Role of Nitric Oxide in the Angiogenic Response In Vitro to Basic Fibroblast Growth Factor

From the Terrence Donnelly Heart Centre, Division of Cardiology, St. Michael's Hospital, Department of Medicine, University of Toronto, Ontario, Canada.

Correspondence to D.J. Stewart, Director, Division of Cardiology, University of Toronto, and Head, Division of Cardiology, St. Michael's Hospital, 30 Bond St, Toronto, Ontario, M5B 1W8, Canada. E-mail stewartd{at}smh.toronto.on.ca

	Abstract

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Abstract—Angiogenesis is a complex process that involves the activation of quiescent endothelial cells (ECs) to a proliferative and migratory phenotype and, subsequently, their redifferentiation to form vascular tubes. We hypothesized that NO contributes to angiogenesis by terminating the proliferative action of angiogenic growth factors and initiating a genetic program of EC differentiation. Human umbilical vein ECs (HUVECs) and calf pulmonary artery ECs (CPAECs) were grown directly on plastic dishes or on three-dimensional fibrin matrices. In the absence of fibrin, treatment with NO-donor compounds, such as S-nitroso-N-acetylpenicillamine (SNAP, 0.1 and 0.4 mmol/L), produced a dose-dependent inhibition of proliferation in both cell lines, whereas the inhibition of endogenous NO production using N^G-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L) or N^G-monomethyl-L-arginine (L-NMMA, 1 mmol/L) significantly increased proliferation of the CPAECs. The addition of basic fibroblast growth factor (bFGF, 30 ng/mL) increased the expression of endothelial NO synthase mRNA and the production of NO in both cell types when cultured on three-dimensional fibrin gels and produced profound morphological changes characterized by the appearance of extensive capillary-like vascular structures and the loss of EC monolayers. These changes were quantified by measuring total tube length per low-power field (x100), and a differentiation index was derived using the ratio of tube length over area covered by residual EC monolayer. In the absence of additional angiogenic factors, the differentiation index was low for both HUVECs and CPAECs (control, 1.16±0.19 and 2.07±0.87, respectively). Treatment with bFGF increased the differentiation index significantly in both cell types (10.59±2.03 and 20.02±5.01 for HUVECs and CPAECs, respectively; P<.05 versus control), and the addition of SNAP (0.4 mmol/L) mimicked the angiogenic response to bFGF (8.57±1.34 and 12.20±3.49 for HUVECs and CPAECs, respectively; P<.05 versus control). Moreover, L-NAME inhibited EC tube formation in response to bFGF in a dose-response manner, consistent with a role of endogenous NO production in EC differentiation in this angiogenic model. These findings suggest that NO may act as a crucial signal in the angiogenic response to bFGF, terminating the proliferative actions of angiogenic growth factors and promoting EC differentiation into vascular tubes.

Key Words: nitric oxide • angiogenesis • fibrin gel • proliferation • differentiation

	Introduction

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Angiogenesis, the formation of new capillaries from preexisting vascular structures,¹ is of paramount importance in the maintenance of vascular integrity both in the repair of tissue damage in wound healing and in the formation of collateral vessels in response to ischemia.^{1 2 3} Angiogenesis is a complex process that is orchestrated by a multitude of cytokines and growth factors.² In its broadest sense, angiogenesis cannot be viewed as a single process; it involves a spectrum of cellular events, beginning with stimulation of quiescent nondividing ECs to a state of rapid proliferation and migration and terminating with the redifferentiation of activated ECs into vascular tubes.^{2 4 5 6} Thus, it is likely that different mediators are involved in different phases of angiogenesis. Inflammatory cytokines such as tumor necrosis factor-² and, more recently, soluble E-selectin⁶ promote EC migration and matrix dissolution, which are essential for the initiation of capillary tube formation. During later stages of angiogenesis, various angiogenic growth factors such as VEGF and bFGF promote growth and may mediate the organization of ECs into complex vascular tubes.^{1 2 3 4 7}

