Propionyl-L-carnitineReducesProliferationandPotentiates ... · jury arterial remodeling. Propionyl-L-carnitine (PLC) (24) is a carnitine derivative that has recently been included

Propionyl-L-carnitine Reduces Proliferation and PotentiatesBax-related Apoptosis of Aortic Intimal Smooth Muscle Cellsby Modulating Nuclear Factor-�B Activity*

Received for publication, June 27, 2006, and in revised form, November 22, 2006 Published, JBC Papers in Press, December 17, 2006, DOI 10.1074/jbc.M606148200

Augusto Orlandi‡1, Arianna Francesconi‡, Marcella Marcellini§, Antonio Di Lascio‡, and Luigi Giusto Spagnoli‡

From the ‡Institute of Anatomic Pathology, Tor Vergata University, Rome 00133, Italy and §Sigma-Tau Research Laboratories,Pomezia (Rome) 00040, Italy

Propionyl-L-carnitine (PLC) has been introduced among thetherapeutic approaches of peripheral arterial disease, and morerecently, an increase of intimal cell apoptosis has been demon-strated tocontribute to its effectiveness in rabbit carotidpostinjurymyointimal hyperplasia prevention. How PLC mediates theseeffects on vascular smooth muscle cells (SMCs) remains poorlyunderstood. We investigated the role of NF-�B in PLC-inducedarterial remodeling. In vivo, daily PLC treatment 15 days afterinjury resulted in a reduction of relative rat aortic intimal volume,an increase of apoptosis, Bax up-regulation without changing theBcl-2 level, and a reduction of NF-�B, vascular cell adhesion mol-ecule-1,monocyte chemotactic protein-1, and survivin inmyointi-mal thickening compared with controls. In the presence of 10%serum,areducedG13SphaseprogressionprecededPLC-inducedintimal cell apoptosis; in 0.1% serumcultures, in a dose-dependentmanner, PLC rapidly induced intimal cell apoptosis and reducedp65, p50, IAP-1, and IAP-2 expression. Inhibiting NF-�B activa-tion through SN50 increased apoptotic rate and Bax expression inintimal but not in medial SMCs, and successive PLC treatmentfailed to induce a further increase in apoptotic rate. Bax antisenseoligodeoxynucleotide reduced PLC-induced intimal cell apo-ptosis and cytochrome c release. ThePLC-induced attenuationof NF-�B activity in intimal cells was also due to the increase ofI�B-� bioavailability, as the result of a parallel induction ofI�B-� synthesis and reduction of phosphorylation and degrada-tion. Collectively, these findings document that NF-�B activityinhibition contributes to PLC-induced proliferative arrest andBax-related apoptosis of intimal SMCs.

Intimal smooth muscle cell (SMC)2 accumulation plays acrucial role in the pathogenesis of vascular diseases, which

include postangioplasty restenosis (1, 2), and many studiesaimed at explaining the molecular pathways regulating themyointimal hyperplastic process. Restenosis remains the mostfeared complication following percutaneous transluminalangioplasty (3). Since angioplasty procedures grow exponen-tially (4), even slightly limiting restenosis can be an enormoussocioeconomic benefit. SMC apoptosis plays a relevant role infetal cardiovascular tissue remodeling (5). In normal arterialvessels, postnatal apoptosis is practically absent (6), whereas anincrease is observed in pathological conditions, such as athero-sclerotic plaques and restenosis (7–10). In rat myointimalthickening following vascular injury, a common experimentalmodel of postangioplasty restenosis (11, 12), an apoptoticreduction of neointimal SMCs counteracts the excessive prolif-eration and favors the restoration of vascular homeostasis (6, 8).Consequently, vascular SMC apoptosis control appears to be agoal in strategies aimed at limiting the clinical impact of rest-enosis (13). The NF-�B proteins and their I�B protein inhibi-tory subunits make up a group of regulatory transcriptionalfactors in a variety of physiological functions (14, 15), whichinclude cell survival (16, 17). In regard to vascular SMCs, invitro experiments demonstrated that NF-�B influences bovineSMC proliferation (18) and variably modulates human and ratSMC survival (19), also depending on culture conditions (20).In postinjury rat aortic intimal thickening, increased NF-�Blevels are detected (19, 21), similar to those observed in humanfibroatheromatous plaques (22). Balloon angioplasty-inducedNF-�B activation seems to contribute to lumen loss in rabbitiliac arteries via induction of an inflammatory response and adecrease in apoptotic rate (23). All together, these data suggestthat NF-�B-regulated apoptosis plays a critical role in postin-jury arterial remodeling. Propionyl-L-carnitine (PLC) (24) is acarnitine derivative that has recently been included amongpharmacological approaches to peripheral vasculopathy (25).PLC has a higher affinity for the plasma membrane transportsystem, being more lipophilic and penetrating better thanL-carnitine (26). We previously documented that daily PLCadministration reduces thickening and increases SMC apopto-sis of rabbit carotid neointima 3 weeks after injury (27). Sincepropyonic acid is a precursor of some nonsteroidal anti-inflam-matory drugs (28), it is still unclear whether the proapoptotic

* This study was supported in part by a grant from Spedali Civili of Brescia(Protocol 20906055) and Sigma Tau (Pomezia) for animal maintenance.The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) NM_023987, AF_190020,X63722, NM_031530, X02231, XM_343065, NM_199267, and XM_342346.

1 To whom correspondence should be addressed: Anatomic Pathology Insti-tute, Dept. Biopathology and Image Diagnostics, Tor Vergata University,Via Montpellier 1, 00133 Rome, Italy. Tel.: 39-06-20903960; Fax: 39-06-20902209; E-mail: [email protected].

2 The abbreviations used are: SMC, smooth muscle cell; PLC, propionyl-L-carni-tine; SO, sham-operated; CTR, control; bw, body weight; �-actin, �-smoothmuscle actin; BrdUrd, bromodeoxyuridine; TUNEL, TdT-mediated dUTP-biotin

nick-end labeling; FCS, fetal calf serum; ODN, oligodeoxynucleotide; EMSA,electrophoretic mobility shift assay; RT-PCR, reverse transcription-PCR; Q-PCR,real time PCR; IAP, inhibitor of apoptosis protein; VCAM-1, vascular cell adhe-sion molecule-1; MCP-1, monocyte chemotactic protein-1.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 7, pp. 4932–4942, February 16, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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effect derives from a direct PLC-SMC interaction or dependson an aspecific reduction of parietal inflammation. In the pres-ent study, we investigated the mechanisms through which PLCmodulates intimal SMC apoptosis and influences arterialremodeling in vivo and in vitro. Our results document that PLCspecifically induces growth inhibition and an increase in theapoptotic rate of intimal SMCs by inhibiting NF-�B activationand regulating I�B-� inhibitory protein expression.

