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Originally published In Press as doi:10.1074/jbc.M500322200 on January 28, 2005

J. Biol. Chem., Vol. 280, Issue 15, 14700-14708, April 15, 2005
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Blockade of Nuclear Factor of Activated T Cells Activation Signaling Suppresses Balloon Injury-induced Neointima Formation in a Rat Carotid Artery Model*

Zhimin Liu{ddagger}, Chunxiang Zhang{ddagger}, Nagadhara Dronadula, Quanyi Li, and Gadiparthi N. Rao§

From the Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, January 10, 2005 , and in revised form, January 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously reported that nuclear factor of activated T cells (NFATs) play an important role in the regulation of vascular smooth muscle cell migration and proliferation by receptor tyrosine kinase and G protein-coupled receptor agonists, platelet-derived growth factor-BB and thrombin, respectively. To understand the role of NFATs in vascular disease, we have now studied the involvement of these transcription factors in neointima formation in a rat carotid artery balloon injury model. The levels of NFATc1 in injured right common carotid arteries were increased at 72 h, 1 week, and 2 weeks after balloon injury compared with its levels in uninjured left common carotid arteries. Intraperitoneal injection of cyclosporine A (CsA), a pharmacological inhibitor of the calcineurin-NFAT activation pathway, suppressed balloon injury-induced neointima formation by 40%. Similarly, adenoviral-mediated expression of GFPVIVIT, a competent peptide inhibitor of the calcineurin-NFAT activation pathway, in injured arteries also reduced neointima formation by about 40%. Furthermore, CsA and GFPVIVIT attenuated balloon injury-induced neointimal smooth muscle cell proliferation as determined by bromodeoxyuridine staining. Platelet-derived growth factor-BB induced the expression of COX-2 in cultured VSMC in a time- and NFAT-dependent manner. COX-2 expression was also increased in the right common carotid artery in a time-dependent manner after balloon injury as compared with its levels in uninjured left common carotid artery and both CsA and GFPVIVIT negated this response. Together these results for the first time demonstrate that NFATs play a critical role in neointima formation via induction of expression of COX-2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is believed that sustained inflammation following vascular injury is a triggering event in the pathogenesis of vessel wall diseases (1, 2). The dysfunctional endothelial cells and inflammatory cells at the site of vascular injury produce a variety of molecules possessing various biological activities (1–3). A majority of these molecules appear to be mitogenic, chemotactic, or both to vascular smooth muscle cells (VSMC)1 (4–8). The availability of these molecules at the site of vascular injury may stimulate VSMC dedifferentiation (9, 10). The dedifferentiated VSMC may cease their ability to contract and relax and acquire their embryonic non-contractile synthetic phenotype state. These SMC via their migration from media to intima and multiplication in intima contribute to the progression of lesions such as restenosis after angioplasty (10). It was demonstrated that inhibition of expression and/or bioactivity of molecules such as peptide growth factors that are produced at the site of vascular injury ameliorate the lesion progression (11, 12). Because many molecules are involved in arterial wall lesions, identifying the unifying mechanisms of these substances may eventually lead to development of better therapeutic approaches against these vascular diseases.

