Dynamic induction of pro-angiogenic milieu after transplantation of marrow-derived mesenchymal stem cells in experimental myocardial infarction.

IJCA-17700; No of Pages 14 International Journal of Cardiology xxx (2014) xxx–xxx

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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

Dynamic induction of pro-angiogenic milieu after transplantation of marrow-derived mesenchymal stem cells in experimental myocardial infarction☆ Reza Rahbarghazi a,b,c,1, Seyed Mahdi Nassiri a,1,⁎, Seyed Hossein Ahmadi d,1, Elham Mohammadi a,1, Shahram Rabbani d,1, Atefeh Araghi e,1, Hossein Hosseinkhani f,1 a

Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Umbilical Cord Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran c Biotechnology Research Center, Tabriz Branch Azad University, Tabriz, Iran d Tehran Heart Center, Tehran University of Medical Sciences, Tehran, Iran e Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran f Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology (TAIWAN TECH), Taipei, 10607, Taiwan b

a r t i c l e

i n f o

Article history: Received 8 June 2013 Received in revised form 18 January 2014 Accepted 9 March 2014 Available online xxxx Keywords: Angiogenesis Acute myocardial infarction Mesenchymal stem cells Myocardial regeneration

a b s t r a c t Background: Cell-based pro-angiogenic therapy by bone marrow mesenchymal stem cells (MSCs) has been touted as a means to reducing the adverse effects of cardiac remodeling after myocardial infarction (MI). Milieu-dependent regulation of pro-angiogenic potential of MSCs after infarction remains to be elucidated. In this study, the effects of marrow-derived MSCs on the kinetics of angiogenesis signaling factors were investigated in a rabbit model of MI. Methods: MI was induced in rabbits, and the animals were randomized into two groups (cell transplantation and control, each group with 21 animals). 1 × 106 autologous marrow-derived MSCs were injected into the myocardium of the border zone after transfection with a green fluorescent protein (GFP) lentiviral reporter vector. Control animals received PBS vehicle only. Effect of the transplanted cells on the hearts was evaluated over time by pathological, immunofluorescence, western blotting, immuno-electron microscopy, and echocardiographic analyses. Results: Transplanted GFP-positive MSCs were enriched with time in the peri-infarct border zone with differentiation potential into three major cell types of the heart, including cardiomyocytes, endothelial cells, and smooth muscle cells, and there was significant augmentation of microvascular density. The transplanted cells could change the milieu of the injured myocardium to increase the expression levels of VEGF as well as the ratio of Ang-2 to Ang-1, and to reduce the ratio of phosphorylated Tie2 to Tie2. Conclusion: An angiogenesis-promoting milieu was induced after the transplantation of marrow MSCs in the injured myocardium. Compared with the resident cells, the transplanted cells had a greater rate of cellular kinetics in the infarcted myocardium. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction In recent years, cardiovascular-related illnesses, most likely coronary heart disease (CHD), are assumed to be the major cause of heart failure in both developed and developing countries [1]. Long-term blood deprivation will eventually lead to biological responses such as tissue ☆ Acknowledgment of grant support: This study was supported by a research grant from the University of Tehran and a grant from Tehran Heart Center. ⁎ Corresponding author at: University of Tehran, Faculty of Veterinary Medicine, P.O. Box: 14155-6453, Qareeb St., Azadi Ave., Tehran, Iran. Tel.: +98 2161117128; fax: +98 2166438327. E-mail addresses: [email protected], [email protected] (S.M. Nassiri). 1 This author takes responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.

necrosis, inevitable production of collagen, and replacement of the cardiomyocytes by inefficient contractile myofibroblasts in the infarcted area that contribute to adverse ventricular remodeling and unfavorable outcomes [2]. Angiogenesis, the formation or recruitment of new blood vessels, has been the focus of many therapeutic approaches during myocardial infarction (MI) [3]. Intricate mechanisms are involved in the development of the blood flow in the infarcted area, in which angiogenic growth factors, cytokines and different cells act in an orchestrated manner [4]. Understanding the different aspects of the underlying mechanisms at molecular level will strengthen our knowledge on angiogenesis pathways and help to consider as much related phenomena as possible during the development of new methods for angiogenesisoriented therapy [5]. It seems that there is a crucial and concerted regulation between an unidentified number of genes, in time-, dose-,

http://dx.doi.org/10.1016/j.ijcard.2014.03.008 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Rahbarghazi R, et al, Dynamic induction of pro-angiogenic milieu after transplantation of marrow-derived mesenchymal stem cells in experimental myocardial infarction, Int J Cardiol (2014), http://dx.doi.org/10.1016/j.ijcard.2014.03.008

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R. Rahbarghazi et al. / International Journal of Cardiology xxx (2014) xxx–xxx

and spatial-dependant manner, and the neo-angiogenesis process [6, 7]. Elucidation of the kinetics of signaling molecules influencing angiogenesis in physiologic and pathologic conditions is currently under close scrutiny [8]. Tie2 and Tie1, the endothelial-selective tyrosine kinase receptors, interact with ligands such as Angiopoietin1 (Ang1) and Angiopoietin2 (Ang2) and act in a coordinated fashion with VEGF (vascular endothelial growth factor) and VEGF receptors (VEGFR1, VEGFR2) during blood vessel morphogenesis and maturation [9]. During the recent years, stem cell therapy – in particular bone marrow mesenchymal stem cell (BMSC) therapy – has been deemed a modality for both stimulating and inhibiting angiogenesis [10,11]. However, there is a controversy on the mechanistic basis of stem cell–endothelial cell interactions. Recent experimental studies conducted to prevent the unfavorable remodeling and to induce myocardial regeneration are based on cell therapy, angiogenic gene delivery, or administration of angiogenesis-related peptides into the damaged myocardium [5,12]. It is thought that the production of MSC-mediated proangiogenic cytokines and their capacity for trans-differentiation into cardiomyocyte-like cells are two major

mechanisms whereby MSCs contribute to the functional improvement of the infarcted myocardium [13]. We have recently demonstrated that MSCs interact with endothelial cells in juxtacrine and paracrine manners in vitro [14]. In this study, we sought to investigate whether autologous rabbit BMSC (rBMSC) transplantation can modify the kinetics of angiogenesis signaling molecules and their cognate receptors simultaneously in the infarcted myocardial tissue. Indeed, we aimed to answer the following question: by which mechanisms do rBMSCs ameliorate neo-angiogenesis after experimental myocardial infarction? 2. Material and methods 2.1. Animals In this study, 42 adult male New Zealand White Rabbits, weighing 1.6–3.2 kg, were treated in accordance with the published guideline for the “Care and Use of Laboratory Animals” (NIH Publication No. 85-23, revised 1996). All the implementation phases of this study were approved by the Animal Care Committee of Tehran Heart Center.

