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(Circulation. 1995;92:88-95.)
© 1995 American Heart Association, Inc.

Articles

Angiotensin II Type 1 Receptor Blockade Inhibits the Expression of Immediate-Early Genes and Fibronectin in Rat Injured Artery

Shokei Kim, MD; Masaki Kawamura; Hideki Wanibuchi, MD; Kensuke Ohta, MD; Akinori Hamaguchi; Takashi Omura, MD; Tokihito Yukimura, MD; Katsuyuki Miura, MD; Hiroshi Iwao, MD

From the Department of Pharmacology (S.K., K.O., A.H., T.O., T.Y., K.M., H.I.) and First Department of Pathology (H.W.), Osaka City University Medical School; and Pharmaceutical Research Division (M.K.), Takeda Chemical Industries Ltd, Osaka, Japan.

Correspondence to Shokei Kim, MD, Department of Pharmacology, Osaka City University Medical School, 1-4-54 Asahimachi, Abeno, Osaka 545, Japan.

	Abstract

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Background Vascular injury activates various kinds of genes, including proto-oncogenes, growth factors, and extracellular matrix proteins. However, the significance of activation of these genes in neointimal formation is poorly understood. Angiotensin II type 1 (AT₁) receptor antagonist is shown to prevent neointimal formation after vascular injury, although the mechanism is unclear. To understand the molecular mechanism of vascular thickening, we examined the effects of AT₁ receptor blockade on the gene expression of proto-oncogenes, transforming growth factor–ß₁ (TGF-ß₁), and extracellular matrix proteins after vascular injury.

Methods and Results Endothelial denudation of the left common carotid artery in Sprague-Dawley rats was performed with a Fogarty 2F balloon catheter. TCV-116 (10 mg · kg^-1 · d^-1), a selective nonpeptide AT₁ receptor antagonist, or vehicle was administered orally to rats from 1 day before to 14 days after balloon injury. Injured left and uninjured right common carotid arteries were removed from rats at 1, 6, and 24 hours and 3, 7, and 14 days after balloon injury. Tissue mRNA levels were measured with Northern blot analysis using specific cDNA probes and corrected for 18S ribosomal RNA value. Arterial mRNAs for c-fos, c-jun, jun B, jun D, and Egr-1 increased significantly at 1 hour after balloon injury and decreased rapidly. At 6 hours, ornithine decarboxylase (ODC) mRNA expression reached the maximal levels. TGF-ß₁ and fibronectin mRNA levels started to increase at 6 hours after injury and remained enhanced until 7 days after injury. On the other hand, collagen types I, III, and IV and laminin mRNA levels were not significantly increased over 7 days. Treatment with TCV-116 significantly inhibited the induction of mRNAs for c-fos, c-jun, Egr-1, ODC, and fibronectin in injured artery, whereas the increase in TGF-ß₁ gene expression after injury was not prevented by TCV-116. Immunohistological studies indicated that TCV-116 decreased not only the intimal thickening but also the amount of these extracellular matrix proteins in the intima.

Conclusions The results indicate that AT₁ receptor blockade inhibits the induction of immediate-early genes, ODC, and fibronectin in rat injured artery. Thus, inhibition of intimal thickening by AT₁ receptor blockade may be mediated at least in part by suppression of multiple genes related to cell growth and migration in the very early phase after vascular injury.

Key Words: TCV-116 • genes • angiotensin

	Introduction

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Arterial injury produced by balloon angioplasty causes proliferation and migration of medial smooth muscle cells, leading to progressive neointimal thickening and narrowing of the vascular lumen.^{1 2} This arterial repair process has been shown to be associated with significant induction of various kinds of genes, including immediate-early genes,^{3 4} growth factors,^{5 6} and extracellular matrix components,⁶ thereby suggesting the importance of these genes in the formation of neointima. However, the in vivo role of these activated genes in injured artery remains to be elucidated.

Angiotensin II type 1 (AT₁) receptor antagonist^{7 8 9} as well as angiotensin-converting enzyme (ACE) inhibitors¹⁰ prevent neointimal formation after vascular injury of the carotid artery in rats. On the other hand, a calcium channel antagonist, with a blood pressure–lowering effect similar to AT₁ receptor antagonist, fails to prevent neointimal formation after vascular injury.¹⁰ Furthermore, exogenously infused angiotensin II induces smooth muscle cell proliferation in both normal and injured arteries.¹¹ These findings, taken together with higher expression of AT₁ receptor in the neointima than in the media,¹² indicate that angiotensin II, via AT₁ receptor, plays an important role in vascular thickening in this model. Thus, investigation of the mechanism of prevention of neointimal thickening by AT₁ receptor blockade is essential in elucidating the mechanism of injury-induced vascular smooth muscle cell proliferation and migration. However, the molecular mechanism by which AT₁ receptor blockade in vivo inhibits vascular thickening remains to be determined. In the present study, we examined the effect of AT₁ receptor antagonist on the gene expression of immediate-early genes, transforming growth factor–ß₁ (TGF-ß₁), and extracellular matrix components in injured rat carotid artery. We obtained evidence for specific suppression of expression of immediate-early genes, ornithine decarboxylase (ODC), and fibronectin by AT₁ receptor antagonist in injured artery.

	Methods

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Drug
TCV-116 was synthesized by Takeda Chemical Industries, Ltd. TCV-116 is a highly potent and selective nonpeptide AT₁ receptor antagonist without agonistic action and has long-acting antagonistic activity in vivo.^{9 13}

Balloon Injury
All procedures were done in accordance with institutional guidelines for animal research. The present study was performed on 291 male Sprague-Dawley rats (age range, 9 to 12 weeks; Clea Japan). Rats were separated into two groups: the vehicle-treated group (control group, n=146) and the TCV-116–treated group (n=145). TCV-116 (10 mg · kg^-1 · d^-1) suspended in gum arabic was administered to rats by gastric gavage once a day from 1 day before balloon injury to the end of the experiments. The control group of rats (vehicle-treated rats) were administered an equal volume of gum arabic in the same manner. For balloon injury, rats were anesthetized with sodium pentobarbital (50 mg/kg IP), and the endothelium of the left common carotid artery was denuded by three passages of a Fogarty 2F balloon catheter (Baxter Healthcare) as previously described.⁹

For Northern blot analysis, rats were anesthetized with pentobarbital at various time points after balloon injury, and the injured left common carotid artery and noninjured right common carotid artery (control) were rapidly isolated from adherent tissues, frozen in liquid nitrogen, and stored at -80°C until use. The number of rats examined at 1, 6, and 24 hours and 3, 7, and 14 days was 29, 28, 30, 29, 14, and 14, respectively, for the vehicle-treated group and 29, 29, 27, 29, 14, and 15, respectively, for the TCV-116–treated group. Northern blot analysis was performed on four different pools of samples for each group at 1, 6, and 24 hours and 3 days after injury and on one pool of samples for each group at 7 and 14 days after injury.

