Introduction

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Nitric oxide (NO) plays an important role in neuronal cell death during cerebral ischemia. It has been demonstrated that cortical NO levels increase severalfold after middle cerebral artery occlusion (MCAO) (Malinski et al., 1993) and a significantly higher level of nitric-oxide synthase (NOS) activity is sustained over an extended period (Samdani et al., 1997). The initial burst of NO generation is apparently mediated by the constitutively expressed neuronal nitric-oxide synthase (nNOS) (Kader et al., 1993; Malinski et al., 1993). This calcium-dependent isoform of NOS is stimulated in response to N-methyl-D-aspartate (NMDA) receptor ion channel activation (Dawson et al., 1993). Subsequent and sustained NO production during ischemic injury is apparently attributed to an increased expression of nNOS (Gotoh et al., 1996) and induction of inducible NOS gene (Iadecola et al., 1995). NO and its oxidative metabolites, e.g., peroxinitrite have been implicated in the initiation and promotion of neuronal cell death and ischemic brain injury (Eliasson et al., 1999). Inhibition of NO synthesis by NOS inhibitors, e.g., nitro-L-arginine methylester (Coert et al., 1999), L-nitroarginine, and 7-nitroindazole significantly reduces infarct size in various models of cerebral ischemia (Yoshida et al., 1994). In nNOS gene knockout mice wherein NOS activity was reduced to less than 5% of normal, the size of cerebral infarct in response to MCAO was significantly reduced (Hara et al., 1996). Similarly, the cerebral lesions in response to NMDA microinjection were 45% smaller in nNOS knockout mice compared with the wild type (Huang et al., 1994). Cortical cultures derived from the nNOS knockout mice were resistant to NMDA-mediated cytotoxicity (Dawson et al., 1996). These data strongly support the role of NO in neuronal cell damage in vitro and in vivo.

Basic fibroblast growth factor (bFGF) also designated as FGF-2 was initially discovered as a potent stimulator of fibroblast, smooth muscle, mesenchymal, and endothelial cell proliferation (Gospodarowicz et al., 1974). Subsequent studies have shown that FGF also acts as a trophic factor for neuronal cells, promoting cell survival (Finklestein et al., 1993), growth, and differentiation in vitro (Gurney et al., 1992). In human brain, FGF-2 receptors are predominantly expressed in the central nervous system neurons and in cerebellar Purkinje cells (Cordon-Cordo et al., 1990). Expression of FGF-2 is significantly increased during neuronal injury (Kiyota et al., 1991). Similarly, the expression of FGF receptors is enhanced after cerebral ischemia (Kiyota et al., 1991). Recent studies have shown that FGF-2 reduces infarct size in experimental models of cerebral ischemia. Intravenous infusion of FGF-2, at the onset of reperfusion after a 3-h ischemia in rats, produced a significant reduction in infarct size and improvement in neurological performance (Koketsu et al., 1994; Fisher et al., 1995; Jiang et al., 1996; Ren and Finklestein, 1997). Similar reduction in infarction was obtained in a model of permanent MCAO in rats (Tanaka et al., 1995), cats (Bethel et al., 1997), and mice (Huang et al., 1997). FGF-2 was also found to reduce neuronal injury in response to microinjection of NMDA or ischemic insult in neonatal rats (Nozaki et al., 1993) and neuronal cell death in global model of ischemia in gerbil (Nakata et al., 1993). The acute effects of FGF in the models of cerebral ischemia suggest that FGF may directly or indirectly affect mechanisms involved in ischemic neuronal damage. The molecular mechanism of neuroprotective action of FGF-2, however, is not understood. Because NO plays an important role in neuronal damage after cerebral ischemia, we have studied the effect of FGF on NO-mediated cell death. Human neuroblastoma SHSY-5Y cells were used for the investigation of the effect of FGF family of growth factors on NO-mediated cell death. These cells undergo apoptosis in response to NO and exhibit various activities of apoptosis pathway, including the expression of bcl-2, bax, capases, and activation of poly(ADP-ribose) polymerase. Our results show that NO donors produce a dose-dependent death of SHSY-5Y cells. Cell death induced by NO was apoptotic in nature. Treatment with FGF-2 offered significant protection from NO-mediated apoptosis. FGF-2-mediated protection was observed even when its addition was delayed for 6 h after treatment with SNAP. Among the members of FGF family tested, FGF-2 was the most effective protector of NO-mediated cell death.

