Intracoronary delivery of DNAzymes targeting human EGR-1...

ORIGINAL PAPERJournal of PathologyJ Pathol (2012)Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/path.2991

Intracoronary delivery of DNAzymes targeting human EGR-1reduces infarct size following myocardial ischaemia reperfusionRavinay Bhindi,1 – 4* Roger G Fahmy,1 Aisling C McMahon,3 Levon M Khachigian1 and Harry C Lowe1 – 3

1 Centre for Vascular Research, University of New South Wales, Sydney, NSW 2052, Australia2 Department of Cardiology, Concord Repatriation General Hospital, Concord, NSW 2139, Australia3 ANZAC Research Institute, Concord, NSW 2139, Australia4 North Shore Heart Research Group, University of Sydney, St Leonards, NSW 2065, Australia

*Correspondence to: Ravinay Bhindi, Centre for Vascular Research, University of New South Wales, NSW 2052, Australia.e-mail: [email protected]

AbstractDespite improvements in treatment, myocardial infarction (MI) remains an important cause of morbidity andmortality. Inflammation arising from ischaemic and reperfusion injury is a key mechanism which underpinsmyocardial damage and impairment of cardiac function. Early growth response-1 (Egr-1) is an early immediategene and a master regulator that has been implicated in the pathogenesis of ischaemia-reperfusion (IR) injury. Thisstudy sought to examine the effect of selective inhibition of Egr-1 using catalytic deoxyribonucleic acid molecules(DNAzymes, DZs) delivered via the clinically relevant coronary route in a large animal model of myocardial IR.It was hypothesized that Egr-1 inhibition with intracoronary DZ would reduce infarction size by modulatingits downstream effector molecules. Egr-1 DZs inhibited the adherence of THP-1 monocytes to IL-1β-activatedendothelial cells in vitro and retained its catalytic activity up to 225 min after in vivo administration. In a porcinemodel of myocardial IR (45 min ischaemia/3 h reperfusion), DZ was taken up in the cytoplasm and nuclei ofcardiomyocytes and endothelial cells in the myocardium after intracoronary delivery. Egr-1 DZs reduced infarctsize and improved cardiac functional recovery following intracoronary delivery at the initiation of IR in this largeanimal model of MI. This was associated with inhibition of pro-inflammatory Egr-1 and ICAM-1 expression, andthe reduced expression of TNF-α, PAI-1, TF, and myocardial MPO activity in tissue derived from the border zoneof the infarct. Taken together, these data suggest that strategies targeting Egr-1 via the intracoronary route afterIR injury in pigs have potential therapeutic implications in human MI.Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: myocardial infarction; gene modulation; DNAzymes; Egr-1

Received 4 February 2011; Revised 18 July 2011; Accepted 23 August 2011

No conflicts of interest were declared.

Introduction

Despite improvements in therapies optimizing epi-cardial coronary patency, acute myocardial infarction(AMI) and its sequelae present an increasing globalhealth burden [1]. Understanding and controlling thetranscriptional processes involved in myocardial injury,particularly in large animal models, is critical to therational development of novel treatments, such thatacute targeting represents an exciting new strategypotentially providing myocardial protection in thissetting [2–4].

A candidate gene target is early growth response-1 (Egr-1). Egr-1 is a prototype member of the zincfinger group of transcription factors and a key im-mediate-early master-regulator gene [2]. It has beenimplicated in the pathogenesis of ischaemia–reper-fusion (IR) injury in the heart [3], lung [4], gut [5], andkidney [6], as well as in neointima formation following

vascular injury [2,7]. Egr-1 is also known to positivelymodulate inflammation, thrombosis, and apoptosis byboth direct and indirect mechanisms [4,8].