Nuclear factor of activated T cells (NFATs) are members of a multigene family of transcription factors that belong to the Rel group (13). These transcription factors are named as NFATc1 (also known as NFATc or NFAT2), NFATc2 (also known as NFATp or NFAT1), NFATc3 (also known as NFAT4 or NFATx), and NFATc4 (also known as NFAT3) and each of these molecules appeared to be expressed as several isoforms by alternative splicing (13–15). In recent years, an additional member of the NFAT family of transcription factors, namely NFAT5, was cloned and characterized (16). NFAT5 differs from the rest of the four members of the NFAT family by its lack of co-operativity with the Fos and Jun proteins (16). In regard to expression and functional aspects of these transcription factors, earlier studies have reported the presence of NFATc1, NFATc2, and NFATc3 mainly in T cells regulating the expression of cytokine genes such as interleukin-2 (IL-2) (17). However, later studies have demonstrated the presence of all five NFAT proteins in non-immune cells as well (13, 18, 19). Studies with knock-out mice also established a role for NFATs in non-immune cells as observed from: 1) knock-out mice for NFATc1 failed to develop normal cardiac valves (20, 21); 2) knock-out mice for NFATc2 and NFATc3 exhibited reduced skeletal muscle size; and 3) mice with disruption of NFATc3/c4 genes died around E11 with defects in vessel wall assembly (22–24). One of the members of the NFAT family transcription factors, namely NFATc4, was also reported to play an important role in cardiac hypertrophy (25). In contrast to NFATc1 to NFATc4, NFAT5 was shown to be involved in the regulation of tonicity responsive genes (26). NFATc1 to NFATc4 exist in the cytoplasm as phosphoproteins in resting state and their activation requires dephosphorylation. A calcium/calmodulin-dependent serine/threonine phosphatase, known as calcineurin, has been reported to specifically dephosphorylate these NFATs leading to their activation (17). In regard to NFAT5, both calcineurin-dependent and -independent mechanisms were reported to be involved in its activation (27, 28). Activated NFATs translocate to the nucleus, bind to consensus DNA sequence GGAAAAT present in the promoter regions of genes as monomers or dimers via their Rel homology domain, and influence their transcription (13). However, other than the phenotypic observations made from the knock-out mice models (20–24), the functional aspects of NFATs in non-immune cells are largely unclear. Toward understanding the functional role of NFATs in non-immune cells, we have previously reported that their activation is required for RTK and GPCR agonist-induced VSMC migration and proliferation (29, 30). Here, we tested their role in neointima formation in a rat carotid artery balloon injury model. The present data show for the first time that NFATs play an essential role in neointima formation via their involvement in balloon injury-induced expression of COX-2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Aprotinin, phenylmethylsulfonyl fluoride, sodium orthovanadate, sodium deoxycholate, leupeptin, HEPES, and dithiothreitol were purchased from Sigma. Bromodeoxyuridine (BrdUrd) in situ detection kit (550803) was bought from BD Biosciences (San Diego, CA). Cyclosporin A (CsA) was obtained from Biomol (Plymouth Meeting, PA). [{gamma}-32P]ATP (3000 Ci/mmol) was obtained from PerkinElmer Life Sciences. Monoclonal NFATc1 antibodies (MA3-024) were bought from Affinity Bioreagents (Golden, CO). Rabbit polyclonal COX-2 antibodies (160106) were purchased from Cayman Chemical Company (Ann Arbor, MI). Recombinant human PDGF-BB was from R&D Systems Inc. (Minneapolis, MN). Consensus double-stranded NFATc binding oligonucleotides, 5'-CGCCCAAAGAGGAAAATTTGTTTCATA-3' and 3'-GCGGGTTTCTCCTTTTAAACAAAGTAT-5' (SC-2577), and mouse monoclonal anti-GFP antibodies (SC-9996) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). T4 polynucleotide kinase and the luciferase assay kit were procured from Promega (Madison, WI). FuGENE 6 transfection reagent was obtained from Roche Molecular Biochemicals (Indianapolis, IN).

Cell Culture—VSMC were isolated from the thoracic aorta of 100 to 150-g male Sprague-Dawley rats by enzymatic dissociation as described earlier (29). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures were maintained at 37 °C in a humidified 95% air and 5% CO2 atmosphere. Cells were quiesced by incubating in Dulbecco's modified Eagle's medium containing 0.1% calf serum for 72 h and used to perform the experiments unless otherwise stated.

Carotid Artery Balloon Injury—All the animal protocols were performed in accordance with the relevant guidelines and regulations approved by the Internal Animal Care & Use Committee of the University of Tennessee Health Science Center. Balloon injury was performed essentially as described by us previously (31). Briefly, rats weighing 250–300 g were anesthetized by injecting (via intraperitoneal) ketamine (60 mg/kg) and xylazine (5 mg/kg). Under a stereomicroscope, the right common, external and internal carotid arteries were exposed by a longitudinal midline cervical incision and blood flow was temporarily interrupted by ligation of the common and internal carotid arteries using vessel clips. External carotid artery was ligated permanently. A 2F Fogarty arterial embolectomy catheter was introduced through an arteriotomy in the external carotid artery just below the ligature and advanced to the common carotid artery. To produce carotid artery injury, the balloon was inflated with saline and passed 6 times with rotation from just under the proximal edge of the omohyoid muscle to the carotid bifurcation. After this, the balloon was deflated and the catheter was withdrawn. The external carotid artery was ligated with a 6-0 silk suture and the blood flow restored by removing the clips at the common and internal carotid arteries. After inspection to ascertain adequate pulsation of the common carotid artery, the surgical incision was closed and the rats were allowed to recover from anesthesia in a humidified and warmed chamber for 2 to 4 h. At different time points after balloon injury, the animals were sacrificed with an overdose of pentobarbital (200 mg/kg) and the carotid arteries were collected for RNA and protein isolation, and morphometric analysis. For morphometric analysis, carotid arteries were fixed in 10% formalin, dehydrated, and embedded in paraffin. Sections (5 µm thick) obtained at equally spaced intervals in the middle of injured and control common carotid artery segments were stained with hematoxylin and eosin. The intimal (I) and medial (M) areas were measured using NIH Image 1.62 program and the I/M ratios were calculated.

