Skeletal myoblast based delivery of angiogenic growth factors:a comparison between angiopoietin-1 and VEGF gene delivery for therapeutic angiogenesis in the heart
Lei YeHusnain Kh HaiderShujia JiangRu San TanIn-Chin SongRuowen GePeter K. LawEugene KW SimShujia Jiang Husnain Kh Haider
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Objectives This study investigated the efficacy of human skeletal myoblasts (SkM) mediated either human vascular endothelial growth factor-165 (hVEGF165) or angiopoietin-1 (Ang-1) on vascular development and myocardial regional perfusion. Methods A porcine heart model of chronic infarction was created in 28 female swine by coronary artery ligation. The animals were randomized into: (1) group-1, DMEM injected (n=6), (2) group-2, Ad-null transduced SkM transplanted (n=6), (3) group-3, Ad-hVEGF165 transduced SkM transplanted (n=8), and (4) group-4, Ad-Ang-1 transduced SkM (n=8). Three weeks later, 5 ml DMEM containing 3×108 SkM carrying exogenous genes were intramyocardially injected into 20 sites in left ventricle in groups-2, -3 and -4. Animals in group-1 were injected 5 ml DMEM without cells. Animals were kept on 5 mg/kg cyclosporine per day for 6 weeks. Regional blood flow was measured using fluorescent microspheres. The heart was explanted at 2, 6 and 12 weeks after transplantation for histological studies. Results Histological examination showed survival of lac-z expressing myoblasts in host tissue. Capillary density based on Von Willebrand factor-VIII (vWF-VIII) at low power field (×100) was 57.13±11.85 in group-3 at 6 weeks and declined to 32.1±5.21 at 12 weeks, while it was 39.9±10.26 at 6 weeks and increased to 45.14±6.54 at 12 weeks in group-4. The mature blood vessel index was highest in group- 4 at 6 and 12 weeks after transplantation. The regional blood flow in the center and peri-infarct area was significantly increased in animals of groups-3 and -4. Conclusions SkM carrying either hVEGF165 or Ang-1 induced neovascularization with increased blood flow. Ang-1 overexpression resulted in mature and stable blood vessel formation and may be a more potent arteriogenic inducer for neovascularization.Keywords:
Therapeutic angiogenesis
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To investigate the possible mechanisms of improving ischemic myocardial function by transplantation of bone marrow cells in a rat ischemic heart model.Myocardial infarction was induced in inbred Lewis rats by left anterior descending artery ligation. Bone marrow cells were injected into an ischemic zone (BMI group). On days 1, 3, 7, 14 and 28 post-transplantation, the expressions of vascular endothelial growth factor (VEGF) and its receptor (Flk-1) and the differentiation of transplanted cells were determined by immunofluorescence and RT-PCR. The number of vessels was examined by immunohistochemistry. The cardiac function was evaluated by hemodynamics.Immunofluorescence microscopy of hearts from BMI group revealed that expressions of VEGF and Flk-1 were promoted within cardiomyocytes in the infarction zone, the peri-infarct zone and in some transplanted bone marrow cells. RT-PCR also showed that mRNA expression levels of VEGF and Flk-1 in BMI group were significantly higher than those of the control group, reached the highest level on days 3 and 14 post-transplantation, and then gradually declined. On days 7, 14 and 28, the vessel count showed the number of blood vessels in the BMI group was greater than that of the control group at the same time, and the cardiac function in the BMI group was improved more significantly than that of the control group. After day 14, the specific markers for myocardium or vascular endothelial cells were detected in the transplanted bone marrow cells.BMI improved the acute ischemic cardiac function by both upregulating the expressions of VEGF and Flk-1 in transplanted BMCs and recipient endogenous cardiomyocytes that enhanced angiogenesis.
