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Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair without Stem Cell Transplantation *

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 13, pp. 9949 –9961, March 26, 2010 Author’s Choice © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair without Stem □ S Cell Transplantation Received for publication, November 20, 2009, and in revised form, January 15, 2010 Published, JBC Papers in Press, January 22, 2010, DOI 10.1074/jbc.M109.087254 ‡§1 ¶1 ¶ ¶ ¶ Vincenzo Lionetti , Silvia Cantoni , Claudia Cavallini , Francesca Bianchi , Sabrina Valente**, Irene Frascari , ¶ § ¶ ¶ ‡‡ ¶ Elena Olivi , Giovanni D. Aquaro , Francesca Bonavita , Ignazio Scarlata , Margherita Maioli , Valentina Vaccari , ¶ §§ ‡¶¶ ¶2 Riccardo Tassinari , Antonietta Bartoli , Fabio A. Recchia , Gianandrea Pasquinelli**, and Carlo Ventura ‡ ¶ From the Sector of Medicine, Scuola Superiore S. Anna, I-56124 Pisa, Italy, the Laboratory of Molecular Biology and Stem Cell Engineering, Cardiovascular Department-National Institute of Biostructures and Biosystems, S. Orsola-Malpighi Hospital, University of Bologna, I-40138 Bologna, Italy, the Bioscience Institute, RSM-47891 Falciano, Republic of San Marino, the Institute ‡‡ of Clinical Physiology, Consiglio Nazionale delle Ricerche Fondazione G. Monasterio, I-56124 Pisa, Italy, the Department of §§ Biomedical Sciences, University of Sassari, I-07100 Sassari, Italy, the Department of Physics, University of Pisa, I-56124 Pisa, Italy, ¶¶ the Department of Physiology, New York Medical College, Valhalla, New York 10595, and the **Department of Hematology, Oncology, and Clinical Pathology, University of Bologna, I-40138 Bologna, Italy Possible cardiac repair by adult stem cell transplantation is therapy can be afforded by HBR without the need of stem cell currently hampered by poor cell viability and delivery efficiency, transplantation or vector-mediated gene delivery. uncertain differentiating fate in vivo, the needs of ex vivo cell expansion, and consequent delay in transplantation after the onset of heart attack. By the aid of magnetic resonance imaging, Cardiomyocyte loss during myocardial infarction (MI) is positron emission tomography, and immunohistochemistry, we associated with dysfunction of underperfused myocardium, show that injection of a hyaluronan mixed ester of butyric and eventually progressing toward heart failure. Analysis of the res- retinoic acid (HBR) into infarcted rat hearts afforded substantial cuing potential associated with transplantation of human mes- cardiovascular repair and recovery of myocardial performance. enchymal stem cells (hMSCs) in animal models of MI has HBR restored cardiac [ F]fluorodeoxyglucose uptake and recently led to the conclusion that paracrine actions exerted by increased capillary density and led to the recruitment of endog- adult stem cells through the release of soluble factors might be enous Stro-1-positive stem cells. A terminal deoxynucleotidyl- important mechanisms of tissue repair and functional improve- transferase-mediated dUTP nick end labeling assay demon- ment (1, 2). To this end, we have recently shown that hMSCs strated that HBR-treated hearts exhibited a decrease in the isolated from fetal membranes of human term placenta number of apoptotic cardiomyocytes. In isolated rat cardiomyo- (FMhMSCs) secreted large amounts of angiogenic, mitogenic, cytes and Stro-1 stem cells, HBR enhanced the transcription of antiapoptotic, and antifibrotic factors, as compared with vascular endothelial growth factor, hepatocyte growth factor, hMSCs isolated from the human bone marrow, significantly kdr, akt, and pim-1. HBR also increased the secretion of vascular contributing to improved cardiovascular function in infarcted endothelial growth factor and hepatocyte growth factor, sug- rat hearts (3). It is noteworthy that ex vivo preconditioning of gesting that the mixed ester may have recruited both myocardial FMhMSCs with a mixed ester of hyaluronan with butyric and and Stro-1 cells also. An increase in capillarogenesis was induced retinoic acid (HBR) acted transcriptionally to increase both the in vitro with medium obtained from HBR-exposed cells. In the commitment to cardiovascular lineages and the secretion of infarcted myocardium, HBR injection increased histone H4 acety- trophic mediators, remarkably enhancing stem cell-mediated lation significantly. Acetyl-H4 immunoreactivity increased in rat improvement in vivo (3). cardiomyocytes and Stro-1 cells exposed to HBR, compared with Here, we directly injected HBR into the myocardium of untreated cells. In conclusion, efficient cardiac regenerative infarcted rat hearts and provide evidence that the mixed ester afforded substantial recovery of myocardial performance with- out the need of stem cell transplantation. The HBR action was * This work was supported by Regione Emilia Romagna, Programma di also associated with an increase in the number of Stro-1-posi- Ricerca Regione-Universita` 2007/2009, Area 1b “Medicina Rigenerativa,” Italy; Fondazione Luisa Fanti Melloni, Bologna, Italy; Sintofarm S.p.A. (Guastalla, Reggio Emilia), Italy; Tavola Valdese, Rome, Italy; and and “Com- The abbreviations used are: MI, myocardial infarction; HBR, hyaluronan pagnia di San Paolo,” Torino, Italy. mixed ester of butyric and retinoic acid; VEGF, vascular endothelial growth Author’s Choice—Final version full access. factor; HGF, hepatocyte growth factor; hMSC, human mesenchymal stem □ S The on-line version of this article (available at http://www.jbc.org) contains cell; MRI, magnetic resonance imaging; PET, positron emission tomogra- 18 18 supplemental Fig. 1 and Movies 1 and 2. phy; mPET, small animal positron emission tomography; [ F]FDG, [ F]fluo- Both authors contributed equally to this work. rodeoxyglucose; RCm, rat neonatal cardiomyocyte(s); RAOECs, rat aortic To whom correspondence should be addressed: Laboratory of Molecular endothelial cells; HUVEC, human umbilical vein endothelial cell(s); DS, Biology and Stem Cell Engineering, Cardiovascular Dept.-National Institute degree of substitution; LV, left ventricular; vWF, von Willebrand factor; TBS, of Biostructures and Biosystems, S. Orsola-Malpighi Hospital, University of Tris-buffered saline; Ab, antibody; GAPDH, glyceraldehyde-3-phosphate Bologna, Via Massarenti 9, I-40138 Bologna, Italy. Fax/Tel.: 39-051340339; dehydrogenase; HA, hemagglutinin; BU, butyric acid; RA, retinoic acid; PBS, E-mail: [email protected] or [email protected]. phosphate-buffered saline; PDGF, platelet-derived growth factor. MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9949 This is an Open Access article under the CC BY license. Endogenous Cell Therapy with a Synthetic Molecule tive cells within the injected myocardium. These responses apical) and six circumferential regions (i.e. anterior, anterior- probably involved the activation of a gene program of paracrine lateral, inferolateral, inferior, inferoseptal, and anterior-septal). patterning for myocardial protection and angiogenesis and the LV regions were selected as described previously (5). Briefly, the enhanced survival of locally recruited stem cells. infarcted area comprised segments with more than 25% of their area occupied by scar tissue, and the border zone comprised EXPERIMENTAL PROCEDURES segments containing less than 25% of scar tissue area and was Synthesis of HBR—The procedure for the synthesis and char- immediately contiguous (either circumferentially or longitudi- acterization of HBR, and the related chemical structure are nally) to the infarcted area. The remote segments that did not reported in detail elsewhere (4). The primary hydroxyl group in contain scar tissue were those located outside the border zone. position 6 of the N-acetyl-D-glucosamine residues in the To assess the relationship between in vivo measurements of polysaccharide backbone is the most reactive toward esterifica- myocardial contractility and metabolism, mPET was per- tion. Briefly, we prepared a double salt of tetrabutylammonium formed within 24–36 h after MRI. with two functional groups of hyaluronan, specifically its car- MRI Measurements—MRI protocol was performed with a boxyl and 6-hydroxyl, in order to achieve good solubility in 1.5-tesla clinical whole body scanner (Signa CVI, GE Medical polar aprotic organic solvents and to increase the nucleophilic- Systems) by using a phased array send-receive coil for a human ity of the oxygen atom at C-6. Retinoylation with retinoyl chlo- knee (knee PA coil), as described previously (6). An electrocar- ride, which is the rate-limiting step, was carried out before diograph-triggered SSFP (FIESTA) pulse sequence was ac- butyrylation by means of butyric anhydride and 4-(dimethyl- quired to assess LV function (parameters: 200-mm field of view, amino)pyridine as a hypernucleophilic acylation catalyst (4). 3-mm slice thickness, no gap, number of excitations 5, two The degree of substitution (DS) was considered as the number views per segment, echo time/repetition time 1.6/3.2, flip angle of the esterified OH groups for each repeating unit of hyalu- 45°, matrix 192 192, and reconstruction matrix 256 256). In ronic acid (GlcNAc-GlcUA dimer). The weight-average molec- each rodent, a total of four LV short axis (to completely cover ular weight of HBR, referred to as the weight-average of sodium ventricular main axis in end diastole) and two LV long axis hyaluronate, was determined by high performance size exclu- views (vertical and horizontal long axis) were acquired. For sion chromatography (4). each view, 10 cine frames were acquired. For detection of non- Myocardial Infarction (MI)—Studies were performed on viable myocardium, which appears hyperintense, and for male Wistar rats (n  30, 250–300 g in size). Animals were assessment of LV infarct size, delayed enhancement images sedated (xylazine 14 mg/kg, intraperitoneally), anesthetized were obtained 3 min after bolus injection of Gadobutrol (Zoletil100, 40 mg/kg, intraperitoneally), and ventilated with a (Gadovist; 0.02 mmol per 100 g of body weight) via the tail mixture of air and oxygen (1:1), and MI was induced as vein; images were acquired in the same short axis and long axis described previously (3). Briefly, a thoracotomy was performed slices as used for cine MRI. An electrocardiograph-triggered in the left fourth intercostal space, and a permanent surgical fast gradient echo inversion recovery pulse sequence was uti- ligation was placed around the left anterior descending coro- lized with the following parameters: TR 4.2 ms, TE minimum, nary artery near its origin with a 6-0 silk suture, during electro- flip angle 20°, matrix 192  192, NEX 5, field of view 20–20 cardiographic monitoring for ST changes and arrhythmias. The mm, slice thickness 3 mm, no interslice gap, 1 R-R interval. The chest was closed in layers, and pneumothorax was reduced. inversion time was fixed to 100 ms and eventually optimized to Experimental protocols were approved by the Animal Care null signal from the normal myocardium when appropriate. Committee of the Italian Ministry of Health, in accordance with Image Processing—To assess the infarct size, the extent of Italian law (DL-116, January 27, 1992). delayed enhanced areas was measured using a semiautomatic Healthy animals were randomly divided into three experi- software, previously validated by us (7). For this purpose, we mental groups: 1) MI treated with 100 l of sterile phosphate- used all short axis images and two additional long axis images buffered saline (PBS) as vehicle (control group, MI  PBS, n  for the analysis of the cardiac apex. In each image, the bound- 10), 2) MI treated with 100 l of HBR solution (0.2 mg of HBR aries of contrast-enhanced areas were traced and manually cor- per 100 g of rat weight) (treated group, MI HBR, n 10), and rected when needed. Contrast-enhanced regions, namely the 3) sham-operated rats (SHAM, n  10), in which left anterior infarcted regions, were expressed in grams as well as in percent- descending coronary artery was not occluded. The sterile solu- age of the entire left ventricle. Left ventricle end diastolic and tions were injected into the viable myocardium bordering the end systolic volumes, LV mass, and ejection fraction were mea- infarct zone and the infarcted site 45 min after the coronary sured from the cine images using previously validated software ligation by a syringe with a needle of 24 gauge. The infarct zone (Mass, MEDIS). was identified by the pale color of the myocardium. Small ani- The infarcted region was detected in cine images by compar- mal positron emission tomography (mPET) and 1.5-tesla mag- ison with the respective delayed enhancement image. Then netic resonance imaging (MRI) were performed 4 weeks after regional end diastolic and end systolic wall thickness was mea- coronary ligation. sured in the core of the infarcted area, in the