Vascular Endothelial Growth Factor– Expressing Mesenchymal Stem Cell Transplantation for the Treatment of Acute Myocardial Infarction ___________________________________ Vascular Endothelial Growth Factor– Expressing Mesenchymal Stem Cell Transplantation for the Treatment of Acute Myocardial Infarction 1. Ryo Matsumoto, 2. Takashi Omura, 3. Minoru Yoshiyama, 4. Tetsuya Hayashi, 5. Sakiko Inamoto, 6. Ki-Ryang Koh, 7. Kensuke Ohta, 8. Yasukatsu Izumi, 9. Yasuhiro Nakamura, 10. Kaname Akioka, 11. Yasushi Kitaura, 12. Kazuhide Takeuchi, 13. Junichi Yoshikawa + Author Affiliations 1. From the Departments of Internal Medicine and Cardiology (R.M., T.O., M.Y., Y.N., K.A., K.T., J.Y.), Clinical Hematology and Clinical Diagnostics (K.-R.K., K.O.), and Pharmacology (Y.I.), Osaka City University Medical School, Osaka, Japan; and the Third Department of Internal Medicine (T.H., S.I., Y.K.), Osaka Medical College, Osaka, Japan. 1. Correspondence to Takashi Omura, Department of Internal Medicine and Cardiology, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan. E-mail [email protected] Abstract Objective— Vascular endothelial growth factor (VEGF) plays an important role in inducing angiogenesis. Mesenchymal stem cells (MSCs) may have potential for differentiation to several types of cells, including myocytes. We hypothesized that transplantation of VEGF-expressing MSCs could effectively treat acute myocardial infarction (MI) by providing enhanced cardioprotection, followed by angiogenic effects in salvaging ischemic myocardium. Methods and Results— The human VEGF165 gene was transfected to cultured MSCs of Lewis rats using an adenoviral vector. Six million VEGF-transfected and LacZ-transfected MSCs (VEGF group), LacZ-transfected MSCs (control group), or serum-free medium only (medium group) were injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. At 1 week after MI, MSCs were detected by X-gal staining in infarcted region. High expression of VEGF was immunostained in the VEGF group. At 28 days after MI, infarct size, left ventricular dimensions, ejection fraction, E wave/A wave ratio and capillary density of the infarcted region were most improved in the VEGF group, compared with the medium group. Immunohistochemically, α-smooth muscle actin–positive cells were most increased in the VEGF group. Conclusions— This combined strategy of cell transplantation with gene therapy could be a useful therapy for the treatment of acute MI. Key Words: angiogenesis gene therapy myocardial infarction stem cell transplantation Cell transplantation has become a promising novel therapy for ischemic heart disease and heart failure. Recent studies have revealed that various types of cells are effective in cell transplantation after myocardial infarction (MI), such as skeletal myoblasts,1,2 smooth muscle cells,3 and bone marrow mononuclear cells.4 Bone marrow mononuclear cells are especially useful because they contain, among various lineage cells, hematopoietic cells and endothelial progenitor cells; therefore they have the ability to induce angiogenesis in ischemic tissue. A reported clinical trial of cell transplantation with skeletal myoblasts and mononuclear bone marrow cells showed that such therapies can have cardioprotective and angiogenic effects after MI.5,6 However, selection of the most appropriate cell types for transplantation is controversial. Mesenchymal stem cells (MSCs) are isolated from bone marrow mononuclear cells and can be expanded ex vivo. Under appropriate culture conditions, MSCs have the potential to terminally differentiate into osteocytes, chondrocytes, adipocytes, tenocytes, myotubes, astrocytes, hematopoietic supporting stroma, and endothelial cells.7 MSCs have also been used in a model of cell transplantation,8,9 showing that these cells could differentiate into myogenic cells. Therefore, MSCs have many characteristics that make them useful for cellular therapy. Vascular endothelial growth factor (VEGF) is a strong therapeutic reagent for treating ischemia by inducing angiogenesis.10 It has been reported that direct intramyocardial gene transfer results in localized enhancement of VEGF levels and successful angiogenesis in animal models of MI.11 Furthermore, recent human trials of angiogenesis gene therapy using naked plasmid DNA or an adenoviral vector coding for VEGF have shown favorable results.12,13 Cell-mediated gene transfer may also be useful for sustained local