Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Jan 1.
Published in final edited form as: Biol Reprod. 2007 Sep 26;78(1):143–150. doi: 10.1095/biolreprod.107.064477

Activation of Multiple Signaling Pathways Is Critical for Fibroblast Growth Factor 2- and Vascular Endothelial Growth Factor-Stimulated Ovine Fetoplacental Endothelial Cell Proliferation1

Jing Zheng 3,2, YunXia Wen 3, Yang Song 3, Kai Wang 3,6, Dong-Bao Chen 7, Ronald R Magness 3,4,5
PMCID: PMC2441762  NIHMSID: NIHMS53828  PMID: 17901071

Abstract

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) are two key regulators of placental angiogenesis. The potent vasodilator nitric oxide (NO) could also act as a key mediator of FGF2- and VEGF-induced angiogenesis. However, the postreceptor signaling pathways governing these FGF2- and VEGF-induced placental angiogenic responses are poorly understood. In this study, we assessed the role of endogenous NO, mitogen-activated protein kinase 3/1 (MAPK3/1), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) in FGF2- and VEGF-stimulated proliferation of ovine fetoplacental endothelial (OFPAE) cells. Both FGF2 and VEGF time-dependently stimulated (P < 0.05) NO production and activated AKT1. Both FGF2- and VEGF-stimulated cell proliferation was dose-dependently inhibited (P < 0.05) by NG-monomethyl-L-arginine (L-NMMA; an NO synthase inhibitor), PD98059 (a selective MAPK3/1 kinase 1 and 2 [MAP2K1/2] inhibitor), or LY294002 (a selective phosphatidylinositol 3 kinase [PI3K] inhibitor) but not by phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (PTIO, a potent extracellular NO scavenger). At the maximal inhibitory dose without cytotoxicity, PD98059 and LY294002 completely inhibited VEGF-induced cell proliferation but only partially attenuated (P < 0.05) FGF2-induced cell proliferation. PD98059 and LY294002 also inhibited (P < 0.05) FGF2- and VEGF-induced phosphorylation of MAPK3/1 and AKT1, respectively. L-NMMA did not significantly affect FGF2- and VEGF-induced phosphorylation of either MAPK3/1 or AKT1. Thus, in OFPAE cells, both FGF2- and VEGF-stimulated cell proliferation is partly mediated via NO as an intracellular and downstream signal of MAPK3/1 and AKT1 activation. Moreover, activation of both MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for FGF2-stimulated cell proliferation, whereas activation of either one pathway is sufficient for mediating the VEGF-induced maximal cell proliferation, indicating that these two kinase pathways differentially mediate the FGF2- and VEGF-stimulated OFPAE cell proliferation.

Keywords: AKT1, endothelial cell proliferation, FGF2, growth factors, kinases, MAPK3/1, nitric oxide, pregnancy, vascular endothelial growth factor

INTRODUCTION

Angiogenesis and vasodilatation are two critical processes controlling the dramatic increases in placental blood flow, which is directly correlated with fetal growth, fetal survival, and neonatal birth weight [1, 2]. We and other researchers have obtained solid evidence showing that expressions of fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) positively correlate to the placental vascular growth during pregnancy [1–4]. Meanwhile, expression of endothelial nitric oxide (NO) synthase (NOS3) in placental tissues and cord blood NO concentrations increase in parallel with these angiogenic factors and placental blood flow [1–4]. These data implicate that interplays between FGF2, VEGF, and NOS-derived NO play an important role in the integral regulation of placental angiogenesis and vasodilatation [5].

Apart from being a potent vasodilator, it is of note that NO is capable of directly promoting endothelial mitosis and migration (two essential steps of angiogenesis). With bovine coronary venular endothelial cells, Ziche et al. [6, 7] have previously reported that exogenous NO stimulates endothelial cellular DNA synthesis, proliferation, and migration in vitro. Exogenous NO also mediates in vivo angiogenic responses induced by a vasoactive agent substance P in a rabbit “cornea pocket assay,” in which angiogenesis could be completely inhibited by the NOS inhibitor L-NG -Nitroarginine methylester (L-NAME) [7]. Morerecently, we observed a direct stimulatory role of NO in ovine fetoplacental endothelial (OFPAE) cell proliferation, in part, via activation of the mitogen-activated protein kinase 1 and 2/mitogen-activated protein kinase 3/1 (MAP2K1/2/MAPK3/1) cascade [8].

Previous studies have shown that VEGF-, but not FGF2-, induced in vivo angiogenesis is mediated via the NO/cGMP pathway [9, 10]. A clear role of NO as a downstream signal in VEGF-induced angiogenesis is further supported by many in vivo observations, including those made in rabbit ischemia models [11], NOS3 knockout mice [12], and NOS3 overexpressed rats [13]. Although the role of NO in the FGF2-induced angiogenesis is less conclusive, Babaei et al. [14] showed that NO functions as a crucial signal in the angiogenic response to FGF2 by promoting endothelial cell differentiation into capillary-like tube structures while terminating the proliferative actions with both human umbilical vein (HUVE) and calf pulmonary artery endothelial cell lines. Recently, we have also shown that FGF2-stimulated cell proliferation is associated with increased NOS3 protein expression in OFPAE cells [15, 16]. These studies, therefore, suggest that NO can function as an intermediate signal, which differentially regulates different steps of the angiogenic process upon FGF2 and VEGF stimulation.

