Therapeutic Potential of a Synthetic Peptide Inhibitor of Nuclear Factor of Activated T Cells as Antirestenotic Agent
Objective— The calcineurin/nuclear factor of activated T cells (NFAT) axis plays a pivotal role in the regulation of critical genes in vascular smooth muscle cell (vSMC) proliferation and inflammation, which makes NFAT inhibition an attractive modality in the prevention of restenosis.
Methods and Results— Synthetic peptide VIVIT potently inhibited NFAT activation in RAW 264.7 macrophages, Ea.Hy.926 endothelial cells and vSMCs, and blocked ionomycin-elicited nuclear import of NFAT. VIVIT, as well as cyclosporine A (CsA) or FK506, completely blunted platelet-derived growth factor-BB (PDGF-BB) and thrombin-induced vSMC proliferation. Moreover, it significantly inhibited PDGF-BB and thrombin-induced interleukin-6, interleukin-8, transforming growth factor-β1, stromal cell-derived factor-1α, and monocyte chemotactic protein-1 expression in vSMCs. Unlike FK506 or CsA, VIVIT did not affect nuclear factor κB reporter gene activation and did only marginally affect endothelial wound healing in vitro. VIVIT did not intervene in phorbol 12-myristate 13-acetate-stimulated extracellular signal-regulated kinase activation, confirming its specificity for NFAT. Furthermore, our data establish that NFAT is a regulator of PDGF-BB induced vSMC proliferation.
Conclusions— VIVIT appears to be a specific and potent inhibitor of NFAT activation and thus of NFAT-mediated proliferation and inflammation. Unlike FK506 or CsA, synthetic VIVIT therapy will not be accompanied by non-NFAT-mediated side effects on calcineurin signaling and constitutes a promising lead in antirestenotic therapy.
Restenosis occurs in 30% to 40% of the treated patients and continues to be the major obstacle limiting the long-term efficacy of coronary angioplasty. It is mainly driven by a vascular inflammatory process, in which a major event is the proliferation and migration of smooth muscle cells (SMCs) from media to intima.1,2 The introduction of drug-eluting stents has been a true breakthrough in the prevention and treatment of restenosis, but the entrapped immunosuppressive and cytotoxic agents are relatively unspecific and can lead to adverse effects in flanking regions and to impaired vessel functionality.3,4 Given the inflammatory nature of restenosis, it is conceivable that more specific anti-inflammatory compounds may display a more favorable activity profile.
One of the potential targets in this regard is calcineurin, a calmodulin-dependent, calcium-activated phosphatase. It plays a key role in the activation of T cells, B cells, NK cells, and mast cells, as well as the major vascular cell types, including SMCs, endothelial cells (ECs), and macrophages.5 In addition, it was shown to be a multifunctional regulator of various downstream signaling pathways.6 One of the downstream effectors, nuclear factor of activated T cells (NFAT), has been indicated in osteoclast differentiation, muscle fiber-type specialization, cardiac valve development, and myocardial hypertrophy.7 Activated calcineurin binds and dephosphorylates NFAT, which then translocates to the nucleus to activate cells and induce cytokine expression. Given the important role of calcineurin-NFAT signaling in various physiological and immunologic processes, its inhibition is considered a powerful therapeutic modality in the treatment of graft transplant rejection and autoimmune diseases. Recently, it has been suggested that NFAT also plays an important role in the regulation of vascular SMC (vSMC) migration and proliferation by platelet-derived growth factor-BB (PDGF-BB) and thrombin, respectively, both crucial processes in restenosis,8 implying that it could be very effective in antirestenotic therapy as well.9 Cyclosporine A (CsA) and FK506 disrupt calcineurin phosphatase activity and thus affect all the downstream signal transduction pathways. This rather nonselective inhibition could lead to undesired side effects and toxicity, which have prompted the need for more selective NFAT inhibitors that do not compromise non-NFAT-mediated calcineurin signaling.10
Recently, an N-terminal consensus motif in NFAT (ie, PXIXIT) was identified as the main docking site for calcineurin on NFAT. Further optimization of this motif led to the discovery of VIVIT, which was shown to selectively and potently inhibit calcineurin-NFAT interaction when expressed intracellular.11 Similarly, the C terminal of NFAT and linker region of calcineurin A were found to contain conserved consensus motifs that facilitate calcineurin docking and NFAT dephosphorylation.12,13 Proof of concept of the potential of VIVIT in antirestenotic therapy has been attained recently using VIVIT-encoding plasmids or virus vectors.14,15 In this study, we therefore sought to address whether VIVIT peptide itself could already be effective in modulating ECs, SMCs, and macrophage function, and thus in antirestenotic therapy.