It is now recognized that in addition to its many other functions, the endothelium produces a number of potent vasoactive factors that play a crucial role in regulating vascular tone and growth. One of the most important endothelial vasoactive factors is the free radical gas NO,⁸ which is produced from the amino acid L-arginine by the action of NOS.⁹ Three isoforms of NOS have been identified: nNOS, iNOS, and eNOS.¹⁰ Both nNOS and eNOS are constitutively expressed, and their activity is tightly regulated by calcium and calmodulin,^{10 11} whereas iNOS is expressed in response to stimulation by cytokines.¹¹ NO not only is a potent vasodilator and antiplatelet factor¹² but also inhibits proliferation and migration of smooth muscle cells and fibroblasts¹³ ; thus, it is thought that NO plays a crucial role in the maintenance of vascular homeostasis.

Angiogenesis is predominantly an EC process and can be initiated in vivo by factors that have nearly complete selectivity of action for ECs, such as VEGF.⁷ Moreover, "angiogenesis" can be demonstrated in vitro in EC monoculture.¹⁴ Thus, the endothelium is both necessary and sufficient for new capillary vessel formation. Not surprisingly, it has been suggested that endothelial factors, such as NO, may play a key role in angiogenesis, although reports to date are conflicting, showing both angiogenic¹⁵ and antiangiogenic activity.¹⁶ We hypothesized that NO might contribute to EC differentiation in response to angiogenic growth factors, possibly by inhibiting proliferation of activated ECs and promoting the formation of vascular tubes. The aim of the present study was to determine the importance of NO in the well-characterized three-dimensional fibrin gel model of in vitro angiogenesis.

	Materials and Methods

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EC Culture
HUVECs and CPAECs were obtained from the American Type Culture Collection or isolated from fresh umbilical veins by 0.2% collagenase digestion.¹⁷ HUVECs were grown to confluence in Ham's/F-12 medium (GIBCO) and supplemented with 15% FBS, penicillin (500 U/mL), streptomycin (50 µg/mL), and heparin (100 µg/mL) (all from GIBCO) and EC growth factor (20 µg/mL, Boehringer Mannheim) equilibrated with 95% air/5% CO₂ at 37°C. CPAECs were grown in DMEM supplemented with 10% FBS and antibiotics as described above. Confluent EC cultures between the 5th and 18th passages were washed with HBSS, harvested using 0.05% trypsin/0.53 mmol/L EDTA, and counted using a hemocytometer. ECs were resuspended in F-12 medium or DMEM and plated on six-well dishes, either directly on plastic or on dishes coated with fibrin matrices. ECs were incubated for 48 hours in the presence or absence of the following agents singly or in combination: bFGF (30 ng/mL, Boehringer Mannheim), L-NAME (0.3 to 3.0 mmol/L), its enantiomer D-NAME (1 mmol/L), L-NMMA (1 mmol/L), SNAP (0.1 and 0.4 mmol/L), SNP (0.1 to 10 µmol/L), GSNO (1 to 100 µmol/L), and SIN-1 (0.1 to 10 µmol/L, Sigma Chemical Co).

Preparation of Fibrin Gels
Endotoxin- and plasminogen-free human and bovine fibrinogen (10 and 5 mg/mL, respectively; Calbiochem NOVAbiochem Corp) was dissolved in serum-free medium and filtered through 0.2-µm filters (Millex GS, Millipore). Fibrin matrices were prepared by polymerizing the fibrinogen solution using a low concentration of -thrombin (2.5 U/mL, Sigma). After polymerization, gels were soaked in culture medium containing 10% FBS for 2 hours at 37°C to inactivate the thrombin. Where appropriate, bFGF was added to both the fibrinogen solution before polymerization as well to the medium bathing the cells. ECs were plated on the surface of the three-dimensional matrix and cultured for periods up to 48 hours, in the presence or absence of study agents as described above.