Both FGF2 and VEGF initiate the cellular responses when they bind to and subsequently activate their respective specific tyrosine kinase receptors, thereby triggering multiple downstream protein kinase pathways [9, 10, 14, 17–19]. Among these pathways, the MAP2K1/2 (also termed MEK1/2)/MAPK3/1) (also termed as ERK1/2) and phosphatidylinositol 3 kinase (PI3K)/v-akt murine thymoma viral oncogene homolog 1 (AKT1) pathways are the best studied. Of interest, activation of these two kinase pathways by FGF2 and VEGF may lead to the same angiogenic response in one endothelial cell type or distinct angiogenic responses in the other endothelial cell type. For example, the NO/cGMP/MAPK3/1 pathway mediates VEGF-, but not FGF2-, induced cell proliferation in bovine coronary venular endothelial cells [9, 10], whereas in HUVE cells, such a cell response could be mediated by the PI3K/NOS3/NO pathway [20]. In other cases, parallel activation of MAP2K1/2 and PI3K pathways is required for maximal FGF2-induced endothelial cell proliferation [18]. Moreover, the signaling control mechanisms of FGF2- and VEGF-induced angiogenesis could be further complicated by cross-talks between these two pathways because activated MAPK3/1 or upstream kinases of MAP2K1/2 are capable of activating the PI3K/AKT1 pathway [21, 22], which, in turn, either inhibits [21–24] or potentiates [25] the MAP2K1/2/MAPK3/1 pathway. Thus, FGF2- and VEGF-activated MAPK3/1 and AKT1 pathways may act in a parallel, synergistic, or antagonistic manner [26].

Relatively little is known about the postreceptor signaling pathways governing FGF2- and VEGF-induced placental angiogenesis. Therefore, in the present study, we investigated the role of intracellular signaling pathways in FGF2- and VEGF-stimulated endothelial proliferation with the OFPAE cell line as a model. We evaluated in OFPAE cells 1) whether and how endogenous NO mediated cell proliferation; 2) whether the MAP2K1/2/MAPK3/1 and PI3K/AKT1 mediated FGF2- and VEGF-stimulated cell proliferation; and 3) whether NO mediated FGF2- and VEGF-induced activation of MAPK3/1 and AKT1.

MATERIALS AND METHODS

Cell Preparation

The OFPAE cell line was established in our laboratory [4]. All OFPAE cells used in this study were at passages 8–10. The Animal Use Protocol was approved by the Research Animal Care and Use Committees of both the School of Medicine and Public Health and the College of Agriculture and Life Sciences of the University of Wisconsin-Madison.

Measurement of Total NO Levels in Culture Media

The concentration of total NO (nitrite and nitrate, NOx) in culture media was determined by chemiluminescence with an NO Analyzer (Sievers Instruments, Boulder, CO) as described previously [27, 28]. After 16 h of serum starvation, cells were treated without or with 10 ng/ml of FGF2 or VEGF for 0, 6, 12, 24, or 36 h. Samples (100 μl/treatment) of media collected were injected into the analyzer. The chemiluminescence signal generated by NOx was recorded and processed. Total NOx was calculated by a standard curve generated with sodium nitrate (100 nM-100 μM; Sigma-Aldrich, St. Louis, MO) as the standard. All data were normalized by the protein content of corresponding wells and corrected by subtracting those from the corresponding medium control (cells were treated with media only).

Cell Proliferation Assay

Cell proliferation assays were carried out as described [8, 15, 28]. We have previously shown that FGF2 and VEGF at 10 ng/ml significantly stimulated OFPAE cell proliferation [15]. Cells were cultured in 96-well plates (2000 cells/well) in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin (all from Gibco-BRL, Gaithersburg, MD) and cultured for 48 h. To examine the effects of endogenous NO on FGF2- and VEGF-stimulated cell proliferation, L-arginine-free DMEM, which was specially formulated by Gibco-BRL, was used. Cells were serum deprived in this DMEM supplemented with 25 μM L-arginine (Gibco-BRL) and 0.1% BSA for 16 h. Cells were treated with bovine FGF2 (R & D Systems, Minneapolis, MN), or human recombinant VEGF165 (PeproTech, Inc., Rocky Hill, NJ) at 10 ng/ml in the absence or presence of NG-monomethyl-L-arginine (L-NMMA) (25, 250, 2500, and 5000 μM; CalBiochem, San Diego, CA), NG-monomethyl-D-arginine (D-NMMA, 2500 and 5000 μM; CalBiochem), or phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (PTIO) (Sigma-Aldrich; a potent extracellular NO scavenger [8]; 6.25–50 μM). In preliminary studies, we observed that a minimum of 12.5 μM L-arginine was required for FGF2-induced cell growth and for maintaining cell viability and normal morphology. To determine the roles of the MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways, cells were treated in a similar fashion, except that during serum starvation and growth factor treatments, cells were cultured in regular DMEM, which contained approximately 400 μM L-arginine and was supplemented with 0.1% BSA. Cells were treated with FGF2 or VEGF165 in the absence or presence of PD98059 (a selective MAP2K1/2 inhibitor, 1-h pretreatment; CalBiochem) and/or LY 294002 (PI3K inhibitor, 1-h pretreatment; CalBiochem). After an additional 48 h of growth factor treatments, the numbers of cells were determined by the crystal violet assay [8, 15, 28]. Wells containing known cell numbers (0, 5000, 10 000, 20 000, or 40 000 cells/well; 6-well/cell density) were treated in a similar fashion to establish standard curves.

