Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells
N. N. Chattergoon,1
F. M. D'Souza,2
W. Deng,1
H. Chen,2
A. L. Hyman,1
P. J. Kadowitz,1 and
J. R. Jeter, Jr.2
Departments of 1Pharmacology and 2Structural and Cellular Biology, Tulane Medical School, New Orleans, Louisiana
Submitted 26 February 2004
; accepted in final form 15 July 2004
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ABSTRACT
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Pulmonary hypertension is characterized by vascular remodeling involving smooth muscle cell proliferation and migration. Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators, and the inhibition of aortic smooth muscle cell (ASMC) proliferation by NO has been documented, but less is known about the effects of CGRP. The mechanism by which overexpression of CGRP inhibits proliferation in pulmonary artery smooth muscle cells (PASMC) and ASMC following in vitro transfection by the gene coding for prepro-CGRP was investigated. Increased expression of p53 is known to stimulate p21, which inhibits G1 cyclin/cdk complexes, thereby inhibiting cell proliferation. We hypothesize that p53 and p21 are involved in the growth inhibitory effect of CGRP. In this study, CGRP was shown to inhibit ASMC and PASMC proliferation. In PASMC transfected with CGRP and exposed to a PKA inhibitor (PKAi), cell proliferation was restored. p53 and p21 expression increased in CGRP-treated cells but decreased in cells treated with CGRP and PKAi. PASMC treated with CGRP and a PKG inhibitor (PKGi) recovered from inhibition of proliferation induced by CGRP. ASMC treated with CGRP and then PKAi or PKGi recovered only when exposed to the PKAi and not PKGi. Although CGRP is thought to act through a cAMP-dependent pathway, cGMP involvement in the response to CGRP has been reported. It is concluded that p53 plays a role in CGRP-induced inhibition of cell proliferation and cAMP/PKA appears to mediate this effect in ASMC and PASMC, whereas cGMP appears to be involved in PASMC proliferation.
vascular smooth muscle cells; nitric oxide; adenovirus
PULMONARY HYPERTENSION (PH) is a disease affecting mainly women in the third to fourth decade of life (26). The pathogenesis of primary pulmonary hypertension (PPH) is unknown, but the initial insult in a predisposed individual is probably at the level of vascular endothelium involving shear forces, viruses, drugs, hypoxia, or autoimmune disease (3). The pathogenesis of PPH appears to involve a group of susceptible genes, including bone morphogenic protein receptor type II (BMPR2) and vasoactive intestinal peptide (VIP), a potent vasodilator. PPH has recently been shown to be associated with mutations in the BMPR2 receptor, and defects in BMPR2 have been reported to underlie at least 50% of cases of familial PPH (22). In another study, VIP receptors have been shown to be upregulated in PPH patients, and administration of VIP results in significant improvement of pulmonary hemodynamics in PPH patients, suggesting a role for VIP in the pathogenesis of PPH (27). When untreated, PH results in a progressive elevation in pulmonary arterial pressure, right heart hypertrophy and failure, and death (33). Vascular remodeling and structural changes resulting from smooth muscle cell proliferation and migration characterize progressive PH (33). Vascular smooth muscle cells (VSMC) in the medial layer of the vasculature are in a "contractile" state in vivo. Under normal conditions, these cells express high levels of smooth muscle-specific
-actin and do not proliferate (11). Injury to the vessel wall increases smooth muscle cell responsiveness to growth factors, resulting in a loss of inhibition of proliferation, and vascular remodeling. An imbalance between the production of contracting and relaxing factors may accompany this event (3).
