Induction of Renin in Medial Smooth Muscle Cells by Balloon Injury
Abstract We studied the expression of each component of the renin-angiotensin system (renin, angiotensin I–converting enzyme, angiotensinogen, and angiotensin II type I receptor) in balloon-injured rat carotid artery. We assessed the expression levels of the respective mRNAs by competitive polymerase chain reaction. Renin mRNA concentration was markedly increased 24 hours after balloon injury and remained higher than that in the control at 7 days after balloon injury. Angiotensin-converting enzyme mRNA concentration was decreased 24 hours after balloon injury and was increased at 14 days after balloon injury. No significant change in angiotensinogen mRNA concentration was observed throughout the study period. Angiotensin type I receptor mRNA concentration was increased beginning 3 days after balloon injury and remained higher than that in the control at 14 days after balloon injury. Immunohistochemical analysis showed that renin was transiently expressed in medial smooth muscle cells after balloon injury. Administration of quinapril markedly reduced neointimal formation and was accompanied by an attenuation of the increase in the concentrations of angiotensin type I receptor and angiotensin-converting enzyme mRNAs. The upregulation of renin mRNA in balloon-injured rat carotid artery preceded and may play a role in neointimal formation.
The best-studied model of neointimal formation is the response of the rat carotid artery to balloon injury.1 2 In the rat, the balloon-injury model begins with complete destruction of the endothelium and extensive death of medial smooth muscle cells, followed by medial smooth muscle cell proliferation.3 4 Medial smooth muscle cells migrate into the intima2 5 6 and then start a transient burst phase of proliferation.7 Finally, smooth muscle cells deposit large amounts of extracellular matrix material.8
Many kinds of growth factors and cytokines, such as basic fibroblast growth factor, Ang II, platelet-derived growth factors, insulin-like growth factor I, and transforming growth factor-β, have been suggested to play important roles in neointimal formation following balloon injury. Blockade of the RAS by ACE inhibitors or AT1R antagonists has been shown to reduce neointimal formation in the rat.5 9 10 Ang II is known to be a growth-promoting factor.11 Several previous studies have indicated that Ang II generation might be elevated in balloon-injured artery. Induction of both ACE gene expression in neointima12 13 and AGT gene expression in the media and neointima14 have been reported. Smooth muscle cells in the neointima have been reported to show a higher expression level of AT1R than those in the media.15 This increased AT1R expression in neointimal smooth muscle cells may in itself be sufficient to enhance the local proliferation of smooth muscle cells caused by Ang II.
However, the increased expression of ACE, AGT, and AT1R were all observed 1 week after balloon injury and did not seem to precede neointimal formation. Recent detailed analyses of the mechanisms of ACE inhibitors in reducing neointimal formation have indicated that the inhibition of smooth muscle cell migration from media to intima is the main mechanism by which neointimal formation is reduced.16 17 The migration of smooth muscle cells from media to intima reportedly occurs 3 to 14 days after balloon injury.2 17 If Ang II is responsible for this migration, enhanced generation of or sensitivity to Ang II should occur before this migration. Therefore, we investigated the time course of the expression of renin, ACE, AGT, and AT1R mRNAs in balloon-injured rat carotid artery.
Carotid Artery Balloon Injury
Male Sprague-Dawley rats (350 to 400 g) were obtained from Clea Japan (Osaka). The rats were anesthetized with sodium pentobarbital (40 mg/kg IP). A 2F Fogarty catheter (Baxter) was inserted through the right iliac artery to the left common carotid artery. The left common carotid artery was exposed for visual confirmation of catheter insertion. The inflated balloon (0.4 mL air) was pulled through the common carotid artery three times, and the right iliac artery was permanently ligated. This study was conducted in accordance with current guidelines for the care and use of experimental animals of Shiga University of Medical Sciences.
