Propylthiouracil, independent of its antithyroid effect ... - Springer Link

Basic Res Cardiol (2010) 105:19–28 DOI 10.1007/s00395-009-0045-z

ORIGINAL CONTRIBUTION

Propylthiouracil, independent of its antithyroid effect, promotes vascular smooth muscle cells differentiation via PTEN induction Wei-Jan Chen Æ Jong-Hwei S. Pang Æ Kwang-Huei Lin Æ Dany-Young Lee Æ Lung-An Hsu Æ Chi-Tai Kuo

Received: 16 December 2008 / Revised: 22 June 2009 / Accepted: 6 July 2009 / Published online: 25 July 2009 Ó Springer-Verlag 2009

Abstract Propylthiouracil (PTU), independent of its antithyroid effect, is recently found to have an antiatherosclerotic effect. The aim of this study is to determine the impact of PTU on phenotypic modulation of vascular smooth muscle cells (VSMCs), as phenotypic modulation may contribute to the growth of atherosclerotic lesions and neointimal formation after arterial injury. Propylthiouracil reduced neointimal formation in balloon-injured rat carotid arteries. In vitro, PTU may convert VSMCs from a seruminduced dedifferentiation state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of SMC differentiation marker contractile proteins, including calponin and smooth muscle (SM)myosin heavy chain (SM-MHC). Transient transfection studies in VSMCs demonstrated that PTU induced the activity of SMC marker genes (calponin and SM-MHC) promoters, indicating that PTU up-regulates these genes expression predominantly at the transcriptional level.

Electronic supplementary material The online version of this article (doi:10.1007/s00395-009-0045-z) contains supplementary material, which is available to authorized users. W.-J. Chen (&) D.-Y. Lee L.-A. Hsu C.-T. Kuo First Cardiovascular Division, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Fu-Shin Road no 5, Kwei-Shan, Tao-Yuan 333, Taiwan e-mail: [email protected] J.-H. S. Pang Graduate Institute of Clinical Medical Sciences, Tao-Yuan, Taiwan K.-H. Lin Department of Biochemistry, Chang Gung University, Tao-Yuan, Taiwan

Furthermore, PTU enhanced the expression of PTEN and inhibition of PTEN by siRNA knockdown blocked PTUinduced activation of contractile proteins expression and promoter activity. In the rat carotid injury model, PTU reversed the down-regulation of contractile proteins and up-regulated PTEN in the neointima induced by balloon injury. Propylthiouracil promotes VSMC differentiation, at lest in part, via induction of the PTEN-mediated pathway. These findings suggest a possible mechanism by which PTU may contribute to its beneficial effects on atherogenesis and neointimal formation after arterial injury. Keywords Atherosclerosis Differentiation Neointimal formation Propylthiouracil PTEN Vascular smooth muscle cells

Introduction Neointimal formation is a common pathological lesion in diverse cardiovascular diseases, such as atherosclerosis and restenosis after balloon angioplasty. Phenotypic modulation of vascular smooth muscle cells (VSMCs) from a differentiated (contractile) to a dedifferentiated (synthetic, noncontractile) state plays a crucial role in the pathogenesis of neointimal formation [20–22]. In the neointima induced by balloon injury, VSMCs modulate phenotype and exhibit marked changes in morphology, proliferation rate, and differentiation marker proteins [6, 20–22, 25]. The increase in VSMCs proliferation and decrease in differentiation marker proteins including smooth muscle a-actin (SM-aactin), calponin, and smooth muscle myosin heavy chain (SM-MHC) expression constitute the main feature of neointimal formation in all balloon-injured animal models [20–22]. When isolated from adult rat aorta and grown in

