Sarco/Endoplasmic Reticulum Ca2+-ATPase Gene Transfer Reduces Vascular Smooth Muscle Cell Proliferation and Neointima Formation in the Rat
http://www.100md.com
循环研究杂志 2005年第9期
the INSERM U621/IFR-14 (L.L., S.Y., L.H., A.-M.L.), UPMC-CHU Pitiee-Salpe簍rieere, Paris, France
the Cardiovascular Research Center (F.d.M., R.J.H.), Massachusetts General Hospital and Harvard Medical School, Charlestown, Mass
INSERM EMI 0228/IFR-118, USTL (T.C.), Villeneuve d’Ascq, France
the Service de Cytometrie IBAIC (M.H.), IFR-FR46, Universitee Paris-Sud, Facultee des Sciences, Orsay, France.
Abstract
Proliferation of vascular smooth muscle cells (VSMC) is a primary cause of vascular disorders and is associated with major alterations in Ca2+ handling supported by loss of the sarco/endoplasmic reticulum calcium ATPase, SERCA2a. To determine the importance of SERCA2a in neointima formation, we have prevented loss of its expression by adenoviral gene transfer in a model of balloon injury of the rat carotid artery. Two weeks after injury, the intima/media ratio was significantly lower in SERCA2a-infected than in injured noninfected or injured -galactosidaseeCinfected carotids (0.29±0.04 versus 0.89±0.19 and 0.72±0.14, respectively; P<0.05), and was comparable to that observed in control carotids (0.21±0.03). The pathways leading to proliferation were analyzed in serum-stimulated VSMC. Forced expression of SERCA2a arrested cell cycle at the G1 phase and prevented apoptosis. SERCA2a inhibits proliferation through inactivation of calcineurin (PP2B) and its target transcription factor NFAT (nuclear factor of activated T-cells) resulting in lowering of cyclin D1 and pRb levels. By using NFAT-competing peptide VIVIT, we showed that NFAT activity is strongly required to promote VSMC proliferation. In conclusion, we provide the first evidence that increasing SERCA2a activity inhibits VSMC proliferation and balloon injuryeCinduced neointima formation.
Key Words: vascular smooth muscle cell proliferation gene transfer SERCA2a calcium signaling nuclear factor of activated T-cells
Introduction
Cell hyper-proliferation is an important etiology factor of cardiovascular diseases such as primary atherosclerosis, restenosis, and vein-graft disease.1,2 The neointimal vascular smooth muscle cell (VSMC) proliferation constitutes a primary cause of vascular disorders1 but, despite increasing knowledge about the cell-cycle regulation of VSMC, the molecular mechanism governing VSMC proliferation remains elusive. Thus, identifying genetic modifiers of VSMC proliferation is a major focus in cardiovascular biology and medicine.3 VSMCs have the ability to transition between quiescent differentiated and proliferating phenotypes. Acquisition of proliferating phenotype by VSMC is associated with alterations in Ca2+ handling supported by modification of Ca2+ transporter expression.4
In quiescent VSMC, the Ca2+ signal consists mainly of localized elementary calcium events. The global increase in Ca2+ concentration is rapidly reduced by calcium pumps, keeping the cytoplasmic Ca2+ concentration low. Spontaneous spark frequency decreases after activation of phosphoinositol-3 kinase (PI3K) and G protein-coupled receptors, probably attributable to inhibition of the ryanodine receptor (RyR).5 Activation of PI3K and G protein-coupled receptors also results in a sustained increase in cytosolic [Ca2+] attributable to the generation of repetitive Ca2+ waves,5,6 inhibition of Ca2+ pump activity,7 and increased Ca2+ entry.8 Chronic increases in cytosolic Ca2+ concentration in proliferating VSMC are supported by long-term alterations in the levels of Ca2+-handling proteins such as loss of RyR and SERCA2a,9 replacement of L-type voltage-operated Ca2+ channels by T-type voltage-operated Ca2+ channels, loss of the plasma membrane Ca2+ pumps, and upregulation of the transient receptor potential Ca2+ channels.4
Any chronic alterations in the spatio-temporal pattern of Ca2+ signals should alter gene expression by activating different kinases and phosphatases, modulating Ca2+-regulated transcription factors such as NFAT (nuclear factor of activated T lymphocytes).
A sustained increase in cytosolic [Ca2+] is necessary to activate calcineurin, a Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase 2B (PP2B) that dephosphorylates many proteins including NFAT, inducing its translocation into the nucleus.10,11 NFAT is exported from the nucleus after phosphorylation by the glycogen synthase kinase, GSK-3,12 or by JNK-2.13 This antagonizes the effect of PP2B. PI3K- or Gq/11-coupled receptor agonists, known to be mitogenic factors, induce NFAT-dependent transcriptional activation in vascular myocytes.6,7,14eC16
Several previous studies have suggested that SERCA2a is involved in the control of proliferation and growth: transgenic mice with only one allele of the ATP2a2 gene (SERCA2) develop numerous cancers of the upper digestive tract and skin, and cardiac hypertrophy17,18; low levels of SERCA2a are associated with cardiac hypertrophy both in humans and animals19eC21; mice in which SERCA2a has been replaced by SERCA2b develop cardiac hypertrophy22; finally, loss of SERCA2a is associated with VSMC proliferation.7,9 Similarly, only SERCA2b is present in proliferating BC3H1 muscle cells whereas SERCA1, SERCA2a, and SERCA2b are present in quiescent differentiated BC3H1 muscle cells.23
We hypothesized that increasing the rate of SR Ca2+ uptake by restoring SERCA2a expression might inhibit VSMC proliferation and prevent neointima formation induced by injury.
Materials and Methods
Injury of the Rat Carotid Artery and Adenoviral Vector-Mediated Gene Delivery
Animals were treated in accordance with institutional guidelines. The left external carotid artery from adult male Sprague-Dawley rats (Charles River, Mass) weighing 400 to 500 g was injured using a 2F Fogarty embolectomy catheter (Baxter Healthcare Corp) that was introduced into the common carotid artery through the external carotid and inflated to 2 atmospheres 3x 15 s. After both the proximal common and the proximal internal carotid arteries were clamped, viral infusion mixtures containing 1x1010 pfu of Ad- SERCA2a/GFP (Ad-S2a), or Ad-Gal/GFP (Ad-Gal)24 diluted to a total volume of 50 e蘈 was instilled between the 2 clamps, and the external carotid artery was then ligated. Perfusion was restored through the internal and the common carotid artery after 30 minutes of instillation, and the neck incision was closed. Two weeks after surgery the animals were heparinized and carotids were collected, flushed with saline, included in cryomatrix and frozen at eC80°C. Intima-to-media thickness ratio was measured from hematoxylin-and eosin-stained cross-sections with a computer-based (Lucia, Nikon) morphometric system. Apoptosis was analyzed by TUNEL staining (ApopTag Red, Serologicals Corp).