RNA Isolation and Analysis
Rats were perfused via the left ventricle with ice-cold phosphate-buffered saline (PBS: 150 mmol/L NaCl in 10 mmol/L Na2HPO4/NaH2PO4, pH 7.4) to remove blood, and the left and right common carotid arteries between the bifurcation of the external carotid artery and aorta were carefully removed. A small portion (5 mm) was cut from the aortic end. For RNA isolation, the adventitia was carefully removed, and three carotid arteries were pooled to make one RNA sample. Total RNA was extracted according to the method of Chomczynski and Sacchi18 with a polytron homogenizer (Kinematica AG). RNA concentration was spectrophotometrically determined at 260 nm, and RNA quality was visually confirmed by agarose gel electrophoresis, as previously reported.19 The expression levels of renin, ACE, AGT, and AT1R mRNAs were determined by a competitive RT-PCR method as previously reported.19 20 21
Briefly, 2 μg of total RNA samples mixed with known amounts (Table⇓) of the deletion- or insertion-mutated cRNA for renin, ACE, AGT, and AT1R underwent RT using random primers. The resulting cDNA mixture was purified by phenol/chloroform extraction and two rounds of ethanol precipitation with ammonium acetate and dissolved in 40 μL water. Five microliters of the cDNA mixture was amplified in a total reaction mixture of 25 μL containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1.5 to 2.0 mmol/L MgCl2, 0.01% (wt/vol) gelatin, 0.2 mmol/L dNTP, 50 nmol/L [α-32P]dCTP (3000 Ci/mmol), 25 pmol of sense and antisense primers, and 0.5 U Taq DNA polymerase (Toyobo). The PCR amplification profile included an initial denaturing step at 94°C for 1 minute and 30 to 35 cycles at 94°C for 1 minute, 58°C for 1 minute, and 74°C for 1 to 2 minutes. The PCR products were electrophoresed on a 1.7% agarose gel for visual inspection and a 5% polyacrylamide gel for precise quantification, as previously reported.19 20 21 The primers and sizes of the PCR products are summarized in the Table⇓. Since the mutated cRNA for ACE has a 4-bp insertion at the Avr II site, the PCR product from the mutated cRNA lacks this Avr II site. The PCR product from native ACE mRNA should liberate 195- and 122-bp fragments by Avr II (New England Biolabs, Inc) digestion. More than 90% of the AT1R mRNA expressed in the control and balloon-injured arteries was AT1aR mRNA (data not shown). Expression levels of the mRNAs were calculated as Expression Level (molecules per microgram)=Amount of Mutated cRNA (molecules)×(IN/IM)×(CM/CN), where IN and IM represent the intensity of the PCR product from native and mutated RNAs, respectively, and CN and CM represent the content of dCTP in the PCR product from native and mutated RNA, respectively.
To confirm whether the PCR products correspond to the native mRNA, the PCR products were directly sequenced by a sequencing kit (Taq Cycle sequencing kit, Takara Shyuzo, Co Ltd).
The rats were deeply anesthetized with sodium pentobarbital (70 mg/kg IP). They were then perfused via the left ventricle, initially with ice-cold PBS (150 mmol/L NaCl in 10 mmol Na2HPO4/NaH2PO4, pH 7.4) and subsequently with a fixative containing 4% paraformaldehyde in 0.1 mol/L phosphate buffer (0.1 mol/L Na2HPO4/NaH2PO4, pH 7.4). The carotid arteries were immersed for 2 days in a postfixative containing 4% paraformaldehyde in phosphate buffer at 4°C. The arteries were then placed in phosphate buffer containing 15% sucrose for 2 days. The arteries were frozen and cut into 20-μm-thick sections with a cryostat. The sections were rinsed for at least 2 days with several changes of PBS containing 0.3% Triton X-100 (PBST) at 4°C before immunohistochemical staining.
Free-floating sections, which were pretreated with 0.5% H2O2 in PBST to destroy intrinsic peroxidase activity, were incubated for 2 days at 4°C with rabbit anti-rat renin antiserum (diluted 1:60 000), for 1 hour at room temperature with biotinylated anti-rabbit IgG (diluted 1:1000), and for 1 hour at room temperature with avidin-biotin-peroxidase complex (diluted 1:4000, ABC Elite, Vector). All sera were diluted with PBST, and sections were always rinsed in PBST after each step. Peroxidase activity was revealed by 0.02% 3,3′-diaminobenzidine (Wakenyaku) in 50 mmol/L Tris-HCl (pH 7.6), 0.005% H2O2, and 0.3% nickel ammonium sulfate. Control experiments included the substitution of primary antiserum with preimmune serum or preabsorbed serum, which showed no specific staining. Rabbit antiserum to rat renin was prepared as previously reported.22 Its specificity was ascertained by the lack of a cross-reaction with human renin and rat cathepsin D, at dilutions greater than 1:500. Used at a dilution of 1:80 000 in the immunohistochemical staining of rat kidney by the method described above, juxtaglomerular cells were stained exclusively.
Effects of Quinapril
Quinapril was dissolved in drinking water. Quinapril (10 mg/kg per day) or placebo (water) was administered orally once a day for 15 days (1 day before and 14 days after balloon injury). For assessment of the intimal area, three cross sections were cut per carotid artery and stained with hematoxylin-eosin. Cross-sectional intimal areas were determined with an image analyzer (Luzex 3, Nikon). The mean value of the intimal or medial areas determined from these three cross sections was considered the intimal or medial area for each rat.
