Effect of APOSEC on chronic ischemic left ventricular dysfunction

Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction q Noemi Pavo a, Matthias Zimmermann b, Dietmar Pils c, Michael Mildner d, Zsolt Petrási e, Örs Petneházy e, János Fuzik a, András Jakab f, Christian Gabriel g, Wolfgang Sipos h, Gerald Maurer a, Mariann Gyöngyösi a,1, Hendrik Jan Ankersmit b, i, *,1 a Department of Cardiology, Medical University of Vienna, Austria b Department of Thoracic Surgery, Medical University of Vienna, Austria c Department of Obstetrics and Gynecology e Molecular Oncology Group, Medical University of Vienna, Austria d Department of Dermatology, Medical University of Vienna, Austria e Institute of Diagnostic Imaging and Radiation Oncology, University of Kaposvar, Hungary f Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Austria g RedCrossTransfusion Service for Upper Austria, Linz, Austria h Clinical Department for Farm Animals and Herd Management, University of Veterinary Medicine, Vienna, Austria i Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Vienna, Austria article info Article history: Received 23 November 2013 Accepted 20 December 2013 Available online xxx Keywords: Cell-free regeneration therapy Gene expression Immunomodulation Myocardial infarction Remodeling Animal model abstract The quantity of cells with paracrine effects for use in myocardial regeneration therapy is limited. This study investigated the effects of catheter-based endomyocardial delivery of secretome of 2.5 /C2 109 apoptotic peripheral blood mononuclear cells (APOSEC) on porcine chronic post-myocardial infarction (MI) left ventricular (LV) dysfunction and on gene expression. Closed-chest reperfused MI was induced in pigs by 90-min occlusion followed by reperfusion of the mid-LAD (day 0). At day 30, animals were randomized to receive porcine APOSEC ( n ¼ 8) or medium solution (control; n ¼ 8) injected intra- myocardially into the MI border zone using 3D NOGA guidance. At day 60, cardiac MRI with late enhancement and diagnostic NOGA (myocardial viability) were performed. Gene expression pro filing of the infarct core, border zone, and normal myocardium was performed using microarray analysis and confirmed by quantitative real-time PCR. Injection of APOSEC signi ficantly decreased infarct size (p < 0.05) and improved cardiac index and myocardial viability compared to controls. A trend towards higher LV ejection fraction was observed in APOSEC vs. controls (45.4 /C6 5.9% vs. 37.4 /C6 8.9%, p ¼ 0.052). Transcriptome analysis revealed significant downregulation of caspase-1, tumor necrosis factor and other inflammatory genes in APOSEC-affected areas. rtPCR showed higher expression of myogenic factor Mefc2 (p < 0.05) and downregulated caspase genes ( p < 0.05) in APOSEC-treated pigs. In conclusion, over- expression of MEF2c and repression of caspase was related to decreased infarct size and improved cardiac function in secretome-treated animals. Altered gene expression 1-month post-APOSEC treatment proved the long-acting effects of cell-free therapy with paracrine factors. /C2112014 The Authors. Published by Elsevier Ltd. All rights reserved. 1. Introduction Although great effort has been made to replace infarcted myocardium with regenerative cells, the current methods have shown marginal efficacy in clinical applications [1e5]. According to the ‘dying stem cell’ hypothesis, regenerative stem cells are already undergoing apoptosis even as they are being delivered into the ischemic myocardium [4]. Thus, the best way to attenuate infarc- tion may be to induce immunomodulatory mechanisms in a q This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. * Corresponding author. Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Department of Thoracic Surgery, Medical University of Vienna, Waehringer Guertel 43 18-20, A-1090 Vienna, Austria. Tel.: þ43 1 40400 6777 (of fice), þ43 1 40400 6857 (lab); fax: þ43 1 40400 6977. E-mail address: [email protected] (H.J. Ankersmit). 1 H.J. Ankersmit and M. Gyöngyösi share the last authorship. Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevi er.com/locate/biomaterials 0142-9612/$ e see front matter /C2112014 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.12.071 Biomaterials xxx (2014) 1 e10 Please cite this article in press as: Pavo N, et al., Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2013.12.071 paracrine manner [5e8]. We have shown previously that the secretome of apoptotic peripheral blood cells (APOSEC), which is derived from large numbers of easily-obtainable peripheral blood mononuclear cells (PBMCs), can be used to therapeutically regen- erate the myocardium after acute ischemic injury [9e12]. The optimal mode of delivering reparative cells or substances into the ischemic myocardium (i.e. intracoronary or percutaneous intramyocardial delivery) is still a matter of debate. Intracoronary delivery of cells increases the risk of temporary occlusion of the infarct-related artery and requires active migration of the cells from the vessel lumen to the injured tissue site [13]. The inherent dis- advantages of cell therapy include the obvious limitations of