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(Circulation. 1997;96:1906-1913.)
© 1997 American Heart Association, Inc.

Articles

Stimulated Activation of Platelet-Derived Growth Factor Receptor In Vivo in Balloon-Injured Arteries

A Link Between Angiotensin II and Intimal Thickening

Jun-ichi Abe, MD; Jun-o Deguchi, MD; Takeo Matsumoto, BS; Noriko Takuwa, MD; Masakuni Noda, PhD; Minoru Ohno, MD; Masatoshi Makuuchi, MD; Kiyoshi Kurokawa, MD; ; Yoh Takuwa, MD

From the Departments of Cardiovascular Biology (J.A., M.N., Y.T.), Surgery (J.D., M.M.), Internal Medicine (M.O., K.K.), and Physiology (N.T.), Faculty of Medicine, University of Tokyo, and the Department of Pathology, National Cancer Center East Hospital, Kashiwa, and Department of Laboratory Medicine (T.M.), Mitsui Memorial Hospital, Tokyo, Japan.

Correspondence to Yoh Takuwa, MD, Department of Cardiovascular Biology, Faculty of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.

	Abstract

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Background Growth factors such as platelet-derived growth factor (PDGF) have been postulated to be important mediators of neointimal formation in balloon-injured artery. Binding of growth factors to their receptors activates intrinsic receptor tyrosine kinase, resulting in tyrosine phosphorylation of receptors themselves and cellular substrate proteins. We investigated in vivo activities of growth factors by determining the extent of tyrosine phosphorylation of growth factor receptors and substrate proteins in injured artery.

Methods and Results Rat balloon-injured carotid artery was analyzed for phosphotyrosine content of PDGF - and ß-receptors, epidermal growth factor (EGF) receptors, and insulin receptor substrate-1 (IRS-1) by immunoprecipitation and anti-phosphotyrosine Western blot. The development of intimal thickening after deendothelializing balloon catheterization of rat carotid artery was accompanied by transient twofold to threefold increases in the extent of tyrosyl phosphorylation of PDGF - and ß-receptors but not EGF receptor or IRS-1. The AT₁ angiotensin II (Ang II) receptor antagonist TCV-116 markedly inhibited both tyrosyl phosphorylation of PDGF - and ß-receptors and intimal thickening. The AT₁ antagonist reduced mRNA levels of both PDGF-A and -B chains in injured arteries.

Conclusions The present study provides direct evidence for increased PDGF activities in injured artery in situ and the involvement of Ang II in stimulated activation of PDGF receptors. These results are consistent with the pathogenetic role for PDGF in intimal thickening.

Key Words: stenosis • growth substances • balloon

	Introduction

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Vascular proliferative disorders underlie 50% of all mortality in developed countries. The major processes that are common to various types of stenotic arterial lesions include insult to the endothelium and reactive proliferation, migration, and accumulation of smooth muscle cells in the intima.¹ The rat carotid artery model of transluminal balloon catheter angioplasty has provided much insight into the pathogenesis of intimal thickening.^{2 3 4} Among growth factors that are capable of stimulating vascular smooth muscle cell proliferation and migration in vitro, PDGF, especially the BB homodimer, has been implicated in the development of intimal smooth muscle cell accumulation after angioplasty.^{1 4 5 6} Indeed, it was reported that daily infusion of neutralizing PDGF antibody into athymic rats during the first 8 days after balloon injury caused a 40% reduction in the intimal lesion size.⁷ However, the effect of anti-PDGF antibody during the late phase, when the bulk of neointima is produced, was not examined. It was also shown that continuous infusion of PDGF-BB homodimer into rats subjected to balloon angioplasty greatly potentiated the intimal thickening of injured artery, mainly because of stimulation of migration of smooth muscle cells from the media to the intima.⁸ These studies may suggest the important role of PDGF in the development of the intimal thickening. However, the actual activation of PDGF receptors in vivo in injured arteries is as yet unknown. Another line of studies indicated that Ang II is involved in intimal thickening at least in the rat model through activation of an AT₁ subtype of Ang II receptors.^{4 9 10 11 12} However, the mechanism of action of Ang II remains elusive, because Ang II is at most a poor mitogen for vascular smooth muscle cells in vitro.^{13 14}