RNA Extraction and Northern Blot Analysis
Total cellular RNA was isolated using the method of Chomczynski and Sacchi.¹⁸ Total RNA (20 µg) from each sample was treated with 2.2 mol/L formaldehyde in 50% formamide and 200 mmol/L HEPES, pH 7, at 65°C for 15 minutes, and RNA was separated by electrophoresis in 1.2% agarose/2.2 mol/L formaldehyde gels in MOPS buffer containing 20 mmol/L 3-(N-morpho)sulfonic acid, 8 mmol/L sodium acetate, 1 mmol/L EDTA, and 5 mmol/L NaOH and transferred by capillary blotting to Gene Screen Plus nylon membranes (NEN Research Products) following the method suggested by the manufacturer. The membrane was optimally cross-linked with UV light (1200 J, UVXL-1000, Fisher Scientific) or baked at +80°C in a vacuum oven. Membranes were hybridized for 24 hours at 42°C with specific cDNA probes radiolabeled with [-³²P]CTP (Amersham) by the random primer technique to a specific activity of at least 1x10⁹ cpm/µg in hybridization buffer containing 50% formamide, 5x SSC (750 mmol/L NaCl and 0.03 mol/L sodium citrate), 10% dextran sulfate, 1% SDS, and 100 µg/mL denatured salmon sperm DNA. After hybridization, the filters were washed under conditions of high stringency in 2x SSC (0.3 mol/L NaCl and 0.03 mol/L sodium citrate 2H₂O, pH 7.0) containing l % SDS according to the manufacturer's instructions. Autoradiography was performed using double intensifying screens (Cronex) and X-OMAT AR film (Eastman Kodak Co). Signal intensity was quantified as integrated areas using scanning densitometry. Densitometric data were standardized against the level of GAPDH mRNA expression in each sample. The cDNA probe for eNOS was produced as described previously.¹⁹ GAPDH cDNA, a constitutively expressed gene, was obtained from the American Type Culture Collection (No. 57091), and a 0.78-kb PstI-XhoI fragment was used as a cDNA probe.

Reverse-Transcription Polymerase Chain Reaction
Total RNA (2 µg) was reverse-transcribed, as previously reported,²⁰ in 20 µL of reaction volume containing 250 ng of random primers, 0.5 mmol/L of each dNTP, 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl₂, 10 mmol/L dithiothreitol, and 200 U Moloney murine leukemia virus reverse transcriptase (GIBCO/BRL). After 1 hour of incubation at 37°C, samples were heated to 95°C for 5 minutes and then chilled on ice. Thermal cycling of 5 µL of the cDNA from the reverse transcriptase mix was performed using specific primers for bFGF: sense, 5'-GCCTTCCCGCCCGGCCACTTCAAGG-3'; antisense, 5'-GCACACACTCCTTTGATAGACACAA-3'. Coamplification of the same cDNA was performed for GAPDH as an internal standard with the following primers: sense, 5'-GGTGAAGGTCGGAGTCAACGGATTTGG-3'; antisense, 5'-GGCCATGAGGTCCACCACCCTGTT-3'. Primers were obtained from the Pharmacia Biotechnology Service Center at the Hospital for Sick Children (Toronto, Canada). Primer (1 µmol/L of each) was added to the reaction mixture containing 50 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl₂, 50 mmol/L KCl, 0.2 mmol/L of each dNTP, and 2.5 U of Taq polymerase (Boehringer Mannheim) in a final volume of 50 µL. Amplification was performed in a thermocycler using the following parameters: for bFGF, 94°C for 1 minute, 63°C for 1 minute, and 72°C for 1 minute (35 cycles); for GAPDH, 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute (35 cycles). The resultant products were separated on a 1.5% agarose gel, together with a 100-bp DNA ladder, and bands were visualized with ethidium bromide. The sizes of the amplification products were 179 and 983 nt for bFGF and GAPDH, respectively, and the intensity of the bands were measured by densitometry.

