Surgical Injury and Ischemia Prime the Adipose Stromal Vascular Fraction and Increase Angiogenic Capacity in a Mouse Limb Ischemia Model
Surgical Injury and Ischemia Prime the Adipose Stromal Vascular Fraction and Increase Angiogenic Capacity in a Mouse Limb Ischemia Model
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DOI：
10.1155/2020/7219149
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发表时间：
2020-05-18
影响因子：
4.3
通讯作者：
Yoshida, Ken-ichiro
中科院分区：
文献类型：
--
作者：
Kishimoto, Satoko;Inoue, Ken-ichi;Yoshida, Ken-ichiro
脂肪源性间质血管成分（SVF）是一种有效的自体细胞移植来源。然而，SVF的质量和数量取决于患者的年龄，并发症和其他因素。在这项研究中，我们开发了一种方法，通过外科手术可重复地增加细胞数量并提高脂肪来源的SVFs的质量，我们称之为“伤口修复启动”。“通过切碎脂肪实质（损伤）和结扎皮下脂肪供血动脉（缺血），对BALB/c小鼠腹股沟区的皮下脂肪进行手术处理（引发）。在预充程序后第0、1、3、5或7天分离SVF。通过对差异表达基因进行的微阵列和途径分析来测量引发的SVF的基因表达水平。通过流式细胞术分析致敏的SVFs的细胞组成的变化。将SVFs移植到同系缺血后肢，以测量其血管生成和再生潜力。用激光多普勒血流灌注成像仪测量后肢血流量，并通过缺血组织的CD 31染色定量毛细血管密度。分别通过荧光免疫染色和Western印迹法测定SVFs中HIF-1 α和VEGF-A合成的稳定性。结果，在引发后第7天，单位脂肪重量的SVF数量显著增加。在差异表达的基因中，有先天免疫相关的信号，在引发后第1天和第3天。在致敏的SVF中，在第7天，CD 45阳性血液单核细胞分数减少，而CD 31-CD 45双阴性间充质细胞分数增加。F4/80阳性巨噬细胞分数在引发后第1天和第7天增加。在第1天和第3天，间充质门控CD 34阳性脂肪祖细胞分数和间充质门控CD 140 A阳性/CD 9阳性前脂肪细胞分数连续降低。移植致敏的SVFs导致毛细血管密度增加和血流量增加，改善缺血肢体的再生。HIF-1 α在引发的皮肤脂肪中原位稳定，引发的SVFs的VEGF-A合成在引发后5天达到峰值。因此，伤口修复引发导致SVF数量增加，血管生成潜力增强。
The adipose-derived stromal vascular fraction (SVF) is an effective source for autologous cell transplantation. However, the quality and quantity of SVFs vary depending on the patient's age, complications, and other factors. In this study, we developed a method to reproducibly increase the cell number and improve the quality of adipose-derived SVFs by surgical procedures, which we term "wound repair priming." Subcutaneous fat from the inguinal region of BALB/c mice was surgically processed (primed) by mincing adipose parenchyma (injury) and ligating the subcutaneous fat-feeding artery (ischemia). SVFs were isolated on day 0, 1, 3, 5, or 7 after the priming procedures. Gene expression levels of the primed SVFs were measured via microarray and pathway analyses which were performed for differentially expressed genes. Changes in cellular compositions of primed SVFs were analyzed by flow cytometry. SVFs were transplanted into syngeneic ischemic hindlimbs to measure their angiogenic and regeneration potential. Hindlimb blood flow was measured using a laser Doppler blood perfusion imager, and capillary density was quantified by CD31 staining of ischemic tissues. Stabilization of HIF-1 alpha and VEGF-A synthesis in the SVFs were measured by fluorescent immunostaining and Western blotting, respectively. As a result, the number of SVFs per fat weight was increased significantly on day 7 after priming. Among the differentially expressed genes were innate immunity-related signals on both days 1 and 3 after priming. In primed SVFs, the CD45-positive blood mononuclear cell fraction decreased, and the CD31-CD45-double negative mesenchymal cell fraction increased on day 7. The F4/80-positive macrophage fraction was increased on days 1 and 7 after priming. There was a serial decrease in the mesenchymal-gated CD34-positive adipose progenitor fraction and mesenchymal-gated CD140A-positive/CD9-positive preadipocyte fraction on days 1 and 3. Transplantation of primed SVFs resulted in increased capillary density and augmented blood flow, improving regeneration of the ischemic limbs. HIF-1 alpha was stabilized in the primed cutaneous fat in situ, and VEGF-A synthesis of the primed SVFs was on a peak on 5 days after priming. Wound repair priming thus resulted in SVFs with increased number and augmented angiogenic potential.