2) effect on rat retinal cells proliferation in vitro. Our data show that treatment with FGF2 increases proliferation in a concentration- and time-dependent manner. The effect of 25 ng/ml FGF2 was blocked by 10 microM genistein, a tyrosine kinase inhibitor and by 25 microM LY294002, a PI3 kinase inhibitor. The concomitant treatment with 0.3 microM chelerythrine chloride, a protein kinase C inhibitor, and 6.25 microM LY294002 also inhibited the effect of FGF2. Our results suggest that the proliferative effect of FGF2 on retinal cell cultures involves the activation of distinct kinases.

Effects of FGF2 and oxygen in the BMP4-driven differentiation of trophoblast from human embryonic stem cells

Human embryonic stem cells (hESC) differentiate into trophoblast when treated with BMP4. Here we studied the effects of either low (4% O 2, L) or atmospheric O 2 (20% O 2, A) in the presence and absence of FGF2 on H1 hESC cultured in the presence of BMP4. Differentiation progressed from the periphery toward the center of colonies. It occurred most quickly in the absence of FGF2 and under A and was slowest in the presence of FGF2 and under L. Chorionic gonadotropin (CG) production required A, while FGF2 suppressed progesterone synthesis under both A and L. FGF2 was then omitted while we examined the trophoblast markers SSEA-1 and cytokeratin-7 and-8, whose expression also progressed inward from the periphery of colonies and occurred more rapidly under A than under L. By day 5, most cells outside central islands of Oct4-positive cells were positive for these antigens under both conditions and many also expressed HLA-G, a marker of extravillous cytotrophoblast. Under A, but not L, CGα and CGβ became prominent in GATA2-positive, peripherally located, multinucleated cells. In conclusion, BMP4 induced conversion of hESC exclusively toward trophoblast; FGF2 slowed differentiation, while O 2 accelerated this process and promoted syncytiotrophoblast formation.

Fibroblast growth factor-2 in hyperplastic pituitaries of D2R knockout female mice

Dopamine D2 receptor (D2R) knockout (KO) female mice develop chronic hyperprolactinemia and pituitary hyperplasia. Our objective was to study the expression of the mitogen fibroblast growth factor (FGF2) and its receptor, FGFR1, comparatively in pituitaries from KO and wild-type (WT) female mice. We also evaluated FGF2 subcellular localization and FGF2 effects on pituitary function. FGF2-induced prolactin release showed a similar response pattern in both genotypes, even though basal and FGF2-stimulated release was higher in KO. FGF2 stimulated pituitary cellular proliferation (MTS assay and [(3)H]thymidine incorporation), with no differences between genotypes. FGF2 concentration (measured by ELISA) in whole pituitaries or cultured cells was lower in KO (P < 0.00001 and 0.00014). Immunofluorescence histochemistry showed less FGF2 in pituitaries from KO females and revealed a distinct FGF2 localization pattern between genotypes, being predominantly nuclear in KO and cytosolic in WT pituitaries. Finally, FGF2 could not be detected in the conditioned media from pituitary cultures of both genotypes. FGFR1 levels (Western blot and immunohistochemistry) were higher in pituitaries of KO. Basal concentration of phosphorylated ERKs was lower in KO cells (P = 0.018). However, when stimulated with FGF2, a significantly higher increment of ERK phosphorylation was evidenced in KO cells (P < or = 0.02). We conclude that disruption of the D2R caused an overall decrease in pituitary FGF2 levels, with an increased distribution in the nucleus, and increased FGFR1 levels. These results are important in the search for reliable prognostic indicators for patients with pituitary dopamine-resistant prolactinomas, which will make tumor-specific therapy possible.

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.

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.

