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Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:182-189
Published online before print October 26, 2006, doi: 10.1161/01.ATV.0000251021.28725.e8
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Right arrow Catheter-based coronary interventions: stents
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:182.)
© 2007 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Pioglitazone Inhibits In-Stent Restenosis in Atherosclerotic Rabbits by Targeting Transforming Growth Factor-ß and MCP-1

Michael Joner; Andrew Farb; Qi Cheng; Aloke V. Finn; Eduardo Acampado; Allen P. Burke; Kristi Skorija; Wendy Creighton; Frank D. Kolodgie; Herman K. Gold; Renu Virmani

From CVPath Institute Inc (M.J., Q.C., F.D.K., E.A., A.P.B., W.C., K.S., R.V.), Gaithersburg, Md; the Center for Devices and Radiological Health, U.S. Food and Drug Administration (A.F.), Rockville, Md; Cardiac Unit (A.V.F., H.K.G.), Department of Internal Medicine, Massachusetts General Hospital, Boston, Mass.

Correspondence to Renu Virmani, MD, Medical Director, CVPath, International Registry of Pathology, 19 Firstfield, Road, Gaithersburg, MD 20878. E-mail rvirmani{at}cvpath.org


*    Abstract
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Objective— Although emerging data from preclinical and clinical studies suggests a reduction of in-stent restenosis with peroxisome proliferator-activated receptor (PPAR)-{gamma} agonists, the reduction of neointimal growth via anti-inflammatory mechanisms has not been explored.

Methods and Results— Hypercholesterolemic New Zealand White rabbits (n=45) received bilateral balloon-expandable stents implanted into atherosclerotic iliac arteries. Animals were randomized to oral pioglitazone 3 (low dose) or 10 mg/kg per day (high dose) started on the day of stent implantation; control rabbits received placebo. Tissue harvest was performed 28 days after stenting, and stented segments underwent histology, morphometry, immunostaining for macrophages, and scanning electron microscopy. In selected animals, stented arterial segments were placed in organoid culture for 48 hours, and the conditioned media was assayed for 23 different cytokines. There was a 21% reduction in neointimal area for high-dose pioglitazone treated versus placebo rabbits (P<0.005), which was associated with a significant reduction of neointimal macrophages. Analysis of conditioned media revealed an 82% and 74% reduction in the release of monocyte chemoattractant protein-1 (MCP-1) (P<0.007) and transforming growth factor (TGF)-ß1 (P<0.01), respectively, in stented segments from animals treated with 10 mg/kg per day pioglitazone versus placebo.

Conclusions— Oral pioglitazone suppresses in-stent neointimal growth by limiting local inflammatory pathways and may be useful as an adjunctive therapy in patients undergoing percutaneous interventions.

Although emerging data from preclinical and clinical studies suggests a reduction of in-stent restenosis with peroxisome proliferator-activated receptor (PPAR)-{gamma} agonists, the reduction of neointimal growth via anti-inflammatory mechanisms have not been explored. Oral pioglitazone suppresses in-stent neointimal growth by limiting local inflammatory pathways and may be useful as an adjunctive therapy in patients undergoing percutaneous interventions.


Key Words: inflammation • pioglitazone • rabbit • restenosis • stent


*    Introduction
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 *Introduction
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Drug-eluting stents (DES) are the standard of care for the percutaneous treatment of obstructive atherosclerotic coronary artery lesions. Whereas DES reduce restenosis rates in selected patients, in-stent restenosis rates remain high in certain patient populations and clinical settings. Inflammation plays an important role in the pathogenesis of atherosclerosis and restenosis, and recent treatment advances have focused on anti-inflammatory pathways. Experimental studies of stenting and analysis of human coronary stent implants show a strong correlation between stent-associated inflammation and neointimal growth.1,2