Western Immumoblot Analysis for MAPK3/1 and AKT1

Immunoblot analysis was performed as described previously [8, 15, 16, 28]. After 16 h of serum deprivation, OFPAE cells were treated for 0–60 min with FGF2 or VEGF (10 ng/ml). In the previous study, we demonstrated that both FGF2 and VEGF rapidly activate MAPK3/1, beginning after 5 min of treatment, reaching maximum levels at 10 min, and decreasing after 20 min [15]. In the present study, time dependence of AKT1 phosphorylation was first determined. Additional cells were treated for 10 min with FGF2 or VEGF in the absence or presence of PD98059 (20 μM, 1-h pretreatment) or LY294002 (5 μM, 1-h pretreatment). Controls included cells cultured with medium and the inhibitors alone. To examine the effects of NO on FGF2- and VEGF-stimulated cell proliferation, additional cells were treated in a similar fashion, except that during serum starvation and growth factor treatment, cells were cultured in the DMEM containing only 25 μM L-arginine in the absence or presence of L-NMMA (5000 μM) or D-NMMA (5000 μM). Cells were washed with ice-cold PBS, harvested, and lysed by sonication in buffer (4 mM sodium pyrophosphate; 50 mM Hepes, pH 7.5; 100 mM NaCl; 10 mM EDTA; 10 mM sodium fluoride; 2 mM sodium orthovanadate [Na3VO4]; 1 mM PMSF; 1% Triton X-100; 5 μg/ml of leupeptin; and 5 μg/ml of aprotinin). The lysates were centrifuged, and protein concentrations of the supernatants were determined. Proteins (2.5 μg/lane for MAPK3/1, 10–15 μg/lane for AKT1) were separated on 12% SDS-PAGE gels, electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA), immunoblotted with either rabbit phospho-specific (1:2000) or total MAPK3/1 (1:2000) or phospho-specific (AKT1 473; 1:1000) or total AKT1 (1:2000) antibody. All of these four antibodies were purchased from Cell Signaling Technology (Beverly, MA). The MAPK3/1 and AKT1 on the membranes were visualized by a chemiluminescence system (Amersham Life Science Inc., Arlington Heights, IL) and quantified by scanning densitometry (model GS 670; Bio-Rad, Hercules, CA).

Statistical Procedures

All data were analyzed by a one-way analysis of variance (SigmaStat; Jandel Scientific, San Rafael, CA). When an F test was significant, data were compared with their respective control by Bonferroni multiple comparisons or the Student t-test. Data are reported as the mean ± SEM.

RESULTS

Both FGF2 and VEGF Increased Total NOx Levels

Both FGF2 and VEGF time-dependently increased NOx levels in the medium (Fig. 1). For VEGF, the stimulatory effect on total NOx levels was detectable (P < 0.05) after 6 h of treatment, continued to increase by 12 h, and remained elevated up to 36 h. For FGF2, the stimulatory effect appeared after 6- and 12-h treatments and reached statistical significance (P < 0.05) after a 24-h treatment. The stimulatory effect of VEGF on NOx levels was more robust (~2- to 4-fold from 6 to 36 h) than that of FGF2.

FIG. 1.

FIG. 1

Open in a new tab

Total NOx production by OFPAE cells in response to FGF2 and VEGF. NOx concentrations in medium samples were determined by nitrite/nitrate chemiluminescence with an NO analyzer, based on a reaction of conversion of nitrite and nitrate to NO. Total NOx levels were calculated by a standard curve generated with sodium nitrate as the standard and normalized by the protein content of corresponding wells. Data are subtracted from those in medium controls and are expressed as means ± SEM from three independent experiments. Within each growth factor treatment, means with different letters (a, b, c) differ (P < 0.05) from the time 0 control. #, means differ (P < 0.07) from FGF2 treatment at the corresponding time point; *, means differ (P < 0.05) from FGF2 treatment at the corresponding time point.

L-NMMA, but not PTIO, Inhibited FGF2- and VEGF-Stimulated Cell Proliferation

L-NMMA at high (2.5 and 5 mM), but not low (25 and 250 μM), concentrations significantly (P < 0.05) inhibited FGF2-and VEGF-induced cell proliferation (Fig. 2). These two inhibitory doses of L-NMMA were 100- and 200-fold above L-arginine concentrations (25 μM), which is a typical excess amount required for competitive inhibition. D-NMMA at 2.5 and 5 mM did not affect the FGF2- and VEGF-induced cell proliferation. Neither L-NMMA alone at all doses studied nor D-NMMA alone at 2.5 and 5 mM significantly changed cell numbers after 2 days of culture (Fig. 2), indicating that L-NMMA and D-NMMA do not reduce cell viability. PTIO, a potent extracellular NO scavenger, from 6.25 to 50 μM did not have a significant effect on the FGF2- and VEGF-stimulated cell proliferation (Fig. 2, C and D). We have recently reported that PTIO at 50 μM exhausted NO released from 1 μM of sodium nitroprusside SNP (a potent NO donor) and greatly reduced NO levels released from 10 μM of SNP on OFPAE cells [8], at which concentrations SNP releases much higher levels of NO than OFPAE cells (J. Zheng et al., unpublished results).

FIG. 2.

FIG. 2

Open in a new tab

Effects of L-NMMA and PTIO on FGF2- and VEGF-stimulated OFPAE cell proliferation. After serum starvation for 16 h, cells were treated with 10 ng/ml of FGF2 or VEGF in the specially formulated DMEM supplemented with 25 μM L-arginine in the absence or presence of L-NMMA, D-NMMA, or PTIO (1-h pretreatment). Cells were counted after 48 h of treatment. Data for each point are averaged from six experiments and expressed as means ± SEM percentage of the controls. The numbers of cells per well in the control were 8665 ± 576.3 and 5593 ± 523.3, respectively, for the L/D-NMMA (A and B) and PTIO (C and D) treatments. Means with different letters (a, b, c, d) differ significantly (P < 0.05).