Construction of Adenoviral Vectors—The GFP and GFPVIVIT DNA fragments were excised from pEGFP-N3 (Clontech) and pEGFPVIVIT (32), respectively, by digestion of the plasmids with SalI and NotI and subcloned into an entry vector, pENTR3C (Invitrogen) producing pENTR3C-GFP and pENTR3C-GFPVIVIT. Both pENTR3C-GFP and pENTR3C-GFPVIVIT were transformed into Escherichia coli DH5{alpha} and the plasmids were amplified. These plasmids were recombinated with pAd/CMV/V5-DEST as described by the manufacturer (Invitrogen) producing pAdGFP and pAdGFPVIVIT plasmids and verified by DNA sequencing. The pAdGFP and pAdGFPVIVIT were linearized with PacI and transfected into HEK293A cells. The resulting adenovirus was further amplified by infection of HEK293A cells and purified by cesium chloride gradient ultracentrifugation (33). The AdGFP and AdGFPVIVIT virus were titrated using standard plaque assay (33).

Delivery of Adenoviruses into Injured Arteries—After balloon injury, solutions of (100 µl) AdGFP (1010 pfu/ml) or AdGFPVIVIT (1010 pfu/ml) were infused into the ligated segment of the common carotid artery for 30 min. The ligatures and catheter were then removed, the external carotid artery was ligated, and the incision closed. To test the effect of CsA on neointima formation, the drug was injected into the animal intraperitoneally (10 mg/kg/day) for the entire duration of the experiment.

Electrophoretic Mobility Shift Assay—Quiescent VSMC were treated with and without PGDF-BB (20 ng/ml) in the presence and absence of CsA (10 µM) for 2 h and nuclear extracts were prepared as previously described (30). Protein-DNA complexes were formed by incubating 5 µg of nuclear protein in a total volume of 20 µl consisting of 15 mM HEPES, pH 7.9, 3 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4.5 µg of bovine serum albumin, 2 µg of poly(dI-dC), 15% glycerol, and 100,000 cpm of 32P-labeled consensus double-stranded NFAT binding oligonucleotide probe for 30 min on ice. Protein-DNA complexes were resolved on a 4% polyacrylamide gel using 1x Tris glycine-EDTA buffer (25 mM Tris-HCl, pH 8.5, 200 mM glycine, 0.1 mM EDTA). Double-stranded oligonucleotides (NFATc, 5'-CGCCCAAAGAGGAAAATTTGTTTCATA-3', 3'-GCGGGTTTCTCCTTTTAAACAAAGTAT-5') were labeled with [{gamma}-32P]ATP using the T4 polynucleotide kinase kit as per the supplier's protocol (Promega). To test the effect of GFPVIVIT on PDGF-BB-induced NFAT-DNA binding activity, cells were first infected with either AdGFP or AdGFPVIVIT and quiesced before they were subjected to treatments with agonists and nuclear extract preparation.

Immunohistochemistry—For BrdUrd immunostaining, rats were injected with BrdUrd (30 mg/kg) 24 and 12 h before sacrificing. Carotid arteries were dissected out from the sacrificed animals and the fat tissue was removed. Vessel tissues were then fixed in 10% formalin and embedded in paraffin. Sections were cut with 5-µm thickness and immunostained with anti-BrdUrd antibodies using the BrdUrd labeling kit (BD Biosciences). Similarly, sections of injured right common carotid artery 2 weeks after balloon injury with and without the indicated regimen were stained with anti-CD45 antibodies to detect inflammation.

RT-PCR for COX-2, GAPDH, and NFATc1—Total cellular RNA from VSMC and tissues was isolated with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The RNA was quantified, and 1 µg of RNA from control and each treatment was used for the RT-PCR. The RT-PCR was performed using the SuperScript III One-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen). The reaction mixture in a total volume of 50 µl consists of 1x reaction buffer, 1 µg of RNA, 0.5 µM each of forward and reverse primers of a specific gene, and 2 µl of SuperScript III RT/Platinum Taq polymerase. RT-PCR amplification was carried out in a Gene Amp 2400 System (PerkinElmer Life Sciences) with an initial first strand cDNA synthesis reaction at 55 °C for 30 min. The PCR was performed at 94 °C for 4 min followed by 25 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The final extension reaction was carried out at 72 °C for 10 min. Fifteen microliters of the PCR products were then separated by electrophoresis on a 2% agarose gel. The PCR products were visualized by ethidium bromide staining, photographed, and quantified by densitometry. The primers used are as follows: rat COX-2 primers: forward, 5'-AATTCAAGACACTCTATCACTG-3' and reverse, 5'-AATTGAGGCAGTGTTGATGTACC-3'; rat GAPDH primers: forward, 5'-ATAGACAAGATGGTGAAGGTCGG-3' and reverse, 5'-TCATGAGCCCTTCCACGATGCC-3'; NFATc1 primers: forward, 5'-TCCACGACGTGGAGGTGGAAGACG-3' and reverse, 5'-TGATGGCTGCCACAATGGCAGAGC-3'. The RT-PCR primers for NFATc1 were designed based on the conserved DNA sequences between mouse and human NFATc1 cDNA and the RT-PCR products derived by the use of these primers, and rat aortic smooth muscle cell and tissue RNAs were confirmed by DNA sequencing and BLAST analysis.

