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A successful experimental model for intimal hyperplasia prevention using a resveratrol-delivering balloon

A successful experimental model for intimal hyperplasia prevention using a resveratrol-delivering balloon
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   A successful experimental model for intimalhyperplasia prevention using a resveratrol-deliveringballoon  Valerio Tolva, MD, FEBVS, a,b Silvia Mazzola, DVM, PhD, c  Pietro Zerbi, MD, d  Renato Casana, MD, a Mariangela Albertini, DVM, c  Laura Calvillo, PhD, e Francesca Selmin, PhD, f  and  Francesco Cilurzo, PhD, f  Milan, Italy  Objective:   Restenosis due to intimal hyperplasia is a major clinical problem that compromises the success of angioplasty and endovascular surgery. Resveratrol (RSV) has demonstrated a bene fi cial effect on restenosis from angioplasty. Un-fortunately, the physicochemical characteristics of RSV reduce the practicality of its immediate clinical application. This work proposes an experimental model aiming to setup an intravessel, elutable, RSV-containing compound. Methods:   A 140  m g/mL RSV sterile injectable solution with a suitable viscosity for intravascular administration by drug-delivery catheter (RSV-c) was prepared. This solution was locally administered in the common iliac artery of adult maleNewZealandWhiterabbitsusingadedicateddevice(Genie;Acrostak,Geneva,Switzerland) afterthe induction ofintimalhyperplasiabytraumaticangioplasty.TheRSVconcentrationsinthewall arterywere determined,andthethicknessoftheharvested iliac arteries was measured over a 1-month period. Results:  TheGeniecatheterwasappliedinrabbitvessels,andthelocaldeliveryresultedinaneffectivereductioninrestenosisafterplainangioplasty.Notably,RSV-cforcedintothearterywallbyballoonexpansionmightaccumulateintheinterstitialareas or within cells, avoiding the washout of solutions. Magni fi cation micrographs showed intimal proliferation wassigni fi cantlyinhibitedwhenRSV-cwasapplied.Moreover,noadverseeventsweredocumentedininvitroorinvivostudies. Conclusions:   RSV can be advantageously administered in the arterial walls by a drug-delivery catheter to reduce the risk of restenosis. (J Vasc Surg 2014; - :1-7.) Clinical Relevance:   The incidence of intimal hyperplasia varies in different risk populations (eg, diabetic patients) up to35% after bare-metal stent implantation and is reduced, but still exists and is problematic, after implantation of drug-eluting stents. Scouting experiences have shown that treatment with antioxidants improves endothelial cell coverage,decreases intimal hyperplasia, and reduces oxidative stress, thus promoting the patency of stents and grafts. In ourexperimental model, we observed that resveratrol locally administrated in the artery by a drug-eluting balloon has thepotentialities to reduce the intimal hyperplasia thanks to a local anti-in fl ammatory response. Restenosis due to intimal hyperplasia is a major clinicalproblem that compromises the success of angioplasty andendovascular surgery. 1,2 The pathogenesis of restenosis ismultifactorial, involving such events as endothelial injury,in fl ammation, platelet activation, and hyperplasia of the in-tima, primarily due to vascular smooth muscle cell (VSMC)replication. 3 The incidence of intimal hyperplasia varies indifferent risk populations (eg, diabetic patients), up to35% of whom require bare-metal stent implantation. Clin-ical evidence has shown that intimal hyperplasia is reducedbut continues to cause problems after the implantation of drug-eluting stents. 