PD98059 and LY294002 Inhibited FGF2- and VEGF-Stimulated Cell Proliferation

Both FGF2- and VEGF-stimulated OFPAE cell proliferation was dose-dependently inhibited (P < 0.05) by PD98059 and LY294002 (Fig. 3). PD98059 decreased (P < 0.05) FGF2-stimulated cell proliferation at 10 and 20 μM but not at 2.5 and 5 μM (Fig. 3A). VEGF-stimulated cell proliferation was inhibited (P < 0.05) by PD98059 at 2.5–10 μM and abolished (P < 0.05) at 20 μM (Fig. 3B). LY294002 significantly reduced (P < 0.05) FGF2-stimulated cell proliferation at 2.5–5.0 μM, whereas it completely blocked (P < 0.05) VEGF-stimulated cell proliferation at 2.5 μM (Fig. 3, C and D). LY294002 at 0.625 and 1.25 μM did not alter either FGF2- or VEGF-stimulated cell proliferation (Fig. 3, C and D). In the absence of FGF2 and VEGF, PD98059 at 40 μM, but not below 20 μM, and LY294002 at 10 μM, but not below 5 μM, significantly (P < 0.05) decreased cell numbers (Fig. 3). These data suggest that toxic effects of these inhibitors on OFPAE cells occurred only at these relatively high concentrations.

FIG. 3.

FIG. 3

Open in a new tab

Effects of PD98059 and LY294002 on FGF2- and VEGF-stimulated OFPAE cell proliferation. After 16 h of serum starvation, cells were treated with 10 ng/ml of FGF2 or VEGF in the absence or presence of PD98059 or LY294002 (1-h pretreatment). Cells were counted after 48 h of treatment. Data for each point are averaged from five experiments and expressed as means ± SEM percentage of the control. The number of cells per well in the control was 6111 ± 276.9 and 6194 ± 442.3, respectively, for the PD98059 (A and B) and LY294002 (C and D) treatments. Means with different letters (a, b, c, d, e, f) differ within each growth factor and kinase inhibitor treatment (P < 0.05).

Because either PD98059 or LY294002 at concentrations studied only partially reduced FGF2-stimulated cell proliferation (Fig. 3, A and C), we determined whether treating cells with both PD98059 (20 μM) and LY294002 (5 μM) simultaneously could completely abrogate FGF2-stimulated cell proliferation. We observed that treatment with a combination of PD98059 and LY294002 did completely inhibit (P < 0.05) FGF2-stimulated cell proliferation but that it also greatly decreased (P < 0.05) cell numbers in the absence of FGF2 (Fig. 4), indicating that the combination of PD98059 and LY294002 at their respective nontoxic doses are toxic for OFPAE cells.

FIG. 4.

FIG. 4

Open in a new tab

Effects of PD98059 and LY294002 combination treatments on FGF2-stimulated OFPAE cell proliferation. After 16 h of serum starvation, cells were treated with 10 ng/ml of FGF2 in the absence or presence of PD98059 (20 μM) and LY294002 (5 μM; 1-h pretreatment). Cells were counted after 48 h of treatment. Data for each point are averaged from five experiments and expressed as means ± SEM percentage of the control. The number of cells per well in control was 9704 ± 405.8. Means with different letters (a, b, c) differ significantly (P < 0.05).

Effects of PD98059, LY294002, and L-NMMA on Phosphorylation of MAPK3/1 and AKT1

We have previously shown that in OFPAE cells, both FGF2 and VEGF at 10 ng/ml time-dependently and rapidly (≤5 min) activated MAPK3/1 without altering total MAPK3/1 levels [15]. In the present study, we demonstrated that FGF2 and VEGF also rapidly (≤10 min) increased phosphorylation levels of AKT1, without changing total AKT1 levels (Fig. 5). Interestingly, unlike FGF2-induced MAPK3/1 phosphorylation, which lasts only ~10 min after activation, FGF2-induced AKT1 phosphorylation was sustained much longer (for up to 60 min after the treatment).

FIG. 5.

FIG. 5

Open in a new tab

Phosphorylation of AKT1 induced by FGF2 and VEGF in OFPAE cells. Cells were treated with 10 ng/ml of FGF2 or VEGF for 0–60 min. Proteins were separated on SDS-PAGE gels and analyzed by immunoblotting with antibodies against phospho-specific (pAKT1) or total AKT1 (tAKT1). Data averaged from three independent experiments are expressed as means ± SEM fold of the control. Means with asterisks and number symbols differ significantly (P < 0.05) from the control for FGF2 and VEGF treatments, respectively.

To examine if there are cross-talks between the MAP2K1/2/MAPK3/1 and PI3K/AKT1 signaling pathways, cells were treated with FGF2 or VEGF in the absence or presence of PD98059 (20 μM) or LY294002 (5 μM). The doses of these inhibitors, generally considered highly selective for the corresponding kinase, were chosen on the basis of their inhibitory effects on cell proliferation as shown in Figure 1. PD98059 and LY294002 greatly decreased (P < 0.05) FGF2-and VEGF-induced phosphorylation of MAPK3/1 (Fig. 6) and AKT1 (Fig. 7), respectively. LY294002 reduced (P < 0.05) VEGF-, but not FGF2-, induced phosphorylation of MAPK3/1 (Fig. 6). PD98059 did not affect FGF2- and VEGF-induced phosphorylation of AKT1 (Fig. 7). To determine if endogenous NO plays a role in the FGF2- and VEGF-induced MAPK3/1 and AKT1 pathways, cells were treated with L-NMMA and then challenged with growth factors. We found that treatment of cells with L-NMMA failed to change FGF2- and VEGF-induced phosphorylation of MAPK3/1 (Fig. 8) and AKT1 (Fig. 9).

FIG. 6.