Transient Transfection and Luciferase Assay—VSMC were plated evenly onto 100-mm dishes and grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. At 50–80% confluence, medium was replaced and cells were transfected with 3 µg/dish of pT8NFAT-Luc plasmid (32) using FuGENE 6 transfection reagent according to the manufacturer's instructions (Roche Molecular Biochemicals). Forty-eight hours after transfection, VSMC were treated with and without PGDF-BB (20 ng/ml) in the presence and absence of CsA (10 µM) for 6 h and cell lysates were prepared. VSMC lysates were normalized for protein and assayed for luciferase activity using the Luciferase Assay System (Promega) and Turner Luminometer (TD-20/20). To test the effect of GFPVIVIT on PDGF-BB-induced NFAT-Luc activity, cells were transfected with pT8NFAT-Luc plasmid and then infected with either AdGFP or AdGFPVIVIT before they were quiesced and subjected to treatments.

Western Blot Analysis—Tissues and/or VSMC were rinsed with cold phosphate-buffered saline (PBS) and frozen immediately in liquid nitrogen. Cells were lysed by thawing in 250 µl of lysis buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate) for 20 min on ice, whereas tissues were homogenized in the same buffer. Cell and/or tissue extracts were transferred into 1.5-ml Eppendorf tubes and cleared by centrifugation at 12,000 rpm for 20 min at 4 °C. Cell or tissue extracts containing an equal amount of protein were resolved by electrophoresis on 0.1% SDS and 10% polyacrylamide gels. The proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond, Amersham Biosciences). After blocking in 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected using a chemiluminescence reagent kit (Amersham Biosciences).

Statistics—All the experiments were repeated at least three times with similar pattern of results. Data are presented as mean ± S.D., and the treatment effects were analyzed by Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PDGF-BB Stimulates NFAT-dependent Transcription in VSMC—NFATs are phosphoproteins and exist in the cytoplasm in an inactive state. These transcription factors are activated via their dephosphorylation. Calcineurin, a calcium/calmodulin-dependent serine/threonine phosphatase specifically dephosphorylates NFATs leading to their translocation from the cytoplasm to the nucleus (12). In the nucleus, they bind as monomers or dimers via their Rel homology domain to a consensus DNA sequence GGAAAAT present in the promoter regions of genes and influence their transcription (24). Previously, we have reported that the RTK and GPCR agonists, PDGF-BB and thrombin, respectively, cause translocation of NFATs, particularly NFATc1, from the cytoplasm to the nucleus in VSMC (29). In addition, we have demonstrated that NFATs are involved in PDGF-BB and thrombin-induced VSMC migration and proliferation (29, 30). To understand the role of NFATs in vascular wall diseases, we have now tested their involvement in neointima formation after angioplasty. To achieve this goal, we first measured NFAT activity in response to PDGF-BB in VSMC. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of CsA (10 µM), a potent inhibitor of calcineurin (17), for 2 h, and nuclear extracts were prepared. Nuclear extracts containing an equal amount of protein from control and PDGF-BB-treated VSMC were incubated with 100,000 cpm of 32P-labeled consensus double-stranded NFAT binding oligonucleotide probe and the protein-DNA complexes were separated by electrophoresis on a polyacrylamide gel. PDGF-BB increased NFAT-DNA binding activity by 2–3-fold as compared with control (Fig. 1A). CsA significantly blocked the NFAT-DNA binding activity induced by PDGF-BB. NFATc1 to NFATc4 possess a highly conserved calcineurin-binding site PxIxIT in their regulatory domain at the NH2 terminus. Based on this information a peptide, namely VIVIT, that specifically competes with NFATs for binding to calcineurin, was developed (32). Expression of GFPVIVIT fusion protein in T cells inhibited only calcineurin-sensitive NFAT-dependent but not calcineurin-sensitive NFAT-independent inducible expression of IL-2, IL-3, IL-13, tumor necrosis factor-{alpha}, granulocyte macrophage-colony stimulating factor, and macrophage inflammatory protein-1{alpha} by phorbol 12-myristate 13-acetate and ionomycin (32). To confirm the effect of CsA on PDGF-BB-induced NFAT-DNA binding activity, VSMC were infected with AdGFP or AdGFPVIVIT at a multiplicity of infection of 40, growth-arrested for 48 h, treated with and without PDGF-BB (20 ng/ml) for 2 h, and nuclear extracts were prepared. An equal amount of nuclear protein from control and each treatment was analyzed for NFAT-DNA binding activity as described above. Consistent with the effect of CsA, adenoviral-mediated expression of GFPVIVIT but not GFP reduced PDGF-BB-induced NFAT-DNA binding activity (Fig. 1A).