4 Overall, however, despite many yearsof clinical experience with drug-eluting balloons, the num-ber of large, high-quality, randomized clinical trials is low,and further data are urgently needed across the spectrum of clinical indications.Taxol (Bristol-Myers Squib, Princeton, NJ) and othercytostatic drugs destroy a cell ’ s ability to use its cytoskeletonin a  fl exible manner, and considering the clinical results,further research on a more physiologic mechanism of actionshould be pursued. Antioxidants are currently under investi-gation due to their protective activitywithinthe vessels. Rose-nbaum et al 5 showed that the endothelialization of prostheticgrafts was signi fi cantly reduced and anastomotic hyperplasia was signi fi cantly increased in rabbits fed a high-cholesteroldiet. Treatment with an antioxidant improves endothelialcell coverage, decreases intimal hyperplasia, and reducesoxidative stress, promoting the patency of prosthetic grafts.Resveratrol (RSV) is a polyphenolic phytoalexinic anti-oxidant that is produced by grapes and other plants inresponsetoinjuriousinfections.Thereareseveralpioneeringreports on RSV, including studies on the inhibition of the From the Surgical Department, Istituto Auxologico Italiano Istituto diRicovero e Cura a Carattere Scienti fi co (IRCCS), Presidio OspedalieroCapitanio a ; the Dipartimento di Scienze della Salute, University of Milan-Bicocca b ; the Dipartimento di Scienze Veterinarie e SanitàPubblica, c Dipartimento di Scienze Biomediche e Cliniche  “ L. Sacco ” , d and Dipartimento di Scienze Farmaceutiche, f  Università degli Studi diMilano; and Department and Laboratory of Cardiac Arrhtmias on Ge-netic Basis, Istituto Auxologico Italiano IRCCS. e  Author con fl ict of interest: none.Reprint requests: Valerio Tolva, MD, Istituto Auxologico Italiano IRCSS,Presidio Ospedalerio Capitanio, Dipartimento Chirurgico, Via G.Mercalli 30, 20121 Milan (e-mail: ).The editors and reviewers of this article have no relevant   fi nancial relationshipstodisclosepertheJVS policythat requires reviewers to decline reviewofany manuscript for which they may have a con fl ict of interest.0741-5214Copyright     2014 by the Society for Vascular Surgery. 1  arachidonate metabolism by interactions with the 5-lipoxygenase and cyclooxygenase pathways in leukocytes. 6-12 However, RSV attracted little interest until 1979, when the “ FrenchParadox ” 13 reportedthepositivebene fi tofadietcon-taining RSV, in particular the moderate consumption of red wine, for coronary heart disease.The molecular structure of RSV, unfortunately, re-duces its immediate clinical application for three main rea-sons: (1) its status as a highly lipophilic molecule; (2) its fast drifting from the  trans  -phase to  cis  -phase, representing anoxidized and inactive state, respectively; and (3) its low circadian bioavailability for rapid hepatic metabolism. Asconsequence of these features, the oral bioavailability of RSV is negligible because it is rapidly conjugated toimprove the solubility of the compound.The disposition of   14 C-labeled RSV, as orally and intra- venously administered in healthy volunteers, was evaluatedtoestimatetheextentoftheoraldoseabsorbed,thebioavail-ability of the unchanged drug, and the drug ’ s metabolicphase. RSV demonstrated high oral absorption but rapidand extensive metabolism, resulting in only trace amountsof unchanged RSV remaining until systemic circulation. 14 Fivemajormetabolitesweredetectedintheurinesamples, 14 plasma, and colorectal cancer tissues, 15 although all wereonly measured qualitatively due to a lack of available refer-ence materials. Metabolite (M) 1 was a RSV monoglucuro-nide. M2, an isomeric RSV monoglucuronide, was found ingreater abundance. M3 was a dihydroresveratrol monoglu-curonide, whereas M4 (resveratrol monosulfate) and M5(dihydroresveratrol sulfate) were two poorly resolved RSV sulfates. Although results on the ef  fi cacy of RSV reportedin the literature are controversial, very recent data obtainedin colon cancer cells have supported the notion that RSV,despite its low bioavailability, is able to act through its me-tabolites, mainly the sulfoconjugate but also the combina-tion of sulfate/glucuronide derivatives. 15,16 Despite the wide literature on RSV, only few preclinicalstudies have demonstrated the ef  fi cacy of RSV in an animalmodel or investigated the possibility of locally adminis-tering this antioxidant by drug-eluting stents. 6,7 On thebasis of our experience, we decided to setup a sterile, inject-able, and hydrophilic RSV-containing compound (RSV-c).This solution was locally administered in the common iliacartery of adult male New Zealand White rabbits using adedicated device. METHODS  All experiments in this study were conducted in accor-dance with the Institutional Guidelines for the Care andUse of Laboratory Animals and in accordance with theUniversità degli Studi di Milano Ethical Committee guide-lines. The Italian Ministero della Salute approved theresearch protocol. In vitro study Cell seeding.  