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Endocrinology Vol. 147, No. 9 4169-4178
Copyright © 2006 by The Endocrine Society

Increase of C-Type Natriuretic Peptide Expression by Serum and Platelet-Derived Growth Factor-BB in Human Aortic Smooth Muscle Cells Is Dependent on Protein Kinase C Activation

Maria C. Mendonça, Sonia Q. Doi, Steven Glerum and Donald F. Sellitti

Department of Medicine, Divisions of Endocrinology and Nephrology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799

Address all correspondence and requests for reprints to: Donald F. Sellitti, Ph.D., Uniformed Services University of the Health Sciences, Department of Medicine, 4301 Jones Bridge Road, A3060, Bethesda, Maryland 20814-4799. E-mail: dsellitti{at}usuhs.mil.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
C-type natriuretic peptide (CNP) is produced by the vascular smooth muscle cells (SMCs) of injured and atherosclerotic arteries, in which it may exert autocrine control over SMCs by binding to its principal receptors, NPR-B and NPR-C, but few studies have examined the factors that regulate CNP expression in human SMCs. In the present report, we show that serum induces significant increases in both CNP and NPR-C transcript levels in human, but not rat SMCs in culture, and that pretreatment with either the general tyrosine kinase inhibitor genistein, the platelet-derived growth factor (PDGF) tyrosine kinase inhibitor AG 1296, or the protein kinase C (PKC) inhibitor GF109203X blocks most of the serum-induced increase in CNP. PDGF-BB also induced significant dose-dependent increases in CNP transcript that correlated temporally with the serum effect on CNP mRNA. Inhibition of several PDGF-BB signaling pathways downstream of receptor activation showed that PKC inhibition with GF109203X was almost as effective as genistein in abolishing the PDGF-BB-induced up-regulation of CNP mRNA. Furthermore, PKC activation by phorbol 12-myristate 13-acetate (PMA) produced an extremely high level of CNP mRNA that was abolished by GF109203X. Immunoreactive CNP was markedly increased in SMCs receiving 10% serum, 20 ng/ml PDGF-BB, or PMA, and was decreased in PDGF-treated and PMA-treated cells by AG 1296 and GF109203X, respectively. This report suggests that in humans, PDGF and other factors signaling through receptor tyrosine kinases and downstream activation of PKC could represent an important control for CNP expression in vascular smooth muscle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NATRIURETIC PEPTIDE system, consisting of the family of natriuretic peptides [atrial natriuretic factor (ANF), brain natriuretic peptide, and C-type natriuretic peptide (CNP)] acting through specific receptors in the vascular wall [guanylyl cyclase natriuretic peptide receptors (NPRs)-A and NPR-B and the nonguanylyl cyclase receptor, NPR-C] regulates vascular tone and may be involved in the etiologies of salt-sensitive hypertension and atherosclerosis (1, 2, 3). Unlike the closely related hormone ANF, CNP does not enter the circulation in significant amounts, and it has come to be regarded as an autocrine or paracrine factor in the tissues and organs in which it is expressed (3). In the vasculature, CNP production in the endothelium and binding to smooth muscle cells via both NPR-B and NPR-C is thought to function in the control of vascular contractility (4) and also to play a significant role in inhibiting vascular remodeling after injury (5, 6).

Vascular smooth muscle cells (SMCs) have been regarded largely as the target of CNP action rather than a source of the peptide, but several recent studies (7, 8, 9, 10) have shown that SMCs in culture or in vivo can express CNP transcript or immunoreactive CNP protein. Most recently Naruko et al. (10) demonstrated that within 2 months after percutaneous coronary intervention, most neointimal SMCs expressed immunoreactive CNP, as did most neointimal SMCs in atherectomy specimens (7). These authors suggest that an autocrine production of CNP by SMCs could be important in controlling the progress of neointimal growth after percutaneous coronary intervention. Altered CNP expression after vascular damage also implies that regulation of CNP production in SMCs may be controlled by factors secreted by injured endothelium, platelets, or inflammatory cells.

However, despite the potential importance of CNP as an autocrine or paracrine regulator of vascular smooth muscle function and disease, not a great deal is known about the serum factors and signaling mechanisms that regulate its expression in SMCs or endothelial cells. Woodard et al. (9) demonstrated using cultured SMCs derived from Wistar rats that basic fibroblast growth factor (bFGF) suppresses CNP transcript levels. In contrast, in SMCs derived from stroke-prone spontaneously hypertensive rats (SPHRs), these authors found that the CNP response to bFGF was opposite that of the normotensive Wistar rats, with bFGF evincing an up-regulation rather than down-regulation of CNP mRNA content. These results not only demonstrate a growth factor regulation of CNP in SMCs at the transcriptional level but also suggest that that regulation could be altered in smooth muscle cells from genetically stroke-prone animals. There have to date been no reports of the transcriptional regulation of CNP by growth factors in SMCs of human origin.

Given the importance of growth factors, present in endothelial cells, platelets, and inflammatory cells and in serum in the development of both atherosclerotic and restenotic lesions (11), we studied the roles of serum and platelet-derived growth factor (PDGF)-BB in regulating CNP and its cognate receptors (NPR-B and NPR-C) in vascular smooth muscle of human origin. We show that the expression of CNP is markedly up-regulated by both serum and PDGF-BB in human but not rat SMC in culture and that protein kinase C (PKC) activation may play a prominent role in this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant human PDGF-BB, recombinant human bFGF, genistein, genistin, LY 294002, GF 109203X-HCl, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP2), PD 98059, and AG 1296 were purchased from Calbiochem-EMD Biosciences (La Jolla, CA). Fetal bovine serum (FBS) for the serum studies was obtained from Life Technologies, Inc. (Grand Island, NY) and was certified mycoplasma, virus, bacteriophage, and endotoxin free. Polyclonal rabbit antiserum against CNP-22 was obtained from Peninsula Laboratories (Belmont, CA).