A potent gene silencing strategy is the use of cat-alytic deoxyribonucleotide molecules—termed DNAenzymes or DNAzymes (DZs) [9]. These agents cleavethe phosphodiester linkage between a selected purineand pyrimidine through specific base pairing of theDZ arms with the target mRNA and a de-esterificationreaction [10]. Delivered locally in vivo, such Egr-1-targeting DZs inhibit endothelial cell and smooth mus-cle cell (SMC) proliferation, and neointima formationin a number of animal models [7,10,11].

More recently, a broader role for Egr-1 in theresponse to injury has been suggested within othercell types within the cardiovascular system [2,3,12].Specifically in myocardial IR injury in the rat, Egr-1 is acutely up-regulated, and we have shown thatDZs targeting rodent Egr-1 selectively reduce theinfarct size following direct intramyocardial injection

Copyright © 2012 Pathological Society of Great Britain and Ireland. J Pathol (2012)Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com

R Bhindi et al

[3]. However, there are well-documented difficultiesin translating small animal studies to achieving mean-ingful results clinically [13]. This is especially so inthe field of myocardial infarction, where differences indrug delivery method, timing, and the type of animalmodel may be of particular importance [13].

The present study was therefore undertaken in a largeanimal model of AMI, delivering DZs via the clinicallyrelevant intracoronary route to address some of thesedifficulties and to form a first step towards the clinicalevaluation of DZs as potential therapeutic agents formyocardial IR injury.

Materials and methods

DNAzyme preparationDZs were commercially synthesized with a 3′-linkedinverted thymidine at the 3′ termini to confer improvedstability and purified by HPLC (Tri Link, San Diego,CA, USA). The sequences used were DZF: 5′-GCGGGGACAGGCTAGCTACAACGACAGCTGCATTi-3′;and DZFScr: 5′-GCCAGCCGCGGCTAGC TACAACGATGGCTCCACTi-3′ (Ti = 3′ 3′-linked inverted T).DZF targets human and swine EGR-1, whilst DZFScris the size-matched control molecule with a preserved10–23 catalytic core [14] but scrambled flanking bind-ing arm sequences.

DNAzyme formulationIn vitro transfections were performed in growth mediaat a final DZ concentration of 0.4 μM with FuGENE6(Roche Diagnostics, Indianapolis, IN, USA) accordingto the manufacturer’s instructions. DZ was deliveredvia the intracoronary route in 2 ml volumes containingPBS, 300 μl of FuGENE6 (Roche Diagnostics), 1 mMMgCl2, and 1 mg of DZ.

DNAzyme cleavage in vivoThe assay was performed as previously describedwith minor modifications [15]. Anaesthetized 6-week-old female Balb/c nude mice received mid-dorsalintradermal injections of 100 mg of Dz. Two hun-dred and twenty-five minutes following the injection,skin around the injection site was carefully excisedand homogenized using Fast Prep FP120 Bio 101(Thermo Savant, Carlsbad, CA, USA) for three cyclesat 20 s/cycle in Lysing Matrix D tubes (Q-BIOgene,Carlsbad, CA, USA) containing 1.2 ml of TRIzol(Invitrogen, Gaithersburg, MD, USA), and DNA wasthen extracted according to the manufacturer’s instruc-tions. DNA was then purified using P30 micro bio-spin columns (Bio-Rad, Hercules, CA, USA). SyntheticRNA substrate (0.5 mg) was 32P-labelled using T4polynucleotide kinase and purified from unincorporatednucleotides using P30 micro bio-spin columns. Twomicrolitres of DNA isolated from tissue was incubatedwith 1 ml of 32P-labelled synthetic RNA substrate for

1 h at 37 ◦C. Two microlitres of the cleavage reactionwas then added to 4 μl of formamide loading dye andloaded onto 12% denaturing PAGE gels. Autoradiog-raphy was used to visualize cleavage products.

Endothelial–monocytic cell adhesion assayThe assay was performed essentially as previouslydescribed [15]. Human microvascular endothelial cells(HMEC-1) (American Type Culture Collection, Manas-sas, VA, USA) were propagated in MCDB 131 medium(Invitrogen) containing 10% fetal bovine serum (FBS)(Invitrogen), 2 mM L-glutamine, 10 ng/mL epidermalgrowth factor, 1 μg/ml hydrocortisone, and 5 U/mlpenicillin–streptomycin (all from Invitrogen).

