Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165.
Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165.
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DOI：
10.1186/s13287-015-0199-6
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发表时间：
2015-10-26
影响因子：
7.5
通讯作者：
Parfyonova YV
中科院分区：
文献类型：
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作者：
Makarevich PI;Boldyreva MA;Gluhanyuk EV;Efimenko AY;Dergilev KV;Shevchenko EK;Sharonov GV;Gallinger JO;Rodina PA;Sarkisyan SS;Hu YC;Parfyonova YV
利用脂肪来源的基质细胞(ADSC)进行细胞治疗是促进血管生成和再生的一种新兴方法。给药技术是至关重要的，其中最小的结构-细胞片(CS)具有一定的优势。CS的输送允许细胞与基质蛋白一起移植，以促进植入。通过病毒转导表达促血管生成因子也可以增加细胞的治疗潜力。在本工作中，我们报道了转导表达人血管内皮生长因子165a/a亚型(VEGF165)的小鼠ADSC的CS的治疗效果，该亚型在肢体缺血模型中显示出恢复血流和保护组织的效力。从C57雄性小鼠分离的小鼠ADSC(MADSC)用于CS形成的扩增，每个CS有106个细胞。通过杆状病毒(BV)系统转导表达人VEGF165。将CS移植到手术诱发肢体缺血的小鼠皮下，然后进行激光多普勒血流灌注测量。在终点处死动物，评估骨骼肌的坏死和血管密度；对CS和下层肌肉进行细胞凋亡、增殖、单核细胞和血管染色。利用BV系统和丁酸钠处理，我们在mADSC中表达了人VEGF165(表达的VEGF165可达 ≈ 25-27 ng/ml/10~5细胞)，并优化了条件以确保转导后的细胞活力。术后2周，与未经处理的动物相比，植入模拟转导CS的动物肢体血流灌注明显改善，毛细血管密度增加，坏死灶减少。在移植表达VEGF165的CS后，观察到血流和血管生成的额外改善，表明转基因构建物的治疗潜力增强。此外，我们发现作为CS的mADSC在灌流和血管生成方面优于同等剂量的悬浮细胞。对提取的CS进行组织学分析，发现移植的mADSC有有限的增殖和约10%的凋亡率。BV转导的CS和对照CS均可见明显的血管化和单核细胞浸润，提示移植物与宿主之间存在相互作用。CS皮下移植ADSC可有效促进肢体缺血后的血管生成和组织保护，转导表达VEGF165的ADSC有可能提高疗效。本文的在线版本(doi：10.1186/s13287-015-0199-6)包含补充材料，授权用户可以使用。
Cell therapy using adipose-derived stromal cells (ADSC) is an intensively developing approach to promote angiogenesis and regeneration. Administration technique is crucial and among others minimal constructs - cell sheets (CS) have certain advantages. Delivery of CS allows transplantation of cells along with matrix proteins to facilitate engraftment. Cells’ therapeutic potential can be also increased by expression of proangiogenic factors by viral transduction. In this work we report on therapeutic efficacy of CS from mouse ADSC transduced to express human vascular endothelial growth factor 165 a/a isoform (VEGF165), which showed potency to restore perfusion and protect tissue in a model of limb ischemia. Mouse ADSC (mADSC) isolated from C57 male mice were expanded for CS formation (106cells per CS). Constructs were transduced to express human VEGF165 by baculoviral (BV) system. CS were transplanted subcutaneously to mice with surgically induced limb ischemia and followed by laser Doppler perfusion measurements. At endpoint animals were sacrificed and skeletal muscle was evaluated for necrosis and vessel density; CS with underlying muscle was stained for apoptosis, proliferation, monocytes and blood vessels. Using BV system and sodium butyrate treatment we expressed human VEGF165 in mADSC (production of VEGF165 reached ≈ 25-27 ng/ml/105 cells) and optimized conditions to ensure cells’ viability after transduction. Implantation of mock-transduced CS resulted in significant improvement of limb perfusion, increased capillary density and necrosis reduction at 2 weeks post-surgery compared to untreated animals. Additional improvement of blood flow and angiogenesis was observed after transplantation of VEGF165-expressing CS indicating enhanced therapeutic potential of genetically modified constructs. Moreover, we found delivery of mADSC as CS to be superior to equivalent dose of suspended cells in terms of perfusion and angiogenesis. Histology analysis of extracted CS detected limited proliferation and approximately 10 % prevalence of apoptosis in transplanted mADSC. Significant vascularization of CS and infiltration by monocytes were found in both – BV-transduced and control CS indicating graft and host interaction after transplantation. Delivery of ADSC by subcutaneous transplantation of CS is effective for stimulation of angiogenesis and tissue protection in limb ischemia with a potential for efficacy improvement by BV transduction to express VEGF165. The online version of this article (doi:10.1186/s13287-015-0199-6) contains supplementary material, which is available to authorized users.