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate genes involved in important cellular functions such as lipid and glucose metabolism.3,4 Several types of PPARs have been described, including PPAR-{alpha}, PPAR-{gamma}, and PPAR-{delta}.5 PPAR-{gamma} is expressed by endothelial cells, macrophages, mononuclear cells and smooth muscle cells (SMCs) in atherosclerotic plaques.6,7 PPAR-{gamma} is known to regulate anti-inflammatory and anti-atherogenic responses as well as cell proliferation and migration.6,7,8,9,10

Thiazolidinediones (TDZs) are insulin sensitizing agents that act as ligands for PPAR-{gamma}. Three TZDs (troglitazone, rosiglitazone, and pioglitazone) have been developed for clinical use in type 2 diabetics. Pioglitazone has been shown to predominantly activate PPAR-{gamma} and weakly activate PPAR-{alpha};11 it is associated with inhibition of carotid intimal thickening in diabetic fatty rats12 and reduction of coronary restenosis after bare metal stenting in humans.13 Although a beneficial effect of pioglitazone after stent implantation has been reported before, the mechanism of this favorable outcome remains unknown.

To date, this is the first systematic evaluation of TZD effects after deployment of bare metal stents in an atherosclerotic animal model. The aim of this study was to investigate the impact of pioglitazone on neointimal growth after bare metal stent implantation and examine its anti-inflammatory effects on arterial wall.


*    Methods
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 *Methods
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This study was approved by the Institutional Animal Care and Use Committee of the Armed Forces Institute of Pathology and conformed to the position of the American Heart Association on use of animals in research.

Rabbit Model of Experimental Atherosclerosis
Forty-five New Zealand white Rabbits (3.0 to 4.0 kg; Hazeltov, Denver, Pa), age 3 to 4 months, were fed an atherogenic diet consisting of 1% cholesterol and 6% peanut oil (F4366-CHL, Bio-Serv, Inc, NJ) for 5 weeks. One week after initiating the high-cholesterol diet, iliac artery atheroma were created by balloon injury using a Fogarty catheter as previously described.14 General anesthesia was induced using ketamine (25 mg/kg), xylazine (2.5 mg/kg), and acepromazine (0.2 mg/kg) and was maintained with isoflurane. Animals were maintained on the same atherogenic diet for an additional 4 weeks, at which time the diet was switched to a reduced cholesterol diet (containing 0.025% cholesterol) for the remainder of the study. Animals were phlebotomized at 0, 7, 35, 56, and 91 days for measurements of serum cholesterol and triglycerides.

Stent Placement, Drug Treatment, and Tissue Harvest
Pre-mounted Penta stents (3.0 mm x18 mm; Guidant Corporation, Calif) were deployed at their nominal pressure (9 to 11 atm, 30-second balloon inflation) in each iliac artery under fluoroscopic guidance 63 days after study initiation. After stent implantation, angiography was performed to document vessel patency. All animals received aspirin, 40 mg/d orally, until euthanasia. In addition, heparin (150 IU/kg) was administered intra-arterially before catheterization procedures. Animals were randomized to either pioglitazone 3 mg/kg per day (low dose, n=15), pioglitazone 10 mg/kg per day (high dose, n=15), or placebo (n=15) for 28 days, given by oral gavage. Drug administration was started on the day of stenting and continued until euthanasia.

Twenty-eight days after stenting, animals were anesthetized, and underwent iliac artery angiography followed by euthanasia and perfusion-fixation. The stented arteries were embedded in methylmethacrylate. Sections were taken from the proximal, middle, and distal portions of the stent, and a 3-mm arterial segment just proximal and distal to the stents was also processed (to evaluate for edge effects). All sections were stained with hematoxylin-eosin, Movat pentachrome, and Carstair’s stain. To assess cellular proliferation, animals received bromodeoxyuridine (BrdU) before euthanasia, and the number of proliferating cells and neointimal cell density were analyzed as previously described.15 Sections were also stained with antibodies to RAM11 (Dako Corp, Calif) and HHF35 (Enzo, NY) to identify macrophages and smooth muscle actin, respectively.

