Introduction

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Progressive arterial thickening after balloon injury results from the migration and proliferation of vascular smooth muscle cells (VSMCs) within the intima and from an excessive production of extracellular matrix (Nikol et al., 1996). Various kinds of genes, such as proto-oncogenes (Miano et al., 1990; Kim et al., 1995), growth factors (Majesky et al., 1990, 1991; Ferns et al., 1991; Wolf et al., 1994), and extracellular matrix (ECM) (Majesky et al., 1991; Kim et al., 1995), participate in the prime stages of the repair process. Several lines of evidence indicate that the renin-angiotensin system (RAS) located in the arterial wall is involved in the vascular thickening that occurs in response to injury. In fact, vascular angiotensinogen (Rakugi et al., 1993), renin (Iwai et al., 1997), angiotensin-converting enzyme (ACE) (Rakugi et al., 1994; Fernandez-Alfonso et al., 1997), and angiotensin II (Ang II) type 1 receptor (AT₁) (Viswanathan et al., 1992; Iwai et al., 1997) all show increases after injury in both their mRNA and protein levels. Moreover, an overexpression of AT₂ in injured arteries achieved by gene transfer causes an attenuation of neointimal formation (Nakajima et al., 1995), and ACE inhibitors (Powell et al., 1989) and AT₁ antagonists (Kauffman et al., 1991; Kawamura et al., 1993) inhibit neointimal formation after injury. Many studies focused on RAS have been performed to clarify the precise mechanisms underlying the vascular thickening that is seen after injury. Kim et al. (1995) reported that the increases in the mRNA levels of the immediate-early genes and fibronectin that occur in the rat balloon-injured artery could be suppressed by AT₁ blockade. Moreover, the ACE inhibitor quinapril attenuated the increase in the mRNA levels for AT₁ and ACE but not for renin in the injured rat carotid artery (Iwai et al., 1997). However, the effect of AT₁ antagonists on the consequences of the overexpression of AT₁ in injured arteries remains an open question.

KRH-594 is a novel synthetic AT₁ antagonist that is orally active (Tamura et al., 1997a,b). KRH-594 inhibits the specific binding of ¹²⁵I-Ang II to the rat liver membrane with a K_i value of 0.39 nM (Tamura et al., 1997b), and it antagonizes the Ang II-induced contractile response in the rabbit aorta with a pK_B value of 10.4 (Tamura et al., 1997a). In vivo studies have demonstrated that KRH-594 produces sustained antihypertensive effects in both spontaneously hypertensive rats and renal hypertensive rats and dogs (Inada et al., 1999) and that it has suppressive effects on experimental cardiac hypertrophy and left ventricular failure (Murakami et al., 1997).

In this study, we examined the effects of KRH-594 on the time-related changes in AT_1A and AT_1B mRNA levels in the balloon-injured rat carotid artery. The levels of the mRNAs for transforming growth factor-₁ (TGF-₁), fibronectin, and collagen types I and III were also measured. In addition, the gene expression of platelet-derived growth factor-receptor  (PDGF-R) was determined because this receptor has been reported to be transactivated by Ang II stimulation through AT₁ (Linseman et al., 1995; Abe et al., 1997) and to participate in the vascular thickening that occurs after balloon injury (Majesky et al., 1990). We report here that KRH-594 prevented the increases in the levels of the mRNA for AT_1A and PDGF-R that normally occur after injury. KRH-594 also attenuated the enhancement of the expression of the genes for TGF-₁ and fibronectin and reduced the levels of the mRNAs for collagen types I and III (which were not significantly affected by injury). Moreover, the neointima observed 14 days after injury was significantly decreased by treatment with KRH-594 or another AT₁ antagonist, TCV-116 (candesartan cilexetil). These findings suggest that a prevention of the up-regulation of AT_1A and PDGF-R may be involved in the inhibitory effect of AT₁ antagonists on neointimal formation after arterial injury.

	Materials and Methods

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Animal Model of Vascular Injury. Endothelial denudation of the left common carotid artery was performed as described previously (Clowes et al., 1983). In brief, male Sprague-Dawley rats (Clea Japan, Tokyo, Japan) aged 9 weeks (weighing 300-320 g) were anesthetized with sodium pentobarbital (50 mg/kg i.p.), and the artery was denuded by three passages of a Fogarty 2F balloon embolectomy catheter (Baxter, Irvine, CA). In the morphological study, KRH-594 (Kissei Pharmaceutical Co., Ltd., Matsumoto, Japan; 3, 10, or 30 mg/kg/day), TCV-116 (candesartan cilexetil, synthesized by Kissei; 1 or 3 mg/kg/day), or hydralazine hydrochloride (Sigma Chemical, St. Louis, MO; 10 mg/kg/day) were administered orally by gastric gavage once a day from 6 days before to 14 days after injury. In the gene expression study, KRH-594 was given at a dosage of 3 or 10 mg/kg/day from 1 day before injury to the day of sacrifice (at various times after the injury). The volume administrated was 5 ml/kg. Control rats received the same volume of vehicle (0.5% carboxymethyl cellulose solution).