Calcitonin gene-related peptide (CGRP) is a 37-amino acid endogenous peptide formed as an alternative splice product of the calcitonin gene (2). This peptide is widely distributed throughout the central and peripheral nervous systems (21). In the cardiovascular system, CGRP-containing nerves are located throughout the heart and lung and surround most arteries (37, 38). CGRP has a broad range of activities, including acting as a sensory neuropeptide and a potent vasodilator (1, 6, 37). Nitric oxide (NO) is another vasodilator of interest, and both agents have been shown to alleviate the symptoms of PH (7, 9, 28, 37). NO has been shown to inhibit proliferation of VSMC, and this inhibition ultimately involves p53 (12, 37). In vivo studies in rats have shown that circulating CGRP levels are associated with protection (higher levels) and exacerbation (lower levels) of vascular smooth muscle remodeling (34, 35) and that the loss of CGRP may also result in endothelial proliferation (35). However, the cellular mechanisms involved are not well understood. Whereas NO is known to signal through a cGMP-protein kinase A (PKA)-p53 pathway in aortic smooth muscle (12, 37), the mechanism by which CGRP elicits its beneficial effect is unknown. It is proposed that CGRP signals through a cAMP pathway (8, 13, 15, 23, 25, 37). In arterial smooth muscle, vasodilator agents increase cAMP and inhibit smooth muscle proliferation by arresting the cells primarily in G1 phase (4, 39). Whether CGRP also inhibits proliferation through a PKA-p53 pathway is unknown. Although there have been some studies on the effects of CGRP on aortic smooth muscle cells (ASMC) from different species (14, 20, 38), there are no studies documenting the effect of CGRP on pulmonary artery smooth muscle cells (PASMC).
Recent studies have focused on the effects of transfection of specific genes delivered locally to an organ to avoid systemic effects (10, 1619). In the present study, the transfection of both ASMC and PASMC with replication-deficient adenoviruses serotype 5 encoding nuclear-targeted
-galactosidase (
-gal; AdCMV ntlacZ) or CGRP (AdCMVCGRP) was used to investigate the inhibitory effect of CGRP. The purpose of this study was to determine whether 1) CGRP inhibits proliferation in PASMC and ASMC, 2) CGRP acts through cAMP or cGMP, and 3) the action of the peptide involves p53 and p21.
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MATERIALS AND METHODS
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Culture of rat ASMC and PASMC.
Rat VSMC were obtained from male Sprague-Dawley rats (n = 10) weighing
350500 g. They were anesthetized intraperitoneally with 140 mg/ml of thiobutabarbital sodium salt (Inactin) according to a protocol approved by the Tulane University Health Sciences Center Animal Use and Care Committee. The aorta and pulmonary artery were removed and placed in medium 199 (Sigma Chemical, St. Louis, MO) (14). The excised vessel was incubated in a collagenase solution (200 U/ml of collagenase type I and 0.4 mg/ml of trypsin inhibitor, Sigma) for 30 min at 37°C. After incubation, the adventitia was removed and the vessels were cut longitudinally. A sterile cotton swab was used to disrupt the endothelial lining of the vessels. Each vessel was then minced into small pieces and placed in a collagenase/elastase solution (200 U/ml of collagenase type I, 0.4 mg/ml of trypsin inhibitor, and 15 U/ml of elastase type III, Sigma) for 2 h at 37°C. The tissue was washed in medium 199 supplemented with 10% FBS containing penicillin, streptomycin, and amphotericin B (PSA; 10,000 U/ml, 10,000 µg/ml, and 25 µg/ml). The tissue pieces were plated on a 25-cm2 cell culture flask. The flask was placed in a humidified incubator (95% air, 5% CO2) at 37°C. The tissue blocks were allowed to attach for 57 days, after which the medium in the flask was aspirated. Fresh medium supplemented with FBS and PSA was added. Upon reaching 70% confluency, cells were passaged by trypsinization (0.25% trypsin and 0.053 mmol/l EDTA; GIBCO Laboratories, Grand Island, NY). The identity of the cells as smooth muscle cells was confirmed by the typical "hill-and-valley" appearance (32). In addition, immunohistochemical studies revealed that >95% of the cells were positive for smooth muscle-specific
-actin (mouse, 1:1,000, Boehringer Mannheim).
Adenoviral vectors.
