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Am J Physiol Heart Circ Physiol 280: H99-H107, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 1, H99-H107, January 2001

ANG II potentiates mitogenic effect of norepinephrine in vascular muscle cells: role of FGF-2

Astrid Parenti, Laura Brogelli, Sandra Donnini, Marina Ziche1, and Fabrizio Ledda

Department of Pharmacology, University of Florence, Florence 50139; and 1 Institute of Pharmacological Science, University of Siena, Siena 53100, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the possible cooperation between norepinephrine (NE) and ANG II on proliferation of cultured vascular smooth muscle cells (VSMCs) and the involved cellular mechanisms. Nanomolar NE concentrations stimulated VSMC proliferation through a prazosin-sensitive effect. The pretreatment of cells with 100 nM ANG II for 24 h significantly potentiated the NE-induced VSMC proliferation; this potentiating effect of ANG II was blocked by losartan but was unaffected by the AT2 receptor antagonist PD-123177. ANG II pretreatment also potentiated the increase in inositol phosphate turnover and upregulated the cell expression of fibroblast growth factor (FGF-2) induced by NE. Anti-FGF-2 neutralizing antibodies prevented the potentiating effect of ANG II on NE-induced cell growth. Both ANG II and NE stimulated extracellular signal-related kinase (ERK1) activation, but an ANG II potentiation of the effect of NE on ERK1 activity was not detectable. Moreover, ANG II significantly increased protein synthesis but did not potentiate the hypertrophic effect of NE. These findings demonstrate that ANG II and NE cooperate in promoting VSMC growth and that FGF-2 upregulation is involved in this effect.

smooth muscle cells; fibroblast growth factor-2; mitogen-activated protein kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR SMOOTH MUSCLE CELLS (VSMC) in adult blood vessels are maintained in a nonproliferative phenotype through the interaction between growth-stimulating and growth-inhibiting factors. This phenotype may be modulated toward cellular hyperplasia or hypertrophy in response to pathological stimuli. Among the variety of substances that are considered as possible VSMC mitogens, the potent vasoconstrictor agents ANG II and norepinephrine (NE) are recognized as able to promote the development of cardiac hypertrophy as well as increase vessel wall thickness (6, 10). In cultured VSMCs, both NE and phenylephrine induce mitogenic effects associated with stimulation of mitogen-activated protein kinase (MAPK) activity with accumulation of protooncogenes such as c-fos, c-jun, and c-myc (13, 35). Moreover, phenylephrine increases protein synthesis in VSMCs in vitro (28) and induces trophic effects on arterial smooth muscle in vivo (21). The alpha 1-adrenoceptor involved in the hypertrophic and growth-promoting effects of sympathomimetic amines has been characterized as belonging to the chloroethylclonidine-sensitive alpha 1B-subtype of adrenoceptors (28, 35). With regard to ANG II, many reports indicate that this peptide is provided with hypertrophic and hyperplastic effects on cultured VSMCs (1, 11) and that the mitogenic effect of ANG II depends, at least in part, on the induction of growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), and transforming growth factor-alpha (TGF-alpha ) (2, 14, 19). In vivo infusion of ANG II promotes DNA synthesis in VSMCs of the normal and balloon-injured arterial wall of the rat (32). The trophic effects induced by ANG II are attributable to stimulation of ANG II type-1 receptors (AT1) because they are inhibited by AT1 receptor antagonists, such as losartan (18, 29); conversely, ANG II type-2 receptors (AT2) induce inhibition of endothelial cell proliferation (29). It has been previously suggested that the growth-promoting effects of ANG II may also be due to an enhanced VSMCs response to sympathetic stimulation; in fact, alpha -adrenoceptor blockade by prazosin inhibits the increase in DNA synthesis induced by ANG II in VSMCs in vivo (32). Moreover, preincubation with ANG II induces transcription and expression of alpha 1-adrenoceptors and potentiates the trophic response to phenylephrine in cultured VSMCs in vitro (12).