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  		FIG. 1.
PDGF-BB transactivates NFATs in VSMC. A, quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of CsA (10 µM) for 2 h and nuclear extracts were prepared. To study the effects of GFP and GFPVIVIT on PDGF-BB-induced NFAT-DNA binding activity, cells were infected first with these adenovirus and quiesced before they were subjected to the indicated treatments. Nuclear extracts containing an equal amount of protein from control and each treatment were analyzed for NFAT-DNA binding activity using 32P-labeled consensus double-stranded NFAT binding oligonucleotide probe. B, quiescent VSMC that were transfected with pT8NFAT-Luc plasmid DNA and quiesced were treated with and without PDGF-BB (20 ng/ml) for 6 h and cell extracts were prepared. Cell extracts containing an equal amount of protein from control and each treatment were assayed for luciferase activity. To study the effects of GFP and GFPVIVIT on PDGF-BB-induced NFAT-mediated reporter gene activity, cells were transfected with pT8NFAT-Luc followed by infection with these adenovirus and quiesced before they were subjected to treatments. *, p < 0.01 versus control or GFP + control; **, p < 0.05 versus PDGF-BB or GFP + PDGF-BB treatment alone.

 
To find whether the increased NFAT-DNA binding activity leads to a corresponding up-regulation in NFAT-dependent transcription, VSMC were transfected with a plasmid, pT8NFAT-Luc, in which a luciferase reporter gene expression is controlled by NFAT-dependent IL-2 promoter (34) and quiesced. Cells were then treated with and without PDGF-BB (20 ng/ml) for 6 h in the presence and absence of CsA (10 µM) and cell extracts were prepared. Cell extracts from control and agonist-treated VSMC normalized for protein were assayed for luciferase activity. Consistent with NFAT-DNA binding activity, luciferase activity was also increased by about 2-fold in PDGF-BB-treated VSMC versus control (Fig. 1B). CsA blocked the PDGF-BB-induced increase in luciferase activity (Fig. 1B). CsA alone had no effect on basal luciferase activity. Similarly, adenoviral-mediated expression of GFPVIVIT but not GFP inhibited PDGF-BB-induced luciferase activity (Fig. 1B). These results are in agreement with our previously published findings (29, 30).

Balloon Injury of Carotid Artery Induces the Expression of NFATc1—Having demonstrated a role for NFATs in RTK and GPCR agonist-induced VSMC migration and proliferation in vitro (29, 30), we have now studied their role in vascular wall diseases. We initially determined NFATc1 levels in carotid arteries at various times after balloon injury. The balloon-injured right common carotid arteries and uninjured left common carotid arteries were dissected out from the sacrificed rats and RNA and protein were isolated. An equal amount of RNA from each sample was analyzed by RT-PCR for NFATc1 using its specific primers. NFATc1 mRNA levels were found increased in the injured arteries starting at 24 h after balloon injury as compared with its levels in uninjured arteries (Fig. 2A). To confirm this result further at the protein level, an equal amount of protein from the balloon-injured and uninjured arteries was analyzed by Western blotting for NFATc1 using its specific antibodies. As shown in Fig. 2B, increases in NFATc1 protein level were observed in the injured arteries at 72 h, peaked at 1 week, and declined at 2 weeks after balloon injury as compared with its levels in uninjured arteries. These results clearly show that balloon injury induces the expression of NFATc1 in rat carotid artery.



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  		FIG. 2.
Balloon injury of carotid artery induces the expression of NFATc1. Total cellular RNA and protein were isolated from balloon-injured right common carotid arteries at various times after angioplasty and uninjured left common carotid arteries. A, an equal amount of RNA from each sample was subjected to RT-PCR using NFATc1 or GAPDH specific primers and the products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining, and quantified by densitometry. B, an equal amount of protein from each sample was analyzed by Western blotting for NFATc1 using its specific antibodies. BI, balloon injury.