	Materials and Methods

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Cell Culture

Human neuroblastoma SHSY-5Y cells (American Type Culture Collection, Manassas, VA) were initiated at passage 21 and maintained in a 1:1 mixture of Eagle's minimum essential medium and Ham's F-12 medium (prepared in endotoxin-free water) containing 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO_2. Cells were passaged by trypsinization at 60% confluence and used up to passage 31. No discernable changes were observed either in the morphology or response to FGF during the 10 passages.

Fibroblast Growth Factors

Human recombinant FGF-2 was purchased from Upstate Biotechnology (Lake Placid, NY) Other FGF homologs were obtained form R&D Systems (Minneapolis, MN).

Measurement of Cell Viability

Cell viability was measured using two different methods as follows.

Cell Counting. Cells were seeded in 48-well plates at a density of 120,000 cells/well in a 1:1 mixture of Eagle's minimum essential medium and Ham's F-12 medium containing 10% FBS and indicated concentrations of FGF. After 1 h, an indicated amount of SNAP was added and cells were incubated for additional 24 h. Effect of SNAP on cell number was determined after removal of cells floating in the medium and then counting the attached cells after trypsinization.

Sodium 3'-[(1-Phenyl-amino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)-benzene Sulfonic Acid Hydrate (XTT) Assay. The conversion of XTT to formazan by metabolically active cells is a commonly used assay for determination of cell viability (Jost et al., 1992). For this assay, cells were cultured in 96-well plates at a density of 20,000 cells/well in a 1:1 mixture of Eagle's minimum essential medium and Ham's F-12 medium containing 10% FBS and then treated with the indicated concentrations of FGF-2. After 1 h, 300 µM SNAP was added and the cells were incubated for 24 h. XTT dye (Boehringer Mannheim, Indianapolis, IN) was then added and the color was developed by incubation for additional 18 h. Absorbance was measured at 405 nm. Percentage of cell survival was determined from the ratio of absorbance obtained from treated cultures to that from control cultures, multiplied by 100.

Determination of Apoptosis

Apoptotic cell death is distinguished from necrotic cell death by nuclear damage, resulting in DNA cleavage into oligonucleosomes by endogenous endonucleases. The characteristic fragmentation of chromatin DNA into nucleosome of about 180 base pairs, produced during apoptosis, can be assayed by either enzymatic labeling of DNA strands (terminal deoxynucleotidyl transferase biotin-mediated dUTP nick end labeling, TUNEL) (Chapman et al., 1995) or by quantifying the oligonucleosomes generated using histone specific antibody (Allen et al., 1997). We have used both these methods to demonstrate NO-induced apoptosis in SHSY-5Y cells. For TUNEL assay, cells were cultured and treated with SNAP and FGF-2 as described in the XTT assay. After 24 h, cells were fixed with zinc-formaldehyde for 30 min and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice, followed by a 60-min incubation with TUNEL labeling mixture (Boehringer Mannheim). Cells were washed with PBS and the labeled DNA fragments were observed by fluorescence microscopy.

For oligonucleosome measurement, cells were cultured as in the XTT assay. After 24-h incubation with 300 µM SNAP in the presence or absence of 10 ng/ml FGF-2, oligonucleosomes formed were measured using the Oligonucleosome assay kit from Boehringer Mannheim. Absorbance was measured at 450 nm.

Determination of DNA Synthesis

SHSY-5Y cells were cultured in 96-well plates at a density of 8000 cells/well. After 48 h, cells were treated with the indicated concentrations of FGF-2 and 1 µCi of [³H]thymidine. After 24 h, cells were fixed with methanol and DNA synthesis was determined by counting radiolabeled thymidine incorporation, using a scintillation counter.