It is well known that many growth factors activate the intrinsic receptor tyrosine kinase upon binding to their receptors, which results in tyrosyl phosphorylation of receptors themselves and cellular substrate proteins for receptor kinases.^{15 16} This leads to the activation of the second messenger pathway necessary for the induction of gene expression, DNA synthesis, and mitogenesis. Therefore, determination of the extent of tyrosyl phosphorylation of growth factor receptors and substrate proteins in cells and tissues will provide a good measure of receptor activation in vivo. In the present study, to know in vivo activities of growth factors, including PDGF, in injured arteries, we measured the extent of tyrosyl phosphorylation of growth factor receptors and a substrate protein. In addition, we tried to discover the link between Ang II and an increase in growth factor activity. We now present direct evidence for PDGF receptor activation in vivo in injured vessels, which closely correlates with the development of neointima. We have also found that PDGF receptor activation is located downstream of the Ang II action.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Preparation of Balloon Injury Model
Male 14-week-old normotensive Wistar rats (400 to 500 g) were anesthetized with sodium pentobarbital (4 mg/100 g) and ethyl ether.^{11 17} The left common carotid artery was cleared of surrounding connective tissue and cannulated through the external carotid artery with a 2F Fogarty catheter (Baxter Healthcare). The balloon was air-inflated to distend the left common carotid artery and passed three times up and down from the aortocarotid junction to the carotid bifurcation to produce deendothelializing injury. The catheter was removed, the external carotid artery was ligated, and the wound was closed. The right common carotid artery, which was used as a normal control, was exposed in the same way but not subjected to angioplasty, and the wound was closed. The selective AT₁ Ang II receptor antagonist TCV-116 (candesartan cilexetil)¹⁸ (10 mg · kg body wt^-1 · d^-1) and amlodipine (10 mg · kg body wt^-1 · d^-1) were orally administered into rats by an intragastric catheter once a day for the indicated time periods. The dose of TCV-116 used in the present study was chosen from the results of a previous study¹² showing that this oral dose of TCV-116 caused the strongest inhibition of intimal thickening in balloon-injured rat carotid arteries among various oral doses tested. The dose of amlodipine was selected from our preliminary experiments in which this dose of amlodipine caused a blood pressure decrease similar to that caused by 10 mg/kg TCV-116. The systolic blood pressures of TCV-116– and amlodipine-treated rats were 141±16 and 142±10 mm Hg, respectively, before drug administration was begun and 111±6 and 111±7 mm Hg, respectively, 4 days after drug administration was begun. Control animals received a gum arabic solution vehicle. In experiments shown in Fig 4, CV-11974, the active form of TCV-116 (10 mg · kg body wt^-1 · d^-1), dissolved in 200 µL of 25% F127 pluronic gel was also topically administered around the injured left carotid arteries immediately after balloon angioplasty as described previously.^{11 17} The uninjured right common carotid artery received F127 pluronic gel vehicle. The combination of oral and topical administration of the AT₁ receptor antagonist was adopted because we observed that it caused a stronger inhibition of intimal thickening than did oral administration alone.¹¹ The number of rats or samples of each treatment group are indicated in the figure. At time points indicated in legends of each figure, the rats were anesthetized as described above and killed for morphometric analysis of neointimal formation, measurement of protein tyrosyl phosphorylation, and determination of mRNA levels by Northern analysis. Anticoagulant was not administered throughout the experimental period. Thrombus formation was observed in <10% of injured arteries, and these vessels were excluded from both morphometric analysis and determination of tyrosyl phosphorylation. For morphometric analysis, the middle one third of the common carotid artery was embedded in paraffin, cross-sectioned at a thickness of 3 µm, and subjected to elastica–van Gieson staining. Intimal and medial cross-sectional areas of five cross sections of the left common artery obtained from each rat were measured, and the I/M cross-sectional area ratio was determined as described previously.¹¹