FIG. 6

Open in a new tab

Effects of PD98059 and LY294002 on FGF2- and VEGF-induced phosphorylation of MAPK3/1 in OFPAE cells. After serum starvation, cells were treated with FGF2 or VEGF for 10 min in the absence or presence of PD98059 (20 μM) or LY294002 (5 μM) (1-h pretreatment). Proteins were separated on SDS-PAGE gels and were analyzed by immunoblotting with antibodies against phospho-specific (pMAPK3/1) or total MAPK3/1 (tMPAK3/1). Data averaged from four independent experiments are expressed as means ± SEM fold of the control. Means with different letters (a, b, c, d) differ significantly (P < 0.05).

FIG. 7.

FIG. 7

Open in a new tab

Effects of PD98059 and LY294002 on FGF2- and VEGF-induced phosphorylation of AKT1 in OFPAE cells. After serum starvation, cells were treated with FGF2 or VEGF for 10 min in the absence or presence of PD98059 (20 μM) or LY294002 (5 μM) (1-h pretreatment). Proteins were separated on SDS-PAGE gels and analyzed by immunoblotting with antibodies against phospho-specific AKT1 (pAKT1), or total (tAKT1). Data averaged from four independent experiments are expressed as means ± SEM fold of the control. Means with different letters (a, b) differ significantly (P < 0.05).

FIG. 8.

FIG. 8

Open in a new tab

Effects of L-NMMA on FGF2- and VEGF-induced MAPK3/1 phosphorylation in OFPAE cells. After cultured in DMEM containing 25 μM L-arginine, cells were treated with FGF2 (A) or VEGF (B) in the absence or presence of L-NMMA (5 mM) or D-NMMA (5 mM) (1-h pretreatment) for 10 min. MAPK3/1 phosphorylation was analyzed as described in Figure 7. Data averaged from four independent experiments are expressed as means ± SEM fold of the control. Means with different letters (a, b) differ significantly within each isoform of MAPK (P < 0.05).

FIG. 9.

FIG. 9

Open in a new tab

Effects of L-NMMA on FGF2- and VEGF-induced AKT1 phosphorylation in OFPAE cells. After cultured in DMEM containing 25 μM L-arginine, cells were treated with FGF2 or VEGF in the absence or presence of L-NMMA (5 mM) or D-NMMA (5 mM) (1-h pretreatment) for 10 min. AKT1 phosphorylation was analyzed as described in Figure 5. Data averaged from four independent experiments are expressed as means ± SEM fold of the control. Means with different letters (a, b) differ significantly (P < 0.05).

DISCUSSION

To better understand the intracellular signaling mechanisms governing FGF2- and VEGF-induced placental angiogenesis, we examined whether endogenous NO and protein kinase pathways (MAPK3/1 and AKT1) participate in mediating FGF2- and VEGF-stimulated cell proliferation with an ovine fetoplacental artery endothelial cell model. The involvement of endogenous NO in both FGF2- and VEGF-stimulated OFPAE cell proliferation is identified. We provide, for the first time, evidence that such involvement of NO is mediated primarily via an intracellular but not an autocrine and/or paracrine mechanism(s) because PTIO, an extracellular NO scavenger, does not alter the FGF2- and VEGF-stimulated cell proliferation. Our findings also demonstrate that activation of the MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for FGF2-stimulated cell proliferation, whereas activation of either pathway is sufficient for mediating the VEGF-induced maximal cell proliferation. Thus, our data indicate that these two kinase pathways differentially mediate the FGF2- and VEGF-stimulated OFPAE cell proliferation. Moreover, our data suggest that NO functions as a downstream signal of MAPK3/1 and AKT1 during the FGF2- and VEGF-stimulated OFPAE cell proliferation, as treatment with L-NMMA had no significant impact on the activation of MAPK3/1 and AKT1 induced by FGF2 and VEGF.

Placental NO production increases during normal sheep pregnancy [27, 29] and is associated with elevations in local expression of FGF2 and VEGF, vascular density, and blood flow to the placentas [30, 31]. Moreover, the increased NO production is highly correlated to protein expression of NOS3, but not NOS2 (also known as inducible NOS), in ovine placentas [27], suggesting that NOS3 is the major NOS isoform responsible for the increased NO. These associations led us to hypothesize that there are close interactions between angiogenic factors and NO in the placentas. This premise is supported by our current data showing stimulatory effects of FGF2 and VEGF on NO production by OFPAE cells, which is consistent with reports showing such actions of FGF2 and VEGF on endothelial cells derived from other nonplacental tissues [11–14, 18, 32–40]. Intriguingly, even though FGF2, but not VEGF, increases NOS3 protein expression in OFPAE cells [15, 16], VEGF is much more potent than FGF2 in stimulating NO production in OFPAE cells (Fig. 1). These data suggest that FGF2 and VEGF stimulate NO production by increasing NOS3 protein expression (FGF2) or directly enhancing enzymatic activity of NOS3 (FGF2/VEGF) in OFPAE cells. However, it is apparent that different mechanisms are involved in FGF2 and VEGF regulation of NO production and NOS3 expression in OFPAE cells, which needs to be investigated. Together, our current data further support the critical role of FGF2 and VEGF in regulation of placental vasodilatation, in addition to their angiogenic activities [1–4].

NO has been reported to mediate VEGF-, but not FGF2-, induced cell proliferation and migration in bovine venular endothelial cells [9, 10]. Babaei et al. [14], however, have demonstrated that NO plays different roles at different steps of the FGF2-induced angiogenesis. They showed that NO terminates FGF2-induced cell proliferation while promoting formation of capillary-like tube structures in both human umbilical core vein and calf pulmonary artery endothelial cells. In keeping with the role of NO as a critical downstream signal for FGF2- and VEGF-induced angiogenesis [9, 10, 14], our current data, however, show that endogenous NO participates in both FGF2- and VEGF-stimulated OFPAE cell proliferation. Thus, NO plays a different role at different steps in the FGF2-and/or VEGF-stimulated angiogenesis (i.e., proliferation/migration vs. formation of capillary-like tube structures), which possibly depends on the origin (vascular bed) or developmental stage of the endothelial cells studied.