Quantification of EC Proliferation
Cell replication was determined by quantifying the incorporation of [methyl-³H]thymidine into trichloroacetic acid–insoluble macromolecules as described previously.^{21 22} ECs were seeded at a density 1 to 2x10⁴ cells per well in 24-well dishes. After cell attachment, bFGF and other study agents were added to the medium, and the cells were incubated for 24 hours. After a preincubation period of 18 hours, a tracer amount of [³H]thymidine (1 µCi per well; specific activity, 1.85 TBq/mmol; Amersham) was added, and the cells were incubated for a 6-hour period. Subsequently, the cells were washed three times with PBS and fixed with ice-cold 10% (wt/vol) trichloroacetic acid for 20 minutes. The resulting precipitate was washed with ice-cold 10% trichloroacetic acid and 95% ethanol and solubilized by mixing with 200 µL of 0.3N NaOH at room temperature for 20 minutes, followed by neutralization with the same volume of 0.3N HCl. ³H radioactivity was measured in a liquid scintillation counter (Liquid Scintillation System, Beckman Instruments Inc).

In addition, CPAECs were seeded as above on culture slides and cultured for 6 hours before being pulse-labeled with 50 µmol/L of BrdU for 1 hour, washed extensively, and then fixed with 4% paraformaldehyde. The slides were then blocked with 0.3% H₂O₂ and permeabilized with 2N HCl at 37°C for 15 minutes. Anti-BrdU monoclonal antibody (mouse IgG, DAKO) at 1:40 dilution in 1% normal horse serum and 1% BSA/PBS was applied to each slide for 60 minutes at 37°C. BrdU staining was detected with a biotinylated horse anti-mouse IgG (dilution, 1:250; Vector Laboratories). Finally, the slides were incubated with avidin-biotin-peroxidase complex (1:100 dilution, Vector Laboratories) in PBS for 45 minutes, followed by incubation with diaminobenzidine in 0.03% H₂O₂ and 50 mmol/L Tris buffer (pH 7.2). The slides were then counterstained with hematoxylin, and the total number of cell nuclei staining positive for BrdU was counted under each condition and expressed as a percentage of total number of nuclei. Cytotoxic effects of NO-donor compounds were determined by trypan blue exclusion in cell suspension after 48 hours of incubation.

Quantification of Endothelial Differentiation
Culture plates containing HUVECs and CPAECs were assessed after 48 hours of incubation under study conditions using an Olympus BX50 inverted microscope (x100). Images were digitized using a Sony CCD-IRIS/RGB camera (Cohu Inc) and analyzed using a computer-assisted morphometric analysis system (C Imaging, Compix Inc) by observers blinded with respect to the experimental conditions. Tubelike structures (30 µm) were identified, and total tube length was derived for each of four randomly chosen fields. At the same time, the total area of the culture surface covered by ECs was determined in the same fields. The differentiation index was calculated as the ratio of the total tube length over cell area for each field, and a mean differentiation index value was obtained for each culture well.

Nitrite Assay
Nitrite, a stable end product of NO, was measured in the culture medium using the colorimetric Griess assay. An aliquot of phenol red–free medium (80 µL) from each culture well was mixed with 20 µL of nitrate reductase for conversion of nitrate to nitrite, followed by 100 µL of the Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine dihydrochloride in 2% phosphoric acid) (Alexis Corp). The mixture was incubated for 10 minutes at room temperature to allow the color to develop, and the absorbance at 540 nm was measured in a microplate reader (Vmax 250, Molecular Devices). Concentrations were determined by comparison with a sodium nitrite standard curve. Results were expressed per 10⁴ cells.

Statistical Analysis
Statistical analysis was performed using SYS-STAT software. Significance of differences between groups was performed using Student t test or a two-tailed Wilcoxon signed rank test and a one-way ANOVA for dose-response experiments as appropriate. Values are presented as mean±SD. Differences were considered statistically significant at P<0.05.

	Results

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The effect of bFGF on EC morphology was dependent on the conditions of cell culture, as shown in Figure 1. In the absence of bFGF (panels a and c), cultured CPAECs exhibited a classical cobblestone appearance regardless of whether they were cultured directly on plastic or on fibrin matrices for 48 hours. The addition of bFGF had little effect on morphology of ECs grown on plastic (panel b), whereas this growth factor induced profound differentiation of ECs grown on fibrin (panel d), with the appearance of capillary-like tubes and loss of area covered by EC monolayers. Similar results were obtained with HUVECs (data not shown).