EXPERIMENTAL PROCEDURES

Rats—Male Wistar rats, weighing 270–290 g and individu-ally housed, were used according to the experimental distribu-tion reported in Table 1. All experiments were performedaccording to guidelines compatible with theNational Institutesof Health Committee on the Care and Use of Laboratory Ani-mals. Rats were anesthetized with Nembutal sodium (Abbott),35 mg/kg body weight (bw) intraperitoneally, and the thoracicaorta endothelium was removed by an embolectomy catheter(2F-Fogarty, Baxter, American Edwards Laboratories, Anasco,Puerto Rico), as previously reported (29). In sham-operated(SO) rats, the carotid was ligated without ballooning. Immedi-ately following ballooning, a group of randomized rats received120 mg/kg bw of PLC (Sigma) dissolved in 1 ml of saline solu-tion as vehicle by oral gavage. The remaining balloonized (CTR)and SO rats only received the saline solution. Treatment wasrepeated daily. Fifteen days later, the animals received 30mg/kgbw of bromodeoxyuridine (BrdUrd) intraperitoneally (Sigma).Two hours later, rats were anesthetized, and vessels werewashed with saline and perfused with buffered formalinthrough a cannula inserted in the left ventricle. After aortictissue sampling (30), 4-�m-thick serial sections were stainedwith hematoxylin and eosin or Movat’s pentachrome oremployed for immunohistochemistry. For the biomolecularstudies, rats were anesthetized and killed by means of cervicaldislocation, and their aortas were isolated in sterile conditions.Small thoracic aortic rings were frozen in cooled isopentane forcryostatic sections. Remaining intimal and medial tissues wereisolated as previously reported (30).Morphometry, Immunohistochemistry, Proliferation, and

Apoptosis in Vivo—To determine the effect of PLC on intimalhyperplasia 15 days after injury, we calculated the relative inti-mal volume as the ratio of intimal area/(intimal � medialarea)� 100 on sections stainedwithMovat’s pentachrome (31).Immunohistochemistry studies involved serial deparaffined

sections immunoreacted at room temperature for 1 hwith anti-smooth muscle actin (�-actin; 1:100), anti-desmin (1:50), anti-vimentin (1:100; Dako, Dakopatts, Denmark), and the anti-p65subunit of NF-�B (1:100; Santa Cruz Biotechnology, Inc., Santa

Cruz, CA) monoclonal or a polyclonal rabbit anti-Bax (1:250),anti-p50 subunit of NF-�B (1:50), anti-I�B-� (1:200), anti-sur-vivin antibodies (1:10), anti-vascular cell adhesion molecule-1(VCAM-1, 1:200; Santa Cruz Biotechnology), or a goat anti-monocyte chemotactic protein-1 (MCP-1, 1:100; Santa CruzBiotechnology) followed by a goat anti-mouse, anti-rabbit, anddonkey anti-goat IgG, respectively.Diaminobenzidinewas usedas the final chromogen. Nonimmune IgG was used as control.For p65, immunohistochemical stainings were repeated with amonoclonal anti-p65 antibody (1:100) (Chemicon, Temecula,CA), which gave similar results. For endothelial cells, ring cryo-static sections were fixed in cold methanol and then incubatedwith a rabbit anti-human VIII (1:100; Dako), and a positivereaction was revealed as reported above.To quantify intimal and medial SMC proliferation, an anti-

BrdUrdmonoclonal antibody (Ylem,Avezzano, Italy) was used,and the percentage of BrdUrd-positive nuclei per total cells(BrdUrd labeling index) was calculated (27).To emphasize SMCapoptosis in vivo, rehydrated sectionswere

stripped fromproteins through incubationwith 300 units/ml pro-teinase K (Sigma) for 15 min at 37 °C, apoptotic nuclei wererevealed by TdT-mediated dUTP-biotin nick-end labeling(TUNEL), and the percentage of positive nuclei per total cells(TUNEL labeling index) was calculated, as previously reported(27). To determine the myocytic nature of proliferating apoptoticcells, double immunohistochemistry was also performed (27).Proliferation, Immunohistochemistry, andApoptosis in Vitro—

Intimal aortic cells 15 days after injury and medial SMCs fromSO rats were isolated by enzymatic digestion and allowed togrow to the fifth passage, as previously reported (29). Cells wereplated in sparse conditions (2.5 � 103 cells/cm2) and synchro-nized in Dulbecco’s modified Eagle’s medium (Invitrogen) sup-plemented with 0.1% fetal calf serum (FCS; Biological Indus-tries, Haemek, Israel) for 24 h. PLC was added at variousconcentrations in the presence of 0.1 or 10% FCS, and themedium was changed after 2 days. Following the treatments,cells were trypsinized, and the counted/seeded cell ratio wasevaluated. Cell viability was calculated using 0.4% trypan blueexclusion in triplicate. [3H]Thymidine incorporation wasdetermined as previously reported (32).For immunofluorescence staining in vitro, cells growing on

glass slides were fixed for 5 min in cold methanol (�20 °C),rinsed twice in phosphate-buffered saline, and incubated withanti �-actin, anti-Bax, anti-p65, and anti-p50 monoclonal orpolyclonal antibodies (32). Cells were photographed using aNikon fluorescent microscope and DNA chromatin morphol-ogy under UV visualization.

TABLE 1Experimental distribution of ratsRats were sacrificed 15 days after surgical procedure and aortic injury by ballooning. Daily treatment with PLC (120 mg/kg body weight dissolved in saline by oral gavage)started immediately after balloon injury.

Group Treatment Days after injury Rats for each methoda

CTR Aortic injury by ballooning 15 a (5), b (7), c (3), d (5)PLC Aortic injury by ballooning � PLC 15 a (5), b (7), c (3), d (5)SO Sham operation 15 a (5), b (7), c (3), d (5)

a The numbers in parentheses represent the number of rats for plasma analysis (a); morphology,morphometry, and electronmicroscopy (b); biochemistry andmolecular biology(c); and cell culture (d).