For pathological examination and immunohistochemistry, the middle third segments of injured left and noninjured right common carotid arteries were excised from vehicle-treated (n=3) and TCV-116–treated (n=3) rats at 14 days after balloon injury and processed as described.

RNA Isolation and Northern Blot Hybridization
Total RNA was extracted from the pooled carotid arteries according to the method of Chomczynski and Sacchi,¹⁴ as previously described.¹⁵ The RNA pellet was dissolved in 0.1% diethyl pyrocarbonate–treated water and stored at -80°C until use. The RNA concentration was spectrophotometrically determined at 260 nm. From each pool of carotid arteries, 10 µg of total RNA was denatured by incubation with 1 mol/L deionized glyoxal and 50% dimethyl sulfoxide at 50°C for 1 hour, electrophoresed on a 1% agarose gel at 50 V, and transferred to a nylon membrane (Gene Screen Plus, EI du Pont de Nemours & Co, NEN Products).¹⁵ Each cDNA probe was labeled with (³²P)-dCTP (specific activity, 3000 Ci/mmol/L; EI du Pont de Nemours & Co) by random primer extension method¹⁶ with a Random Primer DNA Labeling Kit (Takara). Prehybridization and hybridization were performed according to the manufacturer's instructions, as previously described.¹⁵ In brief, the membrane was prehybridized in a solution containing 50% formamide, 5x Denhardt's solution (containing Ficoll, polyvinylpyrrolidone, and bovine serum albumin, 1 mg/mL each), 5x SSPE (containing 0.75 mol/L sodium chloride, 50 mmol/L sodium phosphate, and 5 mmol/L EDTA), 1% sodium dodecyl sulfate (SDS), and 200 µg/mL denatured salmon sperm DNA at 42°C for 4 hours. Then, the membrane was hybridized with ³²P-labeled cDNA (1 to 2x10⁶ dpm/mL) at 42°C for 24 hours in fresh hybridization solution that was identical to the prehybridization solution except for the absence of salmon sperm DNA. After being washed, the membrane was exposed to Kodak XAR-5 film between two intensifying screens at -70°C. The nylon membrane was stripped off by being boiled in 0.1x SSC (1x SSC contains 0.15 mol/L sodium chloride and 0.015 mol/L sodium citrate, pH 7) solution containing 1% SDS and then rehybridized with other cDNA probes.

In addition, to monitor the RNA content of the different lanes, we hybridized the blots with a 24-base oligonucleotide probe (5'-ACGGTATCTGATCGTCTTCGAACC-3') complementary to rat 18S ribosomal RNA.¹⁷ The oligonucleotide probe was labeled with (-³²P)-ATP (6000 Ci/mmol/L) at the 5' end by using T4 polynucleotide kinase (Takara) and was purified by chromatography on a Bio-Spin 6 column (Bio-Rad). The membranes were prehybridized in a solution containing 20 mmol/L NaH₂PO₄ (pH 7.4), 6x SSC, 5x Denhardt's solution, 0.1% SDS, and 200 µg/mL denatured salmon sperm DNA at 42°C for 4 hours and then hybridized in the same solution containing the radiolabeled oligonucleotide probe at 42°C for 24 hours. After hybridization, the membranes were washed in 2x SSC at room temperature for 10 minutes, further washed in 2x SSC containing 1% SDS at 67°C for 60 minutes, and finally washed in 0.1x SSC at room temperature for 20 minutes. Autoradiography was performed as described above.

To evaluate tissue mRNA levels, we used an optical scanner (EPSON GT8000, Seoko) to digitize autoradiograms. The autoradiogram bands in the digitized image were measured for density with the use of the public-domain National Institutes of Health IMAGE program and a computer (Macintosh LC-III, Apple Computer, Inc), as previously described.¹⁵ For all RNA samples, the density of an individual mRNA band was normalized for that of 18S ribosomal RNA to correct for the difference in RNA loading and/or transfer.

cDNA Probes
v-fos cDNA was purchased from Oncor.¹⁸ cDNAs for c-jun,¹⁹ jun B,²⁰ jun D,²¹ and Egr-1²² were purchased from American Type Culture Collection. Rat TGF-ß₁ cDNA (a 1.0-kb HindIII/Xba I fragment) was obtained from Dr S.W. Qian.²³ Rat fibronectin cDNA (a 0.27-kb HindIII/EcoRI fragment) was obtained from Dr R.O. Hynes.²⁴ Rat 1 (I) collagen cDNA (a 1.3-kb Pst I/BamHI fragment) was obtained from Dr D. Rowe.²⁵ Mouse cDNAs for 1 (III) collagen (1.8-kb EcoRI/EcoRI fragment),²⁶ 1 (IV) collagen (0.83-kb Ava I/Pst I),²⁷ and laminin B1 chain (0.65-kb BamHI/EcoRI fragment)²⁸ were obtained from Dr Y. Yamada. Mouse ODC cDNA was provided by Dr P. Coffino.²⁹

Pathological Examination and Immunohistochemistry
For histology, the middle third segment of injured or noninjured common carotid artery was removed from TCV-116– (n=3) or vehicle-treated (n=3) rats at 14 days after balloon injury. For immunohistochemistry, the tissues were immediately embedded in OCT compound (Tissue Tek, Miles, Inc) and frozen in dry ice/acetone. The frozen specimens were cut into 6-µm sections with a cryostat and fixed in cold acetone for 5 minutes at 4°C. The primary antibodies used in the present study were as follows: rabbit anti-rat type I collagen polyclonal antisera (diluted 1:50), rabbit anti-rat type III collagen polyclonal antisera (diluted 1:50), and rabbit anti-rat laminin polyclonal antisera (diluted 1:200) from Chemicon; monoclonal anti-human cellular fibronectin IgG₁ (diluted 1:500) (Upstate Biotechnology, Inc); and rabbit anti-bovine type IV collagen antisera (diluted 1:1000) (LSL, Inc).