Fig. 1. Improvement in echocardiographic parameters and infarcted area after cell transplantation. AWT, PWT, EDD/BW ratio, EF and FS parameters obtained before MI, 2 days, 7 days and 21 days after cell injection (A). The left column is the data from the 2-day groups, the middle column is the data from the 7-day groups, and the right column is the data from the 21-day groups. rBMSCs reduced both left ventricular infarcted area and perimeter in the cell transplantation groups (B). Collagen deposition analysis revealed a significant difference at day 21 post-cell therapy (B). Transverse size of cardiomyocytes was not statistically different in both infarction border zones and remote areas at day 21 post-MI between the celltransplanted and control groups (C). (n = 7 rabbits per group at each time point, Student's t-test (A–C) and Mann–Whitney U-test (B)). Data are expressed as mean ± SD. *P b 0.05.




Fig. 2. Enrichment of the GFP-positive transplanted cells in the injured myocardium. Representative histograms of flow cytometry analysis and percentage of the GFP-positive cells in the LV anterior and posterior walls at three different time points after cell transplantation (A–D). Flow cytometry analysis of the LV anterior and posterior wall single cell suspensions is displayed (A–C). The cells are displayed on side scatter (X axis) versus log fluorescence intensity (FL1, Y axis). Data were collected on 10,000 live cells. The GFP-positive cells, characterized as cells with increased fluorescence intensity, are seen in the cell-transplanted hearts. The percentage of GFP-positive cells is shown in the flow cytometry plots within the gates. The values were normalized based on the controls (D). As is shown, the percentage of the GFP-positive cells increased in the anterior wall by time, while no differences were observed in the posterior wall (n = 3 rabbits per group). Data are expressed as mean ± SD.

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R. Rahbarghazi et al. / International Journal of Cardiology xxx (2014) xxx–xxx 2.2. Cell isolation, characterization, and differentiation All the relevant information on cell isolation and characterization is provided in the online data supplement. 2.3. rBMSC transfection with lentiviral vector To track the cell kinetics after injection in the cardiac tissue, green fluorescent protein (GFP) tagged lentiviral vector was used as previously described [15]. Briefly, the cultured rBMSCs at 70–80% confluence were transfected with a GFP-encoding lentiviral backbone vector, pLV-IRES-GFP. The cells were transduced with pLV-IRES-GFP at the multiplicity of infection of 30 in the presence of 5 mg/mL polybrene, followed by a second transduction after 48 h [16]. The transduced and non-transduced (control) rBMSCs are referred here as rBMSCs-GFP+ and rBMSCs-GFP−, respectively. To assess GFP expression, the transduced as well as non-transduced control cells were trypsinized (Catalog# XC-T1717/20; Biosera, East Sussex, UK) and subjected to flow cytometry assay using a Partec PAS II flow cytometer (Partec® GmbH, Münster, Germany). The output data were processed with the FloMax Ver. 2.4 f (Partec® GmbH, Münster, Germany). 2.4. Myocardial infarction and cell transplantation In order to induce MI, the rabbits were premedicated with a combination of ketamin (50 mg/kg i.m.) and xylazine (5 mg/kg i.m.). After adequate anesthetic depth, the animals were placed in a dorsal recumbent position and then intubated orally. Tracheal ventilation was done using a respiratory ventilator and anesthesia machine (Sulla 909 V; Dräger, Germany). Throughout the procedure, the rabbit's heart rate, peripheral O2 saturation, pulse rate and electrocardiogram were also monitored (Model: 90485; SpaceLabs Medical Inc., USA). To occlude the left anterior descending coronary artery (LAD), the hearts were exposed through left anterior thoracotomy and the LAD was ligated with a 6-0 polypropylene, just below the tip of the left auricle [17]. Blanching of the myocardium and electrocardiography were employed to evaluate myocardial ischemia [18]. The animals were randomized into two major groups: control (C) group and cell transplantation (T) group, with 21 animals in each group. In order to evaluate the influence of cell transplantation on the kinetics of the tissue proteins involved in angiogenesis signaling in vivo and to assess myocardial pathology in a temporal manner after cell therapy, the animals in the C and T groups were divided into the following three groups: C2 and T2, which were sacrificed 2 days after the operation; C7 and T7, which were sacrificed 7 days after the operation; and C21 and T21, which were sacrificed 21 days after the operation. A total volume of 200 μL of PBS without or with 1 × 106 autologous rBMSCs was injected at four sites bordering the infarcted area 20–30 min after the LAD ligation in the C and T groups, respectively. Prior to thoracic wall closure, blood discharges and accumulated fluids were drained via a chest tube drainage system to reduce pericardial fibrosis. 2.5. Assessment of cardiac performance by echocardiography To assess and compare the cardiac performance pre- and post-MI, the rabbits were submitted to a transthoracic echocardiography analysis using an echocardiographic system (Toshiba SSA-380A) fitted with a 7.5 MHz linear transducer. Left ventricular (LV) end-diastolic dimensions (LVEDd, mm), LV anterior wall thickness (LVAWT), LV posterior wall thickness end-diastolic (LVPWd, mm), LV ejection fraction (EF, %) and fractional shortening (FS, %) were calculated [19]. The C and T groups were imaged at four time points: before MI (for all groups), 2 days post-MI (for all groups), 7 days post-MI (for the C7, T7, C21, and T21 groups), and 21 days post-MI (for the C21 and T21 groups). 2.6. Myocardial infarct size Prior to sacrifice, the rabbits in each group were treated intravenously with heparin (500 U/kg) and then killed with an overdose of pentobarbital®. Afterward, the hearts were removed rapidly and LV weights were measured. The LV was then cut into eight transverse slices (each slice = 1.5 mm) from base to apex (S1 to S8 from base to apex). S1, S5 and S7 were fixed in 10% neutral-buffered formalin and paraffin embedded for histopathology. S3, S6 and S8 were used for immunofluorescence assay. In slices S2 and S4, the infarcted tissue, border zone tissue (surviving myocardial tissue areas within 1 mm of infarcted area) and the remote normal tissue were separated and immediately snapfrozen for western blotting. For histopathology, two 5 μm serial sections prepared from paraffin-embedded slices were subjected to stain with hematoxylin-eosin and Masson's trichrome [20]. The Masson's trichrome-stained sections were scanned with a HP Scanjet G3110 apparatus (Hewlett-Packard Company, CA, USA) at 600 dpi and used for measuring infarct size. For this purpose, both the area (pixel2) and the perimeter (mm) of the infarcted and non-infarcted regions were measured for transversely sectioned preparations using AxioVision Version Rel 4.8 (Carl Zeiss Microimaging Inc., Germany) and Image J Version 1.44p (NIH, USA) software as previously described with some modifications [21]. Moreover, in the Masson's trichrome-stained sections, the infarcted area was divided