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conventional culture conditions, VSMCs may undergo a similar phenotypic transit from a differentiated to a dedifferentiated state [3, 8, 13, 19]. Serum in the culture medium has been recognized as a key factor to induce VSMC dedifferentiation [3, 8, 13, 19]. Because blood serum may contact directly with VSMCs in the medial layer when endothelial cells are denuded by balloon injury, this study utilized a serum deprivation and restoration culture model to mimic phenotypic reversion in vivo. Propylthiouracil (PTU) is used routinely in the treatment of hyperthyroidism. Propylthiouracil exerts its antithyroid effect by inhibiting iodide oxidation, monoiodotyrosine iodination and coupling steps in thyroxine production, as well as the peripheral conversion of thyroxine (T4) to triiodothyronine (T3) [15]. In addition to its antithyroid effect, PTU has been shown to inhibit the development of atherosclerosis in the aortas of rabbits fed with a high cholesterol diet [1]. The antiatherosclerotic effect of PTU is attributed to its inhibitory effect on VSMC proliferation and migration [1, 2]. Because VSMC proliferation and phenotypic modulation are the main causes of neointimal formation after arterial injury, it is hypothesized that PTU may have an effect on neointimal formation and phenotypic modulation of VSMCs. The aim of this study is, first, to investigate whether PTU has a protective effect on neointimal formation in balloon-injured rat carotid arteries. The second aim is to determine whether PTU exerts a direct action on modulating VSMC differentiation, focusing on the expression of contractile proteins. Third, the underlying signal transduction pathway responsible for the modulation was also investigated.

Materials and methods Rat model of balloon injury The animal study was followed as described previously [2] and approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital. Adult male Sprague-Dawley rats weighing 350 to 400 g were anesthetized by intraperitoneal injection of ketamine 100 mg/kg and xylazine 5 mg/kg. Angioplasty of the left external carotid artery was performed using an inflated 2F Forgarty embolectomy catheter. Propylthiouracil (5 mg/100 g/day) was administered daily by gavage, and balloon injury was performed on day 4. Propylthiouracil treatment was continued until rats were euthanized at 7 or 14 days after balloon injury. The uninjured and injured carotid arteries were removed and frozen for confocal immunohistochemical or Western blot analysis.

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Basic Res Cardiol (2010) 105:19–28

Cell culture Rat VSMCs were prepared by enzymatic digestion of the thoracic aortic media from adult Sprague-Dawley rats and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as described [1, 2]. Cells used in all experiments were between the 5th and 10th passage and were cultured on conventional uncoated dishes. Most chemicals were purchased from Sigma (St. Louis, MO). Propylthiouracil was dissolved in dimethylsulphoxide (DMSO). Cells were treated with vehicle (DMSO) or drugs as indicated in the figure legends. Immunohistochemistry and cytochemistry Immunohistochemistry and cytochemistry were performed using anti-calponin (Dako, Carpinteria, CA), anti-SMMHC (SM2, Seikagaku, Tokyo, Japan), and anti-PTEN (Cell signaling, Beverly, MA) antibodies as primary antibodies followed by FITC (green color) or Cy3 (red color, Chemicon, Temecula, CA)-conjugated secondary antibody and were visualized by confocal immunofluorescent microscope. Western blot analysis For Western blot, immunoblotting was performed using anti-SM-a-actin, anti-tubulin antibodies (Santa Cruz, Delaware Avenue, CA), anti-calponin (Dako), anti-SM-MHC (SM2, Seikagaku), and anti-PTEN (Cell signaling) as primary antibodies. Bolts were developed with 4CN PLUS (Perkin Elmer, Boston, MA) for chromogenic detection of horseradish peroxidase-conjugated secondary antibody or Western LightningTM (Perkin Elmer) for chemiluminescent detection. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) Total cellular RNA was extracted using TRIzol reagent (Life Technologies, Rockville, MD) and real-time quantitative RT-PCR was performed, as described previously [11]. Oligonucleotide sequences for the specific primers used in the PCR reaction are shown in the supplementary online Table. Transient transfection and luciferase Assays The transcriptional regulation of contractile proteins gene was assessed with a luciferase reporter gene expressed under the control of rat SM-MHC (-4,200 to ?11,600 bp) promoters within the pGL3-basic vector as described (generous gift of Dr. Gary K. Owens, University of


Virginia) [4]. For preparation of reporter construct in rat calponin gene, the 50 -flanking DNA from the rat calponin gene was created by PCR from VSMCs DNA using a forward primer (50 -GACGCACGCGTTGAGGCAAGCACTG TCCATTC-30 ) and a reverse primer (50 -TCACTCTCGA GTCGGTTAAAGTGTGCGGAAGA-30 ) (GenBank accession no. AF123268). The resulting PCR product was approximately 578 bp. The insert was then digested and inserted in the reporter vector pGL3 Basic (Promega, Madison, WI) using the restriction sites MluI and XhoI. For transient transfection assays, VSMCs at 50–60% confluence were transfected with indicated plasmids using a liposome technology (FuGENE 6 [Roche, Indianapolis]) according to manufacturer’s protocol. The transfection efficiency by this method was approximately 60–70%. After an additional 36 h, samples were sent for measurement of luciferase activity with an assay system (Dual-LuciferaseÒ Reporter, Promega). Luciferase activities were measured with a luminometer (Luminoskan TL PMS, Thermo Labsystems) and normalized by cellular protein concentrations.