Cells were transfected with replication-deficient recombinant adenoviruses encoding either nuclear-targeted
-gal (AdCMV ntlacZ) or prepro-CGRP (AdCMVCGRP). Both adenoviruses are driven by the cytomegalovirus (CMV) promoter. The adenoviruses were prepared by the University of Iowa, Gene Transfer Vector Core (Iowa City, IA).
Proliferation studies in transfected cells.
Cells at passage 2 were used in all experiments. ASMC were plated at a density of 6 x 104 cells/well in a six-well cell culture plate (Costar), whereas PASMC were plated at a density of 3 x 104 cells/well. Cells were transfected with 150, 300, or 600 multiplicities of infection (MOI) of AdCMVCGRP from a stock of 1 x 1010 pfu/ml. MOI is defined as pfu/cell. Cell numbers were obtained over the course of 4 days after treatment. Once the optimal dose for CGRP transfection was determined, subsequent experiments were conducted using 300315 MOI of AdCMVCGRP from the stock. Cell numbers of ntlacZ-transfected cells were compared with nontransfected cells to ensure that the CMV promoter itself had no effect on proliferation. Each treatment dose was done in triplicate per experiment.
Detection of
-gal.
For histochemical analysis of
-gal localization, passage 2 VSMC were plated as previously described and transfected with AdCMV ntlacZ. Twenty-four hours later, the cells were fixed with 10% formalin and incubated in X-Gal stain [in PBS: 20 mmol/l K4Fe(CN)6·3H2O, 20 mmol/l K3Fe(CN)6, 2 mmol/l MgCl2, and 1 mg/ml of X-Gal (Sigma)] for 2 h at 24°C. Cells were examined for positive
-gal staining under a light microscope. Because the adenovirus encoded nuclear-targeted
-gal, nuclei stained blue were indicative of positive staining of
-gal. The number of cells infected was determined by counting the number of blue nuclei within a microscopic field at a final magnification of x100. Six random fields were counted, and each field contained 80100 cells.
Western blot analysis of CGRP.
Passage 2 cells were plated as described above. Nontransfected cells and cells transfected with either ntlacZ or CGRP were used in these experiments. Cells were allowed to grow for 4 days following transfection. Cell counts were obtained daily. Cells were harvested by trypsinization with 0.25% trypsin-EDTA. Media from the experimental treatments were also collected. Harvested cells were lysed using a hypotonic buffer (10 mM Tris, 1.5 mM MgCl2, 1 mM PMSF, protease inhibitor cocktail, Sigma). Protein levels for media and cell lysates were quantified using the BCA Protein Assay (Pierce, Rockford, IL). Equivalent quantities of protein (25 µg) from each sample were electrophoresed on a 420% gradient gel (Jule, Milford, CT). After electrophoresis, the gel was transferred onto a nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 5% milk in Tris-buffered saline (TBS) + 0.1% Tween 20 (TBST) for 1 h. The membrane was washed in TBST three times for 10 min each. Subsequently, it was incubated in polyclonal rabbit anti-CGRP antibody (Peninsula Laboratories, San Carlos, CA) at a dilution of 1:2,000 for 1 h. After the membrane was washed in TBST, the bound antibody was detected with anti-rabbit IgG-horseradish peroxidase (HRP) secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:4,000. CGRP was visualized using the ECL chemiluminescence kit (Amersham) and BioMax MR-1 film (Eastman Kodak).
Immunohistochemistry for CGRP.