border regions, and Functional Assessment—Regional left ventricular (LV) myo- in the remote myocardium. Regional contractility was assessed cardial glucose uptake was measured by mPET to assess oxida- by employing the end systolic wall thickening in three short- tive metabolism, whereas regional contractility and infarct size axis segments (basal, middle, and apical) for correlations with were quantified by conventional MRI. For both types of imag- matched PET slices. Absolute regional wall thickening was cal- ing, we used three cross-sectional planes (i.e. basal, middle, and culated in the same regions by the difference between end sys- 9950 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule tolic and end diastolic wall thickness. Relative regional wall thickening was calculated with the formula, end systolic/end diastolic wall thickness  100, and expressed as a percentage. PET Measurements—Each sedated rat was placed on a scan- ner bed in prone position and received an intravenous injection 18 18 of [ F]fluorodeoxyglucose ([ F]FDG; 45 MBq) via the tail vein in a 0.15-ml volume. The residual dose in the syringe was mea- sured to verify the effective injected dose. The heart was cen- tered on the field of view. PET was performed with a small animal positron emission tomograph (GE eXplore Vista DR, GE Healthcare) (8). The dynamic list mode acquisition was immediately started for a total time of 30 min. Once the scan was finished, animals were placed in a recovery box with a warm temperature until complete recovery. All images were recon- structed with iterative reconstruction on OSEM 2D (Siemens) and visualized frame by frame with dedicated software in three planes (axial, sagittal, and coronal), as described previously (9). To perform viability analysis in each experimental condition, dynamic scans were reformatted by adding all frames of the last 10 min of acquisition in order to obtain a static acquisition image. Semiquantitative analysis of regional LV [ F]FDG uptake was performed with specific software adapted for ani- mals (ECTB) (9, 10), using resting scores of 0–4 (0, normal; 1, equivocal; 2, moderately reduced; 3, severely reduced; 4, absent). Tissue Immunohistochemistry—Hearts were arrested in dias- tole, and 3-mm-thick transverse slices were cut through the short axis of both ventricles at midseptal level. Macroscopic FIGURE 1. MRI-derived measures of left ventricular function. Global (A) infarct size was assessed by nitro blue tetrazolium staining. and regional (B) LV function are shown. Values are means  S.E.; n  11 animals for each experimental condition. LVEF, LV ejection fraction; CO, car- Samples were fixed in 10% buffered formalin and embedded in diac output; LVEDV, LV end diastolic volume; LVESV, LV end systolic volume; paraffin, and 4-m-thick sections were used for histological, ED, end diastolic; ES, end systolic. The HBR-treated group received 100 lof immunohistochemical, and immunofluorescence analysis. For HBR solution (0.2 mg of HBR/100 g of rat weight). *, p  0.05 versus SHAM; †, p  0.05 versus MI  PBS (one-way analysis of variance with subsequent conventional histopathological analysis, sections were stained Bonferroni test). with hematoxylin and eosin. Picro-Mallory trichrome staining was used for determining the degree of fibrosis. Images were digitized through a video camera (JVC 3CCD video camera, nol absolute for 10 min at room temperature in the dark; KY-F55B) connected with a Leitz diaplan light microscope; sections were then processed for immunohistochemistry TM original images were taken at 10 and analyzed using Image- with a non-biotin-amplified method (NovoLink Polymer Pro Plus 6 software (Media Cybernetics, Inc.). Detection System, Novocastra Laboratories Ltd.). After Four-m-thick dewaxed sections were used for immunohis- washing with 1 TBS, the slides were incubated with Novo- TM tochemical studies. Capillary density was assessed by a poly- castra protein block for 5 min in a wet chamber to reduce clonal antibody against von Willebrand factor (vWF). Stem cell the nonspecific binding of primary antibody and polymer recruitment (mesenchymal cells or mononuclear cells) into the reagent and rinsed twice with 1 TBS. Tissue sections were infarcted myocardium was assessed using antibodies directed subsequently stained using monoclonal antibodies against against Stro-1 and c-Kit antigens, respectively. Cycling cells Stro-1 (1:100; R & D Systems, Inc.), Ki-67 (1:230, clone were identified with an antibody against nuclear transcription MM1; Novocastra), NG2 (1:100; R&D Systems, Inc.), factor Ki-67. The accumulation and the spatial distribution of PDGF-R (1:100; R&D Systems, Inc.), and polyclonal anti- acetyl-histones was investigated with an antibody directed bodies against acetyl-histone H4 (1:150, Lys8; Upstate Bio- against acetyl-histone H4. Perivascular cells were characterized technology), c-Kit (1:200, DakoCytomation), VEGF (1:50, using antibodies against NG2 and PDGF-R, and expression of JH121 clone; Abcam), vWF (1:2000; DakoCytomation) in 1% vascular endothelial growth factor (VEGF) was detected with bovine serum albumin in PBS overnight at 4 °C. After wash- an antibody recognizing all VEGF isoforms. Specimens were ing, slides were incubated for 30 min at room temperature TM deparaffinated with xylene, rehydrated through decreasing with Novocastra post-primary block to enhance penetra- concentrations of ethanol, rinsed in distilled water, and sub- tion of the next polymer reagent, rinsed in 1 TBS, and TM jected to an antigen retrieval treatment. Antigens were incubated with NovoLink Polymer for 30 min at room unmasked with citrate buffer, pH 6.0, at 120 °C, 1 atm for 21 temperature. After washing, the sections were exposed to min. After cooling and washing, endogenous peroxidase the substrate/chromogen 3,3-diaminobenzidine prepared TM activity was neutralized using a 3% H O solution in metha- from Novocastra 3,3-diaminobenzidine chromogen and 2 2 MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9951 Endogenous Cell Therapy with a Synthetic Molecule formed as described above. Then sections were incubated for 30 min at room temperature in a wet cham- ber with blocking solution contain- ing sheep serum (1:10) in 1% bovine serum albumin to reduce nonspe- cific staining. Tissue sections were labeled with the Stro-1 monoclonal antibody (1:100; R&D Systems) at 37 °C for 45 min in a wet chamber. After rinses, slides were incubated with a Cy3-conjugated sheep anti- mouse secondary antibody (1:1000; Sigma), in 1% bovine serum albumin in PBS for 45 min at 37 °C; sections were then treated with goat serum (1:10) or 2% rabbit serum in 1% bovine serum albumin in PBS for 30 min at room temperature and stained with anti-CD45 (1:100; Santa Cruz Biotechnology, Inc.) or anti -sarcomeric actin monoclonal antibody (1:500; Sigma) or anti-c- Kit (1:200; DakoCytomation) or anti-vWF polyclonal antibodies (1:2000; DakoCytomation); all im- munostainings were performed for 45 min at 37 °C in the dark. The slides were then incubated in polyclonal goat anti-rabbit fluo- rescein isothiocyanate-conjugated (fluorescein isothiocyanate, 1:500; Sigma) or rabbit anti-mouse fluo- rescein isothiocyanate-conjugated (fluorescein isothiocyanate, 1:250; DakoCytomation) antibodies for FIGURE 2. MRI delayed contrast enhancement of myocardial scar. Top, representative LV short and long axis 45 min at 37 °C in the dark. Finally, gadolinium-delayed contrast-enhanced MRI images for each experimental condition. Bottom, regional LV after several rinses, the samples delayed enhancement in infarcted LV treated with 100 l of HBR solution (0.2 mg of HBR/100 g of rat weight) were coverslipped with ProLong or PBS. Values are means S.E.; n 11 animals for each experimental condition. SA, short axis; LVDE, LV delayed contrast enhancement. *, p 0.05 versus SHAM; †, p 0.05 versus MI PBS (one-way analysis of variance with antifade reagent with 4,6-dia- subsequent Bonferroni test). midino-2-phenylindole (Molecu- lar Probes). For negative control, TM NovoLink 3,3-diaminobenzidine substrate buffer, rinsed sections were processed omitting the primary antibody. in distilled water, and counterstained with Gill’s hematoxy- Gene Expression—Total RNA was extracted using TRIzol lin. Then samples were dehydrated, coverslipped, and reagent (Invitrogen), and 1 g was reverse-transcribed into TM viewed by light microscopy using the Image-Pro Plus pro- cDNA in a 21-l reaction volume with SuperScript III gram. Negative control was obtained by omitting the pri- reverse transcriptase. To assess gene expression, 2 l of cDNA mary Abs. Assessment of apoptotic programmed cell death were used for Real Time PCR performed with a Lightcycler by terminal deoxynucleotidyltransferase-mediated dUTP system (Roche Applied Science) and with the SYBR Green I nick end labeling assay is described in the supplemen- FastStart kit (Lightcycler FastStart DNA MasterPLUS SYBR tal material. Green I) following the manufacturer’s instructions. Primer Immunofluorescence—To investigate the co-expression of sequence is reported in the supplemental material. different antigens in the same cell, a double immunofluores- Data were normalized using GAPDH as an index of cDNA cence procedure was used. We aimed to demonstrate whether content after reverse transcription. Amplification included ini- Stro-1-positive cells co-expressed c-Kit, vWF, CD45, and tial denaturation at 95 °C for 10 min, 50 cycles of denaturation -sarcomeric actin, respectively. Four-m-thick dewaxed sec- at 95 °C for 10 s, annealing at 59–63 °C for 6–10 s, and exten- tions were rehydrated with decreasing concentrations of etha- sion at 72 °C for 10 s, performed at a temperature transition rate nol and rinsed in distilled water. Antigen retrieval was per- of 20 °C/s. Fluorescence was measured at the end of each exten- 9952 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule RNA (about 5 10 cpm) were then subjected to a solution hybridiza- tion RNase protection assay and were hybridized for 12 h at 55 °C in the presence of unlabeled antisense VEGF and pim-1 mRNA. To gener- ate these cRNA probes, cDNA frag- ments of rat VEGF (597 bp), pim-1 (609 bp), or GAPDH (574 bp) genes were inserted into a pCRII-TOPO vector. Transcription of plasmids linearized with BamHI generated antisense strands of Pim-1 and GAPDH mRNA, whereas transcrip- tion of plasmids linearized with XbaI produced an antisense strand of VEGF mRNA. Samples were then incubated with a combination of RNase A and T1 and exposed to proteinase K. The protected frag- ments were recovered after phenol chloroform extraction and electro- phoretically separated in a poly- acrylamide non-denaturing gel. Auto- radiographic exposure was for 48 h. In Vitro Vasculogenesis Assay— Analysis of capillary-like tube for- mation was performed using extra- cellular matrix gel (Sigma). Fifty l of gel matrix solution, diluted 1:2 with Dulbecco’s modified Eagle’s medium, was applied to each well on a 96-well plate and incubated for 1 h at 37 °C. Human umbilical vein endothelial cells (HUVECs) (Lonza) and rat aortic endothelial cells (RAOECs) (Cell Applications, Inc.) FIGURE 3. Myocardial glucose uptake measured by [ F]FDG. Top, representative LV polar map, short and were cultured in endothelial growth long axis mPET images for each experimental condition; Bottom, mean score values of myocardial [ F]FDG of infarcted LV treated with 100 l of HBR solution (0.2 mg of HBR/100 g of rat weight) or PBS. n  11 animals for medium (EGM-2 from Lonza and each experimental condition. SA, short axis; LA, long axis. a.u., arbitrary units. RAOEC growth medium from Cell Applications, Inc., respectively) sion step. Specificity of the product was determined by a until confluence. They were then trypsinized and seeded on the melting curve analysis, conducted after completion of the preprepared polymerized gel. For each well, 1  10 cells were cycling process with the aid of a temperature ramp (from 55 suspended in 50 l of conditioned medium obtained from rat to 95 °C at 0.1 °C/s) and continuous fluorescence monitor- neonatal cardiomyocytes (RCm) (Cell Applications, Inc.) or rat ing. Samples were run in duplicate, and the average thresh- Stro-1-positive cells (ScienCell) cultured for 24 h in the absence old cycle (Ct) value was used for calculations. Relative quan- or presence of HBR (2 mg/ml) and incubated at 37 °C. Capil- tification of mRNA expression was calculated with the lary-like structures were observed after 2 h and at regular inter- comparative Ct method using the “delta-delta method” for vals during the following 24 h and photographed using an comparing relative expression results between treatments in inverted optical microscope equipped with a digital sight cam- real time PCR (11). era (Nikon). Nuclear Run-off Transcription Assay—Isolation of nuclei and Enzyme-linked Immunosorbent Assay—VEGF and hepato- assessment