Cell Culture
VSMC were isolated from the media of the thoracic aorta from male Wistar rats and cultured as described.7 Cells were used between passages 2 to 8. Cells were infected with adenovirus for 48 hours at 1 to 10 pfu/cell. Proliferation was measured by using CellTiter96 Cell Proliferation Assay kit (Promega). Cell cycle was analyzed using a FACS Vantage (Becton-Dickinson) flow cytometer.
For transient transfection and reporter gene assay, cells were infected with Ad-S2a for 48 hours, then cotransfected using FuGene 6 (Roche) with NFAT- promoter-luciferase construct (NFAT-Luc, Stratagene) and CMV-promoter--galactosidase construct (pCMV, clontech). The luciferase activity was measured by using "the luciferase assay kit" (Promega) and normalized to the -galactosidase activity. It was expressed as percent of control in relative luciferase units.
Intracellular [Ca2+]i Measurements
Fluorescence images of FURA-2/AM loaded cells were collected at 100 ms or 1.5 s intervals by a Sensicam QE CCD camera (PCO Computer Optics GmbH), digitized, and integrated in real time by an image processor (Metafluor). Results (F/F) are expressed as ratios between 340 and 380 fluorescence signals measured during a response divided by the ratios measured in resting conditions.
Protein Analysis
The sources of primary antibodies and protein analysis protocols are detailed in expanded methods.
Confocal Microscopy
Slides were examined using a Zeiss LSM-150 confocal scanning laser microscope with a Plan Apochromat 63X objective (NA 1.40, oil immersion). All settings were kept constant to allow comparison.
Statistical Analysis
All quantitative data are presented as means of at least 3 independent experiments ±SEM. An unpaired t-test was used to calculate differences between means. Differences were considered significant when P<0.05.
An expanded Materials and Methods is available in the online data supplement at http://circres.ahajournals.org.
Results
SERCA 2a Gene Transfer Prevents Balloon Angioplasty-Associated Restenosis
SERCA2a and SERCA2b are coexpressed in the media of control carotid arteries. SERCA2a is not expressed in the media or intima 2 weeks after injury whereas SERCA2b is still present (Figure 1A). We prevented SERCA2a loss in the injured carotid arteries by infection with Ad-SERCA2a/GFP (Ad-S2a). The same concentration of Ad-Gal/GFP (Ad-Gal) was used as a control. Transfection efficacy was evaluated by using an anti-GFP antibody. Representative SERCA2a, SERCA2b, and GFP immunolabelings are presented in Figure 1B and 1C. Fluorescence of GFP and SERCA are not exactly superimposed for 2 possible reasons: (1) after 14 days the virus and GFP may already be gone whereas SERCA2a is still present because proliferation has been blocked, (2) Ad-S2a could infect not only proliferating cells, but also neighboring cells and prevent loss of SERCA2a in the entire cell population. However, in online Figure IIS, we show that Ad-S2a restores expression only in infected cells. The morphometric analysis was performed on hematoxylin/eosin stained cross-sections (Figure 1D). Neointima formation was observed in injured noninfected and injured GaleCinfected carotids whereas there was no sign of neointima formation in S2a-infected carotids. The degree of restenosis was determined by measuring the intima and media thickness and calculating the intima/media (I/M) thickness ratio (Figure 1E). I/M ratios of injured, noninfected, and -Gal infected arteries were 0.89±0.19, n=7 and 0.72±0.14, n=6, respectively (NS). The I/M ratio of S2a-infected carotids was not different from that of control arteries (0.29±0.04, n=6 and 0.21±0.03, n=9, respectively; NS), but I/M ratios from both control and S2a-infected arteries were significantly lower than that of injured noninfected or -Gal-infected carotids (P<0.05). An increase of the adventitial layer was clearly visible as a consequence of injury as already reported.25
SERCA2a Prevents Proliferation-Induced Changes in Phenotype and Apoptosis
Proliferation of VSMC is characterized by decreased expression of smooth muscle myosin heavy chains 1 and 2 (SM1 and SM2) and increased nonmuscle myosin heavy chain B (NM- B).26 SM1 and SM2 were present in injured S2a-infected and in control arteries, but only at low level in injured noninfected or Gal-infected arteries where a high level of NM-B was detected (Figure 2). No apoptosis was detected in the media of S2a-infected arteries, whereas apoptosis was observed in neointima, media and adventia of injured noninfected or -Gal-infected arteries (Figure 2).
To decipher the mechanism involved in inhibition of proliferation and apoptosis by SERCA2a we have used cultured rat aortic VSMC. Serum (10%) was used to induce proliferation because neointima formation is promoted by various mitogens released into the serum by the endothelium and blood cells. Freshly dissociated quiescent VSMC display "contractile" phenotype27 associated with expression of smooth muscle-specific myosins SM1 and SM2 (online Figure IS). Both SERCA2a and SERCA2b were expressed in these cells, as described.9 Proliferating cells did not contain SERCA2a and only low levels of SM1 and SM2, but still expressed NM-B and smooth muscle cell specific markers such as calponin and caldesmon (Figure 1S). Thus, at the time of infection, VSMCs were in the synthetic phenotype. SERCA2a expression was restored by adenovirus infection, but the level of SERCA2b did not change after infection (Figure 3A).
Measuring GFP fluorescence allowed monitoring the efficacy of adenoviral infection that was increased significantly 4 days after infection (online Figure IIS). Thus, experiments were performed on day 4 after infection. At this stage, flow cytometry showed that 60% to 90% of cells were GFP-positive.