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into four equal parts by using AutoCAD 2012 software (Autodesk Inc., USA), and the longest diameter was drawn for each part. Then, the midpoint was marked for each diameter and finally, the blue color intensity was measured by Photoshop software Ver. CS5 (Adobe Systems Incorporated, USA) (Supplementary Fig. 1). Shortest transverse size of at least 100 cardiomyocytes at ten serial sections per heart in the peri-infarct and remote areas of the LV sections was measured using AxioVision Version Rel 4.8, and data were compared between the control and the cell transplantation groups. 2.7. Tracking rBMSCs injected into the peri-infarct zone To assess the successful transplantation of GFP-positive rBMSCs in the myocardial tissue undergoing post-MI remodeling, the existence of GFP-positive cells in the myocardium was analyzed by flow cytometry on days 2, 7 and 21 post-injection [22,23]. For this purpose, LV tissue samples were obtained from the posterior and anterior walls (1 mg each), transferred into 15 mL conical tubes and digested by tissue digest solution consisting of 0.2% collagenase type 1 (Catalog# C0130; Sigma-Aldrich, USA) and 0.5% trypsin-EDTA (Catalog# XC-T1717/20; Biosera, UK) at 37 °C in water bath for 20 min [24]. After adding the same volume of 20% FBS in DMEM-low glucose medium, the suspension was collected and centrifuged at 3500 rpm for 10 min at 4 °C. Uniformity of the cells was assessed by observing on a Neubauer slide before submitting the samples for flow cytometry analysis. Flow cytometry was performed using the Partec PAS II flow cytometer (Partec® GmbH, Münster, Germany). The output data were processed with the FloMax Ver. 2.4 f (Partec® GmbH, Münster, Germany). 2.8. Immunohistochemical analysis To assess the effects of the rBMSCs on angiogenesis in myocardial tissue, 5 μm tissue sections from the T and C paraffin-embedded cardiac tissues at three time points after transplantation were immunostained using CD31 (1:200, Catalog# M0823; Dako) or anti alpha-smooth muscle actin (α-SMA) (1:100, Catalog# M0851, Dako, Denmark) antibodies [4,25]. Briefly, the sections were deparaffinized, incubated in 3% H2O2 solution for 30 min, and then autoclaved at 15 psi in citrate buffer (pH, 6.0) for 15 min for antigen retrieval. After cooling, the slides were incubated with the primary antibodies for 30 min, washed with PBS, and colored with the EnVision+ Dual Link System HRP kit (Dako, Denmark). 3, 3′-Diaminobenzidine (DAB) was used as the chromogen. All the slides were finally counterstained with Mayer's hematoxylin solution. Per section, the number of CD31 positive-capillaries was counted in border (peri-infarct), infarct (anterior wall) and remote (posterior wall) areas in each of the 10 serial high-power fields (HPFs) by light microscopy. Alpha-SMA positive-small arteries as well as α-SMA-positive myofibroblasts were also counted in the remote and infarct areas. 2.9. Immunofluorescence analysis To investigate the fate of the rBMSCs-GFP+ in the cardiac tissue after transplantation, immunofluorescence assay was performed as previously described [26]. Slices S3, S6, and S8 from the C and T hearts were maintained in a 20% sucrose solution overnight at 4 °C, then embedded in Tissue Freezing Media (Cat# TFM-5; Triangle Biomedical Sciences, Inc., USA), snap-frozen and sectioned at 7 μm with MC 4000 Cryostat (Histo-Line Laboratories, Italy). The sections were thawed at room temperature and dried for 30 min, washed twice (5 min each) in PBS with 0.1% Triton X-100, rinsed in PBS three times (5 min each), blocked with 1% BSA solutions (Catalog# A2153; Sigma-Aldrich) for 5 min and then with 5% goat serum for 30 min, and subsequently incubated with FITC-conjugated goat antiGFP IgG (1:200, Catalog# ab6662; abcam), mouse anti-cardiac troponin T (1:200, Catalog# ab33589; abcam), mouse anti-CD31 (ready solution, Catalog# AM232; BioGenex, USA), or mouse anti-alpha Smooth muscle actin (α-SMA) (1:100, Catalog# M0851; Dako, Denmark) antibodies for 2 h. The sections were, thereafter, washed three times with PBS, incubated with goat anti-mouse IgG-Texas red (TR) (1:1000, Catalog# ab6787; abcam) for 1 h at room temperature, and washed three times (5 min each) with PBSTween 20 (0.1%). In the double-stained section, 4′, 6-diamidino-2-phenylindol (DAPI, 1 μg/mL, Catalog# D9542; Sigma-Aldrich) was used for nuclear counterstaining. Microscopic examinations (Olympus) of the sections were performed, and the images were processed by DP2-BSW software Ver.2.2. 2.10. Assessment of cell fusion by flow cytometry In order to evaluate the probability of cell fusion after transplantation of rBMSCs, the cell transplanted hearts at the three time points along with passage 3 rBMSCs were subjected to the assessment of the DNA content during cell cycle analysis by flow cytometry as described previously with some modifications [27]. Briefly, for each heart, equal amounts of tissue pieces were transferred to 15-mL falcon tubes containing 3 mL of 0.5% trypsin-EDTA (Catalog No. XC-T1717/20; Biosera) and 0.3 mg/mL collagenase type I (Catalog No. C0130; SigmaAldrich) and incubated at 37 °C in the water bath for 15 min. After adding the same volume of 20% FBS, the cell suspension was centrifuged at 3500 rpm for 10 min at 4 °C into a cell

Fig. 3. Effects of rBMSC transplantation on capillary density and small artery formation. Representative images of CD31 positive capillaries at the peri-infarct and infarcted zones (A). The number of capillaries was counted in each of the 10 serial microscopic fields. Data analysis revealed that cell transplantation resulted in a significant increase in capillary density at days 7 and 21 post-transplantation. α-SMA positive arterioles and myofibroblasts in the infarcted areas of the cell-transplanted and control groups 21 days post-injection (B). rBMSCs augmented the density of α-SMA positive small arteries in the infarcted area at day 21 after cell transplantation. (n = 6 rabbits per group; three sections were examined for each heart; Mann–Whitney U-test (A and B)). Data are expressed as mean ± SD. *P b 0.05.


