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Cardiovascular Research 1998 38(1):263-271; doi:10.1016/S0008-6363(97)00296-4
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Thrombin receptor expression is increased by angiotensin II in cultured and native vascular smooth muscle cells

Beate Fisslthaler*, Valerie B Schini-Kerth, Ingrid Fleming and Rudi Busse

Institut für Kardiovaskuläre Physiologie, Klinikum der Johann Wolfgang Goethe Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany

* Corresponding author. Tel.: +49 (69) 6301-6995; Fax: +49 (69) 6301-7668; E-mail: fisslthaler@em.uni-frankfurt.de

Received 19 June 1997; accepted 28 October 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The factors involved in restenosis after balloon angioplasty are poorly characterized but the local concentration of the potent mitogens angiotensin II (AII) and thrombin is known to be increased at sites of vascular injury. We investigated the possibility of a synergistic interaction between AII and thrombin by studying the effects of AII on the expression of the thrombin receptor in rat aortic smooth muscle cells (VMSC). Methods: Thrombin receptor mRNA expression was studied by Northern blot analysis and RT-PCR using total RNA extracted from VMSC or from endothelium-denuded rat aortae. As a measure of thrombin receptor protein expression, we assessed either the thrombin-stimulated release of 6-keto-prostaglandin F1{alpha} from VMSC or the contraction of endothelium-denuded rat aortic rings. Results: The thrombin receptor mRNA was expressed at a low level in both cultured and native VMSC. AII concentration- and time-dependently increased expression of thrombin receptor mRNA in VSMC and augmented the thrombin-induced release of 6-keto-prostaglandin F1{alpha} as well as the thrombin-induced contraction. Blockade of the angiotensin subtype 1 (AT1) receptor by EXP3174 or D8731 prevented the AII-mediated increase in thrombin receptor expression. The effect of AII on the increase in thrombin receptor mRNA expression was enhanced by the protein kinase C inhibitor Ro 31-8220, but was unaffected by prolonged incubation with phorbol myristate acetate or the tyrosine kinase inhibitors genistein and erbstatin A. Conclusion: These results demonstrate that AII enhances the expression of thrombin receptor in cultured and native VMSC. In cultured cells, this effect is mediated by the activation of the AT1 receptor subtype. This synergistic effect between AII and the thrombin receptor may promote the extensive proliferation of smooth muscle cells in response to vascular injury.

KEYWORDS Rat aortic smooth muscle cell; Restenosis; Angiotensin II; Thrombin receptor; Tyrosine kinase inhibitor; AT1 receptor; Protein kinase C


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Vascular smooth muscle cell (VSMC) proliferation is influenced by a variety of cytokines and growth factors generated at sites of injury. Indeed, extensive growth and migration of VSMC occurs following the mechanical injury of arteries and the resulting intimal thickening represents a major complication following balloon angioplasty. Similar events have been described during atherogenesis and following coronary artery bypass surgery. Balloon angioplasty of human coronary or canine carotid arteries results in an increase in the expression of angiotensin-converting enzyme (ACE), the chymostatin-sensitive AII-generating enzyme [1, 2]and angiotensinogen in the media and neointima [3], all of which lead to an increase in the local concentration of angiotensin (AII). AII, besides its function as potent vasoconstrictor, has been shown to induce hypertrophy in cultured VSMC [4], and to promote the proliferation and migration of VSMC in the rat arterial wall [5]. Inhibition of ACE, or the application of angiotensin subtype 1 (AT1) receptor antagonists prior to, or shortly after, angioplasty of the carotid artery has been reported to attenuate intimal thickening [6, 7]. Moreover, an increased expression of AT1 receptors has been reported in the media and neointima of balloon-injured rat carotid arteries, supporting the concept that the AII-induced changes are mediated by the activation of this receptor subtype [8].