Scanning Electron Microscopy
Selected stents from each treatment group (n=6) were processed for scanning electron microscopy (SEM) for evaluation of endothelialization as previously described.15

Organoid Culture
In selected animals (4 stents per treatment group), freshly harvested (nonfixed) arteries were segmented into stented and nonstented portions and incubated in serum-free DMEM at 37°C in humidified 5% CO2 and 95% air. The conditioned media was removed, concentrated, and analyzed for various cytokines using a Cytokine Array I (RayBiotech, Inc) kit. Cytokine quantification was performed by densitometry using Gel-Pro®-Analyser software (Version 4.5 for Windows, Media Cybernetics®). Values for each cytokine were derived by extrapolation to a standard curve and normalization to the density of a provided negative control. Corresponding values from individual cytokines were compared among groups within proximal, stented, and distal sections.

Cell Culture
J774 cells obtained from ATCC (Manassas, VA) were maintained in DMEM containing 10% fetal bovine serum. The cells were seeded (1x105 cells/cm2) in 6-well plates and incubated for 24 hours in DMEM containing 10% fetal bovine serum. The next day the cells were serum starved in DMEM containing 0.25% bovine serum albumin. Cells were then stimulated with tumor necrosis factor (TNF)-{alpha} in the presence of varying concentrations of pioglitazone (1 µmol/L, 5 µmol/L, and 10 µmol/L pioglitazone, respectively). Additional studies were performed with 15-deoxy-prostaglandin (PG) J2 (pure PPAR-{gamma} agonist) or bezafibrate (PPAR-{alpha} agonist) (Sigma-Aldrich, St. Louis, Mo) for 24 hours. Media were changed to serum-free DMEM containing 0.1% bovine serum albumin the next day and kept for 48 hours. After this incubation period, media were harvested for cytokine detection.

Cell Lysis and Western Blot Analysis
Cells were treated as previously described and solubilized in a modified Radioimmunoprecipitation (RIPA) lysis buffer containing 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS).16 SDS-Page and Western Blotting were performed as described.17

Antibodies and Reagents
Monoclonal antibodies against MCP-1 (clone 20521 D), polyclonal antibodies against TGF-ß1 (ab9758), and ß-actin (ab8227) were obtained from PharMingen, BD Biosciences (San Jose, Calif) and Abcam (Cambridge, Mass), respectively. Secondary peroxidase-conjugated goat anti-rabbit antibodies (ab6721) were obtained from Abcam and peroxidase-conjugated rabbit anti-mouse antibodies (AP124P) from Chemicon (Temecula, Calif), respectively. Pioglitazone powder was kindly provided by Takeda Pharmaceuticals North America (Lincolnshire, Ill).

Data Analysis
All arterial segments were examined with the observer blinded to the treatment group. Stents were evaluated for thrombus formation, inflammation, and cellular proliferation. Computerized planimetry was performed (IP Laboratory software for Macintosh, Scanalytics, BD Biosciences, Rockville, Md) on all stented sections as previously described.15 The neointimal cell proliferation index (percent proliferating cells) was defined as the ratio of BrdU-positive neointimal cells to total neointimal cells. Fibrin was identified on H&E and Carstair’s stained sections and semiquantified.15 The percentage of the neointima occupied by RAM11-positive macrophages was determined by digital color (intensity) threshold imaging (IP Laboratory software for Macintosh, Scanalytics, BD Biosciences, Rockville, Md).

Statistical Analysis
Numerical data are presented as mean±SD. Continuous variables were first checked for normal-distribution by Shapiro-Wilk goodness-of-fit test and analysis of variance (ANOVA) or Wilcoxon rank-sum test performed where suitable. Dunnett’s post hoc adjustment was used to determine significant differences, and P<0.05 was considered statistically significant. The neointimal and plaque area were analyzed with linear regression to derive a slope, intercept, and correlation coefficient to determine relations.