of nuclear purity were performed as detailed else- cyte growth factor (HGF) were determined in supernatants where (3). Only freshly isolated nuclei were used in each ex- harvested from RCm and Stro-1-positive cells cultured in the periment. Nuclear run-off experiments were carried out as presence or absence of HBR (2 mg/ml) at different times (12 described previously (3). Nuclear RNA was isolated by using h, 24 h, 3 days, and 6 days) by using commercially available guanidine thiocyanate and acid phenol extraction, followed by kits (rat VEGF enzyme-linked immunosorbent assay kit TM 32 purification on RNAMATRIX . Equal counts of P-labeled (R&D Systems) and rat HGF enzyme-linked immunosorbent MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9953 Endogenous Cell Therapy with a Synthetic Molecule Western blotting detection reagents (GE Healthcare) according to the manufacturer’s instructions. Statistical Analysis—Statistical analysis was performed using GraphPad Prism, version 4. Data were evaluated by using a two- tailed, unpaired Student’s t test and analysis of variance as appropriate, with the Bonferroni post hoc test, assuming a p value less than 0.05 as the limit of significance. RESULTS The glycoconjugate HBR is an ester between the hydroxyl groups of hyaluronan (HA) and the car- boxyl groups of both butyric acid (BU) and retinoic acid (RA). All of the synthesized HBR exhibited a DS with BU (DS ) ranging between BU 0.05 and 1.5, whereas the DS with RA (DS ) was between 0.002 and RA 0.1. The DS /DS ratio was at BU RA least 6. In the HBR used in the pres- ent study, DS and DS were 1.44 BU RA and 0.032, respectively. The weight- average molecular weight ranged between 10,000 and 30,000 daltons. HBR Induced No Adverse Effects in Healthy Rats—No adverse reac- tions were observed during and after intramyocardial injection of HBR in healthy rats. Within 4 FIGURE 4. HBR increased the number of capillary vessels, Stro-1-positive cells, and perivascular ele- weeks, the animals exhibited no ments. A–C, 4 weeks following myocardial infarction. Transversally cut left ventricular myocardium from HBR- cardiovascular complications, such treated (100 l of HBR solution) hearts showed a reduced scar, compared with PBS-treated animals (A, upper as arrhythmias, pulmonary edema, images, arrows demarcate the infarcted area). Picro-Mallory stains in blue the area of scar and in red the myo- cardium parenchyma. In the border zone of HBR-treated hearts, scar reduction was associated with fewer ascites, or thrombosis. Animal apoptotic cardiomyocytes (A, lower images; scale bars,20 m) and increased number of capillary vessels (B). behavior was normal. Histological vWF expression highlights endothelial cells (arrows) lining the capillary inner wall (B, scale bars, 300 m(upper analysis did not show interstitial images) and 50m(lower images). C, in HBR-treated samples, Stro-1 positive cells (arrows) increased in number and were closely associated with the outer capillary wall, whereas c-Kit-positive cells did not vary significantly, edema and inflammatory infiltrates. compared with untreated animals. Scale bars,20 m. D–F, 24 h following myocardial infarction. D, the number HBR Injection into the Infarcted of Ki-67-positive cells significantly increased in the HBR-treated animals (arrows); scale bars,50 m. E, cells expressing NG2 and PDGF-R (arrows); scale bars,30 m(upper images) and 100 m(lower images). F, VEGF Myocardium Enhanced Myocardial expression (arrows). Scale bars, 100 m(left and middle images) and 50 m(right image). *, significantly differ- Performance—MRI analysis showed ent from PBS-treated hearts, p 0.05 (statistical test: two-tailed, unpaired Student’s t test). IHC, immunohisto- a marked recovery of cardiac per- chemistry. Full-size images of each individual panel are presented in supplemental Fig. 1. formance in infarcted rats treated with HBR, compared with un- assay kit (B-Bridge International)), according to the user treated animals (supplemental Movies 1 and 2). The ejection manual. All samples were assayed at least in duplicate. fraction and cardiac output remarkably recovered in infarcted Western Blotting—Total heart lysate and total lysate of RCm rats receiving HBR, with significant reduction in LV end dia- and Stro-1-positive cells were subjected to SDS-PAGE. Acetyl- stolic volume 4 weeks after MI (Fig. 1A). No major recovery of histone H4 and GAPDH were detected by incubation with a LV global function was observed 1 or 2 weeks following the polyclonal rabbit Lys8-acetyl-histone H4-specific antibody injection of HBR (data not shown). LV end systolic wall thick- (1:1000 dilution; Upstate Biotechnology) and a monoclonal ening, an index of regional contractile function, and LV end rabbit GAPDH-specific antibody (1:1000 dilution; Cell Signal- diastolic thickness, an index of regional mass, were preserved in ing), respectively, followed by incubation with horseradish per- the LV border zone of infarcted HBR-treated hearts (Fig. 1B). oxidase-conjugated antibody to rabbit IgG (Cell Signaling). Conversely, no increase in contractility and mass was observed Antigen-antibody complexes were visualized by using ECL in the remote zone (Fig. 1B). Cardiac MRI was performed to 9954 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule FIGURE 5. HBR affects gene expression and secretion of trophic mediators. A and B, VEGF, kdr, HGF, akt, and pim-1 gene expression was assessed by real-time PCR. RCm (A) or Stro-1 cells (B) were cultured for 24 h (black bar), 3 days (dark gray bar), and 6 days (light gray bar) in the absence or presence of HBR (2 mg/ml). The abundance of each mRNA in untreated cells was defined as 1, and the amounts of VEGF, kdr, HGF, akt, and pim-1 mRNA from HBR-treated cells were plotted relative to that value (mean S.E.; n 6). C–F, time course analysis of VEGF and HGF, respectively, released by RCm (C and E) or Stro-1 cells (D and F) cultured in the absence (black bar) or presence (gray bar) of HBR (mean  S.E.; n  6). *, significantly different from untreated cells (controls), p  0.05 (GraphPad Prism version 4 (available on the World Wide Web), two-tailed, unpaired Student’s t test). G, in vitro capillarogenesis was assessed in HUVECs (a and c) and RAOECs (b and d) exposed to a conditioned medium obtained from RCm or Stro-1 cells cultured for 24 h in the absence (black bar) or presence (gray bar) of HBR. Morphological characteristics of capillary-like networks were evaluated by using NIS-Elements D Nikon software (version 3.06). Data are the mean S.E.; n  3. *, significantly different from HBR; #, significantly different from HBR; p  0.05. MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9955 Endogenous Cell Therapy with a Synthetic Molecule Immunohistochemical analysis of vWF expression revealed that the density of capillary vessels was sig- nificantly increased at the infarct border zone of animals injected with HBR, as compared with in- farcted tissue receiving saline as a placebo (Fig. 4B). Unlike control samples, in the HBR-treated tissue sections, the regeneration of vWF- positive vascular structures was associated with the presence of loose connective tissue in which scattered round and spindle ele- ments were seen embedded; in some samples, this “vascular front” extended from the subepicardial ventricular myocardium and spread into adjacent clusters of “viable” car- diomyocytes. Immunohistochemis- try revealed an increased number of FIGURE 6. Comparative analysis of the effect of HBR, HA, BU, and RA on the secretion of VEGF from perivascular Stro-1-positive cells rat Stro-1 positive cells. Cells were incubated for the indicated times in the absence or presence of 2.0 near newly formed capillaries in mg/ml HBR, 1.5 mg/ml HA, 2.5 mM BU, or 10 M RA alone or exposed to a hydrolyzed (HY) HBR solution (2.0 mg/ml), obtained from a 2-h basic HBR hydrolysis followed by pH neutralization. This procedure has been HBR-treated hearts after 4 weeks, shown to afford a complete release of each single HBR grafted moiety (C. Ventura et al., unpublished significantly exceeding the few Stro- observations). Data are the mean  S.E. of four separate experiments. *, significantly different from HBR alone (GraphPad Prism version 4 (available on the World Wide Web), two-tailed, unpaired Student’s t test). 1-positive cells detected in the non- injected group (Fig. 4C). Interest- ingly, most of the Stro-1-positive detect scar tissue, which appears hyperintense, and for infarct cells lacked staining with vWF (data not shown). On the con- size assessment in LV injected with PBS or HBR (Fig. 2, top). It trary, some Stro-1-positive cells coexpressed cardiac -sarco- is noteworthy that we found a significant reduction in the meric actin (data not shown). On the whole, differently from extension of the delayed enhancement of the LV infarct zone in the Stro-1 expressing elements, the number of c-Kit-positive HBR-injected hearts compared with the untreated group (Fig. cells did not differ significantly among treated and untreated 2, top and bottom). animals. This discrepancy may suggest that, at 4 weeks, Stro-1- HBR Primed Recovery of Cardiac [ F]FDG Uptake—Fig. 3 resident perivascular mesenchymal cells but not c-Kit bone displays a polar map of regional distribution and short and long marrow-derived mononuclear cells were selectively embedded axis images of [ F]FDG uptake in each experimental condition. in the site of tissue repair following HBR injection. The ischemic injury in the anterior wall significantly recovered The presence of Ki-67 was used to determine whether there in infarcted hearts receiving HBR (Fig. 3). Glucose metabolism were cycling cells in the myocardium at the time of sacrifice; after assessed by [ F]FDG uptake, an index of myocardial viability, 24 h, a significant increase in Ki-67 expression was found in the was also preserved in the border and remote regions of examined sections from HBR-injected animals, as compared with infarcted, HBR-treated hearts (Fig. 3). the untreated group (Fig. 4D). In the HBR-treated hearts, the Ki-67 HBR Increased Capillary Density and Induced Recruitment of nuclear antigen was markedly expressed by perivascular spindle- Stro-1-positive Cells—Gross pathologic examination of ische- and round-shaped stromal cells located between cardiomyocytes. mic myocardium after nitro blue tetrazolium staining revealed These cells expressed NG2 and PDGF-R (Fig. 4E), a set of mark- that HBR injection substantially decreased the percentage of ers that have been previously shown to represent a phenotype indi- LV occupied by fibrosis. Picro-Mallory trichrome staining and cator of pericyte/perivascular identity (12). The same cells lacked quantitative analyses showed that the infarct area was signifi- expression of hematopoietic cell markers (data not shown). Other cantly smaller in animals receiving HBR than in the untreated Ki-67-positive cytotypes included endothelial cells and polymor- group (Fig. 4A, upper images). Interestingly, in many of the phonuclear leukocytes. VEGF cytoplasmic expression also signifi- HBR-treated samples, the blue-stained areas, reflecting infarct cantly increased in the cardiomyocytes following HBR injection scarring, were mainly confined to a limited area in the sub- (Fig. 4F). endocardial zone, which also exhibited regions of viable, red- HBR Enhanced the Gene Expression and Secretion of Angio- stained tissue. A terminal deoxynucleotidyltransferase-me- genic, Antiapoptotic, and Antifibrotic Factors in Both Rat Ven- diated dUTP nick end labeling assay kit demonstrated that tricular Cardiomyocytes and Stro-1-positive Stem Cells—The HBR-treated hearts exhibited a decreased number of apo- infarcted myocardium has been shown to release a number of ptotic cardiomyocytes when compared with untreated factors, including the granulocyte colony-stimulating factor hearts (Fig. 4A, lower images). and the granulocyte-macrophage colony-stimulating factor, 9956 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule involved in bone marrow cell mobilization and homing (13, jected to MI in the absence or presence of HBR (data not 14). No significant difference in plasma levels of each factor shown). was observed throughout a 24-h period between rats sub- Real-time PCR analysis revealed that in vitro exposure to HBR of both RCm and rat Stro-1-positive stem cells signifi- cantly enhanced the gene expression of VEGF, kdr (encoding a major VEGF receptor), HGF, akt, and pim-1 (Fig. 5, A and B). In RCm, HBR-mediated stimulation of pim-1 gene expression occurred in a time-dependent, biphasic fashion. Increased pim-1 mRNA was detected within 24 h after HBR stimulation and then declined toward basal values after 3 days. A second elevation in pim-1 gene expression was observed after 6 days of RCm exposure to the mixed ester (Fig. 5A). The HBR treatment enhanced the secretion of VEGF and HGF in the culture medium from both RCm