The VEGF can induce endothelial cell proliferation and migration via activation of the NO/cGMP/MAPK3/1 [9, 10] or PI3K/NOS3/NO [20, 38] pathway, suggesting that NO can act as either an upstream or downstream signal in angiogenic factor-induced activation of protein kinases in endothelial cells. Our current data that the NOS inhibitor, L-NMMA, fails to affect the activation of MAPK3/1 or AKT1 induced by FGF2 and VEGF support the notion that NO lies downstream of MAPK3/1 and AKT1 activation [15, 16, 32, 33]. Alternatively, it is plausible, but highly unlikely, that NO functions in parallel of MAPK3/1 and AKT1 activation. Moreover, it is also possible that, in turn, FGF2- and VEGF-increased NO induces activation of these protein kinases, if the increased NO can reach relatively high levels comparable to those released from exogenous NO donors [8–10, 15, 16, 41].

Both FGF2- and VEGF-induced cellular responses are mediated via activation of multiple protein kinases, including MAPK3/1 and AKT1 [14, 17–19]. Our present study clearly demonstrates that the MAP2/K1/2/MAPK3/1 and PI3K/AKT1 pathways are critical in mediating both FGF2- and VEGF-stimulated OFPAE cell proliferation. Of note is that VEGF-stimulated OFPAE cell proliferation is much more sensitive to PD98059 and LY294002 than FGF2-stimulated cell proliferation. For example, the minimal concentration of PD98059 effectively inhibiting VEGF-stimulated cell proliferation is 4-fold (≤2.5 vs. 10 μM) lower than that inhibiting the FGF2-induced action. Moreover, PD98059 at 20 μM, a maximal concentration without affecting the cell number after 2 days of treatment in the absence of FGF2 and VEGF, inhibits FGF2-stimulated cell proliferation only by approximately 38% while reducing FGF2-induced phosphorylation of MAPK3/1 by approximately 85% and 68%. Similarly, LY294002 at 5 μM decreases FGF2-stimulated cell proliferation by approximately 45% while abolishing FGF2-induced AKT1 phosphorylation. These observations indicate that FGF2-stimulated cell proliferation is mediated partially via activation of the MAP2/K1/2/MAPK3/1 and PI3K/AKT1 pathways. However, it remains to be elucidated whether synergic activation of both pathways is required for the complete FGF2 induction of OFPAE cell proliferation because of cytotoxic effects of the combined PD98059 and LY294002 on OFPAE cells, as shown in Figure 4. In contrast to FGF2-induced actions, PD98059 at 20 μM and LY294002 at 5 μM block both VEGF-stimulated cell proliferation and phosphorylation of MAPK3/1 and AKT1, indicating that a complete VEGF-induced cell proliferation could be mediated via activation of either of these two pathways. Thus, in OFPAE cells, the MAP3K1/MAPK3/1 and PI3K/AKT1 pathways differentially mediate FGF2- and VEGF-stimulated cell proliferation.

The integration of the intracellular signaling pathways through complex cross-talks is poorly understood, particularly in the endothelium. Parallel activation of both MAPK3/1 and PI3K by FGF2 has been reported in bovine choriocapillary endothelial cells [18]. AKT1, however, upon its activation by MAPK3/1 or the upstream kinases of MAP2K1/2, could either inhibit [21–24] or synergistically enhance [25] the MAP2K1/2/MAPK3/1 pathway in certain nonendothelial cells. In this study, we found that PD98059 failed to alter either FGF2- or VEGF-induced phosphorylation levels of AKT1 in OFPAE cells. These data suggest that the MAP2K1/2/MAPK3/1 pathway does not mediate the PI3K/AKT1 pathway in OFPAE cells. However, we cannot exclude the possibility that the upstream kinases of MAP2K1/2 participate in such cross-talk. In contrast, LY294002 reduced VEGF-, but not FGF2-, induced phosphorylation levels of MAPK3/1. The inhibitory effect was modest but reached statistical significance. These data suggest that the activated PI3K/AKT1 pathway promotes VEGF-induced MAPK3/1 activation in OFPAE cells, but the underlying mechanism awaits further investigation.

In conclusion, we have demonstrated in the present study that increased intracellular NO and activation of multiple protein kinases are critical for both FGF2- and VEGF-stimulated fetoplacental endothelial cell proliferation. Our data suggest that interactions among FGF2, VEGF, and NO play an important role in the integral regulation of placental angiogenesis and vasodilatation. Thus, these data advance our understanding of the complex signaling mechanism controlling placental angiogenesis and vasodilatation. Our data may also provide clues for developing useful means for modulating placental vasculature functions and blood flows by altering the signaling pathways activated by the angiogenic factors.

Footnotes

1

Supported, in part, by NIH grants HL64703 (to J.Z.), HD38843 (to R.R.M. and J.Z.), HL49210 (to R.R.M.), and HL74947 and HL70562 (to D.-B.C.)