View larger version (156K): [in this window] [in a new window]   Figure 1. CPAECs cultured for 48 hours directly on plastic dishes in the absence (a) or presence (b) of bFGF formed a typical cobblestone monolayer. A similar appearance was seen when ECs were plated on a fibrin matrix in the absence of the angiogenic factor (c), whereas the addition of bFGF to ECs on three-dimensional fibrin gels (d) produced dramatic changes in morphology characterized by the appearance of abundant tubelike endothelial structures. Original magnification x100.

Figure 2A shows representative Northern blots demonstrating the changes in endothelial gene expression in response to bFGF in both HUVECs and CPAECs grown on plastic or on fibrin matrices. Under nonangiogenic conditions (ie, culture on plastic), bFGF had little effect on eNOS mRNA expression (lanes 1 and 2). ECs cultured on fibrin alone demonstrated a consistent decrease in the basal expression of eNOS (lane 3); however, under conditions that favored vascular tube formation, addition of bFGF resulted in a significant increase in the expression of eNOS in both cell types (lane 4). These experiments were repeated three times with similar results. Thrombin at the concentration used to polymerize fibrinogen (2.5 U/mL) had no direct effect on eNOS mRNA expression (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 2. Alterations in endothelial gene expression were evaluated by Northern analysis. A, Representative blots for eNOS (4.2 kb) compared with GAPDH (1.7 kb) to correct for differences in RNA loading. Each lane was loaded with 20 µg of total RNA from HUVECs or CPAECs, grown on plastic or fibrin (ie, nonangiogenic and angiogenic conditions, respectively) in the presence or absence of bFGF (30 ng/mL) for 48 hours as indicated. B, Relative density of bands from ECs on fibrin (lanes 3 and 4, highlighted in the box) normalized for GAPDH and expressed as a ratio of the density of bFGF vs control for HUVECs (shaded bars) and CPAECs (solid bars) (mean±SD for three experiments).

The effect of bFGF on NO production by HUVECs and CPAECs is shown in Figure 3. Addition of bFGF to ECs on plastic had no significant effect on the accumulation of the stable degradation products, nitrite and nitrate. However, under angiogenic conditions (ie, ECs grown on fibrin matrices), NO production was significantly increased by bFGF in both cell types. Addition of L-NAME (1 mmol/L) to the culture medium significantly attenuated nitrite accumulation in response to bFGF.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effects of bFGF on NO release from HUVECs and CPAECs. Cells were incubated for 48 hours on plastic or fibrin matrices with phenol-free medium in the presence or absence of bFGF (30 ng/mL) and L-NAME (1 mmol/L). After the incubation, medium was collected and stored at -20°C until assay. Nitrite concentration in the medium was determined as described in "Materials and Methods." Values are presented as mean±SD of three different experiments.

The effect of various manipulations of NO production on rates of EC proliferation is shown in Figure 4. Under control conditions in the presence of FBS, both CPAECs and HUVECs demonstrated relatively high basal levels of thymidine incorporation, which were further increased by the addition of bFGF (Figure 4A). Inhibition of endogenous NO production using L-NAME also caused a slight increase in proliferation rates, which achieved statistical significance for CPAECs. Identical results were obtained for L-NMMA (data not shown). In contrast, the pharmacological generation of NO using SNAP produced a dose-dependent inhibition of proliferation in both cell lines. Identical results were observed in CPAECs using BrdU nuclear staining as a marker of cells engaged in the DNA synthesis (Figure 4B). A direct cytotoxic effect of the NO-donor compounds on EC viability was ruled out by in vitro experiments showing no difference in trypan blue exclusion between treated and control cells after 48 hours of incubation at the concentrations shown in Table 2 and Figure 6.

View larger version (56K): [in this window] [in a new window]   Figure 4. A, The effect of bFGF and various manipulations of the NO pathway on [3H]thymidine incorporation in ECs cultured on plastic dishes was determined in CPAECs (shaded bars, left ordinate) and HUVECs (solid bars, right ordinate). Cells were cultured on plastic in the presence or absence of bFGF (30 ng/mL), L-NAME (1 mmol/L), and SNAP (0.1 and 0.4 mmol/L) for 24 hours. B, BrdU labeling is shown for CPAECs cultured for 48 hours in the presence or absence of test agents as described in panel A.