The aim of the present study was to evaluate the possible cooperation between NE and ANG II on proliferation of rat aortic smooth muscle cells in culture. The mechanisms involved in this kind of cooperation were also investigated by studying the postreceptor signaling events activated by NE stimulation after ANG II pretreatment. Because there is accumulating evidence that the biological activity of ANG II is mediated by endogenous growth factors such as FGF-2 (14), we investigated whether autocrine expression of FGF-2 was involved in the cooperation between ANG II and NE in VSMCs. Finally, because MAPKs represent a transduction mechanism involved in the cellular response to growth factors and stress stimulation (25) and are possibly involved in the pathogenesis of hypertension (3), we also investigated MAPK-extracellular signal-regulated kinase (ERK1) activation. It has been shown that VSMCs isolated from spontaneously hypertensive rats (SHR) display a higher MAPK activity than that found in cells of normotensive rats (20, 34), and that MAPKs are activated after balloon injury through an effect that is mediated, at least in part, by AT1 receptors (16), thus suggesting an important role of this intracellular pathway in injured vascular tissue.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Cultures

We isolated VSMCs from the thoracic aorta of male Wistar rats weighing between 200 and 250 g. The vessels were aseptically excised and placed in DMEM. Adhering fat and connective tissue were removed by blunt dissection. The aorta was then opened longitudinally and preincubated with 0.1% collagenase for 30 min at 37°C in 95% O2-5% CO2. The adventitia was carefully removed, and the luminal surface was scraped with forceps to remove endothelial cells and then minced into 1-mm pieces and incubated again with 0.1% collagenase for 20 min. After centrifugation, the pellet was resuspended in medium containing 10% calf serum (CS). Cells were cultured in DMEM supplemented with 10% CS, 100 U/ml penicillin, and 100 µg/ml streptomycin, kept in a humidified incubator at 37°C in 5% CO2, and split two times a week 1:2 with the use of a trypsin-EDTA solution. Cells were characterized by immunohistochemical assay with a monoclonal anti-alpha -actin antibody (Sigma Chemical, St. Louis, MO): 90% of the cells expressed alpha -actin. Cells between passages 2 and 8 were used in these experiments.

Cell Proliferation

VSMC proliferation was quantified by the total cell number and by DNA synthesis. Experiments were performed using DMEM supplemented with 1% CS.

Evaluation of total cell number. Cells (5 × 103/700 µl) suspended in 5% CS medium were seeded in 48-multiwell plates and allowed to adhere overnight. Cells were kept in starving conditions in 0.1% CS for 48 h. The media were then removed and replaced with 1% CS medium containing the test substances. Proliferation was evaluated after 96 h of exposure to test substances. The effect of NE was compared with the control condition in 1% CS medium and to the effect produced by 10 ng/ml PDGF-BB or 10% CS. Receptor antagonists were added 30 min before adding the mitogenic substances. Pretreatment with ANG II was performed for 24 h, and NE was then added. Cells were fixed with methanol and stained with Diff-Quik (Merz, Allschwil, Switzerland). The number of cells was counted in 10 random fields of each well at ×200 with the aid of a 21-mm2 ocular grid.

Evaluation of DNA synthesis. Cells (104/1,000 µl) suspended in 5% CS medium were seeded in 24-multiwell plates and allowed to adhere overnight. The cells were then kept in starving conditions (0.1% CS) for 48 h. Media were removed and replaced with 1% CS medium containing test substances for 8 and 48 h and pulsed for 4 h with 0.5 µCi [3H]thymidine/well. DNA was precipitated with 5% trichloroacetic acid, and extracted with 0.3 M NaOH, and the recovered radioactivity was measured. Data were expressed as recovered counts per minute per well.

Protein Synthesis

VSMC hypertrophy was quantified by evaluation of protein synthesis and of total protein content per 105 cells. Experiments were performed using DMEM supplemented with 1% CS. Cells (104/1,000 µl) suspended in 5% CS medium were seeded in 24-multiwell plates and allowed to adhere overnight. The cells were then kept in starving conditions (0.1% CS) for 48 h. Media were removed and replaced with 1% CS medium containing test substances for 8 and 24 h. During the last 3 h of stimulation, cells were pulsed with 1 µCi [3H]leucine/well. Labeled proteins were precipitated with 10% trichloroacetic acid and extracted with 0.3 M NaOH/1% sodium dodecyl sulfate (SDS), and the recovered radioactivity was measured. Data were expressed as recovered counts per minute per well.