Four groups of HMEC-1 cells grown in 24-wellplates were serum-arrested for 6 h. Three groups ofcells were transfected at 80–90% confluence with0.4 μM DZF, vehicle (VEH) or DZFScr. After 18 h,the cells were washed with PBS and fresh serum-free medium was added. A second transfection wasperformed using the same treatments and in addition,20 ng/ml IL-1β was added to the same groups. Sixhours later, THP-1 monocytic cells (American TypeCulture Collection) were added to each well at a den-sity of 2.5 × 105 cells per well. Alternatively, THP-1cells were transfected with 0.4 μM DZF, DZFScr orVEH via the same protocol and added to cytokine-treated endothelial cultures in 24-well plates at a den-sity of 2.5 × 105 cells per well. In both experiments,30 min after the addition of THP-1 cells, the wells werewashed thrice with PBS to remove non-adherent cells,and monocytic cells adherent to the endothelium werequantitated and expressed as the number of translucentcells per field using the 100× objective of a phase-contrast Olympus microscope. In separate experiments,Egr-1 and ICAM-1 protein expression was assessedvia western blotting in HMEC-1 cells subjected to thesame treatments described above but not co-culturedwith THP-1 cells.

Western blot analysisWestern blot analysis was performed on HMEC-1total cell extracts prepared as previously described[16] using rabbit polyclonal Egr-1 and ICAM-1 anti-bodies (Santa Cruz Biotechnology, Santa Cruz, CA,USA). The secondary antibody used was horseradishperoxidase-conjugated swine anti-rabbit IgG (1 : 1000;DAKO, Glostrup, Denmark). Immunoreactivity wasdetected using a Western Lightning chemiluminescencekit (PerkinElmer Life Sciences, Boston, MA, USA).

Pig model of IRProcedures were performed in accordance with theUniversity of New South Wales Animal Care EthicsCommittee guidelines. Five groups of male land-racepigs (n = 5 per group, 30–40 kg) were sedated withan intramuscular injection of ketamine (i.m. 6 mg/kg)xylazine (i.m. 0.4 mg/kg) and then anaesthesia was


Intracoronary delivery of DZs targeting human EGR-1 reduces infarct size following myocardial IR

induced and maintained using inhaled 3–5% isofluranevia an endotracheal tube.

All groups underwent left coronary angiography viathe right femoral artery using a 7Fr Hockey stick guid-ing catheter (Boston Scientific, Boston, MA, USA).One group underwent a sham operation. The remain-ing four groups were subjected to IR by endoluminalocclusion of the left anterior descending artery (LAD)immediately beyond the first diagonal branch for aperiod of 45 min, followed by reperfusion for a periodof 3 h. LAD occlusion was achieved by the inflation ofan appropriately sized coronary balloon and stent overa 0.014-inch coronary wire with subsequent deflationleaving the stent in situ to define the level of coronaryocclusion. Immediately prior to LAD occlusion, threegroups of pigs received an intracoronary bolus of eitherVEH, DZF or DZFScr into the LAD via a multifunctionprobing coronary catheter (Schneider, Bulach, Switzer-land) placed just distal to the first diagonal branch. Allpigs received aspirin prior to the procedure and i.v.heparin (100 units/kg) immediately after insertion ofthe femoral sheath and then hourly. Metoprolol (i.v.5 mg boluses) and lignocaine (i.v. 3 mg/kg boluses)were administered at the onset of ischaemia, following20 min of ischaemia, and immediately prior to reper-fusion.