and Stro-1 cells. Time course analyses revealed that the stimulatory action pro- gressively increased during the first 24 h, persisting throughout FIGURE 7. Analysis of VEGF and pim-1 gene transcription in isolated a 6-day period (Fig. 5, C–F). HBR did not appreciably affect the nuclei. A and B, nuclei were isolated from rat Stro-1-positive cells cultured for secretion of VEGF or HGF from HUVECs or RAOECs; nor did 24 h (VEGF gene transcription) or 6 days (pim-1 gene transcription) in the absence or presence of 2.0 mg/ml HBR, respectively. From lanes C–H, nuclei it induce these cells to form capillary-like structures in a semi- were isolated from untreated cells and then directly incubated for 12 h with- solid medium in vitro (data not shown). However, a remarkable out any drug (C) or in the presence of 2.0 mg/ml HBR (D), 1.5 mg/ml HA (E), 2.5 mM BU (F), 10 M RA (G), or with a combination of BU and RA (H). Autoradio- increase in capillarogenesis was observed when either HUVECs graphic exposure was for 2 days on Kodak X-Omat film with an intensifying or RAOECs were cultured with medium obtained from car- screen. The right side of each panel shows the position of radiolabeled DNA diomyocytes or Stro-1-positive cells exposed to the mixed ester markers, showing that the single protected fragments migrated with a molec- ular size comparable with that of VEGF (597 bases), pim-1 (609 bases), or (Fig. 5G). The amount of VEGF released by Stro-1 cells was GAPDH (574 bases) mRNA. Due to the similar size of VEGF-, pim-1-, and slightly increased by BU but not HA alone, being significantly GAPDH-protected fragments, P-labeled nuclear RNA was hybridized sepa- augmented by 10 M all-trans-RA, and further enhanced fol- rately with cRNA probes, and the corresponding hybrids were run onto dif- ferent gels. Autoradiograms are representative of six separate experiments. lowing exposure to hydrolized HBR, resulting in the release of all moieties grafted within the mixed ester (Fig. 6). Nevertheless, under these experimental condi- tions, the yield of growth factor secretion was considerably lower than that detected in HBR-ex- posed cells (Fig. 6). Cumulatively, these findings suggest the activa- tion of prominent paracrine effects by HBR on both cell popu- lations, enhancing the expression of cytokines and genes with a cru- cial role in cell survival and angiogenesis. HBR Acted at the Transcriptional Level—To further dissect the cellu- lar response to HBR, nuclear run-off experiments were designed to assess whether HBR may have affected the rate of gene transcrip- tion and whether, in the affirmative, it may have acted as a unit or after hydrolysis of hyaluronan grafted moieties. Fig. 7 shows that nuclei isolated from HBR-treated Stro-1- positive cells exhibited a consistent FIGURE 8. Time course analysis of the effect of HBR on myocardial histone acetylation. Western blot increase in the transcription rate of analysis of histone H4 acetylation was performed in total tissue extracts from border (BZ) and remote zone (RZ) VEGF and pim-1 genes, as com- of infarcted hearts (MI)6or16h(A)or24h(B) after injection in the absence (PBS) or presence of 100 lofHBR solution (0.2 mg of HBR/100 g of rat weight). GAPDH was used as a loading control. Acetylation level was estimated pared with nuclei isolated from bydensitometricquantification(n 3).*,significantlydifferentfromthesamecontroltimepointwithintheMIBZ(A) untreated cells. In separate experi- or from MI BZ or MI RZ (B). p  0.05 (GraphPad Prism version 4 (available on the World Wide Web), two-tailed, unpaired Student’s t test). All Western blots were performed at least three times with similar results. ments, nuclei were isolated from MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9957 Endogenous Cell Therapy with a Synthetic Molecule 9958 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule untreated cells and subsequently incubated with HBR or ated with a reverse myocardial remodeling (decrease of fibrosis exposed to HA, BU, or RA administered alone or in combina- and dilatation) and consistent increase in myocardial perform- tion. Although nuclear exposure to HBR or HA failed to trigger ance. A possible explanation may result from in vivo studies a transcriptional response, the incubation with BU or RA demonstrating that HBR-treated hearts had a significant enhanced gene transcription (Fig. 7). The transcription rate was decrease of apoptotic cardiomyocytes, as revealed by a terminal further enhanced when nuclei were exposed to a combination deoxynucleotidyltransferase-mediated dUTP nick end labeling of BU and RA (Fig. 7). assay, as well as from in vitro experiments showing enhanced HBR Increased Histone Acetylation—Western blot analysis gene expression and secretion of VEGF and HGF in both RCm performed in tissue extracts revealed that, consonant with pre- and Stro-1-positive cells exposed to HBR. Growth factor secre- vious observations (15, 16), histone H4 acetylation decreased in tion may have primed a proangiogenic context (18), reinforced the infarcted hearts. Six h after HBR injection, acetyl-histone by the stimulatory effect of HBR on the expression of the kdr H4 signal rose in the border zone, progressively increasing up to gene, encoding a VEGF receptor involved in autocrine/para- 16 h, as compared with the untreated group (Fig. 8A). After crine amplification of VEGF secretion and angiogenic signaling 24 h, histone H4 acetylation increased both in the border and (19, 20). Moreover, HGF gene transfer into the myocardium remote zone of infarcted HBR-injected hearts, as compared improved myocardial function and geometry (21), due to anti- with the non-injected tissue (Fig. 8B). Following HBR treat- fibrotic effects through inhibition of transforming growth fac- ment, a more pronounced signal was detected in the border tor- expression. HBR-mediated recruitment of both myocar- zone, compared with the remote area. Akin to Western blot dial and Stro-1 cells into a proangiogenic paracrine circuitry of studies, immunohistochemical analyses showed an overall cardiac repair is further supported by the finding that (i) an increase in the signal of acetyl-histone H4 in hearts of HBR- increase in capillarogenesis was induced in vitro in both treated animals, as compared with untreated hearts (Fig. 9A). HUVECs and RAOECs with a medium obtained from HBR-ex- The number of cardiomyocyte nuclei stained with acetyl-his- posed cells; (ii) no secretion of VEGF or HGF or in vitro angiogen- tone H4 antibody was significantly increased by HBR treatment esis was found following a direct HUVEC or RAOEC exposure to in the border zone (Fig. 9A). In vitro experiments provided evi- HBR; and (iii) in vivo, a large number of capillary vessels in HBR- dence that acetyl-H4 immunoreactivity was also higher in injected hearts were “decorated” by Stro-1-positive cells, lacking HBR-exposed RCm and Stro-1-positive cells, as compared with the expression of vWF, an endothelial marker. Although some unexposed cells (Fig. 9B). Stro-1-positive cells in HBR-injected hearts expressed heart-spe- cific -sarcomeric actin, these few elements appeared to lack DISCUSSION mature sarcomeric organization, and their role in rescuing The present study shows for the first time that acute myocar- infarcted myocardium remains questionable. dial injection of HBR, a hyaluronan ester previously shown to The observation that exposure of RCm and Stro-1 cells to enhance hMSC-mediated cardiac repair in vivo (3), rescued HBR increased VEGF, HGF, akt, and pim-1 gene expression infarcted rat hearts by increasing vascularization, promoting may have further relevant implications. These genes are deeply cardiomyocyte survival, and restoring normal tissue function. involved in cardioprotective signaling, including antiapoptotic The HBR action involved an early increase in the number of and antifibrotic responses (21–25). Enhanced expression of pericytes, a reserve of progenitor cells that may be integral to both akt and pim-1 promotes cardiomyocyte survival and the origin of MSCs and other related adult stem cells (12), sug- growth in transgenic mice subjected to acute myocardial infarc- gesting a role of perivascular cells in capillary density enhance- tion (26, 27). Here, we show that in RCm pim-1 gene expression ment and tissue repair elicited by the mixed ester. was augmented in a biphasic manner by the addition of HBR. It is now evident that an extremely limited percentage of Biphasic kinetics in pim-1 gene expression have been previ- stem cells will engraft and survive within the recipient myocar- ously demonstrated in other cell types exposed to agents regu- dium following intracoronary infusion or transendocardial lating cell growth and differentiation (28, 29) and appeared to injection (17). Moreover, the use of injectable scaffolds to aug- involve a complex interplay between cell signaling networks, ment stem cell engraftment is not devoid of harmful decrease in cell cycle progression, gene transcription, and/or mRNA stabil- myocardial perfusion. Hence, the development of deliverable ity (29). Cardiac control of Pim-1 gene expression is still poorly molecules promoting paracrine mechanisms of cardiac repair understood, and future work is required to dissect the molecu- and survival of endogenously recruited stem cells, without lar mechanisms underlying Pim-1 regulation by HBR in myo- immediate need of stem cell transplantation, would have remarkable biomedical implications. Here, we show that HBR cardial cells. may fulfill both requirements. Four weeks after HBR injection, HBR may also have increased the survival and rescuing a significant increase in perivascular Stro-1-positive cells could potential of Stro-1-positive cells recruited to the injured myo- be detected within the infarcted hearts. This effect was associ- cardium. In this regard, both VEGF and HGF have been shown FIGURE 9. HBR increased histone acetylation in infarcted myocardium, isolated cardiomyocytes and Stro-1 cells. A, immunohistochemistry of acetyl- histone H4. Acetylation level was estimated in infarcted hearts by intensity quantification and counting of stained cardiomyocyte nuclei 24 h after injection in the absence (PBS) or presence of HBR solution (0.2 mg of HBR/100 g of rat weight). Scale bar,30 m. *, significantly different from PBS-treated, p  0.05. B, Western blot analysis of total cell extracts from RCm and Stro-1-positive cells cultured in the absence (CTR) or presence of HBR (2 mg/ml) (n  3). *, significantly different from the same control time point, p 0.05 (GraphPad Prism version 4 (available on the World Wide Web), two-tailed, unpaired Student’s t test). All Western blots were performed at least three times with similar results. MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9959 Endogenous Cell Therapy with a Synthetic Molecule to promote MSC survival and therapeutic potential (30), practice; and (iii) analytical methodologies for control of good enhancing stem cell-mediated cardioprotection in infarcted manufacturing practice bioprocessing and differentiation effi- hearts (31, 32). Up-regulation of both akt and pim-1 gene ciencies. Therefore, the timing for cell culture and expansion expression in HBR-treated Stro-1 cells is also worthy of consid- within a good manufacturing practice setting will involve a sub- eration, due to the prominent role of these serine/threonine stantial delay (several weeks) in autologous stem cell transplan- kinases in stem cell survival and differentiation (30, 33). MSCs tation with respect to the acute phase of a heart attack. Mean- overexpressing akt prevent remodeling and restore perform- while, the cardiac repair afforded by a myocardial injection of ance of infarcted hearts (1, 34). Moreover, pim-1 has been HBR may serve as first aid to rescue a damaged heart. This shown to be required for endothelial and mural cell differenti- intervention may be followed by delayed transplantation of ation in vitro (35). autologous stem cells, eventually preconditioned ex vivo with It is relevant that a “program” of genes involved in cardiac the same molecule, to enhance the long term potential for car- protection and stem cell survival can be chemically induced by diovascular cell therapy. a synthetic molecule, without the need of viral vector-mediated gene transfer technologies. The molecular dissection of mech- Acknowledgment—We thank Dr. Cristina Nanni (Department of anisms underlying HBR-mediated responses in vitro and in vivo Nuclear Medicine, S. Orsola-Malpighi Hospital, Bologna, Italy) for technical support in the PET studies. remains to be fully elucidated. 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Cell 6, 1049–1058 MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9961 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair without Stem Cell Transplantation *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2010 Elsevier Inc.