Immunohistochemical staining was performed using an avidin-biotin-peroxidase kit (Vectastain Elite ABC kit, Vector Laboratories, Inc). Peroxidase activity was visualized using 3,3'-diaminobenzidine as chromogen, and the sections were counterstained with hematoxylin. As a negative control, the primary antibody was omitted and replaced by an equal concentration of nonimmune rabbit sera or mouse monoclonal IgG.

For quantitative analysis, immunostained sections were examined with a light microscope connected to the image-analysis system IPAP (Sumika Technos Corp).³⁰ All microscopic images were measured at a magnification of x100, at which the artery filled a given field. A binary digitized image of the arterial wall was obtained automatically by the programmed segmentation procedure. The intimal region was then separated from the tunica media by interactively tracing from each measurement the borderline using a computer system with a mouse. The color density of the immunostained sections was measured as the optical density (OD) by the IPAP system to determine the relative density of each antigen in the intima and the media. For each immunostained section, the total antigen score of the intima and the media was calculated as OD multiplied by the intimal area and OD multiplied by the medial area, respectively.

For hematoxylin and eosin staining, arterial tissues were fixed in phosphate-buffered 4% paraformaldehyde, embedded in paraffin, and sliced into 4-µm-thick sections. The intimal and medial areas were calculated as previously described.⁹ Furthermore, the number of intimal cells was counted in each section at a magnification of x600 and then divided by the intimal area.

Statistical Analysis
For data on mRNA at 1, 6, and 24 hours and 3 days and on immunostaining at 14 days after balloon injury, results are expressed as mean±SEM. Data on mRNA were analyzed by two-way ANOVA, and the differences between vehicle-treated and TCV-116–treated groups were determined by the least-squares mean test (SuperANOVA, Abacus Concepts). Statistical significance of the difference in immunohistochemical data between vehicle-treated and TCV-116–treated groups was determined by the unpaired Student's t test because immunohistochemical study was performed on carotid arteries only at 14 days after balloon injury. A value of P<.05 was considered statistically significant.

	Results

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Effects of TCV-116 on Expressions of Immediate-Early Genes and ODC
As shown in Figs 1 and 2, arterial c-fos and Egr-1 mRNAs were not detected in noninjured artery by Northern blot analysis, whereas mRNAs for the jun family (c-jun, jun B, and jun D) could be readily detected in noninjured artery, and slight ODC mRNA was detected. c-fos and Egr-1 mRNA expressions increased significantly to detectable levels at 1 hour after balloon injury and decreased to undetectable levels at 6 hours. mRNA levels for c-jun, jun B, and jun D also increased to maximal levels (10.5-, 6.8-, and 3.5-fold, respectively, compared with noninjured artery) at 1 hour after injury. The expression of ODC mRNA reached a peak (4.8-fold compared with noninjured artery) at 6 hours and remained increased by 1.5-fold at 24 hours.

View larger version (78K): [in this window] [in a new window]   Figure 1. Autoradiographs of mRNAs for c-fos, c-jun, jun B, jun D, Egr-1, ornithine decarboxylase (ODC), and 18S ribosomal RNA (18S rRNA) in carotid artery at 1, 6, and 24 hours after balloon injury. Sizes of mRNA were 2.2 kb for c-fos, 2.7 and 3.2 kb for c-jun, 2.1 kb for jun B, 2.1 kb for jun D, 3.4 kb for Egr-1, and 2.2 and 2.7 kb for ODC. Cont indicates noninjured right carotid artery; Inj, injured left carotid artery; V, vehicle-treated group; and T, TCV-116–treated group.

View larger version (19K): [in this window] [in a new window]   Figure 2. Bar graphs of mRNA levels for c-fos, c-jun, jun B, jun D, Egr-1, and ornithine decarboxylase (ODC) in carotid artery at 1, 6, and 24 hours after balloon injury. Ordinates show each mRNA value, corrected for 18S ribosomal RNA value. For c-jun, jun B, jun D, and ODC, the mRNA levels in noninjured right carotid artery at each time point were regarded as 1. For c-fos and Egr-1, the mean value of mRNA in the injured artery of vehicle-treated group at 1 hour was regarded as 100. Each bar represents mean±SEM (n=4). P<.05, *P<.01 vs vehicle-treated group. Vehicle indicates vehicle-treated group; TCV-116, TCV-116–treated group.

As shown in Fig 2, treatment with TCV-116 inhibited the increase in c-fos, c-jun, and Egr-1 mRNAs at 1 hour after injury by 30% (P<.01), 41% (P<.01), and 19% (P<.01), respectively. On the other hand, jun B and jun D gene expressions were not significantly suppressed by TCV-116. TCV-116 inhibited the induction of ODC expression by 40% (P<.01) and 53% (P<.05) at 6 and 24 hours, respectively.

Effects of TCV-116 on TGF-ß₁ Gene Expression
As shown in Fig 3, arterial TGF-ß₁ mRNA levels were increased to maximal levels (by 2.2-fold) at 6 hours after balloon injury and remained elevated for 14 days. However, there was no significant inhibition of TGF-ß₁ gene expression by TCV-116 from 1 hour to 7 days after balloon injury.