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Aim To treat myocardial infarction(MI) with bone marrow mesenchymal stem cell(MSC) transplantation combined with vascular endothelial growth factor(VEGF) gene therapy in rabbits and to study its mechanisms. Methods Forty-eight rabbits were randomly divided into MI group(n=12),MSC group(n=12),VEGF group(n=12),MSC+VEGF group(M+V group,n=12).Rabbit myocardial infarction models were founded by the ligation of left anterior descending artery.10~7 MSC were injected into the infarct-zone in four sites 2 weeks later in MSC and M+V group.phVEGF gene were injected in infarct-zone in VEGF group and MSC transfected with phVEGF gene were injected in M+V group.Heart function including LVEDP,LVSP,LVDP,-dp/dtmax,+dp/dtmax,were measured in vivo.The hearts were harvested at 4 weeks after transplantation and sectioned for HE stain,immunohistochemical stain of BrdU and Ⅷ factor antigen. Results The left ventricular hemodynamics parameters showed that heart function were improved more in M+V group than MSC group,MI group and VEGF group. The numbers of BrdU posivtive cell in M+V group(61.24±8.51)were more than in MSC group(44.21±7.68,P0.01).The numbers of vessels in infarcted zone were more in M+V group(48.75±7.96) than in MSC group(33.08±6.12,P0.01),VEGF group(29.98±8.04,P0.01)and MI group(18.32±3.88,P0.01). Conclusions VEGF gene transfected MSC transplantation could improve heart function after myocardial infarction,and they were more effective than sole MSC transplantation.Keeping more MSC survival and ameliorating the blood supply of infarct-zone might be involved in the mechanisms.
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To determine the improvements of post-infarction heart function after transplantation of autologous skeletal myoblasts transfected with VEGF165 in rabbits.Myocardium infarction was induced in rabbits by left anterior descending coronary artery ligation. At 2 weeks, 1.75×10(7) autologous skeletal myoblasts transfected with pcDNA3.1-VEGF165 were infused into the region of MI via direct intramuscular injection; pcDNA3.1 served as a control.The DAPI-labeled and Desmin-positive immunostained skeletal myofibers were found throughout the infracted areas and border zones, and the density of blood capillary in the MI region transplanted by myoblasts with VEGF165 was increased (measured 4 weeks later and compared with controls). Heart function was examined by the Buxco system and demonstrated that maximum dp/dt [(1607.23±102.67) mmHg/s vs (1217.77±89.91) mmHg/s] and minimum dp/dt [(-1535.09 ± 81.34) mmHg/s vs (1174.58 ± 91.5) mmHg/s] were improved in the heart transplanted with the transfected myoblasts(P<0.05).Autologous skeletal myoblasts transfected with VEGF165 could ameliorate the blood supply in the MI region, and aid recovery of heart function more quickly in post-infarction hearts. This suggests an effective treatment for myocardium infarction.
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The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (VEGF(165)) gene transfer into skeletal myoblasts (SkMs) for treatment of acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid-VEGF(165) (CD-pVEGF(165)) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non-transfected SkM (group 2) or CD-pVEGF(165) transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF(165) up to day 18 with peak at day 2. SkMs were observed in all cell-transplanted groups, as visualized with 6-diamidino-2-phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 +/- 2.0) compared with group 2 (21.6 +/- 1.1%, P < 0.001) and group 1 (16.9 +/- 1.1%, P < 0.001). Immunostaining for CD31 showed significantly increased capillary density in group 3 (14.88 +/- 0.9) compared with group 2 (8.5 +/- 0.49, P < 0.001) and group 1 (5.69 +/- 0.3, P < 0.001). Improved blood flow (ml/min./g) was achieved in animal group 3 (0.173 +/- 0.04) as compared with animal group 2 (0.122 +/- 0.016; P= 0.047) and group 1 (0.062 +/- 0.012; P < 0.001). In conclusion, CD liposome mediated VEGF(165) gene transfer with SkMs effectively induced neovascularization in the ischaemic hind limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.
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Objective: To evaluate the protective effect of bone marrow stromal cells (BMSCs) transferred by Ad·ANG ex vivo on ischemic myocardium. Methods: ELISA method was used to assay the expression and secretion of angiogenin (ANG) after Ad·ANG transfection of BMSCs ex vivo. Then BMSCs with Ad·ANG were transplanted into ischemic myocardium of isogenic Lewis rats. 4 weeks later, the parameters of heart function, such as EF and EDLV, were examined by echocardiography. Survival and differentiation of transplanted BMSCs and angiogenesis were appraised by histology and transmission electron micrography. Results: ANG was found in both lysate and culture medium after transfection of BMSCs. A maximum expression of ANG was observed at 4-7 days after transfection and could still be assayed 15 days later. 4 weeks later after transplantation in the BMSCs with Ad·ANG group, heart function improved better than the single BMSCs group(P0.05), Ad·ANG group (P0.01) and blank control group (P0.01). Angiogenesis in test group was more obvious than three control groups(P0.01). Conclusion: Satisfactory expression of ANG can be obtained Ad·ANG was transferred into BMSCs ex vivo. After transplantation of BMSCs with Ad·ANG into ischemic myocardium, improvement of heart function and angiogenesis are more obvious than single BMSCs transplantation and Ad·ANG injection.