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0021-9258
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1083-351X
DOI
10.1074/jbc.m109.087254
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 13, pp. 9949 –9961, March 26, 2010 Author’s Choice © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair without Stem □ S Cell Transplantation Received for publication, November 20, 2009, and in revised form, January 15, 2010 Published, JBC Papers in Press, January 22, 2010, DOI 10.1074/jbc.M109.087254 ‡§1 ¶1 ¶ ¶ ¶ Vincenzo Lionetti , Silvia Cantoni , Claudia Cavallini , Francesca Bianchi , Sabrina Valente**, Irene Frascari , ¶ § ¶ ¶ ‡‡ ¶ Elena Olivi , Giovanni D. Aquaro , Francesca Bonavita , Ignazio Scarlata , Margherita Maioli , Valentina Vaccari , ¶ §§ ‡¶¶ ¶2 Riccardo Tassinari , Antonietta Bartoli , Fabio A. Recchia , Gianandrea Pasquinelli**, and Carlo Ventura ‡ ¶ From the Sector of Medicine, Scuola Superiore S. Anna, I-56124 Pisa, Italy, the Laboratory of Molecular Biology and Stem Cell Engineering, Cardiovascular Department-National Institute of Biostructures and Biosystems, S. Orsola-Malpighi Hospital, University of Bologna, I-40138 Bologna, Italy, the Bioscience Institute, RSM-47891 Falciano, Republic of San Marino, the Institute ‡‡ of Clinical Physiology, Consiglio Nazionale delle Ricerche Fondazione G. Monasterio, I-56124 Pisa, Italy, the Department of §§ Biomedical Sciences, University of Sassari, I-07100 Sassari, Italy, the Department of Physics, University of Pisa, I-56124 Pisa, Italy, ¶¶ the Department of Physiology, New York Medical College, Valhalla, New York 10595, and the **Department of Hematology, Oncology, and Clinical Pathology, University of Bologna, I-40138 Bologna, Italy Possible cardiac repair by adult stem cell transplantation is therapy can be afforded by HBR without the need of stem cell currently hampered by poor cell viability and delivery efficiency, transplantation or vector-mediated gene delivery. uncertain differentiating fate in vivo, the needs of ex vivo cell expansion, and consequent delay in transplantation after the onset of heart attack. By the aid of magnetic resonance imaging, Cardiomyocyte loss during myocardial infarction (MI) is positron emission tomography, and immunohistochemistry, we associated with dysfunction of underperfused myocardium, show that injection of a hyaluronan mixed ester of butyric and eventually progressing toward heart failure. Analysis of the res- retinoic acid (HBR) into infarcted rat hearts afforded substantial cuing potential associated with transplantation of human mes- cardiovascular repair and recovery of myocardial performance. enchymal stem cells (hMSCs) in animal models of MI has HBR restored cardiac [ F]fluorodeoxyglucose uptake and recently led to the conclusion that paracrine actions exerted by increased capillary density and led to the recruitment of endog- adult stem cells through the release of soluble factors might be enous Stro-1-positive stem cells. A terminal deoxynucleotidyl- important mechanisms of tissue repair and functional improve- transferase-mediated dUTP nick end labeling assay demon- ment (1, 2). To this end, we have recently shown that hMSCs strated that HBR-treated hearts exhibited a decrease in the isolated from fetal membranes of human term placenta number of apoptotic cardiomyocytes. In isolated rat cardiomyo- (FMhMSCs) secreted large amounts of angiogenic, mitogenic, cytes and Stro-1 stem cells, HBR enhanced the transcription of antiapoptotic, and antifibrotic factors, as compared with vascular endothelial growth factor, hepatocyte growth factor, hMSCs isolated from the human bone marrow, significantly kdr, akt, and pim-1. HBR also increased the secretion of vascular contributing to improved cardiovascular function in infarcted endothelial growth factor and hepatocyte growth factor, sug- rat hearts (3). It is noteworthy that ex vivo preconditioning of gesting that the mixed ester may have recruited both myocardial FMhMSCs with a mixed ester of hyaluronan with butyric and and Stro-1 cells also. An increase in capillarogenesis was induced retinoic acid (HBR) acted transcriptionally to increase both the in vitro with medium obtained from HBR-exposed cells. In the commitment to cardiovascular lineages and the secretion of infarcted myocardium, HBR injection increased histone H4 acety- trophic mediators, remarkably enhancing stem cell-mediated lation significantly. Acetyl-H4 immunoreactivity increased in rat improvement in vivo (3). cardiomyocytes and Stro-1 cells exposed to HBR, compared with Here, we directly injected HBR into the myocardium of untreated cells. In conclusion, efficient cardiac regenerative infarcted rat hearts and provide evidence that the mixed ester afforded substantial recovery of myocardial performance with- out the need of stem cell transplantation. The HBR action was * This work was supported by Regione Emilia Romagna, Programma di also associated with an increase in the number of Stro-1-posi- Ricerca Regione-Universita` 2007/2009, Area 1b “Medicina Rigenerativa,” Italy; Fondazione Luisa Fanti Melloni, Bologna, Italy; Sintofarm S.p.A. (Guastalla, Reggio Emilia), Italy; Tavola Valdese, Rome, Italy; and and “Com- The abbreviations used are: MI, myocardial infarction; HBR, hyaluronan pagnia di San Paolo,” Torino, Italy. mixed ester of butyric and retinoic acid; VEGF, vascular endothelial growth Author’s Choice—Final version full access. factor; HGF, hepatocyte growth factor; hMSC, human mesenchymal stem □ S The on-line version of this article (available at http://www.jbc.org) contains cell; MRI, magnetic resonance imaging; PET, positron emission tomogra- 18 18 supplemental Fig. 1 and Movies 1 and 2. phy; mPET, small animal positron emission tomography; [ F]FDG, [ F]fluo- Both authors contributed equally to this work. rodeoxyglucose; RCm, rat neonatal cardiomyocyte(s); RAOECs, rat aortic To whom correspondence should be addressed: Laboratory of Molecular endothelial cells; HUVEC, human umbilical vein endothelial cell(s); DS, Biology and Stem Cell Engineering, Cardiovascular Dept.-National Institute degree of substitution; LV, left ventricular; vWF, von Willebrand factor; TBS, of Biostructures and Biosystems, S. Orsola-Malpighi Hospital, University of Tris-buffered saline; Ab, antibody; GAPDH, glyceraldehyde-3-phosphate Bologna, Via Massarenti 9, I-40138 Bologna, Italy. Fax/Tel.: 39-051340339; dehydrogenase; HA, hemagglutinin; BU, butyric acid; RA, retinoic acid; PBS, E-mail: [email protected] or [email protected]. phosphate-buffered saline; PDGF, platelet-derived growth factor. MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9949 This is an Open Access article under the CC BY license. Endogenous Cell Therapy with a Synthetic Molecule tive cells within the injected myocardium. These responses apical) and six circumferential regions (i.e. anterior, anterior- probably involved the activation of a gene program of paracrine lateral, inferolateral, inferior, inferoseptal, and anterior-septal). patterning for myocardial protection and angiogenesis and the LV regions were selected as described previously (5). Briefly, the enhanced survival of locally recruited stem cells. infarcted area comprised segments with more than 25% of their area occupied by scar tissue, and the border zone comprised EXPERIMENTAL PROCEDURES segments containing less than 25% of scar tissue area and was Synthesis of HBR—The procedure for the synthesis and char- immediately contiguous (either circumferentially or longitudi- acterization of HBR, and the related chemical structure are nally) to the infarcted area. The remote segments that did not reported in detail elsewhere (4). The primary hydroxyl group in contain scar tissue were those located outside the border zone. position 6 of the N-acetyl-D-glucosamine residues in the To assess the relationship between in vivo measurements of polysaccharide backbone is the most reactive toward esterifica- myocardial contractility and metabolism, mPET was per- tion. Briefly, we prepared a double salt of tetrabutylammonium formed within 24–36 h after MRI. with two functional groups of hyaluronan, specifically its car- MRI Measurements—MRI protocol was performed with a boxyl and 6-hydroxyl, in order to achieve good solubility in 1.5-tesla clinical whole body scanner (Signa CVI, GE Medical polar aprotic organic solvents and to increase the nucleophilic- Systems) by using a phased array send-receive coil for a human ity of the oxygen atom at C-6. Retinoylation with retinoyl chlo- knee (knee PA coil), as described previously (6). An electrocar- ride, which is the rate-limiting step, was carried out before diograph-triggered SSFP (FIESTA) pulse sequence was ac- butyrylation by means of butyric anhydride and 4-(dimethyl- quired to assess LV function (parameters: 200-mm field of view, amino)pyridine as a hypernucleophilic acylation catalyst (4). 3-mm slice thickness, no gap, number of excitations 5, two The degree of substitution (DS) was considered as the number views per segment, echo time/repetition time 1.6/3.2, flip angle of the esterified OH groups for each repeating unit of hyalu- 45°, matrix 192 192, and reconstruction matrix 256 256). In ronic acid (GlcNAc-GlcUA dimer). The weight-average molec- each rodent, a total of four LV short axis (to completely cover ular weight of HBR, referred to as the weight-average of sodium ventricular main axis in end diastole) and two LV long axis hyaluronate, was determined by high performance size exclu- views (vertical and horizontal long axis) were acquired. For sion chromatography (4). each view, 10 cine frames were acquired. For detection of non- Myocardial Infarction (MI)—Studies were performed on viable myocardium, which appears hyperintense, and for male Wistar rats (n  30, 250–300 g in size). Animals were assessment of LV infarct size, delayed enhancement images sedated (xylazine 14 mg/kg, intraperitoneally), anesthetized were obtained 3 min after bolus injection of Gadobutrol (Zoletil100, 40 mg/kg, intraperitoneally), and ventilated with a (Gadovist; 0.02 mmol per 100 g of body weight) via the tail mixture of air and oxygen (1:1), and MI was induced as vein; images were acquired in the same short axis and long axis described previously (3). Briefly, a thoracotomy was performed slices as used for cine MRI. An electrocardiograph-triggered in the left fourth intercostal space, and a permanent surgical fast gradient echo inversion recovery pulse sequence was uti- ligation was placed around the left anterior descending coro- lized with the following parameters: TR 4.2 ms, TE minimum, nary artery near its origin with a 6-0 silk suture, during electro- flip angle 20°, matrix 192  192, NEX 5, field of view 20–20 cardiographic monitoring for ST changes and arrhythmias. The mm, slice thickness 3 mm, no interslice gap, 1 R-R interval. The chest was closed in layers, and pneumothorax was reduced. inversion time was fixed to 100 ms and eventually optimized to Experimental protocols were approved by the Animal Care null signal from the normal myocardium when appropriate. Committee of the Italian Ministry of Health, in accordance with Image Processing—To assess the infarct size, the extent of Italian law (DL-116, January 27, 1992). delayed enhanced areas was measured using a semiautomatic Healthy animals were randomly divided into three experi- software, previously validated by us (7). For this purpose, we mental groups: 1) MI treated with 100 l of sterile phosphate- used all short axis images and two additional long axis images buffered saline (PBS) as vehicle (control group, MI  PBS, n  for the analysis of the cardiac apex. In each image, the bound- 10), 2) MI treated with 100 l of HBR solution (0.2 mg of HBR aries of contrast-enhanced areas were traced and manually cor- per 100 g of rat weight) (treated group, MI HBR, n 10), and rected when needed. Contrast-enhanced regions, namely the 3) sham-operated rats (SHAM, n  10), in which left anterior infarcted regions, were expressed in grams as well as in percent- descending coronary artery was not occluded. The sterile solu- age of the entire left ventricle. Left ventricle end diastolic and tions were injected into the viable myocardium bordering the end systolic volumes, LV mass, and ejection fraction were mea- infarct zone and the infarcted site 45 min after the coronary sured from the cine images using previously validated software ligation by a syringe with a needle of 24 gauge. The infarct zone (Mass, MEDIS). was identified by the pale color of the myocardium. Small ani- The infarcted region was detected in cine images by compar- mal positron emission tomography (mPET) and 1.5-tesla mag- ison with the respective delayed enhancement image. Then netic resonance imaging (MRI) were performed 4 weeks after regional end diastolic and end systolic wall thickness was mea- coronary ligation. sured in the core of the infarcted area, in the border regions, and