LiPing Su

Transplantation of Nanoparticle Transfected Skeletal Myoblasts Overexpressing Vascular Endothelial Growth Factor-165 for Cardiac Repair

Background— We investigated the feasibility and efficacy of polyethylenimine (PEI) based human vascular endothelial growth factor-165 (hVEGF165) gene transfer into human skeletal myoblasts (HSM) for cell based delivery to the infarcted myocardium. Methods and Results— Based on optimized transfection procedure using enhanced green fluorescent protein (pEGFP), HSM were transfected with plasmid-hVEGF165 (phVEGF165) carried by PEI (PEI- phVEGF165) nanoparticles. The transfected HSM were characterized for transfection and expression of hVEGF165 in vitro and transplanted into rat heart model of acute myocardial infarction (AMI): group-1=DMEM injection, group-2= HSM transplantation, group-3= PEI-phVEGF165–transfected HSM (PEI-phVEGF165 myoblast) transplantation. A total of 48 rats received cyclosporine injection from 3 days before and until 4 weeks after cell transplantation. Echocardiography was performed to assess the heart function. Animals were sacrificed for molecular and histological studies on the heart tissue at 4 weeks after treatment. Based on optimized transfection conditions, transfected HSM expressed hVEGF165 for 18 days with >90% cell viability in vitro. Apoptotic index was reduced in group-2 and group-3 as compared with group-1. Blood vessel density (×400) by immunostaining for PECAM-1 in group-3 was significantly higher (P=0.043 for both) as compared with group-1 and group-2 at 4 weeks. Regional blood flow (ml/min/g) in the left ventricular anterior wall was higher in group-3 (P=0.043 for both) as compared with group-1 and group-2. Improved ejection fraction was achieved in group-3 (58.44±4.92%) as compared with group-1 (P=0.004). Conclusion— PEI nanoparticle mediated hVEGF165 gene transfer into HSM is feasible and safe. It may serve as a novel and efficient alternative for angiomyogenesis in cardiac repair.

Angiomyogenesis using liposome based vascular endothelial growth factor-165 transfection with skeletal myoblast for cardiac repair

We aim to investigate the feasibility and efficacy of cholesterol (Chol)+DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (hVEGF(165)) gene transfer into human skeletal myoblasts (hSkM) for cardiac repair. The feasibility and efficacy of CD liposome for gene transfer with hSkM was characterized using plasmid carrying enhanced green fluorescent protein (pEGFP). Based on the optimized transfection procedure, hSkM were transfected with CD lipoplexes carrying plasmid-hVEGF(165) (CD-phVEGF(165)). The genetically modified hSkM were transplanted into rat heart model of acute myocardial infarction. Flow cytometry revealed that about 7.99% hSkM could be transfected with pEGFP. Based on the optimized transfection condition, transfected hSkM expressed hVEGF(165) up to day-18 (1.7+/-0.1ng/ml) with peak at day-2 (13.1+/-0.52ng/ml) with >85% cell viability. Animal studies revealed that reduced apoptosis, improved angiogenesis with blood flow in group-3 animals heart were achieved as compared to group-1 and 2. Ejection fraction was best recovered in group-3 animals. The study demonstrates that though gene transfection efficiency using CD liposome mediated hVEGF(165) gene transfer with hSkM was low; hVEGF(165) gene expression efficiency was sufficient to induce neovascularization, improve blood flow and injured heart function.

Nonviral Vector-Based Gene Transfection of Primary Human Skeletal Myoblasts

Low-level transgene efficiency is one of the main obstacles in ex vivo nonviral vector–mediated gene transfer into primary human skeletal myoblasts (hSkMs). We optimized the cholesterol:N-[1-(2, 3-dioleoyloxy)propyl]-N, N, N-trimethylammonium methylsulfate liposome (CD liposome) and 22-kDa polyethylenimine (PEI22)– and 25-kDa polyethylenimine (PEI25)–mediated transfection of primary hSkMs for angiogenic gene delivery. We found that transfection efficiency and cell viability of three nonviral vectors were cell passage dependent: early cell passages of hSkMs had higher transfection efficiencies with poor cell viabilities, whereas later cell passages of hSkMs had lower transfection efficiencies with better cell viabilities. Trypsinization improved the transfection efficiency by 20% to 60% compared with adherent hSkMs. Optimum gene transfection efficiency was found with passage 6 trypsinized hSkMs: transfection efficiency with CD lipoplexes was 6.99 ± 0.13%, PEI22 polyplexes was 18.58 ± 1.57%, and PEI25 polyplexes was 13.32 ± 0.88%. When pEGFP (a plasmid encoding the enhanced green fluorescent protein) was replaced with a vector containing human vascular endothelial growth factor 165 (phVEGF165), the optimized gene transfection conditions resulted in hVEGF165 expression up to Day 18 with a peak level at Day 2 after transfection. This study demonstrated that therapeutic angiogenic gene transfer through CD or PEI is feasible and safe after optimization. It could be a potential strategy for treatment of