Three hours after reperfusion, a lethal injection ofKCl followed by phenobarbital was administered toinduce euthanasia. Ten-millimetre transverse sectionsof the left ventricle distal to the implanted stent werethen incubated in 1% triphenyltetrazolium chloride(TTC) for 20 min at 37 ◦C and photographed usinga Canon IXUS camera. Infarct size measurementswere made by two independent investigators blindedto the groups. Sections were then placed in 4%phosphate-buffered formalin for subsequent histologyand immunohistochemistry. In addition, tissue from theborder zone of the infarcted area was snap-frozen inliquid nitrogen for subsequent biochemical and geneexpression analyses.

Pilot experiments to establish the efficacy of intra-coronary drug delivery used the same IR protocol butanimals received an intracoronary injection of fluores-cein isothiocyanate (FITC)-labelled DZ.

Echocardiography

2D transthoracic echocardiography was performedusing a Vivid 4 GE machine and a 2.5 Hz probe withparasternal short axis images at the mid-papillary levelbeing acquired in the right parasternal window priorto ischaemia and then every 15 min of the procedure.Left ventricular (LV) internal dimensions were mea-sured in end-systole (ESD) and end-diastole (EDD) inthe standard fashion on three consecutive beats andaveraged using an off-line analysis system (EchoPACPC). LV function was assessed using fractional short-ening (FS), which was derived as follows: FS = (EDD

− ESD)/EDD × 100 [17,18]. Echocardiography mea-surements were made by two independent investigatorsblinded to the groups.

Myeloperoxidase (MPO) activityTissue obtained from the infarct zone was stored inBuffer A at −80 ◦C. Tissue was homogenized usingLysing Matrix D (Qbiogene Inc, Carlsbad, USA) andthe BIO101 Thermo Savant FastPrep FP120 (QbiogeneInc) for 40 s at a power setting of 4.0. MPO activitywas assessed by separately measuring chlorination andperoxidation activity using the EnzCheK Myeloperox-idase Activity Assay Kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s instructions.

RT-polymerase chain reaction (PCR)Total RNA was extracted from myocardial tissueobtained from the infarct region in the area at riskof each group using the Total RNA Mini Kit (Bio-Rad, CA, USA). This was followed by homogenizationusing Lysing Matrix D (Qbiogene Inc) and the BIO101Thermo Savant FastPrep FP120 (Qbiogene Inc) for40 s at a power setting of 4.0. cDNA was generatedusing Superscript III and Oligo dT primer (Invitro-gen) according to the manufacturer’s instructions. Theamplification conditions used, with variable annealingtemperature and cycle number (shown in the Sup-porting information, Supplementary Table 1 with theprimer sequences), were as follows: 94 ◦C for 2 min;30 cycles of 94 ◦C for 30 s; annealing temperature asindicated in Supplementary Table 1 for 30 s; 72 ◦C for1 min; and an extension at 72 ◦C for 4 min. A list ofthe primers used may be found in the Supporting infor-mation, Supplementary Table 1.

ImmunohistochemistryImmunohistochemistry was performed on unstainedsections using specific antibodies to the transcriptionfactor Egr-1 (Santa Cruz Biotechnology), the cell adhe-sion molecule ICAM-1 (R&D Systems, MN, USA),and the oxidative stress marker nitrotyrosine (06–284;Upstate, Lake Placid, NY, USA). The secondary anti-body was diaminobenzidine (DAB)-labelled. Stainingintensity was graded by two blinded reviewers.

StatisticsData were analysed using the non-parametricWilcoxon–Mann–Whitney test in PRISM software(version 3; GraphPad, Inc, San Diego, CA, USA), withsignificance defined as p < 0.05 and data expressed asmean ± standard error of the mean.