Total RNA Isolation from Vascular Tissue. For the gene expression study, carotid endothelial denudation was performed as described above. Five to eight rats were sacrificed for each time point, namely, at 0, 0.25 (6 h), 1, 3, 7, and 14 days. After anesthetization, the rat was perfused with ice-cold phosphate-buffered saline (PBS). The isolated carotid artery was excised for a length of approximately 20 mm (starting 5 mm away from both the internal-external branch and the aortic arch), immediately frozen by soaking it in liquid nitrogen, and stored at 80°C until use. The vascular tissue was homogenized in ISOGEN (Nippon Gene, Tokyo, Japan) and total RNA was extracted according to the manufacturer's instructions. The total RNA was then treated with 10 U/ml RQ1 RNase-Free DNase (Promega, Madison, WI) in the presence of 120 U/ml RNase inhibitor (rRNasin; Promega) in a 100-µl reaction to remove contaminating genomic DNA. The DNase-treated RNA was purified using an RNeasy Mini Kit (QIAGEN, Hilden, Germany), and the concentration of RNA was then determined from the absorbance at 260 nm.

Competitive Reverse Transcription-Polymerase Chain Reaction. Competitive reverse transcription-polymerase chain reaction (RT-PCR) was performed as described (Gilliland et al., 1990) with minor modifications. One microgram of total RNA was reverse-transcribed in a 20-µl reaction, using oligo(dT)₁₆ as a primer, at 42°C for 15 min. The resulting cDNA mixture was divided into aliquots and used to measure all parameters in a single RT reaction. Specific PCR primers for each target were designed as shown in Table 1. The primers for TGF-₁ have been reported (Nadeau et al., 1995). We checked the PCR products as a target sequence by restriction enzyme mapping. To synthesize the homologous, deletion-mutated competitors, the RT-PCR products derived from the total RNA from rat artery or liver were subcloned into a pCR 2.1 vector (InVitrogen, La Jolla, CA). The plasmids carrying each PCR product were digested by the restriction enzyme(s) listed in Table 1 and self-ligated after blunting the recessed ends with T4 DNA polymerase (New England Biolabs, Beverly, MA). The competitors used in this study were mostly deletion-mutated cDNAs amplified by PCR using the plasmid-carrying deletion-mutated cDNA as a template and its corresponding primers. The exception was the competitor for TGF-₁, which was a heterologous DNA produced with a Competitive DNA PCR Kit (Takara Shuzo, Tokyo, Japan) using primers as mentioned above (Abe et al., 1995). These competitors were purified using a PCR Purification Kit (QIAGEN). The concentrations were determined from the absorbance at 260 nm. Competitors of four serial dilutions with 3-fold steps were mixed with the cDNA mixture derived from the total RNA from the carotid artery and then subjected to the PCR. The PCR was performed using a Gene Amp RNA PCR kit (Perkin-Elmer) and Gene Amp PCR system (model 9700; Perkin-Elmer). The amplification sequence consisted of an initial denaturation at 95°C for 2 min followed by 28 to 38 cycles (indicated in Table 1) of 95°C for 15 s and 60°C for 30 s, with the final step performed at 72°C for 7 min according to the manufacturer's protocol. PCR products were electrophoresed in a 2% agarose gel and photographed after visualization by ethidium bromide staining. The density of the bands was analyzed using NIH Image computer software on a Macintosh computer (Becker et al., 1996). A given mRNA level was expressed as a ratio with respect to the level of mRNA for GAPDH (determined concomitantly).

Morphological Measurements. Seventy-nine rats were used for the morphological study. Rats were anesthetized with sodium pentobarbital and perfused with PBS followed by fixation solution (1% paraformaldehyde and 2% glutaraldehyde in PBS). The perfusion-fixed carotid arteries were excised and immersed in neutralized formalin. After fixation, the artery was embedded in paraffin, and Elastica van Gieson-stained cross sections were prepared. Neointimal and medial areas were measured with the aid of a semiautomatic digitizing system (System Supply, Nagano, Japan). The mean intimal and medial areas for each artery were determined from eight sections obtained from the isolated portion of the artery.