Cell culture
Primary cultures of human (catalog no. CC-2571) and rat (catalog no. AC-7009) aortic SMCs (AOSMCs) were obtained from Cambrex BioScience (Walkersville, MD) at passage 3 and grown in smooth muscle basal medium (SmBM; Cambrex) modified by the addition of the following components to constitute smooth muscle growth medium: human epidermal growth factor (0.5 ng/ml), human fibroblast growth factor (2 ng/ml), insulin (5 µg/ml), 5% FBS, and 50 µg/ml gentamicin sulfate. Assays were performed on cells between passages 6 and 8 plated into plastic 6- or 24-well plates as described below. For RNA extraction studies, AOSMCs were allowed to grow to confluence in six-well plates and then washed with serum-free medium and incubated for an additional 48 h in serum-free SmBM before the addition of serum or reagents and subsequent RNA extraction. In one study, cultured AOSMCs were induced to differentiate by plating into a 24-well plate containing BIOCOAT growth factor reduced Matrigel matrix (Becton Dickinson Labware, Bedford, MA) in a serum-free medium (SMC-D-STIM; Becton Dickinson) designed for smooth muscle differentiation. AOSMCs grown in Matrigel acquired a nodular morphology within 24 h (12).

Human aortic endothelial cells (HAECs; catalog no. CC-2635) were also obtained from Cambrex at passage 3 and were certified by the supplier as Factor VIII-related antigen positive and {alpha}-actin negative. These cells were subcultured in endothelial growth medium (Cambrex) containing endothelial basal medium (EBM; Cambrex) to which was added bovine brain extract, hydrocortisone, human epidermal growth factor, 5% FBS, and gentamicin/amphotericin-B. For RNA extraction, HAECs were allowed to grow to confluence in 6-well culture dishes in endothelial growth medium and then washed and exposed to serum-free EBM for 24 h before the addition of test reagents and subsequent RNA extraction.

Immunocytochemistry
AOSMCs were seeded onto round glass coverslips placed in 24-well plates and were grown in smooth muscle growth medium before switching medium to serum-free SmBM for 48 h. Cells then received treatment media (serum-free SmBM, 10% FBS in SmBM, 20 ng/ml PDGF-BB, or 1 µM PMA in SmBM with or without preincubation with inhibitors) for 24 and 48 h before staining. Cells were transferred to a new 24-well plate in PBS (pH 7.4) and washed three times in PBS before fixation for 5 min in 70% ethanol/1% glacial acetic acid at room temperature. After fixation, cells were washed twice with PBS for 3 min each and then blocked with PBS containing 3% BSA and incubated for 1 h at room temperature with 225 µl rabbit polyclonal anti-CNP (Peninsula) diluted 1:200 in PBS/3% BSA. After three washes of 3 min each in PBS, cells were incubated for 30 min at 37 C with fluorescein isothiocyanate-conjugated goat antirabbit IgG (MP Biomedical, Irvine, CA) diluted 1:50 in PBS. Cells were washed again (three 3-min washes in PBS) and then mounted on glass slides and coverslipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and observed and photographed using a fluorescence microscope (Eclipse E800; Nikon, Tokyo, Japan) linked to a high-resolution 3.3- and 5.0-megapixel digital camera (Q Imaging, Burnaby British Columbia, Canada). No light or color modifications were made in any of the photographs; all of them were taken at same exposure, gain and offset.

Real-time PCR
For real-time PCR, 100 ng total RNA were used for reverse transcription and amplification of target cDNA in the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). TaqMan one-step RT-PCR master mix reagents were purchased from Applied Biosystems. Primers and 5'FAM-labeled probe for human and rat CNP, NPR-A, NPR-B, NPR-C, and ß-actin were designed from NCBI GenBank sequences using Primer Express software (version 1.5; Applied Biosystems) and synthesized by Epoch Biosciences (San Diego, CA). Primer and probe sequences are listed in Table 1Go.


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  		TABLE 1. Sequences of primers and Taqman probes used in real-time PCR

 
For human and rat ß-actin, reaction mix contained 0.25 U/µl MultiScribe enzyme, 0.4 U/µl RNase inhibitor, 300 nM sense and antisense primer, 150 nM probe, and 100 ng RNA in a total volume of 50 µl. Reaction mix for all other target genes was identical except for a TaqMan probe concentration of 250 nM. Thermal cycling parameters were as follows: 48 C, 30 min; 95 C, 10 min; and 40 cycles of denaturing at 95 C for 15 sec, and annealing/extension for 1 min at 60 C. Threshold cycle (Ct), the cycle at which emission rises above baseline, was determined for both the target gene (e.g. CNP) and ß-actin for each sample, and relative quantitation of target gene transcript was determined by a modification of the comparative Ct method as described in the ABI Prism 7700 sequence detection system manual, user bulletin 2 (Applied Biosystems). In this procedure, target (e.g. CNP) Ct values were determined for each sample (n = 3 or 4 for each treatment) as was the corresponding ß-actin Ct value for each sample determined in a separate assay. A {Delta}Ct for each sample was then calculated by subtracting from each target Ct value the mean ß-actin Ct averaged from all ß-actin Ct values in that particular assay. This was done to avoid the possibility that the contribution of small intergroup differences in ß-actin Ct could affect calculation of {Delta}Ct for the target gene. Relative quantitation of target transcript levels and significant differences in these levels among treatment groups were determined as described below.

Statistical analysis
Differences between transcript levels among treatment groups in real-time PCR assays were evaluated by calculating mean {Delta}Ct values for each group and comparing these using InStat software (GraphPad Software, Inc., San Diego, CA; ANOVA followed by the Tukey-Kramer test) to determine significance. Relative quantitation of transcript levels (compared with control) was determined by evaluating the expression 2–{Delta}{Delta}Ct, where {Delta}{Delta}Ct represents the subtraction of the {Delta}Ct determined for control from the {Delta}Ct determined for each treatment group. Error bars represent positive and negative error of each calculated value.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum (10% FBS) up-regulates CNP and NPR-C but not NPR-B and NPR-A mRNAs in human AOSMCs
The effect of serum addition on transcript levels of CNP and the three natriuretic peptide receptors in 48 h serum-starved human AOSMCs is shown in Fig. 1Go. CNP mRNA levels responded to serum with an almost 45-fold increase by 3 h, reaching a maximum level at 8 h and declining by 24 h to a level that was still approximately 14-fold greater than its matched serum-free control. NPR-C transcript was also significantly up-regulated within 3 h by exposure to 10% FBS; however, it required a 24-h exposure to serum to attain its maximal (~5-fold) value, compared with its matched serum-free control. In contrast, mRNA levels of the guanylyl cyclase receptors NPR-B and NPR-A were not increased above control by serum addition at any time point examined. Instead, NPR-B transcript showed a transient reduction in the serum-treated cells at 8 h, compared with control, and NPR-A was significantly reduced by serum at 24 h (Fig. 1Go).