View larger version (30K): [in this window] [in a new window]   Figure 3. A, Autoradiographs of transforming growth factor–ß1 (TGF-ß1) mRNA and TGF-ß1 mRNA levels corrected for 18S ribosomal RNA levels in carotid artery at 1, 6, and 24 hours and 3, 7, and 14 days after balloon injury. Size of mRNA for TGF-ß1 was 2.5 kb. Cont indicates noninjured right carotid artery at 1 hour after injury; V, vehicle-treated group; T, TCV-116–treated group; and 18S rRNA, 18S ribosomal RNA. B, Bar graphs of TGF-ß1 mRNA values, corrected for 18S ribosomal RNA values at 1, 6, and 24 hours and 3, 7, and 14 days after balloon injury. Such values in noninjured right carotid artery at each time point were regarded as 1. For 1, 6, and 24 hours and 3 days, each bar represents mean±SEM (n=4). For 7 and 14 days, each bar represents a single datum from one pool of 14 or 15 rat arteries. Vehicle indicates vehicle-treated group; TCV-116, TCV-116–treated group.

Effects of TCV-116 on Gene Expression of Extracellular Matrix and Basement Membrane Components
As shown in Figs 4 and 5, arterial fibronectin mRNA levels started to increase by 1.4-fold at 6 hours and reached a peak (2.9-fold) at 3 days. TCV-116 suppressed the elevation of fibronectin mRNA levels by 43% (P<.05), 47% (P<.01), 41% (P<.01), and 57% at 6 hours, 24 hours, 3 days, and 7 days, respectively, after injury. In contrast to fibronectin, arterial types I and III collagen mRNA levels were decreased at 6 and 24 hours after injury and were not significantly changed by treatment with TCV-116. mRNA levels for collagen type IV and laminin B1, which are the main basement membrane components, were not significantly altered throughout the experiments and were not suppressed by TCV-116.

View larger version (79K): [in this window] [in a new window]   Figure 4. Autoradiographs of mRNAs for extracellular matrix components in injured left carotid artery at 1, 6, and 24 hours and 3, 7, and 14 days. Sizes of mRNA were 7.9 kb for fibronectin, 4.7 and 5.7 kb for type I collagen (collagen I), 6.0 kb for type III collagen (collagen III), 6.8 kb for type IV collagen (collagen IV), and 6.0 kb for laminin B1 chain. V indicates vehicle-treated group; T, TCV-116–treated group. In the present study, the density of mRNA band cannot be compared among samples from different time points because Northern blot analysis and autoradiography of RNA samples from different time points were not carried out at the same time.

View larger version (31K): [in this window] [in a new window]   Figure 5. Bar graphs of mRNA levels for extracellular matrix components, corrected for 18S ribosomal RNA levels, in carotid artery at 1, 6, and 24 hours and 3, 7, and 14 days after balloon injury. Vehicle indicates vehicle-treated group; TCV-116, TCV-116–treated group. Each mRNA value, corrected for 18S ribosomal RNA, in noninjured right carotid artery at each time point was regarded as 1. For 1, 6, and 24 hours and 3 days, each bar represents mean±SEM (n=4). For 7 and 14 days, each bar represents a single datum from one pool of 14 or 15 rat arteries. P<.05, *P<.01 vs vehicle-treated group.

Immunohistochemistry
As shown in Fig 6, TCV-116 significantly prevented neointimal formation 14 days after balloon injury, confirming a previous report.⁹ In all sections examined, the densities of fibronectin; of types I, III, and IV collagen; and of laminin were higher in the intima than in the media, as indicated by Fig 6 and by OD in the Table. TCV-116 did not significantly affect the density of these five extracellular matrix proteins in the intima or media, although their densities in the intima tended to increase in response to TCV-116 treatment. However, the antigen score (OD multiplied by the area) of fibronectin, types I and III collagen, and laminin in the intima was significantly decreased by TCV-116, because the intimal area was significantly decreased by TCV-116. There was no significant difference in the number of intimal cells per intimal area between vehicle- (n=3) and TCV-116– (n=3) treated groups (0.0113±0.0019 versus 0.0111±0.0006 per µm²). On the other hand, there was no difference in the antigen score of the above five extracellular matrix proteins in the media between vehicle- and TCV-116–treated groups.

View larger version (0K): [in this window] [in a new window]   Figure 6. Immunostaining of injured left carotid arterial sections at 14 days after ballooning. Vehicle (left 4 panels) indicates vehicle-treated group; TCV-116 (right 4 panels), TCV-116–treated group; FN, immunostaining with anti-fibronectin antibody; Col III, immunostaining with anti-type III collagen antibody; and Col IV, immunostaining with anti-type IV collagen antibody. Original magnification x60. Second left and right panels from the top indicate the border of the intima and the media in the carotid arterial sections immunostained with anti-fibronectin antibody. Yellow and blue areas indicate the intima and media, respectively.

View this table: [in this window] [in a new window]   Table 1. Quantitative Analysis of Extracellular Matrix Proteins in Injured Carotid Artery

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
It has been established that both AT₁ receptor antagonist and ACE inhibitor prevent the formation of neointima after balloon injury of the carotid artery in rats, which is in contrast to the lack of a preventive effect of calcium channel antagonist.^{7 8 9 10} Accumulating evidence indicates that a local renin-angiotensin system exists in vascular tissues.^{31 32 33}

Furthermore, vascular injury of rat carotid artery with a balloon catheter dramatically induces the expression of both angiotensinogen³⁴ and AT₁ receptor¹² in the neointimal cells as well as medial cells. These findings, taken together with evidence for the stimulation of vascular smooth muscle cell proliferation by angiotensin II in vivo,¹¹ support the concept that via AT₁ receptor, a local renin-angiotensin system may play a central role in neointimal thickening after balloon injury in rats. In vitro studies with cultured rat vascular smooth muscle cells show that angiotensin II can stimulate the expression of immediate-early genes,^{35 36 37} growth factors,^{37 38 39} and extracellular matrix proteins.⁴⁰ These gene expressions appear to be involved in the proliferation or migration of various cells in vitro^{38 41 42} and are shown to be significantly increased in rat injured artery.^{3 4 5 6} However, the role of these gene expressions in neointimal thickening is poorly understood. Thus, investigation of the effect of TCV-116 on these gene expressions is extremely important in understanding not only the significance of a local renin-angiotensin system in vascular injury but also the molecular mechanism of neointimal thickening.