the relatively small number of autologous adult cells that are available and the relatively large injection volume of the cells, especially for intramyocardial application [14,15]. Using APOSEC, which contains a mixture of cytokines and growth factors, might avoid the disad- vantages of cell-based therapies and be more feasible while also enhancing therapeutic ef ficiency. Due to the early death of many regenerative cells when injected into the necrotic core, intramyocardial regenerative therapies instead target the infarct border zone, which shows signs of hi- bernating myocardium. Interestingly, some reports have described the metabolic, proteomic, and genomic differences between the infarct core and remote (normal) myocardium, but there is no genomic information about this special border zone of infarction [16,17]. Furthermore, no studies have investigated the effects of secretome-based therapies on gene expression in the core and border zone of myocardial infarction (MI). We hypothesized that percutaneous intramyocardial delivery of APOSEC would be safe and effective in a clinically relevant porcine model of chronic left ventricular (LV) dysfunction after MI. We further hypothesized that the trophic effect of APOSEC could cause structural changes, leading to improved hemodynamic function post-MI via altered gene expression. In our experiments we have injected APOSEC or placebo locally intramyocardially into the border zone of infarction using the catheter-based 3D NOGA- guided intramyocardial application method and investigated the LV function, infarct size and gene expression of the ischemic injured myocardium. 2. Materials and methods 2.1. Porcine model of acute MI and APOSEC production This study used a porcine closed-chest reperfused acute MI model. Domestic pigs ( n ¼ 22) underwent percutaneous 90-min balloon occlusion of the mid-left anterior descending coronary artery (LAD) followed by reperfusion ( Fig. 1 and Supplementary Materials and Methods ). APOSEC was produced from pig PBMCs (Supplementary Materials and Methods ). Animal investigations were carried out in accordance with the “Position of the American Heart Association on Research Ani- mal Use,” as adopted by the AHA on November 1 1, 1984. The study was approved by the Ethics Committee on Animal Experimentation at the University of Kaposvar, Hungary. Fig. 1. Study design. A. Schematic showing how APOSEC is prepared from peripheral blood mononuclear cells (PBMCs). B. Study design. C. 3D views of the left ven tricle derived by NOGA endocardial mapping and NOGA-guided injection into the border zone of chronic infarction. Brown dots mark the locations of the intramyocardial injections in the border zone of infarction. Color-coded 3D image (upper) were converted to a 2D polar map (below). Red represents non-viable infarcted myocardium, while gre en and yellow indicated the border zone of infarction. Normal viable myocardium is colored blue and purple. N. Pavo et al. / Biomaterials xxx (2014) 1 e102 Please cite this article in press as: Pavo N, et al., Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2013.12.071 2.2. Percutaneous intramyocardial delivery of APOSEC or medium solution using 3D NOGA guidance One month after MI (day 30; development of scar and LV remodeling; Fig. 1), the animals were sedated, intubated, and the patency of the infarct-related artery was confirmed by coronary angiography. LV hemodynamic parameters were measured via pigtail catheters. NOGA endocardial mapping was performed using a percuta- neous NOGA Star catheter (Cordis, Johnson & Johnson, Miami Lakes, FL, USA) as described previously [18]. After creation of the 3D shape of the LV infarction, the diagnostic NOGA Star catheter was replaced by a NOGA MyoStar injection catheter (Cordis, Johnson & Johnson). The pigs were randomized to receive either APOSEC (derived from 2.5 /C2 109 PBMCs, resuspended in 4 ml physiologic saline, 300- ml al- iquots) or medium as a control (resuspended lyophilized Cell Growth medium) in 11 /C6 1 locations of the border zone of infarction using 3D NOGA-guided percuta- neous intramyocardial delivery. The animals were allowed to recover. At 60 days post-infarction (30 days post-injection), control coronary angiography and NOGA endocardial mapping were performed while the pigs were under general anesthesia. 2.3. Cardiac magnetic resonance imaging (MRI) Cardiac MRI with late enhancement (LE) was performed 3 days and 60 days after the reperfused acute MI procedure using a 1.5-T clinical scanner (Avanto, Siemens, Erlangen, Germany) with a phased-array coil and a vector ECG system (Supplementary Materials and Methods ). The LV end-diastolic (EDV) and end- systolic (ESV) volumes, ejection fraction (EF), and infarct size were measured (Supplemental Materials and Methods ) [19]. 2.4. LV hemodynamic measurements LV hemodynamic measurements were performed via pigtail catheter and included systolic aortic pressure, LV systolic and end-diastolic pressures (EDP), LV contractility expressed as dP/dt and dP/dt/P, and LV end-diastolic stiffness (LV EDP/ EDV). 