Requirement for p38 Mitogen-Activated Protein Kinase Activity in Neointima Formation After Vascular Injury
Background— Angioplasty and stent delivery are performed to treat atherosclerotic vascular disease but often cause deleterious neointimal lesion formation. Previously, growth factor receptor-bound protein 2 (Grb2), an intracellular linker protein, was shown to be essential for neointima formation and for p38 mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (SMCs). In this study, the role of vascular SMC p38α MAPK in neointimal development was examined.
Methods and Results— Compound transgenic mice were generated with doxycycline-inducible SMC-specific expression of dominant-negative p38α MAPK (DN-p38α). Doxycycline treatment resulted in the expression of DN-p38α mRNA and protein in transgenic arteries. Doxycycline-treated compound transgenic mice were resistant to neointima formation 21 days after carotid injury and showed reduced arterial p38 MAPK activation. To explore the mechanism by which p38α MAPK promotes neointima formation, an in vitro SMC culture system was used. Inhibition of p38α MAPK in cultured SMCs by treatment with SB202190 or small interfering RNA blocked platelet-derived growth factor–induced SMC proliferation, DNA replication, phosphorylation of the retinoblastoma protein, and induction of minichromosome maintenance protein 6.
Conclusions— SMC p38α MAPK activation is required for neointima formation, perhaps because of its ability to promote retinoblastoma protein phosphorylation and minichromosome maintenance protein 6 expression.
Received August 17, 2008; accepted May 28, 2008.
The proliferation of arterial smooth muscle cells (SMCs) contributes to several pathological conditions, including restenosis after angioplasty, posttransplantation coronary artery disease, and hypertensive vasculopathy. Elaboration of neointima made up of extracellular matrix and SMCs that proliferate and migrate from the tunica media is known to contribute to the development of restenosis.1,2
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SMC proliferation may be triggered by the action of extracellular peptide growth factors and ligands that are released locally in blood vessels and that stimulate intracellular signaling cascades. In particular, platelet-derived growth factor (PDGF) and basic fibroblast growth factor are thought to be involved in the neointimal hyperplasia that develops after vascular injury.3 Other growth factors and ligands, including epidermal growth factor and thrombin, are released locally in vessels after injury.4,5 These ligands bind to transmembrane receptors on the surface of SMCs that initiate intracellular signaling cascades.
In previous work, the role of the intracellular linker protein growth factor receptor-bound protein 2 (Grb2) in the development of neointima after vascular injury was evaluated.6 Grb2 is an Src homology 2 and Src homology 3 domain-containing protein that facilitates the activation of the small GTPase ras and downstream mitogen-activated protein kinases (MAPKs) by growth factor receptor tyrosine kinases. Haploinsufficiency for Grb2-rendered mice resistant to neointimal lesion development after carotid injury.6 In addition, Grb2+/− cultured murine aortic SMCs were resistant to PDGF-induced cell proliferation. Reduction of Grb2 protein in cultured rat arterial SMCs caused defective p38 MAPK and c-jun N-terminal kinase (JNK) MAPK activation in response to PDGF stimulation. These findings suggest that p38 MAPK or JNK MAPK, or both in combination, is a critical intermediary in growth factor signaling cascades downstream of Grb2 that are responsible for SMC proliferation and neointima development.