We found a dramatic increase in c-fos and c-jun mRNAs at 1 hour after injury, which is in good agreement with previous data.³ Of note is the observation that the induction of these genes was significantly suppressed by treatment with TCV-116. We also examined the effect of TCV-116 on expression of two other jun family genes (jun B and jun D), which are suggested to be the negative regulators of c-jun and to inhibit the activation of TPA response element (TRE) promoter by c-jun.^{21 43 44} In contrast to c-jun, the gene expression of jun B and jun D was not suppressed by TCV-116, thereby suggesting that the mechanism of induction of jun B and jun D after vascular injury may differ from that of c-jun. Thus, of three jun family genes, TCV-116 appears to selectively inhibit c-jun expression in injured artery. c-fos/c-jun heterodimer (AP-1 complex) binds to specific DNA elements called AP-1 binding sites or TRE, stimulating the transcription of various target genes containing this responsive element in their 5' promoter region.⁴⁵ Recently, AP-1 complex has been shown to stimulate DNA replication as well as transcription.⁴¹ Furthermore, recent investigation with rat vascular smooth muscle cells transfected with an expression vector containing TRE linked to the reporter gene (chloramphenicol acetyl transferase) provides direct evidence that angiotensin II can regulate gene expression by the TRE sequence.⁴⁶ These findings, taken together with the fact that the source of increased AP-1 complex after injury is vascular smooth muscle cells,⁴ suggest that the inhibition of AP-1 complex by AT₁ receptor blockade may play a role in the suppression of vascular smooth muscle cell proliferation after injury. In addition to the suppression of AP-1 complex, AT₁ receptor blockade inhibited the induction of Egr-1, which is a transcriptional factor with three zinc finger domains and is often coregulated with c-fos, although the function is unclear.⁴⁷ Thus, it is likely that AT₁ receptor antagonist in vivo may significantly affect the immediate response of vascular smooth muscle cells to injury.

In the present study, ODC mRNA was measured as a marker of smooth muscle cell entry into the prereplicative (G1) phase.⁴⁸ Rapid and transient induction of ODC mRNA was observed with a peak at 6 hours after injury, which is consistent with a previous report.⁵ TCV-116 suppressed ODC mRNA induction particularly at the peak time point (6 hours), different than the in vivo effect of heparin, which prevents neointimal thickening without suppression of ODC induction.⁴⁹ Thus, TCV-116 in vivo may inhibit, to some extent, the entry of smooth muscle cell into the G1 phase from the quiescent (G0) phase and prevent neointimal thickening by a mechanism different than that of heparin.⁴⁹

Fibronectin mRNA levels also were increased after balloon injury, which is consistent with a previous report.⁶ Fibronectin, one of the main extracellular matrix proteins, stimulates modulation of vascular smooth muscle cells from a contractile to a synthetic phenotype, thereby contributing to vascular smooth muscle cell proliferation.⁵⁰ Furthermore, in vitro studies show that fibronectin participates in cell migration.⁵¹ Thus, the specific induction of fibronectin in injured artery may play an important role in neointimal thickening after vascular injury. Interestingly, TCV-116 significantly inhibited fibronectin mRNA expression from 6 hours to 7 days after injury. Furthermore, Prescott et al,⁸ who examined the effect of losartan (another AT₁ receptor antagonist) on vascular smooth muscle cell proliferation and migration in balloon-injured rat carotid artery, demonstrated that AT₁ receptor antagonist inhibits intimal thickening by inhibiting both vascular smooth muscle cell proliferation and migration. These observations, taken together with the fact that the migration of smooth muscle cells from the media to the intima starts at approximately 4 days after injury,¹ suggest that the inhibition of fibronectin expression by AT₁ receptor antagonist may be in part responsible for the inhibition of vascular smooth muscle cell proliferation, migration, or both. In contrast, the gene expression of types I and III collagen and basement membrane components (type IV collagen and laminin) was not significantly increased after injury, and there was no significant effect of TCV-116 on these expressions. Furthermore, we have previously found that in vivo infusion of either subpressor or pressor dose of angiotensin II, via AT₁ receptor, stimulates the gene expression of vascular fibronectin but not of types I and III collagen.⁵² Thus, angiotensin II appears to be a specific regulator of rat vascular fibronectin gene expression in vivo.

The antigen score, as determined by immunohistochemistry, suggests that the accumulation of fibronectin protein may be decreased in the intima by treatment with TCV-116. Thus, the inhibition of fibronectin gene expression by TCV-116 appears to lead to the decrease in fibronectin protein. Interestingly, the antigen score of types I and III collagen and laminin in the intima was also reduced by TCV-116, despite no significant decrease in mRNA levels occurring with TCV-116. These observations indicate that accumulation of collagen and laminin in injured artery may be more affected by the translational rate, posttranslational modification, or degradation rate than by the transcriptional rate. The reduction of total extracellular matrix protein content in the intima by TCV-116 appears to be due to the decreased total number of smooth muscle cells in the intima rather than to the decreased production of extracellular matrix per intimal cell, because there was no significant difference in the intimal cell density (cell number per the intimal area) between vehicle- and TCV-116–treated groups.

TGF-ß₁, a multifunctional growth factor, plays an important role not only in cell proliferation and hypertrophy but also in the synthesis of extracellular matrix such as fibronectin and collagen.⁵³ A previous report on rat injured carotid artery indicates that the gene expression of TGF-ß₁ is significantly increased after vascular injury, that TGF-ß₁ protein is mainly localized in neointimal smooth muscle cells, and that infusion of TGF-ß₁ into rats with preexisting neointima produces significant stimulation of neointimal smooth muscle cell DNA synthesis.⁶ In situ hybridization analysis on human atheromata obtained in vivo showed that TGF-ß₁ mRNA expression is increased in human vascular restenosis lesions.⁵⁴ These findings indicate that TGF-ß₁ may be responsible for neointimal thickening after vascular injury. Furthermore, angiotensin II stimulates TGF-ß₁ gene expression in vitro in cultured rat vascular smooth muscle cells, which plays an important role in the regulation of smooth muscle cell proliferation by angiotensin II.³⁹ Thus, whether TCV-116 in vivo can inhibit TGF-ß₁ gene expression in injured artery is an important question. In the present study, TGF-ß₁ gene expression was significantly increased from 6 hours after vascular injury, which is consistent with a previous report.⁶ However, interestingly, TCV-116 did not inhibit TGF-ß₁ gene expression in injured artery throughout the experiments. Thus, in vivo inhibition of vascular smooth muscle cell proliferation and fibronectin gene expression by AT₁ receptor antagonist appears to not be mediated by TGF-ß₁. These observations, taken together with our previous report that exogenously administered angiotensin II in vivo does not stimulate vascular TGF-ß₁ gene expression,⁵² indicate that there is a significant difference in vivo versus in vitro in the effect of angiotensin II on vascular TGF-ß₁ expression.