2.5. Myocardial viability, infarct transmurality and regional functional assessment by NOGA Using the 12-segment polar map analysis principles, the mean unipolar, bipolar, and local linear shortening values (UPV, BiP, and LLS, respectively) of the injected areas were calculated and reported as mV or %, respectively. 2.6. Histology The hearts were explanted at day 60 and sliced, and representative specimens of normal tissue and of the border zone and infarcted areas were fixed in 10% neutral buffered formalin, embedded in paraf fin, sectioned, and stained for hematoxylin- eosin, angiogenic marker of CD31 and stem cell factor CD1 17 ( Supplemental Materials and Methods ). 2.7. Gene expression analysis using RNA microarrays and real-time PCR (rtPCR) Tissue samples for RNA extraction were obtained directly from the infarct core, border zone, and remote myocardium and stored in RNAlater (Qiagen, Germany) at /C0 20 /C14 C. Total RNA was isolated from tissue samples using the RNeasy Microarray Tissue Mini Kit (Qiagen, Germany). RNA quality was checked on RNA Nano chips using the Agilent 2100 Bioanalyzer platform (Agilent Technologies). 2.7.1. RNA microarrays Microarray gene analysis was performed by Miltenyi (Miltenyi Biotec GmbH, Germany) ( Supplemental Materials and Methods ). The fluorescence signals of the hybridized Agilent Microarrays were measured using Agilent ’s Microarray Scanner System (Agilent Technologies) and analyzed using Agilent Feature Extraction Soft- ware (FES). Raw hybridization values were loaded into R (GNU R) using the R-package limma (v3.14.4)[20,21]. Microarray data have been uploaded to the Gene Expression Omnibus database ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc¼GSE47397). Biological interpretation of signi ficant gene lists was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6 [22]. Data were reported in annotational clusters or as single gene expression, heat maps, and Venn diagrams. 2.7.2. rtPCR of selected genes The mRNA of myocardial probes of all 3 regions from both groups of pigs was reverse transcribed to cDNA (Qiagen, Germany) and expression was quanti fied by rtPCR (Applied Biosystems 7500 Real-Time PCR System, Life Technologies, USA). The primers for the target sequences were designed using Primer3 software ( http:// primer3.wi.mit.edu/primer3web_help.htm; Microsynth, Switzerland) (Supplemental Table 1). The expression rates of the target genes were normalized to the geometric means of the housekeeping genes GAPDH, HPRT1, and PPIA, which were selected as endogenous controls due to their stable expression. The relative gene expression level was calculated using the DCt method (i.e. expression level relative to an endogenous control). The expression changes were calculated relative to expression levels in the normal myocardium of the same animal. 2.8. Statistics Continuous data of the two groups of pigs are reported as mean/C6 standard deviation. Differences between the groups were tested using the Student’s t test for continuous variables and thec2 test for categorical variables. A difference was considered statistically significant at p < 0.05. Data analyses and interpretations were performed by an experi- enced observer who was blinded to the randomization and to all study results. Statistical analysis was performed using SPSS 18.0 (SPSS Inc, USA) software. For gene array analysis, to test for all comparisons (i.e. differences between re- gions of interest: normal, border, and infarct and APOSEC vs. control) a linear model for each gene was fitted and the estimated coef ficients and standard errors for these contrasts were computed. Subsequently, moderated t-statistics, moderated F-sta- tistics, and the log-odds of differential expression were calculated by empirical Bayes shrinkage of the standard errors towards a common value. False discovery rates (FDR) below 5% were considered statistically signi ficant for each comparison. Higher FDR cut-offs were used for some contrasts, especially for the use of the gene lists for biological interpretation. Unsupervised hierarchical clustering was per- formed with the Euclidean distance as the distance function and the Ward algorithm in R, using centered and scaled log2 expression values. Principal Component Anal- ysis (PCA) was also performed for centered and scaled values. 3. Results 3.1. Effect of APOSEC on infarct size and hemodynamics Six pigs died during the reperfused MI procedure ( n ¼ 5) or 2 days later ( n ¼ 1). The remaining 16 pigs were randomized to the APOSEC group ( n ¼ 8) or to the control group ( n ¼ 8). Figs. 2 and 3 show the 3-day and final MRI data. Compared to control animals, the APOSEC-treated animals had signi ficantly smaller infarcts (13.92 /C6 1.34% vs. 21.58 /C6 2.09%, p < 0.05) and a significantly higher cardiac index (4.40 /C6 3.94 l/min/m 2 vs. 3.07 /C6 2.35 l/min/m 2, p < 0.05). There was also a trend towards a higher EF (45.4 /C6 5.9% vs. 37.4 /C6 8.0%, p ¼ 0.052) and signi ficant changes in LV EF from baseline to follow-up (FUP) in the APOSEC- treated vs. control animals. The area at risk as measured by MRI at day 3 was similar in the two groups (46 /C6 9% vs. 45 /C6 10% of the entire LV myocardium). The baseline, pre-injection, and post-injection hemodynamic data was not different between the groups (data not shown). Significantly lower EDP and end-diastolic stiffness was measured in the APOSEC group at the final FUP ( Fig. 3). Infarct transmurality, as assessed by MRI and NOGA bipolar maps, showed smaller trans- mural infarctions in the APOSEC-treated pigs ( Fig. 2). The APOSEC- treated animals had signi ficantly higher UPV and BiP values for the treated area with a trend towards higher LLS ( Fig. 4 ). 