The p38 MAPK family of serine/threonine kinases consists of 4 members (p38α, p38β, p38γ, and p38δ); p38α MAPK is ubiquitously expressed in mammalian tissues.7 The activation and function of p38α MAPK have been studied in more detail than the other family members, and in some cell types, activation of p38α MAPK antagonizes cell cycle progression, promotes inflammatory responses, and may also contribute to apoptosis.7 In particular, p38α MAPK activity in cardiomyocytes blocks cell cycle progression and cytokinesis and induces the expression of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6.8,9 Furthermore, in immature thymocytes, p38α MAPK activation by V(D)J-mediated double-strand DNA breaks induces a G2/M cell cycle checkpoint via the phosphorylation and accumulation of p53.10 However, the role of p38α MAPK in cell proliferation and cell survival depends on cell type and physiological context.7
The biological response of cultured mammalian SMCs to p38α MAPK activation contrasts with that observed in cardiomyocytes and immature thymocytes. In particular, recently published studies support the hypothesis that p38α MAPK plays an important role in growth factor–stimulated SMC proliferation. Work by Chen et al11 demonstrated that endothelin-dependent rat aortic SMC (RAOSMC) proliferation depends on p38α/β MAPK activity. Specifically, endothelin-1–stimulated p38α/β MAPK activation led to c-Myc and E2F gene expression, which contributed to cell cycle progression. In another study, Jacob et al12 demonstrated that human cultured vascular SMC proliferation in response to injury depends on p38α/β MAPK activation. Furthermore, Kavurma and Khachigian,13 using SB202190, showed that p38α/β MAPK is important for cultured SMC proliferation. However, the specific requirement for p38α MAPK in neointima formation in vivo has not been determined. In this work, the role of p38α MAPK in the pathogenesis of neointima formation was analyzed by use of compound transgenic mice with inducible SMC-specific expression of DN-p38α.
Generation of SM/TRE-DN-p38α Compound Transgenic Mice
SM22α-rtTA (SM) mice were originally generated in the C57Bl/6J×CBA mixed genetic background with DNA encoding the reverse tetracycline transactivator (rtTA) linked to a 455–base pair fragment of the SM22α promoter as previously described.14 More than 10 original transgenic lines were generated, and 1 line that exhibited persistent arterial expression of rtTA in adult mice was used in these studies. SM mice were crossed into the C57Bl/6J background for >7 generations.
TRE-flag-DN-p38α (TRE-DN-p38α) mice were generated with DNA containing a dominant-negative form of murine p38α MAPK in which the TxY activation loop is mutated to AxF.15 In addition, the DNA is linked to an N-terminus flag tag sequence and tetracycline response element (TRE). A linearized form of this DNA construct was injected into the male pronucleus of 1-cell murine pure C57Bl/6J embryos to generate TRE-DN-p38α mice by the Diabetes Research and Training Center Transgenic Core Facility at Washington University in St Louis, School of Medicine. One founder was obtained and was bred with C57Bl/6J nontransgenic mice.
TRE-DN-p38α mice were bred with SM mice to generate SM22α-rtTA×TRE-flag-DN-p38α (SM/TRE-DN-p38α) mice in the C57Bl/6J genetic background. SM/TRE-DN-p38α mice were obtained in the expected Mendelian ratio, appeared normal, and were fertile. SM/TRE-DN-p38α compound transgenic mice were compared with SM single transgenic littermates for all experiments described in this study. All studies involving the use of mice were performed in strict accordance with protocols approved by the Animal Studies Committee of Washington University in St Louis School of Medicine.
All statistical analysis was performed with Sigma Stat 3.1 (Systat Software, Inc, San Jose, Calif). For animal lesion area data, Mann–Whitney rank-sum tests were performed. For cell culture data, Kruskal-Wallis 1-way ANOVA on ranks tests with all pairwise multiple comparison procedures, conducted via the Student-Newman-Keuls method, were performed to assess statistical significance. Proliferating cell nuclear antigen (PCNA) index data were analyzed with a t test. A value of P<0.05 was considered statistically significant.