PLC Enhances Intimal SMC Apoptosis by Reducing NF-�B Activity

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Apoptosis of adherent cells was investigated by calculatingthe percentage of nuclei showing apoptotic features usingHoechst staining in triplicate (32). Assigning a serial number toeach slide ensured the objectivity of all measurements.To determine the role of NF-�B in the apoptotic process,

SMCs were cultured in the presence of 0.1% FCS and SN50(Bio-Mol), a peptide that specifically inhibits nuclear transloca-tion of NF-�B (33), at a 5 �M concentration for 24 h; in someinstances, PLC at a 50 �M concentration was added 1 h afterSN50; alternatively, after 24 h of treatment with PLC, SN50wasadded for 1 h.As a control, we repeated the experimentswith anequimolar concentration of SN50M (Bio-Mol), an inactive syn-thetic analogwith amutated nuclear localization sequence (33).In other experiments, gliotoxin (Sigma) at 5 �M was used for6 h, followed by 24 h of treatment with 50 �M PLC.

A phosphorothionate-modified antisense ODN 5�-TGCTC-CCGGACCCGTCCST-3�, specific for the translation initiationregion of rat Bax mRNA (34), and a control scrambled ODNwere commercially synthesized (Invitrogen), purified, andtransfected into cultures using oligofectamine (Invitrogen) for45 min and then replaced with a new medium plus ODN aloneor in association with 50 �M PLC.Flow Cytometry and Cell Cycle Analysis—Flow cytometry

was performed as previously reported using a fluorescence-ac-tivated cell sorting scan flow cytometer (BD Biosciences) and aLysis II program (32). The number of cells in the sub-G1 (DNAcontent �2N), G0/G1, S, and G2/M phases was expressed as apercentage of total events (10,000 cells).Western Blotting—Cytoplasmic extracts were prepared

according to standard protocols (35), and SDS-PAGE andWestern blotting were performed with 2–50 �g of proteins, aspreviously reported (29). Nitrocellulose filters (0.45 mm;Schleicher&Schuell) were incubatedwith amouse anti-vimen-tin (1:200; YLEM), anti-� actin (1:500, DAKO), anti-cyto-chrome c and anti-caspase-3 (1:500 and 1:100; Pharmingen),followed by a goat anti-mouse IgG (1:105), or alternatively witha rabbit anti-total actin (kindly provided by Prof. G. Gabbiani;1:100), anti-p50, anti-I�B-� (1:100 and 1:50) and anti-Bax pro-tein (1:200) or a goat anti-p65 (1:500), followed by a goat-anti-rabbit or donkey anti-goat IgG (1:105). Detection and quantifi-cation of Kodak X-Omat films were performed as previouslyreported (29). In order to consider protein loading, the densi-tometric value of each proteinwas normalized to that of vimen-tin after stripping. Western blots were repeated in triplicate.Electrophoretic Mobility Shift and I�B-� Phosphorylation

Assays—To perform the electrophoretic mobility shift assay(EMSA), nuclear extracts were prepared by standard protocols(35). Briefly, 2 � 106 cells were collected into 1.5-ml centrifugetubeswith 1ml of phosphate-buffered saline, pelleted for 1min,and resuspended in 400�l of cold buffer (10mMHEPES, pH7.9,1.5mMMgCl2, 10mMKCl, 0.5mM dithiothreitol, 0.2mM phen-ylmethylsulfonyl fluoride). Samples were incubated on ice for10min, vortexed for 10 s, pelleted, and resuspended in 100�l ofstorage buffer (20 mM HEPES, pH 7.9, 25% glycerol, 420 mMNaCl, 1.5 mMMgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and0.2 mM phenylmethylsulfonyl fluoride). Samples were thenincubated on ice for 20min and centrifuged. Supernatants werecollected for use as nuclear extracts. Protein concentrations

were determined (29), and EMSA analysis and supershift assayswere performed using an NF-�B-specific oligonucleotide(Invitrogen). The sequence was as follows: 5�-AGTTGAGGG-GACTTTCCCAGGC-3�. The double-stranded nucleotideswere end-labeled with [�-32P]ATP using T4 polynucleotidekinase; 5�g of nuclear extract was used in each assay for NF-�BDNA binding using standard protocols (35). NF-�B antibodiesused for supershift EMSA were anti-p50 and anti-p65 (SantaCruz Biotechnology).I�B-� phosphorylation was determined by overnight immu-

noprecipitation of 250 �l of cytoplasmic extracts using 2 �g ofI�B-� antibody at 4 °C. After washing, agarose beads wereresuspended in SDS-PAGE, and Western blotting was per-formed using an antibody recognizing phosphotyrosine resi-dues (1:500), as reported above.RNA Extraction and Reverse Transcription (RT)-PCR—Total

RNAwas extracted fromcells cultured in 10-cmdishes by usingTRIzol reagent (Invitrogen), as previously reported (36). Thefirst strand of cDNA was produced from 2 �g of total RNAusing 200 units of Superscript reverse transcriptase (Invitro-gen) and random primers (Roche Applied Bioscience). The fol-lowing primer pairs were used: for rat inhibitor of apoptosisprotein-1 (IAP-1;GenBankTMaccession numberNM_023987),5�-TGG CTA CTT CAG TGG CTC CT-3� (forward) and5�-GCA AAG CAG GCC ACT CTA TC-3� (reverse); for ratinhibitor of apoptosis protein-2 (IAP-2; GenBankTM accessionnumber AF_190020), 5�-CCAGCCTGCCCTCAAACCCTCT-3� (forward) and 5�-GGG TCA TCT CCG GGT TCC CAAC-3� (reverse); for rat VCAM-1 (GenBankTM accession numberX63722), 5�-GAACACTCTTACCTGTGTACAGC-3� (for-ward) and 5�-CCA TCCTCATAGCAATTAATGTGAG-3�(reverse); for rat MCP-1 (GenBankTM accession numberNM_031530), 5�-TTC TGG GCC TGT TGT TCA CA-�3 (for-ward) and 5�-GGTCACTTCTACAGAAGTCC-�3 (reverse).A semiquantitative analysis was performed in triplicate bydensitometric methods, and the intensity of each band wasexpressed in units of optical density; rat glyceraldehyde3-phosphate dehydrogenase was used as the control gene(GenBankTM accession number X02231): 5�-ATG GTGAAGGTCGGTGTGAACG-3� (forward) and 5�-GTCATCGAT GAC CTT GGC CAG-3� (reverse).Real Time PCR Conditions—For real time PCR (Q-PCR)