Materials and Methods
FK506 was obtained from Fujisawa GmbH, CsA, phorbol 12-myristate 13-acetate (PMA), ionomycin, and thrombin were from Sigma, PDGF-BB was from Biosource International Inc, and anti-phosphor-p44/p42 mitogen-activated protein kinase (MAPK) polyclonal antibody was from Cell Signaling Technology. U0126 and SB203580 were from Promega.
The murine macrophage cell line RAW 264.7, human ECs Ea. Hy. 926, and murine vSMCs, isolated from thoracic aortas of male C57BL/6 mice as described,16 were grown in DMEM supplemented with 10% (v/v) heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cultures were maintained at 37°C in humidified 95% air and 5% CO2. Unless otherwise stated, vSMCs were growth arrested before the experiments by incubating in DMEM containing 0.1% normal calf serum for 72 hours.
NFAT reporter plasmid (pNFAT-luc) was a kind gift from Dr L.J. De Windt (Hubrecht Laboratory, Interuniversity Cardiology Institute, the Netherlands). pRL-TK was from Promega. pNFAT-green fluorescent protein-1 (GFP-1) plasmid was a kind gift from Dr Anjana Rao (Harvard Medical School, Boston, Mass), and nuclear factor κB (NF-κB) reporter plasmid (pNF-κB-luc) was kindly provided by Dr Onno Meijer (Leiden/Amsterdam Center for Drug Research, Leiden, the Netherlands).
Solid Phase Peptide Synthesis
VIVIT (MAGPHPVIVITGPHEE) and VEET control (MAGPPHIVEETPHVIG) peptides were synthesized using Fmoc solid-phase peptide synthesis on a Multisyntech Syro Multiple Peptide Synthesizer. Crude peptides were purified on a preparative C18 RP high-performance liquid chromatography column (Alltech) using a BIOCAD VISION automated purification system. Purified peptides were characterized by liquid chromatography/mass spectrometry (LC-MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and found to be ≥least 95% pure.
Transient Transfection and Dual Luciferase Assay
Cells were seeded in 24-well plates at a density of 5×104 cells per well, and 24 hours later, cells were cotransfected with pNFAT-Luc reporter and pRL-TK reference plasmid (encoding Renilla luciferase) with ExGen-500 transfection reagent (MBI Fermentas). One day after transfection, cells were treated with and without PMA (200 nmol/L)/ionomycin (500 nmol/L) or PDGF-BB (20 ng/mL) in the presence and absence of FK506 (10 μmol/L) or VIVIT (100 μmol/L) for 12 hours; cell lysates were prepared and simultaneously assayed for renilla and firefly luciferase activity by the Dual Luciferase Assay System (Promega) and Turner Luminometer.
Endothelial Wound Healing Assay
Murine H5V ECs were cultured in 24-well plates as a confluent monolayer. The monolayer was incubated in the absence of serum for 16 hours and wounded in a line across the well with a 200-μL pipette tip, then washed 2× with serum-free medium and incubated with 10% FCS in the presence or absence of FK506 (10 μmol/L) or VIVIT (100 μmol/L) for 24 hours. Photographs were taken at 0- and 24-hour incubation period at the marked wound location. The wound healing effect was measured using the NIH ImageJ program and expressed as migrated distance.