Following disruption of the endothelium, sub-endothelial cell layers are exposed which promote the activation of platelets and the initiation of the coagulation cascade resulting in the formation of thrombin. This serine protease is present in balloon-injured vessels several weeks after injury [9]and, as it is a potent mitogen in fibroblasts and VSMC [10, 11], has been implicated in the development of atherosclerotic lesions and restenosis. Indeed, neointimal thickening after balloon angioplasty in rabbits was significantly decreased by treatment with heparin or hirudin [12, 13].

Cellular responses to thrombin are mediated largely, if not solely, by a receptor which belongs to the superfamily G-protein coupled receptors. Thrombin activates this receptor by proteolytical cleavage of the N-terminal region and the new amino terminus is a tethered ligand [8, 14]. Stimulation is followed by rapid desensitization via phosphorylation, subsequent internalization and degradation of the receptor [15]. Consequently, in the absence of an intracellular store of thrombin receptors which can rapidly replenish the plasma membrane, the expression of the functional thrombin receptor is dependent on its de novo synthesis.

In human atherosclerotic lesions and in balloon-injured rat carotid arteries, an increased expression of the thrombin receptor has been demonstrated using in situ hybridization and immunohistochemical techniques [16, 17]. This enhanced receptor expression is most probably regulated by factors produced by the vascular wall and by activated platelets in the vicinity of the lesion. Indeed, basic fibroblast growth factor (bFGF), platelet-derived growth factorAA (PDGFAA) [17]and serotonin (5-HT) [18]can increase the expression of thrombin receptor mRNA. Given the apparent importance of AII in instigating proliferation in injured vessels, we investigated whether AII is also able to increase in expression of the thrombin receptor, which is a potent mitogen in VSMC.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Angiotensin II was purchased from Bachem, 2-N-((butyl-4-chloro-1-[(2-)1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazol-5-carboxylic acid (EXP3174) from Merck Sharp and Dohme (Munich, Germany), 2-ethyl-4-[(2'-(1H-1,2,3,4-tetrazol-5-yl)biphenyl-4-yl)methoxy]quinoline (D8731) from ICI Pharmaceuticals (Macclesfield, UK), 1-(4-amino-3-methyl-phenyl)methyl-5-diphenyl-acetyl-4,5,6,7-tetrahydro-1H-imidazol[4,5-C]pyridine-6-carboxylic acid (PD123177) and nicotinic acid–Tyr–N{alpha}-benzyl-oxycarbonyl–Arg–Lys–His–Pro–Ile–OH (CGP42112A) were kindly provided by Hoechst Marion Roussel (Frankfurt, Germany), 2'-amino-3'-methoxyflavone (PD98059) was from Biomol (Hamburg, Germany), genistein, erbstatin A and PP1 were from Calbiochem (Bad Soden, Germany), Ro 31-8220 was from Roche Products Ltd. (Welwyn Garden City, UK), PDGFAB and staurosporine were from Boehringer Mannheim (Mannheim, Germany). Minimum essential medium (MEM) containing Earle's salts; penicillin and streptomycin from Gibco Life Sciences (Heidelberg, Germany), trypsin, and fetal calf serum (FCS) from PAN systems (Aidenbach, Germany), [{alpha}-32P]desoxycytosintriphosphate ([{alpha}-32P]dCTP) from Hartmann Analytic (Braunschweig, Germany). {alpha}-Thrombin (specific clotting activity, 3488.6 U/mg) was kindly provided by Dr J.W. Fenton II (Albany, NY, USA). All other chemicals were purchased from Sigma (Heidelberg, Germany) and of the highest purity available.

2.2 Cell culture
Vascular smooth muscle cells (VSMC) were isolated from the thoracic aortae of male Wistar rats (Charles River, Wiga) by elastase and collagenase digestion and cultured in MEM containing Earle's salts, glutamine (2 mmol/l), N-Tris (hydroxylmethyl) methyl-2-aminoethane sulfonic acid (TES; 5 mmol/l), N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES; 5 mmol/l), penicillin (50 U/ml), streptomycin (50 µg/ml) and FCS (10%). After reaching confluence, the cells were serially passaged and all experiments were performed using cells from passage 9 and higher. Two days prior to each experiment, the culture medium was replaced with medium containing bovine serum albumin (BSA, 0.1%).