Statistical Analysis

The results are expressed as the mean ± S.E.M. Significance level was evaluated by one-tailed Student's t test and tested at P values specified in the figure legends.

	Results

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Effect of NO and FGF on Cell Survival. Effect of NO on the survival of SHSY-5Y neuroblastoma cells was determined by culturing cells in 48-well plates and then treating with varying concentrations of SNAP (0-600 µM) in the absence or presence of 10 ng/ml FGF-2. After 24 h, cell survival was determined by cell counting using a Coulter counter. Figure 1 shows that treatment of SHSY-5Y cells with SNAP produced a concentration-dependent decrease in cell number. In the presence of 10 ng/ml FGF-2, loss of cells was greatly reduced. For example, in the presence of 100 µM SNAP, cell number in culture was reduced by 28 ± 7.3%. Addition of FGF-2 produced complete protection from NO-mediated cell death. At higher concentrations of SNAP where cell death was more than 70%, protection by FGF-2 was less effective. Figure 1 also shows that the total number of cells in the FGF-2-treated cultures (0 µM SNAP) were significantly higher than the control cultures, suggesting that FGF-2 may have either increased cell proliferation or enhanced survival of SHSY-5Y cells.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Effect of FGF-2 on NO-mediated cell death. SHSY-5Y cells were plated in 48-well plates (120,000 cells/well) in the absence () or presence () of 10 ng/ml FGF-2. After 1 h, indicated concentrations of SNAP were added. The cells were then incubated for 24 h. Surviving cells were trypsinized and counted. Values are mean ± S.E.M. of triplicates. *P < .005.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent inhibition of SHSY-5Y cell death by FGF-2. Cells (20,000 cells/well) were plated in 96-well microtiter plate in the presence of indicated concentrations of FGF-2. SNAP (300 µM) was then added and the cells were further incubated for 22 h. Cell survival was determined using XTT assay. Values are mean ± S.E.M. of triplicates. *P < .005.

Effect of FGF-2 on Survival and Proliferation of SHSY-5Y Cells. An increase in cell number observed in the presence of FGF-2 (Fig. 1, 0 µM SNAP) could have been due to a stimulation of cell proliferation and/or reduction in cell death. These effects of FGF-2 were further investigated by examining its effect on DNA synthesis in SHSY-5Y cells. Figure 3A shows that inclusion of FGF-2 in cell culture produced a concentration-dependent increase in cell number over a 72-h incubation period. In the presence of optimum concentration of FGF (10 ng/ml), the increase in cell number was about 2-fold over the control cultures. To determine whether the increase in cell number was due to a mitogenic effect of FGF-2, the effect of FGF on DNA synthesis was determined after the cells were plated for 48 h. Figure 3B shows that treatment of cells with FGF-2 did not significantly alter DNA synthesis. Under similar experimental conditions, FGF-2 produced a 3- to 4-fold increase in DNA synthesis in endothelial cells (data not shown). These data suggest that FGF-2 acts as a survival factor, and also protects SHSY-5Y cells from NO-mediated cell death.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   FGF-2 increases cell number without stimulation of DNA synthesis. A, SHSY-5Y cells were incubated in the absence or presence of indicated concentrations of FGF-2 for 72 h. The change in cell number was determined by cell counting after trypsinization. Results are the means ± S.E.M. of triplicates. *P < .005. B, SH-SY5Y cells were plated in 96-well microtiter plates (10,000 cells/well) in medium containing 2% FBS. After 48 h, indicated concentrations of FGF-2 and 1 µCi of [3H]thymidine were added. The cells were incubated for an additional 24 h. [3H]Thymidine incorporation in DNA during 24 h was determined after fixing the cells in methanol and counting radioactivity using a scintillation counter. The data are normalized for [3H]thymidine incorporation per 8000 cells.