3.2. Angiogenesis and homing of endogenous c-kit positive cells in the border zone of experimental MI There was a significant increase in the number of CD31 þ cells in the APOSEC group in the border zone of infarction, indicating enhanced angiogenesis ( Fig. 5 ). The CD31 þ cell density per DAPI positive cells in the infarct and border zone were 15.3 /C6 1.9% vs. 12.6 /C6 4.5% and 19.9 /C6 1.8% vs. 1 1.3/C6 3.2% in the APOSEC vs. con- trols. Significantly (p < 0.05) higher numbers of CD1 17þ cells were found in infarct core (2.0 /C6 0.9% vs. 0.6 /C6 0.7%) and border area (3.0 /C6 1.4% vs. 0.9 /C6 0.8%) in APOSEC-pigs suggesting enhanced accumulation of endogenous cardiac stem cells as compared with medium-treated animals. 3.3. Gene expression differences between APOSEC- and medium- treated animals Differential gene expression of infarcted, border zone, and remote myocardium in the chronic failing hearts before treatment is N. Pavo et al. / Biomaterials xxx (2014) 1 e10 3 Please cite this article in press as: Pavo N, et al., Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2013.12.071 shown in the Supplemental Results. PCA and hierarchical clustering of the gene expression patterns of the corresponding myocardial areas in APOSEC- and medium-treated animals revealed 10 genes in the infarct core and 23 genes in the globally treated areas (infarct core and border zone) that showed significantly different expression compared to remote myocardium ( Fig. 6 , Table 1). Systematic and integrative gene analysis using DAVID Bioinformatics Resources identified and classi fied only a portion of the signi ficantly up and downregulated genes (1 1 of 23 genes, 47.8%). Due to the expression of single genes in several pathways, these genes could not be clas- sified into unique clusters. There was signi ficant downregulation of apoptosis-, inflammation-, and lipid metabolism-associated genes. This included repression of caspase-1 (apoptosis-related cysteine peptidase and interleukin 1-beta-convertase), tumor necrosis factor alpha (TNF-alpha), stromal derived factor (SDF)2-like protein 1 (endoplasmic reticulum stress-inducible gene), arachidonate 15- lipoxygenase (lipid metabolism), uroplakin 1B (coding a trans- membrane protein), claudin 3 (tight junction protein), and other genes that were not characterized further. Using less-stringent criteria (FDR <10%), additional 53 genes were detected that showed different expression levels in the APOSEC group vs. the control group. Of these, 24 genes were identified by the functional annotation analysis ( Table 1). Six of the 24 genes were overexpressed, while 18 were repressed. The APOSEC-affected myocardial area showed upregulation of insulin-like growth factor (an angiogenic growth factor), Kruppel- like factor (a regulator of vascular tone and homeostasis and an anti-atherogenic), myozenin (a gamma- filamin and alpha-actinin- binding protein in skeletal muscles), and a gene similar to glycer- onephosphate O-acyltransferase (thought to protect cells from oxidative stress). In contrast, some of the downregulated genes belonged to the in flammatory and immune system superfamily: killer cell lectin-like receptor subfamily K, cystatin F, ectonucleoside triphosphate diphosphohydrolase 1, CD2, complement component 1 q subcomponent, A-chain, C-X-C motif ligand 9 and 10, C-type lectin domain family 5, member A, CD247, and beta-2 micro- globulin. Other downregulated genes included some involved in lipid metabolism (a scavenger receptor for phosphatidylserine and oxidized low density lipoprotein), a histocompatibility antigen (MHC class II DR-alpha), a structural matrix protein (ameloblastin), and other less well characterized genes (suppression-inducing transmembrane adapter 1). If FDR <20% was used (data available at the GEO homepage), additional annotated overexpressed genes in the border area of Fig. 2. Left ventricular function and infarct transmurality assessed by MRI and NOGA. A. Short axis views of follow-up (FUP) late enhancement (LE) magnetic r esonance images show end-diastolic (EDV) and end-systolic (ESV) contours with LE 30 days after percutaneous intramyocardial injection of APOSEC or medium solution . Note the reduced ventricular volumes and infarct area in an APOSEC-treated pig. B. Bull ’s eye plots of LE-MRI data demonstrating treatment effects in representative APOSEC-treated (top row) and medium- treated (control) pigs (bottom row). Note that segmental infarct transmurality is reduced in the FUP images of an APOSEC-treated pig, while slight en largement of the infarct area is seen in a medium solution-treated pig. The following segments are affected: middle anterior, middle anteroseptal, apical