BACKGROUND Critical limb ischemia (CLI) constitutes the most aggressive form of peripheral arterial occlusive disease, characterized by the blockade of arteries supplying blood to the lower extremities, significantly diminishing oxygen and nutrient supply. CLI patients usually undergo amputation of fingers, feet, or extremities, with a high risk of mortality due to associated comorbidities. Circulating angiogenic cells (CACs), also known as early endothelial progenitor cells, constitute promising candidates for cell therapy in CLI due to their assigned vascular regenerative properties. Preclinical and clinical assays with CACs have shown promising results. A better understanding of how these cells participate in vascular regeneration would significantly help to potentiate their role in revascularization. Herein, we analyzed the initial molecular mechanisms triggered by human CACs after being administered to a murine model of CLI, in order to understand how these cells promote angiogenesis within the ischemic tissues. METHODS Balb-c nude mice (n:24) were distributed in four different groups: healthy controls (C, n:4), shams (SH, n:4), and ischemic mice (after femoral ligation) that received either 50 μl physiological serum (SC, n:8) or 5 × 105 human CACs (SE, n:8). Ischemic mice were sacrificed on days 2 and 4 (n:4/group/day), and immunohistochemistry assays and qPCR amplification of Alu-human-specific sequences were carried out for cell detection and vascular density measurements. Additionally, a label-free MS-based quantitative approach was performed to identify protein changes related. RESULTS Administration of CACs induced in the ischemic tissues an increase in the number of blood vessels as well as the diameter size compared to ischemic, non-treated mice, although the number of CACs decreased within time. The initial protein changes taking place in response to ischemia and more importantly, right after administration of CACs to CLI mice, are shown. CONCLUSIONS Our results indicate that CACs migrate to the injured area; moreover, they trigger protein changes correlated with cell migration, cell death, angiogenesis, and arteriogenesis in the host. These changes indicate that CACs promote from the beginning an increase in the number of vessels as well as the development of an appropriate vascular network.

中文翻译：

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识别在严重肢体缺血中对循环血管生成细胞介导的治疗反应的初始分子变化。

背景技术严重肢体缺血（CLI）构成外周动脉闭塞性疾病的最激进形式，其特征是阻塞了向下肢供应血液的动脉，显着减少了氧气和营养供应。CLI患者通常会进行手指，脚或四肢的截肢，由于相关的合并症，死亡风险很高。循环血管生成细胞（CAC），也称为早期内皮祖细胞，由于具有指定的血管再生特性，因此有望在CLI中进行细胞治疗。用CAC进行的临床前和临床分析已显示出令人鼓舞的结果。对这些细胞如何参与血管再生的更好理解将大大有助于增强其在血运重建中的作用。在这里 为了了解这些细胞如何促进缺血组织中的血管生成，我们分析了将人类CACs给药至CLI鼠模型后触发的初始分子机制。方法将Balb-c裸鼠（n：24）分为四个不同的组：健康对照组（C，n：4），sha鼠（SH，n：4）和缺血小鼠（股骨结扎后）分别接受50μl生理血清（SC，n：8）或5×105人CAC（SE，n：8）。在第2天和第4天（n：4 /组/天）处死缺血小鼠，并进行Alu-人特异性序列的免疫组织化学测定和qPCR扩增以进行细胞检测和血管密度测量。另外，进行了无标签的基于MS的定量方法以鉴定相关的蛋白质变化。结果与缺血未治疗的小鼠相比，在缺血组织中诱导的CAC的给药增加了血管数量以及直径大小，尽管CAC的数量在一定时间内减少了。最初的蛋白质变化是响应于缺血而发生的，更重要的是，显示了在将CLI给予CAC后立即发生的变化。结论我们的结果表明CAC迁移到了受伤区域。此外，它们触发与宿主细胞迁移，细胞死亡，血管生成和动脉生成相关的蛋白质变化。这些变化表明，CAC从一开始就促进了血管数量的增加以及适当血管网络的发展。尽管CAC的数量在一段时间内减少了。最初的蛋白质变化是响应于局部缺血而发生的，更重要的是，显示了在对CLI小鼠施用CAC后立即发生的变化。结论我们的结果表明CAC迁移到了受伤区域。此外，它们触发与宿主细胞迁移，细胞死亡，血管生成和动脉生成相关的蛋白质变化。这些变化表明，CAC从一开始就促进了血管数量的增加以及适当血管网络的发展。尽管CAC的数量在一段时间内减少了。最初的蛋白质变化是响应于局部缺血而发生的，更重要的是，显示了在对CLI小鼠施用CAC后立即发生的变化。结论我们的结果表明CAC迁移到了受伤区域。此外，它们触发与宿主细胞迁移，细胞死亡，血管生成和动脉生成相关的蛋白质变化。这些变化表明，CAC从一开始就促进了血管数量的增加以及适当血管网络的发展。它们触发与宿主细胞迁移，细胞死亡，血管生成和动脉生成相关的蛋白质变化。这些变化表明，CAC从一开始就促进了血管数量的增加以及适当血管网络的发展。它们触发与宿主细胞迁移，细胞死亡，血管生成和动脉生成相关的蛋白质变化。这些变化表明，CAC从一开始就促进了血管数量的增加以及适当血管网络的发展。