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by unpaired t test. For multiple groups, one-way ANOVA with post hoc Scheffe test was used to compare data among groups. Correlations between variables were calculated by Pearson’s coefficient. A value of P 2 0.05 was considered statistically significant.

Results Effect of PTU on neointimal formation The effect of PTU on neointimal formation by using a rat carotid artery balloon-injury model was examined. Treatment with PTU reduced neointimal formation, as indicated by intima/media area ratio, in the arteries 14 days after balloon injury (Fig. 1a and b). The contribution of the antithyroid effect of PTU to the reduction of neointimal formation may be neglected by normal thyrotropin (TSH) levels in all rats treated with PTU for 14 days (1.8 ± 0.9 lIU/mL, normal range: 0.3–5.5 lIU/mL). Serum PTU concentration after 14-day treatment was 3.21 ± 1.68 lg/mL.

PTEN knockdown using small interfering (si) RNA Morphological changes induced by PTU in VSMCs Chemically synthetic siRNA for PTEN and control siRNA were purchased from Dharmacon (Lafayette, CO, catalog number J-080104). The siRNAs were transfected into VSMC using DharmaFECT 1 (Dharmacon) according to the manufacturer instructions.

Serum PTU and TSH levels were measured by HPLC and chemiluminescence, respectively, as described previously [1, 2].

Adult rat VSMCs (70–80% confluence) cultured in 10% FBS-containing medium rapidly dedifferentiate to a synthetic phenotype, characterized by a flattened morphology (Fig. 2a). These cells transformed to a highly elongated spindle shape after the addition of PTU to FBS-containing medium (Fig. 2b–d). Furthermore, the PTU-treated VSMCs formed dense, multilayered cell bundles that appeared as organization of VSMCs in the vessel wall (Fig. 2d). These changes induced by PTU were evident 8 h after addition of PTU and were completed by 24 h.

Statistical analysis

Effects of PTU on contractile proteins expression

Mean and standard deviation (SD) were used to describe the data. Differences between two groups were determined

To explore whether PTU induces the differentiated phenotype, the effects of PTU on the expression of

Fig. 1 Effect of PTU on neointimal formation in rat carotid arteries after balloon injury. a Representative cross-section of the injured carotid arteries in control and PTU-treated rats 14 days after balloon injury. b Quantitative analysis of neointimal formation by measuring intima/media area ratios in injured carotid arteries of control and

PTU-treated rats. Each value was normalized to the levels of control, which was set at 1.0, and represented the mean ± SD of five rats. P \ 0.05; *: the different symbols represent the significant differences among groups

Serum PTU and thyrotropin (TSH) levels

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Fig. 2 Phase-contrast photomicrographs showing rat VSMCs cultured in DMEM with 10% FBS at basal (a), 8 h (b), 16 h (c), and 24 h (d) after the addition of 5 mmol/L propylthiouracil (PTU). The arrow indicates the organization of VSMCs

contractile proteins (SM-a-actin, calponin, and SM-MHC) were determined. Previous studies have demonstrated that VSMCs in the quiescent media display their differentiated characters, with increased expression of contractile proteins [3, 8, 13, 19]. Consistently, using Western blot, the expression of calponin and SM-MHC in VSMCs increased in the serum-deprived condition compared with cells treated with serum. However, this difference could not be found in the expression of SM-a-actin, an observation consistent with previous report showing that serum has no effect on SM-a-actin expression [20]. It is likely that SM-a-actin is the earliest marker of SMC differentiation, and its expression is not completely suppressed during the dedifferentiation process in subcultured VSMCs [20]. Administration of PTU dose-dependently induced the expression of calponin and SM-MHC in serum-treated VSMCs. In comparison, administering PTU into serum-treated VSMCs did not affect SM-a-actin expression at any concentration (Fig. 3a). The effect of PTU on contractile proteins (SM-a-actin, calponin, and SM-MHC) expression at the mRNA level in real-time RT-PCR paralleled that of PTU on the expression of their corresponding proteins in Western blot (Fig. 3b). These findings indicate that PTU may modulate VSMCs from the serum-induced dedifferentiated state to the differentiated state as indicated by increased contractile proteins