Figure 1
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  		FIG. 1. Time course of change in CNP and NPR mRNA levels after serum (10% FBS) addition to 48 h serum-starved human AOSMCs. Confluent cells in 6-well plates received either serum-free SmBM medium or SmBM containing 10% FBS at 0 h and after incubation at 37 C, 5% CO2 were harvested for total RNA at the times indicated. A control plate (0 h) received fresh serum-free SmBM and was harvested immediately. CNP, NPR-C, NPR-B, and NPR-A transcript levels were determined using real-time PCR and normalized to a 0 h control value of 1 as described in Materials and Methods. Data are presented as means ± positive and negative error; n = 3 wells/group. **, P < 0.01; ***, P < 0.001 vs. time-matched serum-free control.

 
To determine whether altering the differentiation state of the cultured human AOSMCs could change the response of CNP transcript levels to serum, CNP mRNA levels were determined in AOSMCs grown on the differentiation substrate, Matrigel, and exposed to either serum-free medium or medium containing 10% FBS for 8 h before assay. Even though much of the culture assumed a nodular form, 10% serum still elicited a significant (14-fold) increase in CNP mRNA over serum-free control (data not shown), although the level of increase was lower than that seen in monolayer culture (48-fold above control in Fig. 1Go).

CNP response to serum in rat AOSMCs and human endothelial cells differs from human AOSMCs
To examine the species specificity of the effects of serum on CNP and NPR in AOSMCs, transcript levels of CNP, NPR-C, NPR-B, and NPR-A were also determined in cultures of rat AOSMCs after 48 h serum starvation and readdition of serum for 24 h (Fig. 2Go). In marked contrast to the results with human SMCs, exposure of 48 h serum-starved rat SMCs to 10% FBS for 24 h resulted in an 80% reduction in CNP transcript levels and an approximately 65% reduction in NPR-C transcript, whereas NPR-A was significantly elevated in the presence of serum. Similar to the results with human cells, NPR-B mRNA was not altered, compared with control after 24 h of exposure to serum (Fig. 2Go).


Figure 2
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  		FIG. 2. The effect of serum (10% FBS) on CNP and NPR mRNA levels in rat AOSMCs after 48 h serum starvation. Confluent cells in 6-well plates received either serum-free SmBM medium or SmBM containing 10% FBS and were harvested for total RNA extraction after a 24-h incubation at 37 C. CNP, NPR-C, NPR-B, and NPR-A transcript levels were determined using real-time PCR with rat-specific primers and normalized to the serum-free value for each transcript as described in Materials and Methods. Data are presented as means ± positive and negative error; n = 3 wells/group. **, P < 0.01; ***, P < 0.001 vs. control.

 
Because endothelial cells have been cited as a major source of CNP in the normal vasculature, we examined the time course of serum addition on CNP and NPR mRNA levels in HAECs. Results showed clearly that CNP is regulated differently by serum in these cells, compared with the smooth muscle cells (Fig. 3Go). In contrast to the human AOSMCs, HAECs showed a much lower response in CNP mRNA to serum addition, reaching a level of only 3-fold control value at 3 h and declining toward time-matched control values at 8 and 24 h after serum addition. NPR-C mRNA also increased after serum addition, maintaining a level of approximately 4 times that of age-matched control through 24 h (although the difference at 8 h did not attain significance). In addition, NPR-B transcript levels in HAECs were unaffected by serum addition at any time during the 24-h period of study, and NPR-A (in a time course similar to that of CNP mRNA levels) exhibited a significant peak at 3 h after serum but declined to control value at 24 h.


Figure 3
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  		FIG. 3. Time course of change in CNP and NPR mRNA levels in HAECs after serum (10% FBS) addition to 24 h serum-starved cells. Confluent cells in 6-well plates received either serum-free EBM or EBM containing 10% FBS at 0 h and after incubation at 37 C, 5% CO2 were harvested for total RNA at the times indicated. A control plate (0 h) received fresh serum-free EBM and was harvested immediately. CNP, NPR-C, NPR-B, and NPR-A transcript levels were determined using real-time PCR and normalized to the 0 h control value of 1 as described in Materials and Methods. Data are presented as means ± positive and negative error; n = 3 wells/group. *, P < 0.05 vs. time-matched serum-free control.

 
Tyrosine kinase activation by serum and growth factors leads to increased CNP mRNA in human AOSMCs
The results of the above survey of the regulation of the natriuretic peptide system in different vascular-derived cell types suggest that the CNP gene in human SMCs represents the component of this system most responsive to changes in serum concentration. Because many of the major biological effects of serum in cell growth and transcription are due to the presence of growth factors (bFGF, PDGF, vascular endothelial growth factor, and others) signaling through receptor tyrosine kinases, we tested the effect of the general tyrosine kinase inhibitor genistein on the serum-up-regulation of CNP transcript in human AOSMCs. Results showed that most (mean of 87% in three separate studies, one of which is shown here) of the positive effect of serum on CNP mRNA was abolished by tyrosine kinase inhibition, supporting a major role for tyrosine kinase-linked signaling in this process (Fig. 4AGo). In light of previous studies showing the suppression of CNP transcript by bFGF (9) and NPR-C transcript by PDGF-BB in rat vascular smooth muscle (13), we examined the effects of these two growth factors on CNP in human AOSMCs (Fig. 4BGo). Each growth factor induced a significant up-regulation in CNP transcript (9.2- and 5.5-fold, respectively, for PDGF and bFGF in this study), albeit less than that induced by 10% serum. As expected for ligand signaling through tyrosine kinase-linked receptors, the PDGF- and bFGF-induced increases in CNP mRNA were completely abolished in the presence of genistein (Fig. 4BGo). The failure of genistin, the inactive analog of genistein, to suppress either the serum- or PDGF-induced increase in CNP mRNA confirmed the specificity of genistein as a tyrosine kinase inhibitor (data not shown).


