过表达糖氧还蛋白-1 的脂肪来源干细胞衍生的外泌体保护内皮细胞并增强 2 型糖尿病小鼠肢体缺血模型中的血管生成。

BACKGROUND: Diabetic limb ischemia is a clinical syndrome and refractory to therapy. Our previous study demonstrated that adipose-derived stem cells (ADSCs) overexpressing glyoxalase-1 (GLO-1) promoted the regeneration of ischemic lower limbs in diabetic mice, but low survival rate, difficulty in differentiation, and tumorigenicity of the transplanted cells restricted its application. Recent studies have found that exosomes secreted by the ADSCs have the advantages of containing parental beneficial factors and exhibiting non-immunogenic, non-tumorigenic, and strong stable characteristics. METHODS: ADSCs overexpressing GLO-1 (G-ADSCs) were established using lentivirus transfection, and exosomes secreted from ADSCs (G-ADSC-Exos) were isolated and characterized to coculture with human umbilical vein endothelial cells (HUVECs). Proliferation, apoptosis, migration, and tube formation of the HUVECs were detected under high-glucose conditions. The G-ADSC-Exos were injected into ischemic hindlimb muscles of type 2 diabetes mellitus (T2DM) mice, and the laser Doppler perfusion index, Masson's staining, immunofluorescence, and immunohistochemistry assays were adopted to assess the treatment efficiency. Moreover, the underlying regulatory mechanisms of the G-ADSC-Exos on the proliferation, migration, angiogenesis, and apoptosis of the HUVECs were explored. RESULTS: The G-ADSC-Exos enhanced the proliferation, migration, tube formation, and anti-apoptosis of the HUVECs in vitro under high-glucose conditions. After in vivo transplantation, the G-ADSC-Exo group showed significantly higher laser Doppler perfusion index, better muscle structural integrity, and higher microvessel's density than the ADSC-Exo and control groups by Masson's staining and immunofluorescence assays. The underlying mechanisms by which the G-ADSC-Exos protected endothelial cells both in vitro and in vivo might be via the activation of eNOS/AKT/ERK/P-38 signaling pathways, inhibition of AP-1/ROS/NLRP3/ASC/Caspase-1/IL-1β, as well as the increased secretion of VEGF, IGF-1, and FGF. CONCLUSION: Exosomes derived from adipose-derived stem cells overexpressing GLO-1 protected the endothelial cells and promoted the angiogenesis in type 2 diabetic mice with limb ischemia, which will be a promising clinical treatment in diabetic lower limb ischemia.

背景：糖尿病肢体缺血是一种临床综合征，对治疗有抗性。我们之前的研究表明，过表达糖氧还蛋白 1（GLO-1）的脂肪干细胞（ADSCs）促进了糖尿病小鼠缺血下肢的再生，但移植细胞的存活率低、分化困难和致瘤性限制了其应用。最近的研究发现，ADSCs 分泌的外泌体具有包含亲代有益因子的优势，并且表现出非免疫原性、非致瘤性和强稳定性的特点。

方法：使用慢病毒转染建立过表达 GLO-1 的 ADSCs（G-ADSCs），并分离和表征 ADSCs 分泌的外泌体（G-ADSC-Exos），然后与人脐静脉内皮细胞（HUVECs）共培养。在高糖条件下检测 HUVECs 的增殖、凋亡、迁移和管形成。将 G-ADSC-Exos 注射到 2 型糖尿病（T2DM）小鼠的缺血后肢肌肉中，采用激光多普勒灌注指数、Masson 染色、免疫荧光和免疫组织化学检测评估治疗效果。此外，还探讨了 G-ADSC-Exos 对 HUVECs 增殖、迁移、血管生成和凋亡的潜在调节机制。

结果：G-ADSC-Exos 在高糖条件下增强了 HUVECs 的增殖、迁移、管形成和抗凋亡作用。体内移植后，与 ADSC-Exo 组和对照组相比，G-ADSC-Exo 组的激光多普勒灌注指数更高，Masson 染色和免疫荧光检测显示肌肉结构完整性更好，微血管密度更高。G-ADSC-Exos 在体外和体内保护内皮细胞的潜在机制可能是通过激活 eNOS/AKT/ERK/P-38 信号通路、抑制 AP-1/ROS/NLRP3/ASC/Caspase-1/IL-1β，以及增加 VEGF、IGF-1 和 FGF 的分泌。

结论：过表达 GLO-1 的脂肪干细胞衍生的外泌体可保护内皮细胞，促进 2 型糖尿病伴肢体缺血小鼠的血管生成，有望成为糖尿病下肢缺血的一种有前途的临床治疗方法。