Functional Assessment—Regional left ventricular (LV) myo- in the remote myocardium. Regional contractility was assessed cardial glucose uptake was measured by mPET to assess oxida- by employing the end systolic wall thickening in three short- tive metabolism, whereas regional contractility and infarct size axis segments (basal, middle, and apical) for correlations with were quantified by conventional MRI. For both types of imag- matched PET slices. Absolute regional wall thickening was cal- ing, we used three cross-sectional planes (i.e. basal, middle, and culated in the same regions by the difference between end sys- 9950 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule tolic and end diastolic wall thickness. Relative regional wall thickening was calculated with the formula, end systolic/end diastolic wall thickness  100, and expressed as a percentage. PET Measurements—Each sedated rat was placed on a scan- ner bed in prone position and received an intravenous injection 18 18 of [ F]fluorodeoxyglucose ([ F]FDG; 45 MBq) via the tail vein in a 0.15-ml volume. The residual dose in the syringe was mea- sured to verify the effective injected dose. The heart was cen- tered on the field of view. PET was performed with a small animal positron emission tomograph (GE eXplore Vista DR, GE Healthcare) (8). The dynamic list mode acquisition was immediately started for a total time of 30 min. Once the scan was finished, animals were placed in a recovery box with a warm temperature until complete recovery. All images were recon- structed with iterative reconstruction on OSEM 2D (Siemens) and visualized frame by frame with dedicated software in three planes (axial, sagittal, and coronal), as described previously (9). To perform viability analysis in each experimental condition, dynamic scans were reformatted by adding all frames of the last 10 min of acquisition in order to obtain a static acquisition image. Semiquantitative analysis of regional LV [ F]FDG uptake was performed with specific software adapted for ani- mals (ECTB) (9, 10), using resting scores of 0–4 (0, normal; 1, equivocal; 2, moderately reduced; 3, severely reduced; 4, absent). Tissue Immunohistochemistry—Hearts were arrested in dias- tole, and 3-mm-thick transverse slices were cut through the short axis of both ventricles at midseptal level. Macroscopic FIGURE 1. MRI-derived measures of left ventricular function. Global (A) infarct size was assessed by nitro blue tetrazolium staining. and regional (B) LV function are shown. Values are means  S.E.; n  11 animals for each experimental condition. LVEF, LV ejection fraction; CO, car- Samples were fixed in 10% buffered formalin and embedded in diac output; LVEDV, LV end diastolic volume; LVESV, LV end systolic volume; paraffin, and 4-m-thick sections were used for histological, ED, end diastolic; ES, end systolic. The HBR-treated group received 100 lof immunohistochemical, and immunofluorescence analysis. For HBR solution (0.2 mg of HBR/100 g of rat weight). *, p  0.05 versus SHAM; †, p  0.05 versus MI  PBS (one-way analysis of variance with subsequent conventional histopathological analysis, sections were stained Bonferroni test). with hematoxylin and eosin. Picro-Mallory trichrome staining was used for determining the degree of fibrosis. Images were digitized through a video camera (JVC 3CCD video camera, nol absolute for 10 min at room temperature in the dark; KY-F55B) connected with a Leitz diaplan light microscope; sections were then processed for immunohistochemistry TM original images were taken at 10 and analyzed using Image- with a non-biotin-amplified method (NovoLink Polymer Pro Plus 6 software (Media Cybernetics, Inc.). Detection System, Novocastra Laboratories Ltd.). After Four-m-thick dewaxed sections were used for immunohis- washing with 1 TBS, the slides were incubated with Novo- TM tochemical studies. Capillary density was assessed by a poly- castra protein block for 5 min in a wet chamber to reduce clonal antibody against von Willebrand factor (vWF). Stem cell the nonspecific binding of primary antibody and polymer recruitment (mesenchymal cells or mononuclear cells) into the reagent and rinsed twice with 1 TBS. Tissue sections were infarcted myocardium was assessed using antibodies directed subsequently stained using monoclonal antibodies against against Stro-1 and c-Kit antigens, respectively. Cycling cells Stro-1 (1:100; R & D Systems, Inc.), Ki-67 (1:230, clone were identified with an antibody against nuclear transcription MM1; Novocastra), NG2 (1:100; R&D Systems, Inc.), factor Ki-67. The accumulation and the spatial distribution of PDGF-R (1:100; R&D Systems, Inc.), and polyclonal anti- acetyl-histones was investigated with an antibody directed bodies against acetyl-histone H4 (1:150, Lys8; Upstate Bio- against acetyl-histone H4. Perivascular cells were characterized technology), c-Kit (1:200, DakoCytomation), VEGF (1:50, using antibodies against NG2 and PDGF-R, and expression of JH121 clone; Abcam), vWF (1:2000; DakoCytomation) in 1% vascular endothelial growth factor (VEGF) was detected with bovine serum albumin in PBS overnight at 4 °C. After wash- an antibody recognizing all VEGF isoforms. Specimens were ing, slides were incubated for 30 min at room temperature TM deparaffinated with xylene, rehydrated through decreasing with Novocastra post-primary block to enhance penetra- concentrations of ethanol, rinsed in distilled water, and sub- tion of the next polymer reagent, rinsed in 1 TBS, and TM jected to an antigen retrieval treatment. Antigens were incubated with NovoLink Polymer for 30 min at room unmasked with citrate buffer, pH 6.0, at 120 °C, 1 atm for 21 temperature. After washing, the sections were exposed to min. After cooling and washing, endogenous peroxidase the substrate/chromogen 3,3-diaminobenzidine prepared TM activity was neutralized using a 3% H O solution in metha- from Novocastra 3,3-diaminobenzidine chromogen and 2 2 MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9951 Endogenous Cell Therapy with a Synthetic Molecule formed as described above. Then sections were incubated for 30 min at room temperature in a wet cham- ber with blocking solution contain- ing sheep serum (1:10) in 1% bovine serum albumin to reduce nonspe- cific staining. Tissue sections were labeled with the Stro-1 monoclonal antibody (1:100; R&D Systems) at 37 °C for 45 min in a wet chamber. After rinses, slides were incubated with a Cy3-conjugated sheep anti- mouse secondary antibody (1:1000; Sigma), in 1% bovine serum albumin in PBS for 45 min at 37 °C; sections were then treated with goat serum (1:10) or 2% rabbit serum in 1% bovine serum albumin in PBS for 30 min at room temperature and stained with anti-CD45 (1:100; Santa Cruz Biotechnology, Inc.) or anti -sarcomeric actin monoclonal antibody (1:500; Sigma) or anti-c- Kit (1:200; DakoCytomation) or anti-vWF polyclonal antibodies (1:2000; DakoCytomation); all im- munostainings were performed for 45 min at 37 °C in the dark. The slides were then incubated in polyclonal goat anti-rabbit fluo- rescein isothiocyanate-conjugated (fluorescein isothiocyanate, 1:500; Sigma) or rabbit anti-mouse fluo- rescein isothiocyanate-conjugated (fluorescein isothiocyanate, 1:250; DakoCytomation) antibodies for FIGURE 2. MRI delayed contrast enhancement of myocardial scar. Top, representative LV short and long axis 45 min at 37 °C in the dark. Finally, gadolinium-delayed contrast-enhanced MRI images for each experimental condition. Bottom, regional LV after several rinses, the samples delayed enhancement in infarcted LV treated with 100 l of HBR solution (0.2 mg of HBR/100 g of rat weight) were coverslipped with ProLong or PBS. Values are means S.E.; n 11 animals for each experimental condition. SA, short axis; LVDE, LV delayed contrast enhancement. *, p 0.05 versus SHAM; †, p 0.05 versus MI PBS (one-way analysis of variance with antifade reagent with 4,6-dia- subsequent Bonferroni test). midino-2-phenylindole (Molecu- lar Probes). For negative control, TM NovoLink 3,3-diaminobenzidine substrate buffer, rinsed sections were processed omitting the primary antibody. in distilled water, and counterstained with Gill’s hematoxy- Gene Expression—Total RNA was extracted using TRIzol lin. Then samples were dehydrated, coverslipped, and reagent (Invitrogen), and 1 g was reverse-transcribed into TM viewed by light microscopy using the Image-Pro Plus pro- cDNA in a 21-l reaction volume with SuperScript III gram. Negative control was obtained by omitting the pri- reverse transcriptase. To assess gene expression, 2 l of cDNA mary Abs. Assessment of apoptotic programmed cell death were used for Real Time PCR performed with a Lightcycler by terminal deoxynucleotidyltransferase-mediated dUTP system (Roche Applied Science) and with the SYBR Green I nick end labeling assay is described in the supplemen- FastStart kit (Lightcycler FastStart DNA MasterPLUS SYBR tal material. Green I) following the manufacturer’s instructions. Primer Immunofluorescence—To investigate the co-expression of sequence is reported in the supplemental material. different antigens in the same cell, a double immunofluores- Data were normalized using GAPDH as an index of cDNA cence procedure was used. We aimed to demonstrate whether content after reverse transcription. Amplification included ini- Stro-1-positive cells co-expressed c-Kit, vWF, CD45, and tial denaturation at 95 °C for 10 min, 50 cycles of denaturation -sarcomeric actin, respectively. Four-m-thick dewaxed sec- at 95 °C for 10 s, annealing at 59–63 °C for 6–10 s, and exten- tions were rehydrated with decreasing concentrations of etha- sion at 72 °C for 10 s, performed at a temperature transition rate nol and rinsed in distilled water. Antigen retrieval was per- of 20 °C/s. Fluorescence was measured at the end of each exten- 9952 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule RNA (about 5 10 cpm) were then subjected to a solution hybridiza- tion RNase protection assay and were hybridized for 12 h at 55 °C in the presence of unlabeled antisense VEGF and pim-1 mRNA. To gener- ate these cRNA probes, cDNA frag- ments of rat VEGF (597 bp), pim-1 (609 bp), or GAPDH (574 bp) genes were inserted into a pCRII-TOPO vector. Transcription of plasmids linearized with BamHI generated antisense strands of Pim-1 and GAPDH mRNA, whereas transcrip- tion of plasmids linearized with XbaI produced an antisense strand of VEGF mRNA. Samples were then incubated with a combination of RNase A and T1 and exposed to proteinase K. The protected frag- ments were recovered after phenol chloroform extraction and electro- phoretically separated in a poly- acrylamide non-denaturing gel. Auto- radiographic exposure was for 48 h. In Vitro Vasculogenesis Assay— Analysis of capillary-like tube for- mation was performed using extra- cellular matrix gel (Sigma). Fifty l of gel matrix solution, diluted 1:2 with Dulbecco’s modified Eagle’s medium, was applied to each well on a 96-well plate and incubated for 1 h at 37 °C. Human umbilical vein endothelial cells (HUVECs) (Lonza) and rat aortic endothelial cells (RAOECs) (Cell Applications, Inc.) FIGURE 3. Myocardial glucose uptake measured by [ F]FDG. Top, representative LV polar map, short and were cultured in endothelial growth long axis mPET images for each experimental condition; Bottom, mean score values of myocardial [ F]FDG of infarcted LV treated with 100 l of HBR solution (0.2 mg of HBR/100 g of rat weight) or PBS. n  11 animals for medium (EGM-2 from Lonza and each experimental condition. SA, short axis; LA, long axis. a.u., arbitrary units. RAOEC growth medium from Cell Applications, Inc., respectively) sion step. Specificity of the product was determined by a until confluence. They were then trypsinized and seeded on the melting curve analysis, conducted after completion of the preprepared polymerized gel. For each well, 1  10 cells were cycling process with the aid of a temperature ramp (from 55 suspended in 50 l of conditioned medium obtained from rat to 95 °C at 0.1 °C/s) and continuous fluorescence monitor- neonatal cardiomyocytes (RCm) (Cell Applications, Inc.) or rat ing. Samples were run in duplicate, and the average thresh- Stro-1-positive cells (ScienCell) cultured for 24 h in the absence old cycle (Ct) value was used for calculations. Relative quan- or presence of HBR (2 mg/ml) and incubated at 37 °C. Capil- tification of mRNA expression was calculated with the lary-like structures were observed after 2 h and at regular inter- comparative Ct method using the “delta-delta method” for vals during the following 24 h and photographed using an comparing relative expression results between treatments in inverted optical microscope equipped with a digital sight cam- real time PCR (11). era (Nikon). Nuclear Run-off Transcription Assay—Isolation of nuclei and Enzyme-linked Immunosorbent Assay—VEGF and hepato- assessment of nuclear purity were performed as detailed else- cyte