Thymosin β4 attenuates early diabetic nephropathy in a mouse model of type 2 diabetes mellitus.

The chronic inflammatory processes and endothelial dysfunction play important roles in the development of diabetic nephropathy (DN); the study aims to evaluate the effect of thymosin &bgr;4 (T&bgr;4), which has apparent anti-inflammatory properties and is capable of improving endothelial dysfunction, in early DN in a mouse model of type 2 diabetes mellitus. KK Cg-Ay/J (KK) mice, aged 12–14 weeks, were divided into the following groups: KK control group that was treated with saline; KK T&bgr;4 group that was treated with T&bgr;4 100 ng/10 g of intraperitoneal injection once a day. Nondiabetic age-matched C57BL mice were used as additional normal control and also treated with T&bgr;4. The urinary albumin/creatinine ratio (ACR), plasma urea nitrogen and creatinine, body weight, fasting blood glucose and 2-hour blood glucose during oral glucose tolerance testing, blood hemoglobin A1c, cholesterol, and triglyceride were determined at baseline time and 12 weeks after T&bgr;4 treatment for phenotypic characterizations. The KK T&bgr;4 group had reduced the mean fasting blood glucose, 2-hour blood glucose during oral glucose tolerance testing, hemoglobin A1c, and triglyceride levels compared with that in the KK control group (P < 0.05). T&bgr;4 treatment markedly reduced ACR (KK T&bgr;4 = 328.54 ± 46.14 mg/g vs. KK control = 540.34 ± 50.31 mg/g, P < 0.05). T&bgr;4 also significantly ameliorated renal pathological changes of KK T&bgr;4 mice as compared with that in KK control mice. T&bgr;4 treatment did not affect glucose homeostasis and urinary ACR and glomeruli of C57BL mice. These data in a novel mouse model of DN suggest that T&bgr;4 may ameliorate renal damage. This peptide may be a novel potential alternative agent for treatment of DN.

Liposome-based vascular endothelial growth factor-165 transfection with skeletal myoblast for treatment of ischaemic limb disease

The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor‐165 (VEGF165) 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‐VEGF165 (CD‐pVEGF165) 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‐pVEGF165 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 VEGF165 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 VEGF165 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.

Skeletal myoblast transplantation on gene expression profiles of insulin signaling pathway and mitochondrial biogenesis and function in skeletal muscle

AIM The study aims to investigate the gene expression profiling of insulin signaling pathway and mitochondrial biogenesis and function in the skeletal muscle of KK mice. METHODS KK mice were divided into the following groups: KK control group, basal medium (M199) only; KK fibroblast group, with human fibroblast transplantation; KK myoblast group, with human skeletal myoblast transplantation. C57BL mice received hSkM transplantation as a normal control. Cells were transplanted into mice hind limb skeletal muscle. All animals were treated with cyclosporine for 6 weeks only. The mice were sacrificed in a fasting state at 12 weeks after treatment. Hind limb skeletal muscle was harvested and used for study of gene expression profiling. RESULTS hSkMs survived extensively in mice skeletal muscle at 12 weeks after cell transplantation. Glucose tolerance test showed a significant decrease of blood glucose in the mice of KK myoblast group compared to the KK control and fibroblast groups. Transcriptional patterns of insulin signaling pathway showed alterations in KK myoblast as compared with KK control group (23 genes), KK fibroblast group (7 genes), and C57BL group (8 genes). Transcriptional patterns of mitochondrial biogenesis and function also had alterations in KK myoblast as compared with KK control group (27 genes), KK fibroblast group (9 genes), and C57BL group (6 genes). CONCLUSIONS These data demonstrated for the first time that hSKM transplantation resulted in a change of gene transcript in multiple genes involved in insulin signaling pathway and mitochondrial biogenesis and function.