Total proteins. Cells (50 × 104/2,000 µl) suspended in 5% CS medium were seeded in 6-multiwell plates and allowed to adhere overnight. The cells were then kept in starving conditions (0.1% CS) for 48 h. Media were removed and replaced with 1% CS medium containing test substances for 4 days. At the end of incubation, cells were lysed in a buffer containing 50 mM Tris (pH 7.4), 1% Triton X-100, 1 mM EGTA, 100 mM NaCl, 1 mM Na3VO4, 200 mM phenylmethylsulfonyl fluoride (PMSF), 25 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 mM NaF. Protein contents were determined by bicinchoninic acid (BCA) assay according to the manufacturer's protocol (Pierce, Rockford, IL). Data were expressed as total proteins per 105 cells.

Inositol Phosphate Turnover

Inositol monophosphate [Ins(1)P] levels were measured as described previously (23). Briefly, we labeled VSMCs with myo-[3H]inositol (2 µCi/ml) in DMEM without cold inositol for 48 h. Then cells were incubated for 10 min with 20 mM LiCl to block inositol polyphosphate 1-phosphatase and inositol monophosphatase and then with test compounds for 15 min. Cell-associated inositols were extracted by chloroform-methanol (1:1). Water-soluble fractions were applied to anion-exchange columns (Resin AG-X8, 200-400 mesh, formiate form), and Ins(1)P levels were measured as recovered radioactivity.

ERK1 Assay

The MAPK ERK1 activity was measured as previously described (23). Briefly, we stimulated the VSMCs with NE and ANG II for 5 min. In the experiments in which cooperation between ANG II and NE was evaluated, cells were preincubated for 24 h with ANG II and then stimulated with NE for 5 min. Cells were lysed, and 50 µg of proteins of each sample were used to immunoprecipitate ERK1 with anti-ERK1 polyclonal antibody. Each immunoprecipitate was used to measure ERK1 activity with a kinase assay, which was carried out at 30°C for 10 min in 30 µl of assay buffer containing 5 µg of myelin basic protein (MBP), 20 µM ATP, and 3 µCi of [gamma -32P]ATP. The samples were resolved by 12% SDS-PAGE, and the incorporation of [gamma -32P]ATP was visualized by autoradiography. Gel slices of the 20-kDa MBP bands were also cut out in most of the experiments, and their radioactivity was measured by liquid scintillation counting.

RT-PCR Analysis

After overnight starvation, VSMCs were pretreated for 24 h with 100 nM ANG II and then incubated for 8 h with test substances. At the end of incubation, total RNA was isolated by ultrapure TRIzol (GIBCO BRL). The cDNA solution was then diluted 1:10, divided into aliquots in amplification tubes, and stored at -20°C until PCR analysis. A typical PCR reaction mixture was prepared as follows: 1.5 U of Taq DNA polymerase (5,000 U/ml; Pharmacia) was added to 5 µl of reaction buffer 10× (Pharmacia), 1 µl of dNTP (10 mM each), 2 µl primers (5 mM each): sense 5'-GCC TTC CCG CCC GGC CAC TTC AAG G-3', antisense 5'-GCA CAC ACT CCT TTG ATA GAC ACA A-3' (5), 5 µl of cDNA dilution, and water to 50 µl final volume. Amplification was performed in sequential cycles including 30 s of denaturation at 94°C, 30 s of annealing at 55°C, and a 2-min extension at 72°C. Calibration was performed by coamplification of the same cDNA samples with primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sense 5'-CTA CTG GCG CTG CCA AGG CTG T-3'; antisense 5'-GCC ATG AGG TCC ACC ACC GTG TTG-3' (27). For PCR amplification we used a GeneAmp PCR System (model 2400, Perkin-Elmer) (36). The sizes of the amplification products were 358 and 179 bp for GAPDH and FGF-2, respectively. After amplification, triplicate 20-µl aliquots were electrophoresed in 2.5% agarose gel and PCR products highlighted by ethidium bromide. The intensities of the bands corresponding to amplificates were quantified by densitometric analysis. We accessed the bands with a scanner (UMAX MagicScan, version 2.3.1) by using the Adobe Photoshop (version 3.0) and then analyzed them with IMAGE (version 1.60b5).

Western Blot Analysis

FGF-2 expression was measured as described previously (36). Briefly, ANG II-pretreated VSMCs were stimulated with 0.1 nM NE for 15 h. The cell lysate was run on 12% SDS-PAGE, blotted onto polyvinylidene difluoride membrane (Millipore), and immunostained with mouse monoclonal anti-FGF-2 antibody (1:1,000). The antigen-antibody complexes were visualized with the use of appropriate secondary antibodies and the ECL detection system, as recommended by the manufacturer (Amersham).