Results

We first sought to determine whether intracoronaryadministration of DZs would be likely to have an


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Figure 1. (A) In an in vitro co-culture model, DZF transfection of endothelial cells demonstrated cytokine-induced up-regulation ofEgr-1 and ICAM-1 protein expression, which was selectively attenuated in the DZF-treated cohort. (B) In the same in vitro co-culturemodel, transfection of endothelial cells with DZF, but not (C) THP-1 monocytes with DZF, significantly attenuated the adhesion of THP-1monocytes to cytokine-activated endothelial cells. (D) DZF injected in vivo intradermally retained its ability to cleave its substrate afterbeing re-extracted from tissue 225 min later. This compared with no cleavage of substrate when either no DZF or DZFScr was added to thereaction. DZF showed robust cleavage of its substrate when added in vitro.

effect locally by observing the effects of local DZadministration on human microvascular endothelialcells (HMEC-1). In an in vitro co-culture model, fol-lowing endothelial cell (HMEC) activation with IL-1β,we observed both ICAM-1 and Egr-1 protein expres-sion to be up-regulated at 4 h. This up-regulation wasattenuated by transfection of HMECs with DZF but notDZFScr (Figure 1A). Importantly, Egr-1 and ICAM-1 suppression was associated with reduced adhesionof added THP-1 monocytes to the activated endothe-lial cells (Figure 1B). This effect was cell-specific andnot observed when THP-1 cells were transfected witheither DZF or DZFScr and added to activated endothe-lial cells (Figure 1C). These observations suggest thatlocally administered DZs are capable of transfectioninto endothelial cells, achieving biological efficacy,within a short time frame, and that DZ modulation of

monocytic adhesion to endothelial cells is influencedin large part by its effects on endothelial cells, ratherthan monocytes. Moreover, the observation of specificEgr-1 abrogation occurring with ICAM-1 protein inhi-bition suggests that ICAM-1 may be involved in theseeffects in endothelial cells.

We then assessed whether DZF remained catalyti-cally active in vivo over a time frame to allow in vivotherapeutic use. Following subdermal injection into theskin of nude mice for 225 min, DZF retained its abilityto cleave the substrate after being extracted from tissue(Figure 1D).

Next, the feasibility of intracoronary DZ deliveryto the porcine microvasculature and myocardium wasexplored using FITC-tagged DZ. Intracoronary FITC-DZ was delivered directly into the LAD via a multi-function probing catheter prior to the onset of 45 min



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Figure 2. (A) Intracoronary delivery of FITC-labelled DZ (upper panel) resulted in both endothelial cell (arrows) and cardiomyocyte uptake,in contrast to no uptake following the delivery of non-FITC-labelled DZ control (lower panel). (B) Egr-1, ICAM-1, TNF-α, PAI-1, and TFmRNA were up-regulated in myocardial tissue taken from the area at risk (AAR) following IR. These effects were selectively abrogated inthe DZF-treated cohort. (C) MPO activity was reduced in tissue obtained from the AAR in the DZF-treated cohort compared with the othergroups. (D) Nitrotyrosine expression was up-regulated in cardiomyocytes in the AAR following IR and was selectively reduced in animalstreated with DZF (scale bar = 50 μm). (E) The DZF-treated cohort had a significant improvement in myocardial function measured byechocardiography at the end of reperfusion, compared with the other cohorts after equivalent degrees of myocardial dysfunction inducedby myocardial IR. (F) Intracoronary DZF significantly reduced the infarct size by more than 40% compared with the other cohorts.

of ischaemia. Diffuse cytoplasmic and nuclear uptakeof DZ was demonstrated in endothelial cells and car-diomyocytes 3 h after reperfusion (Figure 2A).

The effects of bolus administration of 1 mg of intra-coronary DZ were then examined. Following 45 minof ischaemia and 3 h of reperfusion injury, Egr-1 andICAM-1 mRNA and protein expression were both up-regulated in the myocardium within the area at risk(AAR) (Figures 2B and 3). Egr-1 mRNA expressionand cytoplasmic and nuclear protein expression in car-diomyocytes was selectively inhibited within the AARin the DZF-treated group compared with the othercohorts (Figures 2A and 3). Likewise, ICAM-1 mRNAexpression and ICAM-1 protein ablation were observedin both cardiomyocytes and endothelial cells within the