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expression. Furthermore, exogenous supplementation of thyroid hormone did not abrogate the response induced by PTU, suggesting that the effect of PTU on contractile protein expression is unrelated to its extra-thyroidal effects (Fig. 3c). Because PTU was dissolved in DMSO, maximal concentration of DMSO (0.2%) in culture medium did not affect contractile protein expression (Fig. 3c). Immunocytochemical localization of contractile proteins In agreement with the results obtained from Western Blot, immunocytochemistry showed abundant expression of calponin and SM-MHC in the cytoplasm of VSMCs during the serum-deprived condition and in PTU-treated VSMCs. In contrast, little expression was observed in serum-treated VSMCs (Fig. 4). As described previously, in addition to quantitative changes in contractile proteins expression, phenotypic modulation of VSMCs is associated with other qualitative changes, particularly reorganization of contractile proteins within the cell [24]. Immunocytochemistry showed the organization of calponin into randomly distributed bundles throughout the cell in serum-deprived and PTU-treated VSMCs (Fig. 4). However, this change could not be demonstrated in SM-MHC-staining cells (Fig. 4).


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Fig. 3 Effect of PTU on contractile proteins expression in VSMCs a After 48 h of serum deprivation, VSMCs at 60–70% confluence were treated with the indicated conditions for 36 h. The expression of SM-a-actin, calponin, SM-MHC (SM2), PTEN, and tubulin was evaluated by Western blot. The relative expression levels of each protein were quantified by densitometry and normalized to the levels under quiescent condition, which was set at 1.0. Each value represents the mean ± SD of four independent experiments. b Total RNA was prepared from VSMCs treated with indicated conditions as described in Fig. 2a. Real-time RT-PCR was performed as described in ‘‘Methods’’. Each value represents the mean ± SD of four independent experiments. P \ 0.05; *, , : the different symbols represent the significant differences among groups. c Growtharrested VSMCs were treated with indicated conditions for 36 h. T3 = triiodothyronine

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Fig. 4 Confocal immunofluorescent micrographs show the distribution of contractile proteins in VSMCs. Rat VSMCs at 50% confluence were treated with the indicated conditions for 48 h. Immunocytochemical analysis was performed using antibodies against calponin or SM-MHC (SM2), and FITC-conjugated secondary antibodies. Background image obtained in the absence of primary antibody is also shown

Fig. 5 Propylthiouracil increased transcriptional activity of contractile proteins promoter–reporter constructs. Growth-arrested VSMCs were transfected with indicated plasmids containing contractile proteins promoter and treated with indicated conditions for 36 h. The luciferase activity was assayed as described in ‘‘Methods’’. Each

value (mean ± SD; n = 4) is expressed as a fold of luciferase activity relative to the control condition. a Calponin, b SM-MHC; P \ 0.05; *, : the different symbols represent the significant differences among groups

Effects of PTU on contractile proteins gene transcription

and apoptosis [5, 9, 10, 17, 18, 23]. Recent study from our laboratory demonstrated that PTU inhibited VSMC proliferation and migration via induction of PTEN-mediated pathway [1]. This study further investigated whether PTU induces VSMC differentiation through a PTEN-related mechanism. Consistent with our previously reported data, PTU up-regulated the expression of PTEN dose-dependently by Western blot and RT-PCR at the protein and mRNA levels, respectively (Fig. 3a and b). To further document the involvement of PTEN in PTU-induced contractile proteins expression, this study used siRNA to inhibit PTEN signaling in VSMCs. Western blot analysis showed that transfection of VSMCs with PTEN siRNA attenuated PTU-induced activation of calponin (Fig. 6a). The transfection efficiency of PTEN siRNA into VSMCs was confirmed by the lack of PTEN expression in these cells (Fig. 6a). In co-transfection experiments, cells were co-transfected with plasmids containing contractile proteins promoter region and PTEN siRNA. As expected, the

The next experiments were designed to determine whether the up-regulation of contractile proteins in PTU-treated VSMCs is caused by increased transcriptional activity of their genes. Accordingly, two recombinant plasmids containing rat calponin and SM-MHC promoters linked to luciferase reporter gene were transfected into VSMCs. As shown in Fig. 5, cells treated with PTU showed a dosedependent increase in calponin and SM-MHC promoter activity compared with control cells. These experimental results were consistent with those obtained from Western blot and RT-PCR. Involvement of PTEN in PTU-induced contractile proteins expression The tumor suppressor gene, PTEN, is thought to play a critical role in cell proliferation, differentiation, migration,