Between days 0 and 4, control and Ad-Gal-infected cell numbers increased 5-fold, whereas the number of Ad-S2a-infected cells increased less (P<0.001) (Figure 3B). More than 50% of the cells in Ad-S2a-infected cultures were arrested in G1, whereas most cells in control and Ad-Gal-infected cultures were in the S phase (Figure 3C). The percentage of apoptotic cells, measured by DNA fragmentation (TUNEL) in control, Ad-S2a-infected, Ad-Gal-infected cells, and cells treated with staurosporine (1 e/mL, 4 hours) as positive a control, were 4.3±3.3, 0.8±0.3, 8.6±2.3, and 85±5.2, respectively (average of 3 independent experiments; 50 to 200 cells/experiment). The absence of caspase 9 cleavage (online Figure IIIS) and of a sub-G1 peak in ad-S2a-infected cells also confirmed the absence of apoptosis. Thus, SERCA2a expression inhibits progression into the cell cycle at the G1 phase and does not induce apoptosis.
SERCA2a Expression Alters Ca2+ Patterns
We analyzed the effect of SERCA2a on the kinetics of the Ca2+ transient by measuring ATP-induced Ca2+ levels in Ad-S2a- or Ad-Gal-infected cells and in noninfected cells (Figure 4). The amplitude of the [Ca2+]i increase was dependent on the ATP concentration in both SERCA2a-expressing and control cells. Graded [Ca2+]i increases were observed after successive applications of 10 and 100 eol/L ATP on the same cell in control and SERCA2a-expressing cells in Ca2+-free medium. An increase from 10 to 100 eol/L ATP evoked a 2-fold increase in the amplitude of the Ca2+ signals, but the amplitude of the [Ca2+]i increase in SERCA2a-expressing VSMC was 6-fold lower than that in control cells after stimulation with either 10 (DF/F: 0.19±0.04, n=6 versus 1.27±0.21, n=17; P<0.05) or 100 eol/L ATP (0.41±0.13, n=6 versus 2.54±0.32, n=15; P<0.05). However, the responses to 20 eol/L tBuBHQ were larger in SERCA2a-expressing cells (0.40±0.03, n=5 versus 0.15±0.02, n=5; P<0.01), suggesting increased Ca2+ store. Two features were suggestive of a reduction in the global Ca2+ signals in SERCA2a-expressing cells. First, the oscillations observed in controls were totally abolished in SERCA2a-expressing cells (Figure 4). Second, video-imaging showed that Ca2+ signals in SERCA2a-expressing cells were restricted to the perinuclear region at 10 eol/L ATP and slowly spread (5 to 7 s after the onset of the response) to the entire cell at 100 eol/L ATP, whereas the increases observed at both ATP concentrations in control cells were immediate in the whole cell (see online videos 1 and 2). Thus, the apparent shift in ATP sensitivity not only reflects a decrease in the amplitude of Ca2+ signals but also a defect in the propagation of the signal in SERCA2a-expressing cells. Therefore, the fast recapture of the Ca2+ released from the intracellular stores on ATP stimulation in SERCA2a-expressing cells greatly reduced Ca2+ signals.
SERCA2a Inhibits PP2B
As a sustained increase in cytosolic [Ca2+] favors the formation of calmodulin/PP2B complexes, protein extracts were subjected to anti-calmodulin precipitation followed by anti-PP2B specific immunoblot (Figure 5). PP2B did not bind to calmodulin in SERCA2a-expressing cells or when PP2B had been inhibited by cyclosporin A (CsA). PP2B was associated with calmodulin in control cells, in Ad-Gal-infected cells and when SERCA had been inhibited by thapsigargin (Tg). The absence of the complex was not attributable to the absence of either PP2B or calmodulin because the absolute levels of these 2 proteins remained constant, as shown by immunoblot of total extracts.
In the same way we analyzed the activity of the Ca2+/calmodulin-dependent kinase II (CaMKII). The amount of calmodulin/CaMKII complexes did not differ between control, Ad S2a-infected, or -Gal-infected cells but was increased by Tg. Neither the total amount of CaMKII nor the amount of its phosphorylated (autonomously active) form was affected by SERCA2a expression (Figure 5). The maximal CaMKII activity measured in vitro was not different in control, Ad S2a-infected and Ad -Gal-infected cells: 11.01±0.26; 12.59±6.02; 11.06±0.72 pmoles ATP/min/e proteins, respectively (NS).
Thus, the activity of SERCA2a is particularly important to maintain PP2B in its inactive form, but has little influence on CaMKII activity.
SERCA2a Controls NFAT Activity
One important role of PP2B is to dephosphorylate and translocate NFAT into the nucleus. Only NFATc3 was detected by RT-PCR in passaged VSMC (not shown). The same isoform has been reported in freshly dissociated cells and in aortic tissue7 as well as in cerebral arteries.6 Immunofluorescence demonstrated the presence of NFAT in the cytosol and in the nuclei of proliferating control and Ad-Gal-infected cultures (Figure 6A). NFAT was present mainly in the cytosol of Ad-S2a-infected cells, but inhibition of SERCA activity by Tg (1 eol/L, 1hour) induced its translocation to the nucleus. These results were confirmed by immunoblot of cytosolic and nuclear fractions (Figure 6B). Only one band of 115 kDa, corresponding to the predicted apparent mass of NFATc3, was detected and the total amount of NFAT was not altered by infection. Despite a high level of NFAT in the cytosolic fraction of Ad-S2a-infected cells, the nuclear fraction contained little NFAT. The amount of phosphorylated GSK 3 (inactive form) was not altered by SERCA expression or activity. Furthermore, NFAT-binding activity was analyzed by electromobility shift assays (Figure 6C). High NFAT-binding was detected in proliferating control cells. The binding was specific, because it was inhibited by a 50-fold excess of NFAT cold probe. CsA inhibited DNA-protein complex, as expected. We detected low NFAT-binding in Ad-S2a-infected cells, but inhibition of SERCA by Tg restored DNA-protein interaction. The results from NFAT-driven luciferase reporter gene assay also confirmed that Ad-S2a inhibits the transcriptional activity of NFAT (Figure 6D). Tg increased luciferase activity in both control and Ad-S2a-infected cells, but NFAT-driven luciferase activity was lower in Ad-S2a-infected cells. Greater increase in luciferase activity in control can be explained by increased number of transient receptor potential Ca2+ channels in proliferating cells.8 As expected, CsA completely blocked NFAT promoter activity. These results demonstrate that SERCA2a activity controls NFAT transcriptional activity.