Figure 4
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  		FIG. 4. Effects of tyrosine kinase inhibition on serum and growth factor enhancement of CNP mRNA in human AOSMCs. A, Serum ± genistein. B, PDGF-BB and bFGF ± genistein. C, PDGF-BB ± AG 1296. D, Serum ± AG 1296. Confluent cells in 6-well plates were serum starved in SmBM medium for 48 h and then received 1.2 ml SmBM medium containing either 200 µM genistein in dimethylsulfoxide (DMSO) or only DMSO for 1 h before addition of 1.2 ml of the following: 1) SmBM medium (control), 2) 20% FBS, 3) 40 ng/ml PDGF-BB, or 4) 40 ng/ml bFGF. Cells were quickly mixed to achieve final concentrations of half these stated values (i.e. 100 µM genistein, 10% FBS, and 20 ng/ml PDGF-BB and bFGF). In a separate study, confluent, 48 h serum-starved AOSMCs in 1.2 ml SmBM received either 40 µM AG 1296 in DMSO or DMSO only for 1 h before the addition of 1.2 ml of: 1) SmBM (control), 2) 20% FBS, or 3) 40 ng/ml PDGF-BB. The media were quickly mixed. After an additional 8 h incubation at 37 C, 5% CO2 cells were harvested for total RNA extraction, and CNP transcript levels were determined using real-time PCR and normalized to the 0 h control value of 1. Data are presented as means ± positive and negative error; n = 3 wells/group. ***, P < 0.001 vs. time-matched serum-free control; #, P < 0.001 vs. FBS- or growth factor-treated group; {alpha}, P < 0.05, compared with FBS-treated group.

 
To further assess the role of PDGF signaling in CNP regulation, we measured CNP transcript levels after 8 h exposure to either serum or PDGF-BB in the presence or absence of the tyrphostin AG 1296, a specific inhibitor of PDGF receptor tyrosine kinase. As expected, AG 1296 abolished the PDGF-BB-induced up-regulation of CNP mRNA (Fig. 4CGo), but it also significantly inhibited the serum-induced increase in CNP transcript by almost 60% in this study (Fig. 4DGo).

PDGF-BB up-regulation of CNP and NPR-C transcript occurs in human but not rat AOSMCs
Because PDGF receptor signaling appears to play a major role in the regulation of CNP transcription in SMCs, we analyzed the role of PDGF-BB in this process in greater detail (Figs. 5Go and 6Go). Examination of the time course of CNP mRNA up-regulation in human AOSMCs in response to PDGF-BB (Fig. 5AGo) revealed a profile very similar to that elicited by serum, with attainment of maximal levels (compared with 0 h control) at 8 h after treatment and the maintenance of significant increases above control through 24 h of treatment. The dose relatedness of the regulation of CNP mRNA by PDGF-BB is illustrated in Fig. 5BGo and shows a rather abrupt increase in response between 2 and 10 ng/ml and a plateau at a dose of 20 ng/ml. NPR-C transcript response to PDGF-BB was also similar to its response to serum (Fig. 1Go) in that the maximal value, compared with time-matched control, was relatively low (about 2.4-fold control) and obtained only after a 24-h exposure (Fig. 5CGo). However, unlike serum, PDGF-BB did not induce significant increases in NPR-C vs. time-matched controls at earlier time points.


Figure 5
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  		FIG. 5. Time- and dose-dependent effects of PDGF-BB on CNP and NPR-C transcript levels in human AOSMCs. A, Time course of change in CNP mRNA levels after PDGF-BB addition to 48-h serum-starved human AOSMCs. Confluent cells in 6-well plates received either serum-free SmBM medium or SmBM containing 20 ng/ml human recombinant PDGF-BB at 0 h and after incubation at 37 C, 5% CO2 were harvested for total RNA at the times indicated. A control plate (0 h) received fresh serum-free SmBM and was harvested immediately. CNP transcript levels were determined using real-time PCR and normalized to the 0 h control value of 1. B, Effect of PDGF-BB concentration on CNP mRNA levels in human AOSMCs. Confluent, 48-h serum-starved human AOSMCs in 6-well plates received 0, 0.5, 2, 10, 20, or 50 ng/ml PDGF-BB for 8 h before cell harvesting and RNA extraction. Real-time PCR for CNP was performed as described above as was the determination of relative change in CNP mRNA levels. C, Time course of change in NPR-C mRNA levels after PDGF-BB addition to 48-h serum-starved human AOSMCs using cells described in Fig. 5AGo. Data are presented as means ± positive and negative error; n = 3 wells/group. **, P < 0.01 and ***, P < 0.001 vs. time-matched control (A and C) or no PDGF control (B).

 

Figure 6
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  		FIG. 6. Time course of change in CNP and NPR-C transcript levels in rat AOSMCs after addition of PDGF-BB. Confluent cells in 6-well plates were serum starved for 48 h and then received either serum-free SmBM medium or SmBM containing 20 ng/ml human recombinant PDGF-BB at 0 h and after incubation at 37 C, 5% CO2 were harvested for total RNA at the times indicated. A control plate (0 h) received fresh serum-free SmBM and was harvested immediately. CNP (A) and NPR-C (B) transcript levels were determined using real-time PCR and normalized to the 0 h control value of 1. Data are presented as means ± positive and negative error; n = 3 wells/group. *, P < 0.05; ***, P < 0.001 vs. serum-free control.

 
In marked contrast to the results with human cells, PDGF-BB treatment of rat AOSMCs resulted in an early (3 h) reduction in CNP transcript to 40% of the time-matched control value that continued to fall to about 7% of control at 8 h and 2% at 24 h (Fig. 6AGo). PDGF-BB-induced changes in NPR-C transcript levels followed a very similar pattern, with a downward trend evident at 3 h and significant reductions, compared with time-matched controls at both 8 and 24 h (Fig. 6BGo). These results suggest that PDGF signaling may underlie at least part of the observed difference between human and rat in CNP and NPR-C expression in response to serum.

PKC activation is a major component of serum and PDGF-BB regulation of CNP mRNA
To address the contribution of downstream signaling pathways of PDGF to their ultimate effect on CNP mRNA levels, we examined the ability of PDGF-BB (20 ng/ml) to up-regulate CNP transcript in the presence of specific inhibitors of several PDGF protein kinase signaling mechanisms that represent previously defined pathways of PDGF action in the cell (Fig. 7Go). The results showed that in addition to the expected negation of PDGF-BB effect by blocking PDGF receptor tyrosine kinase activity with genistein, inhibition of src family kinases, phosphatidylinositol 3-kinase (PI 3-kinase), MAPK kinase (MEK)/MAPK, and PKC all suppressed by a significant extent the up-regulation of CNP transcript by PDGF. At the doses administered, however, of the pathways downstream of tyrosine kinase, PKC inhibition by GF 109203X resulted in the greatest reduction of CNP mRNA in PDGF-treated cells, reducing this transcript to the level of untreated control (Fig. 7Go). Because activation of PKC appeared to be critical for up-regulation of CNP mRNA by PDGF-BB, we tested the effect of the PKC activator PMA on CNP transcript levels in the presence or absence of GF 109203X (Fig. 8AGo). PMA alone induced an almost 450-fold increase in CNP over serum-free control that was completely abolished in the presence of PKC inhibitor. Similarly, preincubation of FBS-treated AOSMCs with GF 109203X resulted in a significant (71%) reduction in FBS-stimulated CNP transcript, suggesting that like PDGF-BB, serum exerts much of its transcriptional effect on CNP through PKC-using pathways (Fig. 8BGo).