In contrast to the preventive effect of ACE inhibitor on neointimal formation after balloon injury in rats, a recent multicenter clinical trial (MERCATOR study⁵⁵ ) showed that ACE inhibition with cilazapril does not prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA), thereby suggesting that the rat carotid artery balloon injury model may not be a good model for similar injuries in human arteries and that angiotensin II may not be responsible for human arterial restenosis. However, chymase, which is a potent and specific angiotensin II–forming serine protease that is not inhibited by ACE inhibitor, has been identified in human vascular tissues, thereby indicating the existence of an alternative pathway of angiotensin II production in the human vascular wall.⁵⁶ Therefore, it is also possible that the lack of prevention of restenosis after PTCA by ACE inhibition may be explained by the inability of ACE inhibitor to suppress the alternative pathway of angiotensin II production. These findings indicate that the use of AT₁ receptor antagonist is essential to elucidate the role of angiotensin II in restenosis after PTCA.

In conclusion, the present study shows that AT₁ receptor blockade inhibits the gene expression of c-fos, c-jun, Egr-1, ODC, and fibronectin after vascular injury in rats. These observations suggest that the prevention of neointimal formation in rats by AT₁ receptor antagonist may be due in part to the inhibition of multiple kinds of growth-related genes activated very early after injury. However, there is no direct evidence that the growth and migration of vascular smooth muscle cells after balloon injury are dependent on AP-1 complex and fibronectin. Furthermore, in vitro data suggest that other genes, such as c-myc and platelet-derived growth factor, which were not examined in the present study, also may be involved in the regulation of vascular smooth muscle cell proliferation by angiotensin II.³⁷ Thus, further research is needed to elucidate the molecular mechanism of prevention of intimal thickening by AT₁ receptor antagonist.

	Acknowledgments

 
This work was supported in part by Grant-in-Aid for Scientific Research 05770067 from the Ministry of Education, Science, and Culture. We are grateful to Eriko Gomi for technical assistance.

Received December 27, 1994; revision received January 4, 1995; accepted January 9, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res. 1985;56:139-145.[Abstract/Free Full Text]

2. Schwartz SM, Stemerman MB, Benditt EP. The aortic intima, II: repair of the aortic lining after mechanical denudation. Am J Pathol. 1975;81:15-42. [Abstract]

3. Miano JM, Tota RR, Vlasic N, Danishefsky KJ, Stemerman MB. Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal. Am J Pathol. 1990;137:761-765. [Abstract]

4. Miano JM, Vlasic N, Tota RR, Stemerman MB. Localization of Fos and Jun proteins in rat aortic smooth muscle cells after vascular injury. Am J Pathol. 1993;142:715-724. [Abstract]

5. Majesky MW, Reidy MA, Bowen-Pope DF, Hart CE, Wilcox JN, Schwartz SM. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol. 1990;111:2149-2158. [Abstract/Free Full Text]

6. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor ß1 during repair of arterial injury. J Clin Invest. 1991;88:904-910.

7. Osterrieder W, Muller RKM, Powell JS, Clozel JP, Hefti F, Baumgartner HR. Role of angiotensin II in injury-induced neointima formation in rats. Hypertension. 1991;18(suppl II):II-60-II-64.

8. Prescott MF, Webb RL, Reidy MA. Angiotensin-converting enzyme inhibitor versus angiotensin II, AT1 receptor antagonist. Am J Pathol. 1991;139:1291-1296. [Abstract]

9. Kawamura M, Terashita Z, Okuda H, Imura Y, Shino A, Nakao M, Nishikawa K. TCV-116, a novel angiotensin II receptor antagonist, prevents intimal thickening and impairment of vascular function after carotid injury in rats. J Pharmacol Exp Ther. 1993;266:1664-1669. [Abstract/Free Full Text]

10. Powell JS, Clozel JP, Muller RKM, Kuhn H, Hefti F, Hosang M, Baumgartner HR. Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury. Science. 1989;245:186-188. [Abstract/Free Full Text]

11. Daemen MJAP, Lombardi DM, Bosman FT, Schwartz SM. Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall. Circ Res. 1991;68:450-456. [Abstract/Free Full Text]

12. Viswanathan M, Stromberg C, Seltzer A, Saavedra JM. Balloon angioplasty enhances the expression of angiotensin II AT1 receptors in neointima of rat aorta. J Clin Invest. 1992;90:1707-1712.