Human coronary artery smooth musclecells (HSMCs; C-017-5C, Gibco-Invitrogen, Carlsbad,Calif) were seeded, according to the kit  ’ s instructions(2.5  10 3 cells/cm 2 to reach 80% con fl uence in 7-9 days)and maintained in culture at a lower density in six-wellplates to determine the proliferation rate. Experiments were conducted on cells between passages 4 and 6. Cell proliferation study.  HSMCs were divided inthree experimental groups:1. Control: medium (Gibco-Invitrogen Medium 231 with Supplement; n  ¼  8).2. RSV (Biotivia Italia, Verona, Italy): 25 mM dilutionin ethanol,  fi nal concentration 100  m M in medium(n  ¼  12).3. Vehicle (VEH): ethanol 0.4  m L/mL in medium(n  ¼  12). All groups were incubated for 48 h at 37  C. The me-dium and treatments were changed every 24 h to prevent RSV degradation. On day 2, the cells were washed twice with phosphate-buffered saline (PBS), harvested by trypsi-nization, and counted with a Z2 Coulter counter (Beck-man Coulter Inc, Fullerton, Calif). Preparation of the RSV-c  Toobtainasterile,injectablesolution,weoptimizedthesolubility of RSV and its viscosity. RSV was dissolved in a0.40% w/v tamarind seed polysaccharide and 2.50% w/v Kolliphor HS15 (BASF, Florham Park, NJ) solution that had been previously sterilized by vapor steam under pres-sure. Afterwards, the solution was  fi ltered by disposable,sterile, and pyrogen-free polyether sulfone  fi lters at thenominalporosityof0.22 m mintoasterileambertypeIglasscontainer at a laminar air fl ow workbench. A media  fi ll pro-gram assured the validation of the aseptic process. The con-tainers were stored at room temperature until use. A placebo formulation was prepared according to theprocedure reported above.To avoid the fast removal of RSV by the blood streamafter intimal administration, a high-viscosity solution was pre-pared. Tamarind seed polysaccharide, a well-known biocom-patible polymer, was used to confer a suitable cinematic viscosity to the vehicle for administration by drug-elutingballoon. According to our previous experience, 12 the viscosity of the preparation was based on the nonionic contrast me-dium iomeprol (Iomeron; Bracco, Milan, Italy), with kine-matic viscosity at 37  C of 5.617 6  0.034 mm 2 /s. The  fi nalkinematic viscosity of the RSV-c was very close to that of the reference solution (6.097  6  0.0379 mm 2 /s). KolliphorHS15 is a surfactant used for parenteral preparations and was added to the formulation to dissolve 140  m g/mL RVS. In vivo study  Animals.  Thirty-six male New Zealand White rabbits, weighing from 2.8 to 3.6 kg, were assigned randomly andin equal numbers to different study subgroups (Table). Theanimals were housed in a dedicated facility and fed withstandard diet with free access to water.  Angioplasty and delivery procedures.  Preoperativecolor Doppler ultrasound (Titan; SonoSite Inc, Bothell, JOURNAL OF VASCULAR SURGERY  2  Tolva et al   --- 2014   Wash) was used to scan all animals to measure their meanarterial size and femoral artery peak systolic velocity (PSV)and end-diastolic velocity (EDV) to obtain preoperativemorphologic and velocimetric data. The mean right iliacartery diameter was 3 6 0.6 mm. The right femoral artery PSV was 90 cm/s and EDV was 60 cm/s.The animals were treated with an anesthetic protocolto ensure the full unconsciousness during the surgical pro-cedures and an excellent level of perioperative analgesia.Rabbits were premedicated with a subcutaneous injectionof dexmedetomidine (80  m g/kg; Dexdomitor, OrionCorp, Milan, Italy), ketamine (25 mg/kg; Ketavet, Inter- vet Productions SRL, Latina, Italy), and buprenorphine(20  m g/kg; Temgesic, Schering Plough Spa, Milan, Italy). After the induction, a steady depth of anesthesia was main-tained during the experimental protocol by the continuousinfusion of a dilute solution of propofol (1-3 mg/kg/h;Fresenius Kabi, Isola della Scala, Italy), into the auricular vein. All animals