Figure 7
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  		FIG. 7. Effect of PDGF signaling pathway inhibitors on up-regulation of CNP mRNA by PDGF-BB. Confluent cells in 6-well plates were serum starved in SmBM medium for 48 h and then received 1.2 ml SmBM medium in DMSO for 1 h containing: 1) dimethylsulfoxide (DMSO) only (control and PDGF without inhibitor), 2) PP2 (2 µM), 3) genistein (200 µM), 4) GF109203X HCl (4 µM), or 5) LY294002 (20 µM) and PD98059 (60 µM). Then came the addition of 1.2 ml of the following: 1) SmBM medium (control plate only) or SmBM containing 40 ng/ml human recombinant PDGF-BB (all other plates) and immediate mixing of the contents to give a final concentration of 20 ng/ml PDGF-BB and half of the above-stated inhibitor concentrations. After an additional 8 h incubation at 37 C, 5% CO2, cells were harvested for total RNA extraction and CNP transcript levels were determined using real-time PCR and normalized to the serum-free control value of 1. Con, Control; Gen, genistein; GF, GF109203X; LY, LY294002; and PD, PD98059. Data are presented as means ± positive and negative error; n = 3 wells/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. PDGF only.

 

Figure 8
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  		FIG. 8. The role of PKC activation in serum-induced up-regulation of CNP mRNA. Confluent cells in 6-well plates were serum starved in SmBM medium for 48 h and then received 1.2 ml SmBM medium containing either dimethylsulfoxide (DMSO) only (control, PMA, and 10% FBS without inhibitors) or GF109203X HCl (GFX; 4 µM; PMA and 10% FBS with inhibitor) for 1 h before addition of 1.2 ml of the following: 1) SmBM medium (control plate only), 2) 2 µM PMA, or 3) 20% FBS SmBM. This was followed by immediate mixing of the contents to reduce FBS, PMA, and inhibitor concentrations to half of the values cited above. After additional 8 h incubation at 37 C, 5% CO2, cells were harvested for total RNA extraction, and CNP transcript levels were determined using real-time PCR and normalized to the serum-free control value of 1. Data are presented as means ± positive and negative error; n = 3 wells/group. Due to differences in scale of CNP mRNA increase with PMA, compared with FBS, data are graphed as separate figures: A, Effect of PMA on CNP transcript and inhibition with GF 109203X. B, Effect of 10% FBS on CNP transcript and inhibition with GF109203X.

 
To assess the effect of both serum and PDGF-BB treatment on intracellular CNP protein levels in human AOSMCs, cells were grown on coverglass in serum-free medium for 48 h and then received serum-free medium (control), 10% FBS, or 20 ng/ml PDGF-BB for an additional 24 (Fig. 9Go, A–D) and 48 h (Fig. 9Go, E–H) before fixation and staining with anti-CNP using indirect immunofluorescence. Results (Fig. 9Go) showed a higher intensity of immunoreactive-CNP (ir-CNP) staining in the FBS- (Fig. 9Go, B and F) and PDGF-BB-treated cells (Fig. 9Go, C and G), compared with serum-free controls (Fig. 9Go, A and E), with much of the ir-CNP in the form of a diffuse cytoplasmic staining together with discrete granules of greater staining intensity. The positive-CNP staining in FBS- and PDGF-treated cells appeared to attain its highest intensity after 48 h exposure. Control slides of PDGF-BB-treated cells from which anti-CNP antibody was omitted showed no appreciable fluorescence (Fig. 9Go, D and H).


Figure 9
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  		FIG. 9. Effects of serum and PDGF-BB on ir-CNP protein. Ir-CNP in human AOSMCs exposed to serum-free medium (A and E), 10% FBS (B and F), or 20 ng/ml PDGF-BB (C and G) for 24 (A–D) and 48 h (E–H), and stained by indirect immunofluorescence with a rabbit polyclonal antibody against CNP-22. Negative controls (D and H) are cells that received PDGF-BB for 24 (D) and 48 h (H) but received no primary anti-CNP antibody.

 
The effect on intracellular ir-CNP of blocking PDGF signaling with AG 1296 is illustrated in Fig. 10Go. Intense ir-CNP staining was observed in cells exposed to 20 ng/ml PDGF-BB for 24 h (Fig. 10CGo) but not in PDGF-treated cells coincubated with AG 1296 (Fig. 10DGo) or control cells receiving only serum-free medium (Fig. 10AGo) or AG 1296 alone (Fig. 10BGo). The addition of AG 1296 to PDGF-treated cells also appeared to influence cell morphology, inducing the cells to assume a less fibroblast-like form that those treated with PDGF only.


Figure 10
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  		FIG. 10. Inhibition of PDGF-BB-induced increase in immunoreactive CNP in human AOSMCs by preincubation with the PDGF tyrosine kinase inhibitor, AG 1296 (A–D) and inhibition of PMA-induced ir-CNP by preincubation with the PKC inhibitor GF 109302X (E–H). A–D, Seventy-two-hour serum-starved cells received either serum-free medium (A and B) or 20 ng/ml PDGF-BB (C and D) in either the absence (A and C) or presence of 20 µM AG 1296 (B and D), and after a 24-h incubation were fixed and stained for CNP. E–H, Serum-starved human AOSMCs received PMA either serum-free medium (E and F) or 1 µM PMA (G and H) in either the absence (E and G) or presence (F and H) of 2 µM GF 109203X and after a 24-h incubation were fixed and stained for CNP.