2.3 RNA extraction and Northern blotting
Total RNA from cultured VSMC and endothelium-denuded rat aortae was isolated using acid guanidium isothiocyanate as described [19]. RNA was spectrophotometrically quantified by the absorbance at 260 nm and equal amounts of total RNA (20–30 µg) were separated on a 1% formaldehyde agarose gel. After transfer onto nylon membranes (Amersham, Braunschweig, Germany), the RNA was fixed by UV-crosslinking and baking at 80°C for 2 h. Thereafter, filters were prehybridized in Tris/HCl pH 7.5 (20 mmol/l), NaCl (800 mmol/l), sodium citrate (80 mmol/l), Ficoll (0.1%), polyvinylpyrrolidone (0.1%), BSA fraction V (0.1%), (5xDenhardt's solution), sodium dodecylsulfate (SDS, 0.02%), and denatured salmon sperm DNA (250 µg/ml) for 4–16 h at 60°C. A PstI-restriction fragment from a cDNA clone of the Chinese hamster thrombin receptor (kindly provided by E. Van Obberghen-Schilling, Nice, France) was radioactively labeled with [{alpha}-32P]dCTP by a random-prime labeling kit (Pharmacia, Freiburg, Germany) and used as probe for hybridization. Hybridization for 16–20 h was performed in the same solution as prehybridization containing the radioactive labeled probe. The final washing was performed with NaCl (300 mmol/l), sodium citrate (30 mmol/l), SDS (0.1%) at 55°C. The blots were exposed to X-ray films for 1–4 days at –70°C and the optical density of the specific signals was quantified by densitometric analysis (ImageMaster 1D, Pharmacia Freiburg, Germany). The RNA content in each lane was normalized by measuring the intensity of either the ethidium bromide staining or the hybridization signal from the 18S RNA.

2.4 RT-PCR
Equal amounts of total RNA (2 µg) from rat aortae were used for reverse transcription (Superscript, Gibco, Life Technology, Heidelberg, Germany) using random hexamers (pdN6, Pharmacia) as primers. One tenth of the reverse transcription reaction was subjected to PCR analysis (30 cycles, 52°C annealing) in which the thrombin receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were co-amplified. The thrombin receptor-specific primers were derived from rat and human sequences (Genbank accession numbers M81642 [GenBank] and U63331 [GenBank] , respectively: upstream primer, 5'-CAGCCAGAATCTGAGA(T/G)GAT-3'; downstream primer, 5'-GACCCGAACTGCCAATCGGT-3') the sequences for GAPDH were derived from the cDNA sequence from rat (Genbank accession number X02231 [GenBank] and X00972: upstream primer, 5'-TATGACAACTCCCTCAAGAT-3'; downstream primer, 5'-AGATCCACAACGGATACATT-3') and the amplified fragments had an expected length of 420 and 320 bp, respectively. The PCR reaction products were subjected to non-denaturing agarose gel electrophoresis, stained with ethidium bromide and the intensity of the fluorescence of the specific bands at 340 nm was quantified by densitometric analysis.

2.5 Release of 6-keto-prostaglandin F1{alpha}
Confluent VSMC were incubated in the presence or absence of different concentrations of AII for 6 h. Thereafter, the cells were washed with a HEPES-modified Tyrode's solution and stimulated with either thrombin (5 U/ml, 20 min) or PDGFAB (30 ng/ml, 20 min). 6-Keto-prostaglandin F1{alpha} (6-keto-PGF1{alpha}), the stable metabolite of prostacyclin, released into the supernatant was determined using a specific radioimmunoassay (Dupont, Dreieich, Germany).