were heparinized with 80 IU/kg heparinsulfate (Phararepa; PharmaTex, Milan, Italy) 2 minutesbefore the introducer sheath was inserted. Afterthesurgicalincision,thesuper fi cialfasciaandmus-cles were separated bluntly, layer-by-layer, until the right common femoral artery was exposed. Proximal and distal vascular controls were assured with two 2-mm silicon liga-tures to minimize bleeding. Using the modi fi ed Seldingertechnique, we directly inserted a 4F Cook Micropuncturesheath(WilliamCookEuropeApS,Bjaeverskov,Denmark).To induce and establish intimal hyperplasia in the rabbit  ’ siliac artery, we performed a traumatic angioplasty with aBantam Alfa balloon catheter (diameter: 3.0 mm, length:2cm;ClearStream TechnologiesLtd,Enniscorthy,Ireland) with Doppler ultrasound monitoring. Injury was created by in fl ating the balloon to 8 atm with a manometer syringe for3min.Afterwards,thecatheterwasremoved,andtheGeniedrug-delivery catheter (Acrostak, Geneva, Switzerland) was introduced into the aorta-iliac bifurcation by colorDopplerultrasoundmonitoringtodeliverthe20mLcarrieror RSV-c (Table).The Genie balloon catheter is designed to dilate andtreat arteries by the local delivery of the proposed solution(RSV-c) and reference compounds (vehicle), ensuring afully controlled release to the vessel wall. Clinical experi-ences with Genie suggested delivery of RSV in 2 minutes,maintaining a mean in fl ating pressure of 6 atm. 17  At theend of the procedure, the right common femoral artery  was sutured with 7-0 polypropylene interrupted stitches.The blood supply of the leg was not affected by this surgi-cal procedure.Pain control and antibiotic coverage were achievedthrough the subcutaneous administration of buprenor-phine (15 mg/kg; Temgesic) plus meloxicam (0.2 mg/kg; Mobic, Boehringer Ingelheim, Milan, Italy) plus enro- fl oxacin (10 mg/kg, Baytril; Bayer SpA, Rome, Italy). At the time points indicated from the experimental protocol(Table), the balloon-treated aorta-iliac bifurcation was sur-gically explanted, and the animals were euthanized with ananesthetic overdose. Tissue and serum measurements of RSV-c.  Before,immediately after the delivery procedure, and at predeter-mined times ranging from 15 min to 90 min, blood sam-ples (2 mL) were collected from the auricular vein.Samples were immediately centrifuged at 3500 rpm for15 min at 4  C, and the serum was frozen and storedat   80  C until further processing. The serum was thawed,250  m L was added to 1 mL methanol, vortexed 1 minute,and centrifuged at 5000 rpm for 15 minutes at 15  C. Thesupernatant was analyzed by the high-performance liquidchromatography (HPLC) method reported below.The animals were euthanized at 2, 6, and 24 h afteradministration (Table). Segments of the iliac artery wereremoved and washed with a physiologic solution, and any  visible blood coagula or residual fat tissues were carefully removed. The tissue was cut into a small specimen, placedin a vial containing 0.2 mL methanol, and sonicated by aMicroson ultrasonic cell disruptor (Qsonica, Newton,Conn) in an ice bath for 30 minutes.The concentration of RSV was assayed using thefollowing HPLC method:RSV was analyzed by HPLC (ChemStation HP 1100, Agilent, Santa Clara, Calif), as previously described. 14 Brie fl  y, an ODS Hypersil analytical column (Thermo Sci-enti fi c, Waltham, Mass) was used as the stationary phase(4.6    100 mm; 3  m m particle size), and a combinationof MilliQ (Millipore Corp, Billerica, Mass) water/meth-anol/tri fl uoroacetic acid (65%/35%/0.3%, v/v/v) wasused as the mobile phase. The  fl ow rate was controlled at 0.9 mL/min. The ef  fl uent was monitored at 304 and286 nm for the determination of   trans  -RSV and  cis  -RSV (or isomeric RSV), respectively. The injection volume was10  m L, and the analysis was performed at 30  C. Becauseauthentic RSV metabolites were not available as referencematerials, the amounts of the metabolites were calculatedas  “ RSV equivalents, ”  using the assumption that the recov-ered characteristics and relationship between peak area ra-tio and concentration were the same as for the parent RSV. Histologic measurements The harvested iliac arteries of sham, carrier, and RSV-cgroups were  fi xed in 10% buffered formalin; then, cross-sections were cut and embedded in paraf  fi n. A morpho-logic evaluation of the vessel wall thickness and intimal Table.  