For a description of other methods used in this study, please see the online-only Data Supplement.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Generation of Compound Transgenic Mice With Inducible SMC-Specific Expression of DN-p38α
In previous work, Grb2+/− mice were demonstrated to be resistant to neointima formation in response to carotid injury, and arterial p38 MAPK activation was found to be reduced in these animals.6 To directly test the role of p38α MAPK in neointima formation in vivo, we generated compound transgenic mice with doxycycline-inducible expression of dominant-negative p38α MAPK in arterial SMCs. A DNA construct was generated that contained a tetracycline response element (TRE) linked to cDNA encoding DN-p38α where the TxY kinase activation loop amino acid sequence is mutated to AxF.15 This DNA construct was linearized and injected into the male pronucleus of 1-cell murine C57Bl/6J embryos and used to generate TRE-DN-p38α founder mice. One line of TRE-DN-p38α transgenic mice in the C57Bl/6J genetic background was created by this method. TRE-DN-p38α transgenic mice were crossed with SM transgenic mice that were previously demonstrated to exhibit arterial SMC-specific expression of rtTA.14
To induce expression of DN-p38α MAPK, SM/TRE-DN-p38α compound transgenic mice and control single transgenic SM mice were treated with doxycycline. After 1 week of doxycycline treatment, aortas were isolated for reverse-transcriptase polymerase chain reaction (RT-PCR) assays. RT-PCR with aortic RNA demonstrated that DN-p38α MAPK mRNA was expressed in the SM/TRE-DN-p38α compound transgenic background but not in the SM single transgenic background (Figure 1A). Doxycycline-inducible expression of DN-p38α MAPK mRNA in the carotid arteries of SM/TRE-DN-p38α compound transgenic mice also was assessed. After 1 week of doxycycline, induction of DN-p38α MAPK mRNA was observed (Figure 1B). In addition, after 2 weeks of doxycycline treatment, carotid arteries, aortas, and other tissues were obtained for protein analysis. Western blot analysis of protein lysates revealed that DN-p38α protein was expressed in the aorta in the SM/TRE-DN-p38α compound transgenic background but not in the control single transgenic SM background (Figure 1C). Furthermore, 2 weeks of doxycycline induction resulted in decreased activation of native p38 MAPK in the aorta and carotid arteries of SM/TRE-DN-p38α compound transgenic mice compared with arterial tissues from SM mice (Figure 1D). In contrast, DN-p38α induction did not block extracellular signal-regulated kinase (ERK) MAPK activation (Figure 1D).
Requirement of p38α MAPK for Response to Carotid Arterial Injury in Mice
To evaluate the role of SMC p38α MAPK activity in the pathogenesis of neointima formation, doxycycline-treated compound transgenic SM/TRE-DN-p38α and single transgenic SM mice were subjected to carotid injury by the epoxy resin beaded-probe method. Both groups of mice tolerated the procedure well, and no postoperative mortality was observed. Evans Blue dye and von Willebrand factor staining demonstrated that our carotid injury method produces consistent endothelial denudation and that the degree of denudation is similar from mouse to mouse (Figure I of the online-only Data Supplement).
Histological examination of carotid cross sections performed 3 weeks after injury revealed robust neointimal lesion formation in single transgenic SM mice (Figure 2A and 2C). In SM mice, the neointimal lesions were at least partially occlusive, and sometimes the lesions completely occluded the vessel lumen. In contrast, SM/TRE-DN-p38α compound transgenic mice exhibited markedly reduced lesion formation. An 84% reduction was noted in the mean neointima-to-media ratio of SM/TRE-DN-p38α mice compared with SM mice (Figure 2B). Specifically, the mean±SE neointima-to-media ratio of SM mice was 2.23±0.80 but was only 0.36±0.10 in SM/TRE-DN-p38α mice (P=0.029; Figure 2B). In addition, the mean neointimal area was less for SM/TRE-DN-p38α mice compared with SM mice (P=0.017; Figure 2C). However, the mean medial areas of SM and SM/TRE-DN-p38α mice were similar (Figure 2D).
The SMC component of neointimal lesions was assessed by immunohistochemical staining with an anti–SMC-actin primary antibody. Immunohistochemical analysis revealed that SMCs were a major component of neointimal lesions in SM mice (Figure 3). Although SMCs were present in the tunica media and intima of SM/TRE-DN-p38α mice, SMC-actin staining was markedly reduced compared with SM mice (Figure 3). Vascular signaling also was evaluated in carotid cross sections from mice. Immunohistochemical staining with anti–phospho p38 MAPK antibody showed that p38 MAPK activation was present in both the tunica media and neointima of single transgenic SM mice (Figure 3). In contrast, p38 MAPK activation was dramatically reduced in the tunica media and intima of SM/TRE-DN-p38α mice (Figure 3).