 
Inhibitors of Calcineurin-NFAT Activation Signaling Suppress Balloon Injury-induced Neointima Formation—We next studied the effect of CsA and GFPVIVIT on balloon injury-induced neointima formation. Right common carotid artery was injured with an inflated balloon catheter and 2 weeks after injury the injured right and uninjured left common carotid arteries were dissected out, fixed, stained with hemotoxylin and eosin (H & E), and the intimal area to medial area ratios were determined by morphometric analysis. As shown in Fig. 3, A and B, substantial neointimal formation occurred in injured right common carotid artery as compared with uninjured left common carotid artery. Administration of CsA (10 mg/kg/day) reduced neointima formation by about 40% (Fig. 3A). To confirm this result further, immediately after balloon injury, adenovirus expressing either GFPVIVIT or GFP (1010 pfu/ml) was infused into the injured right common carotid artery and 2 weeks after injury arteries were isolated and used for Western blotting detection of GFP and/or morphometric analysis as described above. Infusion of adenovirus harboring GFPVIVIT or GFP (1010 pfu/ml) into injured arteries lead to a 12-fold increase in the expression of GFP and/or GFPVIVIT in balloon-injured arteries as compared with their levels in uninfected control arteries (data not shown). Adenoviral-mediated expression of GFPVIVIT but not GFP also inhibited neointima formation by about 40% (Fig. 3B). These results suggest that NFATs are involved in vascular wall remodeling in response to injury and play a role in neointima formation.



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  		FIG. 3.
CsA and GFPVIVIT inhibit balloon injury-induced neointima formation. Balloon injury was performed in the right common carotid artery and the animals were divided into four groups. One group received vehicle only, and the second group received CsA (10 mg/kg/day) intraperitoneally for 2 weeks (A). The third and fourth groups received adenovirus containing GFP or GFPVIVIT, respectively, by infusion into the injured arteries (B). Two weeks after balloon injury, arteries were isolated, fixed, sectioned, stained with H & E, morphometry analysis was performed, and the I/M ratios were calculated. *, p < 0.05 versus vehicle-BI or GFP-BI (n = 6 rats). BI, balloon injury.

 
Inhibitors of Calcineurin-NFAT Activation Signaling Suppress Balloon Injury-induced SMC Proliferation—Increased SMC proliferation is a hallmark in neointima formation (3–5). Previously, we have demonstrated that NFATs play a role in the regulation of VSMC proliferation by both RTK and GPCR agonists (29). To extend these in vitro observations to in vivo, we have now tested the effect of CsA and GFPVIVIT on neointimal SMC proliferation. Twenty-four and 12 h before the end of 2 weeks after balloon injury rats were injected with BrdUrd, sacrificed, and arteries were isolated, fixed, and stained with anti-BrdUrd antibodies. BrdUrd-positive cells were counted. About 7% of cells were found to be BrdUrd-positive in the neointimal region 2 weeks after balloon injury (Fig. 4A). In contrast, only 1 to 2% of cells were found to be BrdUrd-positive in the neointimal region of injured arteries of rats that received CsA (10 mg/kg/day) for 2 weeks (Fig. 4A). Similarly, adenoviral-mediated expression of GFPVIVIT but not GFP into injured arteries reduced BrdUrd-positive cells in the neointimal region from 8 to 2% (Fig. 4B). These results demonstrate that blockade of calcineurin-NFAT activation signaling inhibits neointimal SMC proliferation by about 70%.



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  		FIG. 4.
CsA and GFPVIVIT inhibit balloon injury-induced neointimal SMC proliferation. Balloon injury was performed in the right common carotid artery and the animals were divided into four groups. One group received vehicle only, and the second group received CsA (10 mg/kg/day) intraperitoneally for 2 weeks (A). The third and fourth groups received adenovirus containing GFP or GFPVIVIT, respectively, by infusion into the injured arteries (B). Twenty-four and 12 h before the end of 2 weeks after balloon injury, rats were injected with BrdUrd, sacrificed, arteries were isolated, fixed, and stained with anti-BrdUrd antibodies. BrdUrd-positive cells were counted in 10 randomly selected fields and values are expressed as % of total number of cells counted. *, p < 0.05 versus vehicle-BI or GFP-BI (n = 6 rats). BI, balloon injury.

 
PDGF-BB Induces COX-2 Expression in a Time- and NFAT-dependent Manner in VSMC—NFATs have been shown to play a role in the regulation of COX-2 (35). In addition, a large number of studies have demonstrated that COX-2 plays a role in the development of various carcinomas (36, 37). In view of these observations, we envisioned that COX-2 might be a target gene of NFATs in VSMC. To test this, quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) for the indicated times and total cellular RNA was isolated. An equal amount of RNA from control and PDGF-BB-treated VSMC was analyzed by RT-PCR for COX-2 using its specific primers. PDGF-BB induced COX-2 expression in a time-dependent manner with a maximum effect of 20-fold at 2 h and declining thereafter (Fig. 5A). To confirm this result further, extracts of control and various times of PDGF-BB-treated (20 ng/ml) VSMC were prepared and an equal amount of protein from each condition was analyzed by Western blotting for COX-2 using its specific antibodies. Consistent with its effect on the COX-2 mRNA level, PDGF-BB also induced COX-2 expression at the protein level in a time-dependent manner (Fig. 5B). To understand the role of NFATs in PDGF-BB-induced COX-2 expression, VSMC were infected with AdGFPVIVIT or AdGFP at a multiplicity of infection of 40, quiesced, treated with and without PDGF-BB (20 ng/ml) for 2 h, and total cellular RNA and protein were isolated and analyzed for COX-2 mRNA and protein levels as described above. Adenoviral-mediated expression of GFPVIVIT but not GFP substantially inhibited PDGF-BB-induced COX-2 expression at both mRNA and protein levels (Fig. 6, A and B). These results indicate that PDGF-BB induces COX-2 expression in VSMC in a time- and NFAT-dependent manner.