SMC Proliferation Assay
Growth-arrested vSMCs were treated with 5 U/mL thrombin or 20 ng/mL PDGF-BB in the absence or presence of VIVIT, CsA/FK506, or U0126/SB203580. After 4 hours, 1 μCi/mL of [3H]-thymidine was added to the vSMCs and left to incubate for another 20 hours. Cells were washed 3 times with ice-cold PBS and lysed in 500 μL 0.1 mol/L NaOH, transferred to a liquid scintillation vial, and 4.5 mL Emulsifier-Safe was added (Packard-Biosciences), after which the radioactivity was measured in a liquid scintillation counter.
vSMC Apoptosis Assay
vSMCs were seeded evenly in 24-well plates at a density of 5×104 cells per well, and after 24 hours, they were treated with VIVIT at a concentration ranging from 0.01 to 30 μmol/L. Twelve hours later, cells were detached by mild trypsination, stained with Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit I (BD Biosciences), then subjected to fluorescence-activated cell sorter analysis (FACScalibur; BD Biosciences).
RT-PCR and Inhibition of Transcription
Growth-arrested vSMCs were treated for 2 hours with or without PDGF-BB (20 ng/mL) or thrombin (5 U/mL) in the presence of CsA (10 μmol/L), VIVIT (100 μmol/L), or FK506 (10 μmol/L). Total cellular RNA was then isolated with Trizol Reagent (Invitrogen). Quantitative analysis of gene expression was performed on the ABI PRISM 7700 Taqman apparatus (Applied Biosystems). Murine hypoxanthine phosphoribosyltransferase (mHPRT) was used as standard housekeeping gene, and nonreverse-transcribed RNA samples were used as control to determine genomic DNA contamination. Relative gene expression was calculated to that of mHPRT on the basis of ΔCt values. Primer sequences are given in online supplement I (available at http://atvb.ahajournals.org).
Interleukin-6 Secretion by vSMCs
Growth-arrested vSMCs were treated with and without PDGF-BB (20 ng/mL) or thrombin (5 U/mL) in the presence of CsA (10 μmol/L), VIVIT (100 μmol/L), or FK506 (10 μmol/L). After 24 hours, interleukin-6 (IL-6) that had been released into the culture medium was measured by ELISA (BD Biosciences).
Inhibition of GFP-NFAT1 Nuclear Translocation
Cultured vSMCs were transfected with pGFP-NFAT1 by electroporation using the Amaxa nucleofector kit V according to manufacturer instructions (Amaxa GmbH). After 24 hours, cells were stimulated with 500 nmol/L ionomycin for 20 minutes in the presence of VIVIT (100 μmol/L) or FK506 (10 μmol/L). Cellular GFP localization was assessed by fluorescence microscopy.
NFAT Activation and MAPK Activation
Activated extracellular signal-regulated kinase (ERK) was detected by Western blotting against the biphosphorylated protein p44/42. vSMCs were treated with and without PMA (20 nmol/L) or PDGF-BB (20 ng/mL) in the presence of series concentrations of VIVIT, CsA, U0126, or SB203580 for 15 minutes. Lysates were prepared by suspending cells in lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.5% Triton X-100, 1% sodium deoxycholate, 0.5 mol/L phenylmethylsulfonyl fluoride, 2 mmol/L Na3VO4, 50 mmol/L NaF, 1 mmol/L EGTA, 50 μg/mL aprotinin, 50 μg/mL chymostatin, and 25 μg/mL pepstatin) and centrifuged (13 000 rpm; 10 minutes). The protein content of the supernatant was measured by bicinchoninic acid assay (Pierce). Equal amounts of protein were subjected to 10% SDS-PAGE, and proteins were transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA in PBST (0.1% Tween 20 in PBS) for 1 hour at room temperature and probed with anti-phospho-p44/42 antibody (1:1000) diluted in 1% BSA/PBST for 1 hour at room temperature, followed by incubation for 1 hour with horseradish peroxidase-conjugated secondary swine anti-rabbit horseradish peroxidase (DAKO; 1:2000) diluted in 1% BSA/PBST. Protein was visualized by the ECL-plus detection system according to manufacturer instructions. The blots have been evaluated densitometrically by using the Image-Quant software and normalized with control group as arbitrary density.