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Objectives This study investigated the efficacy of human skeletal myoblasts (SkM) mediated either human vascular endothelial growth factor-165 (hVEGF165) or angiopoietin-1 (Ang-1) on vascular development and myocardial regional perfusion. Methods A porcine heart model of chronic infarction was created in 28 female swine by coronary artery ligation. The animals were randomized into:(1) group-1, DMEM injected (n=6), (2) group-2, Ad-null transduced SkM transplanted (n=6), (3) group-3, Ad-hVEGF165 transduced SkM transplanted (n=8), and (4) group-4, Ad-Ang-1 transduced SkM (n=8). Three weeks later, 5 ml DMEM containing 3× 108 SkM carrying exogenous genes were intramyocardially injected into 20 sites in left ventricle in groups-2, -3 and -4. Animals in group-1 were injected 5 ml DMEM without cells. Animals were kept on 5 mg/kg cyclosporine per day for 6 weeks. Regional blood flow was measured using fluorescent microspheres. The heart was explanted at 2, 6 and 12 weeks after transplantation for histological studies. Results Histological examination showed survival of lac-z expressing myoblasts in host tissue. Capillary density based on Von Willebrand factor-Ⅷ (vWF-Ⅷ) at low power field (× 100) was 57.13+11.85 in group-3 at 6 weeks and declined to 32.1±5.21 at 12 weeks, while it was 39.9±10.26 at 6 weeks and increased to 45.14±6.54 at 12 weeks in group-4. The mature blood vessel index was highest in group4 at 6 and 12 weeks after transplantation. The regional blood flow in the center and peri-infarct area was significantly increased in animals of groups-3 and -4. Conclusions SkM carrying either hVEGF165 or Ang- 1 induced neovascularization with increased blood flow. Ang- 1 overexpression resulted in mature and stable blood vessel formation and may be a more potent arteriogenic inducer for neovascularization.(J Geriatr Cardiol 2006;3:152-60.)
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Abstract Stem cells transplanted to an injured heart affect the host myocardium indirectly. The cytokine hepatocyte growth factor (HGF) may play a key role in this paracrine activity. We hypothesized that HGF‐overexpressing stem cells would restore cardiac function after myocardial infarction (MI). Because there is a high rate of cell death when injecting the cells intramyocardially, we used scaffold‐based cell transfer. Skeletal myoblasts (SkMs) were isolated and expanded from newborn Lewis rats. Cells were transfected with pcDNA3‐huHGF and seeded on polyurethane (PU) scaffolds or diluted in medium for cell injection. The seeded scaffolds were transplanted in rats two weeks after MI (group: PU‐HGF‐SkM) or the infection solution was intramyocardially injected (group: Inj‐HGF‐SkM). Two groups (Inj‐SkM and PU‐SkM) have been prepared with untransfected cells and sham group without any cell therapy served as control ( n = 10 each group). At the beginning of treatment (baseline) and six weeks later, hemodynamic parameters were assessed. At the end of the study, histological analysis was employed. In sham animals we detected a decrease in systolic and diastolic function during the observation time. Treatment with untransfected myoblasts did not lead to any significant changes in hemodynamic parameters between the intervention and six weeks later. In group PU‐HGF‐SkM, systolic parameters like dP/dt(max), dP/dt(min) and isovolumic contraction improved significantly from baseline to study end. Some diastolic parameters were inferior as compared to baseline (SB‐Ked, pressure half time [PHT], Tau). In group Inj‐HGF‐SkM, only PHT was impaired as compared to preinterventional values. Histological analysis showed significantly more capillaries in the infarction border zone in groups PU‐HGF‐SkM than in sham and Inj‐SkM group. The infarction size was not affected by the therapy. Transplanting HGF‐transfected myoblasts after MI can limit the development of ventricular dysfunction. Scaffold‐based therapy in combination with gene therapy accelerates this capacity. This hemodynamic amelioration is accompanied by neovascularization, but not by smaller infarction sizes.
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