anterior, and apical sept al. Left and middle panels: segmental infarct transmurality in 3-day and 30 days after injection (FUP, day-60) MR images. Transmurality was measured as the percentage of the LE a rea (i.e. the scar) relative to the volume of the corresponding myocardial segment. Right panels: treatment effects as visualized by differences between FUP and day-3 segmental me asurements. Positive values refer to growing segmental infarct transmurality (red) while negative values indicate reduced infarct transmurality (blue). C. Bull ’s eye plots of NOGA bipolar (transmurality) maps at baseline (injection) and FUP corresponding to the MRI polar plots (B) in the same APOSEC- and control pigs. Red represents greater infarct transmur ality. N. Pavo et al. / Biomaterials xxx (2014) 1 e104 Please cite this article in press as: Pavo N, et al., Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2013.12.071 APOSEC-treated animals belonged to 42 clusters (an additional 686 genes). These clusters included genes involved in purine metabolism (phosphodiesterase 3A, 4A, and 4B, pyruvate kinase), in the cell cycle (e.g. fizzy/cell division cycle 20 related 1), in the calcium signaling pathway (adrenergic beta-1 receptor, nitric oxide synthase 2, inducible, calmodulin), and in regulation of the a c t i nc y t o s k e l e t o n(fibronectin receptor, vinculin/cell adhesion related gene/). The clusters also included other cardiac-related genes, such as myocardin (important for cardiomyocyte survival and maintenance), GATA-binding protein 4 (transcription factor needed for normal heart development), and adrenergic beta-1 receptor. The main clusters of downregulated genes in the border zone of the APOSEC-treated animals included genes responsible for toll-like receptor pathways (CD40 molecule, CD86 molecule, chemokine ligands, interleukin-1 receptor-associated kinase 4, and toll-like receptor 3 and 7, which are responsible for the innate immune response and mediation of ischemic/reper- fusion injury), and genes involved in cardiomyopathy (trans- forming growth factor beta). 3.4. rtPCR rtPCR analysis of mRNA expression in the border zone of infarction in the APOSEC-treated animals con firmed that there was significant overexpression of the cardiac myogenesis and vascular development gene, myocyte-speci fic enhancer factor 2C (MEF2c), and repression of the apoptosis regulator caspase-3 ( Fig. 6 ). In the border zone, there was a trend towards higher expression ( p < 0.1) of angiogenic regulatory factor hypoxia-inducible factor 1-alpha and transcription factor GATA-4 in the APOSEC group compared with the control group. There were no signi ficant differences be- tween the groups in terms of the expressions of genes involved in stem cell homing (CXCR12), the transcription factor myogenin, and the extracellular matrix proteins matrix metalloproteinase-9 and transforming growth factor beta. 4. Discussion Here we demonstrated that 3D NOGA-guided injection of APOSEC is safe, feasible, and effective. APOSEC injection was asso- ciated with an enhanced vascular density and homing of c-kit þ endogenous stem cells in the peri-infarct and infarcted area, with a reduction of infarct size, and with a signi ficant increase in LV EF contractile function. Gene pro filing analysis of the APOSEC-treated myocardial areas revealed that there was downregulation of in- flammatory and apoptotic genes. Post-hoc validation of gene expression by rtPCR showed higher expression levels of myogenic factor and Mefc2 and robust downregulation of apoptosis regulator caspase-3. We previously showed that similar to the secretome of mesenchymal stem cells (MSCs) [23], APOSEC contains anti- apoptotic and regenerative factors that promote angiogenesis and myocardial repair [1 1,12]. In contrast to MSCs, PBMCs contain different types of mononuclear cells. However, one big advantage of PBMCs is that the available autologous cells are numerous. Even if ex vivo cell culture expansion of MSCs might provide suf ficient quantities of secretome, the biological characteristics of the MSC secretome depend on the number of passage of the cells with possibility to chromosome variability after passage 4, and on the cell donor, making it dif ficult to standardize secretome production Fig. 3. MRI results. Ejection fraction (EF) at day 3 (BASE) and at day 60 (follow-up; FUP) showing a signi ficant increase in EF at FUP in the APOSEC-treated pigs ( n ¼ 8) compared to the control pigs ( n ¼ 8). The APOSEC-treated pigs showed signi ficantly smaller infarct size and better cardiac indexes. There was signi ficantly lower end-diastolic pressure and end- diastolic stiffness in the APOSEC-treated group. * p < 0.05. N. Pavo et al. / Biomaterials xxx (2014) 1 e10 5 Please cite this article in press as: Pavo N, et al., Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2013.12.071 in large amount. Notably, a pre-de fined mixture of known regen- erative factors did not exert effects that were similar to those of the naturally derived secretome [23]. Our experiments investigated changes in the gene expression profile of the host tissue (ischemically injured myocardial