Drugs

DMEM, a penicillin-streptomycin-glutamine solution, a trypsin-EDTA solution, gelatin, ascorbic acid, NG-nitro-L-arginine, phenylephrine, ANG II, prazosin, anti-rabbit IgG agarose beads, and MBP were purchased from Sigma Chemical. CS was purchased from Hyclone (Logan, UT), PDGF was from Boehringer Mannheim (Mannheim, Germany), and losartan and PD-123177 were supplied by Menarini Pharmaceuticals (Florence, Italy). Rabbit anti-ERK1 polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-FGF-2 monoclonal antibody clone bFM-1 (neutralizing) and mouse anti-FGF-2 monoclonal antibody clone bFM-2 were from UBI (Lake Placid, NY). Acrylamide, N,N,N',N'-tetramethylethylenediamine, ammonium persulfate, Coomassie brilliant blue, anion-exchange columns prepared with AG-K8 resin (200-400 mesh, formiate form) were from Bio-Rad Laboratories (Richmond, CA). PD-98059 (2-[2'-amino-3'-methoxyphenyl]-oxanaphthalen-4-1) and recombinant human FGF-2 were from Calbiochem- Novabiochem (San Diego, CA). [gamma -32P]ATP was from NEN- DuPont (Boston, MA), myo-[2-3H] inositol (18 Ci/mmol specific activity) and L-[3H]arginine were from Amersham (Buckinghamshire, UK). Prazosin was dissolved in ethanol + HCl, whereas all of the other substances were dissolved in distilled water and further diluted in DMEM. The cells were grown on sterile plastic (Costar Europe, The Netherlands).

Statistical Evaluation

The data are reported as means ± SE. Each experiment was run in duplicate. Statistical analysis was performed by using Student's t-test for unpaired data and ANOVA followed by Scheffé's test. A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Potentiation of Growth-Promoting Effect of NE by ANG II

VSMC proliferation was stimulated in a concentration-dependent manner by exposure for 96 h to NE in the range of concentrations between 0.1 and 100 nM. The maximum stimulating effect, observed at 100 nM NE, amounted to ~80% of the maximum effect obtained with 10 ng/ml PDGF (Fig. 1). The increase in cell number was associated with an increase in DNA synthesis, as detected by [3H]thymidine incorporation: increases of 54.3 ± 5% and of 28.3 ± 4.2% of counts per minute above baseline were detected in the presence of 10 nM NE after 8 and 24 h, respectively (n = 3 experiments). Pretreatment for 30 min with prazosin (1 µM) did not interfere with either basal growth or PDGF-induced VSMC proliferation (n = 3). The NE-induced stimulation of cell proliferation was inhibited by pretreatment with the alpha -adrenoceptor antagonist, thus confirming that the NE effect was mediated by alpha -adrenoceptor activation (Fig. 1).


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  		Fig. 1.   Concentration-dependent stimulation of proliferation induced by noreprinephrine (NE) alone (open bars) and in the presence of 1 µM prazosin (solid bars). The effect of NE is compared with that elicited by 10 ng/ml platelet-derived growth factor BB (PDGF-BB). Data are expressed as total cells counted/well after 4 days. Values are means ± SE of 6 experiments in duplicate. *P < 0.05, **P < 0.01 vs. basal unstimulated cells; #P < 0.05 vs. NE alone.

ANG II, at the concentration of 100 nM, slightly stimulated VSMC proliferation, inducing an approximate 8% increase over baseline (n = 6). However, cell preincubation for 24 h with the same concentration of ANG II significantly potentiated stimulation of VSMC proliferation induced by concentrations of NE ranging from 0.01 to 1 nM (Fig. 2A). The potentiating effect of the peptide was more evident at the lowest NE concentration used (0.01 nM). In fact, this concentration of NE, which alone was not able to increase cell proliferation, significantly increased the number of VSMCs after pretreatment with 100 nM ANG II. Concentrations of ANG II <100 nM did not potentiate NE-induced cell proliferation (data not shown). The AT1 receptor antagonist losartan (1 µM) did not modify the basal growth rate of VSMCs. However, when losartan was added 30 min before exposure to ANG II, it completely inhibited the ANG II potentiation of the growth-promoting effect of 0.01 nM NE. On the contrary, the ANG II-induced potentiation of NE response was not influenced by the AT2 receptor antagonist PD-123177 at a concentration of 1 µM (Fig. 2B).