2.6 Organ bath experiments
Experiments were performed using aortae obtained from Wistar Kyoto rats which were housed in conditions conforming to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). The descending thoracic aorta was removed under sterile conditions from anesthetized rats (60 mg/kg sodium pentobarbitone i.p.), cleaned of connective tissue and dissected into rings (3 mm in length). Aortic rings were denuded of endothelium and then incubated in MEM containing 0.1% BSA and polymyxin B (1 µg/ml) and in the absence and presence of AII (100 nmol/l) for 8 h. Thereafter, the aortic rings were washed thoroughly and mounted between force transducers (Scaime, France) and a rigid support for measurement of isometric force and placed in organ baths containing warmed (37°C), oxygenated (95% O2, 5% CO2) Krebs–Henseleit solution (pH 7.4) of the following composition (mmol/l): NaCl, 118.4; NaHCO3, 25; KCl, 4.7; CaCl2, 1.6; KH2PO4 1.2; MgSO4, 1.2; glucose 11.1. Passive tension was adjusted over a 30-min equilibration period to 2 g; thereafter the segments were exposed to KCl (80 mmol/l) until stable contractions were obtained. A concentration response curve to phenylephrine (1 nmol/l to 1 µmol/l) was determined and the effective removal of the endothelium was assessed by the lack of response to acetylcholine (1 µmol/l). After washout, the thrombin-induced (5 and 10 U/ml) increase in vascular tone was measured.

2.7 Statistical analysis
Data are expressed as mean±s.e.m. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by a Bonferroni t-test or the two-tailed Student's t-test for paired and unpaired data, where appropriate. A value of P<0.05 was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of AII on thrombin receptor mRNA expression
Levels of mRNA encoding the thrombin receptor were determined by Northern blot analysis using total RNA isolated from confluent cultures of VSMC. Treatment of VSMC with AII (1 µmol/l) increased expression of thrombin receptor mRNA (Fig. 1). Maximal expression of thrombin receptor mRNA (3-fold over basal level) was observed 2 h after stimulation with AII and returned to baseline levels within 8 h. Prolonged incubation (up to 24 h) with AII showed no secondary increase in the thrombin receptor mRNA level (data not shown). Therefore, all of the following Northern blot experiments were performed using cells incubated with AII for 2 h. The effect of AII on the expression of the thrombin receptor was concentration-dependent with an increase in thrombin receptor mRNA expression first being apparent in response to 1 nmol/l AII (Fig. 2).


Figure 1
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  		Fig. 1 Time course of the AII-induced increase in thrombin receptor mRNA expression in cultured VSMC. Quiescent VSMC were treated with 1 µmol/l AII for the times indicated. Specific Northern blot signals were quantified by densitometry of the autoradiographs and normalized by comparison with the intensity of the ethidium bromide-stained 18S RNA. Results are presented ±s.e.m. of 4 independent experiments. *P<0.05.

 

Figure 2
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  		Fig. 2 Concentration-dependency of the AII-induced increase of thrombin receptor mRNA expression. Confluent cultures of quiescent VSMC were incubated with either solvent (Co) or AII (0.1 nmol/l to 1 µmol/l) for 2 h. Results are presented as the thrombin receptor-specific signals after AII treatment normalized by comparison with 18S RNA and are the mean±s.e.m. of 7 independent experiments. *P<0.05.

 
3.2 Effect of AII receptor antagonists
To characterize the AII receptor mediating the AII-induced increase in thrombin receptor mRNA quiescent VSMC were treated with preferential AT1- (EXP3174; 100 nmol/l and D8731; 1 µmol/l), AT2- (PD123177; 100 nmol/l) receptor antagonists or the AT2-selective ligand (CGP42112A; 1 µmol/l) 20 min prior the incubation with AII (1 µmol/l). Blockade of the AT1 receptor using EXP3174 or D8731 significantly inhibited the effect of AII, whereas the AT2 receptor antagonist PD123177 and the selective ligand CGP42112A, failed to affect the AII-mediated increase in thrombin receptor mRNA (Fig. 3). Incubation of VSMC with the AII receptor antagonists in the absence of AII was without effect on the expression of thrombin receptor mRNA (Fig. 3, upper panel).