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Fig. 6 Effect of PTEN on PTUinduced contractile proteins expression. a After 48 h of serum deprivation, VSMCs at 50–60% confluence were transfected with indicated siRNAs and treated with or without 5 mmol/L PTU in 10% FBS-containing media for 36 h. The expression of calponin, PTEN, and tubulin was evaluated by Western blot. The data are representative of three independent experiments. P \ 0.05; *, : the different symbols represent the significant differences among groups. b Growth-arrested VSMCs were cotransfected with indicated plasmids containing contractile proteins promoter and siRNAs followed by treatment with or without 5 mmol/L PTU in 10% FBScontaining media for 36 h. Each value (mean ± SD; n = 4) is expressed as a fold of luciferase activity relative to the control condition. The symbol (*) represents the significant difference. c Rat VSMCs were transfected with PTEN siRNA and treated with or without 5 mmol/L PTU in 10% FBScontaining media for 48 hours. Green fluorescence indicates PTEN expression, whereas red staining represents cells expressing SM-MHC (SM2) in confocal microscope. Relations between density staining for PTEN and SM-MHC using scatter plot were shown

up-regulation of contractile proteins promoter activity in PTU-treated cells was dose-dependently suppressed by co-transfection of PTEN siRNA (Fig. 6b). These experimental findings provide further evidence that PTEN is

required for transcriptional regulation of contractile protein genes by PTU. In VSMCs transfected with PTEN siRNA, confocal immunocytochemical analysis showed a fair correlation

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Fig. 7 Effect of PTU on contractile proteins and PTEN expression in rat carotid arteries after balloon injury. A Confocal immunohistochemical analysis of calponin, SM-MHC (SM2), and PTEN at 7 or 14 days after carotid injury. Propylthiouracil up-regulates PTEN (a, b), calponin (c, d), and SM-MHC (SM2) (e, f) in the neointima induced by balloon injury. Arrows denote the internal elastic lamina, and the arrowheads denote the external elastic lamina. B The levels of calponin, SM-MHC (SM2), and PTEN were detected by Western blot. C The relative expression levels of each protein were quantified by densitometry and normalized to the levels of control, which was set at 1.0. Each value represents the mean ± SD of five rats. P \ 0.05; *: the different symbol represents the significant differences among groups

between the density staining for PTEN and SM-MHC (SM2) (Fig. 6c). After 48 h of PTU treatment, the expression of PTEN and SM-MHC increased with the same correlation. That is, cells expressing low PTEN in response to siRNA proportionally decreased SM-MHC expression (Fig. 6c). These data indicate that PTU up-regulates the expression of PTEN and may thereby result in a PTUinduced increase in differentiation proteins expression. Effect of PTU on contractile proteins and PTEN expression in balloon-injured rat carotid arteries This study further evaluated whether the conclusion drawn from the present in vitro model system demonstrating the differentiation-promoting effect of PTU could be applied to an in vivo condition. In agreement with previous studies [21], the expression of VSMC differentiation markers, including calponin and SM-MHC, decreased in the arteries

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7 and 14 days after balloon injury (Fig. 7A c, e, B, and C). Propylthiouracil treatment opposed the down-regulation of calponin and SM-MHC induced by balloon injury. Immunohistochemical analysis indicated that staining for calponin and SM-MHC was qualitatively increased in the neointima of injured arteries of 14-day PTU-treated rat compared with control (Fig. 7A c–f). However, the increase in calponin and SM-MHC could not be demonstrated in the injured arteries of 7-day PTU-treated rat, possibly due to the late onset of PTU effect (Fig. 7B and C). Although initial experiments with staining for PTEN in the neointima of 14-day PTU-treated arteries were unrevealing, an increase in PTEN was noted in the neointima of 7-day PTU-treated arteries (Fig. 7A a and b). The changes in the immunohistochemistry could be quantitatively documented by the Western blot analysis (Fig. 7B and C). Conceivably, the findings obtained from the balloon injury system demonstrate that PTU activated PTEN preceding the up-regulation


of contractile proteins in injury-stimulated VSMCs, which were consistent with those from an in vitro condition.