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  		FIG. 5.
PDGF-BB and balloon injury induce COX-2 expression in VSMC and carotid artery, respectively. A and B, quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) for various times and total cellular RNA and protein were isolated. A, an equal amount of RNA from each sample was subjected to RT-PCR using rat COX-2-specific primers and the products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining, and quantified by densitometry. B, an equal amount of protein from each sample was analyzed by Western blotting for COX-2 using its specific antibodies. C and D, RNA and protein were isolated from the right common carotid arteries that were dissected out at various times after balloon injury and uninjured left common carotid arteries and analyzed for COX-2 and GAPDH mRNA and COX-2 protein levels as described above. BI, balloon injury.

 



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  		FIG. 6.
Blockade of calcineurin-NFAT activation signaling by CsA and GFPVIVIT suppresses PDGF-BB and balloon injury-induced COX-2 expression in VSMC and carotid arteries, respectively. Total cellular RNA and protein were isolated from VSMC treated with and without PDGF-BB (20 ng/ml) in the presence and absence of CsA (10 µM) or GFPVIVIT for 2 h or from the right common carotid arteries that were dissected out at 72 h after balloon injury and uninjured left common carotid arteries. A and C, an equal amount of RNA from each sample was subjected to RT-PCR using rat COX-2- and GAPDH-specific primers and the products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining, and quantified by densitometry. B and D, an equal amount of protein from each sample was analyzed by Western blotting for COX-2 using its specific antibodies. BI, balloon injury.

 
Balloon Injury of Carotid Artery Induces the Expression of COX-2 in a Time- and NFAT-dependent Manner—To determine whether mechanical injury to vessel wall leads to induction of expression COX-2, if so, the role of NFATs in its up-regulation, we first studied the time course effect of balloon injury on COX-2 mRNA and protein levels in the right common carotid artery. RNA and protein were isolated from the injured right common carotid arteries at various times after balloon injury and uninjured left common carotid arteries and analyzed by RT-PCR and Western blotting, respectively, for COX-2 mRNA and protein levels using its specific primers and antibodies. As shown in Fig. 5C, an increase in the expression of COX-2 mRNA was observed at 24 h after balloon injury, peaked at 72 h after balloon injury, and declined thereafter as compared with its levels in uninjured vessels. A similar pattern of up-regulation in the levels of COX-2 protein was observed in the injured arteries with a maximum increase at 72 h after balloon injury as compared with its levels in uninjured arteries (Fig. 5D). Administration of CsA (10 mg/kg/day) or adenoviral-mediated expression of GFPVIVIT but not GFP suppressed COX-2 expression induced by balloon injury (Fig. 6, C and D).

Balloon Injury of Carotid Artery Induces the Expression of CD45 in NFAT-dependent Manner—NFATs and COX-2 play an important role in inflammation (38, 39). To find whether NFATs via their role in balloon injury-induced COX-2 expression modulates inflammation, 2 weeks after balloon injury the injured and uninjured arteries were isolated, fixed, sectioned, and stained with anti-CD45 antibodies. The CD45-positive cells were counted. Substantial levels of CD45 were detected in the neointimal region 2 weeks after balloon injury, and administration of CsA (10 mg/kg/day) or adenoviral-mediated expression of GFPVIVIT but not GFP inhibited this response by 50% (Fig. 7, A and B).