References

  • 1.Reynolds LP, Redmer DA. Utero-placental vascular development and placental function. J Anim Sci. 1995;73:1839–1851. doi: 10.2527/1995.7361839x. [DOI] [PubMed] [Google Scholar]
  • 2.Magness RR, Zheng J. Maternal cardiovascular alterations during pregnancy. In: Gluckman PD, Heymann MA, editors. Pediatric and Perinatal Perspectives: The Scientific Basic. London: Edward Arnold Publishers; 1996. pp. 762–772. [Google Scholar]
  • 3.Mayhew TM, Charnock-Jones DS, Kaufmann P. Aspects of human fetoplacental vasculogenesis and angiogenesis. III Changes in complicated pregnancies. Placenta. 2004;25:127–139. doi: 10.1016/j.placenta.2003.10.010. [DOI] [PubMed] [Google Scholar]
  • 4.Zheng J, Bird IM, Chen DB, Magness RR. Angiotensin II regulation of ovine fetoplacental artery endothelial functions: interactions with nitric oxide. J Physiol. 2005;565:59–69. doi: 10.1113/jphysiol.2004.082420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Sladek SM, Magness RR, Conrad KP. Nitric oxide and pregnancy. Am J Physiol. 1997;272:R441–463. doi: 10.1152/ajpregu.1997.272.2.R441. [DOI] [PubMed] [Google Scholar]
  • 6.Ziche M, Morbidelli L, Masini E, Granger HJ, Geppetti P, Ledda F. Nitric oxide promotes DNA synthesis and cycle GMP formation in endothelial cells from postcapillary venules. Biochem Biophys Res Commun. 1993;192:1198–1203. doi: 10.1006/bbrc.1993.1543. [DOI] [PubMed] [Google Scholar]
  • 7.Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, Ledda F. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest. 1994;94:2036–2044. doi: 10.1172/JCI117557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Zheng J, Wen YX, Austin JL, Chen DB. Exogenous nitric oxide stimulates cell proliferation via activation of a mitogen activated protein kinase pathway in ovine fetoplacental artery endothelial cells. Biol Reprod. 2006;74:375–382. doi: 10.1095/biolreprod.105.043190. [DOI] [PubMed] [Google Scholar]
  • 9.Ziche M, Morbidelli L, Mchoudhuri R, Zhang HT, Donnini S, Granger HJ, Maggi CA, Bicknell R. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997;99:2625–2634. doi: 10.1172/JCI119451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Parenti A, Morbidelli L, Cui XL, Douglas JG, Hood JD, Granger HJ, Ledda F, Ziche M. Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase 1/2 activation in postcapillary endothelium. J Biol Chem. 1998;273:4220–4226. doi: 10.1074/jbc.273.7.4220. [DOI] [PubMed] [Google Scholar]
  • 11.Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney M, Chen D, Chen D, Symes JF, Fishman MC, Huang PL, et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest. 1998;101:2567–2578. doi: 10.1172/JCI1560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Fukumura D, Gohongi T, Kadambi A, Izumi Y, Ang J, Yun CO, Buerk DG, Huang PL, Jain RK. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc Natl Acad Sci U S A. 2001;98:2604–2609. doi: 10.1073/pnas.041359198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Namba T, Koike H, Murakami K, Aoki M, Makino H, Hashiya N, Ogihara T, Kaneda Y, Kohno M, Morishita R. Angiogenesis induced by endothelial nitric oxide synthase gene through vascular endothelial growth factor expression in a rat hindlimb ischemia model. Circulation. 2003;108:2250–2257. doi: 10.1161/01.CIR.0000093190.53478.78. [DOI] [PubMed] [Google Scholar]
  • 14.Babaei S, Teichert-Kuliszewska K, Monge JC, Mohamed F, Bendeck MP, Stewart DJ. Role of nitric oxide in the angiogenic response in vitro to basic fibroblast growth factor. Circ Res. 1998;82:1007–1015. doi: 10.1161/01.res.82.9.1007. [DOI] [PubMed] [Google Scholar]
  • 15.Zheng J, Bird IM, Melsaether AN, Magness RR. Activation of the mitogen activated protein kinase cascade is necessary but not sufficient for basic fibroblast growth factor and epidermal growth factor stimulated expression of endothelial nitric oxide synthase in ovine fetoplacental artery endothelial cells. Endocrinology. 1999;140:1399–1407. doi: 10.1210/endo.140.3.6542. [DOI] [PubMed] [Google Scholar]
  • 16.Li Y, Zheng J, Bird IM, Magness RR. Mechanisms of shear stress-induced endothelial nitric-oxide synthase phosphorylation and expression in ovine fetoplacental artery endothelial cells. Biol Reprod. 2004;70:785–796. doi: 10.1095/biolreprod.103.022293. [DOI] [PubMed] [Google Scholar]
  • 17.Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, Ferrara N. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem. 1998;273:30336–30343. doi: 10.1074/jbc.273.46.30336. [DOI] [PubMed] [Google Scholar]
  • 18.Zubilewicz A, Hecquet C, Jeanny JC, Soubrane G, Courtois Y, Mascarelli F. Two distinct signalling pathways are involved in FGF2-stimulated proliferation of choriocapillary endothelial cells: a comparative study with VEGF. Oncogene. 2001;20:1403–1413. doi: 10.1038/sj.onc.1204231. [DOI] [PubMed] [Google Scholar]
  • 19.Eriksson K, Magnusson P, Dixelius J, Claesson-Welsh L, Cross MJ. Angiostatin and endostatin inhibit endothelial cell migration in response to FGF and VEGF without interfering with specific intracellular signal transduction pathways. FEBS Lett. 2003;536:19–24. doi: 10.1016/s0014-5793(03)00003-6. [DOI] [PubMed] [Google Scholar]
