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      Effects of Myocardial Transplantation of Marrow Mesenchymal Stem Cells Transfected with Vascular Endothelial Growth Factor for the Improvement of Heart Function and Angiogenesis after Myocardial Infarction

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          Abstract

          Objective: To establish the transfection method of vascular endothelial growth factor (VEGF) gene into mesenchymal stem cells (MSCs), to investigate the effect of this gene-transfected MSCs for heart function restoration and angiogenesis after myocardial infarction, and to compare the therapeutic differences among cell therapy, gene therapy, and combined therapy. Methods: Ischemic heart models were constructed in inbred Wistar rats by ligation of the left anterior descending coronary artery. MSCs of Wistar rats were isolated by density gradient centrifugation and purified on the basis of their ability to adhere to plastic, and identified by checking the surface markers and their differentiation capacity, and then followed by transfection of pcDNA<sub>3.1</sub>-hVEGF<sub>165</sub> using the liposome-mediated method. The expression of hVEGF<sub>165</sub> in the transfected cells was detected by Enzyme-Linked Immunosorbent Assay, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western Blot Analysis. The ligated animals were randomly divided into four groups (12 in each) and, after 2 weeks, were injected at the heart infarct zone with hVEGF<sub>165</sub>-transfected MSCs (Combo group), MSCs (Cell group), liposome-hVEGF gene plasmid (Gene group), or medium (Control group). And other six ligated rats (without any injection) were used as Model-assessment group for the baseline heart infarcted size evaluation, and other 12 non-ligated rats (Non-ischemic group) were used as the normal control. Four weeks after the injection, the rats’ cardiac function was measured by the Buxco system. Brdu and Troponin-T double labeling and factor VIII were identified by immunohistochemical staining to demonstrate the survival and differentiation of engrafted cells or to evaluate the angiogenesis in the injured heart area; heart infarcted size was calculated by Evan’s blue staining. VEGF expression was evaluated by RT-PCR. Results: MSCs can be successfully isolated and cultured by density gradient centrifugation followed by adherence-separation. The cultured MSCs were CD34–, CD45–, CD44+ and SH+. They can differentiate into osteoblasts and adipocytes successfully. The expression of hVEGF<sub>165</sub> in the transfected MSCs was demonstrated with Enzyme-Linked Immunosorbent Assay, RT-PCR and Western Blot Assay. Four weeks after the cells were transplanted, among all groups but the Non-ischemic group, the Combo group had the smallest heart infarcted size and the best heart function. The capillary density of the Combo group was significantly greater than those of both Cell and Control groups. The heart infarcted size, heart function and capillary density of both Cell and Gene groups were similar with each other and smaller, better and greater than those of the Control group, respectively. Brdu and Troponin-T double staining detected a varied increase in the number of survived cardiomyoctyes at the heart infarcted area, some of which were double stain positive. RT-PCR showed that the hVEGF<sub>165</sub> gene was expressed in the Combo and Gene groups, and that the former was higher than the latter. Conclusions: Eukaryotic expression vector pcDNA<sub>3.1</sub>-hVEGF<sub>165</sub> can effectively be expressed in MSCs. Transplantation of VEGF gene-transfected MSCs can bring better improvement in myocardial perfusion and in restoration of heart function than either cellular or gene therapy alone.

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          Most cited references 14

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          Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms.

          We recently demonstrated that marrow stromal cells (MSCs) augment collateral remodeling through release of several cytokines such as VEGF and bFGF rather than via cell incorporation into new or remodeling vessels. The present study was designed to characterize the full spectrum of cytokine genes expressed by MSCs and to further examine the role of paracrine mechanisms that underpin their therapeutic potential. Normal human MSCs were cultured under normoxic or hypoxic conditions for 72 hours. The gene expression profile of the cells was determined using Affymetrix GeneChips representing 12 000 genes. A wide array of arteriogenic cytokine genes were expressed at baseline, and several were induced >1.5-fold by hypoxic stress. The gene array data were confirmed using ELISA assays and immunoblotting of the MSC conditioned media (MSC(CM)). MSC(CM) promoted in vitro proliferation and migration of endothelial cells in a dose-dependent manner; anti-VEGF and anti-FGF antibodies only partially attenuated these effects. Similarly, MSC(CM) promoted smooth muscle cell proliferation and migration in a dose-dependent manner. Using a murine hindlimb ischemia model, murine MSC(CM) enhanced collateral flow recovery and remodeling, improved limb function, reduced the incidence of autoamputation, and attenuated muscle atrophy compared with control media. These data indicate that paracrine signaling is an important mediator of bone marrow cell therapy in tissue ischemia, and that cell incorporation into vessels is not a prerequisite for their effects.
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            Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines.