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  		Fig. 2.   A: effect of cell pretreatment with 100 nM ANG II for 24 h on vascular smooth muscle cell (VSMC) proliferation induced by different concentrations of NE. NE alone (open bars), ANG II alone (solid bar), NE + 100 nM ANG II (hatched bars). #P < 0.05, ##P < 0.01 vs. NE alone. B: effect of losartan (1 µM, solid bar) and PD-123177 (1 µM, stippled bar) on 100 nM ANG II-induced potentiation of VSMC growth stimulated by NE (0.01 nM, open bar). ##P < 0.01 vs. NE alone; *P < 0.05 vs. NE + ANG II. Values are means ± SE of at least 4 experiments in duplicate.

Potentiation by ANG II of NE-Induced Increase in Inositol Turnover

Because the results of the above-mentioned experiments indicated that the proliferative effect of NE is mediated by alpha -adrenoceptors and that AT1-receptors are involved in the potentiating effect of ANG II, the InsP turnover activation was investigated to confirm that the ANG II-induced potentiation of NE effects is receptor mediated. A concentration-dependent increase in Ins(1)P level was induced by a 15-min exposure of VSMCs to increasing concentrations of NE (1-1,000 nM), with statistically significant effects detected starting from the 10 nM concentration of the amine. The maximal effect obtained with 1 µM NE was quantitatively similar to that obtained after the same incubation time with 100 nM ANG II alone (Fig. 3A). The pretreatment of cells with 100 nM ANG II for 24 h potentiated the NE-induced Ins(1)P formation, without affecting the basal level. This effect was mainly evident at the lowest concentration of NE (1 nM), at which the Ins(1)P level accumulation, detected after ANG II incubation, was significantly higher than that obtained without ANG II pretreatment (Fig. 3B).


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  		Fig. 3.   A: concentration-dependent inositol monophosphate [Ins(1)P] level accumulation in response to NE (open bars) and to ANG II (100 nM, hatched bar) after 15-min stimulation. *P < 0.05, **P < 0.01 vs. basal unstimulated cells. B: effect of the cell pretreatment for 24 h with 100 nM ANG II on IP1 accumulation induced by the exposure to NE for 15 min; NE alone (open bars), NE plus ANG II (solid bars). Data are expressed as percent increase over basal unstimulated cells. Values are means ± SE of 3 experiments in duplicate. #P < 0.05 vs. NE alone.

Effect of ANG II and NE on FGF-2 Expression

Because endogenous FGF-2 is involved in proliferation of many cell types (24, 36), the hypothesis that the potentiation by ANG II of the growth-promoting effect of NE is mediated by upregulation of endogenous FGF-2 was tested. For this purpose, the expression of FGF-2 mRNA and protein was evaluated in subconfluent and serum-starved VSMCs pretreated for 24 h with 100 nM ANG II and then exposed to a concentration of NE (0.1 nM); the effect has been previously shown to be significantly potentiated by ANG II. As shown in Fig. 4A, 0.1 nM NE alone did not modify FGF-2 mRNA expression, measured after 8 h of stimulation. Conversely, ANG II pretreatment slightly increased basal FGF-2 mRNA expression and significantly upregulated the growth factor in NE-stimulated cells.


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  		Fig. 4.   Effect of ANG II on fibroblast growth factor (FGF-2) mRNA expression in VSMCs evaluated by differential RT-PCR analysis after 8 h of exposure to NE alone (open bars), and to NE after a 24-h preincubation with 100 nM ANG II in the absence (cross-hatched bars) and in the presence (hatched bars) of 1 µM losartan. Data are means ± SE (n = 3) of densitometrical analysis (OD) of the PCR bands and are expressed as OD ratio (FGF-2/GAPDH). **P < 0.01 vs. NE alone, #P < 0.05 vs. ANG II + NE. Inset: representative differential RT-PCR showing FGF-2 and GAPDH expression. C, basal unstimulated cells; A, 100 nM ANG II; L, 1 µM losartan; A + N, 100 nM ANG II + 0.1 nM NE; A + N + L, 100 nM ANG II + 0.1 nM NE + 1 µM losartan.