Measurement of Blood Pressure and Heart Rate. In the rats used for the morphological study, 1 day before the isolation of the artery (i.e., on day 13 after the injury), systolic blood pressure (SBP) and heart rate (HR) were measured by the tail cuff method (Indirect Blood Pressure Meter, BP-98A; Softron, Tokyo, Japan). Measurements were taken at 6 h (for KRH-594 and TCV-116) or 2 h (for hydralazine hydrochloride) after drug administration (the times at which the maximal effects of these drugs on blood pressure were observed).

Statistical Analysis. All data are expressed as mean ± S.E. Statistical significance was determined by a one-way analysis of variance followed by Dunnett's two-sided multiple comparison test. Student's t test was used when comparisons were made between two groups. P values less than .05 were considered significant.

	Results

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Effects of KRH-594 on Gene Expression. To determine the levels of various mRNAs in the carotid artery, we performed competitive RT-PCR using the appropriate deletion-mutated cDNA as a competitor. This strategy enabled us to detect a low level of mRNA in the small pieces of tissue and to measure several mRNAs stably during the quantification period. In a preliminary study, we found we could detect changes of at least 2-fold in the level of each mRNA by our quantification protocol using a serially diluted cDNA mixture as a template. The relationship between the dilution constant of the cDNA mixture and the obtained values gives a good fit (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effect of KRH-594 on the levels of mRNA for AT1A, AT1B, and PDGF-R in the rat carotid artery at several time points after balloon injury. The left carotid artery was isolated at the indicated time after the injury. Rats were orally treated either with KRH-594 at 3 (hatched bar) or 10 (solid bar) mg/kg/day or with vehicle (open bar). Total RNA was isolated, and each mRNA level was measured by competitive RT-PCR and normalized by the GAPDH mRNA level. Each point represents the mean ± S.E. for five to eight rats. *p < .05, **p < .01 versus vehicle-treated control. p < .05, p < .01 versus day 0.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Effect of KRH-594 on the levels of mRNA for fibronectin, TGF-1, and type I and type III collagens in the rat carotid artery after balloon injury. The left carotid artery was isolated at the indicated time after the injury. Rats were orally treated either with KRH-594 at 3 (hatched bar) or 10 (solid bar) mg/kg/day or with vehicle (open bar). Total RNA was isolated, and each mRNA level was measured by competitive RT-PCR and normalized by the GAPDH mRNA level. Each point represents the mean ± S.E. for five to eight rats. *p < .05, **p < .01 versus vehicle-treated control. p < .05, p < .01, p < .001 versus day 0.

Morphology of the Balloon-Injured Carotid Artery. Under the light microscope, obvious neointima formation could be seen in the cross sections of the control carotid artery 14 days after balloon injury (Fig. 3A, above the internal elastic lamina). KRH-594 at 10 and 30 mg/kg/day (Fig. 3, B and C), and 3 mg/kg/day dosage of another AT₁ antagonist, TCV-116 (Fig. 3E), markedly suppressed this neointimal formation. The quantitative analysis shown in Fig. 4 demonstrated that both the intimal area and the intima-to-media (I/M) ratio were reduced by KRH-594 and TCV-116 in a dose-dependent manner. In fact, KRH-594 at 10 and 30 mg/kg/day and TCV-116 at 3 mg/kg/day significantly reduced the I/M ratio by 53.8%, 58.8%, and 44.2%, respectively. Hydralazine hydrochloride (10 mg/kg/day) depressed it by 34.7% of control, but the decrease was not statistically significant.

View larger version (97K): [in this window] [in a new window]   Fig. 3.   Typical vertical sections of the rat carotid artery 2 weeks after balloon injury. A, vehicle-treated control. B, 10 mg/kg/day KRH-594. C, 30 mg/kg/day KRH-594. D, 1 mg/kg/day TCV-116. E, 3 mg/kg/day TCV-116. F, 10 mg/kg/day hydralazine. Open arrows indicate the internal elastic lamina. Elastica van Gieson stain; scale bar, 100 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 4.   Intimal area and I/M ratio for the carotid artery on day 14 after injury. Intimal and medial areas in Elastica van Gieson-stained specimens were measured under a light microscope using a semiautomatic digitizing system. Each column represents mean ± S.E. for 8 to 10 rats. *p < .05, **p < .01, ***p < .001 versus the corresponding vehicle-treated control. 1, vehicle-treated control-1. 2, 3 mg/kg/day KRH-594. 3, 10 mg/kg/day KRH-594. 4, 30 mg/kg/day KRH-594. 5, 1 mg/kg/day TCV-116. 6, 3 mg/kg/day TCV-116. 7, vehicle-treated control-2. 8, 10 mg/kg/day hydralazine. Control-2 is for hydralazine treatment, which was performed separately from KRH and TCV treatments.