Immunocytochemistry was performed using the Immunohistochemistry Staining Kit (Peninsula Laboratories). The medium was aspirated from the chamber slides and washed briefly in 1x PBS. The cells were fixed in methanol at 10°C for 5 min. The cells were washed in PBS three times and allowed to air dry. To quench endogenous peroxidase activity, the fixed cells were incubated in a 0.5% solution of hydrogen peroxide in PBS for 7 min. The cells were then washed twice in PBS for 5 min each. To block nonspecific binding, the cells were incubated in serum block for 20 min. Cells were treated with the primary antibody anti-CGRP (rabbit monoclonal, Peninsula) at three dilutions, 1:100, 1:200, and 1:500, for 2 h. The slides were rinsed and washed in PBS twice and then exposed to biotinylated secondary antibody for 30 min. The cells were again rinsed and washed in PBS before a 30-min treatment of HRP-streptavidin complex. After again being washed in PBS, the cells were exposed to a substrate-chromogen mixture for
3 min until the desired stain intensity was visualized under a light microscope. The reaction was stopped by addition of deionized water. The slides were counterstained with Mayer's hematoxylin for 3 min. The slides were washed in tap water and then put into PBS until they turned blue, for
30 s, and then rinsed well again with distilled water. The slides were dehydrated using 95% and 100% ethanols and xylene. The slides were then coverslipped and observed under a light microscope.
Western blot analysis for
-tubulin.
The same membrane for the CGRP Western blot was reprobed for
-tubulin to verify that protein samples were loaded equally in all lanes. The membrane was washed two times for 20 min each in 25 ml of TBST before being blocked again in 5% milk in TBST for 1 h. Western blot analysis was conducted as previously described, using rabbit monoclonal anti-
-tubulin antibody (Boehringer Mannheim).
Measurement of cAMP and cGMP.
Cyclic nucleotide levels (cAMP and cGMP) were determined to ascertain their role in inhibiting proliferation. ASMC and PASMC were plated at a density of 1 x 105 cells/well in a 96-well plate as per instructions. The cells were allowed 24 h to attach before they were treated with 150, 300, and 600 MOI of AdCMVCGRP or 600 MOI of AdCMV ntlacZ. The cultures were assayed for total cAMP and cGMP production after a 4-day incubation with the adenoviral vectors. The nonacetylation Biotrak cAMP EIA system for cAMP measurement was used. The acetylation protocol of Biotrak cGMP EIA system was used for cGMP measurement (Amersham Life Science, Arlington Heights, IL).
Effect of PKA inhibitor and PKG inhibitor on rat ASMC proliferation.
To determine the role of PKA and protein kinase G (PKG) in CGRP-induced inhibition of proliferation, CGRP-transfected cells were treated with Rp-8-Br-cAMPs [PKA inhibitor (PKAi)] at 25, 50, and 100 µM or Rp-8-Br-cGMPs [PKG inhibitor (PKGi)] at 12.5, 25, and 50 µM (Alexis Biochemicals, San Diego, CA). From dose-response studies, 100 µM PKAi and 50 µM PKGi were determined to be the effective concentrations. AdCMVCGRP was administered with the PKAi or the PKGi. Cell numbers were obtained daily for 4 days, after which cells were harvested for further studies.
Effect of CGRP transfection on expression of p21 and p53.
Passage 2 VSMC were untreated and transfected with
-gal or CGRP. CGRP-transfected cells were treated with increasing concentrations of PKAi or PKGi as described above. Cells were treated for 4 days, harvested, and lysed. Protein levels were quantified, and equivalent amounts of protein from each group were run on a 420% gradient gel. Western blot analyses were performed to detect the levels of p21 or p53. Monoclonal mouse anti-p21 antibody (Santa Cruz Biotechnology) was used as the primary antibody for the detection of p21, whereas polyclonal rabbit anti-p53 antibody (Santa Cruz Biotechnology) was used to detect p53. Bound antibody was detected with anti-mouse IgG-HRP and anti-rabbit IgG-HRP secondary antibody, respectively. Bound antibody was visualized using the ECL chemiluminescence kit (Amersham).
Determination of cell viability.
Viability of nontransfected and transfected cells was determined using the trypan blue exclusion test. Cells were exposed to the treatment for 4 days, after which cells were trypsinized and studied for viability using the trypan blue test. Cell numbers were determined using a hemocytometer and calculated as percentage of viable cells per total cells.
Data analysis.