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R. Rahbarghazi et al. / International Journal of Cardiology xxx (2014) xxx–xxx pellet. The supernatant was discarded and then 1 mL of the Tris-base solution (pH, 7) was added. To achieve efficient cell dissociation, the cell suspension was triturated gently using a syringe. Furthermore, 500 μL of the RNase solution (0.025g/1 mL PBS; Catalog No. R6513; Sigma) and then 100 μL of 1% Triton-X100 was added and incubated for 15 min and 10 min, respectively. Finally, the cell suspension was exposed to 0.5 mL of propidium iodide (PI) [0.005g/5 mL Tris-base] at 37 °C in the water bath for 30 min to label all DNA content. To compare the DNA content between the pre-transplanted and post-transplanted cells, the rBMSC suspension was also subject to PI staining as mentioned above. Flow cytometry analysis was then performed using FACSCalibur (BD Bioscience), and the output data were processed with the FlowJo software. For each heart, the cell population was shown as scatter plots with forward scatter (FSC) on the x-axis and side scatter (SSC) on the y-axis. Then, the GFP-positive transplanted cells were displayed on the x-axis in a log mode versus SSC on the y-axis. To distinguish between single cells and aggregates, the pulse-width (FL3-W; x-axis) versus pulse-area (FL3-A; y-axis) plots were drawn. Finally, the single cells were subject to cell cycle analysis by FlowJo. Cell fusion was then assessed as the percentage increase in the G2 phase in the GFP-positive cells in the transplanted hearts compared with rBMSCs before injection. 2.11. Western blotting To evaluate the kinetics of angiogenic signaling molecules in the myocardial tissue after cell transplantation, slices S2 and S4 from each heart were submitted to western blotting assay. For each rabbit, western blotting was performed on three tissue areas (infarct, border, and the remote normal) of the LV. For each area, 50 mg of the tissue was homogenized in 600 μL of PRO-PRE™ Protein Extraction Solution (Catalog# 17801; iNtRON Biotechnology, Seongnam, Korea) using a Dounce homogenizer. Then the samples were sonicated on ice for 30 s (Catalog# Ultrasonic Processor UP50H; Hielscher Ultrasound Technology, Germany). Highly efficient cell lysis was achieved by incubating the mixture for 20 min in a freezer at −20 °C according to the manufacturer's instructions. Finally, the extracts were centrifuged at 13,000 rpm for 15 min at 4 °C. Supernatants were then collected and protein concentration was determined using SMART™ Micro BCA Protein Assay Kit (Catalog# 21071; iNtRON Biotechnology, Korea) with a Smartspec Plus spectrophotometer (Bio-Rad). Subsequently, 50 μg of cardiac tissue protein extract (under reducing condition) was loaded on SDS-PAGE gels (5% stacking and 10% separating gels) after a 5 min boiling and transferred to 0.2 μm immune-Blot™ polyvinylidene difluoride (PVDF) membranes (Catalog# 162-017777; Bio-Rad laboratories, CA, USA). The membranes were blocked with 3% non-fat dry milk (Catalog# 1.15363.0500; Merck KGaA, Darmstadt, Germany) or 5% BSA (Catalog# A-7888; Sigma-Aldrich, Mo, USA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h in accordance with the manufacturer's recommendations. They were thereafter incubated with individual primary antibodies for 1 h at room temperature and washed three times (3 × 10 min) with TBST. The membranes were then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, followed by incubation in a solution containing 0.006% 3, 3′-diaminobenzidine (DAB) (Catalog# D5637; Sigma-Aldrich, Mo, USA) to visualize immunoreactive bands. Western blot analysis was performed with a panel of antibodies, including mouse antiVEGF (5 μg/mL, Catalog# ab1316; Abcam), rabbit anti-VEGF receptor 1 (1:10,000, Catalog# ab32152; Abcam), rabbit anti-VEGF receptor 2 (1 μg/mL, Catalog# ab39256; Abcam), rabbit anti-Angiopoietin 1 (1 μg/mL, Catalog# ab95230; Abcam), rabbit antiAngiopoietin 2 (1 μg/mL, Catalog# ab65835; Abcam), mouse anti-Tie1 (2 μg/mL, Catalog# ab27851; Abcam), mouse anti-Tie2 (1 μg/mL; Catalog# ab24859; Abcam), rabbit antiphospho-Tie2 (1:1000, Catalog# ab78142; Abcam), mouse anti-beta actin-loading control (1 μg/mL, Catalog# ab8224; Abcam), rabbit anti-mouse IgG-HRP (1:4000; Catalog# ab6728; Abcam) and sheep anti-rabbit IgG-HRP (1:5000, Catalog# ab6795; Abcam). The membranes were scanned using an HP Scanjet G3110 apparatus (Hewlett-Packard Company, CA, USA). Finally, quantification of each band was accomplished by the corresponding densitometry of the actin band using Image J Version 1.44p software (NIH, USA) as described previously [28]. The area (%) under the curve of each band was divided by the corresponding percentage of the area under the curve of the actin band, and the calculated values were compared statistically between the C and T groups.

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7.4) at 4 °C overnight, post-fixed in 1% osmium tetraoxide for 2 h, and then washed again in PBS for 1 h. After the dehydration process through acetone concentration gradient, the tissues were infiltrated and embedded in resin. Semi-thin sections (600 nm) were then generated from the resin-embedded samples by an ultra-microtome system (Leica Ultracut UCT Microtome; Leica Microsystems) with diamond knife (Micro Star Technologies Inc., Huntsville, TX), stained with 0.5% toluidine blue and observed by a light microscope to detect the infarcted zone margin. The ultrathin sections (60 nm) from the target areas were incubated in 50 μL of 5% Na-metaperiodate (Merck, Darmstadt, Germany) for 20 min to remove excess fixatives, washed three times (3 × 5 min) with distilled water (50 μL) and then with a drop of PBS containing 0.1% Triton X-100 (10 min). Afterwards, the grids were blocked with 1% BSA solutions for 10 min. The sections were then incubated with a droplet of primary rabbit anti-GFP antibody (1:100, Catalog# ab6556; Abcam) for 1 h, washed three times with PBS, blocked with second blocking solution containing 5% goat serum for 30 min, and then incubated in a droplet of goat anti-rabbit IgG conjugated to 10-nm gold particles (1:100, Catalog# ab27234; Abcam) for 30 min. After washing three times in PBS containing 0.1% Tween20 (2 × 5 min) and twice in distilled water (2 × 10 min), the grids were allowed to dry at room temperature. Then, the sections were conventionally doubled-stained with aqueous uranylacetate and lead citrate. To search for immunogold-labeled cells, the sections were ultrastructurally examined and photographed at 50 kV by a Zeiss EM109 electron microscope. 2.13. Statistical analysis Statistical analysis was carried out by SPSS software Ver.16 (SPSS Inc., Chicago, IL, USA) and the data were presented as mean ± standard deviation (SD). Both Mann– Whitney U test and the Student's t-test were used for nonparametric analysis and parametric data analysis, respectively. Mean difference was significant at P b 0.05. In histograms, statistical difference between the groups is shown by brackets with *P b 0.05 and **P b 0.005.

3. Results 3.1. Mortality Totally, 6 deaths were recorded after MI. Of these, 2 rabbits died immediately after ligation of the LAD due to cardiac arrest. The other 4, (two animals in each cell transplanted and control group) died within the first 6 to 18 days after MI induction due to unknown causes. Subsequently, the dead rabbits were replaced, so a total number of 42 animals were included in this study. 3.2. Flow cytometry analysis for transgene expression (GFP) in rBMSCs transfected with lentiviral vector A GFP-expressing reporter vector was used to monitor the rBMSC engraftment in the myocardial tissue in the cell transplantation groups [30]. Previous studies by us and others demonstrated that GFP expression by the lentiviral vector does not alter the differentiation or paracrine potential in mesenchymal stem cells [16,31]. By the flow cytometry as well as immunofluorescence analyses for the quantitative measurement of transduction efficiencies in rBMSCs, we found that more than 70% of the transfected rBMSCs showed high levels of GFP transgene expression (Supplementary Fig. 2).