13. Shibouta Y, Inada Y, Ojima M, Wada T, Noda M, Sanada T, Kubo K, Kohara Y, Naka T, Nishikawa K. Pharmacological profile of a highly potent and long-acting angiotensin II receptor antagonist, 2-ethoxy-1-((2-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-1H-benzimidazole-7-carboxylic acid (CV-11974), and its prodrug, (±)-1-(cyclohexyloxycarbonyloxy)-ethyl-2-ethoxy-1-((2-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-1H-benzimidazole-7-carboxylate (TCV-116). J Pharmacol Exp Ther. 1993;266:114-120. [Abstract/Free Full Text]

14. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]

15. Kim S, Ohta K, Hamaguchi A, Omura T, Yukimura T, Miura K, Inada Y, Wada T, Ishimura Y, Chatani F, Iwao H. Role of angiotensin II in renal injury of deoxycorticosterone acetate-salt hypertensive rats. Hypertension. 1994;24:195-204. [Abstract/Free Full Text]

16. Feinberg AP, Vogelstein B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983;132:6-13. [Medline] [Order article via Infotrieve]

17. Mendez RE, Pfeffer JM, Ortola FV, Bloch KD, Anderson S, Seidman JG, Brenner BM. Atrial natriuretic peptide transcription, storage and release in rats with myocardial infarction. Am J Physiol. 1987;253:H1449-H1455. [Abstract/Free Full Text]

18. Curran T, Peters G, Beveren CV, Teich NM, Verma IM. FBJ murine osteosarcoma virus: identification and molecular cloning of biologically active proviral DNA. J Virol. 1982;44:674-682. [Abstract/Free Full Text]

19. Ryder K, Nathans D. Induction of protooncogene c-jun by serum growth factors. Proc Natl Acad Sci U S A. 1988;85:8464-8467. [Abstract/Free Full Text]

20. Ryder K, Lau LF, Nathans D. A gene activated by growth factors is related to the oncogene v-jun. Proc Natl Acad Sci U S A. 1988;85:1487-1491. [Abstract/Free Full Text]

21. Ryder K, Lanahan A, Oerez-Albuerne E, Nathans D. Jun-D: a third member of the Jun gene family. Proc Natl Acad Sci U S A. 1989;86:1500-1503. [Abstract/Free Full Text]

22. Christy BA, Lau LF, Nathans D. A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with `zinc finger' sequences. Proc Natl Acad Sci U S A. 1988;85:7857-7861. [Abstract/Free Full Text]

23. Qian SW, Kondaiah P, Roberts AB, Sporn MB. cDNA cloning by PCR of rat transforming growth factor ß-1. Nucleic Acids Res. 1990;18:3059. [Free Full Text]

24. Schwarzbauer JE, Tamkun JW, Lemischka IR, Hynes RO. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell. 1983;35:421-431.[Medline] [Order article via Infotrieve]

25. Genovese C, Rowe D, Kream B. Construction of DNA sequences complementary to rat 1 and 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxy vitamin D. Biochemistry. 1984;23:6210-6216. [Medline] [Order article via Infotrieve]

26. Liau G, Yamada Y, Crombrugghe BD. Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts. J Biol Chem. 1985;260:531-536. [Abstract/Free Full Text]

27. Oberbaumer U, Laurent M, Schwartz U, Sakurai Y, Yamada Y, Vogell G, Voss T, Siebold B, Glanville RW, Kuhn K. Amino acid sequence of the non-collagenous globular domain (NC1) of the 1 (IV) chain of basement membrane collagen as derived from complementary DNA. Eur J Biochem. 1985;147:217-224. [Medline] [Order article via Infotrieve]

28. Sasaki M, Kato S, Kohno K, Martin GR, Yamada Y. Sequence of the cDNA encoding the laminin B1 chain reveals a multidomain protein containing cysteine-rich repeats. Proc Natl Acad Sci U S A. 1987;84:935-939. [Abstract/Free Full Text]

29. McConlogue L, Gupta M, Wu L, Coffino P. Molecular cloning and expression of the mouse ornithine decarboxylase gene. Proc Natl Acad Sci U S A. 1984;81:540-544. [Abstract/Free Full Text]

30. Watanabe T, Katsura Y, Yoshitake A, Masataki H, Mori T. IPAP: image processor for analytical pathology. J Toxicol Pathol. 1994;7:353-361.

31. Dzau VJ. Circulating vs local renin-angiotensin system in cardiovascular homeostasis. Circulation. 1988;77(suppl I):I-1-I-3.

32. Dzau VJ, Gibbons GH, Pratt RE. Molecular mechanism of vascular renin-angiotensin system in myointimal hyperplasia. Hypertension. 1991;18(suppl II):II-100-II-105.

33. Naftilan AJ, Zuo WM, Ingelfinger J, Ryan TJ, Pratt RE, Dzau VJ. Localization and differential regulation of angiotensinogen mRNA expression in the vessel wall. J Clin Invest. 1991;87:1300-1311.

34. Rakugi H, Jacob HJ, Krieger JE, Ingelfinger JR, Pratt RE. Vascular injury induces angiotensinogen gene expression in the media and neointima. Circulation. 1993;87:283-290. [Abstract/Free Full Text]

35. Naftilan AJ, Pratt RE, Eldridge CS, Lin HL, Dzau VJ. Angiotensin II induces c-fos expression in smooth muscle via transcriptional control. Hypertension. 1989;13:706-711. [Abstract/Free Full Text]

36. Taubman MB, Berk BC, Izumo S, Tsuda T, Alexander RW, Nadal-Ginard B. Angiotensin II induces c-fos mRNA in aortic smooth muscle. J Biol Chem. 1989;264:526-530. [Abstract/Free Full Text]

37. Naftilan AJ, Pratt RE, Dzau VJ. Induction of platelet-derived growth factor A-chain and c-myc gene expressions by angiotensin II in cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1419-1424.

38. Schelling P, Fischer H, Ganten D. Angiotensin and cell growth: a link to cardiovascular hypertrophy. J Hypertens. 1991;9:3-15. [Medline] [Order article via Infotrieve]

39. Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs hyperplasia. J Clin Invest. 1992;90:456-461.