Protein assays were read using the Labsystems Multiskan MS plate reader. Western blot autoradiographs were scanned using Adobe Photoshop 7.0. The data were analyzed statistically using one-way ANOVA followed by post hoc analysis with Tukey's test.
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RESULTS
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Effect of AdCMVCGRP on VSMC proliferation.
The effect of AdCMVCGRP at MOI of 150, 300, and 600 on PASMC and on ASMC numbers was investigated over a 4-day period, and these data are summarized in Fig. 1. Control ASMC and PASMC numbers increased over the 4-day period in which growth curves were measured. The reporter gene AdCMV ntlacZ, which served as a control adenoviral vector, had no significant effect on ASMC or PASMC proliferation when administered at an MOI of 600, the greatest MOI concentration of AdCMVCGRP used (Fig. 1). AdCMVCGRP at an MOI of 150600 caused a significant dose-dependent decrease in cell number. The decrease in cell number on day 4 is shown in Fig. 1. Cells treated with AdCMVCGRP >500 MOI showed signs of decreased cell viability.

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Fig. 1. Dose-response curves for AdCMVCGRP in pulmonary artery smooth muscle cells (PASMC; A) and aortic smooth muscle cells (ASMC; B), respectively. Right panels show the decrease in cell number on day 4. ntlacZ, AdCMVntlacZ; #P < 0.05; *P < 0.001. CGRP, calcitonin gene-related peptide; MOI, multiplicities of infection.
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The results from trypan blue exclusion analysis indicate >95% viability at 300 MOI in ASMC and 315 MOI in PASMC. These are the concentrations used in subsequent experiments. Addition of the CGRP receptor antagonist CGRP837 restored proliferation inhibited by AdCMVCGRP alone by >50% (Fig. 2).

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Fig. 2. Effect of the CGRP837, a CGRP receptor antagonist, on CGRP-transfected smooth muscle cells. Proliferation over a 4-day period after treatment with increasing concentrations of the CGRP receptor antagonist is shown. In both ASMC (A) and PASMC (B), proliferation is restored to at least 50% of the number of control cells.
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Measurement of CGRP.
Western blot analysis was used to detect the formation of CGRP in control VSMC, cells transfected with the reporter gene, and cells transfected with AdCMVCGRP. CGRP was measured in smooth muscle cell lysates and in the culture media from the cells, and these results are shown in Fig. 3. Equivalent amounts of protein were loaded in all wells. ASMC and PASMC transfected with AdCMVCGRP at MOI of 150, 300, and 600 show dose-dependent increases in CGRP protein in the cell lysates (Fig. 3). CGRP was also detected in the media of cells transfected with AdCMVCGRP at MOI of 300 and 600. CGRP was not detected in cells transfected with AdCMV ntlacZ at 600 MOI (Fig. 3). The Western blots were also probed for
-tubulin to show that protein was run in all lanes. The results of these experiments indicate that CGRP is formed and is secreted into the media.

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Fig. 3. Western blot analysis for CGRP in cell lysates and media of ASMC and PASMC treated with AdCMVCGRP.
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Immunohistochemical staining for CGRP was performed, and VSMC transfected with AdCMVCGRP at an MOI of 300 revealed the presence of CGRP staining on day 4 (Fig. 4). Control and treated cells were counterstained with hematoxylin. AdCMVCGRP-transfected cells revealed staining in the cytoplasm compared with nontransfected control smooth muscle cells (Fig. 4). The results of these experiments show that CGRP staining is localized to the cell cytoplasm and is similar to the localization of endothelial nitric oxide synthase (eNOS) (12).

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Fig. 4. Immunohistochemical stains showing the localization of CGRP 2 days after transfection in vascular smooth muscle cells. Left: nontransfected cells; right: cells transfected with the adenovirus encoding prepro-CGRP. Both treatment groups were initially exposed to anti-CGRP antibody, stained with diaminobenzidine, and followed by a hematoxylin counterstain. Arrows point to the cytoplasm of the cells where CGRP-positive staining is found in the AdCMVCGRP group and not in the control group. Magnification, x100.