2.12. Immunoelectron microscopy

3.3. rBMSCs improve cardiac function after transplantation into the myocardium

Transmission electron microscopy processes and immunogold labeling were done as previously described with some modification [29]. Briefly, the harvested cardiac tissues from the border zone of the infarcted zone (21 days post-MI cell transplantation group and control group, N = 2 each) were fixed in 2.5% glutaraldehyde, washed in PBS (PH =

Echocardiographic analysis was performed to assess the cardiac function over time. None of the echocardiographic parameters were statistically significant between the cell transplantation and

Fig. 4. Representative images of GFP-CD31, -α-SMA, and -troponin T double-positive cells in the peri-infarct areas. As is shown, capillary, small arterial, and myogenic differentiation of transplanted rBMSCs is evident in the injured myocardium (A–C). At day 2, aggregates of GFP-positive cells are seen in the peri-infarct border zone (A). The GFP-expressing rBMSCs are found to contribute to capillary formation at the peri-infarct zone (arrows) at days 7 and 21 after cell transplantation. The microenvironment where marrow MSCs are located has an important role in trans-differentiation. Differentiated cells are seen as yellow cells using the multiple fluorescene filter. As is shown, the GFP-positive cells were integrated into the structure of small arteries (arrows) at days 7 and 21 after transplantation (B). Moreover, our analyses showed that double GFP-cardiac troponin T positive cells are evident in the peri-infarct zone at day 21 after cell injection (C). The GFP positive cells co-expressed cardiac-specific troponin T. The nuclei were stained with DAPI. Immuno-electron labeling for GFP reveals an immuno-labeled MSC with trans-differentiation into ventricular cardiomyocytes (D). As is seen, well-developed myofibrils with parallel arrangements are organized to form sarcomers. Nascent Z discs (asterisk) and developing T tubules (arrows) are seen. Immuno-gold staining against GFP in the non-cell transplanted hearts was used as control. Percentages of GFP+ cells that co-expressed CD31, α-SMA, or troponin T at days 2, 7, and 21 after transplantation in the injured myocardium (E). In 3 hearts, the total number of GFP+ cells was counted in 10 sections per heart and the percentages of double-positive cells were determined. Data are expressed as mean ± SD (E). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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Fig. 5. Flow cytometric DNA content histograms from the pre-transplanted (control) and post-transplanted GFP+ rBMSCs at days 2, 7, and 21 after transplantation. The x-axis is the PI fluorescence intensity, which corresponds to the DNA content in the cells. The fusion event was considered as the percentage increase in the G2 phase in the GFP+ transplanted rBMSCs compared with the control pre-transplanted cells. As is shown, almost all of rBMSCs, either in vitro or in vivo, were in the G0/G1 phase (area under the green curve) and a very low percentage of the cells were in the G2 phase (representative data from three samples in each group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


control groups pre-MI and 2 days post-MI. A significant increase was observed in the AWT in the cell transplantation hearts compared to the control hearts at 7 and 21 days post-MI (Fig. 1A), indicating that cell transplantation prevented more adverse LV remodeling as early as 7 days after transplantation. In contrast, as was expected, no significant changes of the PWT were evident pre- and post-MI in all the groups at all times (Fig. 1A). A significant decrease in the EDD/BW ratio was also obtained at 21 days after cell transplantation in the T21 as compared to the C 21 hearts (P = 0.049). In addition, EF and FS parameters significantly improved in the T21 group compared to the C 21 group (P = 0.033 and P = 0.042, respectively), but these values were less than the pre-MI baseline values at all times post-MI. 3.4. rBMSCs diminish infarct expansion and infarct collagen deposition Seven and twenty-one days post-injection, we observed scar tissue consisting of collagen fibers, blood vessels, leukocytes, fibroblasts, fibrocytes, and fatty tissue in the infarcted zone of the all groups [32]. Our analysis revealed that the infarcted size was statistically reduced in the cell transplantation groups as compared to their control groups (Fig. 1B and Supplementary Fig. 3). No significant difference was observed between the control and the cell transplantation groups at 7 days post-MI in the color intensity of collagen deposition in the infarcted myocardium (PC7–T7 = 0.528), whereas collagen deposits were significantly reduced 21 days post-MI in the cell transplantation groups when compared with the control groups (PC21–T21 = 0.043) (Fig. 1B). Our analysis showed that the perimeter of the infarcted segment relative to the total perimeter of the LV was decreased statistically at 7 and 21 days after cell transplantation (PC7–T7 = 0.037 and PC21–T21 = 0.034) (Fig. 1B). In order to exclude the possibility of the hypertrophy of the cardiomyocytes for the observed reduction of the infarct size, transverse size of the cardiomyocytes in the peri-infarct zone and the remote area of the LV sections were compared between the control and the cell transplantation groups 21 days post-MI; no significant results were observed between groups (Premote = 0.912, Pborder = 0.832) (Fig. 1C). 3.5. The enrichment of rBMSCs after injection into the peri-infarct zone Percentage of the GFP-positive cells in the tissue samples was quantified by flow cytometry after digesting the tissue into a single cell suspension. We found that the number of the GFP-positive cells in the LV anterior wall increased by time, whereas no clear difference was observed in the posterior wall (Fig. 2A–D). By flow cytometry, GFPpositive cells constituted 0.090% of the cells in the cell transplanted hearts 2 days after transepicardial cell injection and this reached 0.164% and 0.295% at 7 and 21 days post-injection, respectively (Fig. 2D). It seems that at early days after cell injection into the peri-infarct zone, the percentage of the injected cells is reduced as a result of metabolism reduction [33] or apoptosis [34]. However, the remaining GFP-positive cells might enter the cell cycle [35] or increase cellular metabolism following adaptation to the new niche, which ultimately led to the enrichment of GFP-positive cells in the injured myocardium. In addition, the flow cytometry results showed that the enrichment of the GFP-positive cells was restricted to the injured tissue, as no GFP positive cells were detected in the remote areas of the posterior wall. 3.6. Effect of rBMSCs on microvascular density after myocardial infarction Capillary density was evaluated in histological sections in the remote, border, and infarct zones, and the density of the α-SMA positive small arteries was assessed in the infarcted and remote areas (Fig. 3A–B). Immunohistochemical analysis using anti-CD31 and anti-α-SMA antibodies showed that the number of CD31 positive capillaries as

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well as α-SMA positive small arteries had significant differences in the cell transplanted groups when compared with the controls. CD31 positive capillary density increased statistically in the peri-infarct (PC7–T7 = 0.026, PC21–T21 = 0.046) and infarcted zones (PC7–T7 = 0.033, PC21–T21 = 0.049) in the cell transplanted groups 7 and 21 days post-injection (Fig. 3A). It can be inferred from the P values that the microvascular growth rate was higher in the peri-infarct zone of the cell-transplanted groups than in the infarcted zone. In the remote areas, vascular density was similar between the groups at all times (Fig. 3A). Alpha-SMA positive small artery density was significantly enhanced at the infarcted zone after 21 days (PC21–T21 = 0.047), although no significant differences were evident 2 and 7 days post-injection (PC2–T2 = 0.324, PC7–T7 = 0.814) (Fig. 3B). Again, in the remote areas, arteriolar density was comparable between the groups at all times. Also, by α-SMA immunohistochemical staining, we found that – after injecting the rBMSCs – the number of myofibroblasts was not significantly different between the cell-transplanted and control groups at the infarcted and remote zones at any times.