Systolic Blood Pressure (SBP) and HR. As shown in Table 2, KRH-594 (3, 10, and 30 mg/kg/day) significantly decreased SBP in a dose-dependent manner. TCV-116 (1 and 3 mg/kg/day) and hydralazine (10 mg/kg/day) also reduced SBP. Although the time point for the maximal hypotensive effect was different for each drug, all three drugs exerted a significant hypotensive effect for at least 8 h after drug administration (data not shown). Neither of the AT₁ antagonists changed heart rate significantly, but hydralazine significantly increased it (Table 2).

Comparison among Effects of Drugs on AT_1A mRNA Level. Finally, we made a quantitative comparison between the effects of KRH-594, TCV-116, and hydralazine on the up-regulation of AT_1A. Figure 5 shows the effects of KRH-594 (3 and 10 mg/kg/day; data from Fig. 1), TCV-116 (3 mg/kg/day), and hydralazine (10 mg/kg/day) on the AT_1A mRNA level 3 days after injury, a time when the gene expression for AT_1A was greatly elevated in control rats. We chose these doses of the three drugs because they produced similar SBP-lowering effects (as shown in Table 2). KRH-594 (10 mg/kg/day) significantly suppressed the mRNA level by 76.5%. TCV-116 (3 mg/kg/day) and hydralazine hydrochloride (10 mg/kg/day) reduced the mRNA level by 60.3% and 25.9%, respectively, but the reductions were not statistically significant.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Effects of KRH-594, TCV-116, and hydralazine on the AT1A mRNA level in the balloon-injured carotid artery. Three days after injury, the artery was isolated. Then the AT1A mRNA level was measured by competitive RT-PCR and normalized with respect to the GAPDH mRNA level. Each column represents mean ± S.E. for 6 to 12 rats. *p < .05 versus vehicle-treated control. 1, vehicle-treated control. 2, 3 mg/kg/day KRH-594. 3, 10 mg/kg/day KRH-594. 4, 3 mg/kg/day TCV-116. 5, 10 mg/kg/day hydralazine hydrochloride. 6, noninjured control.

	Discussion

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Balloon injury induces an up-regulation of RAS components (Rakugi et al., 1993, 1994; Fernandez-Alfonso et al., 1997; Iwai et al., 1997), including AT₁ (Viswanathan et al., 1992; Fernandez-Alfonso et al., 1997), located in the arterial wall. Moreover, ACE inhibitors (Powell et al., 1989) and AT₁ antagonists (Kauffman et al., 1991; Kawamura et al., 1993) prevent neointimal formation after such injury. For this reason, it has been considered that local RAS is involved in the production of vascular thickening after injury. Here, we report that a selective AT₁ antagonist KRH-594 inhibited the up-regulation of its target receptor, AT_1A, after balloon injury. Many factors regulate AT₁ gene expression in vitro (Nickenig and Murphy, 1994; Lassegue et al., 1995; Ullian et al., 1996; Nickenig et al., 1997). However, the regulators of AT₁ gene expression in injured artery are still unknown. The prevention of the up-regulation of AT_1A by KRH-594 indicates that AT_1A expression in the injured artery is at least in part regulated by Ang II, which is probably produced by a local RAS. This seems to be confirmed by the previous findings that Ang II, when infused after balloon injury, induced an increase in AT₁ receptor levels that correlated with increased DNA replication in the neointima (deBlois et al., 1996). In addition, the prevention of AT_1A up-regulation by KRH-594 almost certainly dose not result from a decrease in the size of the neointima because neointimal formation (Fernandez-Alfonso et al., 1997), and the associated enhancement of DNA replication (Clowes et al., 1983) had not even taken place at 3 days after the injury (the time when the raised AT_1A mRNA level was found to be reduced by KRH-594 in the present study). Further studies are needed to clarify exactly how an AT₁ antagonist regulates AT_1A gene expression.