Next, we examined whether SERCA is involved in the regulation of expression or activity of proteins required for passage of G1/S checkpoint and known to be regulated by NFAT.28 The amounts of cyclin D1 and phosphorylated retinoblastoma protein (pRb) were decreased in SERCA2a-expressing cells as well as in cells treated with CsA (Figure 6E). Finally, we infected proliferating VSMC (in the presence of 10% FCS) with the NFAT-competing peptide, VIVIT. Four days after infection, the number of cells in Ad-VIVIT-infected cultures was significantly less than in controls (24.6±1.6% versus 100±4.7%, P<0.001) and similar to cells cultured in the presence of 0.5% FCS (22.8±1.7%). These results demonstrate that NFAT transcriptional activity is required for VSMC proliferation.
Discussion
We demonstrate that SERCA2a is a powerful regulator of VSMC proliferation in vivo and in vitro. This conclusion is based on several lines of evidence. First, SERCA2a expression is markedly downregulated in highly proliferating VSMCs in vivo in a model of balloon injury of the rat carotid artery. Second, SERCA2a is repressed in vitro in serum-induced proliferation9 and in proliferation induced by PDGF9 or very low density lipoproteins.7 Finally, we show here that overexpression of SERCA2a inhibits mitogenic stimuli mediated-proliferation of VSMC and blocks balloon injury-induced neointimal VSMC proliferation in vivo. These observations suggest that SERCA2a expression and activity are necessary to maintain a nonproliferating state of VSMC and that deregulation of SERCA activity and thus abnormal Ca2+ dynamics results in proliferation.
SERCA2a and SERCA2b are both expressed in VSMC from the carotid artery but the relative amount of each isoform was not determined. We have previously shown by S1 nuclease assay that, in control aorta, the mRNA encoding SERCA2a and SERCA2b represents 30% and 70% of total SERCA2 mRNA respectively.29 Because Western blot was not possible on injured arteries because of the lack of tissue and immunofluorescence is not quantitative, we conclude that SERCA2a disappears after balloon-injury, but whether SERCA2b increases is not clear. In culture, Western blot (Figure 3A) shows that SERCA2a gene transfer did not affect SERCA2b expression. Balloon injury probably alters expression of other SR Ca2+-handling proteins, but this has not been analyzed yet.
We have previously shown that induction of VSMC proliferation is associated with prolongation of ATP-induced Ca2+ waves because of a slower rate of Ca2+ clearance.7 This prolongation of Ca2+ waves or sustained increase in cytosolic Ca2+ level is associated with the activation of the PP2B signaling pathway.7 Here, we demonstrate that reexpression of SERCA2a in passaged VSMC reduces both the amplitude and propagation of IP3-evoked Ca2+ signals. The decrease in the amplitude of the Ca2+ wave is in agreement with results observed in SERCA2a-overexpressing CHO cells30 and is consistent with the rapid reuptake of Ca2+ in the endoplasmic reticulum resulting in a higher endoplasmic reticulum Ca2+ level.30,31
We found that reducing cytosolic Ca2+ by means of SERCA2a expression inhibited the activity of PP2B without affecting CaMKII activity. PP2B activity appears to be important for proliferation and hyperthrophic growth because: (1) inhibition of PP2B by VIVIT or CsA arrested the proliferation of VSMC (present study and Yellaturu et al15); (2) PP2B can activate hypertrophic signals both in vitro in neonatal myocytes and in vivo in transgenic mice32 as well as in the myocardium of patients with pressure overload.33 Inactivation of PP2B by SERCA2a expression has at least 1 major consequence that is inhibition of NFAT transcriptional activity. SERCA2a does not affect the CaMKII in agreement with the notion that this pathway is more dependent on Ca2+ influx.34 Yet, we cannot exclude the possibility that other Ca2+-dependent signaling pathways are altered by SERCA2a.
At least 3 members of the NFAT family (NFATc1, NFATc3, and NFATc4) appear to induce proliferation (reviewed by Chen4). Experiments using mice with targeted disruption of NFATc3 or NFATc3/c4 have shown that these isoforms are essential for myogenesis,35 ventricular myocyte proliferation,36 and vascular wall assembly.37 NFAT family members have been implicated in both cardiac hypertrophy and insulin-like growth factor 1-induced skeletal myocyte hypertrophy.32,38 The targets of NFAT, responsible for induction of proliferation, have not yet been clearly identified. Neal et al reported that NFATc1 controls cell cycle progression in 3T3-L1 preadipocytes by inducting the cell cycle-related genes encoding cyclin D1, cyclin D2, c-myc, and increasing Rb phosphorylation, all of which are required for passage of the G1/S checkpoint.28 Here we show that suppression of NFAT activity in VSMC decreases the amount of cyclin D1 and pRb.
Apoptosis has been described in both neointima, media and adventitia39,40 with a maximum in neointima and media between 7 and 14 days after surgery40 We demonstrate here that apoptosis was present at 7 days in Ad-Gal-infected arteries and was prevented by infusion of Ad-S2a.
Restoring SERCA2a levels or ablating its inhibitor phospholamban by gene transfer improves function, metabolism, and survival in a rat model of heart failure and in myocytes isolated from patients with end-stage heart failure41eC44 and clinical trials of SERCA2a and phospholamban gene43,45 therapies for heart failure are impending. This study provides new evidence that SERCA2a and its effect on calcium handling also induces a beneficial effect on VSMC proliferation and vascular remodelling. SERCA could be a new target for therapeutic efforts of VSMC disorders.
The carotid injury model used in this study has limitations because it addresses only one part of the whole spectrum of coronary artery disease. However, abnormal regulation and proliferation of vascular smooth muscles at atherosclerotic sites in coronary arteries is an important manifestation of the disease-state.
In conclusion, we provide the first evidence that increasing SERCA2a activity inhibits balloon injury-induced neointima formation. By lowering the cytosolic [Ca2+], SERCA2a inactivates PP2B and its downstream signaling cascade, resulting in decreased NFAT transcriptional activity.