*    Results
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 *Results
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Ninety stents were implanted in the iliac arteries of 45 rabbits. Early deaths included 1 animal from each group caused by arterial dissection within 24 hours after stent implantation. One rabbit from the placebo group could not be stented because of bilateral total occlusion. An additional rabbit from the 3 mg/kg per day pioglitazone group was excluded because this animal demonstrated liver failure likely attributed to excessive circulating cholesterol (>3000 mg/dL). The remaining rabbits appeared healthy with no signs of drug toxicity identified by weight loss, lethargy, or icterus. Analysis was therefore, performed on the stents from the remaining 40 rabbits to include: 3 mg/kg per day pioglitazone (n=13), and 10 mg/kg per day pioglitazone (n=14), and placebo rabbits (n=13) (Figure 1).


Figure 1
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  		Figure 1. Study flow chart.

Repeat angiography at euthanasia showed widely patent stents in all treatment groups without stent migration or aneurysm formation. Stent to artery ratios were {approx}1:1.2 and were similar among groups.

Circulating cholesterol levels were maximal at 35 days (2518±1204 mg/dL for the 10 mg/kg per day group, 2192±932 mg/dL for the 3 mg/kg per day group and 1787±1133 mg/dL for the placebo group, P=NS), whereas serum triglyceride levels were essentially unchanged from baseline (180±127 mg/dL for the 10 mg/kg per day group, 125±51 mg/dL for the 3 mg/kg per day group and 158±83 mg/dL for the placebo group, P=NS). At the time point of stent implantation and euthanasia, serum cholesterol and triglyceride levels were not significantly different among groups.

Morphometry
Eighteen stents from 10 mg/kg per day pioglitazone treated rabbits were compared with 16 stents from 3 mg/kg per day pioglitazone-treated animals and to 16 stents from the placebo group. There were no differences among treatment groups with respect to external elastic lamina area, stent area, and plaque area (Table 1). Arterial injury was mild and similar in all treatment groups (mean injury scores <1) (data not shown). Treatment with 10 mg/kg per day pioglitazone resulted in a significant 21% reduction of neointimal area and a 32% reduction in intimal thickness compared with placebo (Table 1, Figure 2). Neointimal area and thickness in the 3 mg/kg per day group were similar to placebo. Percent stent stenosis was significantly reduced in animals treated with 10 mg/kg per day pioglitazone compared with placebo (32.8±6.2% versus 39.5±10.3%, P<0.04); the 3 mg/kg per day pioglitazone was similar to placebo (Table 1).


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  		TABLE 1. Cross-Sectional Vessel Areas and Neointimal Response Comparison Between Treatment Groups


Figure 2
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  		Figure 2. A, Iliac artery morphology at 28 days following stent placement is shown at low power (20x) and high power (200x) magnification after staining with Movat Pentachrome. Note, neointimal coverage of stent struts is least in the 10 mg/kg per day vs 3 mg/kg per day pioglitazone and placebo treated animals. B, The bar graph shows a significant reduction in neointimal area (mm2) between placebo and 10 mg/kg per day pioglitazone treated rabbits, respectively.

Quantitative assessment of fibrin deposition, inflammation, and cellular proliferation were similar among groups, whereas neointimal cell density was significantly reduced only at the higher 10 mg/kg per day pioglitazone group (Table 2). Overall, there was 80% to 90% luminal surface coverage of endothelial cells in all stents observed by SEM, with some struts in the high dose pioglitazone group showing accumulated fibrin with scattered inflammatory cells. The nonendothelialized stent strut surfaces predominantly contained macrophages and minute platelet aggregates.


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  		TABLE 2. Comparison of Inflammatory Reaction and Cellular Proliferation Between Treatment Groups

The relationship of underlying plaque size to neointimal growth showed a significant correlation in placebo animals (r2=0.25, P<0.05). In contrast, neointimal thickening was attenuated in animals treated with 10 mg/kg per day pioglitazone, which poorly correlated with plaque area (r2=0.03, P=NS) despite the greater plaque size in these animals.