analysis, 2 �g of total RNAwere reverse-transcribed using ran-dom primers (Roche Applied Science) and Superscript reversetranscriptase (Invitrogen). ThemRNA levels of I�B-�, p65, andp50 were measured by Q-PCR using gene-specific primers: forrat I�B-� (GenBankTM accession number XM_343065),5�-TGG CCA GTG TAG CAG TCT TG-3� (forward) and5�-GACATCAGCACCCAAAGTCA-3� (reverse); for rat p65(GenBankTM accession number NM_199267), 5�-GGA CGATCTGTTTCCCCTCAT-3� (forward) and 5�-TGATCTCCACATATGGCCCAG-3� (reverse), and for rat p50 (GenBankTMaccessionnumberXM_342346), 5�-AGCACCAAGACCGAAGCA A-3� (forward) and 5�-TCT CCC GTA ACC GCG TAGTC-3� (reverse). For PCR amplification, Power Syber Greenmaster mix was used (Applied Biosystems). For each sample,PCR fluorescent signals were normalized to that of the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase. All




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Q-PCR analyses were carried out using the Mx3005P PCR sys-tem (Stratagene).DNA Isolation and Ligation-mediated PCR—In order to bet-

ter identify and quantify apoptosis-associated DNA fragmenta-tion, DNA was extracted, and ligation-mediated PCR was per-formed (32). The nucleosomal ladder was quantified in 1.2%agarose gels stained with ethidium bromide (32).Statistical Analysis—Results were expressed as the mathe-

matical mean of single experiments � S.E. Results were statis-tically analyzed by Student’s t test. The differences were con-sidered statistically significant with a value of p � 0.05.

RESULTS

PLCReduces Intimal Thickening and Potentiates Intimal CellApoptosis in Vivo—In rat aortas 15 days following endothelialinjury, a diffuse neointimal thickening was clearly evident andmade up of rounded or elongated cells embedded in an abun-dant extracellular matrix. Ultrastructural analysis demon-strated abundant cytoplasmic synthetic organelles and fewperipheral myofilament bundles in intimal cells of both PLCand CTR groups, characteristic of a dedifferentiated vascularSMC phenotype (37). A morphometric analysis demonstratedthat PLC treatment induced a reduction in relative intimal vol-

ume and an increase in neointimalbut not medial TUNEL labelingindex compared with the CTRgroup (p � 0.02 and p � 0.01,respectively; Fig. 1, A and B). TheTUNEL labeling index of the PLCand CTR tunica media was ex-tremely low and less than the re-spective overlying intimal value (p �0.01) and similar to that of the SOgroup. On the other hand, theneointimal BrdUrd labeling indexdid not differ when comparing PLCand CTR groups, although both didincrease compared with the respec-tive underlying and SO tunicamedia (p� 0.001; Fig. 1C). The PLC,CTR, and SO tunica media BrdUrdlabeling index did not differ fromeach other. Double immunohisto-chemistry showed slight cytoplas-mic positivity for �-actin in mostBrdUrd (70.7� 2.1 and 72.6� 2.2%)and TUNEL (73.3 � 2.5 and 74.6 �2.2%) positive intimal cells in PLCand CTR group, respectively. Inorder to confirm apoptotic rate dif-ferences, ligation-mediated PCRwas performed on DNA samplesextracted from freshly isolated aor-tic tissue. Densitometric analysisshowed a more evident apoptoticladder in PLC compared with CTRrat neointimal tissue (p� 0.001; Fig.1D). A very small amount of frag-

mented DNA was observed in PCR products from SO aortictissue. Finally, scattered groups of endothelial cells were focallyobserved in re-endothelialized areas of the neointimal surface,but their number and extent did not vary when comparingCTR(20.8 � 4.2 cells/mm2) and PLC groups (27.1 � 6.2 cells/mm2).PLCModulates Intimal Cell Expression of NF-�B, I�B-�, and

Apoptosis-related Proteins in Vivo—In order to demonstratethe differences in survival- and apoptosis-related proteinexpression in vivo, serial aortic sections were stained by immu-nohistochemistry (Fig. 2). In SO tunica media, p65, p50, I�B-�and Bax protein immunostainings were practically negative.Extensive cytoplasmic immunopositivity for p65 and p50 wasobserved in most intimal cells; a smaller percentage of nuclearpositivity for p65 was also present in CTR intimal cells (6.8 �1.8). It was reduced in PLC neointima (3.0 � 0.8; p � 0.01) andabsent in SO tunica media. In addition, a more extensive posi-tive cytoplasmic survivin reaction was observed in CTR com-pared with PLC neointimal cells. The opposite was true forI�B-�, Bax, and �-actin immunostaining, their expressionbeing greater in PLC compared with CTR neointimal cells.Moreover, a reduction in immunostaining for VCAM-1 andMCP-1, two NF-�B-regulated genes (23), was observed in theneointima of PLC-treated rats. Underlying and SO tunica

FIGURE 1. Effects of PLC on rat aortic neointima development, proliferation and apoptosis in vivo. A–C,bar graphs representing the differences in neointimal development, aortic tissue apoptosis, and proliferationamong experimental groups; rat aortic tissue was obtained 15 days after injury by ballooning with (PLC) orwithout (CTR) daily PLC treatment (120 mg/kg bw dissolved in saline by oral gavage) starting immediately afterballooning or after sham operation (SO). A, intimal relative volume morphometrically calculated on aorticsections stained with Movat’s pentachrome, according to a stereological formula. B, TUNEL labeling indexcalculated as a percentage of positive nuclei � S.E. C, Bride labeling index as a percentage of immunohisto-chemical positive nuclei � S.E. D, agarose gel under UV light after staining with ethidium bromide shows theladder production after blunt end linker ligation and 25 cycles of PCR of 1 �g of genomic DNA. Quantificationof optical density value after densitometric analysis confirms the higher level of apoptotic DNA fragmentationafter treatment with PLC compared with control (CTR) and SO aortic tissue. First lane, �X174 DNA marker fromSigma; * and **, p � 0.02 and 0.01, respectively.