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (HL-057623 and HL 071763 to R.J.H.), by INSERM and Association Franaise contre les Myopathies (grant 9087 to A.-M.L. and L.L.), and from the Association pour la Recherche contre le Cancer (grants 5621 and 4615 to T.C.). R.J.H. is a Paul Beeson Scholar of American Federation of Aging Research. We thank Valeerie Nicolas for help with the microscopy data.
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the Cardiovascular Research Center (F.d.M., R.J.H.), Massachusetts General Hospital and Harvard Medical School, Charlestown, Mass
INSERM EMI 0228/IFR-118, USTL (T.C.), Villeneuve d’Ascq, France
the Service de Cytometrie IBAIC (M.H.), IFR-FR46, Universitee Paris-Sud, Facultee des Sciences, Orsay, France.
Abstract
Proliferation of vascular smooth muscle cells (VSMC) is a primary cause of vascular disorders and is associated with major alterations in Ca2+ handling supported by loss of the sarco/endoplasmic reticulum calcium ATPase, SERCA2a. To determine the importance of SERCA2a in neointima formation, we have prevented loss of its expression by adenoviral gene transfer in a model of balloon injury of the rat carotid artery. Two weeks after injury, the intima/media ratio was significantly lower in SERCA2a-infected than in injured noninfected or injured -galactosidaseeCinfected carotids (0.29±0.04 versus 0.89±0.19 and 0.72±0.14, respectively; P<0.05), and was comparable to that observed in control carotids (0.21±0.03). The pathways leading to proliferation were analyzed in serum-stimulated VSMC. Forced expression of SERCA2a arrested cell cycle at the G1 phase and prevented apoptosis. SERCA2a inhibits proliferation through inactivation of calcineurin (PP2B) and its target transcription factor NFAT (nuclear factor of activated T-cells) resulting in lowering of cyclin D1 and pRb levels. By using NFAT-competing peptide VIVIT, we showed that NFAT activity is strongly required to promote VSMC proliferation. In conclusion, we provide the first evidence that increasing SERCA2a activity inhibits VSMC proliferation and balloon injuryeCinduced neointima formation.
Key Words: vascular smooth muscle cell proliferation gene transfer SERCA2a calcium signaling nuclear factor of activated T-cells
Introduction
Cell hyper-proliferation is an important etiology factor of cardiovascular diseases such as primary atherosclerosis, restenosis, and vein-graft disease.1,2 The neointimal vascular smooth muscle cell (VSMC) proliferation constitutes a primary cause of vascular disorders1 but, despite increasing knowledge about the cell-cycle regulation of VSMC, the molecular mechanism governing VSMC proliferation remains elusive. Thus, identifying genetic modifiers of VSMC proliferation is a major focus in cardiovascular biology and medicine.3 VSMCs have the ability to transition between quiescent differentiated and proliferating phenotypes. Acquisition of proliferating phenotype by VSMC is associated with alterations in Ca2+ handling supported by modification of Ca2+ transporter expression.4
In quiescent VSMC, the Ca2+ signal consists mainly of localized elementary calcium events. The global increase in Ca2+ concentration is rapidly reduced by calcium pumps, keeping the cytoplasmic Ca2+ concentration low. Spontaneous spark frequency decreases after activation of phosphoinositol-3 kinase (PI3K) and G protein-coupled receptors, probably attributable to inhibition of the ryanodine receptor (RyR).5 Activation of PI3K and G protein-coupled receptors also results in a sustained increase in cytosolic [Ca2+] attributable to the generation of repetitive Ca2+ waves,5,6 inhibition of Ca2+ pump activity,7 and increased Ca2+ entry.8 Chronic increases in cytosolic Ca2+ concentration in proliferating VSMC are supported by long-term alterations in the levels of Ca2+-handling proteins such as loss of RyR and SERCA2a,9 replacement of L-type voltage-operated Ca2+ channels by T-type voltage-operated Ca2+ channels, loss of the plasma membrane Ca2+ pumps, and upregulation of the transient receptor potential Ca2+ channels.4
Any chronic alterations in the spatio-temporal pattern of Ca2+ signals should alter gene expression by activating different kinases and phosphatases, modulating Ca2+-regulated transcription factors such as NFAT (nuclear factor of activated T lymphocytes).
A sustained increase in cytosolic [Ca2+] is necessary to activate calcineurin, a Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase 2B (PP2B) that dephosphorylates many proteins including NFAT, inducing its translocation into the nucleus.10,11 NFAT is exported from the nucleus after phosphorylation by the glycogen synthase kinase, GSK-3,12 or by JNK-2.13 This antagonizes the effect of PP2B. PI3K- or Gq/11-coupled receptor agonists, known to be mitogenic factors, induce NFAT-dependent transcriptional activation in vascular myocytes.6,7,14eC16
Several previous studies have suggested that SERCA2a is involved in the control of proliferation and growth: transgenic mice with only one allele of the ATP2a2 gene (SERCA2) develop numerous cancers of the upper digestive tract and skin, and cardiac hypertrophy17,18; low levels of SERCA2a are associated with cardiac hypertrophy both in humans and animals19eC21; mice in which SERCA2a has been replaced by SERCA2b develop cardiac hypertrophy22; finally, loss of SERCA2a is associated with VSMC proliferation.7,9 Similarly, only SERCA2b is present in proliferating BC3H1 muscle cells whereas SERCA1, SERCA2a, and SERCA2b are present in quiescent differentiated BC3H1 muscle cells.23
We hypothesized that increasing the rate of SR Ca2+ uptake by restoring SERCA2a expression might inhibit VSMC proliferation and prevent neointima formation induced by injury.
Materials and Methods
Injury of the Rat Carotid Artery and Adenoviral Vector-Mediated Gene Delivery
Animals were treated in accordance with institutional guidelines. The left external carotid artery from adult male Sprague-Dawley rats (Charles River, Mass) weighing 400 to 500 g was injured using a 2F Fogarty embolectomy catheter (Baxter Healthcare Corp) that was introduced into the common carotid artery through the external carotid and inflated to 2 atmospheres 3x 15 s. After both the proximal common and the proximal internal carotid arteries were clamped, viral infusion mixtures containing 1x1010 pfu of Ad- SERCA2a/GFP (Ad-S2a), or Ad-Gal/GFP (Ad-Gal)24 diluted to a total volume of 50 e蘈 was instilled between the 2 clamps, and the external carotid artery was then ligated. Perfusion was restored through the internal and the common carotid artery after 30 minutes of instillation, and the neck incision was closed. Two weeks after surgery the animals were heparinized and carotids were collected, flushed with saline, included in cryomatrix and frozen at eC80°C. Intima-to-media thickness ratio was measured from hematoxylin-and eosin-stained cross-sections with a computer-based (Lucia, Nikon) morphometric system. Apoptosis was analyzed by TUNEL staining (ApopTag Red, Serologicals Corp).