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Measurement of cAMP and cGMP.
Enzyme inmmunoassays were performed to measure the formation of cAMP and cGMP in VSMC transfected with AdCMVCGRP, and these data are summarized in Figs. 5 and 6. The values for cAMP and cGMP have been normalized to the concentrations measured in nontransfected cells and represent increases in cyclic nucleotide levels per well. AdCMVCGRP transfection at MOI of 150, 300, and 600 caused a significant increase in cAMP concentration in ASMC and in PASMC at day 4, whereas transfection with AdCMV ntlacZ at a MOI of 600 had no significant effect (Fig. 5).

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Fig. 5. Results of cAMP enzyme immunoassay on day 4 of treatment. A: ASMC treated with 300 MOI of AdCMVCGRP shows significant increase in cAMP production. B: 315 MOI of AdCMVCGRP significantly increases cAMP production in PASMC. *P < 0.05 vs. control.
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Fig. 6. Results of cGMP enzyme immunoassay on day 4 of treatment. A: ASMC treated with 300 MOI of AdCMVCGRP. B: PASMC show a significant increase in cGMP production at 315 MOI of AdCMVCGRP. *P < 0.05 vs. control; #P < 0.05 vs. ntlacZ.
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Transfection with AdCMVCGRP had no significant effect on cGMP concentration in ASMC on day 4 but increased cGMP concentration at MOI of 300 and 600 on day 4 in PASMC (Fig. 6).
Effect of Rp-8-Br-cAMPS and Rp-8-Br-cGMPS.
The role of PKAi and PKGi on the inhibitory effect of AdCMVCGRP at MOI of 300 on proliferation of VSMC was investigated, and these data are summarized in Figs. 710. The inhibitory effect of AdCMVCGRP on proliferation of ASMC and PASMC in the presence of increasing concentrations of the PKA inhibitor is shown in Figs. 7 and 8. Transfection with AdCMVCGRP at 300 MOI significantly inhibited proliferation in ASMC and in PASMC on day 4 compared with cell numbers in nontransfected cells and in ntlacZ-transfected cells (Figs. 7 and 8). After transfection with AdCMVCGRP and treatment with PKAi, there is attenuation of the inhibitory effect of AdCMVCGRP on proliferation (Figs. 7 and 8). As the concentration of PKAi increases from 25 to 100 µM, there is an increase in cell number of ASMC and PASMC on day 4. Treatment with PKAi alone had no significant effect on cell number (data not shown). Western blot analysis for p53 and p21 shows that expression of these proteins increases in ASMC and PASMC treated with AdCMVCGRP at an MOI of 300 on day 4 (Figs. 7 and 8). However, the addition of PKAi decreases the expression of p53 and p21 (Figs. 7 and 8).

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Fig. 7. Effect of increasing concentrations of the PKA inhibitor (PKAi; Rp-8-Br-cAMPS) on CGRP-treated ASMC. A: cell proliferation in the presence of CGRP and PKAi. B: cell number at day 4 following treatment. C: p53 and p21 concentrations decrease with addition of PKAi. #P < 0.05 vs. control; *P < 0.001 vs. control.
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Fig. 10. Effect of increasing doses of PKGi (Rp-8-Br-cGMPs) on CGRP-treated ASMC. A: cell proliferation in the presence of CGRP and the PKGi. B: cell number at day 4 following treatment. C: p53 and p21 protein expression are upregulated in the presence of GCRP and PKGi. *P < 0.001 vs. control; #P < 0.001 vs. ntlacZ.
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Fig. 8. Effect of increasing concentrations of the PKAi (Rp-8-Br-cAMPs) on CGRP-treated PASMC. A: cell proliferation in the presence of CGRP and the PKAi. B: cell number at day 4 following treatment. C: p53 and p21 concentrations decrease with addition of PKAi. *P < 0.001; #P < 0.05 vs. control; P < 0.01 vs. ntlacZ.