Neointimal Macrophage and SMC Content
Neointimal macrophages identified by RAM11 and expressed as a percentage of total neointima were significantly decreased (47%) in the 10 mg/kg per day group compared with placebo, whereas the reduction in the 3 mg/kg per day group did not reach statistical significance (Table 2, Figure 3). Neointimal smooth muscle cell content as identified by specific {alpha}-actin staining revealed a reduction of neointimal SMCs after pioglitazone treatment only in animals treated with 10 mg/kg per day pioglitazone (please see http://atvb.ahajournals.org).


Figure 3
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  		Figure 3. Photomicrographs showing macrophages after staining with antibody against RAM11 in placebo (A), 3 mg/kg per day pioglitazone (B), and 10 mg/kg per day pioglitazone-treated (C) animals. Bar graph (D) showing a gradual reduction of neointimal macrophages after treatment with low-dose and high-dose pioglitazone, respectively. Only high-dose pioglitazone treatment resulted in significant reduction of neointimal macrophages.

Cytokine Array of Organoid Cultures and Stimulated J774 Mouse Macrophages
Analysis of conditioned media from stented arteries maintained in organoid culture confirmed the release of several immunogenic and chemoattractant cytokines to include (IL-6, IL-9, IL-10, IL-12) and growth factors (GCSF, granulocyte macrophage-colony stimulating factor), RANTES, sTNFRI, TNF-{alpha}) irrespective of treatment group. In the 3 and 10 mg/kg per day pioglitazone groups, however, there was a selective reduction in MCP-1 and TGF-ß1 compared with animals given placebo (Figure 4).


Figure 4
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  		Figure 4. Cytokines were analyzed in conditioned media of iliac arteries and quantified by densitometry. Significant reduction was seen for MCP-1 (A), TGF-ß1(B) and TGF-ß (C), respectively, in rabbits treated with 3 mg/kg per day and 10 mg/kg per day pioglitazone compared with placebo (D).

To further explore the dose-response relationship of pioglitazone on cytokine release, cultured J774 mouse macrophages were stimulated with TNF-{alpha} with and without varying concentrations of pioglitazone and the conditioned media assayed. Concentrations of pioglitazone between 1 µmol/L and 10 µmol/L were effective in reducing MCP-1 levels with a maximum decrease of 92% at the highest (10 µmol/L) dose (please see http://atvb.ahajournals.org). The inhibitory effect on MCP-1 was less pronounced in the presence of the natural PPAR-{gamma} agonist 15-deoxy-PG J2, whereas the PPAR-{alpha} agonist, bezafibrate was without effect (please see http://atvb.ahajournals.org).

Western Blot Analysis
Immunoblotting for MCP-1 and TGF-ß1 revealed markedly attenuated protein expression levels in J774 macrophages after treatment with 10 µmol/L pioglitazone. The reduction seen after treatment with 5 µmol/L pioglitazone was to a lesser degree (please see http://atvb.ahajournals.org).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, oral pioglitazone (PPAR-{gamma} agonist) was effective in reducing neointimal growth after stenting in an animal model of atherosclerotic disease. A dose-response effect with a dependent threshold is suggested as only the higher 10 mg/kg per day dose was associated with a significant reduction of in-stent neointimal area and restenosis while the benefits of 3 mg/kg per day were limited. The reduction in neointimal growth attributed to 10 mg/kg per day pioglitazone was accompanied by decreased numbers of neointimal macrophages and SMCs within the restenotic lesion. Importantly, oral pioglitazone did not appear to interfere with arterial healing post stenting (similar degrees of endothelialization and accumulated fibrin compared with placebo animals) and was without signs of systemic toxicity. Cytokine analysis of conditioned media from 48-hour organoid cultures showed a selective decrease in the levels of MCP-1 and TGF-ß in stented arteries from animals treated with 10 mg/kg per day pioglitazone when compared with animals given placebo. The selective dose-dependent reduction of MCP-1 and TGF-ß by pioglitazone was further confirmed in TNF-{alpha} stimulated J774 cells in culture. Collectively, these studies suggest that threshold concentrations of oral pioglitazone may be clinically effective in reducing in-stent restenosis, in part mediated by its inhibitory effects on selective proatherogenic cytokines. Taken further, the mechanisms supported in the present study may help explain the reduction in neointimal volume after coronary stent implantation in a recent clinical trial of non-diabetic patients treated with pioglitazone.13