3.7. Transplanted rBMSCs directly contributed to both angiogenesis and myogenesis Immunofluorescence analysis demonstrated that the transfected rBMSCs had a strong potency for multiple trans-differentiation (e.g. angiogenic and myogenic) after injection into the injured myocardial tissue. GFP-positive rBMSCs were found in the structure of the capillaries as GFP-CD31-double positive cells were detected in the peri-infarct areas of the cardiac tissues at 7 and 21 days post-transplantation (Fig. 4A). At day 2, aggregates of GFP-positive cells could be found in the peri-infarct border zone without trans-differentiation into myogenic or vasculogenic cells (Fig. 4A–C). Concurrently, a fraction of the transplanted cells underwent differentiation into α-SMA positive cells in the wall of the vessels. These GFP-α-SMA-double positive cells were detectable at 7 and 21 days after transplantation (Fig. 4B). Meanwhile, immunofluorescence double-staining demonstrated a fraction of the transplanted GFPpositive rBMSCs expressed cardiac troponin T, indicating that a number of the transplanted rBMSCs could differentiate into troponin T positive cardiomyocytes at day 21 (Fig. 4C). No GFP-troponin T-double positive cells were found at days 2 and 7. To quantitatively assess the differentiation potential of the transplanted MSCs in the heart, we determined the percentage of double-positive cells per total GFP-positive cells at the three time points (Fig. 4E). At day 21, average percentages of 18.22%, 24.45%, and 32.33% of double-positive cells were quantified for CD31, α-SMA, and troponin T, respectively (Fig. 4E), indicating the differentiation of more than 70% of the transplanted cells in the injured myocardium within 3 weeks. Further, we performed immunoelectron labeling for GFP to detect GFP-positive cells in two cell transplanted hearts. GFP-positive cells were detected in the periinfarct border zone using an antibody tagged with nanogold particles. Ultrastructural analysis of the immuno-labeled cells revealed the trans-differentiation of the transplanted MSCs-GFP+ into ventricular cardiomyocytes. By day 21 post-injection, the grafted cells exhibited well-developed myofibrils with organization into a sarcomeric pattern within the cytoplasm. Developing T-tubules were seen in the vicinity of the nascent Z discs, indicating differentiation of the cells into ventricular cardiomyocytes (Fig. 4D) [36]. To illuminate the potential of cell fusion after transplantation of rBMSCs in the injured myocardium, the DNA content of the GFPpositive cells in the transplanted hearts was compared with that of the pre-transplanted rBMSCs. In average, the cell fusion was measured as 0.2%, 1.7%, and 4% of the cell transplanted GFP+ rBMSCs 2, 7 and 21 days post-transplantation, respectively, indicating that cell fusion did not play a significant role in myocardial regeneration after cell therapy, which is consistent with some previous reports (Fig. 5) [37].


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Fig. 7. Changes in Tie2-pho/Tie-2 and Ang-2/Ang-1 ratios in the infarct, peri-infarct border, and remote areas 2, 7, and 21 days after cell injection. Data are expressed as mean ± SD. *P b 0.05 (Student's t-test).

3.8. Effect of transplanted rBMSCs on expression of factors involving in angiogenesis signaling Expression of important factors involving in angiogenesis signaling evaluated by Western blot analysis revealed that levels of VEGF, Ang-1, Ang-2, Tie-2, Tie2-pho, and VEGFR-2 expression in the injured cardiac tissue were changed by transplanted rBMSCs. Western blot analysis showed no Tie-1 or VEGFR-1 immuno-reactive bands in the control and cell-transplanted myocardium. The expression level of all proteins in the remote areas was similar between the celltransplanted and control groups at all times, except for phosphorylated Tie2 (Tie2-pho), for which significant changes were observed even in the remote zone in addition to the infarct and peri-infarct zones at day 2 after cell transplantation (Fig. 6A–B).

3.8.1. VEGF Western blotting showed that VEGF expression was enhanced in the cell-transplanted groups compared to controls (Fig. 6A–B). In the peri-infarct region, the mean expression of VEGF factor was statistically greater in the cell transplantation groups than in the control groups at day 2 (P = 0.029). At days 7 and 21, while the mean VEGF expression in the cell-transplanted hearts was higher than that in the control groups, it did not reach the significant level (P7 = 0.114, P21 = 0.486). In the infarcted area, the mean expression of VEGF factor was also greater in the cell transplantation group than in the control groups and significant changes were observed during 7 and 21 days after cell injection (P7 = 0.038, P21 = 0.027).

3.8.2. Ang-2 Like VEGF, Ang-2 expression level was greater in the cell transplanted groups at 2, 7, and 21 days after cell transplantation and a significant change was observed in the peri-infarct zone at 21 days (P = 0.048). Our results showed that a concomitant elevation of VEGF and Ang-2 expression level occurred after the transplantation of marrow-derived MSCs in the injured myocardium (Fig. 6A–B). 3.8.3. Ang-1 In contrast to VEGF and Ang-2 factors, we found that Ang-1 expression clearly decreased in the cell-treated peri-infarct zone at all-time points, and at day 21, its level was significantly decreased in the cell-transplanted hearts compared with the control hearts (P 2 = 0343, P 7 = 0.486, P 21 = 0.047). No significant changes were observed in Ang-1 at the infarct zone between the groups (Fig. 6A–B). 3.8.4. Tie-2 Injection of the rBMSCs into the injured myocardium increased Tie-2 expression level compared to the non-cell transplanted hearts, which was not statistically significant (Fig. 6A–B). 3.8.5. Tie2-pho However, Western blot analysis revealed that the mean of Tie2pho level was decreased in the cell-transplanted groups compared to the controls at days 2, 7 and 21 post-injection in the periinfarct, infarct, and even remote zones. Reduction of phosphorylated form of Tie-2 was significant 2 days post-injection in all the three

Fig. 6. Effects of marrow MSCs on the expression of key angiogenesis signaling proteins in the injured myocardium by Western blotting analysis. As is illustrated, the immunoreactive bands are evident at days 2, 7, and 21 in the peri-infarct, infarct, and remote zones in the cell-transplanted (T) and control (C) groups (n = 7 rabbits per group) (A). Molecular weights of the immunoreactive bands are as follows: VEGF ≃ 42kD, Ang-2 ≃ 63, Ang-1 ≃ 53, Tie-2 ≃ 126, Tie-pho ≃ 126, and VEGFR-2 ≃ 150. In part B, the histograms show the rBMSC effects on VEGF, Ang-2, Ang-1, Tie-2, Tie2-pho, and VEGFR-2 expression levels in the infarct, peri-infarct, and remote areas at days 2, 7, and 21 post-injection in the injured myocardium. Y-axis stands for arbitrary units. After band densitometry, the area under the curve of each band was divided by the area under the curve of the corresponding β-actin band, and the calculated values were compared between the groups. Data are expressed as mean ± SD. *P b 0.05 (Student's t-test (B)).