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media immunostainings for p65, p50, I�B-�, survivin, Bax,VCAM-1, and MCP-1 were almost negative.Phenotypic Heterogeneity Influences Proliferation and PLC-

induced Apoptosis of SMCs—In order to determine the role ofphenotypic differences in the proliferative and apoptoticbehavior of SMCs in vitro, aortic intimal cells obtained 15 daysafter ballooning were compared with SOmedial SMCs. Intimalcells appeared characteristically epithelioid with a tendency togrow in small groups. When confluent, they conserved theirepithelioid appearance and grew in a single layer (Fig. 3A), dif-ferently from medial SMCs, which were spindle-shaped with acharacteristic “hill and valley” pattern (Fig. 3B). When culturedin the presence of 10% FCS, intimal cells proliferated morecomparedwithmedial SMCs (p� 0.01; Fig. 3C). The 6-day PLCtreatment (Fig. 3D) resulted in a dose-dependent reduction ofthe counted/seeded cell ratio in intimal (p � 0.01 for all exam-ined concentrations) but not in medial SMCs. [3H]thymidineincorporation correlated with the growth curves of the twopopulations (Fig. 3E).

Cytometric analysis (Table 2) demonstrated that in the pres-ence of 10% FCS, the percentage of medial SMCs in G0/G1 washigher comparedwith intimal cells. The 2-day 50�MPLC treat-ment inhibited intimal cell G13 S progression, as shown by thehigher percentage in theG0/G1 phase, and only a slight increaseof the percentage of subdiploid intimal cells was observed. Cellviability was also unchanged at this time. After 4 days of treat-ment, the percentage of subdiploid apoptotic cells alsoincreased in intimal compared withmedial SMCs (p� 0.01). In0.1% FCS cultures, the 2-day PLC treatment induced a rapidincrease of subdiploid cells in the intimal population comparedwith control and medial SMCs (p � 0.01). PLC did not modifythe cell cycle in medial SMC cultures. Cell viability corre-sponded with the cytometric analysis. To confirm the presenceof apoptotic cells, we calculated the percentage of condensed orfragmented nuclei in 0.1% FCS sparse adherent cultures. Twodays later, the percentage of Hoechst-stained apoptotic cells

FIGURE 2. Immunohistochemical detection of NF-�B-related, survival,and apoptosis-related proteins in rat aortas neointima with or withoutPLC treatment. Formalin-fixed serial sections of aortic tissue from sham-op-erated rats (left column), 15 days after endothelial injury by ballooning (centralcolumn) or ballooning plus treatment with PLC (120 mg/kg bw die-dissolvedsaline by oral gavage starting immediately after injury; right column) wereimmunostained with antibodies against p65, p50, I�B-�, survivin, Bax,�-smooth muscle actin, VCAM-1, and MCP-1, using appropriate concentra-tions and diaminobenzidine as chromogen. Original magnification was�250.

FIGURE 3. Vascular SMCs phenotype, cell growth, and the antiprolifera-tive effect of PLC. Rat aortic intimal cells and sham-operated medial SMCs 15days after injury were allowed to grow to fifth passage. A, intimal cells appearepithelioid and grow in monolayer. B, medial SMCs show the classic hill-and-valley growth pattern. C and D, growth curves of intimal and medial SMCs.Cells were seeded at 2.5 � 103 cells/cm2 density in 60-mm dishes, synchro-nized in Dulbecco’s modified Eagle’s medium plus 0.1% FCS for 24 h and thentreated or not with PLC at various concentrations in the presence of 10% FCS.E, for [3H]thymidine incorporation, after synchronization, fresh medium plus10% FCS containing 0.1 mCi/ml [3H]thymidine (5 Ci/(mmol/liter) specificactivity) was added for 20 h in the presence of different PLC concentrations.Incorporation was expressed as the ratio between counts/min (cpm) and thenumber of cells; values are expressed as mean � S.E. of three independentexperiments.




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was higher in 50 �M PLC-treated intimal (6.9 � 0.8) than inmedial SMCs (1.2 � 0.5; p � 0.01).NF-�B Inhibition Regulates Intimal Cell Apoptosis—In order

to determine the role of NF-�B in the apoptotic cascade ofintimal cells, Western blotting and EMSA were performed incytoplasmic andnuclear extracts, respectively. Intimal cells cul-tured for 4 days with 10% FCS or for 2 days with 0.1% FCSshowed a higher constitutive and active NF-�B expression thanmedial SMCs (Fig. 4, A and B).To confirm whether NF-�B plays a role in intimal cell apo-

ptosis, we cultured the cells for 24 h in 0.1% FCS and a nontoxicconcentration of SN50, a synthetic peptide that specificallyblocks nuclear NF-�B translocation (38). Cytometry (Fig. 5A)and Hoechst staining (Fig. 5B) showed that SN50, which inhib-its NF-�B nuclear translocation, induces an increase in the per-centage of apoptosis in intimal (7.9 � 0.9; p � 0.01) but not inmedial SMCs (2.0� 0.5) compared with respective controls, con-firming that the reduction of NF-�B activity is an important pre-requisite for intimal cell apoptosis.The inactiveSN50Manalogdidnot induce intimal cell apoptosis. Ligation-mediated PCR con-firmed differences in SN50-induced apoptotic response (Fig. 5D).Densitometric blot analysis also demonstrated that adding SN50resulted in a Bax up-regulation (188 � 16.5%), cytochrome crelease (180 � 20%), and reduced surviving expression (60.2 �10.5%) in the cytosolic fraction of intimal cell cultures.NF-�B Inhibition Associates with PLC-induced Intimal Cell

Apoptosis—In order to determine the role of NF-�B in the PLC-induced apoptotic cascade of intimal SMCs, EMSA andWesternblotting were performed. In a dose-dependent manner, PLCreducedNF-�Bactivity in intimal cells (Fig. 4C).At 50�Mconcen-tration, PLC induced a significant reduction in both constitutiveandactiveNF-�B intimal cell expression (p�0.01; Fig. 4,AandB).These changes were not evident in medial SMC cultures.To confirm that NF-�B inhibition contributes to PLC-in-

duced apoptosis, we cultured intimal cells for 1 h with SN50 at5 �M concentration. The sequential treatment with 50 �M PLCfor 24 h failed to induce a further increase of intimal cell apo-

ptosis by cytometry (Fig. 5A) andHoechst staining (7.0� 1.0%).Similarly, SN50 did not further significantly increase the 50 �MPLC-induced intimal cell apoptosis (8.1 � 1.0% by Hoechststaining). These data strongly suggest a link between the pro-apoptotic action of PLC and NF-�B inhibition.PLC Modulates Intimal Cell Expression of Apoptosis-related