 
The contribution of PKC activation to CNP accumulation was also evaluated using indirect immunofluorescence (Fig. 10Go, E–H). As with serum and PDGF-BB, a 24-h treatment with PMA resulted in a greater intensity of ir-CNP staining (Fig. 10GGo) compared with control cells receiving only serum-free medium (Fig. 10EGo). Moreover, coincubation of PMA-treated cells with the PKC inhibitor GF 109203X (Fig. 10HGo) reduced ir-CNP to a level similar to serum-free control and control cells receiving only GF 109203X (Fig. 10FGo). The results of these immunofluorescence studies support a hypothesis that changes in CNP transcript levels induced by serum and PDGF-BB and the PKC signaling pathway may result in long-term changes in intracellular CNP protein that could alter biologic activities of the natriuretic peptide system.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that CNP mRNA is expressed in cultured human vascular SMCs and that this transcript is dramatically increased by both serum and PDGF. This finding stands in marked contrast to human endothelial cells in which CNP was only moderately and transiently up-regulated by serum and to rat SMCs, which responded to both PDGF and serum with a reduction in CNP mRNA. The serum and growth factor regulation of CNP in the human vasculature therefore appears to be both species and phenotype dependent.

A consensus view of CNP as a vasoprotective agent has arisen after in vitro studies showing inhibition of SMC proliferation (14, 15) and migration (16, 17) and in vivo studies describing a CNP-induced suppression of neointimal formation after vascular injury (5, 6, 18). Most often the source of the CNP in the vasculature is cited as the endothelium, in which immunostaining has shown the peptide to be expressed at high levels (7). However, ir-CNP has also been identified in vascular SMCs, particularly those associated with atherosclerotic plaques (7, 8) and restenotic lesions (10). Therefore, in areas of vascular damage, autocrine production is likely to be an important source of CNP affecting smooth muscle function. The present study showing as much as a 20-fold increase in SMC levels of CNP transcript within 8 h of exposure to PDGF-BB suggests that a principal mechanism underlying the up-regulation of CNP at sites of vascular injury could involve PDGF, which is known to be produced by activated macrophages, endothelial cells, SMCs, and platelets in developing atherosclerotic lesions (19). Because secretion of PDGF from injured endothelium constitutes a crucial event in the formation of an atherosclerotic lesion by inducing migration of medial SMCs to the intima and causing these cells to proliferate (19), PDGF-induced up-regulation of CNP production and secretion by the SMCs could conceivably represent an autocrine mechanism for suppressing the vascular remodeling leading to atheroma or restenosis.

An important role for PDGF signaling in the serum-induced up-regulation of CNP is suggested by our finding that the specific PDGF tyrosine kinase inhibitor AG 1296 suppressed as much as 60% of the serum effect on CNP mRNA. Some of this effect is likely due to the presence of PDGF in serum (20); however, because the PDGF content of serum is probably too low to account entirely for the greater effect of serum than recombinant PDGF-BB on CNP mRNA, these data suggest that the transactivation of PDGF receptor tyrosine kinase by other serum factors (e.g. lysophosphatidic acid) (21) may be responsible for much of the observed serum effect on CNP transcription. PDGF receptors therefore conceivably could regulate CNP by not only binding PDGF but also cross talk with other factors such as bioactive lipids present both in plasma and in damaged vessels.

Intracellular signaling pathways downstream of PDGF-BB-induced dimerization and autophosphorylation of PDGF receptors have been examined in considerable detail (19, 22). Most are initiated by transduction molecules that interact with phosphorylated tyrosine residues through specific src homology 2 domains, and in the present study we examined the contribution of four of the major src homology 2 domain transduction pathways (PI 3-kinase, phospholipase C{gamma}/PKC, Grb2/Sos1-Ras-MEK-MAPK, and src kinase) to the regulation of CNP mRNA in human AOSMCs using the PI-3 kinase inhibitor LY294002, the PKC inhibitor GF 109203X, the MEK inhibitor PD 098059, and src kinase inhibitor PP2, respectively (22, 23). Results showed that blocking of each of these four pathways significantly inhibited the PDGF-induced up-regulation of CNP transcript, with PKC and MEK inhibition having the greatest effect on CNP mRNA, and PI 3-kinase and src kinase inhibition having the least effect. Almost identical results were obtained in a separate study (data not shown).

Each of these signaling pathways has been shown to mediate the PDGF stimulation of cell growth (22), and all have been shown to be linked by extensive cross talk upstream of the mitogenic effect. For example, phospholipase C{gamma}, whose downstream effector in part is the generation of diacylglycerol and subsequent activation of the classical PKCs, is regulated by phosphatidylinositol-3,4,5-trisphosphate, a product of PI-3 kinase activity (24). Although our results indicate that signaling through any of these pathways downstream of PDGF receptor activation might result in increased smooth muscle CNP, they also point to a particular importance of PKC-signaling in this role because at the inhibitor doses used, PKC inactivation was almost as effective as tyrosine kinase inhibition in abolishing the increase of CNP mRNA in PDGF-treated cells. Therefore, PKC activation can be considered a principal signaling mechanism linking growth factor binding and CNP up-regulation in human SMCs.

This hypothesis is further supported by the observation that the PKC activator PMA induces an almost 450-fold increase in CNP transcript that is blocked by coincubation with the general PKC inhibitor GF 109203X and in addition an increase in ir-CNP that is diminished in the presence of GF 109203X. The PKC arm of PDGF signaling has been identified previously with several transcriptional actions of PDGF in smooth muscle, including the regulation of angiopoitein 1, a protein that controls vascular remodeling by binding to an endothelium-specific receptor tyrosine kinase (25).

The present study was not designed to address the question of which of the approximately 12 isoforms of PKC [classical ({alpha}, ß1, ß2, and {gamma}), novel ({delta}, {epsilon}, {eta}, {theta}, and µ), and atypical ({zeta} and {lambda}/{iota})] (26) transduces the effect of PDGF-BB on CNP transcript levels; however, the ability of the phorbol ester PMA to up-regulate CNP mRNA suggests that the atypical forms are not involved. Regarding the remaining PKC isoforms, it will be of interest to determine specifically which ones transmit the signal for CNP regulation, especially in light of the findings that several PKC subtype signaling pathways are up-regulated by atherosclerotic risk factors (e.g. PKCß isoform up-regulation by hyperglycemia) (27).

A surprising finding of this study is that cultured rat aortic SMCs differ from their human counterpart in their response to both serum and PDGF, exhibiting a decrease rather than an increase in CNP transcript on exposure to either agent. Woodard et al. (9) have shown in cultured rat AOSMCs that bFGF reduces CNP transcript levels to approximately one third of their control value after 24 h, consistent with our observation of a significant decrease in CNP transcript by PDGF in rat AOSMCs within 8 h and suggesting that growth factor signaling in the rat may result in an inhibition of the natriuretic peptide action in the vasculature. However, these authors also noted that cultured SMCs from SPHRs instead showed an increase in CNP mRNA in response to bFGF, similar to human SMCs in the present study. Several genetically determined differences in cell signaling exist between cultured SMCs derived from stroke-prone SPHRs and normotensive rats, including enhanced phospholipase D activity (28) in the SPHRs, and it is tempting to speculate that SMCs from the hypertensive rat and human may share growth factor-related signal pathways leading to CNP up-regulation.