View larger version (50K): [in this window] [in a new window]   Figure 6. The effect of bFGF and various manipulations of the NO pathway on the differentiation of ECs grown on fibrin gels, HUVECs (A), and CPAECs (B). EC differentiation was quantified by both the total length of tubelike structures per low-power field (LPF) (solid bars, ordinates on right) and the differentiation index (shaded bars, ordinates on left), as described in "Materials and Methods." Bars represent mean±SD for three separate experiments.

Figure 5 shows representative fields from CPAECs (panels a through d) and HUVECs (panels e through h) depicting the effect of altering the production of NO on EC differentiation in the fibrin matrix model using a similar pharmacological approach. Compared with cells cultured under control conditions (panels a and e), the addition of bFGF resulted in the extensive formation of capillary-like structures (panels b and f). In the absence of bFGF, the exposure of cells to the NO-donor compound, SNAP, resulted in identical morphological changes (panels c and g), whereas the inhibition of endogenous NO production by L-NAME (1 mmol/L) (panels d and h) partially prevented EC differentiation in response to bFGF in both cell types. Summary data for three experiments are shown in Figure 6. The differentiation index and tube length were very low for both HUVECs (Figure 6A) and CPAECs (Figure 6B) grown on fibrin under control conditions. Addition of bFGF produced significant increases in differentiation index and tube length in both cell types, although the magnitude of differentiation was greater for bovine than for human ECs. Inhibition of endogenous NO production using L-NAME significantly reduced differentiation in response to bFGF. The inhibition of EC differentiation in response to bFGF by L-NAME was dose dependent (Table 1) and nearly complete at the higher concentrations of the inhibitors. In contrast, D-NAME (1 mmol/L) had no effect on the angiogenic response to bFGF, suggesting that the effects of L-NAME were related to the inhibition of NO production. Similar results were obtained using another inhibitor of NO synthase, L-NMMA (1 mmol/L) (Table 1). Addition of exogenous NO (ie, SNAP [Figure 6] or SNP, SIN-1, and GSNO [Table 2]) produced a dose-dependent increase in indices of EC differentiation to levels not different from those observed in response to bFGF.

View larger version (138K): [in this window] [in a new window]   Figure 5. Photomicrographs of CPAECs (a through d) and HUVECs (e through h) cultured on fibrin gels for 48 hours in the absence of angiogenic factors (a and e), in the presence of bFGF (30 ng/mL, b and f) or SNAP (0.4 mmol/L, c and g), or with the combination of bFGF (30 ng/mL) and L-NAME (1 mmol/L) (d and h). Original magnification x100.

The effect of the angiogenic mediators on expression of bFGF mRNA was studied in HUVECs by RT-PCR. Bands of the expected size were obtained for bFGF (179 nt) and GAPDH (983 nt) as shown in Figure 7. The density of the bands normalized for GAPDH was not significantly altered relative to control by exposure to bFGF (30 ng/mL) and SNAP (0.4 mmol/L) or the combination. This experiment was repeated twice with an identical result.

View larger version (48K): [in this window] [in a new window]   Figure 7. The effects of exogenous NO production on bFGF expression was investigated by RT-PCR. HUVECs were exposed to SNAP (0.4 mmol/L) or bFGF (30 ng/mL) singly or in combination for 48 hours under angiogenic conditions (ie, on fibrin matrices), and bFGF mRNA expression was quantified by RT-PCR analysis.

	Discussion

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In the present study, bFGF produced differentiation of ECs cultured on three-dimensional fibrin matrices into tube-like structures while increasing the expression of eNOS mRNA and the production of NO. The pharmacological generation of NO inhibited the proliferation of ECs on plastic and promoted differentiation of ECs in the fibrin matrix model. Moreover, inhibition of endogenous NO synthesis significantly reduced EC differentiation in response to bFGF in this model. These results are compatible with a central role of endogenous NO in angiogenesis.