Proteins and NF-�B-regulated Genes in Vitro—Densitometricanalysis of blots (Fig. 4A) showed that p65 expression reduction(28.3 � 4.1%) was parallel to the increase of Bax expression(169.0 � 16%) after 2 days of PLC treatment, whereas the Bcl-2level was almost unchanged compared with control intimalcells. Bax, Bcl-2, and cytochrome c expression in medial SMCswas low. A PLC-induced increase in cytochrome c and caspase3 expressionwas also detected in intimal (190.5� 15 and 165�12%, respectively) but not in PLC-treatedmedial SMCs. Densi-tometric analysis confirmed the lower level of �-actin (20.5 �4%) in intimal compared with medial SMCs (32). PLC induceda slight increase of �-actin expression in intimal (146.2 � 6%;p� 0.02) but not inmedial SMCs (118.5� 12%) comparedwithrespective controls. RT-PCR showed a dose-dependent reduc-tion of IAP-1 and, less markedly, IAP-2 in PLC-treated intimalcells compared with controls (Fig. 4D). RT-PCR also confirmedthe PLC-induced dose-dependent reduction of the transcriptsfor VCAM-1 and MCP-1 in intimal cells in vitro.NF-�B Mediates Bax Up-regulation and Cytochrome c

Release inApoptotic Intimal SMCs—Todetermine if Bcl-2 fam-ily member proteins are NF-�B-regulated, an antisense ODNagainst Bax up-regulation was added to intimal cell cultures.Bax antisense ODN, added to intimal cell cultures, blocked Baxup-regulation in a dose-dependent manner (Fig. 4E), whereasBcl-2 was unchanged. Control scramble ODN had no effect.The absence of Bax up-regulation paralleled the absence ofcytochrome c release in the cytosol, suggesting thatNF-�B con-trols cytochrome c release through Bax protein expression. Baxantisense ODN also prevented, at least in part, both SN50 andPLC-induced apoptosis and Bax up-regulation, as documentedby flow cytometry (Fig. 5E).

TABLE 2Cell cycle analysis and viability of intimal and medial SMCsExperiments were performed in the presence of 10 and 0.1% FCS. Cells were plated in sparse conditions (2.5� 103 cells/cm2) and synchronized in 0.1% FCS for 24 h. 50mMPLC was added or not to the medium and changed after 2 days. Following the treatments, cells were trypsinized, fixed, and stained with propidium iodide. Apoptosis(�G0/G1) and cell cycle were analyzed by flow cytometry (10,000 events). Cell viability was calculated using 0.4% trypan blue exclusion in triplicate, and results are expressedas mathematical mean � S.E.

10% FCSMedial SMCs Intimal cells Medial SMCs Intimal cells Medial SMCs Intimal cells

PLC (50 �M) � � � � � �Days 2 2 2 2 4 4�G0/G1 0.57 0.56 0.44 2.57 1.61 6.26G0/G1 84.36 74.73 84.96 84.88 73.07 68.59S 7.12 13.07 7.37 5.01 13.77 9.65G2/M 7.95 11.64 7.23 7.54 11.55 15.5Cell viability 99.3 � 0.67 99.7 � 0.33 99.0 � 0.58 96.3 � 0.88 96.0 � 1.53 90.0 � 1.15

0.1% FCSMedial SMCs Intimal cells Medial SMCs Intimal cells

PLC (50 �M) � � � �Days 2 2 2 2�G0/G1 0.26 1.77 1.33 7.09G0/G1 77.59 79.22 77.39 78.55S 10.35 8.25 10.40 6.03G2/M 11.80 10.76 10.88 5.33Cell viability 99.7 � 0.33 95.3 � 1.76 86.0 � 2.08 89.0 � 1.15




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PLC Inhibits I�B-� Phosphorylation and Degradation—Todetermine if theNF-�B inhibitory activity of PLCwas due to theinhibition of I��-� degradation, we performed Western blot-ting on cytoplasmic extracts of intimal cells. After 24 h at 50�M

PLC concentration in the presenceof 0.1% FCS (Fig. 4A), the I�B-�level increased compared with con-trol cultures (189 � 10%; p � 0.01).Western blotting also revealed aparallel decrease of phosphorylatedI�B-� (Fig. 4F). Altogether, thesedata suggest that PLC inhibitsNF-�B nuclear translocation andtranscriptional activity by prevent-ing phosphorylation and degrada-tion of I�B-�.To better elucidate the mecha-

nism by which PLC reduces NF-�Bactivation in intimal cells, we alsoinvestigated the dose-dependenteffect of PLC on I�B-�, p65, and p50mRNA levels. Our results showedan induction of I�B-� mRNA (Fig.6A, 50-fold versus control at 100 �MPLC after 6 h of treatment; p �0.01). In contrast, inhibition of bothp65 and p50 (55 and 75% inhibitionversus control at 100 �M PLC after24 h of treatment, respectively; p �0.01) was found.Finally, the 6-h 5 �M treatment

with gliotoxin, a fungal toxin thatspecifically inhibits the degradationof I�B-� (39), in the presence of0.1% FCS induced intimal cell apo-ptosis (6.3 � 1.0% by Hoechst).Adding 50 �M PLC for 24 h failed tofurther increase apoptotic rate(7.0 � 1.3%) compared with controlcultures (6.8 � 1.0%). Cytometry(Fig. 6D) confirmed these results.

DISCUSSION

We previously documented (27)that PLC induces a reduction inmyointimal thickening and anincrease in intimal SMC apoptosisin rabbit carotids 3 weeks afterinjury. In the present study, wedemonstrated that PLC-inducedNF-�B inhibition in intimal but notin medial SMCs determines an up-regulation of Bax-related apoptosis,along with a survivin reduction andan I�B-� increase. The presentresults clarify some of the biomo-lecular mechanisms through whichPLC influences vascular SMC sur-

vival and document the close link between phenotypic hetero-geneity and apoptosis during postarterial injury remodeling.Previous reports describe PLC as specific for cardiac and skel-etal muscle and the positive anaplerotic mitochondrial effect of