protein delivery.14 In addition to its angiogenic effect, VEGF may provide myocardial protection against ischemic injury.15,16 Considering these findings, we hypothesized that cell transplantation using VEGF-expressing MSCs could enhance the cardioprotective effects of MSCs, followed by angiogenesis effects in salvaging host myocardium. The results of this study indicate a key role for transplantation of VEGF-expressing MSCs as a strategy for cellular cardiomyoplasty after MI. Methods Cell Isolation and Culture Both donors and recipients were inbred male Lewis rats (SLC, Shizuoka, Japan) weighing 250 to 300 grams. Bone marrow was extruded from tibias and femurs. Bone marrow mononuclear cells were isolated using density gradient centrifugation (Nyco Prep 1.077 Animal; AXIS-SHIELD PoC, AS, Oslo, Norway). Then, the mononuclear cells were cultured in low-glucose Dulbecco’s modified eagle medium containing 10% fetal bovine serum for MSC outgrowth. The nonadherent cells were removed by a medium change at 48 hours and every 4 days thereafter. Adenoviruses and Gene Transfer A cDNA fragment containing the full-length coding regions of human VEGF165 was obtained from mRNA of human umbilical vascular endothelial cells by a RT-PCR method, using a reverse-transcription PCR kit (Toyobo Co, Ltd, Osaka, Japan). The recombinant adenovirus, expressing either β-galactosidase (LacZ) or human VEGF165, was generated using cosmid cassettes and the adenovirus DNA-terminal protein complex method (COS/TPC method), with an Adenovirus Expression Vector Kit (Takara, Osaka, Japan). For adenovirus-mediated gene transfer, MSCs were exposed to adenoviral vectors at a multiplicity of infection (MOI) of ≈10 to ≈20 for 12 hours. An In Vitro Characterization of MSCs For immunofluorescence studies, 15-day cultured MSCs were washed once and fixed with 4% (v/v) paraformaldehyde in phosphate-buffered saline. Primary antibodies were anti–α smooth muscle actin, anti-vimentin (Sigma), and secondary antibodies were anti-gout polyvalentfluorescein isothiocyanate (FITC) conjugate, anti-gout polyvalent-Cy3 conjugate (Sigma), which were incubated for 30 minutes each at room temperature. Two days after LacZ-expressing adenovirus transfection, the cells were fixed with 0.2% glutaraldehyde for 10 minutes, and X-gal staining was performed.17 To evaluate the lineage and surface marker phenotype of the cultured MSCs, cells were detached and incubated in phosphate-buffered saline containing 1% bovine serum albumin with the following fluorescent antibodies: anti–CD45-FITC as a panleukocyte maker; anti-CD11b (Mac- 1)-FITC as monocyte/macrophage maker; anti–HLA-DR-FITC as activated T lymphocytes or natural killer cells maker; anti-CD90 (Thy-1)-FITC as pan T cells or early bone marrow progenitor cells maker; and anti-CD31 (PECAM)-FITC as endothelial maker (Beckman Coulter). Cells were analyzed on a fluorescence-activated cell sorter Calibur Instrument (Becton- Dickinson) with 10 000 events stored. Human VEGF Expression The VEGF-transfected MSCs were metabolically labeled with 35S methionine and 35S cysteine. After 48 hours, both culture medium and cell lysates were prepared for immunoprecipitation with anti-human VEGF antibody (Santa Cruz Biotechnology). After SDS-gel electrophoresis using 15% polyacrylamide gels, the labeled proteins were analyzed using a phosphoimager (FUJIX BAS 2000; Fuji). To confirm the level of secreted VEGFs, the culture medium was collected from MSCs (n=6 in each group) at 1, 3, 5, 7, 10 days after gene transfection. VEGF levels in the medium were quantified using a human VEGF immunoassay kit (R&D Systems Inc). MI and Cell Transplantation Male Lewis rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) and mechanically ventilated. After the heart was exposed through a lateral thoracotomy, myocardial infarction was produced by transient ligation of the left coronary artery for 1 hour.18 Ischemia and reperfusion were confirmed by visible discoloration. Although this procedure was being performed, gene-transferred MSCs were harvested using trypsin and resuspended in serum-free Dulbecco’s modified eagle medium just before grafting to the heart. After left coronary artery reperfusion, the VEGF-transfected and LacZ-transfected MSCs (VEGF group), or LacZtransfected MSCs (control group), or serum-free Dulbecco’s modified eagle medium only (medium group) were injected into the anterior and lateral border zone