Angiogenesis is a highly complex process that involves at least two distinct phases.^{1 4} The initial phase is characterized by activation of quiescent ECs to a proliferative and migratory phenotype.^{1 5 6} Among the mediators of this transition are inflammatory cytokines such as tumor necrosis factor- and interleukin 8²³ and, possibly, soluble adhesion molecules such as E-selectin.⁶ Growth factors such as bFGF and VEGF are also potent stimulators of EC proliferation and migration^{1 2 3} and likely contribute to EC activation in the initial stages of the angiogenic response. A later phase of new vessel formation involves the redifferentiation of the migrating and proliferating ECs into vascular tubes. This necessitates a reversal of EC phenotype, back to a more quiescent state typical of mature vascular structures.¹ It is perhaps somewhat of an enigma that the same mediator, ie, bFGF, could be involved in distinct phases of the angiogenic process, which appear to require opposite actions. The present data suggest that NO, produced in response to bFGF, may act as a molecular "switch," counteracting the growth-promoting actions of this angiogenic mediator and initiating a program of EC differentiation. This is in agreement with very similar observations in a neuroblastoma cell line²⁴ in which NO was reported to mediate termination of the proliferative response to nerve growth factor.

In the present study, we did not see changes in eNOS mRNA expression in response to bFGF in ECs grown on plastic (Figure 2), although another group has reported that bFGF increased eNOS expression in these conditions.²⁵ In contrast, in the angiogenic conditions with ECs cultured on a fibrin matrix, bFGF did increase the expression of eNOS and the production of NO in differentiating ECs. Under these conditions, inhibition of NOS activity prevented tube formation, confirming the importance of increased endogenous NO production in mediating the angiogenic response to bFGF. Interestingly, the fibrin matrix alone (ie, in the absence of bFGF) reduced basal eNOS expression. This is in keeping with a further "activation" of ECs grown on the fibrin matrix, which may be an important element of this in vitro model, possibly mediated by matrix–adhesion molecule interactions. Indeed, specific interaction of endothelial _v/ß₃ integrin with fibrin may be critical for EC differentiation in this model.²⁶ We ruled out the possibility that the very low concentrations of thrombin used to polymerize the fibrinogen solution had direct effects on eNOS expression, since in separate experiments, thrombin at these concentrations had no effect of endothelial factor gene expression. Although the highest quality fibrinogen was used in the present experiments, it is possible that there was low-level contamination of the fibrinogen solution with cytokines. We have previously demonstrated that tumor necrosis factor-, even at concentrations as low as 10 U/mL, could profoundly downregulate expression of this gene¹⁹ by a posttranscriptional mechanism involving the destabilization of eNOS mRNA. Despite the lower levels of eNOS mRNA expression in cells cultured on fibrin matrices, there was little change in basal NO production, suggesting an increased level of NOS activity under these conditions.

Previous studies of the role of NO in the angiogenic response have provided conflicting results. Ziche et al¹⁵ have reported that NO mediated the angiogenic effects of substance P in the rat cornea. In another report, tumor cells transfected with inducible NOS grew more slowly in vitro but exhibited increased metastatic spread in vivo,²⁷ associated with evidence of increased tumor vascularity. However, it has also been reported that the NO-donor compound SNP inhibited angiogenesis in the chick chorioallantoic membrane^{16 28} and reduced vascular tube formation of HUVECs grown on Matrigel.¹⁶ One can only speculate about the reasons for these apparent discrepancies. Certainly, there may be important differences between the Matrigel and fibrin matrix models of angiogenesis. ECs grown on Matrigel form cords spontaneously; this occurrence is likely due to contamination of the tumor basement membrane components with cytokines and growth factors.^{14 29} In contrast, in the fibrin gel model used in the present study, exogenous angiogenic factors must be added for full EC differentiation to be apparent. Furthermore, in fibrin gels it has been shown that ECs form tubes with identifiable lumina,^{14 30} and this process requires new gene expression,¹⁴ which does not always appear to be the case in the Matrigel model.¹⁴