AAR in the same groups, with the effects on ICAM-1protein expression observable in both cardiomyocytesand endothelial cells (Figures 2A and 3). The mRNAexpression of TNF-α, PAI-1, and TF, known Egr-1-dependent targets, was inhibited in myocardial tissuederived from the AAR only in the DZF-treated animals(Figure 2B). Surprisingly, a change in TNF-α, PAI-1,and TF protein expression was not observed at the timepoints examined. MPO activity, a marker of tissue neu-trophil (PMNL) density, was similarly suppressed onlyin the DZF-treated group, as shown by measures ofchlorination (Figure 2C) with similar results for mea-sures of peroxidation (data not shown). Nitrotyrosine,a marker of oxidative stress, was strongly expressed incardiomyocytes within the AAR following myocardial


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Figure 3. (A) Egr-1 protein expression was up-regulated in cardiomyocytes in the AAR following IR and was selectively reduced in theDZF-treated group. Representative sections are shown in the left panels (scale bar = 50 μm), and % of total cells staining > 2+ intensityis shown in the right panel. (B) ICAM-1 protein expression was up-regulated in both cardiomyocytes and endothelial cells (inset) in theAAR following IR and was reduced selectively in the DZF-treated group. Representative sections are shown in the left panels (scalebar = 50 μm), and % of total cells staining > 2+ intensity is shown in the right panel.

IR. This expression was selectively reduced in ani-mals treated with DZF, suggesting that Egr-1 inhibitionattenuates myocardial IR-induced myocardial oxidativestress (Figure 2D).

These changes in Egr-1 and ICAM-1 mRNA andprotein expression; TNF-α, PAI-1, and TF mRNAexpression; and MPO activity were accompanied byfunctional changes. Assessed by echocardiography,there was improved FS at the end of reperfusionin the DZF-treated group alone following equivalentdegrees of myocardial dysfunction during ischaemia(Figure 2E). Furthermore, the DZF-treated group had amore than 40% reduction in infarct size (IS) comparedwith the other cohorts (Figure 2F). There was nosignificant difference in haemodynamic conditions,arrhythmia rate, or anti-arrhythmic medication dosesbetween the cohorts (data not shown). These datatogether indicate that DZs targeting EGR-1 deliveredlocally via the coronary artery reduce the infarct sizeafter myocardial IR injury.

Discussion

These studies indicate the utility of specific genemodulatory therapy delivered via a clinically rele-vant method for the treatment of myocardial infarc-tion. They add to recent observations that Egr-1 isof biological importance in AMI in three important

ways. First, they show that Egr-1 plays a key rolein the pathogenesis of myocardial IR injury follow-ing temporary balloon occlusion of the coronary arteryin the pig, a model of AMI with clear analogy tothe human; second, that DNAzymes targeting humanEGR-1—delivered via the clinically relevant intracoro-nary route—have biological efficacy in this contextby reducing the infarct size and protecting cardiacfunction; and third, that DZ-mediated Egr-1 attenua-tion is associated with selective ICAM-1 suppression,a reduction in leukocyte accumulation both in vitroand in vivo, and a decrease in cardiomyocyte oxida-tive stress, suggesting a possible mechanism by whichefficacy is achieved.

Egr-1 has received recent recognition as a keyupstream activator controlling a variety of injuryresponses within a cardiovascular context [2]. While ithas been previously observed that Egr-1 up-regulationoccurs early following myocardial ischaemia [19], onlyour own recent work has identified this up-regulation tobe of biological importance using a rat model [3]. Thepresent studies add greatly to these initial observations,in that an in vitro and in vivo biological effect wasachievable using a DZ targeting the human sequenceEgr-1. In this regard, the present findings thereforeform a pre-clinical evaluation of such an approach.Similarly, the data provided here support the notion thatthese agents may provide a clinically applicable ther-apy, in that effective intracoronary delivery is achieved.In the context of acute interventional therapy for AMI,



such a route is clearly readily available. The data sug-gest that an intracoronary delivery route may even bepreferential, given the local effects of DZs observedin vitro and the local suppression of endothelial cellICAM-1 expression seen in vivo.