            Bone marrow implantation (BMI) was shown to enhance angiogenesis in a rat ischemic heart model. This preclinical study using a swine model was designed to test the safety and therapeutic effectiveness of BMI. BM-derived mononuclear cells (BM-MNCs) were injected into a zone made ischemic by coronary artery ligation. Three weeks after BMI, regional blood flow and capillary densities were significantly higher (4.6- and 2.8-fold, respectively), and cardiac function was improved. Angiography revealed that there was a marked increase (5.7-fold) in number of visible collateral vessels. Implantation of porcine coronary microvascular endothelial cells (CMECs) did not cause any significant increase in capillary densities. Labeled BM-MNCs were incorporated into approximately 31% of neocapillaries and corresponded to approximately 8.7% of macrophages but did not actively survive as myoblasts or fibroblasts. There was no bone formation by osteoblasts or malignant ventricular arrhythmia. Time-dependent changes in plasma levels for cardiac enzymes (troponin I and creatine kinase-MB) did not differ between the BMI, CMEC, and medium-alone implantation groups. BM-MNCs contained 16% of endothelial-lineage cells and expressed basic fibroblast growth factor>vascular endothelial growth factor>angiopoietin 1 mRNAs, and their cardiac levels were significantly upregulated by BMI. Cardiac interleukin-1beta and tumor necrosis factor-alpha mRNA expression were also induced by BMI but not by CMEC implantation. BM-MNCs were actively differentiated to endothelial cells in vitro and formed network structure with human umbilical vein endothelial cells. BMI may constitute a novel safety strategy for achieving optimal therapeutic angiogenesis by the natural ability of the BM cells to secrete potent angiogenic ligands and cytokines as well as to be incorporated into foci of neovascularization.
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              Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT).

              Catheter-based intracoronary vascular endothelial growth factor (VEGF) gene transfer is a potential treatment for coronary heart disease. However, only limited data are available about local VEGF gene transfer given during angioplasty (PTCA) and stenting. Patients with coronary heart disease (n=103; Canadian Cardiovascular Society class II to III; mean age, 58+/-6 years) were recruited in this randomized, placebo-controlled, double-blind phase II study. PTCA was performed with standard methods, followed by gene transfer with a perfusion-infusion catheter. Ninety percent of the patients were given stents; 37 patients received VEGF adenovirus (VEGF-Adv, 2x10(10) pfu), 28 patients received VEGF plasmid liposome (VEGF-P/L; 2000 microg of DNA with 2000 microL of DOTMA:DOPE [1:1 wt/wt]), and 38 control patients received Ringer's lactate. Follow-up time was 6 months. Gene transfer to coronary arteries was feasible and well tolerated. The overall clinical restenosis rate was 6%. In quantitative coronary angiography analysis, the minimal lumen diameter and percent of diameter stenosis did not significantly differ between the study groups. However, myocardial perfusion showed a significant improvement in the VEGF-Adv-treated patients after the 6-month follow-up. Some inflammatory responses were transiently present in the VEGF-Adv group, but no increases were detected in the incidences of serious adverse events in any of the study groups. Gene transfer with VEGF-Adv or VEGF-P/L during PTCA and stenting shows that (1) intracoronary gene transfer can be performed safely (no major gene transfer-related adverse effects were detected), (2) no differences in clinical restenosis rate or minimal lumen diameter were present after the 6-month follow-up, and (3) a significant increase was detected in myocardial perfusion in the VEGF-Adv-treated patients.
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                Author and article information

                Journal
                CRD
                Cardiology
                10.1159/issn.0008-6312
                Cardiology
                S. Karger AG
                0008-6312
                1421-9751
                2007
                December 2006
                31 May 2006
                : 107
                : 1
                : 17-29
                Affiliations
                aDepartment of Cardiothoracic Surgery, Second Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China; bSection of Vascular Surgery, Department of Surgery, University of Chicago, Chicago, Ill., USA
                Article
                93609 Cardiology 2007;107:17–29
                10.1159/000093609
                16741354
                © 2007 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 13, References: 26, Pages: 13
                Categories
                Original Research

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