Recognizing that all SMCs lose their contractile elements and acquire a so-called synthetic phenotype (increased secretion and proliferation reminiscent of SMCs in the neointima) on establishment of in vitro culture, we assessed the effect of the redifferentiation substrate Matrigel on the regulation of CNP by serum and found little difference from cells grown on plastic. This could be taken as evidence that human cells respond in vivo to serum and PDGF as we have shown here; however, because the nodular form of SMCs induced by Matrigel cannot be considered the functional equivalent of SMCs in the vascular media, caution must be exercised in equating the present findings to the regulation of SMCs in intact arteries.

Also differing between rat and human SMCs in the present study was the regulation of NPR-C mRNA levels, which have been shown to be down-regulated in rat arterial SMCs by both FGF-2 (bFGF) and PDGF-BB via a MAPK-dependent pathway (13). PDGF-BB and serum were also shown to suppress NPR-C transcription in the rat mesangial cell, a type of modified smooth muscle located between the glomerular capillary loops (29). The up-regulation of NPR-C transcript by both serum and PDGF-BB in human SMCs, in contrast, suggests a different intracellular pathway to NPR-C regulation in these two species. A coordinated increase of CNP and NPR-C has recently been observed in human SMCs in sites of restenosis (10), and our present results suggest that PDGF in these areas could play a role in this parallel response. The consequences of up-regulated NPR-C in areas of vascular damage are still unclear. This receptor has long been assumed to act as a clearance receptor, and in this role its up-regulation would reduce local concentrations of both ANF and CNP (30). However, NPR-C has recently been identified as the receptor responsible for transducing a hyperpolarizing activity of CNP (2, 4), and it has also been shown to mediate the inhibition of smooth muscle proliferation (31), suggesting that CNP signaling through this receptor, in addition to the NPR-B, could contribute to the antihypertensive and antiatherosclerotic actions of the natriuretic peptides in the vasculature.

The serum and growth factor regulation of NPR-B was not a major emphasis of the present study, but it seems very likely that this receptor in human SMCs is not up-regulated by serum components and undergoes instead a transient down-regulation at 8 h (Fig. 1Go). Rahmutula and Gardner (32) recently reported the transcriptional down-regulation of NPR-B by its principal ligand CNP in rat SMCs in culture, and others have noted the posttranscriptional desensitization of NPR-B by CNP in these same cells via receptor dephosphorylation (33). If a similar CNP/cGMP effect on NPR-B expression and activity exists in human SMCs, it is possible that up-regulation of CNP in smooth muscle of developing atheromas and restenotic lesions could contribute to the progressive down-regulation of NPR-B expression that has been observed at these sites. Because of such possible inhibitory interactions between CNP and its receptors, the consequences of long-term up-regulation of CNP in the vasculature remain difficult to assess.

In summary this study demonstrates that both serum and PDGF mediate the up-regulation of CNP transcription in the human vascular SMC by activation of tyrosine kinase and PKC pathways. These findings suggest that PDGF and other serum factors signaling through receptor tyrosine kinases or downstream PKC activation could be important contributors to CNP expression in the media of injured vessels. Further investigation will be required to determine the role that growth factor-induced activation of the vascular natriuretic peptide system plays in the progression of human atherosclerotic and restenotic lesions.


   Acknowledgments
 
We express our appreciation for the advice and assistance of Mr. James Masterson and Dr. Cristina Semino-Mora.


   Footnotes
 
This work was supported by Grant H083QZ from the Uniformed Services University of the Health Sciences.

First Published Online June 15, 2006

Abbreviations: ANF, Atrial natriuretic factor; AOSMC, aortic SMC; bFGF, basic fibroblast growth factor; CNP, C-type natriuretic peptide; Ct, threshold cycle; EBM, endothelial basal medium; FBS, fetal bovine serum; HAEC, human aortic endothelial cell; ir-CNP, immunoreactive-CNP; MEK, MAPK kinase; NPR, natriuretic peptide receptor; PDGF, platelet-derived growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SmBM, smooth muscle basal medium; SMC, smooth muscle cell; SPHR, spontaneously hypertensive rat.

Received February 22, 2006.