NO Mediates Apoptosis in SHSY-5Y Cells. To determine whether the cell death observed in response to SNAP (as determined by XTT and cell number) was apoptotic in nature, a TUNEL assay on SNAP and FGF-2-treated cells was performed. Figure 4 shows that in control cultures only a few cells were TUNEL positive. Treatment with 300 µM SNAP resulted in 58 TUNEL-positive cells per field. In the presence of 10 ng/ml FGF-2, the TUNEL-positive cells in SNAP-treated cultures were reduced to six per field. These results show that NO-mediated cell death was predominantly due to apoptosis. The TUNEL assay further confirmed the protective effect of FGF-2.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Effect of FGF-2 on NO-mediated apoptosis. Apoptosis in SHSY-5Y cells in the absence (A) or presence (B) of 300 µM SNAP, or SNAP + 10 ng/ml FGF-2 (C) was determined using TUNEL assay. Cell nuclei exhibiting fluorescence in the field of view shown here are counted as apoptotic cells. A phase contrast image of control cells (D) indicates that the lack of fluorescence is not simply due to the absence of cells in the plates. The phase contrast images of other cultures (A-C) were similar to that shown in D.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Inhibition of NO-induced oligonucleosome generation by FGF-2. SHSY-5Y cells were treated with 150 µM SNAP in the presence or absence of varying concentrations of FGF-2. After 24 h, DNA fragmentation was quantified by measuring oligonucleosome content in cell lysates using an enzyme-linked immunosorbent assay. Values are mean ± S.E.M. of triplicates. P < .005.

Delayed Addition of FGF Induces Cytoprotection. In the above-mentioned studies, FGF-2 was added to the cells 1 h before the treatment with SNAP. To determine whether FGF-2 affected early or late events of NO-mediated apoptosis, FGF was added at various times after treatment with SNAP. Cell survival was then determined using XTT assay. As shown in Fig. 6, FGF-2 inhibited NO-mediated cell death even when its addition was delayed up to 6 h. These results suggest that the site of FGF-2 action may reside at events that occurs 6 h after NO treatment.

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Effect of delayed addition of FGF-2 on NO-mediated apoptosis. SHSY-5Y cells were plated as described in Fig. 2 legend. FGF-2 (10 ng/ml) was added at the indicated times after the addition of 300 µM SNAP. Cell survival after 24 h was determined by XTT assay.

Effect of FGF Homologs on NO-Mediated Cell Death. Figure 7 shows the effect of different homologs of FGF on NO-mediated cytotoxicity in SHSY-5Ycells. Cells were plated in the absence or presence of 10 ng/ml of the indicated growth factor. SNAP (300 µM) was then added and the cultures were incubated for 24 h. Cell survival was determined by XTT assay. As shown here, FGF-2 produced the highest effect on NO-mediated cell death. Significant effect was also observed with FGF-4. Other homologs of FGF neither enhanced cell survival nor protected from NO-mediated cytotoxicity.

View larger version (44K): [in this window] [in a new window]   Fig. 7.   Effect of FGF homologs on NO-mediated cell death. SHSY-5Y cells were cultured in the absence or presence of 10 ng/ml FGF homologs for 1 h and then treated with 300 µM SNAP. After 24 h, cell survival was determined using XTT assay. Values are mean ± S.E.M. of triplicates. P < .005.

	Discussion

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In experimental models of myocardial and cerebral ischemia, treatment with FGF family of polypeptides significantly reduces infarct size (Koketsu et al., 1994; Fisher et al., 1995; Jiang et al., 1996; Ren and Finklestein, 1997). FGF induces hypotension and increases cerebral blood flow (Regli et al., 1994). A part of the acute effect of FGF on infarction could be attributed to its vasodilatory activity. FGF treatment also supports the survival of neurons in central nervous system (Cuevas et al., 1991). Other studies suggest that FGF protects neuronal cell survival in vitro (Finklestein et al., 1993). Thus, the mechanism of the neuroprotective action of FGF is not fully understood. A variety of studies have suggested that NMDA receptor activation and production of nitric oxide play an important role in ischemia-induced cerebral neuronal cell death (Garthwaite et al., 1989). The aim of the present study was to determine whether FGF-2 treatment offered protection of neuronal cells from NO-mediated apoptosis. Our results show that in cultures of human neuroblastoma SHSY-5Y cells, NO donor SNAP produced a dose-dependent reduction in viable cells. Addition of FGF-2 to these cultures produced a dramatic reversal of cell death. Protection from NO-mediated cell death was demonstrated by counting actual number of viable cells as well as by XTT assay. Using TUNEL and oligosome assays, we have demonstrated that at lower concentrations of SNAP (50-150 µM) NO-induced cell death predominantly occurred via the apoptotic pathway. At these concentrations of SNAP, FGF treatment effectively reduced cell death. At high concentrations of SNAP, where cell death probably occurs through a combination of apoptosis and necrosis, the protective action of FGF-2 was significantly reduced. These results suggest that the effect of FGF-2 may be on the mechanisms of apoptosis and not simply on the generation or degradation of NO. We also found that in steady-state cultures of SHSY-5Y cells, addition of FGF-2 produced a significant increase in cell number over time. FGF-2 treatment did not significantly enhance DNA synthesis in SHSY-5Y cells, suggesting that the increase in cell number was due to an increased cell survival and not due to an increase in cell proliferation.