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  		FIG. 7.
CsA and VIVIT inhibit balloon injury-induced CD45 expression in neointimal region. Balloon injury was performed in the right common carotid artery and the animals were divided into four groups. One group received vehicle only, and the second group received CsA (10 mg/kg/day) intraperitoneally for 2 weeks (A). The third and fourth groups received adenovirus containing GFP or GFPVIVIT, respectively, by infusion into the injured arteries (B). Two weeks after balloon injury, arteries were isolated, fixed, sectioned, and stained with anti-CD45 antibodies. The CD45-positive cells were counted in 10 randomly chosen fields and expressed as % of total number of cells counted. *, p < 0.05 versus vehicle-BI or GFP-BI (n = 6 rats). BI, balloon injury.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The important findings of the present study are as follows. 1) NFATc1 levels were increased in the arteries after balloon injury as compared with its levels in uninjured arteries. 2) Pharmacological and competent peptide-based inhibition of calcineurin-NFAT activation signaling substantially reduced neointimal SMC proliferation and neointima formation. 3) PDGF-BB, a potent mitogen and chemoattractant for VSMC, induced COX-2 expression in a time- and NFAT-dependent manner in these cells in culture. 4) COX-2 expression was also increased in the arteries after balloon injury as compared with its levels in uninjured arteries. 5) Inhibition of NFATs by CsA and GFPVIVIT significantly blocked COX-2 expression induced by balloon injury. 6) Substantial levels of CD45, an inflammation marker, were observed in the neointimal region and blockade of calcineurin-NFAT activation signaling by CsA and GFPVIVIT markedly reduced its expression.

Many studies have shown that NFATs play a critical role in cardiac development and hypertrophy (25, 40–42). It was also demonstrated that NFAT function is essential for vessel wall assembly (22–24). Previously, we have reported that NFATs play a role in the regulation of VSMC migration and proliferation by both RTK and GPCR agonists (29, 30). In addition, work from other laboratories indicated that forced expression of a constitutively active NFATc1 in murine 3T3-L1 pre-adipocyte fibroblasts leads to their transformation (43). Together, these results clearly suggest that in addition to their role in the regulation of cytokine genes in immune cells, NFATs are involved in mediating several other biological aspects including cardiovascular development, cell proliferation, and cell migration. Migration of VSMC from media to intima and their proliferation in intima play a contributing role in neointima formation after angioplasty (4–8). The findings that NFATs are involved in the regulation of VSMC migration and proliferation (29, 30) and inhibition of calcineurin-NFAT activation signaling suppresses neointima formation after balloon injury (present study) reveal another hitherto undiscovered role for these transcription factors in vascular diseases, particularly in restenosis. A large body of data suggests a role for COX-2 in inflammation (38, 44). Similarly, NFATs via their involvement in the regulation of cytokine genes have been implicated in the mediation of inflammation (39). Because inflammation is a critical player in the vessel wall diseases (1, 2), and both NFATc1 and COX-2 are increased in the vessel wall after balloon injury and inhibition of calcineurin-NFAT activation signaling suppressed balloon injury-induced COX-2 and CD45 expression and neointima formation, it is likely that NFATs via their role in injury-induced COX-2 expression leads to a sustained inflammation at the site of injury and thereby influences restenosis. A role for COX-2 in the vessel wall diseases was also suggested by the findings that COX-2 expression was increased in VSMC in response to serum and in rat aorta in response to balloon injury (45), and COX-2-specific drugs inhibited both atherosclerosis and neointima formation (44, 46). In addition, a role for NFATs, particularly NFATc1, in the induction of expression of COX-2 in VSMC in response to PDGF-BB and in rat carotid artery in response to balloon injury can be supported by the presence of its putative binding site in rat COX-2 promoter (47). Earlier studies have indicated that bone marrow cells contribute, at least to a certain extent, to neointima formation in some types of vascular injury (48). Because NFATs have been demonstrated to be involved in vessel wall assembly (22–24), it is tempting to speculate that these transcription factors may be involved in neointima formation via influencing, at least to some level, the migration of bone marrow-derived stem cells to the site of vascular injury.

Although the findings that NFATc1 is translocated from the cytoplasm to the nucleus in response to RTK and GPCR agonists in VSMC and induced in balloon-injured arteries support a role for it in neointima formation, an involvement for other members of the NFAT family of transcription factors in this vessel wall disease cannot be ruled out by the present study. Regardless of the type of NFAT, the present work demonstrates for the first time a role for these transcription factors in balloon injury-induced COX-2 expression and neointima formation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL69908 and HL64165 (to G. N. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both authors contributed equally to this work. Back

§ To whom correspondence should be addressed: Dept. of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-7321; Fax: 901-448-7126; E-mail: grao{at}physio1.utmem.edu.

1 The abbreviations used are: VSMC, vascular smooth muscle cells; SMC, smooth muscle cells; COX-2, cyclooxygenase-2; CsA, cyclosporine A; GPCR, G protein-coupled receptor; NFATs, nuclear factor of activated T cells; RTK, receptor tyrosine kinase; BrdUrd, bromodeoxyuridine; IL, interleukin; PDGF, platelet-derived growth factor; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Anjana Rao and Michael P. Bell for providing pGFPVIVIT and pT8NFAT-Luc plasmids, respectively.



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