View larger version (0K): [in this window] [in a new window]   Figure 4. AT1 Ang II antagonist TCV-116 but not Ca2+ channel antagonist amlodipine inhibits tyrosyl phosphorylation of both PDGF - and ß-receptors and neointima formation. A, Anti-phosphotyrosine immunoblots of PDGF - and ß-receptors immunoprecipitated from injured (I) and uninjured (U) carotid arteries from rats treated with either TCV-116 or amlodipine and nontreated rats at 14 days. B, Quantitative summary of tyrosyl phosphorylation of PDGF receptors. Values are expressed as multiples of basal value in uninjured control artery and mean±SEM. C, Inhibition of neointima formation by AT1 Ang II antagonist TCV-116. Values are I/M ratio and mean±SEM. Either TCV-116 or amlodipine (both 10 mg · kg body wt-1 · d-1) was orally administered into rats from 4 days before to 14 days after balloon injury. Also, CV-2961 (10 mg/kg body wt), active form of TCV-116 dissolved in 25% F127 pluronic gel, was locally applied to injured left common carotid artery. Fourteen days after injury, carotid arteries were removed and analyzed for tyrosyl phosphorylation of PDGF receptors and neointima formation. Numbers in parentheses denote number of samples (B) and rats (C).

Measurement of Tyrosyl Phosphorylation
Left injured and right uninjured common carotid arteries were excised at times indicated in legends of each figure and immediately homogenized at 4°C in a buffer containing 50 mmol/L Tris-HCl (pH 8.0), 120 mmol/L NaCl, 0.5% Nonidet-P 40, 100 mmol/L NaF, 1 mmol/L Na₃VO₄, 0.1% SDS, 2 mmol/L EGTA, 0.19 mmol/L leupeptin, 370 U/L aprotinin, and 0.6 mmol/L phenylmethylsulfonyl fluoride. Homogenates were cleared by centrifugation at 10 000 rpm for 5 minutes. The supernatants from three arteries were combined, and 800 µg protein was subjected to immunoprecipitation with either specific anti–PDGF -receptor antibody raised against the carboxyl-terminal 110 amino acids of mouse PDGF -receptor fused to glutathione S-transferase expressed bacterially, specific anti-PDGF ß-receptor antibody raised against the peptide (1013 to 1025) of human PDGF ß-receptor, anti-EGF receptor antibody against the protein encoded by exon 2 of human EGF receptor gene fused to glutathione S-transferase, or anti–IRS-1 antibody raised against recombinant rat IRS-1 produced in insect cells by baculovirus. Immunoprecipitates were recovered by incubation with protein A sepharose for anti–PDGF receptor antibodies and anti–IRS-1 antibody or protein G–sepharose for anti-EGF antibody, solubilized in Laemmli's SDS sample buffer, and resolved in 8% SDS-PAGE. Separated proteins were electrotransferred onto an Immobilon-P (Millipore) membrane, probed by anti-phosphotyrosine antibody or anti–PDGF-ß receptor antibody, and visualized by the ECL system (Amersham) or an alkaline phosphatase–conjugated second antibody. The densities of the bands were quantified by densitometry with a scanning densitometer (PDI). Differences in background intensity were corrected by subtraction of a background level, which was determined in the area just above a position of a band of interest, from raw values of band intensity. The results were expressed as multiples over a value in uninjured control carotid artery from rats that received no drug.

RNA Isolation and Northern Blot Analysis
Frozen arterial tissue was ground to a fine powder under liquid nitrogen, and total cellular RNA was extracted by the acid guanidinium isothiocyanate–phenol-chloroform method.¹⁹ Total RNA was separated by formaldehyde–1.0% agarose gel electrophoresis and transferred onto a nylon membrane (Hybond N, Amersham). Blots were hybridized as previously described²⁰ with cDNA probes labeled with [³²P]dCTP by the random priming method. DNA probes used for RNA blot hybridization were as follows: PDGF-A, a 1.3-kb EcoRI human cDNA fragment from pD1; PDGF-B, a 3.0-kb EcoRI rat cDNA from pBS–rPDGF-B(3-4a); PDGF -receptor, a 6.4-kb EcoRI rat cDNA from p802E/B5; and PDGF ß-receptor, a 4.7-kb EcoRI–Xba I human cDNA fragment from phPDGF-R (gifts from Dr H. Okazaki).²¹