Cell Culture
VSMC were isolated from the media of the thoracic aorta from male Wistar rats and cultured as described.7 Cells were used between passages 2 to 8. Cells were infected with adenovirus for 48 hours at 1 to 10 pfu/cell. Proliferation was measured by using CellTiter96 Cell Proliferation Assay kit (Promega). Cell cycle was analyzed using a FACS Vantage (Becton-Dickinson) flow cytometer.
For transient transfection and reporter gene assay, cells were infected with Ad-S2a for 48 hours, then cotransfected using FuGene 6 (Roche) with NFAT- promoter-luciferase construct (NFAT-Luc, Stratagene) and CMV-promoter--galactosidase construct (pCMV, clontech). The luciferase activity was measured by using "the luciferase assay kit" (Promega) and normalized to the -galactosidase activity. It was expressed as percent of control in relative luciferase units.
Intracellular [Ca2+]i Measurements
Fluorescence images of FURA-2/AM loaded cells were collected at 100 ms or 1.5 s intervals by a Sensicam QE CCD camera (PCO Computer Optics GmbH), digitized, and integrated in real time by an image processor (Metafluor). Results (F/F) are expressed as ratios between 340 and 380 fluorescence signals measured during a response divided by the ratios measured in resting conditions.
Protein Analysis
The sources of primary antibodies and protein analysis protocols are detailed in expanded methods.
Confocal Microscopy
Slides were examined using a Zeiss LSM-150 confocal scanning laser microscope with a Plan Apochromat 63X objective (NA 1.40, oil immersion). All settings were kept constant to allow comparison.
Statistical Analysis
All quantitative data are presented as means of at least 3 independent experiments ±SEM. An unpaired t-test was used to calculate differences between means. Differences were considered significant when P<0.05.
An expanded Materials and Methods is available in the online data supplement at http://circres.ahajournals.org.
Results
SERCA 2a Gene Transfer Prevents Balloon Angioplasty-Associated Restenosis
SERCA2a and SERCA2b are coexpressed in the media of control carotid arteries. SERCA2a is not expressed in the media or intima 2 weeks after injury whereas SERCA2b is still present (Figure 1A). We prevented SERCA2a loss in the injured carotid arteries by infection with Ad-SERCA2a/GFP (Ad-S2a). The same concentration of Ad-Gal/GFP (Ad-Gal) was used as a control. Transfection efficacy was evaluated by using an anti-GFP antibody. Representative SERCA2a, SERCA2b, and GFP immunolabelings are presented in Figure 1B and 1C. Fluorescence of GFP and SERCA are not exactly superimposed for 2 possible reasons: (1) after 14 days the virus and GFP may already be gone whereas SERCA2a is still present because proliferation has been blocked, (2) Ad-S2a could infect not only proliferating cells, but also neighboring cells and prevent loss of SERCA2a in the entire cell population. However, in online Figure IIS, we show that Ad-S2a restores expression only in infected cells. The morphometric analysis was performed on hematoxylin/eosin stained cross-sections (Figure 1D). Neointima formation was observed in injured noninfected and injured GaleCinfected carotids whereas there was no sign of neointima formation in S2a-infected carotids. The degree of restenosis was determined by measuring the intima and media thickness and calculating the intima/media (I/M) thickness ratio (Figure 1E). I/M ratios of injured, noninfected, and -Gal infected arteries were 0.89±0.19, n=7 and 0.72±0.14, n=6, respectively (NS). The I/M ratio of S2a-infected carotids was not different from that of control arteries (0.29±0.04, n=6 and 0.21±0.03, n=9, respectively; NS), but I/M ratios from both control and S2a-infected arteries were significantly lower than that of injured noninfected or -Gal-infected carotids (P<0.05). An increase of the adventitial layer was clearly visible as a consequence of injury as already reported.25
SERCA2a Prevents Proliferation-Induced Changes in Phenotype and Apoptosis
Proliferation of VSMC is characterized by decreased expression of smooth muscle myosin heavy chains 1 and 2 (SM1 and SM2) and increased nonmuscle myosin heavy chain B (NM- B).26 SM1 and SM2 were present in injured S2a-infected and in control arteries, but only at low level in injured noninfected or Gal-infected arteries where a high level of NM-B was detected (Figure 2). No apoptosis was detected in the media of S2a-infected arteries, whereas apoptosis was observed in neointima, media and adventia of injured noninfected or -Gal-infected arteries (Figure 2).
To decipher the mechanism involved in inhibition of proliferation and apoptosis by SERCA2a we have used cultured rat aortic VSMC. Serum (10%) was used to induce proliferation because neointima formation is promoted by various mitogens released into the serum by the endothelium and blood cells. Freshly dissociated quiescent VSMC display "contractile" phenotype27 associated with expression of smooth muscle-specific myosins SM1 and SM2 (online Figure IS). Both SERCA2a and SERCA2b were expressed in these cells, as described.9 Proliferating cells did not contain SERCA2a and only low levels of SM1 and SM2, but still expressed NM-B and smooth muscle cell specific markers such as calponin and caldesmon (Figure 1S). Thus, at the time of infection, VSMCs were in the synthetic phenotype. SERCA2a expression was restored by adenovirus infection, but the level of SERCA2b did not change after infection (Figure 3A).
Measuring GFP fluorescence allowed monitoring the efficacy of adenoviral infection that was increased significantly 4 days after infection (online Figure IIS). Thus, experiments were performed on day 4 after infection. At this stage, flow cytometry showed that 60% to 90% of cells were GFP-positive.