growth factor (HGF) were determined in supernatants where (3). Only freshly isolated nuclei were used in each ex- harvested from RCm and Stro-1-positive cells cultured in the periment. Nuclear run-off experiments were carried out as presence or absence of HBR (2 mg/ml) at different times (12 described previously (3). Nuclear RNA was isolated by using h, 24 h, 3 days, and 6 days) by using commercially available guanidine thiocyanate and acid phenol extraction, followed by kits (rat VEGF enzyme-linked immunosorbent assay kit TM 32 purification on RNAMATRIX . Equal counts of P-labeled (R&D Systems) and rat HGF enzyme-linked immunosorbent MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9953 Endogenous Cell Therapy with a Synthetic Molecule Western blotting detection reagents (GE Healthcare) according to the manufacturer’s instructions. Statistical Analysis—Statistical analysis was performed using GraphPad Prism, version 4. Data were evaluated by using a two- tailed, unpaired Student’s t test and analysis of variance as appropriate, with the Bonferroni post hoc test, assuming a p value less than 0.05 as the limit of significance. RESULTS The glycoconjugate HBR is an ester between the hydroxyl groups of hyaluronan (HA) and the car- boxyl groups of both butyric acid (BU) and retinoic acid (RA). All of the synthesized HBR exhibited a DS with BU (DS ) ranging between BU 0.05 and 1.5, whereas the DS with RA (DS ) was between 0.002 and RA 0.1. The DS /DS ratio was at BU RA least 6. In the HBR used in the pres- ent study, DS and DS were 1.44 BU RA and 0.032, respectively. The weight- average molecular weight ranged between 10,000 and 30,000 daltons. HBR Induced No Adverse Effects in Healthy Rats—No adverse reac- tions were observed during and after intramyocardial injection of HBR in healthy rats. Within 4 FIGURE 4. HBR increased the number of capillary vessels, Stro-1-positive cells, and perivascular ele- weeks, the animals exhibited no ments. A–C, 4 weeks following myocardial infarction. Transversally cut left ventricular myocardium from HBR- cardiovascular complications, such treated (100 l of HBR solution) hearts showed a reduced scar, compared with PBS-treated animals (A, upper as arrhythmias, pulmonary edema, images, arrows demarcate the infarcted area). Picro-Mallory stains in blue the area of scar and in red the myo- cardium parenchyma. In the border zone of HBR-treated hearts, scar reduction was associated with fewer ascites, or thrombosis. Animal apoptotic cardiomyocytes (A, lower images; scale bars,20 m) and increased number of capillary vessels (B). behavior was normal. Histological vWF expression highlights endothelial cells (arrows) lining the capillary inner wall (B, scale bars, 300 m(upper analysis did not show interstitial images) and 50m(lower images). C, in HBR-treated samples, Stro-1 positive cells (arrows) increased in number and were closely associated with the outer capillary wall, whereas c-Kit-positive cells did not vary significantly, edema and inflammatory infiltrates. compared with untreated animals. Scale bars,20 m. D–F, 24 h following myocardial infarction. D, the number HBR Injection into the Infarcted of Ki-67-positive cells significantly increased in the HBR-treated animals (arrows); scale bars,50 m. E, cells expressing NG2 and PDGF-R (arrows); scale bars,30 m(upper images) and 100 m(lower images). F, VEGF Myocardium Enhanced Myocardial expression (arrows). Scale bars, 100 m(left and middle images) and 50 m(right image). *, significantly differ- Performance—MRI analysis showed ent from PBS-treated hearts, p 0.05 (statistical test: two-tailed, unpaired Student’s t test). IHC, immunohisto- a marked recovery of cardiac per- chemistry. Full-size images of each individual panel are presented in supplemental Fig. 1. formance in infarcted rats treated with HBR, compared with un- assay kit (B-Bridge International)), according to the user treated animals (supplemental Movies 1 and 2). The ejection manual. All samples were assayed at least in duplicate. fraction and cardiac output remarkably recovered in infarcted Western Blotting—Total heart lysate and total lysate of RCm rats receiving HBR, with significant reduction in LV end dia- and Stro-1-positive cells were subjected to SDS-PAGE. Acetyl- stolic volume 4 weeks after MI (Fig. 1A). No major recovery of histone H4 and GAPDH were detected by incubation with a LV global function was observed 1 or 2 weeks following the polyclonal rabbit Lys8-acetyl-histone H4-specific antibody injection of HBR (data not shown). LV end systolic wall thick- (1:1000 dilution; Upstate Biotechnology) and a monoclonal ening, an index of regional contractile function, and LV end rabbit GAPDH-specific antibody (1:1000 dilution; Cell Signal- diastolic thickness, an index of regional mass, were preserved in ing), respectively, followed by incubation with horseradish per- the LV border zone of infarcted HBR-treated hearts (Fig. 1B). oxidase-conjugated antibody to rabbit IgG (Cell Signaling). Conversely, no increase in contractility and mass was observed Antigen-antibody complexes were visualized by using ECL in the remote zone (Fig. 1B). Cardiac MRI was performed to 9954 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule FIGURE 5. HBR affects gene expression and secretion of trophic mediators. A and B, VEGF, kdr, HGF, akt, and pim-1 gene expression was assessed by real-time PCR. RCm (A) or Stro-1 cells (B) were cultured for 24 h (black bar), 3 days (dark gray bar), and 6 days (light gray bar) in the absence or presence of HBR (2 mg/ml). The abundance of each mRNA in untreated cells was defined as 1, and the amounts of VEGF, kdr, HGF, akt, and pim-1 mRNA from HBR-treated cells were plotted relative to that value (mean S.E.; n 6). C–F, time course analysis of VEGF and HGF, respectively, released by RCm (C and E) or Stro-1 cells (D and F) cultured in the absence (black bar) or presence (gray bar) of HBR (mean  S.E.; n  6). *, significantly different from untreated cells (controls), p  0.05 (GraphPad Prism version 4 (available on the World Wide Web), two-tailed, unpaired Student’s t test). G, in vitro capillarogenesis was assessed in HUVECs (a and c) and RAOECs (b and d) exposed to a conditioned medium obtained from RCm or Stro-1 cells cultured for 24 h in the absence (black bar) or presence (gray bar) of HBR. Morphological characteristics of capillary-like networks were evaluated by using NIS-Elements D Nikon software (version 3.06). Data are the mean S.E.; n  3. *, significantly different from HBR; #, significantly different from HBR; p  0.05. MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9955 Endogenous Cell Therapy with a Synthetic Molecule Immunohistochemical analysis of vWF expression revealed that the density of capillary vessels was sig- nificantly increased at the infarct border zone of animals injected with HBR, as compared with in- farcted tissue receiving saline as a placebo (Fig. 4B). Unlike control samples, in the HBR-treated tissue sections, the regeneration of vWF- positive vascular structures was associated with the presence of loose connective tissue in which scattered round and spindle ele- ments were seen embedded; in some samples, this “vascular front” extended from the subepicardial ventricular myocardium and spread into adjacent clusters of “viable” car- diomyocytes. Immunohistochemis- try revealed an increased number of FIGURE 6. Comparative analysis of the effect of HBR, HA, BU, and RA on the secretion of VEGF from perivascular Stro-1-positive cells rat Stro-1 positive cells. Cells were incubated for the indicated times in the absence or presence of 2.0 near newly formed capillaries in mg/ml HBR, 1.5 mg/ml HA, 2.5 mM BU, or 10 M RA alone or exposed to a hydrolyzed (HY) HBR solution (2.0 mg/ml), obtained from a 2-h basic HBR hydrolysis followed by pH neutralization. This procedure has been HBR-treated hearts after 4 weeks, shown to afford a complete release of each single HBR grafted moiety (C. Ventura et al., unpublished significantly exceeding the few Stro- observations). Data are the mean  S.E. of four separate experiments. *, significantly different from HBR alone (GraphPad Prism version 4 (available on the World Wide Web), two-tailed, unpaired Student’s t test). 1-positive cells detected in the non- injected group (Fig. 4C). Interest- ingly, most of the Stro-1-positive detect scar tissue, which appears hyperintense, and for infarct cells lacked staining with vWF (data not shown). On the con- size assessment in LV injected with PBS or HBR (Fig. 2, top). It trary, some Stro-1-positive cells coexpressed cardiac -sarco- is noteworthy that we found a significant reduction in the meric actin (data not shown). On the whole, differently from extension of the delayed enhancement of the LV infarct zone in the Stro-1 expressing elements, the number of c-Kit-positive HBR-injected hearts compared with the untreated group (Fig. cells did not differ significantly among treated and untreated 2, top and bottom). animals. This discrepancy may suggest that, at 4 weeks, Stro-1- HBR Primed Recovery of Cardiac [ F]FDG Uptake—Fig. 3 resident perivascular mesenchymal cells but not c-Kit bone displays a polar map of regional distribution and short and long marrow-derived mononuclear cells were selectively embedded axis images of [ F]FDG uptake in each experimental condition. in the site of tissue repair following HBR injection. The ischemic injury in the anterior wall significantly recovered The presence of Ki-67 was used to determine whether there in infarcted hearts receiving HBR (Fig. 3). Glucose metabolism were cycling cells in the myocardium at the time of sacrifice; after assessed by [ F]FDG uptake, an index of myocardial viability, 24 h, a significant increase in Ki-67 expression was found in the was also preserved in the border and remote regions of examined sections from HBR-injected animals, as compared with infarcted, HBR-treated hearts (Fig. 3). the untreated group (Fig. 4D). In the HBR-treated hearts, the Ki-67 HBR Increased Capillary Density and Induced Recruitment of nuclear antigen was markedly expressed by perivascular spindle- Stro-1-positive Cells—Gross pathologic examination of ische- and round-shaped stromal cells located between cardiomyocytes. mic myocardium after nitro blue tetrazolium staining revealed These cells expressed NG2 and PDGF-R (Fig. 4E), a set of mark- that HBR injection substantially decreased the percentage of ers that have been previously shown to represent a phenotype indi- LV occupied by fibrosis. Picro-Mallory trichrome staining and cator of pericyte/perivascular identity (12). The same cells lacked quantitative analyses showed that the infarct area was signifi- expression of hematopoietic cell markers (data not shown). Other cantly smaller in animals receiving HBR than in the untreated Ki-67-positive cytotypes included endothelial cells and polymor- group (Fig. 4A, upper images). Interestingly, in many of the phonuclear leukocytes. VEGF cytoplasmic expression also signifi- HBR-treated samples, the blue-stained areas, reflecting infarct cantly increased in the cardiomyocytes following HBR injection scarring, were mainly confined to a limited area in the sub- (Fig. 4F). endocardial zone, which also exhibited regions of viable, red- HBR Enhanced the Gene Expression and Secretion of Angio- stained tissue. A terminal deoxynucleotidyltransferase-me- genic, Antiapoptotic, and Antifibrotic Factors in Both Rat Ven- diated dUTP nick end labeling assay kit demonstrated that tricular Cardiomyocytes and Stro-1-positive Stem Cells—The HBR-treated hearts exhibited a decreased number of apo- infarcted myocardium has been shown to release a number of ptotic cardiomyocytes when compared with untreated factors, including the granulocyte colony-stimulating factor hearts (Fig. 4A, lower images). and the granulocyte-macrophage colony-stimulating factor, 9956 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule involved in bone marrow cell mobilization and homing (13, jected to MI in the absence or presence of HBR (data not 14). No significant difference in plasma levels of each factor shown). was observed throughout a 24-h period between rats sub- Real-time PCR analysis revealed that in vitro exposure to HBR of both RCm and rat Stro-1-positive stem cells signifi- cantly enhanced the gene expression of VEGF, kdr (encoding a major VEGF receptor), HGF, akt, and pim-1 (Fig. 5, A and B). In RCm, HBR-mediated stimulation of pim-1 gene expression occurred in a time-dependent, biphasic fashion. Increased pim-1 mRNA was detected within 24 h after HBR stimulation and then declined toward basal values after 3 days. A second elevation in pim-1 gene expression was observed after 6 days of RCm exposure to the mixed ester (Fig. 5A). The HBR treatment enhanced the secretion of VEGF and HGF in the culture medium from both RCm and Stro-1 cells. Time course analyses revealed that the stimulatory action pro- gressively