Experiments in RASM Cells
RASM cells were obtained from aorta of an 18-week-old Wistar rat by the explant method.²² Cells were grown in DMEM supplemented with 10% FCS (Commonwealth Serum Laboratory), 10⁵ U/L penicillin G, and 137 µmol/L streptomycin (Wako) under a 95% air/5% CO₂ atmosphere. Immunoprecipitation of PDGF receptors, EGF receptors, and IRS-1 from RASM cells was performed as described above for carotid arteries after cells were lysed in the homogenization buffer described above. ¹²⁵I-labeled PDGF binding studies were performed as described.²³ Briefly, cells grown in a 24-well plate were incubated in Hanks' balanced salt solution with 0.2% BSA containing 120 pmol/L ¹²⁵I-labeled human PDGF-BB (Amersham) in the presence or absence of 8 nmol/L PDGF-BB at 4°C for 3 hours. After cells were washed three times with ice-cold Hanks' solution, the cell-bound radioactivity was counted with a -counter (Aloka). Specific binding was determined as total binding minus nonspecific binding in the presence of an excessive amount of unlabeled PDGF-BB.

Statistics
The data are presented as the mean±SEM. The statistical significance of differences between two groups (Figs 4B, 6B, and 6C) was determined by the Mann-Whitney test. Changes over time (Figs 2B and 2C and 3B and 3C) and multiple comparisons (Figs 1B, 4C, and 5) were analyzed by Scheffé's test.

View larger version (0K): [in this window] [in a new window]   Figure 6. Administration of AT1 Ang II antagonist TCV-116 for last 4 days inhibits tyrosyl phosphorylation of both PDGF - and ß-receptors. A, Anti-phosphotyrosine and anti–PDGF ß-receptor immunoblots of PDGF receptors immunoprecipitated from injured (I) and uninjured (U) carotid arteries from rats treated with TCV-116 from 10 to 14 days after injury and nontreated rats at 14 days. B, Quantitative summary of effects of 4 days' administration of TCV-116 on tyrosyl phosphorylation of PDGF receptors. Values are expressed as multiples of basal value in uninjured control artery and mean±SEM. C, Quantitative summary of PDGF ß-receptor protein level. Values are multiples of basal value in uninjured control artery and are mean±SEM. TCV-116 (10 mg · kg body wt-1 · d-1) was orally administered into rats from 10 to 14 days after balloon injury. Fourteen days after injury, carotid arteries were removed and analyzed for tyrosyl phosphorylation of PDGF receptors and protein levels of PDGF ß-receptor. Numbers in parentheses denote number of samples.

View larger version (0K): [in this window] [in a new window]   Figure 2. Tyrosyl phosphorylation of PDGF - and ß-receptors in balloon-injured rat carotid artery. A, Time-dependent changes in extents of tyrosyl phosphorylation of PDGF - and ß-receptors and expression of PDGF ß-receptors. PDGF - and ß-receptors were immunoprecipitated from homogenates of both injured and uninjured carotid arteries by specific antibodies and analyzed by Western blotting with anti-phosphotyrosine antibody (anti-PY) or specific anti–PDGF ß-receptor antibody. B, Quantitative summary of PDGF receptor phosphorylation. C, Quantitative summary of PDGF ß-receptor expression. Values are expressed as multiples of basal value in uninjured control artery and are mean±SEM (B and C). Numbers in parentheses denote number of samples. *Significant (P<.05) difference vs values at day 0.

View larger version (0K): [in this window] [in a new window]   Figure 1. Tyrosyl phosphorylation of PDGF - and ß-receptors in cultured RASM cells. A, Nonpretreated RASM cells were unstimulated (lanes 1 and 7) or stimulated with 2 nmol/L of either PDGF-AA (lanes 2 and 8) or PDGF-BB (lanes 3 and 9) for 10 minutes. RASM cells pretreated with 2 nmol/L of either PDGF-AA or PDGF-BB for 3 hours were also stimulated with 2 nmol/L of either PDGF-AA (lane 4) or PDGF-BB (lanes 5, 6, and 10) for 10 minutes. PDGF - and ß-receptors were immunoprecipitated from cell lysate by specific anti–PDGF -receptor antibody or anti–PDGF ß-receptor antibody and analyzed by Western blotting with anti-phosphotyrosine antibody (anti-PY). IP indicates immunoprecipitation; WB, Western blot. Arrowheads indicate positions of PDGF - and ß-receptors. Numbers on left denote molecular masses (kD). B, 125I-labeled PDGF-BB binding to RASM cells. Cells were nonpretreated or pretreated with 2 nmol/L PDGF-AA or PDGF-BB for 3 hours before binding experiment. Values denote specific binding of 125I-labeled PDGF-BB and are mean±SEM of three determinations.