cells) after direct contact with the injected mixture of paracrine factors (APOSEC). Even if reparative cells in cell-based therapies that are delivered to the ischemic area produce numerous signaling mole- cules, such as matrix proteins and anti-in flammatory and angio- genic substances, the host tissue response is the crucial factor in myocardial tissue regeneration. Changes in gene expression in ischemic cells depend on the severity of the injury. Interestingly, APOSEC mainly caused gene silencing in the pro-in flammatory cascade along with, to a lesser extent, some gene induction. However, it is important to note that almost half of the downregulated genes, and none of the upregu- lated genes, had currently known functions. These latter genes might belong to metabolic, angiogenic, or myogenic functional clusters that cause synergistic regenerative processes as demon- strated by infarct size reduction and prevention of ventricular remodeling. 4.1. APOSEC induces long-lasting differences in gene expression Although the studies were exploratory, previous analyses of the gene expression pro files of chronically infarcted, reperfused, and remodeled ischemic myocardium identi fied functional uni- or bimodal genes with known functions [24e30]. The published data, which are mostly from studies conducted in rodent MI models, are heterogeneous in terms of the number and type of differentially- Fig. 4. NOGA results and representative NOGA images. A. Baseline (BASE) and follow-up (FUP) unipolar voltage (an index of myocardial viability) of the injec ted area of APOSEC- and medium-treated (control) pigs. At FUP (day-60), the APOSEC group had signi ficantly higher unipolar voltage values (left) and bipolar voltage (index of infarct transmurality) values. There were trends towards higher local linear shortening voltage values (an index of segmental wall motion) at FUP. B. Representative NOGA images sho w the injection points around the infarcted area in APOSEC- and medium-treated pigs. The infarcted area is visibly smaller at FUP in the APOSEC-pigs, indicating that ventri cular remodeling was reduced in these animals. Color coding is as de fined in Fig. 1. N. Pavo et al. / Biomaterials xxx (2014) 1 e106 Please cite this article in press as: Pavo N, et al., Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2013.12.071 expressed genes. To our knowledge, this is the first report on gene expression profiles in closed-chest reperfused MI and also the first report to describe molecular biological changes in remote and infarcted myocardium and in the border zone of infarction (Supplemental Discussion). Locally injected APOSEC contains paracrine factors proven to be anti-inflammatory, leading to repression of active pro-inflammatory genes, such as genes coding for TNF-alpha, SDF2-like protein 1, and caspase-1. However, several genes that showed signi ficantly changed expression are currently not assigned to known function. The expansion of the FDR <5% cut-off to a FDR <10% cut-off gave more information about changes in gene expression in APOSEC- treated areas. Speci fically, this less-stringent cut-off identi fied several relevant genes with altered expression. There was upregu- lation of angiogenic factors and other vascular tone and homeostasis regulators and repression of pro-in flammatory genes such as com- plement components and histocompatibility antigens. 4.2. Comparison with literature data In contrast to the large animal model of reperfused MI used here, Jameel et al. surgically ligated the first and second diagonal branches of LAD of mini-swine and immediately injected 50 million bone marrow-derived multipotent progenitor cells [31]. This sur- gical cell transplantation resulted in a marked improvement in EF (51.2% vs. 35.7%) and in a decrease in infarct size (4.6% vs. 8.6%) at the 4-month FUP in treated vs. control groups, respectively, without engraftment of the cells. Gene expression analysis revealed 41 downregulated and 1 1 upregulated genes in the cell trans- plantation group; the authors attributed these changes to the paracrine effects of the injected cells. There was also down- regulation of genes in the in flammatory pathway, similar to what was seen in our experiments. The differences between the gene expression pathways identi fied in the two studies may due to the use of different MI models and treatments as well as to the pres- ence of different paracrine mediators. Similar to the MSC secretome [23], the secretome of apoptotic PBMCs (i.e. APOSEC) has the potential to block apoptosis and local inflammation, enhancing neoangiogenesis and limiting infarct expansion and remodeling. Through overexpression of regulatory genes at the FDR <10% level, such as Kruppel-like factor and myozenin, it is possible that APOSEC acts to restore vascular and muscle homeostasis. We did not see changes in any genes that are known to contribute to endogenous regeneration via the mobili- zation and homing of cardiac or bone marrow stem cells, such as genes coding for SDF-1 or for chemotactic cytokines. There is also the question of the importance of isolated gene expression i.e. expression of genes that