In vivo study design a Design Group PK Sham Carrier RSV-c  Subgroup b 2, 6, 24 3, 30 3, 30 3, 30Time 2, 6, and 24 h 3 and 30 d 3 and 30 d 3 and 30 dCompound RSV-c None Carrier RSV-cProcedure DDC Simple PTA PTA  þ DDC PTA  þ DDC DDC,  Drug-delivery catheter;  PK,  pharmacokinetic;  PTA,  percutaneoustransluminal angioplasty;  RSV-c,  resveratrol compound. a  Administration of 20 mL carrier or RSV-c by DDC. b n  ¼  4 for each subgroup.JOURNAL OF VASCULAR SURGERY  Volume - , Number -  Tolva et al   3  hyperplasia was performed on each tissue block, cutting 4- m m sections. Hematoxylin and eosin staining clearly show the internal elastic lamina, the external elastic lamina, theintimal thickness, and the cells in the vessel wall. Each his-tologic section was scanned, the intimal and external andinternal elastic lamina were manually identi fi ed. Intimallayer thickness (ie, distance between the lumen and internalelastic lamina), medial layer thickness (ie, distance betweeninternal and external elastic lamina), lumen area, intimallayer area, and medial layer area were measured usingImageJ image analysis software (National Institutes of Health, Bethesda, Md;, 1997-2014).Thickness was measured at four points (ie, cardinalpoints: north, east, south, west), and the mean value wastaken. Then, to compare different treatment groups, theintimal/medial thickness ratio, the intimal/medial area ra-tio, and the intimal proliferation index (ratio of intimal areato [intimal þ medial] area) were used for statistical analysis.For group RSV-c30 and sham30, proliferating cells in theintimal layer were identi fi ed by immunohistochemistry, us-ing antibodies against Ki-67 protein (Clone MIB-1;DAKO, Glostrup, Denmark). Results of Ki-67-positivecells count were normalized for Ki67/ m m 2  All histological analysis were performed in a blindedfashion. Statistical analysis Data are reported as the means  6  standard deviation.The data from the proliferation studies were analyzed by taking the means of three counts for each well and thenconsidering each of the independent wells as a separatedata point. Comparisons among the groups were per-formed by analysis of variance with Bonferroni correction. P   <  .05 was considered signi fi cant. RESULTSIn vitro evaluation.  The number of cells wasexpressed as cells/mL. Cell proliferation in the presenceof the vehicle was similar to that in the control group(25  6  5 cells/mL in VEH group vs 25  6  4 cells/mL incontrol group;  P   ¼  .8, not signi fi cant [NS]). RVS signi fi -cantly inhibited HSMC proliferation (Fig 1) compared with the VEH and control group (20 6 3 cells/mL in RSV  vs 25  6  5 cells/mL in VEH and 25  6  4 cells/mL incontrol,  P   <  .01). Tissue measurements of RSV-c.  The RSV wasdetectable and quanti fi able in serum only immediately afteradministration ( < 0.2  m g/mL), and none of its metabolites were found.Only traces of unchanged RSV were detectable in thearterial samples from the iliac vessel until 6 h after theballooning procedure (Fig 2). However, RSV M1 andM4 metabolites were qualitatively identi fi ed # 2 h and per-sisted over the considered time period. After prolonged pe-riods of time, the concentrations of these two metabolitesdecreased and the M2 and M3 metabolites became detect-able. From these data, we posit that the RSV-c infused in asolution at the site of the artery by a drug-delivery catheter was retained in the tissue and then underwent an extensiveand rapid metabolism. In vivo evaluation and histologic measurements. No procedure-related deaths were documented during thehousing period. Before euthanasia, all rabbits underwent color Doppler ultrasound imaging to test for patency and velocimetric patterns. The RSV-c30 group always showedthe patency of their iliac vessels, which was associated withan average value of PSV and EDV of 70  6  7 and 40  6 5 cm/s, respectively. In the sham30 and carrier30 groups, we found an average value of PSV and EDV of 30  6  20and 20  6  15 cm/s, respectively. When we analyzed thedata together, we found a signi fi cant difference ( P   <  .05)between the RSV-c30 and the sham30 or carrier30 groups. A literature review showed a lack of comparisons of the Fig 1.  