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zones (P2-remote = 0.027, P2-infarct = 0.027, P2-border = 0.029) and at 7 day post-transplantation in the infarct (P 7-infarct = 0.031) and peri-infarct (P7-border = 0.029) areas (Fig. 6A–B). 3.8.6. VEGFR-2 For VEGFR-2, an upward trend was observed after cell transplantation although the changes did not reach the significant level (Fig. 6A–B). 3.8.7. Tie2-pho/Tie-2 ratio Tie2-pho/Tie-2 ratio was decreased significantly at days 2 and 7 after cell transplantation in the peri-infarct border (P2-border = 0.046; P7-border = 0.029) and infarct (P2-infarct = 0.49; P7-infarct = 0.028) zones (Fig. 7); however, no significant changes were observed for this ratio after 21 days. No significant results were detected in the remote area at any times (Fig. 7). 3.8.8. Ang-2/Ang-1 ratio Ang-2/Ang-1 ratio was also increased by cell transplantation at days 7 and 21 post-injection in the peri-infarct zone (P 7 = 0.114, P 21 = 0.030). In the infarcted area, however, the changes in the Ang-2/Ang-1 ratio following cell transplantation did not reach a significant level (Fig. 7). 4. Discussions Angiogenic stimulation for the treatment of the infarcted myocardium is being vigorously pursued by cell-based therapy, in particular, mesenchymal stem cell. In the present study, the effect of marrow-derived mesenchymal stem cell transplantation on the angiogenesis signals was investigated over time in a rabbit model of acute MI. Indeed, we focused on the influence of marrow MSCs on the injured cardiac milieu as regards angiogenesis-related signaling molecules. We also studied the effect of cardiac milieu on marrowderived mesenchymal stem cell trans-differentiation potency and growth kinetics. Enhancement of angiogenesis and decrease of the infarct size achieved after cell transplantation were associated with the prevention of LV dilatation and improvement of cardiac function. rBMSCs prevented infarct expansion in the first few days following injection, and after a while accelerated healing processes. The reduction of the infarct size in the cell-transplanted groups can be ascribed to the reduction in collagen synthesis [38] and/or generation of new cardiomyocytes [37]. Since the transverse sizes of the cardiomyocytes were similar in all the hearts, hypertrophy of the pre-existing cardiomyocytes could not be responsible for the reduction in the myocardial infarct size in the cell-transplanted hearts compared to the controls [39]. A variety of beneficial effects, including reduction of the apoptosis in cardiomyocytes adjacent to the infarct, increase of blood flow, trans-differentiation into cardiomyocytes, decrease in collagen-related inflammatory cytokines, and immune and inflammatory modulation, has been elucidated by different groups of authors as the underlying mechanisms for improvement of cardiac function by mesenchymal stem cells [40–42]. Soluble factors secreted by MSCs such as VEGF, Ang-1, IGF-1, EGF, SDF-1α, and bFGF have also been reported to enhance the tissue regeneration, vascular density, and cardiomyocyte karyokinesis, leading to a decrease in the scar tissue [14,37]. In this study, we affirmed the potency of transplanted MSCs in the milieu of the infarcted myocardium to differentiate into major cells of the cardiac tissue, including cardiomyocytes, endothelial cells, and smooth muscle cells within a few days after cell transplantation, which is independent of cell fusion consistent with previous findings [37,43,44]. In the present study, more than 70% of the transplanted cells were found differentiated into myogenic and vasculogenic cells within 3 weeks after transplantation. Similar to this rate of differentiation,

culture of MSCs in a hydrogel with a comparable modulus to that of native heart tissue resulted in cardiac differentiation of more than 76% of MSCs [45]. Also, in a sex-mismatched cell transplantation model in mice, the authors showed that a majority of Y chromosome-positive transplanted cells expressed cardiac markers within 4 weeks after injection in recipient female hearts [46]. In addition, Davani et al. demonstrated that approximately 22% of total number of engrafted MSCs acquired CD31+ phenotype after 30 days of injection in a rat model of MI, which is comparable to 18% in the current study [47]. In another study of allogeneic MSC transplantation into chronically scarred myocardium via catheter-based transendocardial injection in the miniswine, however, the trilineage differentiation rate of MSCs was calculated as 24% [48]. In the present study, we used autologous cells, which eliminated undesirable immunologic response, leading to a higher rate of cell survival after transplantation [49]. In addition, autologous transplanted cells possess an extremely higher potential for differentiation than allogeneic transplantation [50]. Moreover, a GFP-encoding lentiviral vector was used to track the transplanted cells in this study. Genome integration of lentiviral vectors allows transfected cells to express the reporter protein during cell transdifferentiation, yielding an efficient technique for tracing transplanted cells that undergo post-transplantation differentiation [51,52]. We also demonstrated that the transplanted GFP-positive cells were enriched with time in the peri-infarct area, indicating that compared with the resident cells of the tissue, the transplanted cells possess a higher rate of cellular kinetics in the hostile milieu of the injured myocardium. The transplanted cells were also found to have a great tendency to remain in the peri-infarct border zone, the active site of regeneration. rBMSCs strongly augmented microvascular density after transplantation into the myocardium affected by MI. By probing molecules involving in angiogenesis signaling, we found that the transplanted rBMSCs at the injured myocardium enhanced simultaneously the expression levels of VEGF, Ang-2, Tie-2, and VEGFR-2 and reduced Tie2pho and Ang-1 levels. We showed that the concentration of VEGF signal protein started to increase at day 2 and, then reached a maximum level at day 7 and was maintained up to 21 days after cell transplantation. Moreover, the protein level of Ang-2 in the cell-transplanted groups continuously increased over time when compared with the non-celltransplanted control groups within 21 days of cell transplantation. Taken together, these findings suggest that the increase in Ang-2 in the cell transplantation groups, which could, at least partially, be secreted directly from rBMSCs [53], was associated with both time- and concentration-dependent increase in VEGF. As VEGF and Ang-2 exert their angiogenic effects synergically [54], co-stimulation of these factors may play a key role for the induction of pro-angiogenic milieu by marrow MSCs. In addition, considering the dual pro- and anti-angiogenic function of Ang-2 in different physiopathological conditions, a potential role has been suggested for VEGF in the Ang-2 pro-angiogenic function against its anti-angiogenic effects [55]. In contrast to some earlier reports [56], the increase in the VEGF level was associated with the decrease of Ang-1 at the peri-infarct border zone. We presumed that the sustained increase of Ang-2, coupled with reciprocal decrease in Ang1 at the peri-infarct border zone, can induce kinetics of angiogenic changes such as proliferation and migration of endothelial cells, and stimulation of sprouting angiogenesis in response to MI [9,55], Indeed, a rise in the cardiac ratio of Ang-2 to Ang-1, as we found after MSC transplantation, has been described to have a pivotal role for establishing a pro-angiogenic milieu after MI [57]. The other important finding of this study was the reduction in Tie2-pho receptor level after cell transplantation [58], which is in accordance with increased Ang-2 to Ang-1 ratio since it has been shown that phosphorylation of Tie-2 is induced by the stimulation of Ang-1, whereas Ang-2 acts – in opposite manner – as negative regulator of Tie-2 phosphorylation [59,60]. Nonetheless, some recent findings show that agonist or antagonist action of Ang-2 on Tie-2 receptor is context dependant [61]. Abundant evidence has shown that phosphorylation of Tie2 activates various intracellular