Values are expressed as mean±SD. A 2-tailed unpaired Student t test was used to compare individual groups. A level of P<0.05 was considered significant.
VIVIT Selectively and Potently Inhibits Calcineurin-Mediated NFAT Activation in ECs, SMCs, and Macrophages
The inhibitory effect of synthetic VIVIT was tested on endothelial, SMCs, and macrophages by a dual luciferase reporter assay. As expected, coactivation of cells with ionomycin and PMA resulted in a significant upregulation of NFAT-dependent gene expression. FK506 (10 μmol/L) was found to block the ionomycin/PMA-induced luciferase activity to basal levels in ECs (Figure 1A) and murine RAW 264.7 macrophages (Figure 1B). VIVIT (100 μmol/L) had a similar inhibitory capacity in both cell types, whereas a VIVIT analogue (VEET) appeared to be inactive. Previously, it had been reported that CsA could inhibit the PDGF-BB- and thrombin-induced NFAT activation8 in thoracic aorta of rats. Indeed, we show that PDGF-BB is able to activate NFAT in murine vSMCs. FK506 and VIVIT, but not the inactive analogue VEET, were able to completely counteract this activation (Figure 1C). Importantly, although VIVIT inhibited NFAT activation to a similar extent as FK506, it did not interfere with PMA/ionomycin-induced NF-κB activation (Figure 1D).
Next, we determined the inhibitory capacity of synthetic VIVIT in various cell types via the NFAT dual luciferase assay. The IC50 of VIVIT in murine macrophage RAW 264.7 cells, vSMCs, and human ECs were very comparable (ie, 29.1, 30.2, and 45.7 μmol/L, respectively; Figure 2), suggesting that the anti-inflammatory activity of VIVIT is not cell type dependent.
vSMC Proliferation Inhibition and Apoptosis Assay
In the next stage, we assessed the capacity of VIVIT functionally. Compared with the untreated control, CsA/FK506 treatment by itself already led to a significant inhibition in vSMC proliferation and completely abrogated the mitogenic effects of PDGF-BB and thrombin (Figure 3A; P<0.001). VIVIT significantly inhibited both PDGF-BB- and thrombin-induced vSMC proliferation but did not affect baseline proliferation of unstimulated vSMCs (Figure 3A). In contrast, VEET, the inactive control peptide, did not impair the proliferative responses to PDGF-BB and thrombin at all, establishing the specificity of VIVIT. Interestingly, a similar antiproliferative effect on PDGF-BB or thrombin-stimulated vSMCs was observed for the mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK-ERK) inhibitor U0126 (10 μmol/L) as well as the p38 inhibitor SB203580 (10 μmol/L; Figure 3B), tentatively suggesting that part of the effects of the NFAT inhibitors can be attributed to the inhibition of MAPK activation. Previously, we have shown that low-dose FK506 can inhibit vSMC apoptosis and block collar-induced atherosclerotic plaque development in apolipoprotein E-deficient mice.17 Here, we show that VIVIT, up to 30 μmol/L, does not significantly affect vSMC apoptosis (see online supplement II.)