However, it is possible that the apparent disagreement may be related not only to differences in the various models of angiogenesis but also to differences in the actions of NO, depending on the preexisting conditions. Thus, the direct effect of NO in most cell systems is that of growth inhibition, as was observed in both HUVECs and CPAECs grown in the absence of the fibrin matrix. In the early phase of angiogenesis, which is characterized by EC activation, this effect of NO could reduce the initial proliferative response. However, during the later stages of the angiogenic process, the present data would suggest that increased production of NO may be a differentiating signal acting to terminate the proliferation of activated ECs and facilitate the initiation of a program of EC differentiation. Recently, Papapetropoulos et al³¹ have demonstrated that inhibitors of NOS largely prevented differentiation of ECs in response to another angiogenic factor, transforming growth factor-ß, in a collagen matrix model of angiogenesis in which EC proliferation was greatly inhibited. These findings are in agreement with those of the present study and suggest a general role of NO in the formation of vascular tubes in different angiogenic models and in response to different angiogenic factors.

Of particular interest are the observations of Ziche's group^{15 32 33} suggesting that rather than inhibiting EC proliferation, NO might actually mediate the mitogenic effects of VEGF on postcapillary coronary ECs. Again, this is in contradistinction to the present study, which found that NO inhibited EC proliferation in response in two different EC types, using two different methods to quantify mitogenic activity. It is possible that NO may have opposite effects in microvascular compared with macrovascular ECs. However, others have reported consistent growth inhibition by a variety of NO-donor compounds in both macrovascular^{34 35} and microvascular³⁴ EC lines. In coronary venular microvascular ECs, Ziche et al³⁶ have recently reported that the proliferative effects of NO are indirect, mediated by increased expression of bFGF. However, such a mechanism cannot account for the angiogenic effects of NO described in the present report, as there was no increase in bFGF expression induced by SNAP. Moreover, inhibitors of NOS reduced EC differentiation in response to exogenous bFGF, suggesting that in our system NO mediated the effects of this angiogenic growth factor rather than the other way around. The same group has also recently reported data suggesting that NO may mediate the angiogenic response to VEGF but not bFGF.³⁷ However, these results are not directly comparable to those of the present report. Ziche et al³⁷ studied proliferation and migration of coronary venular ECs cultured on plastic, which likely reflect events of the early stages of angiogenesis involving EC activation, whereas in the present study we quantified EC vascular tube formation in the three-dimensional fibrin gels, which is more representative of the later stages of the angiogenic response.

Taken together, our results are consistent with the conclusion that increased production of NO might act as a crucial molecular "signal" in the angiogenic response to bFGF, terminating EC proliferation and initiating the formation of vascular tubes in the fibrin matrix model of angiogenesis. The observations that capillary-like tube formation in our model can be largely prevented by inhibition of endothelial NOS activity and that exogenous NO from NO donor compounds reproduced the angiogenic effects on bFGF strongly support a central role of NO in the initiation of EC differentiation.

	Selected Abbreviations and Acronyms

 

bFGF = basic FGF BrdU = 5'-bromo-2'-deoxyuridine CPAEC = calf pulmonary artery EC EC = endothelial cell eNOS, iNOS, nNOS = endothelial, inducible, and neuronal NOS FGF = fibroblast growth factor GSNO = S-nitrosoglutathione HUVEC = human umbilical vein EC L-NAME = NG-nitro-L-arginine methyl ester L-NMMA = NG-monomethyl-L-arginine NOS = NO synthase RT-PCR = reverse-transcription polymerase chain reaction SIN-1 = 3-morpholinosydnonimine SNAP = S-nitroso-N-acetylpenicillamine SNP = sodium nitroprusside VEGF = vascular endothelial growth factor

	Acknowledgments

 
This study was supported by a grant from the Medical Research Council of Canada. Dr Stewart is the Dexter Man Chair of Cardiology, University of Toronto. Dr Bendeck is a Scholar of the Heart and Stroke Foundation of Canada.

Received December 17, 1997; accepted March 2, 1998.

	References

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