signaling pathways such as the Akt, ERK1/2, and p38 MAPK pathways, and inhibits the JNK/SAPK pathway, leading to vascular hyporeactivity [62–64]. Increase in the Ang-2: Ang-1 ratio, resulting in the dominant role of Ang-2 against Ang-1, and decrease in the Tie2-pho: Tie-2 ratio in the presence of VEGF and VEGFR-2, resulting in the activation of VEGF signaling, can induce an angiogenesis-promoting milieu after rBMSC injection into the peri-infarct area. Finally, our results revealed that rBMSCs contributed to increased VEGF, VEGFR-2, Tie-2, and Ang-2 protein levels in the injured myocardium, although a reduction in Ang-1 and Tie2-pho levels was observed. Angiogenic signaling factors investigated in this study should have crosstalk with each other in a time- or concentration-dependant manner. The changes seen in these angiogenic factors may be imposed directly by the transplanted cells, or cells transplanted in the injured myocardium may influence the milieu to regulate the endogenous pro-angiogenic molecules in a manner that strengthens the angiogenic microenvironment [65]. 5. Limitations In the present study, the rodent animal model of acute MI was used. However, future studies should be conducted to scrutinize MSC-based angiogenesis signaling in large animal models of MI which have more similarities to the human heart. Also, dynamic induction of angiogenic milieu over a period of 21 days after the transplantation of marrow MSCs was investigated in this study. Be that as it may, it is appropriate that the beneficial effect of MSCs in the injured myocardium be assessed through long-term monitoring of MSC-derived angiogenesis and tracing the fate of the transplanted cells. 6. Conclusions In conclusion, the present study claims that transplantation of marrow-derived MSCs into the infarcted myocardium augments neovascularization in both paracrine and trans-differentiation and manners. Inter-regulated balance between the pro-angiogenic molecules whose expression levels were changed by the bone marrow MSCs could result in angiogenic switching on. Also, the transplanted cells were enriched over time in the injured myocardium. These findings denote that marrow-derived MSCs, as angiogenesis inducer, possess immense therapeutic powers for decreasing the adverse effects of myocardial remodeling and increasing cardiac performance after MI. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2014.03.008. Acknowledgments We extend our appreciation to Dr. Y. Gheisari for kindly providing us with GFP-encoding lentiviral vector. We thank Mrs. Leila Aghoush, Leila Haghighi, Dr. Mohammad Taheri, Mr. Mohammad Jaffar Ebrahimian, and Mrs. Maryam Zakipour for their technical assistance. The authors of this manuscript hereby certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Mitsos S, Katsanos K, Koletsis E, et al. Therapeutic angiogenesis for myocardial ischemia revisited: basic biological concepts and focus on latest clinical trials. Angiogenesis 2012;15:1–22. [2] van den Borne SW, Diez J, Blankesteijn WM, Verjans J, Hofstra L, Narula J. Myocardial remodeling after infarction: the role of myofibroblasts. Nat Rev Cardiol 2010;7:30–7. [3] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000;6:389–95. [4] Tang J, Xie Q, Pan G, Wang J, Wang M. Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion. Eur J Cardiothorac Surg 2006;30:353–61.

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Dynamic Tracking of Injected Mesenchymal Stem Cells after Myocardial Infarction in Rats: A Serial 7T MRI Study.

Timing of transplantation of autologous bone marrow derived mesenchymal stem cells for treating myocardial infarction.

Mesenchymal stem cells for treatment of myocardial infarction.

Proangiogenic Features of Mesenchymal Stem Cells and Their Therapeutic Applications.

Myocardial transfection of hypoxia-inducible factor-1α and co-transplantation of mesenchymal stem cells enhance cardiac repair in rats with experimental myocardial infarction.

Bone marrow mesenchymal stem cells improve myocardial function in a swine model of acute myocardial infarction.

Intracoronary Transplantation of Mesenchymal Stem Cells with Overexpressed Integrin-Linked Kinase Improves Cardiac Function in Porcine Myocardial Infarction.

Stem cells for myocardial infarction.

Multifactorial Experimental Design to Optimize the Anti-Inflammatory and Proangiogenic Potential of Mesenchymal Stem Cell Spheroids.

Transplanted Human Amniotic Epithelial Cells Secrete Paracrine Proangiogenic Cytokines in Rat Model of Myocardial Infarction.

Effect of transplantation of bone marrow stem cells on myocardial infarction size in a rabbit model.

Mesenchymal stem cell characteristics of dental pulp and periodontal ligament stem cells after in vivo transplantation.

Intravenous administration of atorvastatin-pretreated mesenchymal stem cells improves cardiac performance after acute myocardial infarction: role of CXCR4.

A randomized, open-label, multicenter trial for the safety and efficacy of adult mesenchymal stem cells after acute myocardial infarction.

The optimization of cell therapy by combinational application with apicidin-treated mesenchymal stem cells after myocardial infarction.

Amelioration of experimental autoimmune encephalomyelitis through transplantation of placental derived mesenchymal stem cells.

PROTECTIVE EFFECT OF Ailanthus excelsa ROXB IN MYOCARDIAL INFARCTION POST MESENCHYMAL STEM CELL TRANSPLANTATION: STUDY IN CHRONIC ISCHEMIC RAT MODEL.

Human umbilical cord tissue-derived mesenchymal stromal cells attenuate remodeling after myocardial infarction by proangiogenic, antiapoptotic, and endogenous cell-activation mechanisms.

Mesenchymal stem cells overexpressing integrin-linked kinase attenuate left ventricular remodeling and improve cardiac function after myocardial infarction.

Effects of captopril on contractility after myocardial infarction: experimental observations.

Proangiogenic compositions of microvesicles derived from human umbilical cord mesenchymal stem cells.

Activation of NRG1-ERBB4 signaling potentiates mesenchymal stem cell-mediated myocardial repairs following myocardial infarction.

Intramyocardial Adipose-Derived Stem Cell Transplantation Increases Pericardial Fat with Recovery of Myocardial Function after Acute Myocardial Infarction.

Comparison of human induced pluripotent stem-cell derived cardiomyocytes with human mesenchymal stem cells following acute myocardial infarction.