Effect of VIVIT on Endothelial Wound Healing
Endothelial wound healing is a critical process in restenosis and should preferably not be impaired in antirestenotic therapy. We show that exposure of ECs to serum induced wound healing by &7-fold. Importantly, FCS-induced wound healing was found to be attenuated (−68%; P<0.001) in the presence of the nonspecific NFAT inhibitor FK506 but only marginally by VIVIT (−15%; P<−0.03) and not by VEET (−4%; Figure 3C)
VIVIT Inhibits PDGF-BB- and Thrombin-Induced Inflammatory Responses in vSMCs
VIVIT was found to profoundly reduce PDGF-BB-induced IL-6, IL-8 monocyte chemotactic protein-1 (MCP-1), and stromal cell-derived factor-1α (SDF-1α) expression in vSMCs but only mildly reduced that of transforming growth factor-β1 (TGF-β1). Although CsA/FK506 displayed an even stronger capacity than VIVIT to inhibit IL-6, IL-8, SDF-1α, and MCP-1 expression, they also completely ablated TGF-β1 induction by both stimuli. Apparently, VIVIT and the established immunosuppressants do differ in their anti-inflammatory profile (Figure 4). (A similar pattern of thrombin-induced group is shown in online supplement III.) In addition, PDGF-BB and thrombin not only induced IL-6 expression at an mRNA level but also at a protein level, and this induced production was not observed after pretreatment of vSMCs with VIVIT and CsA/FK506 (Figure 5A). Also in this case, FK506 and CsA did reduce IL-6 secretion below baseline levels, whereas VIVIT did not.
GFP-NFAT1 Nuclear Translocation Assay
Ionomycin (500 nmol/L) induced complete dephosphorylation of NFAT1, as can be derived from fluorescent studies showing nuclear translocation of fluorescent GFP-NFAT1 fusion protein in vSMCs (Figure 5B) and HEK293 cells (data not shown). Nuclear translocation of NFAT1–GFP in ionomycin stimulated cells could be almost completely prevented by FK506 (−92%; P<0.01) as well as VIVIT (−88%; P<0.01; Figure 5B and 5C).
Effects of VIVIT on ERK Activation in vSMCs
PMA and PDGF-BB treatment were both seen to activate the ERK pathway in vSMCs. Neither VIVIT nor CsA/FK506 showed inhibition in PMA-induced ERK activation (Figure 6A). However, VIVIT dose-dependently inhibited PDGF-BB-induced ERK activation, and the IC50 essentially concurred with that of NFAT activation in the NFAT reporter assay (Figure 6B). At 20 μmol/L, VIVIT already quenched PDGF-BB-stimulated ERK activation, and a similar level of inhibition was seen for CsA and the specific MEK-ERK inhibitor U0126 but not for the p38 inhibitor SB203580. We further show that U0126 and SB203580 completely inhibit PMA/ionomycin-stimulated NFAT activity in RAW 264.7 cells (Figure 6C).
NFAT is regarded as an important transcriptional regulator of cytokine and growth factor expression in T cells and cardiomyocytes.7 In this study, we show that NFAT also regulates cellular activation of macrophages, vSMCs, and ECs, suggesting a key role of this transcription factor in various vasculopathies such as atherosclerosis and restenosis. Both established nonspecific NFAT inhibitors (CsA and FK506) and a synthetic peptide antagonist of NFAT-calcineurin interaction, VIVIT, were found to inhibit NFAT activation at a DNA, mRNA, and protein as well as functionally, hereby influencing cell proliferation. An inert VIVIT analogue (VEET) appeared to be ineffective. In these studies, the activity profile of the new peptide antagonist VIVIT contrasted favorably to that of the conventional immunosuppressants, which may probably be linked to its more selective mode of action.
As NFAT responsive inflammatory genes, we selected IL-6, IL-8, MCP-1, and TGF-β1 because they all are deemed relevant to restenosis. IL-6 regulates SMC motility, IL-8 and MCP-1 are chemokines predominantly involved in neutrophil or monocyte recruitment,18 whereas TGF-β1 is a profibrogenic growth factor that can stimulate extracellular matrix formation and suppress extracellular matrix-degrading protease production.19 Coordinate inhibition of these and other targets, as effected by NFAT inhibition, is expected to be a very effective strategy to quench the inflammatory process of restenosis. Indeed, we found in this study that although the conventional inhibitors do interfere with NF-κB signaling, translating into undesired side effects, this was not observed with VIVIT.