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The role of PKG in the inhibition of VSMC proliferation with AdCMVCGRP transfection was investigated, and these data are summarized in Figs. 9 and 10. Transfection with AdCMVCGRP at 300 MOI decreased ASMC and PASMC number on day 4 (Figs. 9 and 10). Treatment with Rp-8-Br-cGMPS in concentrations of 12.5, 25, and 50 µM attenuates the inhibitory effect of AdCMVCGRP on proliferation of PASMC (Fig. 9). The Western blots in Fig. 8 show increasing p53 and p21 expression in AdCMVCGRP-transfected PASMC (Fig. 9). The expression of p53 and p21 decreases as the concentration of PKGi increases from 12.5 to 50 µM (Fig. 9).

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Fig. 9. Effect of increasing concentrations of the protein kinase G inhibitor (PKGi; Rp-8-Br-cGMPs) on CGRP-treated PASMC. A: cell proliferation in the presence of CGRP and the PKGi. B: cell number at day 4 following treatment. C: p53 and p21 concentrations decrease with addition of PKGi. #P < 0.05; *P < 0.001 vs. control; P < 0.001 vs. CGRP at 300 MOI.
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The effect of PKGi on AdCMVCGRP transfection on ASMC cell number is shown in Fig. 10. In contrast to the release of inhibition induced by the PKG inhibitor in PASMC, PKGi in concentrations of 12.5 to 50 µM did not attenuate or reverse the inhibition of proliferation induced by AdCMVCGRP in ASMC (Fig. 10). The expression of p53 and p21 increased in AdCMVCGRP-treated ASMC; however, these levels remained elevated at day 4 following treatment with PKGi (Fig. 10). The data with cGMP levels and with PKGi suggest a role for cGMP and PKG in mediating the inhibitory effect of AdCMVCGRP on PASMC proliferation but not on ASMC proliferation.
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DISCUSSION
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There is a paucity of information in the literature with regard to the effects of CGRP in cell culture models. This study is the first to describe the effect of CGRP on PASMC in vitro. The results of this study show that the vasodilator CGRP inhibits vascular smooth muscle proliferation at a concentration of 300 MOI in rat aortic VSMC and a concentration of 315 MOI in pulmonary artery VSMC. The inhibition of proliferation also appears to be sustained throughout the course of the experiment. An MOI >500 appears to be toxic and induces cell death. Transfection with the reporter gene coding for
-gal had minimal effect on VSMC proliferation, as does the viral vector, suggesting that the effect on proliferation observed is due to the gene of interest. Immunohistochemical experiments reveal that CGRP protein is localized to the cytoplasm. The Western blot data provide support for the hypothesis that when formed, CGRP is released into the media. CGRP then acts in an autocrine/paracrine manner on cell surface receptors to inhibit proliferation (34, 36). The binding of CGRP presumably activates a signaling pathway mediated by cyclic nucleotides that results in inhibition of cell proliferation. CGRP is known to stimulate two receptor subtypes, CGRP1 and CGRP2, which are G protein-coupled receptors (34). VSMC have CGRP1 receptors that mediate vasodilation (30, 34). The restoration of proliferation by CGRP837, the CGRP receptor antagonist, suggests that inhibition of proliferation observed in the present study was due to activation of a CGRP1 membrane receptor. There are receptor-modifying proteins, when associated with a generic receptor, that can modify the generic receptor to act like a CGRP receptor. Receptor activity-modifying proteins (RAMP) biological functions are transporting calcitonin receptor-like receptors (CRLR) to the cell membrane and determining the glycosylation state to define the receptor pharmacology. An association between RAMP1 and CRLR has been found to create a CGRP1 receptor (24, 29, 30).