Western blot analysis in unstimulated VSMCs revealed three immunoreactive bands with an apparent mass of 18, 22, and 24 kDa, the latter being the most expressed. NE (0.1 nM) did not modify FGF-2 expression after 15 h stimulation, whereas ANG II (100 nM) slightly upregulated FGF-2 when given alone and potentiated the NE effect on FGF-2 expression (Table 1).

                              
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  		Table 1.   Effect of ANG II on FGF-2 expression in vascular smooth muscle cells

Involvement of endogenous FGF-2 in the ANG II-induced potentiation of VSMC growth induced by NE. The observation that a significant upregulation of FGF-2 occurred in VSMCs stimulated by ANG II plus NE prompted us to investigate whether the growth factor was involved in the observed potentiation of NE-induced cell proliferation induced by the pretreatment with ANG II. Therefore, VSMCs pretreated for 24 h with ANG II (100 nM) were stimulated with NE in the presence or in the absence of a neutralizing anti-FGF-2 monoclonal antibody (2 µg/ml). This antibody did not affect basal proliferation but significantly prevented the ANG II-induced potentiation of VSMC proliferation in response to NE (Fig. 5), thus definitely showing that endogenous FGF-2 is the mediator of the cooperation between NE and ANG II on VSMC growth.


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  		Fig. 5.   Effect of anti-FGF-2 antibody (neutralizing) on the ANG II-mediated potentiation of VSMC proliferation induced by 0.01 and 0.1 nM NE. Data are expressed as percent increase in cell number evaluated 4 days after NE stimulation alone (open bars), and NE stimulation after 24-h pretreatment with 100 nM ANG II in the absence (solid bars) and in the presence (hatched bars) of 2 µg/ml of mouse anti-FGF-2 monoclonal antibody. Values are means ± SE of 3 experiments in duplicate. **P < 0.01 vs. NE alone, #P < 0.01 vs. ANG II + NE.

Effects of ANG II and NE on ERK1 Activation

It is known that both ANG II and NE are able to stimulate ERK1 activity in VSMCs (7, 35). We then investigated whether the MAPK cascade was involved as an intracellular pathway between the receptor signaling (i.e., InsP turnover) and nuclear message (i.e., FGF-2 upregulation and cell duplication). Both NE and ANG II alone were able to significantly increase ERK1 activity in untreated cells within 5 min (Fig. 6). After 24 h of preincubation with 100 nM ANG II, the basal ERK1 activity was still significantly higher than that in unstimulated cells. The exposure of ANG II pretreated cells to 0.1-1 nM NE for 5 min did not induce a further increase in ERK1 activity (Fig. 6), thus suggesting that ERK1 activation was not directly involved in the potentiating effect of ANG II on NE effect.


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  		Fig. 6.   Extracellular signal-related kinase (ERK1) activity in response to NE and ANG II in VSMCs exposed for 5 min to 100 nM ANG II and NE (0.1-1 nM), or pretreated with ANG II for 24 h and then exposed for 5 min to NE. ERK1 was immunoprecipitated, and its activity was measured with an in vitro kinase assay. Values are means ± SE of counts per minute (cpm) recovered from cut bands in 2 experiments in duplicate. **P < 0.01 vs. basal unstimulated cells. Inset: representative autoradiography. C, control unstimulated cells; A, 100 nM ANG II; NE, 1 nM NE; A + NE, ANG II for 24 h + NE for 5 min.

Effect of ANG II and NE Treatment of VSMCs in Hypertrophy

We first assessed whether NE stimulation was able to induce hypertrophy of VSMCs and then whether ANG II potentiated the NE effect. NE, in concentrations ranging from 0.1 to 10 nM, was unable to significantly increase either the [3H]leucin incorporation (Fig. 7A) or the total protein content measured after 4 days (Fig. 7B). The exposure to 100 nM ANG II for 8 h significantly increased the protein synthesis of untreated VSMCs by 30 ± 3% (n = 4). The pretreatment of cells with 100 nM ANG II for 24 h significantly increased both basal protein synthesis and total protein content of VSMCs but did not modify the effect of NE (Fig. 7, A and B).


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  		Fig. 7.   A: protein synthesis, measured by [3H]leucine incorporation, after 6 h of exposure to different concentrations of NE in untreated cells (open bars) and in cells preincubated with ANG II for 24 h (hatched bars). Data are reported as radioactivity recovered/well. B: hypertrophy, assessed as total proteins/105 cells recovered after 4 days in culture, in response to different concentrations of NE in untreated cells (open bars) and in cells preincubated with ANG II for 24 h (hatched bars). Values are means ± SE of 4 experiments in duplicate. **P < 0.01 vs. basal unstimulated cells.