Accepted for publication June 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Silberbach M, Roberts CT 2001 Natriuretic peptide signaling: molecular and cellular pathways to growth regulation. Cell Signal 13:221–231[CrossRef][Medline]
  2. Ahluwalia A, MacAllister RJ, Hobbs AJ 2004 Vascular actions of natriuretic peptides. Cyclic GMP-dependent and -independent mechanisms. Basic Res Cardiol 99:83–89[CrossRef][Medline]
  3. Scotland RS, Ahluwalia A, Hobbs AJ 2005 C-type natriuretic peptide in vascular physiology and disease. Pharmacol Ther 105:85–93[CrossRef][Medline]
  4. Chauhan SD, Nilsson H, Ahluwalia A, Hobbs AJ 2003 Release of C-type natriuretic peptide accounts for the biological activity of endothelium-derived hyperpolarizing factor. Proc Natl Acad Sci USA 100:1426–1431[Abstract/Free Full Text]
  5. Ohno N, Itoh H, Ikeda T, Ueyama K, Yamahara K, Doi K, Yamashita J, Inoue M, Masatsugu K, Sawada N, Fukunaga Y, Sakaguchi S, Sone M, Yurugi T, Kook H, Komeda M, Nakao K 2002 Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide. Circulation 105:1623–1626[Abstract/Free Full Text]
  6. Qian JY, Haruno A, Asada Y, Nishida T, Saito Y, Matsuda T, Ueno H 2002 Local expression of C-type natriuretic peptide suppresses inflammation, eliminates shear stress-induced thrombosis, and prevents neointima formation through enhanced nitric oxide production in rabbit injured carotid arteries. Circ Res 91:1063–1069[Abstract/Free Full Text]
  7. Naruko T, Ueda M, van der Wal AC, van der Loos CM, Itoh H, Nakao K, Becker AE 1996 C-type natriuretic peptide in human coronary atherosclerotic lesions. Circulation 94:3103–3108[Abstract/Free Full Text]
  8. Casco VH, Veinot JP, Kuroski de Bold ML, Masters RG, Stevenson MM, de Bold AJ 2002 Natriuretic peptide system gene expression in human coronary arteries. J Histochem Cytochem 50:799–809[Abstract/Free Full Text]
  9. Woodard GE, Rosado JA, Brown J 2002 Expression and control of C-type natriuretic peptide in rat vascular smooth muscle cells. Am J Physiol Regul Integr Comp Physiol 282:R156–R165
  10. Naruko T, Itoh A, Haze K, Ehara S, Fukushima H, Sugama Y, Shirai N, Ikura Y, Ohsawa M, Ueda M 2005 C-type natriuretic peptide and natriuretic peptide receptors are expressed by smooth muscle cells in the neointima after percutaneous coronary intervention. Atherosclerosis 181:241–250[CrossRef][Medline]
  11. Ross R 1993 The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801–809[CrossRef][Medline]
  12. Li X, Tsai P, Wieder ED, Kribben A, Van Putten V, Schrier RW, Nemenoff RA 1994 Vascular smooth muscle cells grown on Matrigel. A model of the contractile phenotype with decreased activation of mitogen-activated protein kinase. J Biol Chem 269:19653–19658[Abstract/Free Full Text]
  13. Sun J-Z, Oparil S, Lucchesi P, Thompson JA, Chen Y-F 2001 Tyrosine kinase receptor activation inhibits NPR-C in lung arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 281:L155–L163
  14. Furuya M, Yoshida M, Hayashi Y, Ohnuma N, Minamino N, Kangawa K, Matsuo H 1991 C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells. Biochem Biophys Res Commun 177:927–931[CrossRef][Medline]
  15. Doi K, Itoh H, Ikeda T, Hosada K, Ogawa Y Igaki T, Yamashita J, Chun TH, Inoue M, Masatsuga K, Matsuda K, Ohmori K, Nakao K 1997 Adenovirus-mediated gene transfer of the C-type natriuretic peptide causes G1 growth inhibition of cultured vascular smooth muscle cells. Biochem Biophys Res Commun 239:889–894[CrossRef][Medline]
  16. Ikeda M, Kohno M, Yasunari K, Yokokawa K, Horio T, Ueda M, Morisaki N, Yoshikawa J 1997 Natriuretic peptide family as a novel antimigration factor of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 17:731–736[Abstract/Free Full Text]
  17. Kohno M, Yokokawa K, Yasunari K, Kano H, Minami M, Ueda M, Yoshikawa J 1997 Effect of natriuretic peptide family on the oxidized LDL-induced migration of human coronary artery smooth muscle cells. Circ Res 81:585–590[Abstract/Free Full Text]
  18. Shinomiya M, Tashiro J, Saito Y, Yoshida S, Furuya M, Oka N, Tanaka S, Kangawa K, Matsuo H 1994 C-type natriuretic peptide inhibits intimal thickening of rabbit carotid artery after balloon catheter injury. Biochem Biophys Res Commun 205:1051–1056[CrossRef][Medline]
  19. Heldin C-H, Westermark B 1999 Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79:1283–1316[Abstract/Free Full Text]
  20. Huang JS, Huang SS, Deuel TF 1983 Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma binding protein. J Cell Biol 97:383–388[Abstract/Free Full Text]
  21. Wang L, Cummings R, Zhao Y, Kazlauskas A, Sham JKS, Morris A, Georas S, Brindley DN, Natarajan V 2003 Involvement of phospholipase D2 in lysophosphatidate-induced transactivation of platelet-derived growth factor receptor-ß in human bronchial epithelial cells. J Biol Chem 278:39931–39940[Abstract/Free Full Text]
  22. Heldin C-H, Ostman A, Ronnstrand L 1998 Signal transduction via platelet-derived growth factor receptors. Biochim Biophys Acta 1378:F79–F113
  23. Kamimura M, Bea F, Akizawa T, Katus HA, Kreuzer J, Viedt C 2004 Platelet-derived growth factor induces tissue factor expression in vascular smooth muscle cells via activation of Egr-1. Hypertension 44:944–951[Abstract/Free Full Text]
  24. Falasca M, Logan SK, Lehto VP, Baccante G, Lemmon MA, Schlessinger J 1998 Activation of phospholipase C{gamma} by PI3-kinase-induced PH domain-mediated membrane targeting. EMBO J 17:414–422[CrossRef][Medline]
  25. Nishishita T, Lin PC 2004 Angiopoietin 1, PDGF-B, and TGF-ß gene regulation in endothelial cell and smooth muscle cell interaction. J Cell Biochem 91:584–593[CrossRef][Medline]
  26. Campbell M, Trimble E 2005 Modification of PI3K-and MAPK-dependent chemotaxis in aortic vascular smooth muscle cells by protein kinase CßII. Circ Res 96:197–206[Abstract/Free Full Text]
  27. Ishii H, Koya D, King GL 1998 Protein kinase C activation and its role in the development of vascular complications in diabetes mellitus. J Mol Med 76:21–31[CrossRef][Medline]
  28. Kondo T, Inui H, Konishi F, Inagami T 1994 Enhanced phospholipase D activity in vascular smooth muscle cells derived from spontaneously hypertensive rats. Clin Exp Hypertens 16:17–28[Medline]
  29. Paul RV, Wackym PS, Budisavljevic M, Everett E, Norris JS 1993 Regulation of atrial natriuretic peptide clearance receptors in mesangial cells by growth factors. J Biol Chem 268:18205–18212[Abstract/Free Full Text]
  30. Maack T, Okolicany J, Koh GY, Price DA 1993 Functional properties of atrial natriuretic factor receptors. Semin Nephrol 13:50–60[Medline]
  31. Cahill PA, Hassid A 1991 Clearance receptor-binding atrial natriuretic peptides inhibit mitogenesis and proliferation of rat aortic smooth muscle cells. Biochem Biophys Res Commun 179:1606–1613[CrossRef][Medline]
  32. Rahmutula D, Gardner DG 2005 C-type natriuretic peptide down regulates expression of its cognate receptor in rat aortic smooth muscle cells. Endocrinology 146:4968–4974[Abstract/Free Full Text]
  33. Potter LR 1998 Phosphorylation-dependent regulation of the guanylyl cyclase-linked natriuretic peptide receptor B: dephosphorylation is a mechanism of desensitization. Biochemistry 37:2422–2429[CrossRef][Medline]




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