PPAR-{gamma} Agonists and Arterial Inflammation
Several lines of evidence suggest that PPAR-{gamma} agonists possess anti-inflammatory properties that may be responsible for in-stent neointima suppression. In a recent study by Seki et al, human macrophages exposed to PPAR-{gamma} activators demonstrated a significant reduction of MCP-1 with diminished adhesion to endothelial monolayers.18 MCP-1, a member of the CC chemokine subfamily, has been identified as a specific chemotactic factor for monocytes and/or macrophages in various pathologic disorders.19,20 Secreted by macrophages, endothelial and vascular SMCs, MCP-1 importantly contributes to the early inflammatory response associated with arterial injury in normolipemic and hyperlipemic animal models.21–23 It is also reported that MCP-1 is markedly increased in atherosclerotic lesions24,25 and that its prolonged production is associated with higher risk of restenosis after angioplasty in humans.26,27

As demonstrated in the present study, diminished MCP-1 release in stented atherosclerotic arteries is likely a key factor of reduced neointimal growth following pioglitazone treatment. The reduction of MCP-1 associated with pioglitazone appears to be selective for the TNF-{alpha} pathway because stimulation of J774 macrophages with interferon-{gamma} results in a reciprocal increase in MCP-1 (data not shown). However, the inhibitory property of pioglitazone does not appear to be specifically targeted at TNF-{alpha} receptor downregulation because J774 cells were stimulated and treated with drug concomitantly. Studies in the literature, in contrast, do support the notion that pioglitazone lowers the expression of TNF-{alpha} receptors. In a report by Hofmann et al, in a mouse model of obesity-linked diabetes, TNFR2 receptor mRNA in the fat of insulin-resistant mice was reduced {approx}25% after pioglitazone treatment at doses of 20 mg/kg per day.28

Previous animal studies of the effects of thiazolidinediones on inflammation of the arterial wall are limited. In Watanabe Heritable Hyperlipidemic rabbits, oral troglitazone at 100 mg/kg per day was shown to reduce the extent of luminal macrophages.29 In vitro demonstration of attenuated inflammatory cell proliferation and migration by PPAR-{gamma} agonists was associated with neointimal growth suppression in hypercholesterolemic rabbits after balloon angioplasty.29 Another study reported a significant decrease of TNF-{alpha} production in fatty rats following pioglitazone treatment at 3 mg/kg per day. However, no data on neointimal growth were provided in this study.30 In this regard, the present study is an extension of previous studies and is to our knowledge the first to demonstrate the efficacy of PPAR activators on suppression of neointimal growth after stenting in a model of pre-existing atheroma. This evaluation is unlike the majority of previous studies where nonatherosclerotic (normal) arteries are typically stented. In the present model, atherosclerotic arteries were rich in resident inflammatory cells and extracellular matrix before catheter-based intervention, which more closely resembles the clinical setting. The use of an atherosclerotic animal model may be particularly useful to study novel interventions that directly target inflammatory responses to stenting.

PPAR-{gamma} Agonist and Smooth Muscle Responses
The rationale that pioglitazone directly influences SMC proliferation is certainly valid since the reduction in inflammatory cytokines may not entirely explain the anti-restenotic effect of PPAR-{gamma}. In this respect, we investigated whether pioglitazone alters neointimal SMC content in representative tissue sections from stented atherosclerotic iliac arteries using a monoclonal antibody directed against smooth muscle specific actin together with SMC density measurements. Antibody staining revealed a marked decrease in neointimal SMC content in stented atherosclerotic arteries in rabbits treated with 10 mg/kg per day pioglitazone. A dose-dependent reduction in neointimal SMC density was also observed with increasing concentrations of pioglitazone with statistical significance achieved only in rabbits treated at the higher 10 mg/kg per day dose.