Discussion Our recent study has demonstrated that PTU has an inhibitory effect on VSMC proliferation, which is independent of its antithyroid effect. The antiproliferative effect of PTU on VSMCs has been thought to be responsible for its antiatherosclerotic effect in cholesterol-fed rabbits [1]. This study further confirmed that PTU induced VSMC differentiation as determined by cell morphology and high expression of contractile proteins. Notably, several studies have shown a poor correlation between VSMC proliferation rates and their modified differentiated phenotype in either in vitro or in vivo studies, these processes are under distinct regulatory control [20]. That is, cessation of proliferation is not always associated with differentiated phenotype of VSMC. For instance, VSMCs within advanced atherosclerotic lesions show a low rate of proliferation but marked reductions in VSMC differentiation marker genes expression. Propylthiouracil induced the expression of differentiation marker contractile proteins and also inhibited the proliferation of VSMCs in a similar fashion, raising the possibility that PTU promotes coordinated regulation of proliferation and contractile proteins expression to induce the differentiated VSMC phenotype. This study evaluated the role of PTEN in producing PTUinduced VSMC differentiation. The experimental results presented here demonstrate that PTU induces VSMC differentiation in a PTEN-dependent manner. This conclusion is drawn on the basis of two findings. First, the up-regulation of contractile proteins by PTU and the responsiveness of PTEN expression to PTU are of similar kinetics. Second, the differentiation markers induced by PTU can be abrogated by PTEN inhibitor. The contribution of PTEN to tumor cell differentiation has been the subject of intense investigation [5]. In contrast to tumor cells, little is known about the role of PTEN in promoting VSMC differentiation. Earlier studies have demonstrated that PTEN activity is increased in differentiated SMCs, whereas vascular injury results in PTEN inactivation [7, 12, 17, 18]. This study provides the first demonstration of a drug that induces VSMC differentiation via PTEN-dependent pathway. Furthermore, some studies from our and other laboratories have found that PTEN is also associated with VSMC proliferation [1, 9, 10]. Because PTU inhibits VSMC proliferation and induces VSMC differentiation through the same mechanism, this study has identified a critical checkpoint in regulating VSMC proliferation and differentiation simultaneously.

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Propylthiouracil belongs to a class of structures known as ‘‘thionamides’’. Thionamides are characterized by a thiocarbamide backbone in the chemical structure. Notably, unlike other thionamides, PTU possesses an additional effect to block extra-thyroidal conversion of T4 to T3 by noncompetitive inhibition of type I 50 -monodeiodinase in liver, kidney, brain, and pituitary gland [15]. Therefore, the involvement of this extra-thyroidal effect in producing VSMC differentiation in vitro should be taken into account. This possibility can be virtually excluded by an additional experiment showing that exogenous supplementation of thyroid hormone in PTU-treated VSMCs did not abrogate the changes induced by PTU. The concentration of PTU presented here to induce phenotypic modulation of VSMCs exceeds the therapeutic range reported in humans given the standard dose of 300– 600 mg daily. The clinically relevant concentration of PTU in blood after a normal intake of therapeutic dose may be 1 to 10 lg/mL (&0.006–0.06 mmol/L) [15]. Although the levels of PTU used in this in vitro study exceed those expected in humans, the blood concentration of PTU (3.21 ± 1.68 lg/mL) in the balloon-injured rats was within the therapeutic range. Conceivably, PTU may induce VSMCs differentiation in the vessel wall during clinical application. The relatively high effective concentration in vitro may arise from the difference between blood and tissue level concentrations of PTU. Neointimal formation is an important clinical problem associated with vascular intervention. Proliferation VSMC, migration to the intima, and dedifferentiation determine the extent of neointimal formation, thus leading to restenosis after angioplasty and stent placement. Recently, drugeluting stent coated with rapamycin is proved to be a promising approach for preventing restenosis [16]. Rapamycin, an inhibitor of TOR, inhibits the development of neointimal formation by its ability to inhibit VSMC proliferation, migration, protein synthesis, and promote differentiation [14]. Interestingly, PTEN may interfere with the target of rapamycin (TOR), which is the main therapeutic target for treating restenosis. In combination with our findings reported previously [1, 2], this study confirmed that PTU, via PTEN induction, has the ability to inhibit VSMC proliferation, migration, and to induce VSMC differentiation. Future studies may explore the potential clinical implication by coating PTU on the stent to prevent restenosis. Acknowledgments This work was supported by grants from the Chang Gung Research Grant Foundation [G32102, and G331023] and National Science Council Grant [NSC-94-2314-B-182A-198]. We thank Mr. Chih-Chun Chen for his technical assistance in confocal microscope.

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