Data are expressed as mean±SD. Statistical analyses were performed with one-or two-way ANOVA. When Bartlett’s test for the homogeneity of variances suggested that within-group variance was not homogeneous among the groups, a logarithmic transformation was performed to allow for the use of ANOVA.
Validity of the Competitive RT-PCR Method
The validity of the competitive RT-PCR method has been confirmed in previous studies.19 20 21 Fig 1⇓ illustrates the validity of the competitive RT-PCR method for the quantification of ACE mRNA level. Synthetic ACE cRNA combined with various amounts of synthetic point-mutated ACE cRNA was reverse-transcribed and amplified by PCR. The observed molar ratio was highly correlated with the molar ratio of ACE cRNA to point-mutated ACE cRNA (R2=.984, P<.0001) within the range examined.
The PCR products from the carotid arteries 48 hours after balloon injury were directly sequenced and confirmed to correspond to the native mRNAs (renin, AGT, ACE, and AT1aR) (data not shown).
Time Course of RAS mRNA Expression in Balloon-Injured Carotid Artery
Histological examination at 14 days after balloon injury revealed marked neointimal formation only in the balloon-injured (left) carotid artery, which confirmed the validity of our procedures (Fig 2⇓).
Fig 3⇓ shows a typical assessment of the expression levels of RAS mRNA, and Fig 4⇓ summarizes the time course of the expression levels of RAS mRNA after balloon injury. The renin mRNA concentration was markedly increased 24 hours after balloon injury, and an increased renin mRNA concentration was still evident 7 days after balloon injury. The renin mRNA concentration at 14 days after balloon injury was not significantly higher than that in control intact carotid artery (Fig 4A⇓). The AT1R mRNA concentration was significantly increased beginning 3 days after balloon injury and remained higher than that in the control at 14 days after balloon injury (Fig 4B⇓). More than 90% of the AT1R mRNA expressed in the control and balloon-injured arteries consisted of AT1aR mRNA (data not shown). The ACE mRNA concentration was decreased 24 hours after balloon injury and increased 14 days after balloon injury (Fig 4C⇓). No significant change in AGT mRNA concentration was observed throughout the study period (Fig 4D⇓).
Immunohistochemical analysis revealed that medial smooth muscle cells showed renin-like immunoreactivity after balloon injury (Fig 2B⇑). This immunoreactivity was evident 24 hours and 3 days after balloon injury and was not evident 14 days after balloon injury (Fig 2⇑). No significant renin-like immunoreactivity was detected in neointima (Fig 2C⇑). Occasionally, renin-like immunoreactivity was detected in adventitia between 1 and 3 days after balloon injury (Fig 2F⇑). Some of these cells in the adventitia were stained with OX-42, a monoclonal antibody to macrophage/monocyte cells (data not shown). This time course of renin-like immunoreactivity paralleled that of renin mRNA expression (Figs 2⇑ and 4⇑).
Effects of ACE Inhibitor
On the basis of the above observations, we hypothesized that the increased renin expression in medial smooth muscle cells after balloon injury might be responsible for Ang II generation in situ and for the subsequent events leading to neointimal formation. Administration of the ACE inhibitor quinapril for 14 days markedly reduced neointimal formation (Fig 5⇓). Although quinapril administration did not modify renin mRNA expression in balloon-injured artery, it attenuated the increase in AT1R mRNA expression at 3, 7, and 14 days after balloon injury (Fig 6⇓). Likewise, quinapril administration significantly attenuated the increase in ACE mRNA expression at 14 days after balloon injury (Fig 6⇓).
Blockade of the RAS by ACE inhibitors or AT1R antagonists reduces neointimal formation after balloon injury.5 9 10 Moreover, neointimal formation after balloon injury was exaggerated by prolonged Ang II infusion.23 Therefore, it is evident that Ang II is involved in neointimal formation. Induction of gene expression for ACE,12 13 AGT,14 and AT1R15 in the neointima has been reported. The present study reports for the first time that renin expression is transiently induced in the medial smooth muscle cells of balloon-injured carotid artery.