Role of Retinoblastoma Protein and Minichromosome Maintenance Protein 6 in p38α MAPK–Induced SMC Proliferation
To explore the molecular mechanism(s) by which p38α MAPK promotes carotid artery neointima formation in response to injury, an in vitro model system with a cultured rat vascular SMC-derived cell line was used. Subconfluent A10 cells were pretreated with a pharmacological inhibitor of both p38α and p38β MAPK, with SB202190, or with control diluent. PDGF treatment of A10 cells caused robust phosphorylation of the p38 MAPK activation loop (Figure 4A). Activation of p38 MAPK, but not ERK MAPK, was blocked by SB202190 (Figure 4A). When quiescent A10 cells were pretreated with control diluent and then stimulated with PDGF for 20 hours, a 1.85-fold increase in cell number was observed (P<0.05; Figure 4B). However, when A10 cells were pretreated with SB202190, PDGF treatment did not result in increased cell numbers (Figure 4B). [3H]-thymidine incorporation assays were performed to determine whether p38 MAPK function is required for PDGF-induced DNA synthesis. PDGF treatment of A10 cells resulted in an 11.4-fold increase in [3H]-thymidine incorporation (P<0.05; Figure 4C), but SB202190 dramatically reduced [3H]-thymidine incorporation (Figure 4C). Similarly, SB202190 blocked PDGF-induced primary RAOSMC proliferation and reduced [3H]-thymidine incorporation (online-only Data Supplement Figure IIA and IIB).
To specifically reduce p38α MAPK protein levels and activity in cultured cells, a short interfering (siRNA) approach was used. Subconfluent A10 cells were transfected with a rat p38α MAPK siRNA construct or with control, nontargeting siRNA. After 24 hours of transfection in serum-free media, A10 cells were stimulated with PDGF or control diluent for 20 hours. After stimulation, some A10 cells were used to generate lysates for Western blotting. Western blot analysis revealed that p38α MAPK protein levels were dramatically reduced in p38α MAPK siRNA–transfected A10 cells compared with control siRNA–transfected cells (Figure 5A). Specifically, densitometric analysis demonstrated an ≈76% reduction in total p38α MAPK protein (Figure 5B). When stimulated with PDGF for 20 hours, A10 cells transfected with p38α MAPK siRNA proliferated at a much slower rate than control siRNA–transfected cells (Figure 5C). A10 cell number increased by 1.9-fold after PDGF stimulation in control siRNA-transfected cells (P<0.05). An increase in A10 cell number was blocked with p38α MAPK siRNA. After 20 hours of PDGF stimulation, [3H]-thymidine incorporation was reduced ≈56% in p38α MAPK siRNA–transfected A10 cells compared with control siRNA–transfected cells (data not shown; P=0.007).
To address the downstream target(s) of p38α MAPK action responsible for growth factor–stimulated SMC proliferation, the phosphorylation status of a key regulator of mammalian cell cycle progression, retinoblastoma protein (Rb), was evaluated.16 In nonproliferating cells, Rb is hypophosphorylated and is able to bind to and sequester members of the E2F family of transcription factors. Activation of cyclin-dependent kinases results in the hyperphosphorylation of Rb, the subsequent release of E2F proteins, and the transcription of genes involved in cell cycle progression.17,18 In previous work, inhibition of human vascular SMC proliferation by salicylate administration correlated with reduced Rb hyperphosphorylation.19 PDGF treatment of A10 cells for 20 hours resulted in a robust increase in Rb phosphorylation that was blocked by pretreatment of cells with SB202190 (Figure 6A). In addition, PDGF induced hyperphosphorylation of Rb in RAOSMCs that was blocked by SB202190 pretreatment (online-only Data Supplement Figure III).
One target of E2F activity during cell cycle progression is minichromosome maintenance protein 6 (MCM6). MCM6 is a regulator of DNA replication and DNA helicase activity.20 In previous work, liver X receptor ligand inhibition of human vascular SMC proliferation correlated with reduced expression of MCM6.21 PDGF treatment of subconfluent A10 cells for 24 hours resulted in a robust increase in MCM6 protein levels that was blocked by pretreatment of cells with SB202190 (Figure 6B).