are not functionally related to each other or that are in different pathways. However, small changes in the function of a key gene can act as a signal that triggers a biologically relevant effect. rtPCR of selected genes con firmed the signi ficant repression of the apoptosis regulator caspase-3. The increase in expression of hypoxia-inducible factor 1-alpha and GATA-4 in the border zone of Fig. 5. Angiogenic effect of APOSEC. A eD. H&E stained porcine myocardium 60 days after induction of myocardial infarction and treated with intramyocardial injection of APOSEC or medium (infarct core A and C, /40 /C2 magnification/, and border zone of infarction B and D, /10 /C2 magnification/, respectively). E eH. Immuno fluorescence CD31þ staining of the infarct core (E and G) and border areas (F and H) of an APOSEC or medium-treated pig, respectively. Red indicates CD31 þ cells, counterstaining with DAPI. I eL. CD1 17 (c-kitþ) immunofluorescence staining of the infarct core (I and K) and border areas (J and L) of an APOSEC or medium-treated pig, respectively. Green color represents CD 117þ cells, counterstained with DAPI. M and N. APOSEC-treated pigs show a higher density of CD31 þ and CD1 17þ cells both in infarct core and border areas, indicating enhanced level of microvascularization and homing of endogenous c-kit þ cardiac stem cells (* p < 0.05). N. Pavo et al. / Biomaterials xxx (2014) 1 e10 7 Please cite this article in press as: Pavo N, et al., Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2013.12.071 Fig. 6. Gene array analysis of differential expression of gene clusters in APOSEC-treated and medium-treated pigs in the infarct core and treated region (in farct core and border zone). A. Comparison of genes with signi ficantly different expression levels [false discovery rate (FDR) 5%] in infarct (left) and treated regions (right; infarct core and border zone) of APOSEC- and medium-t reated pigs (clustered and reported as a heat map). Name of genes: 1 ¼ similar to LOC513955 protein; 2 ¼ CD209 molecule; 3 ¼ n.i.; 4 ¼ claudin 3; 5 ¼ n.i.; 6 ¼ similar to trichohyalin; 7 ¼ arachidonate 15-lipoxygenase; 8 ¼ epididymal secretory protein; 9 ¼ CD209 molecule; 10 ¼ uroplakin 1B; 1 1¼ n.i.; 12 ¼ n.i.; 13 ¼ n.i.; 14 ¼ similar to trichohyalin; 15 ¼ similar to Uncharacterized protein CXorf21 homolog; 16 ¼ n.i.; 17 ¼ caspase 1. apoptosis-related cysteine peptidase (interleukin 1. beta. convertase); 18 ¼ n.i.; 19 ¼ n.i.; 20 ¼ tumor necrosis factor (ligand) superfamily member 13b; 21 ¼ similar to Stromal cell-derived factor 2-like protein 1 precursor (SDF2-like protein 1) (PWP1-interacting protein 8); 22 ¼ similar to LOC513955 protein; 23 ¼ arachidonate 15-lipoxygenase; 24 ¼ n.i.; 25 ¼ epididymal secretory protein; 26 ¼ uroplakin 1B; 27 ¼ n.i.; 28 ¼ n.i.; 29 ¼ n.i.; 30 ¼ n.i.; 31 ¼ claudin 3; 32 ¼ similar to Protein S100-A2 (S100 calcium-binding protein A2) (Protein S-100L); 33 ¼ n.i. n.i. ¼ not identified genes. B. Results of real-time PCR analysis of the expression of selected genes in APOSEC- and medium-treated pigs: HIF1- a: hypoxia-inducible factor 1- a; MMP-9: matrix metalloproteinase-9, CXCL12: chemokine (C_X_C motif) ligand-12; MEF2c: myocyte-specific enhancer factor 2c; MYOG: myogenin; GATA-4: zinc finger transcription factor that binds to the DNA sequence “GATA”-4; TGF-beta3: transforming growth factor-beta3. Y axis: expression level normalized to gene expression of the normal myocardium. Each group: n ¼ 8. * p < 0.05. N. Pavo et al. / Biomaterials xxx (2014) 1 e108 Please cite this article in press as: Pavo N, et al., Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2013.12.071 Table 1 Up and downregulated genes of the infarct core and combined treated (infarct core and border zone) areas in the APOSEC-treated animals. Systematic name Log fold changes Adjusted P value Name of gene Regulation in APOSEC group Infarct core FDR5% NM_001129972 /C0 1.426 0.029 CD209 molecule Down NM_213931 /C0 3.367 0.038 Arachidonate 15-lipoxygenase Down ENSSSCT00000010283 /C0 0.675 <0.001 Similar to LOC513955 protein Down NM_001160075 /C0 0.798 <0.001 Claudin 3 Down ENSSSCT00000013027 /C0 2.047 0.005 n.i. Down NM_001123212 /C0 1.081 0.001 Uroplakin 1B Down TC533994 /C0 1.573 0.043 n.i. Down ENSSSCT00000008105 /C0 1.249 0.016 Epididymal secretory protein Down XM_001927650 /C0 0.847 0.002 Similar to trichohyalin Down Combined treated areas FDR5% NM_214162 /C0 1.887 0.029 Caspase 1-apoptosis-related cysteine peptidase (interleukin 1-beta-convertase) Down NM_001097498 /C0 0.753 0.015 Tumor necrosis factor (ligand) superfamily member 13b Down ENSSSCT00000010283 /C0 0.815 0.003 Similar to LOC513955 protein Down NM_001160075 /C0 0.949 0.005 Claudin 3 Down ENSSSCT00000013346 /C0 1.18 0.007 Similar to uncharacterized protein CXorf21 homolog Down NM_001123212 /C0 1.287 0.01 Uroplakin 1B Down ENSSSCT00000011046 /C0 1.623 0.014 Similar to stromal cell-derived factor 2-like protein 1 precursor (SDF2-like protein 1) (PWP1-interacting protein 8) Down ENSSSCT00000007208 /C0 2.936 0.015 Similar to Protein S100-A2 (S100 calcium-binding protein A2) (Protein S-100L) Down ENSSSCT00000008105 /C0 1.249 0.016 Epididymal secretory protein