Most of the functions of Ang II are mediated by AT₁, especially the AT_1A subtype, in the rat (Inagami and Kitami, 1994). Rat AT_1A and AT_1B show a high homology with each other in terms of their amino acid sequence (96%); however, there is very little homology between their promoter regions (~36%), which suggests a possible difference in the mechanisms regulating receptor expression (Inagami and Kitami, 1994). In the present study, AT_1B, unlike AT_1A, was not up-regulated in response to injury. The subtype recognition of KRH-594 for these two receptors is still unknown. However, the finding that neither balloon injury nor this AT₁ antagonist significantly affected AT_1B expression in the injured artery may indicate a differential regulation of these two subtypes of AT₁.

For the following reasons, PDGF and its receptors are also considered to be major factors in the vascular thickening that occurs after injury. An antibody to PDGF has been reported to reduce the neointimal smooth muscle accumulation after angioplasty (Ferns et al., 1991). Both PDGF-R mRNA (Majesky et al., 1990) and protein (Sirois et al., 1997) are induced in the artery in response to injury (after transient down-regulation; Majesky et al., 1990), especially in the neointima. In addition, PDGF-R and -R phosphorylations are enhanced in the injured artery (Panek et al., 1997). Moreover, a recent report demonstrated that transactivation of PDGF-R occurred on Ang II stimulation through AT₁ in cultured VSMCs (Linseman et al., 1995). Ang II-mediated tyrosyl phosphorylation of PDGF-R and -R is also observed in the balloon-injured rat artery, although the PDGF-R protein level is not affected (Abe et al., 1997). In the present study, the PDGF-R mRNA level increased in response to injury, and KRH-594 suppressed this increase. Taken together with our data on AT_1A, we think that Ang II may regulate gene expression for both AT_1A and PDGF-R in the injured artery and that the suppressive effect of KRH-594 on the neointimal formation that occurs after balloon injury is mediated in part by a prevention of PDGF-R up-regulation.

It is well known that the accumulation of ECM, after its synthesis by neointimal SMC, causes vascular thickening after injury and that Ang II stimulates ECM production in cultured VSMCs (Kato et al., 1991). As representatives of the many ECM components, we measured the mRNA levels for fibronectin and type I and type III collagen, whose expression is well characterized in this model (Majesky et al., 1991; Kim et al., 1995). In addition, the mRNA for TGF-₁ was measured because TGF-₁ has been reported to up-regulate the ECM components mentioned above (Chen et al., 1987) and to contribute to neointima formation after arterial injury (Wolf et al., 1994). In the present study, mRNA levels for fibronectin and TGF-₁ increased, and these for the two types of collagen were unchanged after injury. The expression profiles for these genes were consistent with those in the previous reports (Majesky et al., 1991; Kim et al., 1995). KRH-594 at 10 mg/kg/day reduced the level of fibronectin mRNA throughout the 14-day study period, and that of TGF-₁ mRNA from day 3 to day 14, and down-regulated both collagens from 6 h to 7 days. However, KRH-594 failed to decrease the enhanced TGF-₁ mRNA level at 6 h and 1 day after the injury, although it down-regulated the levels of the mRNA for fibronectin and two collagens at these time points. Although the situation is complicated because active TGF-₁ is post-translationally formed by a complex process, these results seem to suggest that the gene expression of the ECM components measured in this study is regulated by Ang II rather than by TGF-₁, at least in the first day or so after injury.

In the present study, KRH-594, TCV-116, and hydralazine all significantly decreased SBP on day 13 after the injury in a dose-dependent manner. The decrease in SBP seemed to correlate with the reduction in neointimal formation. However, the effect of hydralazine on the suppression of neointimal formation was less than that of the AT₁ antagonists when doses with similar blood pressure-lowering effects were compared in the present study. Similar results for hydralazine were indicated by others compared with an ACE inhibitor (Powell et al., 1991). Moreover, 3 mg/kg/day KRH-594 and 1 mg/kg/day TCV-116 significantly decreased SBP but not the I/M ratio. On the basis of these results, it is suggested that AT₁ antagonists are able to reduce vascular thickening after injury, partly via their suppressive effect on AT_1A up-regulation, which can be distinguished from their blood pressure-lowering effects.

In summary, we demonstrated that the AT₁ antagonist KRH-594 prevents the up-regulation of AT_1A, PDGF-R, and ECM-related genes that normally occurs after arterial injury and that it decreases the subsequent neointimal formation. These findings are consistent with the ideas that the up-regulation of AT_1A and PDGF-R that occurs in the balloon-injured rat carotid artery is at least in part an AT₁-mediated phenomenon and that a prevention of the up-regulation of this receptor may contribute to the effects of AT₁ antagonists in reducing neointimal formation after arterial injury.