Role of ERK1 on Hypertrophic Effect Induced by ANG II

The hypothesis that the ERK1 activation was linked to other cellular events promoted by ANG II, such as cell hypertrophy, was tested. For this purpose, VSMCs were exposed to ANG II in the presence of 10 µM of PD-98059, a selective inhibitor of MEK1, which is the kinase upstream of ERK1. PD-98059 was added together with ANG II for 24 h in a first set of experiments and during the last 6 h of exposure to ANG II in a second set. PD-98059 time dependently inhibited both the basal and the ANG II-induced increase in protein synthesis (Fig. 8). The inhibition of ANG II effect by PD-98059 amounted to ~60% when it was added together with ANG II, and to ~30% when it was added during the last 6 h of stimulation. During the 24-h stimulation with PD-98059, the cell number was not changed (72 ± 7, n = 3) compared with unstimulated cells (70 ± 10, n = 3).


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  		Fig. 8.   Effect of mitogen-activated protein kinase kinase (MAPKK) inhibitor PD-98059 on protein synthesis in untreated cells and in cells exposed to 100 nM ANG II. Protein synthesis was assessed as [3H]leucine incorporation after a 24 h stimulation. PD-98059 (10 µM) was added for 24 h (hatched bars) or during the last 6 h of incubation (solid bars). Values are means ± SE of 3 experiments in duplicate. **P < 0.01 vs. basal unstimulated cells; #P < 0.05, ##P < 0.01 vs. ANG II.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The increase in vascular wall thickness that occurs in pathological conditions such as atherosclerosis, hypertension, and restenosis, results from a phenotypic change in VSMCs leading to inappropriate cell proliferation. Thus it is important to understand the mechanisms implicated in VSMC growth regulation. The potent vasoactive agents ANG II and NE are recognized as endogenous mitogenic substances involved in pathological vascular remodeling (30). The present results demonstrate that NE has a proliferative effect on cultured VSMCs at concentrations much lower than those previously indicated (35). In fact, our findings show that NE exerts a mitogenic effect at concentrations as low as 0.1 and 1 nM; it is of interest to note that such low concentrations of the amine have no effect on vascular smooth muscle tone because contractile activity in vascular preparations is detectable only at NE concentrations above 10 nM (personal unpublished observations, and 8). Thus by extrapolating these results to the in vivo vascular effects of the sympathetic transmitter, it may be suggested that prolonged contact with concentrations of the amine unable to elicit any effect on vascular tone could induce a shift in VSMC phenotype toward the proliferative state. The observation that the stimulating effect of NE was blocked by prazosin represents a further demonstration of the involvement of alpha -adrenoceptors in the mitogenic effect of NE (35).

However, the major finding of the present study consists of the first-time observation that ANG II is able to significantly potentiate the proliferative effect of NE on VSMCs when used at a concentration (100 nM) unable to significantly promote VSMC proliferation. The potentiation of NE-induced VSMC proliferation by ANG II, which is more than additive, was blocked by the AT1 receptor antagonist losartan, but was left unaffected by the AT2 receptor antagonist PD-123177, thus confirming the lack of functional antiproliferative AT2 receptors in rat aortic smooth muscle cells (29). To further demonstrate that the ANG II-induced potentiation on NE effects are receptor mediated, the effects on InsP turnover were assessed. Our observation that a significant increase in InsP turnover occurred in cells exposed to NE after pretreatment with ANG II may be interpreted as due to a change in affinity of NE for its receptor or to an increased rate of alpha -adrenoceptor synthesis in VSMCs induced by ANG II, as previously reported (12). The mechanism by which ANG II potentiates the mitogenic effect of NE was investigated by measuring FGF-2 expression, because an important role of FGF-2 in the autocrine control of cell growth has been demonstrated for many cell types, including fibroblasts, endothelial cells, and smooth muscle cells (9, 15, 36). FGF-2 is a potent growth factor for many cell types including smooth muscle cells (4), and it is under the control of growth factors, cytokines, and hormones (17, 33). Our data clearly show that ANG II upregulates FGF-2 expression, and we demonstrate for the first time that it potentiates NE effect on either mRNA or protein expression of growth factor. The importance of FGF-2 as an autocrine control for the cooperation between the catecholamine and ANG II on VSMC growth is demonstrated by the lack of the potentiating effect of ANG II on NE-induced VSMC proliferation when cells are stimulated with the substances in the presence of a neutralizing antibody anti-FGF-2. The involvement of endogenous FGF-2 as a mediator of ANG II effect has been previously identified as one of the mechanisms necessary for ANG II activity (14). Moreover, it has been previously shown that ANG II can promote cellular hypertrophy and/or hyperplasia in VSMCs through a mechanism consisting, at least in part, in the induction of growth factors such as PDGF, FGF-2, and transforming growth factor (2, 14, 19). The present data comprise the first report of the involvement of FGF-2 in the cooperation between ANG II and NE on cellular growth. Su and co-workers (31) have reported that the use of an anti-FGF-2 antibody is able to block vascular remodeling of large vessels in vivo, thus strengthening the important role of endogenous FGF-2 in pathological vascular remodeling.