cAMP has been implicated in several studies as the primary intracellular signaling molecule used by CGRP (8, 13, 15, 23, 25, 37). A significant increase in cAMP following AdCMVCGRP transfection has been observed in both cell types. We have also been able to show that after PKA is blocked, there is a recovery from inhibition of proliferation caused by CGRP gene transfer. These results suggest that cAMP appears to play a role in the CGRP-induced inhibition of proliferation in rat VSMC. The effect of the PKGi in PASMC treated with CGRP was unexpected because there was a sustained inhibition in aortic cells treated with CGRP in the presence of PKGi. Li et al. (20) described the antiproliferative effect of CGRP in rat and rabbit aortic cells in the second and fifth passages. In their study, a significant decrease in proliferation was not observed in the second passage and is different from the effect reported in the present study. The reason for the difference is uncertain. They also reported an increase in cAMP levels as observed in the present study. However, there are no studies in the literature showing the effects of direct CGRP inhibition on VSMC. Current studies looking at the effect of CGRP on cell proliferation have coupled it with adrenomedullin, VIP, NO, or another vasodilator. Future studies focusing on the effects of CGRP alone are needed. There is a lack of information in the literature about the role of PKG in the signaling events induced by CGRP. One possibility could be that PASMC may have higher PKG expression than ASMC. The measurement of cGMP levels, however, showed a significant increase in this nucleotide in PASMC and not ASMC, which correlates with the effects of PKGi in CGRP-treated VSMC. These data suggest a role for cGMP in CGRP-mediated inhibition in PASMC, and this effect does not occur in ASMC.
Although the cells used in these studies are both from rat, there were definite differences between aortic and pulmonary VSMC. ASMC appear to be more sensitive than PASMC to the inhibitory effects of CGRP on proliferation. This difference may be related to vascular function or the presence or absence of certain proteins. Differences in concentration of AdCMVCGRP required to inhibit proliferation and other cellular features were observed. PASMC have a faster cell cycle time than ASMC (18 vs. 21 h). Although that may not appear to be a notable difference, there was a clear difference in cell density observed by day 4 of experiments in control cells. Pulmonary artery cells were plated at one-half the density of aortic cells because, after allowing 24 h for attachment before treatment, PASMC numbers would be almost twofold that of ASMC. It has been shown that VSMC respond differently to stimuli depending on the vascular bed from which they were derived and may be different with regard to proliferation (31).
In the present study, the CGRP pathway has been linked to increasing levels of p21 and p53 and cell cycle inhibition. CGRP appears to signal through a cAMP pathway and not a cGMP pathway in ASMC. The more interesting finding is that CGRP appears to signal through both cAMP and cGMP pathways in PASMC. Most of the literature involving cGMP in the CGRP pathway also links these effects of CGRP to NO release (5, 14). The studies by Gray and Marshall (14) involved rat aortic rings with intact endothelium and the release of NO. However, this study is different in that endothelial cells are not present in the experiments, including experiments on cyclic nucleotide measurement and inhibitor studies involving PKAi and PKGi. The vasodilatory effects of CGRP have been studied and have been linked to increased cAMP and, sometimes, a NO/cGMP pathway. There is no distinct pathway in the literature for a mechanism by which CGRP directly inhibits smooth muscle cell proliferation in the absence of other vasodilators or related peptides. It is not known why PASMC recover from inhibition of proliferation in the presence of the PKGi. This effect can be due to a difference between cell types and/or the expression of PKG protein in the cells.
The results of the current study offer further insight into the mechanism by which CGRP affects VSMC proliferation. cAMP and PKA have been directly linked to the inhibitory actions of CGRP. cGMP has also been linked to the inhibitory effects, but the role of cGMP still requires further investigation in these cells. Downstream molecules, p53 and p21, have been shown to play a significant role in previous studies involving eNOS and in the present study with CGRP (12). The ability to inhibit proliferation of this cell type can be beneficial in the treatment of proliferative vascular disorders including restenosis and PH.
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GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-62000 and the National Cancer Institute Grant CA-65600.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. R. Jeter, Jr., 1430 Tulane Ave., Dept. of Structural and Cellular Biology SL 49, New Orleans, LA 70112 (E-mail: jjeter{at}tulane.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES
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