Figure 3
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  		Fig. 3 Effect of angiotensin receptor antagonists on the AII-mediated thrombin receptor mRNA expression. Serum-deprived VSMC were stimulated for 2 h with AII (100 nmol/l), in the absence or presence of either EXP3174 (100 nmol/l, EXP) or PD123177 (100 nmol/l, PD). The upper panel shows an autoradiograph of a Northern blot for thrombin receptor mRNA and the middle panel shows the corresponding ethidium bromide-stained 18S RNA. The bar graph shows the statistical analysis of densitometrically quantified specific signals normalized for loading variation with the 18S RNA band intensities from 7 independent experiments. The signal intensities are expressed relative to control values as mean±s.e.m. *P<0.05; #significant inhibitory effect of EXP3174.

 
In order to verify the existence of functional AT1 receptors in cultured VSMC, the expression of the immediate early gene c-fos was determined by Northern blot analysis. Under basal conditions no c-fos mRNA expression was detectable. Incubation with AII (1 µmol/l, 30 min) resulted in an induction of the c-fos mRNA expression, which was abrogated by co-incubation with AT1, but not by AT2 receptor antagonists (Fig. 4).


Figure 4
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  		Fig. 4 Inhibition of AII-mediated induction of c-fos mRNA by AT1 receptor antagonists. Serum-deprived VSMC were stimulated with either solvent or AII (1 µmol/l, 30 min) in the absence or presence of either the AT2 selective ligand CGP42112A (CGP, 1 µmol/l), the AT2 receptor antagonist PD123177 (PD, 100 nmol/l), or the AT1 receptor antagonists D8731 (D87, 1 µmol/l) or EXP3174 (EXP, 100 nmol/l). The upper panel shows an autoradiograph of a Northern blot for c-fos. The lower panel shows the corresponding ethidium bromide-stained 18S RNA bands.

 
3.3 Effect of AII on thrombin-induced responses
Due to the known difficulties in quantifying thrombin receptor protein levels in non-transfected VSMC using standard techniques, such as Western blotting, FACS analysis, or immunohistochemistry, we determined the thrombin-induced release of 6-keto-PGF1{alpha} as an index of functionally active thrombin receptors in VSMC. In unstimulated VSMC, the basal release of 6-keto-PGF1{alpha} was below 0.1 ng/106 cells and pretreatment of the cells with AII (10 and 100 nmol/l, 6 h) resulted in a slight increase in 6-keto-PGF1{alpha} production. Thrombin (30 nmol/l), induced an 3-fold increase in the release of 6-keto-PGF1{alpha} from cells incubated in the absence of AII (Fig. 5A). Thrombin-induced production of 6-keto-PGF1{alpha} was concentration-dependently augmented (up to 30-fold) in cells pretreated with AII (Fig. 5B). This enhanced responsiveness to thrombin could not be attributed to a general activation of cell metabolism by AII since the PDGFAB (30 ng/ml)-induced production of 6-keto-PGF1{alpha} was not affected by pretreatment with AII (data not shown).


Figure 5
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  		Fig. 5 Effect of AII pretreatment on the thrombin-induced release of 6-keto-PGF1{alpha} from VSMC. Serum-deprived VSMC were incubated for 6 h in serum-free medium in the presence of the indicated concentrations of AII. After replacing the medium, the cells were treated with either (A) solvent or (B) thrombin (5 U/ml). After 20 min, the supernatant was removed and the concentration of 6-keto-PGF1{alpha} therein was analyzed by radioimmunoassay. Results are presented as the mean±s.e.m. from 4 independent experiments, *Significant increase in the thrombin-stimulated release of 6-keto-PGF1{alpha} from AII-treated cells.