Between days 0 and 4, control and Ad-Gal-infected cell numbers increased 5-fold, whereas the number of Ad-S2a-infected cells increased less (P<0.001) (Figure 3B). More than 50% of the cells in Ad-S2a-infected cultures were arrested in G1, whereas most cells in control and Ad-Gal-infected cultures were in the S phase (Figure 3C). The percentage of apoptotic cells, measured by DNA fragmentation (TUNEL) in control, Ad-S2a-infected, Ad-Gal-infected cells, and cells treated with staurosporine (1 e/mL, 4 hours) as positive a control, were 4.3±3.3, 0.8±0.3, 8.6±2.3, and 85±5.2, respectively (average of 3 independent experiments; 50 to 200 cells/experiment). The absence of caspase 9 cleavage (online Figure IIIS) and of a sub-G1 peak in ad-S2a-infected cells also confirmed the absence of apoptosis. Thus, SERCA2a expression inhibits progression into the cell cycle at the G1 phase and does not induce apoptosis.
SERCA2a Expression Alters Ca2+ Patterns
We analyzed the effect of SERCA2a on the kinetics of the Ca2+ transient by measuring ATP-induced Ca2+ levels in Ad-S2a- or Ad-Gal-infected cells and in noninfected cells (Figure 4). The amplitude of the [Ca2+]i increase was dependent on the ATP concentration in both SERCA2a-expressing and control cells. Graded [Ca2+]i increases were observed after successive applications of 10 and 100 eol/L ATP on the same cell in control and SERCA2a-expressing cells in Ca2+-free medium. An increase from 10 to 100 eol/L ATP evoked a 2-fold increase in the amplitude of the Ca2+ signals, but the amplitude of the [Ca2+]i increase in SERCA2a-expressing VSMC was 6-fold lower than that in control cells after stimulation with either 10 (DF/F: 0.19±0.04, n=6 versus 1.27±0.21, n=17; P<0.05) or 100 eol/L ATP (0.41±0.13, n=6 versus 2.54±0.32, n=15; P<0.05). However, the responses to 20 eol/L tBuBHQ were larger in SERCA2a-expressing cells (0.40±0.03, n=5 versus 0.15±0.02, n=5; P<0.01), suggesting increased Ca2+ store. Two features were suggestive of a reduction in the global Ca2+ signals in SERCA2a-expressing cells. First, the oscillations observed in controls were totally abolished in SERCA2a-expressing cells (Figure 4). Second, video-imaging showed that Ca2+ signals in SERCA2a-expressing cells were restricted to the perinuclear region at 10 eol/L ATP and slowly spread (5 to 7 s after the onset of the response) to the entire cell at 100 eol/L ATP, whereas the increases observed at both ATP concentrations in control cells were immediate in the whole cell (see online videos 1 and 2). Thus, the apparent shift in ATP sensitivity not only reflects a decrease in the amplitude of Ca2+ signals but also a defect in the propagation of the signal in SERCA2a-expressing cells. Therefore, the fast recapture of the Ca2+ released from the intracellular stores on ATP stimulation in SERCA2a-expressing cells greatly reduced Ca2+ signals.
SERCA2a Inhibits PP2B
As a sustained increase in cytosolic [Ca2+] favors the formation of calmodulin/PP2B complexes, protein extracts were subjected to anti-calmodulin precipitation followed by anti-PP2B specific immunoblot (Figure 5). PP2B did not bind to calmodulin in SERCA2a-expressing cells or when PP2B had been inhibited by cyclosporin A (CsA). PP2B was associated with calmodulin in control cells, in Ad-Gal-infected cells and when SERCA had been inhibited by thapsigargin (Tg). The absence of the complex was not attributable to the absence of either PP2B or calmodulin because the absolute levels of these 2 proteins remained constant, as shown by immunoblot of total extracts.
In the same way we analyzed the activity of the Ca2+/calmodulin-dependent kinase II (CaMKII). The amount of calmodulin/CaMKII complexes did not differ between control, Ad S2a-infected, or -Gal-infected cells but was increased by Tg. Neither the total amount of CaMKII nor the amount of its phosphorylated (autonomously active) form was affected by SERCA2a expression (Figure 5). The maximal CaMKII activity measured in vitro was not different in control, Ad S2a-infected and Ad -Gal-infected cells: 11.01±0.26; 12.59±6.02; 11.06±0.72 pmoles ATP/min/e proteins, respectively (NS).
Thus, the activity of SERCA2a is particularly important to maintain PP2B in its inactive form, but has little influence on CaMKII activity.
SERCA2a Controls NFAT Activity
One important role of PP2B is to dephosphorylate and translocate NFAT into the nucleus. Only NFATc3 was detected by RT-PCR in passaged VSMC (not shown). The same isoform has been reported in freshly dissociated cells and in aortic tissue7 as well as in cerebral arteries.6 Immunofluorescence demonstrated the presence of NFAT in the cytosol and in the nuclei of proliferating control and Ad-Gal-infected cultures (Figure 6A). NFAT was present mainly in the cytosol of Ad-S2a-infected cells, but inhibition of SERCA activity by Tg (1 eol/L, 1hour) induced its translocation to the nucleus. These results were confirmed by immunoblot of cytosolic and nuclear fractions (Figure 6B). Only one band of 115 kDa, corresponding to the predicted apparent mass of NFATc3, was detected and the total amount of NFAT was not altered by infection. Despite a high level of NFAT in the cytosolic fraction of Ad-S2a-infected cells, the nuclear fraction contained little NFAT. The amount of phosphorylated GSK 3 (inactive form) was not altered by SERCA expression or activity. Furthermore, NFAT-binding activity was analyzed by electromobility shift assays (Figure 6C). High NFAT-binding was detected in proliferating control cells. The binding was specific, because it was inhibited by a 50-fold excess of NFAT cold probe. CsA inhibited DNA-protein complex, as expected. We detected low NFAT-binding in Ad-S2a-infected cells, but inhibition of SERCA by Tg restored DNA-protein interaction. The results from NFAT-driven luciferase reporter gene assay also confirmed that Ad-S2a inhibits the transcriptional activity of NFAT (Figure 6D). Tg increased luciferase activity in both control and Ad-S2a-infected cells, but NFAT-driven luciferase activity was lower in Ad-S2a-infected cells. Greater increase in luciferase activity in control can be explained by increased number of transient receptor potential Ca2+ channels in proliferating cells.8 As expected, CsA completely blocked NFAT promoter activity. These results demonstrate that SERCA2a activity controls NFAT transcriptional activity.