Common and distinct factors regulate expression of mRNA for ETV5 and GDNF, Sertoli cell proteins essential for spermatogonial stem cell maintenance

Ets variant gene 5 (ETV5) and glial cell-derived neurotrophic factor (GDNF) are produced in Sertoli cells and required for maintenance and self-renewal of spermatogonial stem cells (SSCs) in mice. Fibroblast growth factors (FGFs) have been reported to stimulate Etv5 mRNA expression, and FSH was shown to stimulate Gdnf mRNA in Sertoli cell cultures, but there is no other information on factors that regulate these key Sertoli cell proteins necessary for stem cell maintenance. In this study, we investigated regulation of ETV5 and GDNF using the TM4 murine Sertoli cell line. FGF2 stimulated a time- and dose-dependent increase in Etv5 mRNA expression, with a maximal 8.3-fold increase at 6 h following 25 ng/ml FGF2 treatment. This FGF2 dose also stimulated Gdnf mRNA at 48 h. FGF2 effects on Etv5 and Gdnf mRNA were partially mediated through mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-signaling cascades. Specific inhibitors of MAPK (PD98059) and PI3K (wortmannin) pathways reduced Etv5 and Gdnf mRNA expression in FGF2-treated cells. Epidermal growth factor (EGF) stimulated Etv5 mRNA but not Gdnf mRNA. TNFα and IL-1β stimulated Gdnf mRNA, but had no effect on Etv5 mRNA. Other hormonal regulators of Sertoli cells such as testosterone, triiodothyronine and activin A did not affect Etv5 or Gdnf mRNA expression. Results with primary Sertoli cell cultures confirmed findings obtained with the TM4 cell line, validating the use of the TM4 model to examine regulation of Etv5 and Gdnf mRNA expression. In conclusion, we have identified common and unique pathways that regulate Etv5 and Gdnf mRNA in Sertoli cells, and FGFs are emerging as key regulators of the Sertoli cell proteins that control SSCs.

FGF2 effects in periosteal fibroblasts bearing the FGFR2 receptor Pro253 Arg mutation

Aim: A growing number of mutations mapped in the receptor gene for fibroblast growth factor have been implicated in several cranial development disorders including the Apert and Crouzon syndromes. The present paper investigated cellular mechanisms underlying Apert phenotype, by analyzing the effects of FGF2 in primary cultures of Apert periosteal fibroblasts carrying the FGFR2 Pro253Arg mutation. Results: FGF2 administration significantly decreased extracellular matrix production in mutant cells by stimulating degradative enzymatic activities. Gene expression analysis revealed that decorin and biglycan, two proteoglycans involved in collagen fibrillogenesis, were more expressed in mutant cells and down-regulated by FGF2. FGF2 receptor binding showed little differences in high affinity receptor counts between mutant and wild-type cells, while we showed for the first time that low affinity receptors are significantly fewer in mutant cells. Differences were found in Crouzon syndrome, where both high and low affinity receptor counts were up-regulated. Conclusions: The different mutation and low affinity receptor regulation in mutant receptors support the hypothesis that the impact on the activity of the ligand–receptor complex could allow distinct modes of FGF2 activation in Apert and Crouzon syndromes, which interfere with the FGFR2 signalling cascade.

Fibroblast Growth Factor-2 Maintains the Differentiation Potential of Nucleus Pulposus Cells In Vitro

To investigate effects of FGF-2 on nucleus pulposus cell growth and differentiation. To elucidate the phenotypic changes that occur during expansion of nucleus pulposus cells in monolayer culture, and to investigate the effects of fibroblast growth factor (FGF)-2 on cell growth and differentiation. Nucleus pulposus cells would have a limited application for autologous cell transplantation if phenotypic dedifferentiation takes place during culture expansion. FGF-2 has been shown to retain the differentiation potential of monolayer expanded chondrocytic cells. However, its effect on nucleus pulposus cells is not known. Bovine nucleus pulposus cells were serially passaged in the presence or absence of FGF-2 (1 and 10 ng/mL). After passage numbers 1 and 7, cells were immobilized in alginate beads and treated with transforming growth factor (TGF)-beta1 for 1 week to assess their differentiation. During culture expansion in monolayer, nucleus pulposus cells maintained the expression of aggrecan messenger ribonucleic acid (mRNA). However, mRNA levels of collagen type I, collagen type II, Sox-9, and versican decreased with increasing passage number for both control (untreated) cells and FGF-2 treated cells. When grown in alginate with TFG-beta1, passage 7 cells that received FGF-2 during culture expansion restored the mRNA expression of type II collagen, Sox-9, COMP, chondroadherin, and fibromodulin. Moreover, FGF-2 treatment resulted in increased sulfated proteoglycan synthesis and lower aggrecan turnover compared to untreated controls under identical culture conditions. FGF-2 treated cells continued to express HIF-1alpha protein till passage 7, while MMP-2 expression was evident in cells treated with TGF-beta1. In addition, cells pretreated with FGF-2 showed higher induction of phospho ERK1/2 after treatment with TGF-beta1. Also, FGF-2 maintained smad 2/smad 3 mediated signaling in cells after TGF-beta treatment. FGF-2 action resulted in reduced actin stress fiber formation and migratory cell morphology, with no effect on cell proliferation. The presence of FGF-2 during culture expansion of nucleus pulposus cells in monolayer can sustain a differentiated cell phenotype by maintaining responsiveness to TGF-beta1. Our results suggest that FGF-2 should be tested for its ability to maintain the reactivity of the nucleus pulposus cells to other morphogenic factors that may be used for cell-based transplantation therapy.