 
3.4 Effect of PKC and tyrosine kinase inhibitors
Using inhibitors of various protein kinases, we investigated the signalling pathway involved in the AII-induced increase in thrombin receptor mRNA expression in VSMC. The AII (100 nmol/l, 2 h)-mediated increase in thrombin receptor mRNA expression was enhanced by the PKC inhibitor Ro 31-8220 (100 nmol/l) and the non-specific kinase inhibitor staurosporine (10 nmol/l, Fig. 6A). Neither of the inhibitors used exerted significant effects on thrombin receptor mRNA levels in the absence of AII (data not shown).


Figure 6
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  		Fig. 6 Effects of kinase inhibitors on the expression of thrombin receptor mRNA in serum-deprived VSMC. The upper panels show the autoradiographs of Northern blots for the thrombin receptor, middle panels show the corresponding ethidium bromide-stained 18S RNA representing the amount of RNA loaded in each lane. The bar graphs represent thrombin receptor mRNA signal normalized vs. the 18S RNA signal. Prior to stimulation with AII (100 nmol/l, 2 h), in all experiments VSMC were incubated for 15 min with the following inhibitors: (A) Ro 31-8220 (Ro, 100 nmol/l) and staurosporine (Stauro, 10 nmol/l); (B) genistein (Gen, 10 µmol/l) and erbstatin A (Erb, 10 µmol/l); (C) PP1 (100 nmol/l) and PD98059 (PD, 50 µmol/l). Results are presented as the mean±s.e.m. of 3–4 independent experiments. *P<0.05 for the stimulatory effect of AII; #significant influence of the inhibitors on the AII response.

 
Prolonged incubation of VSMC with phorbol myristate acetate (PMA, 0.3 µmol/l, 24 h) to down-regulate PKC enzymes, did not alter the AII-induced increase in thrombin receptor mRNA expression (data not shown). The tyrosine kinase inhibitors genistein (10 µmol/l) and erbstatin A (10 µmol/l, Fig. 6B), the specific pp60c-src inhibitor PP1 (100 nmol/l) and the mitogen activated protein (MAP) kinase inhibitor PD98059 (50 µmol/l) also failed to affect the AII-mediated increase in thrombin receptor mRNA (Fig. 6C).

3.5 Effect of AII on thrombin receptor expression in the rat aorta
The effect of AII on the expression of thrombin receptor mRNA in endothelium-denuded rat aortic rings was determined using semi-quantitative RT-PCR. Under the experimental conditions used, AII (100 nmol/l, 2 or 6 h) increased the thrombin receptor mRNA level by 1.6- and 1.3-fold, respectively (Fig. 7). The expression of GAPDH was taken as an internal standard for the PCR as AII was shown not to affect GAPDH mRNA levels in Northern blot experiments (data not shown).


Figure 7
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  		Fig. 7 Effect of AII on the expression of the thrombin receptor in endothelium-denuded rat aortic rings. Bar graph summarizing the results of PCR experiments using total RNA from endothelium-denuded aortic rings incubated for 2 or 6 h in the presence or absence of AII (100 nmol/l). The ethidium bromide-stained band intensities specific for thrombin receptor were normalized by comparison with the signals obtained for GAPDH-specific bands. Results are presented as the mean±s.e.m. from 5 separate experiments. *P<0.05.

 
To verify that the increase in thrombin receptor mRNA expression was associated with an increase in the expression of functional thrombin receptors, the contractile response of endothelium-denuded aortic rings to thrombin was assessed. While preincubation of rat aortic rings with AII (100 nmol/l; 8 h) did not affect contractions obtained to either KCl (80 mmol/l) or phenylephrine (1 nmol/l to 1 µmol/l; not shown) the response to thrombin (5 and 10 U/ml) was enhanced by 290 and 410%, respectively (Fig. 8). Similar, although less pronounced, effects of AII pretreatment were observed in rings incubated with AII for 6 h (data not shown).