Based on the literature, direct influence of PPAR-{gamma} agonist on SMCs is debatable as some studies report profound anti-proliferative and anti-migratory effects of pioglitazone on human and rat vascular SMCs,31–33 whereas others suggest an absence of response.34 The discrepancy may remain in the fact that thiazolidinediones may exert anti-proliferative effects independent of PPAR-{gamma},35 specifically at higher concentrations. For example, high-dose pioglitazone has been shown to weakly activate PPAR-{delta}, which may add to inhibition of SMC proliferation and migration.36 Based on the present findings, pioglitazone, particularly at higher concentrations may directly influence SMC proliferation, which might in part reflect the observed decrease in neointimal growth. The significance of these findings, however, better serve as a proof of concept, without necessarily aiming at finding effective dosages for application in humans.

PPAR-{gamma} Agonist and TGF-ß
TGF-ß is a common pathogenic indicator of atherosclerosis and in-stent restenosis because it is a potent inducer of extracellular matrix production. In the present study, pioglitazone selectively attenuated the release of TGF-ß, which in turn may limit the synthesis of extracellular matrix proteins critical to increased neointimal growth. In human mesangial cells, pioglitazone attenuates TGF-ß-induction of fibronectin synthesis,37 which may have relevant implications to the present study because fibronectin serves as a provisional matrix for inflammation and subsequent wound repair. In addition, macrophages produce and are highly responsive to TGF-ß, thereby regulating SMCs depending on the local cytokine environment.38 Upregulation of the TGF-ß system has been reported after balloon catheter injury39 and pharmacological inhibition significantly attenuated neointimal growth and remodeling after stenting.40 These findings contribute to the notion that inhibition of the TGF-ß pathway results in reduced neointimal growth following arterial injury. However, others report that decreased TGF-ß signaling may play a pathogenic role in reinforcing the proliferation and migration of vascular SMCs. Because of these inconsistent findings of TGF-ß on vascular SMCs, further studies are needed to clarify the influence of TGF-ß in arterial wound repair.

In summary, oral pioglitazone treatment in 28-day rabbits with pre-existing atheroma caused a significant decrease in the amount of in-stent neointimal growth when compared with animals receiving placebo. As applied in the present study, daily doses of the drug were well-tolerated with no evidence of systemic toxicity. A threshold concentration of pioglitazone is likely necessary for the reduction of neointimal growth because beneficial effects were specifically appreciated at higher doses. The marked decrease in neointimal macrophages, SMCs and relevant pro-atherogenic inflammatory cytokines are likely the underlying cause of neointimal suppression associated with oral pioglitazone. The mechanistic findings in our study together support the hypothesis that thiazolidinediones may reduce restenosis rates associated with percutaneous interventions and may have favorable effects on de novo atherosclerotic plaques.


*    Acknowledgments
 
We thank Rosalind Mathew, Lila Adams, Hedwig Avallone, and Hazel M. Jenkins (CVPath) for their excellent technical assistance.

Sources of Funding

This work was supported in part by research grants from Takeda Pharmaceuticals North America (Lincolnshire, Ill) and the International Registry of Pathology. Company sponsored research support was received from Medtronic AVE, Guidant, Abbott, W.L. Gore, General Electric, diaDexus, Takeda, Atrium Medical Corporation, Invatec, ev3, TopSpin Medical (Israel) Ltd., Boston Scientific, NDC Cordis Corporation, Novartis, Paracor Medical, Inc., C.R. Bard Inc, Orbus Medical Technologies.

Disclosures

R.V., F.K., and A.B. received company sponsored research support from Medtronic AVE, Guidant, Abbott, W.L. Gore, Atrium Medical Corporation, Boston Scientific, NDC Cordis Corporation, Novartis, Orbus Medical Technologies, Biotronik, BioSensors, Alchimer, and Terumo, and are consultants for Medtronic AVE, Guidant, Abbott Laboratories, W.L. Gore, Terumo, and Volcano Therapeutics Inc.


*    Footnotes
 
Original received May 16, 2006; final version accepted October 11, 2006.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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