We assessed the requirement for MCM6 in RAOSMC proliferation and DNA replication by transfecting RAOSMCs with MCM6-specific siRNA. Subconfluent RAOSMCs were transfected with a rat MCM6 siRNA construct or with control, nontargeting siRNA. After 2 days of transfection in serum-free media, RAOSMCs were stimulated with PDGF or control diluent for 15 hours. After stimulation, some RAOSMC lysate was generated for Western blotting. Western blot analysis revealed that MCM6 protein levels were dramatically reduced in MCM6 siRNA–transfected RAOSMCs compared with control siRNA–transfected cells (Figure 7A and 7B). In RAOSMCs transfected with control siRNA, overnight stimulation with PDGF induced a 2-fold increase in MCM6 protein (P<0.05; Figure 7A and 7B). MCM6-specific siRNA reduced baseline MCM6 protein to ≈55% compared with control siRNA–transfected RAOSMCs (P<0.05). In addition, MCM6-specific siRNA blocked PDGF induction of MCM6 protein (Figure 7A and 7B, P<0.05). Moreover, MCM6-specific siRNA reduced both PDGF-induced proliferation (Figure 7C) and thymidine incorporation (data not shown). Specifically, in control siRNA–transfected RAOSMCs, cell number increased by 2.5-fold after 40 hours of PDGF stimulation (P<0.05; Figure 7C). However, the increase in RAOSMC number was blocked with MCM6 siRNA.
MCM6 expression was evaluated in carotid cross sections of SM and SM/TRE- DN-p38α mice by immunohistochemical staining. Immunohistochemical analysis revealed that carotid cross sections from SM mice isolated 3 weeks after injury had increased MCM6 expression compared with sections from compound transgenic SM/TRE-DN-p38α mice (Figure 8A). Sections also were stained for PCNA. Carotid cross sections from SM mice had increased medial PCNA staining compared with SM/TRE-DN-p38α mice (Figure 8B). Specifically, 6.69±0.45% of SM medial nuclei were PCNA positive. However, this value was reduced to 1.04±1.80% for SM/TRE-DN-p38α mice (P=0.006; Figure 8B).
To evaluate whether increased apoptosis was responsible for the phenotype observed in SM/TRE-DN-p38α mice 3 weeks after carotid injury, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed on carotid cross sections. Infrequent TUNEL-positive cells were detected in the media and neointima of SM carotid arteries (n=3), but no TUNEL-positive cells were detected in the media or neointima of SM/TRE-DN-p38α carotid arteries (n=3; data not shown).
The MAPKs are important effectors of growth factor and ligand action in eukaryotic cells. The p38 family of MAPKs consists of 4 proteins encoded by separate genes that regulate important aspects of cell physiology, including cell growth, proliferation, survival, and differentiation.7 In this work, the role of p38α MAPK in the formation of neointima after arterial injury was analyzed. In previous work, the role of the ubiquitously expressed intracellular linker protein Grb2, which links growth factor receptor activation to MAPK activation, was analyzed.6 Grb2-haploinsufficient mice were resistant to neointima formation after carotid injury. Furthermore, Grb2-haploinsufficient cultured SMCs exhibited reduced p38 and JNK MAPK activation in response to PDGF stimulation.
To address the specific role of p38α MAPK in neointima formation, SM/TRE-DN-p38α mice were generated in the present study. Doxycycline treatment of these animals resulted in the expression of DN-p38α mRNA and protein in aortas and carotid arteries. Next, doxycycline-treated SM/TRE-DN-p38α compound transgenic mice were found to be resistant to carotid injury-induced neointima formation compared with control SM mice. Furthermore, immunohistochemical analysis revealed that p38 MAPK activation was dramatically reduced in the tunica media and intima of SM/TRE-DN-p38α mice after carotid injury. These results strongly suggest that p38α MAPK activity is required for neointima formation after arterial injury.
To determine the molecular mechanism(s) that contribute to p38α MAPK-mediated neointima formation, an in vitro SMC culture system was used. PDGF-induced cell proliferation was blocked in cultured SMCs after treatment with SB202190, an inhibitor of p38α/β MAPK. To examine more specifically the role of p38α MAPK in SMC proliferation in vitro, an siRNA approach was used. PDGF-induced SMC proliferation was blocked in cells treated with p38α MAPK siRNA.