FIGURE 4. Vascular SMC phenotype influences the expression of apoptosis-related proteins in vitro. A, rataortic intimal cells obtained 15 days after injury and sham-operated medial SMCs were seeded at 2.5 � 103

cells/cm2 in 60-mm dishes, synchronized for 24 h, and cultured in the presence or absence of PLC (50 �M) and0.1% FCS for 48 h. Cytoplasmic extracts (5–20 �g/lane) were used for Western blotting to identify apoptosis-related proteins. Vimentin was used as loading control. One of three similar experiments is shown. B, EMSAanalysis of NF-�B activity in 5 �g of rat aortic intimal and medial SMC nuclear extracts; a marked reduction inNF-�B activity (upper arrow, p65/50 heterodimer; lower arrow, p50/50 homodimer) was observed after 24 h oftreatment with 5 �M SN50 as well as 50 �M PLC. C, EMSA analysis of NF-�B activity in 5 �g of rat aortic intimaland medial SMC nuclear extracts after 24 h of treatment with the indicated concentrations of PLC. In intimalcells, a dose-dependent significant reduction of NF-�B activity is observed (upper arrow, p65/50 heterodimer;lower arrow, p50/50 homodimer). D, detection of rat IAP-1, IAP-2, VCAM-1, and MCP-1 by RT-PCR. Rat aorticintimal cells obtained 15 days after injury were cultured at 2.5 � 103 cell/cm2 density in the presence of 0.1%FCS for 12 h and different PLC concentrations; total RNA was isolated using TRIzol reagent, and RT-PCR wasperformed as described under “Experimental Procedures.” Glyceraldehyde 3-phosphate dehydrogenase wasused as the control gene. First lane, pGEM DNA marker from Promega. The results are representative of threedifferent experiments. E, SN50-induced inhibition of NF-�B nuclear translocation modulates Bax expression.An antisense ODN specific for the translation initiation region of rat Bax mRNA and a control scrambled ODNwere purified and transfected into intimal cell cultures using oligofectamine for 45 min and then replaced witha new medium plus ODN alone or in association with 50 �M PLC. Bax, Bcl-2, and cytochrome c expression wereanalyzed by Western blotting on cytoplasmic extracts using �-actin control of loading. All results are repre-sentative of three separate experiments. F, I�B-� phosphorylation in rat aortic intimal and medial SMCs eval-uated by overnight immunoprecipitation of 250-�l cytoplasmic extracts with 2 �g of I�B-� antibody at 4 °C.Washed agarose beads were resuspended in SDS-PAGE, and Western blotting was performed using an anti-body recognizing I�B-� phosphotyrosine residues.




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this molecule (40, 41). Successively, an anti-ischemic effect ofPLC on endothelial cells (42) and an endothelium-dependentvasodilatation in small intact arteries that vanishes in de-endo-thelialized ones have also been described (43). These pharma-cological effects are confirmed from the improved walkingcapabilities in PLC-treated peripheral arterial disease patients(25, 44). Although these findings suggest a beneficial more thana cytotoxic effect of PLC on endothelial cells (43), we did notobserve a difference in the extent of the re-endothelialized sur-face between PLC and CTR aortas. In rabbits, the PLC-inducedreduction in restenosis percentage was not accompanied bysignificant changes in the carotid mean diameter, excluding avasodilatative effect (27). As a consequence, PLC seems to exerta direct effect on vascular SMCs.Our study documents that a PLC-induced down-regulation

of constitutive and active NF-�B fuels intimal SMC apoptosis,

as observed in other cell types (16).This strongly suggests that theswitching function of NF-�Bbetween vascular survival and apo-ptosis in response to microenviron-mental changes is critically regu-lated by a defined gene subsetexpression that characterizes differ-ent vascular SMC phenotypes (45).In fact, intimal SMCs isolated 15days after injury show a dedifferen-tiated phenotype, with low �-actinand high constitutive NF-�B andBax levels (32, 37), and maintain anepithelioid appearance and highproliferative capacity in vitro (29,46). The same is not observed inmedial SMC cultures, which displaya contractile and elongated pheno-type with low NF-�B and Bax levelsin vivo and in vitro (20, 21, 32). Inti-mal SMCs, unlike those of tunicamedia, show an increased suscepti-bility to apoptosis induced by all-trans-retinoic acid in vivo and invitro (32, 47). Two months afterinjury, rat aortic intimal SMCsrevert to a fully differentiated phe-notype (48). After this phenotypicswitch, intimal SMCs are similar tomedial SMCs and show low NF-�Bexpression along with reduced sus-ceptibility to apoptotic stimuli (29,32). These results highlight the cru-cial role of phenotypic heterogene-ity and help to explain apparentlycontrasting data concerning therole of NF-�B in arterial SMCpathobiology (17, 18, 20, 49). Thetarget cell phenotype criticallyinfluences the effects on prolifera-tion and apoptosis induced by

NF-�B modulation (50), similar to what has been reported forother variables (20).Our results document that the apoptotic rate increase fol-

lowing PLC-induced NF-�B inhibition in intimal cells wasaccompanied by Bax and cytochrome c up-regulation. SN50-induced NF-�B inhibition also determined Bax up-regulationand successive treatment, whereas PLC failed to induce a fur-ther increase of apoptotic rate, suggesting a similar apoptoticpathway. TheBax to Bcl-2 ratiowas increased in intimal cells byBax overexpression, as reported in human radial artery intimalhyperplasia (51). However, as for other cell types (52), Bax over-expression per se is not lethal but, on the contrary, compatiblewith proliferation in vitro. We also documented that the PLC-induced increase of the apoptotic rate is associated with areduction of survivin expression in vivo. Survivin belongs to thefamily of genes known as apoptosis inhibitors and counteracts a

FIGURE 5. NF-�B nuclear translocation inhibition induces intimal SMC apoptosis. A, flow cytometric anal-ysis of rat aortic intimal cells cultured 15 days after injury and sham-operated medial SMCs in sub-G1 (DNAcontent �2N) was calculated as a percentage of total events (10,000 cells). After 24 h of serum starvation, SN50and its inactive analog SN50M were added at a 5 �M concentration in the presence of 0.1% FCS, and cells wererecovered after 24 h. In some experiences, PLC at 50 �M concentration was added after 1 h; in other experi-ments, after 24-h treatment with PLC, SN50 was added for 1 h at 5 �M concentration; B and C, Hoechst stainingof intimal cells after 24 h in the presence of 0.1% FCS alone (B) or plus SN50M at a 5 �M concentration (C),respectively. Hoechst staining (C) reveals nuclear condensation and fragmentation after NF-�B nuclear trans-location inhibition. D, DNA laddering after blunt end ligation PCR. Agarose gel under UV light after stainingwith ethidium bromide shows the different ladder production after blunt end linker ligation and 25 cycles ofPCR of 1 �g of genomic DNA in rat aortic medial and intimal SMCs after 24 h in the presence of 0.1% FCS aloneor plus SN50 at a 5 �M concentration; first lane, �X174 DNA marker from Sigma (D-0672). E, flow cytometricanalysis of rat aortic intimal cells cultured 15 days after injury, serum-starved for 24 h, and transfected with anantisense ODN specific for the translation inhibition region of rat Bax mRNA. After 45 min, SN50 at a 5 �M

concentration was added for 1 h, or, alternatively, PLC was added at a 50 �M concentration for 24 h with freshmedium.