Figure 8
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  		Fig. 8 Effect of AII on the contractile response to thrombin in endothelium-denuded rat aortic rings. A: original tracings showing the contractile response to thrombin (5 U/ml) in solvent- and AII (100 nmol/l)-treated aortic rings. B: statistical summary of the stable thrombin (5 and 10 U/ml)-induced increase in tone in endothelium-denuded rat aortic rings. The rings were incubated in the absence (open columns) and presence (shaded columns) of AII (100 nmol/l, 8 h). Results are presented as the mean±s.e.m. from 5 separate experiments. *P<0.05; **P<0.01.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the present study, we have demonstrated that in both native and cultured VSMC, AII increases the expression of thrombin receptor mRNA. In cultured VSMC, this effect is mediated by activation of AT1 receptors and an intracellular signalling pathway negatively regulated by PKC. Moreover, despite the lack of a direct evidence that thrombin receptor protein was increased, we were able to show that AII significantly increased the thrombin-induced release of 6-keto-PGF1{alpha}, and the thrombin-induced contraction of endothelium-denuded rings. Both findings are indicative of an enhanced expression of functional thrombin receptors.

Using molecular cloning and sequencing techniques, three different AII receptor subtypes have been identified to-date. The effects of AII on vasoconstriction and mitogenesis in vascular cells has been ascribed, almost exclusively, to the activation of the AT1 receptor [8, 20], whereas the AT2 receptor, which is reportedly coupled to an increase in tyrosine phosphatase activity, appears to be mainly involved in developmental and neuronal responses to AII [21]. Somewhat paradoxically, an anti-proliferative action of AII mediated by the AT2 receptor has been described in microvascular endothelial cells [22]. No physiological role for the AT3 receptor subtype has been described to date [23]. Since, in the present study, the AII-induced increase in thrombin receptor, c-fos mRNA expression and the tyrosine phosphorylation of MAP kinase (data not shown) were unaffected by either the AT2 receptor antagonist PD123177 or the specific ligand CGP42112A, but were significantly attenuated in the presence of the AT1 receptor antagonists EXP3174 or D8731, the responses observed are most probably the consequence of AT1 receptor activation. Indeed, during the preparation of this manuscript, the prolonged administration of AII in vivo was reported to enhance the expression of the thrombin receptor in the aorta and to enhance thrombin-induced contraction of endothelium-denuded aortic rings [24]. These effects were also reversed by the AT1 antagonist, losartan.

The AT1 receptor is a G-protein-coupled receptor and the binding of AII activates phospholipases and subsequently PKC (for reviews see [25, 26]). There is an increasing amount of evidence suggesting that following their occupancy, G-protein-coupled receptors, which are devoid of inherent protein tyrosine kinase activity, can also rapidly induce tyrosine phosphorylation of cellular substrate proteins as well as of the receptors themselves. For example, mutation of conserved intramembrane tyrosine residues in the AT1A receptor prevents its coupling to G-proteins as well as the subsequent activation of phospholipase C (PLC) [27, 28]. In VSMC, in which AII is able to activate tyrosine kinase pathways and cause contraction [29], a fusion protein, comprising the intracellular tail of the AT1 receptor, was found to be an excellent substrate for the src family of protein tyrosine kinases [30]. This phenomenon, at least in the case of the AT1 receptor, was shown to be linked to the tyrosine phosphorylation of PLC-{gamma}1 and to a subsequent increase in the formation of inositol 1,4,5-trisphosphate [31]. Despite evidence suggesting a role for tyrosine kinases in the intracellular signalling pathways activated following occupation of the AT1 receptor, the AII-mediated increase in thrombin receptor mRNA was insensitive to genistein, erbstatin A and PP1 and therefore does not appear to involve tyrosine kinase activation. However, it cannot be excluded that tyrosine kinases, which are insensitive to the inhibitors used, are involved in mediating the AII-induced response. The lack of effect of genistein on this pathway also implies that neither the activation of the Janus-like kinase (JAK) nor the STAT-pathway, both of which are highly sensitive to genistein [32], are likely to be involved in transducing the effect of AT1 receptor activation under the experimental conditions studied. Moreover, although a PD98059-sensitive tyrosine phosphorylation and activation of MAP kinases was observed in VSMC following stimulation with AII (data not shown), this compound failed to influence the AII-induced increase in thrombin receptor mRNA. Thus, the activation of MAP kinases cannot be implicated in the signalling pathway leading to enhanced thrombin receptor expression.