increased during the first 24 h, persisting throughout FIGURE 7. Analysis of VEGF and pim-1 gene transcription in isolated a 6-day period (Fig. 5, C–F). HBR did not appreciably affect the nuclei. A and B, nuclei were isolated from rat Stro-1-positive cells cultured for secretion of VEGF or HGF from HUVECs or RAOECs; nor did 24 h (VEGF gene transcription) or 6 days (pim-1 gene transcription) in the absence or presence of 2.0 mg/ml HBR, respectively. From lanes C–H, nuclei it induce these cells to form capillary-like structures in a semi- were isolated from untreated cells and then directly incubated for 12 h with- solid medium in vitro (data not shown). However, a remarkable out any drug (C) or in the presence of 2.0 mg/ml HBR (D), 1.5 mg/ml HA (E), 2.5 mM BU (F), 10 M RA (G), or with a combination of BU and RA (H). Autoradio- increase in capillarogenesis was observed when either HUVECs graphic exposure was for 2 days on Kodak X-Omat film with an intensifying or RAOECs were cultured with medium obtained from car- screen. The right side of each panel shows the position of radiolabeled DNA diomyocytes or Stro-1-positive cells exposed to the mixed ester markers, showing that the single protected fragments migrated with a molec- ular size comparable with that of VEGF (597 bases), pim-1 (609 bases), or (Fig. 5G). The amount of VEGF released by Stro-1 cells was GAPDH (574 bases) mRNA. Due to the similar size of VEGF-, pim-1-, and slightly increased by BU but not HA alone, being significantly GAPDH-protected fragments, P-labeled nuclear RNA was hybridized sepa- augmented by 10 M all-trans-RA, and further enhanced fol- rately with cRNA probes, and the corresponding hybrids were run onto dif- ferent gels. Autoradiograms are representative of six separate experiments. lowing exposure to hydrolized HBR, resulting in the release of all moieties grafted within the mixed ester (Fig. 6). Nevertheless, under these experimental condi- tions, the yield of growth factor secretion was considerably lower than that detected in HBR-ex- posed cells (Fig. 6). Cumulatively, these findings suggest the activa- tion of prominent paracrine effects by HBR on both cell popu- lations, enhancing the expression of cytokines and genes with a cru- cial role in cell survival and angiogenesis. HBR Acted at the Transcriptional Level—To further dissect the cellu- lar response to HBR, nuclear run-off experiments were designed to assess whether HBR may have affected the rate of gene transcrip- tion and whether, in the affirmative, it may have acted as a unit or after hydrolysis of hyaluronan grafted moieties. Fig. 7 shows that nuclei isolated from HBR-treated Stro-1- positive cells exhibited a consistent FIGURE 8. Time course analysis of the effect of HBR on myocardial histone acetylation. Western blot increase in the transcription rate of analysis of histone H4 acetylation was performed in total tissue extracts from border (BZ) and remote zone (RZ) VEGF and pim-1 genes, as com- of infarcted hearts (MI)6or16h(A)or24h(B) after injection in the absence (PBS) or presence of 100 lofHBR solution (0.2 mg of HBR/100 g of rat weight). GAPDH was used as a loading control. Acetylation level was estimated pared with nuclei isolated from bydensitometricquantification(n 3).*,significantlydifferentfromthesamecontroltimepointwithintheMIBZ(A) untreated cells. In separate experi- or from MI BZ or MI RZ (B). p  0.05 (GraphPad Prism version 4 (available on the World Wide Web), two-tailed, unpaired Student’s t test). All Western blots were performed at least three times with similar results. ments, nuclei were isolated from MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9957 Endogenous Cell Therapy with a Synthetic Molecule 9958 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 13 •MARCH 26, 2010 Endogenous Cell Therapy with a Synthetic Molecule untreated cells and subsequently incubated with HBR or ated with a reverse myocardial remodeling (decrease of fibrosis exposed to HA, BU, or RA administered alone or in combina- and dilatation) and consistent increase in myocardial perform- tion. Although nuclear exposure to HBR or HA failed to trigger ance. A possible explanation may result from in vivo studies a transcriptional response, the incubation with BU or RA demonstrating that HBR-treated hearts had a significant enhanced gene transcription (Fig. 7). The transcription rate was decrease of apoptotic cardiomyocytes, as revealed by a terminal further enhanced when nuclei were exposed to a combination deoxynucleotidyltransferase-mediated dUTP nick end labeling of BU and RA (Fig. 7). assay, as well as from in vitro experiments showing enhanced HBR Increased Histone Acetylation—Western blot analysis gene expression and secretion of VEGF and HGF in both RCm performed in tissue extracts revealed that, consonant with pre- and Stro-1-positive cells exposed to HBR. Growth factor secre- vious observations (15, 16), histone H4 acetylation decreased in tion may have primed a proangiogenic context (18), reinforced the infarcted hearts. Six h after HBR injection, acetyl-histone by the stimulatory effect of HBR on the expression of the kdr H4 signal rose in the border zone, progressively increasing up to gene, encoding a VEGF receptor involved in autocrine/para- 16 h, as compared with the untreated group (Fig. 8A). After crine amplification of VEGF secretion and angiogenic signaling 24 h, histone H4 acetylation increased both in the border and (19, 20). Moreover, HGF gene transfer into the myocardium remote zone of infarcted HBR-injected hearts, as compared improved myocardial function and geometry (21), due to anti- with the non-injected tissue (Fig. 8B). Following HBR treat- fibrotic effects through inhibition of transforming growth fac- ment, a more pronounced signal was detected in the border tor- expression. HBR-mediated recruitment of both myocar- zone, compared with the remote area. Akin to Western blot dial and Stro-1 cells into a proangiogenic paracrine circuitry of studies, immunohistochemical analyses showed an overall cardiac repair is further supported by the finding that (i) an increase in the signal of acetyl-histone H4 in hearts of HBR- increase in capillarogenesis was induced in vitro in both treated animals, as compared with untreated hearts (Fig. 9A). HUVECs and RAOECs with a medium obtained from HBR-ex- The number of cardiomyocyte nuclei stained with acetyl-his- posed cells; (ii) no secretion of VEGF or HGF or in vitro angiogen- tone H4 antibody was significantly increased by HBR treatment esis was found following a direct HUVEC or RAOEC exposure to in the border zone (Fig. 9A). In vitro experiments provided evi- HBR; and (iii) in vivo, a large number of capillary vessels in HBR- dence that acetyl-H4 immunoreactivity was also higher in injected hearts were “decorated” by Stro-1-positive cells, lacking HBR-exposed RCm and Stro-1-positive cells, as compared with the expression of vWF, an endothelial marker. Although some unexposed cells (Fig. 9B). Stro-1-positive cells in HBR-injected hearts expressed heart-spe- cific -sarcomeric actin, these few elements appeared to lack DISCUSSION mature sarcomeric organization, and their role in rescuing The present study shows for the first time that acute myocar- infarcted myocardium remains questionable. dial injection of HBR, a hyaluronan ester previously shown to The observation that exposure of RCm and Stro-1 cells to enhance hMSC-mediated cardiac repair in vivo (3), rescued HBR increased VEGF, HGF, akt, and pim-1 gene expression infarcted rat hearts by increasing vascularization, promoting may have further relevant implications. These genes are deeply cardiomyocyte survival, and restoring normal tissue function. involved in cardioprotective signaling, including antiapoptotic The HBR action involved an early increase in the number of and antifibrotic responses (21–25). Enhanced expression of pericytes, a reserve of progenitor cells that may be integral to both akt and pim-1 promotes cardiomyocyte survival and the origin of MSCs and other related adult stem cells (12), sug- growth in transgenic mice subjected to acute myocardial infarc- gesting a role of perivascular cells in capillary density enhance- tion (26, 27). Here, we show that in RCm pim-1 gene expression ment and tissue repair elicited by the mixed ester. was augmented in a biphasic manner by the addition of HBR. It is now evident that an extremely limited percentage of Biphasic kinetics in pim-1 gene expression have been previ- stem cells will engraft and survive within the recipient myocar- ously demonstrated in other cell types exposed to agents regu- dium following intracoronary infusion or transendocardial lating cell growth and differentiation (28, 29) and appeared to injection (17). Moreover, the use of injectable scaffolds to aug- involve a complex interplay between cell signaling networks, ment stem cell engraftment is not devoid of harmful decrease in cell cycle progression, gene transcription, and/or mRNA stabil- myocardial perfusion. Hence, the development of deliverable ity (29). Cardiac control of Pim-1 gene expression is still poorly molecules promoting paracrine mechanisms of cardiac repair understood, and future work is required to dissect the molecu- and survival of endogenously recruited stem cells, without lar mechanisms underlying Pim-1 regulation by HBR in myo- immediate need of stem cell transplantation, would have remarkable biomedical implications. Here, we show that HBR cardial cells. may fulfill both requirements. Four weeks after HBR injection, HBR may also have increased the survival and rescuing a significant increase in perivascular Stro-1-positive cells could potential of Stro-1-positive cells recruited to the injured myo- be detected within the infarcted hearts. This effect was associ- cardium. In this regard, both VEGF and HGF have been shown FIGURE 9. HBR increased histone acetylation in infarcted myocardium, isolated cardiomyocytes and Stro-1 cells. A, immunohistochemistry of acetyl- histone H4. Acetylation level was estimated in infarcted hearts by intensity quantification and counting of stained cardiomyocyte nuclei 24 h after injection in the absence (PBS) or presence of HBR solution (0.2 mg of HBR/100 g of rat weight). Scale bar,30 m. *, significantly different from PBS-treated, p  0.05. B, Western blot analysis of total cell extracts from RCm and Stro-1-positive cells cultured in the absence (CTR) or presence of HBR (2 mg/ml) (n  3). *, significantly different from the same control time point, p 0.05 (GraphPad Prism version 4 (available on the World Wide Web), two-tailed, unpaired Student’s t test). All Western blots were performed at least three times with similar results. MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9959 Endogenous Cell Therapy with a Synthetic Molecule to promote MSC survival and therapeutic potential (30), practice; and (iii) analytical methodologies for control of good enhancing stem cell-mediated cardioprotection in infarcted manufacturing practice bioprocessing and differentiation effi- hearts (31, 32). Up-regulation of both akt and pim-1 gene ciencies. Therefore, the timing for cell culture and expansion expression in HBR-treated Stro-1 cells is also worthy of consid- within a good manufacturing practice setting will involve a sub- eration, due to the prominent role of these serine/threonine stantial delay (several weeks) in autologous stem cell transplan- kinases in stem cell survival and differentiation (30, 33). MSCs tation with respect to the acute phase of a heart attack. Mean- overexpressing akt prevent remodeling and restore perform- while, the cardiac repair afforded by a myocardial injection of ance of infarcted hearts (1, 34). Moreover, pim-1 has been HBR may serve as first aid to rescue a damaged heart. This shown to be required for endothelial and mural cell differenti- intervention may be followed by delayed transplantation of ation in vitro (35). autologous stem cells, eventually preconditioned ex vivo with It is relevant that a “program” of genes involved in cardiac the same molecule, to enhance the long term potential for car- protection and stem cell survival can be chemically induced by diovascular cell therapy. a synthetic molecule, without the need of viral vector-mediated gene transfer technologies. The molecular dissection of mech- Acknowledgment—We thank Dr. Cristina Nanni (Department of anisms underlying HBR-mediated responses in vitro and in vivo Nuclear Medicine, S. Orsola-Malpighi Hospital, Bologna, Italy) for technical support in the PET studies. remains to be fully elucidated. 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Cell 6, 1049–1058 MARCH 26, 2010• VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9961

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Mar 26, 2010

Keywords: Gene Expression; Glycoconjugate; Growth Factors; Hyaluronate; Ischemia; Angiogenesis; Reperfusion; Survival

References