Down XM_001927650 /C0 0.908 0.022 Similar to trichohyalin Down NM_213931 /C0 3.367 0.038 Arachidonate 15-lipoxygenase Down TC520240 /C0 1.61 0.047 n.i. Down ENSSSCT00000013027 /C0 2.617 0.013 n.i. Down AK233548 /C0 1.097 0.015 n.i. Down ENSSSCT00000011046 /C0 1.297 0.015 n.i. Down AK230687 /C0 2.509 0.015 n.i. Down ENSSSCT00000011934 /C0 1.895 0.016 n.i. Down A_72_P409998 0.929 0.007 n.i. Up TC601625 1.154 0.013 n.i. Up TC591961 0.592 0.015 n.i. Up TC540937 2.522 0.015 n.i. Up TC526711 3.032 0.029 n.i. Up AK232497 2.857 0.04 n.i. Up Combined treated areas FDR between 5% and 10% NM_214037 /C0 1.42 0.093 Ameloblastin Down DQ845172 /C0 1.5 0.097 Beta-2-microglobulin Down NM_213990 /C0 1.51 0.093 C-type lectin domain family 5, member A Down NM_213776 /C0 1.26 0.093 CD2 molecule Down NM_214155 /C0 1.18 0.093 CD247 molecule Down NM_001008691 /C0 3.35 0.093 Chemokine (C-X-C motif) ligand 10 Down NM_001114289 /C0 3.97 0.097 Chemokine (C-X-C motif) ligand 9 Down NM_001003924 /C0 1.89 0.093 Complement component 1, q subcomponent, A-chain Down NM_214153 /C0 1.32 0.093 Ectonucleoside triphosphate diphosphohydrolase 1 Down NM_214000 /C0 0.95 0.008 Haptoglobin Down NM_213813 /C0 2.46 0.063 Killer cell lectin-like receptor subfamily K, member 1 Down NM_001097415 /C0 0.81 0.089 Lymphocyte antigen 86 Down NM_001113706 /C0 1.64 0.093 MHC class II DR-alpha Down NM_213811 /C0 2.36 0.076 Scavenger receptor for phosphatidylserine and oxidized low density lipoprotein Down ENSSSCT00000007803 /C0 1.72 0.081 Similar to cystatin F Down AK232017 /C0 2.63 0.097 Similar to signaling threshold-regulating transmembrane adapter 1 precursor (suppression-inducing transmembrane adapter 1) (SHP2-interacting transmembrane adapter protein) (gp30/40) Down NM_001001632 /C0 1.45 0.08 Tropomyosin 3 Down NM_213883 3.05 0.063 Insulin-like growth factor 2 (somatomedin A) Up NM_001134346 1.41 0.093 Kruppel-like factor 11 Up NM_001025222 1.39 0.097 Myozenin 1 Up ENSSSCT00000007708 0.92 0.093 Prion protein Up ENSSSCT00000011141 1.01 0.093 Similar to glyceronephosphate O-acyltransferase Up AY609888 0.67 0.081 Similar to NAD(P) dependent steroid dehydrogenase-like Up AY609888 0.78 0.097 Similar to NAD(P) dependent steroid dehydrogenase-like Up Further not identi fied genes at FDR 5-10%: downregulated: EW261758, NM_001243319, AK397775, DN121339, TC533994, TC544302, L21161, ENSSSCT00000000690, NM_001161643, ENSSSCT00000005170, A_72_P418159, A_72_P418159, AK394244, TC594134, TC569821, CK451290, TC608152, AK230519, TC591665, TC5448 51, NM_001256770, TC555560, ENSSSCT00000017072, A_72_P773461, TC611786, AK239173; and upregulated in APOSEC-treated animals: TC543654, TC614889, BX923234, AK349090. FDR: false discovery rate, n.i.: not identi fied genes. N. Pavo et al. / Biomaterials xxx (2014) 1 e10 9 Please cite this article in press as: Pavo N, et al., Long-acting bene ficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2013.12.071 infarction showed trend towards higher expression in APOSEC- treated animals. MEF2c expression was induced and showed sig- nificant overexpression in the gene array when FDR<20% was used. 4.3. Limitations Control animals received local intramyocardial injections of medium solution, therefore the gene expression might be different from that in non-treated infarcted animals. Therefore it is not appropriate to conduct a direct comparison of our “ medium-solu- tion-treated” control group with literature data that describe gene patterns in rodent ischemic/reperfusion hearts [30] or in rodent or pig chronic ischemia/infarction models using placebo, sham oper- ation or no treatment in control animals [25e28,31]. Macroscopic identi fication of the real border zone of the infarction at day 60 that corresponds to the injected area at day 30 is crucial for obtaining valuable information from the experiments. In this study, localizing the LAD segment close to the origin of the second diagonal branch where the occlusion was made previously and identifying the margin of the infarcted area proved technically feasible. In addition, the differences in the gene expression pro files in the normal, border, and infarct areas con firmed that these areas were sampled correctly. 5. Conclusions APOSEC attenuates chronic myocardial ischemia-induced ven- tricular remodeling in a clinically relevant large animal model. Specifically, APOSEC induced long-lasting differences in gene expression that led to silencing of genes involved in apoptosis and inflammation. The gene pro filing results identi fied genes that represent targets for anti-remodeling and anti-ischemic therapy. Conflict of interest The authors declare competing financial interests. The Medical University of Vienna has a claimed financial interest (patent num- ber EP2201954, WO2010070105-A1, filed 18 Dec 2008). HJA is a shareholder of Aposcience AG, which owns the rights to commer- cialize APOSEC for therapeutic use. Funding This study was funded by the Christian Doppler Research As- sociation (Vienna, Austria), Aposcience AG (Vienna, Austria), the Ludwig Boltzmann Institute, and the Medical University of Vienna. Appendix A. 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