Lastly, the hypothesis that Egr-1 targeting mayachieve efficacy, at least in part, by ICAM-1 sup-pression and reduction in leukocyte accumulation isproposed. This mechanism is consistent with our ownprior observations [3] and is supported by Zhou et al[20]. The notion of neutrophil infiltration in AMI beingcontributory to rather than simply a result of infarc-tion is, however, controversial [21]. As has been welldocumented and discussed, clinical trials of antago-nists to leukocyte–endothelial cell interaction have notachieved benefit clinically [22].

It is therefore likely that mechanisms in additionto attenuation of ICAM-1-dependent inflammation areoperational following Egr-1 inhibition in this contextthrough other Egr-1-dependent molecules such as TNF-α, VCAM-1, TF, PAI-1, and p53 [4,23], and processessuch as coagulation and apoptosis. Selective inhibitionby DZF of TNF-α, TF, and PAI-1 mRNA, but not ofprotein, at the same time point may simply representthe time lag in translation of mRNA and protein. Itmay also reflect a threshold effect whereby pre-existingprotein masks a reduction in protein caused by the DZin immunohistochemical analysis.

The pro-coagulant and pro-inflammatory moleculePAI-1, known to be Egr-1-regulated, was shown inthe present study to be inhibited by selective Egr-1 targeting. Xiang et al have previously shown thatDNAzymes targeting PAI-1 enhance infarct neovas-cularization and reduce cardiomyocyte apoptosis [24].Similarly, inhibition of functional TF, VCAM-1, andTNF-α has been shown to reduce the infarct size inexperimental models of myocardial ischaemia reper-fusion [25]. We have previously shown that the pro-apoptotic factor p53 is inhibited by Egr-1 targetingin this context, with associated reductions in apopto-sis and infarct size [3]. Calcium mal-handling is alsoknown to contribute to ischaemia–reperfusion injury,and Egr-1 has been implicated in this process [26].Finally, ischaemic preconditioning [27], which reducesinfarction size, is known to significantly attenuate Egr-1 expression, lending support to the argument that Egr-1 is of importance in myocardial ischaemia reperfusion.These and other potential pathways will require furtherexamination.

The demonstration that DZ remains catalyticallyactive in vivo for at least 225 min, the duration of thepresent in vivo experiment, represents a key advancein highlighting the durability and potency of actionof this class of agent over a time period critical tothe development of IR injury. Moreover, whilst notspecifically addressed in the present study, we havepreviously demonstrated rapid potent effects in vivo ofDZ on leukocyte trafficking within the microvascula-ture [15], supporting the concept that these drugs can

exert their effects within the window of opportunitythat exists in the setting of IR injury.

These observations have the important limitations ofdelivery prior to a defined period of ischaemia andthe absence of an atherothrombotic infarction model,which could lead to an underestimation of the degreeof inflammation. Despite this, they provide key proof-of-principle support for the hypothesis that Egr-1 andits regulated genes are of crucial importance in thepathogenesis of human acute myocardial infarction.Furthermore, we have demonstrated that acute intra-coronary delivery of gene modulatory molecules is aviable clinical option for delivery and lends supportto the further development of DZs as potential clinicaltherapeutic agents in this context where unmet needexists.

Author contribution statement

RB conceived and performed experiments, interpreteddata, and wrote the manuscript. RGF and ACM per-formed experiments and interpreted data. LMK andHCL conceived experiments, interpreted data, andwrote the manuscript.

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SUPPORTING INFORMATION ON THE INTERNET

The following supporting information may be found in the online version of this article.

Table S1. Primer sequences, annealing temperature, cycle number, and expected amplicon size in base pairs (bp).

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Intracoronary delivery of DNAzymes targeting human EGR-1...

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