Our results show that FGF-2 was effective in antagonizing NO-mediated apoptosis even when added 6 h after NO treatment. These data concur with the in vivo studies wherein the infarct size was significantly reduced when FGF treatment was initiated 2 h after the onset of ischemia. These data suggest that FGF-2 affects late-stage events in the apoptosis pathway. The in vivo (Fisher et al., 1995; Jiang et al., 1996; Ren and Finklestein, 1997) and in vitro data suggest that FGF-2 therapy may offer a wider time window for the treatment of stroke patients.

The FGF gene family consists of at least 19 different genes with varying sequence homology (Ohyabashi et al., 1998; Xie et al., 1999). FGF genes are expressed in a tissue-selective manner (Cordon-Cordo et al., 1990). To determine whether, neuroprotection was a common feature of all members of FGF gene family or a selective property of FGF-2, we tested the effect of homologs of FGF on NO-mediated cell death. Our results suggest that a significant protection was achieved only by FGF-2 and FGF-4. The selective effect of FGF-2 and FGF-4 on cell survival observed here is likely due to the nature of the FGF receptors expressed by SHSY-5Y cells. FGF receptors are encoded by four different genes (Johnson and Williams, 1993). The different FGF gene products exhibit varying degree of affinity for these receptors. Furthermore, the existence of splice variants of FGF receptors with varying ligand-binding properties could lead a cell selective action of FGF homologs (Werner et al., 1992). Based on the known receptor binding profile of FGF homologs, our data suggest that the effect of FGF-2 in SH-SY5Y cells is mediated through FGF receptor 1.

There has been a significant interest in understanding the mechanism of neuroprotection by peptide growth factors. In neuronal and non-neuronal cells, NO has been shown to affect several key steps of the apoptosis pathway. For example, NO has been shown to increase caspase 3 (Uehara et al., 1999), down-regulate bcl-2 (Tamatani et al., 1998), and increase cellular accumulation of p53 (Kitamura et al., 1998; Glockzin et al., 1999). Recent studies have also shown that NO inhibits proteosome activity that may play role in the inhibition of p53 and bax degradation (Glockzin et al., 1999). Because FGF has been shown to induce bcl-2 expression in hippocampal neurons (Tamatani et al., 1998), it may be that the expression of bcl-2 is an important step in protecting against NO-induced death in SHSY-5Y cells. Overexpression of bcl-2 gene has been shown to reduce apoptosis in rat sympathetic neuronal cultures deprived of neurotrophic factors (Garcia et al., 1992). In blc-2 transgenic mice, neuronal cells are protected from ischemia-induced injury (Martinou et al., 1994). Our data indicating that neuroprotection by FGF is observed even up to 6 h after SNAP treatment are consistent with the involvement of late event such as bcl-2 expression. In hippocampal neurons, a significant decline in NO-induced bcl-2 occurs at 6 to 8 h after NO treatment (Tamatani et al., 1998). Thus, although the molecular target for FGF action in NO-induced neuroprotection remains to be understood, our studies suggest that SHSY-5Y cells could serve a useful model for delineating the FGF-mediated signaling pathway for inhibition of apoptosis.