Next, we examined whether SERCA is involved in the regulation of expression or activity of proteins required for passage of G1/S checkpoint and known to be regulated by NFAT.28 The amounts of cyclin D1 and phosphorylated retinoblastoma protein (pRb) were decreased in SERCA2a-expressing cells as well as in cells treated with CsA (Figure 6E). Finally, we infected proliferating VSMC (in the presence of 10% FCS) with the NFAT-competing peptide, VIVIT. Four days after infection, the number of cells in Ad-VIVIT-infected cultures was significantly less than in controls (24.6±1.6% versus 100±4.7%, P<0.001) and similar to cells cultured in the presence of 0.5% FCS (22.8±1.7%). These results demonstrate that NFAT transcriptional activity is required for VSMC proliferation.
Discussion
We demonstrate that SERCA2a is a powerful regulator of VSMC proliferation in vivo and in vitro. This conclusion is based on several lines of evidence. First, SERCA2a expression is markedly downregulated in highly proliferating VSMCs in vivo in a model of balloon injury of the rat carotid artery. Second, SERCA2a is repressed in vitro in serum-induced proliferation9 and in proliferation induced by PDGF9 or very low density lipoproteins.7 Finally, we show here that overexpression of SERCA2a inhibits mitogenic stimuli mediated-proliferation of VSMC and blocks balloon injury-induced neointimal VSMC proliferation in vivo. These observations suggest that SERCA2a expression and activity are necessary to maintain a nonproliferating state of VSMC and that deregulation of SERCA activity and thus abnormal Ca2+ dynamics results in proliferation.
SERCA2a and SERCA2b are both expressed in VSMC from the carotid artery but the relative amount of each isoform was not determined. We have previously shown by S1 nuclease assay that, in control aorta, the mRNA encoding SERCA2a and SERCA2b represents 30% and 70% of total SERCA2 mRNA respectively.29 Because Western blot was not possible on injured arteries because of the lack of tissue and immunofluorescence is not quantitative, we conclude that SERCA2a disappears after balloon-injury, but whether SERCA2b increases is not clear. In culture, Western blot (Figure 3A) shows that SERCA2a gene transfer did not affect SERCA2b expression. Balloon injury probably alters expression of other SR Ca2+-handling proteins, but this has not been analyzed yet.
We have previously shown that induction of VSMC proliferation is associated with prolongation of ATP-induced Ca2+ waves because of a slower rate of Ca2+ clearance.7 This prolongation of Ca2+ waves or sustained increase in cytosolic Ca2+ level is associated with the activation of the PP2B signaling pathway.7 Here, we demonstrate that reexpression of SERCA2a in passaged VSMC reduces both the amplitude and propagation of IP3-evoked Ca2+ signals. The decrease in the amplitude of the Ca2+ wave is in agreement with results observed in SERCA2a-overexpressing CHO cells30 and is consistent with the rapid reuptake of Ca2+ in the endoplasmic reticulum resulting in a higher endoplasmic reticulum Ca2+ level.30,31
We found that reducing cytosolic Ca2+ by means of SERCA2a expression inhibited the activity of PP2B without affecting CaMKII activity. PP2B activity appears to be important for proliferation and hyperthrophic growth because: (1) inhibition of PP2B by VIVIT or CsA arrested the proliferation of VSMC (present study and Yellaturu et al15); (2) PP2B can activate hypertrophic signals both in vitro in neonatal myocytes and in vivo in transgenic mice32 as well as in the myocardium of patients with pressure overload.33 Inactivation of PP2B by SERCA2a expression has at least 1 major consequence that is inhibition of NFAT transcriptional activity. SERCA2a does not affect the CaMKII in agreement with the notion that this pathway is more dependent on Ca2+ influx.34 Yet, we cannot exclude the possibility that other Ca2+-dependent signaling pathways are altered by SERCA2a.
At least 3 members of the NFAT family (NFATc1, NFATc3, and NFATc4) appear to induce proliferation (reviewed by Chen4). Experiments using mice with targeted disruption of NFATc3 or NFATc3/c4 have shown that these isoforms are essential for myogenesis,35 ventricular myocyte proliferation,36 and vascular wall assembly.37 NFAT family members have been implicated in both cardiac hypertrophy and insulin-like growth factor 1-induced skeletal myocyte hypertrophy.32,38 The targets of NFAT, responsible for induction of proliferation, have not yet been clearly identified. Neal et al reported that NFATc1 controls cell cycle progression in 3T3-L1 preadipocytes by inducting the cell cycle-related genes encoding cyclin D1, cyclin D2, c-myc, and increasing Rb phosphorylation, all of which are required for passage of the G1/S checkpoint.28 Here we show that suppression of NFAT activity in VSMC decreases the amount of cyclin D1 and pRb.
Apoptosis has been described in both neointima, media and adventitia39,40 with a maximum in neointima and media between 7 and 14 days after surgery40 We demonstrate here that apoptosis was present at 7 days in Ad-Gal-infected arteries and was prevented by infusion of Ad-S2a.
Restoring SERCA2a levels or ablating its inhibitor phospholamban by gene transfer improves function, metabolism, and survival in a rat model of heart failure and in myocytes isolated from patients with end-stage heart failure41eC44 and clinical trials of SERCA2a and phospholamban gene43,45 therapies for heart failure are impending. This study provides new evidence that SERCA2a and its effect on calcium handling also induces a beneficial effect on VSMC proliferation and vascular remodelling. SERCA could be a new target for therapeutic efforts of VSMC disorders.
The carotid injury model used in this study has limitations because it addresses only one part of the whole spectrum of coronary artery disease. However, abnormal regulation and proliferation of vascular smooth muscles at atherosclerotic sites in coronary arteries is an important manifestation of the disease-state.
In conclusion, we provide the first evidence that increasing SERCA2a activity inhibits balloon injury-induced neointima formation. By lowering the cytosolic [Ca2+], SERCA2a inactivates PP2B and its downstream signaling cascade, resulting in decreased NFAT transcriptional activity.
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (HL-057623 and HL 071763 to R.J.H.), by INSERM and Association Franaise contre les Myopathies (grant 9087 to A.-M.L. and L.L.), and from the Association pour la Recherche contre le Cancer (grants 5621 and 4615 to T.C.). R.J.H. is a Paul Beeson Scholar of American Federation of Aging Research. We thank Valeerie Nicolas for help with the microscopy data.
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