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constitutive pathway that induces apoptosis duringmitosis (53,54). Serum or platelet-derived growth factor-AB stimulatesSMC survivin expression and prevents caspase activation (53).It is worth noting that intimal SMCs showed high constitu-tive IAP-1 and IAP-2 levels, and their PLC-induced reduc-tion was accompanied by an increase in apoptotic rate. IAP-1and IAP-2 are members of an antiapoptotic protein familythat binds and inhibits caspase 3, 7, and/or 9 (55). IAP-1 and-2 reductions seem to favor intimal SMC apoptosis followingNF-�B inhibition and Bax up-regulation. The relevance ofNF-�B in the apoptotic pathway of intimal SMCs was sup-

ported by the observation thatNF-�B down-regulation does notinvolve changes in p53 expression,a transcription factor involved inapoptosis of other cell types (56).Since serum deprivation favorsintimal SMC apoptosis, it is likelythat a PLC-induced arrest of DNAsynthesis in the G0-G1 phase is aprerequisite for the enhanced sus-ceptibility of intimal cells to invitro apoptosis. As a matter of fact,other reports document high con-stitutive NF-�B expression asessential for vascular SMC prolif-eration (49), and its inhibitionreduces proliferation and migra-tion in vitro (18).Apoptosis also depends on the

balance between NF-�B and I�Binhibitory proteins that bind andmaintain NF-�B in an inactive cyto-plasmic form (21), preventing itsrelease and nuclear translocation(15, 21, 57). Consequently, I�B lev-els regulate NF-�B bioavailability(58). Specific NF-�B activity inhibi-tion by either proteosome inhibitorsthat prevent I�B degradation, byantisense and decoy oligonucleo-tide, or by I�B-� overexpressionpromotes apoptosis induced byphysical or chemical agents (59).Our results indicated that PLC acts,at least in part, by increasing I�B-�bioavailability in intimal cells. Morein detail, PLC determined anincrease of synthesis and a parallelreduction of I�B-� degradation, anearly event in injured vessels (21,49), in part by reducing its phospho-rylation. Therefore, the I�B-�expression increase contributed toPLC-induced inhibition of NF-�Bactivity. This is indirectly confirmedby the observation that pretreat-ment with gliotoxin, which specifi-

cally inhibits NF-�B activation by reducing I�B degradation(39), prevents further increase of PLC-induced apoptosis. Asimilar process has been reported for other drugs interferingwith spontaneous or pharmacologically induced NF-�B activa-tion (60–62). The PLC effect coincides with previouslyreported data showing that I�B-� overexpression reduceslumen loss in a rabbit iliac artery restenosis model (23). As forSMCs, it has been previously reported that in low density orhighly proliferating conditions, NF-�B inhibition by 1-chloro-3-tosylamido-7-amino-2-heptanone, a proteolytic I�B-� deg-radation inhibitor, results in proapoptotic stimulus in arterial

FIGURE 6. PLC-induced NF-�B inhibition is mediated by I�B-�, p65, and p50 modulation. A, PLC reducesNF-�B activation by modulating I�B-� and subunit-p65 and p50 mRNA levels. Intimal cells were treated withdifferent PLC concentrations for 6 (left column) and 24 h (right column). I�B-�, p65, and p50 mRNA levels weremeasured using Q-PCR as described under “Experimental Procedures.” The results are expressed as means �S.E. of two independent experiments, each performed in triplicate (*, p � 0.02; **, p � 0.01). B, gliotoxinspecifically inhibits the degradation of I�B-�. Intimal cells after serum starvation were treated for 6 h with 5 �M

gliotoxin in the presence of 0.1% FCS, followed by 50 �M PLC for 24 h. Flow cytometric analysis shows that PLCfailed to further increase the apoptotic rate.




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SMC cultures, whereas in high density or low proliferating con-ditions, this effect was not detected (20).Our results strongly support PLC as a possible therapeutic

adjuvant in preventing postangioplasty restenosis. For the lat-ter, various approaches have been suggested (63), includingadenoviral gene-based (23) and oligonucleotide-basedmolecu-lar therapy (64) that targets the platelet-derived growth factorreceptor or Rho-kinase (65). In general, one limit to the phar-macological approach of preventing restenosis is the trouble inmaintaining a local or tissue concentration able to reproduce inhumans those beneficial effects documented in experimentalmodels without high or toxic drug concentrations. A desirablepharmacological agent would have to be anti-inflammatory, beable to inhibit SMC proliferation, be tolerable, and be free fromsignificant adverse effects (66). PLCoffersmany of these advan-tages, including safe clinical practice (25, 44). The effects onmyointimal thickening we describe here follow other previ-ously reported protective properties of PLC for blood vessels(64). Future trials should aim to determine PLC effectiveness asan adjuvant systemic pharmacological approach or in drug-eluting stents to prevent human restenosis.In conclusion, our findings provide new insights into the pos-

itive arterial remodeling induced byPLC.Moreover, we providenew data on the susceptibility of SMCs from different layers ofthe arterial wall to apoptosis, reinforcing the main role of phe-notypic heterogeneity in the apoptotic cascade of vascularSMCs.

Acknowledgments—We thank Dr. A. Valentini for Q-PCR analysisand A. Colantoni, S. Cappelli, L. Santangelo, and A. Volpe for techni-cal assistance.

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Luigi Giusto SpagnoliAugusto Orlandi, Arianna Francesconi, Marcella Marcellini, Antonio Di Lascio and

B Activityκof Aortic Intimal Smooth Muscle Cells by Modulating Nuclear Factor-Propionyl-L-carnitine Reduces Proliferation and Potentiates Bax-related Apoptosis

doi: 10.1074/jbc.M606148200 originally published online December 17, 20062007, 282:4932-4942.J. Biol. Chem.

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