Recombinant basic fibroblast growth factor inhibits the airway hyperresponsiveness, mucus production, and lung inflammation induced by an allergen challenge

IL-13 is believed to be a central mediator of asthma, and TGF-beta1 is a key downstream mediator in the development of IL-13-mediated asthma phenotypes. To evaluate the biological roles of basic fibroblast growth factor (FGF2) in phenotype expression in transgenic (TG) mice overexpressing lung-specific TGF-beta1, and the therapeutic effects of recombinant FGF2 in the development of asthma phenotypes. To evaluate the roles of FGF2 in airway hyperresponsiveness (AHR) expression induced by high levels of TGF-beta1, TGF-beta1 TG (+) mice were bred with FGF2-deficient mice. To evaluate the therapeutic effects of recombinant FGF2 (rFGF2) in the development of asthma, mice were given 10 mug of rFGF2 subcutaneously once a day, 1 hour before the allergen challenge in an asthma mouse model. AHR was evaluated using noninvasive whole-body plethysmography, mucus production by diastase-resistant periodic acid Schiff (DPAS) staining, and lung inflammation using bronchoalveolar lavage (BAL) cellularity and lung histology. AHR decreased in TGF-beta1 TG (+) mice and was accompanied by the upregulation of FGF2 mRNA expression in lung tissues, when compared with littermate wild-type control mice. Interestingly, AHR was enhanced markedly in TGF-beta1 (+) mice with homozygous FGF2 gene disruption. In an asthma mouse model, AHR, mucus production, and lung inflammation were inhibited markedly by rFGF2 treatment. This inhibition was accompanied by downregulation of the allergen-induced proliferation of T cells from regional lymph nodes. FGF2 seems to be a key inhibitor in the development of AHR, and rFGF2 treatment constrains the development of asthma phenotypes.

The Effects of FGF-2 on the Production of Glycosaminoglycan by Human Dental Pulp Cells

The Effects of FGF-2 on the Production of Glycosaminoglycan by Human Dental Pulp Cells

FGF-2 Supplementation Enhances Hematopoietic Differentiation of Rhesus ESC's.