surrounding the infarct area (total 6.0×106 cells in 0.1 mL) with a 32-gauge needle. Because high expression of LacZ and/or VEGF were preliminarily observed at ≈7 days after MI, and because it was better to evaluate the cardiac remodeling at least 4 weeks after MI, we euthanized these rats at 7 and 28 days after MI. A total of 174 rats were used in the present experiments. The level of circulating human VEGFs were quantified using a human VEGF immunoassay kit (R&D Systems Inc) at 3, 7, and 14 days after MI. mRNA levels of platelet-derived growth factor-B, angiopoietin-1, and transforming growth factor-β in infarct area were quantified by reverse-transcriptase PCR using Ready-To-Go You-Prime First-Strand Beads (Amersham Biosciences) and TaqMan quantitative PCR analysis with the ABI PRISM 7700 Detection System. Doppler Echocardiographic Studies At 1 and 28 days after cell transplantation, transthoracic echocardiograph studies were performed using an echocardiographic system equipped with a 12.0-MHz phased-array transducer (SONOS 5500; Philips Medical System, Best, the Netherlands) as previously described.19 The axial resolution provided by a 12.0-MHz transducer is ≈0.18 mm. Light and Electron Microscopic Study For the light microscopic study, the specimens were fixed in 10% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. The tissue sections were stained with Elastica van Gieson and Mallory-azan. Sections from all slices were projected onto a screen for computerassisted planimetry. The ratio of infarct area to left ventricular circumferences of the endocardium and epicardium was expressed as a percentage to define infarct size. For the transmission electron microscopic study, the specimens were fixed in 4% paraformaldehyde containing 0.25% glutaraldehyde and 4.5% sucrose, and ultrathin sections obtained from the embedded blocks were examined with a Hitachi H-7000 electron microscope. Immunohistochemical Analysis For immunohistochemical examination of VEGF, CD31, and α-SM actin, the heart embedded in OCT compound (Tissue Tek, Miles, Inc) was frozen and cut into 5-μm sections. Antibodies for human VEGF (Santa Cruz Biotechnology), CD31 (DAKO, Kyoto, Japan), and α-smooth muscle actin (Sigma) were used as the primary antibody, and the secondary antibody was peroxidase conjugate (Nichirei, Japan). Peroxidase activity was visualized using 3,3′-diaminobenzidine as chromogen, and nuclei were counterstained with methyl green. The VEGF-positive area was calculated by computer-assisted planimetry. The number of capillaries and α-smooth muscle actin–positive cells were counted in randomly selected 5 high-power fields of each section and averaged. To identify LacZ expression, the sections (5-μm-thick) were stained with X-gal. For immunofluorescent staining, the sections were incubated with a polyclonal rabbit FITCconjugated anti–β-galactosidase antibody (Abcam). Then, anti–α-smooth muscle actin (Sigma) or anti-cardiac troponin T (TnT) (Santa Cruz Biotechnology) followed by anti-goat rhodamineconjugated antibody (Santa Cruz Biotechnology) were applied. The number of LacZ-positive cells and LacZ-positive cells stained by α-smooth muscle actin or TnT were counted in randomly selected 5 high-power fields of each section and averaged. Statistics Results were expressed as mean±SEM. Statistical significance was determined using ANOVA and the Student-Newman-Keuls test. Differences were considered statistically significant at P50), we used adenovirus vectors for gene transfection at 10 to 20 MOI. Approximately 10% of MSCs were stained blue by X-gal staining. Figure 1. A, In vitro characterization of MSCs at 15 days of culture. Immunofluorescence staining of α-SM actin (a) and vimentin (b) were observed in all cells. Approximately 10% of cells were stained by X-gal in LacZ-transfected MSCs (c). B, Flow cytometric analysis of MSCs. MSCs expressed CD90 (Thy-1), and not CD45, CD11b (Mac-1), HLA-DR, or CD31 (PECAM). C, In vitro VEGF expression in MSCs. Metabolically labeled VEGF was observed mainly in the culture medium (upper panel). MSCs were transfected with VEGF165-adenoviral (Ad/VEGF) or LacZ-adenoviral vectors. VEGF levels in the culture medium were markedly higher in VEGF– transfected MSCs than LacZ-transfected MSCs. Five day after transfection, the VEGF level was increased ≈700-fold (lower graph). *P
Posted on: Mon, 16 Sep 2013 10:55:55 +0000
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