One potential effector of p38α MAPK action that is involved in SMC proliferation is Rb, a master regulator of cell cycle progression.16 PDGF-induced hyperphosphorylation of Rb was blocked in SMCs after SB202190 treatment. Previous work by Marra et al19 showed that the antiproliferative effect of salicylates on growth factor–stimulated SMCs was correlated with reduced Rb hyperphosphorylation. It is unclear whether p38α MAPK directly phosphorylates Rb in PDGF-stimulated SMCs or if p38α MAPK indirectly affects Rb phosphorylation via activation of a cyclin-dependent kinase or other protein kinase.
Another potential effector of p38α MAPK in SMCs is MCM6, a protein required for DNA synthesis during the S phase of the cell cycle.20 MCM6 gene expression in SMCs is induced by the E2F transcription factor after it is released from hyperphosphorylated Rb.21 PDGF stimulation of control SMCs promoted the expression of MCM6, but this response was blocked in cells treated with SB202190. Furthermore, siRNA reduction of MCM6 blocked PDGF-induced SMC proliferation and DNA replication.
Taken together, our results show that p38α MAPK activity is required for SMC proliferation in vitro and neointima formation in vivo. This activity of p38α MAPK contrasts with its role in cardiomyocytes, in which in certain situations p38α MAPK blocks cell proliferation, promotes apoptosis, and antagonizes hypertrophic growth.8,22–25 However, one of the hallmarks of MAPKs is that the biological effects of kinase activation are highly dependent on the cell type and physiological context.7 One molecular mechanism of p38α MAPK action in SMCs may be to promote the hyperphosphorylation of Rb, the subsequent activation of E2F family members, and the stimulation of MCM6 expression that results in cell proliferation and arterial neointima formation.
The results of this study provide preliminary evidence that p38α MAPK inhibitors may be useful for the treatment of arterial restenosis and transplant vasculopathy. Our previous work and the work of other groups also support the use of p38α MAPK inhibitors to limit pathological cardiac remodeling after myocardial infarction.24–26 Although numerous p38α MAPK inhibitors have been developed, none are currently approved for use in human patients, and clinical trials evaluating the efficacy of these agents in cardiovascular disease have not been published.27
Sources of Funding
This work was supported by grants from the Burroughs Wellcome Fund, the American Heart Association, and the National Institutes of Health (HL061567, HL076770) (Dr Muslin). We acknowledge the assistance of the Diabetes Research and Training Center Transgenic Core Facility at Washington University in St Louis, School of Medicine, directed by Dr Burton Wice (DRTC P60 DK020579). We also acknowledge the assistance of the DDRCC Morphology Core Facility at Washington University in St Louis, School of Medicine (DDRCC P30 DK52574). Dr Proctor is a recipient of an American Heart Association, Heartland Affiliate predoctoral fellowship.
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Atherosclerotic vascular disease is commonly treated by percutaneous arterial interventions. Neointima formation often occurs at the site of intervention and may lead to significant restenosis. The use of drug-eluting stents to treat atherosclerotic vascular disease results in a markedly reduced incidence of restenosis, but delayed in-stent thrombosis can occur that leads to myocardial infarction. A cause of delayed thrombosis may be inadequate endothelialization of the inner surface of the drug-eluting stent resulting from the toxic effects of the embedded agents, sirolimus or taxol. This issue has led to a search for agents that inhibit the smooth muscle cell migration and proliferation required for neointima formation without inhibiting endothelialization. In this work, mice expressing a dominant-inhibitory form of p38 mitogen-activated protein kinase (p38 MAPK) only in smooth muscle cells were resistant to neointima formation after carotid artery injury. Knockdown of p38 MAPK in cultured smooth muscle cells blocked cell proliferation. In previous work, inhibition of p38 MAPK in endothelial cells promoted cell survival and proliferation. Therefore, p38 MAPK inhibition is predicted to inhibit smooth muscle cell proliferation while promoting endothelial cell survival and growth. The use of agents that block p38 MAPK may block restenosis without preventing stent endothelialization.
The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.734848/DC1.