Progress toward clinical application of embryonic stem cells (ESC) derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model such as the rhesus macaque. However, in contrast to human ESC's (hESC's), efforts to induce conclusive hematopoietic differentiation from rhesus ESC's (rESC's) have been unsuccessful. Despite their close phylogenetic relationship, subtle differences exist between the hematopoietic differentiation of rESC's and hESC's. We recently reported that although rESC's have the potential for hematopoietic differentiation; they exhibit an arrest at the hematoendothelial precursor stage of hematopoietic development in culture conditions developed for hESC's. One possible difference may be in the requirement for fibroblast growth factor (FGF) signaling. Despite documentation of its contribution to the maintenance ESC's in an undifferentiated state, the role for FGF-2 in the hematopoietic differentiation of hESC's and rESC's has not been similarly examined. Given its critical role for the formation and subsequent hematopoietic differentiation of murine ESC-derived hemangioblasts, we wondered if enhanced hematopoietic differentiation from rESC's could be achieved by culture supplementation with FGF-2. To answer this question, undifferentiated rESC's were subjected to embryoid body (EB) differentiation with daily FGF-2 supplementation of the cytokine-rich media. Cultures were analyzed by flow cytometry after 16 days of EB culture. We found that the FGF-2 supplemented cultures appeared more robust with an overall higher numbers of cells. More importantly, a dramatic expansion of hematoendothelial precursors (Flk1hi+ VE-cadherin- CD45−), committed hematopoietic progenitors (CD34+CD45+Lin−), and hematopoietic cells (CD45+) was seen in FGF-2 supplemented cultures when compared to controls. These effects were consistent in two separate lines of rESC's (R420 and R456). Next we wondered if the observed effect of FGF-2 on hematopoietic development was concentration-dependent. Therefore, we compared serial increases in FGF-2 concentration (0, 10, 50 and 100 ng/ml) of the EB differentiation media and found the effect to be concentration-dependent. From these results, we conclude that FGF-2 appears to play a critical role in the hematopoietic differentiation of rESC's. Both the development of hematoendothelial precursors and the differentiation of committed hematopoietic cell types are augmented. To study this further, the significance of FGF signaling at various stages of rESC-derived hematopoietic differentiation must be evaluated. A better understanding of the requirements for FGF-2 in EB development will likely lead to improved protocols for the production of human and rhesus ESC-derived hematopoietic progenitors.

Amelioration of Diabetes in Mice after Single-Donor Islet Transplantation Using the Controlled Release of Gelatinized FGF-2

Fibroblast growth factor (FGF)-2 has been recognized to be a key element involved in angiogenesis and a putative factor involved in stem cell-mediated islet regeneration. However, the usefulness of FGF-2 in an islet transplantation setting has not yet been explored. We therefore evaluated the effect of FGF-2 on both islet culture and islet transplantation. Isolated islets were cultured in the presence of 100 ng/ml FGF-2 for a week and then the glucose-responding insulin secretion and insulin contents were measured. Gelatinized FGF-2 (100 ng), which allowed the controlled release of FGF-2, was used for islet transplantation of streptozotocin-induced diabetic mice. Islets (150 IEQ), obtained from a single donor, mixed with gelatinized FGF-2, were transplanted into the subrenal capsule of the mice and the animals were observed for 30 days. Revascularization around the islet grafts was examined. The blood glucose levels were measured and the intraperitoneal glucose tolerance test (IPGTT) was performed. The supplementation of FGF-2 maintained proper insulin secretion and insulin contents in an in vitro culture. The use of gelatinized FGF-2 facilitated revascularization and favorable islet engraftment, thus resulting in an amelioration of the blood glucose levels in diabetic mice. The utilization of FGF-2 showed increased contents of insulin in the islet grafts and revealed a similar pattern as that of normal healthy mice in IPGTT. In contrast, the transplantation of islets without FGF-2 supplementation showed poor revascularization and failed to control the blood glucose levels in the diabetic mice.

Antiangiogenic Antithrombin Blocks the Heparan Sulfate-dependent Binding of Proangiogenic Growth Factors to Their Endothelial Cell Receptors

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.

Trophic factors counteract elevated FGF-2-induced inhibition of adult neurogenesis

The dentate gyrus of adult mammalian brain contains neural progenitor cells with self-renewal and multi-lineage potential. The lineage and maturation of the neural progenitors are determined by the composition and levels of the trophic factors in their microenvironment. In Alzheimer disease (AD) brain, especially the hippocampus, the level of basic fibroblast growth factor (FGF-2) is markedly elevated. Here we show that elevated FGF-2 enhances the division and nestin levels of cultured adult rat hippocampal progenitors but impairs neuronal lineage determination and maturation of these cells in culture. The trophic factors ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), and insulin-like growth factors-1 and -2 (IGF-1, IGF-2) as well as an Alzheimer peptidergic drug, Cerebrolysin® (CL), in which we found these neurotrophic activities, counteract the effect of FGF-2 in inducing neuronal lineage (early neurogenesis). Whereas CNTF is the most active of the neurotrophic factors studied in promoting neurogenesis, CL, probably because of a combined effect of these factors, induces similar changes but without inhibiting cell proliferation. These findings suggest that CNTF, GDNF, IGF-1, and IGF-2 are promising therapeutic targets for AD and other diseases in which neurogenesis is probably inhibited.