Expression of Renin Gene in Medial Smooth Muscle Cells
Renin mRNA expression was prominently induced in balloon-injured carotid artery, and renin-like immunoreactivity was detected in medial smooth muscle cells. We cannot exclude the possibility that renin may be synthesized in as yet unidentified cells in balloon-injured artery, and renin may be taken up and concentrated in medial smooth muscle cells. In fact, cells with renin-like immunoreactivity were occasionally detected in adventitia between 1 and 3 days after balloon injury (Fig 2F⇑). We recently reported that monocyte/macrophage cells infiltrating necrotic myocardium can express renin.24 Indeed, some of the cells with renin-like immunoreactivity in the adventitia were positively stained with OX-42, a monoclonal antibody to monocyte/macrophage cells. However, infiltration of monocyte/macrophage cells was not a consistent finding in the samples examined, while the induction of renin mRNA in balloon-injured carotid artery was observed consistently. It is well known that, unlike in arteries in larger animals, thrombus formation and leukocyte infiltration are minimal in balloon-injured rat artery.25 Moreover, the expression level of renin mRNA in the adventitia of balloon-injured carotid artery was not significantly higher than that in the control carotid artery (data not shown).
Although renin mRNA could not be detected in cultured vascular smooth muscle cells,26 we should not be surprised to find renin gene expression in vascular smooth muscle cells in vivo. Juxtaglomerular cells are known to be modified smooth muscle cells,27 and reninlike immunoreactivity has been detected in vascular smooth muscle cells of fetal intrarenal arteries.28 29
Possible Pathophysiological Significance
Neointimal formation following balloon injury in rat carotid artery consists of four steps.2 16 17 The first step is replication of smooth muscle cells in the media, which occurs 0 to 3 days after balloon injury. The second step is migration of smooth muscle cells from the media to the intima, which occurs 3 to 14 days after balloon injury. The third and fourth steps are proliferation of smooth muscle cells and deposition of extracellular matrix in the neointima, which begins to occur 7 days after balloon injury. The main mechanism of the inhibition of neointimal formation by blockade of the RAS involves inhibition of the first two steps of neointimal formation; ie, medial smooth muscle replication and migration of medial smooth muscle cells into the intima.16 17 Thus, it is reasonable to hypothesize that Ang II generation in situ or Ang II sensitivity in situ may be enhanced during these two steps.
An increase in the AT1R mRNA concentration was evident beginning 3 days after balloon injury. Viswanathan et al15 reported that neointimal smooth muscle cells expressed fourfold more AT1R than medial smooth muscle cells. The gradual increase in the AT1R mRNA concentration may reflect neointimal formation and/or medial smooth muscle cell replication. In either case, this increase was observed beginning 3 days after balloon injury and could not precede the first step of neointimal formation; ie, medial smooth muscle cell replication.
After denudation of endothelium, the ACE mRNA concentration in the balloon-injured artery was decreased, probably because of a high ACE mRNA concentration in endothelial cells. The ACE mRNA concentration gradually increased with neointimal formation. The site of ACE expression is reportedly vascular smooth muscle cells in the neointima.12 13
On the other hand, the induction of renin gene expression in balloon-injured artery preceded neointimal formation. The time course of renin mRNA expression indicated that renin gene expression was induced in medial smooth muscle cells during the first and second steps of neointimal formation. As described above, blockade of the RAS reduces neointimal formation through inhibition of these two steps.16 17 Therefore, it is conceivable that induced renin in the medial smooth muscle cells may be rate limiting and a key to Ang II generation in situ in balloon-injured rat carotid artery. Assessment of the time course of the Ang II level in balloon-injured carotid artery may be necessary for a definitive answer. This issue requires further investigation.
The increase in the AT1R and ACE mRNA concentrations in balloon-injured artery may be interpreted as a reflection of neointimal formation. Attenuation of this increase by quinapril indicated that this ACE inhibitor could reduce neointimal formation. Since vascular smooth muscle cells in neointima express higher levels of AT1R and ACE mRNAs, reduction of the neointimal area leads to reduced concentrations of AT1R and ACE mRNAs.
Quinapril administration did not cause a further induction of renin mRNA in balloon-injured artery. This indicates that Ang II has no significant effects on renin gene expression in these phenotypically modified vascular smooth muscle cells. It is generally accepted that the expression of a gene is regulated in a tissue-specific manner. Thus, it should be no surprise that the renin gene expression in the medial smooth muscle cells is regulated differently than that in juxtaglomerular cells. The precise molecular mechanisms for the induction of renin gene expression in medial smooth muscle cells remain to be determined. Clarification of these mechanisms may be helpful for identifying new strategies for inhibiting the first two steps of neointimal formation.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1R||=||angiotensin II type 1 receptor|
|PCR||=||polymerase chain reaction|
This study was supported in part by a grant-in-aid from the Japanese Ministry of Education, Science, and Culture.
- Received May 17, 1996.
- Revision received June 13, 1996.
- Accepted October 14, 1996.
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