Zernecke et al recently showed that SDF-1α blockade reduces neointimal formation by attenuating the recruitment of peripheral PDGF-R+ progenitor cells to the injured vessel wall.,20 SDF-1α is overexpressed by SMCs in response to vascular injury,21 and we now show that NFAT inhibition reduces PDGF-BB- and thrombin-induced SDF-1α expression, which may translate into a reduced progenitor cell recruitment to the injured vessel. Interestingly, sequence analysis revealed the presence of a potential NFAT response element (AGGAAACAC) at −919 upstream of the mouse SDF-1α transcription initiation site.
Evidence has been gained that calcineurin-NFAT signaling regulates cardiac hypertrophy in coordination with MAPKs.22 Moreover, Zhan et al have recently shown that ERK but not p38 or JNK plays a dominant role in PDGF-BB-stimulated vSMC proliferation.23 In our study, we consistently found ERK to be the key regulator of MAPK-dependent proliferation because p38 and JNK activation could barely be detected under the same treatment (data not shown). CsA and VIVIT did not affect ERK activation after PMA stimulation. Similar results were obtained with a selective NFAT inhibitor,24 implying that general intracellular signals other than calcineurin-NFAT will not be impaired by CsA or VIVIT. On PDGF-BB stimulation, ERK activation was accompanied by increased calcineurin-NFAT signaling and could be inhibited by VIVIT/CsA. The impaired ERK activation may thus directly result from NFAT inhibition. The fact that VIVIT (NFAT-specific) and CsA (calcineurin-specific) are both able to inhibit PDGF-BB-stimulated ERK activation and vSMC proliferation strongly points to a direct cross-talk between NFAT and ERK. In the next step, we addressed whether, vice versa, MEK-ERK signaling modulates NFAT activity. In other words, are both signaling partners aligned in a parallel or sequential fashion? p38 inhibition was reported previously to result in selective inactivation of NFAT in T cells.25 Both MEK-ERK and p38 inhibitors blunted PDGF-BB and thrombin-stimulated NFAT activation and vSMC proliferation, indicative of cross-talk between MEK-ERK and NFAT. Interestingly, both N-terminal dephosphorylation by calcineurin and C-terminal phosphorylation by ERK were suggested recently to lead to NFAT activation.26 The antiproliferative effect of U0126 and SB203580 in vSMCs may thus partly result from NFAT inactivation. Summarizing, NFAT seems to be essential for PDGF-BB-induced vSMC proliferation, and NFAT-ERK but not calcineurin-ERK act in concert as a parallel fashion in PDGF-BB-triggered vSMC proliferation (for an illustration of the proposed mode of action, see online supplement IV).
In conclusion, selective NFAT inhibition appears to be an effective strategy to coordinately quench the proinflammatory and proliferative responses that underlie restenosis. Compared with U0126 or CsA/FK506, the specific NFAT peptide inhibitor VIVIT displays a favorable therapeutic profile because it neither affects protein kinase C-mediated extracellular signal nor calcineurin-mediated NF-κB activity, but it is almost equally active in interdicting NFAT signaling. In addition, previously described small organic molecules as NFAT-calcineurin inhibitor were found to be toxic, which will limit their use in vivo.24 Drug-eluting stents coated with VIVIT (derivatives) or local administration for sustained peptide release may lead to more specific approaches in the antirestenosis therapy.
Sources of Funding
This study was financially supported by grants LFA5952 from Technology Foundation STW. This study was also supported by grants 2003T.201 from the Netherlands Heart Foundation. The authors belong to the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community’s Sixth Framework Program for Research Priority 1 (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254).
Original received December 1, 2005; final version accepted April 18, 2006.
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