Inhibition of PKC using either Ro 31-8220 or staurosporine enhanced the AII-mediated increase in thrombin receptor mRNA suggesting that PKC negatively regulates this signalling process. This observation is not unusual in AII-stimulated cells since PKC has also been reported to be a negative feedback regulator of PLC in VSMC [33]and signal transduction from the receptor to the G-protein in mesangial cells [34]. The lack of effect of PKC down-regulation by long-term treatment with PMA does not preclude a role for PKC in the AII-mediated regulation of thrombin receptor expression since atypical isoforms (e.g. PKC-{lambda} and -{zeta}) do not bind diacylglycerol or phorbol esters [35].

We have previously demonstrated that 5-HT increases expression of the thrombin receptor in VSMC via a pathway sensitive to genistein and erbstatin A as well as inhibitors of PKC [18]. It would therefore appear that the initial components of the signalling pathways which ultimately culminate in enhanced thrombin receptor expression are highly dependent on the type of receptor activated. Whether distal signalling pathways initiated by AII and 5-HT converge to enhance the transcription of the gene encoding the thrombin receptor or to change mRNA stability remains to be clarified. Some experimental observations suggest, that repressor sequence(s) may be present within the distal 5'-region of the thrombin receptor gene enhancer. Indeed, functional analysis demonstrated a markedly lower transcriptional activity of the full-length thrombin receptor promoter than a 5'-truncated form [36].

In certain cell systems, the growth-promoting effects of AII have been shown to be mediated via the increased expression of autocrine growth factors, such as PDGFAA, transforming growth factor-β1 or bFGF [37, 38], factors which also enhance the expression of the thrombin receptor mRNA in VSMC [17, 39]. However, the effect of AII on the expression of thrombin receptor mRNA described in the present study is unlikely to involve the production of growth factors. Indeed, AT1 receptor activation increased thrombin receptor mRNA expression within 1 h whereas the AII-stimulated production of PDGF etc. is generally regarded as a delayed response [40, 41].

As the effect described here was observed in native and cultured VSMC, it is more than likely that AII is able to contribute to the enhanced expression of the thrombin receptor in smooth muscle cells within the human atherosclerotic plaques [16]. This increase in expression of the thrombin receptor could facilitate the mitogenic activity of thrombin. Moreover, since the activity of thrombin results not only in acute thrombus formation, but also in the stimulation of the secretion of platelet-derived vasoactive factors [42], it is conceivable that the effects of vascular injury on the up-regulation of the thrombin receptor are maintained partly as a consequence of the co-operative activity of a series of growth factors. Such effects could theoretically result from either the amplification of intracellular signalling pathways or the cross-talk between/co-activation of certain receptors. For example, the activation of the AII and thrombin receptors has been reported to induce a transactivation of the PDGF-β receptor [43].

In summary, we have demonstrated that AII, via activation of the AT1 receptor on both native and cultured VSMC, enhances expression of thrombin receptor mRNA and potentiates the functional response to thrombin receptor activation via a signalling pathway negatively regulated by a PMA-insensitive isoform of PKC. Although the concentrations of AII used here on cultured VSMC were relatively high, it is tempting to speculate that as the local concentration of AII and the thrombin receptor are known to increase at sites of vascular injury as well as within the atherosclerotic plaque [16, 17]the mechanism herein described may promote the proliferation of VSMC by ensuring the de novo generation of the thrombin receptor and thereby facilitating the mitogenic activity of thrombin.

Time for primary review 25 days.


   Acknowledgements
 
The authors are indebted to Isabel Winter and Mechtild Piepenbrock for expert technical assistance. This study was financially supported by grants from the Deutsche Forschungsgemeinschaft (Bu 436/6-1 and Schi 389/1-1) and from the Institute de Recherches International Servier (Paris, France).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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