View larger version (0K): [in this window] [in a new window]   Figure 5. The 4-day administration of TCV-116 inhibits neointima formation. TCV-116 (10 mg · kg body wt-1 · d-1) was orally administered into rats from 10 to 14 days after balloon injury, as described in legend for Fig 7. Values are mean±SEM. Numbers in parentheses denote number of rats.

Reagents
Specific anti–PDGF ß-receptor antibody, anti-phosphotyrosine antibody, and anti–IRS-1 antibody were purchased from Upstate Biotechnology. Specific anti–PDGF -receptor antibody and anti-EGF receptor antibody were bought from Seikagaku and Gibco/BRL, respectively. Alkaline phosphatase–conjugated second antibody was purchased from Zymed. Protein A sepharose and protein G sepharose were bought from Pharmacia and Sigma, respectively. TCV-116 and CV-11974 were gifts from Takeda Pharmaceutical. Amlodipine was a gift from Pfizer. Human PDGF-AA and PDGF-BB and human EGF were purchased from R&D and Wakunaga Pharmaceutical, respectively. Human IGF-1 was a gift from Fujisawa Pharmaceutical. All other chemicals were of reagent-grade purity.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Balloon Vascular Injury Causes Stimulation of Tyrosyl Phosphorylation of Both PDGF - and ß-Receptors but Not of EGF Receptors or IRS-1
We first examined the specificity of antibodies for PDGF - and ß-receptors that were used in the present study. RASM cells were stimulated with PDGF-AA or PDGF-BB for 10 minutes, and PDGF - and ß-receptors were immunoprecipitated by use of anti–PDGF -receptor antibody and anti–PDGF ß-receptor antibody, respectively, followed by Western blotting with anti-phosphotyrosine antibody. As shown in Fig 1A, both PDGF-AA and PDGF-BB increased the extent of tyrosyl phosphorylation of a 180-kD protein immunoprecipitated with anti–PDGF -receptor antibody (lanes 2 and 3). Conversely, when immunoprecipitation was performed with anti–PDGF ß-receptor antibody, PDGF-BB but not PDGF-AA increased the extent of tyrosyl phosphorylation of a 180-kD protein (lane 8 and 9). When cells were pretreated with PDGF-AA or PDGF-BB for 3 hours, the PDGF binding sites on the cell surface were reduced by 15% and 87%, respectively, as a result of downregulation of PDGF receptors (Fig 1B). Pretreatment of cells with PDGF-AA markedly inhibited stimulation of tyrosyl phosphorylation of the 180-kD protein immunoprecipitated with anti–PDGF -receptor antibody in response to the subsequent addition of either PDGF-AA or PDGF-BB (lanes 4 and 5 in Fig 1A). Pretreatment with PDGF-BB also inhibited stimulation of tyrosyl phosphorylation of anti–PDGF -receptor immunoprecipitates in response to PDGF-BB (lane 6). Similarly, pretreatment of cells with PDGF-BB reduced PDGF-BB–induced increase in tyrosyl phosphorylation of the 180-kD protein immunoprecipitated with anti–PDGF ß-receptor antibody (lane 10). These results indicate that the anti–PDGF - and ß-receptor antibodies used in the present study can specifically immunoprecipitate respective isoforms of rat PDGF receptors and detect tyrosyl phosphorylation of the PDGF receptors.

The expression level of PDGF ß-receptor protein for the same amount of cell protein in carotid arteries was fairly constant for up to 4 weeks after balloon injury (Fig 2C). The expression level of PDGF -receptor protein was not quantitatively evaluated because the antibody used could detect only faint bands on Western blots. Interestingly, considerable extents of tyrosyl phosphorylation of PDGF - and ß-receptors were constantly detected in uninjured arteries (Fig 2A). More importantly, the extent of tyrosyl phosphorylation of both PDGF - and ß-receptors rose 7 days after injury (2.2- and 3.3-fold increase over the basal value of uninjured arteries, respectively), stayed at similar levels until 2 weeks, and then declined to the basal levels by 4 weeks (Fig 2A and 2B).

We also studied changes in tyrosyl phosphorylation of EGF receptors and IRS-1 immunoprecipitated from injured and uninjured carotid arteries. IRS-1 is a substrate protein for the receptor tyrosine kinases activated by insulin and IGF-1.^{24 25} As for PDGF receptors, a significant extent of basal tyrosyl phosphorylation was detected for both EGF receptor and IRS-1 in uninjured arteries (Fig 3A). However, unlike PDGF receptors, the levels of tyrosyl phosphorylation of EGF receptor and IRS-1 did not show any detectable increase over basal levels for up to 2 weeks after injury (Fig 3B and 3C).

View larger version (0K): [in this window] [in a new window]   Figure 3. Tyrosyl phosphorylation of EGF receptors and IRS-1. A, Time-dependent changes in extents of tyrosyl phosphorylation of EGF receptors and IRS-1 in rat carotid arteries. B, Quantitative summary of EGF receptor tyrosyl phosphorylation in rat carotid arteries. C, Quantitative summary of IRS-1 tyrosyl phosphorylation in rat carotid arteries. EGF receptors and IRS-1 immunoprecipitated from RASM cells or carotid arteries excised at indicated times after injury were analyzed by Western blotting using anti-phosphotyrosine antibody (anti-PY). Values are multiples of basal values in uninjured control artery and are mean±SEM of values. Numbers in parentheses denote number of samples.

These results demonstrate for the first time in situ activation of both PDGF - and ß-receptors in balloon catheter–injured carotid arteries. The kinetics of PDGF receptor activation correlates tightly with that of intimal smooth muscle cell accumulation, which starts to occur after a lag period of 3 days and rapidly develops to reach a maximal level 14 days after injury.^{2 11 17} By contrast, EGF, heparin-binding EGF,²⁶ insulin, and IGF-1 are not likely to participate in intimal thickening in injured rat carotid artery.

AT₁ Ang II Receptor Antagonist TCV-116 Inhibits Both PDGF Receptor Tyrosyl Phosphorylation and Intimal Thickening
We examined the effect of the AT₁ receptor antagonist TCV-116²⁷ on activation of both subtypes of PDGF receptors in balloon-injured arteries. Administration of TCV-116 from 4 days before to 14 days after balloon injury nearly completely inhibited tyrosyl phosphorylation of both PDGF - and ß-receptors (1.0- versus 3.6-fold increase over the basal value of uninjured arteries for -receptor and 1.3- versus 2.4-fold for ß-receptor in TCV-116–treated and control animals, respectively), with a concomitant 94% reduction in the neointimal size (I/M ratios of 0.08 versus 1.05) (Fig 4). In addition, TCV-116 suppressed basal levels of tyrosyl phosphorylation of PDGF ß-receptors in uninjured arteries (Fig 4A). In sharp contrast, administration of the Ca²⁺ channel blocker amlodipine, at a dose that caused a blood pressure decrease similar to that achieved with TCV-116, had no inhibitory effect on either PDGF receptor phosphorylation (Fig 4A and 4B) or neointimal size (Fig 4C). These results clearly indicate that Ang II is involved in activation of both - and ß-subtypes of PDGF receptors in rat balloon-injured carotid arteries.

In animals given continuous TCV-116, neointima formation was nearly completely suppressed (Fig 4C). Therefore, it is possible that suppression of PDGF receptor tyrosyl phosphorylation by the AT₁ receptor antagonist would not be the result of the decreased PDGF activity but might merely be a reflection of the very small size of neointima. To explore whether this was true, the administration of TCV-116 was started 10 days after injury, when 70% of the maximal intimal lesion had already developed (Fig 5). Four days later, the effect of TCV-116 on tyrosyl phosphorylation of PDGF receptors was examined. With this protocol as well, TCV-116 inhibited tyrosyl phosphorylation of both PDGF - and ß-receptors (1.5- versus 2.6-fold over the basal value for -receptor and 1.4- versus 3.0-fold for ß-receptor in TCV-116–treated and control animals, respectively) (Fig 6A and 6B). The protein level of PDGF ß-receptor in injured artery was not detectably affected by the AT₁ antagonist (Fig 6C). This treatment also prevented an increase in the neointimal size (Fig 5).

To explore the mechanism for the inhibition by the AT₁ antagonist of PDGF - and ß-receptor tyrosine phosphorylation in injured arteries, we examined mRNA levels of PDGF-A and -B chains and PDGF - and ß-receptors in carotid arteries from rats that received the AT₁ antagonist from 4 days before to 14 days after injury and in control rats. As shown in Fig 7, balloon injury by itself slightly increased mRNA levels of PDGF-A chain in carotid arteries at 14 days but not of PDGF-B chain or PDGF - and ß-receptors. The AT₁ antagonist reduced mRNA levels of PDGF-A, PDGF-B, and PDGF -receptors but not of PDGF ß-receptors in injured arteries. GAPDH mRNA levels were not altered by balloon injury or administration of the AT₁ antagonist. Therefore, the AT₁ antagonist–induced reduction in PDGF-A and -B chain mRNAs may contribute in part to the suppression of injury-induced PDGF receptor activation.

View larger version (0K): [in this window] [in a new window]   Figure 7. Effects of AT1 Ang II antagonist TCV-116 on mRNAs of PDGFs and PDGF receptors in carotid arteries. TCV-116 was administered and balloon arterial injury was performed as described in legend for Fig 4. Total RNA was isolated from carotid arteries and analyzed by Northern blotting. Arrowheads denote positions of PDGF-A transcripts (2.9, 2.3, and 1.7 kb), PDGF-B transcript (3.5 kb), PDGF -receptor transcript (6.5 kb), and PDGF ß-receptor transcript (5.7 kb).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Although PDGF has for years been considered to be a key mitogen and a chemoattractant for smooth muscle cell proliferation after vascular injury,^{1 3 4 5 7 8 28} the activation of PDGF receptors in injured vessels in vivo has been unknown. The present observations for the first time demonstrate that the activation of PDGF - and ß-receptors was indeed stimulated during the growing phase of neointima (the initial 2 weeks) in balloon catheter–injured rat carotid artery. The results of the present study are consistent with the notion that increased activities of endogenous PDGF in injured artery have an important role in intimal smooth muscle accumulation after balloon injury.

PDGF -receptor binds either PDGF-A chain or PDGF-B chain, and PDGF ß-receptor binds only PDGF-B chain.^{6 29} Hence, the activation of both - and ß-receptors of PDGF observed in injured artery could be brought about by an increase in the local activity of either PDGF-B chain alone or both PDGF-A chain and PDGF-B chain. In either case, an increase in the PDGF-B activity in injured artery is of particular importance because PDGF-BB is much more potent in vitro both as a mitogen and as a chemoattractant for smooth muscle cells than PDGF-AA.^{8 30 31 32} Vascular smooth muscle cells in injured arteries may be exposed to PDGF-B chain derived from at least three sources: platelets adhering to denuded artery,³³ neointimal smooth muscle cells,³² and regenerated arterial endothelial cells.^{1 34} In the rat carotid balloon injury model, infiltration into injured arterial wall of monocytes and macrophages that are capable of producing PDGF-B chain was previously found to be very little.³⁵ It was reported that platelet adherence to exposed subendothelium of injured carotid artery was observed for only the first 7 days.³³ Hence, stimulation of PDGF receptors by PDGF released from adhering platelets might be limited to the initial few days. Very recently, Lindner et al³⁶ reported that a fraction (10%) of the most superficial cells in the neointima express PDGF-B chain by in situ hybridization. It was also previously shown that the neointima expresses PDGF ß-receptor relatively more abundantly than the media.²⁹ Lindner and Reidy³⁴ also reported that both PDGF-B chain and PDGF-A chain are expressed in endothelial cells at the wound edge of injured artery. Endothelial regeneration starts at 3 or 4 days after injury in the denuded artery and is nearly complete by 14 days.^{37 38} Therefore, stimulated secretion of PDGF from the regenerated endothelium may also contribute to the increased PDGF-B activity in injured artery.³⁵ In this regard, it is interesting to note that the production of endothelium-derived relaxing factor (NO) is decreased in regenerated endothelium of injured artery.³⁹ Because NO was previously shown to suppress PDGF-B chain expression in vascular endothelial cells,⁴⁰ the decreased ability of the regenerated endothelium to produce NO might also contribute to increased PDGF-B chain expression in the neointima and the endothelium. Two previous studies^{7 8} that examined the effects of infusion of neutralizing PDGF antibody and PDGF-BB homodimer on neointima formation have concluded that PDGF promotes intimal smooth muscle cell accumulation largely by stimulating smooth muscle cell migration into the intima. If we hypothesize that PDGF-B chain secretion from the inner neointima and/or the regenerating endothelium is increased, two events that occur in injured artery, ie, the migration of medial smooth muscle cells into the intima and stimulated activation of PDGF ß-receptors that are largely distributed in intimal smooth muscle cells, could be easily understood.

In the present study, we observed that the extent of tyrosyl phosphorylation of both PDGF - and ß-receptors peaked at 1 to 2 weeks after injury, then declined back to the basal level by 4 weeks (Fig 2A and 2B). In the rat carotid model, the neointima ceases growing at 2 weeks,^{2 4 11 17} despite continued activation of PDGF receptors as shown in the present study. This apparent paradox might be explained by the fact that the regenerated endothelium, which covers nearly the entire surface of the neointima by 2 weeks, also produces antiproliferative activities, including NO, prostacyclin, and C-type natriuretic peptide.⁴¹ These antiproliferative substances could antagonize further growth of the neointima. Alternatively, apoptotic cell death that occurs in injured vascular wall^{42 43} might balance smooth muscle proliferation, causing no overall accumulation of smooth muscle cells at this time after injury.

In recent years, Ang II has attracted much interest because of its suspected role in intimal smooth muscle accumulation after injury⁴ ; inhibitors of ACE and AT₁-selective Ang II receptor antagonists potently suppress intimal smooth muscle cell accumulation in the rat,^{3 4 9 10 11 12} indicating that Ang II is involved in intimal thickening after injury in this animal. It was also shown that Ang II infusion stimulated smooth muscle replication and intimal thickening in balloon-injured artery.⁴⁴ However, conflicting results were reported concerning a direct mitogenic activity of Ang II on cultured rat vascular smooth muscle cells in vitro; several groups found that Ang II neither stimulates smooth muscle cell proliferation by itself nor enhances the mitogenic effects of other growth factors,⁴⁵ whereas others claim that Ang II slightly or moderately stimulates their proliferation.^{13 14} In cultured vascular smooth muscle cells, Ang II was also reported to stimulate expression of PDGF-A chain but not B-chain.⁴⁶ However, PDGF-A chain does not support smooth muscle cell proliferation, at least in vitro.^{30 32} The present observations demonstrate that the AT₁ receptor antagonist potently inhibits the activation of both - and ß-receptors of PDGF in injured artery. Because PDGF acts as a chemoattractant and mitogen for vascular smooth muscle cells, it is very likely that previously reported suppressive actions of AT₁ antagonists on neointimal formation are due at least in part to inhibition of PDGF receptor activation. A precise molecular link between Ang II and PDGF receptor activation is not well understood presently. Our observations in the present study (Fig 7) indicate that Ang II is involved in the regulation of mRNA levels of PDGF-A and -B chains in vivo. This action of Ang II may partly account for AT₁ antagonist–induced inhibition of PDGF receptor activation. Linseman et al⁴⁷ recently demonstrated that stimulation of RASM cells with Ang II via the AT₁ receptor caused tyrosine phosphorylation of PDGF ß-receptors without stimulating autocrine PDGF release, indicating that a cross talk exists between AT₁ Ang II receptor and PDGF ß-receptor. It is an interesting possibility that the cross-talk mechanism between Ang II receptor and PDGF receptor might underlie Ang II–dependent PDGF receptor activation in injured carotid arteries. Further studies are required to define in more detail the mechanism of Ang II-mediated stimulation of PDGF receptor activation in injured artery.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II EGF = epidermal growth factor I/M = intimal-to-medial IGF = insulin-like growth factor IRS = insulin receptor substrate PDGF = platelet-derived growth factor RASM = rat aortic smooth muscle

	Acknowledgments

 
This work was supported by grants from the Ministry of Education, Science, and Culture of Japan, funds for cardiovascular research from Tsumura Co, and funds from the Japan Heart Foundation and Japan Research Foundation for Clinical Pharmacology.

Received February 10, 1997; revision received April 7, 1997; accepted April 18, 1997.

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