In the present study, the ERK1 activity was also measured as an intracellular pathway possibly involved between receptor signaling and FGF-2 upregulation. ERK1 is the MAPK that is chiefly involved in the response to mitogenic stimuli; both NE and ANG II have been reported to be able to stimulate ERKs in VSMCs (7, 35). It is of interest to note that MAPKs have been proposed as modulators of Na+ homeostasis, suggesting that they may be involved in the pathogenesis of hypertension (3, 34). In fact, ERK activation is a major mediator of growth factor-induced activation of the Na+/H+ exchanger in smooth muscle cells (3), thus influencing Na+ homeostasis. Therefore, we decided to test whether ERK1 activation is involved in the potentiating effect of ANG II on NE effects. Although an increase in ERK1 activity was detected in response to ANG II plus NE compared with the effect of NE alone, it was not possible to correlate the potentiating effects of ANG II with ERK1 activation in VSMCs, because the combined effect of ANG II plus NE stimulation was lower than that observed in cells stimulated with ANG II alone. Because these data demonstrate that ERK1 is not a mediator of the observed potentiating effect of ANG II on NE activity, and because we did not observe a potentiation on VSMC hypertrophy in response to ANG II plus NE, we decided to investigate the possible role of ERK1 in the activity of ANG II alone. An ANG II-induced increase in VSMC protein synthesis was indeed detected in the present study, in agreement with previous reports by other groups (1, 26). The finding that the MAPKK inhibitor PD-98059 significantly and in a time-dependent manner inhibited the ANG II-induced increase in protein synthesis demonstrates that ERK1 activation is involved in the hypertrophic effect of ANG II. Thus our data show that ERK1 is involved in postreceptor effect of both NE and ANG II when each one acts alone on VSMCs, but that this kinase does not mediate the potentiating effect of ANG II on NE activity.

In conclusion, this study demonstrates that ANG II, at a concentration endowed with hypertrophic effects but unable to induce a mitogenic effect, consistently potentiates the growth-promoting effect of NE in VSMCs. These data strengthen the hypothesis that ANG II can effectively potentiate the stimulating effect of NE on pathological cell proliferation. The observation that AT1 receptor stimulation is responsible for the potentiating effect of the peptide may be of therapeutic interest, because it suggests that AT1 receptor antagonists may influence not only the vascular tone, but also the inappropriate VSMC growth, which occurs in pathological cardiovascular conditions. This study also shows that the mechanisms involved in this growth-potentiating effect of ANG II are receptor mediated and consist of FGF-2 upregulation. ERK1 activation is significantly activated by the two factors acting independently, but it is not implicated in their cooperation. Interestingly, ERK1 activation is linked to the hypertrophic activity of ANG II when acting alone. These results show that differing effects of ANG II on VSMCs, i.e., cell hypertrophy and the cooperation with NE in cell growth, may be a consequence of specific signal transduction events inside the cells.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research and the National Research Council of Italy (CNR), project no. 97.004472.CT04.


    FOOTNOTES

Address for reprint requests and other correspondence: F. Ledda, Dept. of Pharmacology, Univ. of Florence, V. le G. Pieraccini 6, 50139 Florence, Italy (E-mail: ledda{at}ds.unifi.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 December 1999; accepted in final form 7 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(1):H99-H107
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