40. Kato H, Suzuki H, Tajima S, Ogata Y, Tominaga T, Sato A, Saruta T. Angiotensin II stimulates collagen synthesis in cultured vascular smooth muscle cells. J Hypertens. 1991;9:17-22. [Medline] [Order article via Infotrieve]

41. Murakami Y, Satake M, Yamaguchi-Iwai Y, Sakai M, Muramatsu M, Ito Y. The nuclear protooncogenes c-jun and c-fos as regulators of DNA replication. Proc Natl Acad Sci U S A. 1991;88:3947-3951. [Abstract/Free Full Text]

42. Newmark P. Oncogenes and cell growth. Nature. 1987;327:101-102. [Medline] [Order article via Infotrieve]

43. Chiu R, Angel P, Karin M. Jun-B differs in its biological properties from, and is a negative regulator of, c-Jun. Cell. 1989;59:979-986. [Medline] [Order article via Infotrieve]

44. Schutte J, Viallet J, Nau M, Segal S, Fedorko J, Minna J. Jun-B inhibits and c-fos stimulates the transforming and trans-activating activities of c-jun. Cell. 1989;59:987-997. [Medline] [Order article via Infotrieve]

45. Curran T, Franza BR. Fos and jun: the AP-1 connection. Cell. 1988;55:395-397. [Medline] [Order article via Infotrieve]

46. Takeuchi K, Nakamura N, Cook NS, Pratt RE, Dzau VJ. Angiotensin II can regulate gene expression by the AP-1 binding sequence via a protein kinase C-dependent pathway. Biochem Biophys Res Commun. 1990;172:1189-1194. [Medline] [Order article via Infotrieve]

47. Sukhatme VP, Cao X, Chang LC, Tsai-Morri CH, Stamenkovich D, Ferreira PCP, Cohen DR, Edwards SA, Shows TB, Curran T, Beau MML, Adamson ED. A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell. 1988;53:37-43. [Medline] [Order article via Infotrieve]

48. Heby O, Gray JW, Lindl PA, Marton LJ, Wilson CB. Changes in L-ornithine decarboxylase activity during the cell cycle. Biochem Biophys Res Commun. 1976;71:99-105. [Medline] [Order article via Infotrieve]

49. Majesky MW, Schwartz SM, Clowes MW, Clowes AW. Heparin regulates smooth muscle S phase entry in the injured rat carotid artery. Circ Res. 1987;61:296-300. [Abstract/Free Full Text]

50. Hedin U, Bottger BA, Forsberg E, Johansson S, Thyberg J. Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol. 1988;107:307-319. [Abstract/Free Full Text]

51. Ruoslahti E. Fibronectin and its receptors. Annu Rev Biochem. 1988;57:375-413. [Medline] [Order article via Infotrieve]

52. Kim S, Ohta K, Hamaguchi A, Omura T, Tominaga K, Yukimura T, Miura K, Tanaka M, Iwao H. AT1 receptor-mediated stimulation by angiotensin II of rat aortic fibronectin gene expression in vivo. Br J Pharmacol. 1994;113:662-663. [Medline] [Order article via Infotrieve]

53. Barnard JA, Lyons RM, Moses HL. The cell biology of transforming growth factor-ß. Biochim Biophys Acta. 1990;90:1-7.

54. Nikol S, Isner JM, Pickering JG, Kearney M, Lederc G, Weir L. Expression of transforming growth factor-ß1 is increased in human vascular restenosis lesions. J Clin Invest. 1992;90:1582-1592.

55. The MERCATOR Study Group. Does the new angiotensin converting enzyme inhibitor cilazapril prevent restenosis after percutaneous transluminal coronary angioplasty? Results of the MERCATOR Study: a multicenter, randomized, double-blind, placebo-controlled trial. Circulation. 1992;86:100-110. [Abstract/Free Full Text]

56. Urata H, Strobel F, Ganten D. Widespread tissue distribution of human chymase. J Hypertens. 1994;12(suppl 9):S17-S22.

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				Y. Yonemitsu, Y. Kaneda, S. Tanaka, Y. Nakashima, K. Komori, K. Sugimachi, and K. Sueishi Transfer of Wild-Type p53 Gene Effectively Inhibits Vascular Smooth Muscle Cell Proliferation In Vitro and In Vivo Circ. Res., February 9, 1998; 82(2): 147 - 156. [Abstract] [Full Text] [PDF]

				M. R. Ward, A. Agrotis, P. Kanellakis, R. Dilley, G. Jennings, and A. Bobik Inhibition of Protein Tyrosine Kinases Attenuates Increases in Expression of Transforming Growth Factor-ß Isoforms and Their Receptors Following Arterial Injury Arterioscler Thromb Vasc Biol, November 1, 1997; 17(11): 2461 - 2470. [Abstract] [Full Text]

				S. Kim, H. Wanibuchi, A. Hamaguchi, K. Miura, S. Yamanaka, and H. Iwao Angiotensin Blockade Improves Cardiac and Renal Complications of Type II Diabetic Rats Hypertension, November 1, 1997; 30(5): 1054 - 1061. [Abstract] [Full Text]

				A. Sabri, B. I. Levy, P. Poitevin, L. Caputo, E. Faggin, F. Marotte, L. Rappaport, and J. L. Samuel Differential Roles of AT1 and AT2 Receptor Subtypes in Vascular Trophic and Phenotypic Changes in Response to Stimulation With Angiotensin II Arterioscler Thromb Vasc Biol, February 1, 1997; 17(2): 257 - 264. [Abstract] [Full Text]

				N. Bardy, R. Merval, J. Benessiano, J.-L. Samuel, and A. Tedgui Pressure and Angiotensin II Synergistically Induce Aortic Fibronectin Expression in Organ Culture Model of Rabbit Aorta: Evidence for a Pressure-Induced Tissue Renin-Angiotensin System Circ. Res., July 1, 1996; 79(1): 70 - 78. [Abstract] [Full Text]

				K. Tamura, Y. E. Chen, M. Lopez-Ilasaca, L. Daviet, N. Tamura, T. Ishigami, M. Akishita, I. Takasaki, Y. Tokita, R. E. Pratt, et al. Molecular Mechanism of Fibronectin Gene Activation by Cyclic Stretch in Vascular Smooth Muscle Cells J. Biol. Chem., October 27, 2000; 275(44): 34619 - 34627. [Abstract] [Full Text] [PDF]

				Y. Izumi, S. Kim, M. Namba, H. Yasumoto, H. Miyazaki, M. Hoshiga, Y. Kaneda, R. Morishita, Y. Zhan, and H. Iwao Gene Transfer of Dominant-Negative Mutants of Extracellular Signal-Regulated Kinase and c-Jun NH2-Terminal Kinase Prevents Neointimal Formation in Balloon-Injured Rat Artery Circ. Res., June 8, 2001; 88(11): 1120 - 1126. [Abstract] [Full Text] [PDF]

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