  • 20.Souttou B, Raulais D, Vigny M. Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways. J Cell Physiol. 2001;187:59–64. doi: 10.1002/1097-4652(2001)9999:9999<00::AID-JCP1051>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
  • 21.Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J. 2000;351:289–305. [PMC free article] [PubMed] [Google Scholar]
  • 22.Kolch W. Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat Rev Mol Cell Biol. 2005;6:827–837. doi: 10.1038/nrm1743. [DOI] [PubMed] [Google Scholar]
  • 23.Reusch HP, Zimmermann S, Schaefer M, Paul M, Moelling K. Regulation of Raf by Akt controls growth and differentiation in vascular smooth muscle cells. J Biol Chem. 2001;276:33630–33637. doi: 10.1074/jbc.M105322200. [DOI] [PubMed] [Google Scholar]
  • 24.Moelling K, Schad K, Bosse M, Zimmermann S, Schweneker M. Regulation of Raf-Akt cross-talk. J Biol Chem. 2002;277:31099–31106. doi: 10.1074/jbc.M111974200. [DOI] [PubMed] [Google Scholar]
  • 25.Sheng H, Shao J, DuBois RN. Akt/PKB activity is required for Ha-Ras-mediated transformation of intestinal epithelial cells. J Biol Chem. 2001;276:14498–14504. doi: 10.1074/jbc.M010093200. [DOI] [PubMed] [Google Scholar]
  • 26.Yashima R, Abe M, Tanaka K, Ueno H, Shitara K, Takenoshita S, Sato Y. Heterogeneity of the signal transduction pathways for VEGF-induced MAPKs activation in human vascular endothelial cells. J Cell Physiol. 2001;188:201–210. doi: 10.1002/jcp.1107. [DOI] [PubMed] [Google Scholar]
  • 27.Zheng J, Li Y, Weiss AR, Bird IM, Magness RR. Expression of endothelial and inducible nitric oxide synthase and nitric oxide production in the ovine placental and uterine tissues during late pregnancy. Placenta. 2000;21:516–524. doi: 10.1053/plac.1999.0504. [DOI] [PubMed] [Google Scholar]
  • 28.Zheng J, Wen YX, Chen DB, Bird IM, Magness RR. Angiotensin II elevates nitric oxide synthase 3 expression and nitric oxide production via a mitogen-activated protein kinase cascade in ovine fetoplacental artery endothelial cells. Biol Reprod. 2005;72:1421–1428. doi: 10.1095/biolreprod.104.039172. [DOI] [PubMed] [Google Scholar]
  • 29.Kwon H, Wu G, Meininger CJ, Bazer FW, Spencer TE. Developmental changes in nitric oxide synthesis in the ovine placenta. Biol Reprod. 2004;70:679–686. doi: 10.1095/biolreprod.103.023184. [DOI] [PubMed] [Google Scholar]
  • 30.Zheng J, Vagnoni KE, Bird IM, Magness RR. Expression of basic fibroblast growth factor, endothelial mitogenic activity, and angiotensin II type-1 receptors in the ovine placenta during the third trimester of pregnancy. Biol Reprod. 1997;56:1189–1197. doi: 10.1095/biolreprod56.5.1189. [DOI] [PubMed] [Google Scholar]
  • 31.Reynolds LP, Borowicz PP, Vonnahme KA, Johnson ML, Grazul-Bilska AT, Wallace JM, Caton JS, Redmer DA. Animal models of placental angiogenesis. Placenta. 2005;26:689–708. doi: 10.1016/j.placenta.2004.11.010. [DOI] [PubMed] [Google Scholar]
  • 32.Michell BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, Bozinovski S, de Montellano PO, Kemp BE, Pearson RB. The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol. 1999;9:845–848. doi: 10.1016/s0960-9822(99)80371-6. [DOI] [PubMed] [Google Scholar]
  • 33.Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999;399:597–601. doi: 10.1038/21218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999;399:601–605. doi: 10.1038/21224. [DOI] [PubMed] [Google Scholar]
  • 35.Kadota O, Ohta S, Kumon Y, Sakaki S, Matsuda S, Sakanaka M. Role of basic fibroblast growth factor in the regulation of rat basilar artery tone in vivo. Neurosci Lett. 1995;199:99–102. doi: 10.1016/0304-3940(95)12040-b. [DOI] [PubMed] [Google Scholar]
  • 36.Cuevas P, Carceller F, Ortega S, Zazo M, Nieto I, Gimenez-Gallego G. Hypotensive activity of fibroblast growth factor. Science. 1991;254:1808–1810. doi: 10.1126/science.1957172. [DOI] [PubMed] [Google Scholar]
  • 37.Kostyk SK, Kourembanas EL, Medeiros WD, McQuillan LP, D’Amore PA, Braunhut SJ. Basic fibroblast growth factor increases nitric synthase production in bovine endothelial cells. Am J Physiol. 1995;269:H1583–1589. doi: 10.1152/ajpheart.1995.269.5.H1583. [DOI] [PubMed] [Google Scholar]
  • 38.Morales-Ruiz M, Fulton D, Sowa G, Languino LR, Fujio Y, Walsh K, Sessa WC. Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt. Circ Res. 2000;86:892–896. doi: 10.1161/01.res.86.8.892. [DOI] [PubMed] [Google Scholar]
  • 39.Rousseau S, Houle F, Landry J, Huot J. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene. 1997;15:2169–2177. doi: 10.1038/sj.onc.1201380. [DOI] [PubMed] [Google Scholar]
  • 40.Rousseau S, Houle F, Kotanides H, Witte L, Waltenberger J, Landry J, Huot J. Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase 2 (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase. J Biol Chem. 2000;275:10661–10672. doi: 10.1074/jbc.275.14.10661. [DOI] [PubMed] [Google Scholar]
  • 41.Kawasaki K, Smith RS, Jr, Hsieh CM, Sun J, Chao J, Liao JK. Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis. Mol Cell Biol. 2003;23:5726–5737. doi: 10.1128/MCB.23.16.5726-5737.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES