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Circulation Research. 1999;85:565-574

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(Circulation Research. 1999;85:565-574.)
© 1999 American Heart Association, Inc.

Molecular Medicine

Angiotensin II Stimulates Platelet-Derived Growth Factor-B Chain Expression in Newborn Rat Vascular Smooth Muscle Cells and Neointimal Cells Through Ras, Extracellular Signal–Regulated Protein Kinase, and c-Jun N-Terminal Protein Kinase Mechanisms

Jun-o Deguchi, Masatoshi Makuuchi, Takashi Nakaoka, Tucker Collins, Yoh Takuwa

From the Departments of Molecular and Cellular Physiology (J.D.), Surgery (J.D., M.M.), and Internal Medicine (T.N.), Graduate School of Medicine, University of Tokyo, Japan; Department of Physiology, Kanazawa University School of Medicine (Y.T.), Kanazawa, Japan; and Vascular Research Division, Department of Pathology, Brigham and Women's Hospital (T.C.), Boston, Mass.

Correspondence to Yoh Takuwa, MD, Department of Physiology, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan. E-mail ytakuwa{at}med.kanazawa-u.ac.jp


*    Abstract
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 *Abstract
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Abstract—Platelet-derived growth factors (PDGFs) have been implicated in the pathogenesis of vascular proliferative disorders. Vascular smooth muscle cells (VSMCs) are one of the cell types that produce PDGF-B chain in proliferative lesions, although the mechanism of regulation of PDGF-B chain production in these cells is not well understood. In the present study, we demonstrate that angiotensin II (Ang II), which is also implicated in vascular stenosis after angioplasty and atherosclerosis, markedly stimulates PDGF-B chain mRNA expression in cultured newborn rat medial VSMCs and neointimal VSMCs via an AT1, but not in adult rat VSMCs. In newborn rat VSMCs, Ang II activates extracellular signal–regulated protein kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 mitogen–activated protein kinase. The mitogen-activated protein/ERK (MEK) inhibitor PD98059, but not the p38 inhibitor SB203580, abrogates Ang II–induced PDGF-B mRNA expression. Transient transfection analysis using a PDGF-B promoter-luciferase gene reporter construct reveals that Ang II induces transcriptional activation of PDGF-B chain gene, which is abolished by the expression of a dominant negative form of either ERK or JNK, but not of p38. The expression of a dominant negative form of Ras abolishes the stimulatory effects of Ang II on ERK activity and PDGF-B mRNA expression. In adult rat VSMCs, Ang II activates ERK and JNK, but weakly induces Egr-1, a transcription factor implicated in PDGF-B chain gene expression, compared with newborn VSMCs. These data indicate that Ang II activates PDGF-B chain gene expression in VSMCs through mechanisms involving Ras-ERK and JNK.


Key Words: angiotensin II • platelet-derived growth factor B chain • vascular smooth muscle • Ras • mitogen-activated protein kinase


*    Introduction
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 up arrowAbstract
 *Introduction
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Platelet-derived growth factors (PDGFs) are potent mitogens and chemoattractants for vascular smooth muscle cells (VSMCs).1 2 These growth factors consist of homo- or heterodimers of 2 polypeptides, A and B chains.1 The receptors for the PDGFs consist of {alpha}- and ß-isoforms. The {alpha}-receptor binds either PDGF-A chain or -B chain, and the ß-receptor binds only B chain. PDGF-B chain is generally more potent than PDGF-A chain for VSMCs,3 indicating that activation of the ß-receptor rather than the {alpha}-receptor leads to more pronounced activation of VSMCs. It is believed that PDGFs are involved in the pathogenesis of atherosclerosis and stenosis after balloon angioplasty.4 5 In a rat carotid balloon injury model, procedures to suppress the action of PDGFs, such as repeated injections of neutralizing anti-PDGF antibody6 and local application of antisense oligonucleotides to PDGF ß-receptors,7 suppress neointima formation. In situ hybridization analysis reveals that transcripts of PDGF ß-receptor and PDGF-B chain are both detected in the neointima.8 9 These findings suggest a paracrine/autocrine role for PDGF in lesion formation. In addition, we have recently observed that the extent of PDGF ß-receptor tyrosine phosphorylation is increased in balloon-injured rat carotid arteries.10 These observations are consistent with an essential role for PDGF-B chain and its receptor in neointima formation in balloon-injured arteries. Despite the potential importance of PDGF-B chain in the formation of occlusive lesions in injured arteries, relatively little is known about the regulation of PDGF-B chain expression in neointimal cells.

Angiotensin II (Ang II) has also been implicated in the pathogenesis of neointima formation after balloon injury, especially in the rat model, as demonstrated by the observations that angiotensin-converting enzyme inhibitors11 and antagonists for AT112 effectively suppress neointima formation. Several in vitro studies using cultured VSMCs demonstrate that Ang II induces expression of growth factors including transforming growth factor-ß,13 14 PDGF-A,15 16 and basic fibroblast growth factor,17 suggesting the possibility that Ang II may stimulate growth of VSMCs via an autocrine mechanism. We recently showed that the administration of an AT1 antagonist abolished balloon injury-induced stimulation of PDGF ß-receptor tyrosine phosphorylation, with a reduction in PDGF-B mRNA expression, in rat carotid arteries.10 These observations raise the interesting possibility that Ang II may be involved in PDGF-B gene expression in vivo in VSMCs, especially in neointimal VSMCs, of injured blood vessels.

Previous studies have demonstrated that neointimal VSMCs are phenotypically distinct from medial VSMCs isolated from adult animals, but rather resemble newborn rat medial VSMCs.18 19 20 21 22 For example, adult rat medial VSMCs do not express a detectable level of PDGF-B chain, whereas newborn rat medial VSMCs, like neointimal VSMCs, express a readily detectable level of PDGF-B mRNA.19 20 22 23 In the present study, we demonstrate for the first time that Ang II potently stimulates PDGF-B chain mRNA expression in both neointimal VSMCs and newborn rat VSMCs. We also studied the signaling mechanisms underlying Ang II–induced PDGF-B gene expression and found that it is mediated via the AT1 in a manner dependent on Ras, extracellular signal–regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Thus, the present results indicate that PDGF-B chain gene is one of the target genes regulated by the Ras–mitogen-activated protein kinase (MAPK) signaling pathway in VSMCs.


*    Materials and Methods
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 *Materials and Methods
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Materials
Polyclonal rabbit anti-ERK2 antibody (C-14), polyclonal rabbit anti-JNK1 antibody (C-17), and polyclonal goat anti-p38 MAPK antibody (C-20) were purchased from Santa Cruz Biotechnology. Ang II and myelin basic protein were bought from Sigma. Glutathione-Sepharose and PD98059 were purchased from Pharmacia Biotechnology and Calbiochem, respectively. [{gamma}-32P]ATP and [{alpha}-32P]dCTP were purchased from DuPont-NEN. pGL2-Basic and pSV-ßgal were purchased from Promega. pGEX-cJun(5-89), pactEF–dominant negative (DN) ERK, pGEM-ATF2, pMT-Asn17-H · Ras, pBS-rPDGF-B, human Egr-1 cDNA, human Sp1 cDNA, and AxCALacZ were provided by Dr A.S. Kraft (University of Alabama School of Medicine, Tuscaloosa, Ala), Dr K. Okazaki (Kurume University Institute of Life Sciences, Kurume, Japan), Dr M.R. Green (University of Massachusetts Howard Hughes Medical Institute. Worcester, Mass), Dr J.M. Cooper (Harvard Medical School, Cambridge, Mass), Dr D. Katayose (Tohoku University Medical School, Sendai, Japan), RIKEN Gene Bank (Tsukuba, Japan), Dr R. Tjian (University of California, Berkeley), and Dr I. Saito (University of Tokyo Institute of Medical Sciences, Tokyo, Japan), respectively. Monoclonal anti-Ras antibody, monoclonal anti-ERK antibody, polyclonal anti-phospho-JNK antibody, and monoclonal anti-calponin antibody were purchased from Transduction Laboratory, Zymed, Promega, and Sigma, respectively. Specific polyclonal anti-ERK2 antibody, anti-JNK1 antibody, and anti-p38 antibody were purchased from Santa Cruz Biotechnology. Monoclonal anti-PDGF-B–specific antibody was a kind gift from Mochida Pharmacy. CV11974 (Candesartan) and PD123319 were kindly donated by Takeda Chemical Industries and Parke-Davis, respectively.

Cell Culture
Aortic VSMCs were isolated by the explant method from 1-week-old and 18-week-old Wistar male rats as described.10 24 To obtain neointimal VSMCs, left carotid arteries were removed from an 18-week-old male Wistar rat at 14 days after deendothelializing balloon injury10 and cut open longitudinally. Fine pieces (width <0.5 mm) of the neointima were excised by using fine forceps and scissors under a microscope and transferred to tissue culture dishes for the explant culture. VSMCs were maintained in DMEM supplemented with 10% FCS (Equitech-Bio), 105 U/L penicillin G, and 137 µmol/L streptomycin under an atmosphere of 95% air plus 5% CO2 at 37°C. Before each experiment, confluent cells were serum-deprived by incubation in serum-free DMEM for 48 hours. Cells between the 5th and 20th passages were used in the present study. VSMCs from an 18-week rat media, which were spindle-shaped and grew multilayered with characteristic hill-and-valley formation, were positive for anti–smooth muscle {alpha} actin staining, expressed a detectable amount of calponin as evaluated with Western blotting, and were responsive to vasoactive peptides including Ang II and endothelin-1 as evaluated with the intracellular Ca2+ measurement. In contrast to adult VSMCs, VSMCs obtained from a neonate and neointimal cells were polygonal in shape and grew to a monolayer at confluence, as described in previous reports.18 19 20 21 22 Western analysis using anti-calponin antibody did not detect expression of calponin in newborn VSMCs. These cells also responded to Ang II with an increase in the [Ca2+]i. Both newborn rat VSMCs and neointimal VSMCs were negative for von Willebrand factor as evaluated by immunocytochemical staining using anti–von Willebrand antibody (Dako), which excludes the possibility that VSMC cultures were contaminated with endothelial cells.

Northern Blot Analysis
Ten micrograms of total RNA, isolated from VSMCs by the acid-guanidinium isothiocyanate/phenol/chloroform method, was separated by formaldehyde-1.0% agarose gel electrophoresis and transferred onto a nylon membrane (Hybond N, Amersham) as described.24 Blots were hybridized with cDNA probes labeled with [{alpha}-32P]dCTP by the random priming method. PDGF-B probe is a 530-bp rat cDNA fragment from pBS-rPDGF-B encoding exons 1 to 5.25 Mouse AT1 cDNA was obtained by polymerase chain reaction (PCR).24 A membrane was rehybridized with 32P-labeled GAPDH cDNA probe. The radioactivity of corresponding bands was quantified by a Fuji BAS 2000 Bio-Image Analyzer (Fuji Film).10 24 mRNA levels were corrected for GAPDH mRNA level by calculating the ratio of PDGF-B mRNA or other mRNAs/GAPDH mRNA radioactivity for each sample.

Production of the Recombinant PDGF ß-Receptor Extracellular Domain (XR) Protein
The cDNA of the extracellular region (corresponding to amino acids 1 to 531) of human PDGF ß-receptor, with a stop codon TAG followed by an EcoRI site at the 3' end and an EcoRI site at the 5' end, was obtained by PCR using phPDGF-R (a gift from Dr H. Okazaki, Kirin Brewery, Yokohama, Japan) as a template and was ligated onto pVL1393 (PharMingen) at the EcoRI site downstream of the polyhedron promoter to create pVL1393-XR. Sf9 cells were cotransfected with pVL1393-XR and Baculogold baculovirus DNA (PharMingen) by the lipofection method, and the recombinant baculovirus encoding the PDGFXR cDNA was recovered. PDGFXR protein in serum-free conditioned medium of Sf9 cells infected with the baculovirus carrying the PDGFXR cDNA was purified by wheat germ agglutinin Sepharose 6 MB column as described.26 27

Activation of ERK, JNK, and p38
Each MAPK was immunoprecipitated by using respective specific polyclonal antibodies, and the MAPK activities were assayed in vitro by using myelin basic protein, glutathione S-transferase–cJun(5-89) and glutathione S-transferase–ATF2(1-109) as substrates for ERK, JNK, and p38, respectively, as described previously.28 The radioactivity in the bands corresponding to substrate proteins was measured by using a Fuji BAS 2000 BioImage Analyzer, as described.28

Transient Transfection Assay Using the PDGF-B Promoter-Luciferase Reporter Plasmid
A 1.0-kb fragment (–956 to +45 as indicated as the number of base pairs upstream of the TATA box) of the mouse PDGF-B promoter29 was isolated by PCR and cloned in sense orientation into the NheI-HindIII sites of pGL2-Basic to create Sis-Luc. Human full-length p38 MAPK cDNA was cloned by PCR from the HEL cell cDNA library, and point mutations were created by a PCR-based method.25 28 The nucleotide sequences of the cDNAs obtained by the PCR method were confirmed by sequencing with an ALFred DNA sequencer (Pharmacia Biotechnology). VSMCs were cotransfected by using LipofectAMINE (GIBCO) with Sis-Luc and either of the vectors encoding DN-ERK (pactEF-ERK-D170A),30 DN-JNK1(pME18S-JNK1-T183A, Y185F),30 or DN-p38(pME18S-p38-T180A, Y182F)31 or an empty vector (pME18S or pactEF). Cells were allowed to recover after transfection for 3 hours in DMEM containing 10% FCS and then serum-deprived for 48 hours. Cell lysates were prepared, and luciferase activity was measured with a Lumat LB95001 luminometer (Berthold) using the luciferase assay system (Promega), as described.30 Luciferase activity was normalized for ß-galactosidase activity measured in parallel cultures cotransfected with the ß-galactosidase expression plasmid pSV-ßgal and either of the expression vectors for DN forms of the MAPKs or empty vectors.

Western Blot Analysis of PDGF-B Protein in the Conditioned Media
Serum-deprived VSMCs were incubated with or without Ang II (100 nmol/L) for 36 hours. The conditioned media (8 mL) were collected, concentrated to 1 mL with a centrifugal concentrator (Centriprep 10; Amicon), and acid-precipitated with trichloroacetic acid (a final concentration of 10%) for 1 hour at 4°C. The precipitates were solubilized in Laemmli SDS sample buffer and separated on SDS–17.5% PAGE under reducing conditions, followed by electrotransfer onto Immobilon-P membrane (Millipore). Separated proteins were probed by monoclonal anti-PDGF-B–specific antibody, which was raised against amino acids 73 to 97 of human PDGF-B chain peptide and reactive for rat PDGF-B chain.

Construction of an Adenovirus and Gene Transfer
We constructed a recombinant replication-deficient adenovirus containing each of the genes of DN Ras (Asn17-H · Ras) and DN Rac (Asn17-Rac1), driven by the CAG promoter that consists of the cytomegalovirus 1E enhancer and chicken ß-actin promoter, by use of homologous recombination, as described in detail previously.32 VSMCs were infected with adenoviruses at a multiplicity of infection of {approx}50, allowed to recover in DMEM with 10% FCS for 3 hours, and then serum-deprived for 48 hours before experiments.

Statistics
The data are presented as mean±SE of 3 or more determinations or means of 2 determinations. The statistical significance of differences between the 2 groups was determined by Student t test, whereas multiple comparisons were analyzed by Scheffé test.


*    Results
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 *Results
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Ang II Stimulates Expression of PDGF-B mRNA and Protein in Newborn Rat VSMCs and Neointimal VSMCs
VSMCs from a newborn rat expressed a low level of PDGF-B mRNA at quiescent states (Figure 1ADown). Stimulation of VSMCs with Ang II (10 nmol/L) induced a marked increase in PDGF-B mRNA, which peaked at 4 hours and then gradually declined to an unstimulated level by 24 hours (Figure 1ADown). Ang II stimulation did not change GAPDH gene expression. Neointimal VSMC also responded to Ang II with an induction of PDGF-B mRNA, which became detectable at 2 hours, peaked at 6 hours, and then gradually declined, but was still at a detectable level at 24 hours (Figure 1BDown). In adult rat medial VSMCs, by contrast, PDGF-B mRNA was not at all detectable under the basal unstimulated conditions, and Ang II failed to induce PDGF-B gene expression (Figure 1CDown). The stimulatory effect of Ang II on PDGF-B mRNA in newborn rat VSMCs increased dose dependently with half-maximally and maximally effective concentration values of 700 pmol/L and 10 nmol/L, respectively (Figure 2ADown and 2BDown). This stimulatory effect of Ang II was abolished by the addition of the AT1 receptor-specific antagonist CV11974,10 but not of the AT2 antagonist PD123319 (Figure 3Down), indicating that the AT1 receptor mediates Ang II–induced PDGF-B gene expression.



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  		Figure 1. Ang II stimulates time-dependent expression of PDGF-B mRNA in newborn rat medial VSMCs and neointimal VSMCs, but not in adult rat medial VSMCs. The newborn rat VSMCs (A), neointimal VSMCs (B), and adult rat VSMCs (C) were stimulated with 10 nmol/L Ang II for the indicated time periods. mRNAs of PDGF-B and GAPDH were analyzed by Northern blotting. Arrowheads denote positions of PDGF-B transcript (3.5 kb). Each value in the lower panels of A and B represents a mean of duplicate determinations. The experiments were repeated 2 times with similar results.



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  		Figure 2. Ang II stimulates PDGF-B mRNA expression in a dose-dependent manner in newborn rat VSMCs. The cells were stimulated with varied concentrations of Ang II for 4 hours. A, Autoradiograms. B, Quantitative summary. Each value represents a mean of duplicate determinations.



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  		Figure 3. Ang II stimulates PDGF-B mRNA expression via an AT1. Newborn rat VSMCs were stimulated with 100 nmol/L Ang II in the presence and absence of 1 µmol/L CV11974 or 1 µmol/L PD123319 for 4 hours. A, Autoradiograms. B, Quantitative summary. Each value represents a mean of duplicate determinations.

To examine whether Ang II stimulates the production of PDGF-B protein, the conditioned media from Ang II-stimulated and nonstimulated newborn rat VSMCs were analyzed for PDGF-B protein by Western blotting (Figure 4Down). An anti-PDGF-B–reactive band with a molecular mass of 16 kDa was detected in both nonstimulated and stimulated conditioned media. The amount of the anti-PDGF-B–reactive protein was much larger in the stimulated conditioned medium than in the nonstimulated medium. Preincubation of anti-PDGF-B antibody with PDGF-B abolished this band, which indicates that the observed anti-PDGF-B–reactive protein represents PDGF-B peptide.



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  		Figure 4. Ang II stimulates production of PDGF-B protein. The newborn rat VSMCs were stimulated with 100 nmol/L Ang II or were left unstimulated for 36 hours. The conditioned media were analyzed by Western blotting, as described in Materials and Methods. Each lane was probed with anti–PDGF-B antibody or anti-PDGF-B antibody that had been preincubated with PDGF-B chain for 3 hours. Arrowhead denotes position of PDGF-B chain.

Blocking PDGF-B Action Partially Inhibits Ang II–Induced DNA Synthesis in Newborn VSMCs
Ang II stimulates DNA synthesis of serum-deprived newborn VSMCs {approx}2-fold at 48 hours, whereas Ang II does not affect DNA synthesis of adult VSMCs at either 24 or 48 hours (TableDown). The addition of recombinant extracellular domain of PDGF ß-receptor (XR), which has the activity to bind to and block the action of PDGF-B chain,26 27 significantly attenuates Ang II–induced DNA synthesis in newborn VSMCs. Both VSMC types show the comparable degrees of mitogenic responses to PDGF-BB (0.4 nmol/L) and to epidermal growth factor (0.2 nmol/L) (3.8- and 4.3-fold stimulation for PDGF-BB and 3.9- and 3.3-fold stimulation in newborn and adult VSMCs, respectively). The addition of XR abolishes DNA synthesis induced by PDGF-BB, but not by epidermal growth factor, in either cell type.


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  		Table 1. Ang II–Induced DNA Synthesis in Newborn and Adult VSMCs and the Inhibitory Effect of Blocking PDGF-B Action

Ang II–Induced PDGF-B Gene Expression Is Dependent on ERK and JNK
Stimulation of newborn rat VSMCs with Ang II (100 nmol/L) activated ERK/MAPK in a time-dependent manner, with a maximal 7-fold stimulation at 7 minutes (Figure 5ADown and 5BDown). Ang II also activated JNK and p38, the other 2 members of the MAPK family, although less strongly than ERK. Ang II–induced activation of ERK, JNK, and p38 are all abrogated by the addition of the AT1 antagonist CV11974, but not of the AT2 antagonist PD123319 (data not shown). PD98059 (30 µmol/L), which is an inhibitor of mitogen-activated protein/ERK (MEK, the upstream activating kinase of ERK), nearly totally abolished Ang II–induced PDGF-B mRNA expression (Figure 6ADown). PD98059 at the same dose also strongly inhibited Ang II–induced ERK activation (Figure 6BDown). By contrast, the p38-specific inhibitor SB203580 (10 µmol/L) did not inhibit Ang II–induced PDGF-B gene expression (Figure 6ADown). This dose of SB203580 inhibits a variety of p38-mediated responses in many cell types.33



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  		Figure 5. Ang II activates ERK, JNK, and p38 in a time-dependent manner. Newborn rat VSMCs were stimulated with 100 nmol/L Ang II for the indicated time periods, and the activities of ERK, JNK, and p38 were determined, as described in Materials and Methods. A, Autoradiograms. Arrowheads denote the positions of substrate proteins. B, Quantitative summary. Each value represents mean±SE of 4 determinations.



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  		Figure 6. The MEK inhibitor PD98059, but not the p38 inhibitor SB203580, inhibits Ang II–induced PDGF-B mRNA expression and ERK activities. Newborn rat VSMCs were stimulated with 100 nmol/L Ang II for 4 hours (for determination of PDGF-B mRNA) or for 7 minutes (for determination of ERK activities) in the presence or absence of 30 µmol/L PD98059 or 10 µmol/L SB203580. A, Autoradiograms and a quantitative summary of PDGF-B mRNA expression. B, Autoradiograms and a quantitative summary of ERK activities. Each value represents a mean±SE of 3 determinations. * and § denote a statistical significance (P<0.05) compared with "None" and "Ang II," respectively.

We next studied whether Ang II regulates PDGF-B gene expression at the level of transcription, and if so, whether the MAPKs mediate this, by examining the effects of expression of DN forms of the MAPKs and the addition of the chemical MAPK inhibitors on the promoter activity of PDGF-B gene. We constructed a PDGF-B gene promoter–luciferase fusion vector (Sis-Luc). We transfected newborn rat VSMCs with Sis-Luc and either an expression vector for a DN form of each MAPK or an empty vector, stimulated VSMCs with Ang II (100 nmol/L) for 4 hours, and determined luciferase activity in cell lysates. Ang II induced {approx}2-fold stimulation of the luciferase activity (Figure 7ADown), indicating that Ang II activates the transcription of PDGF-B gene. The expression of a DN form of ERK abolishes Ang II–induced stimulation of luciferase activity. Similarly, the expression of a DN form of JNK abolishes Ang II–induced stimulation of luciferase activity. By contrast, the expression of a DN form of p38 is without effect on Ang II–induced stimulation of the luciferase activity. Consistent with the above results, treatment of the cells with PD98059, but not with SB203580, inhibits Ang II–induced stimulation of luciferase activity (Figure 7BDown). All of these results indicate that ERK and JNK, but not p38, are involved in Ang II–induced transcriptional activation of PDGF-B gene.



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  		Figure 7. The expression of a DN form of ERK or JNK and the addition of PD98059 abolish Ang II–induced PDGF-B promoter activation. A, Newborn rat VSMCs were cotransfected with Sis-Luc and either empty vector or the expression vector for a DN form of each MAPK. B, Newborn VSMCs received PD98059 (30 µmol/L) or SB203580 (10 µmol/L) 30 minutes before Ang II addition. The cells were stimulated ({blacksquare}) with 100 nmol/L Ang II or left unstimulated ({square}) for 4 hours, and the luciferase activity in cell lysate was measured as described in Materials and Methods. Each value represents a mean±SE of 4 determinations. * and § denote statistical significance (P<0.05) compared with no stimulation and Ang II stimulation of the "Vector" group or the nonpretreated group, respectively. The experiments were repeated 3 times with similar results.

Ang II–Induced PDGF-B mRNA Expression and ERK Activation Are Dependent on Ras
To explore the Ras dependence of Ang II–induced MAPK activation and PDGF-B gene expression, we studied the effect of expression of DN-Ras (Asn17-H · Ras) by using an adenoviral vector. We infected VSMCs with an adenovirus encoding DN-Ras or a control adenovirus encoding Escherichia coli ß-galactosidase (LacZ). In newborn VSMCs infected with an adenovirus containing DN-Ras, a prominent band with a molecular mass of 21 kDa corresponding to DN-Ras protein was detected by Western analysis (Figure 8ADown). A faint band detected in VSMCs infected with an adenovirus containing LacZ represents endogenous Ras. In VSMCs infected with the control adenovirus containing LacZ, Ang II stimulated ERK and JNK (Figure 8CDown) and PDGF-B mRNA expression (Figure 8BDown), as in noninfected VSMCs. In VSMCs infected with an adenovirus encoding DN-Ras, Ang II–induced PDGF-B mRNA expression (Figure 8BDown) and ERK activation (Figure 8CDown) are strongly inhibited. By contrast, JNK activation by Ang II is not inhibited by the expression of DN-Ras (Figure 8CDown). Some studies34 previously demonstrated that G protein–coupled receptor agonist–induced JNK activation is dependent on the small G protein Rac. However, the expression of DN-Rac does not inhibit Ang II–induced JNK activation (data not shown).



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  		Figure 8. The expression of a DN-Ras mutant (Asn17-Ras) inhibits Ang II–induced PDGF-B mRNA expression and activation of ERK, but not of JNK. The newborn rat VSMCs were infected with either an adenovirus encoding Asn17-Ras or a control adenovirus encoding LacZ. The cells were stimulated with 100 nmol/L Ang II for 7 minutes (for determination of ERK activities), 15 minutes (for determination of JNK activities), or 4 hours (for determination of PDGF-B mRNA levels), respectively. A, Anti-Ras Western blots of cell lysate proteins from the cells infected with either adenovirus. B, Autoradiograms and a quantitative summary of PDGF-B mRNA expression. Each value represents a mean of duplicate determinations. Experiments were repeated 2 times with similar results. MBP indicates myelin basic protein. C, Autoradiograms and a quantitative summary of ERK activities. Each value represents a mean±SE of 3 determinations. *Statistical significance (P<0.05) compared with no stimulation of the LacZ group.

Adult VSMCs Show Expression of AT1 mRNA and Activation of ERK and JNK in Response to Ang II
Unlike newborn VSMCs, adult VSMCs do not respond to Ang II with an induction of PDGF-B chain mRNA (Figure 1CUp). We examined the Ang II–signaling pathway in adult VSMCs. Adult VSMCs as well as newborn VSMCs expressed AT1 mRNA, as evaluated with Northern analysis (Figure 9ADown). Adult VSMCs responded to Ang II with the activation of ERK and JNK (Figure 9BDown). Adult VSMCs also responded to Ang II with an increase in the [Ca2+]i (data not shown). Thus, like newborn VSMCs, adult VSMCs express functional AT1.



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  		Figure 9. Expression of AT1 mRNA in VSMCs, and activation of ERK and JNK in adult VSMCs. A, Total RNA (10 µg each) was analyzed for AT1 mRNA by Northern blotting. B, Adult VSMCs were stimulated with 100 nmol/L Ang II for the indicated time periods, and the activities of ERK and JNK were determined as described in Materials and Methods. Autoradiograms showing 32P-phosphorylation of substrates are displayed. MBP indicates myelin basic protein.

Ang II Induces a Greater Stimulation of Egr-1 Expression in Newborn VSMCs Than in Adult VSMCs
To explore a mechanism underlying the difference in PDGF-B mRNA response to Ang II between newborn VSMCs and adult VSMCs, we examined possible differences in the signaling events downstream of the MAPKs. We compared the expression of the immediate-early transcription factor Egr-1 and the zinc finger transcription factor Sp1, which are implicated in inducible and basal PDGF-B gene expression, respectively.23 36 37 38 The basal expression level of Egr-1 was similar in both cell types, but Ang II–induced Egr-1 expression was greater in newborn VSMCs (the maximal 5.8-fold stimulation over basal) than in adult VSMCs (the maximal 2.5-fold stimulation) (Figure 10Down). Compared with Egr-1, Sp1 is expressed at a slightly higher level in newborn VSMCs than in adult VSMCs and is not so prominently induced in either cell type.



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  		Figure 10. Time-dependent changes of Egr-1 and Sp1 mRNAs in newborn VSMCs and adult VSMCs. VSMCs were stimulated with 100 nmol/L Ang II at the indicated time periods. mRNAs of Egr-1 and Sp1 were analyzed by Northern blotting. Membranes of both VSMC types were hybridized in the same hybridization solution. Each value in panel B represents a mean of duplicate determinations.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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The present study demonstrates for the first time that Ang II induces PDGF-B mRNA expression in neointimal VSMCs and newborn rat medial VSMCs. Both PDGF-B chain and Ang II have been implicated in atherosclerosis and stenosis after balloon injury.4 5 6 7 11 12 39 The present results are consistent with our recent observation10 that an AT1 antagonist inhibits PDGF ß-receptor activation in vivo in balloon-injured carotid arteries. Collectively, these findings reveal a direct link between Ang II and PDGF-B and suggest that Ang II may stimulate the local production of PDGF-B chain found at sites of vascular proliferative lesions.

A number of studies suggest a role for Ang II in VSMC growth that occurs as a consequence of balloon vascular injury,11 12 hypertension,40 and atherosclerosis.39 Despite our recent observation implicating PDGF-B as a mediator of the Ang II action in vivo,10 previous in vitro studies15 16 failed to demonstrate stimulation of PDGF-B gene expression in response to Ang II. These previous studies used VSMCs derived from adult rats. It is well established that the phenotype of adult rat VSMCs in culture differs from that of neointimal cells.20 21 22 It is also known that neointimal VSMCs phenotypically resemble newborn rat medial VSMCs, in terms of both cell morphology and gene expression.18 19 20 21 22 It was demonstrated that enhanced expression of tropoelastin and {alpha}1-procollagen genes in vivo in the neointima compared with underlying media was maintained in cultures of neointimal cells.22 Similar phenotypes were also observed in newborn rat aorta and cultures of newborn rat medial VSMCs.22 These observations suggested that neointimal cells and newborn medial VSMCs in culture expressed phenotypes characteristic of earlier stages of artery wall development.22 Both newborn rat VSMCs and neointimal VSMCs express PDGF-B gene, as well as PDGF-A gene, and secrete increased amounts of PDGF activities compared with adult rat VSMCs.19 20 21 22 The present results reveal an additional, important property (ie, responsiveness to Ang II with induction of PDGF-B gene expression), which is shared by both neointimal VSMCs and newborn rat VSMCs. We recently observed that an AT1 antagonist reduced the expression level of PDGF-B chain mRNA in balloon-injured rat carotid arteries.10 In situ hybridization analysis revealed that a portion of neointimal cells in balloon-injured carotid arteries expressed PDGF-B chain.9 The present results together with these previous observations suggest that Ang II is involved in PDGF-B gene expression in the neointima in vivo of injured arteries. A number of recent studies provide evidence for the activation of the local renin-angiotensin system in the rat carotid balloon-injury model. It was demonstrated previously that mRNA expression of renin, angiotensinogen, angiotensin-converting enzyme, and AT1 mRNA were upregulated in rat balloon-injured arteries.41 42 43 Thus, these observations suggest that enhancement of the local renin-angiotensin system augments the AT1-mediated signaling in injured arteries, leading to upregulation of PDGF-B gene expression in the neointima.

In addition to the suggested pathological role for the Ang II–PDGF-B chain axis in the formation of vascular proliferative lesions, high constitutive23 and induced expression of PDGF-B chain (the present study) in newborn VSMCs may also suggest that PDGF-B chain plays a developmental role in blood vessels, because phenotypes of newborn VSMCs and neointimal cells in culture are shown to be similar to those of arterial VSMCs in vivo at earlier stages of blood vessel development.22 44 The observation of a thinner wall of aorta in PDGF-B chain–null mice lends support to a role for PDGF-B chain in blood vessel development.45 It is an interesting possibility that Ang II might be involved in PDGF-B expression in the vessel wall during prenatal and postnatal early developmental stages.

The present study also defined the signaling mechanisms underlying Ang II–induced PDGF-B mRNA expression. Ang II activates all 3 members of the MAPKs (ie, ERK, JNK, and p38) in newborn rat VSMCs (Figure 6Up), as in adult VSMCs. The MAPK cascades are important signaling systems that convey signals into the nucleus to initiate cellular responses, including gene expression.46 Therefore, we examined whether the MAPK cascades are involved in Ang II–induced PDGF-B gene expression, by using expression vectors for a DN form of each MAPK, as well as known specific inhibitors for the MAPK cascades. The present results demonstrate that both ERK and JNK, but not p38, are involved in Ang II–induced PDGF-B mRNA expression by positively regulating transcription of PDGF-B gene (Figures 7Up and 8Up). We do not know at present whether or not the activity of the MAPK cascades influences the stability of PDGF-B mRNA. Recent studies22 35 36 37 reveal that the interaction of the immediate-early gene product Egr-1 with a cis-acting element in the proximal PDGF-B promoter is essential for phobol ester- or injury-induced transcriptional activation of PDGF-B gene in vascular endothelial cells. We demonstrate in the present study that Ang II induces Egr-1 mRNA expression in VSMCs (Figure 10Up). Because Ang II and a phorbol ester induce activation of ERK, it is likely that ERK mediates Egr-1 expression, which contributes to the subsequent activation of the secondary responsive PDGF-B gene. We, in fact, observed that Ang II induces Egr-1 mRNA expression in VSMCs in a PD98059-sensitive manner (J.D., Y.T., unpublished data, 1998). It is presently unknown whether the JNK cascade converges onto Egr-1 gene expression or exists as a parallel, independent pathway necessary for activating PDGF-B gene transcription.

Several recent studies47 48 49 demonstrate that Ang II activates Ras, which is a known activator of the Raf-ERK cascade. In the present study, we tested a role for Ras in Ang II–induced MAPK activation and PDGF-B gene expression. The expression of a DN-Ras mutant by using an adenoviral vector efficiently suppresses Ang II–induced ERK activation, indicating that Ras is required for Ang II–induced ERK activation (Figure 9CUp). This is the first direct demonstration of Ras dependence of Ang II–induced ERK activation in VSMCs. Consistent with the requirement of ERK for Ang II–induced PDGF-B gene expression, the expression of a DN-Ras abolishes Ang II–induced PDGF-B gene expression (Figure 9BUp). Thus, PDGF-B gene is the target gene that is under the control of the Ras-MAPK signaling pathway in VSMCs. In contrast to the ERK pathway, Ang II–induced JNK activation is not dependent on Ras (Figure 9CUp).

Like newborn VSMCs, adult VSMCs express AT1 and display Ang II–induced activation of ERK and JNK (Figure 9AUp and 9BUp), which are signals necessary for Ang II–induced PDGF-B chain gene expression (Figures 6Up and 7Up). We found a difference in the Ang II–induced signaling at a site downstream of ERK; Egr-1 induction is much greater in newborn cells compared with adult cells (Figure 10Up). We also observed that the Egr-1 response to Ang II was enhanced in neointimal cells (unpublished data, 1999). These observations, together with the recent knowledge that Egr-1 plays an important stimulatory role in PDGF-B gene transcription,23 36 37 38 implicate a role for Egr-1 in Ang II–induced PDGF-B chain gene expression in newborn VSMCs and neointimal cells. However, adult VSMCs, which exhibit a less prominent but significant Egr-1 mRNA response to ANG II, do not at all show an induction of PDGF-B chain mRNA. This suggests that some additional signals besides the activation of ERK-Egr1 and JNK are indispensable for Ang II–induced PDGF-B chain gene expression in newborn VSMCs. Newborn VSMCs generally show reduced levels of expression of various smooth muscle–specific proteins, including calponin and smooth muscle {alpha}-actin,20 compared with adult VSMCs. On the other hand, they express higher levels of PDGF-B chain, tropoelastin, and osteopontin than adult VSMCs. It is not yet known whether Egr-1 contributes to upregulation of tropoelastin and osteopontin expression and also to downregulation of smooth muscle–specific protein expression indirectly via other transcriptional regulators. An especially interesting avenue to be explored is how these features of gene expression in newborn cells, many of which are shown to be shared by neointimal cells,22 are determined.


*    Acknowledgments
 
This work was supported by grants from the Ministry of Education, Science and Culture of Japan and by the Japan Society for the Promotion of Science "Research for the Future" Program. We thank Rieko Suzuki, Masako Katoh, and Nobuko Yamaguchi for preparing the manuscript.

Received September 28, 1998; accepted July 26, 1999.


*    References
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