In the human coronary artery smooth muscle cells(HSMCs) proliferation study, resveratrol ( RSV  ) slowed cellgrowth in the absence of pathologic stimuli after 48 hours of treatment. Vehicle ( VEH  ) did not alter proliferation compared with the control. * P   <  .01 RSV vs VEH and control. Fig 2.  Recovery of resveratrol ( RSV  ) and its metabolites in artery tissue after the administration of RVS solution (10 mL) by a drug-eluting balloon. Four major metabolites were identi fi ed as RSV monoglucuronide ( M1 ), isomeric RSV monoglucuronide ( M2  ),dihydroresveratrol monoglucuronide ( M3  ), RSV monosulfate( M4  ). Because standards are not commercially available, RSV metabolites are expressed as the peak area at 286 nm corrected forthe conversion factor 1.5 normalized for the artery sample weight.The correction factor was calculated as the ratio of peak area of RSV and photodegraded RSV at 304 and 286 nm. JOURNAL OF VASCULAR SURGERY  4  Tolva et al   --- 2014  data on velocimetry and vessel diameters in the rabbit;nevertheless, we considered the  fl ow reduction (PSV from90 to 30 cm/s) in the femoral artery in the sham30 andcarrier30 groups to be a result of a tight stenosis. Autopticspecimens showed no macroscopic differences among thegroups.Those animals euthanized at the early time point (sham3, carrier3, and RSV-c3) showed hyaline degenera-tion as a result of the percutaneous transluminal angio-plasty (PTA), irrespective of the treatment. However, wenoted some peculiar differences in the disposition of ialindegeneration in RSV-c3. Complete circumferential lesions were present in groups sham3 and carrier3, but not inRSV-c3. Despite the presence of ialin lesions in RSV-c3, we did not observe the same homogenous ialin intimallayer as was found in sham3 and carrier3. Micrographs were able to spot some cellularized areas in RSV-c3(Fig 3). Regardless of whether an empirical evaluation of this early result could be interpreted as a positive effect of RSV-c after PTA, we considered this phenomenon only as a part of the modulatory effects of RSV-c on the local in- fl ammatory response after PTA.The morphologic data at day 30 are summarized inFig 4. The intimal hyperplasia was signi fi cantly greater insham30 group compared with the other treated groups( P   <  .05 by one way analysis of variance with Bonferronicorrection). The comparison between the carrier30 andRSV-c30 groups evidenced the bene fi cial effect of RSV.Indeed, the mean intimal proliferation index value ob-tained with the RSV-c was w 25% lower than that obtained with the carrier. Nevertheless, this difference did not result signi fi cance according to our statistical evaluations.Differences between RSV-c30 and sham30 were statis-tically signi fi cant ( P   <  .05) when counting Ki-67-positive Fig 3.  Hematoxylin and eosin-stained histologic cross-sections of rabbit iliac artery after angioplasty (srcinalmagni fi cation   40).  A,  Group RSV-c3 (resveratrol compound at 3 days) d intimal layer denudation with patchy cel-lularized medial layer and diffuse ialin degeneration of vessel wall.  B,  Group RSV-c30 (resveratrol compound at 30 days) d moderate reduction in lumen size due to slight intimal hyperplasia.  C,  Group carrier30 (group that receivedcarrier at 30 days) d marked reduction in lumen size and asymmetrical thickening of the wall, mainly due to intimalhyperplasia.  D,  Group sham30 (group that received sham treatment at 30 days) d marked intimal hyperplasia, resultingin high intima/media ratio. Fig 4.  Parameters calculated from histologic measurements:comparison of sham30 (sham group at 30 days), carrier30 (carriergroup at 30 days), and RSV-c30 (resveratrol compound at 30 days). Data are shown as mean 6 standard error.  IRI  , Intimalproliferation index (ratio of intimal area to [intimal  þ  medial]area). * P   <  .05 compared with sham30 group. JOURNAL OF VASCULAR SURGERY  Volume - , Number -  Tolva et al   5
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